Antifungal Activity of Selected Malassezia Indolic Compounds Detected in Culture

This is a repository copy of Antifungal activity of selected Malassezia indolic compounds detected in culture.

White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/143331/

Version: Accepted Version

Article: Gaitanis, G, Magiatis, P, Mexia, N et al. (4 more authors) (2019) Antifungal activity of selected Malassezia indolic compounds detected in culture. Mycoses, 62 (7). pp. 597-603. ISSN 0933-7407 https://doi.org/10.1111/myc.12893

© 2019 Blackwell Verlag Gmb. This is the peer reviewed version of the following article:Gaitanis, G, Magiatis, P, Mexia, N, et al. Antifungal activity of selected Malassezia indolic compounds detected in culture. Mycoses. 2019; 62: 597– 603, which has been published in final form at https://doi.org/10.1111/myc.12893. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Uploaded in accordance with the publisher's self-archiving policy.

Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item.

Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request.

[email protected] https://eprints.whiterose.ac.uk/ This article is protected by copyright. All rights reserved. articleThis rightsAll protected is reserved. bycopyright. 10.1111/myc.12893 doi: lead differences to version between this cite Please of the and a Record. Version this been and may which proofreading typesetting, through copyediting, the process, pagination Accepted articleThis undergone has but has for and not accepted review been peer full publication ArticletypeArticle : Original Article PROFESSOR GEORGIOS GAITANIS (Orcid ID : 0000-0001-548 Georgios Gaitanis,* selected activity of Antifungal Malassezia detected culture indolic compounds in Efstratiou, 4 4 3 Athens, Greece. Pharmacy,Kapodistrianof Athens, andUniversity Panepistimioupolis, National 2 Ioannina, Ioannina,Greece Sciences, University of 1 Short Title:Short 5 of Microbiology, National Athens School, University Kapodistrian and of Medical DepartmentVenereal Health and ofSkin of School Faculty Diseases, ofMedicine, Bioiatriki Athens, SA, Greece Mycology Departmen LaboratoryCollection, Culture UOA/HCPF Research and Department Sciences, ofMarine Aegean, University ofthe Mytilene, Greece Department ofPharmacognosyNaturalof Chemistry, and Faculty Products 3 Ioannis Bassukas, D. Antifungal activity of 1 Prokopios Magiatis, 1 Aristea Velegraki Malassezia Malassezia 2 Nikitia Mexia, Nikitia indoles indoles 2-1304) 4 ,5

2 Eleni Melliou, Eleni 2 Maria A. rticle as t t

This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article spp.(N=10) Key words Georgios Gaitanis CorrespondingAuthor: were against 40fungal [yeasts: tested strains hydroxymethyl-indolo[3,2-b]carbazole and and 6-methyl-indolo[3,2-b]carbazole) b]carbazole, tryptanthrin, 6- indolo[3,2b]carbazole, formyl-indolo[3,2 and potential the for InhibitorywasMinimum fungi,opportunistic determined the (MIC) Concentration againstoftryptophanwascommens theseagar.panelA substances tested Malassezia Background Abstract Fax: 00302651007031 [email protected] Email: Tel:00302651007530 Ioannina, Sciences,ofMedicine, Ioannina, ofHealth School University Greece DepartmentVenereal“S. Niarchou” and ofSkin Faculty Avenue, Diseases, of Eight indoles were included ( Eight were indoles included Materials Methods and yeasts produce bioactive indolic substances when grown on L-yeasts on producegrown bioactive substances when indolic ; ; : : Candida Malassezia

in loco spp.(N=7) , indoles, tryptophan, indirubin, antifungal , indoles, tryptophan, antifungal indirubin, antifungal activity on the skin was skin the assessed. antifungal on activity ma ; ; lassezin, pityriacitrin, indirubin, indirubin, pityriacitrin, lassezin, Y arrowia lipolytica Yarrowia Malassezia (N=1) spp.(N=9 ; ; Exophialla );

