IN VITRO ACTIVITY OF LOQUAT LEAF EXTRACT AGAINST OXIDATIVE DAMAGE IN NEURONAL CELL

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1 CURRENT TOPICS IN NUTRACEUTICAL RESEARCH Vol. 11, No. 3, pp , 2013 ISSN print, Copyright 2013 y New Century Helth Pulishers, LLC All rights of reproution in ny form reserve IN VITRO ACTIVITY OF LOQUAT LEAF EXTRACT AGAINST OXIDATIVE DAMAGE IN NEURONAL CELL 1 Sun Jin Hur, 2 Young Il Be, 3 YoungChn Kim, 3 Inwook Choi n 4 Chng Ho Jeong 1 Deprtment of Bioresoures n Foo Siene, Konkuk University, Seoul, , Kore; 2 Bio 21 Center, Jinju , Kore; 3 Kore Foo Reserh Institute, Seongnm, , Kore; n 4 Reserh Center, Wooyng Frozen Foo Co., Lt., Seoheon, , Kore. [Reeive April 13, 2013; Aepte July 14, 2013] [Communite y Prof. Rotimi Aluko] ABSTRACT: This stuy ws onute to investigte the in vitro neuroprotetive n ntioxitive tivities of queous loqut extrt (LLE). The loqut lef extrts inhiite lipi peroxition n showe protetive effets ginst H 2 inue neurotoxiity in oseepenent mnner. In the neuronl ell (PC 12) viility ssy, LLE showe protetive effet ginst H 2 inue neurotoxiity, n the relese of ltte ehyrogense (LDH) into the meium ws lso inhiite ( %). The totl phenoli ontent of LLE ws mg/glli i equivlent (GAE) g, n the mjor phenoli ompouns were rutin, epitehin, n tehin. These t suggest tht n LLE tht inlues the ove phenolis my e useful s nturl ntioxint sustne. KEY WORDS: Epitehin, Loqut lef, Neuroprotetive tivity, Queritrin, Rutin Corresponing Author: Dr. Chng Ho Jeong, Reserh Center, Wooyng Frozen Foo Co., Lt., Seoheon, , Kore: Phone , Fx; ; Emil ifo5479@fookore.om INTRODUCTION The retive oxygen speies (ROS) generte within living orgnisms n y exogenous soures initite retions tht mge iologil moleules n ply n importnt ustive role in isese initition (Aruom, 1996). The formtion of exess ROS n retive nitrogen speies (RNS) y UV irrition, smoking n rug metolism re likely to mge severl ellulr omponents, suh s lipis, proteins, nulei is, n DNA, through oxition or nitrtion proesses (Sw, Akike & Me, 2000). In ition, ROS use inflmmtion or lesions on vrious orgns n re ssoite with vrious egenertive iseses, inluing ner, ging, rterioslerosis, n rheumtism (Choi, Choi, Hn, Be & Chung, 2002; Squrito & Pryor, 1998). Alzheimer s isese (AD), ommon type of ementi, is progressive neuroegenertive isorer hrterize y the umultion senile plques ontining myloi β (Aβ) n neurofirillry tngles in the rin ompose of phosphorylte tu. The use of AD is unertin, ut severl reent stuies hve implite retive oxygen speiesinue oxitive stress in its pthogenesis (Behl, Dvis, Lesley & Shuert, 1994; Hlliwell, 2001). The estknown ntioxints re tritionl nutrients, suh s βrotene, sori i, n αtoopherol. However, there is growing eviene tht signifint portion of the ntioxint pity of mny eile nturl prouts is ue to phytohemils other thn the tritionl vitmins (Co, Sofi & Prior, 1996). Reently, reserhers hve sought to isolte powerful n nontoxi nturl ntioxints from eile plnts to prevent utoxition n lipi peroxition n to reple syntheti ntioxints (Liu et l., 2012). Reserh into the ientifition of nturl ntioxint soures is importnt to promote puli helth. Loqut (Eriootry jponi Linl.) elongs to the Rosee fmily, ut it is one of the few sutropil representtives. It most likely h