Received September 3, 2010; accepted October 8, 2010; published online January 15, 2011.
Full-text LinkOut at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao.
Correspondence: Nilesh Kumar Jain; Tel: +91-9926504077; E-mail: email@example.com
| || |
Chemical-induced liver injury depends mostly on the oxidative stress in hepatic tissue and underlies the pathology of numerous diseases, including cancer. There is still a lack of effective therapeutics; hence, a treatment with antioxidants has been proposed for prevention and/or attenuation of injury［1］. Carbon tetrachloride (CCl4)-induced acute liver damage is the best characterized system of xenobiotic-induced hepatotoxicity and is a commonly screening model to evaluate the hepatoprotective potential of drugs with antioxidant properties［2］. Silymarin, used as reference standard in present study, provides significant protection against CCl4-induced alterations of hepatic markers involved in normal function, oxidative stress and liver histopathology［3］.
The plants of genus Phyllanthus (Euphorbiaceae) have long been used in traditional systems of medicine to treat chronic liver diseases［4］. More than 35 species of this genus have been reported as endemic to India, which are predominantly used as a remedy for hepatic disorders［5］. A number of experimental studies have demonstrated the liver protective potential of Phyllanthus plants in different in vitro and in vivo systems［6-9］. However, several plants of this genus remain unexplored. Systematic investigation of such plants might yield fruitful results in our quest to discover new and promising hepatoprotective agents, which may be developed as pharmaceutical entities or as simple adjuncts to existing therapies. With this view, we selected Phyllanthus acidus (L.) Skeels (P. acidus) for our present study.
P. acidus, commonly known as “harfarauri”, “star gooseberry” or “mayom”, is a small tree and is cultivated as a fruit tree in many Asian countries. The fruit of P. acidus has been reported to be a rich source of ascorbic acid, fibers and carotenes［10］. Several parts of this plant have been used in folk medicine. The roots and seeds are cathartic. The fruit is a liver tonic and a blood purifier and is used in several vitiated conditions of jaundice, bronchitis, constipation, vomiting, biliousness, urinary concretions and piles in Ayurvedic system of medicine［11］. The leaves are useful to treat fever, piles, small pox, blood vomiting, itching and gum infection［12］. Several therapeutic properties including antiviral［13］, antibacterial［14］, neuroprotective［15］, antifibrosis［16］, and anticancer［17］ activities have also been reported for P. acidus. An aqueous extract of P. acidus has been reported to be effective against woodchuck hepatitis virus-DNA polymerase［18］. The hepatoprotective activity of P. acidus leaves against CCl4-induced acute liver damage in rat model has been reported［19］.
In the ethnobotanical claims, the fruit of P. acidus is used for the treatment of different diseases including hepatopathy［11］. To the best of our knowledge there is no scientific report available in support of its hepatoprotective activity. Therefore, to justify the traditional claims we have assessed the hepatoprotective effect of P. acidus fruit on CCl4-induced hepatotoxicity rat model.
| || |
1 Materials and methods
1.1 Experimental animals Wistar albino rats (200 to 220 g) and Swiss albino mice (20 to 25 g) of either sex were used for the studies. The animals were grouped and housed in polyacrylic cages with not more than 6 per cage and maintained under standard laboratory conditions of temperature (25±2) ℃ and relative humidity (55±5)% with dark and light cycle (12/12 h). They were acclimatized to laboratory conditions for 7 d before commencing the experiment and allowed free access to standard pellet diet (H.P. Agro Industries, India) and water ad libitum. Animal studies were approved by the Institutional Animal Ethics Committee (379/01/ab/CPCSEA) and conducted as per the regulations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
1.2 Plant materials and extraction The fresh P. acidus fruits were collected in May 2008 from University campus, Sagar (India) and authenticated by the taxonomist of Botany Department of the Dr. H. S. Gour University, Sagar (India). A voucher specimen (No.Bot/H/4322) has been deposited in the same department for future reference. The fruits were cut into small pieces, dried at room temperature and then subjected to size reduction to a coarse powder by using grinder and stored for further use.
The fruit powder (500 g) was extracted with 70% ethanol by cold maceration (10 d)［20］. After filtration, the extracts were concentrated under reduced pressure in a rotary evaporator to obtain dark brownish green semisolid extracts (yield: 12.4% w/w). For dosing, the crude extracts were uniformly suspended in 2% gum acacia and given orally at 100, 200 and 400 mg/kg body weight to animals.
