Antioxidant and Neuronal Cell Protective Effects of Aqueous Extracts from Lotus Leaf Tea

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1 농업생명과학연구 46(2) pp Journl of Agriculture & Life Science 46(2) pp Antioxidnt nd Neuronl Cell Protective Effects of Aqueous Extrcts from Lotus Lef Te Chng-Ho Jeong 1 Hee-Rok Jeong 2 Sung-Gil Choi 2 Ho Jin Heo 2,3* 1 Wooyng Frozen Food Co., Ltd., Seocheon , Kore 2 Dept. of Food Sci. & Techno., Gyeongsng Ntionl Univ.(Insti. of Agric. & Life Sci.), Jinju , Kore 3 Division of Applied & Life Sci.(BK21), Gyeongsng Ntionl Univ., Jinju , Kore Received: JAN , Revised: MAR , Accepted: APR ABSTRACT Antioxidnt nd neuronl cell protective effects of queous extrct from lotus (Nelumo nucifer) lef te (LLTE) were investigted. The 2,2'-zino-is (3-ethylenzthizoline-6-sulfonic cid) rdicl scvenging effect, ferric reducing ntioxidnt power, nd mlondildehyde inhiition of LLTE were incresed in dose dependent mnner. Intrcellulr rective oxygen species ccumultion resulting from hydrogen peroxide (H 2O 2) tretment ws significntly reduced when LLTE were present in the medi compred to PC12 cells treted with H 2O 2 only. In neuronl cell viility ssy using 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyl- tetrzoliumromide (MTT), LLTE showed protective effect ginst H 2O 2-induced neurotoxicity. In ddition, lctte dehydrogense relese into medium ws lso inhiited y LLTE ( %). Totl phenolics of LLTE were mg/g nd quercetin ws identified s mjor phenolics ( mg/100g). Therefore, ove these dt suggest tht LLTE including quercetin my e useful in the nturl ntioxidnt sustnce, nd my reduce the risk of neurodegenertive disese. Key words - Nelumo nucifer, Quercetin, Antioxidnt, Neuronl cell protection I. INTRODUCTION Accumulted intrcellulr H 2O 2 induces the peroxidtion of memrne lipids nd poptotic cell deth y ctivtion of cspses (Behl et l., 1997; Vlenci & Morán, 2004). Alzheimer s disese (AD) s neurodegenertive disese is one of the most serious threts to humn helth in ged societies of developed countries. Mny studies hve demonstrted tht the rins of ptients with AD re sujected to n increse of oxidtive stress due to free rdicl dmge (Mrkesery & Crney, 1999). Mny phenolics protect neuronl cells from the oxidtive stress induced y ROS or myloid-β (Aβ) protein, which my e relted to the pthogenesis of AD (Kim et l., 2005; Kou et l., 2009). Some phytochemicls from nturl plnt sources such s fruits nd vegetle my reduce the risk of AD ecuse of their ntioxidtive properties diminishing oxidtive insults (Youdim & Joseph, 2001). Epidemiologicl oservtion hs reveled tht the increse of ntioxidnt uptke is inversely ssocited with the risk of AD incidence (Crundmn & Delney, 2002). Antioxidnts re vitl sustnces which posses the ility to protect the ody from dmge cused y free rdicl induced oxidtive stress (Ozsoy et l., 2008). There is n incresing interest in nturl ntioxidnts (e.g. polyphenols), present in medicinl nd dietry plnt, which might help prevent oxidtive dmge (Silv et l., 2005). Phenolics possess idel structurl chemistry for free rdicl-scvenging ctivity, nd hve een shown to e more effective ntioxidnt in vitro thn vitmin C. A few phenolics hve lso *Corresponding uthor: Ho Jin Heo Tel: Fx: E-mil: hjher@gnu.c.kr

