Anti-Inflammatory Activity of Methanol Extract and Fractions from Alchemilla kiwuensis Engl. on LPS Activated Macrophages

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1 Aville online on Interntionl Journl of Phrmognosy nd Phytohemil Reserh 217; 9(4); DOI numer: /phyto.v9i Reserh Artile ISSN: Anti-Inflmmtory Ativity of Methnol Extrt nd Frtions from Alhemill kiwuensis Engl. on Ativted Mrophges Kmthueng M O 1,2, Blyn R 2, Mouokeu R S 3, Tume C 1*, Bnerjee C 2, Singh Chwl A 2, Oumr M 4, Kuite J R 1 1 Lortory of Miroiology nd Antimiroil Sustnes, Fulty of Siene, P.O. ox 67, Dshng, Cmeroon 2 Ntionl Institute of Immunology, Arun Asf Ali Rod, New Delhi 1167, Indi 3 Institute of Fisheries nd Aquti Sienes, University of Doul, P.O. ox 7236, Doul, Cmeroon 4 Deprtment of Animl Biology, Fulty of Siene, University of Bmend, Cmeroon Reeived: 28 th Jn, 17; Revised 17 th Mrh, 17, Aepted: 1 th April, 17; Aville Online:25 th April, 217 ABSTRACT Mediinl plnts hve een used to tret different diseses over the world nd they onstitute n inexhustile soure of iotive metolites. Alhemill kiwuensis is Cmeroonin mediinl plnt used for the tretment of vrious diseses. The present study investigtes the nti-inflmmtory properties of the methnol extrt nd frtions on tivted mrophges. The extrt ws prepred y mertion of dry whole plnt powder in methnol, following y frtioning using hexne nd ethyl ette. The tivity ws evluted on RAW ell nd one mrrow differentited mrophges y mesuring their effet on nitri oxide prodution, phgoytosis, ytokine prodution nd ell surfe mrkers. The rude extrt nd its frtions signifintly (P<.5) inhiited nitri oxide prodution y tivted mrophges. As shown y RT-PCR, inos enzyme ws inhiited in the presene of extrt nd its frtions. They lso signifintly (P<.5) inhiited phgoytosis of E. oli nd prodution of TNF-α nd IL-6 y tivted mrophges. The extrts redued the expression of the o-stimultory moleule, CD8. The findings indite tht Alhemill kiwuensis hs potent nti-inflmmtory properties. Keywords: Alhemill kiwuensis; nti-inflmmtory tivity; o-stimultory moleules; nitri oxide; phgoytosis. INTRODUCTION Inflmmtion is phenomenon tht involves omplex iologil response of the ellulr mhinery ginst foreign prtiles. Inflmmtion ts s min protgonist in the pthogenesis of vrious diseses inluding rheumtoid rthritis, rterioslerosis, infetions, nd sthm 1. Plnt extrts hve gined inresed importne s nti-inflmmtory drugs nd ontriute to primry helthre in signifint world popultion. Literture studies hve onlusively suggested the nti-inflmmtory effet of severl plnt extrts in vrious model systems 2-4. Here, the methnoli extrt of whole hereous perennil plnt, Alhemill kiwuensis ws used. This plnt is used in Cmeroon for the tretment of nemi, hemorrhge, dirrhe nd dietes y lol popultions. The genus Alhemill (Rosee fmily) is reported to omprise of more thn one thousnd speies 5. Mjorly, it is distriuted in Western Eursi, ut is lso prevlent in mountin slopes of Afri. Mrophges re the typil immune ells whih re ritilly involved in inflmmtion. During ytokine surge, teril invsion, PAMP reognition or in response to ny hemil meditors, mrophges respond nd then grdully get detivted to resolve inflmmtion. Inresing evidene suggests tht lssilly tivted mrophges, lso lled M1 ells, re the one involved in the inflmmtion. M1 mrophges re tivted y inflmmtory ytokines or y miroil produts suh s lipopolyshrides 6 nd ply n essentil role in the defense of the orgnism. They reognize pthogen ssoited moleulr ptterns (PAMPs) on teri through pttern reognition