Research Article Toxicity Profile of a Nutraceutical...

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Research Article Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel Perna viridis Kajal Chakraborty, Deepu Joseph, and Selsa J. Chakkalakal Marine Biotechnology Division, Central Marine Fisheries Research Institute, Ernakulam North P.O., PB No. 1603, Cochin, Kerala 682018, India Correspondence should be addressed to Kajal Chakraborty; kajal [email protected] Received 26 February 2014; Accepted 12 May 2014; Published 9 June 2014 Academic Editor: Sanyog Jain Copyright © 2014 Kajal Chakraborty et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e short-term (acute) and long-term (subchronic) toxicity profile, mean lethal dose 50 (LD 50 ), and no-observed-adverse-effect level (NOAEL) of a nutraceutical formulation developed from green mussel Perna viridis, which showed in vitro and in vivo anti- inflammatory properties, were evaluated in the present study. e formulation was administered to the male and female Wistar rats at graded doses (0.5, 1.0, and 2.5 g/kg body weight) for two weeks of acute toxicity study and 0.5, 1.0, and 2.0 g/kg body weight for 90 days in subchronic toxicity study. e LD 50 , variations in clinical signs, changes in body weight, body weight, food/water consumption, organ weight (liver, kidney, spleen, and brain), hematology, serum chemistry, and histopathological changes were evaluated. e LD 50 of the formulation was 5,000 mg/kg BW. No test article related mortalities as well as change in body weight, and food and water consumption were observed. No toxicity related significant changes were noted in renal/hepatic function, hematological indices, and serum biochemical parameters between the control and treated groups. Histopathological alterations were not observed in the vital organs of rats. e subchronic NOAEL for the formulation in rats is greater than 2000mg/kg. is study demonstrated that the green mussel formulation is safe to consume without any adverse effects in the body. 1. Introduction Bivalves are considered vital next to fish and prawns from the nutritive point of view. Bivalve molluscs were reported to contain bioactive lipids, which include fatty acids: sph- ingolipids, phytosterols, diacylglycerols, and so forth. And many of these can influence human health and disease linked to alleviating the symptoms of inflammatory conditions [1]. e green mussel Perna viridis (family: Mytilidae) is a bivalve mollusc native of the Indian coast and throughout the Indo- Pacific and Asia-Pacific [2]. It forms a significant fishery and contributes nearly 50% to the total bivalve production of the area [3]. Among the marine invertebrates, the molluscs are a potential source of bioactive substances with antitumor, antil- eukaemic, anti-inflammatory, antibacterial, and antiviral activities [4, 5]. Traditionally, indigenous people, notably in Western Mexico and throughout the South Pacific, use shellfish supplements as a remedy for arthritis [6]. e com- mercially available products, namely, freeze-dried extract (Seatone) and CO 2 extracted oil (Lyprinol), obtained from Perna canaliculus were reported to inhibit inflammation in the treatment of rheumatoid arthritis and osteoarthritis [7]. Okinawan mollusc Pinna muricata contains aconstituent, pinnatoxin A, which is reported to have Ca 2+ channel acti- vating and anti-inflammatory properties [8]. New Zealand green-lipped mussel P. canaliculus and the Tasmanian blue mussel Mytilus galloprovincialis have been reported to possess anti-inflammatory components [9]. P. canaliculus is restricted to the temperate waters around New Zealand, whereas Perna viridis occurs widely in tropical waters throughout the Indo- Pacific region [10]. ere are several drugs like NSAIDs (aceclofenac, diclofenac, etc.), steroids (glucocorticoid), DMARDs (me- thotrexate and cyclosporin A), and coxibs (celecoxib and rofecoxib) for managing moderate to severe cases of arthritic pain, stiffness, and inflammation [11]. However, the side effects of these drugs are oſten deleterious, which include gastrointestinal ulcers, cardiovascular diseases, and reported toxic effects on the vital organs in the body [12]. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 471565, 14 pages http://dx.doi.org/10.1155/2014/471565

Transcript of Research Article Toxicity Profile of a Nutraceutical...

Page 1: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

Research ArticleToxicity Profile of a Nutraceutical Formulation Derived fromGreen Mussel Perna viridis

Kajal Chakraborty Deepu Joseph and Selsa J Chakkalakal

Marine Biotechnology Division Central Marine Fisheries Research Institute Ernakulam North POPB No 1603 Cochin Kerala 682018 India

Correspondence should be addressed to Kajal Chakraborty kajal cmfriyahoocom

Received 26 February 2014 Accepted 12 May 2014 Published 9 June 2014

Academic Editor Sanyog Jain

Copyright copy 2014 Kajal Chakraborty et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The short-term (acute) and long-term (subchronic) toxicity profile mean lethal dose 50 (LD50) and no-observed-adverse-effect

level (NOAEL) of a nutraceutical formulation developed from green mussel Perna viridis which showed in vitro and in vivo anti-inflammatory properties were evaluated in the present study The formulation was administered to the male and female Wistarrats at graded doses (05 10 and 25 gkg bodyweight) for two weeks of acute toxicity study and 05 10 and 20 gkg bodyweightfor 90 days in subchronic toxicity study The LD

50 variations in clinical signs changes in body weight body weight foodwater

consumption organ weight (liver kidney spleen and brain) hematology serum chemistry and histopathological changes wereevaluated The LD

50of the formulation was 5000mgkg BW No test article related mortalities as well as change in body weight

and food and water consumption were observed No toxicity related significant changes were noted in renalhepatic functionhematological indices and serum biochemical parameters between the control and treated groups Histopathological alterationswere not observed in the vital organs of rats The subchronic NOAEL for the formulation in rats is greater than 2000mgkg Thisstudy demonstrated that the green mussel formulation is safe to consume without any adverse effects in the body

1 Introduction

Bivalves are considered vital next to fish and prawns fromthe nutritive point of view Bivalve molluscs were reportedto contain bioactive lipids which include fatty acids sph-ingolipids phytosterols diacylglycerols and so forth Andmany of these can influence human health and disease linkedto alleviating the symptoms of inflammatory conditions [1]The greenmussel Perna viridis (family Mytilidae) is a bivalvemollusc native of the Indian coast and throughout the Indo-Pacific and Asia-Pacific [2] It forms a significant fishery andcontributes nearly 50 to the total bivalve production of thearea [3]

Among the marine invertebrates the molluscs are apotential source of bioactive substanceswith antitumor antil-eukaemic anti-inflammatory antibacterial and antiviralactivities [4 5] Traditionally indigenous people notablyin Western Mexico and throughout the South Pacific useshellfish supplements as a remedy for arthritis [6] The com-mercially available products namely freeze-dried extract

(Seatone) and CO2extracted oil (Lyprinol) obtained from

Perna canaliculus were reported to inhibit inflammation inthe treatment of rheumatoid arthritis and osteoarthritis [7]Okinawan mollusc Pinna muricata contains aconstituentpinnatoxin A which is reported to have Ca2+ channel acti-vating and anti-inflammatory properties [8] New Zealandgreen-lipped mussel P canaliculus and the Tasmanian bluemusselMytilus galloprovincialishave been reported to possessanti-inflammatory components [9]P canaliculus is restrictedto the temperate waters around New Zealand whereas Pernaviridis occurs widely in tropical waters throughout the Indo-Pacific region [10]

There are several drugs like NSAIDs (aceclofenacdiclofenac etc) steroids (glucocorticoid) DMARDs (me-thotrexate and cyclosporin A) and coxibs (celecoxib androfecoxib) for managing moderate to severe cases of arthriticpain stiffness and inflammation [11] However the sideeffects of these drugs are often deleterious which includegastrointestinal ulcers cardiovascular diseases and reportedtoxic effects on the vital organs in the body [12]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 471565 14 pageshttpdxdoiorg1011552014471565

2 BioMed Research International

The in vitro and in vivo anti-inflammatory studies ofthe green mussel derived nutraceutical formulation showedthat green mussel Perna viridis contains anti-inflammatoryingredients which can be useful against inflammatory painWith the interesting pharmacological properties of thesaid formulation it has become imperative that the anti-inflammatory preparation is evaluated for its toxicity profileAs a part of the safety evaluation of this nutraceuticalformulation the present study was carried out to determinethe changes in body weight food and water consump-tion hematological parameters serum biochemistry andhistopathological changes as indices of toxicosis with the aimof providing guidance for selecting a safe dose of its useThe acute oral toxicity study in 14 days was carried out ata very high dose whereas the repeated dose 90-day oraltoxicity study was performed to establish the no-observed-adverse-effect level (NOAEL) of the extract as parts of asafety assessment according to the internationally acceptedguidelines

2 Materials and Methods

21 Animals The toxicity studies and anti-inflammatorystudy were conducted in adult Wistar rats (both malesand females 180ndash300 g 7-8 weeks old) purchased from SriVenkateshwara Enterprises Bangalore The animals werehoused in well ventilated polypropylene cages under con-trolled temperature (22ndash25∘C) pressure relative humidity(60ndash80) and lightdark cycle of 12 h under normal labo-ratory conditions (24ndash26∘C and 60ndash75 RH) under a 12 hlightdark cycle by fasting with distilled water They wereprovided with animal feed (Sai Durga Feeds and Foods Ban-galore India) and water ad libitum All animal experimentswere conducted after getting prior permission from the Insti-tutional Animal Ethics Committee and as per the instructionsprescribed by the Committee for the Purpose of Control ofSupervision of Experiments on Animal (CPCSEA) Ministryof Environment and Forest Government of India

22 Test Article and Evaluation of Anti-Inflammatory Activi-ties The test article is a nutraceutical formulation preparedfrom the green mussels (Perna viridis) and the detailedcollection of the raw material processing method(s) usedto assure stability under storage conditions and chemicalanalysis demonstrating the composition of the material havebeen described elsewhere [5] Briefly the meat (3 kg) fromthe samples of green mussel (P viridis) (10 kg) collectedfrom their natural habitat at Elathur (Lat 11054101584011610158401015840N Long75012101584021810158401015840E) in the southwest coast of India (Kerala state)has been sucked homogenized and lyophilized to get thefreeze-dried green mussel extract (214 g yield 713) Thecontent thus prepared has been added with lysolecithinsubstituted polysaccharides and phenolic derivatives isolatedfrom Perna viridis In order to enhance the stability and activ-ity of the freeze-dried green mussel extract several naturalsources of antioxidant additives oleoresins of R officinalis(04) and C longa (08) trace amounts of other additivesnamely aqueous freeze-dried extracts of marine macroalgae

(Turbinaria conoides and Sargassum myriocystum 0025ww) Zingiber officinale Tamarindus indica Emblica offici-nalis Citrus limon and Ananas comosus (005 ww) wereselected and added to the freeze-dried extract to make thegreen mussel nutraceutical formulation

The in vitro anti-inflammatory activities of the greenmussel formulation have been carried out in this studyusing cyclooxygenase (COXI and COXII) inhibition assays by27-dichlorofluorescein method [13] and the 5-lipoxygenase(LOXV) inhibition assay [14] For COXI andCOXII inhibitionassays leuco-27-dichlorofluorescein diacetate (5mg) washydrolysed at RT in 1M NaOH (50 120583L) for 10min then 1MHCl (30 120583L) was added to neutralise the excess of NaOHbefore the resulting 1- dichlorofluorescein (DCF) was dilutedin 01M Tris-buffer (pH 8) COX enzyme (COXI and COXII)was diluted in 01M Tris-buffer (pH 8) so that a knownaliquot gave an absorbance change of 005min in the testreaction Test samples (or the equivalent volume of MeOH20120583L) were preincubated with the enzymes at RT for 5minin the presence of hematin Premixed phenol 1-DCF andarachidonic acid were added to the enzyme mixture tobegin the reaction and to give a final reaction mixture ofarachidonic acid (50 120583M) phenol (500 120583M) 1-DCF (20 120583M)and hematin (1 120583M) in 1mL final volume of 01M Tris-buffer (pH 8) The reaction was recorded spectrophoto-metrically over 1min at 502 nm A blank reaction mixture(without enzyme) was analysed in the spectrophotometerreference cell against each test reaction to account for anynonenzymatic activity attributed to the test sample For 5-lipoxygenase (LOXV) inhibition assay an aliquot of the stocksolution (50 120583L in DMSO and tween 20 mixture 29 1ww) of each sample was placed in a 3mL cuvette followedby prewarmed 01M potassium phosphate buffer (295mLpH 63) and linoleic acid solution (48 120583L) Thereafter ice-cold buffer (potassium phosphate) (12120583L) was mixed withLOXV enzyme (100 U) The mixture was then transferred tothe cuvette shaken and placed into the spectrophotometerbefore the absorbance was recorded at 234 nm It is impor-tant to note that prior to testing the sample two sampleswere prepared as mentioned above but only with DMSOand Tween 20 mixtures to serve as controls (no enzymeinhibition)

The in vivo anti-inflammatory activity of the greenmusselformulation was carried out using the carrageenan-inducedrat paw edema as described elsewhere [15] Thirty minutesafter oral administration of the samples (250mgkg animal)and reference drug (aspirin 200mgkg animal) in normalsaline an injection of 01mL of carrageenan (1 in normalsaline) wasmade into the subcutaneous portion of right handpaw of each animal The paw thickness was measured usingan electronic micrometer (aerospace 0ndash25mm range leastcount 0001mm) immediately before carrageenan injectionand 2 3 4 5 and 6 h after carrageenan injection Percentage( difference in paw edema compared to control group) wasobtained using the following formula (119879

119905minus 1198790) times 100119879

0

where 119879119905is the average thickness obtained for each group

before any treatment (0th h) and 1198790is the average paw

thickness for each group after treatment in different timeintervals (2 3 4 5 and 6 h)

BioMed Research International 3

23 Lethal Dose 50 (LD50) of Green Mussel Formulation

Fifteen animals were divided randomly into three groupscontaining 5 animals each After being fasted for 16 h theanimals were administered different doses of green musselformulation suspended in distilled water (5000 2500 and1500mgkg BW) and administered as a single dose throughoral gavage The animals were monitored for 14 days formortality clinical and behavioral symptoms and any adversereaction

24 Acute Oral Toxicity Study of Green Mussel FormulationForty animals (20 males and 20 females) were divided into 4groups each consisting of 5 male and 5 female rats and threedoses (25 10 and 05 gkg) of the green mussel formulationwere administered orally (once daily) for 14 daysThe controlreceived 1mL of water as vehicle every day The animals weremonitored for mortality clinical symptoms and any adversereaction of the test material The body weight and foodconsumption were determined in different time intervals (014 42 70 and 91 days) After 14 days the animals weresacrificed under mild ether anesthesia and the blood wascollected by direct heart puncture method Necropsy wasperformed and observations were recorded Selected organssuch as the liver kidney brain and spleen were dissected outweights were recorded and histopathological analyses wereperformed

25 Subchronic Oral Toxicity Study of Green Mussel Formula-tion Forty animals (20 males and 20 females) were dividedinto 4 groups each consisting of 5 male and 5 female ratsand three doses (20 10 and 05 gkg) of the green musselformulation (1 g suspended in 6mL double distilled water)were administered orally (once daily) for 90 days [16] Thecontrol received 1mL of water as vehicle every day The testanimals were monitored during this period for any type ofclinical symptoms mortality and adverse reactionThe bodyweight and food consumption were determined every sevendays On the 91st day the animals were sacrificed undermild ether anesthesia Blood was collected by direct heartpuncturemethod Necropsywas performed and observationswere recorded Selected organs such as the brain kidney liverand spleen were dissected out weights were recorded andhistopathological analyses were performed

26 Hematology and Clinical Chemistry Parameters Bloodcollected in EDTA tubes was analyzed for hematologicalparameters [17] Red blood cell (RBC) total white blood cellcount (WBC) platelet count and hemoglobin (HGB) weredetermined using a haematology analyzer (Model-Diatron 9Wein Austria) Total white blood cells were measured afterdiluting the blood in Turkrsquos fluid and counting them usinga hemocytometer [18] For differential counts (lymphocyteseosinophils and neutrophils) blood was spread on a cleanslide and treated with Leishmanrsquos stain before being countedmanually with a microscope (100x) [19]

A part of the bloodwas collected in nonheparinized tubesand serum was separated after centrifugation at 5000 rpmfor 10min which was used for the following investigations

Serumglutamic oxaloacetic transaminase (SGOT) and serumglutamic pyruvic transaminase (SGPT) were assayed accord-ing to themethoddescribed byBergmeyer et al [20] Alkalinephosphatase (ALP) was estimated by p-nitrophenyl phos-phate (PNPP) hydrolysis [21] Total bilirubin was determinedby Jendrassik-Diazotized sulphanilic acid method [22] Thetotal protein concentrationwas determined by biuretmethod[18] Albumin was determined based on its reaction withbromocresol green Markers of kidney function such ascreatinine and blood urea nitrogen were estimated by Jaffe-Kinetic and urease method respectively [23] Serum sodiumpotassium and bicarbonate were estimated using Flamephotometer 129 ion selective electrolyte analyzer Chloridewas estimated by mercuric thiocyanate method using a kitfrom Raichem Lifesciences Pvt Ltd India Total choles-terol was estimated by CHODndashPAP (cholesterol oxidasendashphenol + aminophenazone) enzymatic method [24] Triglyc-eride was estimated by GPOndashPAP (glycerol-3-phosphateoxidasendashphenol + aminophenazone) method [25] and high-density lipoprotein (HDL)was determined after precipitationwith phosphotungstic acid Very low-density lipoprotein(VLDL) was estimated by the Friedewald equation (VLDL =triglyceride5) and low-density lipoprotein (LDL) by calcula-tion LDL = total cholesterol minus (HDL + VLDL) [16]

27 Histopathological Analysis A portion of the selectedorgans (brain kidney liver and spleen) of control and treatedgroup (high dose groups) were fixed in 10 neutral bufferedformalin Embedded organs tissue samples were cut intoslices of 2ndash4120583m and stained with hematoxylin-eosin and thesections were observed under light microscope (40x)

