ANTIOXIDANT CAPACITY AND TOTAL PHENOLIC CONTENT OF LEMONGRASS (CYMBOPOGON CITRATUS) LEAVE

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1 ANTIOXIDANT CAPACITY AND TOTAL PHENOLIC CONTENT OF LEMONGRASS (CYMBOPOGON CITRATUS) LEAVE Sin Yen Sh 1, Chiw Mei Si 1, Sui Kit Chng 2, Yee Kwng Ang 1, Hip Seng Yim 1* 1 Deprtment of Food Siene nd Nutrition, Fulty of Applied Sienes, UCSI University No. 1, Jln Menr Gding, UCSI Heights, Kul Lumpur, Mlysi. 2 Deprtment of Nutrition nd Dietetis, Fulty of Mediine nd Helth Sienes, Universiti Putr Mlysi UPM Serdng, Selngor, Mlysi. *Emil: hsyim@usi.edu.my Astrt The im of the study ws to investigte the effet of drying method, solvent onentrtion, extrtion time nd temperture on ntioxidnt tivities nd totl phenoli ontent (TPC) of lemongrss leve extrt. The ntioxidnt tivities were evluted using DPPH rdil svenging ility, ferri reduing ntioxidnt power (FRAP), nd β- rotene lehing inhiition (BCB) ssys; while TPC ws determined using Folin-Ciolteu method. The optimum extrtion onditions otined were oven dried lemongrss leve extrted using 40% ethnol t 25 C for 300 min with smple to solvent rtion of 1:10 in wter th shker (150 rpm). Lemongrss leve extrt possesses resonly ntioxidnt potentil with EC 50 (DPPH) of 192 µg/ml, ntioxidnt tivity (s mesured y BCB inhiition) of 67%, nd FRAP of 129 mg TE/g; s well s TPC vlue of 67 mg GAE/g. However, lemongrss leve extrt exhiited lower ntioxidnt tivity in omprison with BHA nd α-toopherol. TPC ws positively orrelted with ntioxidnt tivities of lemongrss leve extrt ssessed y FRAP (r = 0.995), BCB ssy (r = 0.932), nd DPPH (r = 0.777); inditing the presene of phenoli ompounds ontriuted to the ntioxidnt tivities of lemongrss leve. In onlusion, lemongrss leve ould e soure of nturl ntioxidnts with nutreutil potentil for food industry pplition. Keywords: Antioxidnt tivity, Extrtion onditions, Lemongrss leve, Totl phenoli Sumitted: Reviewed: Aepted: INTRODUCTION Mlysi is well-known for its rih iodiversity of mny indigenous fruits nd vegetles grown in the wild. There re more thn 120 speies representing vrious fmilies of trditionl vegetles of the Mlys in Mlysi, lolly lled s ulm (Mnsor, 1988). Mny lol hers re ooked or eten rw s sld y dipping in shrimp nd hili pste or penut sue to enhne flvour while some re eing oiled nd the extrts re used for onsumption mong Mlysins. Vrious studies reported tht the onsumption of these vegetles hs een ssoited with lower risk of degenertive diseses relted to oxidtive stress like ner, dietes, Alzheimer s disese nd rtheroslerosis (Kuol nd Sirimornpun, 2008). Antioxidtive ompounds tht re enefiil to humn helth ommonly found in fruits nd vegetles re phenolis, etlins, nd rotenoids (Norshzil et l., 2010). Among the phytohemils, phenoli ompounds re reported to e the min ontriutor of ntioxidnt tivity in plnt extrts (Yoo et l., 2008). Lemongrss is tropil plnt ommonly found in Southest Asi, whih its origin n e trked from Indi. Now it is eing ultivted in mny ples, inluding tropil nd sutropil ountries. Lemongrss elongs to the fmily of Poee, nd the genus, Cymopogon. The ommon nmes re lemongrss, fever grss or seri in Mly. There re round 30 speies of lemongrss found ntively in Mlysi; however, there re two generl types, whih re the Cymopogon itrtus nd Cymopogon nrdus. The ltter is more ommonly used s romti plnts for extrtion of itronell essentil oils (FRIM, 2004). Citrtus is more ommon for humn onsumption or s ooking ingredients. Due to its romti nd lemon sented property, lemongrss is ommonly inorported in Asin ooking. The leves, on the other hnd re Aville on-line t 150