its origin in southestern Chin, n it hs een ultivte in Chin n Jpn sine nient times. Now it is lso ultivte in other regions, nmely in the Meiterrnen, Austrli, South Afri, South Ameri, Cliforni, n Ini. The plnt is n evergreen shru or smll tree, with nrrow leves tht re rk green on the upper surfe n hve lighter woolly uner surfe. Its white flowers give rise to pleyellow or eepornge pomes (Vughn & Geissler, 1997). The prinipl ojetive of the eonomi exploittion of the loqut is the proution of its fruits, whih n e eten fresh or proesse into jm

2 104 In vitro neuroprotetive effet of loqut lef extrt n jelly. Furthermore, loqut leves hve een use to tret skin iseses n ietes (De Tommsi, Aquino, De Simone & Pizz, 1992), hroni ronhitis, oughs, phlegm, ulers, n ner (Ito et l., 2000), n they possess ntioxitive tivity (Ferreres et l., 2009). Although it hs lrey een emonstrte tht fresh loqut hs physiologil tivity ue to its ontents of phytohemils, little is known out its protetive effets ginst oxitive stress in neuronl ell, suh s PC12 ells. Therefore, the ojetives of this stuy were to exmine the protetive effets of n queous extrt of loqut lef (LLE) ginst oxitive stress in neuronl ell n ientify the min phenolis s tive ompouns. This work shoul help to prevent vrious iseses tht re of interest in humn helth. MATERIALS AND METHODS Plnt mteril The lef of loqut (Eryootry jponi) ws ollete in Geoje, Gyeongnm, Kore. The smples were wshe with running tp wter efore eing hoppe into piees. They were then ovenrie t 45 o C for 2 ys n groun to power. These smples were store t 20 o C until use for nlysis. Preprtion of extrts Aqueous extrts of freezerie loqut lef power were otine s follows. Loqut lef (50 g) power ws suspene n extrte with 1,000 ml of wter t 100 o C for 2 h. The extrts were filtere through Whtmn No. 2 filter pper (Whtmn Interntionl Limite, Kent, Engln) n evporte to ryness. The queous extrts were onentrte in vuum evportor t 40 o C. The finl extrts were ple in glss ottle n store t 20 o C until use for nlysis. The lyophilize extrts were reissolve in wter to onentrtion of 1,000 μg/ml. Mlonilehye (MDA) ssy using mouse rin homogentes Twenty Bl/ mie (4 months ol, verge 50 ± 5 g oy weight) were otine from Dr. Jeong s lortory t Gyeongsng Ntionl University. After 1 week of ption, the mie were srifie y C gs. The skull ws opene, n the rins were ollete. The mlonilehye (MDA) ssy ws rrie out oring to the metho esrie in previous stuy to etermine the lipi oxition vlue (Chng, Wu, Wng, Kng, Yng & Shyur, 2001). The rins of the Bl/ mie were homogenize in ieol TrisHCl uffer (20 mm, ph 7.4) to proue 1/10 homogente. The homogente ws entrifuge t 12,000 g for 15 min t 4 o C. Then, 1mL liquots of the superntnt were inute with the test smples in the presene of 10 μm FeSO 4 n 0.1 mm sori i t 37 o C for 1 hr. The retion ws terminte y the ition of 1.0 ml trihloroeti i (28%, w/v) n 1.5 ml thiorituri i (TBA, 1%, w/v) in suession, n then the solution ws hete t 100 o C. After 15 min, the olor of the mlonilehye (MDA)TBA omplex ws mesure t 532 nm. (+)Ctehin, wellknown ntioxint, ws use s positive ontrol. The inhiition rtio (%) ws lulte s follows: [1 (Smple sorne / Control sorne)] 100 The smple olletion n euthnsi protools were pprove y the Animl Cre Committee of Konkuk