2 116 Journl of Agriculture & Life Science 46(2) een reported tht prevent decrese in the ctivities of ntioxidnt enzymes, superoxide dismutse (SOD), glutthione peroxidse (GSH-Px), nd prevent significnt deletion of glutthione (GSH) (Yu et l., 2009). Therefore, the importnce nd role of non-nutrient compounds prticulrly phenolics s nturl ntioxidnts hve gretly incresed (Hgermn et l., 1998). To fine new nturl sources of physiologicl compounds, we studied ntioxidnt nd neuronl cell protective effects of LLTE. Lotus (Nelumo nucifer) is perennil, rhizomtous nd qutic plnt, which ws distriuted throughout Asi. All prts of lotus, including the rhizome, lef, stmen, nd seed, hve een used s food-stuffs s well s trditionl medicines in Chin nd Indi (Goo et l., 2009). In Kore, lotus lef, root nd seed re usully consumed s te, or in rised dishes or soups. According to trditionl knowledge, medicinl uses of different prt of lotus plnts re common in the tretment of dirrhe, tissue inflmmtion, nd hemostsis (H et l., 2010). Lotus lef hs een reported to hve eneficil effects on ntioxidnt (Wu et l., 2003), nticteril (Li & Xu, 2008), nti-hyperlipidemic (L Cour et l., 1995), nti-hiv (Kshiwd et l., 2005), nd nti-oesity effects (Ono et l., 2006). Recently, Lin et l. (2008) reported tht the flvonoids (ctechin, quercetin, quercetin-3-oglycoside, nd kempferol-3-o-gluco pyrnoside) hve een isolted s ntioxidnt from the leves. Although it hs lredy een demonstrted tht lotus lef contin phenolic compounds, little is known out effect of lotus lef te on oxidtive stress-induced neurotoxicity. Since the protective effect on neuronl cell of LLTE hs not previously een reported, the ojectives of this study were to determine the ntioxidnt nd protective effect on neuronl cell of LLTE. In ddition, ctive compounds on ntioxidnt nd neuronl cell protection were identified y high performnce liquid chromtogrphy (HPLC) nlysis. Ⅱ. Mterils nd methods 2.1 Chemicls Folin-Cioclteu s phenol regent, 2,2'-zino-is(3- ethylenzthizoline-6-sulfonic cid) (ABTS), potssium persulfte, 2,4,6-tripyridyl-S-trizine (TPTZ), trichlorocetic cid (TCA), thiorituric cid (TBA), vitmin C, α -tocopherol, ctechin, 2-[4-(2- hydroxyethyl) piperzin- 1-yl]ethnesulfonic cid (HEPES), sodium icronte, penicillin, streptomycin, myricetin, quercetin, kempferol, ferrous sulfte (FeSO 4), hydrogenperoxide (H 2O 2), dimethyl sulfoxide (DMSO), penicillin, streptomycin, 3-[4,5- dimethythizol-2-yl]-2,5- diphenyl tetrzolium romide (MTT) ssy kit, 2',7'-dichlorofluorescein dicette (DCF-DA), lctte dehydrogense (LDH) ssy kit, nd ll solvents used were of nlyticl grde nd purchsed from Sigm Chemicl Co (St. Louis, MO, USA). RPMI 1640 medium nd fetl ovine serum ws otined from Gico BRL (Grnd Islnd, NY, USA). 2.2 Extrction from the lotus lef te Lotus (Nelumo nucifer) lef te ws purchsed from locl mrket in Hmyng of Kore in Octoer These smples were stored t -20 until use. Freeze-dried smples from lotus lef te were otined s follows. Powdered lotus lef te (50 g) ws suspended nd extrcted with 500 ml of wter t 10 0 for 2 hr. The extrcts were filtered through Whtmn No. 2 filter pper (Whtmn Interntionl Limited, Kent, Englnd) nd evported to dryness. The queous extrct ws concentrted in vcuum evportor t 40. Wter filtrte ws frozen nd lyophilized. The lyophilized extrcts were plced in glss ottle nd stored t -20 until used. The lyophilized extrcts were re-dissolved in wter to concentrtion of 1,000 μg/ml. 2.3 ABTS rdicl scvenging ctivity ABTS ws dissolved in wter to mke concentrtion of 7 mmol/l. ABTS ws produced y recting the ABTS stock solution with 2.45 mm/l potssium persulfte (finl concentrtion) nd llowing