reeptors (PRRs) expressed on immune ells nd trigger the prodution of inflmmtory meditors whih ssist the host in elimintion of infetious gents. However, hyper-indution of suh meditors y dysregulted innte immune ells leds to the systemi inflmmtory response syndrome (SIRS), severe tissue dmge nd septi shok 7,8. Mrophges hve rod funtions in the mintenne of tissue homeostsis through the lerne of miroorgnisms nd repir of tissues fter inflmmtion 9. Phgoytosis, y mrophges, is one of the importnt strtegy used y innte defense to eliminte the dnger from the host system 1. When tivted, mrophges exhiit greter pity for phgoytosis nd kill ingested miroes y produing retive oxygen intermedites nd retive nitrogen intermedites suh s nitri oxide (NO) through the tion of induile nitri oxide synthse (inos). NO is known to e one of the mjor effetor moleule in mrophge-medited ytotoxiity. It n omine with the superoxide nion to yield even more potent ntimiroil moleules 11.Upon stimultion y endotoxins suh s, tivted *Author for Correspondene: tumehrist@yhoo.om

2 Kmthueng et l. / Anti-inflmmtory Ativity of mrophges serete ytokines suh s TNF-α, IL-1β nd IL-6 tht indues erly inflmmtory retions to infetious gents 12. Due to their role in using inflmmtion nd phgoytosis, mrophges re lso involved in mny hroni inflmmtory diseses of immune system, rnging from grnulom formtion in tuerulosis to myordil infrtion to ner progression, ll re used y persistent tivtion mrophges. Anti-inflmmtory drugs suh s nonsteroidl nti-inflmmtory drug (NSAIDs) re urrently used for tretment of inflmmtion. These drugs re known to use severe side effets in the ody suh s hert ttks nd strokes. Mny plnt extrts hve een shown to hve nti-inflmmtory properties without using ny side effets. In the pst, the immunomodultory tivity of A. kiwuensis methnol extrt ws explored on immune system omponents, oth in-vitro in tissue ulture nd in vivo in mie 13 ; herein, nti-inflmmtory properties of this extrt nd its hexne nd ethyl ette frtions were investigted on -tivted mrophges. MATERIALS AND METHODS Preprtion of plnt extrts Alhemill kiwuensis ws olleted from the Bmoutous Mountin in the West region of Cmeroon in Novemer 214. It ws identified y otnil expert t the Ntionl Herrium in Youndé, Cmeroon, y ompring to the vouher speimen registered s 35613/HNC. The whole plnt ws ir-dried, grounded with lortory lender nd merted in methnol (5g in 3 l) for 48 hours. The rude extrt ws otined fter evportion of the solvent under redued pressure t 4 C. This extrt ws further prtitioned in hexne nd ethyl ette. The hexne frtion ws otined fter dissolution of forty grms of methnol extrt in 1.5 l hexne solvent, the mixture ws then filtered nd onentrted. The residue otined ws soked in 1.5 l ethyl ette solvent nd 2 ml wter ws dded to otin two distint phses. The upper phse ws olleted nd onentrted for ethyl ette frtion. The rude extrt, hexne nd ethyl ette frtions were dissolved in RPMI to otin onentrtions of nd nd filtered using.2 µm filter. Cell line nd ulture onditions RAW 264.7, murine mrophge ell line ws otined from the Amerin Type Culture Colletion. Cells were grown in ulture flsks in Roswell Prk Memoril Institute medium (RPMI-164) supplemented with 1 % fetl ovine serum (FBS), 1 U/ml peniillin nd 1 µg/ml streptomyin t 37 C in 5% CO 2. They were further seeded t ells/well in 24 well tissue ulture pltes or ells/well in 96 well tissue ulture pltes, stimulted with (1µg/ml) nd inuted with methnol extrt, hexne or ethyl ette frtions (128 nd ) for 24 hours. Untreted ells nd