Multiple Activities of Punica granatum Linne against Acne Vulgaris

12 Pages·2017·2.45 MB·English

International Journal o f
Molecular Sciences
Article
Multiple Activities of Punica granatum Linne against
Acne Vulgaris
Chia-JungLee1,Lih-GeengChen2,Wen-LiLiang3andChing-ChiungWang1,3,4,*
1 PhDProgramforClinicalDrugDiscoveryofChineseHerbalMedicine,CollegeofPharmacy,
TaipeiMedicalUniversity,Taipei11031,Taiwan;[email protected]
2 DepartmentofMicrobiology,ImmunologyandBiopharmaceuticals,CollegeofLifeSciences,
NationalChiayiUniversity,Chiayi60004,Taiwan;[email protected]
3 SchoolofPharmacy,CollegeofPharmacy,TaipeiMedicalUniversity,Taipei11031,Taiwan;
[email protected]
4 OrthopedicsResearchCenter,TaipeiMedicalUniversityHospital,Taipei11031,Taiwan
* Correspondence:[email protected];Tel.:[phone](ext.6161)
AcademicEditors:PaulaAndradeandPatríciaValentão
Received:31October2016;Accepted:13December2016;Published:12January2017
Abstract: Acne is a common skin condition with sebum overproduction, hyperkeratosis,
Propionibacterium acnes (P. acnes) and Staphylococcus aureus, and inflammation. Punica granatum
(pomegranate)iswell-knownforitsanti-inflammatoryeffects;however,fewstudieshavediscussed
the anti-acne effects of pomegranate. In this study, we found that pomegranate extract (PG-E)
significantlyreducedP.acnes-inducededemainWistarratears. Therefore,anevaluationplatform
usingmultiplepathogenicmechanismsofacnewasestablishedtoexploretheanti-acneeffectsof
pomegranate. ResultsshowedthatPG-Einhibitedbacterialgrowthandlipaseactivity. Througha
bioguided-fractionation-isolationsystem,fourhydrolysabletannins,punicalagin(1),punicalin(2),
strictinin A (3), and granatin B (4), were isolated. Compounds 1 and 2 had greater anti-bacterial
activitiesandanti-testosterone-inducedHaCaTproliferativeeffectsthantheothers. Compounds1,
3,and4displayedlipaseinhibitoryeffects. Compound4decreasedcyclooxygenase-2expression
and downregulated prostaglandin E production in heat-killed P. acnes-treated RAW 246.7 cells.
2
Inconclusion,PG-Eisabundantinhydrolysabletanninsthatdisplaymultipleanti-acnecapacities,
includinganti-bacterial,anti-lipase,anti-keratinocyteproliferation,andanti-inflammatoryactions.
Hence, PG-E has great potential in the application of anti-acne and skin-care products, and
punicalagin(1),themosteffectivecomponentinPG-E,canbeemployedasaqualitycontrolmarker.
Keywords: acne;Punicagranatum;anti-inflammation
1. Introduction
Acne vulgaris (acne), a common skin disease, usually appears in young adolescents with a
hormoneimbalance[1,2]. Morethan85%ofteenagersareaffectedbyacne,andmostofthemcontinue
tobeaffectedintoadulthood. IntheUnitedStates,acnetherapiescostapproximately$1billionper
year,whilemorethan$100millionisspentonover-the-counteracneproducts[3,4]. Majorcausesof
acneformationarefourfold: (1)increasingsebumproductionbyoveractiveoilglands;(2)retention
hyperkeratosis,whichblocksskinpores;(3)activitiesofnormalskinbacteria(Propionibacteriumacnes
andStaphylococcusaureus);and(4)skininflammation[5].
Insebumproduction,humansebumiscomposedoffattyacids,triglycerides,waxesters,andso
on. Skinareasrichinsebaceousglandsarepositivelycorrelatedwithacnelesions[2]. Nowadays,one
therapeuticstrategyfortheincreasedsebumproductionistopicaluseofazelaicacid,adicarboxylicacid
foundinrye,wheat,andbarley,whichcanblockthesynthesisoffattyacidstoavoidtheoverproduction
Int.J.Mol.Sci.2017,18,141;doi:10.3390/ijms18010141 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2017,18,141 2of12
ofsebum. However,long-termuseofazelaicacidresultsinskindepigmentation[6]. Evidenceshave
shown that Chamaecyparis obtuse (C. obtuse) extract and tea tree oil were usually applied for the
reduction of sebum productions. However, the therapeutic effect of C. obtuse is suboptimal and
toxicityissueshouldbeconcernedintheuseofteatreeoils[7]. Inhyperkeratosis,androgens,such
as testosterone, cause hyperresponsiveness that stimulates sebocytes and follicular keratinocytes,
resulting in hyperplasia of the sebaceous glands [1]. Artemisia capillaries (A. capillaries) is reported
foritsanti-inflammatoryeffectsagainsthepaticinjury. TopicalapplicationofA.capillariesreduced
hyperkeratosis of the epidermis, also suggesting the potential therapeutic activity to treat atopic
dermatitis [8]. Retinoic acid and salicylic acid are typically use to treat hyperkeratinization, but
thesechemicalagentsalsoleadtoskinirritationanddesquamation[9]. Inskinbacterialinfections,
oral anti-biotics such as tetracycline, erythromycin, and clindamycin are the most popular drugs
fortreatingbacteria-inducedacneatpresent. Overuseofantibioticsisahugeproblemformodern
medicine,becauseitresultsinanti-bioticresistance. Moreover,manylimitationsofanti-bioticswere
found when used as acne treatment. For example, tetracycline should be used in a fasting status,
andoneshouldavoidtakingitwithmilk. Pregnantwomenandchildrenarealsodiscouragedfrom
using tetracycline [10]. Several botanical extracts displayed the anti-skin bacterial effects, such as
Coriandrumsativumleafextract,butnoneofthemdiscussedthemultipletherapeuticeffectsagainst
acne[11]. Inanti-inflammation,non-steroidanti-inflammatorydrugs(NSAIDs),suchasibuprofen,
diclofenac, and ponstan, are typically used to inhibit biosynthesis of prostaglandin, which is an
important mediator in inflammatory reactions. NSAIDs can rapidly decrease the inflammation
of acne skin, but the commonest side effects of NSAIDs are peptic ulcers and liver toxicity [12].
Inaccordancewithpreviousresearch,weexaminedtheanti-acneeffectsofpomegranateextract(PG-E)
invivoandusedmultiplepathogenicmechanismsofacnevulgaristoestablishananti-acnesystem
model. First,weusedHaCaTcells,animmortalhumankeratinocytecellline,asatargettosimulate
testosterone-induced epithelial cells and keratin accumulation. Increased bacterial lipase activity
facilitatesthesynthesisoffattyacidsandbacterialgrowth.Inaddition,Propionibacteriumacnes(P.acnes)
and Staphylococcus aureus (S. aureus) are the major pathogenic and most abundant bacteria on the
skinsurfaceofacne-affectedskin. Eventually,thecombinationofbacteriawiththeabove-described
situationswillresultininflammationoftheskin. Hence,weusedTHP-1,ahumanmonocyticcell
line,asthetargettosimulateanti-inflammatoryactivitiesagainstdifferentinducers. Thepathogenic
mechanismofacneandexperimentaldesignofourplatformissummarizedinTable1.
Table1.Thepathogenicmechanismofacneandexperimentaldesignofourplatform.
