Anti-Acne Activity of Italian Medicinal Plants Used for Skin Infection

14 Pages·2016·0.72 MB·English

by  Cassandra L. Quave

ORIGINALRESEARCH
published:10November2016
doi:10.3389/fphar.2016.00425
Anti-Acne Activity of Italian Medicinal
Plants Used for Skin Infection
KateNelson1,JamesT.Lyles2,TracyLi2,AlessandroSaitta3,EugeniaAddie-Noye2,
PaulaTyler4andCassandraL.Quave1,2,5*
1DepartmentofDermatology,EmoryUniversitySchoolofMedicine,Atlanta,GA,USA,2CenterfortheStudyofHuman
Health,EmoryUniversityCollegeofArtsandSciences,Atlanta,GA,USA,3DepartmentofAgriculturalandForestSciences,
UniversitàDegliStudidiPalermo,Palermo,Italy,4DepartmentofChemistry,EmoryUniversityCollegeofArtsandSciences,
Atlanta,GA,USA,5AntibioticResistanceCenter,EmoryUniversity,Atlanta,GA,USA
Propionibacterium acnes is implicated in the pathogenesis of acne vulgaris,
which impacts >85% of teenagers. Novel therapies are in high demand and
an ethnopharmacological approach to discovering new plant sources of anti-acne
therapeutics could contribute to filling this void in effective therapies. The aims of our
study were two-fold: (1) To determine if species identified in ethnopharmacological
field studies as having traditional uses for skin and soft tissue infection (SSTI) exhibit
significantlymoreactivityagainstP.acnesthanspecieswithnosuchreporteduse;and(2)
Chemicallycharacterizeactiveextractsandassesstheirsuitabilityforfutureinvestigation.
Extracts of Italian medicinal (for acne and other skin infection) and randomly collected
Editedby: plantsandfungiwerescreenedforgrowth-inhibitoryandanti-biofilmactivityinP.acnes
AdolfoAndrade-Cetto, usingbrothmicrodilutionmethods.Bioactiveextractswerechemicallycharacterizedby
NationalAutonomousUniversityof
HPLC and examined for cytotoxicity against human keratinocytes (HaCaTs). Following
Mexico,Mexico
evaluationof157extractsfrom10fungiand58plants,weidentifiedcrudeextractsfrom
Reviewedby:
TomCoenye, seven species exhibiting growth inhibitory activity (MICs 64–256 µg mL−1). All active
GhentUniversity,Belgium
extracts were examined for cytotoxicity against HaCaTs; extracts from one fungal and
AdeyemiOladapoAremu,
UniversityofKwaZulu-Natal, oneplantspeciesweretoxic(IC50256µgmL−1).HPLCanalysiswithchemicalstandards
SouthAfrica revealed many of these extracts contained chlorogenic acid, p-coumaric acid, ellagic
*Correspondence: acid,gallicacid,andtannicacid.Inconclusion,speciesusedintraditionalmedicinefor
CassandraL.Quave
theskinexhibitedsignificantlygreater(p<0.05)growthinhibitoryandbiofilmeradication
[email protected]
activity than random species, supporting the validity of an ethnobotanical approach to
Specialtysection: identifying new therapeutics. The anti-acne activity of three extracts is reported for the
Thisarticlewassubmittedto
first time: Vitis vinifera leaves, Asphodelus microcarpus leaves, and Vicia sativa aerial
Ethnopharmacology,
asectionofthejournal parts.
FrontiersinPharmacology
Keywords: biofilms, antimicrobials, antibiotics, acne, medicinal plants, Propionibacterium acnes,
Received:30August2016
ethnopharmacology
Accepted:26October2016
Published:10November2016
Citation: INTRODUCTION
NelsonK,LylesJT,LiT,SaittaA,
Addie-NoyeE,TylerPandQuaveCL
Acne vulgaris is a common skin disorder affecting more than 85% of teenagers in the United
(2016)Anti-AcneActivityofItalian
States (James et al., 2009). While it is not a lethal or debilitating disorder, it can be painful
MedicinalPlantsUsedforSkin
Infection.Front.Pharmacol.7:425. and disfiguring, causing significant physiological distress, and heavy economic burden. Acne is
doi:10.3389/fphar.2016.00425 associatedwithincreasesinanxiety,depression,andsuicidalideation(Dunnetal.,2011).In2001,
FrontiersinPharmacology|www.frontiersin.org 1 November2016|Volume7|Article425
Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
areportestimatedthatover$1billionisspenteachyearintheUS plant species of which 31 had an ethnobotanical use and
on acne related health care visits and acne therapies (Lehmann 32 were random collections. The random collection extracts
et al., 2001). However, these therapies are not cures, but rather had no vasorelaxation activity, but 19% of ethnobotanical
waysofmanagingthisfolliculardisorder. extracts did (Slish et al., 1999). Another study focused on
Current treatments fall into either two categories: topical the search for antifungal activity compared 114 extracts with
or oral. Common topical treatments include benzoyl peroxide, antifungal ethnobotanical use and 183 extracts with either no
retinoids, and antibiotics (i.e., erythromycin or clindamycin), ethnobotanical indication or non-fungal ethnobotanical use. In
and common oral treatments include retinoids and antibiotics a series of bioassays, extracts in the ethnobotanical indication
(i.e.,tetracyclineandmacrolides;Layton,2016;Zaengleinetal., group consistently had higher hit rates with the antifungal
2016).Incasesofsevereacne,combinationaltreatmentsareused, indicated extracts; 40% had antifungal activity against at least
usuallyemployingbenzoylperoxide,retinoids,and/orantibiotics onefungusvs.21%ofplantswithnoanti-fungalethnobotanical
together(Layton,2016;Zaengleinetal.,2016).Antibioticshave use (Svetaz et al., 2010). Similarly, a study comparing three
beenusedforover50yearstotreatacneandtodayonecourseof groups,skinandsofttissueinfection(SSTI)botanicals,non-skin
topicalorsystemictreatmenttypicallylasts3–6months(Walsh botanicals, and random plants, for their Staphylococcus aureus
et al., 2016). Antibiotics are thought to inhibit inflammation antibacterial and anti-biofilm actions found no difference
indicativeofacnewhenusedtopicallyandsystemically,aswellas between the three groups for antibacterial activity, but SSTI
targetP.acneswhenusedtopically(Walshetal.,2016).Likemany ethnobotanicals had more anti-biofilm potential in S. aureus
other bacteria, P. acnes is also subject to emerging antibiotic models than the random collections (Quave et al., 2008a).