3 Jeong et l. : Antioxidnt nd Neuronl Cell Protective Effects of Aqueous Extrcts from Lotus Lef Te 117 the mixture to stnd in the drk t room temperture for hr efore use. For the study of smples, the ABTS stock solution ws diluted with phosphteuffered sline 5 mm/l, ph 7.4 to n sornce of 0.70 t 734 nm. After the ddition of 980 μl of diluted ABTS to 20 μl of smple, the sornce reding ws tken 5 min fter the initil mixing (Jeong et l., 2010). This ctivity is given s percent ABTS scvenging tht is clculted s: % ABTS scvenging ctivity = [(control sornce smple sornce) / (control sornce)] Ferric reducing ntioxidnt power (FRAP) The FRAP ssy developed y Jeong et l. (2010). In short, 1.5 ml of working, prewrmed 37 FRAP regent (10 volumes 300 mm/l cette uffer, ph vol of 10 mm/l 2,4,6-tripyridyl-S-trizine in 40 mm/l HCl + 1 vol of 20 mm/l FeCl 3) ws mixed with 50 μl of LLTE nd stndrds. This ws vortex mixed nd sornce t 593 nm ws red ginst regent lnk t predetermined time fter smpleregent mixing. The test ws performed t 37 nd the 0-4 min rection time window ws used. 2.5 Mlondildehyde (MDA) ssy using mouse rin homogentes This ssy ws crried out to the method descried y Chng et l. (2001). The rin of young dult mle Bl/c mice ws dissected nd homogenized in ice-cold Tris-HCl uffer (20 mm, ph 7.4) to produce 1/10 homogente. The homogente ws centrifuged t 12,000 g for 15 min t 4. One ml liquots of the superntnt were incuted with the test smples in the presence of 10 μm FeSO 4 nd 0.1 mm vitmin C t 37 for 1 hr. The rection ws terminted y ddition of 1.0 ml TCA (28%, w/v) nd 1.5 ml TBA (1%, w/v) in succession, nd then the solution ws heted t 100. After 15 min, the color of the MDA-TBA complex ws mesured t 532 nm. (+)-Ctechin, well-known ntioxidnt, ws used s positive control. The inhiition rtio (%) ws clculted s follows: % inhiition = [(control sornce smple sornce) / control sornce] Neuronl cell culture The PC12 cell line ws derived from trnsplntle rt pheochromocytom. The cells respond reversily to nerve growth fctor (NGF) y induction of the neuronl phenotype. PC12 cells (KCLB 21721, Kore Cell Line Bnk, Seoul, Kore) were propgted in RPMI 1640 medium contining 10% fetl ovine serum, 25 mm HEPES, 25 mm sodium icronte, 50 units/ml penicillin, nd 100 μg/ml streptomycin. 2.7 Mesurement of intrcellulr oxidtive stress Levels of intrcellulr rective oxygen species (ROS) were determined y DCF-DA (fluorescent proe) ssy (Chng et l., 2001). In rief, cells (10 4 cells/well on 96-well) were treted for 10 min with the indicted concentrtions of the LLTE or vitmin C. The cells were then treted with or without 200 μm hydrogen peroxide for 2 hr. At the end of the tretment, cells were incuted in the presence of 50 μm DCF-DA in phosphte uffered sline (PBS). Fluorescence ws then quntified using TECAN SER-NR fluorometer (Sn Jose, CA, USA) using 485 nm excittion nd 530 nm emission filters. 2.8 Determintion of cell viility MTT reduction ssy ws determined using the in vitro toxicology ssy kit (TOX-1, Sigm Co.). Neuronl PC12 cells were plted t density of 10 6 cells/well on 96-well pltes in 100 μl of RPMI. The cells were pre-incuted with LLTE for 48 hr efore the 200 μm H 2O 2 ws dded. The cells were treted with or without H 2O 2 for 2 hr. The mount of MTT formzn product ws determined y mesuring sornce using microplte reder (680, Bio-Rd, Tokyo, Jpn) t test wvelength of 570 nm nd reference wvelength of 690 nm. Neuronl PC12 cells were precipitted y

