Cardioprotective effect of Gracilaria corticata in ... · 1*V.Prabha 1R.Divya, and 2V .Sivakumar 1...

Prabha, et al., 2019/ Cardioprotective effect of Gracilaria corticata

International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 5 (3) 1

RESEARCH ARTICLE

International Research Journal of Pharmaceutical and Biosciences

Pri -ISSN: 2394 - 5826 http://www.irjpbs.com e-ISSN: 2394 - 5834

Cardioprotective effect of Gracilaria corticata in

Experimental Rats

1*V.Prabha 1R.Divya, and 2V .Sivakumar

1 PG and Research Department of Biochemistry, DKM College for Women, Vellore, Tamilnadu.

2 PG and Research Department of Biochemistry, Adhiparasakthi College of Arts and Science, Kalavai, Vellore District, Tamilnadu.

Article info Abstract

Article history:

Received 01 AUG 2019

Accepted 05 SEP 2019

*Corresponding author:

[email protected].

Copyright 2019 irjpbs

Oxidative stresses resulted from free radicals and are associated with many diseases. Isoproterenol induces oxidative stress by generation of highly cytotoxic free radicals, thus increases cell permeability. Increased cell permeability causes cell damage, induces cell necrosis in liver and kidneys and myocardial infarction (MI) in heart. Thus, the present study was designed to investigate the total protective effect of Gracilaria corticata (GC) in isoproterenol (ISO) induced toxicity. Adult male wistar albino rats were pre-treated with Gracilaria corticata in various concentration such as 200mg, 400mg and 600mg /kg bw. daily for 16days after the treatment period, ISO (85mg/kg bw) was subcutaneously injected into the rats at 24 hrs. intervals for 2 days. ISO-induced toxicity was indicated by increased serum markers, significantly increase in cholesterol, LDL, triglyceride and decreased in HDL concentration. The activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase are decreased and altered the hematological parameters and decreased the protein levels and increased blood glucose levels are due to oxidative stress created by ISO. However, pre-treatment with Gracilaria corticata to protect the biochemical and haematological parameters to normal to indicating the total protective effect of Gracilaria corticata against ISO induced toxic in experimental rats. Histopathological findings of the heart, liver and kidney tissues further confirmed that Gracilaria corticata has total protective effect against ISO induced toxicity.

Key words: Oxidative stress, Isoproterenol, Gracilaria corticata, Protective effect.



INTRODUCTION

Isoproterenol (ISO) is a synthetic catecholamine and β-adrenergic agonist documented to produce severe stress in the myocardium and to result in MI if administered in supramaximal doses [1]. ISO produces necrosis that leads to increased lipid peroxidation and increased levels of lipids and altered enzyme and antioxidant activities. The proposed mechanisms to explain ISO-induced toxicity include generation of highly cytotoxic free radicals through the autoxidation of catecholamines [2].

As a consequence of an increasing demand for the biodiversity in screening program, seeking therapeutic drugs from natural products there is now a greater interest in the marine organism especially marine algae [3,4]. The nutritional value of marine algae has long been recognized in the orient than in western world with limited use as a dietary part [5]. Seaweeds are low in fats but contain vitamins and bioactive compounds like terpenoids and sulphated polysaccharides a potential natural antioxidant, which are not found in land plants [6].

Gracilaria corticata (G.corticata) is a predominant red microalga species found in coastal regions of Indian subcontinent, belonging to the family Gracilariacea. It possesses several biomedical properties such as antibacterial, antiviral, antifungal, antiprotozoal, anti- inflammatory, antioxidant, cytotoxic, contraception, gastrointestinal, cardiovascular, hypoglycemia, antienzymes, spasmolytic and allelophatic effects [7]. The main functional groups involved in Gracilaria corticata uptake are Carboxyl, Sulfydryl and Hydroxyl which are the prime constituents of Gracilaria corticata. The estimation of trace elements in Gracilaria corticata is of great value so that the seaweed can be treated with necessary nutrient materials in order to ensure proper growth, yields and make them resistant to disease and toxicity. In this study the ethanolic extract of Gracilaria corticata was used to exhibit the overall protective effect in oxidative stress created in rats using isoproterenol.