treted ells were used s negtive nd positive ontrols respetively. Animls BALB/ mie (6 to 8 weeks old, weight 25 to 3 g) were otined from Ntionl Institute of Immunology, New Delhi. Breeding nd mintenne were done t the niml fility of this institute where ll nimls were mintined in pthogen-free room. Bone mrrow ells isoltion nd ulture Mie were srified using ron dioxide (CO 2) nd pelvi nd femorl ones were disseted nd ll the remining tissue on the ones ws removed. Eh one end ws ut off nd one mrrow ells were olleted from femorl shfts y flushing with 3 ml of old sterile RPMI 164 supplemented with 1 % fetl ovine serum (FBS), 1 U/ml peniillin nd 1 µg/ml streptomyin. The ell suspension ws pssed through sieve to remove lrge lumps nd ell suspension ws wshed 2 3 times with sterile RPMI 164. Cells from one mrrow were ultured for 7 dys with 1 ng/ml M-CSF t 37 C in n inutor ontining 5% CO 2; ulture medium ws repled every three dys. At the end of dy 7, ulture superntnt ws disrded nd dherent ells were dethed using EDTA. These differentited ells (BMDM) were then seeded t ells/well in 24 well tissue ulture pltes or ells/well in 96 well tissue ulture pltes, stimulted with (1µg/ml) nd inuted with methnol extrt, hexne or ethyl ette frtions (128 nd ) for 24 hours. Untreted ells nd treted ells were used s negtive nd positive ontrols respetively. Nitri oxide quntifition Nitri oxide ws estimted from the umultion of nitrite ion (NO - 2 ) using the Griess regent. Cell superntnt ws inuted with Griess regent (1% sulphnilmide nd.1% α-nphthylmine (v/v) in 2.5% phosphori id) for 1 min in drk t room temperture. The sorne vlues were mesured t 54 nm using miroplte reder. The onentrtions of nitrites were determined y stndrd urve prepred with seril dilutions of sodium nitrite s stndrd 14. RNA isoltion, reverse trnsription nd quntittive rel time (qrt) PCR nlysis RAW mrophges (2 1 6 ells/well) were seeded in 6 well pltes nd were treted s desried ove for 24 hours. Totl RNA ws isolted from treted ells using Trizol regents nd RNA onentrtion nd purity were determined y spetrophotometry. One mirogrm of totl RNA from eh smple ws reverse trnsried using first strnd DNA synthesis kit s permnufturer s instrutions (MBI Ferments). Rel-Time PCR for quntifition ws done using SYBR green PCR Mster Mix (Applied Biosystems) ording to the mnufturer s instrutions (ABIViiA, Applied Biosystems) using gene speifi primers inos, 5 - ACATGCAGAATGAGTACCGG-3 nd 5 - TCAACATCTCCTGGTGGAAC-3 ; L7, 5 - AGCTCATCTATGAGAAGGC-3 nd 5 - AAGACGAAGGAGCTGCAGAAC-3. Amplifition nd detetion of ll genes ws performed with ABI ViiA using the following therml yling onditions: one yle of 95 C for 1 min, 4 yles of 95 C for15 s, 6 C for 1 min. Retions were performed with DNAs from three independent experiments nd the expression of eh trnsript ws quntified y the omprtive ΔΔCT method nd normlized to those of L-7 hosen s endogenous ontrol. IJPPR, Volume 9, Issue 4: April 217 Pge 474

3 BMDM RAW ells Kmthueng et l. / Anti-inflmmtory Ativity of Tle 1: Effet of A. kiwuensis methnol extrt nd its frtions on some ell surfe mrkers. Cell surfe mrkers (MFI) CD CD CD CD ells 1.25 ± ± ± ±. A ± ± ± ± 3.77 A1 1.7 ± ± ± ± 3.3 A2 1.9 ± ± ± ± 3.78 A ± ± ± ± 1.33 A ± ± ± ± 4.54 A ± ± ± ± µg/ml 1.8 ± ± ± ±.65 ells.16 ± ±.23 d 3.6 ± ±.43 A1.14 ± ±.3 d.87 ± ±.39 A1.11 ± ± ± ±.85 A2.15 ± ±.1 d 2.11 ± ±.95 A2.14 ± ±.21 d 1.47 ± ± 1.72 A3.15 ± ±.17 d 4.1 ± ±.51 A3 1,52 ±, ± ± ±.19 1µg/ml.21 ± ± ± ±.2 The ells were stimulted with 1 µg/ml nd treted with 128 nd extrt nd frtions. Cell surfe