INTRODUCTION:

Hepatic fibrosis is associated with chronic viral, toxic, autoimmune or cholestatic liver injuries. This process of liver fibrosis which resembles the mechanism of wound healing is represented by three phases called, acute inflammation, synthesis of collagen and noncollagenous extra cellular matrix (ECM) components, and tissue remodelling (scar formation). Majority of people with chronic Hepatitis C virus (HCV) infection develops liver fibrosis that progresses to cirrhosis within 10-20 years. It has been established that Human Immuno Deficiency virus (HIV) co-infection in HCV infected patients accelerate the progression of fibrosis, likewise there are evidences of numerous chemicals and drugs that harm the liver^1-4. These explain the need of antifibrotic agents and this work is designed for antifibrotic effect of a natural product Cyclea peltata (H.f and T) belonging to menispermaceae family.

Cyclea peltata was originally identified by Hooker f and Thomson as described in Flora of Bombay, and the genus Cyclea was originally proposed and characterized by Arnott and adopted by Mr. Miers. It is a slender twining shrub with sparingly pilose stems and branches, the leaves are 3 - 6 with 2 - 4.5 inches long, deltoid or ovate, acute, truncate or slightly sinuate at the base with rounded angles, mucronate, more or less hairy on the nerves and veins, margins are often ciliate. Flowers are minute, smaller than the preceding with axillary male panicles, slender but much branched, branches are remote and divaricate, the lower the longer and gradually becomes shorter upwards, the uppermost part are very short or obsolete, flowers are subsessile, interruptedly spicate or collected into heads. Calyxes are campanulate and divided nearly to the base into 4 (rarely 5) segments without pilose and four fid corolla. Female panicles are racemose and much shorter than the male, sepals are oblong and glabrous, petals are orbicular and shorter than the sepals, ovary and drupe are often pilose⁵.

Medicinal values of Cyclea peltata (H.F and T):

The medicinal property of Cyclea peltata has already been reported in many studies. It was reported that folklore people uses Cyclea peltata Lam in skin diseases, allergies, burns, cuts, wounds, inflammation, leprosy, leucoderma, scabies, smallpox and certain sexually transmitted diseases (STD) along with other medicinal plants⁶.The roots of Cyclea peltata have been proved for their inhibitory properties on nephrolithiasis induced by treating with 1% ethylene glycolated water by A.J.M.Christina, et al⁷.

The website of ROOTS (Research Organization for Oriental Traditions and Sciences) of India shows Cyclea peltata as one of the ingredients in an ayurvedic preparation called diabe drink to manage diabetes mellitus. A review article by S.A.Dahanukar et al, on pharmacology of medicinal plants suggests the CNS activity of Cyclea peltata⁸. This plant belongs to Menispermaceae family which is recognized for its medicinal properties and one of the most famous compounds isolated from this family is “Curare”. It is used as an arrow poison by South American Amazonian tribes of Brazil, Colombia, Ecuador and Peru⁹. This poison was first reported in 1548 during the exploration of Lake Marcaibo region in Venezuela by Alonso Pérez de Tolosa, historical evidence in Bisset’s article shows that Sir Walter Ralegh did not bring this poison to Europe during 1595 and it was Keymis in 1596, while exploring the Orinoco basin, came across the word ‘ourari’ Scientific studies and pharmacology of this poison “curare” was revealed during the 18^th century which ended up in isolating an alkaloid called tubocurarine which causes relaxation of skeletal muscles through blockade of the acetylcholine receptor on the motor endplates^10-11.