1.3 Chemicals and reagents CCl4, 2,2-diphenyl-1-picrylhydrazil (DPPH), thiobarbituric acid (TBA) and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). Silymarin was obtained as a gift sample from Serum International Ltd., Pune, India. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and bilirubin estimation kits were procured from Span Diagnostics, Surat, India. All other chemicals and reagents used were of analytical grade.
1.4 Preliminary phytochemical screening Preliminary phytochemical analysis of P. acidus extracts was performed to identify the nature of phytoconstituents［21］.
1.5 Acute toxicity studies Healthy adult male albino mice (18 to 22 g) were used for acute toxicity studies as per Organization for Economic Cooperation and Development (OECD) guidelines (Guideline 423: Acute Toxic Category Method)［22］. On the basis of these studies, the doses of 100, 200 and 400 mg/kg extracts per os (p.o.) were selected for in vivo experiments.
1.6 CCl4-induced hepatotoxicity
1.6.1 Experimental protocol The experiment was conducted according to the method procedures described previously［23］. Rats were randomly divided into six groups, each consisting of six rats. Group Ⅰ (normal control) rats received distilled water (1 mL/kg, p.o.) daily for 5 d and olive oil (1 mL/kg, subcutaneously (s.c.)) on days 2 and 3. Group Ⅱ (CCl4 control) rats received distilled water (1 mL/kg, p.o.) daily for 5 d and CCl4︰olive oil (1︰1, 2 mL/kg, s.c.) on days 2 and 3. Group Ⅲ rats were treated with the reference drug silymarin (100 mg/kg, p.o.) daily for 5 d and received CCl4︰olive oil (1︰1, 2 mL/kg, s.c.) on days 2 and 3, 30 min after administration of silymarin. Groups Ⅳ to Ⅵ were treated with ethanolic extracts of P. acidus at 100, 200 and 400 mg/kg, p.o., respectively, for 5 d and received CCl4︰olive oil (1︰1, 2 mL/kg, s.c.) on days 2 and 3, 30 min after administration of the extracts.
On the sixth day, under ether anesthesia, blood and liver samples of rats were collected. The blood samples were allowed to clot for 30 min and the serum was separated by centrifugation at 3 000×g at 4 ℃. The livers were immediately taken out and washed with ice-cold normal saline, stored at -80 ℃ and processed for determination of oxidative stress markers and histopathological studies.
1.6.2 Determination of liver function markers Activities of the serum enzymes ALT, AST［24］ and ALP［25］ were determined. Total bilirubin (TB)［26］ and serum total protein (TP) contents［27］ were also determined to assess the CCl4-induced acute liver injury.
1.6.3 Determination of oxidative stress markers Fresh liver samples were perfused with ice-cold normal saline to completely remove all the red blood cells followed by homogenization with 0.2 mol/L phosphate buffer using homogenizer. Then the homogenate was centrifuged at 2 500×g for 10 min at 4 ℃ and used for the analysis of oxidative stress markers. Lipid peroxidation was assayed by measuring the malondialdehyde (MDA) content［28］ in the tissue homogenate. Levels of hepatic reduced glutathione (GSH)［29］ and antioxidant enzymes such as superoxide dismutase (SOD)［30］, catalase (CAT)［31］, and glutathione peroxidase (GPx)［32］ in the homogenate were measured accordingly.
1.7 DPPH scavenging assay DPPH was used in this assay to assess the free radial-scavenging (antioxidant) property of P. acidus extracts［33］. Briefly, methanol solution of P. acidus extracts (50 to 300 μg/mL) was mixed with 0.1 mmol/L DPPH methanol solution at a ratio of 3︰1. The contents were mixed vigorously and allowed to stand at 20 ℃ for 30 min. Then the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher radical-scavenging activity. The inhibitory concentration (IC50) value (the concentration required to scavenge 50% DPPH free radicals) was calculated. Ascorbic acid, a known antioxidant, was used as a positive control. All the tests were carried out in triplicate.
