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Original Experimental Research
Journal of Chinese Integrative Medicine: Volume 9   January, 2011   Number 1

DOI: 10.3736/jcim20110109
Effects of Phyllanthus acidus (L.) Skeels fruit on carbon tetrachloride-induced acute oxidative damage in livers of rats and mice
1. Nilesh Kumar Jain (Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar 470003, India E-mail: nilesh_jain414@yahoo.com)
2. Santram Lodhi (Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar 470003, India )
3. Avijeet Jain (Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar 470003, India )
4. Alok Nahata (Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar 470003, India )
5. Abhay K. Singhai (Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar 470003, India )

Objective: The present study was undertaken with a view to validate the traditional use of Phyllanthus acidus (L.) Skeels fruit as a hepatoprotective agent.
Methods: The 70% ethanolic extract of P. acidus fruit (100, 200 and 400 mg/kg, p.o.), and reference drug silymarin (100 mg/kg, p.o.) were given to rats of different groups respectively once a day for 5 d and the carbon tetrachloride (CCl4) (2 mL/kg, subcutaneously) was given on days 2 and 3. Serum levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin (TB) and total protein (TP) were assessed along with liver histopathological examination. The effects on oxidative stress markers such as lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were also assessed in liver tissue homogenate to evaluate in vivo antioxidant activity. In addition, the effects on hexobarbitone-induced sleeping time were observed and the free radical-scavenging potential was determined by using 2,2-diphenyl-1-picrylhydrazil (DPPH) in mice.
Results: P. acidus extracts and silymarin exhibited a significant hepatoprotective effect as evident from the decreases of serum AST, ALT and ALP levels and LPO and increases in the levels of TP, GSH, SOD, CAT, and GPx compared with control group (P<0.01 or P<0.05). The biochemical results were supplemented with results of histopathological sections of the liver tissues. P. acidus extracts considerably shortened the duration of hexobarbitone-induced sleeping time in mice compared with control group (P<0.01) and showed remarkable DPPH-scavenging activity.
Conclusion: The present findings suggest that the hepatoprotective effect of P. acidus against CCl4-induced oxidative damage may be related to its antioxidant and free radical-scavenging potentials.

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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: nilesh_jain414@yahoo.com

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     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 injury1. 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 properties2. 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 histopathology3.
     The plants of genus Phyllanthus (Euphorbiaceae) have long been used in traditional systems of medicine to treat chronic liver diseases4. More than 35 species of this genus have been reported as endemic to India, which are predominantly used as a remedy for hepatic disorders5. A number of experimental studies have demonstrated the liver protective potential of Phyllanthus plants in different in vitro and in vivo systems6-9. 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 carotenes10. 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 medicine11. The leaves are useful to treat fever, piles, small pox, blood vomiting, itching and gum infection12. Several therapeutic properties including antiviral13, antibacterial14, neuroprotective15, antifibrosis16, and anticancer17 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 polymerase18. The hepatoprotective activity of P. acidus leaves against CCl4-induced acute liver damage in rat model has been reported19.
     In the ethnobotanical claims, the fruit of P. acidus is used for the treatment of different diseases including hepatopathy11. 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 phytoconstituents21.
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 previously23. 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, AST24 and ALP25 were determined. Total bilirubin (TB)26 and serum total protein (TP) contents27 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) content28 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 extracts33. 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 recorded34.
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.

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2  Results
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.

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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 model35.
     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 death36.
     The elevated levels of AST, ALT, ALP and serum bilirubin are diagnostic indicators of acute liver injury37. 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 toxicity38. 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 radicals39. Treatment with P. acidus extracts considerably prevented lipid peroxidation, indicating its antioxidant action. Scavenging of DPPH is related to the inhibition of lipid peroxidation23. 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 CCl440.
     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 hexobarbitone41. Treatment with drugs that stimulates hepatic drug metabolizing enzymes considerably shortens the duration of hexobarbitone-induced sleeping time42. 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 carotenes10. The hepatoprotective and antioxidant action of ascorbic acid against acetaminophen-induced toxicity has already been reported43. 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 animals44. These compounds also exert antioxidant or in vitro free radical-scavenging effects45-47. 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 compounds6, 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.

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4  Acknowledgements
     Authors are thankful to All India Council for Technical Education (AICTE), New Delhi, India for providing the financial assistance.

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