resistance and novel therapies are in high demand worldwide Investigating antibacterial bioactivities beyond bacteriostatic or
(Sardanaetal.,2015;DelRossoandZeichner,2016;Walshetal., bactericidal action, such as pathogenesis factors, could reveal
2016).Pathogenesistargets,suchasquorumsensingandbiofilms, other mechanisms through which plant secondary metabolites
are organism specific and could limit resistance development impact pathogen survival and fitness, increasing the chance of
(LaSarreandFederle,2013). discoveringnewtherapeutics.Forthepurposesofclarity,herewe
Biofilms contribute to the recalcitrant nature of acne by useSSTItorefertoinfectionsoftheskinandsofttissuesranging
presentingaphysicalbarriertowhichfewmoleculesarecapable from mild to severe, including those that are purulent (acne,
ofpenetrating,allowingthebacteriawithintopersistandcause furuncles, abscesses, carbuncles) to non-purulent (necrotizing
infection(Donlan,2001;Vlassovaetal.,2011).Biofilmshavebeen infection,cellulitis,erysipelas)innature.
implicated in implant-related and chronic infections (Donlan, Our research group has already demonstrated interesting
2001; Aubin et al., 2014), accounting for an estimated 80% of anti-staphylococcal activity (in terms of growth, biofilm, and
microbialinfections inthebody(Sun etal.,2013).P. acneshas quorumsensinginhibitoryaction)intheQuaveNaturalProducts
beendetectedinbiofilmsassociatedwithbone,jointandspinal Library(QNPL),whichincludesacollectionofplantandfungal
prosthetics, breast implants, external ventricular shunts, and, extractsderivedfromnaturalingredientsusedspecificallyinthe
in rare instances, cardiac devices (Aubin et al., 2014). P. acnes treatment of SSTIs—caused by a number of skin pathogenic
biofilmsareassociatedwithpathogenesisofacne,withextensive bacteria, including Propionibacterium acnes—and other health
biofilm growth being associated with acne vulgaris (Jahns and concerns in the Mediterranean and other parts of the world
Alexeyev,2014).Thehighincidenceofantibioticresistanceseen (Quave et al., 2012, 2015). Extracts from Italian species in
in P. acnes worldwide is thought to be due to biofilms, as well the QNPL are uniquely tied to ethnopharmacological data
asthehighuseofantibiotics(Sardanaetal.,2015;Walshetal., documentedinfieldstudiesconductedbyQuaveandcolleagues,
2016).OnlyafewstudieshaveevaluatedpotentialP.acnesanti- and the detailed reports of ethnopharmacological uses of these
biofilm compounds (Brackman et al., 2014; Sivasankar et al., species have been previously published (Pieroni et al., 2002,
2016) and to the best of our knowledge only one study has 2004a,b;PieroniandQuave,2005;Quaveetal.,2008b).
evaluatedmedicinalplantsforanti-biofilmactivity(Coenyeetal., TheQNPLhasonlybeentestedagainstalimitednumberof
2012). Medicinal plants, in particular, may present a unique pathogensresponsibleforSSTIs,andhasneverbeenscreenedfor
source of new therapeutic options. Many studies have been activityagainstP.acnes.Inthisstudy,wecomparetheactivityof
dedicated to documentation of traditional uses of medicinal ItalianplantsandfungiintheQNPLthatwereidentifiedasbeing
plants for managing dermatological conditions, and these may usedintraditionalmedicineforthetreatmentofacneandother
representastrongstartingpointfordrugdiscoveryresearchon skininfectionstoarandomcollectionofItalianspecieswithno
thistopic(Pieronietal.,2004b;Quaveetal.,2008b;Caveroetal., reportedtopicalapplicationsforskininfectionorinflammation.
2013;MabonaandVanVuuren,2013;Afolayanetal.,2014;Lall The overall aim of this study is to compare activity of these
andKishore,2014). two groups through examination of their anti-acne potential
An ethnobotanical approach for drug discovery uses against planktonic and biofilm-associated P. acnes and evaluate
traditional medicine, historical texts or traditional healers, active extracts for future investigation. We hypothesize that
as a guide toward potentially medicinal plant species. A extracts from medicinal plants or fungi used for the traditional
number of studies addressing different biological targets have treatmentofSSTIswilldemonstrategreateranti-bacterialactivity
demonstrated that an ethnobotanical approach to identifying against P. acnes (in terms of killing planktonic cells and
bioactivecompoundsinplantsismoreefficaciousthanarandom eradicatingbiofilms)thanthosewithnoreportedapplicationto
search. More than 15 years ago, Slish et al. (1999) compared theskin.