University. Neuronl Cell Culture The PC12 ell line ws erive from trnsplntle rt pheohromoytom. The ells respon reversily to nerve growth ftor (NGF) y inution of the neuronl phenotype. PC12 ells (KCLB 21721, Kore Cell Line Bnk, Seoul, Kore) were propgte in RPMI1640 meium ontining 10% fetl ovine serum, 25 mm HEPES, 25 mm soium ironte, 50 units/ml peniillin n 100 μg/ml streptomyin. Neuroprotetive effet on oxitive stress An MTT reution ssy ws performe using the in vitro toxiology ssy kit (TOX1, Sigm Co., St. Louis, MO, USA). Neuronl PC12 ells were plte t ensity of 10 6 ells/well on 96well pltes in 100 μl of RPMI. The ells were preinute with queous extrt from loqut lef for 48 hr efore the 200 μm hyrogen peroxie ws e. The ells were trete with or without hyrogen peroxie for 3 hr. The mount of MTT formzn prout ws etermine y mesuring sorne using miroplte reer (680, Bio R, Tokyo, Jpn) t test wvelength of 570 nm n referene wvelength of 690 nm. Neuronl PC12 ells were preipitte y entrifugtion t 250 g for 4 min t room temperture, 100 μl of eh superntnt ws trnsferre into new well, n ltte ehyrogense (LDH) ontent ws etermine using the in vitro toxiology ssy kit (TOX 7, Sigm Co., St. Louis, MO, USA). Dmge of the plsm memrne ws evlute y mesuring the mount of the intrellulr LDH enzyme relese into the meium. Determintion of totl phenolis The totl phenolis were etermine y spetrophotometri metho (Kim, Lee, Lee & Lee, 2002). The phenoli ompouns in the queous extrt of loqut lef were mesure t 280 nm using phenoli ompoun stnr solution y ioe rry etetor (Agilent 1100 series, Agilent Co., Snt Clr, CA, USA). Seprtion ws hieve with Shisheio C 18 olumn (250 mm 4.6 mm i, 5 μm, Shisheio Co., Tokyo, Jpn). The elution solvents were (A) 0.01 Mpotssium phosphte uffer juste to ph 3.0 y phosphori i n (B) methnol. The solvent grient elution progrm use ws s follows: initil 90% (A), hol for 9.5 min; liner grient to 68% (A) in 3.5 min; liner grient to 67% (A) 17 min; liner grient to 20% (A) 1 min; liner grient to 90% (A) 1 min, n hol for 10 min. The flow rte ws 1.5 ml/min. The phenolis were ientifie y omprison of their retention time (RT) vlues n UV spetr with those of known stnrs, n they were quntifie y the pek res from

3 In vitro neuroprotetive effet of loqut lef extrt 105 the hromtogrms. All nlyses were run in triplite, n the men vlues were lulte. The ontent of the phenoli ompouns ws expresse s μg/100 g extrt. Quntifition y HPLC Loqut lef (2 g) ws extrte with oiling wter (100 ml) for 2 h. The otine filtrte ws frtionte with ethyl ette (100 ml) to give n ethyl ette frtion. The frtiontion ws repete 3 times. The ethyl ette frtion ws then evporte to ryness uner reue pressure fter the resiul wter ws remove using nhyrous soium sulfte. Next, the ethyl ette frtions were reissolve in 10 ml of 10% DMSO. The phenoli ompouns in the ethyl ette frtion of the loqut lef were mesure t 280 nm using stnr solution of phenoli ompouns y ioe rry etetor (Agilent 1100 series, Agilent Co., Snt Clr, CA, USA). Seprtion ws hieve with Shisheio C 18 olumn (250 mm 4.6 mm i, 5 μm, Shisheio Co., Tokyo, Jpn). The elution solvents were (A) 0.01 Mpotssium phosphte uffer juste to ph 3.0 y phosphori i n (B) methnol. The solvent grient elution progrm use ws s follows: initil 90% (A), hol for 