mrkers were nlysed y FACS. Results re expressed s the men ± SD (n=3). Vlues of onentrtions ering different supersript letters re signifintly different ording to Wller-Dunkn test (t P<.5) when ompred to the treted ultures. A1= Methnol extrt, A2= hexne frtion, A3= ethyl ette frtion. Tle 2: Effet of A. kiwuensis methnol extrt nd its frtions on mrophges viility. Cell viility (%) Control Methno l extrt Hexne Ethyl ette frtion frtio n ± ± RAW246.7 ells 1. ± ± BMDM 1. ± 15. ± ± 1. ± 1.5 The ells were treted with 128 nd extrt nd frtions. Cell exlusion were nlysed y FACS. Results re expressed s the men ± SD (n=3). Vlues of ell viility ering different supersript letters re signifintly different ording to Wller-Dunkn test (t P<.5) when ompred to the treted ultures. Phgoytosis ssy Phgoytosis of teri y mrophges ws determined y dding 1 fold of fluoresently leled E. oli to the ells. Overnight teril ulture ws wshed nd suspended in PBS, followed y leling with PKH dye for 2 min t room temperture. Exess PKH ws removed y wshing nd the leled ells were stored t 4 C until used. For phgoytosis, mrophges treted with plnt extrts nd 1 µg/ml for 24 h were inuted t 37 C with leled E. oli for 1 hour. Cells were hrvested nd then wshed thrie with PBS to remove non ingested teri. The mount of teri phgoytosed ws determined y mesuring the fluoresene intensity using flow ytometer (FACSVerse, BD Biosienes) nd dt were nlyzed using FlowJo softwre (Treestr, Ashlnd, OR). Quntifition of ytokines Mrophges were stimulted with 1 µg/ml in the presene of plnt extrts. Superntnts were hrvested fter 24 h nd stored t -2 C until nlysis. TNF-α, IL-1β, IL-6 nd IL-1 levels were mesured y ELISA ording to the mnufturer s instrutions (e-biosienes, Sn Diego, CA, USA). Flow ytometry nlysis for ell surfe mrkers Antiodies speifi for CD8, CD86, MHC II, CD44, CD14, Gr-1, CD11, CD11 (BD Biosienes) s diret onjugtes to FITC, PE, APC, APC-Cy7 were used. Cells were inuted with stining ntiodies on ie for 3 min. Control smples were inuted in stining uffer lone (PBS ontining 1% FCS nd.1% sodium zide). Cells were then wshed with PBS. Ded ells were exluded using Sytox dye (Invitrogen) stining. Smples were nlyzed on FACSVerse (BD Biosienes) nd dt nlyzed with FlowJo softwre (Treestr, Ashlnd, OR). Sttistil nlysis The results re expressed s men ± S.E.M. For omprison of mens etween groups, one wy ANOVA ws performed. Sttistil signifine mong different onentrtions ws nlyzed y Wller Dunn s multiple rnge test t.5 using SPSS 16. softwre. RESULTS Methnol extrt nd frtions of A. kiwuensis inhiit nitri oxide prodution of tivted mrophges The formtion of NO y mrophges in response to teril lipopolyshride is hllmrk of inflmmtion. Herein RAW ells nd BMDM were stimulted with in the presene or sene of A. Kiwuensis extrts. The nitri oxide prodution of tivted mrophges ws signifintly inhiited (p<.5) in the presene of A. kiwuensis methnol extrt nd its frtions. The inhiition rnge ws of to % t nd to IJPPR, Volume 9, Issue 4: April 217 Pge 475

