International Journal of Pharmaceutical Sciences

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1 International Journal of Pharmaceutical Sciences INT.J.PH.SCI.,JAN-APRIL 21;2(1): ISSN Original Research Manuscript IN-VITRO HEPATOPROTECTIVE ACTIVITY OF MORINGA OLEIFERA LAM. LEAVE ON ISOLATED RAT HEPATOCYTES. Patel Rameshvar K. *, Manish M. Patel, Nilesh R. Kanzariya, Kirit R. Vaghela, Dr. R.K. Patel, Dr. N. J. Patel Department of Pharmacology, Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva, North Gujarat, India *Corresponding Author: Phone and Fax No.: , ID: ABSTRACT Study was conducted to test the hypothesis that Moringa oleifera Lam. Leave has hepatoprotective activity. For this purpose study was designed using different concentration of crude aqueous and ethanol (alcoholic) extract of Moringa oleifera Lam. leaves to investigate protection against CCl 4 (1 mm) induced hepatocytes injury of rats in vitro and compared with standard silymarin concentrations of.1,.5 and.1 mg/ml. Results shows that extract was effective in the reducing CCl 4 induced enhanced activities of GPT, GOT, lipid peroxidation and % viability. As per results obtained from the study it is evident that low concentration of alcoholic extract has significant hepatoprotective activity at even.1 mg/l while that of aqueous extract was found at.1 mg/l and higher concentration of the drug. This shows hepatocellular damage caused by CCl 4 and its recovery by pretreatment with the crude extract of leaves, suggest that it might be considered as a potential source of natural hepatoprotective agent, which could be related to the free radical scavenging properties of various components present in varying concentration in the extract which is evident from the free radical measurement. Key Words: Carbon tetra chloride, Moringa oleifera., Hepatoprotective activity, Silymarin. INTRODUCTION Herbs play a major role in the management of various liver disorders along with other system associated diseases. Liver is a key organ which regulating homeostasis within the body by various functions and no one reliable liver protective drugs in allopathic medical practices is there. Liver injury caused by toxic chemicals and certain drugs has been recognized as a toxicological problem. Hepatotoxicity is one of very common aliment resulting into serious debilities ranging from severe metabolic disorders to even mortality. Various plants have been 457 reported to possess hepatoprotective property. Some well known plants include Picrorrhiza kurroa, [1] Andrographis paniculate, [2] Eclipta alba, [3] Phyllanthus emblica, [4] etc. because of their hepatoprotective plant constituents are identified and tested on various animal models. Epidemiological survey indicates that occurrence of hepatic and kidney disorders are increasing as life style changes, causing serious problems in the area of public health. The British Liver Trust (BLT) estimates that the disease could kill more than 6, people in the next decade. It is estimated that there are

