December 30, 2015 Archives 2015 vol.3 149-159 EVALUATION OF THE ANTIOXIDANT CAPACITY AND CHARACTERIZATION OF PHENOLIC COMPOUNDS OBTAINED FROM TEA (CAMELLIA SINENSIS) FOR PRODUCTS OF DIFFERENT BRANDS SOLD IN COLOMBIA. Rmiréz-Aristizbl, L.S.*; Ortiz, A.; Ospin-Ocmpo, L.F. Universidd Tecnológic de Pereir, Colombi *lurmire@utp.edu.co Abstrct The ctive compounds from nturl sources, hve become n interest within the scientific community, especilly phenolic compounds due to their effects on helth nd nutrceuticl use s food,[1]. The consumption of green te in Colombi is recent trend nd the mrket is continuously growing, then the most common commercilly vilble types of green te were tested in this study; Orientl, Lipton, Hindu nd Jibel. The objective of this work ws to determine the totl polyphenol content nd in-vitro ntioxidnt cpcity of green te commercilized in Colombi. The ntioxidnt cpcity ws determined by the 1,1- diphenyl-2-picrylhydrzyl (DPPH) free-rdicl scvenging ssy nd the oxygen rdicl bsorbnce cpcity (ORAC) ssy. A similr profile ws observed for the ntioxidnt cpcity determined by both methods; 748.09 ± 39.76 to 1138.45 ± 78.03 μmol Trolox equivlent / g dry smple (µmol TE/g smple) for DPPH ssy, nd 740.83± 37.81 to 1588.05± 21.72 03 μmol Trolox equivlent / g dry smple for ORAC ssy. The totl polyphenol concentrtion in green te ws found to vry 93.76 ± 1.38 to 127.74 ± 3.32 mg gllic cid equivlents (GAE) / g smple, the totl flvonoid content (TFC) vry 10.42 ± 0.52 to 29.15 ± 2.04 mg ctechin equivlent/g smple. The ntioxidnt ctivities were well correlted with the totl polyphenol content (r 2 =0.9911) for the ORAC method nd totl flvonoids (r 2 = 0.997) with DPPH ssy. In generl the behvior of ll smples ws Orientl Lipton> Hindu> Jibel both TPC nd TFC s the biologicl ctivity. A method for identifying the ctechins present, cffein, orgnic nd phenolic cids in green te ws developed by high performnce liquid chromtogrphy (HPLC); the chromtogrphic profiles showed the presence of ten compounds including the most bundnt were, gllic cid, cffeine nd epiglloctechin gllte (EGCG) to which they rettributes his ntioxidnt cpcity. This is the first systemtic screening for the identifiction of polyphenols nd ntioxidnt ctivity in te commercilized in Colomib. Keywords: Antioxidnt ctivity, green te, high-throughput, phenolic content.
