Research Article In Vitro Study on the Antioxidant Potentials of the Leaves and Fruits of Nauclea latifolia

Transcription

1 e Sientifi World Journl Volume 214, Artile ID 43781, 8 pges Reserh Artile In Vitro Study on the Antioxidnt Potentils of the Leves nd Fruits of Nule ltifoli Ademol O. Ayeleso, 1 Oluwfemi O. Oguntibeju, 1 nd Niole L. Brooks 2 1 Oxidtive Stress Reserh Centre, Deprtment of Biomedil Sienes, Fulty of Helth & Wellness Sienes, Cpe Peninsul University of Tehnology, Bellville 7535, South Afri 2 Deprtment of Wellness Sienes, Fulty of Helth & Wellness Sienes, Cpe Peninsul University of Tehnology, Cpe Town 8, South Afri Correspondene should be ddressed to Ademol O. Ayeleso; demol.yeleso@gmil.om Reeived 3 April 214; Revised 13 My 214; Aepted 19 My 214; Published 11 June 214 Ademi Editor: Okezie I. Aruom Copyright 214 Ademol O. Ayeleso et l. This is n open ess rtile distributed under the Cretive Commons Attribution Liense, whih permits unrestrited use, distribution, nd reprodution in ny medium, provided the originl work is properly ited. Thisstudywsrriedouttoinvestigtethein vitro ntioxidnt potentils of the leves nd fruits of Nule ltifoli,strggling shruborsmlltree,ntivetotropilafrindasi.hotwterextrtsoftheleves ndfruitsofnule ltifoli were ssessed for their totl polyphenoli, flvnol, nd flvonol ontents s well s 1-diphenyl-2-pirylhydrzyl (DPPH) svenging bility, ferri reduing ntioxidnt power (FRAP), Trolox equivlene ntioxidnt pity (TEAC), nd oxygen rdil bsorbne pity (ORAC) ssys. The queous extrt of the leves ws found to ontin higher level of totl polyphenols (11.63 ±.23 mg GAE/g), flvnol (1.45 ±.1 mg CE/g), nd flvonol (2.22 ±.37 mg QE/g) thn the extrt of the fruits with vlues of 1.75 ±.2 mg GAE/g (totl polyphenol),.15 ±.1 mg CE/g (flvnol), nd 1. ±.13 mg QE/g (flvonol). Similrly, the queous extrt of the leves lso exhibited higher DPPH (IC mg/ml), FRAP (86.1 ± 3.46 μmol AAE/g), TEAC (94.83 ± 3.57 μmol TE/g), nd ORAC ( ±.73 μmol TE/g) thn the extrt of the fruits with DPPH (IC mg/ml), FRAP (12.23 ±.4 μmol AAE/g), TEAC (12.48 ±.21 μmol TE/g), nd ORAC (58.88 ±.73 μmol TE/g). The present study showed tht Nule ltifoli hs strong ntioxidnt potentils with the leves demonstrting higher in vitro ntioxidnt tivities thn the fruits. 1. Introdution Free rdils, whih belong to group of retive oxygen speies (ROS), re produed through endogenous soure, tht is, the humn body itself, nd exogenous soures suh s tobo smoke, burning of fossil fuels, nd ozone [1]. The imblne between the prodution of ROS nd the tivity of the ntioxidnt defenes is referred to s oxidtive stress [1]. The inhibiting or preventive effets of herbs or spies ginst the deleterious onsequenes of oxidtive stress re due to the presene of nturl ntioxidnts in them [2]. Drug formultions tht re ntioxidnt bsed re used in the prevention nd tretment of omplex diseses whih inlude theroslerosis, stroke, dibetes, Alzheimer s disese, nd ner [2, 3]. Nule ltifoli Smith (fmily: Rubiee) is strggling, evergreen, multistemmed shrub or smll tree ntive to tropil Afri nd Asi. It normlly produes interesting flowers, edible, but not ppeling, lrge red bll fruits with long projeting stmens [4]. Commonly used prts of Nule ltifoli inlude the leves, roots, stem, nd fruits. The fruits serve s key soure of food for the bboons, livestok, reptiles, birds, nd mn [4]. Phytohemils mjorly found in Nule ltifoli inlude indole-quinolizidine, lkloids (glyolkloids), nd sponins [5]. Nkfmiy et l. [6] lso reported tht the fruits of Nule ltifoli ontin opper, iron, oblt, lium, mgnesium, zin, phosphorus, nd vitmins (A, B 1,B 2,C,ndE). Throughout reorded history, Nule ltifoli herbl remedies hve been frequently seen in vrious ples nd re

