The aim of this study is to determine the antilipase and antioxidant activity of Phyllanthus niruri methanolic extract. The inhibitory activity against porcine pancreatic lipase (PPL) was determined by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at 405 nm. The crude extract exhibited strong lipase inhibitory activity with an IC50 value of 27. 65 µg/ mL. The inhibition mode study disclosed that P. niruri could act as noncompetitive inhibitor. Antioxidant activity of P. niruri extract was measured by 2, 2, diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assay. P. niruri showed high antioxidant activity with an EC50 value of 7.471 µg/mL. The results suggested that P. niruri has shown potential as a source of natural antilipase and antioxidant

Ahmad Tajudin, Asilah

Alias, Norsyuhada

Leow, Adam Thean Chor

Mohamad Ali, Mohd Shukuri

Raja Abdul Rahman, Raja Noor Zaliha

Salleh, Abu Bakar

Raja Abd. Rahman, Raja Noor Zaliha

Thean, Chor Leow

Mohamad Ali, Mohd. Shukuri

The International Islamic University Malaysia Repository

Australian Journal of Basic and Applied Sciences, 9(7) April 2015, Pages: 133-136   ISSN:1991-8178  Australian Journal of Basic and Applied Sciences    Journal home page: www.ajbasweb.com     Corresponding Author: Raja Noor Zaliha Raja Abd. Rahman, Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang UPM 43400, Selangor, Malaysia. Antilipase and Antioxidant Activity of Phyllanthus niruri Methanolic Extract  Raja Noor Zaliha Raja Abd. Rahman, Norsyuhada Alias, Adam Thean Chor Leow, Mohd. Shukuri Mohamad Ali, Asilah Ahmad Tajudin and Abu Bakar Salleh  Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang UPM 43400, Selangor, Malaysia  A R T I C L E  I N F O   A B S T R A C T  Article history: Received 12 October 2014  Received in revised form 26 December 2014  Accepted 17 January 2015  Available online 28 February 2015  Keywords: Phyllanthus niruri, antilipase, antioxidant, DPPH   The aim of this study is to determine the antilipase and antioxidant activity of Phyllanthus niruri methanolic extract. The inhibitory activity against porcine pancreatic lipase (PPL) was determined by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at 405 nm. The crude extract exhibited strong lipase inhibitory activity with an IC50 value of 27. 65 µg/ mL. The inhibition mode study disclosed that P. niruri could act as noncompetitive inhibitor. Antioxidant activity of P. niruri extract was measured by 2, 2, diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assay. P. niruri showed high antioxidant activity with an EC50 value of 7.471 µg/mL. The results suggested that P. niruri has shown potential as a source of natural antilipase and antioxidant.   © 2015 AENSI Publisher All rights reserved. To Cite This Article: Raja Noor Zaliha Raja Abd. Rahman, Norsyuhada Alias, Adam Thean Chor Leow, Mohd. Shukuri Mohamad Ali, Asilah Ahmad Tajudin and Abu Bakar Salleh., Antilipase and Antioxidant Activity of Phyllanthus niruri Methanolic Extract. Aust. J. Basic & Appl. Sci., 9(7): 133-136, 2015  INTRODUCTION  Natural products provide a vast collection of pancreatic lipase inhibitors with potential for being developed into clinical products to treat obesity. Birari and Bhutani (Birari, R.B., K.K. Bhutani, 2007) reviewed a variety of plant extracts and secondary metabolites that have inhibitory activity against pancreatic lipase. The main phytochemicals contributed towards pancreatic lipase inhibition consisted of polyphenols, flavonoids, saponins and caffeine (Kim, H.Y., M.H. Kang, 2005; Han, L., et al., 2006; Moreno, D.A., et al., 2006; Shimoda, H., et al., 2006). On the other hand, consumption of edible vegetables with high antioxidant content is essential in combating degenerative diseases, such as coronary artery diseases, stroke, rheumatoid arthritis, diabetes and cancer  (Harish, R., T. Shivanandappa, 2006). There is a great deal of interest in edible plants that contain antioxidants and health – promoting phytochemicals.  