Research Article Correlations between Antioxidant Activity and Alkaloids and Phenols of Maca (Lepidium meyenii)

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1 Hinwi Journl of Foo Qulity Volume 217, Artile ID 31894, 1 pges Reserh Artile Correltions etween Antioxint Ativity n Alklois n Phenols of M (Lepiium meyenii) Jin Gn, Ying Feng, Zho He, Xin Li, n Hong Zhng Reserh Institute of Resoure Insets, Chinese Aemy of Forestry, Bilongsi, Kunming, Yunnn 6224, Chin Corresponene shoul e resse to Jin Gn; Reeive 28 April 217; Revise Septemer 217; Aepte 16 Septemer 217; Pulishe 1 Otoer 217 Aemi Eitor: Ángel A. Cronell-Brrhin Copyright 217 Jin Gn et l. This is n open ess rtile istriute uner the Cretive Commons Attriution Liense, whih permits unrestrite use, istriution, n reproution in ny meium, provie the originl work is properly ite. The ntioxint pity of m hs een onsiere to e the sis for other iotivities, n reveling the tive ntioxint ompouns woul help to eluite vriety of iotive ompouns. In this stuy, the orreltion etween the ntioxint tivity of m n seonry metolites, inluing ferri reuing ntioxint potentil (FRAP), hyroxyl ril svenging ility (HRSA), lipi peroxition inhiition ility (LPIA), totl phenoli ontents (TPCs), totl lkloi ontents (TACs), n totl sterol ontents (TSCs), ws investigte y mesuring. Chloroform ws selete to e n effiient extrtion solvent for ntioxint ompouns in m y polrity frtions test. The results showe tht TPC exhiite signifint liner orreltions (P <.) to FRAP n LPIA, while TAC h signifint liner orreltions (P <.) to FRAP, HRSA, n LPIA. These results suggeste tht lklois n phenols were the most importnt sustnes for the ntioxition of m, of whih the ntioxint effet of lklois seeme to e higher thn tht of phenols. 1. Introution M (Lepiium meyenii) is plnt in the Brssiee fmily tht grows t n ltitue of pproximtely 4 to 4 meters in the Anen region of Peru. The unergroun prt of the plnt, tuerous root, is the min prout of m. The ultivtion of m hs een foun to te k s fr s the 16th entury [1]. M hs een tritionlly use for foo n to inrese nutrition n humn fertility. In reent yers, m hs gine signifint interest ue to its vriety of iotivities, suh s fertility-enhning properties [2, 3], sexul funtion improvement [4], ntiftigue [, 6], inresing immunity funtion [7], inhiition of prostte hyperplsi [8], n reuing menopuse synrome [9] tht hve een emonstrte y previous stuies. The ntioxition of m in vitro n in vivo hs een onfirme y severl reserh stuies. From n in vitro metho, the reserh of Snovl et l. [1] showe tht queous extrts of m hve the pity to svenge free rils n protet ells ginst oxitive stress. In stuy of the in vitro ntioxint tivity of ethnol extrts of m ultivte in Yunnn, ethnol extrts from 3 olore types (white, yellow, n purple) of m were shown to hve ose-epenent totl ntioxint n free ril svenging effets, s mesure y the ferri reuing ntioxint potentil (FRAP), hyroxyl ril svenging ility (HRSA), n 2,2-iphenyl-1-pirylhyrzyl (DPPH) free ril svenging pity [11]. In vivo, Večeř et l. [12] isovere tht the tivity of SOD (superoxie ismutse) in the liver, GSH-Px (glutthione peroxise) in the loo, n the level of GSH (glutthione) in the liver were ll signifintly inrese fter rts were fe foer ontining 1% m for 2 weeks. Mie from nturl ging moel were orlly given ethnol extrts of m for 6, n the tivity of GSH-Px in the liver n hert s well s SOD in the ser n liver inrese, while MDA (mlonilehye) in the ser n liver erese, suggesting tht ethnol extrts of m possess ntisenility effets y improving free ril metolism [13]. Both m stok n its ethnol extrt hve emonstrte ntioxint tivity. However, numer of reserhers hve stuie vriety of m iotivities, inluing improve growth rte [14], reue menopuse synrome [9], ntiprolifertive funtions [1], inrese swimming enurne pity [16], n enhne sperm quntity n qulity [17, 18]. These iotivities hve