Pathogenesisof EpithelialCell& Sebum
BacterialGrowth SkinInflammation
AcneVulgaris KeratinAccumulation Accumulation
Heat-killedP.acnes-induced
Invitro Testosterone-induced Propionibacteriumacnes RAW264.7
Anti-lipaseactivity
experimentaldesigns HaCaTcellproliferation Staphylococcusaureus Heat-killedP.acnes-induced
THP-1
Effective
Punicalagin(1) Punicalin(2) StrictininA(3) GranatinB(4)
hydrolysabletannins
P.acnes:Propionibacteriumacnes.
PunicagranatumLinne(pomegranate)belongstothePunicaceaefamily. IntheMediterranean
area,pomegranateiscommonlyusedasafruitfortherapeuticagents,foods,andcosmetics. However,
intraditionalChinesemedicine,pomegranatewasrecordedasanastringentagent[13]. Evidencehas
shown that the pharmacological effects of pomegranate are its anti-inflammatory, anti-oxidative,
anti-lipoperoxidative, anti-bacterial, and anti-tumor activities [13–17]. In our previous study, four
hydrolysabletannins,punicalagin(1),punicalin(2),strictininA(3),andgranatinB(4),wereisolated
frompomegranateusingcolumnchromatographycombinedwithinvitroanti-inflammatory-guided
fractionation[13]. Thesefourcompoundsandthe70%acetonepomegranateextractdisplayedboth
invitroandinvivoanti-inflammatoryeffects. However,fewresearchershavediscussedtheanti-acne
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Int. J. Mol. Sci. 2017, 18, 141 3 of 12
eofff epcotmsoefgpraonmaeteg.r aInn aatded. Tithioenre, ftohree ,parninecvipaalul aatniotin-apcnlaet fcoormmpwonasenutsse doft pooemxpelgorraentahtee aanrtei -aalcsnoe deifsfceucstsseodf
pino mtheisg sratundayte. . Inaddition,theprincipalanti-acnecomponentsofpomegranatearealsodiscussedin
thisstudy.
2. Results and Discussion
2. ResultsandDiscussion
2.1. Pomegranate Attenuate P. acnes-Induced Wistar Ear Edema
2.1. PomegranateAttenuateP.acnes-InducedWistarEarEdema
In vivo anti-acne effects are difficult to assess because of a lack of animal models. We set up an
animIanl vmivoodealn ttoi- asicmneuleaftfee catcsnaer efodrimffiactuioltnt boya sdsieressctblye cianujescetionfga Pl.a accknoesf ianntiom aa rlamt’os deealrs .anWde esveatluupataend
athneim ina lvmivood ealnttoi-saicmnue laetfefeacctns eoffo PrmGa-Eti.o Wnbisytadri rreactt leyairnsj eecxtihnigbiPt .eadcnemesain wtohaenra tl’isvee aPr.a ancdneesv aislu iantjeedcttehde.
iAnsv sivhoowannt ii-nac Fniegeufrfee c1ts, oraftP eGa-rE e.dWeimstaar irna ttheea rPsGex-hEi boiitnetdmeemnat wgrhoeunpl iwveasP .saigcnneisfiicsainntj elcotwede.r Athsasnh otwhant
iinn Fthigeu vreeh1i,crlea tceoanrtreodle gmroauinp.t hIne PthGis- Emooidnteml, elinvteg Pro. uapcnwesa wssaisg ninifijeccatendt lionwtoe rWthisatnart hraatt ienartsh.e Wveeh fiicrlset
csoungtgreosltg trhoaut pP.GIn-Et hcaisnm inohdiebli,t ltihvee gPr.oawcntehs owf aPs. iancnjeecst,e rdesinutlotinWgi isnta ar lroawteearr isn.fWlamefimrsattosruyg sgteasttutsh aantdP Gle-sEs
ceaanr eindheimbiat. thegrowthofP.acnes,resultinginalowerinflammatorystatusandlessearedema.

FFiigguurree 11.. AAnnttii--eeaarr eeddeemmaa eeffffeeccttss ooff ppoommeeggrraannaattee eexxttrraacctt ((PPGG--EE)) ooiinnttmmeenntt aaggaaiinnsstt aa PP.. aaccnneess iinnjjeeccttiioonn;;
** pp << 00..0055,, ccoommppaarreedd ttoo tthhee vveehhiiccllee ggrroouupp oonn tthhee ssaammee ddaayy;; DDaattaa aarree pprreesseenntteedd aass tthhee mmeeaann ±± SSDD;;
SSiiggnniifificcaanncceew waassc caalclcuulalateteddu usisnigngS tSutduednetn’st’ts- tte-stetsbty bSyP SSPSSsoS fstwofatwreavr.e1 5v;.E15a;c hEagcrhou gprocuonpt acoinnetdaieniegdh teriagthst.
rats.
2.2. PomegranateDisplayedNoSkinIrritation
2.2. Pomegranate Displayed No Skin Irritation
Asshowninpreviousstudies,amoderntherapeuticstrategyforacneistouseretinoicacidor
As shown in previous studies, a modern therapeutic strategy for acne is to use retinoic acid or
salicylicacid. However,skinirritationanddesquamationareobviousside-effectsofthesechemical
salicylic acid. However, skin irritation and desquamation are obvious side-effects of these chemical
agents. Inthisstudy,asingle-doseskinirritationtestwasperformedbymodifyingtheDraizetest[18].
agents. In this study, a single-dose skin irritation test was performed by modifying the Draize test
PG-EdisplayednoskinirritationinWistarskinat0.1–10mg/sitefor24–48h,indicatingtheexcellent
[18]. PG-E displayed no skin irritation in Wistar skin at 0.1–10 mg/site for 24–48 h, indicating the
safetyofPG-E(datanotshown).
excellent safety of PG-E (data not shown).
2.3. PomegranateSignificantlyInhibitedP.acnesandS.aureusGrowth
2.3. Pomegranate Significantly Inhibited P. acnes and S. aureus Growth
BecausePG-EsignificantlyattenuatedP.acnes-inducedWistarearedema,wecontinuedtoexplore
the inBevciatruosea cPtiGvi-tEie ssiganndifitcraynttloy daitstceonvueartetdh ePm. aeccnheasn-iinsmdusc.ePdG W-Eisdtaisr pelaayr eeddesmtroan, gweer acnotni-tbinaucetedr itaol
aecxtpivloitrye athgea inins tvPit.raoc naecstitvhiatinesa gaanidn sttryS .taou dreiuscs.ovTehre tdhiea mmeetcehraonfistmhes.i nPhGib-Eit iodnispzolanyeedo fsPtrGo-nEgearg aainntsit-
Pb.acatcenreiasl raacntigveidty 1a1g.3ai–n1s7t. 1P.m acmne.s tMhaonre aogvaeirn,stth Se. ainuhreiubsit. iTohnez doinaemoetfePr Gof- Ethae ginaihnisbtitSio.na uzroenues orfa nPgGe-dE
1a2g.a6i–n1s5t .P9. macmne.s Wraengceadlc u11la.3te–d17t.h1 emrmat.i oMoofrethoeveinr,h tihbei tiinohnibziotinoen czoomnep aorfe PdGt-oE paegnaiicnisllti Sn.. aRuraetuioss raonfgthede
i1n2h.6ib–i1t5io.9n mzomn.e WofeP cGa-lEcualgaateinds tthSe. aruartieou soaf ntdheP .inahcnibesitwioenr ezo0n.4e5 1coamndpa0r.5e4d4 ,tore pspenecictiivlleinly. R(Taatbioles 2o)f. the
inhibition zone of PG-E against S. aureus and P. acnes were 0.451 and 0.544, respectively (Table 2).
Int.J.Mol.Sci.2017,18,141 4of12
Table2.Anti-P.acnesandS.aureuseffectsofpomegranateextract(PG-E).
