Antioxidant activities of various extracts of lotus (Nelumbo nuficera Gaertn) rhizome

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1 Asi P J Clin Nutr 27;16 (Suppl 1): Originl Artile Antioxint tivities of vrious extrts of lotus (Nelumo nufier Gertn) rhizome Dongmei Yng ME 1, Qiushung Wng ME 1, Leqin Ke BE 1,2, Jinmei Jing BE 1 n Tiejin Ying PhD 1 1 Deprtment of Foo Siene n Nutrition, Zhejing University, Hngzhou, Chin 2 Deprtment of Chemistry n Siene, Lishui College, Lishui, Chin Lotus rhizome power ws extrte with solvents of ifferent polrities. Antioxint tivities of the extrts were evlute y 2, 2 -iphenylpirylhyrzyl (DPPH) ssy n β-rotene lehing ssy, n ompre with tht of utylte hyroxynisole (BHA) n sori i. Methnol showe the highest extrt yiel mong ll of solvents. Although etone extrt h the highest totl phenolis ontent, methnol extrt h the highest totl phenolis reovery from lotus power (2.1 mg tehin equivlents/1g lotus power). Extrt of either methnol or etone emonstrte the highest DPPH svenging tivity t oth 66.7 mg/l n mg/l. All extrts exhiite higher ntioxint tivity oeffiient (AAC) thn tht of sori i, furthermore, ihloromethne n petroleum extrts h omprle AAC with BHA y the β-rotene lehing ssy. The properties of the extrting solvents signifintly ffete the yiel, totl phenolis ontent n ntioxint tivity of lotus rhizome extrts. Key Wors: lotus rhizome extrts, totl phenolis ontent, ntioxint tivity, solvent polrity Introution There hve een strong evienes initing tht free rils use oxitive mge to lipis, proteins, n nulei is. 1 Though ntioxint enzymes suh s superoxie ismutse (SOD), tlse (CAT) n glutthione peroxise (GPX) ply n importnt role in svenging oxints n preventing ell injury, these efene mehnisms re not equte. Consequently, ellulr mromoleules re esily sujet to oxitive mge. 2 Severl epiemiologil stuies suggest tht high intke of foo rih in nturl ntioxints inreses the ntioxint pity of the plsm n reues the risk of ertin, ut not ll, ners, hert iseses, n stroke. A gret numer of romti, spiy, meiinl n other plnts ontin hemil ompouns exhiiting ntioxint properties n emonstrte suh protetive effets. 3 In ition to α- toopherol, sori i n rotenois, plnt tissues synthesize wie vriety of phenoli ompouns. 4 Severl stuies hve revele tht mjor prt of the ntioxint tivity my e from ompouns suh s flvonois, isoflvones, flvones, nthoynin, tehin n other phenolis 5 with mehnisms involving oth free rilsvenging n metl heltion. 6 For instne, the so-lle Frenh prox hs een ttriute in prt to the routine onsumption of re wine rih in polyphenolis. 7 Lotus (Nelumo nuifer Gertn) is perennil quti rop with stout reeping yellowish white olore rhizomes. It is oth n ornmentl plnt n ietry stple in Estern Asi, prtiulrly in Chin. 8 All prts of N. nufier re use for vrious meiinl purposes in orientl meiine. 9 The see of N. nuifer is use in folk remeies s iureti, ooling gent, ntiemeti n ntiote in the tretment of tissue inflmmtion, ner, 1,11 skin isese, leprosy n poisoning. 1 Experimentl stuies emonstrte tht see of N. nufier hs heptoprotetive 12 n ntifertility tivities 13 s well s free ril svenging tivity. Liu et l. (24) 11 reporte tht see of N. nufier oul suppress ell yle progression, ytokine genes expression, n ell prolifertion in humn peripherl loo mononuler ells. The lef of N. nuifer is onsiere est for over-oming oy het, n stopping leeing. 14 It is use s rug for hemtemesis, epistxis, hemoptysis, hemturi n metrorrhgi 15 in tritionl Chinese meiine. The lef of N. nufier ontins severl flvonois n lklois, n is effetive in the tretment of hyperlipiemi in roents. 16,17 The 95% ethnol extrt of the lef of N. nuifer ws foun to isply signifint nti-hiv tivity. 9 The stlk extrt of N. nuifer showe nti-pyreti effet, 18 while lef n stmen extrts showe nti-oxint effet n strong ril svenging tivity. 19,21 In Chin, lotus rhizome is ommon vegetle, it n e ooke into ifferent ishes or eten rw. Espeilly, it hs een pplie in Chinese herl presriptions to llevite tissue inflmmtion, ner, n liver irrhosis for long time. 22 Compre with lef n see of N. nuifer, phytohemils in lotus rhizome n their funtionl properties were still little stuie, though it ws notie for rih ntioxints ontent n strong ntioxint tivity. 8,22 Corresponing Author: Professor Tiejin Ying, Dept of Foo Siene n Nutrition, Zhejing University, Hngzhou, Chin, 3129 Tel: ; Fx: Emil: tjying@hzn.om

