Sineewan Phitaktim 1, Mullika Chomnawang 2, Kittipot Sirichaiwetchakoon 1, Benjawan Dunkhunthod 1, Glyn Hobbs 3 and Griangsak Eumkeb 1*

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1 Phitktim et l. BMC Miroiology (2016) 16:195 DOI /s RESEARCH ARTICLE Open Aess Synergism n the mehnism of tion of the omintion of α-mngostin isolte from Grini mngostn L. n oxillin ginst n oxillin-resistnt Stphyloous sprophytius Sineewn Phitktim 1, Mullik Chomnwng 2, Kittipot Sirihiwethkoon 1, Benjwn Dunkhuntho 1, Glyn Hos 3 n Gringsk Eumke 1* Astrt Bkgroun: Glolly, stphylooi hve evelope resistne to mny ntiiotis. New pprohes to hemotherpy re neee n one suh pproh oul e to use plnt erive tives with onventionl ntiiotis in synergesti wy. The purpose of this stuy ws to isolte α-mngostin from the mngosteen (Grini mngostn L.; GML) n investigte ntiteril tivity n mehnisms of tion whenusesinglynwhenomine with oxillin ginst oxillin-resistnt Stphyloous sprophytius (ORSS) strins. The isolte α-mngostin ws onfirme y HPLC hromtogrm n NMR spetrosopy. The minimum inhiitory onentrtion (MIC), hekeror n killing urve were etermine. The moes of tion of these ompouns were lso investigte y enzyme ssy, trnsmission eletron mirosopy (TEM), onfol mirosopi imges, n ytoplsmi memrne (CM) permeiliztion stuies. Results: The MICs of isolte α-mngostin n oxillin ginst these strins were 8 n 128 μg/ml, respetively. Chekeror ssys showe the synergisti tivity of isolte α-mngostin (2 μg/ml) plus oxillin (16 μg/ml) t frtionl inhiitory onentrtion inex (FICI) of The kill urve ssy onfirme tht the viility of oxillin-resistnt Stphyloous sprophytius DMST (ORSS-27055) ws rmtilly reue fter exposure to isolte α-mngostin (2 μg/ml) plus oxillin (16 μg/ml). Enzyme ssys emonstrte tht isolte α-mngostin h n inhiitory tivity ginst β-ltmse in ose-epenent mnner. TEM results lerly showe tht these ORSS ells trete with this omintion use peptioglyn n ytoplsmi memrne mge, irregulr ell shpes n verge ell res were signifintly lrger thn the ontrol. Clerly, onfol mirosopi imges onfirme tht this omintion use onsierle peptioglyn mge n DNA lekge. In ition, the CM permeility of ORSS ws lso inrese y this omintion of tives. (Continue on next pge) * Corresponene: gring@sut..th 1 Shool of Phrmology, Institute of Siene, Surnree University of Tehnology, Nkhon Rthsim 30000, Thiln Full list of uthor informtion is ville t the en of the rtile 2016 The Author(s). Open Aess This rtile is istriute uner the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me. The Cretive Commons Puli Domin Deition wiver ( pplies to the t me ville in this rtile, unless otherwise stte.

2 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 2 of 14 (Continue from previous pge) Conlusions: These finings provie eviene tht isolte α-mngostin lone hs not only some tivity ut lso shows the synergisti tivity with oxillin ginst ORSS The hromone n isoprenyl strutures oul ply signifint role in its tion. This synergisti tivity my involve three mehnisms of tion. Firstly, potentil effets of ytoplsmi memrne isruption n inreses permeility. Seonly, inhiit β-ltmse tivity. Finlly, lso mge to the peptioglyn struture. We proposes the potentil to evelop novel junt phytophrmeutil to oxillin for the tretment of ORSS. Future stuies require linil trils to estlish if the synergy reporte n e trnslte to nimls n humns. Keywors: α-mngostin, Grini mngostn, Oxillin, Oxillin-resistnt S. sprophytius, Synergisti tivity, Mehnism of tion Arevitions: CAMHB, Ction-juste Meuller-Hinton roth; CM, Cytoplsmi memrne; FIC, Frtion inhiitory onentrtion; FICI, Frtion inhiitory onentrtion inex; GML, Grini mngostn L.; MHA, Meuller-Hinton gr; MHB, Meuller-Hinton roth; MIC, Minimum inhiitory onentrtion; ONPG, O-nitrophenol-β-D-gltosie; ORSS, Oxillin-resistnt Stphyloous sprophytius; ORSS-20755, Oxillinresistnt Stphyloous sprophytius DMST 20755; TEM, Trnsmission eletron mirosopy. Bkgroun Antiioti resistne in stphylooi hs een glolly oumente [1]. The resistne of these strins to or spetrum β-ltm ntiiotis, suh s methiillin, oxillin, n fluloxillin, hve emerge rpily only few yers fter the introution of the first rug in this lss n there hs een stey risen in the iniene of methiillin-resistnt S. ureus (MRSA) in linil isoltes [1, 2]. A previous stuy of 12 hospitls in Virgini, USA foun tht overll 53 % of S. ureus isoltes were resistnt to oxillin [3]. In ition, to pthogeni S. ureus, urrentlys. sprophytius, ogulse- negtive stphyloous tht frequently uses ommunity-ssoite unomplite urinry trt infetion (UTI) in young n mile-ge women, hs eome resistnt to β-ltm ntiiotis, suh s methiillin y quisition of stphylool ssette hromosome me (SCCme) element [4, 5]. These prolems present n urgent nee to serh for new ntiiotis n novel pprohes to treting these teril infetions. Plnt-erive ntimiroils re potentil soure of novel therpeutis euse plnts re known to proue vrious ntimiroil moleules to protet themselves from plnt or environmentl pthogens [6]. Furthermore, rug omintion strtegies, in prtiulr, phytohemil n ntiioti omintion pprohs hve een reommene in severl stuies to omt multiple rug-resistnt teri [7 9]. Mngosteen (the queen of fruit), elonging to the fmily Guttifere, is tropil evergreen tree tht is wiely ultivte throughout Ini, Mynmr, Mlysi, Philippines, Sri Lnk, n Thiln [10]. The perirp (peel, rin, n hull) or the ripe fruit of GML hs een tritionlly use for the tretment of irrhe, inflmmtion, ominl pin, ysentery, woun infetion, suppurtion n hroni ulers [11]. The α-mngostin, xnthone erivtive, hs een foun to possess severl enefiil iologil tivities, suh s ompetitive ntgonist of the histmine H 1 reeptor n wek ntioxint properties [12], ntiteril tivity ginst Helioter pylori, ntiinflmmtory tivities, inhiition of oxitive mge y humn low-ensity lipoproteins (LDL), ntimiroil tivity ginst methiillin-resistnt Stphyloous ureus [13]. However, the reent stuies hve not reporte on the ntiteril tivity of α-mngostin n synergism with oxillin ginst oxillin-resistnt S. sprophytius. To this en, the present stuy ws instigte to eluite ntiteril n synergisti tivity of α-mngostin isolte from the GML perirp n oxillin either lone or in omintion ginst this strin. The ntiteril tions n ell line toxiity of these ompouns were lso investigte. Methos Plnt mterils, β-ltm ntiiotis, teril strins, n ell line The rie fruit hulls of mngosteen were purhse lolly in Nkhon-Rthsim, Thiln. The smples were ientifie y Dr. Pul J. Grote, Surneree University of Tehnology. The vouher speimens (SGM0804U) were eposite in the Shool of Phrmology, Institute of Siene, Surnree University of Tehnology, Nkhon-Rthsim, Thiln. The mture fruit ws lene. The fruit rins were ut into smll piees, rieinhotovent50 Cfor72hngrouninto power, psse through sieve (20 mesh). The powere smple ws kept in n irtight ontiner protete from light until use. All linil isoltes of S. sprophytius were otine from the Deprtment of Meil Siene, Ntionl Institute of Helth, Ministry of Puli Helth, Bngkok, Thiln. The suseptile strin S. ureus ATCC 29213, referene strin, ws otine from the Amerin Type Culture

3 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 3 of 14 Colletion (ATCC). Oxillin, Nisin, o-nitrophenol-β- D-gltosie (ONPG), α-mngostin stnr, n β- ltmse type IV isolte from E. loe were otine from Sigm-Alrih, UK. Meuller-Hinton roth (MHB) n Mueller-Hinton gr (MHA) were purhse from Oxoi (Bsingstoke, UK). The 3T3-L1 mouse emryoni firolsts n ovine lf serum (CBS) were purhse from the Amerin Type Culture Colletion (ATCC, USA). 3-(4,5-Dimethylthizol-2-yl)- 2,5-iphenyltetrzolium romie (MTT), Duleo s Moifie Egle s meium (DMEM), Fetl ovine serum (FBS), Peniillin-Streptomyin, N-2-hyroxyethylpiperzine-N-2- ethne sulfoni i (HEPES) were otine from Gio Invitrogen (Grn Isln, NY, USA). Isoltion n purifition of α-mngostin α-mngostin from the perirp of GML ws isolte n purifie oring to previous methos with some moifitions [12, 14]. Briefly, 1 kg of rie perirp power of GML ws extrte suessively with n-hexne, ihloromethne (CH 2 Cl 2 ), ethnol, n etone using Soxhlet extrtor. The filtere extrts were then onentrte using rottory evportor to provie yellowish power for n-hexne (84 g) n ihloromethne (106 g), rown stiky semi-soli for ethnol (262 g) n etone (130 g) respetively. The CH 2 Cl 2 rue extrt ws further seprte y sili gel olumn hromtogrphy to yiel 11 frtions. These frtions were sujete to HPLC (C18 olumn, moile phse of methnol-wter (85:15) n flow rte of 1.0 ml/min, photoioe rry etetor) n purifie using preprtive thin lyer hromtogrphy to otin isolte α-mngostin. The α-mngostin ws nlyze y HPLC n its hemil struture ws eluite y 1 HNMRn 13 C NMR. The spetrum struture t of this ompoun ws ompre with those previously reporte [15]. Stnrise teril