PG-ESamples DiameterofInhibitionZone(mm)
PG-E(mg/disc) P.acnes
0.25 11.3±0.6
0.5 15.1±0.5
1.0 17.1±1.4
PG-E(mg/disc) S.aureus
2.5 12.6±0.3
5.0 15.4±0.9
10.0 15.9±0.7
BoththeMIC(minimuminhibitoryconcentration)andMBC(minimumbactericidalconcentration)
of PG-E against P. acnes and S. aureus were 62.5 µg/mL. Several researchers showed that
pomegranatepeelextractdisplayedsignificantanti-bacterialeffectsagainstLactobacillusacidophilus,
Staphylococcus epidermidis, Escherichia coli, Salmonella enteritidis, and Listeria monocytogenes [19–21].
However,fewresearchersdiscussedtheanti-bacterialeffectsofpomegranateagainsttheskin-related
bacterium,P.acnes. Thisisthefirststudytoexploretheanti-P.acneseffectsandtrytoidentifythe
activecomponentsofpomegranate.Throughabioguided-fractionation-isolationsystem,anti-microbial
activities of four hydrolysable tannins in PG-E, punicalagin (1), punicalin (2), strictinin A (3), and
granatinB(4),weretested. ResultsshowedthatCompounds1and2havesignificantanti-bacterial
abilitiesagainstP.acnesandS.aureus,whiletheMICof1againstP.acnesandS.aureuswere6.25µg/mL
(1.3µM)and12.5µg/mL(2.2µM);MICof2againstP.acnesandS.aureuswere6.25µg/mL(1.3µM)and
12.5µg/mL(2.2µM),respectively. Compounds3and4werelesseffectiveagainstP.acnesandS.aureus
(Table3). InReddyetal.’sstudy[15],Compounds1and2werealsoisolatedfrompomegranatejuice.
Compound1displayedsignificantanti-bacterialeffectsagainstPseudomonasaeruginosa,Candidaalbicans,
andCryptococcusneoformans. Anti-oxidativeeffectswerealsofoundfor1and2,suggestingthatPG-E
canacttoaugmenthuman’santi-bacterialandanti-oxidativecapacities[15]. Anti-acnebacteriaeffects
oftannincompoundsinherbalextractsarealsowell-reported,i.e.,tannincomponentsfromCassiatora,
Garcinia mangostana, Momordica charantia, Phyllanthus emblica, and Terminalia arjuna [22]. However,
hydrolysabletanninsinpomegranatearefirstlyreportedfortheirmultipleactivitiesagainstacnein
thisstudy.
Table3.Anti-P.acnesandS.aureuseffectsofpomegranateextract(PG-E).
Anti-BacterialActivity
MIC/MBC(µg/mL) 1 2 3 4
P.acnes 6.25/12.5 6.25/12.5 12.5/25 100/-
S.aureus 12.5/25 12.5/25 25/50 12.5/25
MIC:minimuminhibitoryconcentration;MBC:minimumbactericidalconcentration.
2.4. PomegranatePolyphenolsCausedShrinkageandDamageinP.acnesandS.aureus
Morphologicalchangesareaclearindicatorformonitoringtheprocessofbacterialdeath. SEM,
a powerful electron microscope, can be used to investigate diversification of a bacterium’s shape.
DifferencesbetweentreatmentswithfourhydrolysabletanninsinP.acnesandS.aureusareillustrated
in Figure 2. As shown in Figure 2A, outer membranes of the control group were smooth and
rod-shaped. However,aftertreatmentwith1or2(100µg/mL)for12h,S.aureusproducedaround
grainsandexhibitedbulgingordeformationofitssphericalshape. Intra-inclusionswerefoundtohave
effluxedoutside(representativesareindicatedbyarrows),andthebacteriawereobviouslyswollen.
InRabieetal.’sstudy,thebacterialstructureisanimportantpointforitsnormalphysiology,suchas
theoutermembraneintegrity[23]. Wesuggestedthathydrolysabletanninsinpomegranatedamaged
Int.J.Mol.Sci.2017,18,141 5of12
the structural integrity and bacterial proteins, leading to changes in the integrity and function of
Int. J. Mol. Sci. 2017, 18, 141 5 of 12
bacteria. Compounds1and2alsosignificantlyinhibitedthegrowthofP.acnes(Figure2B).However,
PP.. aaccnneess oorriiggiinnaallllyy hhaadd aa sspphheerriiccaall sshhaappee aanndd ddiissppllaayyeedd aa ssmmooootthh,, ccoommpplleettee ssuurrffaaccee.. TThhee bbaacctteerriiaall
ssuurrffaaccee eexxhhiibbiitteedd oobbvviioouuss sshhrriinnkkaaggee, ,ddaammaaggee ttoo tthhee ssuurrrroouunnddiinnggss,, aanndd iinnccrreeaasseedd ggrraannuulleess aafftteerr
ttrreeaattmmeenntt wwiitthh 11 aanndd 22 ffoorr 1122 hh.. BBiiooffiillmmss ooff bbaacctteerriiaa ppllaayy aann iimmppoorrttaanntt rroollee iinn tthheeiirr vviirruulleennccee aanndd
ppaatthhooggeenniicciittyy.. AAnnootthheerr aannttii--bbaacctteerriaial lssttrraatteeggyy iiss ttoo iinnhhiibbiitt bbiiooffiillmm ffoorrmmaattiioonn.. IItt wwaass rreeppoorrtteedd tthhaatt
ppoommeeggrraannaattee ssiiggnniiffiiccaannttllyy ddeeccrreeaasseess bbiiooffiillmm ffoorrmmaattiioonn bbyy SS.. aauurreeuuss [[2244]].. AAnnttii--bbiiooffiillmm ffoorrmmaattiioonn
ccaappaacciittiieess ooff ppoommeeggrraannaattee wwiillll bbee aann iimmppoorrttaanntt ttaarrggeett iinn ffuuttuurree ssttuuddiieess..

FFiigguurree 22.. SShhrirninkkaaggeea nadndda mdaamgeagofe So.af uSre. uasu(Are)uas n(dAP) .aacnnde sP(B. )aacfnteesr tr(Bea)t mafetnert wtritehatfmouernht ywdriothly sfaobulre
htayndnroinlyssiasbollea tteadnnfrionms ipsoolmateegdr afnroamte epxotmraecgtr(aPnGa-tEe ):expturancict a(lPagGi-nE)(:1 )p,upnuincaiclaagliinn (2(1),), stpruicntiincainlinA ((23)), ,
satnridctignriann Aat i(n3)B, a(n4)d; Agrlalnpahtiont oBs (w4)e; reAltla kpehnotwosi twhesrcea ntankineng weleitcht rsocnanmniicnrgo secloepctyr;onC :mciocnrotrsoclopgyro; uCp: ;
cTohnetrcooln gcreonutpra; tTiohne ocofn1cteon4trwataiosn1 0o0f 1µ gto/ m4 wL;aDs 1a0ta0 aµrge/fmroLm; Dthartae earsee pfraormate therxepee sreimpaernattse, tehxepepriicmtuernetso,f
tohnee poicftwurhei cohf iosnseh oowf wn;hTichhe iasr rsohwowinnd; iTcahtee sararodwis riunpdtiicoantessi tae odfisbraucptteiroina; sSictea loefb baarcotefrcioan; tSrcoallge rboaurp oifs
c5oµnmtro;lS cgarloeubpa riso f5f oµumr;h Sycdarloel ybsaarb olef tfaonunri nhsyadgroailnysstaSb.lea utraenunsiannsd aPg.aaincnste sSi.s a5uµremusa nandd2 Pµ.m a,crneessp iesc t5i vµemly.
and 2 µm, respectively.