MATERIALS AND METHODS

Collection of Plant material The red seaweed Gracilaria corticata plant was collected from mandabam region of Rameshwaram, Tamilnadu, India. The plant authenticated by Dr. P. Jayaraman, Professor, Institute of Herbal Botany Plant Anatomy Research Center, Chennai. A voucher specimen no: PARC/2018/ 2047 was deposited in center for further analysis. Preparation of Plant Extract Ethanol Extract After the collection of Gracilaria corticata they were placed in clean tray and allowed for shade drying. The Gracilaria corticata was subjected to surface sterilization using ethanol and then dried in shade. The dried whole plant was subjected to size reduction to a coarse powder by using dry grinder and passed through sieve (20 mesh). The medicinal plant Gracilaria corticata powdered (100 g) were defatted by treating with pet-ether and then extracted with ethanol solvent by using soxlet apparatus. The solvent was removed under vacuum to get the solid



mass. The residue was weighed and stored in air and water proof containers, kept in refrigerator at 4oC. From this stock, fresh preparation was made whenever required. Experimental Animals The whole experiment was done according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India, and approved by the Animal Ethical Committee of Annamalai University, marine oval no.556: 20.3.2008). The study was conducted on 30 male albino Wistar rats (Rattus norvegicus) weighing 170 –200 g, obtained from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Institute of Health Sciences, Annamalai University, Tamil Nadu, India. They were housed in polypropylene cages (47×34×20 cm; four rats per cage) lined with husk, renewed every 24 h under a 12:12 h light– dark cycle at around 22±2°C and had free access to tap water and food. The rats were fed on a standard pelleted diet (Pranav Agro Industries Limited, Maharashtra, India). The diet provided metabolizable energy of 3,600 kcal. Experimental design In the present study, a total of thirty six rats were used. The rats were divided into six groups of six rats each. Group I: Normal: Rats received 3ml Saline through oral until the end of the experiment. Group II: Standard: Rat is pretreated with verapamil 5μmol/kg body weight (i.v) for 16 days+ ISO administered (85mg/kg bw., subcutaneously , twice an interval of 24 h, i.e., on 17th and 18th day). Group III: Inducer: Rats administered with ISO treated rats (85mg/kg bw. subcutaneously, twice an interval of 24 h, i.e., on 17th and 18th day). Group IV: 200mg/ Gracilaria corticata : Rats are pretreated with G.corticata (200mg/kg bw.) for 16 days+ ISO administered (85mg/kg bw., subcutaneously , twice an interval of 24 h, i.e., on 17th and 18th day). Group V: 400mg/ Gracilaria corticata: Rats are pretreated with G.corticata (400mg/kgbw.) for 16 days + ISO administered (85mg/kg bw, subcutaneously, twice an interval of 24 h, i.e., on 17th and 18th day). Group VI: 600mg/ Gracilaria corticata: Rats are pretreated with G.corticata (600mg/kgbw.) for 16 days + ISO administered (85mg/kg bw, subcutaneously, twice an interval of 24 h, i.e., on 17th and 18th day). Biochemical Estimation Total cholesterol is estimated using CHOD/POD method, HDL fraction is assayed using autozyme cholesterol diagnostic kit, Triglyceride is assayed using GOD/POD method, VLDL and LDL were calculated using the Friedewald method, Total protein is estimated using Biuret method, Glucose is estimated by Trevelyan and Harrison method, Sodium was estimated by the method of Trinder, Potassium was estimated by the method of Jocabs and Hoffmann, Calcium was estimated by the method of Tietz using Robonik Diagnostic Kit, Creatine phosphokinase was estimated by the method of Rosalki, Lactate dehydrogenase (LDH) was estimated by the method of Teitz using Agappe diagnostic kit, Troponin T and Troponin I was assayed using Boehringer Mannheim ES 300 analyser, The Superoxide dismutase was estimated by the



method of Misra and Fridovich, Catalase was estimated by the method of Sinha, and Glutathione peroxidase was estimated by the method of Rotruck. Hematological Estimation The haematological parameters were analysed on the day of blood Collection. White blood corpuscles cells, Red blood corpuscles cell, Heamoglobin, Platelet, Haematocrit or packed cell volume, absolute eosinophil count, MCV, MCH, MCHC, differential count are analysed by blood samples using the standard procedures [19]. Histopathological Examination