In this study, we aim to analyze the antilipase and antioxidant property of Phyllanthus niruri (family: Euphorbiaceae), a perennial herb distributed in tropical area. Previous studies reported that the whole plant, fresh leaves and fruits are used to treat various ailments, including hepatitis (Harish, R., T. Shivanandappa, 2006), antitumor and anticarcinogenic activities (Rajeshkumar, N.V., et al., 2002). Other medicinal properties, such as hypolipidaemic (Khanna, A.K., et al., 2002) and antiviral (Liu, S., et al., 2004) activities of P. niruri have also been shown.   MATERIALS AND METHODS  Preparation of Plant Extracts: The whole plant material was air-dried and ground into fine powder. Hundred g of the powdered material was extracted by maceration in 1 L of 80 % methanol for 3 days at ambient temperature (25 – 30 ⁰C). The extract was concentrated in a rotary vacuum evaporator at 45 ⁰C and kept in a 4 ⁰C chiller for long-term storage.   Lipase Preparation: Porcine pancreatic lipase (PPL) was used as a model enzyme because of its high homology to human pancreatic lipase (85 % identity) and similar enzyme kinetics and behaviour (Lowe, M.E., et al., 1989). Crude lipase was dissolved in 50mM phosphate buffer pH 6 (1 mg/ml) and centrifuged at 12, 000 x g for 5 min to remove insoluble (Lewis, R., D. Direct, 2012; Lehner, R., R. Verger, 1997). Enzyme concentration was set at 0.1 mg/ ml. PPL, pNPB and crude extracts were freshly prepared prior to enzyme assay.   Lipase Inhibition Assay: 134                                                         Raja Noor Zaliha Raja Abd. Rahman et al, 2015 Australian Journal of Basic and Applied Sciences, 9(7) April 2015, Pages: 133-136 The inhibition towards porcine pancreatic lipase was performed according to the modified method by Lewis and Liu (Lewis, R., D. Direct, 2012). The lipase activity was quantified by calculating the conversion of p-nitrophenyl butyrate to p-nitrophenol at 405 nm using UV-transparent 96-well plates on a microplate reader (BIO-TEK, Synergy HT, U.S.A.). Lipase assays were performed by incubating the plant extracts (final concentration 500 µg/ml) with PPL and pNPB in reaction buffer (50 mM potassium phosphate buffer, pH 7.2, 0.5 % Triton X-100) for 15 min. pNPB was first solubilised with dimethylsulfoxide (DMSO), then diluted with the reaction buffer to a final concentration of 2.5 mM in a 100 µl reaction.  The concentration of DMSO in the reaction (1 %) did not affect the enzyme activity. All assays were run at 37 ⁰C and the results are the average of three replicates that were blank subtracted. Orlistat was used as a positive control. The activity was examined with and without the inhibitor. Inhibition of the lipase activity is defined as the decrease in percentage of activity when PPL is incubated with the crude extracts. Lipase inhibition (%) is determined by using the formula: Inhibitory activity (I %) = 100 – [(B − b)/ (A − a) × 100] Where, A = the activity without inhibitor, a = the negative control without inhibitor, B = the activity with inhibitor and b = the negative control with an inhibitor. The concentrations of extracts, giving 50 % lipase inhibition (IC50) were calculated from the least squares regression line of the semi-logarithmic plot against percentage inhibition curves using the GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, USA).  Kinetic Study: The inhibition mode was determined by Hanes-Woolf plot analysis resulted from the enzyme assay data containing increasing concentrations of pNPB (0.25, 0.5, 1.0, 2.0, 4.0 and 6.0 mM) with the absence and presence of difference concentration of extract (10 and 50 µg/ml). All samples were analyzed in triplicate. Hanes-Woolf plot was constructed by plotting the ratio of the substrate concentration to the reaction velocity ([S]/v) against the substrate concentration [S]. Hanes-Woolf plot and nonlinear regression analysis for determination of Michaelis–Menten constant, Km and maximal velocity, Vmax were done using GraphPad Prism 4.0 software.  