2 2 Journl of Foo Qulity een hypothesize s result of ntioxition properties of m, whih re se on the pity of svenging free rils, inhiition of lipi peroxition, improve tivity of ntioxint enzymes, n ttenution of oxitive stress. Bseonthepreviouslymentionequlitiesofm, the ntioxint tivity of m seems to e highly orrelte with its other iotivities; thus, the results of stuies on the ntioxint tivity of m ompouns woul e vlule to isover vriety of iotive ompouns. M ontins seonry metolites, suh s lklois (inluing mmies), phenols, sterols, n gluosinoltes, whih re regre s its min iotive ompouns. However, to te there is lk of informtion onerning the ext iotivities of ompouns tht orrespon to prtiulr funtion. Therefore, the ojetive of this stuy ws to investigte the orreltion etween the ntioxint tivity in vitro n seonry metolites, inluing lklois, phenols, n sterols, using the ntioxint tivity guie frtiontion metho to revel the ntioxitive sustnes of m. 2. Mterils n Methos 2.1. Plnt Mterils. M (yellow) plnts were ollete in Jnury fter seven months of growth in testing grouns elonging to the Reserh Institute of Resoure Insets of Chinese Aemy of Forestry, whih is lote in the Wumeng re in Yunnn provine (Chin) t 4 m ove se level. The smples (hypootyls) were thoroughly wshe with wter n then store t 4 C until extrtion Chemils. Mtrine, glli i, β-sitosterol, n mpesterol were of stnr gre n were purhse from the Ntionl Institutes for Foo n Drug Control (Beijing, Chin). Hexne ws of hromtogrphi pure gre n ws purhse from Thermo Fisher Sientifi (Chin) Co. Lt. (Shnghi, Chin). 4,6-Tripyriyl-trizine (TPTZ) ws of iohemil regent gre n ws purhse from Sigm-Alrih (Shnghi) Tring Co. Lt. (Shnghi, Chin). Ethnol, petroleum ether, hloroform, ethyl ette, n n-utnol were of nlytil gre n were purhse from Kunming Senghu Chemil Regent Co. Lt. (Kunming, Chin). FeSO 4,FeCl 3, sliyli i, utylte hyroxytoluene, linolei i, thiorituri i, Folin Ciolteu regent, n romothymol lue were of nlytil gre n were purhse from Sinophrm Chemil Regent Co. Lt. (Shnghi, Chin) Extrtion n Seprtion. The ntioxint tivity guie frtiontion metho ws employe. The refrigerte m hypootyls were ut into slies n soke in queous ethnol (7%, v/v) t room temperture for 24 hours with soli to solvent rtio of 1 : 1 (w/v) in triplite, n the omine filtrte ws onentrte using rotry evportor (Tokyo, Jpn) uner vuum t 4 Ctoonstntweightto otin the hyroloholi extrt (HE) of m. The extrt ws suspene in eionize wter n prtitione using ifferent solvents in the following orer: petroleum ether, hloroform, ethyl ette, n n-utnol. The resiue ws the queous frtion. The prtitione extrts were onentrte to onstnt weight to otin the petroleum ether (PF), hloroform (CF), ethyl ette (EF), n-utnol (BF), n queous (AF) frtions n store t 4 C prior to nlysis of ntioxint tivities n seonry metolite ontents. The hloroform frtion ws further seprte using sili gel olumn. Briefly, the hloroform frtion ws issolve in hloroform, sili gel ws e t rtio of 1 : 1. (w/w), nthemixturewspkeintothesiligelolumnfter rying nturlly. The ompouns were elute from the sili gel olumn in the orer of inresing polrity using 6 elution solvents with ifferent polrities (mixe with petroleum n ethylettetrtiosof1:1,:1,1:1,:1,swells1%etone n 1% methnol) through C62 BUCHI meium pressure system (BUCHI Lortory Equipment Tring (Shnghi) Lt., Shnghi, Chin). The orresponing eluents were onentrte uner vuum t 4 Ctoonstnt weights to otin the onstituents lele C1, C2, C3, C4, C, n C6 n store t 4 C prior to nlysis of ntioxint tivities n etermintion of seonry metolite ontents Anlysis of Antioxint Ativities Ferri Reuing Antioxint Potentil Assy (FRAP Assy). The FRAP ssy ws performe oring to the metho of Benzie n Strin [19] with slight moifitions. Briefly, the FRAP regent ws freshly prepre y mixing 1 mm 4,6-tripyriyl-trizine (TPTZ) in 4 mm HCl, 2 mm FeCl 3, n 3 mm ette uffer (ph 3.6) t rtio of 1 : 1 : 1 (y volume) prior to evlution. A totl of 12 μl of extrts (3.2 mg/ml), 8 μl of istille wter, n 3.8 ml of FRAP regent were trnsferre into tue n inute t 37 C for 1 min. Asornes were mesure t 93 nm using Thermo Vrioskn Flsh (Thermo Fisher Sientifi, USA) reltive to regent lnk tht ws lso inute t 37 C. The FRAP t for smples were nlyze oring to stnr urve of FeSO 4 (.1.8 mmol/ml). The results were expresse s mg FeSO 4 /g extrt. Eh extrt ws ssye in triplite Hyroxyl Ril Svenging Aility Assy (HRSA Assy). Hyroxyl ril svenging ility ws mesure oring to the metho of Xu n Qin [2] with some moifitions. The retion system ontine. ml of 9 mm FeSO4,. ml of 9 mm sliyli i-ethnol, 2.8 ml of eionize wter, n 2 μl of extrts (3.2 mg/ml). The retion ws strte y ing. ml of 8.8 mm hyrogen peroxie. After inuting for 3 min t 37 C, the sorne of the retion solution ws mesure t 1 nm y Thermo Vrioskn Flsh (Thermo Fisher Sientifi, Wlthm, Msshusetts, USA) using utylte hyroxytoluene (BHT) s positive ontrol. Eh extrt ws ssye in triplite. The hyroxyl ril svenging ility ws lulte using the following formul: Ao Ai Svenging ility (%) = 1%, (1) Ao where Ai ws the sorne of the extrts or BHT n Ao ws the sorne of the ontrol group with. ml of eionize wter inste of extrts.

