Australian Journal of Basic and Applied Sciences

Transcription

1 AENSI Journals Australan Journal of Basc and Appled Scences ISSN: Journal home page: Antoxdant and Antdabetc Actvtes of Ethanolc Ctrus Hystrx Peel Extract: Optmzaton of Extracton Condtons Wenny Irawaty, Felyca E. Soetaredjo, Anng Ayuctra, Martnus E. Santo, Kevn Jonathan, Cyntha D., Ccla Setyabud, Stefan Tanda Chemcal Engneerng Department, Faculty of Engneerng, Wdya Mandala Catholc Unversty Surabaya, Kaljudan 37 Surabaya 114. East Java. Indonesa A R T I C L E I N F O Artcle hstory: Receved 25 June 2014 Receved n revsed form 8 July 2014 Accepted 10 August May 2014 Avalable onlne September 2014 Keywords: Ctrus hystrx, peel, extracton, antoxdant, antdabetes A B S T R A C T The benefts of ctrus fruts have been well documented. As food ngredent, only leaf part of Ctrus hystrx has been utlzed as food ngredent. The ctrus peel may contan hgher antoxdant compounds than the edble part. The objectve of ths study was to optmze the extracton condtons of antoxdant, presented as Total Phenolc Compounds (RSM), from Ctrus hystrx peel usng Response Surface Methodology. In addton, n-vtro study to nvestgate the antdabetc effect of the extract was also performed. Two ndependent varables (tme and ethanol concentraton) were optmzed for maxmzng the total phenolc compounds extracted from the Ctrus hystrx peel usng RSM based on central composte desgn. The optmum condtons of polyphenolc compounds extracton from Ctrus hystrx peel were obtaned at 7.8 h of extracton tme by employng ethanol 41% as the solvent. The extract also exhbted the ant-dabetc actvty AENSI Publsher All rghts reserved. To Cte Ths Artcle: Wenny Irawaty, Felyca E. Soetaredjo, Anng Ayuctra, Martnus E. Santo, Kevn Jonathan, Cyntha D., Ccla Setyabud, Stefan Tanda., Antoxdant and Antdabetc Actvtes of Ethanolc Ctrus Hystrx Peel Extract: Optmzaton of Extracton Condtons. Aust. J. Basc & Appl. Sc., 8(14): 85-89, 2014 INTRODUCTION Ctrus frut has been known for ts varous health benefts. Polyphenolc compounds, a complex group of secondary metabolte substances, contans numerous bologcal actve compounds, exhbt wde range of physologcal propertes such as ant-nflammatory (Menchn et al., 2011), ant-mcrobal (Y et al., 2008), cardprotectve (Putr et al., 2013), neuroprotectve (Zbarsky et al., 2005), ant-adpogeness (Km et al., 2012), ant-dabetes (L et al., 2006), hepatoprotectve (Putr et al., 2013), etc. Varous classes of polyphenolc compounds n ctrus frut have been dentfed as flavanone aglycones (hespertn, narngenn), flavone aglycones (acacetn, quercetn, dosmetn), polymethoxyflavones (quercetogetn, nobletn, tangeretn), flavanone-o-glycosdes (hesperdn, narngn, narrutn, neohesperdn), and flavone-c- and flavone-cglucosdes (Gattuso et al., 2007). The frut peel has been reported to contan hgher antoxdant compounds than the edble part (Gornsten et al., 2002) and therefore, study the antoxdant actvty of the peel becomes a challenge. Ctrus hystrx s one of ctrus cultvars n Indonesa that has not been fully utlzed yet because only leaves used as food ngredent. Therefore, t s an nterestng challenge to nvestgate the antoxdant actvty of Indonesan Ctrus hystrx peel because ts polyphenolc compounds would be dfferent wth the one growth n other areas as reported n lterature. It opens opportunty to further develop Ctrus peel as one of Indonesan natural antoxdant sources. There s no nformaton related to antoxdant actvty of Indonesan Ctrus hystrx peel found n the lterature. Moreover ts antoxdant capacty as ant-dabetes was also another challenge to be nvestgated. In order to extract the ctrus frut, maceraton, hot water extracton, and reflux are commonly used procedures (Putr et al., 2013; Xu et al., 2008; Bocco et al., 1998). The technques are smple but consume large volume of solvents used. Supercrtcal flud extracton, sub-crtcal water extracton, mcrowave-asssted extracton, and ultrasonc treatment are emergng as better alternatves than the former technques (Ruen-ngam et al., 2012; Chegh et al., 2012; Hayat et al., 2010; Ma et al., 2008). However, ther hgh operatng costs may have lmted ther small- or medum-scale ndustral applcatons. In ths study, maceraton extracton was selected to extract antoxdant compounds from Ctrus hystrx peel. Several parameters nfluence the extracton of polyphenolc compounds such as extracton tme, solvent-tosample rato, temperature, solvent type, and the number of repeat extracton. The optmum recovery of phenolc Correspondng Author: Wenny Irawaty, Chemcal Engneerng Department, Faculty of Engneerng, Wdya Mandala Catholc Unversty Surabaya, Kaljudan 37 Surabaya 114. East Java. Indonesa Tel: ; Fax: ; E-mal: wenny..santosa@gmal.com.

