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Enicostemma littorale Blume - A potentialhypolipidemic plant

Natural Product Radiance Vol 3(6) November-December 2004

Keywords: Enicostemma littorale, Chhotachirayta, p­Dimethylaminoazobenzene (p-DAB), Hepatotoxicity, Lipid profile, Lipid

peroxidation, y-Glutamyl transpeptidase.

IPC code; Int. d. 7_ A61K 35n8, A61P 1/16, A61P 3/06

Restoration of liver function by the application of Enicostemma littoraleBlume (aerial part) powder in p-DAB (p-dimethylaminoazobenzene) induced

animals was evaluated by analyzing the serum total cholesterol, triglycerides,

HDL-cholesterol, serum y-glutamyl transpeptidase and liver malondialdehyde

levels. Administration of the chosen plant reduces the hyperlipidemia at significant

level and also reduces the lipid peroxidation and significantly increased HDL­

cholesterol level in serum. When comparing the results with the standard

antilipidemia agent namely, vitamin E, the results obtained using the plant is highly

comparable.

(p-DAB) are reported to induce damage

in hepatic systems (WHO, 1975). p-DAB,

a colouring agent commonly used in foods

is reported to be a powerful liver

carcinogen (Mukai & Goldstein, 1976;

Weinberg, 1996; Fabiana et al, 2001).The electrophilic nature of the metabolites

of p-DAB has the capacity to covalently

bind with RNA, DNA and tissue protein

(Labuc & Blunck, 1979; Ohnishi et al,2001). In addition, reactive oxygen

species such as superoxide, hydroxyl

radicals and hydrogen peroxide (Halliwell

& Gutteridge, 1989; Sies, 1991; Grisham,

1992; Moslen, 1994; Halliwell, 1996) are

released due to the induction of p-DAB,

R GopaP, A GnanamanF*, R Udayakumar and S Sadulla1

ICHORD, Central Leather Research Institute, Adyar, Chennai-20, Tamil Nadu

2Department of Biochemistry,]. ]. College of Arts and Science, Pudukkottai-622404, Tamil Nadu

* Correspondent author, E-mail: agmani_2000@yahoo.com- -which further stimulate the damage of liver

by the metabolized products of lipid

peroxidation, consequently leading to DNA

aberrations (Biswas & Khuda-Bukhsh,

2002). Shamberger et al (1974) reported

that hydroxyl radicals are highly reactive

and are the basis for the generation of lipid

- peroxide products such as

malondialdehyde, which are considered

to be a cause for carcinogenesis.

Several plant products have been

shown to exert a protective role against

the formation of free radicals and playing

a beneficial role in maintaining disease

condition (Ajitha & Rajnarayana, 2001).

Abstract

liver is one of the most important

organs for the metabolism of various

chemicals and is responsible for the major

detoxification. Liver damage/hepato­

carcinoma, in general associated with the

external agents comes under the

classification as carcinogens (Raghuram

et al, 2003). About 80-90% of cancer

related diseases and the hepatic problems

are mainly due to continuous exposure or

contamination of chemical carcinogens in

the food chain (WHO, 1987). Chemical

carcinogens like N-nitrosodiethylamine

(NDEA) and p-dimethylaminoazobenzene

Introduction

, .

Statistical analysis

and serum was separated as per

conventional methods. Liversamples were

separated and was homogenized in 10%

saline. The homogenate was centrifugedat 15,000 x g for 30 minutes at 4°C and

the supernatant was used for lipid

peroxidation analysis. The serum total

cholesterol (TC) was estimated by the

method of Zak's (1957); triglycerides

(TG) was estimated by method of Van

Handel & Zilversmit (1957). High density

lipoprotein cholesterol (HDL-c) was

estimated by the method of Burstein et al(1970). Low-density lipoproteincholesterol (WL-c) was calculated from

the above measurement by using the

equation, LDLC= TC - (HDLC +TG),

suggested by of Friendwald et al (1972).

The y-glutamyl transpeptidase

activity was estimated by the method of

Orloswski & Meister (1965). Lipid

peroxidative effectwas evaluated through

measuring the hepatic content of

thiobarbituric acid reacting substances

(TBARS),expressed as malondialdehyde(MDA) equivalents by the method ofNiehaus & Samuelsson (1968).

Results were statistically

evaluated using one-way analysis of

variance (ANOVA) for repeated

observation. Values of p< 0.05 were

considered to be significant.

Results and DiscussionBiochemical estimations

Animals were divided into four

groups each group containing six animals.

Group I served as normal animals, which

received the vehicle 3% DMSO (5m1Jkg

body wt). Group II animals with

hepatocellular damage induced by

p-dimethylaminoazobenzene (Img/kg

body wt) in 3% DMSO was injectedintraperitoneally (i.p.) once in every 3

days for 15 days. Group 1lI animals with

hepatocellular damage were induced as

per group II and followed by last dosage

were treated with the plant (1g1kg bodywt) in crude form along with physiological

saline twice a day for 15 days by gavage

method. Group IV animals with

hepatocellular damage induced as pergroup II, after the last dose, vitamin E

(30mglkg bodywt) was givenorally in thesuspended form with sunflower oil.

Administration was done for twice a day

for 15 days.

