Hepatoprotective activity of Bridelia retusa  bark extracts  against carbon tetrachloride -induced liver damage rats.

 

Ghawate V. B.^1,2*, Purnima Shrivastava¹, Bhambar R.S.³

¹Department of Pharmaceutical Sciences, Bhagwant University, Ajmer, Rajasthan.

²Department of Pharmacognosy, MES College of Pharmacy, Sonai, Ahmednagar, Maharashtra.

³MGV's College of Pharmacy, Panchwati, Nasik, Maharashtra.

 

ABSTRACT:

The present study is aimed to evaluate the hepatoprotective effect of Bridelia retusa bark extracts against carbon tetrachloride -induced liver damage in rats. The degree of protection was measured by using biochemical parameters such as serum glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and bilirubin. Treatment with Bridelia retusa bark butanol extract (200mg/kg b.w. p.o.)  showed significant reduction in level of SGOT, SGPT, ALP and total bilirubin almost comparable to the Silymarin. The hepatoprotection confirmed by histopathological examination of the liver tissue. From the result it can be concluded that Bridelia retusa bark extract possesses hepatoprotective effect against carbon tetrachloride –induced liver damage in rats.

 

 

 

1. INTRODUCTION:

Liver is one of the important organ of body which play a major role in the metabolism of protein, carbohydrates and lipids. About 20000 deaths found every year due to liver disorders¹. In experimental hepatopathy carbon tetrachloride (CCl₄) has been commonly used because it initiates oxidative damage, decrease the activities of antioxidant enzyme and generation of toxic free radicals^{2, 3} . CCl₄ induce hepatotoxicity in animal models has been shown similar to human liver cirrhosis^{4, 5}.

 

Bridelia retusa (Euphorbiaceae), a small or moderate sized tree, spinus when young and has gray bark. Leaves are used in Ayurvedic formulation for the treatment of urinary tract infections and bark is given orally to women to develop sterility and as contraceptive⁶.The root and the bark are valuable astringent. Stem bark and roots were used for rheumatism and as astringent agents⁷.The paste of leaves of Bridelia retusa, Curculigo orchioides and oil of castor, coconut and gingelly applied externally to cure wounds⁸.The Bridelia retusa in combination with other plants used traditionally to cure hepatitis⁹.

 

The Bridelia retusa bark also used to cure dysentery¹⁰.The isoflavone was isolated from Bridelia retusa leaves extract and its structure was elucidated by spectroscopical study. These Isoflavone showed strong antimicrobial activity against Gram-positive and Gram

negative bacteria¹¹. Liver is vital organ playing important role in metabolism and excretion. In absence of reliable liver protective drug in modern system of medicine, plants from Indian system of medicine are considered to be effective and safe alternative treatment for hepatotoxicity. With this vision the present study was undertaken to investigate the hepatoprotective activity of Bridelia retusa bark extracts against CCl₄ induced toxicity in rats.

 

2. MATERIAL AND METHOD:

2.1 Drugs and Chemicals

Carbon tetrachloride was procured from Merck (India) Ltd. Mumbai. Silymarin was obtained from Cadila Pharma India. All other reagents used for experiment were of high analytical grade.

 

2.2 Plant and Material

Bridelia retusa bark was collected from Western Ghat of Maharashtra and authenticated by J. Jayanthi scientist, Botanical Survey of India, Pune and herbarium was (Voucher No. BRIRVIGI) deposited in BSI Pune.

 

2.3 Preparation of Extracts:

The fresh bark was cleaned, shade, dried and then powdered. Firstly the dried coarse powder of bark was defatted with petroleum ether later on it was subjected to successive extraction in a soxhlet apparatus using chloroform, butanol and 70% acetone. The extracts were subjected to preliminary phytochemical screening.

 

2.4 Phytochemical evaluation of extract.

The preliminary phytochemical screening is done with standard qualitative chemical tests for chloroform, butanol and 7% acetone extracts. The successive extraction process showed presence of different metabolites including alkaloids, glycosides, tannins, steroids,  flavonoids and saponins were confirmed using conventional phytochemical tests.