1.8 Hexobarbitone-induced sleeping time studies Six groups of Swiss albino mice were used for this study (8 per group). Food was withdrawn on the preceding night of the experiment. Normal control and CCl4 control groups received 2% gum acacia for 5 d, p.o.; test group Ⅰ received silymarin (100 mg/kg, p.o.) for 5 d; test groups Ⅱ to Ⅳ received P. acidus extracts at 100, 200 and 400 mg/kg for 5 d. CCl4 (50 μL/kg, p.o.) in vehicle (olive oil) was given to CCl4 control group and test groups Ⅰ to Ⅳ, 1 h after the respective treatment on day 5. All the six groups of animals were given hexobarbitone (60 mg/kg, intraperitoneally (i.p.)), 2 h after CCl4/vehicle treatment. The time between loss of righting reflex and its recovery was recorded［34］.
1.9 Statistical analysis The data of present study were expressed as x±sx. The results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using Instat software (version 2.1). The minimum level of significance was set at P<0.05.
| || |
2.1 Preliminary phytochemical screening On preliminary phytochemical analysis, the P. acidus extracts showed the presence of flavonoids, glycosides, phenolic compounds, proteins, saponins and phytosterols.
2.2 Acute toxicity studies In acute oral toxicity study, the P. acidus extracts did not show any sign and symptoms of toxicity or mortality up to 2 000 mg/kg body weight, which could be considered relatively safe.
2.3 Effects of P. acidus extracts on CCl4-induced hepatotoxicity
2.3.1 Histopathological examination of the liver tissues The hepatoprotective effect of P. acidus extracts was confirmed by histopathological examination of the liver tissues of control and treated animals. The histological architecture of CCl4-treated liver sections showed necrosis, ballooning degeneration, and the loss of cellular boundaries. However, administration of P. acidus extracts and silymarin almost normalized these defects in the histological architecture of the liver tissues, showing marked hepatoprotective effect. Liver sections of normal control rats showed normal hepatic cells with well-preserved cytoplasm, well brought out central vein and prominent nucleus and nucleolus (Figure 1A). Liver section of CCl4 control rats showed massive fatty changes, necrosis, ballooning degeneration, and broad infiltration of the lymphocytes and the loss of cellular boundaries (Figure 1B). Liver section of rats treated with CCl4 and silymarin showed well brought out central vein, hepatic cell with well-preserved cytoplasm, and prominent nucleus and nucleolus (Figure 1C). Liver section of rats treated with CCl4 and P. acidus ethanolic extracts showed well brought out central vein, hepatic cell with well-preserved cytoplasm, and prominent nucleus and nucleolus (Figure 1D).
Figure 1 Histological observations of rat livers with CCl4-induced hepatotoxicity
A: Liver section of normal control rats; B: Liver section of CCl4 control rats; C: Liver section of rats treated with CCl4 and silymarin; D: Liver section of rats treated with CCl4 and P. acidus ethanolic extracts.
2.3.2 Effects of P. acidus extracts on liver function markers The effects of P. acidus extracts on serum enzymes, TB and TP levels in CCl4-induced hepatotoxicity model rats were shown in Table 1. Elevated AST, ALT, ALP and TB and decreased TP level due to CCl4 administration (2 mL/kg, s.c.) were significantly prevented with P. acidus extracts and silymarin treatment compared with CCl4 control group (P<0.05 or P<0.01). There is no statistically significant difference between P. acidus-treated groups and silymarin-treated group.
Table 1 Effects of ethanolic extracts of P. acidus fruit on liver function markers in Wistar rats with CCl4-induced acute hepatotoxicity
**P<0.01, vs group Ⅰ; △P<0.05, △△P<0.01, vs group Ⅱ. Group Ⅰ: normal control; group Ⅱ: CCl4 control; group Ⅲ: silymarin (100 mg/kg); group Ⅳ: P. acidus (100 mg/kg); group Ⅴ: P. acidus (200 mg/kg); group Ⅵ: P. acidus (400 mg/kg).
2.3.3 Effects of P. acidus extracts on oxidative stress markers Reduced activities of enzymatic and non-enzymatic antioxidants (SOD, CAT, GPx and GSH, respectively) and enhanced lipid peroxidation were observed in CCl4 control group (Table 2), whereas the P. acidus extracts- and silymarin-treated groups showed marked rise in antioxidant levels with significant reduction in lipid peroxidation compared with CCl4 control group (P<0.01). There is no statistically significant difference between P. acidus extracts-treated groups and silymarin-treated group.
