Hepatoprotective and in vivo antioxidant effects of Corchorus depressus (L.) Stocks. (Tiliaceae)

Sandeep Kataria¹*, Dilsher Kaur², Shaival Kamalaksha Rao³, Neha Sharma⁴, Ravi K Khajuria⁴

¹Faculty of Pharmaceutical Sciences, Jodhpur National University, Jodhpur, Rajasthan, India.

²Department of Pharmaceutical Chemistry, I. S. F. College of Pharmacy, Moga, Punjab, India

³Department of Pharmacognosy, C.U. Shah College of Pharmacy and Research, Surendranagar, Gujarat, India

⁴Instrumentation Division, Indian Institute of Integrative of Medicine, Jammu, J & K, India

*Corresponding Author E-mail: katariasandy@gmail.com

ABSTRACT:

Objective: To investigate the hepatoprotective and in vivo antioxidant effects of Corchorus depressus in carbon tetrachloride (CCl₄)-induced hepatotoxicity in rats.

Methods: Group allotment in this study included vehicle, CCl₄, Methanol extract of Corchorus depressus 100, 200, and 400 mg/kg + CCl₄ and silymarin 50 mg/kg + CCl₄, and treatment was carried out accordingly. On the 7^th day, rats were sacrificed and blood was withdrawn by cardiac puncture. The levels and activities of serum biochemical parameters and antioxidant enzymes were then assayed using standard procedures.

Results: The results of CCl₄-induced liver toxicity experiment showed that rats treated with the methanol extract of Corchorus depressus, and also the standard treatment, silymarin (50mg/kg), showed a significant decrease in ALT, AST, ALP and total bilirubin levels, which were all elevated in the CCl₄ group (p < 0.01). The results observed after administration of 200mg/kg methanol extract were comparable to those of silymarin at 50mg/kg (p > 0.05). The methanol extract did not show any mortality at doses upto 5000mg/kg body weight. CCl₄ also produced significant (P < 0.05) reductions in the activity of catalase, SOD and GSH, and conversely increased MDA level. Methanol extract of Corchorus depressus produced significant and dose-dependent reversal of CCl₄-diminished activity of the antioxidant enzymes and reduced CCl₄-elevated level of MDA. The standard drug also significantly increased CCl₄-diminished antioxidant enzymes activity and reduced CCl₄-elevated MDA level. In general, the effects of the standard drug were comparable and not significantly different from those of Corchorus depressus.

Conclusion: This study clearly indicates that the methanol extract of Corchorus depressus possesses hepatoprotective and in vivo antioxidant effects. These findings provide scientific support for the traditional use of this herb as an Indian medicine for the treatment of liver diseases.

KEY WORDS: Corchorus depressus; Hepatoprotective; Antioxidant

1. INTRODUCTION

The liver as a vital organ in the body is primarily responsible for the metabolism of endogenous and exogenous agents. It plays an important role in drug elimination and detoxification and liver damage may be caused by xenobiotics, alcohol consumption, malnutrition, infection, anemia and medications [1]. Modern medicines have little to offer for alleviation of hepatic diseases and only limited numbers of drugs are available for the treatment of liver disorders.

The synthetic drugs used for the treatment of liver diseases are inadequate and can have serious side-effects. Traditionally, many of the folk remedies of plant origin have long been used for the treatment of liver diseases [2]. Liver cells possess a number of compensatory mechanisms to deal with reactive oxygen species (ROS) and their effects; among these are the induction of a number of antioxidant proteins such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GSHPx) and the tripeptide glutathione (GSH). Therefore, oxidative stress, caused mainly by ROS, is also associated with hepatic diseases [3]. Despite the fact that hepatic problems are responsible for a significant number of liver transplantations and deaths recorded worldwide, available pharmacotherapeutic options for liver diseases are very limited and there is a great demand for the development of new effective drugs.

Corchorus depressus (Linn.) is a multipurpose herb of Tiliaceae family that is used extensively for the treatment of various diseases/disorders like hepatitis, sexual dysfunction, fevers, tumors, pain, gonorrhea, diarrhea, dysentery, dyspepsia and bleeding piles and is widely distributed in different regions of the world [4-6].

