Phenolic compounds in different parts of young Annona muricata cultivated in Korea and their antioxidant activity

Transcription

1 Appl Biol Chem (2017) 60(5): Online ISSN DOI /s Print ISSN ARTICLE Phenoli ompounds in different prts of young Annon murit ultivted in Kore nd their ntioxidnt tivity Jin-Sik Nm 1,2,3. Seo-Yeon Prk 2. Hye-Lim Jng 2. Young H Rhee 3 Reeived: 10 My 2017 / Aepted: 26 July 2017 / Pulished online: 1 August 2017 Ó The Koren Soiety for Applied Biologil Chemistry 2017 Astrt The ntioxidnt tivities of 80% methnol nd distilled wter extrts of different prts (roots, twigs, nd leves) of young Annon murit were estimted sed on their totl phenol nd flvonoid ontent s well s in vitro 1,1-diphenyl-2-pirylhydrzyl (DPPH) nd 2,2 0 zino-is (3-ethylenzothizoline-6-sulfoni id) (ABTS) rdil svenging tivities, nitrite svenging tivity, Fe 2? - helting tivity, ferri reduing ntioxidnt power (FRAP), nd phenoli ompound ssys. The 80% methnol extrts showed higher ntioxidnt effet thn tht of the wter extrts. The 80% methnol root nd lef extrts showed higher totl phenol ( mg CAE/g) nd flvonoid ( mg RE/g) ontents thn those shown y the other extrts. In ddition, the 80% methnol root extrts showed high DPPH (EC 50 = 0.18 mg/ml) nd ABTS (EC 50 = 0.55 mg/ml) rdil s well s nitrite (EC 50 = 0.21 mg/ml) svenging tivities. The metlhelting effet of the 80% methnol twig extrt ws the highest, ut there were no signifint differenes mong the 80% methnol extrts of the different prts. FRAP vlues of ll extrts inresed in onentrtion-dependent mnner, exept for those of the distilled wter lef extrt, while the 80% methnol root extrts showed the highest & Jin-Sik Nm jsnm@sw..kr Deprtment of Food nd Nutrition, Suwon Women s University, 1098 Juseok-ro, Hwseong, Gyeonggi 18333, Repuli of Kore Food Anlysis Reserh Center, Suwon Women s University, Hwseong, Gyeonggi 18333, Repuli of Kore Deprtment of Miroiology nd Moleulr Biology, Chungnm Ntionl University, Dejeon 34134, Repuli of Kore vlue. In ddition, there ws strong positive orreltion etween the ntioxidnt tivity nd totl phenol ontent (P \ 0.01). A. murit extrts were rih in vrious phytohemils inluding rutin, epitehin, feruli id, nd p-oumri id. These findings indite tht A. murit is potentilly useful soure of sustnes with ntioxidnt effets. Keywords Annon murit Antioxidnt tivity Correltion Grviol Phenoli ompounds Introdution Retive oxygen speies (ROS) generted during metoli proesses in the humn ody re eliminted y enzymti nd non-enzymti ntioxidnt systems tht exist in the ody. However, ntioxidnt system normlities, or exess genertion of ROS due to vrious physil nd hemil ftors, indue oxidtive stress, whih uses tissue dmge nd gene muttion, resulting in vrious hroni diseses, suh s dietes, Alzheimer s disese, nd ging [1]. There is inresing interest in preventing these nd other metoli syndromes. Thus, reserh on the development of funtionl mterils from edile nturl resoures with ntioxidnt effets nd few side effets is tively in progress [2, 3]. Annon murit is tree tht elongs to the Annonee fmily, nd its fruits re used s ingredients in foods nd drinks [4]. In Kore, following the introdution of A. murit in the Siheung region of Gyeonggi in 2014, the fruit is inresingly ultivted owing to the identifition of its iotive sustnes nd iologil effets [5, 6]. A. murit ontins 212 different iotive sustnes inluding etogenins, lkloids, nd phenols. Furthermore,

2 536 Appl Biol Chem (2017) 60(5): it is known to e effetive in relieving ough ssoited with mild skin diseses nd sthm nd hs een used s trditionl mediine in Centrl nd South Ameri, West Afri, nd Southest Asi [7]. The phenoli ompounds in A. murit fruit pulp, s well s the ntioxidnt, ntidieti, nlgesi, nd nti-inflmmtory effets of queous nd ethnoli lef extrts were reently nlyzed [8 11]. Further, ording to reent studies, A. murit is effetive in wound heling, hs ntiteril tivity, redues high lood pressure, nd, in prtiulr, indues ner ell ytotoxiity, therey exerting ntiner effets [12 15]. Although A. murit is useful plnt resoure with vrious iotivities, its ntioxidnt tivity nd tive omponents re not well studied in Kore. Further, most reserh studies hve foused on the leves nd