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366 Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372. e- ISSN 0976 - 3651 Print ISSN 2229 - 7480 International Journal of Biological & Pharmaceutical Research Journal homepage: www.ijbpr.com IJBPR HEPATOPROTECTIVE ACTIVITY OF ROOT BARK OF AZIMA TETRACANTHA LAM. AGAINST CARBON TETRA CHLORIDE (CCL4)-INDUCED HEPATOTOXICITY IN WISTAR MALE ALBINO RATS M. Balakrishnan*, R. Dhanapal1, K.B. Chandra Sekhar2 *l Principal, Rosory College of Pharmacy, yellapur (v), Hasanparthy (M), Warangal, Andhra Pradesh, India.-506 371. 1 Department of Pharmaceutics, Kakatiya Institute of Pharmaceutical Sciences, Pembarthi (V), Hasanparthy (M), Warangal, Andhra Pradesh, India-506 371. 2 Professor of Chemistry, Director of Evaluation, JNT University, Anantapur, Andhra Pradesh, India-515 002. ABSTRACT The present study to evaluate the hepatoprotective activity of ethanol (50%) extract of Azima tetracantha Lam (EEAT) root bark using CCl4 induced hepatic damage in male Wistar albino rats. The EEAT root bark at doses of 40, 80 and 120mg/kg, p.o and the standard drug Liv.52 (40mg/kg,p.o) were administered orally for 7 days in CCl4 intoxicated rats. The hepatoprotective activity was assessed by using various biochemical parameters like SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase (ACP), also total bilirubin along with histopathological studies of liver tissue. The biochemical changes and histopathological studies were observed on 8 th day. There was a significant increase in serum levels of SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase (ACP), also total bilirubin in the CCl 4 treated animals, reflecting liver injury. The administration of EEAT root bark at doses of 40, 80 and 120mg/kg, p.o for 3days resulted in significant decrease (P<0.001) the CCl4-induced elevated levels of the hepatic enzymes SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase (ACP) also total bilirubin in a dose dependent manner. Whereas the extract at tested doses decreases the CCl4-induced elevated level of hepatic enzymes, and also total bilirubin in rats, and its subsequent return towards near normalcy after 7days indicating the recovery of hepatic cells. The histological studies were also carried out to support the above parameters. In the liver sections of the rats treated with EEAT root bark extract for 7 days, the normal cellular architecture was retained as compared to Liv.52, there by further confirming the potent hepatoprotective effect of EEAT root bark. The ethanol (50%) extract of Azima tetracantha Lam (EEAT) root bark afforded significant protection against CCl 4 induced hepatocellular injury. To our knowledge, this report is the first that shows hepatoprotective effect of Azima tetracantha Lam. (Salvodaraceae) root bark against CCl4 induced liver damage. Keywords: CCl4, Hepatotoxicity, Hepatic enzymes, Azima tetracantha, Hepatoprotective, EEAT root bark. Corresponding Author M. Balakrishnan E-mail: [email protected] INRODUCTION Liver is considered as the key organ in the metabolism, detoxification, and secretory functions in the body, and its disorders are numerous with no effective remedies, however, the search for new medicines is still 367 Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372. ongoing. Hepatitis is a common disease in the world especially in the developing countries. Despite, considerable progress in the treatment of liver diseases by oral hepatoprotective agents, search for newer drugs continues because the existing synthetic drugs have several limitations (Liu GT, 1989; Handa SS et al., 2001). Because liver performs many vital functions in the human body, damage of liver causes unbearable problems (Mitra SK et al., 1998; Chattopadhyay RR, 2003). Thus study about hepatoprotective compounds is of importance. Azima tetracantha Lam. (Family: Salvadoraceae) locally known as “Mulsangu”, is a rambling spinous shrub flowering throughout the year found in Peninsular India, West Bengal, Orissa, African Countries and extends through Arabia to tropical Asia. The leaves of the plant are elliptical in shape and are rigid, pale green colored. The flowers are small, greenish white (or) yellow colored, unisexual in axillary fasciles. The berries are white in