2 ommonly used s trditionl remedies. The essentil oil of C. itrtus is lso used in food proesses s food flvouring, perfume nd osmeti industries. There re inresing interests in disovering the ntioxidnt potentil of mediinl plnts sine hers nd mediinl plnt hd een used for tretment sine nient times, efore the development of modern mediinl knowledge. Aumulting evidene suggested tht the reovery, yield nd type of phenolis in n extrt re influened y type nd polrity of extrting solvents, time nd temperture of extrtions s well s physil hrteristis of the smple (Hossin et l., 2010). Thus fr, no speifi or pproprite extrtion solvent is reommended for optiml reovery of polyphenols for most of the plnts studied. This hppens euse of the diverse hemil strutures of phenoli ompounds from simple to polymerize forms tht might ffet their soluility ehvior (Prior et l., 2005). With tht, the ojetive of this study ws to evlute the effet of drying method, solvent onentrtion, extrtion time, nd temperture on ntioxidnt tivity of lemongrss leve. Results from this study would provide deeper understnding on the helth promoting properties of lemongrss leves so tht it would e identified for further investigtions nd hene, developed into vlue-dded foods nd nutreutils for the enefit of mnkind. 2. MATERIALS AND METHODS Smple preprtion Fresh lemongrss (Cymopogon itrtus) ws purhsed from lol mrket in Btu Cves, Selngor, Mlysi. The seletion of leves ws sed on hrvesting riteri where whole plnt, inluding the stem ws hrvested. Fresh lemongrss leves gives pungent itrus rom when rushed or ut. The lemongrss leves were ut 10 m ove the rhizome where the spoilt, injured or worn prt ws removed. The leves were wshed under running tp wter to remove deris nd tpped dry, eqully divided into two portions, one for oven drying in onvetion oven (UFB 500, Memmert, Shwh, Germny) t 40 C for 24 h until onstnt weight ws otined, nd nother one for freeze drying. Smple extrtion Five portions of 5 g dried lemongrss leve powder (oven- nd freeze-dried) were weighed using n nlytil lne (AB204-S, Mettler Toledo, Switzerlnd). With the smple to solvent rtio fixed t 1:10, different onentrtions of ethnol (v/v; 0%, 20%, 40%, 60%, 80%, nd 100%) were prepred. The mixtures were shken for 60 min t 25 C nd 150 rpm in shking inutor. After the extrtion, the extrts were filtered using Whtmn No. 1 filter pper. The filtrte residue ws olleted nd sujeted for seond extrtion with the sme onditions to inrese the extrtion effiieny nd yield. Filtrtes of oth extrtions were omined nd entrifuged (Universl 320R, Hettih Zentrifuge, Germny) t 4500 rpm for 10 min. The superntnt ws onentrted using rotry evportor (Rotvpour R-200, BUCHI, Switzerlnd) t 40 C. The onentrted extrt ws freeze-dried, wrpped with luminium foil, nd stored t -20 C until further nlysis. Determintion of optimum extrtion onditions After the est solvent onentrtion nd drying method were determined, the extrtion ws repeted using the sme proedures, ut with different extrtion time of 60, 120, 180, 240, 300 min followed y different extrtion temperture of 25, 30, 40, 50, nd 60 C. To proeed to the next stge, the est vrile of the prmeter ws hosen sed on the result of 2,2-diphenyl-1-pirylhdrzyl (DPPH) ssy. After determining the optimized extrtion onditions, the ntioxidnt tivities of lemongrss leve were evluted using ferri reduing ntioxidnt power (FRAP) nd β- rotene lehing (BCB) ssys, in ddition to DPPH rdil svenging nd TPC ssys. Totl phenoli ontent The totl phenoli ontent (TPC) ws determined spetrophotometrilly using Folin- Aville on-line t 151