4 inos enzyme Nitrites (µg/ml) Nitrites (µg/ml) Kmthueng et l. / Anti-inflmmtory Ativity of RAW ells MeOH extrt Hex frtion EtA frtion The ells were stimulted with 1 µg/ml nd treted with 128 nd extrt nd frtions. Nitrite ws quntified using Griess regent. Results re expressed s the men ± SD (n=3). Vlues of onentrtions ering different supersript letters re signifintly different ording to Wller-Dunkn test (t P<.5) when ompred to the treted ultures. MeOH extrt: Methnol extrt; Hex frtion: Hexni frtion; EtA frtion: Ethyl ette frtion. Figure 1: Inhiitory effet of A. kiwuensis methnol extrt nd its frtions on NO prodution in stimulted mrophges MeOH extrt BMDM Hex frtion EtA frtion µg/ml MeOH extrt hex frtion EtAC frtion The ells were stimulted with 1 µg/ml nd treted with 128 nd extrt nd frtions. The expression of inos gene ws nlysed y RTqPCR. MeOH extrt: Methnol extrt; Hex frtion: Hexni frtion; EtA frtion: Ethyl ette frtion. Figure 2: Inhiitory effet of A. kiwuensis methnol extrt nd its frtions on inos gene in stimulted RAW ells % t s ompred to positive ontrol, hene inditing towrds nti-inflmmtory properties of this perennil her (fig.1). No differene ws found etween rude extrt nd its frtions. Expression of induile nitri oxide synthse is down regulted y A. kiwuensis extrts Induile nitri oxide synthse is normlly not expressed in ells, ut mrophges when exposed to teril lipopolyshride responds with inresed expression of inos enzyme, leding to genertion of NO. The redution of NO levels in the presene of different extrts of A. kiwuensis led to test whether inos served s trget of this perennil her. To investigte whether A. kiwuensis methnol extrt nd its frtions ffet inos gene expression, RT-PCR ws done using speifi primers for inos. A. kiwuensis methnol extrt nd its frtions signifintly suppressed -indued mrna of inos expression of RAW mrophges when stimulted with (Fig. 2), hene using regultion t trnsriptionl level. IJPPR, Volume 9, Issue 4: April 217 Pge 476

5 P h g o y t o s i s MFI Kmthueng et l. / Anti-inflmmtory Ativity of MeOH extrt Hex frtion EtA frtion 1µg/ml A MeOH extrt B The ells were stimulted with 1µg/ml nd treted with 128 nd extrt nd frtions. Phgoytosis of treted ells ws determined y mesuring the fluoresene intensity of engulfment teri using flow ytometer. Representtive overlid histogrms show the down-regultion of phgoytosis (A). Results re expressed s the men ± SD (n=3). Vlues of onentrtions ering different supersript letters re signifintly different ording to Wller-Dunkn test (t P<.5) when ompred to the treted ultures (B). MeOH extrt: Methnol extrt; Hex frtion: Hexni frtion; EtA frtion: Ethyl ette frtion. Figure 3: Inhiitory effet of A. kiwuensis methnol extrt nd its frtions on phgoytosis in stimulted RAW ells. Hex frtion EtA frtion µg/ml 128µg/ml 512µg/ml Phgoyti ility of Rw ells re inhiited y different frtions of A. kiwuensis Phgoytosis is one of the importnt mehnisms y whih mrophges initite innte immune response. Here, the effet of A. kiwuensis extrts on the phgoyti pility of Rw ells ws investigted. It ws oserved tht extrt nd frtions of A. kiwuensis signifintly inhiited phgoyti tivity of -tivted RAW ells in onentrtion dependent mnner (fig. 3). However, there ws no differene etween rude extrt nd frtions. The levels of inflmmtory ytokines in response to re modulted y A. kiwuensis The prodution of pro-inflmmtory ytokines following stimultion in mrophges is well-estlished mehnism. The inflmmtory response of RAW ells nd BMDM ws ssessed y mesuring the levels of TNF-α, IL1-β nd IL-6 fter inution with nd vrious onentrtions of extrts. Crude extrt of A. kiwuensis nd its ethyl ette frtion inhiited TNF-α nd IL-6 ytokines prodution of RAW ells while hexne frtion did not hve ny effet on these ytokines (fig. 4A). All the study extrts signifintly inhiited -indued seretion of IL-1β (fig 4A). Crude extrt of A. kiwuensis nd its hexne frtion did not hve ny effet on TNF-α of BMDM, ut they inhiited the seretion of IL-1β nd IL-6 (fig 4B). Ethyl ette frtion of A. kiwuensis methnol extrt signifintly inhiited