fruit, ut root- nd twig-sed sustnes re yet to e disovered. Therefore, in the present study, we evluted the extrts of different plnt prts of young A. murit ultivted in Kore to verify their ntioxidnt tivities. In ddition, we seprted the tive omponents using high-performne liquid hromtogrphy (HPLC) to identify sustnes for possile development s new funtionl produts. Mterils nd methods Experimentl mterils nd regents The A. murit used in this study ws ultivted on frms in Gwheon, Gyeonggi. Smples were slied into pproprite sizes, freeze-dried (FD-5512, Ilshin L. Co. Ltd., Gyeonggi, Kore), pulverized, filtered through 40-mesh filter, nd stored t -70 C. Folin Ciolteu s regent, 1,1- diphenyl-2-pirylhydrzyl (DPPH), 2,2-zino-is(3-ethylenzothizoline-6-sulfoni id) dimmonium slt (ABTS), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-trizine-p,p 0 - disulfoni id monosodium slt (ferrozine), 2,4,6 tris(2- pyridyl) 1,3,5-trizine (TPTZ), Griess regent, ethylenedimine tetreti id, hlorogeni id, rutin, glli id, epigllotehin, tehin, ffei id, epitehin, epigllotehin gllte, p-oumri id, gllotehin gllte, feruli id, epitehin gllte, tehin gllte, nringin, nd queretin were purhsed from Sigm- Aldrih Chemil Co. (St. Louis, MO, USA). Other regents used were of nlytil grde. Preprtion of extrts To prepre extrts, 20 g of A. murit powder ws extrted with 200 ml of distilled wter or 80% (v/v) methnol in shking wter th (BS-21, Jeio Teh Co., Gyeonggi, Kore) for 24 h, followed y entrifugtion (Avnti J-26 XPI, Bekmn Coulter, Fullerton, CA, USA) t 11,3259g for 30 min. The superntnt ws filtered through filter pper (Whtmn No. 1, Midstone, Englnd) nd then onentrted using rotry vuum evportor (R-210, Buhi, Flwil, Switzerlnd), followed y freezedrying to remove the solvent. Determintion of totl polyphenol ontent The method of Folin Ciolteu [16] ws used to nlyze the totl polyphenol ontent y treting 50 ll smples of different onentrtions with 25 ll 2 N Folin Ciolteu s regent nd 150 ll 20% sodium ironte (N 2 CO 3 ). This ws followed y inution t 20 C for 15 min, nd then, the optil density ws mesured using n enzymelinked immunosorent ssy (ELISA) plte reder (VersMx Moleulr Devies, Sunnyvle, CA, USA) t 725 nm. The totl polyphenol ontent ws lulted from stndrd lirtion urve onstruted from 0 to 200 lg/ ml hlorogeni id nd ws expressed s milligrm of hlorogeni id equivlents (CAE) per grm of extrt. Determintion of totl flvonoid ontent The method of Zhishen et l. [17] ws used to determine the totl flvonoid ontent y reting 20 ll of the prepred extrts t different onentrtions with 200 ll diethylene glyol nd 20 ll 1 N sodium hydroxide (NOH) t 40 C for 1 h. This ws followed y mesuring sorne t 420 nm using n ELISA plte reder. Rutin ( lg/ml) ws used s stndrd to onstrut lirtion urve, nd the results of the totl flvonoid ontent were lulted s milligrm of rutin equivlents (RE) per grm of extrt. DPPH rdil svenging tivity ssy A modifition of the method of Blois [18] ws used to mesure the DPPH rdil svenging tivity of the extrts. Different onentrtions of the prepred extrts (100 ll) were mixed with 900 ll 0.1 mm DPPH solution nd then were reted in the drk for 30 min. This ws followed y optil density mesurement t 517 nm using n ELISA plte reder. The DPPH rdil svenging tivity (%) is shown s the optil density rtio of the smple- nd non-treted groups, whih ws used to lulte the effetive onentrtion (EC 50 ) vlue, representing the smple onentrtion with 50% rdil svenging tivity. ABTS rdil svenging tivity ssy A prtil modifition of the method of Pellegrini et l. [19] ws used to mesure the ABTS rdil svenging