PhOL Rmiréz Aristizbl et l 150 (149-159) Introduction Te is n infusion of Cmelli sinensis, is one of the most consumed beverge in the word, due to sensory properties, stimulting effects nd potentil helth benefits. The sinesis plnt is originlly from Southest Chin, grdully expnded to Indi, Sri Lnk, nd mny tropicls nd subtropicl countries, [1]. Te cn be divided into three ctegories on the bsis of fermenttion process, green te (non-fermented), blck te (post-fermented) nd oolong te (semi-fermented) [2]. The Americn Medicinl Assocition shows tht green te my reduce cholesterol levels, high blood pressure nd reduce the risk of strokes. The Ntionl Cncer Institute reports tht the ntioxidnts in green te cn prevent vrious types of cncer [3]. Chemicl composition of te is complex: polyphenols(ctechins nd flvonoids), lkloids (cffein, theobromine, etc), mino cids, glucosides, proteins, vltile compounds, minerls nd trce elements [4]. The te components re ffect by diferents vribles like cultivr type, growth conditions, horticulturl prctices (mechnicl-or- hnd plucking ge of leves) nd the technologies used for mnufcturing [5]. The mjor compounds in green te is ctechins, which re flvonols; these in turn re clss of flvonols which re polyphenols [6]. Studies sugest tht the helth benefits of green te re significnt due to the presence of ctechin, becuse re cpble of reducing the mount of free rdicl, nd prevent the formtion of pro-inflmmtory compounds [7]. Green te contins six primry ctechins compounds nmely ctechin (C), glloctechin (GC), epictechin (EC), epiglloctechin (EGC), epictechin gllte (ECG) nd epiglloctechin gllte (EGCG) [3]. The chemicl structure of ctechins hve been relted to their ntioxidnt cpcity, this depend on the number nd position of hidroxyl groups [8]. Therefore the present study ws crried out to determinte totl phenolic compounds nd flvonoids, set HPLC method to identify some cthechins, cffeine, glic cid, ferrulic nd clorogenic cid nd compred ntioxidnt ctivity in different brnds of te sold in Colombi, in order to estblish reltionship between structure nd biologicl ctivity. Methods Te smples Were selected the most representtive green te brnds ccordingly to informtion of the specilly shops. These te smples were purchsed from super- mrket. Four brnds were used; Orientl, Hindu, Lipton nd Jibel. Ech te brnds ws nlysed in triplicte. Extrction of green te compounds A mss of 0.2 g of ech te ws weighed nd mixed with 10 ml of cetic cid. 0.1%, nd reflux extrction method ws used for 30 minutes. Then llowed to cool to room temperture nd centrifugted t 7500 rpm for 15 minutes. The superntnt ws decnted nd brought to 15 ml, liquoted nd stored t 4 C for subsequent nlysis. Totl phenol content (TPC) Totl phenol content in te extrcts ws determinted by high-throughput ssy ccording to the modified Folin-Cioclteu method described by E. Ainsworth nd K. M. Gillespie, [9]. The extrcts were diluted (1:50) in destiled wter, 50 µl of ech smple, stndr or methnol blnk were dded to 96-well-microplte. Then 75 µl Folin regent (1:10) nd 75 µl of N 2 CO 3 7.5% were dded nd homogenized, the bsorbnce ws red t 760 nm fter 60 minutes in microplte reder (Thermo Multiscn Go, serie number 1510-01100, SknIt Softwre 3.2.1.4 RE for Multiskn GO (es)) The phenolic compounds were quntified using clibrtion curves of gllic cid nd expressed s mg Glic cid equivlent / g smple (mg GAE/g smple). Totl flvonoid content. (TFC) Totl flvonoid content in te extrcts ws determinted by high-throughput. Briefly 15 µl of NNO 2 5% ws dded in ech well, except the blnk (15 µl of wter) nd mixed with 100 µl of te extrct (1:25) nd/or Ctechin