4 118 Journl of Agriculture & Life Science 46(2) centrifugtion t 250 g for 4 min t room temperture, 100 μl of the superntnts ws trnsferred into new wells, nd LDH ws determined using the in vitro toxicology ssy kit (TOX-7, Sigm Co.). Dmge of the plsm memrne ws evluted y mesuring the mount of the intr-cellulr enzyme LDH relesed into the medium (Heo et l., 2001). 2.9 Determintion of totl phenolics Totl phenolics were determined y the spectrophotometric nlysis (Jeong et l., 2010). In rief, 1 ml portion of ppropritely diluted extrcts ws dded to 25 ml volumetric flsk contining 9 ml of deionized distilled wter (ddh 2O). A regent lnk using ddh 2O ws prepred. One ml of Folin-Cioclteu s phenol regent ws dded to the mixture nd then shken. After 5 min, 10 ml of 7% N 2CO 3 solution ws dded with mixing. The mixed solution ws then immeditely diluted to volume (25 ml) ddh 2O nd mixed thoroughly. After 90 min t 23, the sornce ws red t 750 nm. The stndrd curve for totl phenolics ws mde using gllic cid stndrd solution (0-100 mg/l) under the sme procedure s ove. Totl phenolics in LLTE were expressed s milligrms of gllic cid equivlents (mg GAE/g) of smple Determintion of flvonols One grm of LLTE ws mixed with 40 ml of wter nd 5 ml of 6 M HCl. After refluxing t 95 for 2 hr, the hydrolyzed solution ws filtered into 50 ml volumetric flsk nd susequently mde up to the volume with wter. Approximtely 1 ml of the finl solution ws llowed to cool under running wter nd filtered through 0.45 µm filter (Nylon Acrodisc 13 Gelmn, Ann Aror, Michigon, USA) prior to injection for Agilent HPLC (1100 series, Agilent Co., Snt Clr, CA, USA) nlysis. The nlysis of flvonols (myricetin, quercetin, nd kempferol) in smple ws crried out y the following HPLC method. The column ws Shiseido RP-18 column (250 mm 4.6 mm i.d., 5 μm, Shiseido Co., Tokyo, Jpn). Moile phse consisted of 30% cetonitrile in M KH 2PO 4 uffer solution (v/v); the ph of the moile phse ws djusted with 6 M HCl to 2.5. The flow rte ws 1.0 ml/min. The column ws operted t 30. The smple injection volume ws 20 µl. UV spectr were recorded from 200 nd 400 nm, nd pek res were mesured t 370 nm (Wng & Helliwell, 2001) Sttisticl nlysis All dt were expressed s men±sd (n=3). Ech experimentl set ws compred with one-wy of vrince (ANOVA) nd Duncn s multiple rnge test (p<0.05) using SAS progrm (Ver. 8.2, SAS Institute, Cry, NC, USA). Ⅲ. Results nd discussion 3.1 ABTS rdicl scvenging ctivities nd FRAP The reduction cpility of ABTS ws determined y the decrese in its sornce t 734 nm, which is induced y ntioxidnts. Positive ABTS test suggests tht the smples were free rdicl scvengers. The scvenging effect of LLTE, vitmin C, nd α -tocopherol on ABTS rdicl ws compred. On the ABTS rdicl, LLTE hd significnt scvenging effects with incresing concentrtion in the rnge of 125-1,000 μg/ml. However, when compred with tht of vitmin C nd α-tocopherol, the scvenging effect of LLTE ws lower. A 1,000 μg/ml of LLTE, vitmin C, nd α-tocopherol exhiited 96.37, 99.86, nd 99.84% inhiition, respectively. The different concentrtions of LLTE (125, 250, 500, nd 1,000 μg/ml) showed ntioxidnt ctivities in dose dependent mnner (19.27, 36.36, 67.86, nd 96.37% inhiition, respectively) on the ABTS rdicl scvenging ssy (Fig. 1A). The IC 50 vlue of vitmin C, α-tocopherol, nd LLTE were 49.38, 81.79, nd μg/ml, respectively. Free rdicl (ABTS) scvenging ctivity of LLTE might e due to the presence of high moleculr phenolics such s flvonoids (Lin et l., 2008).