3 Appl Biol Chem (2017) 60(5): tivity. Briefly, 5 ml 7 mm ABTS nd 88 ll 140mM potssium persulfte were mixed nd kept in the drk t 20 C for 15 h to form rdils. Then, the solution ws diluted to otin n optil density vlue of 0.7 ± 0.02 t 734 nm. One milliliter of this diluted ABTS solution ws dded to 50 ll extrt, whih ws kept in the drk for 10 min, nd the optil density ws susequently mesured t 734 nm. The ABTS rdil svenging tivity (%) ws lulted s the differene in optil densities etween the smple-treted nd non-treted groups to lulte the EC 50 vlue. Nitrite svenging tivity ssy The method of Kto et l. [20] ws used to mesure the nitrite svenging tivity of the extrts. Different onentrtions of the prepred extrts (40 ll) were mixed with 20 ll of 1 mm sodium nitrite (NNO 2 ), nd then, 140 ll 0.1 N HCl ws dded to djust the ph of the retion solution to 1.2. The retion ws inuted t 37 C for 1 h. To the retion solution, 1 ml 5% (v/v) eti id nd 80 ll Griess regent were dded nd reted t 20 C for 15 min. Then, the optil density ws mesured t 520 nm. The nitrite svenging tivity (%) ws expressed s the perentge differene in optil density etween the smple-treted nd non-treted groups to lulte the EC 50. Metl (Fe 21 )-helting tivity ssy The method of Dinis et l. [21] ws modified nd used to mesure the Fe 2? -helting tivity. Briefly, different onentrtions of the prepred extrts (1 ml) were treted with 25 ll 2 mm FeCl 2 nd 5 mm ferrozine t 20 C for 10 min, nd the optil density ws then mesured t 562 nm. The Fe 2? -helting tivity ws expressed s the perentge differene in optil density etween the smple-treted nd non-treted groups to lulte the EC 50 vlue. Ferri reduing ntioxidnt power (FRAP) ssy The FRAP ssy ws performed using the method of Benzie nd Stin [22] to mesure the ntioxidnt tivity. Briefly, 30 mm ette uffer (ph 3.6), 10 mm TPTZ, nd 20 mm FeCl 3 6H 2 O were mixed t 10:1:1 rtio to produe retion solution (700 ll), whih ws used to tret different onentrtions of the formulted extrts (100 ll) for 30 min in the drk. The optil density ws mesured using n ELISA plte reder t 590 nm. The FRAP vlues were lulted s lm trolox equivlents (TEs) using lirtion urves onstruted with trolox s the stndrd. Anlysis of phenoli ompounds using HPLC The extrt ws dissolved in 50% methnol, filtered using syringe filter (0.45 lm). The solution (10 ll) ws injeted into n HPLC system (Agilent 1200 series, Agilent Tehnologies, Plo Alto, CA., USA.) tht ws equipped with Gemini C 18 olumn (3-lm, mm, Phenomenex, Cslehio di Reno, Bologn, Itly). The detetor used for the nlysis ws diode-rry detetor (DAD) t 220 nm, nd 0.2 M phosphori id (solvent A) nd etonitrile (solvent B) were used s the moile phses. The onditions for running the moile phse were 0 20 min B: 0? 15%, min B: 15? 35%, nd min B: 35? 90% t flow rte of 1.0 ml/min. Sttistil nlysis The results re presented s the mens ± stndrd devitions of t lest three independent determintions, nd the SPSS progrm (ver. 10.0, SPSS In., Chigo, IL, USA) ws used for the sttistil nlysis. One-wy nlysis of vrine (ANOVA) ws used to evlute the differenes mong the vrious extrts, nd Dunn s multiple rnge tests were onduted s the post ho nlysis. Differenes were onsidered signifint t P \ The orreltion etween the ntioxidnt sustne nd tivity t P \ 0.01 nd P \ 0.05 levels ws nlyzed y lulting Person s orreltion oeffiient to otin the oeffiient of determintion (r 2 ) vlues. Results nd disussion Yield nd totl polyphenol nd flvonoid ontent The yield, totl polyphenol, nd flvonoid ontents of the 80% methnol nd distilled wter extrts of young A. murit roots, twigs, nd leves re listed in Tle 1. The yield of the 80% methnol extrt ws the highest for the roots (11.00%), ompred to tht for the twigs (5.35%) nd leves (10.30%), nd the yield of the wter extrt ws the highest for the leves (15.50%). The yield ws higher for the wter extrts thn it ws for the 80% methnol extrts for the twigs nd leves, ut not for the roots. George et l. [23] reported tht the yield of the methnol extrt of Indin A. murit leves ws 7.12% nd tht of the distilled wter extrt ws 15.00%, similr trend to the results of our study. El-Chghy et l. [24] extrted Egyptin sugr pple leves with vrious solvents nd otined yields in the order of 50% etone (10.36%) [ distilled wter (10.34%) [ 50% ethnol (6.97%) [ 80% methnol (5.76%), whih shows similr