The histopathological studies show the basic understanding of the structure and function of cells, tissue, organs and organ system, which is also termed as ‘Microscopic Anatomy’. The liver was removed and stored immediately in 10% formalin initially for 48 hours; thereafter the materials were transferred to 70% alcohol and stored. Following fixation, the tissue was subsequently placed in paraffin. 5 µm thick sections were cut using a microtome and then stained with hematoxylin and eosin and mounted using neutral di – styrene - dibutyl propylene (DPX) medium and examined using photomicroscopy.

Statistical Analysis The statistical analysis was performed by ANOVA under one way classification followed by Bonferroni multiple comparison test, changes were considered significant at the P-value of < 0.05 and < 0.01 level of significance. The values were expressed as mean ± SD. RESULTS AND DISCUSSION

In recent years, the biological activities, nutritional value, and potential health benefits of marine algae have been intensively investigated and reviewed. Seaweeds have proved to be an enriched source of vitamins and minerals. Moreover, importance of seaweeds lies in its great food value as they are rich in carbohydrates, protein and Lipids [11].

Several events, such as biochemical, ultra structural changes, histological, electrolyte and membrane changes have been shown to occur within 48 hours after the injection of ISO. Histological changes induced by excessive amounts of ISO include degeneration and necrosis of myocardial fibres, accumulation of inflammatory cells, interstitial edema, lipid droplets and endocardial hemorrhage [12]. All these changes may affect the dietary habits of animal. Loss of body weight could be due to reduced food and water intake. In the present investigation the ISO treated rats are reduced the body weight but Gracilaria corticata pretreated rats are maintaining the body weight due to total protective effects.



TABLE 1: Effect of Gracilaria corticata on animal body weight in normal and isoproterenol induced myocardial toxicity in experimental animals

No. of days

Normal Control

(g)

Standard drug verapamil (5μmol/kg

bw.)

ISO Inducer

(g)

200mg G.C (g)

400mg G.C (g)

600mg G.C (g)

0 days 183 ±0.81 179 ±0.21 185 ±0.06 180 ±0.25 181 ±0.31 180 ±0.07

5 days 186 ±0.09 183 ±0.04 186 ±0.47 187 ±0.05 186 ±0.62 186 ±1.06

10 days 193 ±0.27 189 ±0.32*** 190 ±1.06 192 ±0.68** 192 ±0.04*** 192 ±0.72***

15 days 196 ±0.36 194 ±0.14*** 186 ±1.16 188 ±0.53** 189 ±0.46*** 195 ±0.53***

Values are expressed in mean ± SD of six individual values; One way analysis of variance

followed by Bonferroni multiple comparison test, *** P01, ** P0.05.

Lipid metabolism plays an important role in MI produced by ischemia [24]. The increased concentration of cholesterol could be due to a decrease in HDL, since HDL is known to be involved in the transport of cholesterol from tissues to the liver for its catabolism [13]. ISO induced myocardial necrosis had been shown to elevate plasma TC, TG, LDL, VLDL and decrease HDL levels are indicate the toxicity of ISO [14].

TABLE:2: Effect of Gracilaria corticata on plasma lipid contents levels in normal and isoproterenol induced myocardial toxicity in experimental animals

Parameters Normal control

Standard drug verapamil

(5μmol/kg bw.)

ISO Inducer

200 mg G.C 400 mg G.C

600 mg G.C

Total Cholesterol (mg/dl)

147 ±1.13 143 ±2.31*** 260 ±1.14 156.5 ±1.18**

140.5 ±0.19***

133.5 ±1.06***

HDL (mg/dl) 49.5 ±0.01 47.3 ±0.21*** 25 ±0.6 53 ±0.84** 31.5 ±0.36***

27 ±0.42***

Triglyceride (mg/dl)