suspensions To selet teril suspensions with known vile ount, the metho of Liu et l. [16] ws followe with little moifitions. MHA n Ction-juste Mueller- Hinton roth (CAMHB) were use s meium. Minimum inhiitory onentrtion (MIC) n hekeror etermintions The ntiteril tivity n rug intertion of isolte α-mngostin from the perirp of GML with oxillin were performe y MIC n hekeror ssys, respetively using roth mroilution proeure. These ssys were onute following the methos of Clinil n Lortory Stnr Institute guielines [16, 17]. In summry, 0.25 ml of fu/ml teril suspensions ws e to series of 2.25 ml CAMHB plus 1 in 10 seril ilutions of the α-mngostin plus oxillin omintions to give 5x10 5 fu/ml. Tues of the roth without ntimiroilsl were use s the ontrol. The ultures were inute for 24 h t 37 C. The tests were rrie out in triplite. MICs were etermine for eh ntiteril omintion n the isoologrms were plotte. The intertion etween the two gents ws lulte y the frtionl inhiitory onentrtion (FIC) inex of the omintion. The FIC of eh gent ws lulte y the omplete growth inhiition of miroorgnism in the omintion tue. The following formul ws use for FIC inex lultion: FIC of α- mngostin = MIC α-mngostin in the omintion/mic of α-mngostin lone; FIC of oxillin = MIC of oxillin in the omintion/mic of oxillin lone; therefore, FIC inex = FIC of α-mngostin + FIC of oxillin. When the FIC inex of the omintion is equl to or less thn 0.5, the omintion is efine s synergisti; when the FIC inex flls etween 0.5 n 4.0, it inites no intertion etween the gents n vlue ove four is onsiere to show ntgonism etween the two ompouns [18]. S. ureus ATCC ws use s positive ontrol. The MICs n FIC inex is presente s the mein vlues otine in uplites from three inepenent experiments. Kill urve etermintions (Vile ounts) The experiment ws rrie out to onfirm ntiteril n synergisti tivities of isolte α-mngostin from the perirp of GML when use singly n in omintion with oxillin s previously esrie y Mun et l. n Rihrs et l. [19, 20]. Compouns were use t the hlf miniml inhiitory onentrtion (1/2-MICs) when eh ompoun ws ssesse lone. However, to stuy the effet of the ompouns in omintion, eh ompoun ws use t the MIC tht yiele synergism. Enzyme ssys The ility of isolte α-mngostin from the perirp of GML to inhiit the tivity of β-ltmse type IV isolte from E. loe ws etermine in orne with the methos of Eumke et l. n Rihrs et l. [21, 22]. Conisely, enzylpeniillin, sustrte for β-ltmse type IV, ws juste to onentrtions suffiient to hyrolyze % sustrte within 5 min, β-ltmse t 100 μg/ml ws use. The α-mngostin t 1, 2, 4 n 8 μg/ml were preinute with the enzyme in 50 mm soium phosphte uffer (ph7.0)t37 Cfor5mineforeingsustrte.A time - ourse ssy ws performe t 0, 5, 10, 15 n 20 min using methnol/eti i (100:1) s stopping gent. Aliquots (10 μl) of eh smple were injete onto reverse-phse HPLC (Asentis C18 olumn) to nlyse the remining enzylpeniillin. The moile phse onsiste of 10 mm mmonium ette (ph 4.5 eti i): etonitrile

4 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 4 of 14 (75:25) with flow rte of 1 ml/min, UV etetion of peks ws t 200 nm,, n the olumn mintine t 35 C. The quntity of remining enzylpeniillin ws lulte y ompring the re uner the hromtogrphi urve. Trnsmission eletron mirosopy (TEM) To etermine the ultrstruture morphology of teri fter tretment with isolte α-mngostin from the perirp of GML either lone or in omintion with oxillin, the metho of Rihrs et l. [22] ws followe. To investigte the mehnism of tion of these gents, the hlf-mics of oth ompouns use lone n Su-FICs of the omintion, were hosen for exmintion. To onfirm the effets of these gents either use singly n in omintion on ell size, the ell re from mirogrphs were nlyze y mesuring ell with multiplie y ell length (nm 2 ). The experiment ws performe in triplite, n the ell res re isplye s men ± SEM [23]. Immunofluoresene stining n onfol mirosopy The isruption of peptioglyn fter exposure to α- mngostin either use singly or in onjuntion with oxillin ws performe using immunofluoresene n visulize uner onfol lser snning mirosope following the metho of Teethisong et l. [24]. Shortly, fter the FIC inex ws otine from hekeror, the hlf-mics vlue of isolte α-mngostin or oxillin lone n the 3/4 FIC of this omintion tht showe synergisti FIC inex ws hosen for exmintion. The ells grown without ny ntiteril gent were