colour; usually one seeded and edible. The juice of the leaves is said to relieve the cough phthisis and asthma. In western India juice of the leaves is applied as eardrops against earache and crushed leaves are placed on painful teeth. In India and Sri Lanka the root, root bark and leaves are administered with food as a remedy for rheumatism (Chopra RN et al., 1956; Kritikar KR and Basu BD, 1976; Hebbar SS et al., 2004). The plant is considered as a powerful diuretic and is also used to treat rheumatism, dropsy, dyspepsia, chronic diarrhoea and as a stimulant tonic for women after confinement (Nadkarni KM, 1976). The leaves of this plant have been reported to possess anti-inflammatory, wound-healing activity, diuretic activity and analgesic activity (Syed Ismail T et al., 1997; Jaswanth A et al., 2001; Nandgude TD et al., 2001). Despite the traditional uses, the leaves of this plant have been tested negative in antibacterial and antifungal properties (Vonshak A et al., 2003). A number of chemical constituents have previously been reported from the leaves (Friedelin, Lupeol, Glutinol and beta-sitosterol), the seed oils (fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linoleic acid, eicosenoic acid and flavonoids), the seed, root, stem and young leaves (N-methoxy-3indolylmethyl-glucosinolate), roots and the leaves (terpenoids), also the dimeric piperidine alkaloids azimine, azcarpine and carpaine have previously been isolated from all plant parts (Venkata Rao E and Prasada Rao PRS, 1978; Daulatabad CD et al., 1991; Bennett RN et al., 2004; Rall GJH et al., 1967; Williams UV and Nagarajan S, 1988; Dold AP, 2006). Locally, the traditional healers from Tirunelveli district of Tamilnadu are using root bark (paste with butter milk) of this plant as potent remedy for jaundice (Personal information). However, there are no ethnomedicinal information and scientific findings for the above said traditional claim for jaundice. Therefore, to justify the traditional claims the present study was undertaken to find out if ethanol extract of Azima tetracantha root bark demonstrates any hepatoprotective activity against CCl4induced liver damage in vivo in rats. MATERIALS AND METHODS Collection of plant material Fresh root barks of Azima tetracantha were collected from Wastelands of Kadyanallur, Tirunelveli (District), Tamilnadu, India. The plant specimen was authenticated by Dr.V.Chelladurai, Research Officer Botany, Survey of Medicinal Plants Unit, Tirunelveli. A voucher specimen (V.No: ATC27/08/2005) has been deposited at the herbarium unit of the Department of Botany, Siddha Medical College, Palayamkottai, Tirunelveli (District), Tamilnadu, India. Preparation of plant extract The root barks of Azima tetracantha were chopped, shade-dried and made into a coarse powder. About 500gms of the powdered material was taken and dissolved in two liters of ethanol and distilled water mixture in the ratio of 1:1and subjected to hot maceration process for 24 hours continuously by using Sohxlet apparatus. Then the extract was collected and evaporated under reduced pressure then vacuum-dried. The yield of the extract was 12.4% with reference to dry powder. Preliminary phytochemical screening Various phytochemical tests were carried out on dry extracts obtained to detect the presence of different phytoconstituents by using the methods described by Evans (Evans WC, 1999). Animals Wistar albino rats (150-190) of either sex, procured from National Institute of Nutrition, Hyderabad, were used for the study. The animals were housed in large polypropylene cages in a temperature-controlled room (22±2 ◦C) and provided with standardized pelleted feed (Hindustan Liver, Bangalore) and clean drinking water ad libitum. All procedures compiled with the norms of the Institutional Animal Ethics Committee (Registration No: 129/99/CPCSEA). Acute toxicity studies For toxicity studies the EEAT root bark in the range of doses 50-1000 mg/kg were administered in six groups of ten rats respectively. The percentage survivals were observed after 72 hours. The LD50 was determined using the graphical methods of Reed and Muench (Reed LJ and Muench HA, 1938). In vivo hepatoprotective activity studies The liver protective effect was evaluated using the 368 Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372. carbon tetrachloride (CCl4) model described by Visweswaram et al (Visweswaram D et al., 1994). Thirty six Wistar albino rats (180-200gm) were divided into six groups of six rats each and were subjected to the following treatments: Group-I which served as normal control received distilled water (1 ml/kg; p.o) for 7days. Group II -VI received 0.75 ml/kg CCl4 administered orally as single dose. After 36 hours, Groups III-VI received EEAT root bark with doses of 40, 80 and 120 mg/kg, p.o and the standard drug Liv.52 with dose of 40mg/kg, p.o, respectively once daily for 7days. The blood was collected by puncturing the retro-orbital sinus of three rats from each group on 8th day after the treatment respectively. From the collected blood samples, serum was separated to assess various biochemical parameters. Biochemical estimation The separated serum was subjected to estimate SGOT and SGPT by Reitman and Frankel (Reitman S and Frankel S, 1957) method, alkaline phosphatase (ALP) and acid phosphatase (ACP) by Kind and King (Kind PRN and King EJ, 1954) method, bilirubin by Malloy and Evelyn (Malloy HT and Evelyn KA, 1937) method and urea by Bousquet (Bousquet BF et al., 1971) method. The rats were then sacrificed by bleeding and the liver was carefully dissected, cleaned of extraneous tissue, and part of the liver tissue was immediately processed for histopathological investigation. Histopathological studies The tissues of liver were fixed in 10% formalin and embedded in paraffin wax. Sections of 4-5 microns thickness were made using rotary microtome and stained with haematoxylin-eosin and histological observations were made under light microscope (Luna LG, 1966; Galigher AE and Kozloff EN, 1971). Statistical analysis Results of biochemical estimations are reported as mean ± SEM of six animals in each group. The data were subjected to one-way ANOVA followed by Tukey’s multiple comparison tests. P<0.001 was considered statistically significant. RESULTS Phytochemical Screening The various chemical tests revealed the presence of alkaloids, tannins, proteins, and lipids. (Table -1) Acute toxicity studies The LD of EEAT root bark was found to be 406 50 mg/kg, b.w. The doses selected for this present study were, 10% (40mg), 20 %( 80mg) and 30% (120mg) of LD . 50 Effect of EEAT root bark on CCl4 – induced hepatotoxicity The results of Carbon tetrachloride-induced hepatotoxicity were represented in Table 1. The CCl4 only treated animals exhibited a significant increase (P<0.001) the levels of SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase (ACP) and also total bilirubin when compared to the normal control group on 8 th day, indicating hepatocellular damage. The EEAT root bark at tested doses (group III-V) produced a significant reduction (P<0.001) in the CCl4induced elevated levels of SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase (ACP), also total bilirubin when compared to the CCl4 only treated animals (group-II) after 3days of treatment and reduced furthermore to the normalcy on 8th day although the lowest dose (40 mg/kg) tested could produce significant reduction in enzymatic levels (Table 1). Overall, EEAT root bark at tested doses significantly reduced the levels of hepatic enzymes, total bilirubin in a dose dependent manner. After 7 days, the hepatic enzymes levels were almost restored to the normal after treating with EEAT root bark at the dose of 120mg/kg, p.o. A standard drug, Liv.52 at a dose of 40 mg/kg (group-VI) administered orally produced a significant reduction (p<0. 001) compared to CCl4 only treated animals (group-II) on 8th day and these protective effects almost close to EEAT root bark 120mg/kg,p.o. Effect of EEAT root bark on histolopathological change Histopathological examination of liver sections of control group showed normal cellular architecture with distinct hepatic cells, sinusoidal spaces and central vein on 8th day (Fig.3). Disarrangement of normal hepatic cells with centrilobular necrosis, vacuolization of cytoplasm and fatty degeneration were observed (on 8th day) in CCl4 intoxicated rats (Fig.4). Figure 1. Effect of EEATrb and Liv.52 on CCl4-induced alteration of SGOT, SGPT, ALP and ACP in rat liver after 7 days of treatment 