2 2 The Sientifi World Journl being used s mjor mens of therpeuti medil tretment [7]. Trditionlly, the infusions nd deotions from the stem brk nd leves of the plnt re used for the tretment of mlri, stomh he, fever, dirrhe, nd nemtodes infetions [8 1]. Severl studies hve onfirmed the helth potentils of Nule ltifoli. Some of its mediinl effets inlude ntimlril [11], ntidibeti [8], ntihypertensive [12 14], ntipyreti nd ntinoieptive [15], nd nti-inflmmtory nd nlgesi tivities [7].Itislsoknowntohvestrong ntibteril property [16, 17]. This study ws onduted to investigte the ntioxidnt potentils in the queous extrts of the leves nd fruits of Nule ltifoli s these ould be the ontributing ftors to their helth benefiil effets. 2. Mterils nd Methods 2.1. Preprtion of Plnt Extrts. The plnt ws identified nd uthentited by plnt sientist (Mr Omotyo) nd deposited t the herbrium (E-herbrium UHAE 261) in the Deprtment of Plnt Siene, Ekiti Stte University, Ado Ekiti, Nigeri. The dried nd powdered leves nd fruits (15 g) were extrted with 75 ml of hot (1 C) distilled wter. After 3 min, the extrts were filtered out nd used for the ssys Liquid Chromtogrphy-Mss Spetrometry (LC-MS) Tehnique. LC-MS ws performed on Dionex HPLC system (Dionex Softron, Germering, Germny) equipped with binry solvent mnger nd utosmpler oupled to Bruker ESI Q-TOF mss spetrometer (Bruker Dltonik GmbH, Germny). Constituents of the queous plnt extrts were seprted by reversed phse hromtogrphy on Thermo Fisher Sientifi C18 olumn 5 μm, mm (Bellefonte, USA), using liner grdient of.1% formi id in wter (solvent A) nd etonitrile (solvent B) s solvent t flow rte of.8 ml min 1, n injetion volume of 1 μl, nd n oven temperture of 3 C. MS spetr were quired in negtive mode. Eletrospry voltge ws set to +35 V. Dry gsflowwssetto9lmin 1 with temperture of 3 Cnd nebulizer gs pressure ws set to 35 psi Determintion of Totl Polyphenol, Flvnol, nd Flvonol Contents. The totl polyphenol ontent in the queous plnt extrts ws determined using the Folin-Ciolteu s phenol regent ording to the method desribed by Singleton et l. [18] nd ws determined spetrophotometrilly using miroplte reder nd expressed s mg glli id stndrd equivlents (GAE) per grm smple. The flvnol ontent of the queous plnt extrts ws determined olorimetrilly t 64 nm using the ldehyde DMACA nd expressed s mg tehin stndrd equivlents (CE) per grm smple [19, 2]. The flvonol ontent of the queous plnt extrts ws determined spetrophotometrilly t 36 nm nd expressed smgqueretinstndrdequivlents(qe)pergrmsmple [21]. All determintions were done in triplites DPPH Free Rdil Svenging Ativity. DPPH free rdil svenging tivity of the queous plnt extrts ws rried out ording to method desribed by Zhelev- Dimitrov [22] with slight modifitions. Briefly, 1 μl of the different onentrtions of the queous plnt extrts ws reted with 19 μl of DPPH solution (.625 g DPPH in 5 ml methnol) nd the bsorbne of the smples ws determined fter 3 min using Multiskn Spetrum plte reder (Thermo Fisher Sientifi, USA) t 517 nm. Free rdil svenging tivity of the smples ws expressed ording to the eqution below: Perent (%) inhibition of DPPH tivity = A A A 1, where A is the bsorbne of DPPH in solution without n ntioxidnt nd A is the bsorbne of DPPH in the presene of n ntioxidnt. IC 5 vlue (onentrtion of smple where bsorbne of DPPH dereses 5% with respet to bsorbne of blnk) of the smple ws determined. All determintions were done in triplites Ferri Reduing Antioxidnt Power Assy. The FRAP ssy ws done using the method desribed by Benzie nd Strin [23]. Briefly, 1 μl of the diluted queous plnt extrts ws mixed with 3 μl FRAP regent in 96-well ler plte. The FRAP regent ws mixture (1 : 1 : 1, v/v/v) of ette buffer (3 mm, ph 3.6), tripyridyl trizine (TPTZ) (1 mm in 4 mm HCl), nd FeCl 3 6H 2 O(2mM).Afterinubtion troomtemperturefor3min,thepltewsredt wvelength of 593 nm in Multiskn Spetrum plte reder (Thermo Fisher Sientifi, USA). Asorbi id (AA) ws used s the stndrd nd the results were expressed s μmol AAE/g smple. All determintions were done in triplites Trolox Equivlene Antioxidnt Cpity Assy. The TEAC ssy ws rried out using the priniple of 2,2 - zino-bis(3-ethylbenzothizoline-6-sulphoni id) (ABTS) rdil svenging tivity ording to method desribed by Ou et l. [24]. ABTS + solution ws prepred dy before use by mixing ABTS slt (8 mm) with potssium persulfte (3 mm) nd then storing the solution in the drk until the ssy ould be performed. The ABTS + solution ws further diluted with distilled wter. Twenty five mirolitres (25 μl) of the diluted queous plnt extrts ws mixed with 3 μl ABTS + solution in 96-well ler miroplte. The plte ws red fter 3 min inubtion t room temperture in Multiskn Spetrum plte reder (Thermo Fisher Sientifi, USA) t 734 nm. Trolox ws used s the stndrd nd results were expressed s μmol TE/g smple. All determintions were done in triplites Oxygen Rdil Absorbne Cpity (ORAC) Assy. The ORAC ssy ws onduted ording to the method of Re et l. [25] on96-wellmiroplteusingfluoresene plte reder (Thermo Fisher Sientifi, Wlthm, Mss., USA). The retion onsisted of 12 μl of diluted queous plnt extrts nd 138 μl of fluoresein(14μm), (1)

3 The Sientifi World Journl A MS, 9.1 min number 542 MS, 9.4 min number B MS, 2.2 min number 13 MS, 2.5 min number C MS, 12.9 min number 766 MS, 14. min number MS, 3.5 min number D MS, 6.4 min number E MS, 9.8 min number 586 MS, 1.6 min number F MS, 11.2 min number 666 MS, 11.6 min number Figure 1: LCMS hromtogrm of phenoli id nd flvonoids in the queous extrt of Nule ltifoli leves. (A) Chlorogeni id, (B) tehin, (C) ynroside, (D) tngeritin, (E) rutin/hesperidin, nd (F) hyperoside. whih ws used s trget for free rdil ttk. The retion ws initited by the ddition of 5 μl AAPH (768 μm)ndthefluoresenews(emission538nm,exittion 485 nm) reorded every 1 min for 2 hours. Trolox ws used s the stndrd nd results were expressed s μmol/g smple. All determintions were done in triplites Sttistil Anlysis. Results re expressed s the mens ± stndrd devitions. The signifine of differene between