employe s ontrol [25]. Cytoplsmi memrne (CM) permeiliztion ssys Two methos were use to ssess CM permeiliztion. Firstly; the CM permeiliztion experiment ws performe, with some moifitions, to onfirm results s previously esrie y Shen et l. n Zhou et l. [26, 27]. Shortly fter the FIC inex ws etermine y the hekeror ssy, the hlf-mic vlues for isolte α-mngostin or oxillin lone, n the 3/4 MIC vlues for this omintion tht inite synergisti FIC inex were selete ginst ORSS to mesure CM permeility. This metho ws performe y mesuring the relese of UV-soring mteril (Vrin s Cry 100 UV-Vis spetrophotometer, Vrin, In., Cliforni, USA) [21]. Seonly, the α-mngostin-inue permeiliztion of the CM of ORSS ws etermine essentilly s reently esrie [28]. In rief, to ssy CM permeiliztion, the wells ontine 50 μl ONPG plus either hlf-mic vlues for isolte α-mngostin or oxillin lone n the 3/4 MIC vlues for this omintion tht inite synergisti FIC inex were prepre shortly efore the experiment. Finlly, 50 μl of ell suspension (OD 0.3) ws e to the Tle 1 The 300 MHz 1 H NMR (etone-6) spetrl t of α-mngostin Chemil Shift (δ, ppm) Assignment Chemil Shift (δ, ppm) from referene [15] singlet, OH singlet, H singlet, H triplet, H-12, H oulet, H singlet, 7-OMe oulet, H singlet, H singlet, H singlet, H singlet, H Tle 2 The 300 MHz 13 C NMR (etone-6), spetrl t of α-mngostin Chemil Shift (δ, ppm) Assignment Chemil Shift (δ, ppm) from referene [15] C C C C C C C C C C C C C C C C OCH C C C C C C

5 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 5 of 14 wells to give finl onentrtion of 100 μg/ml ONPG. After wrming to 37 C the pltes were positione in the plte reer t 37 C. ONPG uptke n levge y β- gltosise within the ytoplsm ws hrterize y monitoring sorption over perio of 120 min t 420 nm. Complete permeiliztion ws inue in the presene of 0.5 μg/ml Nisin s positive ontrol n wells lking rugs or isolte α-mngostin test serve s negtive ontrol [29, 30]. In vitro ytotoxiity test (MTT ssys) The 3T3-L1 preipoytes were ulture in Duleo s moifie Egle s meium (DMEM) with high gluose, supplemente with 10 % CBS, 1.5 mg/ml soium ironte, 100 U/ml peniillin n 100 μg/ml streptomyin until onfluent. The ells were mintine t 37 C in 5%CO 2 n 95 % humiity. The ytotoxi effet of α- mngostin, oxillin, either lone or in omintion on ell prolifertion ws etermine using tetrzolium ye (MTT) in olorimetri ssy [31]. Briefly, the ells were seee in 96-well plte t ensity of ells/well. The ells were llowe to here for 48 h n then were trete with vrious onentrtions of three ompouns for 24 h. After inution, the ulture meium ws remove, n 0.5 mg/ml of MTT ws e. Then, ells were further inute for 4 h t 37 C. Formzn rystls forme y vile ells were issolve in DMSO n sorne ws mesure t 540 nm with miroplte spetrophotometer (Benhmrk Plus, Bio-R, Jpn). Sttistil nlysiss The experiments were rrie out in triplite; t were expresse s men ± stnr error of the men (SEM). Signifint ifferenes in the enzyme ssy mong eh of trete groups t the sme time, the ell re of eh trete group, CM permeiliztion, n MTT ssys were nlyse y one-wy ANOVA followe y Sheffe s postho test. The p < 0.01 ws onsiere s the sttistilly signifint ifferene. Results n isussion Isoltion, purifition n ientifition of α-mngostin The isolte α-mngostin, the perent yiel of α- mngostin t % (w/w) of rie power, from the perirp of GML, ws otine n the hemil struture of α-mngostin from 1 HNMRn 13 CNMR (Tles 1 n 2) ompre to the referene s illustrte in Fig. 1. Also, the results from HPLC hromtogrms of this isolte α-mngostin exhiite mjor pek of isolte α-mngostin from the perirp of GML (Fig. 2) whih is prtilly the sme s mjor pek of α-mngostin stnr (Fig. 2). The purity (HPLC) of isolte α-mngostin ws 98.0 %. Fig. 1 Struture of α-mngostin MIC n hekeror etermintions The MIC results for isolte α-mngostin, issolve in 1 % DMSO, from the perirp of GML, oxillin, n nisin ginst ll teste S. sprophytius strins re presente in Tle 3. The results inite tht the MICs for isolte α-mngostin, oxillin, n nisin ginst ll these strins were 8, 128, n 256 μg/ml, respetively. These results inite tht these strins were resistnt to oxillin. Although, the positive ontrol, Stphyloous ureus ATCC 29213, ws suseptile to oxillin t MIC 0.5 μg/ml [32]. The isolte α-mngostin exhiite some inhiitory effet ginst these strins. These results re in greement with the stuies