28 Statistical Analysis Statistical evaluation was carried outwith the Statistical Program for Social Sciences 130 (SPSSInc Chicago USA ver 130) Analyses were carried out intriplicate and the means of all parameters were examinedfor significance by analysis of variance (ANOVA)The valueswere compared with that of untreated control animals

3 Results

31 Anti-Inflammatory Activities of Green Mussel Formu-lation The green mussel formulation (1mgmL) showedinhibiting properties against proinflammatory COXII (50)and LOXV enzymes (47) and the activities were foundto be comparable with standard NSAIDs (Figure 1(a)) Inthis study greenmussel formulation showed lower inhibitionof COXI (41 1mgmL) than synthetic NSAIDs (gt50)Notably the animals challenged with the green mussel for-mulation significantly mitigated (119875 lt 005) the carrageenan-induced paw edema in a time-dependent manner till theend of the 6th h as compared to negative control animalsthroughout the period of study (Figure 1(b))

32 LD50

of Green Mussel Formulation The single doseadministration of the green mussel formulation up to aconcentration of 5000mgkg BW did not produce anymortality after 14 days of observation which indicates that

4 BioMed Research International

100

80

60

40

20

0

05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL

Inhi

bitio

n of

infla

mm

ator

y en

zym

es (

)

COX-I inhibition activity () COX-II inhibition activity () LOX-V inhibition activity ()

IndomethacinAspirin

Green mussel formulation

(a)

0

40

80

120

160

2 3 4 5 6

Diff

eren

ce in

paw

edem

a (

)

Time (h)

Normal salineAspirinGreen mussel formulation

Oral administration of the standard (aspirin) and the test samples

Untreated rat (control)

Reduced volume of paw edema in carrageenan-induced rat

Green mussel extract treated rat

No reduction in paw edemavolume of carrageenantreated rat

Carrageenan (1 in saline 01mLanimal) injection

(subcutaneously into the right hind paw)

thirty minutes after the oral administration of test samples

(b)

Figure 1 (a) In vitro anti-inflammatory activities (COXIII and LOXV inhibition activities) of the green mussel formulation compared withstandard anti-inflammatory drugs aspirin and indomethacin at different concentrations (05 1 and 5mgmL) (b) In vivo anti-inflammatoryactivity ( difference in paw edema compared to control group) of the green mussel formulation compared to standard anti-inflammatorydrug aspirin

themean lethal dose (LD50) of the formulation is greater than

5000mgkg BW The oral toxicity of this formulation can beclassified in category 5 (the lethal acute toxicity is greaterthan 5000mgkg) according to the Globally HarmonizedClassification System of OECD [26]

33 Acute Toxicity Study of Green Mussel Formulation Notreatment-related signs of mortality were observed in theanimals over short-term administration (maximum dose of2500mgkg BW) In addition the administration of thegreen mussel formulation at different doses did not produce

BioMed Research International 5

Table 1 Body weight and food and water consumption during subchronic (90 days) toxicity studies after the administration of green musselformulation

Days Male FemaleControla 20 gkgb 10 gkgb 050 gkgb Controla 20 gkgb 10 gkgb 050 gkgb

Body weight (g)0 2308 plusmn 14 2549 plusmn 25 2636 plusmn 142 2844 plusmn 05 1858 plusmn 15 2185 plusmn 156 1958 plusmn 14 1909 plusmn 52

14 2396 plusmn 45 2618 plusmn 15 2704 plusmn 22 2901 plusmn 14 1924 plusmn 24 2244 plusmn 125 2021 plusmn 26 1968 plusmn 62

42 2524 plusmn 66 2739 plusmn 65 281 plusmn 186 300 plusmn 15 204 plusmn 26 2326 plusmn 116 2121 plusmn 32 2069 plusmn 26

70 2638 plusmn 85lowast2836 plusmn 15

lowast2902 plusmn 195 3084 plusmn 16

lowast2142 plusmn 26

lowast2409 plusmn 114

lowast2207 plusmn 12

lowast2162 plusmn 24

lowast

91 27158 plusmn 142lowast2908 plusmn 16

lowast2967 plusmn 21

lowast3145 plusmn 12

lowast2211 plusmn 09

lowast2466 plusmn 05

lowast2268 plusmn 05

lowast2225 plusmn 29

lowast

Food consumption (g)0 724 plusmn 56 744 plusmn 25 706 plusmn 65 687 plusmn 12 595 plusmn 06 554 plusmn 16 58 plusmn 23 627 plusmn 09

14 664 plusmn 65 733 plusmn 36 626 plusmn 63 613 plusmn 15 625 plusmn 12 493 plusmn 26 546 plusmn 26 577 plusmn 12

42 673 plusmn 69 716 plusmn 39 614 plusmn 42 606 plusmn 25 476 plusmn 11 506 plusmn 27 483 plusmn 15 50 plusmn 13

70 666 plusmn 79 727 plusmn 38 623 plusmn 32 625 plusmn 21 479 plusmn 15 513 plusmn 24 49 plusmn 26 497 plusmn 14

91 683 plusmn 85 747 plusmn 36 632 plusmn 26 655 plusmn 2 487 plusmn 13 508 plusmn 21 497 plusmn 28 50 plusmn 15

Water consumption (mL)0 100 plusmn 4 90 plusmn 2 100 plusmn 2 110 plusmn 3 100 plusmn 2 80 plusmn 2 110 plusmn 4 90 plusmn 2

14 90 plusmn 3 70 plusmn 5 110 plusmn 3 100 plusmn 1 80 plusmn 4 80 plusmn 1 70 plusmn 5 90 plusmn 2

42 90 plusmn 2 110 plusmn 3 90 plusmn 1 100 plusmn 2 100 plusmn 3 80 plusmn 4 90 plusmn 1 90 plusmn 3

70 110 plusmn 3 110 plusmn 1 100 plusmn 5 100 plusmn 3 100 plusmn 2 100 plusmn 1 90 plusmn 2 110 plusmn 3

91 100 plusmn 4 100 plusmn 2 110 plusmn 1 100 plusmn 4 100 plusmn 1 110 plusmn 2 100 plusmn 2 100 plusmn 3

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)

any treatment-related changes in the body weight of theanimals or any differences in the food consumption of maleand female rats when compared to controls No significantchanges were noticed during necropsy and there was nochange in the organ weight

No treatment-related biologically significant effects of thegreen mussel formulation treatment at dose levels of 05ndash25 gkg in hematology parameters such as hemoglobin RBCcount platelet count and total and differential leukocytescounts were apparent in both genders of rats when comparedto untreated animals

The green mussel formulation up to a concentrationof 25 gkg did not produce any change in the hepaticfunction parameters in serum such as SGOT SGPT ALPtotal protein bilirubin albumin and globulinas well as inalbuminglobulin (AG) ratio

The renal function tests such as blood urea and serumcreatinine did not show any variation when compared tocontrols There was also no change in serum electrolytessodium potassium chloride and bicarbonate indicating thatthe green mussel formulation did not produce any change inrenal function

Acute toxicity study of the green mussel formulation didnot show any change in cholesterol triglycerides HDL LDLVLDL and cholesterol levels Histopathological analysis ofthe brain spleen kidney and liver did not show any patho-logical lesions in the organs of animals treated with the greenmussel formulation

The above observations concluded that the green musselformulation did not produce any toxicity toWistar rats whenadministered for two weeks

34 Subchronic Toxicity Study of Green Mussel Formulation

341 General Conditions and Behavior No treatment-relatedsigns of mortality were observed in the animals over theadministration periods (maximum dose of 2000mgkg BW)In addition the administration of the green mussel formu-lation at different doses did not produce any treatment-related changes in clinical signs such as mental state externalappearance and daily activities among the test groups whencompared with the control Any abnormal behavior or casesof diarrhea and soft feceswere not observed during the periodof study No ophthalmological abnormalities were observedin any of the treatment groups prior to study initiation andnear experimental completion In general the experimentalanimals from all treatment groups appeared healthy at theconclusion of the study period and did not induce any clinicalsigns of toxicity in subchronic regimens

342 Body Weight The administration of the green musselformulation during 90 days of long-term subchronic toxicitystudies did not produce any abnormal change in the bodyweight of male and female rats when compared to the control(Table 1) As expected rats gained weight with time In malerats the gain in mean body weights for the treated groups

6 BioMed Research International

0

1

2

3

4O

rgan

wei

ght (

g)

LiverKidney

SpleenBrain

25

gkg

10

gkg

05

gkg

Con

trol

25

gkg

10

gkg

05

gkg

Con

trol

Male Female

(a)

0

1

2

3

4

Org

an w

eigh

t (g)

LiverKidney

SpleenBrain

10

gkg

05

gkg

Con

trol

20

gkg

10

gkg

05

gkg

Con

trol

20

gkg

Males Females

(b)

Figure 2 Mean organ weights (in grams) of male and female rats administered green mussel formulation after (a) acute and (b) subchronictoxicity studies

at 05ndash20 gkg BW was comparable with those in the controlgroup throughout the study Similarly themean bodyweightsof the treated female rats were comparable with those in thecontrol group throughout the studyTherewere no changes inbody weight in the animals attributable to the administrationof the green mussel formulation when compared to the con-trol group Any changes observed were sporadic consideredincidental and unassociated with test article administration

343 Food andWater Consumption The average food intakeof untreated control rats decreased from about 72 g to 68 g(for male rats) and from 60 g to 49 g (for female rats) after 90days of study The same trend was observed for medium andlow dose group (10 and 05 gkg resp) of males and all thedose groups of females Administration of the green musselformulation did not produce any significant difference in thefood consumption of both genders of rats when compared tonormal animals of the high dose group (119875 gt 005) throughoutthe experimental period The summarized food intake of therats recorded after oral administration of the green musselformulation to rats is shown in Table 1 Similarly the waterconsumption did not alter inmale and female rats attributableto administration of the green mussel formulation whencompared to normal animals during chronic and subchronictoxicity studies Changes in the average water consumptionduring the treatment period are presented in Table 1 Spo-radic statistically significant changes in water consumptionwere considered spurious unassociated with the test articleadministration

344 Relative Organ Weight Figure 2 presents the relativeweights of the vital organs (in g) of rats (both genders) Theweights of liver kidney spleen and brain recorded at theend of the subchronic study (day 91) did not show significantdifferences (119875 gt 005) in any of the treatment groups

compared with the control groups (Figure 2) Furthermoregross examination of the vital organs of all rats revealed nodetectable abnormalities

345 Hematological Parameters The effect of the greenmussel formulation on hematological parameters such ashemoglobin (HGB) RBCandWBCcount platelet count anddifferential counts after subchronic toxicity (90 days) studiesis presented in Table 2 No treatment-related biologicallysignificant effects of the green mussel formulation treatmentat dose levels of 05ndash20 gkg in the hemoglobin and RBCand platelet content were apparent in both genders of ratswhen compared to untreated animals (Table 2) (119875 gt 005)and remained within physiological range throughout thetreatment period (90 days) However both male and femalerats administered green mussel formulation (at 10 gkg and05 gkg resp) showed significantly low levels of differentialcounts (lymphocytes eosinophils and neutrophils) com-pared to control animals (119875 lt 005) Similarly femalerats administered 20 and 10 gkg green mussel formulationrecorded significantly low lymphocyte and neutrophil countrespectively (119875 lt 005) No test article-related changes inblood cell morphology were observed during the period ofstudy

346 Serum Biochemical Parameters Table 3 summarizesthe serum biochemical parameters used as the biomark-ers of the liver and renal functioning during the courseof subchronic toxicity studies The serum analysis showedsignificantly low SGOT content for the low dose male rats(05 gkg) and highlow dose female rats (25 and 05 gkg)after 90 days of subchronic study Similarly another markerenzyme of the liver ALP also showed significantly lowvalues for high and low dose group male rats compared tothe control animals after 90 days of subchronic study Theactivity of anothermarker enzyme SGPTwas not significantly

BioMed Research International 7

Normal male

(a)

Normal female

(b) (a1)

Treated male (2gkg body weight)

(b1)

Treated female (2gkg body weight)

Figure 3 The cross section of the male and female rats after subchronic toxicity study of 90 days (a) Normal male (a1) green musselformulation (20 gkg) treated male (b) normal female (b1) green mussel formulation (20 gkg) treated female

Table 2 Hematology analyses data of male and female rats administered the green mussel formulation after subchronic (90 days) toxicitystudies

Treatments Lymphocytes(mm3)

Eosinophils(mm3)

Neutrophils(mm3) HGB (gdL) WBC (mm3) RBC

(106cmm)Platelet

(105cmm)

Male

Controla 582860 plusmn 15914 99820 plusmn 6290 321320 plusmn 1713 1544 plusmn 005 10040 plusmn 586 787 plusmn 007 578 plusmn 006

20 gkgb 463800 plusmn 15450 75260 plusmn 3157 232940 plusmn 8304lowast 1460 plusmn 064 7720 plusmn 2670 741 plusmn 029 464 plusmn 038

10 gkgb 364380 plusmn 18747lowast 61860 plusmn 3324lowast 201760 plusmn 8802lowast 1434 plusmn 073 6280 plusmn 3090 729 plusmn 058 508 plusmn 004

05 gkgb 491220 plusmn 11407 77360 plusmn 1538 275420 plusmn 7898 1464 plusmn 070 8440 plusmn 17470 727 plusmn 008 578 plusmn 006

Female

Controla 522040 plusmn 2310 88880 plusmn 3778 273080 plusmn 8031 1338 plusmn 094 8840 plusmn 1489 637 plusmn 047 606 plusmn 003

20 gkgb 385640 plusmn 16793lowast 64540 plusmn 2894 229820 plusmn 11425 1412 plusmn 044 6800 plusmn 977 697 plusmn 003 578 plusmn 008

10 gkgb 497520 plusmn 26933 77000 plusmn 3175 197480 plusmn 8387lowast 1386 plusmn 083 7720 plusmn 1075 686 plusmn 021 530 plusmn 007

05 gkgb 212280 plusmn 10152lowast 32120 plusmn 1467lowast 109600 plusmn 5093lowast 1354 plusmn 053 3540 plusmn 1017lowast 677 plusmn 007 564 plusmn 006

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)HGB hemoglobin WBC total white blood cell count RBC red blood cell

different (119875 gt 005) in all dose groups of treated rats ascompared to untreated control and albuminglobulin (AG)ratio was not altered in the treated animals of both genders(Table 3)

Subchronic oral administration of the green mussel for-mulation (for 90 days) did not cause any significant changesin hepatic function parameters such as total protein albumintotal bilirubin and globulin in both sexes of rats duringlong-term subchronic toxicity studies The renal functionparameters such as serum creatinine and blood urea did notshow any significant variation (119875 gt 005) in treated animalscompared to controls (Table 3) There were no statisticallysignificant differences in the levels of serum electrolytessuch as chloride potassium sodium and bicarbonate afterthe treatment of green mussel formulation (119875 gt 005)indicating no expressive changes in the general metabolismafter consumption of the green mussel formulation by rats

(Table 4) The green mussel formulation did not produceany significant changes in the total cholesterol HDL LDLand VLDL indicating no expressive changes in the generallipid metabolism after consumption of the test article by rats(Table 4)

347 Histopathological Analysis Necropsy of the treatedanimals after sacrifice did not show any morphologicalchanges of the internal organs or any gross pathologi-cal abnormalities during subchronic toxicity studies Therewere no macroscopic findings considered to be relatedto the treatment of the green mussel formulation (Fig-ure 3) Gross examination of vital organs such as brainkidney spleen and liver of rats and microscopic exami-nation of tissue sections prepared from these organs didnot observe any histopathological alteration in any treatedrats during subchronic toxicity studies The normal and

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 2: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

2 BioMed Research International

The in vitro and in vivo anti-inflammatory studies ofthe green mussel derived nutraceutical formulation showedthat green mussel Perna viridis contains anti-inflammatoryingredients which can be useful against inflammatory painWith the interesting pharmacological properties of thesaid formulation it has become imperative that the anti-inflammatory preparation is evaluated for its toxicity profileAs a part of the safety evaluation of this nutraceuticalformulation the present study was carried out to determinethe changes in body weight food and water consump-tion hematological parameters serum biochemistry andhistopathological changes as indices of toxicosis with the aimof providing guidance for selecting a safe dose of its useThe acute oral toxicity study in 14 days was carried out ata very high dose whereas the repeated dose 90-day oraltoxicity study was performed to establish the no-observed-adverse-effect level (NOAEL) of the extract as parts of asafety assessment according to the internationally acceptedguidelines

2 Materials and Methods

21 Animals The toxicity studies and anti-inflammatorystudy were conducted in adult Wistar rats (both malesand females 180ndash300 g 7-8 weeks old) purchased from SriVenkateshwara Enterprises Bangalore The animals werehoused in well ventilated polypropylene cages under con-trolled temperature (22ndash25∘C) pressure relative humidity(60ndash80) and lightdark cycle of 12 h under normal labo-ratory conditions (24ndash26∘C and 60ndash75 RH) under a 12 hlightdark cycle by fasting with distilled water They wereprovided with animal feed (Sai Durga Feeds and Foods Ban-galore India) and water ad libitum All animal experimentswere conducted after getting prior permission from the Insti-tutional Animal Ethics Committee and as per the instructionsprescribed by the Committee for the Purpose of Control ofSupervision of Experiments on Animal (CPCSEA) Ministryof Environment and Forest Government of India

22 Test Article and Evaluation of Anti-Inflammatory Activi-ties The test article is a nutraceutical formulation preparedfrom the green mussels (Perna viridis) and the detailedcollection of the raw material processing method(s) usedto assure stability under storage conditions and chemicalanalysis demonstrating the composition of the material havebeen described elsewhere [5] Briefly the meat (3 kg) fromthe samples of green mussel (P viridis) (10 kg) collectedfrom their natural habitat at Elathur (Lat 11054101584011610158401015840N Long75012101584021810158401015840E) in the southwest coast of India (Kerala state)has been sucked homogenized and lyophilized to get thefreeze-dried green mussel extract (214 g yield 713) Thecontent thus prepared has been added with lysolecithinsubstituted polysaccharides and phenolic derivatives isolatedfrom Perna viridis In order to enhance the stability and activ-ity of the freeze-dried green mussel extract several naturalsources of antioxidant additives oleoresins of R officinalis(04) and C longa (08) trace amounts of other additivesnamely aqueous freeze-dried extracts of marine macroalgae