82 ±0.07 80 ±0.02*** 220 ±1.01 85.5 ±0.19** 141.5 ±0.07***

80 ±0.27***

LDL (mg/dl) 124.8 ±1.21 126.1 ±2.40*** 131.2 ±1.09

120.9 ±1.2** 83.45 ±1.7***

88.2 ±1.26***

VLDL (mg/dl)

18.55 ±0.45 19.52 ±0.12*** 37.45 ±0.36

16.8 ±1.34** 28.5 ±1.55***

20.8 ±0.93***

LDL/HDL RATIO

2.88 ±1.2 2.68 ±2.1*** 3.23 ±0.94 3.45 ±0.63** 2.77 ±0.25***

2.7 ±0.67***





The present study, indicate the result shown in table 2 to protect the lipid metabolism from ISO toxicity, when compared to the ISO treat groups.

TABLE:3: Effect of Gracilaria corticata on total protein, blood glucose and electrolytes levels in normal and isoproterenol induced myocardial toxicity in experimental animals

Parameters Normal Control

Standard drug verapamil

(5μmol/kg bw.) ISO Inducer 200mgG.C 400mgG.C 600mgG.C

Total Protein (g/dl)

7.05 ±0.33 7.00 ±0.21*** 4.7±0.84 7.6 ±0.42** 7.0 ±0.14***

7.45 ±0.95***

Blood Glucose (mg/dl)

80.63 ±0.96 82.32 ±0.43*** 295.7 ±1.67 192.9 ±1.5**

189.7 ±0.9***

176.49 ±0.52***

Sodium (mmol/l)

130.6 ±0.59 132.1 ±0.21*** 176.5 ±1.04 138.4 ±0.16**

150.2 ±1.15***

143.4 ±0.95***

Potassium (mmol/l)

4.05 ±0.33 4.01 ±0.15*** 6.3 ±0.84 4.5 ±0.28** 4.05 ±0.49***

3.85 ±1.02***

Calcium (mmol/l)

9.2 ±0.84 9.0 ±0.21*** 11 ±0.94 9.9 ±1.04** 7.15 ±0.05***

6.0 ±0.707***



Table :4 Effect of Gracilaria corticata on Hemoglobin, blood cell count levels in normal and isoproterenol induced myocardial toxicity experimental in animals


Standard drug verapamil (5μmol/kg bw.)

ISO Inducer 200 mg G.C 400 mg G.C

600 mg G.C

Hemoglobin (g/dl)

11.8 ±0.25 11.2 ±1.32*** 10.1 ±0.4 12.15 ±1.06**

11.55 ±1.62***

12.25 ±1.06***

W.B.C Count (Cells/cumm)

8015 ±1.7 8021 ±1.2*** 1953 ±0.09 6450 ±1.6**

9725 ±1.4***

5555 ±0.91***

R.B.C Count (Cells/cumm)

4.05 ±0.2 4.00 ±1.4*** 3.0 ±0.56 6.35 ±0.7** 4.71 ±0.57***

6.39 ±0.67***

Platelets Count (Cells/cumm)

3,49,000 ±0.35

3,48,000 ±0.12***

5,39,000 ±1.08

8,00,500 ±0.9**

3,78,000 ±0.3***

6,70,000 ±1.17***

A.E.C (Cells/cumm)

187 ±1.04 180 ±2.21*** 561 ±8.4 186.5 ±1.7**

162.5 ±1.06***

188 ±1.55***





TABLE:5: Table :4 Effect of Gracilaria corticata on Hematological parameter levels in normal and isoproterenol induced myocardial toxicity in experimental animals



ISO INDUCER

200mg G.C

400mg G.C 600mg G.C

PCV (%) 27.1 ±1.02 24.0 ±1.20***

50 ±0.28 38 ±0.24**

31.65 ±0.21***

33.9 ±0.26***

MCV (fl) 59.05 ±0.27

57.03 ±0.24***

87.5 ±0.25 60.9 ±1.1**

61.15 ±0.33***

53.1 ±1.9***

MCH (Pg) 24.85 ±1.04

26.82 ±2.05***

29.25 ±1.06

22.15 ±0.40**

21.05 ±0.37***

18.65 ±0.35***

MCHC (%) 38.9 ±0.38 39.2 ±0.46***

33.9 ±0.8 34.55 ±1.3**

32 ±0.98 35.2 ±0.70***



When cells containing LDH, CK, are damaged or destroyed due to lack of oxygen supply or glucose, the cell membrane becomes permeable or may rupture, which results in the leakage of enzymes [15]. ISO administration in rats is results in producing gross, microscopic myocardial necrosis and depletion of tissue enzymes in the heart. Because of necrosis and leakage, the levels of diagnostic indicators of MI, such as LDH, CK, increase in plasma [16].