In the ethnobotanical claims, the Corchorus depressus is used for the treatment of jaundice and other hepatic diseases [7] by the folk tribes of Rajasthan state, India. To the best of our knowledge there is no scientific report is available in support of the hepatoprotective and antioxidant activity of Corchorus depressus. Therefore, to justify the traditional claims we have assessed the hepatoprotective and in vivo antioxidant effects of the methanol extract of Corchorus depressus in carbon tetrachloride (CCl₄)-induced hepatotoxicity model in rats.

2. MATERIALS AND METHODS

2.1 Plant material

The whole plant of Corchorus depressus L. was collected from Dausa district, Rajasthan, India, in September, 2010, and authenticated by Mr. P. M. Padhye, Botanical Survey of India (BSI), Jodhpur, Rajasthan, India. A voucher specimen of this collection (JNU/JODHPUR/CD/SK-1) has been deposited at the Herbarium of the Botanical Survey of India.

2.2 Preparation of extract

Plant material was washed with distilled water to remove epiphytes and dirt particles and dried at room temperature. The dried plant material was manually ground to a fine powder and stored in airtight bottles. Thirty one hundred grams of dried powder was first defatted with petroleum ether and then extracted with 95% methanol by using Soxhlet apparatus. The solvent was evaporated to dryness and the dried crude extract was stored in air tight bottle at 4°C. The percentage yield of methanol extract was 28%. For testing, the Corchorus depressus methanol (CDM) extract was dissolved in sterile distilled water and diluted to the desired concentrations.

2.3 Test animals

Sprague–Dawley rats (220 ± 20 g) of both sexes were used for the present study. All animal experiments were performed as per the protocols and recommendation of the Institutional Animal Ethics Committee of Pinnacle Biomedical Research Institute, Bhopal, India (Animal Eths Comm/IE/Reg no. 1283/c/09/CPCSEA) and were in accordance with international standard on the care and use of experimental animals. They were kept in a controlled environment at 25 ± 2°C and 30–60% relative humidity with a 12 h light and dark cycle. The animals were fed a standard rodent pellet diet and water ad libitum.

2.4 Acute toxicity study

The acute toxicity test of the CDM was done by up and down method [in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines, 2001] [8]. Overnight fasted albino rat were used for study (three animals in group). The CDM was administered to all the three animals in group at a starting single dose of 5 mg/kg. Animals were observed for a period of 2 h, then occasionally for 4 h for severity of any toxic signs and mortality. When no mortality was observed the same dose would be additionally administered to one more animal. If no mortality is observed at this dose, the same procedure would be repeated for dose levels of 50, 300, 2000 and 5000 mg/kg of extract on separate newer groups. The behavioral changes closely observed for were: hyperactivity, ataxia, tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. The animals were kept under observation up to 14 days after drug administration to find out any delayed mortality.

2.5 Hepatoprotective and antioxidant activity

A total of 36 rats were divided into 6 groups of 6 rats each.

· Group I served as normal control and received only the vehicle (1mL/kg/day of 1% CMC; p.o.).

· Group II received CCl₄ 1mL/kg (1:1of CCl₄ in olive oil) i.p. once daily for 7days.

· Group III received CCl₄ 1mL/kg (1:1of CCl₄ in olive oil) i.p. and silymarin 50mg/kg orally (p.o.) for 7days.

· Groups IV, V, VI were administered methanolic extract of Corchorus depressus at 100, 200, and 400mg/kg body weight p.o., respectively and dose of 1mL/kg i.p. of CCl₄ (1:1 of CCl₄ in olive oil) for 7days.

At the end of the experiment (on day 7), the animals were sacrificed by exsanguinations [1,9]. Liver was dissected out and immediately washed with ice-cold saline to remove blood. Liver homogenate (10.0%, w/v) were prepared with 50 mM cold potassium phosphate buffer (pH 7.4). The resulting suspension was centrifuged at 1000 rpm for 10 min, and the supernatant was collected for further analysis. All treatments were done at 4^◦C.

2.6 Analysis of liver function enzymes

The biochemical parameters like serum enzymes: glutamate pyruvate transaminase (ALT), aspartate aminotransferase (AST) [10], alkaline phosphatase (ALP) [11] and total bilirubin [12] were assayed using assay kits (Span Diagnostic, Surat).

2.7 Measurement of antioxidant enzymes and MDA levels

Measurement of antioxidant enzymes activity and MDA level was done according to standard procedures: SOD [13], catalase [14], reduced glutathione [15] and MDA [16].