indued BMDM seretion of the three ytokines (TNF-α, IL-1β nd IL-6) (fig 4B). To evlute whether the downregultion of inflmmtory ytokines is triggered y n upregultion of IL-1, n nti-inflmmtory ytokine, its level ws mesured. The rude extrt nd its frtions signifintly inhiited -indued IL-1 of RAW nd BMDM (fig 4A nd B), exeption ws noted with rude extrt on BMDM where no effet ws oserved (fig 4B). Results therefore onfirme the modultory effet of A. kiwuensis on ytokine seretion y mrophges in response to stimultion. Extrts of A. kiwuensis up-regulte the expression of reeptors on the surfe of mrophges without ltering the expression of dhesion moleules Mrophges express severl surfe reeptors tht filitte the reognition of miroes ssoited moleulr ptterns, thus enling phgoytosis. The expression of two importnt reeptors; TLR4 nd CD14 ws explored. The flow ytometri nlysis reveled the stimultory effet of methnol extrt nd their frtions of A. kiwuensis on the expression of surfe mrkers of RAW IJPPR, Volume 9, Issue 4: April 217 Pge 477

6 Cytokines (ng/ml) ytokines (ng/ml) Kmthueng et l. / Anti-inflmmtory Ativity of A RAW ells 128µg/ml 512µg/ml TNF-α IL1β IL-6 IL-1 TNF-α IL1β IL-6 IL-1 TNF-α IL1β IL-6 IL-1 MeOH extrt Hex frtion EtA frtion B BMDM TNFα IL-1β IL-6 IL-1 TNFα IL-1β IL-6 IL-1 TNFα IL-1β IL-6 IL-1 MeOH extrt Hex frtion EtA frtion The ells were stimulted with 1µg/ml nd treted with 128 nd extrt nd frtions for 24 hours. Cytokines were quntified in ulture superntnts ording to the mnufturer s instrution. TNF-α, IL-1β, IL-6 nd IL-1 re expressed s the men ± SD (n=3).vlues of onentrtions ering different supersript letters re signifintly different ording to Wller-Dunkn test (t P<.5) when ompred to the treted ultures. MeOH extrt: methnol extrt; Hex frtion: Hexne frtion; EtAC frtion: Ethyl ette frtion. A: RAW se; B: BMDM se. Figure 4: Inhiitory effet of A. kiwuensis methnol extrt nd its frtions on ytokines of stimulted RAW ells ells (Fig. 5). The results therefore suggest tht these extrts ffet the signling pthwy rther thn interfering with the inding of moiety. However, it s not possile to know whether these extrts hve ny influene on other moleules involved in reognition like Binding Protein. It ws oserved tht the expression of ell dhesion moleules - CD11, CD11 nd tivtion mrker CD44 on RAW ells remin unltered in the presene of A. kiwuensis extrts (Tle 1). Though their effet on CD11 expression remins unhnged, they signifintly inhiited the expression of CD11 nd CD44 on BMDM (Tle 1). The expression of o-stimultory moleules nd Clss II MHC gets influened y the extrts of A. kiwuensis In spite of the ft tht methnol extrt nd its frtions did not showed ny effet on CD86 expression of RAW ells nd BMDM (Tle 1), they inhiited CD8 moleules on the surfe of these tivted IJPPR, Volume 9, Issue 4: April 217 Pge 478

7 CD8 MFI CD8 MFI Kmthueng et l. / Anti-inflmmtory Ativity of A RAW ells BMDM d µg/ml 1 B MeOH extrt Hex frtion EtA frtion MeOH extrt Hex frtion EtA frtion 1 µg/ml II MHC The ells were stimulted with 1µg/ml nd treted with 128 nd extrt nd frtions for 24 hours. Cell surfe mrkers were nlysed y FACS. (A) representtive overlid histogrms re shown for CD8 nd results re expressed s the men ± SD (n=3).vlues of onentrtions ering different supersript letters re signifintly different ording to Wller-Dunkn test (t P<.5) when ompred to the treted ultures. Representtive overlid histogrms re shown for MHC II (B). MeOH extrt: Methnol extrt; Hex frtion: Hexni frtion; EtA frtion: Ethyl ette frtion. Figure 5: Inhiitory