Cryptococcus

dermatitidis al and al

This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article (N=2) The MIC range was 0.125-32g/ml and the median log andThe median the range MIC was0.125-32g/ml Results 33strains. in susceptibilityevaluation U-Mann-Whitney and data.published foremployed Kruskal-Wallis were tests Malassezia foundforhighest FICZ. was MIC median The antifungalpotent from the most others. the and agent differed significantly The concentration5 of characterized a compromised barrier. skin variably by andof neck atopic dermatitis. manifestations certa dermatits (SD) dandruff(DF)/seborrheic pityriasis and(PV), versicolor includes 17 species Malassezia with Themicroorganisms an harbors skin human and array species ofmicrobial Introduction significant yeastssignificant and mold Malassezia Conclusions skin. and Aspergillus ; ; molds: molds: with with yeasts principalthe being eukaryotic organisms. yeasts species produceclinically indolic array an that inhibit of Aspergillus and inhibitory this activity was human valid predicted be to also on Candida 3 and in humans has humansin been and of associated the with development /8 of the tested indoles on diseased skin was calculated from wasof skin the on diseased tested calculated indoles strains were more susceptible compared strains were to susceptible more spp.(N=7) s.

; ; Fusarium 4 All All are theseconditions common skin spp.(N=2) 5,6 2 MIC was 4. Indirubin was Indirubin MIC 4. was

; ; 1,2 Rhizopus

Th is genusis currently Cryptococcus

oryzae (N=2)]. (N=2)]. in head in group

This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article Initially it was reported that Initially was it reported that source. L-tryptophannitrogen a substances asexclusive with medium grown the in when M. furfur healthy skin. vitro Malassezia Malassezia M. furfur and patients. PV indolo-[3,2b]-carbazole (ICZ),indolo-[3,2b]-carbazole pityriacitrin (FICZ).Among biologic potential indicate possess indirubin that it it aureus protective at 5%concentration1.7) factor (SPF: damage skin. mediator onhuman (AhR) candidate receptorendogenous ligand ultraviolet and an aryl-hydrocarbon pathogenic fungi. The determined MIC values in g/ml wereg/mlw MICpathogenic in interpreted fungi.The values determined determiningantifungal ofcommensal against activity and a opportunist selection in study screen ofAim this wasdetectable indoles to inflammation and inflammation tumorigenesis biological pathway. this significance of treatment for psoriasis. ascomycetousgastrointestinal truck commensal yeasts ofrestricted the to could against potentiate resis also antibacterial the effectciprofloxacin of significantly larger quantities of indoles compared significantlyquantities ofindoles from isolated those to larger strains 7 Subsequently, strains isolated from SD and PV lesions have strains fromisolated and SD synthesize to lesions the ability PV but is wider distributed among more species within the genus, while wider genus, the amongwhile distributed within is but species more and their respective indoles can be also traced and their respectivealso can indoles be 8 Furthermore, in vitro 9 Importantinclude malassezin, indirubin, molecules of group this . 13 17 it yet sunlightMoreover, has pityriacitrin measurable, small, a L-tryptophan not is The production ligands of these from was found that under the aforementioned culture conditions aforementionedconditions culturewas the foundthat under Malassezia it it M. furfur has become clear that this propertythis has to restricted not become clear that is es some some antifungal 16 15 al and currently is suggestedas potent a topical Regarding indirubin effects of these substances preliminary data substances ofthese effects preliminary genus but has also been described in the been also the in genusdescribed has but strains have the ability the have strains indolic produce to 18