2 86 Wenny Irawaty et al, 2014 compounds would be dfferent from one sample to others that depends on the nature or physcochemcal propertes of the samples. Response Surface Methodology (RSM) was employed to optmze of extracton process condtons of Ctrus hystrx peel n ths study. The method has been wdely used for the optmzaton of extracton condtons such as temperature, solvent concentraton, tme, etc. RSM conssts of mathematcal and statstcal technques to develop a functonal relatonshp between a response of nterest and some ndependent varables. Ths study was amed to optmze the maceraton extracton condtons of antoxdant (phenolc compounds) from Ctrus hystrx peel usng RSM. Addtonally, the most mportant factor contrbuton to the antoxdant actvty of the Ctrus hystrx peel extract was determned. Ant-dabetes actvty of the extract was also nvestgated. MATERIALS AND METHODS Materals used n ths study were Ctrus hystrx, purchased from local market n Surabaya that were collected around January March Ethanol (C 2 H 5 OH), aquades (H 2 O), Foln-Cocalteu reagent, sodum carbonate (Na 2 CO 3 ), gallc acd (C 7 H 6 O 5 ), glucose (C 6 H 12 O 6 ), dntrosalyclcacd (C 7 H 4 N 2 O 7 ), amylum ((C 6 H 10 O 5 ) x ), enzyme α-amylase (from Aspergllus oryzae) was purchased from supplers and used wthout further purfcaton. Methods carred out nvolve the raw materal preparaton, extracton, and analyss steps. Frstly, fresh Ctrus hystrx frut were washed, peeled off and cut nto a sze of 0.5 x 0.5 cm pror to dry t for about 48 h at 35 o C n an ar-oven (Memmert, Germany). The materal was then stored below 0 o C n artght plastc bags for further use. Secondly, the dred sample was weghed and extracted wth 20 ml of solvent n a dark and closed contaner at room temperature and certan extracton tme. After the extracton, the extract was separated and centrfuged to remove the sold part. Thrdly, the total phenolc content (TPC) of Ctrus hystrx peel extract was determned usng the Foln-Cocalteu colormetrc method. The TPC value was expressed as gallc acd equvalents (GAE) per dry mass of Ctrus hystrx peel sample (mg GAE/g) from a standard calbraton curve. The extract was also tested for ant-dabetc actvty by performng n-vtro study usng enzyme α-amylase as the selected model (Nar et al., 2013). Central composte desgn (CCD) was selected to determne the optmum condton of the extracton process by sung MINITAB 14. CCD was constructed wth four axal ponts, four star ponts (± α) and fve replcatons at the center pont. In ths study, 1.41 was used as α for rotatable desgn (Montgomery, 2005). The followng regresson equaton was ftted to the response resulted from CCD by the least square method: k k 2 Y x x x x (1) o 1 1 j j j where Y s the estmated TPC yeld, x and x j are the coded value of an ndependent varables, β 0 s a constant coeffcent, β are lnear coeffcents, β j ( and j) are the nteracton coeffcents and β are the quadratc coeffcents. RESULTS AND DISCUSSION In response surface methodology, natural varables are transformed nto coded varables that have been defned as dmensonless wth a mean zero and the same standard devaton. The expermental values for response (TPC) under dfferent condtons were gven n Table 1. Center pont was set at 6 h of extracton tme and 55% of ethanol concentraton. In all experments, sold-to-solvent rato was fxed at 1: (w/v). The results showed that TPC of local Ctrus hystrx peels ranged from around.02 to mg GAE/g dry weght. By employng multple regresson analyss, the regresson model related the selected ndependent varables and the response of TPC was follows: Y = X X X X X 1 X 2 (2) where Y, X 1, and X 2 are TPC, extracton tme, and ethanol concentraton, respectvely. The ftness and adequacy