9.5 min; liner grient to 68% (A) over 3.5 min; liner grient to 67% (A) over 17 min; liner grient to 20% (A) over 1 min; liner grient to 90% (A) over 1 min, n hol for 10 min. The flow rte ws 1.5 ml/min. The phenolis were ientifie y omprison of their retention time (RT) vlues n UV spetr with those of known stnrs, n they were quntifie y the pek res from the hromtogrms. All nlyses were run in triplite, n the men vlues were lulte. The ontent of the phenoli ompouns ws expresse in µg/100 g extrt (Jeong, Kwk, Kim, Choi, Kim & Heo, 2011). Sttistil nlysis All t were expresse s the men±sd (n=3). Eh experimentl set ws ompre with the onewy nlysis of vrine (ANOVA) n Dunn s multiple rnge test (p<0.05) using SAS (SAS Institute, Cry, NC, USA). RESULTS AND DISCUSSION Inhiition of lipi peroxition There hs een inresing interest in lipi peroxition ue to the formtion of ytotoxi prouts, suh s MDA n 4hyroxynonenl, whih n influene poptosis n severl humn iseses (Sevnin & Ursini, 2000). Therefore, in this ssy, the inhiitory effets of n queous extrt of loqut lef ginst oth ferri ion n sori iinue lipi peroxition in mouse rin homogentes were etermine, n the pink olor of the MDATBA omplex ws etete t 532 nm. The results shown in Fig. 1 revel tht the extrt h suppressing tivities ginst lipi peroxition of the mouse rin homogentes. The LLE h superior inhiitory effets ompre with (+)tehin t ll onentrtions, n more thn 50% of the inhiitory tivity on lipi peroxition ws oserve t the onentrtion of 25 μg/ml. It is lso noteworthy tht (+)tehin, whih hs n EC50 vlue of μg/ml, ws less effetive thn the LLE (EC50 vlues of μg/ml). Severl stuies inite tht rutin, epitehin, tehin, hlorogeni i, n queritrin hve exellent ntioxint n neuronl ell protetive effets (Heo & Lee, 2005; Jeong, Jeong, Choi, Shim & Heo, 2011; RieEvns, Miller & Pgng, 1997). The ove results provie promising seline informtion for the potentil use of LLE in ition to isolte ompouns s n effetive nturl ntioxint. FIGURE 1. Mlonilehye (MDA) ssy of LLE for ferri ion n sori iinue lipi peroxition. : LLE, : Ctehin. 1) The results re presente s the men±sd of 3 inepenent experiments performe in triplite. Different letters etween the sme onentrtions inite signifint ifferenes t p<0.05. Inhiition of lipi peroxition (%) Conentrtions (μg/ml) Neuroprotetive effet ginst H 2 inue ytotoxiity Hyrogen peroxie hs een reporte to inue poptosis in ells of the entrl nervous system. In this stuy, n queous extrt of loqut lef ws selete to investigte the neuroprotetive effets ginst H 2 inue mge euse of the strong ntioxitive tivity. Protetive effets on H 2 inue neuronl ell mge were exmine y the MTT ssy. H 2 use erese in ell viility (50.73%), ut pretretment of the PC12 ells with inresing onentrtions of the LLE inhiite oxitive stressinue ytotoxiity (Fig. 2). The protetive effets of LLE t 500 μg/ml ginst oxitive injury in neuronl ell were similr to the effets of 200 μm vitmin C. This stuy emonstrte tht PC12 poptosis ue to oxitive stress ws suppresse y pretretment with LLE. The MTT ye reution ssy is se on the tlyti tivity of metoli enzymes in intt mitohonri. Hene, this result shows tht the protetion of PC12 ells y LLE re prtilly ue to mitohonril mehnisms. Oxitive stress in AD my