DPPH Free Radical Scavenging Activity Assay: 20 μl of methanolic 2,2-diphenyl-1-picrylhydrazil solution was added to 100 μl of sample solution in 96 well plates. The decrease in absorbance was determined at 515 nm using a microplate reader (BIO-TEK, Synergy HT, U.S.A.) after 30 minutes incubation period at room temperature. The test of each sample and ascorbic acid were conducted in triplicate (n = 3).  Mean, standard deviation and IC50 of the results obtained were determined using GraphPad Prism software (GraphPad, USA). The percentage of the inhibition was calculated using the formula:  Scavenging Percentage % = 100-((AS/AC) x 100) Where,  AS = Absorbance of sample, AC = Absorbance of negative control (DMSO without sample) All samples and readings were prepared in triplicate. The IC50 value of the extract was calculated from the plotted graph of scavenging activity against sample concentration using GraphPad Prism 4.0 software.  RESULTS AND DISCUSSION  Lipase-Inhibitory Activity: Preliminary assay revealed that P. niruri extract inhibited 76.7% pancreatic lipase activity when incubated with PPL at a final concentration of 500 µg/ml for 15 minutes at 37⁰C.  The findings from IC50 value showed that the P. niruri extract markedly inhibited the pancreatic lipase activity with the IC50 value of 27.7 µg/ml. However, the extract was less potent than orlistat (control) in inhibiting pancreatic lipase. Orlistat gave an IC50 value of 1.7 µg/ml, which was about 17 times stronger than P. niruri crude extract (Table 1). This is predictable since the crude extract possesses both active and non-active compounds which may affect the effectiveness. Table 1: PPL inhibitory activity of  P. niruri methanolic extracts (80%).  Scientific name Family Common name Part used Inhibition (%)a IC50 (µg/ml)b Phyllanthus niruri Phyllanthaceae Dukung anak Whole plant 76.7 + 0.4 27. 7 Orlistat (Control)    99.6 + 0.3c 1.7 a Percentage of inhibition represents the ability of extract to inhibit pancreatic lipase activity in the medium; concentration tested was 500 µg/ml in the assay except for c. Determined base on the average of three independent replications. b IC50, concentration causing 50% inhibition; concentration tested were varies from 10-2  to 103 µg/ml in the assay. c Concentration tested was 10 µg/ml in the assay. n  = no inhibition nd = not determined  Inhibition Mode: The inhibition mode of P. niruri extract was visualized using graphical representation of the Michaelis–Menten equation, Hanes-Woolf plot; [S]/v versus [S] as shown in Figure 1. The enzyme kinetic result disclosed that P. niruri exerted an inhibitory effect on pancreatic lipase in a noncompetitive manner. As depicted from the graph, when the 135                                                         Raja Noor Zaliha Raja Abd. Rahman et al, 2015 Australian Journal of Basic and Applied Sciences, 9(7) April 2015, Pages: 133-136 extract concentration was increased, the value for the y-intercept in the equation for each curve increased, whereas the x-intercept remained at fixed point showing these inhibitors do not affect Km but the Vmax decreased.   Fig. 1: Inhibition mode determination. Hanes-Woolf plot; [S]/v versus [S] of kinetic analysis for PPL at 2 different concentrations of P. niruri extract (Abbreviated as DA = dukung anak).  Free radical scavenging activity test: The potential of P. niruri extract as  a free  radical  scavenger was screened using the DPPH scavenging assay. As depicted in Figure 2, the DPPH free radical - scavenging activity of P. niruri extract as well as positive control is concentration dependent. The antioxidant activities of the P. niruri extract increased consistently with the concentration. P. niruri extract showed great ability to inhibit DPPH radical with recorded EC50 value of 7.471 µg/ml. Ascorbic acid was served as the positive control with the estimated EC50 of 2.628 µg/ml.    10-1 100 101 102 103020406080100Phyllanthus niruriVitamin CEC50 (g/ml)2.6287.471 Concentration (g/ml)% DPPH Quenched  Fig. 2: Antioxidant activity of P. niruri extract and positive control at different concentration defined as inhibition percentage of DPPH● in DPPH assay. Values are mean ± standard deviation (n = 3). Ascorbic acid (Vitamin C) was used as positive control.  