Kaempferia parviflora Extract as a Potential Anti-Acne Agent with Anti-Inflammatory, Sebostatic and

14 Pages·2017·3.81 MB·English

International Journal o f
Molecular Sciences
Article
Kaempferia parviflora Extract as a Potential Anti-Acne
Agent with Anti-Inflammatory, Sebostatic and
Anti-Propionibacterium acnes Activity
SoleeJin1andMi-YoungLee1,2,*
1 DepartmentofMedicalScience,CollegeofMedicalScience,SoonChunHyangUniversity,
22SoonChunHyang-ro,Asan,Chungnam31538,Korea;[email protected]
2 DepartmentofMedicalBiotechnology,CollegeofMedicalScience,SoonChunHyangUniversity,
22SoonChunHyang-ro,Asan,Chungnam31538,Korea
* Correspondence:[email protected];Tel./Fax:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:12October2018;Accepted:31October2018;Published:3November2018
Abstract: Kaempferia parviflora, referred to as black ginger, has traditionally been used as a
health-promoting alternative medicine. In this study, we examined the anti-inflammatory,
sebostatic,andanti-PropionibacteriumacnesactivitiesofK.parvifloraextract. Theextractsignificantly
down-regulatedtheexpressionofinducibleNOsynthase(iNOS)andcyclooxygenase-2(COX-2),and
pro-inflammatorycytokinetumornecrosisfactoralpha(TNF-α)level. Moreover,thephosphorylation
ofIkBαandnuclearfactor-kappaB(NF-κB),andtheenhancednucleartranslocationofNF-κBp65in
lipopolysaccharide-stimulatedmurinemacrophage-likecellline(RAW264.7)cellsweremarkedly
decreased by the extract. Notably, the main component of K. parviflora, 5,7-dimethoxyflavone,
alsomodulatedtheexpressionofiNOSandNF-κBsignalmoleculesinP.acnes-stimulatedhuman
keratinocyte(HaCaT)cells. Additionally,K.parvifloraextractinhibitedthelipogenesisofsebocytes,as
evidencedbyareducedleveloftriglycerideandlipidaccumulationinthesebocytes. Thesebostatic
effectwasalsoconfirmedbyareducedexpressionofperoxisomeproliferation-activatingreceptors
(PPAR-γ)andoil-redOstaininginsebocytes.Takentogether,thisstudysuggestsforthefirsttimethat
K.parvifloraextractcouldbedevelopedasapotentialnaturalanti-acneagentwithanti-inflammatory,
sebostatic,andanti-P.acnesactivity.
Keywords: Kaempferiaparviflora;anti-inflammation;sebostaticeffect;anti-P.acneseffect
1. Introduction
Acnevulgarisisoneofthemostcommondermatologicaldiseases,affecting80–85%ofteenagers
globally. It is triggered by several skin flora, including Propionibacterium acnes and Staphylococcus
aureus [1–3]. The pathogenesis of acne includes induction of inflammatory responses, increased
productionofsebum,andhyperplasiaofsebaceousglands,aswellaschangesinthelipidcomposition
ofsebumvialipogenesismodulation[4,5]. Inaddition,theleveloflinoleicacid(LA)insebumand
thesphingolipidlevelinthestratumcorneumofacnepatientswasreportedtobelowerthanthatof
peoplewithnoacne[6].
Oneoftheunderlyingpathogenicmechanismsspecificallyinvolvedinacnepathogenesishasbeen
evidentthroughanelevatedexpressionofcyclooxygenase-2(COX-2)andprostaglandinE2(PGE )
2
associated with an enhanced release of pro-inflammatory cytokines and lipogenesis in sebocytes.
Inflammatory response in aggravated and augmented acne lesions further underlines the role of
peroxisomeproliferation-activatingreceptors(PPARs),alongwithinsulinandaninsulin-likegrowth
factor(IGF-1)[7]. Moreover,theinterplaybetweenlipidsignalsandinflammatoryresponseshasbeen
Int.J.Mol.Sci.2018,19,3457;doi:10.3390/ijms19113457 www.mdpi.com/journal/ijms
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suggestedasacauseofacnedevelopment. Therefore,findingnewtargetswithintheinflammatoryand
sebostaticresponseassociatedwithsebumderegulationmightbeaninnovativetherapeuticstrategy
foracne.