2.8 Histopathological studies

After rats were sacrificed, livers were identified and carefully extracted. Sections were immediately taken from each lobe of the liver. The tissues were fixed in 10% formo-saline, dehydrated in graded alcohol and embedded in paraffin. Sections were prepared and then stained with hematoxylin and eosin dye for photomicroscopic (Motic DMBA 300, New Delhi) observation, including cell necrosis, fatty change, hyaline regeneration, ballooning degeneration [1].

2.9 Statistical analysis

The data were expressed as mean ± S.E.M. Results were analyzed statistically by one-way analysis of variance (ANOVA) followed by Tukey’s test using the Origin version 6.0 (Microcal, Northampton, MA, USA) for Window software. P-value <0.05 was regarded as statistically significant.

3. RESULTS:

3.1 Acute toxicity studies

In acute toxicity study, methanol extract of Corchorus depressus was found to be safe up to 5000 mg/kg. No mortality or toxic symptoms were observed during the entire duration of the study.

3.2 Effect of Corchorus depressus on serum biochemical Parameters

The results of hepatoprotective effect of methanol extract of Corchorus depressus on CCl₄-intoxicated rats are shown in Table 1.The activity of the enzymes ALT, AST, ALP, and the total bilirubin value were significantly increased in the CCl₄ group compared to the control group (p<0.01). The rats treated with Corchorus depressus (100 mg/kg, group IV) showed a significant decrease in ALT and ALP compared to the CCl₄ group (p < 0.05), but the AST and total bilirubin values were comparable to those of CCl₄ group (p > 0.05). The rats treated with Corchorus depressus (200mg/kg, group V and 400mg/kg, group VI), and also the standard treatment Silymarin (50mg/kg, group III), showed a significant decrease in all of the parameters that were elevated in the CCl₄ group (p < 0.01). Overall, the results observed after administration of 200mg/kg Corchorus depressus were comparable to those of Silymarin at 50mg/kg (p > 0.05). Although Silymarin and higher dosages of Corchorus depressus showed a strong hepatoprotective effect against CCl₄-induced liver injury, the levels of liver related biochemical parameters were still significantly higher than those of the control group which did not receive CCl₄ (p < 0.01).

3.3 Effect of Corchorus depressus on in vivo antioxidant enzymes and MDA level

As shown in Table 2, CCl₄ produced significant (P < 0.01) reductions in the activity of SOD, CAT, and GSH. Corchorus depressus (100, 200, and 400 mg/kg) caused significant (P < 0.01, P < 0.05) increases in CCl₄-diminished activity of all antioxidant enzymes assayed with peak effects produced at the highest dose of 400 mg/kg. Enzyme activity values for Corchorus depressus at the different doses were mostly comparable and not significantly different from values obtained for the vehicle group. Silymarin significantly (P < 0.01) reversed CCl₄-diminished antioxidant enzyme activity. Enzyme activity values for silymarin were significantly (P < 0.05) different from vehicle for SOD, CAT and GSH. The effects of Corchorus depressus at its most effective dose were comparable and not significantly different from the effect of silymarin. Carbon tetrachloride significantly (P < 0.01) increased MDA level relative to the vehicle group. Corchorus depressus (100, 200, and 400 mg/kg) significantly (P < 0.01) and dose-dependently diminished CCl₄-elevated MDA level with the value produced at the highest dose (400 mg/kg) being slightly higher but significantly (P < 0.05) different from the vehicle group. Silymarin significantly (P < 0.001) reversed CCl₄-induced MDA level elevation with value being comparable and not significantly different from vehicle. The effect of silymarin was not significantly different from that of Corchorus depressus at 400 mg/kg.

Table 1: Effect of Corchorus depressus on serum biochemical parameters in CCl₄-induced hepatotoxicity in rats.