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Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
MATERIALS AND METHODS thenpreparedatastockconcentrationof10mgmL−1 indH O
2
andfilteredat0.2µmpriortouseinbioassays.Fungalextracts
Plant and Fungal Materials
werepreparedin100%MeOHordH Obydoublesonicationat
2
PlantmaterialswerecollectedinSpring-Summer(May–August)
room temperature for 20 min at a ratio of 1 g:10 mL, followed
over a period of 2006–2013 from the Vulture Alto-Bradano
by filtration, concentration and stock preparation as described
region of Basilicata, Italy and imported to the United States
for plant extracts. In addition to the crude extracts, we also
(US) under US Department of Agriculture permits (PCIP-14-
included bioactive fractions 220D-F2 (from Rubus ulmifolius)
00388; PDEP-09-0228; DP63438). All specimens were collected
and 224C-F2 (from Castanea sativa) in the panel of tested
following the World Health Organization (WHO) guidance
extracts.Themethodsforcreationoftheserefinedfractionshave
on the collection of wild medicinal plants (WHO, 2003).
beenpreviouslypublished(Quaveetal.,2012,2015).
All collections were made on private lands with permission
of landowners. Voucher specimens were deposited at the Microbial Strains and Culture Conditions
Herbarium Lucanum (HLUC) at the Universitá della Basilicata The P. acnes isolate was obtained from the American Type
inPotenza,Italy,andtheEmoryUniversityHerbarium(GEO)in Culture Collection (ATCC(cid:13)R 6919TM). The strain was streaked
Atlanta,GA,USA.Identificationofspeciesfollowedthestandard from freezer stock onto Tryptic soy agar plates (TSA)
Italian flora (Pignatti, 2002), and family assignments followed supplemented with 5% sheep blood (BD, Franklin Lakes, NJ)
the Angiosperm Phylogeny Group III guidance (Tropicos.Org, and incubated at 37◦C for 72 h prior to making liquid cultures
2015).Bulkmaterialswereseparatedbypart(e.g.,leaves,stems, in brain heart infusion (BHI) broth (BD, Franklin Lakes, NJ)
flowers,roots),cutintosmallpieceswithclippers,andthendried supplemented with 1% dextrose (Fisher Scientific, Waltham,
in a plant drier with a heat source (35◦C) and fan for 48–72 h, MA)(additional72hincubation)foruseinthebelowdescribed
untilcompletelydried.Sampleswerepackedwithsilicapackets assays.Allculturesweregrowninanaerobicchambersusingthe
andvacuumsealedbeforeshippingtothelabforextractionand EZAnaerobeChamber(BD,FranklinLakes,NJ)andGasPakEZ
analysis. Upon arrival, plant materials were ground into a fine Anaerobecontainersachets(BD,FranklinLakes,NJ).
powderusingaWileymill(2mmmeshsize;WileyScientific).
FungiwerecollectedinDecember2013andApril–November Minimum Inhibitory Concentration (MIC)
2014 from locations in Sicily and imported to the US under for Growth
USDA permit (P526P-14-02182). Fungal collections were made
There are no established Clinical and Laboratory Standard
in broad-leaved and coniferous forests, preserved in paper
Institute(CLSI)guidelinesforMICtestinginP.acnes.Thus,we
bags and then transported to the laboratory, dried, and
followedpreviouslydescribedmethodswithsomemodifications
identified. Identification of fungi was carried out using fresh
(Tsaietal.,2010).Briefly,liquidculturewasstandardizedto5×
or dried sporomes using European monographs (Ryvarden 107CFUmL−1byopticaldensity(0.05atOD )infreshBHI
590nm
andGilbertson,1993–1994).Speciesnomenclaturefollowedthe
supplemented with 1% dextrose, and this was confirmed with
Index Fungorum (IF, 2015). Bulk materials were dried and
platecounts.OurinitialscreenoftheQNPLwasundertakenin
processed as described above. Retention vouchers of the fungal
96-wellplates(GreinerBio-OneInternational,Monroe,NC)ata
materialarestoredintheQuavelab. finalwell-concentrationof256µgmL−1(<1.28%DMSOinwell)
Extractsfromspeciesincludedinthisstudyweresortedinto inatotalwellvolumeof200µL.TheOD ofplateswasread
600nm
groupsbasedonhistoryoftraditionaluseforornotforacneand
with a Cytation 3 multimode plate reader (BioTek, Winooski,
other SSTI treatments, as documented in previously published
VT)priortoincubatingunderanaerobicconditionsfor72hat
findings from ethnopharmacological field studies conducted in 37◦C. A second OD read was taken at 72 h post-inoculation,
Italy (Pieroni et al., 2002, 2004a,b; Pieroni and Quave, 2005;
andthefinalpercentinhibitionofgrowthwasdeterminedusing
Quave et al., 2008b). Importantly, these prior studies included
apreviouslyreportedformula(Quaveetal.,2008a)whichtakes
reportsofvoucherspecimens,use-citations,anddetaileddataon
intoaccountanyinterferenceduetocolorofthetestextracts.The
localpreparationandapplicationofthenaturalremedies.
mostactiveextracts(%inhibition≥50%)wereretestedbyserial
dilution from 2 to 256 µg mL−1 to determine concentrations
Natural Product Extractions required for MIC and MIC . The MIC and MIC values
50 90 50 90
Crude plant extracts were made as macerations in either 95% weredefinedastheminimumconcentrationnecessarytoachieve
EtOHor100%MeOHwitharatioof1g(plantmaterial):10mL ≥50and≥90%inhibitionofgrowth,respectively.