of Chomnwng et l. n Iinum et l. who reporte ntiteril tivity of iotive ompouns from the perirp of GML extrts ginst MRSA, Stphyloous epiermiis n Propioniterium strins [13, 33]. Furthermore, previous finings foun tht the Grini mngostn extrt exhiite MIC vlues of mg/ml ginst oth Propioniterium nes n Stphyloous epiermiis [34]. The FIC inies for isolte α-mngostin plus oxillin ginst ll teste S. sprophytius strins were These results inite tht these omintions emonstrte synergisti tivity ginst these strins [7, 18]. These finings suggest tht isolte α-mngostin from the perirp of GML extrt not only hs some ntiteril tivity of their own ginst these strins ut lso hve the ility to reverse the resistne of suh teril strins y synergy with oxillin. Kill urve ssys The results for the seprte n omine effets of isolte α-mngostin from the perirp of GML n oxillin on vile ounts of ORSS re presente in Fig. 3. The ontrol showe no reution in the ounts of fu from ontrol inoulum. The vile ounts for the ells trete with isolte α-mngostin t 4 μg/ml were rther lower thn tht of oxillin t 64 μg/ml (etween 1 n 24 h). Clerly, the omintion of 2 μg/ml

6 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 6 of 14 Fig. 2 HPLC hromtogrms of α-mngostin:, Alph-mngostin (Ext.) = α-mngostin extrt from G. mngostn;, Alph-mngostin (St.) = α-mngostin stnr isolte α-mngostin n 16 μg/ml oxillin gretly erese the ell ount to fu/ml fter 4 h to 24 h. These results onfirme the hekeror ssy results, whih inite synergisti tivity tht the omintion proue erese of 2log10fu/ml, ompre with oxillin tretment lone [35]. These results re onsistent with those of Eumke et l. tht glngin plus moxiillin exhiite synergisti tivity ginst peniillin-resistnt S. ureus strins t n FIC inex of 0.05 [9]. Aprt from this, previous finings reporte tht synergisti effet using flvonois n oxillin ginst vnomyin-intermeite S. ureus Tle 3 Minimum inhiitory onentrtion (MIC) of oxillin, α-mngostin, n nisin lone n in omintion Strins MIC (μg/ml) FIC (μg/ml) FIC inex OXA AMT NIS OXA + AMT OXA + AMT S. sprophytius DMST R SI S. sprophytius DMST R SI S. sprophytius DMST R SI S. sprophytius DMST R SI S. sprophytius DMST R SI S. sprophytius DMST R SI S. ureus ATCC P 0.5 S 4 1 N/D N/D S. ureus ATCC P, ws use s positive ontrol S Suseptile, R Resistnt, SI Synergisti intertion, N/D Not etermine OXA Oxillin, AMT α-mngostin, NIS Nisin The MICs re presente s the mein vlues mesure from three inepenent experiments; eh experiment ws performe in triplite

7 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 7 of Vile ount (fu/ml) CON OXA(64) AMT(4) OXA(16) + AMT(2) Time (h) Fig. 3 The effet of oxillin, α-mngostin lone or in omintion on the vile ounts of ORSS CON = ontrol (rugs free); OXA(64) = 64 μg/ml oxillin; AMT(4) = 4 μg/ml α-mngostin; OXA(16) + AMT(2) = 16 μg/ml oxillin plus 2 μg/ml α-mngostin. The vlues plotte re the mens of 4 oservtions, n the vertil rs inite the stnr errors of the mens showe 87.5 % synergism with FIC inies etween [36]. Enzyme ssys Theenzymessyresultsfortheisolteα-mngostin tretment revele tht the levels of enzylpeniillin were signifintly higher ompre with the ontrols (p < 0.01). The levels of enzylpeniillin epene on the isolte α-mngostin onentrtion in oseepenent mnner (p < 0.01) (Fig. 4). Previous finings foun tht 82 % of Stphyloous sprophytius strins proue β-ltmse [37]. Furthermore, Hirno n Byer emonstrte tht in vivo effiy of mpiillin plus sultm oul inhiit oxillinresistnt Stphyloous ureus, whih ommonly proue β-ltmse [38]. In ition, previous finings Benzylpeniillin (ug/ml) 100 CON AMT(1) AMT(2) AMT(4) 80 AMT(8) e Time (min) Fig. 4 The inhiitory tivity of α-mngostin ginst β-ltmse in hyrolysing enzylpeniillin. β-ltmse use from E. loe; CON = ontrol (no testing gent); AMT(1) = 1 μg/ml α-mngostin. The grph shows the remining enzylpeniillin t the sme time. Mens shring the sme supersript re not signifintly ifferent from eh other (Sheffe s test, p < 0.01) e e