Mechanism of hepatic stellate cell activation in liver fibrosis:

In low-blood flow states, hypoxic injury occurs in the pericentral region of hepatic lobules, characterized by a dramatic rise in serum AST levels and subsequently HSC’s undergoes activation. This is mediated by reactive oxygen species, growth factors and inflammatory cytokines produced by hepatocytes, Kupffer cells, leukocytes, platelets and sinusoidal endothelial cells and result in a transition of quiescent vitamin-A-rich HSC’s into proliferative, fibrogenic, contractile myofibroblasts. These cells are devoid of vit-A and have dilated rough endoplasmic reticulum with a prominent golgi complex, which undergoes various phenotypic changes like proliferation, fibrogenesis, chemotaxis, contractility, retinoid loss, leukocyte chemo attraction and cytokine release, and matrix degradation. The activated myofibroblasts produce excess, abnormal matrix as well as matrix metalloproteinase (MMP) inhibitors and results in formation of fibrous tissues. Consequences of fibrosis include cellular signalling dysfunction and architectural and mechanical changes of liver, finally the activated stellate cell, during the resolution of liver injury, undergoes apoptosis (cell death) or reversal to a quiescent HSC^12-13.

In many experimental fibrotic models, CCl₄ and N-nitro dimethyl amine (NNDA) is used to generate liver fibrosis^14-15, likewise bile duct ligation technique was also used in many works to induce fibrosis¹⁶.

MATERIALS AND METHODS:

Animals:

Male albino Wister rats (150-200 g) were purchased from Chellamuthu trust, Madurai and approval from institutional ethical committee, K.M College of pharmacy, Madurai, was obtained prior to the commencement of this work. They were housed in 3 to 4 per cage, maintained at 25 ± 2^oC under a 12-h light and dark cycle and were fed with standard pellet diet with water “Ad libitum”. There was 6 rats in each group and categorised into 4 groups, as normal controls, extract controls, CCL4 treatment group, and CCL4 + extract treatment group.

Phytochemical isolation:

The plant Cyclea peltata was collected from the hills of Palode, Trivandrum, Kerala, India, with the help of natives and colleagues. Voucher specimen was examined and phytosanitary certificate was issued by Directorate of plant protection, Ministry of agriculture, Government of India. The roots was separated and dried in an oven with airflow at 40^o C and powdered. The dried ground material was extracted with non polar, semi polar and polar solvents namely hexane, chloroform, ethanol and 50% ethanol in glass distilled water using a continuous Soxhlet apparatus to exhaustion. Extracts were concentrated under reduced pressure using rotary evaporator (yield was17.5gms) and stored in freezer.

In-vivo induction of liver fibrosis by Carbon tetrachloride:

Liver fibrosis was induced by administering Carbon tetra chloride to rats through oral route (twice a week) for 28 days at the dose of 1 ml/kg body weight mixed with equal volumes of corn oil¹⁷. Three days after the last dose, rats were sacrificed under light ether anaesthesia and blood and liver samples were collected for further evaluation of biochemical parameters and histopathological studies respectively.

Treatment protocol with ethanolic extract of roots of C.peltata:

The ethanolic extract was diluted using distilled water and administered orally for treatment groups using gavages for 28 days, at a daily dose of 100 mg/kg body weight. The control group received equal amount of distilled water for 28 days. For comparison, a group of normal rats and another group treated with extract alone (extract controls) were used, the body weights of animals was monitored every day.

Table 1: Effect of ethanolic extracts C. peltata on the biochemical parameters of rats treated with CCl₄

Treatment	AST (IU/L)	ALT(IU/L)	ALP (IU/L)	Bilirubin (mg/dl)	platelet count (lakhs/cc)
Controls	191.67 ± 2.0	79.16 ± 2.2	577.3 ± 6.8	0.62± 0.04	3.5 ± 0.02
Extract controls	196.17± 7.2	257.3 ± 3.2	659.9 ± 13.8	0.87 ± 0.02	3.2 ± 0.08
CCL4 treatment	365.3 ± 13.3	395.3 ± 3.6*	1124.1 ± 13.8*	2.34 ± 0.12*	1.4 ± 0.12 *
extract treatment	221.8 ± 15.8*^a	91.83 ± 9.9*^a	819.2 ± 13.2*^a	1.64 ± 0.03*^a	2.7 ± 0.07*^a

Data are mean ± SEM. n=6, Newman Keul’s multiple test was used and measured with 95% significance level (P<0.05). (*) indicates that groups are significantly different from normal rats, (*a) indicates that groups are significantly different from CCL4 treated rats.