4 4 The Sientifi World Journl A C B MS, 17.4 min number 134 MS, 18.3 min number 187 MS, 5.5 min number 326 MS, 5.7 min number D E MS, 6. min number 358 MS, 6.2 min number F MS, 1.5 min number 626 MS, 11.1 min number 662 MS, 7.8 min number 462 MS, 7.9 min number Figure 2: LCMS hromtogrm of phenoli id nd flvonoids in the queous extrt of Nule ltifoli fruits. (A) Syringi id, (B) nobiletin, (C) tngeritin, (D) tehin, (E) hrysoeriol, nd (F) isorhmnetin. men vlues of different plnt extrts ws determined by t- test using Grph Pd PRISM Results nd Disussion Essentil soure of new hemil substnes with potentil therpeuti effets is thought to be obtined from mediinl plnts [7, 26, 27]. The ntioxidnt ontents of mediinl plnts my ontribute to protetion ginst diseses [28]. Nturl ntioxidnts hve ttrted gret del of publi nd sientifi ttention beuse of their helth-promoting effets [29]. An imblne between the prodution of retive oxygen speies (ROS) nd the tivity of the ntioxidnt defenes leds to oxidtive stress [1]. In the pthology of severl humn diseses suh s theroslerosis, inflmmtion, ner, rheumtoid rthritis, nd neurodegenertive diseses like Alzheimer s disese nd multiple slerosis, ROS hve been implited [1]. Attention is on ntioxidnt gents of nturl origin due to their bilities to svenge free rdils [28, 3]. Antioxidnt pity is ssoited with ompounds tht n protet biologil system ginst the dmging effet of ROS nd retive nitrogen speies (RNS) [31] Polyphenoli Contents in the Aqueous Extrts of the Leves nd Fruits of Nule ltifoli. Phenoli ompounds re found usully in both edible nd nonedible plnts with severl biologil effets whih inlude ntioxidnt tivity [32]. Figure 1 shows tht the queous extrt of the leves of

5 The Sientifi World Journl (mg/g) b b Totl polyphenol Flvnol Flvonol Polyphenoli ontents of the smples Leves Fruits Figure 3: Totl polyphenol, flvnol, nd flvonol ontents in the queous extrts of the leves nd fruits of Nule ltifoli. All signifint differenes re t P <.5. -, b-b, - Brs hving the sme lphbets re signifintly different from eh other. Nule ltifoli ontins hlorogeni id, tehin, ynroside, tngeritin, rutin/hesperidin, nd hyperoside. Figure 2 shows tht the queous extrt of the fruits of Nule ltifoli ontins syringi id, nobiletin, tngeritin, tehin, hrysoeriol, nd isorhmnetin. In this study, the totl polyphenoli ontents of the queous plnt extrts of the leves nd fruits were determined using the diluted Folin- Ciolteu regent. The totl polyphenoli ontent ws higher in the queous extrt of the leves thn in the extrt of the fruits with men vlues of ±.23 mg GAE/g nd 1.75 mg±.2 mg GAE/g smple, respetively (Figure 3). The flvnolontentwslsohigherinthequeousextrtof the leves (1.45 ±.1 mg CE/g) thn in the extrt of the fruits (.15 ±.1 mg CE/g). In similr vein, flvonol ontent ws signifintly higher in queous extrt of the leves (2.22 ±.37 mg QE/g) thn in the extrt of fruits (1. mg ±.13 mg QE/g) (Figure 3). Overll, the results showed tht queous extrts of both the leves nd the fruits re rih in polyphenols with the leves hving higher polyphenols thn the fruits DPPH Rdil Svenging Ativity in the Aqueous Extrts of the Leves nd Fruits of Nule ltifoli. DPPH rdil is used s stble free rdil to determine the ntioxidnt tivity of nturl ompounds nd the svenging of stble rdil (DPPH) is onsidered vlid nd esy ssy to evlute svenging tivity of ntioxidnts [33 35]. In this ssy, purple olor of DPPH is redued to α,α-diphenyl- β-pirylhydrzine (yellow olored) when neutrlised. The extent of the hnge in olor is proportionl to the onentrtion nd strength of the ntioxidnts [28]. In this study, the queous extrts of both the leves nd the fruits were ble to show free rdil svenging bilities (Figure 4). It ws observed tht the queous extrt of the leves hd higher DPPH tivity thn the extrt of the fruits with the onentrtion of smple where bsorbne of DPPH dereses 5% with respet to bsorbne of blnk (IC 5 )to IC 5 (mg/ml) 1 5 DPPH rdil svenging tivities of the smples Leves Fruits Figure 4: DPPH rdil svenging tivity in the queous extrts of the leves nd fruits of Nule ltifoli. All signifint differenes re t P <.5. - Brs hving the sme lphbets re signifintly different from eh other. be 2.64 mg/ml nd mg/ml, respetively. The effet of ntioxidnts on DPPH ould be due to their hydrogen donting bility [36] Ferri Reduing Antioxidnt Power in the Aqueous Extrts of the Leves nd Fruits of Nule ltifoli. The extent t whih the queous extrt of the leves nd fruits of Nule ltifoli ould redue ferri ions ws done with FRAP ssy. This ssy is mde possible by low moleulr weight ntioxidnts of hydrophili nd/or hydrophobi nture [37]. FRAP ssy hs been used to ompre ntioxidnt tivity in plnts nd mmmls [38 4]. The tion of eletron donting ntioxidnts uses hnge in the bsorbne t 593 nm duetotheformtionofblueoloredfe +2 tripyridyl trizine (TPTZ) ompound from the olorless oxidized Fe +3 form [41, 42]. The queous extrt of the leves showed higher FRAP (86.1 ± 3.46 μmol AAE/g smple) thn the extrt of the fruits (12.23 ±.4 μmol AAE/g smple). It ws lerly shown tht even though the queous extrts of both the leves nd the fruits hve the bilities to redue ferri ions, the tivity of the leves ws higher (Figure 5) Trolox Equivlene Antioxidnt Cpity in the Aqueous Extrts of the Leves nd Fruits of Nule ltifoli. The TEAC ssy is bsed on the suppression of the bsorbne of rdil tions of 2,2-zino-bis(3-ethylbenzothizoline-6- sulfonte) (ABTS) by ntioxidnts in the smple when ABTS inubtes with peroxidse (metmyoglobin) nd H 2 O 2 [43, 44]. It entils tehnique tht produes blue/green ABTS + hromophore through the retion of ABTS nd potssium persulfte [3]. The ssy is prtiulrly interesting in plnt extrts beuse the wvelength bsorption t 734 nm elimintes olor interferene [45]. The results lso showed higher TEACinthequeousextrtofthelevesthnintheextrt

6 6 The Sientifi World Journl 25 2 Conflit of Interests The uthors delre tht there is no onflit of interests regrding the publition of this pper. (μmol/g) b b FRAP TEAC ORAC Antioxidnt tivities (FRAP, TEAC, nd ORAC) of the smples Leves Fruits Figure 5: Antioxidnt tivities (FRAP, TEAC, nd ORAC) in the queous extrts of the leves nd fruits of Nule ltifoli. All signifint differenes re t P <.5. -, b-b, - Brs hving the sme lphbets re signifintly different from eh other. of the fruits with vlues of ± 3.57 μmol TE/g smple nd 12.48±.21 μmol TE/g smple, respetively (Figure 5). In this study, the ntioxidnt ontent of the queous plnt extrts pturedthefreerdil(abts)ndresultedinolorloss nd onsequent redution in bsorbne whih orresponds to the ntioxidnt onentrtion Oxygen Rdil Absorbne Cpity in the Aqueous Extrts of the Leves nd Fruits of Nule ltifoli. The ORAC ssy is used to ssess the ntioxidnt tivity of biologil substrtes whih rnges from pure ompounds, tht is, meltonin nd flvonoids, to omplex mtries suh s vegetbles nd niml tissues [46]. It mesures ntioxidnt inhibition of peroxyl-rdil-indued oxidtions nd shows the rdil hin breking ntioxidnt tivity by H- tom trnsfer [24, 31]. The ORAC ssy uses 2,2 -zobis(2- midinopropne) dihydrohloride (AAPH) for free rdil genertion. AAPH whih is wter soluble hs been widely used s free rdil inititor for biologil studies nd the hemolysis used by AAPH llows studies involving membrne dmge indued by free rdils [47 5]. The results showed the men vlue of ORAC for