The antioxidant activity and nitric oxide production of extracts obtained from the leaves of Chenopodium quinoa Willd

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1 iomedicine (ISSN ) Decemer 217, Vol. 7, No. 4, rticle 24, Pges DOI: 1.151/mdcn/ Originl rticle The ntioxidnt ctivity nd nitric oxide production of extrcts otined from the leves of Chenopodium quino Willd Hsio-Ling Chen 1, Xing-Zhen Ln 1, Yn-Yi Wu 1, Yu-Wen Ou 1, Tsung Chi Chen 2, Wen-Tzu Wu 1, * 1 Deprtment of Food Nutrition nd Helth iotechnology, si University, Tichung 413, Tiwn 2 Deprtment of iotechnology, si University, Tichung 413, Tiwn Received 17 th of pril, 217 ccepted 9 th of My, 217 uthor(s) 217. This rticle is pulished with open ccess y Chin Medicl University Keywords: Chenopodium quino; ntioxidnt ctivity; Nitric oxide STRCT ckground: Most reports hve indicted the ntioxidnt cpcity of quino seeds. However, the leves of Quino (Chenopodium quino Willd.) re usully worthless nd little known out their iologicl ctivities. In this study, the ntioxidnt nd immunomodultory potentil of the quino lef extrcts were explored. Methods: The crude lef extrcts of quino were extrcted using wter, 5% ethnol or 95% ethnol s solvent, denoted WQL, 5% EQL nd 95% EQL, respectively. The ntioxidnt ctivities of quino lef extrcts were ssessed y the ility of 1,1-diphenyl-2-picrylhydrzyl (DPPH) scvenging nd iron chelting. The totl phenolic content ws determined. Inhiition of nitric oxide (NO) production in the lipopolyscchride (LPS)-induced murine mcrophge RW cells ws exmined to guge the nti-inflmmtory ctivity. Results: The 95% EQL showed higher level of totl phenolic content (569.5 mg GE/g extrct) nd etter DPPH scvenging ctivity. The WQL exhiited etter iron chelting cpcity (28.9% t 1 mg/ml). The iron chelting ctivity of the 95% EQL incresed in concentrtion-dependent mnner, which rnged from 1.9% up to 53.9%. The 5% EQL nd 95% EQL significntly inhiited NO production in the LPSstimulted RW cells. Conclusion: We demonstrte tht the extrcts of quino leves possess the iologicl ctivities of ntioxidnt nd nti-inflmmtory. Our finding suggests tht the lef extrct of quino hs potentil to e utilized for nturl helth products. 1. Introduction lrge mount of scientific evidence suggests tht oxidtive stress is involved in the pthogenesis of numerous humn diseses. The nturl ntioxidnts from plnt extrcts re the suject of incresing scientific interest ecuse of their function in the prevention of disese. Most of the reserch on the potentil effects hs een focused on free rdicl scvenging, metl chelting, nti-inflmmtion, nd nticncer ctivities [1, 2]. ll prts of plnts, including their leves, rk, roots, flowers, fruits, nd seeds hve een extensively studied for their ntioxidnt ctivity. The phenolic compounds re widely ville in plnts nd hve een reported s the min contriutors to the ntioxidtive nd diverse iologicl properties [3]. The genus Chenopodium includes more thn 2 species nd mny of these re edile either whole plnts or prts of the plnt. The widely used of Chenopodium species in trditionl medicine hve resulted in its phytochemicls, such s flvonoids [4]. Qui- no (Chenopodium quino Willd.) is known s pseudo-cerel nd hs een found to contin compounds like polyphenols, phytosterols, nd flvonoids, ll with possile nutrceuticl enefits. It is trditionlly consumed y the ntive popultion of the ndes region [5] nd hs een recognized s complete food tht is rich in nutrients [6]. With the incresing worldwide ttention to its nutritionl contents, the quino seeds hve een reported to exhiit eneficil effects on humn