369 Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372. Table 1. EEAT root bark on CCl4-induced alteration of hepatic enzymes, serum bilirubin in rat liver on 8th day observation Design of Treatment Group-I: Normal control (DW-1 ml/kg; p.o) Group-II: CCl4 (0.75 ml/kg; p.o) Group-III: EEAT root bark (40 mg/kg; p.o) Group-IV: EEAT root bark (80 mg/kg; p.o) Group-V: EEAT root bark (120 mg/kg; p.o) Group-VI: Liv.52 (40 mg/kg; p.o) Biochemical parameters ACP(KA ALP (KA Units) Units) total Bilirubin(mg/dl) direct Bilirubin(mg/dl) SGOT(U/ml) SGPT(U/ml) 95.31 ± 1.16 57.61 ± 0.06 142.60 ± 0.92 70.02 ± 1.17 0.62 ± 0.03 c 158.14 ± 1.41* c 255.12 ± 0.91* c 112.02 ± 0.13* c 2.62 ± 0.04* c 1.52 ± 0.02* 260.46 ± 1.15* 92.13 ± 0.25* 191.13 ± 0.04* 105.61 ± 0.04* 2.20 ± 0.05* 1.32 ± 0.03* 161.22 ± .91* 71.07 ± 1.62* 162.01± 0.64* 87.26 ± 0.14* 1.94 ± 0.02* 0.95 ± 0.02* 141.14 ± 1.04* 62.02 ± 0.04* 146.04 ± 0.02* 76.15 ± 0.02* 1.21 ± 0.04* 141.13 ± 0.81* 60.12 ± 0.43* 143.17 ± 0.92* 75.09 ± 0.03* 0.89 ± 0.03* 423.20 ± 1.03* 0.20 ± 0.02* 0.33 ± 0.02* 0.32 ± 0.02* Values are Mean ± SEM of 6 animals each in a group. *P<0.001,when compared group II Vs groupI,III,IV,V and VI EEAT root bark= ethanol (50%) extract of Azima tetracantha Lam (EEAT) root bark, CCl4 = Carbon tetrachloride Figure 2. Effect of EEATrb and Liv.52 on CCl4-induced alteration of total and direct Bilirubin in rat liver after 7 days of treatment Fig 3. Histology of the liver. control group (normal) (Magnification: 100x) Fig 4. Histology of the liver. Treated with only CCl4 on 8th day examination. (Magnification: 100x) Fig 5. Histology of the liver.treated with CCl 4and supplemented with EEATrb (120mg/kg, p.o) on 8 th day examination (Magnification: 100x) 370 Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372. Fig 6. Histology of the liver.Treated with CCl4 and supplemented with Liv.52 (40mg/kg, p.o). examined on 8 th day (Magnification: 100x) The liver sections (on 8th day) of the group-V rats treated with EEAT root bark (120mg/kg, p.o) showed a sign of protection as it was evident by the moderate accumulation of fatty lobules, absence of necrosis and vacuoles (Fig. 5). Almost similar sign of protection was shown in the liver sections of Liv.52 at a dose of 40 mg/kg treated rats (Fig. 6). DISCUSSION AND CONCLUSION The present studies were performed to assess the hepatoprotective activity of ethanol (50%) extract of A.tetracantha root bark in rats against carbon tetrachloride as hepatotoxin to prove its claims in folklore practice against jaundice. It is well documented that carbon tetrachloride-induced hepatic injury is commonly used as an experimental method for the study of hepatoprotective effects of drugs or medicinal plants’ extracts, by in vivo and in vitro techniques (Kiso Y et al., 1983; Allis JW et al., 1990; Cornelius CE, 1993). Carbon tetrachloride (CCl4) is a potent hepatotoxin producing centrilobular hepatic necrosis. It is accumulated in hepatic parenchyma cells and metabolized to CCl3 by liver cytochrome P450-dependent monooxygenases (Recknagel RO et al., 1989). Usually, the extent of hepatic damage is assessed by histopathological evaluation and the level of hepatic enzymes ALT, AST and ALP release in circulation (Plaa G and Charbonneau M, 1994). The administration of CCl4 resulted in a significant increase in the serum SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase (ACP) and also total bilirubin within 36 hours (Wachstein M, 1959; Max Wachstein et al., 1962). The rise in serum levels of AST, ALT, ALP and ACP has been attributed to the damaged structural integrity of the liver, because they are cytoplasmic in location and released into circulation after cellular damages (Sallie R et al., 1991). In our study, the biochemical changes were observed after 7 days. Thereby, it was found that, the administration of EEAT root bark at doses of 40, 80 and 120mg/kg, p.o. These results indicating the production of structural integrity of hepatocytic cell membrane or regeneration of damaged liver cells by the extract. Whereas, the extract at tested doses decreases the CCl4-induced elevated level of hepatic enzymes in rats, and its subsequent return towards near normalcy after 7days. Reduction in the levels of SGOT and SGPT towards the normal