stndrd in 96 wellmicroplte nd left durnig 5 minutes followed by 15 µl of AlCl 3, mixed nd left for 6 minutes t room temperture in the drk; finlly 70 µl of NOH 1M ws dded. The bsorbnce ws red in 5 minutes t 500 nm over microplte reder (Thermo Multiskn Go, serie number 1510-01100, SknIt Softwre 3.2.1.4 RE for Multiskn GO (es)). The flvonoids contents were quntified using clibrtion curves of Ctechin nd expressed s mg ctechin equivlent/g smple. (mg CE/g smple. Antioxidnt Activity DPPH ssy The DPPH ssy ws crried out by high-throughput ccording to the modified method of R. Fernndez- Orozco, [10]. A volume of 25 µl of te extrc (150 ppm in Methnol) nd/or Trolox stndr with the
PhOL Rmiréz Aristizbl et l 151 (149-159) respective controls; Positive control ws Hidroquinone 1000 ppm, negtive control ws Methnol 96%, in 96-well plte ws dded, followed by 100 µl of 50 ppm of DPPH metnolic solution to ech well, except the blnk smple. The mixture ws incubted in the drk t room temperture for 30 minutes, nd the bsorbnce t 517 nm ws mesured by microplte reder (Thermo Multiskn Go, serie number 1510-01100, SknIt Softwre 3.2.1.4 RE for Multiskn GO (es)). The scvenging cpcity ws clculted s: % A.A= [A c (-) A te /Ac (- )]*100, where Ac (-) is the bsorbnce of the control nd A te is the bsorbnce of the tested smple. Trolox ws used s stndr. Free rdicl scvenging cpcities of te were expressed s µmol Trolox Equivlent/ g smple. (µmol TE/g smple) ORAC ssy Antioxidnt ctivity of the diferent tes ws lso ssessed with the ORAC (Oxigen Rdicl Absorbnce Cpcity) ssy cording to the method of K. M. Gillespie [11]. Briefly in ech well of solid white 96 well-microplte, 187 µl of 80 nm florescein disolved in 75 mm PBS (phosphte buffered sline) ws dded followed by 31 µl of te previously diluted 1000 times in PBS. After 15 minutes incubtion in the drk t 37 C, 31 µl of 140 mm AAPH [2,2 -zobis 2- methylpropionmidine) dihydrochloride] were rpidly dded to ech well nd fluorescense recorded from the top every 120 second until fluorescence decyed, using excittion wvelength of 493 nm nd n emmision filter of 515 nm using fluorescence Spectrophotometer (Vrin, Cry Eclipse, version 1.1(135)) The net AUC (re under the fluorescence decy curve) for ech smple/stndrd ws obteined by subtrcting the re of the blnk smple (PBS). Antioxidnt ctivity ws expressed s µmol Trolox equivlent/ g smple (µm TE/g smple) using the liner regression vlue obtined from the trolox clibrtion curve. Determintion of te compounds by High Pressure Liquid Chromtogrphy (HPLC). Te infusions were nlysed on reverse phse high performnce liquid chromtogrphic system (Hitchi LChrom with ultrviolet detector (Hitchi L-2420 UV-Vis) n injection volume of 20 µl, Column Oven L-2300, Pum L-2130. The seprtions were performed using C18 reverse phse column (Ultr AQ C18 150 x 3.2 mm i.d, 3 µm Prticle, Ser# 12031031M, Ct# 9178313, Lot#110836P) nd column temperture ws mintined t 35 C. Softwre used ws EZChrom Elite dt system, version 3.3.1 SP1) the phse mobile consisted of cetic cid (0.5% in wter, solvent A) nd mix of cetonitrile: Etile cette: cetic cid 0.1% in wter (10:2:88 (v/v) solvent B). Grdient elution procedure is presented in tble 1. Peks were identified by compring smple retention times to those of utentic stndrd. UV- detection ws perfromed t 270 nm. Before the HPLC nlysis, the extrcts were filtered through polytetrfuoroethylene (PTFE) membrne crtridge. Experimentl design All mesurements were crried out in triplicte nd the results re sttisticlly nlyzed using the Grhpd prims progrm to determine the verge vlue nd stndrd error nd ANOVA, tukey s Multiple Comprision Test, (significnt