3 Ciolteu regent (FCR) ording to the method desried y Ferreir et l. (2007) with slight modifition. Smple (1 ml) ws dded into test tues, followed y 4.0 ml of FCR (diluted 10 ) nd 5.0 ml of sodium ronte (7.5 g/100 ml). The ontents were mixed thoroughly nd stored in the drk for 30 min. The sorne ws mesured t 725 nm using spetrophotometer (PRIM, Seomm, Alés, Grd, Frne). TPC ws expressed s mg glli id equivlents (GAE) per g of extrt. DPPH rdil svenging ssy The svenging ility ws determined ording to the method of Xu nd Chng (2007) with slight modifition. To prepre DPPH regent, 7.8 mg of DPPH powder ws dissolved in ethnol nd topped up to 100 ml using volumetri flsk. The flsk ws wrpped with luminium foil to redue light exposure towrds DPPH solution. Smple (1 ml) ws dded to 500 µl of ethnoli DPPH solution in test tue nd the mixture ws shken vigorously using vortex, nd then kept in drk ondition for 30 min. After inution, the sorne of the mixture ws mesured t 517 nm ginst ethnol lnk nd distilled wter s negtive ontrol. Svenging ility of smple ws lulted from the eqution elow: DPPH rdil svenging ility (%) = [1 (As smple / As ontrol )] x100 The perentge of svenged DPPH ws plotted ginst logrithm (Log) of smple onentrtion to lulte the EC 50 tht defined s the ility to redue the initil DPPH onentrtion y 50%. BHA nd α-toopherol were used s referene. Ferri reduing ntioxidnt power (FRAP) ssy FRAP ssy ws determined sed on the redution of Fe 3+ -TPTZ to lue oloured Fe 2+ -TPTZ (Biglri et l., 2007). FRAP regent ws prepred from 10 mm 2,4,6-tri(2 pyridyl)- s-trizine (TPTZ) in 40 mm HCl with 20 mm ferri trihloride hexhydrte (FeCl 3.6H 2 O) nd 0.3 M ette uffer (ph 3.6) t rtio of 10:1:1 (v/v/v), nd the strw oloured solution ws kept in wter th t 37 C. Then, FRAP regent (1.5 ml) ws dded with 50 µl smple solution nd the mixture ws shken nd inuted for 4 min. Asorne (593 nm) ws determined reltive to FRAP regent lnk. The result ws lulted from lirtion urve plotted ginst vrious trolox onentrtions ( µg/ml), nd expressed s trolox equivlent ntioxidnt pity (TEAC). β-crotene linolete lehing ssy β-crotene lehing ssy ws onduted using method y Nsim et l. (2008) with slight modifition. To prepre the working regent, 0.2 mg of β-rotene ws dissolved in 1 ml of hloroform, then dded with 0.02 ml linoleni id nd 0.2 ml Tween 40. The hloroform in the mixture ws removed under vuum. Oxygented wter (50 ml) ws dded followed y 0.5 ml smple extrt nd 4 ml regent nd shken until liposome ws formed. Asorne (470 nm) ws red t 0 min nd every intervl of 20 min for 2 h (t = 0, 20, 40, 60, 80, 100, nd 120 min). The nti-lehing rte of smple ws lulted sed on the formul s elow: β-crotene lehing rte (R) = ln (s t = 0 / s t = 20, 40, 60, 80, 100, nd 120 min ) / t Where, ln is nturl log nd t = 0 is the initil sorne t time 0 nd so-forth. Antioxidnt tivity (AA) ws lulted s perentge of inhiition reltive to ontrol using eqution elow: %AA = 100 (R ontrol R smple ) / R ontrol Sttistil nlysis All dt were expressed s men ± stndrd devition nd were sttistilly nlyzed using the SPSS sttistil softwre version 20 (SPSS In, Chigo, Illinois, USA). All nlyses were done in triplite. One-wy nlysis of vrine (ANOVA) nd Tukey s test were used to ompre mens mong groups. Person orreltion test ws used to ssess the Aville on-line t 152