organic solvent for two successive periods of 72 h, with daily
Minimum Biofilm Eradication
agitation. Filtered extracts were combined and concentrated at
reduced pressure with rotary evaporators (<40◦C water bath Concentration (MBEC)
temperature), shell frozen, and then lyophilized. Extracts were Apreviouslydescribedmethodwasfollowedwithmodifications
then suspended in DMSO at a stock concentration of 10mg (Coenyeetal.,2007).Briefly,liquidculturewasstandardizedas
mL−1priortobioassaytesting,withDMSOrepresenting<1.28% described above for MIC testing. Biofilms were established in
of the final well volume for all tests. Aqueous extracts were tissueculturetreatedU-bottom96-wellplates(TPP,Trasadingen,
created by boiling ground plant material in dH O for 20 min Switzerland). Following 24 h incubation under anaerobic
2
(ratio of 1 g:10 mL), followed by filtration, rotary evaporation conditions,mediawasgentlyaspiratedbymanualpipettingand
and lyophilization as described above. Aqueous extracts were extract treated media or control -resveratrol (MP Biomedicals,
FrontiersinPharmacology|www.frontiersin.org 3 November2016|Volume7|Article425
Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
Santa Ana, CA), was added. Initial screens were conducted Santa Clara, CA) with an Agilent ZORBAX Eclipse XDB-
at a final well-concentration of 256 µg mL−1 in a total well C18 (250 mm × 4.6 mm, 5 µm) column (Santa Clara, CA)
volumeof100µL.Establishedbiofilmswereexposedtoextract with compatible guard column at a column temperature of
treatmentfor24hunderanaerobicconditions,andthenmedia 35◦C. Mobile phase reagents were HPLC grade and purchased
was aspirated, wells gently rinsed with PBS, heat-fixed at 37◦C from Fisher Scientific (Waltham, MA), except for the Type 1
for 60 min in an oven and stained with crystal violet (Hardy water, which was obtained from an EMD Millipore MILLI-
Diagnostics, Santa Maria, CA) for 15 min. Excess crystal violet Q water system (Billerica, MA). Mobile phase consisted of a
was washed off the plates in water and the plates were dried linear gradient elution 0.1% formic acid in water (A) and 0.1%
prior to eluting the stain with 10% Tween 80 (2.5% aqueous formic acid in acetonitrile (B) at a flow rate of 2 mL min−1.
preparation) in EtOH. The eluate was diluted at a ratio of 1:10 Initial conditions were 95:5 (A:B) changing to 63:37 (A:B) at
in PBS and the final OD measured with a plate reader. 40 min, to 100% B at 80 min and held until 90 min. Samples
595nm
The most active extracts (% inhibition ≥50%) were retested by were prepared in MeOH and 10 µL or 20 µL injections were
serial dilution from 2 to 256 µg mL−1 to determine MBEC made.Chromatogramsweremonitoredat254nmand280nm.
50
andMBEC .TheMBEC andMBEC valuesweredefinedas Authentic standards were used to aid in peak identification by
90 50 90
theminimumconcentrationnecessarytoachieve≥50and≥90% comparison of retention times and UV profiles. Caffeic acid,
eradicationofattachedbiofilm,respectively. chlorogenicacid,p-coumaricacid,ellagicacid,ferulicacid,and
resveratrol were purchased from MP Biomedicals (Solon, OH)
at≥98%purity.GallicacidwaspurchasedfromAcrosOrganics
Cytotoxicity Analysis
(NJ) at 98% purity. ACS grade tannic acid from Sigma Aldrich
Human immortalized keratinocytes (HaCaT) were used to
(St. Louis, MO) was characterized prior to use by HPLC-PDA
evaluate the potential skin toxicity of the most active extracts
analysis.
identifiedviaMICandMBECtesting.Cellsweremaintainedin
Liquid chromatography-Fourier transform mass
Dulbecco’smodifiedEagle’smediumwithglucoseat4.5gL−1and
spectrometry (LC-FTMS) was performed on the MeOH
L-glutamine(Corning,Corning,NY).Mediawassupplemented
extract of Hapalopilus rutilans using a Shimadzu SIL-ACHT
with10%heat-inactivatedfetalbovineserum(Seradigm,Randor,
(Kyoto, Japan) and Dionex 3600SD HPLC pump (Sunnyvale,
PA) and a solution of 100 IU Penicillin and 100 µg mL−1
CA) with the above described chromatographic conditions.
Streptomycin(Corning,Corning,NY).Cellswereculturedin75
The data was acquired in MS1 mode scanning from m/z 150
cm2 flasksincubatedat37◦Cwith5%CO .Asolutionof0.25%
2 to 1500 on a Thermo Scientific LTQ-FT Ultra MS (Waltham,
trypsin and 0.1% EDTA in HBSS (Corning, Corning, NY) was
MA)innegativeESImodeandprocessedwithThermoScientific
usedtodetachcellsfromthebottomoftheflaskbeforesplitting
Xcalibur 2.2 SP1.48 software (San Jose, CA). The capillary
andplating.
temperatureandvoltagewere275.0◦Cand−19.00V,sheathgas
After reaching at least 80% confluence in the flask, cells
of60,sourcevoltage5.0kV,andsourcecurrent100.0µA.