2 already up to 4, people in the UK who are infected with the hepatitis C virus. It is about 1 times more infectious than HIV. Hepatotoxicity in most cases is due to free radical. Free radicals generated by the metabolism of toxicants initiate the toxicity cascade, [5]. The endogenous free radical scavengers like reduced glutathione, vitamin C, vitamin E, catalase and super oxide dismutase are present in the body. The uninhibited free radicals then initiate cell necrosis and leads hepatotoxicity, [6]. Investigated new drugs have very little to offer for alleviation of these disorders. Free radicals have been implicated to a major extent in pathology of liver disorders. Oxidative damage through free radical generation is one of the mechanisms involved in the hepatotoxic effect of carbon tetrachloride and paracetamol, [7]. Animal models involving the use of antioxidant drugs are useful to study oxidative damage and protection. A plant or test drug having anti-oxidant property proved to be hepatoprotective. The whole Moringa oleifera plant is used in the treatment of psychosis, eye diseases, fever and as an aphrodisiac, [8] the aqueous extracts of roots and barks were found to be effective in preventing implantation, [9] aqueous extracts of fruits have shown significant anti-inflammatory activity, methanolic extracts of leaves have shown anti-ulcer activity, and ethanolic extracts of seeds exhibited anti-tumour activity, [1]. MATERIALS & METHODS PLANT MATERIAL COLLECTION, ISOLATION AND AUTHENTIFICATION: Whole plant of Moringa oleifera was collected from the Botanical garden of Gandhinagar, Sector- 3, Gujarat, India. Authentification of plant Moringa oleifera was done by Dr. Shri Ram, Department of medicinal and aromatic plants, B.A.C.A., Anand agriculture university, Anand. According to method of Alkofahi et al. 1997, [11], aqueous and alcoholic extracts of Moringa oleifera leave was prepared. Lieberman buchard test (Steroids), Dragendorff test, Wagner test (Alkaloids), FeCl 3 test (Tannins) and Shinoda test (Flavanoids) was done for Preliminary Phytochemical screening (Table 2). Alcoholic (A/L) and aqueous (AA) drug extract of various concentration like 1 mg/ml,.1 mg/ml and.1 mg/ml of was prepared in PBS (Phosphate buffer saline) at 7.4 PH. As standard solutins.1 mg/ml,.1 mg/ml and.5 mg/ml concentration of standard Silymarin powder in PBS was made. For preparation of material to induce hepatotoxicity.25 ml of carbon tetrachloride (A.R.) was diluted to 257 ml with PBS (phosphate buffer saline), PH 7.4 was adjusted. ANIMALS: Male Wistar rats (2-25 g) were procured from Central Animal Facility, SKPCPER. They were maintained in controlled temperature ( C), 12 h light/dark cycle as well as humidity (3 7%) and standard diet & water was provided ad libitum. The care and the use of these animals were in accordance with the guidelines of the committee for the purpose of control and supervision on experiments on animals (CPCSEA). Experimental protocols were approved by Institutional Animal Ethic Committee (IAEC). Fresh hepatocytes were isolated from freshly sacrificed normal rat liver. The isolated hepatocytes were devided into five groups. Group 1 received only vehicle and group 2 received only toxin without drug. In other remaining groups three drug concentrations were used. The pre-incubated cells except group 1 were exposed to the mixture of 1 mm carbon tetrachloride and different doses of drug solution in PBS were exposed except group 1 and 2. Both aqueous and alcoholic extracts solution at different dose range were treated in the same manner. For group 3 aqueous extract of leave, group 4 alcoholic extract of leave were used for drug treatment. As standard drug Standard.1 mg/ml,.5 mg/ml and.1 mg/ml silymarin solution were used in group number 5. The tubes were incubated for 1 hr at 37 C. After incubation for 1 hour the cell suspension was centrifuged at low speed (5 gm) and the supernatant solution was used for the estimation of cytosolic enzyme like GOT, GTP. The pellet obtained after centrifugation was resuspended in the HBSS (1ml), & PH adjusted 7.4 and used for Trypan blue exclusion assay. IN VITRO ANTIOXIDANT AND HEPATOPROTECTIVE STUDY: 458