 

2.5 Experimental animals:

Wistar rats weighing 150-200 gm were housed in standard laboratory condition for 12 hr light and dark cycle with standard food and water ad libitum.

 

2.6 Acute oral Toxicity:

As per the OECD revised guideline no 423 and CPCSEA the oral toxicity study was carried out.

 

2.7 Evaluation of Hepatoprotective activity

The method of Mohan G. Kalaskar et al was followed with minor modification¹². Animals were divided into nine groups of rats containing six animals in each group. Group I, which is served as normal control, received 5 % CMC p.o. for 7 days. Group II, received 5 % CMC p.o. for 7 days. Group III, received silymarin suspended in 5% CMC (100 mg/kg, p.o.) for 7 days. Group IV received Bridelia retusa bark chloroform extract (BRBC) 100 mg/kg p.o for 7 days. Group V received chloroform bark extract (BRBC) 200 mg/kg p.o  for 7 days. Group VI received Bridelia retusa bark butanolic extract (BRBB) 100 mg/kg p.o  for 7 days. Group VII received butanolic bark extract (BRBB) 200 mg/kg p.o. for 7 days. Group VIII received Bridelia retusa bark 70% acetone extract (BRBA) 100 mg/kg p.o. for 7 days and Group IX received acetone bark extract (BRBA) 200 mg/kg p.o. for 7 days. On day 7 after 1h administration of the test substances to rats, all animal groups except for normal control were given an intraperitoneal dose of carbon tetrachloride (CCl₄) at 1 ml/kg body weight (1:1 in olive oil). After 24 h of CCl₄ treatment blood was collected from all groups of rats by puncturing the retro-orbital plexus. Serum was separated by centrifugation at 2500 rpm for 10 min and analyzed for various biochemical parameters. All the animals were sacrificed liver was aseptically removed and processed for histological investigation.

 

2.8  Histopathological studies

One animal from each group was utilized for histopathological study. The livers were fixed in 10% formalin for 24 h. The formalin fixed liver stained with haematoxylin-eosin for microscopic observation.

 

3. STATISTICAL ANALYSIS:

Results were expressed as mean ± S.D. Statistical analysis of the data was done using one–way analysis of variance (ANOVA) followed by Dunnett’s ‘t’ test and significance determined using P-values <0.05.

 

4. RESULT:

Table 1: Preliminary phytochemical studies on Bridelia retusa bark extracts.

Tests	Bark
	BRBC	BRBB	BRBA
Alkaloid	-	-	+
Glycoside	-	-	+
Saponins	-	+	+
Steroid	+	+	-
Triterpenoids	+	-	-
Hydrolysable tannin Condensed tannin	-	-	+
Flavonoid	-	+	+
Carbohydrates	-	-	-
Proteins	-	-	-
Fats and lipids	-	-	-

 

 

Table 2: Effects of Bridelia retusa bark extract on rat serum parameters in carbon tetrachloride induced hepatotoxicity

Sr.  no.	Group  (n=6)	SGPT (IU/L)	SGOT (IU/L)	ALP (IU/L)	Total  Bilirubin (mg/dl)
1	Normal	65.33 ± 0.88	74.00 ± 0.66	93.66 ± 1.45	0.70 ± 0.01
2	Toxic control	179.00 ±3.60	199.67 ± 0.88	206.33 ± 4.25	2.36 ± 0.01
3	Standard control	68.66± 0.88**	81.66 ± 0.88**	105.67 ± 4.70**	0.75± 0.01**
4	BRBC100	144.00 ± 9.07*	123.00 ± 1.15*	159.67 ± 0.88*	1.13 ± 0.01*
5	BRBC200	136.67 ± 0.36*	115.33 ±0.88*	150.00 ± 1.52*	1.09 ± 0.01*
6	BRBB100	105.00± 3.60*	121.67 ± 1.45*	139.00± 1.15*	1.06 ± 0.01*
7	BRBB200	71.66 ± 0.88**	85.33 ± 0.88**	110.33 ± 1.76**	0.76 ± 0.01**
8	BRBA100	114.67 ± 3.52*	97.66±0.88*	142.67 ± 1.20*	1.08 ± 0.01*
9	BRBA200	85.00±1.73*	96.00 ± 1.00*	128.00±1.73**	0.95±0.01*