Table 2 Effects of ethanolic extracts of P. acidus fruit on oxidative stress markers in Wistar rats with CCl4-induced acute hepatotoxicity
**P<0.01, vs group Ⅰ; △P<0.05, △△P<0.01, vs group Ⅱ. Group Ⅰ: normal control; group Ⅱ: CCl4 control; group Ⅲ: silymarin (100 mg/kg); group Ⅳ: P. acidus (100 mg/kg); group Ⅴ: P. acidus (200 mg/kg); group Ⅵ: P. acidus (400 mg/kg).
2.4 DPPH-scavenging potential of P. acidus extracts The P. acidus extracts exhibited a maximum DPPH radical-scavenging activity of 87% at a concentration of 155 μg/mL. The IC50 value was found to be 68.2 μg/mL, which is approximately two times of that of ascorbic acid (IC50=32.8 μg/mL) used as reference antioxidant.
2.5 Effects of P. acidus extracts on hexobarbitone-induced sleeping time A significant increase in hexobarbitone-induced sleeping time was observed in mice after CCl4 administration. Pretreatment with P. acidus extracts at different dose levels (100, 200 and 400 mg/kg, p.o.) and silymarin (100 mg/kg, p.o.) considerably shortened the duration of hexobarbitone-induced sleeping time as compared with CCl4 control group, indicating the hepatoprotection (Table 3). There is no statistically significant difference between P. acidus-treated groups and silymarin-treated group.
Table 3 Effects of ethanolic extracts of P. acidus fruit on hexobarbitone-induced sleeping time in CCl4-intoxicated albino mice
▲▲P<0.01, vs normal control group; □P<0.05, □□P<0.01, vs CCl4 control group.
| || |
3 Discussion and conclusion
The present study demonstrates the hepatoprotective action of P. acidus fruit against CCl4-induced liver injury in Wistar rats. Since the biochemical and histological changes associated with CCl4 are almost similar to acute viral hepatitis, CCl4-induced hepatotoxicity was chosen as an experimental model［35］.
It is well established that CCl4 is biotransformed by the cytochrome P-450 (CYP-450), especially by CYP-2E1 to a highly reactive trichloromethyl free radical. This free radical in turn reacts with oxygen to form a trichloromethylperoxy radical, which may attack on the membrane lipids of endoplasmic reticulum more readily than the trichloromethyl free radical. The trichloromethylperoxy radical leads to elicit lipid peroxidation, the disruption of Ca2+ homeostasis, elevation of hepatic enzymes, reduction of protein synthesis and finally results in cell death［36］.
The elevated levels of AST, ALT, ALP and serum bilirubin are diagnostic indicators of acute liver injury［37］. In the present study, administration of CCl4 caused considerable hepatocellular damage as indicated by the massive elevations of AST, ALT, ALP and bilirubin compared with normal control animals. Administration of P. acidus extracts at different dose levels attenuated the increased levels of the serum markers induced by CCl4 and caused a subsequent recovery towards normalization. P. acidus extracts also increased the level of serum TP, which further indicates its hepatoprotective action. The histopathological observations of liver tissues further supported our claims.
It has been hypothesized that one of the principal causes of CCl4-induced hepatopathy is the formation of lipid peroxides by the free radical derivatives of CCl4. Thus, the antioxidant activity or the inhibition of the generation of free radicals is important in the protection against CCl4-induced toxicity［38］. In the present study, significant rise in the MDA level was observed, which suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent the formation of excessive free radicals［39］. Treatment with P. acidus extracts considerably prevented lipid peroxidation, indicating its antioxidant action. Scavenging of DPPH is related to the inhibition of lipid peroxidation［23］. The free radical-scavenging property of P. acidus extracts was further confirmed by DPPH assay.
The body has an effective defense mechanism to prevent and neutralize the free radical-induced damage. This is accompanied by a set of endogenous antioxidant enzymes such as SOD, CAT and GPx. Regarding non-enzymatic antioxidants, GSH is a critical determinant of tissue susceptibility to oxidative damage and the depletion of hepatic GSH has been shown to be associated with an enhanced toxicity to chemicals, including CCl4［40］.
The reduced activities of SOD, CAT and GPx and GSH level as observed in our study point out the hepatic damage in rats administered with CCl4. But the extracts-treated groups showed significant increase in the levels of these antioxidants, which further indicated the antioxidant activity of P. acidus extracts.