3 Journl of Foo Qulity Lipi Peroxition Inhiition Aility Assy (LPIA Assy). Lipi peroxition inhiition ility ws etermine oring to the slightly moifie metho of Ng et l. [21]. The retion system ontine 1. ml of linolei i emulsion,.2 ml of FeSO 4,.2mL of H 2 O 2,.6mL of extrts (3.2 mg/ml), n.4 ml of eionize wter n ws inute t 37 C for 3 min. The retion ws stoppe y ing. ml of trihloroeti i (TCA, 2%, w/w) n 2. ml of thiorituri i (TBA,.8%, w/w), then the solution ws mixe ompletely n hete t 1 Cfor 3 min. After ooling, 4 ml of n-utyl lohol ws e to leh the mlonilehye- (MDA-) TBA omplex, then the mixture ws entrifuge t 3 rpm for 1 min, n the sorne of the superntnt ws mesure t 32 nm using Thermo Vrioskn Flsh (Thermo Fisher Sientifi, USA). Butylte hyroxytoluene (BHT) ws use s positive ontrol. Eh extrt ws ssye in triplite. The inhiition ility (%) ws lulte using the following formul: Ao Ai Inhiition ility (%) = 1%, (2) Ao where Ao ws the sorne of the ontrol with eionize wter inste of extrts n Ai ws the sorne of the teste extrts. 2.. Determintion of Seonry Metolites Determintion of Totl Phenoli Content (TPC). TPC ws etermine oring to the Folin Ciolteu metho [22] with some moifitions. In rief,.2.2 g of eh extrt ws issolve in 2 ml of 7% (v/v) ethnol, nthemixturewsentrifuget3rpmfor1min. The superntnt ws filtere using filter pper, the resiue ws reextrte twie uner the sme onitions, n the omine filtrte ws onentrte using rotry evportor (EYELA, Jpn) uner vuum t 4 C. The extrt volumes were juste to 2 ml with 7% (v/v) ethnol efore they were store in the rk t 4 Cuntilfurtheruse.Anliquot of. ml of extrt prepre ove ws mixe with 4 ml of eionize wter n 1 ml (1 mol/l) Folin Ciolteu regent. After min, 3 ml of 1% (w/w) soium ronte ws e to the previous mixture n fille to 1 ml with eionize wter. The sornes of the retion prouts were mesure using Thermo Vrioskn Flsh (Thermo Fisher Sientifi, USA) t 76 nm fter 2 h of inution t 3 C. The sme proeure ws repete using ifferent onentrtions ( 6μg/mL) of glli i to plot lirtion urves. The totl phenoli ontent of eh extrt ws lulte from the glli i lirtion urve, n the results were expresse s milligrms of glli i equivlents (GAE) per grm of ry weight of extrt (mg GAE/g extrt). Eh extrt ws etermine in triplite Determintion of Totl Alklois Content (TAC). TAC ws nlyze using the metho esrie y Gn et l. [23] with slight moifitions. An liquot of.2.2 g of eh extrt ws issolve in 2 ml of methnol n entrifuge t 3 rpm for 1 min in triplite, then the methnol in the omine superntnt ws reovere using vuum rotry evportion pprtus (EYELA, Jpn) t 4 C. The otine extrt ws issolve in 2% HCl n filtere. The filtrte, whih ws juste to ph 1 using 1% NOH, ws extrte with the sme volume of hloroform 3 times. The omine extrt ws onentrte, fille to 1 ml, n use to etermine totl lklois. A totl of 1 ml of the extrt prepre ove ws mixe with ml of mol/l romothymol lue hromogeni gent (ph 7.) n ml of hloroform in 2 ml olorimetri tue n shken for 2 min. The mixture ws trnsferre into seprting funnel n left to sit for 2 h, then the hloroform lyer lote in the ottom of the funnel ws ollete, of whih 4 ml ws remove n.2 mg of nhyrous soium sulftewsetotheremoveportionnshkentoehyrte the solution. After sitting for 1 min, the sorne of the superntnt ws mesure t 414 nm using Thermo Vrioskn Flsh (Thermo Fisher Sientifi, USA). The TAC of theextrtwslulteusingstnrurveofmtrine (.1.6 mg/ml) n expresse s milligrms of mtrine equivlents (ME) per grm of ry weight of extrt (mg ME/g extrt). Eh extrt ws etermine in triplite Determintion of Totl Sterol Content (TSC). TSC ws etermine oring to the metho of Gn et l. [24] with slight moifitions. Eh extrt (.. g) ws extrte with petroleum ether using soxhlet pprtus for 2ht8 C n onentrte to onstnt weight to otin the lipi omponents. The otine lipi omponents were e to 1 ml of 2. mol/l soium hyroxie n shken strongly for 3 s. The mixture ws hete for sponifition in wter th t 8 Cfor1h,then1mLofsturte soium hlorie solution n 2 ml of n-hexne were e fter ooling to room temperture. The mixture ws shken strongly for 3 min n entrifuge t rpm for 1 min. The queous solution in the ottom of the flsk ws extrte with 2 ml n-hexne 3 times, n 1 g of soium sulfte ws e to the omine hexne extrt to ehyrte the solution. The solution ws filtere with filter pper, onentrte, n fille to 1 ml. The hexne extrt ws filtere through.22 μm filter n injete into the Thermol ITQ9 GC-MS system (Thermo Fisher Sientifi, USA). The nlysis olumn ws TR-MS (3 m 2 μmid.2 μmfilm)pillryolumn (Thermo Fisher Sientifi, USA). The gs hromtogrphi onitions were s follows: the injetion volume ws 2 μl, split rtio ws 2 : 1, inlet temperture ws 33 C, n helium rrier gs flow rte ws 1. ml/min. The temperture ws progrmme s follows: initil temperture of 22 C ws mintine for 1 min, inrese to 3 C t rte of 1 C/min, n mintine for 1 min. The mss soure ws EI, tempertures of the ion soure n trnsmission line were 28 Cn3 C, respetively, snning moe ws full sn, solvent ely ws 3 min, n qulity of the snning rnge ws from 4 to 4. Sterol ompouns were ientifie y the NIST2. stnr mss spetrum tse retrievl n their retention times were ompre with pure stnrs. The sterol ontents were lulte oring to stnr urve plotte using stnr onentrtions (.6.12 mg/ml of β-sitosterol n.4.8 mg/ml of