4 538 Appl Biol Chem (2017) 60(5): Tle 1 Yield, totl phenoli ontent, nd totl flvonoid ontent of different prts of Annon murit Yield 1) (%) TPC 4) (mg CAE/g) TFC (mg RE/g) 80% methnol Roots ± ),,3) ± 1.59 Twigs ± ± 1.63 Leves ± ± 2.35 Distilled wter Roots ± 0.66 e 3.85 ± 0.29 d Twigs ± 0.66 f 3.05 ± 0.21 e Leves ± 0.14 d ± ) Extrtion yield ws lulted s % yield = (weight of smple extrt/initil weight of smple) ) Vlues re men ± SD in triplite 3) Vlues in the sme olumn with different letters re signifintly different t P \ ) TPC totl phenoli ontent, TFC totl flvonoid ontent, CAE hlorogeni id equivlents, RE rutin equivlents trend to the results of our study, lthough the yield ws higher with A. murit thn with sugr pple. Polyphenols re seondry metolites generted through photosynthesis in the plnt. The hydroxyl groups ( OH) in polyphenols, whih hrteristilly ind with proteins nd vrious ompounds, re known to exhiit iotivities, suh s ntioxidnt, ntiner, nd ntioesity effets [25, 26]. Flvonoids re type of polyphenol with si flvone struture, nd they our in high mounts in plnt stems nd fruits. Furthermore, they hve een reported to hve vrious funtionlities, suh s ntioxidnt nd nti-inflmmtory effets [27]. The results of the totl polyphenol nd flvonoid ontent of the different prts of A. murit using different extrtion solvents re listed in Tle 1. The smples extrted with 80% methnol show the highest nd lowest totl polyphenol ontent in the roots nd twigs t ± 0.72 nd ± mg CAE/g, respetively. The distilled wter lef extrts ontined the most polyphenol t ± 0.14 mg CAE/g, while the twigs ontined the lest t ± 0.66 mg CAE/g. When the sme prts were ompred sed on extrtion solvent, the 80% methnol extrt of the roots, twigs, nd leves showed 7.8-, 6.9-, nd 4.9-fold higher ontent, respetively, thn tht of the orresponding distilled wter extrts. El-Chghy et l. [24] ompred the totl phenol ontent in sugr pple lef extrts nd found 353 ± nd 210 ± mg GAE/g in the methnol nd wter extrts, respetively. This trend ws similr to tht oserved in our study, with higher ontent in the methnol thn in the wter extrt, lthough there ws differene in the mounts extrted. Further, the results lso show lrge differene etween the totl phenol ontent of the Indin A. murit methnol lef (36.2 lg GAE/g) nd wter (19.1 lg GAE/g) extrts [23]. This vrition in ontent ould e due to the differene in the limte, suh s temperture, humidity, nd wind, in the res where A. murit ws grown nd ultivted. The totl flvonoid ontent of the 80% methnol nd distilled wter extrts of the leves ws ± 2.35 nd ± 0.12 mg RE/g, respetively, whih ws higher thn tht of roots nd twigs, while the ontent of the wter extrt of the twigs ws the lowest of the six extrts t 3.05 ± 0.21 mg RE/g. When the sme plnt prts were ompred sed on the extrtion solvent, the 80% methnol extrts showed n pproximtely 6-fold higher flvonoid ontent with ll prts thn the distilled wter extrts did. Mriod et l. [28] mesured the totl flvonoid ontent of methnol extrts of sugr pple leves nd roots, nd the results were reported to e nd mg RE/g, respetively, inditing higher flvonoid ontent thn tht of A. murit. However, oth plnt speies hd higher flvonoid levels in the leves thn in the roots. Most studies hve reported high polyphenol nd flvonoid ontent in leves, whih ould e due to the umultion of polyphenols suh s tehin, generted during photosynthesis in the leves [29]. Antioxidnt tivities Plnts ontin vrious ntioxidnt sustnes tht re produed y different mehnisms. Hene, more thn one method needs to e used to verify urtely the ntioxidnt tivity [30]. Therefore, in this study, we used five different ntioxidnt mesurement methods nd lulted the EC 50 in four of them, with the exeption of the FRAP ssy, nd the results re listed in Tle 2. For the DPPH rdil svenging tivity, the EC 50 vlues were the highest for the roots nd leves extrted with 80% methnol t 0.18 ± 0.00 mg/ml for oth, while the mgnitude of the tivity of the distilled wter extrts ws in the order of leves [ roots [ twigs. A previous study showed tht the distilled wter extrt of A. murit leves hd higher DPPH rdil svenging tivity (IC 50 = mg/ml) thn tht of ethnol extrt (IC 50 = mg/ml) [10], whih is similr to tht of the distilled wter extrt of A. murit leves otined in our study (EC 50 = 0.81 ± 0.02 mg/ml). However, the results of our study differed from those of Mriod et l. [28], who reported IC 50 vlues of methnol extrts of oth sugr pple leves nd roots to e 7.81 ± 0.1 lg/ml. Prk et l. [31] mesured DPPH rdil svenging tivities of extrts nd frtions of Ligulri fisheri Turz leves nd found tht the ethnol extrts hd higher tivities thn the wter extrts did.