4 reltionships etween TPC nd ntioxidnt tivities. The signifine level ws set t p < RESULTS AND DISCUSSION Extrtion of lemongrss leve using different ethnol onentrtion (0, 20, 40, 60, 80, nd 100%) ws tested to evlute its free rdil svenging potentil. Figure 1 shows the EC 50 vlue for oth oven-dried nd freeze-dried smples extrted y 40% ethnol onentrtion hd the lowest vlue of 212 nd 229 µg/ml, respetively, ut freeze-dried ws signifintly higher tht oven-dried (p < 0.05). Therefore, 40% ethnol oven-dried smple ws seleted s the est extrtion ondition to proeed with the susequent stge of experiment tht is to determine the optiml extrtion time. DPPH rdil svenging tivity n e influened y extrtion time; the EC 50 of smple extrted with 40% ethnol extrtion for 300 min (253 µg/ml) ws signifintly lower ompred to others (Figure 1). With tht, 300 min ws seleted for the determining of optimum extrtion temperture tht rnges from C. The EC 50 vlue of 25 C showed signifintly lower vlue (288 µg/ml) ompred to other tempertures, with the deresing trend of EC 50 vlue ws 40 C > 60 C > 50 C = 30 C > 25 C. Thus, 25 C ws seleted s the optiml extrtion temperture for high DPPH rdil svenging ility of lemongrss leve. Overll, the optimum extrtion onditions were oven-dried lemongrss leve extrted using 40% ethnol for 300 min t 25 C. With the determined optimum extrtion onditions, n rry of ntioxidnt pity ssys y mens of ferri reduing ntioxidnt power (FRAP), β-rotene lehing ssy, nd DPPH rdil svenging ility, s well s TPC were rried out (Tle 1). The TPC of lemongrss extrt stnds t 67 mg GAE/g extrt. The reduing power n serve s signifint refletion of the ntioxidnt tivity. The reduing power of lemongrss leve extrt ws 129 mg TE/g nd ws signifintly lower ompred to BHA nd α-toopherol. () () () d C * B * Oven-dried A Ethnol (%) e Freeze-dried Time (min) d D * Temperture ( C) Fig. 1 The effet of () solvent onentrtion (nd drying method), () extrtion time nd () temperture on DPPH rdil svenging ility of lemongrss leve extrt () Different smll letters (-d) for oven-dried nd pitl letters (A-E) for freeze-dried smples denote signifintly different (p < 0.05). *Denotes signifintly different (p < 0.05) etween oven- nd freezedried smples, exept for 40% ethnol. () & () Different smll letters (-e) denote signifintly different (p < 0.05). Tle 1 Totl phenoli ontent nd ntioxidnt tivities of Cymopogon itrtus leve extrt t optimized extrtion onditions (40% ethnol, 5 h, 25 C, oven-dried) Smple TPC (GAE, mg/g) FRAP (TEAC, mg/g) d ANT, % (BCB) D * E * DPPH rdil svenging Crude extrt ± ± ± BHA NA ± ± 0.26 < 1 α-toopherol NA ± ± 0.23 < 1 Eh vlue ws expressed s men ± stndrd devition (n=2), exept for DPPH EC 50 vlue. NA: not pplile. TPC=Totl phenoli ontent; FRAP=Ferri reduing ntioxidnt power; ANT (BCB)=Antioxidnt tivity s mesured y β-rotene lehing inhiition (t 500 ppm smple onentrtion). Different supersripts within the olumn (FRAP nd BCB) denote signifintly different (p < 0.05). Defined s effetive onentrtion tht ws le to svenge 50% of the totl DPPH rdils; EC 50 ws lulted y interpoltion of liner regression nlysis (sed on onentrtion-dependent result dt not shown). Aville on-line t 153