were seeded to a 96-well tissue culture plate (Fisher Scientific,
Waltham, MA). Cells were resuspended, counted with a Statistical Analyses
hemocytometer, and standardized to a concentration of 4 ×
Percent inhibition of growth and biofilm formation (screening
104 cells per mL (8 × 103 cells per final 200 µL well volume). concentrationof256µgmL−1)wascomparedforextractssorted
Plateswereincubatedfor48hat37◦Cwith5%CO .Following
2 into two groups: plants used for SSTI and plants those not
incubation, the medium was aspirated, replaced with fresh
documentedforthisuse.GraphPadPrismsoftwarewasusedto
medium, and sterile filtered extracts and the vehicle control
conduct statistical tests. A Student’s t-test (unpaired, 2-tailed),
(DMSO) were serially diluted from 2 to 256 µg mL−1. Plates
wasconductedtocomparepercentinhibitoryactivityofthetwo
wereincubatedfor24hat37◦Cwith5%CO2.Cytotoxicitywas groups,withp<0.05consideredsignificant.Asecondanalysis
determinedwithaLDHAssayKit(G-Biosciences,St.Louis,MO)
ofthedatawasalsoperformedfollowingtheremovalofmultiple
following manufacturer’s protocol. Briefly, after 24 h, 20 µL of
outliersbytheROUTmethod,whichwasmoresuitabletothis
lysis buffer was added in triplicate and the plate was incubated
analysis than the Grubbs’ method as it accounts for multiple
for another 45 min. Plates were centrifuged at 1500 rpm for 4
outliers (Barnett and Lewis, 1994; Motulsky and Brown, 2006).
min.Followingcentrifugation,50µLofsupernatantfromeach
Thefalsediscoveryrate,or“Q,”wassetto1%forthisprocedure.
well was transferred to a new 96-well plate, mixed with 50 µL
ofsubstratemixandincubatedfor20minwhilebeingprotected
RESULTS
from light. After incubation, 50 µL of stop solution was added
toallwells,andabsorbancewasrecordedat490nmwithaplate
A total of 10 fungal species, distributed across 10 genera and 6
reader.
families,and58plantspecies,distributedacross55generaand32
familieswereextractedandevaluatedforgrowthinhibitionand
Characterization of Active Extracts biofilm eradication activity against P. acnes. Seventeen of these
Preliminary characterization of the most active extracts, which species have been documented in the ethnobiological literature
either exhibited a MIC or MBEC , was pursued by HPLC. asbeingusedintherapiesforSSTIs(Pieronietal.,2002,2004a,b;
50 50
TheanalysiswasperformedonanAgilent1260Infinitysystem PieroniandQuave,2005;Quaveetal.,2008b);thesearedenoted
running OpenLab CDS ChemStation (Agilent Technologies, witha“+”intheSSTIcolumnofTable1.
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Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
TABLE1|Minimuminhibitoryconcentrations(MICs)ofplantextracts(µgmL−1)againstPropionibacteriumacnes(ATCC6919).
Taxa[vouchercode] CommonName SSTIuse‡ PartExtracted† ExtractionSolvent MIC50 MIC90
FUNGI
Amanitaceae
Amanitacaesarea(Scop.)Pers.[AS-19] Caesar’smushroom − fb MeOH − −
Ganodermataceae
Ganodermalucidum(Curtis)P.Karst.[AS-10] Lingzhimushroom − fb MeOH − −
H2O − −
Gleophyllaceae
Gloeophyllumsepiarium(Wulfen)P.Karst.[AS-4] − − fb MeOH − −
Hymenochaetales
Fuscoporiatorulosa(Pers.)T.Wagner&M.Fisch.[AS-1] − − fb H2O − −
Trichaptumbiforme(Fr.)Ryvarden[AS-12] − − fb MeOH − −
H2O − −
Meripilaceae
Meripilusgiganteus(Pers.)P.Karst.[AS-11] Giantpolypore − fb MeOH − −
H2O − −
Polyporaceae
Coriolopsisgallica(Fr.)Ryvarden[AS-13] − − fb MeOH − −
H2O − −
Fomesfomentarius(L.)Fr.[AS-4] Tinderfungus − fb MeOH − −
H2O − −
Hapalopilusrutilans(Pers.)Murrill[AS-5] Tendernesting − fb MeOH 128 256
polypore
H2O − −
Trametesversicolor(L.)Lloyd[AS-8] Turkeytail − fb MeOH − −
H2O − −
PLANTAE
Adoxaceae
SambucusebulusL.[CQ-180] DwarfElder + infl EtOH − −
MeOH − −
le EtOH − −
MeOH − −
st EtOH − −
SambucusnigraL.[CQ-151] Elderberry + fr EtOH − −
MeOH − −
le EtOH − −
MeOH − −
wp EtOH − −
infl MeOH − −
Amaryllidaceae
AlliumcepaL.[CQ-206] Onion + le;bu EtOH − −
MeOH − −
Apiaceae
DaucuscarotaL.[CQ-215] QueenAnne’slace − le;st EtOH − −
MeOH − −
infl;infr EtOH − −
FerulacommunisL.[CQ-214] Giantfennel − fr EtOH − −
st EtOH − −
MeOH − −
infl MeOH − −
ro MeOH − −
Foeniculumvulgaresubsp.piperitumCout.[CQ-192] Fennel − le;st EtOH − −
(Continued)
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Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