3 Isolation of rat hepatocytes: The method proposed by Seglen, 1976, [12] was employed for the isolation of liver cells. Hank s balanced salt solution (NaCl 8. g/l, KCl.4 g/l, MgSo 4.7H 2 O.2 g/l, KH2PO4.6 g/l and glucose 1 g/l), PH 7.4 was used for perfusion transport and washing of cells. Fresh hepatocyte preparations and primary cultured hepatocytes were used to study direct antihepatotoxic activity of drugs. Hepatocytes were treated with hepatotoxin and the effect of the plant drug on the same was evaluated. The activities of the transaminases released into the medium were determine. In the present study Isolation of rat hepatocytes, Measuremnt of cell viability and to study the effect of test drug on CCl 4 induced cytotoxicity were taken up. on lipid peroxidation: Ferrous sulphate induced lipid peroxidation scavenging was assayed by estimating the thiobarbituric acid-reactive substances (TBARS) by using the standard method with minor modifications, [15]. Antioxidant study: Method proposed by Molyneux et al 24 was used for Free radical scavenging activity. Statistical method: From the results obtained by practical statistical analysis was done by using Graph pad prism 4, while graphical representation was prepared by using Microsoft office excel 23. Each bar represents is presented as mean±sem. Unpaired t-test was applied for statistical analysis with significant different of P<.5. Trypan blue exclusion assay: According to Baliga et al., 1996, [13] trypan blue exclusion assay was used for measurement of the cell viability. Determination of GOT/GTP level: The hepatocyte suspension after drug treatment was centrifuged at 5 rpm for 1 min and the supernant obtained was used for estimation of GOT and GTP levels, [14]. Evaluation of inhibitory effect of various test extracts RESULTS AND DISCUSSION: Percentage yield of different extracts: In case of alcoholic and aqueous extract of leave greenish brown color was obtained with yield of 6.26% and 21% respectively (Table 1). Phytochemical investigations are shown in Table 2, which shows similar results except flavanoid content, present in petroleum ether extract. Table-1: Physical characteristics and percentage yield of different extracts Sr. No. Name of Plant Extract type Colour Odour % Extractive (w/w) on dry wt basis 1. Moringa Alcoholic Greenish Brown Characteristic Moringa Aqueous Greenish Brown Characteristic 21 Table-2: Analysis of plant extracts for the presence of secondary cell constituents Sr. No. Chemical test Alc. Ext of Moringa Aq. Ext of Moringa 1. Test for steroid Test for Carbohydrates Test for Alkaloids Test for tannins Test for Flavonoids Test for saponins Test for proteins Test for Resins

4 IN VITRO ANTIOXIDANT AND HEPATOPROTECTIVE STUDY: Trypan blue exclusion assay: Around cells/ liver with over 78-8% viability was obtained in our study. The viability of hepatocytes was estimated by Baliga et al., 1996 procedure. Brief exposure of hepatocytes with CCl 4 (1 mm) resulted in to significant reduction in the hepatocyte viability (Graph-4.1). This suggests significant toxic effect of CCl 4 in control. From Graph- 4.1 we can conclude that untreated normal group show average % viability 81.92% while CCl 4 treated control show only 53.2 % viability. Alcoholic extracts of Moringa oleifera leave show good activity at even conc..1 mg/ml which shows potential hepatoprotective activity about 76 % in 1 mg/ml concentration of alcoholic extract. Aqueous extracts of leave show good protection but at high concentration of 1 mg/ml (62.59 %) but at low concentration not significant protection was found. If we compare extract of leave and silymarin we found that silymarin show maximum protection than other treatment at very low concentration. Trypan Blue assay % Viability # Normal Sil.1 Sil.5 Sil.1 AL.1 AL.1 AL 1 AA.1 AA.1 AA 1 ** ** * Conc (mg/ml) Figure 1: Effect of Alcoholic and Aqueous extracts of Moringa oleifera leaf and Silymarin on % viability of hepatocytes. {Each bar represents mean±sem, n=6, Unpaired t-test was applied. * P<.5; ** P<.1; P<.1: Significant different from. # P<.1 significant difference with normal group.} Determination of GOT and GTP level: As shown in graph-4.2 GPT levels are significantly less in alcoholic extracts of leave. For 1 mg/ml concentration of alcoholic extract % protection as compare to untreated control group was found. This clearly suggests that significant protection is offered by alcoholic extracts of leave. In a similar study conducted with aqueous extracts of plant, it was found that the effect on viability is very minimal as compared to alcoholic extracts of leave. Aqueous extracts of leave shows % at concentration of 1 mg/ml. So the effect on GPT release by the aqueous extracts of leave is not as prominent as that observed with alcoholic extracts of leave. Only at concentration of 1 mg/ml of aqueous extracts, the effect GPT levels decrease significantly; suggest weak hepatoprotective activity of aqueous extracts as compared to alcoholic extracts. For GOT graph 4.3 shows that at concentration of.1 mg/ml silymarin shows protection up to 67.34%. If we compare the alcoholic extract of Moringa oleifera leave show 65.7 % protection at 1 mg/ml conc. So, it is clear that alcoholic extracts of leave show prominent hepatoprotective activity as compare to standard but at higher concentration. Aqueous extracts of leave do not show any significant activity at low concentration, but at conc. of 1 mg/ml show % protection. 46