10 and formyl-indolo–[3,2b]-carbazole 14 C. glabrata C. 11 and FICZ a considered is and antiparasitic Malassezia Malassezia it it in mediates functionsas underscoringwider the skin scales from SD SD from scales skin cultures for 12 activity, whilst ith regardith tant tant S. in ic ic This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article indoles previouslyindoles fromdiseased ( isolated skin UOA/HCPFagainst were 8 tested maintainedForty of at the Pathogenic Hellenic fungal Fungi Collection strains Materials Methods and strains included 27 yeasts comprising yeasts strains 27 included carbazole (M)carbazole [3,2-b] (Table 2). The tested (H) and 6-methyl-indolo FICZ)indirubin, ones [3,2-b] and tryptanthrin, 6-hydroxymethyl-indolo 3additional potential potential to (N=10) were10 in maintained ATCC90018 andATCC90018 andrespectively( occasion reference the control strains quality respectivelyeac followed. twicemolds tested Eachwere was on and strain yeastsguidelines and forand method the CLSI M38-A2 microdilution M27-A3 likely dilution issues of the tested indoles to avoid underscoring the MIC value. underscoring of testedthe likely issues avoid to indoles MIC the value. dilution recorded Tablein 2 experiments is 1. was This performed compensate to for order in discarded.results from the the testedMIC Regarding fungal higher the strains, ofthe was experiment the differedresultsfor strains>2 quality dilutions the control dermatitidis MYA-3630 and qualityreference strains control respectively and Rhizopus the concentrations the of respective the order scales in indoles skin in to predict ; ; Candida

in loco oryzae (N=2); and 13 molds (N=2);13 molds and antifungal activity of (N=2). The used indoles were synthesized as previously The used synthesized indoles were (N=2). Aspergillus fumigatus spp. (N=7) Candida albicans Candida -2 M dimethyl sulfoxide stock solutions. Fordimethyl broth the solutions. stock sulfoxide ; ; Y arrowia lipolytica Yarrowia Aspergillus Malassezia Malassezia ATCC90028 for yeasts,for ATCC90028 the molds and for Malassezia ATCC MYA-3626) ATCC were included. Malassezia Malassezia spp. (N=7) indoles (Table 1) including the 5 5 indoles (Table 1) the including ma Paecilomyces variotii spp. (N=9spp. (N=1); the black yeast (N=1); black the related indoles. related indoles. lassezin, pityriacitrin,lassezin, ICZ, ; ; Fusarium );

Cryptococcus Cryptococcus Candida parapsilosis Candida spp. (N=2) ATCC ATCC 8,9, Exophialla h testingh If 10 the the spp. and - ; ;

This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article The concentration of the appliedTheof concentration the published previously.published skin extracts to corresponding g/ml the following formula was employedwas corresponding extractsskin to the g/ml formula following the in transformationFor the ofwas concentration the indoles gr/ml. of taken at 0.9 Cx (mol/mgCx * Molecular extract) weight *10 indirubin (medianindirubin log substances the indolic test fungalarray the presently of strains, employed from Iny analysis results the were statistical from the as 33strains the included analysis Statistical one ofthe differed that from activity in ones any significantly other yeasttested substanceslog strains median indolic and Ove values the forTable fungal 1 tested. strains the compiles MIC the of species Results IL, softwareSPSS USA Chicago, (v22, U and employing tests was Mann-Whitney evaluated the the with Kruskal-Wallis group The number tested. small the ofeach indoles ofstrains susceptibility ofthe lipolytica handMIC the (log highest median other carbazoleandhydroxymethyl-indolo FICZ On the (p=.003) [3,2-b] (p<.001). carbazole(p=.003),(p=.004), malassezin (p=.015), [3,2-b] 6- pityriacitrin Regarding susceptibility with with significantly more sensitive as compared to the latter group over all tested indoles all testedsignificantlyover indoles as group more compared the to latter sensitive Candida and the molds and molds the and Cryptococcus 9 2 2 As the skin extracts mostly consist of skin lipids the of mostlydensity lipids skin extracts skin the consist As MIC=3) was the most potent antifungalMIC=3)agent only andpotent was the the most Rhizopus by fungal generagroups were two fungal discerned: with with

indoles on human skin was calculated from datawas skin onhuman indoles calculated from , 2 Fusarium MIC=5) was found for FICZ (FiFICZ forMIC=5) found was Aspergillus and 9 / 0.9 (g/ml extract)= Cx (g/ml) 0.9(g/ml/ (g/ml) extract)= Cx . The former group. The former was Exophiala 2 MIC was 4 (Fig. 1). BasedMIC (Fig. on 4 was 1). were dueexcluded to : : g. 6-methyl-indolo 6-methyl-indolo 1). 1). Malassezia east east : :