2.5. PomegranatePolyphenolsInhibitedLipaseActivity
2.5. Pomegranate Polyphenols Inhibited Lipase Activity
Lipaseisanenzymethathydrolyzeslipids. Somebacteria,suchasP.acnes,cansecretelipase
Lipase is an enzyme that hydrolyzes lipids. Some bacteria, such as P. acnes, can secrete lipase to
to resolve lipids and facilitate lipid-related nutrient absorption from an external medium. In this
resolve lipids and facilitate lipid-related nutrient absorption from an external medium. In this study,
study,afluorometricassayoflipaseactivitywasadministratedwithandwithoutPG-Eandthefour
a fluorometric assay of lipase activity was administrated with and without PG-E and the four
hydrolysabletannins. Comparedtothecontrol,PG-E(200µg/mL)inhibitedlipaseactivityby20%
hydrolysable tannins. Compared to the control, PG-E (200 µg/mL) inhibited lipase activity by 20% as
as shown by a decrease in lipase substrate degradation. The anti-lipase effects of the essential oil
shown by a decrease in lipase substrate degradation. The anti-lipase effects of the essential oil
compositionsofpomegranatepeelextractshavebeenpreviouslyreported,buttheprincipleactive
compositions of pomegranate peel extracts have been previously reported, but the principle active
principlecomponentswerenotdiscussed[25]. Inthisstudy,threehydrolysabletannins,1,3,and4,
principle components were not discussed [25]. In this study, three hydrolysable tannins, 1, 3, and 4,
at 200 µg/mL significantly inhibited the lipase activity by 39.8%, 34.7%, and 35.2%, respectively
at 200 µg/mL significantly inhibited the lipase activity by 39.8%, 34.7%, and 35.2%, respectively
(Figure3).
(Figure 3).
2.6. PomegranatePolyphenolsInhibitedHaCaTCellProliferation
2.6. Pomegranate Polyphenols Inhibited HaCaT Cell Proliferation
Because keratinocyte over-proliferation is a crucial event in acne formation, testosterone was
usedBaescaaunsei nkdeuracteirnotocysttei mouvelart-eprHolaifCearaTticoenl lips rao lcirfeurcaiatilo env.enRte sinu latscnseh ofworemdatthioant, tteessttoosstteerroonnee twimaes -
uasnedd daos saen-d inepdeuncdere ntotl ystiimnduulacteed HHaaCCaaTT cceelll lpprroolilfieferraatitoionn. .RFeusurtlhtse rs,htoewstoesdt ethroant etessitgonsitfiercoannetl ytimined-u acnedd
dporoseli-fdeerapteinondeonftklye raintidnuocceydte sHaatC1a0Ta ncdel1l 0p0rµogli/femraLtifoonr. 7F2uhrt(hFeigr,u rtees4t)o.sterone significantly induced
proliferation of keratinocytes at 10 and 100 µg/mL for 72 h (Figure 4).
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Int. J. Mol. Sci. 2017, 18, 141 6 of 12
Int. J. Mol. Sci. 2017, 18, 141 6 of 12

FigFuirgeur3e. 3L. ipLaipsaesea caticvtiivtyityi nihnihbibitiitoionno offp poommeeggrraannaattee eexxttrraacctt ((PPGG--EE)) aanndd ffoouur rhhyyddrorolylsyasbalbel etatnanninnisn s
Figure 3. Lipase activity inhibition of pomegranate extract (PG-E) and four hydrolysable tannins
incilnucdleuddepdu pnuicnailcaaglaingi(n1 ()1,)p, upnuinciaclainlin( 2(2),),s strtricicttininiinn AA ((33)),, aanndd ggrraannaattiinn BB (4(4);) ;TT hhe ecocnocnecnetnrtartaiotino onf oPfGP-GE,- E,
included punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4); The concentration of PG-E,
andanCFadoni gCmduo pCrmeoo pmu3o.n puLdonisupdn1asds– e1s4 –1aw4–c 4tawi vswai2tsay0 s2 0 2i0n00µh0 gµi µb/gigmt/i/momLnLL; ;D;o DDfa aapttatoaam a aarrereeeg rfffrarroonommamt e ttt hhherrrxeeeteree ar rcreeetp ppe(ePaeatGastt.s-s E.. ) and four hydrolysable tannins
included punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4); The concentration of PG-E,
and Compounds 1–4 was 200 µg/mL; Data are from three repeats.

Figure 4. Testosterone significantly induced keratinocyte over-proliferation in a dose- and time-
FigFuirgeur4e. 4. TTeessttoosstteerroonnee sisgignnifiicfiacnatnlytl yindinudceudc ekderakteinraotciyntoec yovteer-opvreorl-ifperroaltiif oenr aitni oan dionse-a adnods eti-mae-nd
dependent manner.
timdee-dFpieegnpuderneen d4te. mnTtaemnstnoaesnrtne. reorn.e significantly induced keratinocyte over-proliferation in a dose- and time-
dTehpeernedfoenret ,m tarnenaetmr. ent with testosterone at 10 µg/mL for 72 h was used to assess which
Therefore, treatment with testosterone at 10 µg/mL for 72 h was used to assess which
Thhyedrreofloyrsea,btlree taatnmneinnst cwoiutlhd tienshtoibsitte HroanCeaaTt c1e0llµ pgr/omlifLerfaotiro7n2. Rhewsualst ushsoewdetod athssaet sfsouwrh hicyhdrhoylydsraobllyes able
hydrolyTshaebrleef otraen, ntirnesa tcmoeunldt iwnihthib itte sHtoasCtearTo ncee lla tp r1o0l ifµegra/mtioLn .f oRre s7u2l t hs hwowase du stehda t tofo uars shesysd rwolhyicshab le
tannitnasnncoinusl dcoiunldh isbiigtnHifiacCanatTly cdeellcrperaoseli ftehrea tteisotnos.tRereosnuel-tinsdhuocwede dHtahCaatTf oceullr phryodlirfeorlaytsioanb laet t5a0n µngin/msLc ould
tanhnyidnrso clyosualbdle s itgannnifiincsa ncotluyl dd eincrheiabsite HthaeC ateTs tcoesllt eprroonlief-eirnadtiuocne. dR eHsuaClt asTh ocweleld p trhoalitf eforautri ohny darto 5ly0s µabgl/em L
signifi(Fciagnutrley 5)d. eTcwreoa rseesetahrecht teesatmosst edrisocnues-siendd tuhec epdroHteactCivaeT efcfeeclltsp orfo ploifmereagtriaonnataet ag5a0inµsgt /UmV-Lind(Fuicgeudr e 5).
(Fitgaunrnei n5s) .c Touwldo rseigsneiafriccahn ttelya mdesc dreiascsue stsheed t etshteo spterorotencet-iivned uefcfeedc tHs aoCf apTo mceellg praronlaifteer aagtiaoinn satt U50V µ-ign/dmuLc ed
HaCaT oxidative stress and markers of photoaging [26,27]. Taken together with results of this study,
Two research teams discussed the protective effects of pomegranate against UV-induced HaCaT
Ha(CFiagTu roex 5id).a Ttiwvoe sretrseesasr cahn dte ammasr kdeisrcsu osfs epdh othtoe apgriontgec [t2iv6e,2 e7f]f.e Tctask oefn p toomgeetghrearn wateit hag raeisnusltt Us oVf- itnhdisu scteudd y,
we suggest that pomegranate can act as a UV protector and growth regulator of skin keratinocytes.
oxiwdaeHt siavuCegagsTet srotex stihdsaaattni pvdeo msmtreaegsrrska aennrasdte om cfaaprnkh eaorcstt o oaafs g paih nUogtVo[ a2pg6rio,n2tg7e ][c2.to6T,r2 aa7k]n.e dTn agktroeongw ettothhg eeretrhgweurl iawthtiotrhr e orsfeu ssulktlsitnso okffe ttrhhaiitssin ssottucudydytey, s,. we
suggewste tshuagtgpesotm theagt rpaonmaetegrcaannataec ctaans aactU aVs ap UroVt epcrtootercatonrd agndro gwrothwtrhe greugluatlaotroor fofs kskinink keerraattiinnooccyytteess. .

