Hepatoprotective Activity of Ethanolic and Ethyl Acetate Extract of Ziziphus mauriatiana on Liver Damaged Caused by Paracetamol in Rats

Sawarkar DJ²*, Vijaya C², Turaskar AO², Shende VS¹,Chatap VK¹, Sawant VA¹ and Borkar SN¹

¹Sharadchandra Pawar College of Pharmacy, Pune, Otur, (MS), India.

²Ultra College of Pharmacy, Madurai, (TN), India.

ABSTRACT

The objective of the present study was to investigate the hepatoprotective activity of alcoholic and ethyl acetate leaves extract of Ziziphus mauriatiana against paracetamol induced hepatotoxicity. The material was dried in shade, they were powdred and extracted with alcohol and ethyl acetate. Preliminary phytochemical tests were done in the presence of phytoconstituents like flavonoids, saponins, phenolic compounds and tannins. The present activity on rats shown alteration in the level of biochemical markers of hepatic damage like ALT, AST, ALP, Bilirubin and Protin were tested in both paracetamol treated and untreated groups. Wistar rats were induced hepatotoxicity by oral administration of paracetamol (640mg/kg) for 10 days. The hepatotoxicity induced rats were used for the studies. Ethyl acetate extract (400mg/kg), ethanol extract (300mg/kg) and standard (Silymarin) reduced the elevated levels of enzyme markers such as AST, ALT, ALP, and total bilirubin and increased in total proteins levels. The histopathological investigations also supported above effect of ethyl acetate and ethanolic extract by indicating the reduction in inflammatory collection and absence of fatty vacuoles in liver sections.

KEYWORDS: Ziziphus mauriatiana, Hepatoprotective, Paracetamol, liver

INTRODUCTION

Liver is the vital organ of metabolism and excretion. About 20,000 deaths found every year due to liver disorders. Hepato cellular carcinoma is one of the ten most common tumors in the world with over 2, 50,000 new cases each year¹. Liver diseases remain one of the serious health problems. Conventional drugs used in pharmacotherapy provided a substantial contribution for treatment but may have serious adverse effects. The use of natural remedies for liver disease treatment has been reported long history².

Paracetamol hepatotoxicity is caused by the reaction metabolite N-acetyl-p-benzo quinoneimine (NAPQI), which causes oxidative stress and glutathione (GSH) depletion. It is a well-known antipyretic and analgesic agent, which produces hepatic necrosis at higher doses ³. Paracetamol toxicity is due to the formation of toxic metabolites when a part of it’s metabolized by cytochrome P-450. Introduction of cytochrome ⁴ or depletion of hepatic glutathione is a prerequisite for paracetamol-induced hepatotoxicity. In spite of tremendous strides in modern medicine, there are hardly any drugs that stimulate liver function, offer protection to the liver from damage or help regeneration of hepatic cell ⁵. There are however, members of drugs employed in traditional system of medicine for liver affections ⁶. Many formulations containing herbal extracts are sold in the Indian market for liver disorders. But management of liver disorders by a simple and precise herbal drug is still an intriguing problem. Several Indian medicinal plants have been extensively used in the Indian traditional system of medicine for the management of liver disorder. Some of these plants have already been reported to posses strong antioxidant activity ^{7, 8, 9}.

TABLE – 1 EFFECT OF ZIZIPHUS MAURITIANA LAM LEAF EXTRACT ON PARACETAMOL INDUCED HEPATOTOXICITY IN RATS

SL. NO	GROUP (n)	DOSE (mg/kg)	ALT (IU/L)	AST (IU/L)	ALP (IU/L)	TOTAL PROTEIN (gm %)	TOTAL BILIRUBIN (mol/L)
1	Control	Vehicle	82.66±2.08	130.66±9.71	130.67±3.70	8.15±0.58	1.33±0.05
2	Paracetamol	640	339.66±5.03	502±6.24	294±5.56	4.04±0.34	4.28±0.03
3	Silymarin	100	91.33±2.08**	143±10.58**	152.33±7.02**	7.51±0.41**	1.39±0.04**
4	Ethanol Extract	300	178±2.08**	319.33±13.42*	203.33±4.50*	5.15±0.22**	2.4±0.05**
5	Ethyl acetate Extract	400	123.33±4.72**	175.66±14.66**	173.33±3.78**	6.90±0.28**	1.82±0.05**

