Hepatoprotective activity of fruit extract of Garcinia pedunculata

The objective of this study is to explore the hepatoprotective activity of fruits of Garcinia pedunculata in paracetamol-induced liver toxicity in rats. Paracetamol-induced hepatotoxicity was evaluated by an increase in serum transaminases and alkaline phosphatase activity. Histopathological observation showed extensive disturbance in the liver cytoarchitecture in comparison to normal control liver sections. Pre-treatment with aqueous extract of fruits of G. pedunculata prevented the paracetamol-induced increase in serum transaminases, alkaline phosphatase and histopathological changes. Based on the above observation it can be concluded that G. pedunculata pretreatment exhibited significant hepatoprotective activity against paracetamol-induced hepatotoxicity. Article Info Received: 8 August 2014 Accepted: 1 September 2014 Available Online: 1 October 2014 DOI: 10.3329/bjp.v9i4.19910 Cite this article: Mundugaru R, Varadharajan MC, Basavaiah R. Hepatoprotective activity of fruit extract of Garcinia pedunculata. Bangladesh J Pharmacol. 2017; 12: 483-87. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Hepatoprotective activity of fruit extract of Garcinia pedunculata Ravi Mundugaru1, Madhan Chakkravarthy Varadharajan2 and Ravishankar Basavaiah3 Department of Pharmacology and Toxicology, SDM Centre for Research in Ayurveda and Allied Sciences, Udupi 574 118, India; Department of Research and Development, Biomedical Research Unit and Laboratory Animal Center, Saveetha University Thandalam, Chennai 602 105, India; Department of Pharmacology and Toxicology, SDM Center for Research in Ayurveda and Allied Sciences, Udupi 574 118, India. deposited for further future reference. The powder of G. pedunculata was prepared in the Pharmacy attached to SDM College Ayurveda, Udupi, from authenticated plant material. The powder obtained from a single batch was used throughout the experimental study. Five hundred grams of fruit powder of G. pedunculata was soaked in 2L of distilled water for twenty four hours; it was filtered and concentrated by evaporation. The concentrated extract was used for hepatoprotective activity. Experimental animals Wistar albino rats of either sex weighing 200 ± 50 g were obtained from animal house attached to Department of Pharmacology, SDM Centre for Research in Ayurveda and Allied Sciences, Udupi. The experimental protocol was approved by the Institutional Animal Ethics Committee under the reference no. SDMCRA/ IAEC-2012-13DGM 02ab. The animals were fed with normal rat diet and water ad libitum throughout the study period. The animals were acclimatized for two weeks at the laboratory condition prior to the experimentation. The animals were maintained at controlled lighting of 12:12 hours light and dark cycle, temperature of 25oC and relative humidity of approximately 50 ± 5%. Acute oral toxicity test The acute oral toxicity study was carried out as per OECD guidelines 425 using AOT software. The aqueous extract of fruits of G. pedunculata was made into a suspension in 0.5% gum acacia and dosed in the following order 175, 550, and 2,000 mg/kg body weight. After the dosing the animals were observed for 14 days for mortality. The dose at which the animal dies, that particular dose is repeated two times to determine its toxic potential. If mortality was not observed at 2,000 mg/kg, dosing was stopped and LD50 was determined by using AOT software. Evaluation of hepatoprotective activity Control group were administered with normal tap water at a dose of 5 mL /kg in 0.5% gum acacia, the standard group were administered with silymarin 50 mg/kg as a suspension with 0.5% gum acacia and Group III was administered with aqueous extract of G. padenculata at a dose of 400 mg/kg as a suspension with 0.5% gum acacia, served as test group. The group specific drugs were administered for nine consecutive days. On seventh day one hour after administration of group specific drugs the liver was damaged by intraperitonial injection of paracetamol at a dose of 1 g/ kg in three divided doses. All the animals were sacrificed 48 hours after administration of paracetamol (Yoshigurki et al., 1992). The hepatoprotective effect of extract was evaluated by the assay of serum biochemical markers such as SGOT, SGPT and ALP according to standard methods (Bradly et al., 1972; Wilkinson et al., 1996). Histopathological studies The animals were sacrificed and the abdomen was cut open to isolate the liver. The liver was washed with normal saline and for histopathological study biopsy specimen was fixed in 10% formalin solution and then the liver tissue was embedded in paraffin. The section was cut into 5 μm thickness and stained with hematoxyline-eosin stain and mounted in diphenylxyline. The histopathological changes of liver tissue were observed under compound microscope and their microphotographs were taken (Bancroft et al., 2002).