4 4 Journl of Foo Qulity FRAP (mgfeso4/g) ND NT PF CF EF BF AF HE BTH HRSA (%) ND PF CF EF BF AF HE BTH LPLA (%) f e ND PF CF EF BF AF HE BTH Figure 1: Antioxint tivities of the polrity frtions n hyroloholi extrts from m. The vlues re mens ± stnr evitions of triplite nlyses. Columns lele with ifferent letters re signifintly ifferent (P <.1) y Dunn test. NT mens not teste. ND mens not etete. mpesterol) n orresponing pek res. TSC ws equl to the sum of β-sitosterol n mpesterol ontents. The results were expresse s mg/1 g extrt of the ry weight of the extrt (mg/1 g DW) Sttistil Anlysis. All t were expresse s mens ± stnr evitions. The ifferenes etween the mens were nlyze y Dunn s test of One-Wy ANOVA using SPSS 13. for Winows (SPSS In., Chigo, IL, USA). Correltions n regression nlysis etween the ntioxint tivities of the three inepenent tests (FRAP, HRSA, n LPIA) n ontents of seonry metolites, inluing TPC, TAC, n TSC were onute using the regression progrm in SPSS Results n Disussion 3.1. Antioxint Ativities of Polrity Frtions. Antioxints re usully involve in severl mehnisms of tion, inluing inhiition of free ril genertion, enhnement of svenging pity ginst free rils, n reuing power; thus no single ssy n urtely reflet ll of the ntioxints in mixe or omplex system. Therefore, it is neessry to use t lest two ifferent methos to evlute the ntioxint pities of prouts [2]. In this stuy, three ntioxint ssys, whih were ferri reuing ntioxint potentil (FRAP), hyrogen ril svenging ility (HRSA), n lipi peroxition inhiition ility (LPIA), were pplie to evlute the ntioxint properties of the hyroloholi extrts (HE) of fresh m n five frtions (PF, CF, EF, BF, n AF). The ontents of seonry metolites, inluing TPC, TAC, n SC from five frtions, were lso etermine. The results of the FRAP ssy in Figure 1 show tht vlues from vrious polrity frtions were signifintly ifferent (P <.1), n the FRAP vlue of CF ws the highest (6.81 mg FeSO 4 /g). Similrly, the hyroxyl ril svenging ilities of five polr frtions were lso signifintly ifferent (P <.1). The HRSA of CF ws the highest (31.2%), n the ilities of three frtions (CF, EF, n PF) were mrkely higher thn tht of BHT. The nlysis results of LPIA inite tht the inhiitory ility of BHT ws the highest, while the inhiitory ility of CF ws lower thn tht of BHT (86.7%) ut signifintly higher (73.26%) thn those of the other frtions ( %). Aoring to the ove results, CF h the highest ntioxint tivity, s mesure y FRAP, HRSA, n LPIA Contents of Seonry Metolites of Polrity Frtions. For the ssy of seonry metolite ontents (Figure 2),

5 Journl of Foo Qulity TPC (mg GAE/g) TAC (mg ME/g) PF CF EF BF AF PF CF EF BF AF TSC (mg/1 A) PF CF EF BF AF Figure 2: Contents of seonry metolite of the polrity frtions n hyroloholi extrts from m. The vlues re mens ± stnr evitions of triplite nlyses. Columns lele with ifferent letters re signifintly ifferent (P <.1) y Dunn test. e thetpcofcfwspproximtelythesmesbfwith n 27. mg GAE/g, respetively, n they were oth signifintly higher thn the ontents of the other frtions. The TAC of CF ws 6.6 mg ME/g, whih ws signifintly higher thn the ontents of the other four frtions, while EF h the lowest TAC. The TSCs of the five frtions rnge from.27 to 4.23 mg ME/g n were signifintly ifferent, nthecfvluewsthehighest.theseonrymetolite etermintion results inite tht CF h the highest TPC, TAC, n TSC. No ntioxint tivity ws foun in the hyroloholi extrts (HE) mesure y FRAP, HRSA, n LPIA uner equl onentrtions with polrity frtions (Figure 1), while seonry metolite ontents, inluing TPC, TAC, n TSC, signifintly vrie etween the five frtions. Moreover, some of the frtions showe n even higher ntioxint tivity thn tht of the well-reognize syntheti ntioxint BHT, initing tht the tive ntioxint ompouns were seprte n gthere fter polrity prtitioning. In this stuy, the hloroform frtion onurrently possesse the highest ntioxint tivity n seonry metolite ontents, greeing with reports tht foun hloroform to e n effiient extrtion solvent for ntioxint ompouns from plnt mterils [26, 27]. Unlike this result, the nutnol extrt of m hs een reporte to ontin high phenoli ontents n totl ntioxint tivity, n totl ntioxint tivity is signifintly orrelte with phenoli ontents [28]. However, oring to our stuy, the TPC of then-utnolfrtionwspproximtelythesmesthe hloroform frtion, ut the ntioxint tivity ws muh lower, suggesting tht the ntioxint tivity of m might e relte to not only phenols ut lso other ompouns suh s lklois Antioxint Ativities of Constituents Seprte from Chloroform Frtion. The hloroform frtion, whih h higher ntioxint tivities oring to the ove results, ws further seprte using sili gel olumn to otin vrious onstituents lele C1, C2, C3, C4, C, n C6. The ntioxint tivities (FRAP, HRSA, n LPIA) n seonry metolite ontents (TPC, TAC, n TSC) of the onstituents were nlyze. As inite in Figure 3, the FRAPs of C4, C, n C6 rnge from pproximtely 8.4 to mg FeSO 4 /g, whih were muh higher thn those of the other onstituents ( mg FeSO 4 /g), n ws signifintly higher thn tht of CF (71.1 mg FeSO 4 /g). The HRSA of C6 ws the highest (49.9%) for ll of the onstituents n CF, while other onstituents were lower thn CF (2.%). No LPIA vlues were etete in C1, C2, n C3. The LPIA of C6 ws the highest (71.9%), followe y C4 n C, n CF h the lowest vlue of only 7.7%.