Plant material and chemicals

Experimental Work

Enicostemma littorale Blume purchased from an authorized firm at

(Family - Gentianaceae) a glabrous Bangalore and were housed in spacious

herb commonly used as a bitter tonic and polypropylene cages and maintained

substitute for Swertia chirayita under standard conditions of temperature

(Roxb. ex Flem.) Karst., is also called 27 ±2°C, relative humidity of 60 ± 5%,

as Chhotachirayta. It is also reported 12 hours of lighVdark cycle, and fed with

to possess antitumor (Dash pellet diet manufactured by Pranav Agro

et aI, 2000), antiarthritic (Sahu et aI, Industries Limited, Shangli, Maharashtra

2000), hypoglycaemic (Raviet aI, 2000) under the trade name "Amrutrat and miceand antimalarial activities (Katewa & feed" and had free access to sterilized

Arora, 2001). The anticancer activitYof water.methanolic extract of the plant has been-

evaluated against Dalton's ascetic Experimental designlymphoma in Swissalbino mice (Kavimani

& Manisenthlkumar, 2000). In the present

study authors have evaluated the

hypolipidemic and antilipid peroxidativeactivities of the plant against p-DAB

induced hepatotoxicity in animals.

The plant, E. littorale was

collected from Chennai, TamilNadu in themonth of December and was identified

and authenticated by Dr T. Anandan;Research Officer, Central Research

Institute for Siddha Medicine, Chennai.

The aerial parts were air-dried and ground

well to fine powder, and used as drug in

the crude form along with physiologicalsaline.

p-DAB was purchased from Sigma

Chemical Company (St Louis, Mo, USA).

Allother reagents used were of analyticalgrade.

Animals

Wistar male albino rats having

body weight between 150-200g were

Table 1 emphases the effect of

The animals were fasted for aerial part of E. littorale on total

12 hours after the last dose of the drug cholesterol, triglyceride, HDL-c, WL-c,treatment and were sacrificed by cervical y-Glutamyl. transpeptidase (y-GT) and

decapitation. Blood was collected in tubes Lipid peroxidation levels of p- DAB

402 - Natural Product Radiance Vol 3(6) November-December 2004

induced hepatotoxicity in animals. In the

p-DAB induced animals, the level of total

cholesterol (87.59 ± 5.57 mg/dl),

triglycerides (153.47 ± 6.94 mg/dl), IDL­e (103.23 ± 3.45 mg/dl) and y-GT (8.26

± 1.05 IUIL)were significantlyincreased

and a significantdecrease in HDL-c(10.28

± 1.62mg/dl) in serum was observed

compared to the normal group animals.

Administration of aerial part of the plantsignificantly reduces the level of total

cholesterol (46.79 ± 6.22 mg/dl),

triglycerides (125.73 ± 76.54 mg/dl),

IDL-c (93.59 ± 2.92 mg/dl), y- GT(4.90± 0.48 IUIL)and increases HDL-c(12.25

± 1.65 mg/dl) content in serum samples

analyzed. Malondialdehyde content in theliver was enhanced in the p-DAB induced

hepatotoxicity (168.80 ± 4.70 n moVg)

A·•" ,.

when compared to normal animals. The

plant significantly reduced the

malondialdehyde content in the liver

(90.58 ± 3.18 n mol/g). VitaminEtreated

animals also expressed the similar

reduction of total cholesterol,

triglycerides, LDL-c, y-GT and lipid

peroxides in liver and an increase in

HDL-ccontent in serum samples.

Hepatotoxicity is associated with

biochemical, histological and functional

changes and progressive increase in the

chance of liver damage and death of an

organ. Liverdamage due to increased level

of lipid profile and lipid peroxidation

appears to be an important determinativemechanism of hepatotoxicity studies(Satturwar et al, 2003; Sharma et al,2003). The extract contains several

alkaloids, tlavanoids and reducing sugars

and exhibited hypolipidemia and

antioxidant properties in the present study

similar to the studies carried out byMuraUet al (2002) and Kavimani &

Manisenthlkumar (2000). Animals that

received a subcutaneous implantation ofthe p-DAB displayed cancerous

hyperlipidemia characterized byincreased

serum cholesterol (hyper­scholesterolemia) and triglyceride

(hypertriglyceridemia) levels (Irikura

et al, 1985; Kawasaki et al, 1998;Komatsu et al, 1998). The hyper­

cholesterolemia in the hepatoma-bearing

animals showed a highly atherogenic

lipoprotein profile, that is, a notable

increase in the cholesterol and significantdecrease in the HDLfraction (Irikura et

Table 1: Hypolipidemic activity of Enicostemma littorale Blume

S.No ExperimentTotalTriglyceridesHDL-LDL-y- GlntamylLipid

design

Cholesterol(mg/dl)CholesterolCholesteroltranspeptidase Peroxides(mg/dl)

(mg/dl)(mg/dl)(lUlL)(n molTBARS/g)

1

GroupI 33.57 ± 3.10561.61 ± 4.7416.78 ± 1.0247.48 ± 2.251.11 ± 0.2379.01 ± 2.43Normal animals2

GroupII 87.59± 5.57153.47 ± 6.9410.28 ± 1.62103.25 ± 3.458.26 ± 1.05168.80 ± 4.70DABinduced animals3

Groupill46.79± 6.22 •125.73 ± 6.97 •12.25 ± 1.62'93. 59 ± 2.92'4.90 ± 0.48'90.58 ± 3.18 •DABinduced + E. littoraledrug treated4

GroupIV41.25 ± 4.12 •111.75± 5.15•14.62 ± 1.25'87.4 ± 3.62'3.25 ± 0.25'85.45 ± 2.25'

DABinduced + VitaminE treated

Valuesare expressedas mean ± SD.GroupIII & IV are comparedto GroupII ('p<O.OOl).

Natural Product Radiance Vol 3(6) November-December 2004

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Conclusion

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