Introduction
The liver is a vital organ, supports almost every organ in the body and is vital for survival. Because of its multidimensional function the liver is also prone to many diseases. The most common diseases are heaptitis, alcohol damage, fatty liver, cirrhosis, cancer and drug induced liver damage (Graham et al., 2013). However the liver has great capacity to regenerate and has a large reserve capacity. In most cases the liver only produces symptoms after extensive damage.
Paracetamol is used as an antipyretic and analgesic (Larson et al., 2005;Rumack et al., 1975). At recommended doses and for a limited course of treatment the side effects of paracetamol are mild but the overdose of paracetamol can cause fatal hepatic damage, gastrointestinal complication, renal failure, asthma and skin reactions (Patiri et al., 2007).
Garcinia pedunculata is a medicinal plant commonly known as amlavetasa and available particularly in Assam, Arunachal Pradesh and West Bengal regions. It has been indicated for many ailments such as chronic catarrh, asthma, cough, bronchitis, fever and as a cardiotonic (Kagyung et al., 2010). The phytochemical studies have shown the dried fruit rinds and pericarp of G. pedunculata contains benzophenones, pedenculol, hydroxy citric acid, garcinol and cambogin (Sahu et al., 1989). The plant has been screened for its in vitro antioxidant activity and found to possess an excellent antioxidant property and anti-inflammatory activity (Jayaprakasha et al., 2006;Mundugaru et al., 2014). There are no documented studies available regarding hepatoprotective activity of the aqueous extract of fruits of G. pedunculata. Therefore, the present study has been undertaken to evaluate the hepatoprotective activity. deposited for further future reference. The powder of G. pedunculata was prepared in the Pharmacy attached to SDM College Ayurveda, Udupi, from authenticated plant material. The powder obtained from a single batch was used throughout the experimental study. Five hundred grams of fruit powder of G. pedunculata was soaked in 2L of distilled water for twenty four hours; it was filtered and concentrated by evaporation. The concentrated extract was used for hepatoprotective activity.

Experimental animals
Wistar albino rats of either sex weighing 200 ± 50 g were obtained from animal house attached to Department of Pharmacology, SDM Centre for Research in Ayurveda and Allied Sciences, Udupi. The experimental protocol was approved by the Institutional Animal Ethics Committee under the reference no. SDMCRA/ IAEC-2012-13DGM 02ab. The animals were fed with normal rat diet and water ad libitum throughout the study period. The animals were acclimatized for two weeks at the laboratory condition prior to the experimentation. The animals were maintained at controlled lighting of 12:12 hours light and dark cycle, temperature of 25ºC and relative humidity of approximately 50 ± 5%.

Acute oral toxicity test
The acute oral toxicity study was carried out as per OECD guidelines 425 using AOT software. The aqueous extract of fruits of G. pedunculata was made into a suspension in 0.5% gum acacia and dosed in the following order 175, 550, and 2,000 mg/kg body weight. After the dosing the animals were observed for 14 days for mortality. The dose at which the animal dies, that particular dose is repeated two times to determine its toxic potential. If mortality was not observed at 2,000 mg/kg, dosing was stopped and LD50 was determined by using AOT software.

Evaluation of hepatoprotective activity
Control group were administered with normal tap water at a dose of 5 mL /kg in 0.5% gum acacia, the standard group were administered with silymarin 50 mg/kg as a suspension with 0.5% gum acacia and Group III was administered with aqueous extract of G. padenculata at a dose of 400 mg/kg as a suspension with 0.5% gum acacia, served as test group. The group specific drugs were administered for nine consecutive days. On seventh day one hour after administration of group specific drugs the liver was damaged by intraperitonial injection of paracetamol at a dose of 1 g/ kg in three divided doses. All the animals were sacrificed 48 hours after administration of paracetamol (Yoshigurki et al., 1992). The hepatoprotective effect of extract was evaluated by the assay of serum biochemical markers such as SGOT, SGPT and ALP according to standard methods (Bradly et al., 1972;Wilkinson et al., 1996).

Histopathological studies
The animals were sacrificed and the abdomen was cut open to isolate the liver. The liver was washed with normal saline and for histopathological study biopsy specimen was fixed in 10% formalin solution and then the liver tissue was embedded in paraffin. The section was cut into 5 µm thickness and stained with hematoxyline-eosin stain and mounted in diphenylxyline. The histopathological changes of liver tissue were observed under compound microscope and their microphotographs were taken (Bancroft et al., 2002).