Y. ed, ed, r all

This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article (Figure 1 culturesactivityantifungalhave molecularHereinindolic isolated from demonstrate species we that Discussion (SD3). concentration indirubin the pityriactrin and to one in In(SD2 andcasesthe PV2)respective activity inhibitorycould two attri be would inhibit all including fungalwould inhibit the strains, contain From3 (2SD; 1PV)extracts scales 9 the skin antifungal corresponding the activity susceptibilityon possible skin to data compare to g/ml abovescales (Table the 2) were order in transformed to Theof concentration the carbazole (Figure6-hydroxymethyl-indolo1 [3,2-b] for pityriacitrin, FICZand selected lesional form already treatment the in evaluated as agent is it mentioned topical a ofpsoriasis investigation as significantlyis active most the target the is one. molecule This ofintense of MIC the established with antifungals numerical comparison beStrict, wouldnot as it skin the easily from removed sweat. Furthermore, could indirubin an be on lowwater solubility asset characterizes that detected beand an antifungal the herein additional indication. could properties active ingredient of indigo naturalis,medicine. traditionalactive aindigo popular ingredient Chinese of are presently assessed, they do achieve on the skin concentrationscould that be are theyassessed, achieve presently skin onthe do that context the within Thus, afternot level. or the tissue blood in systemic intake beperformed performedantifungal would be as function the ; ; Panels A, C Panels it it possess ). SD

The pattern of susceptibility was equivalent for eachThe genus pattern for of equivalent was susceptibility Malassezia and es anti-inflammatory anticancer the is it and properties and PV in vitro skin. From the indolic substances tested, indirubin substances indirubin indolic From skin. tested, the associated indoles isolated from isolated skin associated lesional indoles 19 at concentrations detected in scale probesdetected scale in at concentrations of indirubin or the other indoles should not not or of indirubin othershould the indoles Malassezia

indole concentrations that strainstested in loco on the skin and and skin onthe Malassezia Malassezia in this study.this surface. buted to to buted 16 As ). ). 17

y This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article active pathogenic yeastspathogenic( lower than herein the MIC. recorded in vitro producers, i.e. activity their own antifungal against A notable theythat is observation show effectcannotexcluded. existence of this a action to be synergistic adds that does not inhibit does inhibit not Candida resolution to L-tryptophan resolution to use when have these exhausted. been sources sourcesas glycine preferentially the might nitrogen is and ofother explain use colonies Malassezia Another issue by underscored exact the is findings the study role ofthis of and Magiatisdata).from unpublished (Mexia these,tryptanthrin only these observations wewould like suggest to thethat that decisive together, trait array indolic further compounds. of transformationand subsequent the substancewhole ofthe ‘avital’ precursor a into L-tryptophan formation begins ofindolepyruvate from enzyme-mediated an with It their environment.been of these the formation proposed has substances that in by accumulation bioenergetics the optimizing and ofthe of assembly bioproducts their physico-chemical order in exploit improve to milieu their survival competence compounds certain adaptive represents an phenomenon of to ICZ to the action of the omnipresent UV radiation or further oxidative reactions. actionthe of oxidative UV radiationfurther omnipresent the or in vivo 22 by and additional tryptophan be can formed metabolites onthe through skin invivo species, only Malassezia in the accumulation of indolic compounds the in ofindolic accumulation association in yeast with against commensal ( commensal against Malassezia and Malassezia Cryptococcus in vitro species when grown on L-tryptophan agar are 2-3 dilutions L-tryptophan aregrown 2-3 agar when on dilutions species C. glabrata glabrata C. and growth up to a point, yet growth substances the toxicity a ofthese upto point, . 9 Most probably, Most indolic of production the skin onthe Candida ) and ( molds Malassezia has been produce to substances found indolic and 21 8 8