6 6 Journl of Foo Qulity FRAP (mgfeso4/g) HRSA (%) C1 C2 C3 C4 C C6 CF C1 C2 C3 C4 C C6 CF LPLA (%) ND ND ND C1 C2 C3 C4 C C6 CF Figure 3: Antioxint tivities of onstituents seprte from the hloroform frtion. The vlues re mens ± stnr evitions of triplite nlyses. Columns lele with ifferent letters re signifintly ifferent (P <.1) y Dunn test. ND mens not etete Seonry Metolite Contents of Constituents Seprte from Chloroform Frtion. Figure 4 shows tht the seonry metolite ontents, inluing TPC, TAC, n TSC of the vrious onstituents, were signifintly ifferent (P <.1). The TPCs of C4, C, n C6 rnge from 31.6 to mg GAE/g n were higher ompre with the TPCs of the other onstituents n CF (27.29 GAE mg/g). The TAC of C6 ws not only the highest mong ll of the onstituents ut ws lso higher thn tht of CF. The TSC of C2 (171.6 mg/1 g) ws muh higher thn those of the other onstituents n CF, whih rnge from 3.3 to 1.63 mg/1 g n 4.23 mg/1 g, respetively. Signifint ifferenes mong the ntioxint tivities of the vrious onstituents mesure y FRAP, HRSA, n LPIA (P <.1) were oserve. In ition, the FRAPs n LPIAs of C4, C, n C6 were higher thn those of CF, n the HRSA of C6 ws higher thn tht of CF. Similrly to the ntioxint tivity, the TPCs of C4, C, n C6 s well s the TAC of C6 were higher thn those of CF, initing tht the ntioxint tivity ingreients in the hloroform frtion were further seprte n onentrte through sili gel olumn seprtion. Moreover, the ntioxint tivities, TPCs, n TACs of C4, C, n C6 were high, suggesting tht the orreltion etween the ntioxint tivity of m n the TPC s well s the TAC my e ovious. Tle 1: Person s orreltion oeffiients of FRAP, HRSA, n LPIA versus TPC, TAC, n TSC. FRAP HRSA LPIA TPC TAC TSC Correltion is signifint t P <.. Correltion is signifint t P< Correltion etween Seonry Metolites n Antioxint Ativity. Person s orreltion oeffiient ws pplie to evlute the reltionship etween the ntioxint tivity n seonry metolite ontents, inluing TPC, TAC, n TSC from C1 to C6 se on FRAP, HRSA, n LPIA, s shown in Tle 1. The TPC showe signifint positive orreltion to FRAP n LPIA with Person s orreltion oeffiients of.941 n.639, respetively, while high positive orreltions etween TAC versus FRAP, HRSA, n LPIA were foun with Person s orreltion oeffiients of.31,.92, n.88, respetively. However, negtive orreltions etween TSC versus FRAP, HRSA, n LPIA were oserve with Person s orreltion oeffiients of.477,.48, n.396,

7 Journl of Foo Qulity TPC (mg GAE/g) e C1 C2 C3 C4 C C6 CF TAC (mg ME/g) e C1 C2 C3 C4 C C6 CF TSC (mg/1 A) C1 C2 C3 C4 C C6 CF Figure 4: Contents of seonry metolites of onstituents seprte from the hloroform frtion. The vlues re mens ± stnr evitions of triplite nlyses. Columns lele with ifferent letters re signifintly ifferent (P <.1) y Dunn test. Vlues of CF re from Figure 2. Moel Depenent vrile (Y) (1) FRAP Tle 2: Multiple liner regression moels of FRAP, HRSA, n LPIA (Y) with TPCn TAC (X). Inepenent vrile (X) Stnrize oeffiients TPC (X 1 ).861 TAC (X 2 ).227 Regression eqution R 2 Signifint (P vlue) Y = X X (2) HRSA TAC(X).92 Y = X.96. (3) LPIA TPC (X 1 ).37 TAC (X 2 ).747 Y = X X respetively. Moreover, the orreltion oeffiients etween TAC versus HRSA n LPIA were higher thn those etween TPC versus HRSA n LPIA, suggesting tht the ntioxint tivity of m ws epenent on not only phenols ut lso lklois, whih my e ttriute to their synergisti tion. To further nlyze the ontriutions of phenols n lklois to the ntioxint tivity of m s well s their synergisti effets, multiple liner regression ws employe to onstrut multiple liner moels with ntioxint tivity (FRAP, HRSA, n LPIA) s the epenent vrile (Y)n TPC n TAC s inepenent vriles (X). Both stepwise elimintion of vriles (if P >.1) n stepwise insertion of vriles (if P <.1) were performe to estlish the est fit moel tht oul extly represent the influene of TPC n TAC on the ntioxint tivity of m. As inite in Tle 2, moel (1) wstheestfitmoel for FRAP, initing tht FRAP ws signifintly linerly orrelte with TPC n TAC (R 2 =.93, P <.), of whih 93% of FRAP vrition ws epenent on these ftors, while the effet of TPC on FRAP ws higher thn tht of TAC ue to the stnrize oeffiients of TPC n TAC,.861 n.227, respetively. The est fit moel for HRSA (moel (2) in Tle 2) only ontine TAC, suggesting tht TAC (stnrize oeffiient =.92) signifintly linerly orrelte with HRSA lone (R 2 =.93, P <.) without ontriution from TPC, n the 9.6% vrition of HRSA