	ALT (IU/L)	AST (IU/L)	ALP (IU/L)	Total bilirubin (mg/dL)
Group I control	32.41 ± 1.82	142.21 ± 8.12	123.15 ± 1.43	0.87 ± 0.14
Group II CCl₄	293.12 ± 18.34	767.18 ± 98.6	236.28 ± 10.21	4.06 ± 0.78
Group III Silymarin 50 (mg/kg)	158.49 ± 14.67^**	393.14 ± 37.22^*	133.72 ± 6.88^**	1.92 ± 0.7^**
Group IV ME 100 (mg/kg)	132.29 ± 16.98^*	542.08 ± 57.03	186.21 ± 16.09^*	2.18 ± 0.12
Group V ME 200 (mg/kg)	112.24 ± 10.84^**	412.73 ± 62.33^**	119.03 ± 11.6^**	1.94 ± 0.16^**
Group VI ME 400 (mg/kg)	76.64 ± 20.32^**	298.64 ± 46.14^**	102.17 ± 10.87^**	1.56 ± 0.8^**

Values are mean ± S.E.M., n = 6 animals in each group. Symbols represent statistical significance. ME, 95% methanolic extract of Corchorus depressus; ALT, glutamate pyruvate transaminase; AST, aspartate amino- transferase; ALP, alkaline phosphatase

* p < 0.05, as compared to CCl₄-intoxicated group.; ** p < 0.01, as compared to CCl₄-intoxicated group

Table 2: Effect of Corchorus depressus on antioxidant enzymes and malondialdehyde in CCl₄-induced hepatotoxicity in rats.

	SOD (U/mg protein)	CAT (U/mg protein)	MDA (nmol/mg protein)	GSH (mg/g protein)
Group I control	329.68 ± 20.43	54.08 ± 13.32	2.26 ± 0.56	5.43 ± 1.44
Group II CCl₄	158.76 ± 37.65	38.26 ± 7.19	5.13 ± 0.33	2.33 ± 1.04
Group III Silymarin 50 (mg/kg)	262.4 ± 41.34^**	53.18 ± 11.33^*	4.13 ± 1.22^*	5.29 ± 1.34^*
Group IV ME 100 (mg/kg)	183.65 ± 32.45	39.59 ± 14.17	4.55 ± 0.76^*	4.78 ± 1.09^*
Group V ME 200 (mg/kg)	206.49 ± 36.54	43.34 ± 8.97^**	3.89 ± 0.85^**	5.02 ± 1.82^**
Group VI ME 400 (mg/kg)	248.04 ± 34.76^**	57.87 ± 7.66^*	4.02 ± 0.43^**	5.1 ± 0.44^**

Values are mean ± S.E.M., n = 6 animals in each group. Symbols represent statistical significance. ME, 95% methanolic extract of Corchorus depressus; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; GSH, glutathione.

* p < 0.05, as compared to CCl₄-intoxicated group.; ** p < 0.01, as compared to CCl₄-intoxicated group

4. DISCUSSION:

In CCl₄-induced toxicity, CCl₄ is metabolized through the cytochrome P450 monooxygenase system to produce the trichloromethyl radical, which then reacts with oxygen to form the trichloromethylperoxyl radical (CCl₃O₂^•) [17]. These radicals further attack cellular macromolecules such as proteins or lipids, which lead to the lipid peroxidation. Carbon tetrachloride-induced lipid peroxidation increases the permeability of the plasmamembrane to Ca²⁺, leading to severe disturbances of calcium homeostasis and necrotic cell death. In addition, the (CCl₃ radical can directly bind to tissue macromolecules and some of the lipid peroxidation products are reactive aldehydes, e.g., 4-hydroxynonenal, which can form adducts with proteins [18]. In addition to the intracellular events, Kupffer cell activation can contribute to liver injury. Kupffer cells, resident macrophages of the liver which constitute approximately 80% of the fixed macrophages in the body, may enhance liver injury by oxidant stress [19] or TNF-α generation, which may lead to apoptosis categorized CCl₄ as one of the representative toxins for fatty liver, fibrosis and cirrhosis. A large amount of transaminases leakage in the blood, which is always associated with hepatonecrosis [1], is observed in CCl₄- induced hepatotoxicity.

Between ALT and AST, ALT is a better index of liver injury as liver ALT represents 90% of total enzyme present in the body. Alkaline phosphatase activity on the other hand is related to the functioning of hepatocytes, increase in its activity being due to increased synthesis in the presence of increased biliary pressure. Reduction in the levels of ALT and AST towards the respective normal value is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damages caused by CCl₄. Suppression of increased ALP activity with concurrent depletion of raised bilirubin level suggests the stability of biliary dysfunction in rat liver during chronic hepatic injury with CCl₄[20]. The patterns of liver injury has been categorized as hepatocellular (elevated ALT), cholestatic (elevated ALP and total bilirubin) and mixed (elevated ALP and ALT).