Accordingly,can easily transform malassezin This meansThis that production of indoles species. indole concentration The achieved and and Aspergillus Candida ). Furthermore, the Malassezia Malassezia ), opportunistic stains that can that stains 20 23 As for Taken all

in vitro

This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article Inconclusion, skin, substances might be not spatially related the to available conditions.Hence, milieu effectiveconcentrations antifungal ofthese where of further formation subsequent continues onthe molecular species depending availableL-tryptophan indolepyruvate into excrete the environment into it and to compoundsindolic their in environment ability their is and metabolize uptake to controls the variation the in capacity ofdifferent Acknowledgemen healthyand skin. diseased expanding of these ourknowledgeon distribution onthe bioactive substances Futureclinical research elaborate by should onthe significance observation this of array an yeast strains clinically at inhibit mold meaningful of and concentrati None None Interest of Conflict forand technical A. Milioni expert assistance.

thus not actually not thus producer the strains. inhibiting Malassezia ts . The Authors express their Kritikou appreciation wish to . The S. to yeasts species indolic to ability produce the have that Malassezia Malassezia microcolonies onthe strains to accumulate strains to ons.

. HuttenhowerD, Knight C,Gevers et function Structure, al. R, and diversityof the 2. 1. Findley K, Oh J, Yang J, et Findley Yang Topographic al. offungal bacterial and K, Oh J, diversity J, 1. 6. Lee WJ, Kim SongKim CH, JY, Lee et ofbarrier Disruption al. dermatophytosi in WJ, function 6. Lee Park HJ, Choe YB, Characteristics PityriaYW, Patients in with Skin Ahn KJ. 5. GaitanisMayserID. G, Velegraki diseasesSkin P, Bassukas A, associate 4. . TheelenB,ID,Gaitanis Cafarchia Boekhout G, Bassukas C, TL. Dawson T, 3. 7. Mayser P, Wille G, Imkampe G, Mayser P, Wille A, Arnold of W, Thoma N, Monsees Synthesis T. 7. 8. Gaitanis G, Magiatis P, Stathopoulou K, et al. AhR ligands, K, Gaitanis et Stathopoulou AhR al. malassezin, and G, Magiatis P, 8. . Magiatis G, et Pappas Gaitanis al. P, P, 9. This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article healthy microbiome. human communities in human skin. human in skin. communities References and pityriasis versicolor. Versicolor Method, MPA5. Using Non-Invasive Malassezia Malassezia fluorochromesand pigments in nitrogen source. indolo[3,2-b]carbazole areindolo[3,2-b]carbazole selectively produced by human skin.human exceptionally(dioxin) of activators receptor potent ah the detected diseased in from seborrheic dermatitis. yeasts: Facts controversies. and ecology, pathophysiology, and treatment.