8 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 8 of 14 reporte tht α-mngostin ws foun to e tive ginst memrne enzymes of S. mutns UA159 [39]. Furthermore, These results re in greement with those of Denny et l. n Zho et l. tht glngin n epigllotehin gllte, oth shring enzene fuse rings onense with pyrn rings (Chromone or 1,4- enzopyrone) similr to α-mngostin, inhiite metllo-β-ltmse n peniillinse, respetively [40, 41]. These finings suggest tht the isolte α- mngostin from the perirp of GML extrt oul e le to inhiit β-ltmse tivity. These inhiitory results my e s onsequene of the isolte α- mngostin form the omplex with β-ltmse type IV resulting in etivtion of the β-ltmse tivity [9]. Also, the inrese of enzylpeniillin ws oserve in very short time intervl (in minutes; Fig. 9). This result orreltes well with the rpi killing of memrne-trgeting ntimiroil. Thus, reution of the β-ltmse tivity oul e seonry effet fter the memrne is isrupte [42]. TEM The trnsmission eletron mirogrphs of ells from the log phse of growth of ORSS in the presene of isolte α-mngostin, oxillin either lone n in omintion re presente in Fig. 5. The untrete ells ppere norml, peptioglyn, n ytoplsmi memrnes were lerly seen intt. An the morphology of the ells looke norml (Fig. 5). The ORSS ells trete with oxillin re isplye in Fig. 5. This result showe some isruption to oth peptioglyn n ytoplsmi memrne. The verge rosssetionl ell res of these ells were slightly smller thn the ontrols, ut there ws no signifint ifferene (p > 0.01) (Fig. 6). Although, the mirogrph of these ells fter exposure to isolte α-mngostin lone is shown in Fig. 5. Cler mge to peptioglyn n the ytoplsmi memrne ws evient. The verge ell res of these ells were signifintly igger thn those of ontrols (p < 0.01) (Fig. 6). Fig. 5 revels the isolte α-mngostin plus oxillin-trete ells. These Fig. 5 Ultrthin setions of log phse oxillin-resistnt S. sprophytius DMST grown in CAMHB ontining:, ontrol (rug-free);, oxillin t 64 μg/ml;, α-mngostin t 4 μg/ml;, oxillin t 12 μg/ml plus α-mngostin t 1.5 μg/ml; (Mgnifition;, 4000, r = 1 μm;, 6000, r = 0.5 μm;, 10,000, r = 0.5 μm;, 10,000, r = 0.3 μm; Inset mgnifition;,, 38,000 ;, 29,000 ;, 10,000 ; r;,,, 200 nm;, 500 nm)

9 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 9 of 14 previous finings where the omintion of eftziime plus glngin le to mge of the ell ultrstrutures, the integrity of ell wlls n inrese in ell size of eftziime-resistnt S. ureus [9]. Furthermore, this urrent stuy shows similrity to tht of the work of Koh et l. who reporte tht α-mngostin use signifint morphologil effets to S. ureus (MRSA) inluing wll mge n ell lysis [42]. Fig. 6 The effet of either oxillin or α-mngostin on verge ross-setion of ORSS ell res. CON = ontrol (rugs free); OXA(64) = 64 μg/ml oxillin; AMT(4) = 4 μg/ml α-mngostin; OXA(12) + AMT(1.5) = 12 μg/ml oxillin plus 1.5 μg/ml α-mngostin. The men ± SEM for three replites re illustrte. Mens shring the sme supersript re not signifintly ifferent from eh other (Sheffe s test, p < 0.01) ells isplye the gretest mge to the peptioglyn n ytoplsmi memrne resulting in lekge of intrellulr mterils n overll morphologil hnges. Clerly, these verge ell res were signifintly igger thn the ontrol (p < 0.01) (Fig. 6). These results provie eviene tht isolte α-mngostin shows stronger tivity thn oxillin ginst this strin t these onentrtions. These finings gree with Immunofluoresene stining n onfol mirosopy The peptioglyn n DNA-lelle ORSS lerly showe intt ous-shpe n no mge ws oserve in untrete ontrol ells y onfol lser snning imges (Fig. 7). The ells trete with isolte α-mngostin or oxillin lone isplye minor peptioglyn mge n DNA lekge. The omintion of these gents use onsierle peptioglyn mge n DNA lekge ompre to ontrols. The merger of peptioglyn n DNA imges re lso shown. These results re in sustntil greement with the TEM stuy n support preliminry mehnism of tion of this omintion eing trgete t the peptioglyn struture. CM permeiliztion The CM permeility ws mesure y exmining the relese of UV-soring mterils t 260 nm (Fig. 8). After tretment, ORSS ells with isolte α-mngostin, nisin, n the isolte α-mngostin plus oxillin omintion oul inue the relese of 260 nm soring Fig. 7 Shemti representtion of the results of immunofluoresene n onfol lser snning mirosope; Smples of oxillin-resistnt S. sprophytius DMST fter tretment for 4 h with oxillin, α-mngostin either lone or in omintion. CON = ontrol (rugs free); OXA(64) = 64 μg/ml oxillin; AMT(4) = 4 μg/ml α-mngostin; OXA(12) + AMT(1.5) = 12 μg/ml oxillin plus 1.5 μg/ml α-mngostin. The ells were stine for DNA with DAPI (lue, DNA) n lelle for peptioglyn (green, PGC) using respetive ntioies. DNA in ll groups ws lolize in the entrl of the ell n surroune y peptioglyn lyer (MERGED). Sle r = 1 μm