Figure 5. Cytotoxicity of different hydrolysable tannins (A); Anti-keratinocyte over-prolif era tive
effects of hydrolysable tannins (B); B: blank; C: control; * p < 0.05, compared to the testosterone-only
FigFuigrue re5 . 5C. yCtyottootxoixciictiyty o of f ddififfeferreenntt hhyyddrroollyyssaabbllee ttaannnniinnss (A(A);) ; AAnntit-ik-ekreartaintioncoyctye teo voevr-eprr-oplriofelrifaetrivaeti ve
Figurger5o.upC;y Dtoattoa xaircei tpyreosfednitfefde raesn tthhe ymderaonl y±s SaDbl;e Stiganninfiicnasn(cAe )w;aAsn ctail-ckuelraatetidn uosciyntge ao vonere--pwraoyl iafenraalytisvise oeff fects
effeefcftesc tosf ohf yhdyrdorloylsyasbalbel et atannnnininss ( (BB)); ; BB:: bbllaannkk;; CC:: ccoonnttrrooll;; ** p p < < 0 0.0.055, ,c ocommpapraerde dto t oth teh tee stteossttoesrtoenreo-noen-loyn ly
ofhydvarorilaynscaeb (lAeNtaOnVnAin) sv(iaB S);PBSS: bsolaftnwka;rCe :vc.1o5n; tDroatla; *arpe< fr0o.m05 t,hcreoem rpepaeraetds. tothetestosterone-onlygroup;
groguropu; pD; aDtaat aar aer ep rperseesnentetded a ass t hthee m meeaann ±± SSDD;; SSiiggnniiffiiccaannccee w waass c caalcluculaltaetde du suisnign ga oan oen-we-awya ayn aalnyasliys soifs of
D ataarepresentedasthemean±SD;Significancewascalculatedusingaone-wayanalysisofvariance
vavriaarniacnec (eA (NANOOVVAA) v) ivai aS SPPSSSS s sooffttwwaarree vv..1155;; DDaattaa aarree ffrroomm t thhrreeee r ereppeaetast.s .
(ANOVA)viaSPSSsoftwarev.15;Dataarefromthreerepeats.
Int.J.Mol.Sci.2017,18,141 7of12
Int. J. Mol. Sci. 2017, 18, 141 7 of 12
22..77.. PPoommeeggrraannaattee PPoollyypphheennoollss AAtttteennuuaatteedd HHeeaatt--KKiilllleedd PP.. aaccnneess--IInndduucceeddN NOOa annddP PGGEE2 PPrroodduuccttiioonn bbyy RRAAWW
2
226644..77 CCeellllss
IInn oouurr pprreevviioouuss ssttuuddyy,, wwee sshhoowweedd tthhee aannttii--iinnflflaammmmaattoorryy eeffffeeccttss ooff PPGG--EE aanndd ffoouurr hhyyddrroollyyssaabbllee
ttaannnniinnss aaggaaiinnsstt LLPPSS ((lliippooppoollyyssaacccchhaarriiddee))--ttrreeaatteedd RRAAWW 226644..77 cceellllss [[1122]].. MMoorreeoovveerr,, iinn tthhiiss ssttuuddyy,, wwee
uusseedd HHKKPP ((hheeaatt--kkiilllleedd PP.. aaccnneess)), ,ininssteteaadd oof fLLPPS,S t,ot osismimulualtaet tehteh ierriirtraittiaotnio annadn idnfilnaflmammamtioatni ocnaucsaeuds ebdy
bay Pa. Pac.naecsn eisnfiencfteicotnio. nA.sA sshoshwonw inn inFiFgiugruerse 66AA,B,B, ,HHKKPP aatt 2255––220000 µµgg//mmLL ddoossee--ddeeppeennddeennttllyy aanndd
ssiiggnniifificcaannttllyy iinndduucceedd NNOO ((nniittrriicc ooxxiiddee)) aanndd PPGGEE22 ((PPrroossttaaggllaannddiinn EE22)) pprroodduuccttiioonn bbyy RRAAWW 226644..77 cceellllss..
HHoowweevveerr,, aa ggoooodd rreellaattiioonnsshhiipp bbeettwweeeenn ththee ininflflaammmmaatotoryry rersepspoonnses eanadn dthteh edodsoes oefo HfKHPK oPcocuccrurerdre adt
a5t0–5200–02 0µ0gµ/mg/Lm. LU.ltUimltiamtealyte, lyw,ew cehcohsoes e10100 0µµgg/m/mL Lofo fHHKKPP aas soouurr iinnffllaammmmaattiioonn iinndduuccttiioonn ddoossaaggee..
CCoommppoouunnddss 11,, 22,, 33,, aanndd 44 aatt 5500 µµMM ddiissppllaayyeedd oovveerr 5500%% NNOO iinnhhiibbiittoorryy eeffffeeccttss aaggaaiinnsstt HHKKPP--iinndduucceedd
RRAAWW 226644..77 cceellllss ((FFiigguurree 66CC)).. IInn aaddddiittiioonn,, 11 aanndd 44 aatt 5500 µµMM aallssoo ssiiggnniifificcaannttllyy iinnhhiibbiitteedd aabboouutt 5500%% ooff
PPGGEE22 pprroodduuccttiioonn iinn HHKKPP--ttrreeaatteedd RRAAWW 226644..77 cceellllss ((FFiigguurree 66DD))..

Figure 6. Heat-killed P. acnes (HKP) dose-dependently increased nitric oxide (NO) (A) and
Figure 6. Heat-killed P. acnes (HKP) dose-dependently increased nitric oxide (NO) (A) and
pprroossttaaggllaannddiinnE E(2P G(PEG)E(2B) )(pBr)o dpurcotidouncbtiyonR AbWy 26R4A.7Wce ll2s6;4A.7n tic-einllflsa; mAmnatit-oirnyfleafmfemctsatoofrhyy derfofelyctssa bolef
2 2
thayndnrionlsysaagbalien sttanHnKinPs- iangdauincestd HNKOP(-Cin)dauncdedP GNEO ((CD)) apnrdod PuGctEio2 n(Db)y pRrAodWuc2t6io4n.7 bcye llRsA;DWa t2a6a4r.7e fcreolmls;
2
Data are from three repeats.
threerepeats.