5 Appl Biol Chem (2017) 60(5): Tle 2 Hlf-mximl effetive onentrtion (EC 50 ) vlues of different plnt prts of Annon murit EC 50 (mg/ml) DPPH 2) ABTS Nitrite Fe 80% methnol Roots 0.18 ± ),e,4) 0.55 ± 0.01 e 0.21 ± 0.01 d 0.39 ± 0.01 Twigs 0.24 ± 0.01 d 0.89 ± 0.03 d 0.37 ± 0.00 d 0.33 ± 0.00 Leves 0.18 ± 0.00 e 0.84 ± 0.02 d 0.32 ± 0.01 d 0.42 ± 0.01 Distilled wter Roots 1.76 ± ± ± ± 0.39 Twigs 2.15 ± ± ± ± 0.88 Leves 0.81 ± ± ± ± ) EC 50 vlues re expressed s the effetive onentrtion required to otin 50% ntioxidnt effet 2) DPPH DPPH rdil svenging tivity, ABTS ABTS rdil svenging tivity, nitrite nitrite svenging tivity, Fe Fe 2? -helting tivity 3) 4) Vlues re men ± SD in triplite Vlues in the sme olumn with different letters re signifintly different t P \ 0.05 The ABTS rdil svenging tivity results showed tht the EC 50 vlue of the 80% methnol root extrt ws the highest t 0.55 ± 0.01 mg/ml, followed y the leves nd twigs in tht order. The distilled wter extrt of the leves showed n EC 50 of 2.04 ± 0.03 mg/ml, whih ws the highest tivity, while the roots showed the lowest tivity t 5.20 ± 0.06 mg/ml. Ajoye et l. [32] reported tht 200 lg/ml lef wter extrt of ustrd pple of the genus Annon showed ABTS rdil svenging tivity tht ws [60% of the vlue tht we otined. Further, Bskr et l. [33] mesured the ABTS rdil svenging tivity of the ethnol lef extrts of Annonee plnts ultivted in Indi nd reported the IC 50 of A. murit to e 305 lg/ml, whih differed from the results of our study. The EC 50 vlue for the nitrite svenging tivity ws highest for the 80% methnol extrt of the roots t 0.21 ± 0.01 mg/ml, ut this ws not signifintly different from the vlues of the twigs or roots (P [ 0.05). For the distilled wter extrts, the leves nd roots showed the highest nd lowest tivities (EC 50 = 1.84 nd 4.91 mg/ ml), respetively, nd there ws signifint differene mong the prts (P \ 0.05). Bskr et l. [33] reported tht the lef ethnol extrts of Indin A. murit showed n IC 50 vlue of 350 lg/ml, whih is lower thn tht of the 80% methnol extrt nd higher thn tht of the distilled wter extrt of the leves mesured in our study. Further, the nitrite svenging tivity of sme solvent extrts ws higher from the leves of Cudrni triuspidt thn it ws from the stems [34], whih orresponds to the results of our study. The metl ion (Fe 2? )-helting tivity ws the highest in the 80% methnol extrt of the twigs t 0.33 ± 0.00 mg/ml, ut this vlue did not differ signifintly from tht of the roots nd leves (P [ 0.05). Furthermore, the mgnitude of the metl-helting tivity of the distilled wter extrts ws in the following order: leves [ roots [ twigs, nd the differene ws signifint (P \ 0.05). These results differed from those of the study y Öztürk et l. [35], who reported the highest metlhelting tivity in the stems when methnol extrts of rhur roots nd stems were nlyzed. In ddition, the study y Okoko nd Ere [36] reported 40% metlhelting effet of the methnol extrt of ppw tree leves t 2 mg/ml onentrtion. The FRAP ssy, whih is fst nd onvenient, is sed on the priniple tht the ferri tripyridyltrizine (Fe 3? - TPTZ) omplex is redued to ferrous tripyridyltrizine (Fe 2? -TPTZ) in n idi environment y reduing gent [22]. The FRAP vlues of different onentrtions of the 80% methnol nd distilled wter extrts of A. murit roots, twigs, nd leves were mesured, nd the results re shown in Fig. 1. Overll, the 80% methnol extrts showed higher vlues thn those of the distilled wter extrts, nd they exhiited onentrtion-dependent responses, exept t 1000 lg/ml. The 80% methnol root nd