Hepatoprotective activity of Averrhoa bilimbi leaf extract against Alcohol-induced liver damage in wistar rats

 

Jennifer Fernandes1, Raghavendra Prabhu2, Sudhina M3, Ronald Fernandes1*

1Nitte (Deemed to be University), NGSM Institute of Pharmaceutical Sciences (NGSMIPS),

Department of Pharmaceutical Chemistry, Mangalore – 575018.

2Wintac Ltd, Department of Quality Assurance, Bengaluru – 560004.

3Yenepoya (Deemed to be University), Yenepoya Pharmacy College and Research Centre, Department of Pharmaceutical Chemistry, Mangalore – 575018.

*Corresponding Author E-mail: ronaldfernandes@nitte.edu.in

 

ABSTRACT:

Averrhoa bilimbi is one of  the India’s most legendary and sacred trees which belongs to the family Oxalidaceae.The aim of the current study is to examine the hepatoprotective effect of ethanolic leaf extracts of Averrhoa bilimbi against alcohol-induced hepatic damage in albino rats.The degree of protection was determined by evaluating biochemical parameters such as serum Alanine transaminase, Aspartate transaminase,Serum alkaline phosphatase, and total Bilirubin.The study reveals,when compared to the positive control, the leaf extract at dose levels of 100, 200, and 400mg/kg bodyweight p.o. showed a significant (P<0.05) reduction in liver enzyme levels. The histopathological examination of liver sections were supported by these biochemical parameters. The phytochemical analysis of the extract shows the presence of phytosterols and flavonoids, which might be responsible for the hepatoprotective activity.Therefore  it could be concluded that Averrhoa bilimbi leaves extract possess potent hepatoprotective activity against alcohol induced hepatic damage in rats.

 

KEYWORDS: Averrhoa bilimbi, Hepatoprotective activity, Silymarin, liver, Oxalidaceae.

 

 

INTRODUCTION: 

The liver is the major organ responsible for metabolism, detoxification, and secretory functions in the body. Hence, it regulates various important metabolic functions in mammalian systems. Hepatic damage is associated with the distortion of these metabolic functions. The liver tissue is reported to be one of the tissues with a high regenerative capacity. Regeneration of the liver tissues is a result of an organized and controlled response of the liver toward tissue damage induced by toxic agents, trauma, infections, or postsurgery resection. Different chemical agents, including gasoline vapor constituents, are known to be hepatotoxic1. According to an epidemiological study, the severity of hepatic and kidney disorders is growing during recent years as people's lifestyles shift, posing significant public health issues2.

 

 

Hepatotoxicity is a very common ailment that can lead to serious consequences ranging from extreme metabolic disorders to even death. Nowadays many synthetic drugs are available for the treatment of liver ailments, however due to their unfavourable side effects there is a growing impact on the therapeutic evaluation of medicinal plants through systemic research methodology. Several medicinal plants have been reported to have hepatoprotective activities3-5.

 

Averrhoa bilimbi (A. bilimbi) commonly called as bilimbi, is a tropical tree that grows at a height of 5 to 10 meters. It mainly belongs to the family Oxalidaceae. Fruits, leaves are generally used for therapeutic purposes. In traditional medicines A.bilimbi has long been used to treat a wide range of ailments. The leaves infusion and decoctions have been repored to possess various pharmacological activities such as anti-inflammatory, antioxidant and anti-scorbutic, astringent, anti-bacterial, anti-microbial, anti-atherogenic, anti-hyperlipidemic, and postpartum protective properties6.  The flowers and fruits were reported to contain cyanide-3-O-beta-D-glucoside and cyanidin-3,5-O-beta-D-diglucoside7 hexadecanoic acid and (Z)-9-octadecenoic acid, butyl nicotinate, and hexyl nicotinate, respectively8. The hypolipidemic properties of an ethanolic extract of A. bilimbi leaf, as well as the effects on blood glucose and lipids in streptozotocin-diabetic rats, have been reported9. The antibacterial, antifungal and antihypercholesterolemic activity of the fruit juice of A.bilimbi were studied10. The purpose of this study was to determine the efficacy of different doses of ethanolic extract prepared from the leaves of A.bilimbi against hepatic injury caused by alcohol in rats in order to determine the potential use of this plant in preventing hepatic damage.