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Acnetreatmentwithsyntheticchemicalmedicines,suchasantibioticsandsteroids,canresultin
mildtoseveresideeffects[8]. Thus,severalcomplementaryandalternativemedicines,suchasherbal
Acne treatment with synthetic chemical medicines, such as antibiotics and steroids, can result in
extramctisld,p tola snetvoeriels s,iadne defafencttism [8ic].r Tohbuiasl, speevpetriadl ecso,mhpalvemebeneteanryu asnedd awltietrhnaftairvef emweedricsiindees, esfufechct ass. Hheorbwael ver,
theliemxtirtaecdtss, cpielanntti fiocilsa,n adndc lainnitcimalicdraotbaiaol npetphteideefsfi, chaacvye obfetehne useserde mwietdh ifeasr aferewceor nsicdeer nefinfegc,tsa. nHdotwheevyern, eed
tobethcoe mlimplietemd esnciteendtibfiyc afnudrt chleinricreals edaartach o.n the efficacy of these remedies are concerning, and they need
tKoa beem cpofmeripalepmarevnitfleodr ab,ya flusorthkenro rwesneaarcshb. lackgingeror“krachaidum”inThai,isaherbaceousplant
Kaempferia parviflora, also known as black ginger or “krachai dum” in Thai, is a herbaceous plant
belonging to the Zingiberaceae family [9]. It has been traditionally used as a health-promoting
belonging to the Zingiberaceae family [9]. It has been traditionally used as a health-promoting
alternative medicine with anti-inflammatory, anti-allergic, anticholinesterase, adaptogenic, and
alternative medicine with anti-inflammatory, anti-allergic, anticholinesterase, adaptogenic, and anti-
anti-obesity effects [10]. K. parviflora contains several flavonoids, including 5,7-dimethoxyflavone,
obesity effects [10]. K. parviflora contains several flavonoids, including 5,7-dimethoxyflavone, 5-
5-hydroxy-3,7,4(cid:48)-trimethoxyflavone,and5-hydroxy-3,7-dimethoxyflavone[11]. Theextractsofthis
hydroxy-3,7,4′-trimethoxyflavone, and 5-hydroxy-3,7-dimethoxyflavone [11]. The extracts of this
plant have shown efficacies against several disorders, including metabolic, sexual, and cognitive
plant have shown efficacies against several disorders, including metabolic, sexual, and cognitive
disorddiesrosr,daesrsw, easll waseclla nasc ecra[n1c2e]r. H[12o]w. eHvoewr,etvheer,s ptheec isfipceecifffiicc aecfyficoafcKy .opfa rKv.i flpoarravieflxotrraa cetxstroanctsa conne vacunlge aris
remavinulsgtaorisb eredmisacinosv teor ebde. dTishceovperreesde.n Tthset updreysesnutg sgteusdtye dsufgogretshteedfi frosrt tthime feirtsht attimKe. pthaartv iKfl.o praarevtifhloarna olic
extraectthsamnoilgich etxctorancttrsi bmuigtehtt ocotnhteribaumtee ltioo rthaeti aomneolifoaractnioenv oufl gacanreis vbuylgmaroisd buyl amtiondgultahteinggr tohwe tghroowftPh. oafc nes,
sebocPy. atecnleisp, osgebeonceysties ,liapnodgetnheesiisn, flanadm tmhea itnofrlaymremsaptoonrys er.esponse.
2. Re2s.u Rletssults
2.1. A2n.1t.i A-Pn.tai-cPn.e ascAnecst Aivcittiyviotyf Kof. Kp.a prvarivflioflroaraE Exxtrtraacctt
TheTahneti manitcimroibcrioablieaflf eefcftecotf oKf .Kp.a pravrivflioflroarae exxtrtraacctt wwaass iinnvveessttigigaatetded agaagianisnt sttwtow sokisnk filnorflaos,r aP.s ,acPn.easc nes
and San.da uSr.e uaus.reuAs.s Ashs oswhonwinn iFni gFuigruere1 ,12, 52050a nandd5 50000 µμgg//mmLL ooff KK. .ppaarvrivflioflroar aexetxratcrta cintdinudceudc eadlmaolsmt ost
compcloemtepilentaec itnivacattiivoantioonf Po.f aPc. naecsneasn adndS .Sa. uaruerueus,s,r eressppeeccttiivveellyy.. TThheessee reresusultslt ssusguggegsteesdt etdhatth Ka.t pKa.rvpiaflrovriafl ora
extract possessed anti-acne properties, activated via inhibiting the growth of skin bacteria including P.
extractpossessedanti-acneproperties,activatedviainhibitingthegrowthofskinbacteriaincluding
acnes and S. aureus.
P.acnesandS.aureus.

Figure1. AntimicrobialeffectofKaempferiaparvifloraextractagainst(A)Propionibacteriumacnesand
Figure 1. Antimicrobial effect of Kaempferia parviflora extract against (A) Propionibacterium acnes and (B)
(B) Staphylococcus aureus. All data are presented as the mean ± standard deviation (SD) of three
Staphylococcus aureus. All data are presented as the mean ± standard deviation (SD) of three
indepinednedpeenntdeexnpt eexrpimereimntesn.ts.
2.2. Anti-Inflammatory Effect of K. parviflora Extract
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2.2. Anti-InflammatoryEffectofK.parvifloraExtract
The anti-inflammatory effect of K. parviflora was examined by investigating the expression of
The anti-inflammatory effect of K. parviflora was examined by investigating the expression
inflammatory enzymes and their products. The expression pattern of inducible NO synthase (iNOS)
of inflammatory enzymes and their products. The expression pattern of inducible NO synthase
and COX-2 was examined by western blotting analysis (Figure 2A). The expression of iNOS induced
(iNOS)andCOX-2wasexaminedbywesternblottinganalysis(Figure2A).TheexpressionofiNOS
by lipopolysaccharide (LPS) was drastically decreased depending on the concentration of the K.
inducedbylipopolysaccharide(LPS)wasdrasticallydecreaseddependingontheconcentrationofthe
parviflora extracts. COX-2 expression was also reduced at 20 μg/mL of K. parviflora extract. Enhanced
K.parvifloraextracts.COX-2expressionwasalsoreducedat20µg/mLofK.parvifloraextract.Enhanced
production of NO by iNOS was proved to trigger the acute and chronic inflammation involved in
productionofNObyiNOSwasprovedtotriggertheacuteandchronicinflammationinvolvedin
tissue damage [13]. As shown in Figure 2B, K. parviflora extract significantly suppressed the
tissuedamage[13]. AsshowninFigure2B,K.parvifloraextractsignificantlysuppressedtheproduction
production of NO in murine macrophage-like cell line (RAW 264.7) cells in a concentration-
ofdNeOpenindemnut rminaenmnear.c rKo.p phaargveif-lloirkae ecxetlrlalcitn eat( R2A0 Wμg2/m64L.7 d)eccerlelsasineda NcoOn cleenvetrl attoio tnh-adte pobesnedrevnedt mina nthnee r.
