Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (Central University), Bilaspur (C. G.) 495009, India. Email: Original Article EFFECT OF PIPERINE IN COMBINATION WITH 4-HYDROXYISOLEUCINE ENRICHED TRIGONELLA FOENUM-GRAECUM EXTRACT AND SILYMARIN ON PARACETAMOL INDUCED LIVER DAMAGE IN RATS GARIMA JAIN, VINOD D. RANGARI garimasemail@gmail.com Received: 24 Sep 2014 Revised and Accepted: 25 Oct 2014 ABSTRACT Objective: The present study was undertaken to evaluate the hepatoprotective activity of 4-hydroxyisoleucine enriched extract of Trigonella foenum-graecum and silymarin in the presence of piperine, against experimentally induced liver injury. Methods: Wistar albino rats (150-180g) of either sex (n=6) were employed in present study. Experimental hepatotoxicity was induced by the administration of paracetamol (3 g/kg, p. o.). All the test drugs were administered orally in the form of suspension to the animals. The combination of silymarin and piperine, 4-hydroxyisoleucine (28%) enriched T. foenum graecum extract and piperine were given to animals to compare with individual group as well as with toxicant. Results: Administration of paracetamol caused significant increase in of SGOT, AST, ALP, bilirubin and total proteins characterizing experimental hepatotoxicity. 4-Hydroxyisoleucine enriched extract of T. foenum-graecum and silymarin in the presence of piperine, significantly attenuated the toxic effects of hepatotoxicants in liver. Histological study of liver was also carried out to estimate the extent of tissue injury. Conclusion: The combination of piperine and silymarin has shown significant hepatoprotective activity against the injury induced by paracetamol. This was evident from significant reduction in serum enzyme AST, ALT, ALP and total bilirubin. These biochemical observations were also supplemented by histopathological studies of the liver. Keywords: Trigonella foenum-graecum, Hepatoprotective, Silymarin, 4-hydroxyisoleucin, Paracetamol. INTRODUCTION In daily routine, liver continuously come in contact of xenobiotics, hepatotoxins and chemotherapeutic agents that lead to impairment of its functions by induction of lipid peroxidation and other oxidative damages. Toxic chemicals, excess consumption of alcohol, infections and autoimmune disorders etc. may cause liver diseases. Hepatotoxicity is one of the major causes that could give rise to severe metabolic disorders and even mortality [1]. Medicinal plants possess myriads of secondary metabolites that can protect the liver from such types of hepatotoxins. Silymarin is a standardized extract obtained from the seeds of Silybum marianum (L), family Asteraceae (Milk Thistle) which is largely used as a hepato protective agent against poisoning from chemical and environmental toxins. It can be used as a standard natural hepato protective agent for the hepato protective activity studies. 4-Hydroxyisoleucine (4-OH Ile) is a natural nonproteinogenic amino acid with absolute configuration as (2S, 3R, 4S)[2], [3], present in Trigonella foenum-graecum Linn., family Fabaceae (Fenugreek) seeds. It has been reported to be responsible for the insulinotropic activity of fenugreek seeds. It increases glucose-induced release of insulin which is strictly dependent on the glucose concentration [4]. This unique property helps in avoiding undesirable side effects such as hypoglycemia in the therapy of type II diabetes. Thus, 4-OH Ile seems a promising dietary supplement in the treatment and prevention of chronic diseases [5]. Piperine, an alkaloid present in the dried unripe fruits of Piper nigrum Linn, Family Piperaceae, has been well established bioenhancer if used in combination with other drugs. It reduces the drug dose, danger of drug resistance and toxicity of drugs. Piperine has been reported to bring about its bioenhancement effect by different mechanism including, DNA receptor binding [6], modulation of cell signal transduction [7], or by inhibition of drug efflux pump [8]. The major objective of this paper is to explore the bioenhancement effect of piperine on the hepatoprotective activity of 4-hydroxyisoleucine enriched fraction of fenugreek extract, in the experimental animals. MATERIALS AND METHODS Isolation and purification of TF4H (28%) Sugaheal ® Dried mature seeds of Fenugreek, Trigonella foenum graecum, family Fagaceae, were first subjected to screening for the presence of total amino acids and trigonelline using thin layer chromatography on pre-coated silica gel TLC plates using n-butanol: acetic acid: water in a ratio of 12:8:2 and initial scanning using UV at 254 nm for the presence of trigonelline. Ninhydrin reagent was used for colour development of total amino acids. Dried mature Fenugreek seeds in a quantity of 1 Kg were flaked in a flaker to expose the inner core, resulting in flakes of average 15 mm in size. The flakes were then subjected to hydro-alcohol extraction using 6 litres of isopropyl alcohol: water mixture in a ratio of 50:50 at 35 O C for 12 hours. The resultant liquid (about 5500 ml) was concentrated to a final volume of 150 ml under vacuum at 45-50 O C. This liquid was extracted with 3x50 ml of n-hexane to remove fats and lipids. The defatted concentrate was diluted with de-mineralized water to a final volume of 500 ml. This liquid was subjected to fine filtration through 200- mesh size to remove insoluble. The filtered liquid was then passed through a glass column of 500 mm length x 25 mm diameter containing strong acid cation exchange resin in H + The resultant solution was then passed through a glass column of 800 mm length x 25 mm diameter containing 200 ml of freshly regenerated weak acid cation resin in gel form. The eluent from this column was a colourless, neutral liquid having only compounds such as amino acids and trigonelline present in the ratio as in the mother seed. The product was spray dried with the conditions of co-current air flow, inlet temperature, 165 form freshly regenerated with 600 ml of 3% HCl in water, followed by washing to neutral pH. After passing the liquid, the column was washed with de-mineralized water to neutral pH. The loaded amino acid and trigonelline were eluted with 200 ml of 0.5 (N) ammonia solution. The ammonia liquid was circulated in the column until it attained a stable pH of 8.0. O C, outlet temperature 85 O C with automizer revolutions of 30,000 rpm. The resultant granules from International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 7, Issue 2, 2015 Innovare Academic Sciences