Conclusions:  In this paper, we have determined high antilipase and antioxidant activities of P. niruri plant extract.  The results provide preliminary indicator in our searches for the responsible compounds that contribute to the antilipase and antioxidant activities.  REFERENCES  Birari, R.B., K.K. Bhutani, 2007. Pancreatic lipase inhibitors from natural sources: unexplored potential. Drug Discov Today, 12: 879-89. Kim, H.Y., M.H. Kang, 2005. Screening of Korean medicinal plants for lipase inhibitory activity. Phytother Res., 19: 359-61. Han, L., W. Li, S. Narimatsu, L. Liu, H. Fu, H. Okuda, et al. 2006. Inhibitory effects of compounds isolated from fruit of Juglans mandshurica on pancreatic lipase. J Nat Med., 61: 184-6. Moreno, D.A., N. Ilic, A. Poulev, I. Raskin, 2006. Effects of Arachis hypogaea nutshell extract on lipid metabolic enzymes and obesity parameters. Life Sci., 78: 2797-803. Shimoda, H., E. Seki, M. Aitani, 2006.  Inhibitory effect of green coffee bean extract on fat 136                                                         Raja Noor Zaliha Raja Abd. Rahman et al, 2015 Australian Journal of Basic and Applied Sciences, 9(7) April 2015, Pages: 133-136 accumulation and body weight gain in mice. BMC Complement Altern Med., 6: 9. Harish, R., T. Shivanandappa, 2006. Antioxidant activity and hepatoprotective potential of Phyllanthus niruri. Food Chem., 95: 180-5. Rajeshkumar, N.V., K.L. Joy, G. Kuttan, R.S. Ramsewak, M.G. Nair, R. Kuttan, 2002. Antitumour and anticarcinogenic activity of Phyllanthus amarus extract. J Ethnopharmacol., 81: 17-22. Khanna, A.K., F. Riz, R. Chander, 2002. Lipid lowering acti v ity of Phyllanthus niruri in hyperlipemic rats., 82. Liu, S., W. Wei, K. Shi, X. Cao, M. Zhou, Z. Liu, 2014. in vitro and in vivo anti-hepatitis B virus activities of the lignan niranthin isolated from Phyllanthus niruri L. J Ethnopharmacol., 155: 1061-7. Lowe, M.E., J.L. Rosenblum, W. Straussa, 1989. Cloning and characterization of human pancreatic lipase cDNA. J Biol Chem., 264: 20042-8. Lewis, R., D. Direct, 2012. Measurement of Lipase Inhibition by Orlistat Using a Dissolution Linked In Vitro Assay. Clin Pharmacol Biopharm, 01. Lehner, R., R. Verger, 1997. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase. Biochemistry, 36: 1861-8. Singh, D., B. Singh, R.K. Goel, 2011. Traditional uses, phytochemistry and pharmacology of Ficus religiosa: a review. J Ethnopharmacol., 134: 565-83. Sharma, O.P., T.K. Bhat, 2009. DPPH antioxidant assay revisited. Food Chem., 113: 1202-5.   

English

Antilipase and antioxidant activity of Phyllanthus niruri ethanolic extract

Universiti Putra Malaysia Institutional Repository

Antilipase and antioxidant activity of Phyllanthus niruri ethanolic extract   Abstract  The aim of this study is to determine the antilipase and antioxidant activity of Phyllanthus niruri methanolic extract. The inhibitory activity against porcine pancreatic lipase (PPL) was determined by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at 405 nm. The crude extract exhibited strong lipase inhibitory activity with an IC50 value of 27. 65 µg/ mL. The inhibition mode study disclosed that P. niruri could act as noncompetitive inhibitor. Antioxidant activity of P. niruri extract was measured by 2, 2, diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assay. P. niruri showed high antioxidant activity with an EC50 value of 7.471 µg/mL. The results suggested that P. niruri has shown potential as a source of natural antilipase and antioxidant.  Keyword: Phyllanthus niruri; Antilipase; Antioxidant; DPPH    

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Antilipase and antioxidant activity of Phyllanthus niruri ethanolic extract

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