queous extrt of the leves to be ±.73 μmol TE/g smple while tht of the fruits ws ±.73 μmol TE/g smple (Figure 5). The ORAC results lso showed the poteny of the queous plnt extrts to protet ginst oxidtive dmge. In onlusion, the results from this study reveled tht the queous extrts of both the leves nd the fruits of Nule ltifoli hve ntioxidnt potentils with the leves demonstrting stronger tivities. The mediinl properties of this plnt ould be due to its ntioxidnt potentils s evident from this present work. More studies re however needed for investigting its usefulness in the mngement nd tretment of vrious diseses. Aknowledgment This work ws supported through funding from Cpe Peninsul University of Tehnology, Bellville, South Afri. Referenes [1] S. krovánková, L. Mišurová, nd L. Mhů, Antioxidnt tivity nd proteting helth effets of ommon mediinl plnts, Advnes in Food nd Nutrition Reserh,vol.67, pp , 212. [2] N.A.Khlf,A.K.Shky,A.Al-Othmn,Z.El-Agbr,ndH. Frh, Antioxidnt tivity of some ommon plnts, Turkish Biology,vol.32,no.1,pp.51 55,28. [3]T.P.A.Devsgym,J.C.Tilk,K.K.Boloor,K.S.Sne,S. S. Ghskdbi, nd R. D. Lele, Free rdils nd ntioxidnts in humn helth: urrent sttus nd future prospets, Assoition of Physiins of Indi,vol.52,pp ,24. [4] O. Jmes nd H. H. Ugbede, Hypoholesterolemi effets of Nule ltifoli (Smith) fruit studied in lbino rts, Amerin JournlofTropilMediinendPubliHelth,vol.1,no.1,pp , 211. [5]S.D.Krou,T.Thondo,D.P.Ilboudo,ndJ.Simpore, Sub-shrn rubiee: review of their trditionl uses, phytohemistry nd biologil tivities, Pkistn Biologil Sienes,vol.14,no.3,pp ,211. [6] I.I.Nkfmiy,A.J.Mnji,U.U.Modibbo,ndH.A.Umru, Biohemil evlution of Cssipoure ongoensis (Tunti) nd Nule ltifoli (Luzzi) fruits, Afrin Biotehnology, vol.5,no.24,pp ,26. [7] A. D. T. Goji, A. Mohmmed, Y. Tnko, I. Ezekiel, A. O. Okpnhi, nd A. U. U. Dikko, A study of the ntiinflmmtory nd nlgesi tivities of queous extrt of Nule ltifoli leves in Rodent, Asin Medil Sienes,vol.2,no.6,pp ,21. [8] A. Giddo, D. A. Ameh, nd S. E. Atwodi, Effet of Nule ltifoli leves queous extrts on blood gluose levels of norml nd lloxn-indued dibeti rts, Afrin Biotehnology,vol.4,no.1,pp.91 93,25. [9] A. Giddo, D. A. Ameh, S. E. Atwodi, nd S. Ibrhim, Hypoglyemi tivity of Nule ltifoli sm. (Rubiee) in experimentl nimls, Afrin Trditionl, Complementry nd Alterntive Mediines, vol.5,no.2,pp.21 28, 28. [1] O. N. Miter, M. E. Khn, nd T. F. Jmes, Phytohemil nlysis nd the hemotherpeutis of leves nd stem-brk of Nule ltifoli grown in Hong, Admw Stte Nigeri, Asin Plnt Sienes,vol.1,no.3,pp.16 22,211. [11] F. Benoit-Vil, A. Vlentin, V. Courn, Y. Pélissier, M. Mllié, nd J.-M. Bstide, In vitro ntiplsmodil tivity of stem nd root extrts of Nule ltifoli S.M. (Rubiee), Ethnophrmology, vol. 61, no. 3, pp , [12] P. I. Akubue nd G. C. Mittl, Clinil evlution of trditionl herbl prtie in Nigeri: preliminry report, Ethnophrmology,vol.6,no.3,pp ,1982.