5 GPT %GPT release inhibition Normal # ** Sil.1 Sil.5 Sil.1 * ** AL.1 AL.1 AL 1 AA.1 AA.1 AA 1 Conc (mg/ml) Figure 2: Effect of alcoholic and aqueous extract of Moringa. oleifera leaf and Silymarin on CCl 4 induced release of GPT from isolated rat hepatocytes. {Each bar represents mean±sem, n=6, Unpaired t-test was applied. * P<.5; ** P<.1; P<.1: Significant different from. # P<.1 Significant difference with normal group.} GOT %GOT release inhibition Normal # Sil.1 Sil.5 Sil.1 AL.1 AL.1 AL 1 * AA.1 AA.1 AA 1 Conc (mg/ml) Figure 3: Effect of alcoholic and aqueous extracts of Moringa oleifera leaf and Silymarin on CCl 4 induced release of GOT from isolated rat hepatocytes. {Each bar represents mean±sem, n=6, Unpaired t-test was applied. * P<.5; ** P<.1; P<.1: Significant different from. # P<.1 Significant difference with normal group} Antioxidant study: In our study the alcoholic extracts of plant exhibited significant and dose dependant scavenging potency (Graph-4.4). Initial absorbance of DPPH was adjusted to.9. Ascorbic acid was used as standard and it gives protection up to 82.78% at 1.5 mg/ml. Alcoholic extracts of leave show protection up to 8.55 % at 1.5 mg/ml while aqueous extracts show protection up to %. So in leave extracts alcoholic extracts show more free radical scavenging activity but slight less in compair to ascorbic acid. In the study measurement of the effect of drug extracts on 461 malondialdehyde formation via Lipid peroxidation when liver homogenate was exposed to toxicant like FeSO 4. Thus the elevated levels of malondialdehyde are an indication of lipid peroxidation. In our study FeSO 4 exposure to the liver resulted in increased malondialdehyde levels. Pretreatment with alcoholic extracts of plant resulted into significantly lesser malondialdehyde levels than in untreated liver homogenate. Standard silymarin shows significant protection about 37.8% at conc. of.1 mg/ml. Alcoholic extract of leave show % protection for lipid peroxidation. Pretreatment with aqueous

6 extract did not produce significant alterations in malondialdehyde levels as compared to pretreatment with alcoholic extracts as per results presented in graph-4.5. In this study alcoholic extract of leave shows significant hepatoprotective activity by inhibiting the lipidperoxidation induced by FeSO 4. Free radical scavanging of Leaf Asc.1 Asc.5 Asc.1 Asc.5 Asc.1 Asc.15 AL.1 AL.5 AL.15 AL.2 AL.5 AL 1.5 AA.5 AA.1 AA.2 AA.5 % Scavenging AA 2.5 AA 3 Conc. mg/ml Figure 4: Effect of alcoholic and aqueous extract of the Moringa oleifera leaf on free radical scavenging activity using ascorbic acid as a reference standard. {Each bar represents mean±sem, n=6} Figure 5: Effect of Alcoholic and Aqueous Extracts of Moringa oleifera and Silymarin on lipid peroxidative potential using rat liver homogenate. {Each bar represents mean±sem, n=6, Unpaired t-test was applied. * P<.5; ** P<.1; P<.1: Significant different from } Conclusion: Thus it can be concluded that alcoholic extracts of Moringa oleifera produce significant hepatoprotective and antioxidant activity. Aqueous extracts of Leave have much lesser hepatoprotective and antioxidant activity than the alcoholic extract. However direct studies using more fractioned extract could provide better evidence for this claim. 462 REFERENCES 1. Anssari RA, Aswal SS, Chander R, Dhawan BN, Gark NK. Hepatoprotective activity of Kutkin the iridold glycoside mixture of Picrorhiza Kurroa, Indian J Med Res. 1998; 87: Handa SS. Hepatoprotective plants and plant products areport, Indian drugs 1993; 3:

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