 

MATERIALS AND METHODS:

Preparation of ethanolic extract:

The leaves of A.bilimbi were collected from the local areas of Mangalore city , Karnataka. The Authentication of the plant was done by  Botanist Dr. Neoline J. Pinto, Head, Department of Botany, St. Agnes College, Mangalore. The leaves were washed,cleaned, dried,broken down into pieces, and powdered using mechanical grinder. The coarse powder was then passed through sieve no. 40 and extracted with ethanol by maceration process. The extract was  finally concentrated under reduced pressure and controlled temperature by  using a flash evaporator11,12.

 

Phytochemical Screening:

The leaves extractwas thenanalysed for the presence of various phytoconstituentsby using Standard methods as described by Harborne13,14.

 

Animals:

Both male and female wistar rats weighing between 150-200g were collected from central animal house KSHEMA Mangalore. Prior to oral administration, the animals were kept at the animal house for 7 days to allow them to acclimate to laboratory conditions. They were fed with standard diet andwater ad libitum. The animals were maintained according to the guidelines  recommended by the Institutional Animal Ethical Committee (IAEC) constituted under the guidelines of CPCSEA.

 

Acute toxicity studies:

Theacute toxicity test was performed using the "Up and Down" method15,16 and OECD guidelines 42517,18 in adult female wistar rats.The animals were given oral doses of ethanolic extract of A.bilimbi leaves ranging from 100mg/kg of body weight to 2g/kg of body weight.The animals were observed for general activity, neurological,  autonomic profile, and death for a total of 24 hours and 14 days after receiving leaf extracts. There was no mortality, and no signs of harmfulness up to 2g/kg/body weight and discovered to be protected upto 2g/kg body weight.

 

Assessment of hepatoprotective activity19:

Based on their body weight, the wistar rats of either sex were randomly divided into the following six classes, each comprising six male and six female rats. Group- I served as control given 1% carboxy methyl cellulose (CMC) p.o for 30 days. Group –II received the ethanol. The drug silymarin at a dosage of 50mg/kg b.wt, p.o.) was given to Group III served as a control for 30 days. Group – IV, V, and VI received the graded doses of ethanolic extract of A. bilimbi (100, 200, and 400mg/kg b.wt p.o.), respectively, for 30 days.

 

Hepatotoxicity in all groups except group II was induced by alcohol administered orally at a dose of 3ml/100gm/day p.o for 30 days. 24hr after the last dose of alcohol, the blood was collected, and serum obtained after centrifugation (2500rpm for 15min) was used for various biochemical estimation. The animals were sacrificed under deep anesthesia.

 

Biochemical estimation20:

Serum was separated from the blood and subjected to various biochemicalparameters like Aspartate amino transaminase  (AST),  Alkaline phosphatase  (ALP), Alanine transaminase  (ALT),and total  Bilirubin.

 

Histopathological studies21:

The left lobe portion of the liver stored in 10% buffered  formalin for atleast 24 hrs. As per the standard protocol, it was processed and embedded in paraffin. Five µm thick sections cut and transferred into glass slides,  stained with Hematoxylin and eosin (HE) for examination of  histopathological study.

 

Statistical analysis22:

The data obtained in present investigation was subjected to statistical analysis. All results are expressed as mean ± SEM and significance was evaluated by one-way analysis of variance (ANOVA).A value of p < 0.05 was considered to indicate a significant difference between groups.

