Aville online t http://www.ijr.om Interntionl journl of Avne Biologil n Biomeil Reserh Volume 2, Issue 4(2), 2014: 494-500 Antioxint Properties of Vrious Solvent Extrts of Lemon Veren (Lippi Citrioor) Leves Mhnz Choupni *1, Seeeh Arshhi-Delouee 2, Mehrn Almi 3 1 MS. Stuent Deprtment of Foo Siene n Tehnology, Young Reserhers n Elite Clu, Azshhr Brnh, Islmi Az University, Azshhr, Irn 2 Deprtment of Foo Siene n Tehnology, Azshhr Islmi Az University, Azshhr, Irn 3 Deprtment of Foo Siene n Tehnology, Gorgn University of Agriulturl Sienes n Nturl Resoures, Gorgn, Irn Astrt Lemon veren (Lippi itrioor) elongs tothe Verenee (shhpsn) fmily. L.itrioor is hemil omposition of the leves plnt hve ntimiroil properties, refrigernt, nti-hehes onesie, nti- pins nerve n housing, rmintive, helping to igestion, relxtion, izziness, ols therpy, memory Booster n et. In this stuy, phenoli ompouns of L.itrioor leves were extrte with immersion metho y etone, ethnol, methnol n wter. In this metho, solvent type ws foun s n effetive ftor on the extrtion. The highest mount of totl phenol ompouns ws extrte y methnol whih ws 25/94 (mg Glli i per 100 mg of the extrt). Among the solvents,etone extremely h lower effiieny in the extrting phenoli ompouns from the leves of plnt. Three omplementry ssys, reuing power of Fe (III), DPPH ril svenging n totl ntioxint pity were use for nlysis of ntioxint properties of vrious extrts ompre with the syntheti ntioxint, BHT. In ll tests, methnoli extrt, whih h the highest totl phenoli ompouns, showe the highest ntioxint tivity lthough it ws lower tht of BHT. Aoring to the survey results, L.itrioor leves with goo soure of nturl ntioxints for use in foo inustry's. Keywors: Lippi itrioor leves, Reuing power, DPPH svenging tivity, Antioxint tivity Introution Lemon veren is from vereee fmily, Aloysi genesis, n A.troor speies. Extrt of lemon veren ontins phenil perionoies, isoversosies n versosie s the min phenoli omintion. These omintions n e relile sorue of ntioxints, nti-rthritis, ntiner n ntiteril tht n e use for synthesis of noterosotil formultions (Cruz et l., 2003). Strong ntioxint effet of phenol omintions in the extrt of veren n ttenute or stop tivity of superoxie, hyroxyl, n hipohlori rils (Vlento et l., 2002). In ition, ntioxint pity vlues re omprle to those of ommeril ntioxint rink se on green te (Aerrhim et l.,2011).the phenoli ompouns of lemon leves were extrte y wter n ethnol solvents, n the otine extrts were teste on the rte to exmine ntioxint tivity. The results showe tht the ntioxint tivity of ethnol extrt ws higher thn tht of the queous extrt, whih is proly ue to higher ontent of phenoli ompouns extrte y ethnol solvent(el-hwry et l.,2012). The omine supplement ontining stnrize lemon veren extrt n fish oil omeg-3 ftty i reue symptoms of pin Corresponing Author E-mil:Mhz.houpni@yhoo.om 494 Pge
Choupni et l. Int J Av Biol Biom Res. 2014; 2(4):494-500 n rthritis use y free ril mong thletes who use ntioxint rih foos. (Cturl et l.,2011). Exmintion of the ntioxint effet of lemon veren extrtson stuents' lymphoytes uring eroi exerises showe tht phenil n proinoi ontent n versosie ply notle role in reuing gluthtion n reots in lymphoytes nproteting plsm n re loo ells from oxitive mge(crrer-quintnr et l.,2010).consiering sene of stuy on extrtion of phenol omintions from veren using ifferent solvents n mesuring ontent of the phenol omintions n on ntioxint effets of ifferent extrts on reuing Fe(III), ontrolling DPPH rils, n ntioxint hrteristis, the present stuy is n ttempt to etermine phenol ontent n ntioxitive tivity of the extrts n fin sfe n helthy