p< 0.05), were performed to determine significnt differeces. Results Totl phenol content (TPC) The totl phenolic content of the 4 brnds green te re shown in figure 1 nd tble 2, were quntified using clibrtion curves of gllic cid (5-100 mg/l) performed every dy of the ssy. The totl phenolic compounds were found between 93.76-127.74 mg glic cid Equivlent /g smple. The highest levels ws mesured in Orientl brnd (127.74 ±3.32 mg equivlent Glic cid / g smple), similr mounts were lso obtined in Lipton (114.70 ±4.61 mg Glic cid equivlent / g smple) nd Hindu (99.06 ± 2.96 mg Glic cid equivlent / g smple), while Jibel contined the lowest mount (93.76 ± 1.38 mg Glic cid equivlent / g smple). Totl flvonoid content. (TFC) The totl flvonoid content of the te extrct were reltively low compred with TPC, nd rnged from 10.42 ± 0.52-29.6 ± 1.43mg ctechin equivlent / g smple, (see figure 2 nd tble 2) nd quntified using clibrtion curve of ctechin (5-70 mg/l) performe every dy of the ssy. The highest levels ws mesured in Orientl brnd (29.61 ±1.43 mg ctechin equivlent / g smple), similr mounts were lso obtined in Lipton (29.15 ± 2.04 ctechin equivlent / g smple) nd Hindu ( 21.02± 0.77 mg ctechin equivlent / g smple), while Jibel contined the lowest mount (10.42± 0.52 mg ctechin equivlent / g smple). Antioxidnt Activity The ntioxidnt ctivity of the te infusions ws
PhOL Rmiréz Aristizbl et l 152 (149-159) evluted using two independent ssys, DPPH nd ORAC. A clibrtion curve of trolox (6.5-100 um) llowed to compre ntioxidnt ctivity in different brnds of te expressed s μmol Trolox equivlent / g dry smple. The results obtined from DPPH ssy reported in the figure 3 nd tble 2, shown vlues rnged from 748.09 ± 39.76 to 1138.45 ± 78.03 μmol Trolox equivlent / g dry smple, similr to the vlues obtined by ORAC ssy reported in the figure 4, which hve rnge from 740.83± 37.81 to 1588.05± 21.72 03 μmol Trolox equivlent / g dry smple. The Orientl brnd te hd the highest DPPH nd ORAC vlues while the Jibel brnd showed the lowest. Determintion of te compounds by High Pressure Liquid Chromtogrphy (HPLC) The romtic structurl similirity of the green te ctechins mde the seprtion difficult, but HPLC method hs become technique to seprte properly [12]. Compring the retention times estblished by mixing stndrds (see figure 7), llowed to identify nd seprte t lest ten of the eleven compounds in the te extrcts. (See figure 8 to 11). The peks corresponding to gllic cid, cffeine nd EGCG re the most bundnt in ll brnds of te. Jibel presented differences in composition nd ntioxidnt ctivity s it did not lodged ctechin nd ws the only one tht showed even in smll proportions ferulic cid. Discussion Currently, nutrceuticls re becoming prt of the dily diet, becuse the current lifestyle could generte mny diseses which led to the scientific community to the serch for nturl sources of compounds tht help to mintin blnce in the consumer helth. At present, the consumption of green te incresed due to studies tht reported number of helth benefits ssocited with consumption reducing crdiovsculr diseses, ction ginst some cncers, inflmmtory diseses, dibetes nd weight loss [5]. The present study ws crried out to determinte how ntioxidnt ctivity vries from the diferents brnds of green te in order to ssocite the ntioxidnt ctivity with the content of flvonoids nd phenolic compounds to estblish reltionship between the structure nd the bility to remove free rdicls. Te extrction is one of the most fctors ffecting the nlyticl results; the uthors recommend wter s the solvent of choice due to toxicologicl resons. Is sid to temperture of 80 C nd time of 