distilled wter lef extrts showed the highest FRAP tivities ( nd lm TE, respetively). George et l. [23] nlyzed the FRAP tivity of A. murit lef extrts nd reported tht the effets of methnol extrts were higher thn those of distilled wter extrts, whih orresponds to the results of our study. The results of our study re lso in greement with those of Mihel et l. [37], who reported the mgnitude of the FRAP tivity of orgni solvent extrts of se ukthorn prts to e in the following order: roots [ leves [ stems. These results demonstrte tht the ntioxidnt effet of the 80% methnol extrts ws higher thn tht of the distilled wter extrts for ll prts of A. murit. In

6 540 Appl Biol Chem (2017) 60(5): Fig. 1 Ferri reduing ntioxidnt power (FRAP) vlues of (A) 80% methnol nd (B) distilled wter extrts from different prts of Annon murit. Eh vlue represents the men ± SD of triplite mesurements. One-wy nlysis of vrine (ANOVA) ws used to evlute the differenes mong the vrious extrts, nd Dunn s multiple rnge tests were onduted s the post ho nlysis. Vlues with different letters in the sme onentrtion re signifintly different t P \ TE, trolox equivlent (A) 1,400 FRAP (μm TE) (B) FRAP (μm TE) 1,200 1, Roots Twigs Leves ,000 Conentrtion (μg/ml) Roots Twigs Leves ,000 Conentrtion (μg/ml) prtiulr, the 80% methnol extrt of the roots exhiited the highest effets in ll of the ntioxidnt evlution methods, exluding metl-helting tivity. The distilled wter extrts of the leves showed the lowest (2-fold) to the highest ([22-fold) ntioxidnt tivity of ll of the prts. These results ould e due to the different ontents of ntioxidnt sustnes in vrious plnt prts, nd the extrtion effiieny in different solvents. Phenoli ompounds To identify sustnes with ntioxidnt tivity in A. murit extrts ording to the extrtion solvent, HPLC ws used to nlyze the phenoli ompound omposition, nd the results re listed in Tle 3. From 15 potentil sustnes, 14 were deteted (queretin ws not deteted), nd the min phenoli ompounds in the 80% methnol extrt of the roots were feruli id, epitehin, nd ffei id, within the rnge of mg/ 100 g. The phenoli ompounds of the methnol extrt of the twigs were in the following deresing order: epitehin [ p-oumri id [ tehin, nd the ontents of the methnol extrt of the leves were in the order of rutin [ epitehin [ tehin gllte. In prtiulr, the rutin ontent in the methnol extrt of A. murit leves ws mg/100 g, whih ws the highest. The min phenoli ompounds in distilled wter extrts of the roots were p-oumri id, feruli id, nd epitehin gllte, whih were deteted within the rnge of mg/100 g, pproximtely one-tenth of those of the methnol extrts. In the wter extrt of the twigs, only five phenoli ompounds, inluding epitehin (86.91 mg/100 g) were deteted, nd ompounds in the wter extrt of the leves were deteted in the order of rutin [ hlorogeni id [ nringin, inditing mrked differene in the phenoli ompound ontent sed on the solvent nd plnt prt. In the study y George et l. [23], phenoli ompound nlysis of the methnol extrts of Indin A. murit leves deteted queretin, tehin, gllotehin, nd epigllotehin in the wter extrts, in ontrst to our study. Suh differene in the type nd the ontent of phenoli ompounds ould e due to environmentl ftors (i.e., the ultivtion region) nd different eluted mounts of phenoli ompounds (i.e., the extrtion method). Aording to the nlysis of the ethnol extrts of Egyptin A. murit leves y Nwwr et l. [38], phenoli ompounds suh s tehin, epitehin, hlorogeni id, nd rutin were deteted nd were reported to e the min sustnes mediting the ntioxidnt tivity of the plnt. Further, ffei id nd feruli id, whih were deteted in ll of the extrts in this study, exist undntly