2.8. Pomegranate Polyphenols Attenuated Heat-Killed P. acnes-Induced IL-8 and TNF-α Production by
2.8. PomegranatePolyphenolsAttenuatedHeat-KilledP.acnes-InducedIL-8andTNF-αProductionby
TTHHPP--11 CCeellllss
CCyyttookkiinnee,, ssuucchh aass IILL--88 ((iinntteerrlleeuukkiinn 88)) aanndd TTNNFF--αα ((ttuummoorr nneeccrroossiiss ffaaccttoorr--αα)),, rreelleeaasseess aarree aallssoo kkeeyy
ppooiinnttss iinn tthhee iinnflflaammmmaattoorryy ssttaattuuss ooff hhuummaanna accnneel leessiioonnss[ [2288]].. AAss sshhoowwnn iinn FFiigguurree 77,, HHKKPP ((110000 µµgg//mmLL))
oobbvviioouussllyy iinndduucceedd IILL--88 aanndd TTNNFF-α-α prpordoudcutciotino nini na hauhmuamna mnomnooncyotciyc tciecllc elilnlel.i nIne. FIignuFrieg 7uAre, t7hAe ,fothuer
fhoyudrrohlyydsarobllye staabnlneintasn innihnisbiitnehdi bILit-e8d aInLd- 8TaNnFd-αT pNrFo-dαucptrioond uinct idoonsein-dedpoesne-ddeenpt emndaennnterms aant n5e0r–s20a0t
5µ0g–/2m0L0, µagn/dm coLm, panoduncdo m4 hpaodu nthde 4strhoandgethste insthriobnitgoersyt aicnthivibitiyto. rAynatic-tTiNviFty-.α aAcntitvi-iTtiNesF o-fα paocmtievgitriaensaotef
ppoomlyepghreannoaltse arpeo slyhpohwenn ionl sFiagruerseh 7oBw. CnoimnpFoiguunrdes 72B, 3.,C aonmd 4p oexuhnidbsite2d, 3si,gannifdic4anetx ihnihbiibtietdorsyi ganctiifivcitainest
ianghaiibnistot rTyNaFct-iαv iptireosdaugcatiinosnt,T wNiFth-α 4p arolsdou cdtiisopnl,awyiinthg 4thaels ostrdoisnpgleasyti negffethcte. sTtrhoins gise sttheef feficrts.tT thimisei stthhaet
fiCrosmttpimouentdha 4t hCaosm bpeoenu nredp4ohrtaesdb teoe hnarveep oarntteid-intoflahmavmeaatnotriy-i ncyfltaomkimnea teofrfeycctsy.t o kineeffects.
Int.J.Mol.Sci.2017,18,141 8of12
Int. J. Mol. Sci. 2017, 18, 141 8 of 12

FigurFeig7u.rAen 7t.i -Ainnttei-rilnetuerklienuk(IiLn )(-I8L()A-8) (aAn)d antudm tuomrnore cnreocsriossfisa cfatoctro(rT (NTNF)F-)α-α( B(B))r erleeleaasseef rfroommT THHPP--11 cceellllss after
heat-kafitlelerd hPea.ta-kcnilelesd( HP.K aPcn)eisn d(HuKctPio) nin;d*upct<io0n.;0 5* ,pc o< m0.p05a,r ecdomtopathreedH toK Pth-eo nHlyKPg-roonulyp ;gDroautpa; aDreatpar aersee nted
asthepmreeseannte±d SaDs ;thSeig nmiefiacna n±c eSDw;a Ssicganlicfuiclaantceed wusaisn gcaalcounlaet-ewd auysiannga lay soinseo-fwvaayr iaannacleys(iAs NofO vVaAri)anbcye SPSS
(ANOVA) by SPSS software v.15; Data are from three repeats.
softwarev.15;Dataarefromthreerepeats.
3. Materials and Methods
3. MaterialsandMethods
3.1. General
3.1. General
Four active components of pomegranate, punicalagin (1), punicalin (2), strictinin A (3), and
Fgoraunratainc tiBv e(4c),o mwperoen eisnotlsateodf panodm eidgernatnifaieted, ipn uonuicra lpargeivniou(1s) ,stpuudyn ic(Failginure(2 )3,) s[t1r3i]c.t in3-i(n4.5A- (3),
and DgirmanetahtyinlthiBazo(l4-2),-ylw)-2e.r5e-dipishoelnayteltdetraaznodliumid enbtriofimeidde in(MoTuTr), pdreimvieothuysl sstuuldfoyxid(eF ig(uDrMeSO3)), [13].
3-(4.5l-iDpoipmoelythsayclcthhairaizdoe l(-L2P-ySl)), -a2n.5d- dotihpehre cnhyelmteictraalsz owleiurem pubrrcohmasiedde fr(oMmT STi)g,mdaim Inedtuhsytlriseus l(fSoaxinidt eLo(uDiMs, SO),
MO, USA). Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI)
lipopolysaccharide(LPS),andotherchemicalswerepurchasedfromSigmaIndustries(SaintLouis,
medium, fetal bovine serum (FBS), anti-biotics, and glutamine were purchased from GIBCO BRL
MO,USA).Dulbecco’smodifiedEaglemedium(DMEM),RoswellParkMemorialInstitute(RPMI)
(Grand Island, NY, USA). The bacterial culture equipment, including an anaerobic atmosphere by
medium, fetal bovine serum (FBS), anti-biotics, and glutamine were purchased from GIBCO BRL
MGC AnaeroPack-Anaero and MGC AnaeroPack-Jar, were purchased from Mitsubishi Gas Chemical
(GrandIsland, NY,USA).Thebacterialcultureequipment, includingananaerobicatmosphereby
Company (Tokyo, Japan). Blood agar plates (BAPs), Bacto™ tryptic soy broth (TSB), and Bacto™
MGCAnaeroPack-AnaeroandMGCAnaeroPack-Jar,werepurchasedfromMitsubishiGasChemical
tryptic soy agar (TSA) were purchased from Difco (Detroit, MI, USA). The assay kits for tumor
Compnaecnryos(iTs ofakcytoor, (JTaNpaF)n-α)., iBnltoeroldeuakgina r(ILp)l-a1tβe,s a(nBdA ILP-s8) ,wBearec tpou™rchtraysepdt ifcrosmoy eBbiroostchien(TceS B(S)a,na nDdiegBoa, cto™
tryptiCcAso, yUSaAga).r (TSA)werepurchasedfromDifco(Detroit,MI,USA).Theassaykitsfortumornecrosis
factor(TNF)-α,interleukin(IL)-1β,andIL-8werepurchasedfromeBioscience(SanDiego,CA,USA).
3.2. Preparation of Pomegranate Extract (PG-E)
3.2. PreparationofPomegranateExtract(PG-E)
Dried peels of pomegranate were purchased from a traditional Chinese medicine store in Taipei
Danrdie iddepneteiflisedo fbpyo Hmsieegnr-aCnhaatnegw Cehraenpg.u Arc vhoauscehdefrr sopmeciamternad (PitGio-n01a)l wCahsi ndeespeosmiteedd iicni nthees Stochreooinl oTfa ipei
Pharmacy, College of Pharmacy, Taipei Medical University (Taipei, Taiwan). Dried peels were
andidentifiedbyHsien-ChangChang. Avoucherspecimen(PG-01)wasdepositedintheSchoolof
extracted by homogenization with 70% acetone. The extracted solution was filtered, evaporated on a
Pharmacy,CollegeofPharmacy,TaipeiMedicalUniversity(Taipei,Taiwan).Driedpeelswereextracted
rotary evaporator, lyophilized, and called PG-E. PG-E and its active components were dissolved in
by homogenization with 70% acetone. The extracted solution was filtered, evaporated on a rotary
10% DMSO to a concentration of 10 mg/mL for subsequent experiments, stored at 4 °C, and used
evaporator,lyophilized,andcalledPG-E.PG-Eanditsactivecomponentsweredissolvedin10%DMSO
within 1 month. Serial dilutions of test solutions with culture medium were prepared before the in
toacvointrcoe nastrsaaytiso. n of10mg/mLforsubsequentexperiments,storedat4◦C,andusedwithin1month.