Table 2 : Hydroxyproline content of liver, body weight and liver weight following various treatments

Treatment	Hydroxy proline (µgm)	Body weight		Liver weight (gm)
Treatment	Hydroxy proline (µgm)	Day 0	Day 28	Liver weight (gm)
Normal controls	220 ± 11.7	160.1 ± 4.1	161.3 ± 1.44	4.4 ± 0.28
Extract controls	200 ± 5.8	160.16 ± 2.5	162.33± 1.89	4.2 ± 0.30
CCL₄ treated	601 ± 17.5*	163.3 ± 3.3	156.8 ± 1.4*	5.12 ± 0.36*
Extract treated	450 ± 6.1*^a	164.6 ± 4.5	162.5 ± 7.1*^a	4.17± 1.9*^a

Data are mean ± SEM. n=6, Newman Keul’s multiple test was used with 95% significance level (P<0.05). (*), indicates that groups are significantly different from normal rats, (*a), indicates that groups are significantly different from CCL4 treated rats.

Estimation of serum biochemical parameters:

After 28 days the rats were sacrificed under light ether anaesthesia and blood was collected by cardiac puncture. Serum was separated using centrifuge at a speed of 3000 rpm and conducted biochemical estimation. The levels of aspartate transaminase (AST), Alanine transaminase (ALT)¹⁸, alkaline phosphatase (ALP)¹⁹, total bilirubin levels and platelet count were estimated using standard procedures and the results are illustrated in Table 1.

Reitman and Frankel’s method for estimation of AST and ALT

Principle:

Aspartate transaminase catalyses the following reaction,

Keto-glutarate + L-Asparate‎‏ L-Glutamate + Oxalo Acetate

Oxalo acetate is coupled with 2,4 dinitrophenyl hydrazine (2,4-DNPH) to give the corresponding hydrazone, which gives brown color in alkaline medium and this is measured colorimetrically.

Alanine transaminase catalyses the following reaction

Keto glutarate + L-Alanine L-Glutamate + Pyruvate

Pyruvate so formed is coupled with 2, 4 dinitrophenyl hydrazine, which gives brown color in alkaline medium and this can be measured colorimetrically.

Kindkings method for estimation of alkaline phosphatase:

Alkaline phosphatase releases inorganic phosphate from many organic phosphor monomers having pH optimum at 9-10. Phenol is released by enzymatic hydrolysis from phenyl phosphate under defined conditions of time, temperature and pH which is estimated calorimetrically.

Procedure:

Mix 1M buffer solution with 1ml of phenyl phosphate substrate in a test tube and place in a water bath at 37 degree centigrade for 3 minutes. Add 0.1ml of serum, mix gently and incubate for exactly 15 minutes, stop the reaction by the addition of 0.8 ml of .05 N NaOH.

Estimation of Bilirubin:

Principle:

Bilirubin is diazotized only in the presence of dissolving solvent (methanol), which produces red-purple coloured azo-bilirubin. The intensity of red purple colour so developed above is measured calorimetrically and it is proportional to the concentration of the appropriate fraction of bilirubin. This reaction can be represented as

Bilirubin + Diazotized sulfanilic acid Azo-bilirubin (red purple color)

Determination of hydroxyproline content in liver:

The hydroxyproline content of liver was determined by the method followed by Jamall et al²⁰.The specimens of liver were weighed and hydrolysed completely in 6M HCL. A fraction of the sample was treated using Chloramine T solution and Erhlich’s reagent and optical density was measured at 558 nm and the results are shown in Table 2.

Histopathological studies:

Liver samples were weighed and fixed rapidly with 10% neutralized formalin at a PH 7.4. Sections of liver were fixed in paraffin and stained using haematoxylin along with eosin for pathological changes. The architecture of liver was completely intact in normal rats. In CCl₄ treated rats the liver specimens showed parenchyma with sheets of hepatocytes showing hydropic and fatty changes and areas of mild necrosis. These pathological changes were much reduced in liver specimens of rats treated with extract. The rats treated with the extract alone did not show deviation in the architecture of liver. The result of histopathological slides is given in Figure: 2.