K.cpoanrtvriofllo. rTaheexster arecstualtts2 0shµogw/emd Lthdate ctrheea sKe. dpaNrvOiflolerva eelxttoratcht ainthoibbsieterdv etdhei nextphreescsoinontr oolf. iTNhOeSse, wrehsiuchlt s
shsouwbesdeqtuheantttlhye rKed.upacrevdi flthorea perxotdrauccttiionnh iobfi tNedOt,h ae keexyp rmesesdioiantoorf oiNf iOnfSl,awmhmicahtosruy brseesqpuoennsetl.y Inre addudcietdiotnh, e
prothdeu cLtPioSn-inodfuNceOd, aelkeevyatmede dleivaetol roof ftihnefl acmytomkaintoer yTNreFs-pαo nwsaes. Isnigandifdicitainotnly, threedLuPceSd-i nbdyu Kce. dpaerlveivflaotread
leveexltroafctth (Feicgyutroek 2iCne). TNF-αwassignificantlyreducedbyK.parvifloraextract(Figure2C).

FigFuigruer2e .2(.A (A)I)n Ihnihbiibtoitroyrye feffefcetcto fotf hteheK aKeamempfpefreiraiap apravrviflifolorraa eexxttrraacctt oonn tthhee eexxpprreessssiioonn ooff lliippooppoollyyssaacccchhaarriiddee
(LP(LSP)S-i)n-idnuducecdedi ninflfalammmmaattoorryy pprrootteeiinnss,, iinndduuccibiblele NNOO sysynnththasaes e(i(NiNOOS)S a)nadn dcyccyloclooxoyxgyegneansea-s2e -(2CO(CXO-2X),- 2in),
inmmuurirnine emmacarcorpohpahgaeg-leik-lei kceellc elilnlel i(nReA(WR A2W64.72)6 c4e.l7ls). cTehlles .exTphreesesixopnrse ossf iioNnOsSo faniNd OCOSXa-n2d wCerOe Xa-n2alwyzeerde
anwaliythz eImdawgeitJh anIdm naogremJaalnizdedn aograminaslti zβe-dactaigna; i(nBs) tefβfe-catc toifn K;. (pBa)rveifflfoercat eoxtfraKc.t poanr NviOflo praroedxutcrtaicotn oinn LNPSO-
priondduuccteido ninifnlaLmPmSa-itniodnu icne dRAinWfl a2m64m.7a cteiollns; i(nC)R iAnhWibi2to64ry.7 ecffeelclst ;o(fC K). ipnahrvibifiltoorar yexetfrfaecctt oonf LKP.Sp-ainrvdiuflcoerda
exTtrNacFt-oαn leLvPeSl -iinn dRuAcWed 2T6N4.F7- αcellelsv. eAlilnl dRaAtaW ar2e6 4e.x7pcreelslsse.dA lalsd mateaaanr e± eSxDp.r e*s pse <d 0a.s05m ceoamnp±arSeDd. w*pith< 0L.P0S5
cotmrepaaterded cewllist honLlPy.S treatedcellsonly.
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Next, the anti-inflammatory effect of K. parviflora extract on the nuclear factor-kappa
Next, the anti-inflammatory effect of K. parviflora extract on the nuclear factor-kappa B (NF-κB)
B (NF-κB) signaling pathway was investigated. K. parviflora markedly downregulated the
signaling pathway was investigated. K. parviflora markedly downregulated the expression of
expressionofphosphorylatedinhibitorkappaB-alpha(IκBα)andNF-κB,asexaminedbywestern
phosphorylated inhibitor kappa B-alpha (IκBα) and NF-κB, as examined by western blotting (Figure
blotting (Figure 3A). These results suggested that the anti-inflammatory activity of K. parviflora
3A). These results suggested that the anti-inflammatory activity of K. parviflora in LPS-stimulated
in LPS-stimulated RAW 264.7 cells might be due to the suppression of the NF-κB signaling
RAW 264.7 cells might be due to the suppression of the NF-κB signaling pathway. Moreover, K.
pathway. Moreover,K.parviflorasuppressedthenucleartranslocationofp-NF-κBinLPS-stimulated
parviflora suppressed the nuclear translocation of p-NF-κB in LPS-stimulated RAW 264.7 cells, as
RAW 264.7 cells, as shown in the confocal microscopy data (Figure 3B). Nuclear translocation
shown in the confocal microscopy data (Figure 3B). Nuclear translocation of p-NF-κB occurred in
of p-NF-κB occurred in LPS-stimulated RAW 264.7 cells; however, nuclear translocation and
LPS-stimulated RAW 264.7 cells; however, nuclear translocation and accumulation of p-NF-κB were
accumulationofp-NF-κBweredramaticallyreducedbytreatingtheextract. Thesedatashowthat
dramatically reduced by treating the extract. These data show that K. parviflora exerted anti-
K.parvifloraexertedanti-inflammatoryeffectsbymodulatingNF-κBsignalingmoleculesandp-NF-κB
inflammatory effects by modulating NF-κB signaling molecules and p-NF-κB nuclear translocation.
nucleartranslocation.