prout. Mteril n methos Mterils Lemon veren (L. itrioor) leves were hrveste in April 2013 from the tle sefi gon o. The leves were wshe n rie in hot ir oven t 45 C for 6 h. The rie mteril ws groun to fine power, psse through 63-mesh sieve n kept in n ir-tight ontiner t 4 C until furtheruse. All hemils n solvents use in this stuy were otine from the ompnies Merk n Titrhem high purity. Extrtion of ntioxints from Lemon veren leves The rie leves of lemon veren (15 g) were extrte overnight with 100 ml eh of methnol, ethnol, etone or wter, respetively, in mehnil shker t room temperture. Eh extrt ws filtere with Whtmn No. 1 filter pper. The filtrte otine from methnol, ethnol n etone ws evporte to ryness t 40 C in rotry evportor n the wter extrt ws freeze-rie. The rie smple of eh extrt ws weighe to etermine the yiel of solule onstituents n store t 4 C until use. Estimtion of totl phenolis Totl phenoli ontent of eh extrt ws etermine y the Folin-Ciolteu miro-metho (Slinkr & Singleton, 1977). Briefly, 20 µl of extrt solution were mixe with 1.16 ml istille wter n 100µlof Folin Ciolteu regent, followe y ition of 300 µl of N 2 CO 3 solution (20%) fter 1 min n efore 8 min. Susequently, the mixture ws inute in shking inutor t 40 C for 30 min n its sorne ws mesure t 760 nm. Glli i ws use s stnr for lirtion urve. The phenoli ontent ws expresse s glli i equivlents using the following liner eqution se on the lirtion urve: Y= 0.001X+ 0.022 R 2 = 0.9982 where Y is the sorne n X is onentrtion s Gllii equivlents (µg/ml). DPPH ril svenging tivity The ility of extrts to svenge DPPH rils ws etermine oring to the metho of Shim (1992). Briefly, 1 ml of 1 mm methnoli solution of DPPH ws mixe with 3 ml of extrt solution in methnol (ontining 50-300 µg of rie extrt n ). The mixture ws then vortexe vigorously n left for 30 min t room temperture in the rk. The sorne ws mesure t 517 nm n tivity ws expresse s perentge DPPH svenging reltive to ontrol using the following eqution: DPPH svenging tivity (%) = ୱ୭ ୠୟ୬ୡ ୭ୡ୭୬୲ ୭୪ ୠୱ୭ ୠୟ୬ୡ ୭ୱୟ୫୮୪ ୠୱ୭ ୠୟ୬ୡ ୭ୡ୭୬୲ ୭୪ Reuing power of Fe (III) The ility of extrts to reue iron (III) ws ssesse y the metho of (Yilirim et l., 2001). The rie extrt (50-500 µg) in 1 ml of the orresponing solvent ws mixe with 2.5 ml of phosphte uffer (0.2 M, ph 6.6)n 2.5 ml of potssium ferri ynie (K 3 Fe (CN) 6 ;10 gl -1 ), then the mixture ws inute t 50 C 495 Pge
Choupni et l. Int J Av Biol Biom Res. 2014; 2(4):494-500 for 30 min. After inution, 2.5 ml of trihloroeti i (100 g l -1 ) were e n the mixture ws entrifuge t 1650g for 10 min. Finlly, 2.5 ml of the superntnt solution were mixe with 2.5 ml of istille wter n 0.5 ml of FeCl 3 (µg l -1 ) n the sorne ws mesure t 700 nm. High sorne inites high reuing power. Totl ntioxint pity This ssy is se on the reution of Mo (VI) to Mo (V) y the smple n the susequent formtion of green phosphte/mo (V) omplex t ii ph (Prieto et l, 1999). An liquot of 0.1 ml of smple solution (ontining 50-500 µg of rie extrt in orresponing solvent n syntheti ntioxint BHT ws omine in n Eppenorf tue with 1 ml of regent solution (0.6 M sulphuri i,28 mm soium phosphte, n 4 mm mmonium molyte). The tues were ppe n inute in therml lok t 95C for 90 min. After the smples h oole to room temperture, the sorne ws mesure t 695 nm ginst lnk. A typil lnk solution ontine 1 ml of regent solution n the pproprite volume of the sme solvent use for the smple, n it ws inute uner the sme onitions s the rest of the smples. Methoology, t nlysis, smpling The t were ollete through fully-rnom pln n nlyze y ANOVA. Eh test ws onute with 3 replitions. Comprison of the men points ws one y Dunn