(Turbinaria conoides and Sargassum myriocystum 0025ww) Zingiber officinale Tamarindus indica Emblica offici-nalis Citrus limon and Ananas comosus (005 ww) wereselected and added to the freeze-dried extract to make thegreen mussel nutraceutical formulation

The in vitro anti-inflammatory activities of the greenmussel formulation have been carried out in this studyusing cyclooxygenase (COXI and COXII) inhibition assays by27-dichlorofluorescein method [13] and the 5-lipoxygenase(LOXV) inhibition assay [14] For COXI andCOXII inhibitionassays leuco-27-dichlorofluorescein diacetate (5mg) washydrolysed at RT in 1M NaOH (50 120583L) for 10min then 1MHCl (30 120583L) was added to neutralise the excess of NaOHbefore the resulting 1- dichlorofluorescein (DCF) was dilutedin 01M Tris-buffer (pH 8) COX enzyme (COXI and COXII)was diluted in 01M Tris-buffer (pH 8) so that a knownaliquot gave an absorbance change of 005min in the testreaction Test samples (or the equivalent volume of MeOH20120583L) were preincubated with the enzymes at RT for 5minin the presence of hematin Premixed phenol 1-DCF andarachidonic acid were added to the enzyme mixture tobegin the reaction and to give a final reaction mixture ofarachidonic acid (50 120583M) phenol (500 120583M) 1-DCF (20 120583M)and hematin (1 120583M) in 1mL final volume of 01M Tris-buffer (pH 8) The reaction was recorded spectrophoto-metrically over 1min at 502 nm A blank reaction mixture(without enzyme) was analysed in the spectrophotometerreference cell against each test reaction to account for anynonenzymatic activity attributed to the test sample For 5-lipoxygenase (LOXV) inhibition assay an aliquot of the stocksolution (50 120583L in DMSO and tween 20 mixture 29 1ww) of each sample was placed in a 3mL cuvette followedby prewarmed 01M potassium phosphate buffer (295mLpH 63) and linoleic acid solution (48 120583L) Thereafter ice-cold buffer (potassium phosphate) (12120583L) was mixed withLOXV enzyme (100 U) The mixture was then transferred tothe cuvette shaken and placed into the spectrophotometerbefore the absorbance was recorded at 234 nm It is impor-tant to note that prior to testing the sample two sampleswere prepared as mentioned above but only with DMSOand Tween 20 mixtures to serve as controls (no enzymeinhibition)

The in vivo anti-inflammatory activity of the greenmusselformulation was carried out using the carrageenan-inducedrat paw edema as described elsewhere [15] Thirty minutesafter oral administration of the samples (250mgkg animal)and reference drug (aspirin 200mgkg animal) in normalsaline an injection of 01mL of carrageenan (1 in normalsaline) wasmade into the subcutaneous portion of right handpaw of each animal The paw thickness was measured usingan electronic micrometer (aerospace 0ndash25mm range leastcount 0001mm) immediately before carrageenan injectionand 2 3 4 5 and 6 h after carrageenan injection Percentage( difference in paw edema compared to control group) wasobtained using the following formula (119879

119905minus 1198790) times 100119879

0

where 119879119905is the average thickness obtained for each group

before any treatment (0th h) and 1198790is the average paw

thickness for each group after treatment in different timeintervals (2 3 4 5 and 6 h)

BioMed Research International 3

23 Lethal Dose 50 (LD50) of Green Mussel Formulation

Fifteen animals were divided randomly into three groupscontaining 5 animals each After being fasted for 16 h theanimals were administered different doses of green musselformulation suspended in distilled water (5000 2500 and1500mgkg BW) and administered as a single dose throughoral gavage The animals were monitored for 14 days formortality clinical and behavioral symptoms and any adversereaction

24 Acute Oral Toxicity Study of Green Mussel FormulationForty animals (20 males and 20 females) were divided into 4groups each consisting of 5 male and 5 female rats and threedoses (25 10 and 05 gkg) of the green mussel formulationwere administered orally (once daily) for 14 daysThe controlreceived 1mL of water as vehicle every day The animals weremonitored for mortality clinical symptoms and any adversereaction of the test material The body weight and foodconsumption were determined in different time intervals (014 42 70 and 91 days) After 14 days the animals weresacrificed under mild ether anesthesia and the blood wascollected by direct heart puncture method Necropsy wasperformed and observations were recorded Selected organssuch as the liver kidney brain and spleen were dissected outweights were recorded and histopathological analyses wereperformed

25 Subchronic Oral Toxicity Study of Green Mussel Formula-tion Forty animals (20 males and 20 females) were dividedinto 4 groups each consisting of 5 male and 5 female ratsand three doses (20 10 and 05 gkg) of the green musselformulation (1 g suspended in 6mL double distilled water)were administered orally (once daily) for 90 days [16] Thecontrol received 1mL of water as vehicle every day The testanimals were monitored during this period for any type ofclinical symptoms mortality and adverse reactionThe bodyweight and food consumption were determined every sevendays On the 91st day the animals were sacrificed undermild ether anesthesia Blood was collected by direct heartpuncturemethod Necropsywas performed and observationswere recorded Selected organs such as the brain kidney liverand spleen were dissected out weights were recorded andhistopathological analyses were performed

26 Hematology and Clinical Chemistry Parameters Bloodcollected in EDTA tubes was analyzed for hematologicalparameters [17] Red blood cell (RBC) total white blood cellcount (WBC) platelet count and hemoglobin (HGB) weredetermined using a haematology analyzer (Model-Diatron 9Wein Austria) Total white blood cells were measured afterdiluting the blood in Turkrsquos fluid and counting them usinga hemocytometer [18] For differential counts (lymphocyteseosinophils and neutrophils) blood was spread on a cleanslide and treated with Leishmanrsquos stain before being countedmanually with a microscope (100x) [19]

A part of the bloodwas collected in nonheparinized tubesand serum was separated after centrifugation at 5000 rpmfor 10min which was used for the following investigations

Serumglutamic oxaloacetic transaminase (SGOT) and serumglutamic pyruvic transaminase (SGPT) were assayed accord-ing to themethoddescribed byBergmeyer et al [20] Alkalinephosphatase (ALP) was estimated by p-nitrophenyl phos-phate (PNPP) hydrolysis [21] Total bilirubin was determinedby Jendrassik-Diazotized sulphanilic acid method [22] Thetotal protein concentrationwas determined by biuretmethod[18] Albumin was determined based on its reaction withbromocresol green Markers of kidney function such ascreatinine and blood urea nitrogen were estimated by Jaffe-Kinetic and urease method respectively [23] Serum sodiumpotassium and bicarbonate were estimated using Flamephotometer 129 ion selective electrolyte analyzer Chloridewas estimated by mercuric thiocyanate method using a kitfrom Raichem Lifesciences Pvt Ltd India Total choles-terol was estimated by CHODndashPAP (cholesterol oxidasendashphenol + aminophenazone) enzymatic method [24] Triglyc-eride was estimated by GPOndashPAP (glycerol-3-phosphateoxidasendashphenol + aminophenazone) method [25] and high-density lipoprotein (HDL)was determined after precipitationwith phosphotungstic acid Very low-density lipoprotein(VLDL) was estimated by the Friedewald equation (VLDL =triglyceride5) and low-density lipoprotein (LDL) by calcula-tion LDL = total cholesterol minus (HDL + VLDL) [16]

27 Histopathological Analysis A portion of the selectedorgans (brain kidney liver and spleen) of control and treatedgroup (high dose groups) were fixed in 10 neutral bufferedformalin Embedded organs tissue samples were cut intoslices of 2ndash4120583m and stained with hematoxylin-eosin and thesections were observed under light microscope (40x)

28 Statistical Analysis Statistical evaluation was carried outwith the Statistical Program for Social Sciences 130 (SPSSInc Chicago USA ver 130) Analyses were carried out intriplicate and the means of all parameters were examinedfor significance by analysis of variance (ANOVA)The valueswere compared with that of untreated control animals

3 Results

31 Anti-Inflammatory Activities of Green Mussel Formu-lation The green mussel formulation (1mgmL) showedinhibiting properties against proinflammatory COXII (50)and LOXV enzymes (47) and the activities were foundto be comparable with standard NSAIDs (Figure 1(a)) Inthis study greenmussel formulation showed lower inhibitionof COXI (41 1mgmL) than synthetic NSAIDs (gt50)Notably the animals challenged with the green mussel for-mulation significantly mitigated (119875 lt 005) the carrageenan-induced paw edema in a time-dependent manner till theend of the 6th h as compared to negative control animalsthroughout the period of study (Figure 1(b))

32 LD50

of Green Mussel Formulation The single doseadministration of the green mussel formulation up to aconcentration of 5000mgkg BW did not produce anymortality after 14 days of observation which indicates that

4 BioMed Research International

100

80

60

40

20

0

05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL

Inhi

bitio

n of

infla

mm

ator

y en

zym

es (

)

COX-I inhibition activity () COX-II inhibition activity () LOX-V inhibition activity ()

IndomethacinAspirin

Green mussel formulation

(a)

0

40

80

120

160

2 3 4 5 6

Diff

eren

ce in

paw

edem

a (

)

Time (h)

Normal salineAspirinGreen mussel formulation

Oral administration of the standard (aspirin) and the test samples

Untreated rat (control)

Reduced volume of paw edema in carrageenan-induced rat

Green mussel extract treated rat

No reduction in paw edemavolume of carrageenantreated rat

Carrageenan (1 in saline 01mLanimal) injection

(subcutaneously into the right hind paw)

thirty minutes after the oral administration of test samples

(b)

Figure 1 (a) In vitro anti-inflammatory activities (COXIII and LOXV inhibition activities) of the green mussel formulation compared withstandard anti-inflammatory drugs aspirin and indomethacin at different concentrations (05 1 and 5mgmL) (b) In vivo anti-inflammatoryactivity ( difference in paw edema compared to control group) of the green mussel formulation compared to standard anti-inflammatorydrug aspirin

themean lethal dose (LD50) of the formulation is greater than

5000mgkg BW The oral toxicity of this formulation can beclassified in category 5 (the lethal acute toxicity is greaterthan 5000mgkg) according to the Globally HarmonizedClassification System of OECD [26]

33 Acute Toxicity Study of Green Mussel Formulation Notreatment-related signs of mortality were observed in theanimals over short-term administration (maximum dose of2500mgkg BW) In addition the administration of thegreen mussel formulation at different doses did not produce

BioMed Research International 5

Table 1 Body weight and food and water consumption during subchronic (90 days) toxicity studies after the administration of green musselformulation

Days Male FemaleControla 20 gkgb 10 gkgb 050 gkgb Controla 20 gkgb 10 gkgb 050 gkgb

Body weight (g)0 2308 plusmn 14 2549 plusmn 25 2636 plusmn 142 2844 plusmn 05 1858 plusmn 15 2185 plusmn 156 1958 plusmn 14 1909 plusmn 52

14 2396 plusmn 45 2618 plusmn 15 2704 plusmn 22 2901 plusmn 14 1924 plusmn 24 2244 plusmn 125 2021 plusmn 26 1968 plusmn 62

42 2524 plusmn 66 2739 plusmn 65 281 plusmn 186 300 plusmn 15 204 plusmn 26 2326 plusmn 116 2121 plusmn 32 2069 plusmn 26

70 2638 plusmn 85lowast2836 plusmn 15

lowast2902 plusmn 195 3084 plusmn 16

lowast2142 plusmn 26

lowast2409 plusmn 114

lowast2207 plusmn 12

lowast2162 plusmn 24

lowast

91 27158 plusmn 142lowast2908 plusmn 16

lowast2967 plusmn 21

lowast3145 plusmn 12

lowast2211 plusmn 09

lowast2466 plusmn 05

lowast2268 plusmn 05

lowast2225 plusmn 29

lowast

Food consumption (g)0 724 plusmn 56 744 plusmn 25 706 plusmn 65 687 plusmn 12 595 plusmn 06 554 plusmn 16 58 plusmn 23 627 plusmn 09

14 664 plusmn 65 733 plusmn 36 626 plusmn 63 613 plusmn 15 625 plusmn 12 493 plusmn 26 546 plusmn 26 577 plusmn 12

42 673 plusmn 69 716 plusmn 39 614 plusmn 42 606 plusmn 25 476 plusmn 11 506 plusmn 27 483 plusmn 15 50 plusmn 13

70 666 plusmn 79 727 plusmn 38 623 plusmn 32 625 plusmn 21 479 plusmn 15 513 plusmn 24 49 plusmn 26 497 plusmn 14

91 683 plusmn 85 747 plusmn 36 632 plusmn 26 655 plusmn 2 487 plusmn 13 508 plusmn 21 497 plusmn 28 50 plusmn 15

Water consumption (mL)0 100 plusmn 4 90 plusmn 2 100 plusmn 2 110 plusmn 3 100 plusmn 2 80 plusmn 2 110 plusmn 4 90 plusmn 2

14 90 plusmn 3 70 plusmn 5 110 plusmn 3 100 plusmn 1 80 plusmn 4 80 plusmn 1 70 plusmn 5 90 plusmn 2

42 90 plusmn 2 110 plusmn 3 90 plusmn 1 100 plusmn 2 100 plusmn 3 80 plusmn 4 90 plusmn 1 90 plusmn 3

70 110 plusmn 3 110 plusmn 1 100 plusmn 5 100 plusmn 3 100 plusmn 2 100 plusmn 1 90 plusmn 2 110 plusmn 3

91 100 plusmn 4 100 plusmn 2 110 plusmn 1 100 plusmn 4 100 plusmn 1 110 plusmn 2 100 plusmn 2 100 plusmn 3

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)

any treatment-related changes in the body weight of theanimals or any differences in the food consumption of maleand female rats when compared to controls No significantchanges were noticed during necropsy and there was nochange in the organ weight

No treatment-related biologically significant effects of thegreen mussel formulation treatment at dose levels of 05ndash25 gkg in hematology parameters such as hemoglobin RBCcount platelet count and total and differential leukocytescounts were apparent in both genders of rats when comparedto untreated animals

The green mussel formulation up to a concentrationof 25 gkg did not produce any change in the hepaticfunction parameters in serum such as SGOT SGPT ALPtotal protein bilirubin albumin and globulinas well as inalbuminglobulin (AG) ratio

The renal function tests such as blood urea and serumcreatinine did not show any variation when compared tocontrols There was also no change in serum electrolytessodium potassium chloride and bicarbonate indicating thatthe green mussel formulation did not produce any change inrenal function

Acute toxicity study of the green mussel formulation didnot show any change in cholesterol triglycerides HDL LDLVLDL and cholesterol levels Histopathological analysis ofthe brain spleen kidney and liver did not show any patho-logical lesions in the organs of animals treated with the greenmussel formulation

The above observations concluded that the green musselformulation did not produce any toxicity toWistar rats whenadministered for two weeks

34 Subchronic Toxicity Study of Green Mussel Formulation

341 General Conditions and Behavior No treatment-relatedsigns of mortality were observed in the animals over theadministration periods (maximum dose of 2000mgkg BW)In addition the administration of the green mussel formu-lation at different doses did not produce any treatment-related changes in clinical signs such as mental state externalappearance and daily activities among the test groups whencompared with the control Any abnormal behavior or casesof diarrhea and soft feceswere not observed during the periodof study No ophthalmological abnormalities were observedin any of the treatment groups prior to study initiation andnear experimental completion In general the experimentalanimals from all treatment groups appeared healthy at theconclusion of the study period and did not induce any clinicalsigns of toxicity in subchronic regimens

342 Body Weight The administration of the green musselformulation during 90 days of long-term subchronic toxicitystudies did not produce any abnormal change in the bodyweight of male and female rats when compared to the control(Table 1) As expected rats gained weight with time In malerats the gain in mean body weights for the treated groups

6 BioMed Research International

0

1

2

3

4O

rgan

wei

ght (

g)

LiverKidney

SpleenBrain

25

gkg

10

gkg

05

gkg

Con

trol

25

gkg

10

gkg

05

gkg

Con

trol

Male Female

(a)

0

1

2

3

4

Org

an w

eigh

t (g)

LiverKidney

SpleenBrain

10

gkg

05

gkg

Con

trol

20

gkg

10

gkg

05

gkg

Con

trol

20

gkg

Males Females

(b)

Figure 2 Mean organ weights (in grams) of male and female rats administered green mussel formulation after (a) acute and (b) subchronictoxicity studies

at 05ndash20 gkg BW was comparable with those in the controlgroup throughout the study Similarly themean bodyweightsof the treated female rats were comparable with those in thecontrol group throughout the studyTherewere no changes inbody weight in the animals attributable to the administrationof the green mussel formulation when compared to the con-trol group Any changes observed were sporadic consideredincidental and unassociated with test article administration

343 Food andWater Consumption The average food intakeof untreated control rats decreased from about 72 g to 68 g(for male rats) and from 60 g to 49 g (for female rats) after 90days of study The same trend was observed for medium andlow dose group (10 and 05 gkg resp) of males and all thedose groups of females Administration of the green musselformulation did not produce any significant difference in thefood consumption of both genders of rats when compared tonormal animals of the high dose group (119875 gt 005) throughoutthe experimental period The summarized food intake of therats recorded after oral administration of the green musselformulation to rats is shown in Table 1 Similarly the waterconsumption did not alter inmale and female rats attributableto administration of the green mussel formulation whencompared to normal animals during chronic and subchronictoxicity studies Changes in the average water consumptionduring the treatment period are presented in Table 1 Spo-radic statistically significant changes in water consumptionwere considered spurious unassociated with the test articleadministration