5 β-crotene lehing mehnism is sed on hydroperoxides formed from linolei id whih then ttks the highly unsturted β- rotene moleules. The sene of ntioxidnt uses lipid oxidtion to our tht result in the deolourtion of ornge-yellowish β-rotene. The ntioxidnt tivity s mesured y β- rotene lehing inhiition ws found signifintly higher in BHA nd α-toopherol (oth > 95%) ompred to lemongrss leve extrt (67%) (p < 0.05). Lemongrss leve extrt possesses moderte ntioxidnt tivity (60 70%) ording to Kur nd Kpoor (2002). The EC 50 vlue for DPPH rdil svenging ility of lemongrss extrt (192 µg/ml) ws found to e signifintly higher thn tht of BHA nd α-toopherol (oth < 1 µg/ml). Mny uthors hd stressed the need to perform more thn one type of ntioxidnt tivity mesurement to tke into ount the vrious mehnisms of ntioxidnt tion nd limittion of eh method (Rufino et l., 2010). This ours euse different ntioxidnt ssys ville showed different ntioxidnt tivities sed on different mehnisms. Different methods might e mesuring different kinds of ntioxidnt present in the plnts studied. Positive orreltions were oserved etween TPC nd FRAP (r = 0.995); with BCB (r = 0.932); nd DPPH (r = 0.777) (Tle 2). These support the results reported y Leontowiz et l. (2003) tht signifint orreltions etween TPC nd ntioxidnt tivities were found in pple, per peel, nd pulp extrts; s well s in ot (Sere nd Brn, 2011). On the ontrry, no suh orreltions were found in itter gourd extrts, s reported y Kuol nd Sirimornpun (2008). Tle 2 Correltions etween TPC nd DPPH, FRAP, BCB ssys of Cymopogon itrtus leve extrt t optimized extrtion onditions (40% ethnol, 5 h, 25 C, oven-dried) DPPH FRAP BCB All vlues re signifint t p < CONCLUSION The optimized extrtion prmeters of ovendried lemongrss leve were 40% ethnol onentrtion, with 25 C extrtion temperture for 5 h. The optimized extrtion onditions influene the ntioxidnt pity of lemongrss leve extrt. However, the ntioxidnt tivities were found to e lower thn the stndrds (BHA nd α-toopherol). Nonetheless, further studies re needed for the optimiztion of reovery of ntioxidnt ontents nd identifition potent ntioxidnt present in C. itrtus leve. 5. ACKNOWLEDGEMENTS Finnil nd lortory support from UCSI University, Kul Lumpur, Mlysi is very muh ppreited. 6. REFERENCES [1] Biglri, F., AlKrkhi, A.F.M. nd Es, A.M., Antioxidnt tivity nd phenoli ontent of vrious dte plm (phoenix dtylifer) fruits from Irn. Food Chemistry, 2007, 107: [2] Ferreir, I.C.F.R., Bptist P., Vils-Bos, M. nd Brros, L., Free rdil svenging pity nd reduing power of wild edile mushrooms from northest Portugl: Individul p nd stipe tivity. Food Chemistry, 2007, 100: [3] FRIM Commerilistion, FRIM s Herl & Cosmeeutil Produts. Forest Reserh Institute of Mlysi, Kul Lumpur, Mlysi, [4] Hossin, M.B., Brry-Ryn, C., Mrtin-Din, A.B. nd Brunton, N.P., Optimistion of elerted solvent extrtion of ntioidnt ompounds from rosemry (Rosmrinus offiinlis L.), mrjorm (Orignum mjorn L.) nd oregno (Orignum vulgre L.) using response surfe methodology. Food Chemistry, 2010, 126: [5] Kur, C. nd Kpoor, H.C., Antioxidnt tivity nd totl phenoli ontent of some Asin vegetles. Interntionl Journl of Food Siene nd Tehnology, 2002, 37: [6] Kuol, J. nd Sirimornpun, S., Phenoli ontents nd ntioxidnts tivities of itter gourd (Momordi hrnti L.) lef stem nd fruit frtion extrts in vitro. Food Chemistry, 2008, 110: [7] Leontowiz, M., Gorinstein, S., Leontowiz, H., Krzminski, R., Lojek, A., Ktrih, E., et l., Apple nd per peel nd pulp nd their influene on plsm lipids nd ntioxidnt potentils in rts fed holesterol- Aville on-line t 154

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