helth. However, very few studies exist conducted on the iologicl ctivities of quino leves. Quino leves till now hve een thought of s worthless wste ut re ctully edile nd my serve s vlule ingredient in functionl food. The im of this study ws to evlute the potentil ioctivity of quino lef extrcts on ntioxidnt nd nti-inflmmtion ctivities. The ntioxidnt ctivities of these extrcts were determined y using DPPH scvenging nd the cheltion cpcity of ferrous ion. The production of nitric oxide (NO) induced y lipopolyscchride (LPS) in RW cells ws used to ssess the nti-inflmmtory effects. * Corresponding uthor. Deprtment of Helth nd Nutrition iotechnology, si University, No. 5, Lioufeng Rd., Tichung 413, Tiwn. E-mil ddress: irenewu@si.edu.tw (W.-T. Wu). 24

2 2. Mterils nd methods 2.1. Chemicls nd plnt mterils 1,1-Diphenyl-2-picrylhydrzyl (DPPH), Folin-Cioclteu s regent, gllic cid, lipopolyscchride (LPS), ferrozine, sodium cronte, nd dimethyl sulfoxide (DMSO) were purchsed from Sigm-ldrich (St. Louis, MO, US). RPMI 164, fetl ovine serum (FS), ntiiotic ntimycotic solution, phosphte uffered sline (PS), nd 3-[4, 5-dimethylthizole-2-yl]-2,5-diphenyltetrzolium romide (MTT) were purchsed from Gico (Thermo- Fisher Science, Inc.). Ethnol (nlyticl grde) ws purchsed from ECHO Chemicl Co., Ltd. (Mioli, Tiwn). Quino plnts were grown in temperture-controlled greenhouse. Fully expnded leves of quino were hrvested nd freeze-dried for 24-h. The dried smple ws ground into powder using kitchen milling mchine nd stored t -2 C prior to extrction Preprtion of crude extrcts The quino lef powder (1 g) ws extrcted with 5 ml of distilled wter, 5% nd 95% ethnol for 16-h t room temperture, nd the solid-liquid mixture ws filtered through Whtmn No.2 filter pper (DVNTEC qulittive filter pper numer 2). The resulting superntnt ws concentrted under vcuum t 4 C (EYEL rotry evportor N-1, Jpn) nd freeze-dried for 24-h to otin the crude extrcts. The dried wter extrct ws dissolved in PS, denoted WQL. Dried extrcts of quino leves using 5% nd 95% ethnol were dissolved in DMSO nd nmed 5% EQL nd 95% EQL, respectively. The stock solutions of 1 mg/ml were stored t -2 C prior to determining the ntioxidtion ctivity nd the inhiition of NO production Determintion of totl phenolic content The totl phenolic contents of WQL, 5% EQL nd 95% EQL were determined y the Folin-Cioclteu ssy [7]. Ech diluted extrct smple or gllic cid (.5 ml) ws mixed with 2.5 ml of Folin s regent (diluted with distilled wter 1:1) nd 2 ml of 7.5% (w/v) sodium icronte solution. Their sornce ws determined spectrophotometriclly t 7 nm using microplte reder (Epoch 2, iotek, VT, US) fter stnding for min t room temperture. The results were expressed s milligrm gllic cid equivlent (GE) per grm extrct Free rdicl scvenging ssy The DPPH rdicl-scvenging ctivity of ll crude extrcts of quino leves ws determined ccording to the method of Shimd et l. [8] with slight modifictions. The crude extrct (5 μl) with vrying concentrtions (1, 5 nd 1 mg/ml) ws dded to 1 μl of 1 mm DPPH in methnol. This mixture ws shken nd llowed to stnd for min t room temperture in the drk. The sornce ws mesured t 517 nm using microplte reder (Epoch 2, iotek). The percentge inhiition of rdicls ws clculted s [( control - smple ) / control ] 1%, where control is the sornce of DPPH solution without extrct nd smple is the sornce of the smple with DPPH solution Iron chelting ssy The cheltion of ferrous ions y quino lef extrcts ws estimted using the method reported y Dinis et l. [9]. riefly, 25 μl of 2 mm FeCl 2 ws dded to.25 ml of different concentrtions