10 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 10 of 14 OD Time (h) CON OXA(64) AMT(4) OXA(12)+AMT(1.5) NIS(0.5) Fig. 8 Effets of 260 nm soring mteril (DNA, RNA, n metolites) in the ORSS superntnts. These ells were trete with oxillin, α-mngostin either lone or in omintion. CON = ontrol (rugs free); OXA(64) = 64 μg/ml oxillin; AMT(4) = 4 μg/ml α-mngostin; OXA(12) + AMT(1.5) = 12 μg/ml oxillin plus 1.5 μg/ml α-mngostin; NIS(0.5) = 0.50 μg/ml nisin. Nisin t 0.50 μg/ml ws use s positive ontrol, n untrete ells were use s negtive ontrol. The men ± SEM for three replites re illustrte. Mens shring the sme supersript t the sme time re not signifintly ifferent (Sheffe s test, p < 0.01) 0.5 CON OXA(64) AMT(4) NIS(0.5) 0.4 OXA(12)+AMT(1.5) 0.3 OD 420nm Time (min) Fig. 9 Time-epeneny of oxillin plus α-mngostin-inue permeilistion of the ytoplsmi memrne of ORSS The kinetis of α-mngostin-meite ONPG pssge ross the ytoplsmi memrne re monitore s hnge in the optil ensity (OD). The onentrtion of sustrtes ws 100 μg/ml ONPG. CON = ontrol (rugs free); OXA(64) = 64 μg/ml oxillin; AMT(4) = 4 μg/ml α-mngostin; OXA(12) + AMT(1.5) = 12 μg/ml oxillin plus 1.5 μg/ml α-mngostin; NIS(0.5) = 0.50 μg/ml nisin. Nisin t 0.50 μg/ml ws use s positive ontrol, n untrete ells were use s negtive ontrol. Eh tretment performe two times in triplite. The grph shows OD 420nm of eh tretment t the sme time. Mens shring the sme supersript t the sme time re not signifintly ifferent from eh other (Sheffe stest,p < 0.01)

11 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 11 of 14 mterils t signifintly higher levels ompre with the ontrol or oxillin lone (p < 0.01). The CM permeilising ility ws rnke s follows isolte α-mngostin plus oxillin > nisin > isolte α-mngostin > oxillin > ontrol (p < 0.01). These results suggeste tht the synergisti tivity of isolte α-mngostin plus oxillin resulte in inrese ytoplsmi memrne permeility of DNA, RNA, n ellulr metolites [26, 27]. The α-mngostin-inue CM permeilistion of the ORSS y ONPG uptke results re shown in Fig. 9. The ORSS ytoplsmi memrne ws permeilise muh more rpily y the isolte α-mngostin plus oxillin ompre to other groups. Nisin, whih is highly tive ginst the outer memrne, showe CM permeility signifintly lower thn isolte α- mngostin n oxillin omintion (p < 0.01). These Fig. 10 The effet of oxillin, α-mngostin either lone or in omintion on 3T3-L1 preipoytes. 3T3-L1 preipoytes were trete with:. oxillin ( μg/ml);. α-mngostin (0-128 μg/ml); n. oxillin plus α-mngostin (8 + 1 to μg/ml), for 24 h. Results re expresse s perentges of ell viility s ompre with untrete ontrols (n = 8). The men ± SEM for three replites re illustrte. Mens shring the sme supersript re not signifintly ifferent from eh other (Sheffe s test, p < 0.01)

12 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 12 of 14 results re onsistent with the results of CM permeilistion with the UV-soring mteril t 260 nm (Fig. 8). In the sme wy, previous stuies foun tht glngin, whih shres the hromone (or 1,4-enzopyrone) struture with α-mngostin, use CM permeilistion of S. ureus resulting in potssium loss [9, 43]. Our finings len support to previous finings tht α mngostin rpily isrupte the integrity of the ytoplsmi memrne of MRSA ells, leing to losing of intrellulr omponents in onentrtionepenent mnner [42]. The CM permeility results provie eviene tht one of the importnt mehnism of tion of isolte α-mngostin is isruption of the ytoplsmi memrne. This isruption in turn les to etivtion of the β-ltmse tivity. Plnt-erive ntiteril ompouns hve weker ntiteril tivity ompre to tht of syntheti ntiiotis. Therefore, synergisti prigms y omining the onventionl ntiioti with phytohemil ompouns is proven in severl stuies to e n effetive venue to tret infetious iseses use y rugresistnt teri [7 9]. Synergisti intertion omts rug-resistnt teri y hieving multiple synergisti rug trgets, interting with rug-resistnt mehnisms of teri, n neutrlising n eliminting verse effets [7]. Previous stuies foun tht eftziime h synergisti tivity with ilein, luteolin or queretin, whih shre Chromone struture, ginst Streptoous pyogense [23, 44]. In the sme wy, Eumke n oworkers reporte tht ertin β-ltm rugs plus glngin, queretin or ilein, whih lso shre hromone struture, showe synergisti tivity ginst peniillin-resistnt S. ureus [9]. Besies, Rukyi et l. foun tht Pnurtin A, whih possesses enzene ring n n isoprenyl group, isplye n MIC of 1 μg/ml for stphylool linil isoltes n generlly ws more potent thn ommonly use ntimiroils [45]. Also, moleulr ynmi simultions revele tht isoprenyl groups of α-mngostin, whih oupy hromone struture n isoprenyl groups, plye n importnt role in penetrting the lipi ilyer of the MRSA memrne resulting in memrne rekown n inrese permeility [42]. These finings provie eviene tht the enzene ring n the isoprenyl group of oth pnurtin A n α- mngostin ply signifint role in inhiiting the growth of MRSA strins y s iret intertions with the teril memrne [42, 45]. In vitro ytotoxi test (MTT ssys) The results of the MTT ssys re shown in Fig. 10. The α-mngostin or oxillin lone initilly exhiite ytotoxiity ginst 3T3-L1 preipoytes t onentrtions 128 n 1024 μg/ml, respetively, whih is 16 n 8 times higher thn the MICs of α-mngostin n oxillin ginst S. sprophytius respetively (Fig. 10 n ). Moreover, Fig. 10 revele the omintion of α- mngostin n oxillin t onentrtions of 2 n 16 μg/ml, whih showe the synergisti effet of ntiteril tivity ginst this strin, h no ytotoxi effet towrs 3T3-L1 preipoytes fter 24 h of exposure. Moreover, the four times higher thn FICI vlue, 64 μg/ml oxillin plus 8 μg/ml α-mngostin, still i not show the ytotoxi effet on 3T3-L1 preipoytes. Our finings provie eviene tht the MICs n higher osges of gents hve not shown ytotoxi effet on this ell line. Importntly, gretly esire property of ntiteril ompouns is the seletive inhiition ginst teril with less ytotoxi effet to norml ells for voiing sie effets to helthy tissues [46]. The previous stuies reporte tht the IC 50 of α-mngostin on the MRC-5 ell line is t the onentrtion of 7.5 μm [47] n 50 μm on3t3-l1preipoyte ell line [48]. These finings imply tht α-mngostin use lone or in omintion my e useful in eveloping novel junt phytophrmeutil to oxillin for the tretment of ORSS. Conlusions In onlusion, our finings provie eviene tht isolte α-mngostin from the perirp of GML lone hs not only some tivity ginst ORSS ut lso possesses the synergisti tivity with oxillin ginst this strin. The hromone (or 1,4-enzopyrone) struture n isoprenyl groups of α-mngostin oul ply n importnt role in inhiiting this strin. This synergisti tivity of isolte α-mngostin plus oxillin my involve three moes of tion of this xnthone. Firstly, potentil effets of ytoplsmi memrne isruption n inreses permeility. Seonly, inhiition of β-ltmse tivity. Finlly, peptioglyn mge. Our finings provie eviene tht isolte α-mngostin from the perirp of GML hs suffiient mrgin of sfety for therpeuti use. Isolte α-mngostin provies potentil to evelop useful of novel junt phytophrmeutil to oxillin for the tretment of ORSS. Future stuies shoul ress toxiity tests in nimls n humns. Aknowlegement The uthors re highly ppreitive of the One Reserh One Grnt (OROG) sholrship from Surnree University of Tehnology for ssistne in reserh funs support. Funing The uthors re inete n grtefully to the Thiln Reserh Fun n The Crystl Biotehnology Co., Lt. for ssistne in reserh fun support through The Reserh n Reserhers for Inustries Ph.D. sholrships (Grnt No. PHD58I0015 Coe ). Commenting on the report s finings is the responsiility of the grnt reipient, the Thiln Reserh Fun n the Crystl Biotehnology Co., Lt.

13 Phitktim et l. BMC Miroiology (2016) 16:195 Pge 13 of 14 Avilility of t n mteril The rw tsets supporting the onlusions of this rtile n e me ville y emiling the orresponing uthor. Authors ontriutions SP performe the experiments n wrote the report. MC n GS operte some lortories, nlyse t n gve omments. BD i the ytotoxiity test n interprete t. GH supervise, suggeste, revise the reserh n polishe the mnusript. GE esigne the projet, supervise the experiments n wrote the mnusript. All uthors hve re n pprove the finl mnusript. Competing interests The uthors elre tht they hve no ompeting interests. Consent for pulition Not pplile. Ethis pprovl n onsent to prtiipte Not pplile. Author etils 1 Shool of Phrmology, Institute of Siene, Surnree University of Tehnology, Nkhon Rthsim 30000, Thiln. 2 Deprtment of Miroiology, Fulty of Phrmy, Mhiol University, Rjthevi, Bngkok, Thiln. 3 Shool of Phrmy n Biomoleulr Sienes, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK. 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