30 minutes re the optiml conditions, while the wter to te rtio nd the prticle size (round 1 mm) re of importnce[7], [13]. The differents smples were different prticle size nd the extrcts showed diferents colours especilly Jibel tht ws pink nd the other ones were yellow. The Folin-Cioclteu ssy relies on the trnsfer of electrons in lkline medium from phenolic compounds to phosphomolybdic/phophotunsting cid complexes form blue which llows msure of totl phenols nd other oxidtion substrtes. Totl phenolic content (TPC) of green te re presented en tble 2, the highest content ws Orientl (127.74 ± 3.32 mg cid glic equivlent/g smple) nd the lowest ws Jibel (93.76 ± 1.38 mg cid glic equivlent/g smple). In generl the four brnds showed decresing behvior Orientl Lipton>Hindu>Jibel. An nlysis of vrince ANOVA (tukey s Multiple Comprision Test, significnt p< 0.05), identified significnt differeces between Orientl, Hindu nd Jibel. (b, cd,cd) nd no significnt differences were found between Orientl nd Lipton(b,b) (Figure 1). D. Horžić et l compred TPC between herbls infusions nd te, the results showed tht green te hve much higher content of phenols (1380-1830 mg/l gllic cid equivlents) s flvonoids (1070 1280 mg/l gllic cid equivlents) nd these results re comprble with those obtined in this study; if expressed in mg/l gllic cid equivlents (tble 2) rnge between 1250.12 to 1703.22 mg/l cid gllic equivlent (Jibel nd Orientl) is obtined [14]. Another study evluted the TPC in differents brnds mrketed in Chile, showed tht the rnged of totl phenols ws 947.6-1678 mg/l gllic cid equivlents in green te nd 880.7 to 1822.5 mg/l gllic cid equivlents in blck te[15]. In study of severl brnds of the green, of commercilly vilble te in Argentin, lso determined tht generlly green te leves hd higher totl phenol content (14 to 21 g/100 g) [16], these ltest re reltively close to our results. The results of TPC lso were in ccordnce with A. Luximon-Rmm [12] showing content from 62 to 107 mg/g for totl phenols content in nine commercilly blck te, nd 184 ± 36 mg/g in infusions of fresh te leves, however, It is importnt to note the difference in the extrction process. As one possible reson why in our study higher content ws obtined due to temperture conditions nd extrction time. In generl ll smples tested showed high levels of flvonoids nd phenolic compounds. The differences found between brnds my be due to mnufcturing process used by ech industry, prticle size nd other compounds s if Jibel who contins hibiscus nd lemon peel.
PhOL Rmiréz Aristizbl et l 153 (149-159) Totl Flvonoid content (TFC) of green te re presented en tble 2, the highest content ws Lipton (29.15 ± 2.04 mg ctechin equivlent/g smple) nd Orientl (29.61 ± 1.43 mg ctechin equivlent/g smple), while the lowest ws Jibel (10.42 ± 0.52 mg ctechin equivlent/g smple). In generl the four brnds showed decresing behvior Liptonl Orientl >Hindu >Jibel. An nlysis of vrince ANOVA (tukey s Multiple Comprision Test, significnt p< 0.05), identified significnt differeces between Orientl, Hindu nd Jibel. (, b, c) nd no significnt differences were found between Orientl nd Lipton(). (Figure 2). The results of TFC lso were in ccordnce with A. Luximon-Rmm [12] showing content from 15 to 26 mg/g for totl flvonoids in nine commercilly blck te, nd 34± 5 mg/g in infusions of fresh te leves, while in nother study the extrction ws performed with methnol:hcl (50: 1) for 1 hour, nd showed lower content of flvonoids (0,671 ±0.041 g/kg) [17] thn those reported in this study. The ntioxidnt ctivity ws determinted by plictions of the DPPH nd ORAC method. An estimte of the ntioxidnt