344 Relative Organ Weight Figure 2 presents the relativeweights of the vital organs (in g) of rats (both genders) Theweights of liver kidney spleen and brain recorded at theend of the subchronic study (day 91) did not show significantdifferences (119875 gt 005) in any of the treatment groups

compared with the control groups (Figure 2) Furthermoregross examination of the vital organs of all rats revealed nodetectable abnormalities

345 Hematological Parameters The effect of the greenmussel formulation on hematological parameters such ashemoglobin (HGB) RBCandWBCcount platelet count anddifferential counts after subchronic toxicity (90 days) studiesis presented in Table 2 No treatment-related biologicallysignificant effects of the green mussel formulation treatmentat dose levels of 05ndash20 gkg in the hemoglobin and RBCand platelet content were apparent in both genders of ratswhen compared to untreated animals (Table 2) (119875 gt 005)and remained within physiological range throughout thetreatment period (90 days) However both male and femalerats administered green mussel formulation (at 10 gkg and05 gkg resp) showed significantly low levels of differentialcounts (lymphocytes eosinophils and neutrophils) com-pared to control animals (119875 lt 005) Similarly femalerats administered 20 and 10 gkg green mussel formulationrecorded significantly low lymphocyte and neutrophil countrespectively (119875 lt 005) No test article-related changes inblood cell morphology were observed during the period ofstudy

346 Serum Biochemical Parameters Table 3 summarizesthe serum biochemical parameters used as the biomark-ers of the liver and renal functioning during the courseof subchronic toxicity studies The serum analysis showedsignificantly low SGOT content for the low dose male rats(05 gkg) and highlow dose female rats (25 and 05 gkg)after 90 days of subchronic study Similarly another markerenzyme of the liver ALP also showed significantly lowvalues for high and low dose group male rats compared tothe control animals after 90 days of subchronic study Theactivity of anothermarker enzyme SGPTwas not significantly

BioMed Research International 7

Normal male

(a)

Normal female

(b) (a1)

Treated male (2gkg body weight)

(b1)

Treated female (2gkg body weight)

Figure 3 The cross section of the male and female rats after subchronic toxicity study of 90 days (a) Normal male (a1) green musselformulation (20 gkg) treated male (b) normal female (b1) green mussel formulation (20 gkg) treated female

Table 2 Hematology analyses data of male and female rats administered the green mussel formulation after subchronic (90 days) toxicitystudies

Treatments Lymphocytes(mm3)

Eosinophils(mm3)

Neutrophils(mm3) HGB (gdL) WBC (mm3) RBC

(106cmm)Platelet

(105cmm)

Male

Controla 582860 plusmn 15914 99820 plusmn 6290 321320 plusmn 1713 1544 plusmn 005 10040 plusmn 586 787 plusmn 007 578 plusmn 006

20 gkgb 463800 plusmn 15450 75260 plusmn 3157 232940 plusmn 8304lowast 1460 plusmn 064 7720 plusmn 2670 741 plusmn 029 464 plusmn 038

10 gkgb 364380 plusmn 18747lowast 61860 plusmn 3324lowast 201760 plusmn 8802lowast 1434 plusmn 073 6280 plusmn 3090 729 plusmn 058 508 plusmn 004

05 gkgb 491220 plusmn 11407 77360 plusmn 1538 275420 plusmn 7898 1464 plusmn 070 8440 plusmn 17470 727 plusmn 008 578 plusmn 006

Female

Controla 522040 plusmn 2310 88880 plusmn 3778 273080 plusmn 8031 1338 plusmn 094 8840 plusmn 1489 637 plusmn 047 606 plusmn 003

20 gkgb 385640 plusmn 16793lowast 64540 plusmn 2894 229820 plusmn 11425 1412 plusmn 044 6800 plusmn 977 697 plusmn 003 578 plusmn 008

10 gkgb 497520 plusmn 26933 77000 plusmn 3175 197480 plusmn 8387lowast 1386 plusmn 083 7720 plusmn 1075 686 plusmn 021 530 plusmn 007

05 gkgb 212280 plusmn 10152lowast 32120 plusmn 1467lowast 109600 plusmn 5093lowast 1354 plusmn 053 3540 plusmn 1017lowast 677 plusmn 007 564 plusmn 006

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)HGB hemoglobin WBC total white blood cell count RBC red blood cell

different (119875 gt 005) in all dose groups of treated rats ascompared to untreated control and albuminglobulin (AG)ratio was not altered in the treated animals of both genders(Table 3)

Subchronic oral administration of the green mussel for-mulation (for 90 days) did not cause any significant changesin hepatic function parameters such as total protein albumintotal bilirubin and globulin in both sexes of rats duringlong-term subchronic toxicity studies The renal functionparameters such as serum creatinine and blood urea did notshow any significant variation (119875 gt 005) in treated animalscompared to controls (Table 3) There were no statisticallysignificant differences in the levels of serum electrolytessuch as chloride potassium sodium and bicarbonate afterthe treatment of green mussel formulation (119875 gt 005)indicating no expressive changes in the general metabolismafter consumption of the green mussel formulation by rats

(Table 4) The green mussel formulation did not produceany significant changes in the total cholesterol HDL LDLand VLDL indicating no expressive changes in the generallipid metabolism after consumption of the test article by rats(Table 4)

347 Histopathological Analysis Necropsy of the treatedanimals after sacrifice did not show any morphologicalchanges of the internal organs or any gross pathologi-cal abnormalities during subchronic toxicity studies Therewere no macroscopic findings considered to be relatedto the treatment of the green mussel formulation (Fig-ure 3) Gross examination of vital organs such as brainkidney spleen and liver of rats and microscopic exami-nation of tissue sections prepared from these organs didnot observe any histopathological alteration in any treatedrats during subchronic toxicity studies The normal and

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 3: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

BioMed Research International 3

23 Lethal Dose 50 (LD50) of Green Mussel Formulation

Fifteen animals were divided randomly into three groupscontaining 5 animals each After being fasted for 16 h theanimals were administered different doses of green musselformulation suspended in distilled water (5000 2500 and1500mgkg BW) and administered as a single dose throughoral gavage The animals were monitored for 14 days formortality clinical and behavioral symptoms and any adversereaction

24 Acute Oral Toxicity Study of Green Mussel FormulationForty animals (20 males and 20 females) were divided into 4groups each consisting of 5 male and 5 female rats and threedoses (25 10 and 05 gkg) of the green mussel formulationwere administered orally (once daily) for 14 daysThe controlreceived 1mL of water as vehicle every day The animals weremonitored for mortality clinical symptoms and any adversereaction of the test material The body weight and foodconsumption were determined in different time intervals (014 42 70 and 91 days) After 14 days the animals weresacrificed under mild ether anesthesia and the blood wascollected by direct heart puncture method Necropsy wasperformed and observations were recorded Selected organssuch as the liver kidney brain and spleen were dissected outweights were recorded and histopathological analyses wereperformed

25 Subchronic Oral Toxicity Study of Green Mussel Formula-tion Forty animals (20 males and 20 females) were dividedinto 4 groups each consisting of 5 male and 5 female ratsand three doses (20 10 and 05 gkg) of the green musselformulation (1 g suspended in 6mL double distilled water)were administered orally (once daily) for 90 days [16] Thecontrol received 1mL of water as vehicle every day The testanimals were monitored during this period for any type ofclinical symptoms mortality and adverse reactionThe bodyweight and food consumption were determined every sevendays On the 91st day the animals were sacrificed undermild ether anesthesia Blood was collected by direct heartpuncturemethod Necropsywas performed and observationswere recorded Selected organs such as the brain kidney liverand spleen were dissected out weights were recorded andhistopathological analyses were performed

26 Hematology and Clinical Chemistry Parameters Bloodcollected in EDTA tubes was analyzed for hematologicalparameters [17] Red blood cell (RBC) total white blood cellcount (WBC) platelet count and hemoglobin (HGB) weredetermined using a haematology analyzer (Model-Diatron 9Wein Austria) Total white blood cells were measured afterdiluting the blood in Turkrsquos fluid and counting them usinga hemocytometer [18] For differential counts (lymphocyteseosinophils and neutrophils) blood was spread on a cleanslide and treated with Leishmanrsquos stain before being countedmanually with a microscope (100x) [19]

A part of the bloodwas collected in nonheparinized tubesand serum was separated after centrifugation at 5000 rpmfor 10min which was used for the following investigations

Serumglutamic oxaloacetic transaminase (SGOT) and serumglutamic pyruvic transaminase (SGPT) were assayed accord-ing to themethoddescribed byBergmeyer et al [20] Alkalinephosphatase (ALP) was estimated by p-nitrophenyl phos-phate (PNPP) hydrolysis [21] Total bilirubin was determinedby Jendrassik-Diazotized sulphanilic acid method [22] Thetotal protein concentrationwas determined by biuretmethod[18] Albumin was determined based on its reaction withbromocresol green Markers of kidney function such ascreatinine and blood urea nitrogen were estimated by Jaffe-Kinetic and urease method respectively [23] Serum sodiumpotassium and bicarbonate were estimated using Flamephotometer 129 ion selective electrolyte analyzer Chloridewas estimated by mercuric thiocyanate method using a kitfrom Raichem Lifesciences Pvt Ltd India Total choles-terol was estimated by CHODndashPAP (cholesterol oxidasendashphenol + aminophenazone) enzymatic method [24] Triglyc-eride was estimated by GPOndashPAP (glycerol-3-phosphateoxidasendashphenol + aminophenazone) method [25] and high-density lipoprotein (HDL)was determined after precipitationwith phosphotungstic acid Very low-density lipoprotein(VLDL) was estimated by the Friedewald equation (VLDL =triglyceride5) and low-density lipoprotein (LDL) by calcula-tion LDL = total cholesterol minus (HDL + VLDL) [16]

27 Histopathological Analysis A portion of the selectedorgans (brain kidney liver and spleen) of control and treatedgroup (high dose groups) were fixed in 10 neutral bufferedformalin Embedded organs tissue samples were cut intoslices of 2ndash4120583m and stained with hematoxylin-eosin and thesections were observed under light microscope (40x)

28 Statistical Analysis Statistical evaluation was carried outwith the Statistical Program for Social Sciences 130 (SPSSInc Chicago USA ver 130) Analyses were carried out intriplicate and the means of all parameters were examinedfor significance by analysis of variance (ANOVA)The valueswere compared with that of untreated control animals

3 Results

31 Anti-Inflammatory Activities of Green Mussel Formu-lation The green mussel formulation (1mgmL) showedinhibiting properties against proinflammatory COXII (50)and LOXV enzymes (47) and the activities were foundto be comparable with standard NSAIDs (Figure 1(a)) Inthis study greenmussel formulation showed lower inhibitionof COXI (41 1mgmL) than synthetic NSAIDs (gt50)Notably the animals challenged with the green mussel for-mulation significantly mitigated (119875 lt 005) the carrageenan-induced paw edema in a time-dependent manner till theend of the 6th h as compared to negative control animalsthroughout the period of study (Figure 1(b))

32 LD50

of Green Mussel Formulation The single doseadministration of the green mussel formulation up to aconcentration of 5000mgkg BW did not produce anymortality after 14 days of observation which indicates that

4 BioMed Research International

100

80

60

40

20

0

05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL

Inhi

bitio

n of

infla

mm

ator

y en

zym

es (

)

COX-I inhibition activity () COX-II inhibition activity () LOX-V inhibition activity ()

IndomethacinAspirin

Green mussel formulation

(a)

0

40

80

120

160

2 3 4 5 6

Diff

eren

ce in

paw

edem

a (

)

Time (h)

Normal salineAspirinGreen mussel formulation

Oral administration of the standard (aspirin) and the test samples

Untreated rat (control)

Reduced volume of paw edema in carrageenan-induced rat

Green mussel extract treated rat

No reduction in paw edemavolume of carrageenantreated rat

Carrageenan (1 in saline 01mLanimal) injection

(subcutaneously into the right hind paw)

thirty minutes after the oral administration of test samples

(b)

Figure 1 (a) In vitro anti-inflammatory activities (COXIII and LOXV inhibition activities) of the green mussel formulation compared withstandard anti-inflammatory drugs aspirin and indomethacin at different concentrations (05 1 and 5mgmL) (b) In vivo anti-inflammatoryactivity ( difference in paw edema compared to control group) of the green mussel formulation compared to standard anti-inflammatorydrug aspirin

themean lethal dose (LD50) of the formulation is greater than

5000mgkg BW The oral toxicity of this formulation can beclassified in category 5 (the lethal acute toxicity is greaterthan 5000mgkg) according to the Globally HarmonizedClassification System of OECD [26]

33 Acute Toxicity Study of Green Mussel Formulation Notreatment-related signs of mortality were observed in theanimals over short-term administration (maximum dose of2500mgkg BW) In addition the administration of thegreen mussel formulation at different doses did not produce

BioMed Research International 5

Table 1 Body weight and food and water consumption during subchronic (90 days) toxicity studies after the administration of green musselformulation

Days Male FemaleControla 20 gkgb 10 gkgb 050 gkgb Controla 20 gkgb 10 gkgb 050 gkgb

Body weight (g)0 2308 plusmn 14 2549 plusmn 25 2636 plusmn 142 2844 plusmn 05 1858 plusmn 15 2185 plusmn 156 1958 plusmn 14 1909 plusmn 52

14 2396 plusmn 45 2618 plusmn 15 2704 plusmn 22 2901 plusmn 14 1924 plusmn 24 2244 plusmn 125 2021 plusmn 26 1968 plusmn 62

42 2524 plusmn 66 2739 plusmn 65 281 plusmn 186 300 plusmn 15 204 plusmn 26 2326 plusmn 116 2121 plusmn 32 2069 plusmn 26

70 2638 plusmn 85lowast2836 plusmn 15

lowast2902 plusmn 195 3084 plusmn 16

lowast2142 plusmn 26

lowast2409 plusmn 114

lowast2207 plusmn 12

lowast2162 plusmn 24

lowast

91 27158 plusmn 142lowast2908 plusmn 16

lowast2967 plusmn 21

lowast3145 plusmn 12

lowast2211 plusmn 09

lowast2466 plusmn 05

lowast2268 plusmn 05

lowast2225 plusmn 29

lowast

Food consumption (g)0 724 plusmn 56 744 plusmn 25 706 plusmn 65 687 plusmn 12 595 plusmn 06 554 plusmn 16 58 plusmn 23 627 plusmn 09

14 664 plusmn 65 733 plusmn 36 626 plusmn 63 613 plusmn 15 625 plusmn 12 493 plusmn 26 546 plusmn 26 577 plusmn 12

42 673 plusmn 69 716 plusmn 39 614 plusmn 42 606 plusmn 25 476 plusmn 11 506 plusmn 27 483 plusmn 15 50 plusmn 13

70 666 plusmn 79 727 plusmn 38 623 plusmn 32 625 plusmn 21 479 plusmn 15 513 plusmn 24 49 plusmn 26 497 plusmn 14

91 683 plusmn 85 747 plusmn 36 632 plusmn 26 655 plusmn 2 487 plusmn 13 508 plusmn 21 497 plusmn 28 50 plusmn 15

Water consumption (mL)0 100 plusmn 4 90 plusmn 2 100 plusmn 2 110 plusmn 3 100 plusmn 2 80 plusmn 2 110 plusmn 4 90 plusmn 2

14 90 plusmn 3 70 plusmn 5 110 plusmn 3 100 plusmn 1 80 plusmn 4 80 plusmn 1 70 plusmn 5 90 plusmn 2

42 90 plusmn 2 110 plusmn 3 90 plusmn 1 100 plusmn 2 100 plusmn 3 80 plusmn 4 90 plusmn 1 90 plusmn 3

70 110 plusmn 3 110 plusmn 1 100 plusmn 5 100 plusmn 3 100 plusmn 2 100 plusmn 1 90 plusmn 2 110 plusmn 3

91 100 plusmn 4 100 plusmn 2 110 plusmn 1 100 plusmn 4 100 plusmn 1 110 plusmn 2 100 plusmn 2 100 plusmn 3

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)

any treatment-related changes in the body weight of theanimals or any differences in the food consumption of maleand female rats when compared to controls No significantchanges were noticed during necropsy and there was nochange in the organ weight

No treatment-related biologically significant effects of thegreen mussel formulation treatment at dose levels of 05ndash25 gkg in hematology parameters such as hemoglobin RBCcount platelet count and total and differential leukocytescounts were apparent in both genders of rats when comparedto untreated animals

The green mussel formulation up to a concentrationof 25 gkg did not produce any change in the hepaticfunction parameters in serum such as SGOT SGPT ALPtotal protein bilirubin albumin and globulinas well as inalbuminglobulin (AG) ratio

The renal function tests such as blood urea and serumcreatinine did not show any variation when compared tocontrols There was also no change in serum electrolytessodium potassium chloride and bicarbonate indicating thatthe green mussel formulation did not produce any change inrenal function

Acute toxicity study of the green mussel formulation didnot show any change in cholesterol triglycerides HDL LDLVLDL and cholesterol levels Histopathological analysis ofthe brain spleen kidney and liver did not show any patho-logical lesions in the organs of animals treated with the greenmussel formulation

The above observations concluded that the green musselformulation did not produce any toxicity toWistar rats whenadministered for two weeks

34 Subchronic Toxicity Study of Green Mussel Formulation

341 General Conditions and Behavior No treatment-relatedsigns of mortality were observed in the animals over theadministration periods (maximum dose of 2000mgkg BW)In addition the administration of the green mussel formu-lation at different doses did not produce any treatment-related changes in clinical signs such as mental state externalappearance and daily activities among the test groups whencompared with the control Any abnormal behavior or casesof diarrhea and soft feceswere not observed during the periodof study No ophthalmological abnormalities were observedin any of the treatment groups prior to study initiation andnear experimental completion In general the experimentalanimals from all treatment groups appeared healthy at theconclusion of the study period and did not induce any clinicalsigns of toxicity in subchronic regimens

342 Body Weight The administration of the green musselformulation during 90 days of long-term subchronic toxicitystudies did not produce any abnormal change in the bodyweight of male and female rats when compared to the control(Table 1) As expected rats gained weight with time In malerats the gain in mean body weights for the treated groups