s multi-rnge test (P<0.005) in SPSS. The igrms were rwn in EXCEL. Results n isussion Extrt yiel n totl phenolis Tle 1 lists men extrtion performne se on solvent n the effet of the solvent on totl phenol ontent in the extrtion. Vrine nlysis revele tht the solvent h signifint effet (P<0.05) on essene extrtion. Clerly, methnol hs the highest extrt extrtion performne followe y ethnol n etone. Mximum extrtion 10.07(Grms of extrt per 100 g of rie leves) ws one y qu extrt n minimum extrtion 3.43(Grms of extrt per 100 g of rie leves) ws hieve y etone. Type of solvent ws foun to e signifintly effetive (P<0.05) on totl phenol ontent of the extrte extrt. In esening orer, the extrte extrt y methnol, ethnol, etone, n wter h the highest phenol ontent. In spite of high extrtion performne, queous solvent extrte less phenoli ompoun ompring with methnoli solvent. DPPH ril svenging tivity Vrine nlysis results in figure 1 show tht regring pity to ontrol free rils, methnol extrt of Lemon veren is signifintly ifferent from BHT synthesis ntioxint (p<0.05). In ition, type n onentrtion of ethnoli, etoni, n queous essenes were signifintly effetive on ontrolling free rils (p<0.05). Furthermore, this hrteristi inrese long with inrese in onentrtion of the extrts. At onentrtion rnge 50-300µg/ml, highest pity ws oserve in BHT synthesis ntioxint, methnoli extrt, ethnoli, etonei, n queous extrt in esening orer. This is in greement with the reports of Ting & hi-tng (2005), Arshhi-Delouee & Urooj (2007), n Yo & Qing (2007) tht methnol is wiely use n effetive solvent for extrtion of ntioxints.tht methnol is wiely use n effetive solvent for extrtion of ntioxints. Fe +2 (III) reution power Figure 2 illustrtes omprison of men sorption pity of ifferent onentrtions of methnoli, ethnoli, etoni, n queous extrts of Lemon veren n BHT synthesis ntioxint t wvelength 700nm. This hrteristi is n initor of reution power of Fe +2 (III). Vrine nlysis inite 496 Pge
Choupni et l. Int J Av Biol Biom Res. 2014; 2(4):494-500 signifint ifferene (p<0.05) etween vriety of extrts, n BHT synthesis ntioxint regring Fe +2 (III) reution power. As piture, BHT synthesis ntioxint hs the highest Fe +2 (III) reution power t 50-500 µg/ml. There is iret reltionship etween reution power of the extrts n totl phenoli ontent in them, so tht methnoli extrt with highest phenol ontent h the highest reution power n queous essene with lowest phenol ontent lso h lowest reution power. Inrese in onentrtion, in ll ses, inrese sorption power of the extrt solutions. Totl ntioxint pity Men sorption pity of the queous, ethnoli, methnoli, etoni, n BHT synthesis ntioxint re ompre in figure 3. Vrine nlysis results showe tht there is signifint ifferene (p<0.05) mong ifferent extrts of Lemon veren n etween them n BHT synthesis ntioxint regring the sorption pity. As the results showe, regrless of the onentrtion of the extrts (50-50ppm) BHT h the highest ntioxint sorption pity following y methnol extrt. In ition, sorption pity inrese with inrese in onentrtion of the extrt. Conlusions The results otine in this stuy showe tht methnoli extrt emonstrte the most effiient extrtion regring phenoli ompouns, reuing power of Fe (III), DPPH ril svenging, n totl ntioxint pity. Given tht lemon leves ontin lrge mounts of phenoli ompouns, then its ntioxint potentil is high. Stility of eile oils in oxitive environment inreses y ing phenoli extrts of the plnt. Due to the verse effets of syntheti ntioxints on humn helth, lemon juie is reommene s replement for these ompouns in the foos ontining fts. Referene Aerrhim, F., Estrell, S., Susin, C., Arris, S. M., Gonzlez, M. C, Conezo-Hoyos, L.