The damage imposed by CCl4 on hepatocytes causes a loss of drug metabolizing enzymes of the liver which results in the prolongation of sleeping time induced by short acting barbiturates, like hexobarbitone［41］. Treatment with drugs that stimulates hepatic drug metabolizing enzymes considerably shortens the duration of hexobarbitone-induced sleeping time［42］. Thus the reduction of hexobarbitone-induced sleeping time caused by P. acidus extracts in the present study demonstrates its capacity to stimulate hepatic drug metabolizing enzymes.
P. acidus fruit has been reported to be a rich source of ascorbic acid, fibers and carotenes［10］. The hepatoprotective and antioxidant action of ascorbic acid against acetaminophen-induced toxicity has already been reported［43］. In addition, our preliminary phytochemical analysis showed the presence of phenolic compounds and flavonoids in P. acidus extracts. The hepatoprotective property of some phenolics and flavonoids has been reported against xenobiotic-induced hepatotoxicity in animals［44］. These compounds also exert antioxidant or in vitro free radical-scavenging effects［45-47］. Phyllanthus emblica fruit (Indian gooseberry), closely related with P. acidus, is also reported to contain ascorbic acid, flavonoids, tannins and phenolic compounds as chief antioxidant and hepatoprotective compounds［6, 48, 49］. Taking into account of the fact, the presence of such phytoconstituents in P. acidus extracts might be responsible for the offered hepatoprotective and antioxidant effects.
In conclusion, the results of biochemistry and histological studies collectively demonstrate that P. acidus fruit has significant hepatoprotective activity against CCl4-induced acute liver toxicity in rats. The hepatoprotective action of P. acidus is certainly associated with their antioxidant properties acting as a scavenger of free radicals. The improvement in liver injury and liver functions by P. acidus extracts may be due to the presence of flavonoids and phenolic compounds which are reported to offer significant protection against liver toxicity. These preliminary findings on hepatoprotective and antioxidant actions reported herein would lend support to the use of P. acidus fruit as a hepatoprotective agent. Further studies are in progress in our lab to characterize the active principles and for better understanding the mechanism of action.
| || |
Authors are thankful to All India Council for Technical Education (AICTE), New Delhi, India for providing the financial assistance.
| || |
|1. ||Gonzalez CA. Nutrition and cancer: the current epidemiological evidence[J]. Br J Nutr, 2006, 96(Suppl 1) : S42-S45. |
|2. ||Jamshidzadeh A, Fereidooni F, Salehi Z, Niknahad H. Hepatoprotective activity of Gundelia tourenfortii[J]. J Ethnopharmacol, 2005, 101(1-3) : 233-237. |
|3. ||Wills PJ, Asha VV. Protective effect of Lygodium flexuosum (L.) Sw. extract against carbon tetrachloride-induced acute liver injury in rats[J]. J Ethnopharmacol, 2006, 108(3) : 320-326. |
|4. ||Calixto JB, Santos AR, Cechinel Filho V, Yunes RA. Protective effect of Lygodium flexuosum (L.) A review of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic potential[J]. Med Res Rev, 1998, 18(4) : 225-258. |
|5. ||Thyagarajan S, Jayaram S. Natural history of Phyllanthus amarus in the treatment of hepatitis B[J]. Indian J Med Microbiol, 1992, 10(2) : 64-80. |
|6. ||Gulati RK, Agarwal S, Agrawal SS. Hepatoprotective studies on Phyllanthus emblica Linn. and quercetin[J]. Indian J Exp Biol, 1995, 33(4) : 261-268. |
|7. ||Pramyothin P, Samosorn P, Poungshompoo S, Chaichantipyuth C. The protective effects of Phyllanthus emblica Linn. extract on ethanol induced rat hepatic injury[J]. J Ethnopharmacol, 2006, 107(3) : 361-364. |