Figure3. (A)InhibitoryeffectofKaempferiaparvifloraextractonthenuclearfactor-kappaB(NF-κB)
Figure 3. (A) Inhibitory effect of Kaempferia parviflora extract on the nuclear factor-kappa B (NF-κB)
signalingpathwayinLPS-stimulatedRAW264.7cells,examinedbywesternblotting.Theexpressions
signaling pathway in LPS-stimulated RAW 264.7 cells, examined by western blotting. The expressions
of p-IκBα and p-NF-κB were analyzed with ImageJ and normalized against β-actin. * p < 0.05
of p-IκBα and p-NF-κB were analyzed with ImageJ and normalized against β-actin. * p < 0.05 compared
compared with LPS treated cells only; (B) effect of K. parviflora extract on the translocation of
with LPS treated cells only; (B) effect of K. parviflora extract on the translocation of NF-κB p65 in LPS-
NF-κBp65inLPS-inducedRAW264.7cells. ImmunofluorescencestainingforNF-κBp65(red)in
induced RAW 264.7 cells. Immunofluorescence staining for NF-κB p65 (red) in LPS-exposed RAW
LPS-exposedRAW264.7cellswithoutandwith20µg/mLK.parvifloraextract.Nucleiarestainedwith
264.7 cells without and with 20 μg/mL K. parviflora extract. Nuclei are stained with 4,6-diamidino-2-
4,6-diamidino-2-phenylindoledihydrochloride(DAPI)(blue).K.parvifloraextractreducedthenuclear
phenylindole dihydrochloride (DAPI) (blue). K. parviflora extract reduced the nuclear translocation and
translocationandaccumulationofNF-κBp65,whichwasinducedbyLPS.Scalebar=10µm.
accumulation of NF-κB p65, which was induced by LPS. Scale bar = 10 μm.
Next, we evaluated whether 5,7-methoxyflavone, a major active component of K. parviflora,
contNriebxutt,e swteo tehvealaunattie-idn flwamhemthaetro r5y,7e-fmfeectthoofxKy.flpaavrovinfleo,r aa exmtraajocrt aancdtivweh ecothmerpothneenext troafc tKi.s puasrevfiufllotroa,
ctornetartibP.uatecns etso-i nthdeu acnedti-aincnfleamvumlgaatroirsy. Tehffeecptr oesf eKn.c peaorfvi5f,l7o-rda iemxetrtahcotx aynflda vwohneetihnerK .thpea revxifltroarcate ixst ruascetfuwla tso
trideaetn Pti.fi aecdnebsy-inUdPuLcCe-dQ aTcOnFe -vMuSlgianrtise.r mThseo pfrmeasessncaen dofU 5V,7-pdeiamke.tThhoexymflaasvsoonfe2 8in2 K([.M pa+rvHif]l+orma e/zx2tr8a3c)t awnads
idaeUnVtifλied bayt U22P0L,C26-Q3,T3O07F-nMmSc ihna trearcmtesri ostfi cmfaosrss oanmde UflaVv poneaokid. sTwhee rmeafossu nodf ,2i8n2d (i[cMat i+n gHt]h+e mp/rze 2se8n3c) eanofd
max
a5 U,7V-d iλmmeaxt haot x2y2fl0a, v2o63n,e 3i0n7o numre cxhtraarcatc.tIenriasdtidc iftoiorn s,o5m,7e-m fleatvhoonxoyifldasv woneerew faosuanpdp, liineddiocantiPn.ga ctnhees -pinrfeescetnedce
ohf u5m,7a-ndikmeerathtionxoycfyltaev(oHnaeC ianT )ocuerl lsexatsraaccte. llI-nb aasdeddiaticonne, m5,o7d-mel.eTthhoexeyxfplarveossnieo nwoafst haepipNliOedS eonnz yPm. eacanneds-
inthfeecNteFd- κhBumsiganna klienrgatminoolceycutel e(sH,IaκCBaαTa) ncdellNs Fa-sκ Ba ,cwelelr-beainsevde saticgnaet emdobdyewl. eTshteer nexbplorettsisnigo.nA osf sthhoew iNnOinS
eFnizgyumree 4a,ntdh ethPe. aNcnFe-sκ-Bin sdiugcneadlinegx pmreoslseicounleosf, iINκBOαS awnads NsiFg-nκiBfi,c awnetrley irnevdeuscteigdabteyd5 b,7y- mweetshteorxny bfllaovtotinneg..
AMs osrheoowvenr ,ipn hFoisgpuhroe r4y,l attheed PIκ. Bac-αneas-ninddNucFe-dκB ewxperreessniootna bolfy idNoOwSn rwegasu lsaitgendifbicyan5t,7ly-m reedthuocxeydfl bavyo 5n,e7.-
mTehtehsoexryefslauvltosnseu. gMgeosrteeodvtehr,a tpthhoespanhtoir-iynlafltaemd mIκaBto-αry aenfdfe cNtFo-fκKB. pwaerrveifl noroataebxltyr adcotwmnigrehgtubleatdeude btoy t5h,7e-
mpertehsoenxycefloavfo5n,7e-.m Tehtheosex yrfleasvuoltns es,uagngdemsteedd iathteadt tthhreo uagnhti-iinnhfilbamitimonatoofrtyh eefefxepctr eosfs ioKn. opfartvhiefloiNraO eSxatrnadct
might be due to the presence of 5,7-methoxyflavone, and mediated through inhibition of the
expression of the iNOS and NF-κB signaling molecules in P. acnes-stimulated HaCaT cells. Thus, K.
parviflora extract might be useful to treat the inflammatory response of acne vulgaris.