2 159 D Yng, Q Wng, L Ke, J Jing n T Ying Mny ftors n ffet the extrt reovery of ntioxint phytohemils from nturl mterils. Previous stuies hve reporte the influene of some vriles (e.g., temperture, time, solvent-to-soli rtio, et.) on the phenoli yiels of iverse plnt mterils rnging from lmon hulls to pine swust. 23 Solvents suh s wter, ethnol, methnol, etone or their mixture re ommonly use to extrt phytohemils from plnts, n they re nother importnt ftor ffeting oth extrtion yiel n ntioxint tivity of extrts. Due to the omplexities of oth the hemil hrteristis of solvents n the iverse struture n omposition of the plnt mterils, the ehviors of mteril-solvent systems were ifferent from eh other n n hrly e preite. No single solvent oul extrt ll the ntioxints of ifferent polrity n soluility in single plnt. A numer of in vitro methos hve een evelope to mesure the effiieny of ietry ntioxints either s pure ompouns or in foo mixtures. These methos n e lssifie into two mjor types se on the hemistry mehnisms involve: ssys involving hyrogen tom trnsfer retions, inluing ORAC (oxygen ril sorne pity), TRAP (totl ril trpping ntioxint prmeter), Croin lehing ssy, IOU (inhiite oxygen uptke), inhiition of linolei i oxition, inhiition of LDL oxition; n ssys y eletrontrnsfer retion, inluing TEAC (Trolox equivlent ntioxint pity), FRAP (ferri ion reuing ntioxint prmeter), DPPH (2, 2 -iphenylpirylhyrzyl ) svenging tivity, opper (II) reution pity, totl phenols ssy y Folin-Ciolteu regent. 24 Frnkel n Meyer pointe out tht it is prolemti to use oneimensionl methos to evlute multifuntionl foo n iologil ntioxints. 25 In the urrent stuy, we evlute the ntioxint tivity of vrious extrts of lotus rhizome with two methos se on ifferent mehnisms. The ojetive of this stuy ws to investigte the effet of extrting solvents on the yiel, totl phenolis n ntioxint tivity of lotus. Antioxint tivity of lotus extrt ws ompre with tht of sori i n BHA. Mterils n methos Mterils Lotus rhizome ws purhse from lol mrket. It ws wshe y tp wter, hoppe in piees n rie in 6 for 72h, n then groun to pss 1 mm sieve. The smple power ws store t -2 efore experiment. 2, 2 -iphenylpirylhyrzyl (DPPH) n β-rotene were otine from Sigm n ll other hemils use were of nlytil gre. Extrtion Five grms of the lotus rhizome power were weighe n put into 15 ml flsks. Eh flsk ws e with 5 ml of either methnol, ethnol, etone, ihlormethne or petroleum ether. After one week of storge t room temperture, the superntnt n the seiment were seprte y vuum-filtrtion. The solvent in the superntnt ws evporte y vuum-evportor t 35. The rie extrt ws then weighe. Determintion of totl phenoli ontent Totl phenoli ompouns were etermine with Folin Ciolteu regent using glli i s stnr oring to Slinkr n Singleton ml of extrts in volumetri flsk ws ilute with 46 ml of istille wter. 1 ml of Folin Ciolteu regent ws e n the ontent in the flsk ws mixe thoroughly. After 3min, 3 ml of N 2 CO 3 (2%) ws e, then the mixture ws llowe to stn for 2 h with intermittent shking. The sorne ws mesure t 765 nm. The onentrtion of totl phenoli ompouns ws lulte with liner regression eqution otine from glli i stnr grph: Asorne= glli i (μg) An the results were expresse s milligrms of glli i equivlents (GAE) per 1g of ry extrt or per 1g of ry lotus power. Determintion of DPPH ril svenging pity The ntioxint tivity of lotus extrts were mesure in terms of hyrogen onting or ril svenging ility using the stle DPPH metho s moifie y Snhez-Moreno et l ml of the smple t vrious onentrtions ws e to 2.9 ml of DPPH (6μM) solution. When DPPH rets with n ntioxint ompoun onting hyrogen, it is