7 Appl Biol Chem (2017) 60(5): Tle 3 Phenoli ompounds (mg/100 g) of different prts of Annon murit Compounds 80% methnol Distilled wter Roots Twigs Leves Roots Twigs Leves 1 Glli id ± ),,2) 3) 2 Epigllotehin ± ± Ctehin ± ± ± ± 2.40 ef ± 3.11 e ± 2.27 d 4 Chlorogeni id ± ± Cffei id ± ± ± ± 4.18 de ± 7.69 d ± 0.13 e 6 Epitehin ± ± ± ± 5.15 e ± 4.04 d ± 1.45 d 7 Epigllotehin ± ± 0.77 gllte 8 p-coumri id ± ± ± ± 10.1 d ± 2.75 e 9 Gllotehin gllte ± ± ± ± 4.48 d ± 1.15 e 10 Feruli id ± ± ± ± 5.87 d ± 0.53 f ± e 11 Epitehin gllte ± ± ± 2.73 d ± 1.25 e ± Rutin ± ± 8.32 d ± ± Ctehin gllte ± ± ± ± 0.47 d 14 Nringin ± ± ± 1.23 d ± Queretin 1) 2) 3) Vlues re men ± SD in triplite Vlues in the sme row with different letters re signifintly different t P \ 0.05 Not deteted in plnts tht effetively eliminte rdils, suh s ABTS, DPPH, nd superoxide nion. In ddition, ffei nd feruli id re known to exhiit iotivities, suh s redution of lipid oxidtion nd protetion of neurons [39, 40]. Therefore, the ntioxidnt tivity of Koren A. murit extrts is lso likely to e relted to these sustnes. Mriod et l. [28] deteted hlorogeni id (0.022 ± 0.01 mg/100 g) nd feruli id (0.011 ± 0.21 mg/100 g) in the roots, nd hlorogeni id (0.351 ± 0.35 mg/100 g), feruli id (0.014 ± 0.13 mg/ 100 g), nd p-oumri id (0.001 ± 0.01 mg/100 g) in the methnol extrts of sugr pple leves. These vlues re lower thn those of eh of the phenoli ompounds whih re in A. murit. Therefore, Koren A. murit ontins higher levels of phenoli ompounds thn the sugr pple does nd thus ould hve enhned ntioxidnt tivity. Correltion etween ntioxidnt sustne nd tivities The phenoli ompound ontents of plnts re known to e losely orrelted with their rdil svenging nd metl ion reduing tivity [41]. Therefore, the orreltion etween ntioxidnt sustne nd tivity ws evluted in extrts of different prts of the A. murit plnt ording to extrtion solvent, nd the results re represented s the r 2 (Tle 4). The totl polyphenol ontent ws highly orrelted with ll ntioxidnt tivities (P \ 0.01). In prtiulr, the r 2 vlue in the FRAP nlysis ws the highest t (P \ 0.01). The totl flvonoid ontent ws signifintly orrelted with DPPH rdil svenging tivity nd FRAP with r 2 vlues of nd (oth P \ 0.05), respetively, ut these r 2 vlues were low overll. Aordingly, the ntioxidnt tivity of A. murit is onsidered to e more ffeted y the totl polyphenol ontent thn it is y the totl flvonoid ontent. The r 2 vlue of the DPPH rdil svenging tivity ws within the rnge of (P \ 0.01) nd therefore ws highly orrelted with ll ntioxidnt tivities. Further, orreltions with tehin, ffei id, epitehin, feruli id, nd rutin were ll identified, nd in prtiulr, epitehin showed the highest orreltion (r 2 = 0.820, P \ 0.01) of ll of the phenoli ompounds. Tsi et l. [42] reported tht the tehin nd feruli id ontents re highly orrelted with DPPH nd ABTS rdil svenging tivities nd tht tehin hs lose positive reltionship with lipid peroxidtion inhiition. Aordingly, not only epitehin, ut lso tehin nd