Int.J.Mol.Sci.2018,19,3457 5of14
NF-κBsignalingmoleculesinP.acnes-stimulatedHaCaTcells. Thus, K.parvifloraextractmightbe
uInst.e Jf.u Mlotlo. Stcrie. 2a0t1t8h, 1e9i, nx flFaOmR PmEaEtRo RryEVrIeEsWpo nseofacnevulgaris. 5 of 13
Figure4.Inhibitoryeffectof5,7-methoxyflavoneonthePropionibacteriumacnes-inducede xpressionof
inflammatoryproteinsinP.acnes-stimulatedhumankeratinocyte(HaCaT)cells.Theexpressionsof
Figure 4. Inhibitory effect of 5,7-methoxyflavone on the Propionibacterium acnes-induced expression of
iNOS,p-IκBαandp-NF-κBwereanalyzedwithImageJandnormalizedagainstβ-actin.Alldataare
inflammatory proteins in P. acnes-stimulated human keratinocyte (HaCaT) cells. The expressions of
expressedasmean±SD.*p<0.05comparedtoP.acnestreatedcellsonly.
iNOS, p-IκBα and p-NF-κB were analyzed with ImageJ and normalized against β-actin. All data are
2.3. SeexbporsetasstiecdE afsf emcteoafnK ±. SpDar. v* ipfl <or 0a.0E5x ctoramcptsared to P. acnes treated cells only.
2.3. STebhoestsaetbico Estfafetcict oeff fKe.c ptaorfvKifl.opraar Evxifltroarcates xtractsontheleveloftriglyceride,amajorlipidinsebum,
wasexamined. IGF-1andLAwereaddedtosebocytestopromotesebumproduction(Figure5). The
The sebostatic effect of K. parviflora extracts on the level of triglyceride, a major lipid in sebum,
triglyceride(TG)contentinsebocytes,whichwasdoubledbyIGF-1andLA,wassignificantlyreduced
was examined. IGF-1 and LA were added to sebocytes to promote sebum production (Figure 5). The
byK.parvifloraextractat1,2.5and5µg/mL.
triglyceride (TG) content in sebocytes, which was doubled by IGF-1 and LA, was significantly
reduced by K. parviflora extract at 1, 2.5 and 5 μg/mL.
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Figure5.EffectsofKaempferiaparvifloraextracton(A)IGF-1-and(B)linoleicacid-inducedlipogenesis.
FiguTrreig 5ly. cEefrfiedcet(sT oGf) Kleaveemlspwfeerirae panaravlyifzleodrab eyxetnrazcytm oen-l i(nAk)e dIGimF-m1-u naonsdo r(bBe)n ltiansoslaeyic( EaLcIiSdA-i)n.dAullcdeadt aliaproegenesis.
Trigleyxcperreisdseed (TasGm) eleanve±lsS wDe.*rep a<n0a.0ly5zceodm bpayr eedntzoyImGFe--1lionrkliendol eiimcamciduntroeastoedrbceenllst oanslsy.ay (ELISA). All data
are expressed as mean ± SD. * p < 0.05 compared to IGF-1 or linoleic acid treated cells only.
Moreover,theLA-accumulatedintracellularlipidwasreducedinsebocytestreatedwith5µg/mL
K.parvifloraextract(Figure6A),asexaminedbyoilredOstaining. TheIGF-1-inducedexpression
Moreover, the LA-accumulated intracellular lipid was reduced in sebocytes treated with 5
ofperoxisomeproliferator-activatedreceptorgamma(PPAR-γ)insebocyteswasalsosignificantly
μg/mL K. parviflora extract (Figure 6A), as examined by oil red O staining. The IGF-1-induced
inhibitedby5µg/mLK.parvifloraextract(Figure6B).
expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) in sebocytes was also
significantly inhibited by 5 μg/mL K. parviflora extract (Figure 6B).
Int. J. MInolt.. SJ.cMi. o2l0.1S8ci,. 1290,1 8x, F1O9,R34 P5E7ER REVIEW 7of71 4of 13

Figure6.(A)EffectsofKaempferiaparvifloraextractonlipidsynthesisinsebocytes.Thelipidcontent
Figureo f6s. e(bAo)c yEtfefsecwtsa sodf eKteacetmedpfberyiaO pilarrevdiflOorast aeixntirnagcta onnd tlhipeindm syeanstuhreesdisb iynE sLeIbSoAc.yTtehse. sTehboe clyipteisdw ceornetent of
sebocyshteosw wnabsy dmeitcercotsecdop byya tOailm raegdn iOfic sattaioinnionfg× a1n0d0; t(hBe)nef fmecetsasoufrKe.dp abrvyi flEoLraISeAxt.r aTchtoen sethbeoecxypteress swioenreo fshown
by miIcGroFs-1c-oinpdyu acet dap meraogxinsiofmiceatpiroonli foefr a×t1or0-0a;c t(iBva) teefdferecctesp otof rKg.a pmamrvaif(lPoPraA Rex-γtr).aTcth eonex tphrees seixopnrseosfsPioPAn Ro-fγ IGF-1-
inducewda spaenroalxyizseodmwe ipthroImlifaegreaJtoarn-dacntiovramteadli zreedceapgtaoinr sgtaβm-amctian .(PAPlAldRa-tγa).a rTeheex pexrepsrseesdsiaosnms eoafn P±PASRD-.γ was
*p<0.05comparedtoIGF-1orlinoleicacidtreatedcellsonly.
analyzed with ImageJ and normalized against β-actin. All data are expressed as mean ± SD. * p < 0.05
c3o.mDpiasrceuds stoio InGF-1 or linoleic acid treated cells only.