value is an indication of regeneration process. Reduction of ALP levels with concurrent depletion of raised bilirubin level suggests the stability of the biliary function during injury with CCl4. Bilirubin is the conventional indicator of liver diseases (Girish S et al., 2004). The rise in the levels of serum bilirubin is the most sensitive and confirms the intensity of jaundice (Cavin C et al., 2001). These biochemical restorations may be due to the inhibitory effects on cytochrome P450 or/and promotion of its glucuronidation (Drotman RB and Lawhorn GT, 1978). The marked elevation of bilirubin level in the serum of group II CCl4 intoxicated rats were significantly decreased in the groups III-V EEAT root bark treated animals after 7 days of treatment, bilirubin level in the serum CCl4 intoxicated rats subsequently return towards near normalcy in the groups III-V EEAT root bark treated animals. These results further substantiate A.tetracantha as a potent hepatoprotective agent. It has been reported that Liv.52 protects liver from the hepatotoxicity of carbon tetrachloride (Karandikar SM et al., 1963; Meena Kataria and Singh LN, 1997). An appreciable protective effect was observed during 7 days treatment using marketed product (Liv.52). The extent of production by extracts appeared to depend on the duration of treatment. Overall, these results suggest that the extract 371 Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372. could protect the liver against damage induced by CCl 4 when comparable with Liv.52. The attributivity of the observed alterations of SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase (ACP), serum ALT were confirmed by histopathological studies of liver sections which reveal that the normal liver architecture was disturbed by hepatotoxin (CCl4) intoxication. In the liver sections of the rats treated with EEAT root bark extract for 7 days, the normal cellular architecture was retained as compared to Liv.52, there by further confirming the potent hepatoprotective effect of A.tetracantha root bark. Further research is needed to isolate and purify the active principle involved in hepatoprotection of this plant as well as to confirm the mechanisms responsible for hepatoprotective activity. The present finding provides scientific evidence to the ethnomedicinal use of Azima tetracantha root bark in treating jaundice. ACKNOWLEDGEMENT The authors are thankful to Dr.P.Jayaraman, M.Sc., Ph.D, Plant Anatomy Research Centre (PARC), Chennai and Dr.S.Subramaniam.M.B.BS, Histopathalogist, Vijay Clinical Laboratory, Madurai, Tamil Nadu, India, for valuable help in successful completion this research work. REFERENCES Allis JW, Ward TR, Seely JC, Simmons JE. Toxic effects of carbon tetrachloride on rats. Fundamental Appl Toxicol. 1990; 15: 558-570. Bennett RN, Mellon FA, Rosa EA, Perkins L and Kroon PA. Profiling glucosinolates, flavonoids, alkaloids, and other secondary metabolites in tissues of Azima tetracantha L. (Salvadoraceae). J Agri Food Chem. 2004; 52(19): 5856– 5862. Bousquet BF, Julien R, Bon R, Dreux C. Determination of Blood urea. Ann Biol Clin. 1971; 29: 415. Cavin C, Mace K, Offord EA, Schilter B. Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: Mechanisms in rat and human cells. Food Chem Toxicol. 2001; 39: 549–556. Chattopadhyay RR. Possible mechanism of hepatoprotective activity of Azadirachta indica leaf extract: part II. J. Ethnopharmacol. 2003; 89: 217-219. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. Concil of Scientific and Industrial Research (CSIR), New Delhi, 1956: 32, 218. Cornelius CE: Animal Models in Liver Research. San Diego: Academic Press; 1993; 37:341. Daulatabad CD, Desai VA, Hosamani K M and Jamkhandi A M. Novel fatty acids in Azima tetracantha seed oil. J American Oil Chemist’s Soc. 1991; 68(12): 978. Dold AP. Azima tetracantha Lam. In: Schmelzer GH and Gurib-Fakim A (Editors).Prota11: Medicinal plants/Plantes médicinales. Wageningen, Netherlands, 2006. Drotman RB, Lawhorn GT. Serum enzymes as indicators of chemically induced liver damage. Drug Chem Toxicol. 