Toponin-T (cTnT) is a very sensitive and specific indicator in detecting MI. It is a contractile protein that is normally not found in serum, but released when myocardial necrosis occurs cTnT and cTnI levels in blood provided biochemical evidence of myocardial cell injury because these assays have high sensitivity and specificity. Comparison of the diagnostic value of cardiac troponin I and T determinations for detecting early myocardial damage and the relationship with histological findings after isoprenaline-induced cardiac injury in rats. [17]. In table 6 have shown that cTnT is a powerful biomarker in laboratory animals for sensitive and specific detection of cardiac injury arising from various causes.

Free radical scavenging enzymes such as SOD and CAT are known to be the first line cellular defence against oxidative stress, disposing of O2 and H2O2 prior to the interaction to form the more harmful hydroxyl (OH-) radicals [18].



TABLE: 6: Effect of Gracilaria corticata on cardiac marker enzymes levels in normal and isoproterenol induced myocardial toxicity in experimental animals



ISO Inducer 200 mg G.C 400 mg G.C 600 mg G.C

Troponin t -ve -ve +ve -ve -ve -ve

Troponin i -ve -ve +ve -ve -ve -ve

LDH (U/L) 276 ±1.09 265 ±2.04***

576 ±0.62 396 ±0.4** 437 ±0.27***

235 ±0.96***

CPK (U/L) 62.85 ±1.03 64.42 ±1.12***

200.2 ±0.38 74.7 ±0.7** 105.5 ±0.73***

71.4 ±0.06***



These enzymes are critical for defence mechanisms against the harmful effects of ROS and free radicals in biological systems [19]. The activities of these enzymes are lowered due to enhanced LPO in ISO treated group.

TABLE: 7: Effect of Gracilaria corticata on antioxidant enzymes levels in normal and isoproterenol induced myocardial toxicity in experimental animals



ISO Inducer

200mgG.C 400mgG.C 600mgG.C

SOD (U/mg protein)

9.01±0.43

9.13±0.42***

4.56±1.71

10.73±0.81**

12.97±1.81***

16.37±0.7***

Catalase (µmol/mg protein)

86.5±0.210

85.2±0.211***

43.1±0.33

75.5±0.22**

68.3±0.22***

70.3±0.46***

Glutathione peroxidise (U/ kgprotein)

8.25±0.96

8.13±0.93***

6.37±1.86

11.96±0.65**

9.36±1.37***

10.35±0.73***

Values are expressed in mean ± SD of six individual values Values are expressed in mean ± SD of six individual values; One way analysis of variance followed by Bonferroni multiple comparison

test, *** P01, ** P0.05.



TABLE: 8: Effect of Gracilaria corticata on Cardiac, Liver and kidneys cells architecture levels in normal and isoproterenol induced myocardial toxicity in experimental animals

S.NO NORMAL CONTROL

ISO INDUCER 200mg G.C 400mg G.C 600mg G.C

HEART No abnormality detected (Fig.15)

Myocardial degeneration, necrosis(Fig.16), multifocal mononuclear cell infiltration (Fig.17) and mild oedema(Fig.18)

Multifocal myocardial vacuolar degeneration(Fig.19), necrosis and mild oedema. (Fig. 20)

Mild myocardial degeneration (Fig..21), and multifocal mononuclear cell infiltration (Fig.22).

Mild myocardial vacuolar degeneration (Fig.23), necrosis and multifocal mononuclear cell infiltration (Fig.24).

LIVER Hydropic degeneration of hepatocytes (Fig.25)

Congestion, diffuse hydropic degeneration of hepatocytes (Fig.26), milld kupffer cell hyperplasia (Fig..27),andmultifocal mono nuclear cell infiltration.