7 The Sientifi World Journl 7 [13]Z.A.M.Nworgu,D.N.Onwukeme,A.J.Afolyn,F.C. Amehin, nd B. A. Ayinde, Preliminry Studies of blood lowering effets of Nule ltifoli in rts, Afrin Phrmology,vol.2,no.2,pp.37 41,28. [14] F. V. Udoh nd T. Y. Lot, Effets of lef nd root extrts of Nule ltifoli on the rdiovsulr system, Fitoterpi,vol. 69, no. 2, pp , [15] G. S. Tïwe, E. N. Bum, E. Tll et l., Antipyreti nd ntinoieptive effets of Nule ltifoli root deotion nd possible mehnisms of tion, Phrmeutil Biology,vol.49, no. 1, pp , 211. [16] A.M.El-Mhmood,J.H.Doughri,ndF.J.Chnji, Invitro ntibteril tivities of rude extrts of Nule ltifoli nd Dniell oliveri, Sientifi Reserh nd Essys,vol.3,no.3,pp , 28. [17] W.Okiei,M.Ogunlesi,E.A.Osibote,M.K.Binutu,ndM.A. Ademoye, Comprtive studies of the ntimirobil tivity of omponents of different polrities from the leves of Nule ltifoli, Reserh Mediinl Plnt, vol. 5, no. 3, pp , 211. [18] V. L. Singleton, R. Orthofer, nd R. M. Lmuel-Rventós, Anlysis of totl phenols nd other oxidtion substrtes nd ntioxidnts by mens of folin-iolteu regent, Methods in Enzymology, vol. 299, pp , [19] J. A. Delour nd J. D. de Vrebeke, A new olorimetri ssy for flvonoids in pilsner beers, JournloftheInstituteof Brewing,vol.91,no.1,pp.37 4,1985. [2] D. Treutter, Chemil retion detetion of tehins nd pronthoynidins with 4-dimethylminoinnmldehyde, Chromtogrphy A,vol.467,pp ,1989. [21] G. Mzz, L. Fukumoto, P. Delquis, B. Girrd, nd B. Ewert, Anthoynins, phenolis, nd olor of Cbernet Frn, Merlot, nd Pinot Noir wines from British Columbi, Agriulturl nd Food Chemistry,vol.47,no.1,pp , [22] D. Z. Zhelev-Dimitrov, Antioxidnt nd etylholinesterse inhibition properties of Amorph frutios L. nd Phytol merin L, Phrmognosy Mgzine, vol. 9, no. 34, pp , 213. [23] I. F. F. Benzie nd J. J. Strin, The ferri reduing bility of plsm (FRAP) s mesure of ntioxidnt power : the FRAP ssy, Anlytil Biohemistry, vol. 239, no.1,pp. 7 76, [24] B.Ou,M.Hmpsh-Woodill,ndR.L.Prior, Developmentnd vlidtion of n improved oxygen rdil bsorbne pity ssy using fluoresein s the fluoresent probe, Agriulturl nd Food Chemistry,vol.49,no.1,pp , 21. [25] R.Re,N.Pellegrini,A.Proteggente,A.Pnnl,M.Yng,ndC. Rie-Evns, Antioxidnt tivity pplying n improved ABTS rdil tion deoloriztion ssy, Free Rdil Biology nd Mediine,vol.26,no.9-1,pp ,1999. [26] N. R. Frnsworth, Sreening plnts for new mediine, in Biodiversity, E. O. Wilson, Ed., Prt II, pp , Ntionl Ademy Press, Wshington, DC, USA, [27] T. Eisner, Chemil prospeting: ll for tion, in Eology, Eonomi nd Ethis: The Broken Cirles,F.E.BormnndS.R. Kellert,Eds.,YleUniversityPress,199. [28] N. Seed, M. R. Khn, nd M. Shbbir, Antioxidnt tivity, totl phenoli nd totl flvonoid ontents of whole plnt extrts Torilis leptophyll L, BMC Complementry nd Alterntive Mediine,vol.12,rtile221,212. [29] F. Anwr, A. Jmil, S. Iqbl, nd M. A. Sheikh, Antioxidnt tivity of vrious plnt extrts under mbient nd elerted storge of sunflower oil, Grss y Aeites,vol.57,no.2,pp , 26. [3] T. Osw, S. Kvkishi, M. Nmiki, Y. Kurod, D. M. Shnkl, nd M. D. Wters, Antimutgenesis nd Antirinogenesis Mehnisms II, Plenum, New York, NY, USA, 199. [31] A. Krdg, B. Ozelik, nd S. Sner, Review of methods to determine ntioxidnt pities, Food Anlytil Methods,vol. 2, no. 1, pp. 41 6, 29. [32] M. P. Kähkönen,A.I.Hopi,H.J.Vuoreletl., Antioxidnt tivity of plnt extrts ontining phenoli ompounds, Agriulturl nd Food Chemistry, vol.47,no.1,pp , [33] M. Suhj, Spie ntioxidnts isoltion nd their ntirdil tivity: review, Food Composition nd Anlysis, vol. 19, no. 6-7, pp , 26. [34] M. Öztürk, F. Aydoǧmuş-Öztürk, M. E. Duru, nd G. Topçu, Antioxidnt tivity of stem nd root extrts of Rhubrb (Rheum ribes): n edible mediinl plnt, Food Chemistry,vol. 13, no. 2, pp , 27. [35] M. Mizur, A. Aminh, nd W. M. Wn Aid, Totl phenoli ontent nd ntioxidnt tivity of kesum, ginger (Zingiber offiinle) nd turmeri (Curum long) extrt, Interntionl Food Reserh Journl,vol.18,pp ,211. [36] N. Gherrf, S. Ldjel, B. Lbed, nd S. Hmeurline, Evlution of ntioxidnt potentil of vrious extrts of Trgnum nudtumdel, Plnt Sienes Feed,vol.1,no.9, pp ,211. [37] K.Shukl,P.Dikshit,M.K.Tygi,R.Shukl,ndJ.K.Gmbhir, Ameliortive effet of Withni ogulns on dyslipidemi nd oxidtive stress in niotinmide-streptozotoin indued dibetes mellitus, Food nd Chemil Toxiology, vol. 5, no. 1, pp , 212. [38] H. B. Niemeyer nd M. Metzler, Differenes in the ntioxidnt tivity of plnt nd mmmlin lignns, Food Engineering,vol.56,no.2-3,pp ,23. [39] P.-J.Tsi,J.MIntosh,P.Pere,B.Cmden,ndB.R.Jordn, Anthoynin nd ntioxidnt pity in Roselle (Hibisus sbdriff L.) extrt, Food Reserh Interntionl, vol. 35, no. 4, pp , 22. [4] C.Priynk,D.A.Kdm,A.S.Kdm,Y.A.Ghule,ndV.T. Aprdh, Free rdil svenging (DPPH) nd ferri reduing bility (FRAP) of some gymnosperm speies, Interntionl Reserh Botny,vol.3,no.2,pp.34 36,213. [41] Y.Li,C.Guo,J.Yng,J.Wei,J.Xu,ndS.Cheng, Evlutionof ntioxidnt properties of pomegrnte peel extrt in omprison with pomegrnte pulp extrt, Food Chemistry, vol. 96, no. 2, pp , 26. [42] A. D. Gupt, V. Pundeer, G. Bnde, S. Dhr, I. R. Rngnth, nd G. S. Kumri, Evlution of ntioxidnt tivity of four folk ntidibeti mediinl plnts of Indi, Phrmologyonline, vol.1,pp.2 28,29. [43] A. L. Rie nd J. B. Burns, Moving from effiy to effetiveness: red plm oil's role in preventing vitmin A defiieny, Journl of the Amerin College of Nutrition, vol.29,no.1,supplement, pp. 32S 313S, 21. [44] L. Wng, S. Liu, A. D. Prdhn et l., Plsm lyopene, other rotenoids, nd the risk of type 2 dibetes in women, Amerin Epidemiology, vol.164,no.6,pp , 26.

8 8 The Sientifi World Journl [45] H.-B. Li, C.-C. Wong, K.-W. Cheng, nd F. Chen, Antioxidnt properties in vitro nd totl phenoli ontents in methnol extrts from mediinl plnts, LWT Food Siene nd Tehnology,vol.41,no.3,pp ,28. [46] C.-T. Yeh nd G.-C. Yen, Indution of hepti ntioxidnt enzymes by phenoli ids in rts is ompnied by inresed levels of multidrug resistne-ssoited protein 3 mrna expression, Nutrition,vol.136,no. 1,pp.11 15,26. [47] Y.-J. Ci, Q.-Y. Wei, J.-G. Fng et l., The 3,4-dihydroxyl groups re importnt for trns-resvertrol nlogs to exhibit enhned ntioxidnt nd poptoti tivities, Antiner Reserh, vol. 24, no. 2, pp , 24. [48] L. Hou, B. Zhou, L. Yng, nd Z.-L. Liu, Inhibition of humn low density lipoprotein oxidtion by flvonols nd their glyosides, Chemistry nd Physis of Lipids, vol. 129, no. 2, pp , 24. [49] L. Hou, B. Zhou, L. Yng, nd Z.-L. Liu, Inhibition of free rdil initited peroxidtion of humn erythroyte ghosts by flvonols nd their glyosides, Orgni nd Biomoleulr Chemistry,vol.2,no.9,pp ,24. [5] A. A. Fdd, R. E. El-Mekwy, A. El-Shfei, H. S. Freemn, D.Hinks,ndM.El-Fedwy, Design,synthesis,ndphrmologil sreening of novel porphyrin derivtives, Chemistry, vol. 213, Artile ID 3423, 11 pges, 213.

9 Volume Volume Volume 214 Tropil Mediine Sientifi The Sientifi World Journl Autoimmune Diseses Interntionl Antibiotis Volume Volume Volume 214 Toxins Volume 214 Anesthesiology Reserh nd Prtie Volume 214 Submit your mnusripts t Advnes in Phrmologil Sienes Toxiology Volume 214 MEDIATORS of INFLAMMATION Emergeny Mediine Interntionl Volume 214 Pin Reserh nd Tretment Volume Volume 214 Addition Volume 214 Stroke Reserh nd Tretment Volume 214 Vines BioMed Reserh Interntionl Volume 214 Phrmeutis Volume 214 Interntionl Mediinl Chemistry Volume Volume 214 Drug Delivery Volume 214