of the crude extrct (1, 5 nd 1 mg/ml) nd.5 ml of 5 mm ferrozine solution. The mixture ws vigorously shken nd llowed to stnd t room temperture for 1 min. The sornce of the solution ws therefter mesured t 562 nm nd the inhiition percentge of ferrozine Fe 2+ complex formtion ws clculted s the sme s the DPPH method descried ove Cell culture The murine mcrophge RW cell line ws gift from Dr. Yun-Yen Chng. The cells were cultured in RPMI 164 supplemented with 1% FS nd 1% ntiiotic ntimycotic solution t 37 C in humidified incutor under 5% CO Cell viility ssy Cell viility ws determined y the MTT ssy. riefly, cells were seeded in 96-well plte t density of cells/well nd llowed to dhere for 24-h efore tretment. Next, cells were treted with vrious concentrtions of WQL, 5% EQL, nd 95% EQL, respectively. fter 24-h incution, MTT ws dded to finl concentrtion of.5 mg/ml nd the cells were incuted for 2-h t 37 C. The medium ws then removed nd 15 ml of DMSO ws dded to dissolve the formzn precipitte. The sornce ws mesured t 57 nm using microplte reder (Epoch 2, iotek). The cell viility ws normlized to the medium control group nd expressed s % of control. The non-cytotoxic dose of ll crude extrcts ws determined to e 1-1 μg/ml, nd 1, 1 nd 1 μg/ml of ll crude extrcts were used for exposure in the susequent experiments Inhiition of NO production Cells in 96-well pltes (5 1 4 cells/well) were treted with WQL, 5% EQL, nd 95% EQL t finl concentrtion s descried in the previous section. fter 1-h tretment, cells were stimulted with 1 μg/ml of LPS for 24-h. Nitrite content in cell culture medi ws determined y the Griess rection. riefly, 1 μl of the conditioned medi with n equl volume of Griess regent (1% sulfnilmide,.1% NED nd 2.5% H 3 PO 4 ) in 96- well plte ws incuted t room temperture for 1 min. The sornce t 54 nm ws determined y microplte reder (Epoch 2, iotek). The production of nitrite ws clculted from sodium nitrite (NNO 2 ) stndrd curve Sttisticl nlysis ll experiments were performed in triplicte nd results were expressed s mens ± SEM. The sttisticl nlyses were crried out with SPSS version 12. (SPSS, Inc., Chicgo, IL) using onewy NOV followed y Duncn s test. Vlues were considered significntly different when p < Results 3.1. Totl phenolic content The totl phenolic content of the crude extrct of quino leves is shown in Tle 1 nd is expressed s milligrms of gllic cid 25

3 Tle 1 Totl phenolic content of crude extrcts of the quino leves. Smple WQL Totl phenolic content (mg GE/g extrct) 34.4 ± % EQL ± % EQL ± 69.5 WQL = wter extrct of quino leves; 5% EQL = 5% ethnolic extrct of quino leves; 95% EQL = 95% ethnolic extrct of quino leves; GE = gllic cid equivlent. Men vlues followed y different superscripts re significntly different (p <.5). DPPH scvenging ctivity (%) WQL 5% EQL 95% EQL Concentrtion (mg/ml) Iron (II) chelting ctivity (%) WQL 5% EQL 95% EQL C 1 5 Concentrtion (mg/ml) 1 C Fig. 1 - The DPPH scvenging ctivity of the crude extrcts from quino leves. The dt were men ± SEM from three independent experiments. Different letters showed sttisticlly significnt differences (p <.5) mong three extrcts (lower cse letters) nd mong three concentrtions (cpitl letters) s nlyzed y one-wy NOV followed y Duncn s test. WQL = wter extrct of quino leves; 5% EQL = 5% ethnolic extrct of quino leves; 95% EQL = 95% ethnolic extrct of quino leves. Fig. 2 - The iron chelting ctivity of the crude extrcts from quino leves. The dt were men ± SEM from three independent experiments. Different letters showed sttisticlly significnt differences (p <.5) mong three extrcts (lower cse letters) nd mong three concentrtions (cpitl letters) s nlyzed y one-wy NOV followed y Duncn s test. WQL = wter extrct of quino leves; 5% EQL = 5% ethnolic extrct of quino leves; 95% EQL = 95% ethnolic extrct of quino leves. equivlents (GE) per grm of dry extrct. The highest mount of phenolic content ws found in the 95% EQL (569.5 ± 69.5 mg GE/g) DPPH rdicl scvenging ctivity The rdicl scvenging ility of the crude extrcts of quino leves ws mesured using the DPPH scvenging ssy. The results showed tht the 5% EQL nd 95% EQL were le to scvenge DPPH rdicls in concentrtion-dependent mnner (Fig. 1). In the concentrtion of 5 nd 1 mg/ml, the 95% EQL exhiited the highest level of DPPH rdicl scvenging ctivity, which were 5.7 ± 9.7% nd 65.3 ± 4.7%, respectively Iron chelting ctivity In the concentrtion of 1 mg/ml, the WQL showed significntly higher iron chelting ility (28.9 ± 2.8%) thn the 5% EQL nd 95% EQL (Fig. 2). The level of iron chelting ility in the 95% EQL ws oserved in concentrtion-dependent mnner. The WQL nd 5% EQL showed the highest iron chelting ctivity t 5 mg/ml thn their counterprts t 1 mg/ml nd 1 mg/ml Cell viility The effects of pretreted with different concentrtions (1, 1, 1 μg/ml) of WQL, 5% EQL, nd 95% EQL for 1-h nd then exposed simultneously to 1 µg/ml LPS for 24-h, the cell viility ws determined y MTT ssy. The cell viility ws not influenced y the quino lef extrcts nd LPS tretment (Fig. 3). Our result showed tht no significnt cytotoxicity on the RW cells ws oserved even up to 1 μg/ml of ll crude extrcts ws treted Inhiition of NO production in RW cells Pretretment with the WQL, 5% EQL nd 95% EQL t indicted concentrtions (1, 1 nd 1 μg/ml) for 1-h, the inhiition of the LPS-induced NO production rnged from 1.2% to 38.1% (Fig. 4). The WQL showed significntly higher NO inhiition t the concentrtion of 1 μg/ml s compred to its counterprts t 1 nd 1 μg/ml, respectively. In comprison of the three different extrcts t 1 nd 1 μg/ml, the 5% EQL nd 95% EQL showed significntly higher inhiition of NO production in LPS-stimulted RW cells thn WQL. 4. Discussion The ntioxidnt potentil of plnt extrcts hs een widely tested y using vrious ssys in vitro. The rdicl-scvenging ssy nd chelte trnsition metls hve gined cceptnce mong reserchers for their cpcity to rpidly screen mterils of inter- 26

4 12 WQL 5% EQL 95% EQL 5 WQL 5% EQL 95% EQL Cell viility (%) NO inhiition (%) Concentrtion (mg/ml) Concentrtion (mg/ml) 1 Fig. 3 - The cell viility of the crude extrcts in the RW cells. The RW cells were cultured in the presence of WQL, 5% EQL, nd 95% EQL t indicted concentrtions (1, 1 nd 1 µg/ml) for 1-h, nd then 1 µg/ml LPS for 24-h. The cell viility ws mesured vi MTT ssy. Vlues re mens ± SEM (n = 3). WQL = wter extrct of quino leves; 5% EQL = 5% ethnolic extrct of quino leves; 95% EQL = 95% ethnolic extrct of quino leves. est. Iron is well known to e powerful inititor for free rdicl oxidtion nd serves s regent for the Fenton rection nd lipid peroxidtion tht re the cuse of mny diseses, such s cncer [1]. In this study, the DPPH scvenging ctivity showed correltion with the totl phenolic content in the crude extrcts, something tht hs een similrly reported y previous reserches [1, 11]. Similr results hve een reported y jyi et l. in queous lef extrcts of Chenopodium opulifolium leves [12]. These indictes tht phenolic compounds in the lef extrcts my e responsile for the ntioxidnt potentil of Chenopodium species. Phenolics re powerful ntioxidnts nd cn dely or inhiit oxidtive processes y chelting trnsition metls tht ply vitl roles in the