reue, resulting in erese in sorne t 517 nm. The sorne ws reore t 1 min intervls up to 3 min using UV Vis spetrophotometer. The remining onentrtion of DPPH in the retion meium ws lulte with liner regression eqution from lirtion urve: A 517 nm =.123 [DPPH ] (r =.9865). The perentge of remining DPPH ([DPPH ] R ) ws expresse s [DPPH ] R (%) = 1 [DPPH ] T /[DPPH ] T=. where [DPPH ] T is the onentrtion of DPPH t 3 min time n [DPPH ] T= is the initil onentrtion. The perentges of remining DPPH were plotte ginst the smple or stnr onentrtions to otin the mount of ntioxint neessry to reue the initil onentrtion of DPPH.A kineti stuy ws lso onute to evlute the free ril svenging properties of lotus rhizome extrt using stle DPPH. Determintion of ntioxint tivity y β-rotene lehing metho The prevention of oxitive loss of β-rotene in β- rotene/linolei i emulsion ws use to ssess the ntioxint ility of the lotus rhizome extrts. 28 Two milligrms of β-rotene were issolve in 1 ml hloroform n 1 ml of the β-rotene solution ws mixe with 2 mg of purifie linolei i n 2 mg of Tween 4 emulsifier in roun-ottom flsk, hloroform ws then remove in rotry vuum evportor. Distille wter (5 ml) ws e to the flsk n the mixture ws stirre in sonitor. Lotus rhizome extrt or sori i or BHA ws e to 5 ml of the β- rotene/linolei i emulsion to finl onentrtion of 4 mg/l. The ontrol ontine.2 ml wter n 5 ml β-rotene/linolei i emulsion. An sorne t 47 nm ws immeitely reore fter ition of smple. The vils were ppe n ple in wter th t 5. The sorne of the emulsion t 47 nm ws etermine every 2 min until 12 min. A seon emulsion

3 Antioxint tivity of lotus rhizome 16 (B), onsisting of 2 mg of linolei i, 1 mg of Tween 4 n 5 ml of wter, ws lso prepre. Wter (2 μl) with 5 ml emulsion B ws use to zero the spetrophotometer. Antioxint tivity oeffiient (AAC) ws lulte with the following eqution: AAC = (A A(12) - A C(12) ) 1/(A C() - A C (12) ); where A A(12) is the sorne of the ntioxint t 12 min, A C(12) is the sorne of the ontrol t 12 min, n A C() is the sorne of the ontrol t min. Sttistil nlyses All nlyses were run in triplites n results verge. Sttistil nlyses were performe with the Exel n SPSS softwres pkge. Results Yiel The extrtion yiels with ifferent solvents vrie from 4.6 g to.41 g per 1 g lotus power (4.6% to.41%) (Fig 1), n n e rnke from high to low in the following orer: Methnol extrt > ethnol extrt >ethyl ette extrt > etone extrt > petroleum ether extrt > ihloromethne extrt. The yiel of methnol extrt ws 11.1 times of tht of ihloromethne extrt. Our result gree with the yiels from Gevuin velln hulls n the yiels from ukwhet, whih ll showe the following orer: methnol extrt >ethnol extrt >etone extrt. 28,29 Totl phenol ontent Totl phenolis ontents (mg tehin equivlent/1 g extrts), s ffete y the extrting solvents, were rnke from high to low: etone extrt > ethyl ette extrt > ihloromethne extrt > methnol extrt > petroleum ether extrt > ethnol extrt (Tle 1).The totl phenolis ontent of etone extrt ws 3.9 times n 3.3 times of those of ethnol extrt n petroleum ether extrt, respetively. Although totl phenolis in etone extrt ws the highest one when expresse ginst the weight of extrts, methnol extrt h the highest reovery rte ginst lotus rhizome power (2.1 mg tehin equivlent/1 g lotus rhizome power). The orer of reovery from high to low ws: methnol extrt >etone extrt > ethyl ette extrt > ethnol extrt > ihloromethne extrt > petroleum ether extrt. extrts of lotus rhizome(g/1g) Figure 1. Yiel of lotus rhizome extrts (N=3, vlues with ifferent supersripts were signifintly ifferent t p=.5, =the lowest yiel) Methnol Ethnol Aetone Ethyl ette Dihloromethne Tle 1. Totl phenolis in lotus extrts (N=3) Smple Totl phenolis (mg tehin equivlents/1g extrts) Totl phenolis (mg tehin equivlents/1g ry lotus Methnol Ethnol Aetone Ethyl ette Dihloromethne % DPPH remining Time(minutes) Control Methnol Ethnol Aetone Ethyl ette Dihlormethne BHA Figure 2. Retion kinetis of lotus extrts with DPPH ril. The DPPH onentrtion ws 6μM in ll retion mixtures % D PPH rem ining mg/l DPPH ril svenging tivity DPPH is use s free ril to evlute ntioxint tivity of some nturl ompouns. The egree of its isolortion is ttriute to hyrogen onting ility of test ompouns. 