6 BioMed Research International

0

1

2

3

4O

rgan

wei

ght (

g)

LiverKidney

SpleenBrain

25

gkg

10

gkg

05

gkg

Con

trol

25

gkg

10

gkg

05

gkg

Con

trol

Male Female

(a)

0

1

2

3

4

Org

an w

eigh

t (g)

LiverKidney

SpleenBrain

10

gkg

05

gkg

Con

trol

20

gkg

10

gkg

05

gkg

Con

trol

20

gkg

Males Females

(b)

Figure 2 Mean organ weights (in grams) of male and female rats administered green mussel formulation after (a) acute and (b) subchronictoxicity studies

at 05ndash20 gkg BW was comparable with those in the controlgroup throughout the study Similarly themean bodyweightsof the treated female rats were comparable with those in thecontrol group throughout the studyTherewere no changes inbody weight in the animals attributable to the administrationof the green mussel formulation when compared to the con-trol group Any changes observed were sporadic consideredincidental and unassociated with test article administration

343 Food andWater Consumption The average food intakeof untreated control rats decreased from about 72 g to 68 g(for male rats) and from 60 g to 49 g (for female rats) after 90days of study The same trend was observed for medium andlow dose group (10 and 05 gkg resp) of males and all thedose groups of females Administration of the green musselformulation did not produce any significant difference in thefood consumption of both genders of rats when compared tonormal animals of the high dose group (119875 gt 005) throughoutthe experimental period The summarized food intake of therats recorded after oral administration of the green musselformulation to rats is shown in Table 1 Similarly the waterconsumption did not alter inmale and female rats attributableto administration of the green mussel formulation whencompared to normal animals during chronic and subchronictoxicity studies Changes in the average water consumptionduring the treatment period are presented in Table 1 Spo-radic statistically significant changes in water consumptionwere considered spurious unassociated with the test articleadministration

344 Relative Organ Weight Figure 2 presents the relativeweights of the vital organs (in g) of rats (both genders) Theweights of liver kidney spleen and brain recorded at theend of the subchronic study (day 91) did not show significantdifferences (119875 gt 005) in any of the treatment groups

compared with the control groups (Figure 2) Furthermoregross examination of the vital organs of all rats revealed nodetectable abnormalities

345 Hematological Parameters The effect of the greenmussel formulation on hematological parameters such ashemoglobin (HGB) RBCandWBCcount platelet count anddifferential counts after subchronic toxicity (90 days) studiesis presented in Table 2 No treatment-related biologicallysignificant effects of the green mussel formulation treatmentat dose levels of 05ndash20 gkg in the hemoglobin and RBCand platelet content were apparent in both genders of ratswhen compared to untreated animals (Table 2) (119875 gt 005)and remained within physiological range throughout thetreatment period (90 days) However both male and femalerats administered green mussel formulation (at 10 gkg and05 gkg resp) showed significantly low levels of differentialcounts (lymphocytes eosinophils and neutrophils) com-pared to control animals (119875 lt 005) Similarly femalerats administered 20 and 10 gkg green mussel formulationrecorded significantly low lymphocyte and neutrophil countrespectively (119875 lt 005) No test article-related changes inblood cell morphology were observed during the period ofstudy

346 Serum Biochemical Parameters Table 3 summarizesthe serum biochemical parameters used as the biomark-ers of the liver and renal functioning during the courseof subchronic toxicity studies The serum analysis showedsignificantly low SGOT content for the low dose male rats(05 gkg) and highlow dose female rats (25 and 05 gkg)after 90 days of subchronic study Similarly another markerenzyme of the liver ALP also showed significantly lowvalues for high and low dose group male rats compared tothe control animals after 90 days of subchronic study Theactivity of anothermarker enzyme SGPTwas not significantly

BioMed Research International 7

Normal male

(a)

Normal female

(b) (a1)

Treated male (2gkg body weight)

(b1)

Treated female (2gkg body weight)

Figure 3 The cross section of the male and female rats after subchronic toxicity study of 90 days (a) Normal male (a1) green musselformulation (20 gkg) treated male (b) normal female (b1) green mussel formulation (20 gkg) treated female

Table 2 Hematology analyses data of male and female rats administered the green mussel formulation after subchronic (90 days) toxicitystudies

Treatments Lymphocytes(mm3)

Eosinophils(mm3)

Neutrophils(mm3) HGB (gdL) WBC (mm3) RBC

(106cmm)Platelet

(105cmm)

Male

Controla 582860 plusmn 15914 99820 plusmn 6290 321320 plusmn 1713 1544 plusmn 005 10040 plusmn 586 787 plusmn 007 578 plusmn 006

20 gkgb 463800 plusmn 15450 75260 plusmn 3157 232940 plusmn 8304lowast 1460 plusmn 064 7720 plusmn 2670 741 plusmn 029 464 plusmn 038

10 gkgb 364380 plusmn 18747lowast 61860 plusmn 3324lowast 201760 plusmn 8802lowast 1434 plusmn 073 6280 plusmn 3090 729 plusmn 058 508 plusmn 004

05 gkgb 491220 plusmn 11407 77360 plusmn 1538 275420 plusmn 7898 1464 plusmn 070 8440 plusmn 17470 727 plusmn 008 578 plusmn 006

Female

Controla 522040 plusmn 2310 88880 plusmn 3778 273080 plusmn 8031 1338 plusmn 094 8840 plusmn 1489 637 plusmn 047 606 plusmn 003

20 gkgb 385640 plusmn 16793lowast 64540 plusmn 2894 229820 plusmn 11425 1412 plusmn 044 6800 plusmn 977 697 plusmn 003 578 plusmn 008

10 gkgb 497520 plusmn 26933 77000 plusmn 3175 197480 plusmn 8387lowast 1386 plusmn 083 7720 plusmn 1075 686 plusmn 021 530 plusmn 007

05 gkgb 212280 plusmn 10152lowast 32120 plusmn 1467lowast 109600 plusmn 5093lowast 1354 plusmn 053 3540 plusmn 1017lowast 677 plusmn 007 564 plusmn 006

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)HGB hemoglobin WBC total white blood cell count RBC red blood cell

different (119875 gt 005) in all dose groups of treated rats ascompared to untreated control and albuminglobulin (AG)ratio was not altered in the treated animals of both genders(Table 3)

Subchronic oral administration of the green mussel for-mulation (for 90 days) did not cause any significant changesin hepatic function parameters such as total protein albumintotal bilirubin and globulin in both sexes of rats duringlong-term subchronic toxicity studies The renal functionparameters such as serum creatinine and blood urea did notshow any significant variation (119875 gt 005) in treated animalscompared to controls (Table 3) There were no statisticallysignificant differences in the levels of serum electrolytessuch as chloride potassium sodium and bicarbonate afterthe treatment of green mussel formulation (119875 gt 005)indicating no expressive changes in the general metabolismafter consumption of the green mussel formulation by rats

(Table 4) The green mussel formulation did not produceany significant changes in the total cholesterol HDL LDLand VLDL indicating no expressive changes in the generallipid metabolism after consumption of the test article by rats(Table 4)

347 Histopathological Analysis Necropsy of the treatedanimals after sacrifice did not show any morphologicalchanges of the internal organs or any gross pathologi-cal abnormalities during subchronic toxicity studies Therewere no macroscopic findings considered to be relatedto the treatment of the green mussel formulation (Fig-ure 3) Gross examination of vital organs such as brainkidney spleen and liver of rats and microscopic exami-nation of tissue sections prepared from these organs didnot observe any histopathological alteration in any treatedrats during subchronic toxicity studies The normal and

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

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Page 4: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

4 BioMed Research International

100

80

60

40

20

0

05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL 05mgmL 1mgmL 5mgmL

Inhi

bitio

n of

infla

mm

ator

y en

zym

es (

)

COX-I inhibition activity () COX-II inhibition activity () LOX-V inhibition activity ()

IndomethacinAspirin

Green mussel formulation

(a)

0

40

80

120

160

2 3 4 5 6

Diff

eren

ce in

paw

edem

a (

)

Time (h)

Normal salineAspirinGreen mussel formulation

Oral administration of the standard (aspirin) and the test samples

Untreated rat (control)

Reduced volume of paw edema in carrageenan-induced rat

Green mussel extract treated rat

No reduction in paw edemavolume of carrageenantreated rat

Carrageenan (1 in saline 01mLanimal) injection

(subcutaneously into the right hind paw)

thirty minutes after the oral administration of test samples

(b)

Figure 1 (a) In vitro anti-inflammatory activities (COXIII and LOXV inhibition activities) of the green mussel formulation compared withstandard anti-inflammatory drugs aspirin and indomethacin at different concentrations (05 1 and 5mgmL) (b) In vivo anti-inflammatoryactivity ( difference in paw edema compared to control group) of the green mussel formulation compared to standard anti-inflammatorydrug aspirin

themean lethal dose (LD50) of the formulation is greater than

5000mgkg BW The oral toxicity of this formulation can beclassified in category 5 (the lethal acute toxicity is greaterthan 5000mgkg) according to the Globally HarmonizedClassification System of OECD [26]

33 Acute Toxicity Study of Green Mussel Formulation Notreatment-related signs of mortality were observed in theanimals over short-term administration (maximum dose of2500mgkg BW) In addition the administration of thegreen mussel formulation at different doses did not produce

BioMed Research International 5

Table 1 Body weight and food and water consumption during subchronic (90 days) toxicity studies after the administration of green musselformulation

Days Male FemaleControla 20 gkgb 10 gkgb 050 gkgb Controla 20 gkgb 10 gkgb 050 gkgb

Body weight (g)0 2308 plusmn 14 2549 plusmn 25 2636 plusmn 142 2844 plusmn 05 1858 plusmn 15 2185 plusmn 156 1958 plusmn 14 1909 plusmn 52

14 2396 plusmn 45 2618 plusmn 15 2704 plusmn 22 2901 plusmn 14 1924 plusmn 24 2244 plusmn 125 2021 plusmn 26 1968 plusmn 62

42 2524 plusmn 66 2739 plusmn 65 281 plusmn 186 300 plusmn 15 204 plusmn 26 2326 plusmn 116 2121 plusmn 32 2069 plusmn 26

70 2638 plusmn 85lowast2836 plusmn 15

lowast2902 plusmn 195 3084 plusmn 16

lowast2142 plusmn 26

lowast2409 plusmn 114

lowast2207 plusmn 12

lowast2162 plusmn 24

lowast

91 27158 plusmn 142lowast2908 plusmn 16

lowast2967 plusmn 21

lowast3145 plusmn 12

lowast2211 plusmn 09

lowast2466 plusmn 05

lowast2268 plusmn 05

lowast2225 plusmn 29

lowast

Food consumption (g)0 724 plusmn 56 744 plusmn 25 706 plusmn 65 687 plusmn 12 595 plusmn 06 554 plusmn 16 58 plusmn 23 627 plusmn 09

14 664 plusmn 65 733 plusmn 36 626 plusmn 63 613 plusmn 15 625 plusmn 12 493 plusmn 26 546 plusmn 26 577 plusmn 12

42 673 plusmn 69 716 plusmn 39 614 plusmn 42 606 plusmn 25 476 plusmn 11 506 plusmn 27 483 plusmn 15 50 plusmn 13

70 666 plusmn 79 727 plusmn 38 623 plusmn 32 625 plusmn 21 479 plusmn 15 513 plusmn 24 49 plusmn 26 497 plusmn 14

91 683 plusmn 85 747 plusmn 36 632 plusmn 26 655 plusmn 2 487 plusmn 13 508 plusmn 21 497 plusmn 28 50 plusmn 15

Water consumption (mL)0 100 plusmn 4 90 plusmn 2 100 plusmn 2 110 plusmn 3 100 plusmn 2 80 plusmn 2 110 plusmn 4 90 plusmn 2

14 90 plusmn 3 70 plusmn 5 110 plusmn 3 100 plusmn 1 80 plusmn 4 80 plusmn 1 70 plusmn 5 90 plusmn 2

42 90 plusmn 2 110 plusmn 3 90 plusmn 1 100 plusmn 2 100 plusmn 3 80 plusmn 4 90 plusmn 1 90 plusmn 3

70 110 plusmn 3 110 plusmn 1 100 plusmn 5 100 plusmn 3 100 plusmn 2 100 plusmn 1 90 plusmn 2 110 plusmn 3

91 100 plusmn 4 100 plusmn 2 110 plusmn 1 100 plusmn 4 100 plusmn 1 110 plusmn 2 100 plusmn 2 100 plusmn 3

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)

any treatment-related changes in the body weight of theanimals or any differences in the food consumption of maleand female rats when compared to controls No significantchanges were noticed during necropsy and there was nochange in the organ weight

No treatment-related biologically significant effects of thegreen mussel formulation treatment at dose levels of 05ndash25 gkg in hematology parameters such as hemoglobin RBCcount platelet count and total and differential leukocytescounts were apparent in both genders of rats when comparedto untreated animals

The green mussel formulation up to a concentrationof 25 gkg did not produce any change in the hepaticfunction parameters in serum such as SGOT SGPT ALPtotal protein bilirubin albumin and globulinas well as inalbuminglobulin (AG) ratio

The renal function tests such as blood urea and serumcreatinine did not show any variation when compared tocontrols There was also no change in serum electrolytessodium potassium chloride and bicarbonate indicating thatthe green mussel formulation did not produce any change inrenal function

Acute toxicity study of the green mussel formulation didnot show any change in cholesterol triglycerides HDL LDLVLDL and cholesterol levels Histopathological analysis ofthe brain spleen kidney and liver did not show any patho-logical lesions in the organs of animals treated with the greenmussel formulation

The above observations concluded that the green musselformulation did not produce any toxicity toWistar rats whenadministered for two weeks

34 Subchronic Toxicity Study of Green Mussel Formulation

341 General Conditions and Behavior No treatment-relatedsigns of mortality were observed in the animals over theadministration periods (maximum dose of 2000mgkg BW)In addition the administration of the green mussel formu-lation at different doses did not produce any treatment-related changes in clinical signs such as mental state externalappearance and daily activities among the test groups whencompared with the control Any abnormal behavior or casesof diarrhea and soft feceswere not observed during the periodof study No ophthalmological abnormalities were observedin any of the treatment groups prior to study initiation andnear experimental completion In general the experimentalanimals from all treatment groups appeared healthy at theconclusion of the study period and did not induce any clinicalsigns of toxicity in subchronic regimens

342 Body Weight The administration of the green musselformulation during 90 days of long-term subchronic toxicitystudies did not produce any abnormal change in the bodyweight of male and female rats when compared to the control(Table 1) As expected rats gained weight with time In malerats the gain in mean body weights for the treated groups

6 BioMed Research International

0

1

2

3

4O

rgan

wei

ght (

g)

LiverKidney

SpleenBrain

25

gkg

10

gkg

05

gkg

Con

trol

25

gkg

10

gkg

05

gkg

Con

trol

Male Female

(a)

0

1

2

3

4

Org

an w

eigh

t (g)

LiverKidney

SpleenBrain

10

gkg

05

gkg

Con

trol

20

gkg

10

gkg

05

gkg

Con

trol

20

gkg

Males Females

(b)

Figure 2 Mean organ weights (in grams) of male and female rats administered green mussel formulation after (a) acute and (b) subchronictoxicity studies

at 05ndash20 gkg BW was comparable with those in the controlgroup throughout the study Similarly themean bodyweightsof the treated female rats were comparable with those in thecontrol group throughout the studyTherewere no changes inbody weight in the animals attributable to the administrationof the green mussel formulation when compared to the con-trol group Any changes observed were sporadic consideredincidental and unassociated with test article administration

343 Food andWater Consumption The average food intakeof untreated control rats decreased from about 72 g to 68 g(for male rats) and from 60 g to 49 g (for female rats) after 90days of study The same trend was observed for medium andlow dose group (10 and 05 gkg resp) of males and all thedose groups of females Administration of the green musselformulation did not produce any significant difference in thefood consumption of both genders of rats when compared tonormal animals of the high dose group (119875 gt 005) throughoutthe experimental period The summarized food intake of therats recorded after oral administration of the green musselformulation to rats is shown in Table 1 Similarly the waterconsumption did not alter inmale and female rats attributableto administration of the green mussel formulation whencompared to normal animals during chronic and subchronictoxicity studies Changes in the average water consumptionduring the treatment period are presented in Table 1 Spo-radic statistically significant changes in water consumptionwere considered spurious unassociated with the test articleadministration

344 Relative Organ Weight Figure 2 presents the relativeweights of the vital organs (in g) of rats (both genders) Theweights of liver kidney spleen and brain recorded at theend of the subchronic study (day 91) did not show significantdifferences (119875 gt 005) in any of the treatment groups

compared with the control groups (Figure 2) Furthermoregross examination of the vital organs of all rats revealed nodetectable abnormalities

345 Hematological Parameters The effect of the greenmussel formulation on hematological parameters such ashemoglobin (HGB) RBCandWBCcount platelet count anddifferential counts after subchronic toxicity (90 days) studiesis presented in Table 2 No treatment-related biologicallysignificant effects of the green mussel formulation treatmentat dose levels of 05ndash20 gkg in the hemoglobin and RBCand platelet content were apparent in both genders of ratswhen compared to untreated animals (Table 2) (119875 gt 005)and remained within physiological range throughout thetreatment period (90 days) However both male and femalerats administered green mussel formulation (at 10 gkg and05 gkg resp) showed significantly low levels of differentialcounts (lymphocytes eosinophils and neutrophils) com-pared to control animals (119875 lt 005) Similarly femalerats administered 20 and 10 gkg green mussel formulationrecorded significantly low lymphocyte and neutrophil countrespectively (119875 lt 005) No test article-related changes inblood cell morphology were observed during the period ofstudy