(2011). The ntioxint tivity n therml stility of lemon veren (Aloysi triphyll) infusion. Journl Meil Foo, 14(5): 517-527. Arshhi-Delouee,S., Urooj,A. (2007). Antioxint properties of vrious solvent extrts of mulerry(morus ini L.) leves. Foo Chemistry, 102:1233-1240. El-Hwry, S. S., Yousif, M. F., Ael Motl, A. A., A-Hmee, L. M. (2012). Biotivities, phenoli ompouns n in-vitro propgtion of Lippi itrioor Knuth ultivte in Egypt. Foo Chemistry, 50: 1-6. Cturl, N., Funes, L., Perez-Fons, L., Miol, V. (2011). A rnomize Doule- line, pleoontrolle stuy of the effet of omintion of lemon veren extrt n fish oil omeg-3 ftty i on joint mngement. Journl of Alterntive n omplementry Meil, 17(11): 1051-63. Crrer-Quintnr, L., Funes, L., Viues, E., Tur, J., Miol, V., Rohe, E., Pons, A. (2010). Antioxint effet of lemon veren extrts in lymphoytes of trining progrm. Sn Journl Meil Siene Sports, 22(4): 454-61. Cruz, D., Fle, PL., Mourto, A., Vz, PD., Serrlheiro, ML., Lino, AR. 2010.Preprtion n physio hemil hrteriztion of Ag nnoprtiles iosynthesize y lippie itrioor (Lemon veren ). Foo Chemistry. 81(1):67-73 Prieto, P., Pine, M. n Aguilr, M.(1999). Spetrophotometri quntittion of ntoxint pity through the formtion of phosphomolyenum omplex: speifi pplition to the etermintion of vitmin E. Anlytil Biohemistry, 269: 337-341. 497 Pge
Choupni et l. Int J Av Biol Biom Res. 2014; 2(4):494-500 Ting, S., Chi-tng, H. (2005). Antioxint tivites of ukwhet extrts. Journl of Foo Chemistry, 90: 743-749. Shim, K., Fujikw, K., Yhr, K., n Nkmur, T. (1992). Antioxitive properties of xnthin on utoxition of soyen oil in yloextrin emulsion. Journl of Agriulturl n Foo Chemistry. 40: 945-948. Slinkr, K., & Singleton, V. L. (1977). Totl phenol nlysis; utomtion n omprison with mnul methos. Amerin Journl of Enology n Vitiulture, 28: 49-55. Vlento, P., Fernnes, E., Crvlho,F., Brnquinho Anre, P., Ser, R.M., Loues Bstos, M (2002). Stuies on the ntioxint tivity of Lippi itrioor infuion: Svenging effet on Superoxie Ril, Hyroxyl Ril n Hypohlorous Ai. Phrmeutil Soiety of Jpn, 25(10):1324-1327. Yo, H., Qing, L. (2007). Antioxint tivites of rley sees extrts. Journl of Foo Chemistry, 102: 732-737. Yilirim, A., Mvi, A., & Kr, A. A. (2001). Determintion of ntioxint n ntimiroil tivities of Rumex rispus L. extrts. Journl of Agriulturl n Foo Chemistry, 49: 4083-4089. Tle 1. extrt yiel n totl phenoli ontents of ifferent solvent extrts from L.itrioor leves Smple Yiel Totl phenolis Methnol extrt 10.03 ± 0.2 25.94 ± 0.215 Ethnol extrt 9.53 ± 0.5 23.75 ± 0.32 Aetone extrt 3.43 ± 0.37 18.06 ± 0.18 Wter extrt 10.07 ± 0.36 16.58 ± 0.29 Vlues in the sme olumn followe y ifferent letters re signifintly ifferent (p< 0.05).mg of extrt per 100 mg of rie leves; grms of glli i per 100 mg (ry weight) of extrt 498 Pge
Choupni et l. Int J Av Biol Biom Res. 2014; 2(4):494-500 120 DPPH svenging tivity (%) 100 80 60 40 20 0 50 100 250 300 onentrtion(µg/ml) wter eton ethnol methnol BHT Fig1.DPPH svenging tivities of methnol, ethnol, etone n wter extrt of L.itrioor leves. BHT were use s positive ontrols. Perentg ril svenging pity reltive to ontrol. 3.5 Asorne 700 nm 3 2.5 2 1.5 1 0.5 0 0 50 100 150 200 500 Conentrtion ( µg/ml) wter eton ethnol methnol BHT Fig2. Reuing powers of methnol, ethnol, etone n wter extrts of L.itroori leves. 499 Pge
Choupni et l. Int J Av Biol Biom Res. 2014; 2(4):494-500 3 Asorne 695 nm 2.5 2 1.5 1 0.5 0 0 50 100 150 200 500 Conentrtion (µg/ml) etone wter ethnol methnol BHT Fig3. Totl ntioxint tivities of methnol, ethnol, etone n wter extrts of L.itrioor leves. BHT ws use s positive ontrol. 500 Pge