TABLE1|Continued
Taxa[vouchercode] CommonName SSTIuse‡ PartExtracted† ExtractionSolvent MIC50 MIC90
Foeniculumvulgaresubsp.vulgare[CQ-196] Fennel − le;st EtOH − −
MeOH − −
Apocynaceae
VincamajorL.[CQ-139] Bigleafperiwinkle − le;st;ro MeOH − −
Araceae
ArumitalicumMill.[CQ-175] Italianlordsand − st EtOH − −
ladies
fr EtOH − −
sta EtOH − −
le EtOH − −
MeOH − −
Asparagaceae
Leopoldiacomosa(L.)Parl.[CQ-105] Tasselhyacinth − le;infl MeOH − −
bu MeOH − −
Asteraceae
AchilleamillefoliumL.[CQ-176] Commonyarrow + infl EtOH − −
le;st EtOH − −
le;st;fl EtOH − −
MEOH − −
Anacyclustomentosus(Gouan)DC.[CQ-167] Whitebuttons − le;st;fl EtOH − −
CichoriumintybusL.[CQ-161] Chicory − le;st;fl EtOH − −
ScolymushispanicusL.[CQ-199] Common − le;st;fl EtOH − −
goldenthistle
MeOH − −
TussilagofarfaraL.[CQ-202] Coltsfoot − le;st;ro EtOH − −
le;st;fl MeOH − −
Brassicaceae
Cardariadraba(L.)Desv.[CQ-108] Whitetop − fl;st;le;ro MeOH − −
Caprifoliaceae
DipsacusfullonumL.[CQ-201] Fuller’steasel − le;st EtOH − −
MeOH − −
fl EtOH − −
KnautiaarvensisJ.M.Coult.[CQ-190] Fieldscabiosa − le;st;fl EtOH − −
KnautialucanaLacaita&Szabó[CQ-166] − − le;st;fl EtOH − −
MeOH − −
LoniceraalpigenaL.[CQ-213] Alpinehoneysuckle − wp EtOH − −
MeOH − −
le EtOH − −
Caryophyllaceae
SaponariaofficinalisL.[CQ-210] Bouncingbet − le;st;fl EtOH − −
MeOH − −
Dennstaedtiaceae
Pteridiumaquilinum(L.)Kuhn[CQ-211] Western − st MeOH − −
brackenfern
Fabaceae
AcaciadealbataLink[CQ-115] Silverwattle − infl MeOH − −
le MeOH − −
st MeOH − −
AnthyllisvulnerariaL.[CQ-147] Common − le;st;fl EtOH − −
kidneyvetch
MeOH − −
AstragalusmonspessulanusL.[CQ-112] Montpelliermilk − fl;le;ro;st EtOH − −
vetch
MeOH − −
(Continued)
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Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
TABLE1|Continued
Taxa[vouchercode] CommonName SSTIuse‡ PartExtracted† ExtractionSolvent MIC50 MIC90
CoronillaemerusL.[CQ-137] Scorpionsenna − wst EtOH − −
MeOH − −
le;fl MeOH − −
MelilotusalbusMedik.[CQ-193] Sweetclover − le;st;fl EtOH − −
MeOH − −
Trifoliumspp.[CQ-185] − − le;st;fl EtOH − −
MeOH − −
RobiniapseudoacaciaL.[CQ-155] Blacklocust − le MeOH − −
Viciasativasubsp.sativa[CQ-119] Gardenvetch − le;st;fl MeOH 128 256
Fagaceae
CastaneasativaMill.[CQ-191] SweetChestnut + le EtOH 256 −
MeOH 256 −
*Fraction224C-F2 256 256
H2O − −
gle EtOH − −
MeOH 256 −
wp EtOH − −
MeOH 128 −
QuercuscerrisL.[CQ-228;CQ-301] Europeanturkeyoak − st;fr EtOH − −
le MeOH − −
Gentianaceae
Centauriumpulchellum(Sw.)Druce[CQ-217] Branchedcentaury − le;ro;st;fl EtOH − −
Hypericaceae
HypericumperforatumL.[CQ-183] St.John’sWort + le;st;fl EtOH − −
MeOH − −
Juglandaceae
JuglansregiaL.[CQ-181] Walnut + le EtOH − −
MeOH − −
wp EtOH 256 256
MeOH 128 256
imfr MeOH − −
Juncaceae
JuncusarticulatusL.[CQ-216] Jointleafrush − le;fr EtOH − −
Lamiaceae
BallotanigraL.[CQ-160] Blackhorehound + ro MeOH − −
le MeOH − −
st MeOH − −
MarrubiumvulgareL.[CQ-170] Horehound + ro MeOH − −
le;st;fl MeOH − −
MenthaspicataL.[CQ-224] Spearmint − le;st;fl EtOH − −
RosmarinusofficinalisL.[CQ-113] Rosemary + le;st;fl MeOH 128 256
Liliaceae
LiliumcandidumL.[CQ-174] Madonnalily − infl EtOH − −
Malvaceae
MalvasylvestrisL.[CQ-156] Commonmallow + le MeOH − −
st MeOH − −
Moraceae
FicuscaricaL.[CQ-173] Ediblefig + le EtOH − −
wp EtOH − −
MeOH − −
imfr MeOH − −
(Continued)
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Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
TABLE1|Continued
Taxa[vouchercode] CommonName SSTIuse‡ PartExtracted† ExtractionSolvent MIC50 MIC90
Nyctaginaceae
MirabilisjalapaL.[CQ-222] Fouro’clockflower − le;fl;fr EtOH − −
MeOH − −
Oleaceae
OleaeuropaeaL.[CQ-197] Olive − wp EtOH − −
MeOH − −
le MeOH − −
Plantaginaceae
PlantagomajorL.[CQ-225] Boradleafplantain + le;ro;fl;st EtOH − −
MeOH − −
Poaceae
Agropyronrepens(L.)P.Beauv.[CQ-208] Couchgrass − le;st;ro EtOH − −
ArundodonaxL.[CQ-146] Giantreed + le;st MeOH − −
in MeOH − −
Ranunculaceae
DelphiniumfissumWaldst.[CQ-187] − − le;st;fl;fr EtOH − −
RanunculusacrisL.[CQ-135] Meadowbuttercup − le;st;fl MeOH − −
Rosaceae
PrunusspinosaL.[CQ-163] Blackthorn - wp;le EtOH − −
fr MeOH − −
Rosacaninavar.canina[CQ-152] Dogrose wp MeOH − −
80%MeOH − −
ga MeOH − −
le 80%MeOH − −