5 Jeong et l. : Antioxidnt nd Neuronl Cell Protective Effects of Aqueous Extrcts from Lotus Lef Te 119 (A) ABTS rdicl scvenging ctivities (%) c 1) ,000 Concentrtions (mg/ml) (B) Ferric reducing ntioxidnt power (593 nm) ) ,000 Concentrtions (mg/ml) Fig. 1. ABTS rdicl scvenging ctivities (A) nd ferric reducing ntioxidnt power (B) of LLTE. : LLTE, : Vitmin C, : α-tocopherol. 1) Results re presented s the men ± SD of 3 independent experiments in triplicte. Different letters re significntly different t p<0.05.

6 120 Journl of Agriculture & Life Science 46(2) Antioxidnts cn e referred to s reductnts, which inctivte oxidnts. They re involved in redox rections in which rection species (oxidnt) is reduced t the expense of the oxidtion of the ntioxidnt (reductnt). The FRAP ssy mesures the ntioxidnt effect of ny sustnce in the rection medium s reducing ility (Siddhurju & Mnin, 2007). Antioxidnt potentil of LLTE ws estimted from their ility to reduce TPTZ-Fe( ) complex to TPTZ-Fe( ) complex. In the present study, the trend for ferric ion reducing ctivities of LLTE ws shown in Fig. 1B. For LLTE, the sornce clerly incresed, due to the formtion of the Fe 2+ -TPTZ complex with incresing concentrtion. FRAP of LLTE nd positive controls decresed in the order: vitmin C> α-tocopherol>llte, which were 3.35, 3.34, nd 1.12 (sornce), respectively, t the concentrtion of 1,000 μg/ml. Similr to the results otined from the ABTS ssy, LLTE showed reltively strong ferric ion-reducing ctivity. A correltion etween the men vlues of the totl phenolic content nd FRAP deserves detiled ttention, s phenolics in LLTE ws likely cple of reducing ferric ions. According to recent reports, highly positive reltionship existed etween totl phenolics nd ntioxidnt ctivity in mny plnt species (Dsgupt & De, 2004; Dormn & Hiltunen, 2004). Additionlly, mny of the phenolics hve een shown to contin high levels of ntioxidnt ctivities. 3.2 Inhiitory effect of LLTE on lipid peroxidtion There hs een n incresing interest in lipid peroxidtion ecuse formtion of cytotoxic products such s MDA nd 4-hydroxynonenl cn influence cellulr poptosis nd severl humn diseses (Sevnin & Ursini, 2000). Therefore, in this ssy, ntioxidnt ctivities of LLTE on oth ferric ion nd vitmin C-induced lipid peroxidtion on mouse rin homogentes were lso confirmed. Results shown in Fig. 2 reveled tht LLTE hs excellent ctivities in suppressing lipid peroxidtion on mouse rin homogentes. The LLTE hd etter inhiitory effect thn (+)-ctechin t ll concentrtions. More thn 50% of inhiitory ctivity of lipid peroxidtion ws oserved t the concentrtion of 25 μg/ml. It is lso noteworthy tht (+)-ctechin which hs n EC 50 vlue of μg/ml showed less effectiveness thn the LLTE (EC 50 vlue of μg/ml). Therefore, the LLTE might e potentil nd nturl ntioxidnt supplement for food nd phrmceuticl products. It might lso e used to stilize the foods ginst oxidtive deteriortion. 