|8. ||Harish R, Shivanandappa T. Antioxidant activity and hepatoprotective potential of Phyllanthus niruri[J]. Food Chem, 2006, 95(2) : 180-185. |
|9. ||Bhattacharjee R, Sil PC. Protein isolate from the herb, Phyllanthus niruri L. (Euphorbiaceae), plays hepatoprotective role against carbon tetrachloride induced liver damage via its antioxidant properties[J]. Food Chem Toxicol, 2007, 45(5) : 817-826. |
|10. ||Council of Scientific and Industrial Research. The wealth of India: a dictionary of Indian raw materials and industrial products[M]. New Delhi: Publications and Information Directorate, Council of Scientific and Industrial Research, 1998. |
|11. ||Kirtikar KR, Basu BD. Indian medicinal plants[M]. Allahabad: Lalit Mohan Basu, 1987. |
|12. ||Christophe W. Ethnopharmacology of medicinal plants: Asia and the Pacific[M]. New Jersey: Humana Press, 2006. |
|13. ||Direkbusarakom S, Herunsalee A, Yoshimizu M, Ezura Y. Antiviral activity of several Thai traditional herb extracts against fish pathogenic viruses[J]. Fish Pathol, 1996, 31(4) : 209-213. |
|14. ||Meléndez PA, Capriles VA. Antibacterial properties of tropical plants from Puerto Rico[J]. Phytomedicine, 2006, 13(4) : 272-276. |
|15. ||Ingkaninan K, Temkitthawon P, Chuenchom K, Yuyaem T, Thongnoi W. Screening for acetylcholinesterase inhibitory activity in plants used in Thai traditional rejuvenating and neurotonic remedies[J]. J Ethnopharmacol, 2003, 89(2-3) : 261-264. |
|16. ||Sousa M, Ousingsawat J, Seitz R, Puntheeranurak S, Regalado A, Schmidt A, Grego T, Jansakul C, Amaral MD, Schreiber R, Kunzelmann K. An extract from the medicinal plant Phyllanthus acidus and its isolated compounds induce airway chloride secretion: a potential treatment for cystic fibrosis[J]. Mol Pharmacol, 2007, 71(1) : 366-376. |
|17. ||Mahidol C, Prawat H, Prachyawarakorn V, Ruchirawat S. Investigation of some bioactive Thai medicinal plants[J]. Phytochem Rev, 2002, 1(3) : 287-297. |
| [springerlink] |
|18. ||Unander DW, Webster GL, Blumberg BS. Usage and bioassays in Phyllanthus (Euphorbiaceae). Ⅳ. Clustering of antiviral uses and other effects[J]. J Ethnopharmacol, 1995, 45(1) : 1-18. |
|19. ||Lee CY, Peng WH, Cheng HY, Chen FN, Lai MT, Chiu TH. Hepatoprotective effect of Phyllanthus in Taiwan on acute liver damage induced by carbon tetrachloride[J]. Am J Chin Med, 2006, 34(3) : 471-482. |
|20. ||Gopal N, Sengottuvelu S. Hepatoprotective activity of Clerodendrum inerme against CCl4 induced hepatic injury in rats[J]. Fitoterapia, 2008, 79(1) : 24-26. |
|21. ||Harborne JB. Phytochemical methods: a guide to modern technique of plant analysis[M]. 3rd ed. London: Chapman & Hill, 1998. |
|22. ||Organisation for Economic Co-operation and Development. OECD series on testing and assessment: Number 24: Guidance document on acute oral toxicity testing. http://www.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2001)4&doclanguage=en, 2001|
|23. ||Jain A, Soni M, Deb L, Jain A, Rout SP, Gupta VB, Krishna KL. Antioxidant and hepatoprotective activity of ethanolic and aqueous extracts of Momordica dioica Roxb. leaves[J]. J Ethnopharmacol, 2008, 115(1) : 61-66. |
|24. ||Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases[J]. Am J Clin Pathol, 1957, 28(1) : 56-63. |
|25. ||Kind PR, King EJ. Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-antipyrine[J]. J Clin Path, 1954, 7(4) : 322-326. |
|26. ||Mallay HT, Evelyn KA. Estimation of serum bilirubin level with the photoelectric colorimeter[J]. J Biol Chem, 1937, 119: 481-484. |
|27. ||Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent[J]. J Biol Chem, 1951, 193(1) : 265-271. |
|28. ||Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction[J]. Anal Biochem, 1979, 95(2) : 351-358. |
|29. ||Ellman GL. Tissue sulfhydryl groups[J]. Arch Biochem Biophys, 1959, 82(1) : 70-77. |
|30. ||Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels[J]. Anal Biochem, 1971, 44(1) : 276-287. |