8 542 Appl Biol Chem (2017) 60(5): Tle 4 Person s oeffiients of determintion (r 2 ) mong TPC, TFC, nd ntioxidnt tivities of vrious extrts from Annon murit TPC 1) TFC DPPH ABTS Nitrite Fe FRAP Ctehin Cffei id Epitehin Feruli id Rutin TPC * 2) 0.908** 0.893** 0.958** 0.605** 0.976** 0.882** 0.771** 0.931** 0.460** 0.250* TFC * * ** ** DPPH ** 0.859** 0.839** 0.893** 0.835** 0.584** 0.820** 0.317* 0.266* ABTS ** 0.757** 0.811** 0.895** 0.837** 0.790** 0.629** Nitrite ** 0.899** 0.914** 0.876** 0.872** 0.627** Fe ** 0.664** 0.366** 0.483** 0.226* FRAP ** 0.658** 0.872** 0.371* Ctehin ** 0.679** 0.593** Cffei ** 0.837** id Epitehin ** 0.464** Feruli id Rutin 1 1) TPC totl phenoli ontent, TFC totl flvonoid ontent, DPPH DPPH rdil svenging tivity, ABTS ABTS rdil svenging tivity, nitrite nitrite svenging tivity, Fe Fe 2? -helting tivity 2) Signifint t * P \ 0.05; ** P \ 0.01 feruli id ontents re thought to gretly ffet ntioxidnt tivity. However, the study y Thkittirungrod et l. [43] reported tht the totl polyphenol ontent hd high orreltion with reduing power, ut reltively low orreltion with rdil svenging tivity. The results ove suggest tht ntioxidnt tivity does not neessrily inrese with phenol ontent nd intertion with tive sustnes, other thn phenoli ompounds, ould result in slight differenes in ntioxidnt tivity. Therefore, tive sustnes in A. murit, suh s etogenin nd lkloids, should e studied in the future for the further development of enhned funtionl food produts. Referenes 1. Seifried HE, Anderson DE, Fisher EI, Milner JA (2007) A review of the intertion mong dietry ntioxidnts nd retive oxygen speies. J Nutr Biohem 18: Alok S, Jin SK, Verm A, Kumr M, Mhor A, Shrwl M (2014) Herl ntioxidnt in linil prtie: review. Asin P J Trop Biomed 4: Knhev VD, Ksikin OT (2013) Bio-ntioxidnts- hemil se of their ntioxidnt tivity nd enefiil effet on humn helth. Curr Med Chem 20: Srmento CD, Fri JC, Cruz FLD, Brreto WS, Gspr JW, Leite JBV (2003) Crterizçãofísi e quími de frutos de três tipos de grvioleir (Annon murit L.). Rev Brs Fruti 25: Kore Seed nd Vriety Servie (KSVS) (2015) Serhing plnt vriety protetion dtse. sitution/resister_01.jsp 6. Pinto ADQ, Cordeiro MCR, De Andrde SRM, Ferreir FR, Filgueirs HA, Alves RE, Kinpr DI (2005) Annon speies, interntionl entre for underutilised rops. University of Southmpton, Southmpton, pp Asre GA, Afriyie D, Ngl RA, Autite H, Doku D, Mhmood SA, Rhmn H (2015) Antiprolifertive tivity of queous leves extrt of Annon murit L. on the prostte, BPH-1 ells, nd some trget genes. Integr Cner Ther 14: Adewole SO, Cxton-Mrtins EA (2006) Morphologil hnges nd hypoglyemi effets of Annon murit linn. (nnonee) leves queous extrt on pnreti -ells of streptozotointreted dieti rts. Afr J Biomed Res 9: De Sous OV, Vieir GDV, De Pinho JDJR, Ymmoto CH, Alves MS (2010) Antinoieptive nd nti-inflmmtory tivities of the ethnol extrt of Annon murit L. leves in niml models. Int J Mol Si 11: Gvmukuly Y, Aou-Elell F, Wmunyokoli F, AEl-Shemy H (2014) Phytohemil sreening, nti-oxidnt tivity nd in vitro ntiner potentil of ethnoli nd wter leves extrts of Annon murit (Grviol). Asin P J Trop Med 7: Jiménez VM, Grushwitz M, Shweiggert RM, Crle R, Esquivel P (2014) Identifition of phenoli ompounds in soursop (Annon murit) pulp y high-performne liquid hromtogrphy with diode rry nd eletrospry ioniztion mss spetrometri detetion. Food Res Int 65: Adefegh SA, Oyeleye SI, Ooh G (2015) Distriution of phenoli ontents, ntidieti potentils, ntihypertensive properties, nd ntioxidtive effets of soursop (Annon murit L.) fruit prts in vitro. Biohem Res Int 2015: Essm SR, Nyegue MA, Foe CN, Silihe KK, Tmo SB, Eto FX (2016) Antiteril nd ntioxidnt tivities of hydro-ehnol extrts of rks, leves nd stems of Annon murit. Am J Phrmol Si 3: Liu N, Yng HL, Wng P, Lu YC, Yng YJ, Wng L, Lee SC (2016) Funtionl proteomi nlysis revels tht the ethnol extrt of Annon murit L. indues liver ner ell poptosis

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