properties of pure compounds or extrcts re their bility to trp free rdicls, one of the most populr is the method employing stble, 2,20-diphenyl-1- picrylhydrzylrdicl (DPPH. ) [18]. During this ssy, the purple chromogen rdicl is reduced by ntioxidnt/reducing compounds to the corresponding ple yellow hydrzine. The reduction of the purple chromogen rdicl by hydrogen-donting ntioxidnts is monitored by the decrese of opticl density t long wvelengths (515 520 nm)[19]. The oxygen rdicl bsorbnce cpcity (ORAC) ssy hs been widely used to mesure the ntioxidnt ctivity of nutrceuticls, phrmceuticls nd foods, mesure the clssicl bility of n ntioxidnt to quench free rdicls by hydrogen dontion[11]. The results re considered by some to be of biologicl relevnce s reference for ntioxidnt effectiveness. The ntioxidnt ctivity of green te re presented in tble 2, the highest content ws Lipton (1123.39± 147.87 µmol equivlent Trolox/g smple) nd Orientl (1138.45 ± 78.03 µmol equivlent Trolox/g smple) determined by DPPH ssy, while the lowest ws Jibel (748.09 ± 39.76). In generl the four brnds showed decresing behvior Liptonl Orientl >Hindu >Jibel. An nlysis of vrince ANOVA (tukey s Multiple Comprision Test, significnt p< 0.05), identified significnt differeces between Orientl nd Jibel. (, b) nd no significnt differences were found between the rest of smples. See figure 3. The results obtnied by ORAC llowed to see tht the highest ntioxidnt ctivity ws to Orientl te (1588.05 ± 21.72) nd the lowest ws Jibel (740.83 ± 37.81), nd their behvior of ll smples ws Orientl>lipton>Hindu>Jibel. Overll ntioxidnt ctivity of ll smples showed the sme behvior ssessed by two methods; however ORAC dt obtined re slightly higher. The ORAC ssy provides mesure of both the generl nd specific ntioxidnt ction of plnt tissue extrcts nd cn be used in combintion with DPPH ssy to compre nd get better results, both methods re rpid, simple, low cost nd used for food, beverges nd plnts. Jibel showed the lowest levels of phenolic compounds, flvonoids nd ntioxidnt ctivity, possibly due to the ddition of hibiscus nd lemon peel tht could msk other compounds. M. Jeszk-Skowron nd A. Zgoł-Grześkowik showed tht the ntioxidnt ctivity of two out of four pure green te infusions ws 2- or even 3-fold higher thn green te with fruits or quince, green te witht leemon hd good results, these findings could provide informtion tht dditives such s jsmine petls nd lemon skin re excellent ntioxidnts, but it is lso probble tht cheper tes of worse qulity were used for the production of the romtized tes [20]. The ntioxidnt ctivity of Lipton ws compred by other uthor (1000 µmol equivlent Trolox/g smple) who determined the ntioxidnt cpcity of twenty-four commercil green te vrieties give vlues close to those obtined in our study[21]. The different ntioxidnt cpcity exhibited by polypehnols is consistent with their chemicl structure in regrd to number nd position of phenolic hidroxyl groups [22]. The Orientl nd Lipton te extrct showed better ntioxidnt ctivity compred to Jibel nd simultneously these two smples showed the highest nd lowest content of phenolic compounds nd flvonoids respectively, showing corelcion between the ctivity-structure becuse the ntioxidnt ctivity determined by the ORAC method showed correltion with the totl phenolic content (r 2 =0.9911) see figure 5 nd DPPH with totl flvonoids (r 2 = 0.997) see figure 6. Green te is n unfermented te, for this reson is rich in polyphenols. A tipicl chromtogrm of te extrcts is shown in figure 7, while tble 2 shown lists the retention time of the stndrs. Compring the retention times estblished by mixing stndrds, llowed to identify t lest 10 of the 11 compounds in the extrcts. The peks corresponding to gllic cid, cffeine nd EGCG re the most