Values are expressed as Mean ± SEM, n =3 rats in each group. *P < 0.05, **P < 0.01 compared to negative control (group 2)

Fig 1 Control (photos are at 40 x expansion by HE staining)

Even in this modern era, a large extent of Indian population still relies on traditional system of medicine which is mostly plant based, hence it is considered necessary to have experimental evidence and validation. From the literature, it was evident that many plants had already shown the hepatoprotective activity but still the Hepatic diseases comes under schedule ‘J’ which states that “drugs may not claim or cure, to prevent ailments or disease¹⁰.

Ziziphus mauritiana Lam belonging to family Rhamnaceae occurs throughout India. Its leaves are used as astringent, anthelmintic, antipyretic; stomatic.It is also used in wounds, ulcer, diarrhhoea, obesity and in hepatitis^{11, 12}. Leaves of Ziziphus mauritiana was found to contain dammarane saponin, flavonoids-ziziphin, jujubasaponins I-III (jujubogenin glycosides) ¹³.Consequent to the respect of flavonoids possessing hepatoprotective activity ^14. The present study was focused to evaluate the protective effect of Ziziphus mauritiana leaves extracts on paracetamol induced hepatic injury in rats. As there were no studies reported on hepatoprotective effect of the drug in high dose paracetamol induced hepatotoxicity, the same was selected as the model. Ethyl acetate and ethanolic extracts of the leaves are prepared and evaluated for hepatoprotective effect.

MATERIALS AND METHODS:

PLANT COLLECTION AND AUTHENTICATION:

The leaves of Ziziphus mauriatiana were collected from Nellaithalpattu of Madurai District, Tamilnadu in September 2008. The leaves were authenticated by Dr.Stephen, Professor, American collage, Madurai. The leaves were dried under shade for few days and were powdered.

EXTRACTION:

About 800 gms of dry leaves powder material were extracted with petroleum ether (40^oC -60^oC) by continuous hot percolation method using Soxhlet apparatus for 72 hrs for defatting purpose. The marc left after petroleum ether extraction was then dried and extracted with ethyl acetate (76^oC-78^oC) for 72 hrs. The ethyl acetate extract were filtered and concentrated under reduced pressure. A dark black residue was obtained (7.5 gms). The marc left after ethyl acetate extraction were also dried and extracted with ethanol (76^oC-78^oC) for 72 hrs and the ethanol extract were also filtered and concentrated under reduced pressure. The brown residue was obtained (3.5gms). Crude extracts were stored in desiccators^{15, 16}.

PRELIMINARY PHYTOCHEMICAL ANALYSIS:

The extracts obtained by Ziziphus mauriatiana Lam leaves were subjected to qualitative test for the identification of various plant constituents ¹⁷.

ANIMALS:

Wistar rats weighing about (150-180 g) were obtained from the animal house, ultra college of pharmacy, Madurai. They were properly housed in separate cages and fed with standard diet and water ad libitum. The Institutional Animal Ethical committee permitted for the pharmacological activity. (Proposal no.UCP/IAEL/2008/031)

ACUTE ORAL TOXICITY STUDY:

The acute oral toxicity study was done according to OECD 423 guidelines. Administration of the stepwise doses of ethyl acetate extract of leaves of Ziziphus mauritiana from 5 mg/kg upto the dose 3000 mg/kg cure no considerable signs of toxicity in the tested animals while dose of 4000 mg/kg of ethyl acetate extract resulted in 100% mortality¹⁸. For present investigation 1/10^th dose of ethyl acetate extract was selected.