346 Serum Biochemical Parameters Table 3 summarizesthe serum biochemical parameters used as the biomark-ers of the liver and renal functioning during the courseof subchronic toxicity studies The serum analysis showedsignificantly low SGOT content for the low dose male rats(05 gkg) and highlow dose female rats (25 and 05 gkg)after 90 days of subchronic study Similarly another markerenzyme of the liver ALP also showed significantly lowvalues for high and low dose group male rats compared tothe control animals after 90 days of subchronic study Theactivity of anothermarker enzyme SGPTwas not significantly

BioMed Research International 7

Normal male

(a)

Normal female

(b) (a1)

Treated male (2gkg body weight)

(b1)

Treated female (2gkg body weight)

Figure 3 The cross section of the male and female rats after subchronic toxicity study of 90 days (a) Normal male (a1) green musselformulation (20 gkg) treated male (b) normal female (b1) green mussel formulation (20 gkg) treated female

Table 2 Hematology analyses data of male and female rats administered the green mussel formulation after subchronic (90 days) toxicitystudies

Treatments Lymphocytes(mm3)

Eosinophils(mm3)

Neutrophils(mm3) HGB (gdL) WBC (mm3) RBC

(106cmm)Platelet

(105cmm)

Male

Controla 582860 plusmn 15914 99820 plusmn 6290 321320 plusmn 1713 1544 plusmn 005 10040 plusmn 586 787 plusmn 007 578 plusmn 006

20 gkgb 463800 plusmn 15450 75260 plusmn 3157 232940 plusmn 8304lowast 1460 plusmn 064 7720 plusmn 2670 741 plusmn 029 464 plusmn 038

10 gkgb 364380 plusmn 18747lowast 61860 plusmn 3324lowast 201760 plusmn 8802lowast 1434 plusmn 073 6280 plusmn 3090 729 plusmn 058 508 plusmn 004

05 gkgb 491220 plusmn 11407 77360 plusmn 1538 275420 plusmn 7898 1464 plusmn 070 8440 plusmn 17470 727 plusmn 008 578 plusmn 006

Female

Controla 522040 plusmn 2310 88880 plusmn 3778 273080 plusmn 8031 1338 plusmn 094 8840 plusmn 1489 637 plusmn 047 606 plusmn 003

20 gkgb 385640 plusmn 16793lowast 64540 plusmn 2894 229820 plusmn 11425 1412 plusmn 044 6800 plusmn 977 697 plusmn 003 578 plusmn 008

10 gkgb 497520 plusmn 26933 77000 plusmn 3175 197480 plusmn 8387lowast 1386 plusmn 083 7720 plusmn 1075 686 plusmn 021 530 plusmn 007

05 gkgb 212280 plusmn 10152lowast 32120 plusmn 1467lowast 109600 plusmn 5093lowast 1354 plusmn 053 3540 plusmn 1017lowast 677 plusmn 007 564 plusmn 006

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)HGB hemoglobin WBC total white blood cell count RBC red blood cell

different (119875 gt 005) in all dose groups of treated rats ascompared to untreated control and albuminglobulin (AG)ratio was not altered in the treated animals of both genders(Table 3)

Subchronic oral administration of the green mussel for-mulation (for 90 days) did not cause any significant changesin hepatic function parameters such as total protein albumintotal bilirubin and globulin in both sexes of rats duringlong-term subchronic toxicity studies The renal functionparameters such as serum creatinine and blood urea did notshow any significant variation (119875 gt 005) in treated animalscompared to controls (Table 3) There were no statisticallysignificant differences in the levels of serum electrolytessuch as chloride potassium sodium and bicarbonate afterthe treatment of green mussel formulation (119875 gt 005)indicating no expressive changes in the general metabolismafter consumption of the green mussel formulation by rats

(Table 4) The green mussel formulation did not produceany significant changes in the total cholesterol HDL LDLand VLDL indicating no expressive changes in the generallipid metabolism after consumption of the test article by rats(Table 4)

347 Histopathological Analysis Necropsy of the treatedanimals after sacrifice did not show any morphologicalchanges of the internal organs or any gross pathologi-cal abnormalities during subchronic toxicity studies Therewere no macroscopic findings considered to be relatedto the treatment of the green mussel formulation (Fig-ure 3) Gross examination of vital organs such as brainkidney spleen and liver of rats and microscopic exami-nation of tissue sections prepared from these organs didnot observe any histopathological alteration in any treatedrats during subchronic toxicity studies The normal and

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

Page 5: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

BioMed Research International 5

Table 1 Body weight and food and water consumption during subchronic (90 days) toxicity studies after the administration of green musselformulation

Days Male FemaleControla 20 gkgb 10 gkgb 050 gkgb Controla 20 gkgb 10 gkgb 050 gkgb

Body weight (g)0 2308 plusmn 14 2549 plusmn 25 2636 plusmn 142 2844 plusmn 05 1858 plusmn 15 2185 plusmn 156 1958 plusmn 14 1909 plusmn 52

14 2396 plusmn 45 2618 plusmn 15 2704 plusmn 22 2901 plusmn 14 1924 plusmn 24 2244 plusmn 125 2021 plusmn 26 1968 plusmn 62

42 2524 plusmn 66 2739 plusmn 65 281 plusmn 186 300 plusmn 15 204 plusmn 26 2326 plusmn 116 2121 plusmn 32 2069 plusmn 26

70 2638 plusmn 85lowast2836 plusmn 15

lowast2902 plusmn 195 3084 plusmn 16

lowast2142 plusmn 26

lowast2409 plusmn 114

lowast2207 plusmn 12

lowast2162 plusmn 24

lowast

91 27158 plusmn 142lowast2908 plusmn 16

lowast2967 plusmn 21

lowast3145 plusmn 12

lowast2211 plusmn 09

lowast2466 plusmn 05

lowast2268 plusmn 05

lowast2225 plusmn 29

lowast

Food consumption (g)0 724 plusmn 56 744 plusmn 25 706 plusmn 65 687 plusmn 12 595 plusmn 06 554 plusmn 16 58 plusmn 23 627 plusmn 09

14 664 plusmn 65 733 plusmn 36 626 plusmn 63 613 plusmn 15 625 plusmn 12 493 plusmn 26 546 plusmn 26 577 plusmn 12

42 673 plusmn 69 716 plusmn 39 614 plusmn 42 606 plusmn 25 476 plusmn 11 506 plusmn 27 483 plusmn 15 50 plusmn 13

70 666 plusmn 79 727 plusmn 38 623 plusmn 32 625 plusmn 21 479 plusmn 15 513 plusmn 24 49 plusmn 26 497 plusmn 14

91 683 plusmn 85 747 plusmn 36 632 plusmn 26 655 plusmn 2 487 plusmn 13 508 plusmn 21 497 plusmn 28 50 plusmn 15

Water consumption (mL)0 100 plusmn 4 90 plusmn 2 100 plusmn 2 110 plusmn 3 100 plusmn 2 80 plusmn 2 110 plusmn 4 90 plusmn 2

14 90 plusmn 3 70 plusmn 5 110 plusmn 3 100 plusmn 1 80 plusmn 4 80 plusmn 1 70 plusmn 5 90 plusmn 2

42 90 plusmn 2 110 plusmn 3 90 plusmn 1 100 plusmn 2 100 plusmn 3 80 plusmn 4 90 plusmn 1 90 plusmn 3

70 110 plusmn 3 110 plusmn 1 100 plusmn 5 100 plusmn 3 100 plusmn 2 100 plusmn 1 90 plusmn 2 110 plusmn 3

91 100 plusmn 4 100 plusmn 2 110 plusmn 1 100 plusmn 4 100 plusmn 1 110 plusmn 2 100 plusmn 2 100 plusmn 3

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)

any treatment-related changes in the body weight of theanimals or any differences in the food consumption of maleand female rats when compared to controls No significantchanges were noticed during necropsy and there was nochange in the organ weight

No treatment-related biologically significant effects of thegreen mussel formulation treatment at dose levels of 05ndash25 gkg in hematology parameters such as hemoglobin RBCcount platelet count and total and differential leukocytescounts were apparent in both genders of rats when comparedto untreated animals

The green mussel formulation up to a concentrationof 25 gkg did not produce any change in the hepaticfunction parameters in serum such as SGOT SGPT ALPtotal protein bilirubin albumin and globulinas well as inalbuminglobulin (AG) ratio

The renal function tests such as blood urea and serumcreatinine did not show any variation when compared tocontrols There was also no change in serum electrolytessodium potassium chloride and bicarbonate indicating thatthe green mussel formulation did not produce any change inrenal function

Acute toxicity study of the green mussel formulation didnot show any change in cholesterol triglycerides HDL LDLVLDL and cholesterol levels Histopathological analysis ofthe brain spleen kidney and liver did not show any patho-logical lesions in the organs of animals treated with the greenmussel formulation

The above observations concluded that the green musselformulation did not produce any toxicity toWistar rats whenadministered for two weeks

34 Subchronic Toxicity Study of Green Mussel Formulation

341 General Conditions and Behavior No treatment-relatedsigns of mortality were observed in the animals over theadministration periods (maximum dose of 2000mgkg BW)In addition the administration of the green mussel formu-lation at different doses did not produce any treatment-related changes in clinical signs such as mental state externalappearance and daily activities among the test groups whencompared with the control Any abnormal behavior or casesof diarrhea and soft feceswere not observed during the periodof study No ophthalmological abnormalities were observedin any of the treatment groups prior to study initiation andnear experimental completion In general the experimentalanimals from all treatment groups appeared healthy at theconclusion of the study period and did not induce any clinicalsigns of toxicity in subchronic regimens

342 Body Weight The administration of the green musselformulation during 90 days of long-term subchronic toxicitystudies did not produce any abnormal change in the bodyweight of male and female rats when compared to the control(Table 1) As expected rats gained weight with time In malerats the gain in mean body weights for the treated groups

6 BioMed Research International

0

1

2

3

4O

rgan

wei

ght (

g)

LiverKidney

SpleenBrain

25

gkg

10

gkg

05

gkg

Con

trol

25

gkg

10

gkg

05

gkg

Con

trol

Male Female

(a)

0

1

2

3

4

Org

an w

eigh

t (g)

LiverKidney

SpleenBrain

10

gkg

05

gkg

Con

trol

20

gkg

10

gkg

05

gkg

Con

trol

20

gkg

Males Females

(b)

Figure 2 Mean organ weights (in grams) of male and female rats administered green mussel formulation after (a) acute and (b) subchronictoxicity studies

at 05ndash20 gkg BW was comparable with those in the controlgroup throughout the study Similarly themean bodyweightsof the treated female rats were comparable with those in thecontrol group throughout the studyTherewere no changes inbody weight in the animals attributable to the administrationof the green mussel formulation when compared to the con-trol group Any changes observed were sporadic consideredincidental and unassociated with test article administration

343 Food andWater Consumption The average food intakeof untreated control rats decreased from about 72 g to 68 g(for male rats) and from 60 g to 49 g (for female rats) after 90days of study The same trend was observed for medium andlow dose group (10 and 05 gkg resp) of males and all thedose groups of females Administration of the green musselformulation did not produce any significant difference in thefood consumption of both genders of rats when compared tonormal animals of the high dose group (119875 gt 005) throughoutthe experimental period The summarized food intake of therats recorded after oral administration of the green musselformulation to rats is shown in Table 1 Similarly the waterconsumption did not alter inmale and female rats attributableto administration of the green mussel formulation whencompared to normal animals during chronic and subchronictoxicity studies Changes in the average water consumptionduring the treatment period are presented in Table 1 Spo-radic statistically significant changes in water consumptionwere considered spurious unassociated with the test articleadministration

344 Relative Organ Weight Figure 2 presents the relativeweights of the vital organs (in g) of rats (both genders) Theweights of liver kidney spleen and brain recorded at theend of the subchronic study (day 91) did not show significantdifferences (119875 gt 005) in any of the treatment groups

compared with the control groups (Figure 2) Furthermoregross examination of the vital organs of all rats revealed nodetectable abnormalities

345 Hematological Parameters The effect of the greenmussel formulation on hematological parameters such ashemoglobin (HGB) RBCandWBCcount platelet count anddifferential counts after subchronic toxicity (90 days) studiesis presented in Table 2 No treatment-related biologicallysignificant effects of the green mussel formulation treatmentat dose levels of 05ndash20 gkg in the hemoglobin and RBCand platelet content were apparent in both genders of ratswhen compared to untreated animals (Table 2) (119875 gt 005)and remained within physiological range throughout thetreatment period (90 days) However both male and femalerats administered green mussel formulation (at 10 gkg and05 gkg resp) showed significantly low levels of differentialcounts (lymphocytes eosinophils and neutrophils) com-pared to control animals (119875 lt 005) Similarly femalerats administered 20 and 10 gkg green mussel formulationrecorded significantly low lymphocyte and neutrophil countrespectively (119875 lt 005) No test article-related changes inblood cell morphology were observed during the period ofstudy

346 Serum Biochemical Parameters Table 3 summarizesthe serum biochemical parameters used as the biomark-ers of the liver and renal functioning during the courseof subchronic toxicity studies The serum analysis showedsignificantly low SGOT content for the low dose male rats(05 gkg) and highlow dose female rats (25 and 05 gkg)after 90 days of subchronic study Similarly another markerenzyme of the liver ALP also showed significantly lowvalues for high and low dose group male rats compared tothe control animals after 90 days of subchronic study Theactivity of anothermarker enzyme SGPTwas not significantly

BioMed Research International 7

Normal male

(a)

Normal female

(b) (a1)

Treated male (2gkg body weight)

(b1)

Treated female (2gkg body weight)

Figure 3 The cross section of the male and female rats after subchronic toxicity study of 90 days (a) Normal male (a1) green musselformulation (20 gkg) treated male (b) normal female (b1) green mussel formulation (20 gkg) treated female

Table 2 Hematology analyses data of male and female rats administered the green mussel formulation after subchronic (90 days) toxicitystudies

Treatments Lymphocytes(mm3)

Eosinophils(mm3)

Neutrophils(mm3) HGB (gdL) WBC (mm3) RBC

(106cmm)Platelet

(105cmm)

Male

Controla 582860 plusmn 15914 99820 plusmn 6290 321320 plusmn 1713 1544 plusmn 005 10040 plusmn 586 787 plusmn 007 578 plusmn 006

20 gkgb 463800 plusmn 15450 75260 plusmn 3157 232940 plusmn 8304lowast 1460 plusmn 064 7720 plusmn 2670 741 plusmn 029 464 plusmn 038

10 gkgb 364380 plusmn 18747lowast 61860 plusmn 3324lowast 201760 plusmn 8802lowast 1434 plusmn 073 6280 plusmn 3090 729 plusmn 058 508 plusmn 004

05 gkgb 491220 plusmn 11407 77360 plusmn 1538 275420 plusmn 7898 1464 plusmn 070 8440 plusmn 17470 727 plusmn 008 578 plusmn 006

Female

Controla 522040 plusmn 2310 88880 plusmn 3778 273080 plusmn 8031 1338 plusmn 094 8840 plusmn 1489 637 plusmn 047 606 plusmn 003

20 gkgb 385640 plusmn 16793lowast 64540 plusmn 2894 229820 plusmn 11425 1412 plusmn 044 6800 plusmn 977 697 plusmn 003 578 plusmn 008

10 gkgb 497520 plusmn 26933 77000 plusmn 3175 197480 plusmn 8387lowast 1386 plusmn 083 7720 plusmn 1075 686 plusmn 021 530 plusmn 007

05 gkgb 212280 plusmn 10152lowast 32120 plusmn 1467lowast 109600 plusmn 5093lowast 1354 plusmn 053 3540 plusmn 1017lowast 677 plusmn 007 564 plusmn 006

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)HGB hemoglobin WBC total white blood cell count RBC red blood cell

different (119875 gt 005) in all dose groups of treated rats ascompared to untreated control and albuminglobulin (AG)ratio was not altered in the treated animals of both genders(Table 3)

Subchronic oral administration of the green mussel for-mulation (for 90 days) did not cause any significant changesin hepatic function parameters such as total protein albumintotal bilirubin and globulin in both sexes of rats duringlong-term subchronic toxicity studies The renal functionparameters such as serum creatinine and blood urea did notshow any significant variation (119875 gt 005) in treated animalscompared to controls (Table 3) There were no statisticallysignificant differences in the levels of serum electrolytessuch as chloride potassium sodium and bicarbonate afterthe treatment of green mussel formulation (119875 gt 005)indicating no expressive changes in the general metabolismafter consumption of the green mussel formulation by rats

(Table 4) The green mussel formulation did not produceany significant changes in the total cholesterol HDL LDLand VLDL indicating no expressive changes in the generallipid metabolism after consumption of the test article by rats(Table 4)

347 Histopathological Analysis Necropsy of the treatedanimals after sacrifice did not show any morphologicalchanges of the internal organs or any gross pathologi-cal abnormalities during subchronic toxicity studies Therewere no macroscopic findings considered to be relatedto the treatment of the green mussel formulation (Fig-ure 3) Gross examination of vital organs such as brainkidney spleen and liver of rats and microscopic exami-nation of tissue sections prepared from these organs didnot observe any histopathological alteration in any treatedrats during subchronic toxicity studies The normal and

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Page 6: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

6 BioMed Research International

0

1

2

3

4O

rgan

wei

ght (

g)

LiverKidney

SpleenBrain

25

gkg

10

gkg

05

gkg

Con

trol

25

gkg

10

gkg

05

gkg

Con

trol

Male Female

(a)

0

1

2

3

4

Org

an w

eigh

t (g)

LiverKidney

SpleenBrain

10

gkg

05

gkg

Con

trol

20

gkg

10

gkg

05

gkg

Con

trol

20

gkg

Males Females

(b)

Figure 2 Mean organ weights (in grams) of male and female rats administered green mussel formulation after (a) acute and (b) subchronictoxicity studies

at 05ndash20 gkg BW was comparable with those in the controlgroup throughout the study Similarly themean bodyweightsof the treated female rats were comparable with those in thecontrol group throughout the studyTherewere no changes inbody weight in the animals attributable to the administrationof the green mussel formulation when compared to the con-trol group Any changes observed were sporadic consideredincidental and unassociated with test article administration