fr MeOH − −
le;fl 80%MeOH − −
RubusulmifoliusSchott. Elmleafblackberry + ro EtOH − −
*Fraction220D-F2 − −
MeOH − −
wst EtOH − −
Rubiaceae
GaliumverumL.[CQ-177] Yellowbedstraw + le;st;fl EtOH − −
MeOH − −
Scrophulariaceae
VerbascumthapsusL.[CQ-172] Greatmullein − le MeOH − −
Ulmaceae
UlmusminorMill.[CQ-195] Fieldelm − wp EtOH − −
MeOH − −
le MeOH − −
Vitaceae
Vitisviniferavar.aglianico[CQ-209] Grapevine − le MeOH 64 64
Xanthorrhoeaceae
AsphodelusmicrocarpusSalzm.&Viv.[CQ-109] - + infl MeOH − −
le MeOH 128 128
ANTIBIOTICCONTROLS
Clindamycin 0.25 2
Erythromycin 0.063 2
Resveratrol 100 100
PartExtracted†:bu,bulb;fl,flowers;gle,gallinfectedleaves;imfr,immaturefruit;in,internodes;infl,inflorescence;infr,infructescence;fb,fruitingbody;ga,galls;le,leaves;ro,roots;
st,stems;sta,stalk;wp,woodyparts;wst,woodystem.SSTIuse‡:“+”indicatesthatthereisadocumentedethnobotanicaluseforskinandsofttissueinfections(SSTI)orskin
inflammationsforthisspecies;“−“indicatesnodocumenteduseforSSTIs.*Theextractionprotocolfor224C-F2and220D-F2havebeenpreviouslypublished(Quaveetal.,2012,
2015).TheMICforgrowthisrepresentedatMIC50(orat50%inhibitionofthegrowthcontrol)orMIC90for90%inhibition.“−“indicatesthattheMIC50wasnotdetectedinthe
concentrationrangetested(2-256µgmL−1).NoneoftheextractstestedexhibitedMBEC50withinthetestedconcentrationrange,andthusthiscolumnisomittedfromthetable.
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Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
Anti-Bacterial Activity other extracts tested exhibited limited to no toxicity at the
GrowthInhibition concentrationsnecessaryforantibacterialactivity.
MIC and MIC values were detected for one fungus
50 90
(Hapalopilus rutilans) and 6 plants (Vicia sativa subsp. sativa, Chemical Characterization of Active
C.sativa,Juglansregia,Rosmarinusofficinalis,Vitisviniferavar. Extracts
aglianico,andAsphodelusmicrocarpus)atthetestedrange(2–256
We used a panel of ubiquitous chemical standards in effort to
µgmL−1).Theextractionmethodandplantorfungaltissue(s)
provide basic chemical characterization of extracts of interest.
extracted for each species, as well as all MICs for extracts and
The most bioactive extracts were examined for the presence
controlsarereportedinTable1.
of caffeic acid (1), chlorogenic acid (2), p-coumaric acid (3),
Themostactiveextractintermsofgrowthinhibitionincluded
ellagic acid (4), ferulic acid (5), gallic acid (6), resveratrol (7),
aleafextractofV.viniferavar.aglianico,withaMIC andMIC
50 90 and tannic acid (8) by comparison with authentic standards
valueof64µgmL−1.ExtractswithMIC valuesof128µgmL−1
50 by HPLC-DAD. The presence or absence of these standards in
included Hapalopilus rutilans (MeOH extractof fruiting body),
extracts is reported in Table2. Several extracts derived from C.
V. sativa subsp. sativa (MeOH extract of aerial parts), J. regia
sativawereactiveasgrowthinhibitorsforP.acnes.Ellagicacid
(MeOHextractofwoodyparts),R.officinalis(MeOHextractof
(4)andgallicacid(6)werebothdetectedinalloftheC.sativa
aerialparts)andA.microcarpus(MeOHextractofleaves).Several
extracts (SupplementaryFigure1B). More detailed chemical
extracts derived from the leaves and woody parts of C. sativa
characterization of the most active extract fraction, 224C-F2, is
alsoexhibitedMIC valuesrangingfrom128to256µgmL−1.
50 reported in a recent publication concerning its anti-virulence
PositivecontrolsclindamycinanderythromycinexhibitedMIC
50 effectsagainstS.aureus(Quaveetal.,2015).
of0.25µgmL−1and0.063µgmL−1,respectively,andMIC of
90 The H. rutilans extract underwent further characterization
2µgmL−1,whileresveratrolexhibitedanMIC50 andMIC90 of usingnegativeESILC-FTMS.The[M-H]−for9wasdetermined
100µgmL−1. tobe291.06755,providinganempiricalformulaofC H O (1
18 11 4
2.365). Comparison with published literature identified 9 as
BiofilmEradication
the antitumor and dihydroorotate dehydrogenase inhibitor
None of the extracts tested exhibited at least 50% eradication
polyporic acid, which can account for up to 40% of the fungal
of mature biofilm within the tested concentration range. The
fruitingbody’smass(Kraftetal.,1998).
positive control for biofilm eradication (resveratrol) exhibited
MBEC andMBEC of100µgmL−1.