8 8 Journl of Foo Qulity ws ttriute to TAC. Similrly to FRAP, moel (3)showe tht LPIA ws lso signifintly linerly orrelte with oth TPCnTAC(R 2 =.897, P <.); thus TPC n TAC my e responsile for the 89.7% vrition of LPIA. However,theeffetofTACwithstnrizeoeffiient of.747 ws higher thn tht of TPC with stnrize oeffiient of.37. Bse on the urrent stuy, the highest m ntioxint tivity oserve in the FRAP, HRSA, n LPIA ssys ws ue to TPC n TAC, suggesting tht phenols n lklois re the most importnt sustnes. Moreover, lklois seeme to hve higher orreltion with ntioxition thn phenols. Both polyphenols n lklois re mjor ntioxints in nturl prouts, n their ntioxint tivities hve een emonstrte in previous stuies [29 31]. Severl stuies hve shown tht the ntioxint tivity of m ws orrelte with phenols or lklois; Cmpos et l. [32] reporte tht the orreltion etween TPC n ntioxint tivity ws sttistilly signifint (P <.1) withnr 2 vlue of.68 n orreltion oeffiient of.82, initing moertely strong reltionship etween oth vriles. Wng et l. [28] foun signifint orreltion etween TPC n ntioxint tivity with n R 2 vlue of.986. The lipi-solule extrt from m, whih hs reue serum ltte ehyrogense n musle lipi peroxition in weight-loe fore swimming rts s well s inrese hepti n musle totl glutthione, ws foun to ontin mmies, suh s N-enzylhexenmie n N-enzyl--oxo-6E,8E-oteienmie from 7.8 mg/g extrt, suggesting tht the ntioxint effet n e prtly expline y lklois [16]. Aoring to Du [33], lklois extrte from m t onentrtion of 62.4% were onsiere the mjor ntioxint sustne ue to ovious positive orreltions etween lklois with DPPH svenging ility n HRSA. Our stuy not only reinfore these previously mentione results ut lso onfirme tht the ntioxint tivity of m is ttriute to oth lklois n phenols y employing multiple liner regression. TAC h the most influene on ntioxint tivity euse its liner orreltions with FRAP, HRSA, n LPIA were sttistilly signifint with stnrize oeffiients of.227,.92, n.747, respetively, while TPC ws only linerly orrelte with FRAP n LPIA with stnrize oeffiients of.861 n.37, respetively. Moreover, the lklois in m ontine severl speifi ompouns, suh s mmies, menes, n mriine tht hve een regre s the primry tive ingreients of m sine they were first isovere [12, 34, 3]. The ntioxint tivity of m hs lso een hypothesize to e the sis of other iotivities [9, 14, 17, 18]. Thus, these speifi lkloi ompouns my effetively ontriute to the ntioxint tivity of m. However, the phenoli ontent of rw m reporte y previous stuies wslow,rngingfrom2.to9.89mg/g[1,16,36 38].The highest TPCs of m extrts from this stuy n Wng et l. [28] were only n mg/g, respetively, ut the extrt exhiite strong ntioxint tivity with signifint orreltion etween the phenoli ontent n ntioxint tivity. These results my e expline y the hemil struturl ifferenes etween phenoli ompouns. Phenoli ompouns hve ril elimintion pilities ue to their ilities to onte hyrogen n form stle ril intermeites [39]. Rie-Evns et l. [4] stuie the influene of the struturl hemistry of polyphenols on their free ril svenging tivities. In their stuy, glli i (3,4,-trihyroxyenzoi i) showe higher ntioxint tivity orresponing to the three ville hyroxyl groups thn hlorogeni i ( glyosie of 3,4-ihyroxyinnmi i) orresponing to the two ville hyroxyl groups, whihprovethtthentioxinttivityofphenolsepens on the numer of hyroxyl groups in the moleule n hemil struture. Furthermore, the strong ntioxint tivity orresponing to low TPCs my e ssoite with synergisti intertions etween phenoli ompouns n other ntioxint ompouns, suh s lklois. Bse on the ove results, the speifi lklois n phenoli ompouns tht orrelte with the ntioxint tivity of m s well s their synergisti intertions nee to e further investigte. 