343 Food andWater Consumption The average food intakeof untreated control rats decreased from about 72 g to 68 g(for male rats) and from 60 g to 49 g (for female rats) after 90days of study The same trend was observed for medium andlow dose group (10 and 05 gkg resp) of males and all thedose groups of females Administration of the green musselformulation did not produce any significant difference in thefood consumption of both genders of rats when compared tonormal animals of the high dose group (119875 gt 005) throughoutthe experimental period The summarized food intake of therats recorded after oral administration of the green musselformulation to rats is shown in Table 1 Similarly the waterconsumption did not alter inmale and female rats attributableto administration of the green mussel formulation whencompared to normal animals during chronic and subchronictoxicity studies Changes in the average water consumptionduring the treatment period are presented in Table 1 Spo-radic statistically significant changes in water consumptionwere considered spurious unassociated with the test articleadministration

344 Relative Organ Weight Figure 2 presents the relativeweights of the vital organs (in g) of rats (both genders) Theweights of liver kidney spleen and brain recorded at theend of the subchronic study (day 91) did not show significantdifferences (119875 gt 005) in any of the treatment groups

compared with the control groups (Figure 2) Furthermoregross examination of the vital organs of all rats revealed nodetectable abnormalities

345 Hematological Parameters The effect of the greenmussel formulation on hematological parameters such ashemoglobin (HGB) RBCandWBCcount platelet count anddifferential counts after subchronic toxicity (90 days) studiesis presented in Table 2 No treatment-related biologicallysignificant effects of the green mussel formulation treatmentat dose levels of 05ndash20 gkg in the hemoglobin and RBCand platelet content were apparent in both genders of ratswhen compared to untreated animals (Table 2) (119875 gt 005)and remained within physiological range throughout thetreatment period (90 days) However both male and femalerats administered green mussel formulation (at 10 gkg and05 gkg resp) showed significantly low levels of differentialcounts (lymphocytes eosinophils and neutrophils) com-pared to control animals (119875 lt 005) Similarly femalerats administered 20 and 10 gkg green mussel formulationrecorded significantly low lymphocyte and neutrophil countrespectively (119875 lt 005) No test article-related changes inblood cell morphology were observed during the period ofstudy

346 Serum Biochemical Parameters Table 3 summarizesthe serum biochemical parameters used as the biomark-ers of the liver and renal functioning during the courseof subchronic toxicity studies The serum analysis showedsignificantly low SGOT content for the low dose male rats(05 gkg) and highlow dose female rats (25 and 05 gkg)after 90 days of subchronic study Similarly another markerenzyme of the liver ALP also showed significantly lowvalues for high and low dose group male rats compared tothe control animals after 90 days of subchronic study Theactivity of anothermarker enzyme SGPTwas not significantly

BioMed Research International 7

Normal male

(a)

Normal female

(b) (a1)

Treated male (2gkg body weight)

(b1)

Treated female (2gkg body weight)

Figure 3 The cross section of the male and female rats after subchronic toxicity study of 90 days (a) Normal male (a1) green musselformulation (20 gkg) treated male (b) normal female (b1) green mussel formulation (20 gkg) treated female

Table 2 Hematology analyses data of male and female rats administered the green mussel formulation after subchronic (90 days) toxicitystudies

Treatments Lymphocytes(mm3)

Eosinophils(mm3)

Neutrophils(mm3) HGB (gdL) WBC (mm3) RBC

(106cmm)Platelet

(105cmm)

Male

Controla 582860 plusmn 15914 99820 plusmn 6290 321320 plusmn 1713 1544 plusmn 005 10040 plusmn 586 787 plusmn 007 578 plusmn 006

20 gkgb 463800 plusmn 15450 75260 plusmn 3157 232940 plusmn 8304lowast 1460 plusmn 064 7720 plusmn 2670 741 plusmn 029 464 plusmn 038

10 gkgb 364380 plusmn 18747lowast 61860 plusmn 3324lowast 201760 plusmn 8802lowast 1434 plusmn 073 6280 plusmn 3090 729 plusmn 058 508 plusmn 004

05 gkgb 491220 plusmn 11407 77360 plusmn 1538 275420 plusmn 7898 1464 plusmn 070 8440 plusmn 17470 727 plusmn 008 578 plusmn 006

Female

Controla 522040 plusmn 2310 88880 plusmn 3778 273080 plusmn 8031 1338 plusmn 094 8840 plusmn 1489 637 plusmn 047 606 plusmn 003

20 gkgb 385640 plusmn 16793lowast 64540 plusmn 2894 229820 plusmn 11425 1412 plusmn 044 6800 plusmn 977 697 plusmn 003 578 plusmn 008

10 gkgb 497520 plusmn 26933 77000 plusmn 3175 197480 plusmn 8387lowast 1386 plusmn 083 7720 plusmn 1075 686 plusmn 021 530 plusmn 007

05 gkgb 212280 plusmn 10152lowast 32120 plusmn 1467lowast 109600 plusmn 5093lowast 1354 plusmn 053 3540 plusmn 1017lowast 677 plusmn 007 564 plusmn 006

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)HGB hemoglobin WBC total white blood cell count RBC red blood cell

different (119875 gt 005) in all dose groups of treated rats ascompared to untreated control and albuminglobulin (AG)ratio was not altered in the treated animals of both genders(Table 3)

Subchronic oral administration of the green mussel for-mulation (for 90 days) did not cause any significant changesin hepatic function parameters such as total protein albumintotal bilirubin and globulin in both sexes of rats duringlong-term subchronic toxicity studies The renal functionparameters such as serum creatinine and blood urea did notshow any significant variation (119875 gt 005) in treated animalscompared to controls (Table 3) There were no statisticallysignificant differences in the levels of serum electrolytessuch as chloride potassium sodium and bicarbonate afterthe treatment of green mussel formulation (119875 gt 005)indicating no expressive changes in the general metabolismafter consumption of the green mussel formulation by rats

(Table 4) The green mussel formulation did not produceany significant changes in the total cholesterol HDL LDLand VLDL indicating no expressive changes in the generallipid metabolism after consumption of the test article by rats(Table 4)

347 Histopathological Analysis Necropsy of the treatedanimals after sacrifice did not show any morphologicalchanges of the internal organs or any gross pathologi-cal abnormalities during subchronic toxicity studies Therewere no macroscopic findings considered to be relatedto the treatment of the green mussel formulation (Fig-ure 3) Gross examination of vital organs such as brainkidney spleen and liver of rats and microscopic exami-nation of tissue sections prepared from these organs didnot observe any histopathological alteration in any treatedrats during subchronic toxicity studies The normal and

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

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Page 7: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

BioMed Research International 7

Normal male

(a)

Normal female

(b) (a1)

Treated male (2gkg body weight)

(b1)

Treated female (2gkg body weight)

Figure 3 The cross section of the male and female rats after subchronic toxicity study of 90 days (a) Normal male (a1) green musselformulation (20 gkg) treated male (b) normal female (b1) green mussel formulation (20 gkg) treated female

Table 2 Hematology analyses data of male and female rats administered the green mussel formulation after subchronic (90 days) toxicitystudies

Treatments Lymphocytes(mm3)

Eosinophils(mm3)

Neutrophils(mm3) HGB (gdL) WBC (mm3) RBC

(106cmm)Platelet

(105cmm)

Male

Controla 582860 plusmn 15914 99820 plusmn 6290 321320 plusmn 1713 1544 plusmn 005 10040 plusmn 586 787 plusmn 007 578 plusmn 006

20 gkgb 463800 plusmn 15450 75260 plusmn 3157 232940 plusmn 8304lowast 1460 plusmn 064 7720 plusmn 2670 741 plusmn 029 464 plusmn 038

10 gkgb 364380 plusmn 18747lowast 61860 plusmn 3324lowast 201760 plusmn 8802lowast 1434 plusmn 073 6280 plusmn 3090 729 plusmn 058 508 plusmn 004

05 gkgb 491220 plusmn 11407 77360 plusmn 1538 275420 plusmn 7898 1464 plusmn 070 8440 plusmn 17470 727 plusmn 008 578 plusmn 006

Female

Controla 522040 plusmn 2310 88880 plusmn 3778 273080 plusmn 8031 1338 plusmn 094 8840 plusmn 1489 637 plusmn 047 606 plusmn 003

20 gkgb 385640 plusmn 16793lowast 64540 plusmn 2894 229820 plusmn 11425 1412 plusmn 044 6800 plusmn 977 697 plusmn 003 578 plusmn 008

10 gkgb 497520 plusmn 26933 77000 plusmn 3175 197480 plusmn 8387lowast 1386 plusmn 083 7720 plusmn 1075 686 plusmn 021 530 plusmn 007

05 gkgb 212280 plusmn 10152lowast 32120 plusmn 1467lowast 109600 plusmn 5093lowast 1354 plusmn 053 3540 plusmn 1017lowast 677 plusmn 007 564 plusmn 006

lowastData presented as mean plusmn standard deviation (119899 = 5) Significantly different from control 119875 lt 005aControl group received 1mL distilled waterbSample group received three doses of green mussel formulation (20 10 and 05 gkg rat)HGB hemoglobin WBC total white blood cell count RBC red blood cell

different (119875 gt 005) in all dose groups of treated rats ascompared to untreated control and albuminglobulin (AG)ratio was not altered in the treated animals of both genders(Table 3)

Subchronic oral administration of the green mussel for-mulation (for 90 days) did not cause any significant changesin hepatic function parameters such as total protein albumintotal bilirubin and globulin in both sexes of rats duringlong-term subchronic toxicity studies The renal functionparameters such as serum creatinine and blood urea did notshow any significant variation (119875 gt 005) in treated animalscompared to controls (Table 3) There were no statisticallysignificant differences in the levels of serum electrolytessuch as chloride potassium sodium and bicarbonate afterthe treatment of green mussel formulation (119875 gt 005)indicating no expressive changes in the general metabolismafter consumption of the green mussel formulation by rats

(Table 4) The green mussel formulation did not produceany significant changes in the total cholesterol HDL LDLand VLDL indicating no expressive changes in the generallipid metabolism after consumption of the test article by rats(Table 4)

347 Histopathological Analysis Necropsy of the treatedanimals after sacrifice did not show any morphologicalchanges of the internal organs or any gross pathologi-cal abnormalities during subchronic toxicity studies Therewere no macroscopic findings considered to be relatedto the treatment of the green mussel formulation (Fig-ure 3) Gross examination of vital organs such as brainkidney spleen and liver of rats and microscopic exami-nation of tissue sections prepared from these organs didnot observe any histopathological alteration in any treatedrats during subchronic toxicity studies The normal and

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

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Medicinal ChemistryInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

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Autoimmune Diseases

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MEDIATORSINFLAMMATION

of

Page 8: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

8 BioMed Research International

Table3Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

SGOT

SGPT

ALP

Bilirub

inTo

talprotein

Album

inGlobu

linAG

ratio

Urea

Creatin

ine

(UL)

(UL)

(UL)

(mgdL

)(gdL)

(gdL)

(gdL)

(mgdL

)(m

gdL

)

Male

Con

trola18200plusmn3146840plusmn1535620plusmn284020plusmn001782plusmn022350plusmn007432plusmn042

081

4680plusmn096068plusmn008

20g

kgb

18860plusmn157380plusmn105325440plusmn318lowast020plusmn007744plusmn016354plusmn003390plusmn002

091

5080plusmn015062plusmn004

10gkg

b17100plusmn14937840plusmn00233280plusmn249018plusmn004706plusmn006354plusmn026352plusmn034

102

4200plusmn054062plusmn008

050

gkg

b14600plusmn2073lowast6880plusmn04225440plusmn244lowast018plusmn004744plusmn026354plusmn041390plusmn051

093

3200plusmn087lowast062plusmn013

Female

Con

trola19540plusmn10018640plusmn15726740plusmn23022plusmn004752plusmn026414plusmn021338plusmn008

123

7700plusmn594078plusmn003

20g

kgb13260plusmn304lowast6340plusmn19934020plusmn494016plusmn005722plusmn021394plusmn027328plusmn03

120

4900plusmn216lowast056plusmn009

10gkg

b16125plusmn30136400plusmn08134475plusmn501020plusmn001772plusmn002408plusmn02364plusmn018

112

5580plusmn095068plusmn004

050

gkg

b14520plusmn1096lowast6560plusmn13528760plusmn302020plusmn002716plusmn005422plusmn003294plusmn006

145

5620plusmn003068plusmn011

lowastDatap

resented

asmeanplusmnstandard

deviation(119899=5)Sign

ificantlydifferent

from

control119875lt005

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

SGOT

serum

glutam

icoxaloacetic

transaminaseSG

PTserum

glutam

icpyruvictransam

inaseALP

alkalinep

hosphataseA

Gratio

album

inglobu

linratio

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 9: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

BioMed Research International 9

Table4Serum

biochemicalanalysisdataof

malea

ndfemaler

atsa

dministered

theg

reen

musselformulationaft

ersubchron

ic(90days)toxicity

studies

Na+

K+Cl

+HCO3

+Ch

olesterol

Triglycerid

esHDL

LDL

VLD

L(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(msdotm

olL)

(mgdL

)(m

gdL

)(m

gdL

)(m

gdL

)(m

gdL

)

Male

Con

trola14780plusmn066550plusmn03510522plusmn0472580plusmn0847740plusmn01913750plusmn1013280plusmn0421860plusmn0362600plusmn089

20g

kgb

14572plusmn296605plusmn09610488plusmn1192720plusmn1097420plusmn072

13880plusmn18

3460plusmn0141180plusmn0212780plusmn095

10gkg

b14484plusmn034624plusmn08310448plusmn0802640plusmn0147620plusmn07315280plusmn31243320plusmn0301375plusmn0303060plusmn006

050

gkg

b14606plusmn222515plusmn02210448plusmn0442760plusmn0897520plusmn37012600plusmn0223480plusmn0841520plusmn2682520plusmn079

Female

Con

trola13998plusmn068488plusmn01010132plusmn1782560plusmn1147560plusmn04411420plusmn01943220plusmn0302060plusmn6872280plusmn030

20g

kgb

13694plusmn073503plusmn05710438plusmn0792740plusmn0526340plusmn33612340plusmn0213100plusmn071780plusmn0492460plusmn089

10gkg

b13818plusmn066558plusmn06110354plusmn0952660plusmn0897520plusmn06814060plusmn0353280plusmn0081640plusmn0282600plusmn064

050

gkg

b14114plusmn059585plusmn08810484plusmn0832720plusmn0847280plusmn00314560plusmn0702920plusmn1641440plusmn0912920plusmn015

Datap

resented

asmeanplusmnstandard

deviation(119899=5)

a Con

trolgroup

received

1mLdistilledwater

b Sam

pleg

roup

received

threed

oses

ofgreenmusselformulation(2010and

05g

kgrat)

HDLhigh

-densitylip

oproteinLDLlow-densitylip

oproteinV

LDLvery

low-densitylip

oprotein

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 10: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

10 BioMed Research International

Normal brain

(a)

Normal kidney

(b)

(a1)

Treated brain2gkg body weight

(b1)

Treated kidney2gkg body weight

Figure 4 Photomicrograph of histopathological sections of the brain and kidney on day 90 of subchronic toxicity test (a) Normal liver (a1)brain sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing apparently normalglial cells (b) photomicrograph of kidney section from experimental control rats and (b1) kidney sections from experimental rats after 90days of treatment with 20 gkg of the green mussel formulation showing normal glomeruli

treated sections of brain (Figures 4(a) and 4(a1)) kidney(Figures 4(b) and 4(b1)) liver (Figures 5(a) and 5(a1)) andspleen (Figures 5(b) and 5(b1)) showed normal appearancecompared to control rats after subchronic toxicity study Thetreated section of brain showed normal glial cells Astrocytesinterstitial tissue of the brain and the portion of cerebellumalso showed normal appearance compared to control rats(Figure 4(a1)) The treated section of the kidney showednormal glomeruli with normal Bowmanrsquos capsule Glomerulishowed normal cellularity with renal tubules and interstitialtissue demonstrated the normal appearance (Figure 4(b1))The section of liver tissue showed normal portal triads andbiliary duct A few lymphocytic collections were seen inthe portal area which was normal Central venous sys-tems also appeared normal Hepatocytes showed normalmorphology and they were arranged in cords Sinusoidalspace and Kupffer cells also appeared normal (Figure 5(a1))The section of spleen showed normal lymphoid follicleswith germinal centers Sinusoidal spaces are dilated and

they were lined by normal endothelial cells Some areasshowed hemorrhaged congestion with many siderophages(Figure 5(b1)) No other macroscopic or microscopic lesionsin organs examined were observed

4 Discussion

The active principles in the formulation derived from greenmussel P viridis were competitively inhibited inflammatorycyclooxygenases (COXIII) and lipoxygenase (LOXV) in aninflammation and oxidative stress reaction resulting indecreased production of proinflammatory prostaglandinsand leukotrienes In vivo animal model studies revealed thatthe active principles effectively suppressed the carrageenan-induced rat paw edema which indicate that they exhibitits anti-inflammatory action by means of inhibiting eitherthe synthesis release or action of inflammatory mediatorsThe green mussel formulation recorded COXILOXV andCOXIII ratios lower than 10 compared to NSAIDs (gt10)

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 11: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

BioMed Research International 11

Normal liver

(a)

Normal spleen

(b)

(a1)

Treated liver2gkg body weight

(b1)

Treated spleen2gkg body weight

Figure 5 Photomicrograph of histopathological sections of the liver and spleen on day 90 of subchronic toxicity test (a) Normal liver (a1)photomicrograph of liver sections from experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showingapparently normalmorphology of hepatocytes (b) photomicrograph of spleen section fromexperimental control rats and (b1) spleen sectionsfrom experimental rats after 90 days of treatment with 20 gkg of the green mussel formulation showing normal lymphoid follicles withgerminal centers

which indicate their higher selectivity against inflammatoryresponse and lower side effect profiles