3.3 Mesurement of intrcellulr oxidtive stress To exmine intrcellulr ccumultion of ROS in PC12 cells used s neuronl cell model, DCF-DA ws employed. DCF-DA proe, which is freely permele cross cell memrne, is hydrolyzed y cytosolic esterses to non-fluorescent dichlorofluorescein (DCFH). Then, DCFH intercting with ROS is oxidized to highly fluorescent sustnce, 2',7'-dichlorofluorescein (DCF). Exposure of PC12 cells to hydrogen peroxide for 2 hr resulted in % increse of ROS levels compred to control (Fig. 3). Pretretment of PC12 cells y LLTE significntly prevented them from intrcellulr ROS ccumultion in comprison to control PC12 cells tht treted only with hydrogen peroxide. Vitmin C is one of nturlly occurring mjor nutrients hving ntioxidnt ctivity (Kim et l., 2002). At the level of 200 μm vitmin C, s positive control, PC12 cells hd significntly lower oxidtive stress thn PC12 cells with tretments of hydrogen peroxide only (Fig. 3). Oxidtive stress in AD my result from ging, energy deficiency, inflmmtion or excessive production of Amyloid β protein (Aβ). Aβ cn induce cell deth through mechnism involving hydrogen peroxide (Behl et l., 1994). In this respect, this result suggests tht the LLTE with ntioxidnt ctivity my ply n importnt role to reduce the oxidtive stress, which is n importnt risk fctor for neurodegenertive diseses such s AD.

7 Jeong et l. : Antioxidnt nd Neuronl Cell Protective Effects of Aqueous Extrcts from Lotus Lef Te Inhiition of lipid peroxidtion (%) ) Conccentrtions (mg/ml) Fig. 2. Inhiitory effect of LLTE on oth ferric ion nd vitmin C-induced lipid peroxidtion on mouse rin homogentes. : LLTE, : Ctechin. 1) Results re presented s the men ± SD of 3 independent experiments in triplicte. Different letters re significntly different t p< ) Oxidtive stress (%) d e c d f 20 0 Control 200 mm H 2 O mm Vit.C Concenrtion (mg/ml) Fig. 3. Effect of LLTE on ROS production determined in the presence nd sence of H 2O 2 in PC12 cell. 1) Results re presented s the men ± SD of 3 independent experiments in triplicte. Different letters re significntly different t p<0.05.

8 122 Journl of Agriculture & Life Science 46(2) 3.4 Neuronl cell viility of LLTE on H 2O 2-induced neurotoxicity Altertion in mitochondril permeility trnsition pore occurs in cell poptosis (Slet et l., 1997) which is relted to the relese of cytochrome c (Ott et l., 2001). Mitochondri might e one of the min trgets dmged y oxidtive stress cusing neuronl cell deth. Fig. 4 shows the cell viility mesured y MTT ssy s minly eing due to ioctive phenolics derived from LLTE ginst oxidtive stress. MTT is converted to purple formzn y living cells in prt ecuse of mitochondril processes. A significnt protection of neuronl cells ws oserved etween control cells with no tretments y H 2O 2 nd groups treted with LLTE t μg/ml followed y H 2O 2 exposure (Fig. 4). The tretment with H 2O 2 for 2 hr decresed the viility of PC12 cells up to 65.16% compred to the control (100%). At μg/ml, the LLTE provided effective protection for the viility of PC12 cells from oxidtive stress. At 200 μg/ml LLTE, the viility of PC12 cells significntly incresed up to 99.00%. Therefore, these results lso suggested tht neuronl cell protection y LLTE is prtilly due to the mitochondril protective mechnisms. 