Therefore, hepatoprotective activity was evaluated by measuring total bilirubin, measuring activity of the enzymes AST, ALT, and ALP, and by histopathological examination. In our studies, CCl₄-damaged rats that were previously treated with Corchorus depressus showed a significant decrease in serum AST and ALT. This is evidence that both stabilization of the plasma membrane and repair of CC1₄-induced hepatic tissue damage. Hepatic injury by CCl₄ results in raised ALP and total bilirubin levels, which could reflect a pathological alteration in biliary flow. Corchorus depressus treatment reduced the level of both, suggesting that it was able to stabilize CCl₄-induced biliary dysfunction in the rat liver. Histological observations also supported the results from the serum assays. To a large extent, Corchorus depressus administration reversed the hepatic lesions caused by CCl₄ toxicity.

Mammalian cells contain endogenous antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase and catalas. The levels of these enzymes are tightly controlled within all cells to ensure the maintenance of the body’s redox balance. These enzymes are able to detoxify free radicals by converting them back to more stable molecules within the cell, to be used or disposed accordingly [21].

As enzymatic antioxidant systems, both SOD and CAT play important roles in preventing oxidative damage by reactive oxygen species [22]. These enzymes are critical for defense mechanisms against the harmful effects of reactive oxygen species (ROS) and free radicals in biological systems. The SOD converts superoxide radicals (O₂^-) into H₂O₂ and O₂, thus participating with other antioxidant enzymes, in the enzymatic defense against oxygen toxicity. CAT is a key component of the antioxidant defense system. Inhibition of this protective mechanism results in enhanced sensitivity to free radical induced cellular damage. The decrease of CAT may result in a lot of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide [23].

GSH content was another important parameter that revealed oxidative damage in both liver and kidney. GSH, a nonenzymatic antioxidant, decomposes H₂O₂ into molecular oxygen and water, and constitutes the first line of defense against free radicals [24]. The reduction in the intracellular concentration of GSH in the liver of CCl₄-treated rats indicates damage to hepatic cells. Lipid peroxidation is an autocatalytic process, which is a common consequence of cell death. This process may cause peroxidative tissue damage in inflammation, cancer and toxicity of xenobiotics and aging [25]. MDA is a cytotoxic product that is a hallmark of lipid peroxidation. The fact that Corchorus depressus treatment reduced elevated MDA and increased levels of SOD, CAT, and GSH, indicated that Corchorus depressus may prevent the peroxidation of lipids by CCl₄. The CCl₄-induced hepatotoxicity model is extensively used for the evaluation of antioxidant effects of drugs and plant extracts [9].

The results obtained in this study show that CCl₄ significantly reduced the activity of hepatic in vivo antioxidant enzymes — SOD, CAT and GSH; and consequently increased the level of MDA. This effect is expected since as mentioned earlier hepatic damage induced by CCl₄ is mediated by its free radical metabolites, generated from bioactivation, which interact with unsaturated lipid membrane to cause lipid peroxidation and other cellular macromolecules leading to cell damage. Corchorus depressus (100, 200 and 4000 mg/kg) significantly increased CCl₄-diminshed hepatic SOD, CAT and GSH activities and caused diminution of CCl₄-elevated MDA level. The preservation and induction of the activity of in vivo antioxidant enzymes suggest that the methanol extract of Corchorus depressus promotes the scavenging of reactive free radicals thus protecting the histostructural integrity of the liver cells.

5. REFERENCES:

1. Akindele AJ, Ezenwanebe KO, Anunobi CC and Adeyemi OO. Hepatoprotective and in vivo antioxidant effects of Byrsocarpus coccineus Schum. and Thonn. (Connaraceae). Journal of Ethnopharmacology. 129; 2010: 46-52.

2. Ravikumar S and Gnanadesigan M. Hepatoprotective and Antioxidant Properties of Rhizophora mucronata Mangrove Plant in CCl₄ Intoxicated Rats. Journal of Experimental and Clinical Medicine. 4(1); 2012: 66-72.