4. Conlusions In this stuy, the orreltion etween the ntioxint tivity n seonry metolites, inluing TPC, TAC, n TSC, from m ws investigte y FRAP, HRSA, n LPIA, in whih polrity prtition extrtion, further seprtion using sili gel olumn, Person s orreltion oeffiient nlysis, n multiple liner regression were employe. Chloroform ws n effiient solvent for extrting ntioxint ompouns from m, n the tive ntioxint ingreients in the hloroform frtion were further seprte n onentrte using sili gel olumn. The orreltion results from sttistil nlyses exhiite tht the ntioxint tivity ws signifintly orrelte with TPC n TAC se on FRAP, HRSA,nLPIA.Moreover,TPCwssignifintlylinerly orrelte with FRAP n LPIA, while TAC ws signifintly linerly orrelte with FRAP, HRSA, n LPIA, suggesting tht the ntioxint tivity of m is not only ue to phenols utlsolklois.thus,lkloisnphenolsrethemost importnt sustnes tht ontriute to the ntioxition of m, of whih lklois seeme to hve stronger orreltion with ntioxition thn phenols. Further stuies nee to e onute to ientify lklois n phenoli ompouns tht re orrelte with the ntioxint tivity of m s well s their synergisti intertions. These itionl results woul provie new insight into the mehnisms tht le to the speil iotivities of m. Aitionl Points Prtil Applitions. M hs gine signifint interest ue to its vriety of iotivities, suh s fertility-enhning properties, sexul funtion improvement, ntiftigue, n inhiition of prostte hyperplsi. However, there is lk of informtion onerning ext iotivity ompouns tht orrespon to prtiulr funtion. Moreover, some reserhers onsiere tht vrious iotivities of m were ttriute to its ntioxint tivity. Therefore the orreltion etween the ntioxint tivity n seonry metolites

9 Journl of Foo Qulity 9 of m ws investigte in our stuy. The result showe tht oth phenols n lklois re the most importnt sustnes for the ntioxition of m, of whih the ntioxint effet oflkloisseemetoehigherthnthtofphenols,nit ws onsistent with some reports, whih regre lklois in m s primry tivity ingreient. The result of present stuy woul provie new insight into the mehnisms tht le to the speil iotivities of m. Conflits of Interest The uthors elre tht they hve no onflits of interest in this work. Aknowlegments This reserh ws finnilly supporte y the Speil Fun Projet for the Sientifi Reserh of Stte Forest Puli Welfre Inustry, SAF, Chin (21428). Referenes [1] J. León, The M (Lepiium meyenii), little known foo plnt of peru, Eonomi Botny, vol. 18, no. 2, pp , [2] G. F. Gonzles, A. Ruiz, C. Gonzles, L. Villegs, n A. Corov, Effet of Lepiium meyenii (m) roots on spermtogenesis of mle rts, Asin Journl of Anrology, vol. 3, no. 3, pp , 21. [3] A. C. Ruiz-Lun, S. Slzr, N. J. Aspjo, J. Ruio, M. Gso, n G. F. Gonzles, Lepiium meyenii (M) inreses litter size in norml ult femle mie, Reproutive Biology n Enorinology,vol.3,rtile16,2. [4] G.F.Gonzles,A.Córov,K.Vegetl., EffetofLepiium meyenii (MACA) on sexul esire n its sent reltionship with serum testosterone levels in ult helthy men, Anrologi,vol.34,no.6,pp ,22. [] L. J. Yu n W. W. Jin, Stuy on the nutritionl omponents n the nti-ftigue effets of ry power of m (Lepiium meyenii), Foo Siene,vol.2,no.1,pp ,24. [6] T.Miur,H.Motoki,Y.Nito,nI.Suzuki, Antihypoglyemi effet of m in fste n insulin-inue hypoglyemi mie, Journl of Tritionl Meiines,vol.16,no.3,pp.93 96, [7] Y.Z.Zhng,L.J.Yu,J.M.Wn,nW.W.Jin, Effetofethnol extrt of Lepiium meyenii Wlp. (m) on immunologil funtion of mie, Nturl Prout Reserh & Development,vol. 19, no. 2, pp , 27. [8]M.Gso,L.Villegs,S.Yur,J.Ruio,nG.F.Gonzles, Dose-response effet of Re M (Lepiium meyenii) on enign prostti hyperplsi inue y testosterone ennthte, Phytomeiine, vol. 14, no. 7-8, pp , 27. 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Vilhez, Supplementtion of m (Lepiium meyenii) tuer mel in iets improves growth rte n survivl of rinow trout Onorhynhus mykiss (Wlum) levins n juveniles, Aquulture Reserh, vol. 3, no. 3, pp , 24. [1] G. F. Gonzles, V. Vsquez, D. Roriguez et l., Effet of two ifferent extrts of re m in mle rts with testosteroneinue prostti hyperplsi, Asin Journl of Anrology,vol. 9, no. 2, pp , 27. [16] E.H.Choi,J.I.Kng,J.Y.Choetl., Supplementtionofstnrize lipi-solule extrt from m (Lepiium meyenii) inreses swimming enurne pity in rts, Journl of Funtionl Foos,vol.4,no.2,pp.68 73,212. [17] C. Clément, U. Witshi, n M. Kreuzer, The potentil influene of plnt-se fee supplements