3.5 Protective effect of LLTE on H 2O 2-induced memrne dmge The neuronl memrne with polyunsturted ftty cids is vulnerle to oxidtive stress induced y ROS such s H 2O 2. Lipid peroxidtion cn lter the fluidity of the plsm memrne. LDH ssy provided n estimte of the percentge of surviving PC12 cells. The LLTE protected the integrity of the cellulr memrne t ll the concentrtions tested (Fig. 5). With oxidtive tretment with H 2O 2 for 2 hr, the mounts of LDH relese of PC12 cells incresed to 63.88% compred to tht of the control without tretment. However, the vrious levels of LLTE showed protective effects on neuronl cells in dose-dependent mnner from oxidtive stress (Fig. 5). Tretment of PC12 cells with LLTE t μg/ml reveled significnt protection compred to the group treted with oxidtive stress only (Fig. 5). This finding suggests tht the LLTE might ttenute the extent of oxidtive stress from H 2O 2 to insult neuronl PC12 cells nd then protect the cell s iologicl memrne integrity. Our results suggest tht the phenolics of LLTE might e inhiiting the neuronl poptosis which is the ultimte consequence of these cellulr dysfunctions. Therefore, phenolics of LLTE my lso provide n dded helth enefit y reducing the risk of neurodegenertive diseses such s AD. 3.6 Totl phenolics nd flvonol composition of LLTE Phenolic compounds, such s flvonoids, phenolic cid, nd tnnins re considered to e mjor contriutors to the ntioxidnt ctivity of nturl plnts. These ntioxidnts lso possess diverse iologicl ctivities, such s nti-inflmmtory, nti-crcinogenic, nd nti-therosclerotic ctivities. These ctivities my e relted to their ntioxidnt ctivity (Chung et l., 1998). The totl phenolics of LLTE ws presented in Tle 1. The totl phenolics of LLTE ws mg GAE/g. It ws sujected to further nlysis y HPLC. The LLTE contined vriety of phenolic compounds. By compring the retention time nd UV spectr of these compounds with those of stndrds, quercetin s min phenolics ws identified (Fig. 6). Furthermore, the HPLC results indicted tht quercetin ( mg/100 g) ws the predominnt flvonol in this LLTE (Fig. 6 nd Tle 1). Bsed on the results for the phenolic composition of LLTE, we cn conclude tht these compounds (prticulrly quercetin) contriute to the ntioxidnt nd neuronl cell protective effects of LLTE. The results otined in this work re noteworthy, not only with respect to the ntioxidnt nd neuronl cell protective effects of LLTE, ut lso with respect to its content of quercetin. The ctivity of LLTE is ttriuted to these phenolic compounds nd in prticulr to quercetin. The min flvonoids found in green te nd pple fruit is quercetin. They re of

9 Jeong et l. : Antioxidnt nd Neuronl Cell Protective Effects of Aqueous Extrcts from Lotus Lef Te 123 Cell viility (%) ) f c e d c 20 0 Control 200 mm H 2 O mm Vit.C Concentrtion (mg/ml) Fig. 4. Protective effect of LLTE ginst H 2 O 2 -induced cell deth in PC12 cell system. PC12 cells were pretreted for 48 hr with vrious concentrtions. The cells were then treted with 200 μm H 2O 2 for 2 hr. Levels of cell viility were mesured using the MTT ssy kit. Vitmin C (200 μm) ws pplied s positive control. 1) Results re presented s the men ± SD of 3 independent experiments in triplicte. Different letters re significntly different t p< ) LDH relese into medium (%) g f c d d e 0 Control 200 mm H 2 O mm Vit.C Concentrtion (mg/ml) Fig. 5. Inhiitory effect of LDH relese of LLTE on H 2 O 2 -induced memrne dmge in PC12 cells. The cells were then treted with 200 μm H 2O 2 for 2 hr. The LDH ctivity in culture superntnts ws mesured with colorimetric LDH ssy kit. Vitmin C (200 μm) ws pplied s positive control. 1) Results re presented s the men ± SD of 3 independent experiments intriplicte. Different letters re significntly different t p< 0.05.

