Chemical and biological characterization of hepatoprotective efficacy of leaf extracts isolated from Murraya koenigii and Phyllanthus niruri using in vivo and in vitro model systems

Name: Pallavi Shah Id No.: 34087 Year of admission: 2 nd semester 2008-09 Degree: PhD Major: Molecular Biology and Biotechnology Deptt. : M.B. G.E. Advisor: Dr. S.P. Singh Thesis title: Chemical and biological characterization of hepatoprotective efficacy of leaf extracts isolated from Murraya koenigii and Phyllanthus niruri using in vivo and in vitro model systems The present study was undertaken to determine the hepatoprotective efficacy of Murraya koenigii and Phyllanthus niruri hydroethanolic leaf extracts against paracetamol and alcohol induced damage in both in vivo rat and in vitro HepG2 cell line models. Different leaf extracts were prepared and their phytochemical analysis was carried out to determine the active principles present in them. The AWE (Alcohol Water Extract), WE (Water Extract) and AE (Alcoholic Extract) of P. niruri possessed higher content of total phenolics in comparison to that of M. koenigii with AWEs possessing highest content of phenolics for both the plants. The AWEs had the highest total flavonoid content as well i.e. 61.8 and 65 mgCatechin equivalents/g of leaf extract for M. koenigii and P. niruri, respectively. Examination of antioxidant activity by DPPH free radical scavenging assay showed P. niruri extracts had higher antioxidant potential than M. koenigii extracts with AWEs for both the plants to possess the highest scavenging potential. Tannic acid (TA), a known antioxidant was quantified using HPLC in M. koenigii extracts with AE showing the highest content of TA (288ppm). HPLC quantification of phyllanthin, a lignan known for its hepatoprotective potential was done for all P. niruri extracts with WE showing the highest content (223ppm). The GC MS analysis of plant extracts further detected the presence 40 compounds in AWE of M. koenigii and 41 compounds in AWE of P. nruri. Oral administration of alcohol (5mL/kg) for one week followed by paracetamol (1g/kg) for three days produced liver damage in rats as manifested by the significant (P<0.01) rise in serum levels of glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), serum phosphatase (ALP), gamma glutamyl transferase (GGT), total bilirubin and direct bilirubin as compared with respective vehicle control. Post-treatment of rats with the AWEs of Murraya koenigii and Phyllanthus niruri alone (500mg/kg) and in combination (250mg/kg of each) and silymarin for one week significantly (P<0.01) reduced SGOT, SGPT, ALP, GGT, total bilirubin and direct bilirubin levels equivalent to normal. The combination of extracts showed maximum potential in reversal of damage caused by PCM and ethanol. Treatment of rats with alcohol and paracetamol significantly (<0.001) led to increase in lipid peroxidation as measured by malondialdehyde (MDA) in both liver and kidney. This was associated with a significant (<0.001) decrease in reduced glutathione (GSH) and superoxide dismutase (SOD) activity. Biochemical alterations following alcohol and paracetamol administration were ameliorated by post-treatment with M. koenigii and P. niruri leaf extracts. These studies were further supported by histopathological examination of liver and kidney samples. In vitro studies in HepG2 cell line utilized MTT based cytotoxicity assay to determine the doses of toxicants (PCM and ethanol), AWEs of M. koenigii and P. niruri and silymarin to be used for challenging the cells. The cells were subjected to both post treatment aswell as preconditioning with extracts and their effect on leakage of SGOT and SGPT in cell media was assessed. The preconditioning with extracts (in combination) for 24 hrs whereas post treatment for 12 hrs provided maximum protection against increase in SGOT and SGPT levels induced by PCM and ethanol. Intracellular GSH and MDA levels were determined at different time intervals for both preconditioned and post treatment samples. 24 hours of preincubation and 24 hours of post treatment with extracts was able to restore the increased MDA levels and diminished GSH levels to normalcy. Analysis of apoptosis and necrosis revealed that necrosis is the major mode of toxicity in PCM and ethanol induced damage. Gene expression analysis of phase I and phase II metabolizing enzymes revealed that preconditioning influenced CYP1A2 (Cytochrome p4501A2) and GGT (Gamma glutamyl transferase) expression but could not influence the GST (Gltathione S transferase) expression at later stages. Preconditioning thus influenced the initial steps of PCM metabolism where these initially play a significant role. Post conditioning with extracts showed high expression of CYP1A2 gene whereas it could not suppress GST expression showing no interference with initial metabolism of PCM. The GGT expression was suppressed in post treatment samples showing their antioxidant nature. uke % iYyoh “kkg ifjp;ka d la0 % 34087 l= ,oa izos ”k o’kZ % f}rh;] 2008&09 mikf/k % ih0,p0Mh0 eq[; fo’k; % vk.kfod thou foKku foHkkx % vk.kfod tho foKku ,oa tSo izkS|ksfxdh ,oa tSo izkS|ksfxdh xkS.k fo’k; % lw{etho foKku lykgdkj % MkW0 ,l0ih0flag 'kks /k 'kh"kZd % ^^In vivo rFkk In vitro :ikRed ra= ds iz;ksx }kjk Murraya koeingii rFkk Phyllanthus niruri i=lkjks a dh ;dr̀ j{kkRed {kerk dk jlk;fud rFkk tSfod fof'k"Vhdj.k **