effet of A. kiwuensis methnol extrt nd its frtions on CD8 nd II MHC moleules of stimulted mrophges. mrophges (fig 6). The inhiition on RAW ells ws signifint for rude extrt nd its hexne frtion t the onentrtion of with perentge of nd respetively. Ethyl ette frtion showed signifint inhiition t oth used onentrtions with perentge of 87.5 nd 66.1 t nd respetively. For BMDM, inhiition ws oserved t ll the tested onentrtions for rude extrt nd its frtions with perentges rnging from 38.6 to Clss II MHC moleule ws lso inhiited y methnol extrt nd frtions of A. kiwuensis (Fig. 6). Results ppered interesting s expression of Clss II MHC is required for ntigen presenttion for inititing dptive immune response. The extrts hve inhiitory effet on phgoytosis, nd similrly down-regultion of MHC Clss II is n outome prllel to the erlier oservtion. It ws noted following ded ell exlusion stining tht, there ws no signifint ytotoxiity due to the plnt extrt nd its frtions on RAW mrophges nd BMDM in ulture t the higher onentrtion used (512 µg/ml) (Tle 2). When mrophges were treted with extrts without stimultion, they didn t hve ny effet on the different study prmeters (dt not shown), showing tht the extrts y themselves does ffet the ell responses of resting ells ut, t under inflmmtory onditions. DISCUSSION Ativted mrophges exhiit myrid funtions inluding the up-regultion of surfe moleules suh s MHC lss II, nd the memrne moleules of the B7 fmily nd enhne ility to present ntigen nd kill intrellulr IJPPR, Volume 9, Issue 4: April 217 Pge 479

8 Kmthueng et l. / Anti-inflmmtory Ativity of pthogens 15. This killing is rehed y n inrese in the prodution of toxi oxygen speies nd n indution of the induile NO synthse (inos) gene to produe NO 16. These tivted ells lso produe pro-inflmmtory ytokines. However, hyper tivtion of mrophges n use extensive dmge to the host nd leds to sepsis nd septi shok 16,17. The present findings showed tht methnol extrt of A. kiwuensis nd its hexne nd ethyl ette frtions inhiited nitri oxide prodution in stimulted RAW ells nd BMDM. NO is moleule responsile for the nti-tumoriidl nd ntimiroil effets of tivted mrophges. Nevertheless, its exessive prodution is ssoited with ellulr nd tissue dysfuntion nd exertion of inflmmtion through the formtion of peroxynitrite. The formtion of peroxynitrite due to the retion etween NO nd superoxide is involved in the pthogenesis of mny hroni diseses 18,19. up-regultes induile nitri oxide synthse (inos) enzyme responsile of the synthesis of NO. Expression of inos in tivted mrophges is minly responsile for prodution of pthologil onentrtion of NO during inflmmtion 2. The methnoli extrt of A. kiwuensis nd its frtions deresed the level of this enzyme. Ativted mrophges serete numer of importnt ytokines suh s TNF-α, IL-1β nd IL-6. These ytokines promote inflmmtory response to Grm-negtive teri nd some other infetious miroorgnisms. The methnoli extrt of A. kiwuensis nd its frtions signifintly deresed the level of these ytokines in tivted RAW mrophges nd BMDM. These pro-inflmmtory ytokines relesed t the site of inflmmtion filitte oth the dherene of immunesystem ells to vsulr endothelil ells nd their migrtion through the vessel wll into the tissue spes 21. The result is n influx of immune system ells to the site of tissue dmge, where they prtiipte in lerne of the ntigen nd heling of the tissue 22. However, overprodution of pro-inflmmtory ytokines n indue systemi retions tht inlude fever, widespred lood lotting, nd shok 23,24. IL-1 is n nti-inflmmtory