Serialdilutionsoftestsolutionswithculturemediumwerepreparedbeforetheinvitroassays.
3.3. P. acnes-Induced Ear Edema in Wistar Rats
3.3. P.acnes-InducedEarEdemainWistarRats
The animal use protocol was reviewed and approved by the institutional Animal Care and Use
TChoemamniitmteea lour sPeapnreol t(oIAcoClUwCa/sIArCevUiPew), eTdaipaneid Maepdpircoalv Uednibvyertshitey i(nIAstCitUuCtio AnpaplrAonvaiml naol.C LaAreC-a9n7d- Use
Comm01i2tt2e)e. Aolrl Pmaentheold(IsA inCvUolCv/edIA inC aUnPim),aTl aeixppeeirMimeednitcsa wleUrne ipveerrfsoirtmye(dIA inC aUcCcorAdpanpcreo vwailthn toh.eL pAreCv-i9o7u-s0 122).
relevant guidelines and regulations. Female Wistar rats weighing 250 ± 20 g were bought from
Allmethodsinvolvedinanimalexperimentswereperformedinaccordancewiththepreviousrelevant
BioLASCO Taiwan (Yilan, Taiwan) and kept on a 12 h light/12 h dark cycle at 21 ± 2 °C with food and
guidelinesandregulations. FemaleWistarratsweighing250±20gwereboughtfromBioLASCO
water ad libitum. P. acnes (8 × 107 colony-forming units (CFU)/20 µL) was intradermally injected into
Taiwan(Yilan,Taiwan)andkeptona12hlight/12hdarkcycleat21±2◦Cwithfoodandwaterad
the left ear of rats to induce edema (Figure 8). PBS (phosphate-buffered saline) was injected into the
libitum. P.acnes(8×107colony-formingunits(CFU)/20µL)wasintradermallyinjectedintotheleft
earof ratstoinduceedema(Figure8). PBS(phosphate-bufferedsaline)wasinjectedintotheright
earasavehiclecontrol[29]. Onehourbeforetheinjection,PG-Eointment(10%PG-Einhydrophilic
ointment)wasadministered(eightratspergroup)ontheskinsurfacesoftheleftear. Theperimeter
Int.J.IMnt.o Jl.. MScoil.. 2S0ci1. 72,01187,, 1184,1 141 9 of 129 of12
right ear as a vehicle control [29]. One hour before the injection, PG-E ointment (10% PG-E in
of thheyedarorpehdileimc oainitnmcelundt)e wdasd aiadmmeinteisrtearnedd (heiegihgth rtatasf pteerr gPr.oaucpn)e osno trheP sBkSini nsujercfaticoens oaf fttheer l1ef–t4 eadra. Tyhsew ere
measpuerriemdebteyr ocfa tlhipee erasr. eVdoemluam inecolufdeeadr deidamemetaerc aonudl dhebigehct aalfcteurl Pat. eadcnuess oinr gPBmS uinljteicptliiocna taifotenr 1b–y4 ddiaayms eter,
were measured by calipers. Volume of ear edema could be calculated using multiplication by
height, and circular constant. In the vehicle control group, the rat ear edema reduce volume was
diameter, height, and circular constant. In the vehicle control group, the rat ear edema reduce volume
calculatedastheoriginalvolumeofearedemaafterthePBSinjectionminusthevolumeofearedema
was calculated as the original volume of ear edema after the PBS injection minus the volume of ear
afterinjectionondays1,2,3,and4. InthePG-Eointmentgroup,theratearedemareducevolume
edema after injection on days 1, 2, 3, and 4. In the PG-E ointment group, the rat ear edema reduce
wascalculatedastheoriginalvolumeofearedemaaftertheP.acnesinjectionminusthevolumeofear
volume was calculated as the original volume of ear edema after the P. acnes injection minus the
edemaadministratedwithPG-Eointmentafterinjectionondays1,2,3,and4.
volume of ear edema administrated with PG-E ointment after injection on days 1, 2, 3, and 4.

Figure 8. P. acnes-induced ear edema in Wistar rats.
Figure8.P.acnes-inducedearedemainWistarrats.
3.4. Skin Irritation
3.4. SkinIrritation
The skin toxicity of PG-E was evaluated from a single topical application in Wistar rats. Twenty-
fTouhre hsokuirns btoefxoircei ttyheo tfesPt,G th-Ee bwacakss oefv raaltusa wteedre fsrhoamveda frseine golfe hatoirp (i2c.a5 l× a2p.5p climca2 taito enacihn sWitei)s atanrd rats.
Twencthye-cfkoeudr fhoor uanrsy baebfnoorremthaleittieess t(,inthteegbriatyc kasndof arllaetrsgwiese)r.e Asnh aapvpedrofprreieatoe fchonaciren(2tr.5at×ion2 o.5f cPmG-2Ea (t5e0a µcLh site)
andpchere csakmedplfeo) rwaansy smabenaroerdm oanltioti ethse( isnhtaevgerdi tbyaackn dofa al lWerigsiteasr )r.aAt. nThaep ppartocphreisa wteerceo nsecceunrterda tfioorn a o4f hP G-E
(50µeLxppoesrusrae mpeprlieo)dw aansd srmemeaorveedd ownittoh tdhisetislhleadv wedatbear.c Akto 2f4a hW ainsdta 4r8r aht .aTfthere tphae tacphpeslicwateioren soefc PuGre-dE, fora
4hesxkpino siurrrietaptieorni owdaas nodbsreermveodv, eadndw tihthe edxitsetniltl eodf wthae teevr.idAetnt2 4skhina anldler4g8ich raefatcetriotnh ewaaps pelviaclautaiotendo ffoPr G-E,
each test animal. The acute primary irritation test was applied following the classic Draize test [17].
skinirritationwasobserved,andtheextentoftheevidentskinallergicreactionwasevaluatedforeach
testanimal. TheacuteprimaryirritationtestwasappliedfollowingtheclassicDraizetest[17].
3.5. Anti-Bacterial Activities against P. acnes and S. aureus
3.5. Anti-PB.a acctneersi a(lBACRctCiv1i0ti7e2s3a) gaanidn sSt. Pau.arecunse (sBaCnRdCS 1.0a7u8r1eu) ws ere obtained from the Bioresource Collection
and Research Center (Hsinchu, Taiwan). P. acnes was cultured in BAP with an anaerobic atmosphere
P.acnes(BCRC10723)andS.aureus(BCRC10781)wereobtainedfromtheBioresourceCollection
using MGC AnaeroPack-Anaero and MGC AnaeroPack-Jar (Mitsubishi Gas Chemical Company).
and Research Center (Hsinchu, Taiwan). P. acnes was cultured in BAP with an anaerobic
S. aureus was cultured in Bacto™ TSB (Difco). PG-E, punicalagin (1), punicalin (2), strictinin A (3),
atmoasnpdh gerraenuatsinin Bg (M4) GwCereA tnesateerdo aPgaacikns-At Pn. aaecnroes abnyd thMe pGaCpeAr ndiasecr-doiPffaucskio-Jna mr(eMthiotdsu. Abilsl hexipGearismCenhteaml ical
Comppraonceyd)u.reSs. wauerreeu pserwfoarsmceudl tauccroerddiinng Btoa cotuor™ prTevSiBou(sD sitfucdoy). wPitGh -sEli,ghptu mniocdailfaicgaitnion(1 [)1,7p].u Fnreicsahlliyn (2),
strictgirnoiwnnA P. a(3cn),es awnads dgilruatneadt winithB B(a4c)tow™e TrSeB,t easntde d10 amgLa ionfs ptrePp.araecdn ebsacbteyriat h(4e ×p 1a0p8 CerFUd/imscL-)d wifafus sion
methaosedp.tiAcallllyex apdedreidm teon 9t0a lmpLr oofc estdeurirliezsedw mereedpiae (rTfSoArm) aetd 45a c°Cco irnd ai nwgatteor obuatrhp. Trehvei soeuesdesdtu adgyarw miethdisal ight
modwifiecrae tiimonm[e1d7ia].telFyr emsihxleyd garnodw pnouPr.edac innetso wa aPsetdrii lduistehd. PwreipthareBda cstaom™pleT SdBis,csa n(8d m1m0 minL dioafmperteerp) ared
bactewreiare (a4d×ded1.0 I8n CadFdUit/iomn,L 1)0w Ua osf apseenpictiicllainll ywaasd udseedd atos t9h0e pmoLsitoivfe sctoenritlriozle. dPlamteesd wiaer(eT iSnAcu)baatte4d5 ◦C
under anaerobic conditions at 37 °C for 24 h, and inhibition zones in millimeters were measured.
in a water bath. The seeded agar media were immediately mixed and poured into a Petri dish.