3. Dis3c.u1.ssKi.opna rvifloraExtractInhibitstheGrowthofSkinBacteria
The growth of bacterial skin flora depends on the condition of the skin environment which
3.1. K. parviflora Extract Inhibits the Growth of Skin Bacteria
they populate. Propionibacterium species reside predominately in the sebaceous areas, whereas
Staphylococcus species typically populate the dry surface of the skin [14,15]. P. acnes is involved
The growth of bacterial skin flora depends on the condition of the skin environment which they
in an inflammatory response and lipogenesis in acneic skin, contributing to the development and
populate. Propionibacterium species reside predominately in the sebaceous areas, whereas
aggravation of acne because it metabolizes sebum into fuel for its growth in the clogged pores of
Staphylococcus species typically populate the dry surface of the skin [14,15]. P. acnes is involved in an
acnelesions[16,17]. S.aureusisabacteriumcommonlyfoundontheskinsurface. However,itcan
inflammatory response and lipogenesis in acneic skin, contributing to the development and
causemanylife-threateninginfectionswhenitentersthebody’ssystem. Inthisstudy,K.parviflora
aggravation of acne because it metabolizes sebum into fuel for its growth in the clogged pores of acne
extractshowedasignificantantimicrobialeffectagainstthesetwocausativeagentsofacnevulgaris.
lesionOs u[1r6re,1s7u]l.t sSs.u agugreesutes dist haa tbKa.cptearrviuiflmor acoexmtrmacotnmlyig hfotubnedu soefnu lthtoe csoknitnro sluskrfinacdei.s oHrdoewrsevtreigr,g ietr ecdanb ycause
manyt hliefsee-tbharcetaetreian.ing infections when it enters the body’s system. In this study, K. parviflora extract
showed a significant antimicrobial effect against these two causative agents of acne vulgaris. Our
results suggested that K. parviflora extract might be useful to control skin disorders triggered by these
bacteria.
3.2. K. parviflora Extract Inhibits Inflammatory Responses
Inflammation has been suggested as a key factor involved in the development and aggravation of
acne vulgaris [18], although the exact mechanisms underlying the pathogenesis and subsequent
development of acne are not clarified fully. Recently, many efforts have been made to elucidate novel
Int.J.Mol.Sci.2018,19,3457 8of14
3.2. K.parvifloraExtractInhibitsInflammatoryResponses
Inflammationhasbeensuggestedasakeyfactorinvolvedinthedevelopmentandaggravation
ofacnevulgaris[18],althoughtheexactmechanismsunderlyingthepathogenesisandsubsequent
developmentofacnearenotclarifiedfully. Recently,manyeffortshavebeenmadetoelucidatenovel
therapeutictargetsforacne,includingmodulatorsofthesignallingmoleculesintheinflammatory
response. Onthebasisofthisinformation,theinhibitoryeffectofK.parvifloraextractoninflammation
wasinvestigatedinadvancebyusingageneralinflammatorymodelwithLPSandRAW264.7cells.
LPSstimulationinmacrophageshasbeenwidelyknowntoplayacriticalroleininflammatoryresponse
byreleasingpro-inflammatorycytokines,nitricoxideandPGE [19,20].
2
Using Western blotting analysis, we showed that the LPS-induced expression of iNOS was
drastically decreased by K. parviflora extract in a concentration-dependent manner (Figure 2A).
Moreover, K. parviflora extract, which inhibited the expression of iNOS, subsequently reduced the
productionofNO(Figure2B),akeymediatorofinflammatoryresponse. Inaddition, K.parviflora
extracts also reduced the expression of COX-2 and the production of PGE to some extent. These
2
resultssuggestedthattheextractexerteditsanti-inflammatoryactivityviadownregulationofiNOS
andCOX-2expression. Anti-inflammatoryactivitiesassociatedwithiNOSandCOX-2expressionwere
reportedbyavarietyofherbs[21–23]andherbalformulae[24]. COX-2andiNOScanbeinducedby
manyofthesamecytokines,andexpressedtogetherininflamedtissues. Specifically,NOproducedby
iNOSenhancesCOX-2activitythroughperoxynitrite-mediatedactivationoftheperoxidaseactivityof
COX-2[25].
Next,K.parvifloraextractmodulatedNF-κBsignalinginLPS-inducedRAW264.7cells. Activation
ofNF-κBmainlyoccursviaIκBkinase-mediatedphosphorylationofIκBα. PhosphorylatedIκBprotein
is then ubiquitinated and degraded, separating the inactive NF-κB from IκB, leading to an active
state. LPS-activated NF-κB enters the nucleus and binds to DNA to activate the transcription of
severalgenesrelatedtoinflammationandcelldeath. Inthisstudy,theexpressionsofphosphorylated
IκBαandNF-κBp65inRAW264.7cellswereupregulatedinresponsetoLPSstimulation. However,
K.parvifloraextractat20µg/mLdrasticallydownregulatedtheincreasedexpressionofphosphorylated
IκBα and NF-κB (Figure 3A). Therefore, K. parviflora extract exerted an anti-inflammatory effect
through modulating the NF-κB pathway. Moreover, the LPS-induced nuclear translocation and
the accumulation of NF-κB p65 was also markedly reduced by K. parviflora extract (Figure 3B).
In addition, the anti-inflammatory effect of K. parviflora extract might be related to the presence
of5,7-methoxyflavone. Further,5,7-methoxyflavonereducedthelevelsofphosphorylatedIκB-αand
NF-κB,aswellasiNOS,inP.acnes-stimulatedHaCaTcells,whichisacell-basedacnemodel(Figure4).
Inconclusion,K.parvifloraextracts,whichwereexertingananti-inflammatoryeffectdue,mostprobably,
tothepresenceof5,7-methoxyflavone,mightbeeffectiveincontrollingtheinflammatoryacnevulgaris.
3.3. TheAnti-LipogenesisEffectofK.parvifloraExtractinSebocytes
Recently,acnewasdefinedasaninflammatorydiseaseprimarilytriggeredbypro-inflammatory
sebumlipidfractions[26]. Thus,therelationshipbetweeninflammationandlipogenesismightbe
crucial in fully elucidating acne pathogenesis. Sebum is a mixture of lipids composed mainly of
triglycerides,waxesters,squalene,fattyacids,andlowamountsofcholesterol. Excessproduction
ofsebumisattributabletoinflammatorydisordersassociatedwiththeexcessivegrowthofP.acnes.