on sperm quntity n qulity in livestok: A review, Animl Reproution Siene, vol. 132, no. 1-2, pp. 1 1, 212. [18] S. Yur, M. Gso, J. Ruio, J. Nieto, n G. F. Gonzles, Effet of ifferent frtions from hyroloholi extrt of Blk M (Lepiium meyenii) ontestiulrfuntioninultmlerts, Fertility n Sterility,vol.89,no.,pp ,28. [19] I. F. F. Benzie n J. J. Strin, The ferri reuing ility of plsm (FRAP) s mesure of ntioxint power : the FRAP ssy, Anlytil Biohemistry,vol.239,no.1,pp.7 76,1996. [2] H. D. Xu n S. H. Qin, Ultrsoni-ssiste extrtion n in vitro ntioxint tivity evlution of polyshries from Chenomeles sinensis (thouin) koehne, Foo Siene, vol. 31, no. 1, pp , 21. [21] T.B.Ng,F.Liu,nZ.T.Wng, Antioxitivetivityofnturl prouts from plnts, Life Sienes, vol. 66, no. 8, pp , 2. [22] V. L. Singleton, R. Orthofer, n R. M. Lmuel-Rventós, Anlysis of totl phenols n other oxition sustrtes n ntioxints y mens of folin-iolteu regent, Methos in Enzymology, vol. 299, pp , [23] J.Gn,Y.Feng,Z.He,L.F.Xu,H.Zhng,nX.M.Chen, Totl lklois in m (lepiium meyenii) ultivte in Yunnn, Foo Siene,vol.31,no.24,pp ,21. [24] J. Gn, Y. Feng, H. Zhng, Z. He, H. Zheng, n X. Li, Anlysis on omposition n ontent of sterols in three olor types of m, Lepiium meyenii, Journl of Forest Reserh,vol.26,no. 1,pp ,213. [2] M. Antolovih, P. D. Prenzler, E. Ptslies, S. MDonl, n K. Rors, Methos for testing ntioxint tivity, Anlyst, vol. 127, no. 1, pp , 22. [26] S. F. Sulimn, K. L. Ooi, n Supritno, Antioxint n αgluosise inhiitory tivities of uurit fruit vegetles n ientifition of tive n mjor onstituents from phenolirih extrts of lgenri sierri n sehium eule, Journl of Agriulturl n Foo Chemistry, vol.61,no.42,pp.18 19, 213. [27] I. Orhn, M. Krtl, Q. Nz et l., Antioxint n ntiholinesterse evlution of selete Turkish Slvi speies, Foo Chemistry,vol.13,no.4,pp ,27.

10 1 Journl of Foo Qulity [28] L.W.Wng,J.Ling,X.D.Wng,X.F.Yun,B.Zho,nY. W. Yng, High effiient ntioxint tivity of extrts from Lepiium meyenii Wlp, Asin Journl of Chemistry, vol. 24, no. 1, pp , 212. [29] F. Pu, X.-L. Ren, n X.-P. Zhng, Phenoli ompouns n ntioxint tivity in fruits of six Diospyros kki genotypes, Europen Foo Reserh n Tehnology,vol.237,no.6,pp , 213. [3] B. K. Csselst, M. Asenio, P. Conget, H. Speisky, L. A. Viel, n E. A. Lissi, Struture-ntioxitive tivity reltionships in enzylisoquinoline lklois, Phrmologil Reserh,vol. 31,no.2,pp.13 17,199. [31]W.Q.Yin,S.Q.Dun,Y.Zhng,L.Zeng,nX.M.Song, Antioxint tivities of ifferent solvents extrts n lklois of Unri rhynhophyll ( Miq.) Jks, Journl of Gungxi Norml University,vol.28,no.1,pp.31 34,21. [32] D. Cmpos, R. Chirinos, O. Brreto, G. Nortto, n R. Pereshi, Optimize methoology for the simultneous extrtion of gluosinoltes, phenoli ompouns n ntioxint pity from m (Lepiium meyenii), Inustril Crops n Prouts,vol.49,pp ,213. [33] G. X. Du, Stuy on the Isoltion, Purifition n Antioxint Ativities of Alklois in M, South Chin University of Tehnology, Gungzhou, Chin, 211. [34] I.Muhmm,J.Zho,D.C.Dunr,nI.A.Khn, Constituents of Lepiium meyenii m, Phytohemistry, vol. 9, no. 1, pp. 1 11, 22. [3] B. L. Zheng, K. He, C. H. Kim et l., Effet of lipii extrt from Lepiium meyenii on sexul ehvior in mie n rts, Urology, vol., no. 4, pp , 2. [36] L. G. Rnill, Y.-I. Kwon, E. Apostoliis, n K. Shetty, Phenoli ompouns, ntioxint tivity n in vitro inhiitory potentil ginst key enzymes relevnt for hyperglyemi n hypertension of ommonly use meiinl plnts, hers n spies in Ltin Ameri, Bioresoure Tehnology, vol. 11, no. 12, pp , 21. [37] J. Ruio, S. Yur, M. Gso, n G. F. Gonzles, Dose-response effet of lk m (Lepiium meyenii) in mie with memory impirment inue y ethnol, Toxiology Mehnisms n Methos, vol. 21, no. 8, pp , 211. [38] J. Gn, Stuy on ntioxint tivities n ntioxitive sustne sis of m (Lepiium meyenii Wlp.), Chinese Aemy of Forestry, Beijing, Chin, 213. [39] W. Bors n C. Mihel, Chemistry of the ntioxint effet of polyphenols, Annls of the New York Aemy of Sienes,vol. 97, pp. 7 69, 22. [4] C. A. Rie-Evns, N. J. Miller, n G. Pgng, Struturentioxint tivity reltionships of flvonois n phenoli is, Free Ril Biology & Meiine, vol. 2, no. 7, pp , 1996.

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