Each experiment was repeated in triplicate. The experimental protocol of anti-S. aureus (0.5 × 108
Preparedsamplediscs(8mmindiameter)wereadded. Inaddition,10Uofpenicillinwasusedasthe
CFU/mL) activities was the same as that described above with some modifications. The anti-bacterial
positivecontrol. Plateswereincubatedunderanaerobicconditionsat37◦Cfor24h,andinhibition
activity is expressed as the diameter of the inhibition zone against the test microorganisms as follows:
zonesinmillimetersweremeasured. Eachexperimentwasrepeatedintriplicate. Theexperimental
ratio (%) = (diameter zone of sample/diameter zone of penicillin) × 100. An inoculation loop was used
protocolofanti-S.aureus(0.5×108 CFU/mL)activitieswasthesameasthatdescribedabovewith
to collect bacteria from the clear zone on an agar plate. The inoculation loop was enriched in TSB for
some24m ho. Aditfi thcae teinodn so.f Tinhceubaantitoi-nb, aTcStBe rwiaalsa pcutitv ointytoi tsheex spurrefascsee dof ansuttrhieendt iaagmare ptelarteosf atnhde iinnchuibbaitteiodn atz one
again37s t°tCh efoters t24m hic.r oTohreg amniinsimmsumas bfoalcltoewricsi:draal tcioon(c%en)t=ra(tdioina m(MetBeCr)z ownaes oefsstiammaptelde /adsi atmhee tleorwzeostn eof
peniccoilnlicne)nt×rat1io0n0 .oAf nthien osacmulpalteiso nwlhoeorep nwoa vsiusisbeled gtorocwotlhle wctabs aocbteseriravefdro. mThteh meicnleimarumzo ninehoibnitoarny agar
plate. The inoculation loop was enriched in TSB for 24 h. At the end of incubation, TSB was put
ontothesurfaceofnutrientagarplatesandincubatedat37◦Cfor24h. Theminimumbactericidal

concentration(MBC)wasestimatedasthelowestconcentrationofthesampleswherenovisiblegrowth
wasobserved.Theminimuminhibitoryconcentration(MIC)wasestimatedasthelowestconcentration
ofthesampleswherevisiblegrowthwasobserved.
Int.J.Mol.Sci.2017,18,141 10of12
3.6. ScanningElectronMicroscopy(SEM)
P.acnesandS.aureuswereculturedinTSBtotheexponentialphase. Aftercountingbacterial
numbers,P.acnesandS.aureuswereseededin96-wellplatesandincubatedwithsamplesat37◦Cfor
12h. Theculturebrothwascentrifugedat10,000rpmfor10minandwashedwithphosphate-buffered
saline (PBS) twice. Pellets were fixed with 2.5% buffered glutaraldehyde at 4 ◦C for 2 h and then
post-fixedin1%bufferedosmiumtetroxidefor2h.Afterthefixationprocess,bacteriaweredehydrated
inagradedseriesofethanolconcentrations(35%,50%,70%,85%,95%,and100%,w/v),andthese
solventswerereplacedinturnwithliquidcarbondioxidebycriticalpointdrying. Aftermounting
ontocarbonstubsandvacuumsputter-coatingwithgold,thesampleswereanalyzedwithaHitachi
S-2400NSEM(Tokyo,Japan)understandardoperatingconditions.
3.7. Anti-LipaseActivity
Anti-lipaseactivitywasevaluatedbyafluorometricassay. PG-Eanditsactivecomponentswere
reacted with a lipase solution (100 U/mL) and a 4-methylumbelliferyl butyrate solution (10 µM),
and the reaction was terminated using sodium citrate (0.1 M) [30]. Fluorescence was detected in
a fluorometric microplate reader (λex: 365 nm, λem: 445 nm). Each experiment was performed
in triplicate. Results are expressed as the mean ± standard deviation (SD), and the percentage of
inhibitionwascomparedtothecontrol.
3.8. Anti-ProliferativeActivityagainstTestosterone-TreatedHaCaTCells
HaCaT cells, a human keratinocyte cell line, were cultured in DMEM with 10% FBS, 1%
penicillin-streptomycin,and1%L-glutamineat37◦Cand5%CO2. HaCaTcells(2×104cells/well)
wereseededin96-wellplatesandco-treatedwithtestosterone(10µg/mL),andtestsampleswere
incubatedfor18h[31]. CellproliferativeactivitywasdetectedbyanMTTassay.
3.9. Anti-InflammatoryActivityagainstHeat-KilledP.acnes-TreatedRAW264.7Cells
P. acnes (4 × 108 CFU/mL) was incubated at 80 ◦C for 30 min to prepare heat-killed P. acnes
(HKP).TheHKPwasfreeze-driedandstoredat4◦Cuntiluse. RAW264.7cells(BCRC-60001)were
cultured in DMEM with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at 37 ◦C and
5%CO . RAW264.7cells(8×104cells/well)wereseededin96-wellplatesandco-treatedwithHKP
2
(100µg/mL)andtestsamples[32]. After18h,nitricoxide(NO)productionwasdeterminedbymixing
thecellculturemediumwithaGriessreagent,andtheabsorptionwasdetectedinanenzyme-linked
immunosorbent assay (ELISA) reader at 530 nm. Prostaglandin E (PGE ) concentrations in cell
2 2
culturemediaweredeterminedwithaPGE ELISAkit(EnzoLifeSciences,Farmingdale,NY,USA).
2
CellviabilitywasdetectedbyanMTTassay.
3.10. Anti-InflammatoryActivityagainstHeat-KilledP.acnes-TreatedTHP-1Cells
THP-1cells,ahumanmonocyticcellline(BCRC-60430),wereculturedinRPMIwith10%FBS,1%
penicillin-streptomycin,and1%L-glutamineat37◦Cand5%CO2. THP-1cells(1×106 cells/well)
wereseededin24-wellplatesandco-treatedwithHKP(100µg/mL)andtestsamplesfor18h. TNF-α
andIL-8concentrationsofcellculturemediaweredeterminedbyrespectiveenzymeimmunoassaykits.
3.11. StatisticalAnalysis
DataarepresentedasthemeanandSD.SignificancewascalculatedusingStudent’st-testanda
one-wayanalysisofvariance(ANOVA)bySPSSsoftwarev.15(SPSS,Chicago,IL,USA).
4. Conclusions
In this study, we used several approaches to explore the multiple therapeutic effects of PG-E
againstP.acnes. OurresultsshowedthatPG-EdirectlyattenuatedP.acnes-inducedWistarearedema