Research Article Antibacterial and Antioxidant Properties of the Leaves and Stem Essential Oils of Jatropha gossypifolia L.

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1 BioMed Reserh Interntionl Volume 2016, Artile ID , 9 pges Reserh Artile Antiteril nd Antioxidnt Properties of the Leves nd Stem Essentil Oils of Jtroph gossypifoli L. Sundy O. Okoh, 1,2 Benson C. Iwerieor, 1 Omool O. Okoh, 3 Uhehukwu U. Nwodo, 1 nd Anthony I. Okoh 1,2 1 SAMRC Miroil Wter Qulity Monitoring Centre, University of Fort Hre, Alie 5700, South Afri 2 Applied nd Environmentl Miroiology Reserh Group (AEMREG), Deprtment of Biohemistry nd Miroiology, University of Fort Hre, Alie 5700, South Afri 3 Deprtment of Pure nd Applied Chemistry, University of Fort Hre, Alie 5700, South Afri Correspondene should e ddressed to Sundy O. Okoh; sokoh@ufh..z Reeived 6 April 2016; Revised 7 June 2016; Aepted 12 July 2016 Ademi Editor: Cristin Mrtínez-Villlueng Copyright 2016 Sundy O. Okoh et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Antiteril nd ntioxidnt properties of the leves nd stem essentil oils (EOs) of Jtroph gossypifoli nd their effiies ginst infetious nd oxidtive stress diseses were studied in vitro. The EOs otined using Clevenger modified pprtus were hrterized y high resolution GC-MS, while their ntioxidnt nd ntiteril properties were exmined y spetrophotometri nd gr diffusion tehniques, respetively. The EOs exhiited strong ntiteril tivity ginst Esherihi oli, Enteroous feium, nd Stphyloous ureus. The stem essentil oil (SEO) ws more tive thn the lef essentil oil (LEO) ginst test teri with minimum inhiition onentrtion (MIC) rnging from to 0.05 mg/ml nd the LEO from 0.05 to 0.10 mg/ml. The SEO ws teriidl t nd 0.05 mg/ml ginst S. ureus nd E. feium, respetively, nd the LEO ws teriostti ginst the three teri t 0.05 nd 0.10 mg/ml. The SEO IC 50 (0.07 mg/ml) showed tht the ntirdil strength ws superior to LEO (0.32 mg/ml) nd β-rotene (1.62 mg/ml) in svenging 2, 2-diphenyl-1-pirylhydrzyl rdils (DPPH ). The oils effetively redued three other oxidnts to neutrl moleules in onentrtion dependent mnner. Findings from this study suggest tht, prtfromthetrditionlusesoftheplntextrts,theeoshvestrongiotiveompoundswithnoteworthyntiterilnd ntirdil properties nd my e good ndidtes in the serh for led ompounds for the synthesis of novel potent ntiiotis. 1. Introdution The rising hllenge of resistne of teri to mny ntiiotis hs eliited the need for the development of novel therpies with little or no side effet, to effetively mnge mny infetious diseses [1]. Noteworthy phytohemils results in reent yers suggest essentil oil s etter option due its superior properties nd hene my stnd in ple of ntiiotis to overome known infetive teri speies s well s yests nd filmentous fungi [2 4]. Constituents of essentil oil re numerous, omplex, nd known to possess strong ntiteril property, espeilly polyphenol, liphti nd yli terpenes, oxygented terpenes, nd phenylpropenes [5 7]. Essentil oils hve een shown to pssively diffuse ell memrne of teri owing to their permeility properties ross iologil lipid rriers [4, 6]. This memrne intertionnledtomemrneinstilityonsequentlyresulting in the lekge of the teril importnt intrellulr omponents nd ultimtely ell deth ours [6]. Cell wll, ell memrne, intrellulr proteins, nulei ids, enzymes, nd few others re vitl trget sites for drug design nd some essentil oil ompounds hve these speilized prts of the ell s importnt reeptor trgets [8]. Enzymti ntioxidnt defense systems omprising superoxide dismutse (SOD), tlse (CAT), glutthione peroxidse (G-Px), nd other endogenous ntioxidnt moleules, notly glutthione (GSH), do svenge oxygen derived free rdils produed in physiologil nd pthologil proesses. However, the inhiition of suh retive oxygen derived speies suh s superoxide (O 2 ), nitri oxide (NO ),

2 2 BioMed Reserh Interntionl hydroxyl (HO ), nd lipid peroxyl (LP ) generted from odymetolitivitiesswellsenvironmentllyindued rdils overwhelms the odies nturl defense ntioxidnts [9 12]. Furthermore, studies hve shown tht there is deline in viility nd poteny of the humn s ntioxidnts s individul ges [13, 14]. Mn hs used spies, deotions, fruits, vegetles, nd infusions whih re now knowledged s ontining potent seondry metolites ginst diseses eforethepperneofthewrittenword.inthelsttwo dedes, severl studies hve shown seondry metolites inluding phenolis, flvonoids, lkloids, nd essentil oil ompounds s potent ntioxidnts [15 17]. Essentil oil ould funtion s redile option to syntheti ntiiotis due its ility to penetrte miroorgnism ell memrne resulting in inhiition of miroorgnism growth s well s pity to quenh free rdils [6, 17]. In ddition, there re growing onerns on the use of nonnturl preservtives y onsumers nd food proessing industries owing to their reported dverse effets. Studies y Wng et l. [17] on some essentil oil onstituents reveled tht, unlike the syntheti free rdil svengers, the yproduts of nturl ntioxidnts re presumly sfe nd my e preferred in reduing thetotloxidtivestress.plntessentiloilomponents, inluding limonene, linlool, menthol, nd ryophyllene, reported to possess suh signifint iotive properties hve een registered y Europen Commission s flvors for use in food produts [9, 18]. Jtroph gossypifoli (Euphoriee) is trditionl mediinl shru plnt pplied for mngement of skin diseses, dietes, nd ners [19]. In Nigeri, fresh lef queous extrt is utilized in folk mediine for heling of mouth ner nd to terminte skin nd nose leeding while the stem is served s rush for helthy tooth [20, 21]. In Indi leves re used for prevention nd tretment of vriety of diseses inluding dysentery, ezem, dirrhe, nd ithes [22]. Deotion of J. gossypifoli in Trinidd nd Togo ws found potent for treting wound, reduing pin, nd treting snth sores [23]. Phytohemils nlyses hve shown tht different prts of J. gossypifoli ontin phenolis, flvonoids, nd lkloid ompounds [22, 23]. Ao et l. [24] reported phytol, germrene, nd linlool s some of the lef voltile oil onstituents of J. gossypifoli. There hs een dismyed rise of teril resistne to urrently ville ntiiotis; this hs motivted serh for lterntive soures of ntimiroil gents whih re elieved to e found undntly in plnts. There is however derth of informtion on omprtive evlution of the ntimiroil nd ntioxidnt properties s well s the iotive voltile onstituents of the stem nd lef essentil oil of J. gossypifoli; hene this urrent study imed to evlute the ntiteril nd ntioxidnt properties of the leves nd stem essentil oils of Jtroph gossypifoli. 2. Mterils nd Methods 2.1. Anlytil Regents. The hemils nd regents used inluded the following: Mueller-Hinton gr from Oxford Ltd. (Hmpshire, Englnd), dimethyl sulfoxide (DMSO), nd methnol from Fluk Chemils (Buhs, Switzerlnd). 2,2 - Azino-is(3-ethylenzothizoline-6-sulfoni id) dimmonium slt (ABTS), utylted hydroxyl toluene (BHT), nd 2, 2-diphenyl-1-pirylhydrzyl (DPPH) were ought from Sigm-Aldrih (St Louis, USA). All hemils nd regents used were of nlytil grde Plnt Mteril. J. gossypifoli ws otined from Forest Reserh Institute of Nigeri (FRIN), Idn, Oyo Stte, Southwest Nigeri. A plnt txonomist uthentited the plnt nd smples were kept in the Lgos University herrium(luh)withvouherspeimensnumersluh2009nd LUH2011 for the lef nd stem, respetively. The leves were suffiiently ir-dried in 5 dys t the mient room temperture, while the stem ws ut into smller piees nd ir-dried in 7 dys. They were pulverized nd essentil oil ws extrted for 3 h from eh (200 g) using modified Clevenger-type pprtus [25]. The hydrodistilltion experiment ws rried out twie for the lef nd stem seprtely to otin enough oil for iotivity ssys. The extrted essentil oils were dried over nhydrous sodium sulphte, dispensed into tinted vils,ndstoredt4 C. The yield of eh essentil oil ws omputed in w/w% (per grm) of individul hydrodistilled plnt smple Chrteriztion of Essentil Oils y Gs Chromtogrphy-Mss Spetrometry (GC/MS). We employed GC/MS to nlyze nd identify the essentil oil onstituents. The GC- MS onditions were progrmmed s previously desried [26], in whih the mss spetrometer (Hewlett-Pked HP 5973) interfed with n HP 6890 gs hromtogrph. Conditions of the temperture nd olumn were s follows: equilirtion time 3 min, rmp 4 C/min, initil temperture 70 C, nd finl temperture 240 C; inlet: splitless, initil temperture 220 C, pressure 8.27 psi, purge flow 30 ml/min, purge time 0.20 min, nd helium gs; olumn: pillry, 30 m 0.25 mm, internl dimeter 0.25 μm, film thikness 0.7 ml/min, nd verge veloity 32 m/se; MS: EI method t 70 ev. Susequently, identity of eh onstituent ws sertined y using greement of its mss spetr dt (MSD) of Wiley 275, New York referene omputer lirry. In ddition, mthing the retention index (RI) of eh ompound with those in literture ws lso employed in identifying the ompound.thepekreswereusedtootintotlperentge omposition of oil Antiteril Ativity Bteri Suspensions Test. Antiteril tivities of the oils were tested ginst three teril strins omprising two Grm-positive teri referene strins, S. ureus (NCINB 50080) nd E. feium (ATCC19434), nd E. oli O157, s Grm-negtive terium (ATCC ), following the guideline reommended y CLSI (2014). These referene strins were grown in Muller Hinton roth t 37 C for 24 h. Minimum inhiitory onentrtion (MIC) s well s minimum teriidl onentrtion (MBC) potentils ws performedonmullerhintongrpltest37 Cfor24h. Ciprofloxin ws pplied s referene stndrd (RS) or positive ontrol.

3 BioMed Reserh Interntionl MIC nd MBC Evlution. The mirodilution tehniquewsrriedouttoevlutethemics.800,875,900, 950, 975, nd μl of Mueller-Hinton roth (MHB) were dded to eh Eppendorf tue. Five hundred milligrms of othseondleostoksfterevportionofn-hexne ws seprtely dissolved in DMSO (500 μl) nd eh solution ws vortexed. Therefter, liquots of 200 μl, 125μL, 100 μl, 50μL, 25μL, nd 12.5μL were dded, respetively, to eh tue ontining MHB to ring the finl volume in eh to 1 ml nd the mixtures were properly vortexed. The inoulum suspension (20 μl) of eh tested terium (0.5 MFrlnd, fu/ml) ws dded susequently nd vortexed to permit dequte mixing of the essentil oil nd roth. Ciprofloxin nd DMSO served s the positive nd negtive ontrols, respetively. The experiments ove were performed in duplite nd inuted t 37 Cfor24h. Tues with lowest onentrtion without visile growth were reported s the MIC. MBC ws tested y streking out ll wells without visile growth in the MIC tehnique ove onto fresh nutrient gr pltes nd the ulture ws inuted for 24 h t 37 C. The lowest onentrtion of extrts tht did not yield ny growth on the solid medium fter the inution period ws reorded s minimum teriidl onentrtion (MBC) Antioxidnt Property. DPPH, ABTS, nitri oxide, nd lipid peroxyl rdils inhiiting tests were performed to determine the ntirdil property of the two essentil oils DPPH Assy. The DPPH test ws rried out s desried y Liyn-Pthirn nd Shhidi [27]. Briefly in DMSO solution of DPPH (2.7 μm) ws mde; fterwrds 1mLofitwsvortexedwith1mLoftheessentiloildissolved in DMSO whih hs mg/ml of the oil s well s the referene stndrd (RS). Then, the retion mixture ws vortexed nd inuted in the drk for 30 min t mient temperture. The sorne of the retion mixture ws then red t 517 nm ginst referene lnk ontining DMSO. The ssy ws rried in triplite nd DMSO ws used s lnk. Essentil oil s poteny to redue DPPH to neutrl moleule ws omputed s inhiitory perentge using the expression: % inhiition of DPPH y EO or RS = {(As ontrol As smple )} 100, (As ontrol ) where As ontrol isthesorneofthedpphrdil+ DMSO nd As smple isthesorneofdpphrdil+ essentil oil or referene stndrd. The IC 50, tht is, onentrtion of the essentil oil or referene stndrd (positive ontrol) required to redue 50% of the DPPH, ws otined from the stndrd urve produed with vrying onentrtions versus inhiitions nd results ompred to tht of referene stndrd ABTS Rdil Svenging Assy. The ABTS rdil svenging ssy proedure ws rried out following the method of Re et l. [28] with some modifition s desried (1) y Witypn et l. [3] y mixing 1 : 1 volumes of ABTS 7.0 mm nd 4.9 mm potssium persulfte solution. The mixed solution ws kept t room temperture for 12 h in drk hmer. The ABTS rdil tion (ABT + )wsthen diluted with DMSO to equilirte its sorne to (±0.001) t 734 nm. To rry out the ssy, 1000 μlof mg/ml solutions of the test smples (SEO nd LEO) in DMSO ws mixed with 1000 μl ABT + solution, ringing finl volume of eh mixture to 2 ml. The mixture ws llowedtoretfor7min.thesornet760nmws mesured spetrophotometrilly nd the ssy ws rried out in triplite. The rdil svenging tivity of the EO or RC ws expressed in terms of perentge (%) inhiition of ABTS + using expression in (1) desried in Setion Inhiition of Lipid Peroxidtion y TBARS Assy. The inhiition of lipid peroxidtion formtion y the essentil oils ws mesured using n dpttion of the method desried y Bdmus et l. [29] with egg yolk s lipid rih medi. To 10% egg yolk homogente (0.5 ml) ws dded 0.1 ml of the test smples (in DMSO) t vrying onentrtions ( mg/ml) nd the retion mixture mde up to 1 ml. The lipid peroxidtion ws indued y dding 0.05 ml of 0.07 M FeSO 4 nd the mixture ws then inuted for 30 min. Then, 1.5 ml of 10% eti id (ph 3.50) nd 1.5 ml of 0.08% 2- thiorituri id (in 1.1% sodium dodeyl sulphte nd 20% trihloroeti id) were dded nd the mixture ws vortexedndthenhetedt65 C for one hour. Upon ooling, 0.5mLofn-utnolwsddedtoretionmixturend entrifuged for 10 min t 3000 rpm. The upper orgni lyer ws then spirted nd the sorne red t 532 nm. The perentge inhiition of lipid peroxidtion ws lulted using the expression in eqution s desried in Setion Nitri Oxide Rdil Inhiition Assy. The nitri oxide rdil svenging tivities of the essentil oils were rried out ording to the modified method desried y Mkhij et l. [30]. The ompound sodium nitroprusside is known to deompose in queous solution t physiologil ph (7.2) produing nitri oxide rdils (NO ). Under eroi onditions, nitri oxide rdils ret with oxygen to produe stle produts (nitrte nd nitrite) whih n e mesured using Griess regent [31]. To 1 ml of sodium nitroprusside solution (10 mm) ws dded 1 ml of the essentil oil t vrying onentrtions ( mg/ml) nd the mixture ws then inuted t mient temperture for 110 min. After inution, 1 ml of the reting mixture ws dded to Griess regent (1%, sulphnilmide, 1% N-nphthyl-ethylenedimine hydrohloride in 2% o-phosphori id). The sorne of the olor developed ws then mesured t 546 nm ginst the regent lnk. The ssy ws rried out in triplite nd perentge inhiition ws lulted using the expression in (1) Sttistil Anlysis. The results re expressed s the mens ± SD for triplite ssys. Liner regression nlysis ws used to lulte IC 50 vlues while Person s orreltion nlysis nd t-test were used to test for signifine etween

4 4 BioMed Reserh Interntionl onentrtion nd perentge inhiition using SPSS 15.0 for windows (SPSS In.). 3. Results nd Disussion 3.1. Composition of the Essentil Oils Extrted. The gs hromtogrphy-mss spetrometry qulittive nd quntittive nlyses of the essentil oils of J. gossypifoli in our previous report [32] nd the present study reveled tht onstituents of the lef essentil oil (LEO) re predominntly lohols inluding phytol (33.40%) nd linlool (9.81%) presented in Tle 1. Out of the 15 onstituents identified in LEO ounting for 98.70% of the totl oil ontent, four were mong the J. gossypifoli lef oil omponents in Ao et l. [24]. In ddition to phytol (18.05%) nd other terpenoids onstituents, more monoterpenes nd sesquiterpenes inluding limonene (12.40%), germrene D (12.30%), α-opene (12.20%), α-terpinene (10.61%), nd α-romdendrene (10.48%) were identified s mjor ompounds in the stem essentil oil (SEO) thn in the LEO in this study. Lnosterol, humulene, 2, 6-di-utyl-p-resol, heptdenoi id, nd linolei id hve lso een reported s onstituents of LEO of J. gossypifoli [33, 34] ut they were however not found in this study. The disrepny in the omposition of J. gossypifoli essentil oil grown in different regions in Nigeri nd elsewhere my e due to differenes in ftors, suh s limti, sesonl, nd geogrphil onditions,geofplnt,humidityofthehrvestedplntmteril, extrtion tehnique, nd the existene of hemotype [35] Antiteril Ativity of the Essentil Oils. The essentil oils extrted from the leves nd stem of J. gossypifoli strongly exhiited inhiitory tivity ginst the 3 teri strins (Esherihi oli, Enteroous feium, ndstphyloous ureus) investigted. The stem essentil oil (SEO) MIC vlues of ± 0.01, 0.05 ± 0.00, nd0.05 ± 0.00 mg/ml showed tht it is more tive thn the lef essentil oil (LEO) with MIC vlues of 0.05 ± 0.00, 0.10 ± 0.01, nd 0.10 ± 0.01 mg/ml ginst E. feium, S. ureus, nde. oli, respetively (Tle 2). Similrly mg/ml of SEO ws le to kill (teriidl) E. feium, while it requires twie the dose (0.05 mg/ml) to exhiit teriidl tivity ginst S. ureus.unlikethetwogrm-positiveteritested,theoils were less tive ginst Grm-negtive terium (E. oli). However, t 0.10 mg/ml the SEO ws teriidl ginst E. oli whiletheleowsteriosttitthesmeonentrtion (Tle 3). The differenes in ntiteril property ould e due to net repulsion of the two outer omplex memrnes struture(two-lipidilyer)ingrm-negtiveterilell wll whih is sent in Grm-positive teri [36]. These lyers onstitute physil rriers etween miroorgnism nd the environment, preventing intertions of the teril ell with hrmful sustnes. A Grm-positive terium hs only one reltively thik permele memrne, rendering it more suseptile to intertions with the environment [37]. The effets of the stem nd leves oils of J. gossypifoli ginst the teri lso differed; the vrition oserved in the hemils profiles of two oils my possily ount for their vried iotivity [38, 39] in the present study. % inhiition DPPH model p < Conentrtion (mg/ml) log Lef oil Stem oil β-crotene Vitmin C Figure 1: Antirdil effets of lef nd stem oils of J. gossypifoli nd referene stndrd on DPPH rdils:, not signifintly different;, signifintly different (p < 0.05). % inhiition ABTS model p < Conentrtion (mg/ml) log Lef oil Stem oil β-crotene Vitmin C Figure 2: Antirdil effets of lef nd stem oils of J. gossypifoli nd referene stndrd on ABTS rdils;, not signifintly different;, signifintly different (p < 0.05) Antioxidnt Ativity of the Essentil Oils. Antioxidnt properties of the lef nd stem oils of J. gossypifoli were investigted in vitro in four different (DPPH, ABTS, LP, nd NO) rdils models. The perentge inhiitions of these rdils y the oils nd referenes stndrds (vitmin C nd β-rotene) were onentrtion dependent (0.025 to 0.5 mg/ml) expressed in % inhiition versus log( 1.6 to 0.3) s presented in Figures 1 4. The ntirdil effets of LEOndSEO(,)onDPPH were not signifintly different t low onentrtions (0.025 nd 0.05 mg/ml), ut t mg/ml, SEO () exhiited muh higher inhiitory effet thn LEO nd the referene stndrds (RS) nd effets of

5 BioMed Reserh Interntionl 5 Tle 1: Essentil oils onstituents in the lef nd stem of Jtroph gossypifoli. Constituent KI Composition (%) Lef Stem Methods of identifition MS dt QA d α-pinene KI, MSD 93, 79, 41, β-pinene t KI, MSD 93, 69, 41, Cmphene KI, MSD 93, 69, 41, α-terpinene KI, MSD 93, 77, 136, Limonene KI, MSD 68, 93, 107, Linlool KI, MSD 71, 43, 69, Menthol 1142 t 4.87 KI, MSD 79, 43, 41, β-bisolene KI, MSD 204, 69, 41, α-aromdendrene KI, MSD 159, 91, 41, α-cdinene KI, MSD 161, 43, 105, GermreneD KI,MSD 20,161,32, Frnesene KI, MSD 69, 93, 107, γ-cdinene KI, MSD 161, 43, 105, α-muurolene KI, MSD 121, 95, 43, Viridiflorol KI, MSD 32, 79, 55, Pentdeen-5-yne KI, MSD 79, 67, 41, Bisolol KI,MSD 39,204,69,41 90 Germrene B KI, MSD 120, 161, 32, α-copene KI, MSD 105, 119, 161, Dodenoi id KI, MSD 29, 60, 73, Hexdenoi id KI, MSD 60, 73, 43, Otdenl t KI, MSD 41, 57, 82, Phytol KI, MSD 71, 57, 41, ,17-Otdedienl t KI, MSD 280, 265, 279, Totl oil ontent (%) Yield of oil Constituent elution order in olumn HB-5; Kovt s index, some of the m/z for most undnt peks in the mss spetrum, d perentge of GC/MS lirry qulity ssurne of onstituent in SEO/LEO, MSD = mss spetr dt; RI = retention index reltive to C 9 C 23 on the olumn HB-5, t = less thn 0.05%. Tle 2: Minimum inhiitory onentrtion (MIC) vlues (mg/ml) for essentil oils of J. gossypifoli ginst teri strins. Bteri Leves oil Stem oil Stphyloous ureus 0.10 ± 0.01 NG 0.05 ± 0.01 NG Enteroous feium 0.05 ± 0.00 NG ± 0.00 NG Esherihi oli 0.10 ± 0.01 NG 0.05 ± 0.00 NG Signifint differene ws onsidered t level of p < 0.05;NG:nogrowth;VG:visilegrowth. Ciprofloxin Positive ontrol 0.05 ± 0.01 NG 0.05 ± 0.02 NG 0.05 ± 0.01 NG DMSO Negtive ontrol 0.5 ml VG 0.5 ml VG 0.5 ml VG LEO nd RS were similr (). However t 0.5 mg/ml the SEO displyed similr () tivity s tht of the RS (βrotene) while the SEO effet ws signifintly different () from the seond RS (vitmin C) s well s the LEO in svenging DPPH (Figure 1). The DPPH ntirdil ssy is sedonthepremisethtdonorofntomofhydrogenor n eletron is n ntioxidnt or ntirdil nd its strength is demonstrted s DPPH olor hnges (purple to yellow) inthetestsmpleduetoformtionofneutrldpph-h moleule upon sorption of hydrogen from n ntioxidnt [40].However,DPPHtehniqueisnotspeifirdil speies test ut is for generl rdils svenging poteny of n ntioxidnt [40]. Therefore, to evlute the preise ntirdil effiy of LEO nd SEO of J. gossypifoli, we quntittively nd qulittively investigted the presumed ntirdil property using two different speifi rdils speies (LP nd NO ) nd tion rdil (ABTS + ). Overll, in the four experiments the lef nd stem essentil oils of J. gossypifoli exhiited effetive ntirdils potenies ginst the different oxidnts, inditing they re good eletron donors in DPPH nd ABTS tests, nd displyed strong LP nd vlule NO ntioxidnt tivity. Assessed

6 6 BioMed Reserh Interntionl Tle 3: Minimumteriidl onentrtion (MBC) vlues (mg/ml) foressentiloilsofj. gossypifoli ginst teri strins. Bteri Leves oil Stem oil Stphyloous ureus Enteroous feium Esherihi oli Bteriostti t 0.10 ± 0.01 VG Bteriostti t 0.05 ± 0.03 NG Bteriostti t 0.10 ± 0.01 VG Bteriidl t 0.05 ± 0.01 NG Bteriidl t ± 0.00 NG Bteriidl t 0.10 ± 0.00 NG Signifint differene ws onsidered t level of p < 0.05;NG:nogrowth;VG:visilegrowth. Ciprofloxin Positive ontrol Bteriidl t 0.05 ± 0.01 NG Bteriidl t 0.05 ± 0.02 NG Bteriidl t 0.05 ± 0.03 NG DMSO Negtive ontrol 0.5 ml VG 0.5 ml VG 0.5 ml VG % inhiition Lipid peroxidtion model p < 0.05 % inhiition Nitri oxide svenging model p < Conentrtion (mg/ml) log Lef oil Stem oil β-crotene Vitmin C Figure 3: Antirdil effets of lef nd stem oils of J. gossypifoli nd referene stndrds on lipid peroxide rdils:, not signifintly different;, signifintly different (p < 0.05) Conentrtion (mg/ml) log Lef oil Stem oil β-crotene Vitmin C Figure 4: Antirdil effets of lef nd stem oils of J. gossypifoli nd referene stndrds on nitri oxide rdils:, not signifintly different;, signifintly different (p < 0.05). y liner regression nlysis, the IC 50 vlues were lulted while Person s orreltion nlysis nd t-test were used to test signifint differene using SPSS 15.0 for windows (SPSS In.). Both oils redued the DPPH to neutrl DPPH-H moleule ttining 50% derese with IC 50 vlue of 0.07 ± 0.01 mg/ml for SEO nd while tht of LEO is 0.32 ± 0.11 mg/ml (Tle 4). Signifint differene ws onsidered t level of p < The perentges inhiition of the ABTS + y the SEO nd LEO were lower thn results otined in DPPH model, hieving IC 50 vlues of 1.34 ± 0.01 nd 2.35 ± 0.00 mg/ml, respetively (Tle 4). However, unlike in the DPPH ssy, the ntioxidnts ompletely deolorized the lue olor of the oxidnt (ABTS + ) solutions, turning into neutrl moleules (olorless form) from the lowest to highest onentrtions ( mg/ml). This oserved effet ws stronger with SEOthninLEO, β-rotene, nd vitmin C. At mg/ml the effets of LEO nd vitmin C on ABTS + were omprle (), while SEO () exhiited higher effet thn RS nd Tle 4: Antirdil pity of essentil oils extrted from J. gossypifoli [IC 50 (mg/ml)]. Ativity J. gossypifoli Referene ompounds Lef oil Stem oil Vitmin C β-crotene DPPH 0.32 ± ± ± ± 0.12 ABTS ± ± ± ± 0.11 LP 3.31 ± ± ± ± 0.00 NO 2.10 ± ± ± ± 0.10 The IC 50 (mg/ml) ws otined from stndrd urve for eh oil nd referene drugs. The lower the IC 50, the higher the ntirdil strength. Signifint differene ws onsidered t level of p < 0.05.Vluesremen± SD, n=3. LEO (Figure 2). However, s the onentrtions inresed ( mg/ml) the ntirdil effets of the two referene stndrds were similr with oth lower thn SEO () ut higher thn LEO (). At 0.5 mg/ml SEO demonstrted the highest effet, followed y RS nd LEO hving the lowest inhiitory effet on ABTS.Thedisrepnyoservedin tivities of SEO nd LEO ginst the two different oxidnts

7 BioMed Reserh Interntionl 7 (DPPH nd ABTS + )ouldettriutedtomnyftors inluding the omplexity, polrity, nd isomers seletivity of the rdils. In ddition, the ese t whih the oils solvte the rdil s medium my differ nd these vriles hve een reported to influene poteny of voltile onstituents in inhiiting speies of rdils [41]. Thelipidperoxiderdils(LP )inhiitingeffetsofseo nd LEO t different onentrtions re showed in Figure 3. The SEO nd β-rotene exhiited stronger (, ) ntirdil tivities thn the LEO nd vitmin C (, ) ginst lipid peroxide indued y ferri sulphte in homogentes of egg yolk. Interestingly, the IC 50 vlues of 0.55 ± 0.01 nd 0.51 ± 0.00 mg/ml otined for SEO nd β-rotene, respetively (Tle 4), indited no signifint differene (p < 0.05) etween voltile oil (SEO) nd the referene stndrd. The ntirdil tivities of LEO nd vitmin C were wek nd similr (, ) t low onentrtions ( mg/ml), with IC 50 vlues of 3.31 nd 3.01 mg/ml, respetively. However, t mg/ml, their inhiitory tivities ginst lipid peroxide rdils were ove verge. Notle in the lipid peroxidtion model is the signifint differene etween SEO () nd vitmin C s well s similr effets of LEO nd vitmin C (, ) in svenging LP t mg/ml nd 0.5 mg/ml (Figure 3) tht my e sried to the oils terpenoids, whih donte hydrogen toms to H 2 O 2,thusreduingitto2H 2 O. In the nitri oxide ssy, the tivities of LEO nd SEO to inhiit nitri oxide rdil (NO ) produed from red-olored omplex slt of sodium nitroprusside solution [N 2 [Fe(CN) 5 NO] 2H 2 O] t different onentrtions ( mg/ml) re showed in Figure 4. The SEO () demonstrted stronger inhiitory tivity upon NO ompred to LEO s well s the two referene stndrds t nd 0.05 mg/ml, while the tivities of LEO nd vitmin C (RS) re signifintly different (, ) t low onentrtion (0.025 mg/ml). However, with inresing onentrtions ( mg/ml), SEO nd β-rotene displyed high nd omprle ntirdil tivity followed y LEO, while vitminchdthelesteffetinounteringno generted (Figure 4). Interestingly t 0.5 mg/ml the LEO nd β-rotene tivities were similr (, ); however oth displyed lower effet thn SEO (). The IC 50 vlueotinedforseo(1.46± 0.01 mg/ml) ws moderte; however, it ws lower thn tht of LEO (2.10 ± 0.00 mg/ml), rotene (2.03 ± 0.03 mg/ml), nd vitmin C (2.91 ± 0.01 mg/ml) s presented in Tle 4. The high phytol ontent in the EOs in this present study is remrkle nd might hve enhned the iotivity of the oils. Phytol, diterpenoid lohol, hs een reported y Cmill [42] to demonstrte good ntioxidnt effet in vivo nd hs high pity to quenh hydroxyl nd nitri oxide rdils s well s prevent the formtion of lipid peroxides s mesured y thiorituri id retive sustnes (TBARS). The dditive or synergeti effets of identified iotive onstituents in this study (Tle 1) my justify the higher iotivity of the SEO thn the LEO. The ntiteril nd ntioxidnt properties of the SEO might hve een enhned y other terpenoids identified even in little mount, for exmple, menthol (4.87%), γ-dinene (5.49%), nd α-pinene (5.03%), thus suggesting possile synergisti intertion etween the omponents [43, 44]. Some reent studies demonstrted tht some essentil oil ompounds tht were oserved in this present study do possess potent iotive properties [10, 12, 18, 45]. Menthol, for exmple, whih ws found in SEO, hs een reported to demonstrte very high ntimiroil, ntioxidnt, nd nti-inflmmtory tivities [46]. Furthermore, limonene hs een proven in previous studies [47] s strong iotive monoterpene nd its propoptoti effets on humn gstri ner nd its ntitumor nd ntimetstsis tivities hve een demonstrted. Tkhshi [48] reported tht terpinene, nother monoterpene hydroron lso identified in the SEO of J. gossypifoli, hs the ility to inhiit low density lipoprotein oxidtion even in the formtion phse. In ddition, the min omponent phytol, whih ws identified in the two oils, ould hve possily reted with DPPH,ABTS +,LP, nd NO through vrious mehnisms suggested y Foti nd Amorti [49]. The result in this urrent study is in greement with other reports tht hve implited liphti terpene with ntirdil properties, while effet of hydroron monoterpenewhihisyliwithdouleondsissimilrtothe property of phenoli ompounds or α-toopherol [5, 6, 10, 17]. Ativity of SEO ginst E. oli, E. feium, nds. ureus s well s svenging different rdils s oserved in this present study is quite noteworthy. These oservtions my therefore suggest tht SEO of J. gossypifoli ould possily enewpotentndidteintheserhforledompounds for the mngement of infetious nd oxidtive stress-relted disorders suh s Alzheimer s disese (AD), ners, dieti nephropthy, nd rterioslerosis [50 52]. 4. Conlusion This present study indites tht, prt from the lol uses of the lef ndstem of J. gossypifoli, the essentil oil ontined strong iotive phytohemils nd they re good prospet s new ntimiroil gent nd n lterntive to syntheti ntioxidnt nd ould e used s food preservtives on further investigtion. Competing Interests The uthors delre they hve no ompeting interests. Authors Contriutions Sundy O. Okoh nd Benson C. Iwerieor designed the experiments, rried out the nlysis, interpreted the results, nd wrote the mnusript, Omool O. Okoh ssisted with nd supervised the nlysis of results, Uhehukwu U. Nwodossistedwithwritingndproofredingofmnusript, nd Anthony I. Okoh oordinted the reserh nd mnusript preprtion. All uthors hve red nd pproved the finl mnusript. Aknowledgments Authors re grteful to the South Afri Medil Reserh Counil, University of Fort Hre, nd the mngement of FIIRO, Lgos, Nigeri, for finnil support.

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