Congestion (Fig..28)

Congestion, diffuse hydropic degeneration of hepatocytes (Fig.29).

Mild hepatocellular degeneration (Fig.30) and very mild mononuclear cell infiltration.

KIDNEY No abnormalities (Fig.31)

Congestion, multifocal tubular epithelial cell degeneration (Fig.32) and necrosis(fig. 33)

Congestion, multifocal tubular epithelial cell degeneration(Fig.34), and infiltration of mononuclear cells in the interstitium (Fig..35).

Tubular epithelial cell degeneration (Fig.36) ,and interstitial mononuclear cell infiltration.

Tubular epithelial cell degeneration (Fig. 37).



Figure 13

Standard: rat with no abnormities

Figure 14 Inducer: Myocardial degeneration, necrosis

Figure 15

Inducer: heart multifocal mononuclear

cell infiltration 20x

Figure 16

Inducer: mild edema

Figure 17

200 mg G.C: Multifocal myocardial

vacuolar degeneration 20x

Figure 18

200 mg G.C: mild oedema

HEART



Figure 19

400mg G.C: Multifocal myocardial

vacuolar degeneration 10x

Figure 20

400mg G.C: multifocal mononuclear cell

infiltration 10x

Figure 21

600 mg G.C: Mild myocardial vacuolar

degeneration

Figure 22

600mg G.C: Multifocal mononuclear cell

infiltration



Figure 23

Standard: Hydropic degeneration of

hepatocytes

Figure 24

Inducer: Diffuse hydropic degeneration of

hepatocytes

Figure 25

Inducer: Multifocal mono nuclear cell

infiltration

Figure 26

200mg G.C: Congestion

Figure 27

400mg G.C: Congestion

Figure 28

600mg G.C: Mild hepatocellular degeneration

LIVER



Figure 30 Inducer: Congestion, multifocal epithelial cell degeneration10x

Figure 29

Standard: No abnormalities

Figure 31

Inducer: Necrosis 10x

Figure 32 200mg G.C: Congestion, multifocal tubular Epithelial cell degeneration

Figure 33 200mg of G.C: Congestion, infiltration of

mononuclear cells in the interstitium

Figure 34 400mg of G.C: Tubular epithelial cell

Degeneration

KIDNEY



Figure 35 600mg of G.C: Tubular epithelial cell

degeneration

Gracilaria corticata secondary metabolites help in prevent living cells against free radical mediated oxidative stress. The results obtained suggest that Gracilaria corticata has a protective activity against oxidative stress caused by ISO administration. Gracilaria corticata, a potential therapeutic agent, can likewise ensure the tissue from necrosis and the heart from MI. The results of the present study indicate that prior administration of Gracilaria corticata is effective in minimizing all the deleterious effects induced by ISO, thereby justifying its use as a potent antagonist for toxicity therapeutic agent. The beneficial effects of Gracilaria corticata are most likely identified with its capacity to fortify the myocardial cell membrane, to keep up near normal levels of marker enzymes, lipids levels, protein levels, haematological, electrolytes, antioxidants parameters and liver, kidneys and heart tissue architecture. When compared to 200mg/kg, 400mg/kg and 600mg/kg of Gracilaria corticata ethanol extract was highly significantly protect the ISO induced cardio toxicity in experimental animals. The overall results indicate that the total protective property of Gracilaria corticata is due to its antioxidant, hypolipidemic, and membrane stabilizing activities. CONCLUSION

Many synthetic drugs have adverse side effects creating oxidative damage. In living systems, free radicals are constantly generated and they can cause extensive damage to tissues and biomolecules, leading to various disease conditions. The most effective way to eliminate free radicals is with help of antioxidants by promoting their decomposition. An alternative solution to the problem is to consume natural antioxidants from food supplements and traditional medicines. The red seaweed Gracilaria corticata collected from the mandabam region of Rameshwaram Tamilnadu has rich source of secondary metabolites with noticeable biopharmaceutical effects. The study provides evidence for their total protective effect from ISO induced oxidative stress produced myocardial infarction from through biochemical markers, haematological parameters, antioxidant enzymes and histopathological findings of liver, kidneys and cardio cells.



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