Data are presented as Mean±SEM (n=5), one way Annova followed by Dunnett t test.*P<0.05 and **P<0.01vs paracetamol treated group

                                                                                

A)      Normal control rat liver                                  B) Toxic control rat liver

 

             

C)      Silymarin treated rat liver                  D)  BRBC (200mg/kg) treated rat liver

 

                  

E) BRBB (200 mg/kg) treated rat liver                                    F) BRBA (200 mg/kg) treated rat liver

Fig.1 Representative photographs from the liver showing the protective effect of Bridelia retusa on carbon tetrachloride-induced hepatic injury in rats.

 

 

The preliminary phytochemical screening showed the presence of different secondary metabolites like alkaloids, glycosides, tannins, steroids, flavonoids and saponins. In acute toxicity study the extract were found to be safe in dose used and does not showed mortality upto the dose 2000mg/kg,b.w. hence 100 and 200 mg/kg, b.w.dose were selected for activity. Administration of CCl₄ induced a marked increase in the level of serum hepatic biochemical markers, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphate (ALP), Total Bilirubin as compared to normal controls, indicating liver damage. Pretreatment of rats with BRBB and BRBA extract (100 and 200 mg/kg) before CCl₄ administration causes significant reduction in values of SGOT, SGPT, ALP and total Bilirubin in a dose dependent manner. (Table 2) The Hepatoprotective effect of extract was confirmed by histological examination of the liver tissue of control and treated animals. Normal control group showed normal hepatocytes. In CCl₄ treated rat liver section showed necrosis. Pretreatment of Silymarin (100mg/kg) showing normalization of liver architecture. The BRBB and BRBA extracts (200mg/kg) exhibited healing of necrotic lesions. (Fig 1)

 

5. DISCUSSION:

Since the changes associated with CCl₄ induced liver damage are similar to acute viral hepatitis. it has been established that CCl₄  is accumulated in hepatic parenchymal cells and metabolically activated by cytochrome _P450 dependent monooxygenase to form a trichloro methyl free radical which alkylates cellular protein and other macromolecules with a simultaneous attack on polyunsaturated fatty acid in presence of oxygen to produce lipid peroxides¹³. which leads to change in the structures of endoplasmic reticulum and other membrane, loss of metabolic enzymes activation , reduction of protein synthesis and elevation of serum transaminases leading to liver damage¹⁴. a significant increase in the level of SGPT ,SGOT, ALP and bilirubin was observed in CCl₄ treated group indicating considerable hepatocellular injury. Pretreatment with Silymarin and Bridelia retusa bark extracts reduced liver enzymes level and bilirubin. This observation strongly suggested that Bridelia retusa bark extracts provided protection against CCl₄ induced liver injury. Degree of protection was observed maximally with butanol extract (200mg/kg) compared to other extracts. However protection maintained by Silymarin seemed to relatively greater as compared to extracts. The histopathological study revealed that the hepatic cells are almost normal in Bridelia retusa bark butanol extract (200mglkg b.w. p.o.) treated group in contrast with CCl₄ group.

 

6. CONCULSION:

It may be further concluded that the result of this study demonstrated that Bridelia retusa bark extracts protect against carbon tetrachloride-induced liver damage in rats. These result shows that the protective effect of extract may be due to presence flavonoids and steroids. The present study justifies the traditional use of Bridelia retusa in treatment of liver disease. However the exact mechanism(s) and the active compound (s) involved in effect need to clarified in future study.

 

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Received on 22.05.2017          Modified on 25.05.2017

Accepted on 30.05.2017      ©AandV Publications All right reserved