4 106 In vitro neuroprotetive effet of loqut lef extrt result from ging, energy efiieny, inflmmtion n exessive proution of myloi protein (A). A n inue ell eth through mehnism involving hyrogen peroxie (Behl et l., 1994). Our result lso suggests tht the ntioxint tivity of LLE my ply n importnt role in reuing the oxitive stressinue risk in neuroegenertive isese suh s AD. FIGURE 2. Neuroprotetive effet of LLE on H 2 inue ell eth in PC12 ells. Cell viility ws not hnge y vitmin C or the phenolis (t not shown). The results shown re the mens±sd (n=3). A signifint ifferene (p<0.05 vs. vitmin C) ws oserve for H 2 inue ell eth Cell viility (MTT reution : % ontrol) Control H 2 Vit. C 200 μμ 200 μμ ,000 Conentrtions (μg/ml) FIGURE 3. Neuroprotetive effet of the queous extrt of loqut lef on H 2 inue memrne mge in PC12 ell. Cell viility ws not hnge y vitmin C or the phenolis (t not shown). The results shown re the mens±sd (n=3). A signifint ifferene (p<0.05 vs. vitmin C) ws oserve for H 2 inue ell eth. 80 LDH relese into meium : % of ontrol in ll ells, n it is rpily relese into the ell ulture if the plsm memrne is mge y oxints. An inrese in LDH tivity in ulture superntnt inites n inrese in the numer of e or plsm memrnemge ells. After the tretment of PC12 ells with 200 μm H 2 for 3 h, the sorne (490 nm) vlues inrese n LDH tivity inrese mrkely to 257% of tht of the ontrol group, initing tht the ells were impire or tht proportion of the ells were e. Compre with the effets on the orresponing H 2 tretment group, the queous LLE oseepenently erese the sorne vlues t 490 nm. In prtiulr, LLE signifintly ttenute the LDH relese to TABLE 1. Phenoli omposition of loqut lef queous extrt. The vlues re expresse s mens ± SD of three experimentl t. Phenoli ompouns Content (µg/100 g) Glli i Prototehui i Chlorogeni i ±24.36 Ctehin ±28.56 Vnilli i Epitehin ±16.97 Cffei i pcoumri i Feruli i Rutin ±61.08 Queritrin ±18.25 Queretin FIGURE 4. HPLC hromtogrm in the queous extrt of loqut lef. A: stnr, B: loqut lef. Ethyl ette frtion metho ws use for the HPLC nlysis. A B 0 Control H μμ Vit. C 200 μμ ,000 Conentrtions ( μg/ml) Neuroprotetive effet ginst H 2 inue memrne mge To further investigte the protetive effet of LLE, the LDH ssy ws performe. LDH is stle ytoplsmi enzyme

5 In vitro neuroprotetive effet of loqut lef extrt % of the ontrol group t 1,000 μg/ml. These t suggest tht LLE oul protet PC12 ells ginst the lesion inue y H 2 (Fig. 3). Consequently, these results show tht PC12 ell protetion y LLE is prtilly ue to the mitohonril n ell memrne protetive mehnisms ginst H 2 inue neurotoxiity. Totl phenolis n the phenoli omposition of ethyl ette frtion from LLE The FolinCiolteu s ssy is fst n simple metho to rpily etermine the ontent of phenolis in smple. Phenolis re seonry plnt metolites tht re present s nturl prouts. Mny phenolis hve een shown to ontin high levels of ntioxint tivities. The totl phenoli ontent of LLE is presente in Tle 1 n Figure 4. The totl phenoli ontent of LLE ws mg/gae g. To ientify the min phenolis ting s ntioxints n neuroprotetive gents, the ethyl ette frtion of LLE ws sujete to further nlysis y HPLC. The ethyl ette frtion from LLE ontine five importnt phenolis. By ompring the retention times n UV spetr of these ompouns with those of stnrs, five phenolis (rutin, epitehin, tehin, hlorogeni