10 124 Journl of Agriculture & Life Science 46(2) (A) (B) Fig. 6. HPLC chromtogrms of stndrds (A) nd LLTE (B). Retention time min, Myricetin; min, Quercetin; min, Kempferol. interest ecuse they hve wide rnge of phrmceuticl properties including ntioxidnt, nticrcinogenic, ntimicroil ctivities, nd ntineurodegenertive effect (Dreosti et l., 1997; Jnkun et l., 1997; Almjno et l., 2008; Heo & Lee, 2004). In this point, our results with the cell

11 Jeong et l. : Antioxidnt nd Neuronl Cell Protective Effects of Aqueous Extrcts from Lotus Lef Te 125 viility of LLTE were minly due to the quercetin including other ntioxidnt phenolics. Tle 1. Totl phenolics nd flvonols content of Flvonols (mg/100 g) LLTE Totl phenolics (mg GAE/g) Retention time (min) Contents 33.16±1.79 1) Myricetin Quercetin ±4.68 Kempferol Trce 1) The vlues re mens ± SD of three experimentl dt. Finlly, our results verified tht LLTE hs very strong ntioxidnt ctivities, nd suggested tht LLTE cn e utilized s n effective nturl ntioxidnt sources nd chemopreventive gent ginst neurodegenertive disese such s Alzheimer s disese. In dditions, further studies re needed to determine the reltionship etween ntioxidnt nd neuroprotection using severl in vivo tests. Ⅳ. Acknowledgements This work ws supported y the Ntionl Reserch Foundtion of Kore Grnt funded y the Koren Government (KRF F00074, KRF ). Literture Cited Almjno, M. P., R. Cró, J. Jiménez, nd M. H. Gordon Antioxidnt nd ntimicroil ctivities of te infusions. Food Chem. 108: Behl, C., F. Lezoulch, T. Trpp, M. Widmnn, T. Skutell, nd F. Holsoer Glucocorticoids enhnce oxidtive stress-induced cell deth in hippocmpl neurons in vitro. Endocrinology 138: Behl, C., J. B. Dvis, R. Lesley, nd D. Schuert Hydrogen peroxide medites myloid β protein toxicity. Cell 77: Chng, S. T., J. H. Wu, S. Y. Wng, P. L. Kng, N. S. Yng, nd L. F. Shyur Antioxidnt ctivity of extrcts from Acci confuse rk nd hertwood. J. Agr. Food Chem. 49: Chung, K. T., T. Y. Wong, C. I. Wei, Y. W. Hun, nd Y. Lin Tnnins nd humn helth: A review. Crit. Rev. Food Sci. Nutr. 38: Crundmn, M. nd P. Delney Antioxidnt strtegies for Alzheimer s disese. Proc. Nutr. Soc. 61: Dsgupt, N. nd B. De Antioxidnt ctivity of Piper etle L. lef extrct in vitro. Food Chem. 88: Dormn, H. J. D. nd R. Hiltunen Fe( ) reductive nd free rdicl-scvenging properties of summer svory (Sturej hortensis L.) extrct nd sutrctions. Food Chem. 88: Dreosti, I. E., M. J. Wrgovich, nd C. S. Yng Inhiition of crcinogenesis y te: the evidence from experimentl studies. Crit. Rev. Food Sci. Nutr. 37: Goo, H. R., J. S. Cho, nd D. H. N Simultneous determintion of quercetin nd its glycosides from the leves of Nelumo nucifer y reversed-phse high-performnce liquid chromtogrphy. Arch. Phrm. Res. 32: H, J. Y., K. E. Lee, J. M. Prk, A. Y. Dong, nd H. S. Shin Cytoprotective ctivity of lotus (Nelumo nucifer Gertner) lef extrcts on the mouse emryonic firolst cell. Food Sci. Biotechnol. 19: Hgermn, A. E., K. M. Riedl, G. A. Jones, K. N. Sovik, N. T. Ritchrd, P. W. Hrtzfeld, nd R. L. Riechel High moleculr weight plnt polyphenolics (tnnins) s iologicl ntioxidnts. J. Agr. Food Chem. 46: Heo, H. J., nd C. Y. Lee Protective effect of quercetin nd vitmin C ginst oxidtive stressinduced neurodegenertion. J. Agr. Food Chem. 52:

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