Inaddition,alterationsinsebumlipidcompositionalsoplayacrucialroleintheclinicaldevelopment
and aggravation of acne [27]. The ratio of saturated to unsaturated fatty acids was found to have
changed in the sebum of acne patients. In particular, increases in the levels of squalene peroxide
andwaxestersandtheC16:0/C16:1ratio,aswellasdecreasesinLAandvitaminEcontentswere
foundinacnepatients[6]. Low-levellinoleicacidandsphingolipidshavebeeninvolvedinfollicular
hyperkeratosisandlinkedwithcomedoneformation,epidermalbarrierdysfunction,andelevated
permeabilityofthecomedonalwallforenvironmentalstressors[28].
Int.J.Mol.Sci.2018,19,3457 9of14
Inparticular,analteredproportionofmonounsaturatedfattyacidsassociatedwithdesaturation
offattyacidsmayinduceacneonset. Notably,theratiobetween∆6and∆9unsaturatedfattyacids
has been reported to be a biomarker for sebaceous cell maturation [6,29]. In addition, an acne
lesioncontainsanaccumulatedleveloflipidperoxides,specificallysqualeneperoxide. Ahighlevel
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 13
lipid peroxide could activate the peroxisome proliferator activated receptors, thereby stimulating
Inl ipthoixsy gsteundasye, atchteiv iitnyhaibnditosuryb seefqfueecnt tolyf eKn.h paanrcvinifglotrhae eexxtprraecsts ioonn otrfipgrloy-cinerfliadme mleavtoerl yincy stoekbionceystiens was
acne,aswellasprovidingsuitableenvironmentsforP.acnesproliferation[30–32].
examined (Figure 5). IGF-1 and LA, which promote sebaceous lipogenesis and secretion through
In this study, the inhibitory effect of K. parviflora extract on triglyceride level in sebocytes
sebocyte differentiation, were applied to promote sebum production in sebocytes. IGF-1 and LA-
was examined (Figure 5). IGF-1 and LA, which promote sebaceous lipogenesis and secretion
induced an increase in triglyceride level in sebocytes which was significantly decreased by treatment
through sebocyte differentiation, were applied to promote sebum production in sebocytes. IGF-1
with K. parviflora extract. Moreover, the inhibitory effect of K. parviflora extract on lipid accumulation
andLA-inducedanincreaseintriglyceridelevelinsebocyteswhichwassignificantlydecreasedby
in sebocytes was also confirmed by Oil red O staining (Figure 6A).
treatment with K. parviflora extract. Moreover, the inhibitory effect of K. parviflora extract on lipid
Lipogenesis in sebaceous follicles is stimulated by upregulation of PPAR-γ. Thus, specific PPAR-γ
accumulationinsebocyteswasalsoconfirmedbyOilredOstaining(Figure6A).
antagonistsL impoiggehnte sbies inresgeabradceeodu safso lcliacnledsiidsasttiems ufloatre danbtyi-uapcrneeg ualgateionntso. fIPnP AthRi-sγ .sTtuhduys,, stpheeci fiIGcPFP-A1-Rin-γduced
expresasniotang oonfi sPtsPmAiRgh-γt bienr esgeabrdoecdytaess cawnadsi ddatiemsifnoirsahnetdi- abcnye aKg. enptasr.vIinflotrhais estxutrdayc,tt,h epIoGsFtu-1la-itnindugc etdhat K.
parvifloerxap erexstsriaocnt ocfoPuPlAdR b-γe idnesveebloocpyteeds wasa sadni manintiis-ahcendeb yagKe.npat rwviflitohra ae xsterbacots,tpaotsictu elafftiencgt tahsastoKc.iaptaervdi flworiath the
inactiveaxttiroanct ocfo uPlPdAbeRd-γev. eClouprerdenatslayn, saenbtio-asctnaetiacg aegnetnwtist hinahsiebbiotsintagti cseebffuecmta lsispoocigaetendewsisit,h stuhcehin aasc tDivRatMion01, are
ofPPAR-γ. Currently,sebostaticagentsinhibitingsebumlipogenesis,suchasDRM01,areregardedas
regarded as promising candidates for anti-acne agents [33–35].
promisingcandidatesforanti-acneagents[33–35].
In this study, K. parviflora extract effectively suppressed the growth of acne-causing skin bacteria.
Inthisstudy,K.parvifloraextracteffectivelysuppressedthegrowthofacne-causingskinbacteria.
Moreover, the extract downregulated inflammatory responses by regulating the iNOS and NF-κB
Moreover, the extract downregulated inflammatory responses by regulating the iNOS and NF-κB
signaling, and lipogenesis by modulating lipogenesis and PPAR-γ expression (Figure 7). Thus, K.
signaling, and lipogenesis by modulating lipogenesis and PPAR-γ expression (Figure 7). Thus,
parviflora extract might be used as a potential anti-acne agent targeting inflammation and lipogenesis
K.parvifloraextractmightbeusedasapotentialanti-acneagenttargetinginflammationandlipogenesis
triggered by acne-causing bacteria.
triggeredbyacne-causingbacteria.

Figure7. Proposedunderlyingmechanismoftheanti-acneeffectofKaempferiaparviflora. Thet-bar
Figure 7. Proposed underlying mechanism of the anti-acne effect of Kaempferia parviflora. The t-bar
denotesaninhibitoryeffect.
denotes an inhibitory effect.