initition of deleterious free rdicl rections [13]. The results of the present study demonstrted tht the crude lef extrcts of quino exhiited chelting ctivity y reducing the formtion of ferrozine-fe 2+ complex. It hs een known tht iron ws the determining fctor for the initition of lipid peroxidtion [14]. Gwlik-Dziki et l. hve oserved tht the 5% ethnolic extrct from quino leves prevented lipid oxidtion which might e due to its ferrous ion chelting ctivity. esides, the ethnolic extrct of quino leves showed significnt inhiition of prolifertion in prostte cncer cells (MT-LyLu nd T-2) [15]. The quino lef extrcts used in our study cted s effective ferrous ion cheltor which my consequently reduce the oxidtive stress. Interestingly, the WQL showed higher Fe 2+ chelting potentil in spite of its low DPPH ctivity. This oservtion suggests tht the use of different methods is necessry to mesure the ntioxidnt potentils of plnt extrcts. Under norml physiologicl conditions, NO cts s necessry component in the regultion of vrious physiologicl functions such s lood pressure, immune response, nd neurl communiction [16]. However, overproduction of NO cn induce tissue dmge nd is ssocited with inflmmtory diseses including therosclerosis nd hypertension [17, 18]. Therefore, reserchers hve pid more ttention to discovering nturl ntioxidnts tht my ct s potent inhiitors of NO production in reltion to the tretment of chronic inflmmtory diseses [17]. cteril LPS re known to e ctivtors of murine mcrophge RW cells, which re suitle model for evluting the Fig. 4 - The effect of the crude extrcts on the NO production of the RW cells. fter pre-tretment of the cells with different concentrtions of the crude extrcts (1,1 nd 1 μg/ml) for 1-h, then the 1 µg/ml LPS ws dded to the cells for 24-h. Vlues re mens ± SEM (n = 3). Different letters showed sttisticlly significnt differences (p <.5) mong three extrcts (lower cse letters) nd mong three concentrtions (cpitl letters) s nlyzed y one-wy NOV followed y Duncn s test. WQL = wter extrct of quino leves; 5% EQL = 5% ethnolic extrct of quino leves; 95% EQL = 95% ethnolic extrct of quino leves. potentil inhiition of NO production. In this study, the 5% EQL nd 95% EQL were found to evidence n inhiitory ctivity ginst NO production in LPS-stimulted RW cells. These dt re relted to the higher level of totl phenolic content in the ethnolic extrcts when compred to the wter extrct. The phenolic compounds hve long een considered to hve the potentil to inhiit NO nd peroxynitrite production [19], indicting tht the presence of ntioxidnt molecules in ll the crude quino lef extrcts is responsile for their inhiitory effect. Previous epidemiologicl studies hve indicted n inverse reltionship etween the consumption of ntioxidnt-rich food nd the reduction of risk fctors of some humn diseses [2]. In ddition, the secondry metolite of plnts hs lso een reported to ct s excellent nti-inflmmtory gent in oxidtive stress nd inflmmtion [21]. 5. Conclusion The results of the present study indicted tht the 95% EQL contined the highest mount of totl phenolic content nd provided the gretest DPPH scvenging ctivity mong ll extrcts, s well s inhiited NO production in LPS-induced RW cells. Our results suggest tht quino leves re vlule ioctive nturl product for dietry supplementtion. Further studies in vivo re required to confirm these findings. cknowlegements The reserch ws supported y the Ntionl Science Council under Grnt MOST Conflicts of interest The uthors declre no conflicts of interest. 27

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