3. Orhan IE, Sener B and Musharraf SG. Antioxidant and hepatoprotective activity appraisal of four selected Fumaria species and their total phenol and flavonoid quantities. Experimental and Toxicologic Pathology. 64; 2012: 205-209.

4. Kirtikar KR and Basu BD. Indian Medicinal Plants. International book distributors, India, Dehra Dun. 1993.

5. Qureshi R and Bhatti GR. Ethnobotany of plants used by the Thari people of Nara Desert, Pakistan. Fitoterapia. 79; 2008: 468-473.

6. Silori CS and Rana AR. Indigenous Knowledge on medicinal plants and their use in Narayan Sarovar Sanctuary, Kachchh. Ethnobotany. 12; 2000: 1-7.

7. Tripathi YC, Sharma HK and Arya R. Ethnomedicinal appraisal of traditional phytomedicines of arid Rajasthan. Journal of Medicinal and Aromatic Plant Science. 22(4A), 23 (1A); 2000: 487-498.

8. Sharma V, Thakur M and Dixit VK. A comparative study of ethanolic extracts of Pedalium murex Linn. fruits and sildenafil citrate on sexual behaviors and serum testosterone level in male rats during and after treatment. Journal of Ethnopharmacology. 143; 2012: 201-206.

9. Jain A, Soni M, Deb L, Jain A, Rout SP, Gupta VB and Krishna K.L. Antioxidant and hepatoprotective activity of ethanolic and aqueous extracts of Momordica dioica Roxb. Leaves. Journal of Ethnopharmacology. 115; 2008: 61-66.

10. Reitman S and Frankel S. A colorimetric method for the determination of serum glutamate oxaloacetate transaminase. American Journal of Clinical Pathology. 28; 1957: 53-56.

11. King J. The hydrolases-acid and alkaline phosphatases. In: Practical Clinical Enzymology. Nostrand Company Limited, London; 1965: pp. 191-208.

12. Malloy HT and Evelyn KA. The determination of bilirubin with the photometric colourimeter. The Journal of Biological Chemistry. 119; 1937: 481-490.

13. Kakkar P, Das B and Visvanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian Journal of Biochemistry and Biophysics. 21: 1984: 130-132.

14. Cohen G, Dembiec D and Marcus J. Measurement of Catalase activity in tissue extracts. Analytical Biochemistry. 34; 1970: 30-38.

15. Ellaman GL. Tissue sulfhdryl group. Archives of Biochemistry and Biophysics. 82; 1959: 70-72.

16. Ohkawa H, Ohishi N and Yagi K. Assay for Lipid peroxides in animal tissues by thiobarbituric acid rection. Analytical Biochemistry. 95; 1979: 351-358.

17. Shenoy KA, Somayaji SN and Bairy KL. Hepatoprotective effects of Ginkgo biloba against carbon tetrachloride induced hepatic injury in rats. Indian Journal of Pharmacology. 33; 200: 260-266.

18. Weber LW, Boll M and Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Critical Reviews in Toxicology. 33; 2003: 105-136.

19. Elsisi AE, Earnest DL and Sipes IG. Vitamin A potentiation of carbon tetrachloride hepatotoxicity: role of liver macrophages and active oxygen species. Toxicology and Applied Pharmacology. 119; 1993: 295-301.

20. Mukherjee PK. Quality Control of Herbal Drugs. Business Horizons Pharmaceutical Publication, New Delhi. 2002.

21. Saeed SA, Urfy MZS, Ali TM, Khimani FW and Gilani A. Antioxidants: their role in health and disease. International Journal of Pharmacology. 1; 2005: 226-233.

22. Halliwell B and Gutteridge JMC. Role of free radicals and catalytic metal irons in human disease: an overview. Methods in Enzymology. 186; 1990: 59-85.

23. Srinivasan R, Chandrasekar MJN, Nanjan MJ and Suresh B. Antioxidant activity of Caesalpinia digyna root. Journal of Ethnopharmacology. 113; 2007: 284-291.

24. Gultekin F, Delibas N, Yasar S and Kilinc I. In vivo changes in antioxidant systems and protective role of melatonin and a combination of Vitamin C and Vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats. Archives of Toxicology. 75; 2001: 88-96.

25. Halliwell B. Free radicals, antioxidants and human diseases; curiosity, cause, or consequence. Lancet. 334; 1994: 721-724