4. MaterialsandMethods
4. Materials and Methods
4.1. PreparationofK.parvifloraExtractsinSebocytes
4.1. PreparaRtihoinz oomf Ke.s poafrvKi.flpoarrav Eiflxotrraa,cctos lilnec SteedboicnytBeas ngkok, Thailand, were supplied by Biocm Co., Ltd.
(Asan, Korea), andusedfortheexperiment. Thedriedrhizomesweregroundandthensoakedin
Rhizomes of K. parviflora, collected in Bangkok, Thailand, were supplied by Biocm Co., Ltd.
95% ethanol for 24 h at room temperature. K. parviflora filtrate was concentrated under reduced
(Asan, Korea), and used for the experiment. The dried rhizomes were ground and then soaked in
95% ethanol for 24 h at room temperature. K. parviflora filtrate was concentrated under reduced
pressure using a rotary evaporator to obtain K. parviflora extracts with a yield of 15.3%. The presence
of 5,7-dimethoxyflavone in the extract was identified by UPLC-QTOF-MS in terms of mass and UV
peak.
4.2. Microbial Cultivation
Propionibacterium acnes (KCTC 3314) and Staphylococcus aureus subsp. aureus (KCTC 1927) were
obtained from the Korean Culture Center of Microorganisms (Seoul, Korea). P. acnes was grown
anaerobically in reinforced clostridial medium (RCM) broth at 37 °C for 72 h. S. aureus was grown
aerobically in Luria-Bertani (LB) at 37 °C for 12–24 h [36].
Int.J.Mol.Sci.2018,19,3457 10of14
pressureusingarotaryevaporatortoobtainK.parvifloraextractswithayieldof15.3%. Thepresenceof
5,7-dimethoxyflavoneintheextractwasidentifiedbyUPLC-QTOF-MSintermsofmassandUVpeak.
4.2. MicrobialCultivation
Propionibacteriumacnes(KCTC3314)andStaphylococcusaureussubsp. aureus(KCTC1927)were
obtained from the Korean Culture Center of Microorganisms (Seoul, Korea). P. acnes was grown
anaerobicallyinreinforcedclostridialmedium(RCM)brothat37◦Cfor72h. S.aureuswasgrown
aerobicallyinLuria-Bertani(LB)at37◦Cfor12–24h[36].
4.3. BacterialInactivationbyK.parvifloraExtract
Bacterialculturewasstandardizedusinga#0.5McFarlandstandardsolutionaccordingtothe
recommendationsoftheclinicallaboratorystandardinstitute[37]. S.aureusandP.acneswerecultured
andtreatedwitheachconcentrationofK.parvifloraextract. Thebrothmicrodilutionmethodusing
a96-wellmicrotiterplatewasusedtomeasuretheantimicrobialeffectofK.parvifloraextract[38,39].
Bacterial suspensions were diluted to 1.5 × 105 CFU/mL, and K. parviflora extract was added.
Incubationproceededat37◦Cunderanaerobicconditionsfor72horaerobicconditionsfor24h. The
absorbanceofthebacterialsuspensionsat620nmwasmeasuredtoestimatebacterialgrowthinhibition.
4.4. CellCulture
Acell-basedacnemodelwasconstructedaccordingtoourpreviousreports[20,36,40]. TheRAW
264.7andHaCaTwereobtainedfromtheGlobalBioresourceCentre(ATCC,Manassas, USA)and
incubated in complete Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Logan, UT, USA)
containing100U/mLpenicillin,100µg/mLstreptomycin,and10%fetalbovineserum(FBS)at37◦C.
PrimaryhumansebocyteswereobtainedfromCelprogen(SanPedro,CA,USA)andmaintainedin
HumanSebocyteCompleteGrowthMediafromthesamevendor. Cellswereseededandincubated
overnightpriortotreatmentwithK.parvifloraextract[32,40]. Forthestimulationexperiment,HaCaT
cellswereincubatedwithheat-killedP.acnesadjustedattheappropriateconcentrationinserum-free
media for 24 h at 37 ◦C in 5% CO . After stimulation, HaCaT cells were treated with or without
2
5,7-methoxyflavone(Sigma-AldrichCorp.,St. Louis,MO,USA)for48hat37◦Cin5%CO [41].
2
4.5. NitriteDetermination
RAW264.7cellswereseededandincubatedovernightpriortotreatmentwithK.parvifloraextract.
The cells were treated with various concentrations of K. parviflora extract for 18 h with or without
subsequentexposureto1µg/mLLPSat37◦Cinanatmosphereof5%CO inthedark. NOlevel
2
wasdeterminedbymeasuringtheconcentrationoftheendproduct,nitrite,usingtheGriessassay.
Briefly,culturesupernatant(100mL)wasmixedwith150mLofGriesssolution(1:1mixture(v/v)of
1%sulfanilamideand0.1%N-(naphthyl)ethylenediaminedihydrochloridein5%H PO )in96-well
3 4
platesatroomtemperaturefor5min. Absorbanceat570nmwasmeasuredbyamicroplatereader,and
nitriteconcentrationinthecultureswascalculatedusingastandardcurveofsodiumnitrite[32,37].
4.6. CytokineMeasurement
RAW264.7cellswereseededintoa24-wellplateatadensityof2.5×105cells/wellandincubated
overnight prior to the treatments. Cells were pretreated with 0, 5, 10, or 20 µg/mL K. parviflora
extractfor2h. Afterward, 1µg/mLLPSwasaddedtoeachwellandthecellswereincubatedfor
12h. ThesupernatantwastransferredtoanELISAplateandTNF-αlevelintheculturemediumwas
determinedusingacommercialkit(MouseTNF-αELISAkit,BD,Franklinlakes,USA)accordingto
themanufacturer’sinstruction[42].