of the model was further evaluated by the determnaton coeffcent (R-sq). The R-sq, defned as the rato of the explaned varaton to the total varaton, was a measure of the degree of ft. The closer the R-sq value, the better the emprcal model fts the expermental data. The R-sq of the work shown n Table 1 ndcates that 89.2% of the total varaton around the average could be explaned by the regresson model. Ths suggests that the predcted second order polynomal model defned qute well the real behavour of the system. Fgure 1(a, b) llustrates three-dmensonal response surface plot by presentng the response of TPC as a functon of the two selected varables. As seen, hgher amount of phenolc content extracted from the peel n the regon of extracton tme between 7 to 8.5 h and ethanol concentraton between and 45%. Therefore, the yeld of TPC contnues to ncrease wth ethanol concentraton and extracton tme and acheved the optmum condtons at about 7.8 h and 41%, respectvely, before t began to decrease. The predctve TPC value at these condtons s mg GAE/mg. Generally, the polarty of ethanol-water mxture ncreases contnuously wth

3 Ethanol concentraton (%) ethanol concentraton 87 Wenny Irawaty et al, 2014 the water addton nto the system. The optmum ethanol concentraton (41%) observed n ths work ndcates that Ctrus hystrx peel contans more polar compounds. Polar phenolc compounds reported n ctrus are aglycones and glycosdes of flavonods, neohesperdn and neoroctrn (Chan et. al., 2009). The ncrease of TPC yeld wth extracton tme can be explaned by the longer the peel was soaked n the solvent, the longer the contact between the two and thus, more compounds n the sold part wll be dssolved whch the amount and the extracton rate are depend on the solvent polarty. Further verfcaton of the predctve model was performed by repeatng the experment at the optmum condtons and the observed TPC yeld was mg GAE/mg. Ths result ndcates that the expermental result was qute close to the predcted one, ndcatng the hgh ft degree between the observed experment and the response predcted from the regresson model. Table 1: Independent varables and expermental TPC yelds of the CCD. Run Coded varables Real varables TPC X 1 X 2 X 1 X 2 (mg GAE/g) X 1 and X 2 represent extracton tme (h) and ethanol concentraton (%), respectvely. R-sq = (a) TPC (mg GAE/g) TPC tme Tme (h) 80 ethanol Ethanol concentraton concentraton (%) (b) tme Tme (h) Fg. 1: (a) Response of TPC and (b) contour plot of TPC calculated from the model. The extract of Ctrus hystrx peel was also subjected for ant-dabetc actvty test. The study on antdabetes from natural resources becomes nterest because long-term damages, dysfuncton and falure of organs after consumng modern medcnes for a perod of tme. In addton, Indonesa s the fourth country after Inda, Chna, and Unted States wth the largest number of dabetc patents. The mportant am to treat dabetc patence s by stablzng blood glucose level. Glucose or starch absorpton can be elmnated by consumng nhbtor of carbohydrate dgeston such as α-amylase and α-glucosdase nhbtors. The nhbtor wll prevent polysacchardes and/or dsacchardes from beng hydrolyzed to monosacchardes n small ntestne. In ths work, α-amylase was employed as the nhbtor. Fgure 2 shows ant-dabetc actvty of the extract of Ctrus hystrx peel. As seen, the extract nhbts the converson of starch to glucose as shown by lower glucose concentraton observed n soluton than the control one. Ths can be explaned by the presence of polyphenolc compounds (TPC) n the extract promote ts ant-dabetc actvty. The presence of gallc acd, hesperdn, and narngn n ctrus fruts have been suggested to be responsble for ths (Patel and Goyal, 2011; Jung et al., 2004).