ytokine produed y mrophges; it inhiits the prodution of meditors inluding TNF-α nd IL Interestingly, this study reveled tht IL-1 indued y ws inhiited y rude extrt nd its frtions; showing tht this ytokine is not responsile for the effets of plnt extrts on pro-inflmmtory ytokine expression. Upon stimultion, mrophges express higher levels of tivtion mrkers. Clss II MHC moleules re expressed on the ell surfe memrne of the ntigen-presenting ell (APC). They re implited in the reognition of ntigens y T ells 26. Indeed, T helper ells reognize ntigen only when it is ound to lss II MHC moleule. The memrne moleules of B7 fmily (CD8 nd CD86) ind to their reeptors on T ell nd promote theirs tivtion nd prolifertion. CD8 is not onstitutively expressed on the surfe of mrophges ut it is induile y ell tivtion nd it is le to deliver o-stimultory signl tht is neessry for T helper ell tivtion. However, the level of the expression of these o-stimultory moleules my ply n importnt role during the ourse of inflmmtion 27. Thus their redution would e expeted to hve n effet on inflmmtion stte 27. In this study, the methnol extrt of A. kiwuensis nd its frtions signifintly inhiited the expression of CD8 nd lss II MHC moleules of stimulted mrophges. On the other hnd these extrts up regulted CD14 nd TLR4 expression of these ells, showing tht A. kiwuensis extrts didn t interfere with the inding of. is ound t the ell memrne y omplex of proteins tht inludes CD14, MD-2, nd TLR 7. The methnol extrt of A. kiwuensis nd its frtions signifintly inhiited phgoytosis of E. oli y tivted RAW mrophges. It is known tht inreses phgoytosis tivity of -tivted mrophges. At the re of infetion, mrophges reognize the infetious gents, whih re ingested nd killed intrellulrly. During this proess, severl free rdils use to destroy pthogens my e relesed into the extrellulr spe nd my injure host tissues leding to losely orrelted with the pthophysiology of vriety of diseses nd inflmmtion 29. In ddition to inhiit phgoytosis, methnol extrt of A. kiwuensis nd its frtions signifintly inhiited dhesion moleules. Adhesion moleules re implied in the proess of leukoyte umultion t the site of inflmmtion. As well s eing involved in host defense, leukoyteendothelil intertions n e implited in hroniity of pthologi inflmmtion, thus the inhiition of dhesion moleules must e n sset in inflmmtion ondition 3. The oserved differenes etween RAW ells nd mrophges differentited from one mrrow ells my e due to the ft tht ell lines re hrdy mostly euse they hve to pst long time in ulture, nd e ple of surviving multiple rounds of ryopreservtion nd thwing. In onlusion, the methnol extrt of A. kiwuensis nd its hexne nd ethyl ette frtions inhiited phgoytosis of E. oli, ostimultory moleules, ytokines nd nitri oxide prodution y tivted RAW mrophges. Thus, this mediinl plnt my possily e useful s nti-inflmmtory gent. ACKNOWLEDGEMENTS This work ws supported y OWSD Postgrdute Trining Fellowships for Women Sientists from Su-Shrn Afrin nd Lest Developed Countries t Centres of Exellene in the South in 215. REFERENCES 1. Krkuer T. Moleulr therpeuti trgets in inflmmtion, ylooxygense nd NFκB. Current Drug Trgets 24; 3: Lee HJ, Hyun EA, Yoon WJ, Kim BH, Rhee MH, Kng HK, Cho JY, Yoo ES. In vitro nti-inflmmtory nd nti-oxidtive effets of Cinnmomum mphor extrts. Journl of Ethnophrmology 25; 13(2): Leelprksh G, Dss SM. In vitro nti-inflmmtory tivity of methnol extrt of Eniostemm xillre. IJPPR, Volume 9, Issue 4: April 217 Pge 48

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