Many of the allopathic prescriptions including NSAIDsand cyclooxygenase inhibitors used in controlling arthriticconditions have known side effects especially with long-term usage About 25 of the users experience some kindof side effect and 5 develop serious health consequencessuch as stomach bleeding stroke and acute renal failureThegreen mussel formulation proved to be a safer and effectivealternative to these synthetic NSAIDs and other productsavailable in the market

As it is has been observed to be slow acting long-term use of the green mussel formulation may be requiredin treatments of arthritis related diseases In this aspectlong-term toxicity studies of the green mussel formulationare of vital importance for the assessment of its safety inmammalian systems In order to provide safety evidence

for the green mussel formulation as a prospective nutraceu-tical medication for joint pain and arthritis short-termand subchronic toxicity studies were conducted on the ratsto evaluate the possible toxicity The results demonstratea lack of test substance-related general organ or systemictoxicity following oral administration of the green musselformulation at a dose as high as 2500mgkgd in the acutetoxicity study and the repeated oral administration of theformulation at a dose of 2000mgkgday the highest dosetested in the 90-day oral toxicity study According to LoomisandHayes [27] a chemical substance with an LD

50within the

range of 5000ndash15000mgkg is considered as practically non-toxic The calculated LD

50for the green mussel formulation

is found in this range and therefore this nutraceutical for-mulation should be regarded as practically nontoxic in acuteingestion The green mussel formulation too did not causeany toxic symptoms behavioral changes or mortality when

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 12: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

12 BioMed Research International

acutely administered at 5000mgkg to rats and thereforethis formulation can be included under category 5 (low or notoxicity) in accordance with OECD guidelines

Under various regulatory guidelines changes in bodyweight have been used as an integral part of the conventionalsafety evaluation of test materials drugs and chemicals[28 29] The animal behavior feed intake and the normalbody weight changes were not altered during the short-term and subchronic toxicity studies This indicates thatthe food conversion rate was not affected and growth ofrats in treatment groups was comparable to that of controlSince no significant changes were observed in the generalbehavior body weight and food and water intake of rats inthe treated groups as compared to the untreated control afterthe administration of the green mussel formulation duringthe short-term and subchronic toxicity study spanning over90-day period it could be concluded that oral administrationof this test material had no effect on the normal growth of ratsin the concentration studied

Organ weights are widely accepted in the evaluation oftoxicity related studies [30] No significant differences wererecorded in the relative weights of kidney brain spleen andliver indicating that the acutesubchronic oral administrationof the green mussel formulation did not detrimentally affectthe wet weight organ-to-body weight ratio and the color ofthe organs

The hematopoietic system is one of the most sensitivetargets of toxic compounds and is an important index toassess the toxicity of the testmaterial on the physiological andpathological status in human and animals [31] After 90 daysthere were no treatment-related changes in hematologicalparameters between the untreated and the green musselformulation treated groups indicating that the test materialhad no effects on the circulating blood cells nor it inter-fered with their production Some statistically insignificantdifferences were noted in WBC and differential counts whenthe control and treatment groups were compared Howeverthese changes did not appear to be related to the testarticle treatment since they were still within the limits ofnormal biological variation The changes in WBC countswere probably due to the normal responses to foreign bodiesor stress associated with the toxicity studies Decreasedhemoglobin and differential counts were found previouslyin the rats fed with polyunsaturated fatty acids such as 119899-6fatty acid arachidonic acid and 119899-3 fatty acid docosahexaenoicacid containing oils [32] However in the present studyno significant changes in these parameters were apparentTaken together the normal range of hematological indicatorsindicated the absence of hemotoxic potential of the greenmussel formulation

Biochemical determinations of blood parameters inserum serve as an indicator of toxicity of a test material [28]The enzymes namely serum aspartate transaminase (AST)or serum glutamic oxaloacetic transaminase (SGOT) serumalanine transaminase (ALT) or serum glutamic pyruvictransaminase (SGPT) and alkaline phosphatase (ALP) arewell-known enzymes used as good indicators of liver functionand as major markers of hepatic injury [29] In generalaminotransferases AST and ALT are normally contained

within liver cells and their activity in the blood is generallylow If the liver is damaged these enzymes diffuse across thedamaged cell membrane due to altered plasma membranepermeability [33] before being entered into the circulationraising the enzyme levels in the blood and signaling liverdisease In the present study there was no significant differ-ence in ALP ALT and AST in the green mussel formulationadministered animals when compared to control Theseresults suggest that the test material did not alter the hepaticfunction and prevent hepatocyte enzyme from going intothe blood No significant alterations in ALP of the animalsubjects treated with the green mussel formulation indicatedany liver injury [34]Other liver enzyme activities too realizedno significant decrease thereby suggesting that the acuteand subchronic administration of the test material did notalter the hepatocytes and consequently the metabolism of therats

Albumin is synthesized by the hepatocytes and as suchit represents a major synthetic plasma protein and itsdetermination can act as a criterion for assessing the syntheticcapacity of the liver [35] Decrease in plasma proteinstherefore tends to reflect chronic damage The commonpattern seen following significant hepatocellular damage is areduction in albumin accompanied by a relative increase inglobulins which leads to AG ratio reduction [36] Howeverno change in serum proteins and albumin was observedin the acute and subchronic studies which show that thegreen mussel formulation does not inhibit protein synthesisin the ratsThis is supported by the microscopic examinationshowing the normal hepatocytes without any lesions in theliver Bilirubin is a metabolic breakdown product of bloodheme Any course that might induce abnormally increasedlevels of bilirubin accumulating in human serum or plasmausually signifies the presence of a variety of diseases with liverdysfunctions ranging from jaundice to infectious hepatitis[37] Our results proved the report that long-term and highdose administration of the green mussel formulation did notsignificantly alter the bilirubin concentrations which is anindication that it does not interfere with the metabolismof bilirubin in the liver In liver bilirubin also affects theprotein synthesis There was no obvious alteration of proteincontent in the liver at the experimental doses administeredwith the test material in comparison to the control groupIt is of note that under normal circumstances bilirubin-albumin conjugate protects the cells against the potentialtoxicity of bilirubin Any imbalance in the formation of theconjugate results in the decreased protein content in the liverand increased total bilirubin in serum to have a detrimentaleffect leading to injury of the liver Since there was noadverse effect on plasma levels of total bilirubin total proteinand AG ratio in the green mussel formulation treatedanimals when compared to control it may be concludedthat the test article did not alter the renal and hepaticfunction

Serum urea and creatinine are known as the usualmarkers of renal function [35] Any rise in their levelsis only observed if there is marked damage to functionalnephrons [38] Since there was no significant difference inplasma levels of urea and creatinine in the green mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 13: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

BioMed Research International 13

formulation treated animals when compared to control itmay be concluded that the test article did not alter the renalfunctionaries

No significant changes were observed in cholesterolLDL and VLDL levels suggesting that the green musselformulation had no effects on the lipid and carbohydratemetabolism of the rats The microscopic evaluation of theorgans of treated rats group supported the safety of thegreen mussel formulation The liver is the site of cholesteroldisposal and synthesis and glucose synthesis and generatesfree glucose into the blood from hepatic glycogen stores [39]In the present study lowering of triglycerides was observedwhich was not significant The present study also revealedthat serum electrolytes (Na+ K+ Cl+ and HCO

3

minus) did notalter significantly in the treatment groups compared to theuntreated controls throughout the study period

Most importantly the histopathological examination ofselected organs (heart liver brain and kidneys) from treatedand control animals showed normal architecture suggestingthat daily oral administration of the greenmussel formulationfor 90 days caused no detrimental changes or morphologicaldisturbances

The acute and subchronic toxicity studies of the greenmussel formulation using Wistar rats were carried out tounderstand its effect on various parameters such as mortalityweight change food consumption hematological liver andrenal functions serum electrolytes and lipid profile Theresults indicated that the green mussel formulation did notproduce any change in food consumption water consump-tion and body weights in rats indicating that it has notoxicity to these animals Also it did not produce any bio-chemical changes related to hepatic and renal function Thisformulation did not produce any change in hematologicaland serum biochemical parameters Necropsy of the treatedanimals showed normal appearance of various tissues Theno-observed-adverse-effect level (NOAEL) was 2000mgkgBW The toxicological studies demonstrated that the greenmussel formulation is safe to consume without any adverseeffects in the body

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to the Indian Council of Agricul-tural Research (ICAR) New Delhi for providing necessaryfacilities and encouragements to carry out the work Theauthors thank the Director of Central Marine FisheriesResearch Institute for his guidance and support Thanksare due to the Head of Marine Biotechnology DivisionCentral Marine Fisheries Research Institute for facilitatingthe research activity The authors thank Dr RamadasanKuttan Research Director of Amala Cancer Research CentreKerala for help

References

[1] D Li and A J Sinclair ldquoMacronutrient innovations the role offats and sterols in human healthrdquoAsia Pacific Journal of ClinicalNutrition vol 11 no 6 pp S155ndashS162 2002

[2] A J Benson D C Marelli M E Frischer J M Danforth and JD Williams ldquoEstablishment of the green mussel Perna viridis(Linnaeus 1758) (Mollusca Mytilidae) on the West Coast ofFloridardquo Journal of Shellfish Research vol 20 no 1 pp 21ndash292001

[3] P Laxmilatha G S Rao P Patnaik T N Rao M P Rao and BDash ldquoPotential for the hatchery production of spat of the greenmussel Perna viridis Linnaeus (1758)rdquo Aquaculture vol 312 no1ndash4 pp 88ndash94 2011

[4] C Chellaram P T Anand S Kumaran et al ldquoCentral nervoussystem depressant properties of reef associated gastropodsDrupa margariticola and Trochus tentorium from Gulf of Man-nar Southeastern Indiardquo Journal of Chemical and Pharmaceuti-cal Research vol 3 no 1 pp 154ndash159 2011

[5] K Chakraborty S J Chakkalakal and D Joseph ldquoResponse ofpro-inflammatory prostaglandin contents in anti-inflammatorysupplements from Green Mussel Perna viridisL in a timedependent accelerated shelf-life studyrdquo Journal of FunctionalFoods vol 7 pp 527ndash540 2014

[6] M W Whitehouse T A Macrides N Kalafatis W H BettsD R Haynes and J Broadbent ldquoAnti-inflammatory activity ofa lipid fraction (Lyprinol) from the NZ green-lipped musselrdquoInflammopharmacology vol 5 no 3 pp 237ndash246 1997

[7] S McPhee L D Hodges P F A Wright et al ldquoAnti-cyclooxygenase effects of lipid extracts from the New Zealandgreen-lipped mussel Perna canaliculusrdquo Comparative Biochem-istry and Physiology B Biochemistry and Molecular Biology vol146 no 3 pp 346ndash356 2007

[8] D Uemura T Chou T Haino et al ldquoPinnatoxin A a toxicamphoteric macrocycle from the Okinawan bivalve (Pinnamuricata)rdquo Journal of the American Chemical Society vol 117no 3 pp 1155ndash1156 1995

[9] K J Murphy B D Mooney N J Mann P D Nichols and AJ Sinclair ldquoLipid FA and sterol composition of New Zealandgreen lipped mussel (Perna canaliculus) and Tasmanian bluemussel (Mytilus edulis)rdquo Lipids vol 37 no 6 pp 587ndash595 2002

[10] B E Spencer Molluscan Shellfish Farming vol 274 BlackwellScience Malden Mass USA 1st edition 2002

[11] L-D Quan G M Thiele J Tian and D Wang ldquoThe devel-opment of novel therapies for rheumatoid arthritisrdquo ExpertOpinion onTherapeutic Patents vol 18 no 7 pp 723ndash738 2008

[12] T J Schnitzer M Kamin and W H Olson ldquoTramadol allowsreduction of naproxen dose among patients with naproxen-responsive osteoarthritis painrdquoArthritis and Rheumatology vol42 pp 1370ndash1377 1999

[13] L N Larsen E Dahl and J Bremer ldquoPeroxidative oxidationof leuco-dichlorofluorescein by prostaglandin H synthase inprostaglandin biosynthesis from polyunsaturated fatty acidsrdquoBiochimica et Biophysica Acta Lipids and Lipid Metabolism vol1299 no 1 pp 47ndash53 1996

[14] S Baylac and P Racine ldquoInhibition of 5-lipoxygenase byessential oils and other natural fragment extractsrdquo InternationalJournal of Aromatherapy vol 13 no 2-3 pp 138ndash142 2003

[15] C A Winter E A Risley and G W Nuss ldquoCarrageenin-induced edema in hind paw of the rat as an assay for anti-iflammatory drugsrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 111 pp 544ndash547 1962

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 14: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

14 BioMed Research International

[16] M I Ezeja A O Anaga I U Asuzu et al ldquoAcute and sub-chronic toxicity profile of methanol leaf extract of Gouanialongipetala in ratsrdquo Journal of Ethnopharmacology vol 151 pp1155ndash1164 2014

[17] N Chiranthanut S Teekachunhatean A Panthong P Khon-sung D Kanjanapothi and N Lertprasertsuk ldquoToxicity eval-uation of standardized extract of Gynostemma pentaphyllumMakinordquo Journal of Ethnopharmacology vol 149 no 1 pp 228ndash234 2013

[18] DANelson andMWMorrie ldquoBasicMethodologyrdquo inClinicalDiagnosis andManagement By Laboratory Methods J B HenryEd p 589 Saunders Philadelphia Pa USA 1984

[19] G K Pal and P Parvati Textbook of Practical Physiology OrientLongman Private Limited 2003

[20] H U Bergmeyer G N Bowers Jr M Horder and D WMoss ldquoProvisional recommendations on IFCCmethods for themeasurement of catalytic concentrations of enzymes Part 2IFCC method for aspartate aminotransferaserdquo Clinica ChimicaActa vol 70 no 2 pp F19ndashF29 1976

[21] R BMcComb andGN BowersAlkaline Phosphatase PlenumPress New York NY USA 1979

[22] L Jendrassik and P Grof ldquoVereinfachte PhotometrischeMethoden zur Bestimmung des Blutbilirubinsrdquo BiochemischeZeitschrift vol 297 pp 82ndash89 1938

[23] F C Pearlman and R T Y Lee ldquoDetection andmeasurement oftotal bilirubin in serum with use of surfactants as solubilizingagentsrdquo Clinical Chemistry vol 20 no 4 pp 447ndash453 1974

[24] M Jaffe ldquoUber den Niederschlag Welchen Pikrinsaure in Nor-malen Harn Erzeugt und uber neue Reaktion des KreatininesH-S Z Physiolrdquo Chemistry vol 10 pp 391ndash400 2003

[25] T G Cole S G Klotzsch J McNamara et al ldquoMeasurement oftriglyceride concentrationrdquo inHandbook of Lipoprotein TestingN Riafi G R Warnick and M H Dominiczak Eds pp 115ndash126 AACC Press Washington DC USA 1997

[26] A H Betti A C Stein E Dallegrave et al ldquoAcute and repeated-doses (28 days) toxicity study of Hypericum polyanthemumKlotzsch ex Reichardt (Guttiferare) inmicerdquo Food andChemicalToxicology vol 50 no 7 pp 2349ndash2355 2012

[27] T A Loomis and A W Hayes Loomisrsquos Essentials of Toxicologyvol 4 Academic Press San Diego Calif USA 1996

[28] B Schilter C Andersson R Anton et al ldquoGuidance for thesafety assessment of botanicals and botanical preparationsfor use in food and food supplementsrdquo Food and ChemicalToxicology vol 41 no 12 pp 1625ndash1649 2003

[29] J E Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[30] A Wooley in A Guide To Practical Toxicology EvaluationPrediction and Risk vol 465 Informa Healthcare New YorkNY USA 2nd edition 2008

[31] J T Mukinda and P F K Eagles ldquoAcute and sub-chronic oraltoxicity profiles of the aqueous extract of Polygala fruticosa infemale mice and ratsrdquo Journal of Ethnopharmacology vol 128no 1 pp 236ndash240 2010

[32] B A R Lina A P M Wolterbeek Y Suwa et al ldquoSub-chronic (13-week) oral toxicity study preceded by an in uteroexposure phase with arachidonate-enriched triglyceride oil(SUNTGA40S) in ratsrdquo Food and Chemical Toxicology vol 44no 3 pp 326ndash335 2006

[33] H Upur N Amat B Blazekovic and A Talip ldquoProtectiveeffect of Cichorium glandulosum root extract on carbon

tetrachloride-induced and galactosamine-induced hepatotoxi-city in micerdquo Food and Chemical Toxicology vol 47 no 8 pp2022ndash2030 2009

[34] T M Antonelli-Ushirobira E N Kaneshima M Gabriel E AAudi L C Marques and J C P Mello ldquoAcute and subchronictoxicological evaluation of the semipurified extract of seedsof guarana (Paullinia cupana) in rodentsrdquo Food and ChemicalToxicology vol 48 no 7 pp 1817ndash1820 2010

[35] S Muhammad L G Hassan S M Dangoggo et al ldquoAcuteand sub-chronic toxicity studies of kernel extract of Sclerocaryabirrea in ratsrdquo Scientific World Journal vol 6 no 3 pp 1597ndash6343 2011

[36] D D Woodman ldquoAssessment of Hepatotoxicityrdquo in AnimalClinical Chemistry A Primer for Toxicologists G O Evans Edpp 71ndash86 Taylor and Francis London UK 1996

[37] G Yao ldquoBilirubin Metabolismrdquo Chinese Journal of Hepatologyvol 9 p 17 2004

[38] N Lameire W Van Biesen and R Vanholder ldquoAcute renalfailurerdquoThe Lancet vol 365 no 9457 pp 417ndash430 2005

[39] A Kaplan R Jack K E Opheim et al Clinical Chemistry Inter-pretation and Techniques Williams and Wilkins PhiladelphiaPa USA 4th edition 1995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 15: Research Article Toxicity Profile of a Nutraceutical …downloads.hindawi.com/journals/bmri/2014/471565.pdf · Toxicity Profile of a Nutraceutical Formulation Derived from Green Mussel

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of