50 90
DISCUSSION
ComparisonofActivityBasedonTraditionalUse
Initial screening of Italian plants and fungi in the QNPL was The central aim of this study was to determine whether or
conductedat256µgmL−1,resultinginpercentinhibitionvalues not extracts that are derived from plants or fungi used in
for both growth and biofilm. Extracts were sorted into two topical skin therapeutics for acne and other forms of SSTI in
groups, based on history of traditional medicinal use for acne traditionalmedicinewouldhavegreateractivityagainstP.acnes
andotherSSTIsinItaly.Extractsfromplantsusedintraditional than specimens from a random sample. Our hypothesis was
therapies for skin infections exhibited a mean value of 38% supported; we found that extracts of plants used for traditional
inhibitionofgrowth,whichwassignificantlygreater(p<0.001) anti-infectiveskintherapeuticshadasignificantlygreaterimpact
than that of extracts from randomly selected plants and fungi on planktonic growth and on mature biofilms of P. acnes than
(12%), Figure1A. Likewise, there was a significant difference extractsfromrandomsamples(includingmedicinalplantsused
(p < 0.05) in biofilm eradicating activity between both groups forotherpurposesunrelatedtotheskin).Here,wediscusssome
(10% in SSTI versus 5% in random, Figure1B). Outliers in of the specific sources of bioactivity reported in this study and
the data set were also identified and removed by the ROUT makerecommendationsforfutureinvestigation.
methodandthenreassessedusingaStudent’st-test.Fiveoutliers Ofthe157extractsscreenedinthisstudy,12exhibitedmore
were identified and removed from the growth inhibitory data, than50%growthinhibitionatthetestconcentrationrange.These
and the groups were found to be significantly different (p < twelvecamefromonefungusandsixplantspecies.Thebioactive
0.0001),Figure1C.Threeoutlierswereidentifiedandremoved fungal species is a polypore fungus, and is also known by the
inthebiofilmdatasets,alsoreconfirmingasignificantdifference commonnamesoftendernestingpolypore,purpledyepolypore,
betweengroups(p<0.001),Figure1D. orthecinnamonbracket.Whilesomeotherpolyporefungi(i.e.,
Trametes versicolor, also known as Turkey tail) can play an
Mammalian Cytotoxicity important role as cosmetic or cosmeceutical ingredients (Hyde
Extracts with MIC values detected were examined for et al., 2010), to the best of our knowledge, there are no reports
50
cytotoxicityagainstanimmortalizedlineofhumankeratinocytes ofsuchusesforH.rutilans.Althoughnothinghasbeenreported
at a test range of 2–256 µg mL−1 in order to determine concerningtopicaltoxicityorcontactdermatitiswiththisspecies,
whether the observed antibacterial activity was due to specific multiplereportsonpoisoningeventsfollowingconsumptionof
antibacterial action or general toxicity. The only extracts to this fungus have been reported, and include symptoms such
exhibit an IC came from J. regia and Hapalopilus rutilans, as purple discoloration of the urine, nausea, vomiting, blurred
50
both at the highest test dose of 256 µg mL−1 (Figure2). All visions, hallucinations, and even death (Kraft et al., 1998; Villa
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Nelsonetal. Anti-AcneActivityofItalianPlantsandFungi
FIGURE1|Extractswerescreenedforpercentinhibitoryactivityagainst(A)growthand(B)biofilmataconcentrationof256µgmL−1.Outlierdatapoints
wereidentifiedusingtheROUTmethod,removedandthedata-setreanalyzedfor(C)growthand(D)biofilmactivity.*p<0.05;**p<0.01;***p<0.001;****p<
0.0001.
etal.,2013).Suchneurotoxiceffectshavebeenattributedtothe growthinhibitoryactivityobservedastheMICfortheseagainst
high content of polyporic acid in the fungus, which was also anotherstrainofP.acneswasreportedas800,1800,and1000µg
identifiedastheprincipalcomponentoftheextractevaluatedin mL−1,respectively(Patiletal.,2012).While7wasnotdetectedin
thepresentstudy(65.74%ofthecrudeextract,representing5.7% thisleafextract,itisknowntobefoundingrapevine,particularly
ofdrymassofthefruitingbody).Whiletheextractofthisspecies thegrapeskin.Ithasbeenidentifiedashavinggrowthinhibitory
inhibited P. acnes growth (MIC 128 µg mL−1) and inhibited and biofilm-eradicating activity against P. acnes (Coenye et al.,
50
mature biofilm by 45% at a concentration of 256 µg mL−1, it 2012). This is the first report of grape leaf extracts exhibiting
alsoexhibitedcytotoxicitytoskincells(HaCaT)(IC of256µg growth inhibitory effects. The interactions between grapevine
50
mL−1)atornearconcentrationsinhibitorytoP.acnes,suggesting and P. acnes are quite interesting, especially considering recent
thattheactivityobservedisduetogeneraltoxicityoftheextract. phylogenetic and population genetic research that suggests a
While none of the plant species examined were effective at horizontal interkingdom transfer of this human opportunistic
eradicatingmatureP.acnesbiofilm,wedididentifyfivespecies pathogentograpevineasanobligateendophyteduringtheperiod
withgrowthinhibitoryactivityatconcentrationsthatwerenon- inwhichthisspecieswasdomesticatedbyhumans(Campisano
toxic to HaCaTs. The most active was a leaf extract from the etal.,2014).Furtherstudiesonanti-acneactivityofthisextract
aglianicograpevarietal(V.viniferavar.aglianico)withaMIC anditsindividualconstituentsarewarranted.
50
andMIC of64µgmL−1.Nocytotoxicitywasdetectedagainst Another active extract came from V. sativa subsp. sativa
90
HaCaT cells at that concentration and only 40% cytotoxicity (Garden vetch), and it exhibited a MIC and MIC against
50 90
wasreachedatthehighestconcentrationtested(256µgmL−1). P.acnesatconcentrationsof128and256µgmL−1,respectively.
Amongthestandardsincludedinourchemicalcharacterization TheIC forcytotoxicitytoskincells(HaCaT)wasnotdetectable
50
studies, 3, 4, 6, and 8 were all detected in this extract. It is atthistestrange.HPLCanalysisrevealedlowlevelsof6and8,as
unlikely that 4, 6, or 8 were individually responsible for the well as a number of larger peaks that should be prioritized for
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