3 Fig 2 showe the erese in onentrtion of DPPH with time when the onentrtions of ll extrts n BHA were mg/l. Fig 3 illustrte the reltive effetiveness of ifferent extrts of lotus rhizome in reuing onentrtion of DPPH. Signifint DPPH ril svenging tivity ws evient in ll of the teste extrts t mg/l. Extrts of methnol n etone emonstrte higher svenging tivities thn other extrts. However, ll extrts showe lower 133.3mg/l Control Methnol Ethnol Aetone Ethyl ette Dihloromethne BHA Figure 3. Free ril svenging pity of lotus extrts s etermine y the DPPH metho

4 161 D Yng, Q Wng, L Ke, J Jing n T Ying Asorne t 47nm Control Methonl Ethnol Aetone Ethyl ette Dihloromethne VC.7 BHA Time (minute) Figure 4. Chnges of sorne t 47nm with time in β- rotene/linolei i emulsions e with lotus rhizome extrts (4 mg/l). Antioxint tivity oeffiient (AAC) Methne Aetone Dihloromethne Asori i tivity thn BHA n sori i (t not shown) t the sme onentrtion. The orer of ntioxint tivity of lotus rhizome extrts ws: methnol extrt = etone extrt > ihloromethne extrt > petroleum ether extrt >ethnol extrt >ethyl ette extrt. At 66.7 mg/l, lotus rhizome extrts showe similr svenging tivity trens to mg/l exept tht the DPPH svenging tivity of ethyl ette extrt t 66.7 mg/l ws not sttistilly signifint. Antioxint tivity etermine y β-rotene lehing metho In the β-rotene lehing ssy, the ontrol smple without ition of ntioxint oxiize most rpily, while lotus extrts, sori i or BHA showe vrie ntioxint tivities (Fig 4). The sorne erese in emulsion smple e with ihloromethne extrt ws signifintly slower thn tht in smples e with other extrts. In this ssy, ll extrts of lotus rhizome s well s BHA exhiite etter ntioxint tivities thn sori i. Fig.5 showe the ntioxint tivities of the lotus extrts in omprison with tht of sori i n BHA t the onentrtion of 4 mg/l. Their ntioxint powers were rnke in the orer: BHA > ihloromethne extrt > petroleum ether extrt > etone extrt > ethyl ette extrt > methnol extrt > ethnol extrt > sori i. Dihloromethne n petroleum ether extrts emonstrte omprle AAC with BHA t the sme onentrtion. Ethnol Ethyl ette BHA Figure 5. Antioxint tivity of lotus extrts nlyze y β- rotene lehing metho 4 Disussion The ntioxitive phytohemils in grins, vegetles n fruits hve reeive inresing ttention reently for their potentil role in prevention of humn iseses s well s in foo qulity improvement. 31 Lotus rhizome hs een use oth s vegetles n meiine in estern Asi, prtiulrly in Chin. It ws regre to e one of the mny vegetles rih in ntioxint omponents, 32 n mny of its funtionl effets were relte to the ntioxints it ontins. 1 Despite this, lotus rhizome hs not een explore extensively for its phytohemil ttriutes. Severl reserhes using ifferent methos hve prove tht lotus exhiite effetive ntioxint pity. 8,32 In our experiment we heke the effets of solvents on ntioxint tivity n phenoli ontent of lotus rhizome extrts. Extrtion is ritil to the reovery of ntioxint phytohemils. The extrtion yiel epens on solvent, time n temperture of extrtion s well s the hemil nture of the smple. Uner the sme time n temperture onitions, the solvent use n the hemil property of smple re the two most importnt ftors. 3 The extrt yiel of lotus rhizome, rnge from.4% to 4.6% (w/w) ws in generl higher thn tht of potto peel, whih ws.5%, omprle with tht of ukwhet, whih ws 1.5% to 4% 29 epening upon ifferent solvents, ut ws lower thn tht of lentil sees n grpe pome whih were 23.3% n 42% respetively. 