Result
Acute oral toxicity study did not reveal any mortality in any dose up to 2,000 mg/kg of aqueous extract of fruits of the plant G. pedunculata. This indicates that LD50 is much more than 2,000 mg/kg and hence 1/5 th of dose was selected for the present hepatoprotective study.
The damage caused by the treatment of overdose of paracetamol to the structure integrity of liver cells is assessed by the determination of biochemical parameters such as SGOT, SGPT and alkaline phosphatase activities. The levels of SGOT, SGPT and alkaline phosphatase was significantly increased (p<0.001) in the paracetamol treated group in comparison to normal control group. Aqueous extract of fruits of G. pedunculata showed significant reduction in the SGOT,   (Table I).
Microscopic examination of liver sections from paracetamol treated group showed extensive disturbance in the liver cytoarchitecture in comparison to the sections from normal control liver sections ( Figure 1A-D). The changes observed were hepatocyte necrosis, appearance of balloon cells, leukocyte infiltration, micro and macro fatty changes in the hepatocytes, sinusoidal dilatation and appearance of areas of regenerations.
Liver sections from paracetamol pretreated group followed by either extract or silymarin exhibited good protection and almost normal cytoarchitecture in sections from three rats ( Figure 1E-H). Moderate degenerative changes in the form of necrosis and macro fatty changes were observed in sections from two rats. In the remaining sections diffused mild microfatty changes were observed.
Mild fatty changes were observed in G. Pedunculata extract treated group in three rats only. In two sections moderate fatty changes and mild cell infiltration was observed. In one section mild to moderate degenerative changes were observed.

Discussion
In the present study, hepatoprotective activity of aqueous extract of fruits of G. pedunculata was evaluated against paracetamol overdose induced hepatotoxicity in Wistar albino rats.
Ingested paracetamol undergoes metabolism in liver by cytochrome P450, producing most reactive metabolite Nacetyl-p-benzoquinoneimine. At usual doses N-acetylp-benzoquinoneimine is quickly detoxified by conjugation with glutathione (Grosser et al., 2006). While administered at overdose the detoxification pathway remains saturated and as a consequences N-acetyl-pbenzoquinoneimine accumulates causing hepatic damage.
SGOT is a mitochondrial enzyme released from heart, liver, skeletal muscle and kidney. SGOT level may rice in acute necrosis or ischemia of other organs such as the myocardium, besides the liver cell injury. SGPT is a cytosolic enzyme primarily present in the liver tissue. Thus the serum estimation of SGPT is fairly specific for liver tissue is of great value in liver cell injury (Dixon et al., 1971;Schmidt et al., 1975). It is well established that level of serum enzymes such as SGOT and SGPT gets elevated in paracetamol induced hepatotoxicity. In the present study extremely significant elevation was observed in paracetamol treated group. This transaminase activity elevation was found to be significantly reduced by extract and reference standard drug silymarin. This can be considered as the first line of evidence for the presence of significant hepatoprotection in the test drug.
Serum alkaline phosphatase is produced by many tissues, especially bone, liver, intestine and in pregnancy. Elevation in activity of the enzyme can thus be found in disease of bone, liver and in pregnancy. In the absence of bone disease and pregnancy, an elevated serum alkaline phosphatase levels generally reflect hepatobiliary diseases. Alkaline phosphatase is normally excreted in the bile. If it is affected due to liver injury its level gets elevated. This may be one of the reasons for the observed elevation since it is reported that greater elevation occurs in biliary tract obstruction (Rajesh et al., 2001).
In the present study statistically significant increase in the alkaline phosphatase activity was observed after paracetamol injection. This elevation was reversed in significant manner by extract and reference standard drug. Reversal of alkaline phosphatase elevation can be considered as another indicator of hepatoprotection.
Histopathological observation has shown that in three rats only mild fatty changes were observed in extract administered group. In two sections moderate fatty changes and mild cell infiltration was observed. In one section mild to moderate degenerative changes were observed. This indicates extract might have possessed membrane stabilizing action and it may contribute for protection of structural integrity of hepatocytes.
The preliminary phytochemical analysis revealed the presence of flavonoids, saponins, glycosides, steroids, alkaloids and phenols. It is also reported that the fruit of G. pedunculata has good anti-oxidant property and reported in the earlier studies. Thus these phytochemical may be responsible for the hepatoprotective activity of G. pedunculata perhaps through their anti-oxidant activity.

Financial Support
Self-funded

Conflict of Interest
Authors declare no conflict of interest