i, n queritrin) were ientifie (Tle 1). Furthermore, the HPLC results inite tht rutin ( µg/100 g) ws the preominnt phenoli i in the ethyl ette frtion from LLE, followe y epitehin n tehin (Tle 1). Consequently, the physiologil tivities etermine in the ethyl ette frtion from LLE oul e ttriute to phenolis, inluing rutin, epitehin, n tehin. However, further stuies relte to the mehnisms of poptosis, e.g., spse tivtion n gene expression, re neee to unerstn the systemti effets of the min phenolis on ell viility n ell eth. In this stuy, tretment of neuronl ell with LLE revele potent ntioxint pity ginst lipi peroxition. In ition, we foun tht LLE ws effetive for proteting PC12 ells from H 2 inue oxitive stress. Loqut lef ontins physiologil phenolis n my e useful s powerful n nturl ntioxint gent to protet ginst lipi peroxition. Furthermore, LLE ws shown to ontin rutin, epitehin, tehin n their metolites, whih my ount for its neuronl ell protetive effets s well s its ntioxint tivity. Therefore, our results suggest tht the neuronl ell protetive effet of LLE is losely relte to its ntioxint tivity n tht it oul e utilize s nturl ntioxint soure n hemopreventive gent ginst neuroegenertive isorers, suh s Alzheimer s isese. AKNOWLEDGEMENTS This pper ws supporte y the SMART Reserh Professor Progrm of Konkuk University. REFERENCES Aruom O (1996) Assessment of potentil prooxint n ntioxint tions. Journl of the Amerin Oil Chemists Soiety, 73: Behl C, Dvis JB, Lesley R n Shuert D (1994) Hyrogen peroxie meites myloi β protein toxiity. Cell, 77: Co G, Sofi E n Prior RL (1996) Antioxint pity of te n ommon vegetles. Journl of Agriulturl n Foo Chemistry, 44: Chng ST, Wu JH, Wng SY, Kng PL, Yng NS n Shyur LF (2001) Antioxint tivity of extrts from i onfus rk n hertwoo. Journl of Agriulturl n Foo Chemistry, 49: Choi HR, Choi JS, Hn YN, Be SJ n Chung HY (2002) Peroxynitrite svenging tivity of her extrts. Phytotherpy Reserh, 16: De Tommsi N, Aquino R, De Simone F n Pizz C (1992) Plnt metolites. new sesquiterpene n ionone glyosies from eriootry jponi. Journl of Nturl Prouts, 55: Ferreres F, Gomes D, Vlentão P, Gonçlves R, Pio R, Chgs EA, Ser RM n Anre PB (2009) Improve loqut (Eriootry jponi Linl.) ultivrs: Vrition of phenolis n ntioxitive potentil. Foo Chemistry, 114: Hlliwell B (2001) Role of free rils in the neuroegenertive iseses: therpeuti implitions for ntioxint tretment. Drugs & Aging, 18: Heo HJ n Lee CY (2005) Epitehin n tehin in oo inhiit myloi β protein inue poptosis. Journl of Agriulturl n Foo Chemistry, 53: Ito H, Koyshi E, Tkmtsu Y, Li SH, Htno T, Skgmi H, Kusm K, Stoh K, Sugit D, Shimur S, Itoh Y n Yoshi T (2000) Polyphenols from eriootry jponi n their ytotoxiity ginst humn orl tumor ell lines. Chemil & Phrmeutil Bulletin, 48: Jeong CH, Kwk JH, Kim JH, Choi GN, Kim DO n Heo HJ (2011) Neuronl ell protetive n ntioxint effets of phenolis otine from Znthoxylum piperitum lef using in vitro moel system. Foo Chemistry, 125: Jeong CH, Jeong HR, Choi SG, Shim KH n Heo HJ (2011) Neuronl ell protetion n ntioxint tivities of hot wter extrt from ommeril ukwhet te. Koren Journl of Foo Preservtion, 18: Kim DO, Lee KW, Lee HJ n Lee CY (2002) Vitmin C equivlent ntioxint pity (VCEAC) of phenoli

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