1978; 1: 163-171. Evans WC. Trease and Evan’s Pharmacognosy. Thirteenth Edition. English Languagae Book Society/Baillere Tindall. London.1999: 327, 338, 342- 345,346,415,420,443,535-536. Galigher AE and Kozloff EN. Essential Practical Microtechnique, 2nd edn, Lea and Febiger, Philadelphia, 1971: 77-210. Girish S, Achliya, Sudhir, Wadodkar G, Avinash, Dorle K. Evaluation of hepatoprotective effect of Amalkadi Ghrita against carbon tetra chloride induced hepatic damage in rats. J Ethnopharmacol. 2004; 90: 229-232. Handa SS, Sharma A and Chakraborti KK. Natural products and plants as liver protecting drugs. Fitoterapia. 1986; 57:307-45. CCl4 induced hepatic injury in rat. Ind J Pharmacol. 2001; 33: 260-266. Hebbar SS, Harsha VH, Shripathi V and Hegde GR. Ethnomedicine of Dharwad District in Karnataka, India: plants used in oral health care. J Ethnopharmacol. 2004; 94(2–3): 261-266. Jaswanth A, Begum VH, Akilandeswari S, Begum TN, Manimaran S and Ruckmani K. Effects of Azima tetracantha on dermal wound healing in rats. Hamdard Medicus. 2001; 44(3): 13-16. Karandikar SM, Joglekar GV, Chitale GK and Balwani JH. Protection by indigenous drugs against hepatotoxic effects of carbon tetrachloride-a long-term study. Acta Pharmacol Toxicol. 1963; 20: 274-280. Kind PRN, King EJ. Determination of Serum Alkaline Phosphatase. J. Clin. Path. 1954; 7: 132-136. Kiso Y, Tohkin M, Hikino H. Assay method for antihepatotoxic activity using carbon tetrachloride induced cytotoxicity in primary cultured hepatocytes. Planta Med. 1983; 49: 222-225. Kritikar KR and Basu BD. Indian Medicinal Plants.Vol 3, 1976, 600. Liu GT. Pharmacological actions and clinical use of Fructus chizandrae. Chin Med Journal. 1989; 102: 740-749. Luna LG., Manual of Histological Staining. Methods of Armed Forces Institute of Pathology, London, 1966: 1-31. Malloy HT, Evelyn KA, The determination of bilirubin. J Biol Chem. 1937; 119: 481-485. 372 Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372. Max Wachstein, Elizabeth Meisel, and Carmen Falcon. Enzymatic Histochemistry in the Experimentally Damaged Liver. Am J Pathol. 1962; 40(2): 219-241. Meena Kataria and Singh LN. Hepatoprotective Effect of Liv.52 and Kumaryasava on Carbon Tetrachloride induced Hepatic damage in Rats. Indian J Experimental Biol. 1997; 35: 655-657. Mitra SK, Venkataranganna MV, Sundaram R and Gopumadhavan S. Protective effect of HD-03, a herbal formulatin, against various hepatotoxic agents in rats. J. Ethnopharmacol. 1998; 63: 181-186. Nadkarni KM. Indian Meteria Medica, Vol. 1, 3rd. Edn. Popular Prakhasan, Bombay, 1976. Nandgude TD, Bhojwani AP, Kinage Krishna. Analgesic activity of various extracts of leaves of Azima tetracantha Lam. Intl J Green Pharm. 2007; 1(1): 37-38. Plaa G, Charbonneau M. Detection and evaluation of chemically induced liver injury. In: Hayes, A.W. (Ed.), Principles and Methods of Toxicology. Raven Press, New York, 1994: 841-846. Rall GJH, Smalberger TM, Dewaal HL and Arndt RR. Tetrahedron Letters. 1967; 3465. Recknagel RO, Glende EA JR, Dolak JA, Waller RL. Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther. 1989; 43: 139-154. Reed LJ and Muench HA. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27: 493-97. Reitman S, Frankel S. A colorimetric method for the determination of Serum Glutamate Pyruvate Transaminase and Serum Glutamate Oxaloacetate Transaminase. Am J Clin Path. 1957; 28: 56-62. Sallie R, Tredger JM, William R. Drugs and the liver. Part I. Testing liver function. Biopharm Drug Disp. 1991; 12: 251-259. Syed Ismail T, Gopalakrishnan S, Hazeena Begum V and Elango V. Anti-inflammatory activity of Salacia oblonga Wall. and Azima tetracantha Lam. J Ethnopharmacol. 1997; 56(2): 145-152. Venkata Rao E and Prasada Rao PRS. Occurrence of Triterpenoids in Azima Tetracantha. Curr Sci. 1978; 47: 857. Visweswaram D, Rajeswara Rao P, Satyanarayana S. A non-invasive method for screening hepatoprotective drugs against carbon tetrachloride induced hepatotoxicity. Indian J Pharmacol. 1994; 26: 301-303. Vonshak A, Barazani O, Sathiyamoorthy P, Shalev R, Vardy D and Golan-Goldhirsh A. Screening south Indian medicinal plants for antifungal activity against cutaneous pathogens. Phytotherapy Res. 2003; 17(9): 1123-1125. Wachstein M. Enzymatic histochemistry of the liver. Gastroenterol. 1959; 37: 525-37. Williams UV and Nagarajan S. Isorhamnetin 3-O-rutinoside from leaves of Azima tetracantha Lam. Indian J. Chem, B-Org. Chem Incl Med Chem. 1988; 27: 397.