28 In our experiment, methnol ws prove to e the est solvent for extrting ntioxint from lotus rhizome, this grees with reports y Bensky et l 14 n Azizh et l. 33 Among the ifferent groups of nturlly ourring ntioxints from plnts, polyphenolis re perhps one of the most importnt types. Our results showe tht solvent signifintly ffete the phenoli ontent from lotus rhizome extrt. The etone extrt of lotus rhizome showe the highest phenoli ontent mong ll solvents stuie. Aetone ws superior to methnol in seletive extrtion of totl phenolis, this grees with reserh results y Bensky et l.. 14 However, methnol extrt gives the highest totl phenoli reovery from the lotus rhizome euse its extrt yiel is muh higher thn etone extrt. The ntioxint tivity of plnt extrts vry with ssy methos. 29 Therefore, single ssy my e inequte. 34 For this reson, we ross heke ntioxint tivities of vrious extrts of lotus rhizome with two ntioxint tivity ssys se on ifferent mehnisms, nmely DPPH ssy se on eletron-trnsfer retion n β-rotene lehing ssy se on hyrogen tom trnsfer retion. Mny stuies inite tht only polr extrts of plnts showe effetive ntioxint tivity, n some reserhes further prove tht moerte polrity extrts re more potent, even if their totl ntioxint reovery from the plnt is not high. 35 Methnol ppers to perform est in extrting polr ompouns suh s phenolis, flvonois n other polr mteril in erels. 36,37 In the present stuy, methnol extrt of lotus rhizome showe the highest ntioxint tivity y DPPH ssy. However, it ws the ihloromethne extrt whih showe the strongest ntioxint tivity y β-rotene lehing ssy. This isrepny is not

5 Antioxint tivity of lotus rhizome 162 unommon. For exmple, it ws reporte tht though lipophili extrt of oriner emonstrte strong tivity in the 15-LO ssy ( metho to test the tivity of ntioxint ginst linolei i peroxition), it showe low ntioxint tivity in DPPH ssy. 36 The uthor elieve the reson ws tht the involvement of proton ontion y the tive ompouns my e of less importne for 15-LO inhiition thn for DPPH ril svenging. There hve een two opinions on the orreltion etween phenolis n totl ntioxint tivity. Some reports emonstrte positive orreltion etween them, 35,38,39 n the others showe no orreltion. 29,38 Our results gree with the ltter, it seems tht no orreltion exists either etween the perentge inhiition (% svenging effet) ssye y DPPH n totl phenolis (R 2 =.544), or etween the ntioxint tivity (AAC) ssye y β-rotene lenhing metho n totl phenolis (R 2 =.552). Kur n Kpoo (22) thought this phenomenon n e expline on the sis of high ntioxint tivity of some iniviul phenoli units, whih my t s effiient ntioxints rther thn ontriuting to high totl phenolis, 32 while Kähkönen et l. 39 thought tht totl phenolis ontent i not inlue ll the ntioxints, suh s sori i, rotenoi n toopherol. Vinson et l. (21) 4 hel the ie tht the synergism mong the ntioxints in the mixture me the ntioxint tivity not only epenent on the onentrtion of ntioxint, ut lso on the struture n intertion mong the ntioxints. This proly is the reson why smples with similr onentrtions of totl phenolis my vry remrkly in their ntioxint tivity. Different methos se on ifferent mehnisms for mesurement of ntioxint tivity my le to ifferent results even from the sme smple. For exmple, the ntioxint tivity of erry extrt ws minly relte to nthoynins in the metho se on low ensity lipoproteins (LDL) oxition, ut minly orrelte with hyroxyinnmtes in the ssy of liposome oxition. 39 In onlusion, the extrting solvent signifintly ffete the yiel, totl phenoli ontent n ntioxint tivity of lotus rhizome extrts. Methnol extrt h the highest extrt yiel n totl phenoli reovery, s well s the highest ntioxint tivity when etermine y the DPPH ssy, while ihloromethne extrt showe the highest ntioxint tivity when evlute y the β-rotene ssy. Thus, our results inite tht seletive extrtion from nturl mterils, y n pproprite solvent, is importnt for otining frtions with high ntioxint tivity. Methnol oul e the optiml solvent for extrtion of lotus ntioxint. Referenes 1. Shui GH, Leong LP. Anlysis of polyphenoli ntioxints in str fruit using liqui hromtogrphy n mss spetrometry. J hromtogr A 24; 122: Bergeni L, Benes L, Durkov Z, Ferenik M. Chemistry, physiology n pthology of free rils. 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