Statistical Analysis:

The values are expressed as Mean ± SEM and the analysis of data was done using one-way ANOVA and the significance was calculated as per Newman Keul’s multiple range tests. The P < 0.05 was considered significant and ruled out any differences due to chance.

RESULTS AND DISCUSSIONS:

Serum parameters:

The treatment with CCl₄ has altered serum biochemical parameters as well as the architecture of liver. In the fibrotic control group, the serum parameters AST, ALT, and ALP levels as well as bilirubin levels were significantly elevated. However in the treatment group with ethanolic extract of C.peltata the serum levels of these enzymes and bilirubin was significantly lower when

compared to fibrotic control but higher than the normal control groups. There was a reduction in platelet count in fibrotic group but following treatment with the extract the platelet count increased significantly. The figures are represented in Table 1.

Tissue parameters:

The main tissue parameter assessed was hydroxyproline content which was elevated following CCl₄treatment. Treatment with the extract shows significant reduction of this parameter. Liver weight was increased in CCl₄ treated rats which was reduced in the extract treated group and the figures are represented in Table 2.

Fig 3, photographs of liver with fibrosis and fibrotic liver treated with ethanolic extracts of c.peltata.

Liver, the largest internal organ is composed of 6 major cell types viz. hepatocytes, bile duct epithelial cells, Kupffer cells, hepatic stellate cells, sinusoidal epithelium and pit cells²¹. These hepatocytes and bile duct epithelial cells when chronically damaged, gets activated to form fibrous tissues which is due to the synthesis and secretion of collagens from hepatic stellate cells which normally acts as stores of Vitamin A, However when injured repeatedly, they get activated to produce fibrotic neomatrix. Such activated cells lose their Vitamin A content and triggers the activation of extra cellular matrixes including collagens type I and type II, which are fibrillar collagens^22-23. Thus a raised level of collagen content in the extra cellular matrix in fibrosis and the extent of which could be assessed by the hydroxyproline content. Many earlier studies have reported high levels of hydroxyproline content in association with liver fibrosis, which is observed in this study as well, which was reduced by co-treatment with the plant extract.

In our study, CCl₄ is the toxicant that was used to damage liver, which is converted to trichloromethyl radical by the enzyme cytochrome P450, which initiates lipid peroxiadation and liver damage reflected by high levels of serum AST, ALT, ALP and bilirubin (Table: 1).The rats treated with ethanolic extracts of C.Peltata showed significant reduction of AST, ALT, ALP and bilirubin.

A study by Khokhar N et al (2003) reported that the ratio of AST/ALT greater than 1 in combination with a platelet count of less that 150,000 could predict advanced stage of fibrosis²⁴. In our study the ratio of AST/ALT in CCl₄ treated rats was greater than 1 with the platelet count less than 150,000 in CCl₄ treated rats confirming the advanced progression of fibrosis. Both of these parameters were corrected towards normal in C.peltata treated rats.

Hepatomegaly has been reported as a symptom of cirrhosis. As cirrhosis has been defined as the end stage consequence of fibrosis²⁵, this symptom can be the reason for increase in liver weight of CCl₄ treated rats. In the treatment group the weight of liver was reduced and the liver/body weight ratio was also normal, moreover the architecture of the liver was less deranged in the extract treated rats as shown in Figure 2. These observations suggest that the ethanolic extract of C.Peltata is effective against CCl₄ induced liver fibrosis in rats.

CONCLUSION:

This study proves that, ethanolic extract of Cyclea peltata which was first identified by Hooker.F and Thomson, as described in flora of Bombay, shows a significant property of reverting the fibrous tissues (Anti-fibrosis) formed in liver cells due to the repeated injury with CCL₄.There are evidences for alkaloids present in this plant and has to be subjected for further fractionation and structural elucidation. The plant itself or compounds if isolated can be a breakthrough for the future researches on hepatoprotection.

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Received on 31.01.2009 Modified on 28.02.2009

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 201-205