PhOL Rmiréz Aristizbl et l 154 (149-159) bundnt in ll brnds of te (Figure 8-11). Though lso it evidenced the presence of Trigonelline, GC, EGC, chlorogenic cid, ctechin, EC, Ferulic cid nd ECG. The ntioxidnt ctivity my be relted to the high content of these compounds especilly EGCG who hs more hydroxyl groups within the structure, lso compred to the literture reported where higher contents in te. These results cn be compred with those reported by C. Wu, H. Xu, J. Héritier, nd W. Andluer[23] where EGCG ws the mjor ctechin in ll te vrieties, rnging from 44.6% to 53.7% of the totl ctechins, similr to most te vrieties in Chin. On the other hnd the extrction process lso influenced the results becuse the reflux extrction method hs been used most often in the extrction of green te cffeine nd ctechins[24]. 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mg Eq Acid Gllic Equivlent/ g smple) mg Eq Acid Gllic Equivlent/g smple PhOL Rmiréz Aristizbl et l 155 (149-159) 14 12 b cd b cd 10 8 6 4 2 ORIENTAL HINDU LIPTON JAIBEL Figure 1. Totl phenol content (TPC). 35,00 3 25,00 b 2 15,00 1 c 5,00 ORIENTAL HINDU LIPTON JAIBEL Figure 2. Totl flvonoid content. (TFC)
µmoles Trolox Equivlent/g smple µmoles Trolox Equivlent /g smple PhOL Rmiréz Aristizbl et l 156 (149-159) 140 120 100 80 b 60 40 20 ORIENTAL HINDU LIPTON JAIBEL Figure 3. Antioxidnt ctivity by DPPH 180 160 b 140 120 100 80 c c 60 40 20 ORIENTAL HINDU LIPTON JAIBEL Figure 4. Antioxidnt ctivity by ORAC
umoles Trolox Equivlent/ g smple by DPPH umoles Trolox Equivlent/ g smple by ORAC PhOL Rmiréz Aristizbl et l 157 (149-159) 180 160 140 120 100 80 60 40 y = 326,9x - 1654, R² = 0,991 20 0 2 4 6 8 10 12 TPC (mmol/l Acid Gllic Equivlent) Figure 5. Correltion ORAC Vs TPC 120 100 80 60 40 y = 437,7x + 543,9 R² = 0,997 20 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 TFC (mmol/l Ctequin equivlent) Figure 6.Correltion DPPH Vs TFC
PhOL Rmiréz Aristizbl et l 158 (149-159) Figure 7. HPLC Chromtogrm of the stndrs Figure 8. HPLC Chromtogrm of the Jibel green te extrct Figure 9. HPLC Chromtogrm of the Lipton green te extrct Figure 10. HPLC Chromtogrm of the Lipton green te extrct Figure 11. HPLC Chromtogrm of the Hindu green te extrct
PhOL Rmiréz Aristizbl et l 159 (149-159) Tble 1. Grdient elution profile for HPLC nlysis Time Solvent Solvent Flow (min) A B (ml/min) 0 10 90 0,5 15 40 60 0,5 17 50 50 07 20 60 40 0,7 25 40 60 0,7 30 20 80 0,7 35 10 90 0,5 Totl phenol content (TPC) Tble 2. Resume, TPC, TFC, DPPH nd ORAC results of green te Totl flvonoid content. (TFC) DPPH ssy ORAC ssy Smple mg/g eq mg/l eq mg/g Eq µmolestrolox µmolestrolox mg/l eq CAT AG AG Ctechin Eq/g smple Eq/g smple um Trolox Eq Orientl 127.74 ± 3.32 1703 ±10.01 29.61 ± 1.43 394.85±19.03 1138.45 ± 78.03 1588.05 ± 21.72 21,17±0.29 Hindu 99.06± 2.96 1320.80± 7.76 21.02 ± 0.77 280.25± 10.29 982.20 ± 61.57 1339.03 ± 27.90 11.57±0.37 Lipton 114.70 ± 4.61 1529.38 ± 8.99 29.15 ± 2.04 388.63±27.16 1123.39 ± 147.87 867.95 ± 68.10 17.85±0.91 Jibel 93.76 ± 1.38 1250.12 ± 7.35 10.42 ± 0.52 138.69±6.89 748.09 ± 39.76 740.83 ± 37.81 9.88±0.50 Tble 3. Composition of green te, determined by HPLC Stndr Retention Time (tr=min) # Pek Presence/Absence Orientl Lipton Hindu Jibel Trigonelline 2.5 1 X X X X Gllic Acid 3.5 2 X X X X Glloctechin 4.12 3 X X X X Epigloctequin 5.13 4 X X X X Chlorogenic cid 6.42 5 X X X X Ctechin 6.6 6 X X X Cffeine 7.63 7 X X X X Epictechin 8.41 8 X X X X EGCG 12.05 9 X X X X Ferrúlico cid 18.5 10 X ECG 26 11 X X X X