PHARMACOLOGICAL INVESTIGATION:

HEPATOPROTECTIVE ACTIVITY:

Rats were divided into five groups, each of three animals. Group I served as control which received normal saline (10 ml/kg; p.o) Group II served as toxicant control and received paracetamol (640 mg/kg; p.o) in 50 % sucrose solution. Group III received paracetamol (640 mg/kg; p.o) and silymarin (100 mg/kg; p.o) in vehicle. Group IV and V received (300 mg/kg) of ethanolic and (400 mg/kg) ethyl acetate extracts in 50 % sucrose solution, 1h before administration of paracetamol daily for 10 days ^19,20,21.

Blood sample collection:

At the end of the experimental period, the animals were sacrificed after ether anesthesia and blood was collected without the use of anticoagulant for serum preparation. The blood sample were collected by retro arbital puncture and allowed to stand for 10 min before being centrifuged at 2000 rpm for another 10 min and serum were collected using micropipette and used to access liver functions such as AST, ALT, ALP, total protein and bilirubin.

Fig 2-Paracetamol treated (photos are at 40 x expansion by HE staining)

Fig 3-Silymarin treated (photos are at 40 x expansion by HE staining)

Histopathology:

After collection of blood sample, livers were excised, washed with normal saline and processed separately for histological observation. Initially, the tissues were fixed in 10% buffered neutral formalin and histopathological examination was carried out

Statistics and Calculation:

The results are expressed as mean SEM. The comparison between the groups was made by analysis of variance (ANOVA) followed by Dunnett’s‘t’ test as per suitability. p < 0.05 was considered as significant, p < 0.01 was considered as very significant and p < 0.001 was considered as extremely significant.

RESULTS AND DISCUSSION:

During the past several decades, there has been a global trend for the revival of interest in the traditional system of medicine. Simultaneously, the need for the basic scientific investigation of medicinal plants using indigenous medical systems have became more interesting and relevant. Since no effective Allopathic medicine available for the treatment of liver diseases, plant was selected on the basis of traditional use and documentation. The present study was carried out to evaluate the hepatoprotective activities of Ziziphus mauritiana Lam leaves extracts. The preliminary phytochemical investigations of ethyl acetate and ethanolic extract of Ziziphus mauritiana Lam showed the presence of phytoconstituents like flavonoids, saponins, phenolic compounds and tannins.

Fig 4-Ethanolic extract treated (photos are at 40 x expansion by HE staining)

Fig 5-Ethyl acetate extract treated (photos are at 40 x expansion by HE staining)

Wistar rats were induced hepatotoxicity by oral administration of paracetamol (640 mg/kg) for 10 days. The hepatotoxicity induced rats were used for the studies. Ethyl acetate extract (400 mg/kg), ethanol extract (300 mg/kg) and standard (Silymarin 100 mg/kg) reduced the elevated levels of enzyme markers such as AST, ALT, ALP, and total bilirubin and increased in total proteins levels. The histopathological investigations (shown in figure1-5) also supported above effect of ethyl acetate and ethanolic extract by indicating the reduction in inflammatory collection and absence of fatty vacuoles in liver sections.

Silymarin normalized ALT level by 96.62 %, ethanol extract by 62.90%, ethyl acetate extract by 84.17%. AST level by 96.50 % (Silymarin), 49.37 % (ethanol extract) and 87.73 % (ethyl acetate extract). ALP level 86.58 % (Silymarin), 55.48 % (Ethanol extract), 77 % (ethyl acetate extract). Total bilirubin level 97.66 % (Silymarin), 63.72 % (ethanol extract), 83.38% (ethyl acetate extract). Total protein level 84.42% (Silymarin), 57.42% (ethanol extract), 70% (ethyl acetate extract). Thus ethyl acetate extract normalize levels like that of Silymarin. (Shown in table-1)

The hepatoprotective activity may be due to normalizing cell phospholipids synthesis by counteracting fatty liver. This study can be further extended for isolation, characterization and identification of active phytoconstituents responsible for these activities.

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Received on 01.09.2009

Accepted on 05.10.2009

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 1(3): Nov. – Dec. 2009, 194-197