4 88 Wenny Irawaty et al, 2014 Fg. 2: Effect of the addton of Ctrys hystrx peel extract on glucose producton. Conclusons: In the present study, the extracton of total phenolc compounds from Ctrus hystrx peel was optmzed usng response surface methodology based on central composte desgn. The quadratc model for predctng the total phenolc content was obtaned and both factors nvestgated here contrbuted sgnfcantly to the TPC yeld. The optmum extracton condtons of extracton tme and ethanol concentraton were 7.8 h and 41%, respectvely. The most mportant result observed here s that Ctrus hystrx peel extract also exhbted antdabetc actvty. Further study needs to be nvestgated to get further nsght on what specfc phenolc compound was responsble for ths actvty. ACKNOWLEDGMENT Ths work was funded by Drectorate General of Hgher Educaton through Fundamental Research Grant 2014 to Wenny Irawaty, Felyca E. Soetaredjo, and Anng Ayuctra. The authors thank Ms. Luh Jun Asrn for her assstance n statstcs part. REFERENCES Bocco, A., M.E. Cuveler, H. Rchard and C. Berset, Antoxdant actvty and phenolc composton of ctrus peel and seed extract. J Agrc Food Chem, 46: Chan, S.W., C.Y. Lee, C.F. Yap, W.M. Wan Ada and C.W. Ho, Optmsaton of extracton condtons for phenolc compounds from lmau purut (Ctrus hystrx) peels. Inter Food Res J, 16: Chegh, C.I., E.Y. Chung and M.S. Chung, Enhanced extracton of flavanones hesperdn and narrutn from Ctrus unshu peel usng subcrtcal water. J Food Eng, 110: Gattuso, G., D. Barreca, C. Gargull, U. Leuzz and C. Carst, Flavonod composton of Ctrus juces. Molecules, 12: Gornsten, S., O.M. Belloso, A. Lojek, M. Cz, R.S. Fortuny and Y.S. Park, Comparatve content of some phytochemcals n Spansh apples, peaches and pears. J Sc Food Agrc, 82: Hayat, K., X. Zhang, H. Chen, S. Xa, C. Ja and F. Zhong, Lberaton and separaton of phenolc compounds from ctrus mandarn peels by mcrowave heatng and ts effect on antoxdant actvty. Sep Purf Technol, 73: Jung, U.J., Lee, M.K., Jeong, K.S. and Cho, M.S., The hypoglycemc effects of hesperdn and narngn are partly medated by hepatc glucose-regulatng enzymes n C57BL/KsJ-db/db mce. J Nutr, 134(10): Km, G.S., H.J. Park, J.H. Woo, M.K. Km, P.O. Koh, W. Mn, Y.G. Ko, C.H. Km, C.K. Won and J.H. Cho, Ctrus aurantum flavonods nhbt adpogeness through the Akt sgnalng pathway n 3T3-L1 cells. BMC Complementary & Alternatve Medcne, 12: 31. L, R.W., A.G. Therault, K. Au, T.D. Douglas, A. Casasch, E.M. Kurowska and R. Mukherjee, Ctrus polymethoxylated flavones mprove lpd and glucose homeostass and modulate adpocytoknes n fructose-nduced nsuln resstant hamsters. Lfe Scences, 79: Ma, Y.Q., J.C. Chen, D.H. Lu and X.Q. Ye, Effect of ultrasonc treatment on the total phenolc and antoxdant actvty of extracts from ctrus peel. J Food Sc, 73(8): T Menchn, F., M.R. Lozzo, M. Bones, F. Confort, D.D. Luca, G.A. Statt, B. Cndo, F. Menchn and R. Tunds, Phytochemcal profle, antoxdant, ant-nflammantory and hypoglycemc potental of hydroalcoholc extracts from Ctrus medca L. cv Damante flowers, leaves and fruts at two maturty stages. Food Chem Toxcol, 49(7):

5 89 Wenny Irawaty et al, 2014 Montgomery, D.C., Desgn and analyss of experments. John Wley & Sons, Inc., New Jersey. Putr, H., S. Nagad, Y.A. Larasat, N. Wulandar and A. Hermawan, Cardoprotectve and hepatoprotectve effects of Ctrus hystrx peels extract on rats model. Asan Pac J Trop Bomed, 3(5): Nar, S.S., A. Kavrekar and A. Mshra, In vtro studes on alpha amylase and alpha glucosdase nhbtory actvtes of selected plant extracts. Pelaga Research Lbrary, 3(1): Patel, S.S. and Goyal RK., Cardoprotectve effects of gallc acd n dabetes-nduced myocardal dysfuncton n rats. Pharmacognosy Res, 3(4): Ruen-ngam, D., A.T. Qutan, M. Tanaka, M. Sasak and M. Goto, Reacton knetcs of hydrothermal hydrolyss of hesperdn nto more valuable compounds under supercrtcal carbon doxde condtons. J of Supercrtcal Fluds, 66: Xu, G.H., J.C. Chen, D.H. Lu, Y.H. Zhang, P. Jang and X.Q. Ye, Mnerals, phenolc compounds, and antoxdant capacty of Ctrus peel extract by hot water. J Food Sc., 73(1): C11-C18. Y, Z., Y. Yu, Y. Lang and B. Zeng, In vtro antoxdant and antmcrobal actvtes of the extract of Percarpum Ctr Retculatae a new Ctrus cultvar and ts man flavonods. LWT-Food Sc Technol, 41(4): Zbarsky, V., K.P. Datla, S. Parkar, D.K. Ra, O.I. Aruoma, D.T. Dexter, Neuroprotectve propertes of the natural phenolc antoxdants curcumn and narngenn but not quercetn and fsetn n a 6-OHDA model of Parknson s dsease. Free Radc Res, 39: