Influence of Boiling, Steaming and Frying of Selected Leafy Vegetables on the In Vitro Anti-inflammation Associated Biological Activities

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1 plnts Artile Influene of Boiling, Steming n Frying of Selete Lefy Vegetles on the In Vitro Anti-inflmmtion Assoite Biologil Ativities K. D. P. P. Gunthilke 1,2, K. K. D. S. Rnweer 2 n H. P. V. Rupsinghe 3, * ID 1 Deprtment of Foo Siene & Tehnology, Fulty of Livestok, Fisheries & Nutrition, Wym University of Sri Lnk, Mknur, Gonwil 10250, Sri Lnk; gunthilkep@dl.c 2 Deprtment of Foo Siene n Tehnology, Fulty of Applie Sienes, University of Sri Jyewrenepur, Gngowil, Nugego 60170, Sri Lnk; kksrn@yhoo.om 3 Deprtment of Plnt, Foo, n Environmentl Sienes, Fulty of Agriulture, Dlhousie University, Truro, NS B2N 5E3, Cn * Corresponene: vrupsinghe@l.; Tel.: Reeive: 14 Ferury 2018; Aepte: 13 Mrh 2018; Pulishe: 16 Mrh 2018 Astrt: The im of the present stuy ws to evlute the effet of ooking (oiling, steming, n frying) on nti-inflmmtion ssoite properties in vitro of six populrly onsume green lefy vegetles in Sri Lnk, nmely: Centell siti, Cssi uriult, Gymnem ltiferum, Olx zeylni, Sesni grniflor, n Pssiflor eulis. The nti-inflmmtion ssoite properties of methnoli extrts of ooke leves were evlute using four in vitro iologil ssys, nmely, hemolysis inhiition, proteinse inhiition, protein enturtion inhiition, n lipoxygense inhiition. Results revele tht the frying of ll the teste lefy vegetles h reue the inhiition ilities of protein enturtion, hemolysis, proteinse, n lipoxygense tivities when ompre with other foo preprtion methos. Steming signifintly inrese the protein enturtion n hemolysis inhiition in O. zeylni n P. eulis. Steming of leves inrese inhiition tivity of protein enturtion in G. ltiferum (y 44.8%) n P. eulis (y 44%); hemolysis in C. siti, C. uriult, n S. grniflor; lipoxygense inhiition ility in P. eulis (y 50%), C. siti (y 400%), n C. uriult leves (y 250%); proteinse inhiition in C. uriult (100%) when ompre with tht of rw leves. In generl, steming n oiling in ontrst to frying protet the helth-promoting properties of the lefy vegetles. Keywors: plnt-foo; proessing; nutreutils; inflmmtion; helth 1. Introution Mny egenertive humn iseses, suh s ner, inflmmtion, n riovsulr iseses hve een reognize s onsequene of free ril mge. [1] Inflmmtion is prt of the omplex iologil response of vsulr tissues to hrmful stimuli, whih is frequently linke with pin n involves mny iologil ourrenes, suh s n inrese of vsulr permeility, n inrese of protein enturtion, n memrne ltertion [2]. Numerous reent stuies hve shown tht hroni inflmmtion is ssoite with wie rnge of progressive iseses suh s ner, neurologil isese, metoli isorer, n riovsulr isese [3]. Therefore, there hve een mny stuies unertken on how to ely or prevent the onset of these hroni iseses, s these le to glol helth prolems. The most likely n prtil wy to fight ginst egenertive iseses, suh s inflmmtion, is to improve oy ntioxint sttus, whih oul e hieve y higher onsumption of vegetles n fruits [4]. Foos from plnt origin usully ontin nturl ntioxints tht n svenge free rils [5]. Green lefy vegetles re rih soures of minerls n ntioxint Plnts 2018, 7, 22; oi: /plnts

2 Plnts 2018, 7, 22 2 of 10 vitmins [6], s well s other ntioxint ompouns, suh s polyphenols n rotenois [7,8] Among the lefy vegetles tht re ville in Sri Lnk, Cssi uriult, Gymnem ltiferum, Olx zeylni, Sesni grniflor, Pssiflor eulis, n Centell siti re reporte to possess higher ntioxint tivities, s esrie in Gunthilke n Rnweer [7]. Although green lefy vegetles re onsiere s potentil soures of ietry ntioxints n iotives, only few stuies my hve een reporte on evluting the impt of ooking on the nti-inflmmtory properties. Therefore, this stuy ime to investigte the influene of ooking of selete lefy vegetles on their nti-inflmmtion ssoite iologil properties in vitro, whih were mesure y het-inue hemolysis ssy, protein enturtion ssy, proteinse inhiition ssy, n lipoxygense inhiition ssy. 2. Results n Disussion 2.1. Protein Denturtion Denturtion of proteins is well-oumente use of inflmmtion [9]. As prt of the investigtion on the hnges in the nti-inflmmtory tivity, the ility of ooke lef extrts on protein enturtion ws stuie. Figure 1 shows the effet of ooking of lefy vegetles on protein enturtion inhiitory tivity. Protein enturtion inhiition ility signifintly inrese (p < 0.05) in steme leves of O. zeylni (y 44.8%) n P. eulis (y 44%) when ompre with tht of their fresh leves. Interestingly, frying of ll the teste lefy vegetles resulte in the lowest protein enturtion inhiition ility when ompre with other proessing methos. Boile leves of O. zeylni, C. uriult, S. grniflor, n G. ltiferum hve shown signifintly lower (p < 0.05) inhiition ility when ompre with their fresh leves, wheres oile leves of C. siti, n P. eulis hve shown signifintly higher (p < 0.05) protein enturtion inhiition ility. Signifintly lower protein enturtion inhiition ility ws oserve in ll of the ooke leves of C. uriult, S. grniflor, n G. ltiferum when ompre with their fresh leves. In stuy, methnol extrt of whole plnt of Oxlis orniult Linn (Fmily: Oxliee) ws ssesse y Skt et l. [9] for its nti-inflmmtory tivity y in vitro methos n ws reporte tht the extrt showe nti-inflmmtory tivity y inhiiting the het-inue lumin enturtion with the IC 50 vlues of ± 2.78 µg/ml, respetively. Bse on nother stuy, flvonoi-rih frtion of M. myristi hve shown n lumen enturtion in ose-epenent mnner n the mximum inhiition of enturtion of lumin ws foun to roun 75.38% ± 0.56% t 350 µg/ml: IC 50 vlue of 258 µg/ml, while stnr nti-inflmmtory rug (spirin) showe mximum inhiition of 98.41% ± 0.13% t the sme onentrtion [10]. However, the preise mehnism of this memrne stiliztion ws yet to e eluite. It hs een propose tht the plnt extrts might inhiit the relese of the lysosoml ontent of neutrophils t the site of inflmmtion [11]. Aoring to Chou [12], these neutrophils lysosoml onstituents inlue teriil enzymes n proteinses whih upon extrellulr relese use further tissue inflmmtion n mge. Tle 1 shows the orreltion of nti-inflmmtory properties with polyphenols, flvonois, n rotenois of green lefy vegetles. The hnges in protein enturtion ility with the ooking tretments my e relte to the hnges in polyphenols n flvonois ontent. In stuy, it ws foun tht heting of green lefy vegetles reue the vitmin C ontent, thus reuing properties n free rils svenging properties [5]. Tle 1. Person orreltions etween mjor iotives (totl phenolis, totl flvonois, totl rotenois) n % inhiition of protein enturtion, hemolysis enturtion, lipoxygense tivity, n proteinse tivity of ooke lefy vegetles. Prmeters r P Totl phenolis versus protein enturtion Totl phenolis versus hemolysis Totl phenolis versus lipoxygense tivity

3 Plnts 2018, 7, 22 3 of 10 Tle 1. Cont. Plnts 2018, 7, x FOR PEER REVIEW Prmeters r P 3 of 10 Totl Totl flvonois phenolis versus proteinse enturtion tivity Totl Totl flvonois flvonois versus versus proteinhemolysis enturtion Totl flvonois Totl flvonois versus versus lipoxygense hemolysis tivity Totl flvonois versus lipoxygense tivity Totl flvonois versus proteinse tivity Totl flvonois versus proteinse tivity Totl rotenois versus protein enturtion Totl rotenois versus protein enturtion Totl rotenois versus hemolysis Totl rotenois versus hemolysis Totl rotenois versus lipoxygense tivity tivity Totl rotenois versus proteinse tivity MK C MKF MKB MKS PFC PFF PFB PFS GKC GKF GKB GKS RWC RWF RWB RWS KUC KUF KUB KUS KMC KMB KMS % Inhiition Figure 1. Protein enturtion inhiition ilityof of rw n ooke extrtsof of some GLV. MK, O. zeylni; RW, C. C. uriult; KM, KM, S. S. grniflor; KU, KU, G. G. ltiferum; ltiferum; PF, PF, P. eulis; P. eulis; GK, GK, C. siti. C. siti. C-fresh fresh leves; leves; F-frie; F-frie; B-oile; B-oile; S-steme. S-steme. Dt Dt re re presente presente s s mens mens ± ± stnr evitions of three replite etermintions. Columns with ifferent letters for eh vegetle re signifintly ifferent (p < 0.05) Het-Inue Hemolysis Lefy types sujete to ifferent ooking Aoring to Chipp n o-uthors [13], lysosoml enzymes tht re relese uring inflmmtion les to the tissue injury y mging the mromoleules, suh s proteins, lipis, DNA, et. Further, it it mges tissues y y lipi lipi peroxition of of memrnes, whih whih re ssume re ssume to e responsile to e responsile for ertin for ertin pthologil onitions s hert s hert ttks n n rheumtoi rthritis, et. Therefore, the stiliztion of of lysosoml memrne is is vitl in ontrolling the the inflmmtory response y y inhiiting the relese the relese of lysosoml of lysosoml onstituents onstituents of tivte of tivte neutrophil, neutrophil, suh s teriil suh s teriil enzymes enzymes n proteses, n whih proteses, my le whih to further my tissue le to inflmmtion further tissue n mge inflmmtion upon extrellulr n mge relese upon or extrellulr y stilizingrelese the lysosoml or y stilizing memrne the [13]. lysosoml The memrne memrne of the[13]. humn s The memrne re loo ell of the is nlogous humn s re to the loo lysosoml ell is memrne, nlogous to n the itslysosoml stiliztion memrne, implies tht n the its extrt stiliztion my s implies well stilize tht the lysosoml extrt my memrnes. s well The stilize in vitro lysosoml iossy memrnes. tht ws usethe in this in vitro stuyiossy etermines tht thews stiliztion use in this of humn stuy etermines re loo ell the memrne stiliztion yof hypo humn tonilly re loo inue ell memrne y hypo lysis, tonilly n this inue n e tken memrne s lysis, in vitro n mesure this n ofe nti-inflmmtory tken s in vitro tivity mesure of the of nti-inflmmtory mny rugs vrious tivity plnt of extrts the mny [13]. rugs or vrious Theplnt het-inue extrts [13]. hemolysis inhiition ilities of rw n ooke lef smples re shown in Figure The 2. het-inue Similrly, the frying hemolysis proess inhiition signifintly ilities reue of rw (p n < 0.05) ooke the hemolysis lef smples inhiition re shown ility in Figure of O. zeylni, 2. Similrly, C. uriult, the frying S. proess grniflor, signifintly G. ltiferum, reue P eulis, (p < 0.05) nthe C. siti hemolysis y 4.0%, inhiition 5.5%, ility 6.0%, of O. zeylni, C. uriult, S. grniflor, G. ltiferum, P eulis, n C. siti y 4.0%, 5.5%, 6.0%, 6.2%, 7.0%, n 5.1%, respetively. Boile leves of C. siti, C. uriult, n S. grniflor hve shown signifintly higher (p < 0.05) hemolysis inhiition ility hen ompre with tht of rw n their other ooke leves. Steme leves of O. zeylni, P. eulis, n G. ltiferum hve shown the similr or higher hemolyti inhiition ility thn tht of their rw n other ooke leves.

4 Plnts 2018, 7, 22 4 of %, 7.0%, n 5.1%, respetively. Boile leves of C. siti, C. uriult, n S. grniflor hve shown signifintly higher (p < 0.05) hemolysis inhiition ility hen ompre with tht of rw n their Plnts other 2018, 7, ooke x FOR PEER leves. REVIEW Steme leves of O. zeylni, P. eulis, n G. ltiferum hve shown4 of the 10 similr or higher hemolyti inhiition ility thn tht of their rw n other ooke leves. Steme n Steme oilen leves oile of leves S. grniflor of S. grniflor showe signifintly showe signifintly higher (p higher < 0.05) (p hemolyti < 0.05) hemolyti inhiition inhiition ility when ility ompre when ompre with tht with of its tht rw of its leves. rw leves. In previous In previous stuy, the stuy, methnoli the methnoli extrt extrt of the whole of the plnt whole ofplnt Oxlis of orniult Oxlis orniult Linn hslinn eenhs shown een the shown re loo the re ells loo memrne ells memrne stiliztion stiliztion with the IC with 50 vlues the IC50 of vlues ± 9.6 of µg/ml ± 9.6 [9]. µg/ml Tle 1 shows [9]. Tle poor 1 shows orreltion poor of het-inue orreltion of hemolysis het-inue with polyphenols, hemolysis with flvonois, polyphenols, n rotenois flvonois, n of green rotenois lefy vegetles. of green lefy vegetles MK C MKF MKB MKS PFC PFF PFB PFS GKC GKF GKB GKS RWC RWF RWB RWS KUC KUF KUB KUS KMC KMB KMS % Inhiition Lefy types sujete to ifferent ooking Figure 2. Het-inue hemolysis inhiition ility of rw n ooke extrts of some GLV. Vlues represent mens of triplite reings. MK, O. O. zeylni; RW, RW, C. C. uriult; KM, KM, S. S. grniflor; KU, KU, G. G. ltiferum; PF, PF, P. P. eulis; GK, C. C. siti. C-fresh leves; F-frie; B-oile; S-steme. Dt re presente s mens ± stnr evitions of three replite etermintions. Columns with ifferent letters for eh vegetle re signifintly ifferent (p (p < 0.05) Lipoxygense Inhiitory Ativity. The mehnism of nti-inflmmtion involves series of events in whih metolism of Arhioni i plys n importnt role [10] Arhioni i is leve from the memrne phospholipis upon pproprite stimultion of of neutrophils, n n n n e onverte e onverte to leukotrienes to n prostglnins n through through the tion the tion of lipoxygense of lipoxygense n ylooxygense n pthwys, pthwys, respetively respetively [10]. Lipoxygense [10]. Lipoxygense enzymes enzymes tlyze tlyze the the oxition of of Arhioni i i (linolei i) to to proue leukotrienes tht re re importnt meitors in in vriety vriety of inflmmtory of events events [14]. In [14]. previous In previous stuy, it stuy, ws reporte it ws reporte tht the essentil tht the essentil oil of Cymopogon oil of Cymopogon gignteus from gignteus Beninfrom hs potentil Benin hs topotentil e use s to n e nti-inflmmtory use s nti-inflmmtory gent towrs gent lipoxygense towrs lipoxygense inhiition [14]. inhiition Therefore, [14]. use Therefore, of in vitrouse inhiition of vitro of lipoxygense inhiition of oul lipoxygense e goooul moel e for the goo sreening moel for of plnts the sreening with inflmmtory of plnts with potentils. inflmmtory Figure 3 shows potentils. the lipoxygense Figure 3 shows inhiition the lipoxygense ility of rw inhiition n ooke ility lefy of types, rw n nooke the results lefy lerly types, showe n the tht results thelerly lipoxygense showe inhiition tht the lipoxygense ility h reue inhiition uring ility frying h reue in ll of uring the lef frying vrieties in ll when of the ompre lef vrieties with when their ompre fresh, oile, with n their steme fresh, oile, leves. n Boiling steme of leves. inrese Boiling of the leves lipoxygense inrese inhiition the lipoxygense ility signifintly inhiition ility (p < 0.05) signifintly in O. zeylni, (p < 0.05) P. eulis, in O. C. siti, zeylni, n P. C. eulis, uriult C. siti, levesn when C. ompre uriult leves with tht when of their ompre rw leves. with tht However, of their oiling rw leves. hs inrese However, theoiling lipoxygense hs inrese inhiition the ility lipoxygense in S. grniflor inhiition s ility ompre in S. grniflor with its steme s ompre leves, with though its steme it is lower leves, thn though its fresh it is leve. lower Interestingly, thn its fresh ooke leve. Interestingly, leves of G. ltiferum ooke leves hveof shown G. ltiferum signifintly hve shown lower (p signifintly < 0.05) lipoxygense lower (p < inhiition 0.05) lipoxygense ility when inhiition ompre ility with when thtompre of its rw with leves. tht of its rw leves.

5 Plnts 2018, 7, 22 5 of 10 Plnts 2018, 7, x FOR PEER REVIEW 5 of MKC MKF MKB MKS PFC PFF PFB PFS GKC GKF GKB GKS RWC RWF RWB RWS KUC KUF KUB KUS KMC KMB KMS % Inhiition Lefy type sujete to ifferent ooking methos Figure3. 3. Lipoxygense inhiition ilityof of rw n ooke extrtsof of some GLV. Vlues represent mensof of triplite reings. MK, O. O. zeylni; RW, C. C. uriult; KM, S. S. grniflor; KU, G. G. ltiferum; PF, P. P. eulis; GK, GK, C. C. siti. C-fresh C-fresh leves; leves; F-frie; F-frie; B-oile; B-oile; S-steme. S-steme. Dt re Dt presente re presente s mens s ± mens stnr ± stnr evitions evitions of three of replite three replite etermintions. Columns Columns with with ifferent letters for for eh vegetle re signifintly ifferent (p < 0.05) Proteinse Inhiitory Ativity Plnt extrts hve een reporte to inhiit protein enturtion. Although, the the preise mehnismof of this memrne stiliztion ws yetto toe e eluite, utit it hs een propose tht the extrt might inhiit the relese of the lysosoml ontent of of neutrophils t t the the site site of of inflmmtion [9]. [9]. These These neutrophils lysosoml lysosoml onstituents onstituents inlue inlue teriil teriil enzymes enzymes n proteinses, n proteinses, whih, whih, upon upon extrellulr extrellulr relese, relese, use use further further tissue tissue inflmmtion inflmmtion n mge n mge [12]. [12]. Figure 4 emonstrtes the proteinse inhiition ilityof of rw n ooke lefy types. Frie levesof ofp. P. eulis. C. C. siti, siti, n n G. ltiferum G. ltiferum showe showe signifintly signifintly lower lower (p < 0.05) (p < inhiition 0.05) inhiition % when % ompre when ompre with tht with of tht their of rw, their oile, rw, oile, n steme n steme leves. leves. Rw leves Rwof leves G. ltiferum of G. ltiferum n S. grniflor S. grniflor hve shown hvehigher shownproteinse higher proteinse inhiition inhiition ility ompre ility ompre with their with ll their ooke ll ooke forms. However, forms. However, ll of the ll ooke of theleves ooke of leves C. uriult of C. uriult hve shown hve similr shown or higher similrproteinse or higher inhiition proteinse ility inhiition ompre ility ompre with tht of with its tht rw of leves. its rwfor leves. exmples, For exmples, oiling n oiling steming n steming of leves of leves of C. uriult, of C. uriult, inrese inrese the proteinse the proteinse inhiitory inhiitory tivity tivity y 54.2% yn 54.2% 56.7%, nrespetively. 56.7%, respetively. Boile leves Boile of leves O. zeylni of O. zeylni exhiite exhiite similr ility similr of ility proteinse of proteinse inhiition inhiition to its rw leves. to its rw However, leves. frie However, n steme frie nleves steme of O. leves zeylni of O. showe zeylni signifintly showe signifintly lower (p < 0.05) lower proteinse (p < 0.05) inhiition proteinse ility inhiition when ompre ility when with ompre tht of oile with tht n ofrw oile leves. n rw Aout leves. 80% Aout reution 80% in reution proteinse proteinse inhiitory inhiitory tivity ws tivity oserve ws oserve in Gymnem in Gymnem ltiferum. ltiferum. Aoring Aoring to the Tle to1, the nti-inflmmtory Tle 1, nti-inflmmtory ssoite ssoite tivities re tivities orrelte re orrelte (<0.5) with (<0.5) polyphenols, with polyphenols, rotenois, rotenois, n flvonois n flvonois of green of green lefy lefy vegetles. Therefore, the hngesin in proteinse inhiition ility with the ooking tretments mye e relteto to the hngesin in polyphenols, flvonois, n rotenois ontent in these leves. In Inthis stuy, it it ws ws foun foun tht tht oiling oiling n n steming steming proess proess might might inrese inrese or erese or erese the ntiinflmmtory nti-inflmmtory tivities. tivities. The frying The frying proess proess reues reues the nti-inflmmtory the nti-inflmmtory tivity tivity of ll lefy of ll types. lefy the The types. hnges The hnges in nti-inflmmtory in nti-inflmmtory tivities tivities of these ofstuie these stuie lefy types lefyin types the ooking the ooking proess proess my e my ue eto ue hnges to hnges in iotives, in iotives, suh s suh polyphenols, s polyphenols, flvonois, flvonois, n rotenois. n rotenois. Whtel-Glor Whtel-Glor n o-uthors n o-uthors [15] hve [15] hve reporte reporte tht tht n inrese n inrese in polyphenols, suh suh s s flvonois, fter fter susequent oiling or steming, my e e relte to to n enhne enhne vilility vilility for extrtion, for extrtion, to more to effiient more effiient relese relese of polyphenols of polyphenols or flvonois or flvonois ompouns ompouns from intrellulr from intrellulr proteins proteins n ltere n ell ltere wll strutures. ell wll strutures. However, oring However, to oring Plermoto et Plermo l. [16], the et l. more [16], intense the more the ooking intense the tretment, ooking suh tretment, s frying, suh the s frying, the greter the flvonoi egrtion. Further, High frying tempertures, in ft, oul use the oil to proue hyroperoxie free rils n elerte the egrtion of rotenois,

6 Plnts 2018, 7, 22 6 of 10 greter Plnts 2018, the7, flvonoi x FOR PEER egrtion. REVIEW Further, High frying tempertures, in ft, oul use the oil 6 of to 10 proue hyroperoxie free rils n elerte the egrtion of rotenois, s well le to reution s well le in their to iotivity reution [17]. in their Aoringly, iotivity vrition [17]. Aoringly, losses nvrition gins of phenolis in losses like n iotives gins of ue phenolis to ooking like iotives tretments ue in stuie to ooking lefy tretments types oul in stuie e ue to lefy thetypes oul of ooking, e ue the to nture the types of leves, of ooking, n the forms nture of of the leves, iotives n the tht forms re of present the iotives in the plnt tht mtries. re present in the plnt mtries. 35 % Inhiition MKC MKF MKB MKS PFC PFF PFB PFS GKC GKF GKB GKS RWC RWF RWB RWS KUC KUF KUB KUS KMC Figure 4. Proteinse inhiition ility of rw n ooke extrts of some GLV. Vlues represent mens of triplite reings. MK, O. zeylni; RW, C. uriult; KM, S. grniflor; KU, G. ltiferum; PF, P. eulis; GK, C. C. siti. C-fresh leves; leves; F-frie; F-frie; B-oile; B-oile; S-steme. S-steme. Dt re Dt presente re presente s mens s mens ± stnr ± stnr evitions evitions of three of three replite replite etermintions. Columns with with ifferent letters for eh vegetle re signifintly ifferent (p < 0.05). 3. Mterils n Methos Lefy types sujete to ifferent ooking methos 3.1. Mterils Fresh green lefy lefy vegetle vegetle smples; smples; Rnwr Rnwr (Cssi (Cssi uriult), uriult), mell (Olx mell Zeylni), (Olx Zeylni), Gotukol (Centell Gotukol siti), (Centell Ceylon siti), owceylon tree (Gymnem ow tree ltiferum), (Gymnem Kthurumurung ltiferum), Kthurumurung (Sesni grniflor), (Sesni grniflor), Pssion fruit n Pssion (Pssiflor fruit eulis) (Pssiflor were ollete eulis) were in Gmph ollete nin Kurunegl Gmph n istrits Kurunegl in Sri Lnk. istrits All of in the Sri hemils Lnk. All were of the ofhemils nlytil gre were of n nlytil were purhse gre n fromwere Sigm purhse Alrih, from St. Louis, Sigm MO, Alrih, USA through St. Louis, Anlytil MO, USA Instrument through Anlytil Pvt Lt., Instrument Colomo, Sri Pvt Lnk. Lt., Colomo, Sri Lnk Preprtion of of Cooke Smples Clene lef smples were sujete to to ifferent ooking tretments seprtely t t tmospheri pressure. Cooking onitions were selete se on on preliminry trils. trils. The lene n wshe leves were ut into smll piees, n the smples (400 g) were ivie into four prts (100 g eh), keeping one portion s s ontrol (unooke, store t t 4 C C in the refrigertor until use for within 24 h), n the the rest rest ws ws sujete sujete to to ifferent ifferent ooking ooking tretments, tretments, s inite s inite elow. elow. Briefly, Briefly, for oiling, for oiling, lef smples lef smples (100 (100 g) were g) were e e to oiling to oiling tp wter tp wter (150 ml) (150 in ml) overe in overe stinless-steel stinless-steel pot n pot were n ooke were ooke on moerte on moerte flme for flme 5 min, for n 5 min, thenn smples then smples were rine were off rine n oole off n rpily oole onrpily plenty of on ie. plenty For steming, of ie. For lef steming, smpleslef weresmples ple on were perforte ple on try perforte in stinless try steel in stemer stinless overe steel over stemer oiling overe wter over for 5oiling min, n wter thenfor 5 min, were n then rpily smples oolewere on ie. rpily For frying, oole leves on ie. were For e frying, to leves 500 ml were of white e oonut to 500 ml oil of ( Nwhite Joy, oonut Amjioil n( N Lukmngi Joy, Amji pvt Lt., n Colomo, Lukmngi Sri pvt Lnk) Lt., in Colomo, stinless Sri steel Lnk) pnin t 170 stinless C nsteel stirre pn for t three 170 C minutes n stirre until the for smple three minutes emeuntil risp-tener. the smple At the eme en of risp-tener. eh tril, the At the smples en of were eh rine tril, the off smples n e were rine with lotting off n pper e to llow with lotting for the sorption pper to llow of exeeing for the sorption oil. The ooke of exeeing lef smples oil. The were ooke homogenize lef smples n were store homogenize t 18 C. n As store t 18 C. As nti-inflmmtory tivities were lulte oring to ry mtter sis, moisture ontents of the ooke smples were etermine oring to the metho esrie y Turkmen et l. [18].

7 Plnts 2018, 7, 22 7 of 10 nti-inflmmtory tivities were lulte oring to ry mtter sis, moisture ontents of the ooke smples were etermine oring to the metho esrie y Turkmen et l. [18] Preprtion of Methnoli Extrts. Methnoli extrts of leves were prepre oring to the metho esrie y Gunthilke & Rnweer [8]. Briefly, one grm of ooke lefy vegetle smples ws weighe n mixe with 8mL of 70% methnol n vortexe t high spee for thirty minutes, n then entrifuge (Hettih, EBA 20, Hettih GmH & Co., Tuttlingen, Germny) for 10 min t 792 g. The extrts were susequently filtere through filter pper (Whtmn No. 42, Whtmn Pper Lt, Mistone, UK. The solvents tht remine in rue extrts were remove using rotry evportor (HAHNVAPOR, Moel HS-2005 V, HAHNSHIN Sientifi, Seoul, Kore) t 40 C. The prepre onentrte extrts were oven rie t 40 C for 12 h n were store t 18 C in ir-tight srew-ppe ryogeni vils until they ssye within one week. Extrts were issolve in methnol to otin onentrtion of 3 mg/ml for eh ssy Anti-Inflmmtory Properties Het-Inue Hemolysis Erythroytes suspension ws prepre y the metho esrie y Shine et l. [19], with some moifitions [20]. Briefly, loo ws otine from helthy humn volunteer n trnsferre to heprinize entrifuge tues n entrifuge t 3000 rpm for 5 min n wshe three times with equl volume of norml sline (0.9% soium hlorie). The volume of the loo ws mesure n reonstitute s 10% (v/v) suspension with isotoni uffer solution (10 mm soium phosphte uffer ph 7.4, the omposition of the uffer solution (g/l) ws NH 2 PO 4 (0.2), N 2 HPO 4 (1.15) n NCl (9.0). Het-inue hemolysis ws rrie out, s esrie y Okoli et l. [21], with some moifitions. Aout 0.05 ml of loo ell suspension n 0.05 ml extrts of ooke leves were mixe with 2.95 ml phosphte uffer (ph 7.4), n the mixture ws mixe gently n inute t 54 C for 20 min in wter th. At the en of the inution, the retion mixture ws entrifuge t 2500 rpm for 3 min n the sorne of the superntnt mesure t 540 nm using UV/VIS spetrometer (Optim, SP-3000, Tokyo, Jpn). Phosphte uffer solution without smple ws use s the ontrol. The level of hemolysis ws lulte using the following reltion Eqution (1): % inhiition of hemolysis = 100 (1 A2/A1) (1) where, A1 = Asorption of the ontrol smple, A2 = Asorption of test smple solution Effet on Protein Denturtion The test ws performe following the metho esrie y Gmhire et l. [22], with some moifitions [20]. Briefly, 0.2 ml of 1% ovine lumin, ml of phosphte uffere sline (PBS, ph 6.4), n 0.02 ml of ooke lef extrt ws mixe gently, n ws inute t 37 C for 15 min in wter th, n then the retion mixture ws hete t 70 C for 5 min. After ooling, the sorne of the solutions ws mesure t 660 nm using UV/VIS spetrometer. Phosphte uffer solution without smple ws use s the ontrol, n the perentge inhiition of protein enturtion ws lulte y using the following formul Eqution (2) % inhiition of enturtion = 100 (1 A2/A1) (2) where A1 = Asorption of the ontrol smple n A2 = Asorption of the test smple

8 Plnts 2018, 7, 22 8 of Proteinse Inhiitory Ativity The test ws performe oring to the moifie metho of Skt et l. [9], with some moifitions, s suggeste y [20]. Briefly, 0.06 mg trypsin, 1 ml of 20 mm Tris-HCl uffer (ph 7.4), 0.02 ml ooke lef extrt, n ml methnol were mixe, n the retion mixture ws inute t 37 C for 5 min, n then 1 ml of 0.8% (w/v) sein ws e. The mixture ws inute further for n itionl 20 min. Aout 2 ml of 70% perhlori i ws e to terminte the retion. Clouy suspension ws entrifuge, n the sorne of the superntnt ws re t 210 nm ginst uffer s lnk. The perentge of inhiition of proteinse tivity ws lulte. Phosphte uffer solution without smple ws use s the ontrol. The perentge inhiition of protein enturtion ws lulte y using the following Eqution (3): % inhiition of enturtion = 100 (1 A2/A1) (3) where A1 = Asorption of the ontrol smple n A2 = Asorption of the test smple Lipoxygense Inhiition Assy Lipoxygense ws ssye, oring to the metho esrie y Wu [23], with some moifitions eing mentione in Gunthilke [20]. Briefly, 1 ml soium orte uffer (0.1 M, ph 8.8) n 10 µl lipoxygense (8000 U/mL) ws inute with 10 µl ooke lef extrt in 1 ml uvette t room temperture for 5 min. The retion ws strte y the ition of linolei i sustrte (10 µl, 10 mmol). The sorne of the resulting mixture ws mesure t 234 nm, n the phosphte uffer solution without smple ws use s the ontrol, n the perentge inhiition of lipoxygense ws lulte using the following Eqution (4): % Inhiition = 100 (sorne of the ontrol sorne of the smple)/sorne of the ontrol (4) 3.5. Anlysis of Phenolis, Flvonois n Crotenois Anlysis of polyphenols ws mesure, s esrie in Gunthilke n Rupsinghe [24], flvonoi ontent using Gunthilke et l. [6], n the rotenoi ontent y using the metho tht ws esrie in Gunthilke n Rnweer [8]. However, t re not shown in this pper, n the t were use for the orreltion stuies with nti-inflmmtory t Sttistil Anlysis All t re presente s the men ± stnr evition for ll in vitro ssys one. All of the smples were nlyze in triplite, n one-wy nlysis of vrine (ANOVA) ws performe using MINITAB 15 softwre (Minit In, Stte College, PA, USA). When there were signifint ifferenes (p < 0.05), multiple men omprisons were rrie out using LSD metho. Person s orreltion oeffiients (r), with the level of signifine (P 0.05) (2-tile) for totl polyphenols, flvonois, n rotenois versus stuie nti-inflmmtory results were estimte using MINITAB 15 softwre. Polyphenols, flvonois, n rotenois ontent of the extrts tht were use for orreltion stuies re se on the sme stuy; however, t re not shown. 4. Conlusions The present stuy lerly inites tht the in vitro nti-inflmmtory ssoite iologil tivities of stuie green lefy vegetles re moifie, inrese or erese, y oiling, steming, n frying proess, epening upon the vegetle speies. Among the ooking methos, the frying of ll lefy vegetles hs reue the inhiition ilities of protein enturtion, hemolysis, proteinse, n lipoxygense tivities when ompre with other ooking methos tht were stuie. Steming signifintly inrese the protein enturtion n hemolysis inhiition in O. zeylni n P. eulis.

9 Plnts 2018, 7, 22 9 of 10 Boiling of leves inrese the inhiitory tivity of protein enturtion in C. siti n P. eulis; hemolysis in C. siti, C. uriult, n S. grniflor; lipoxygense inhiition ility in O. zeylni, P. eulis, C. siti n C. uriult leves; proteinse inhiition in C. uriult when ompre with tht of rw n their other ooke leves. The results of the stuy n e use s tse, proviing informtion on the effets of ifferent ooking methos on the helth promotion potentil of green lefy vegetles stuie. Aknowlegments: The uthors woul like to knowlege Ntionl Siene Fountion of Sri Lnk for the finnil support uner the Competitive Reserh Grnt Sheme (Projet No.: RG/AG/2014/04). Author Contriutions: K.D.P.P.G. performe ll the experiments, nlyze the t, n wrote the mnusript. All the uthors ontriute to the esigning of the experiments n proofreing the mnusript. Conflits of Interest: The uthors elre no onflit of interest. Referenes 1. Klior, A.C.; Deoussis, G.V.Z.; Shmit, H. Dietry ntioxints in preventing therogenesis A review. J. Atherosler. 2006, 187, [CrossRef] [PuMe] 2. Ferrero-Millni, L.; Nelsen, O.H.; Anerson, P.S.; Girrin, S.E. Chroni inflmmtion: Importne of NOD2 n NALP3 in interleukin-1β genertion. Clin. Exp. Immunol. 2007, 147, [CrossRef] [PuMe] 3. Cler, P.C.; Alers, R.; Antoine, J.M.; Blum, S.; Bouret-Sir, R.; Ferns, G.A.; Folkerts, G.; Friemnn, P.S.; Frost, G.S.; Gurner, F.; et l. Inflmmtory isese proesses n intertions with nutrition. Br. J. Nutr. 2009, 101, [CrossRef] [PuMe] 4. Zhng, M.; Hettirhhy, N.S.; Horx, R.; Knnn, A.; Prisooy, A.; Muhunn, A. Phytohemils, the ntioxint n ntimiroil tivity of Hiisus sriff, Centell siti, Moring oleifer n Murry koenigii leves. J. Me. Plnts Res. 2011, 5, Ooh, G. Effet of lnhing on the ntioxint properties of some tropil green lefy vegetles. LWT Foo Si. Tehnol. 2005, 38, [CrossRef] 6. Gunthilke, K.D.P.P.; Rnweer, K.K.D.S.; Rupsinghe, H.P.V. Chnge of phenolis, rotenois, n ntioxint pity following simulte gstrointestinl igestion n ilysis of selete eile green leves. Foo Chem. 2018, 245, [CrossRef] [PuMe] 7. Gunthilke, K.D.P.P.; Rnweer, K.K.D.S. Antioxitive properties of 34 green lefy vegetles. J. Funt. Foos 2016, 26, [CrossRef] 8. Anrwuln, N.; Kurnisih, D.; Apriy, R.A.; Rhmt, H.; Roto, A.V.; Bolling, B.W. Polyphenols, rotenois, n sori i in unerutilize meiinl vegetles. J. Funt. Foos 2012, 4, [CrossRef] 9. Skt, S.; Juvekr, A.R.; Gmhire, M.N. In vitro ntioxint n nti-inflmmtory tivity of methnol extrt of Oxlis orniult Linn. Int. J. Phrm. Phrm. Si. 2010, 2, Akinwunmi, K.F.; Oyepo, O.O. In vitro Anti-inflmmtory Evlution of Afrin Nutmeg (Monoor myristi) Sees. Eur. J. Me. Plnts 2015, 8, [CrossRef] 11. Govinppg, S.S.; Poojshri, M.N.; Snn, T.S.; Chnrpp, C.P. Antimiroil, ntioxint n in vitrontiinflmmtory tivity of ethnol extrt n tive phytohemil sreening of Weeli trilot (L) Hith. J. Phrm. Phytother. 2011, 3, Chou, C.T. The nti inflmmtory effet of Tripterygium wilforii Hook F on juvnt inue pw eem in rts n inflmmtory meitor relese. Phytother. Res. 1997, 11, [CrossRef] 13. Chipp, S.C.; Vnglpti, M. Antioxint, n nti-inflmmtory n nti-rthriti tivity of Centell siti extrts. J. Chem. Biol. Phys. Si. 2011, 1, Alitonou, G.A.; Avlessi, F.; Sohounhloue, D.K.; Agnniet, H.; Bessiere, J.M.; Menut, C. Investigtions on the essentil oil of Cymopogon gignteus from Benin for its potentil use s n nti-inflmmtory gent. Int. J. Aromther. 2006, 16, [CrossRef] 15. Whtel-Glor, S.; Wong, K.W.; Benzie, I.F. The effet of ooking on Brssi vegetles. Foo Chem. 2008, 110, [CrossRef] 16. Plermo, M.; Pellegrini, N.; Foglino, V. The effet of ooking on the phytohemil ontent of vegetles. J. Si. Foo Agri. 2014, 94, [CrossRef] [PuMe]

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