 

RESULTS AND DISCUSSION:

Alcohol administration in normal rats elevated the serum levels of alanine aminotransaminase, aspartate amino transaminase, alkaline phosphatase, total bilirubin significantly. As compared to the alcohol treated group, the rats in all the  three groups treated with ethanolic extract of A.bilimbi at graded dose levels had significantly lower levels of alanine aminotransaminase, aspartate aminotransaminase, alkaline phosphatase, and total bilirubin (Table 1).

 

Table 1: Effects of the ethanolic extract of leaves of Averrhoa bilimbi on alcohol-induced hepatotoxicity in albino rats.

Group and Treatment

ALT (IU/L)

AST (IU/L)

ALP (IU/L)

Total Bilirubin (mg/dL)

Control

49.29±1.10

69.73±1.23

141.3±1.20

0.13±0.01

Ethanol Treated

97.27±1.17**

136.4±1.20**

244.7±0.98**

1.02±0.01**

 Ethanol+ Silymarin(50mg)

67.65±1.26**

84.75±0.93**

170.7±0.93**

0.205±0.016**

Ethanol + AB (100mg)

87.50±1.2**

124.3±1.15**

225.6±1.18**

0.71±0.01**

Ethanol + AB (200mg)

81.18±1.17**

116.6±1.20**

210.6±0.82**

0.58±0.01**

Ethanol + AB (400mg)

73.35±0.93**

99.40±0.93**

194.5±1.20**

0.4±0.01**

Values  expressed as mean±SEM, n=6 rats in each group. AB-Averrhoa bilimbi**Significant (p˂0.05) compared to control.

 

Histopathological slides of liver

 

Figure 1. Control group

 

Figure 2. Alcoholtreated group

 

Figure 3. Silymarin 50mg/kg/b.wt

 

Figure 4. 100mg/kg/b.wt

 

Figure 5.200mg/kg/b.wt

 

Figure 6. 400mg/kg/b.wt

 

The current study on A.bilimbi leaf extract revealed the presence of plant sterols and flavonoids in the ethanolic extract.The data presented here indicate a significant hepatoprotective effect of A. bilimbi leaf extract, which protected wistar rats from ethanol-induced damage when compared to controls. The levels of ALT, AST, ALP, and bilirubin were significantly reduced after several days of ethanol treatment23. On microscopic examination the liver of group I, had a regular portal triad, sinusoids, and cord arrangement of hepatocytes (Fig 1). Group II's microscopic examination revealed significant liver necrosis and inflammation (Fig 2).The microscopic examination of group VI's liver revealed almost normal-appearing hepatocytes (Fig 6). Thus the findings of this group are comparable with the finding of the silymarin treated group, suggesting the hepatoprotection at this dose.The microscopic examination of the liver of group V reported mild fatty changes (Fig 5). According to the microscopic analysis of the liver of group IV,  the test extract used at 100mg/kg b.wt did not provide much protection against fatty change in the liver, as the parts of the liver at this dose showed moderate fatty change and mild hydropic change(Fig 4). The liver of group III was examined under a microscope and showed hepatocytes that appeared to be almost normal (Fig 3).

 

CONCLUSION

Administration of ethanolic extract of leaves of A.bilimbi at all the dose levels viz., 100, 200, and 400 mg/kg/b.wt, showed significant hepatoprotective activity but the results obtained from the dose level of 400 mg/kg/b.wt statistically comparable with the results obtained from the standard drug silymarin in histopathological support reports also revealed that there is a marked hepatoprotection in group III, IV, V and VI. Though the ethanolic extract of leaves of A.bilimbi, which contains flavonoids, triterpenoids, saponins and steroids, showed significant hepatoprotective effects.

 

CONFLICTS OF INTEREST

The authors declare no conflicts of interestregarding this investigation.

 

ACKNOWLEDGEMENT:

Authors are thankful to the NGSM Institute of Pharmaceutical Sciences and Nitte (Deemed to be University), Mangalore, for providing necessary facilities and support to conduct this study.

 

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Received on 24.05.2021            Modified on 11.03.2022

Accepted on 14.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(4):1727-1730.

DOI: 10.52711/0974-360X.2023.00284