|31. ||Sinha AK. Colorimetric assay of catalase[J]. Anal Biochem, 1972, 47(2) : 389-394. |
|32. ||Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: biochemical role as a component of glutathione peroxidase[J]. Science, 1973, 179(73) : 588-590. |
|33. ||Blois MS. Antioxidant determination by the use of a stable free radical[J]. Nature, 1958, 181: 1199-1200. |
|34. ||Van Puyvelde L, Kayonga A, Brioen P, Costa J, Ndimubakunzi A, De Kimpe N, Schamp N. The hepatoprotective principle of Hypoestes triflora leaves[J]. J Ethnopharmacol, 1989, 26(2) : 121-127. |
|35. ||Suja SR, Latha PG, Pushpangadan P, Rajasekharan S. Evaluation of hepatoprotective effects of Helminthostachys zeylanica (L.) Hook against carbon tetrachloride-induced liver damage in Wistar rats[J]. J Ethnopharmacol, 2004, 92(1) : 61-66. |
|36. ||Clawson GA. Mechanisms of carbon tetrachloride hepatotoxicity[J]. Pathol Immunopathol Res, 1989, 8(2) : 104-112. |
|37. ||Rajesh MG, Latha MS. Preliminary evaluation of the antihepatotoxic activity of Kamilari, a polyherbal formulation[J]. J Ethnopharmacol, 2004, 91(1) : 99-104. |
|38. ||Recknagel RO, Glende Jr EA, Britton RS. Free radical damage and lipid peroxidation. In: Meeks RG, Harrison SD, Bull RJ. Hepatotoxicology[M]. Boca Raton: CRC Press, 1991. |
|39. ||Shenoy KA, Somayaji SN, Bairy KL. Hepatoprotective effects of Ginkgo biloba against carbon tetrachloride induced hepatic injury in rats[J]. Indian J Pharmacol, 2001, 33(4) : 260-266. |
|40. ||Sanmugapriya E, Venkataraman S. Studies on hepatoprotective and antioxidant actions of Strychnos potatorum Linn. seeds on CCl4-induced acute hepatic injury in experimental rats[J]. J Ethnopharmacol, 2006, 105(1-2) : 154-160. |
|41. ||Anand KK, Singh B, Saxena AK, Chandan BK, Gupta VN, Bhardwaj V. 3,4,5-Trihydroxy benzoic acid (gallic acid), the hepatoprotective principle in the fruits of Terminalia belerica — bioassay guided activity[J]. Pharmacol Res, 1997, 36(4) : 315-321. |
|42. ||Chandan BK, Saxena AK, Shukla S, Sharma N, Gupta DK, Suri KA, Suri J, Bhadauria M, Singh B. Hepatoprotective potential of Aloe barbadensis Mill. against carbon tetrachloride induced hepatotoxicity[J]. J Ethnopharmacol, 2007, 111(3) : 560-566. |
|43. ||Lake BG, Harris RA, Phillips JC, Gangolli SD. Studies on the effects of L-ascorbic acid on acetaminophen-induced hepatotoxicity. 1. Inhibition of the covalent binding of acetaminophen metabolites to hepatic microsomes in vitro[J]. Toxicol Appl Pharmacol, 1981, 60(2) : 229-240. |
|44. ||Carini R, Comoglio A, Albano E, Poli G. Lipid peroxidation and irreversible damage in the rat hepatocyte model. Protection by the silybin-phospholipid complex IdB 1016[J]. Biochem Pharmacol, 1992, 43(10) : 2111-2115. |
|45. ||Fraga CG, Martino VS, Ferraro GE, Coussio JD, Boveris A. Flavonoids as antioxidants evaluated by in vitro and in situ liver chemiluminescence[J]. Biochem Pharmacol, 1987, 36(5) : 717-720. |
|46. ||Laughton MJ, Halliwell B, Evans PJ, Hoult JR. Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossypol and myricetin. Effects on lipid peroxidation, hydroxyl radical generation and bleomycin-dependent damage to DNA[J]. Biochem Pharmacol, 1989, 38(17) : 2859-2865. |
|47. ||Sanz MJ, Ferrandiz ML, Cejudo M, Terencio MC, Gil B, Bustos G, Ubeda A, Gunasegaran R, Alcaraz MJ. Influence of a series of natural flavonoids on free radical generating systems and oxidative stress[J]. Xenobiotica, 1994, 24(7) : 689-699. |
|48. ||Jose JK, Kuttan R. Antioxidant activity of Emblica officinalis[J]. J Clin Biochem Nutr, 1995, 19(2) : 63-70. |
|49. ||Ghosal S, Tripathi VK, Chauhan S. Active constituents of Emblica of?ficinalis: PartⅠ. The chemistry and antioxidative effect of two new hydrolysable tannins, emblicanin A and B[J]. Indian J Chem, 1996, 35(9) : 941-948. |
| || |