J InvestJ Dermatol Mycoses J Dermatol J 1998; J InvestJ Dermatol Nature Nature . 2013; 41 Malassezia furfur Malassezia :265-71 . 2012; 2013; . 2011; 133 Malassezia :2023-30 :2023-30 498 486 38 Clin Dermatol Clin 2008; :367-370 :1049-1053 :207-214 Ann Dermatol by tryptophan single as use of the yeasts of produce a collection Malassezia furfur Malassezia 128 Med Mycol :1620-5 . 2013; . 2012; . 2018; 31 :455-463. :455-463. strains isolated 24 56S :444-452. d with with d :10-25 sis sis s 10. Mexia N, Gaitanis Mexia G, Velegraki A, Denison Magiatis MS, A, Soshilov P. 10. 11. Ponnusamy K, Petchiammal C, MohankumarIn C, Ponnusamy Hopper R, antifungal W. K, Petchiammal vitro 11. 12. Krivogorsky B, Grundt P, Yolken R, Jones-Brando L. Krivogorsky Inhibition Yolken B, P, R,Jones-Brando of Grundt 12. 3 PonnusamyI, K, RamasamyIndirubin M, Savarimuthu MG. potentiates Paulraj 13. 14. Gambichler T, Krämer H-J, Boms et Quantification al. S, Gambichler of ultraviolet H-J, protective Krämer T, 14. 15. Park SL, Justiniano R, Williams JD, Cabello CM, Qiao S, Wondrak GT. The GT. Cabello The R,Williams CM, Park SL,JD, Qiao Wondrak S, Justiniano 15. 16. Cheng X, Merz K-H. The Role of Indirubins in InflammationIndirubins and Cheng of Associated in Role K-H. The X, Merz 16. 17. Lin Y-K, See L-C, Huang Y-H, Chi C-C, Hui RC-Y. Comparison of indirubin HuiRC-Y. C-C, Comparison Lin Chi ofL-C, indirubin See Y-H, Y-K, Huang 17. This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article Arch Biochem Biophys Pityriazepin AhR ligands from other and potent isolated tinctoria Indianactivity from a ethnomedicinal plant isolated South ofindirubin gondii Infect Dis ciprofloxacin of pump efflux NorA activity the in 2008; effects humans. ofpityriacitrin in Keratinocytes. IsFormylindolo[3,2-b]Carbazole Epidermal in Photosensitizer a NanomolarUVA Tryptophan-DerivedAryl ReceptorLigand Endogenous Hydrocarbon 6- Tumorigenesis. In:Tumorigenesis. concentrations in indigo naturalis for ointment concentrations psoriasis indigo treatment: in a randomized, 2016:269-290. doi:10.1007/978-3-319-41342-6_12. 2016:269-290. doi:10.1007/978-3-319-41342-6_12. 52 by and indirubin tryptanthrin analogs. :4466-4469. :4466-4469.

R. Br. 2010; J InvestJ Dermatol

J Ethnopharmacol J 42 :500-505 :500-505 Advances Medicine Biology inExperimental and 2015; 571 :16-20. . 2015; Arch Dermatol ResArch Dermatol 2010; 135 132 :1649-1658 :1649-1658 :349-354 :349-354 Antimicrob Agents Chemother Staphylococcus aureus 2007; Malassezia furfur Malassezia 299 :517-520 :517-520 Toxoplasma . Vol 929. ; . Vol929.; Wrightia . yeast. Scand J J Scand

8 MayserBLJ, M, Krämer Kindler P, Haase P, Wenzel Spiteller of H-J, G. Production 18. 9 Lewis Antifungal Current in RE. Concepts Pharmacology. 19. 20. Barchmann T, Hort W, Krämer H-J, Mayser Barchmann Krämer P. Glycine W, regulator tryptopha T, as Hort a H-J, of 20. 1 Zutheret aminotransferase K, The al. U,Tam1 P, Hettwer Mayser tryptophan 21. 22. Wille G, Mayser et Wille W, Malassezin al. P, Thoma 22. 23.

Accepted Article indole pigments by double-blind, dosage-controlled trial. dependent in synthesis pigment 2011; Ustilago maydisUstilago stepcatalyses for synthes pigment tryptophan-dependent biosynthetic single the arylhydrocarbon receptor from the yeastarylhydrocarbon from receptor the endogenous signal substances. tryptophanreceptor be affinityhigh and very with bind the to Ah are to likely derivativesRannug photooxidized of Certain U, Rosenkranz HS,al. et A, Rannug 2001; 86 9 :955-60 :805-817. . Mol Microbiol Candida glabrata Candida J BiolJ Chem Malassezia furfur Malassezia 2008; . Br J Dermatol Br J 68 Me Malassezia furfur Malassezia :152-72 :152-72 d Mycol 1987; -- A novel ofthe agonist . 262 2007; Mycoses 2018; :15422-7 45 178 . :519-524 Mayo Proc Clin 2011; Bioorg Med Chem :124-131 54 :17-22 :17-22

Accepted Article indoles (g/ml). tested Inhibitor Table corresponding the Minimum 1.The includedand the species in study fungal

Molds Yeasts Rhrizopus oryzae oryzae Rhrizopus oryzae Rhrizopus oxysporum Fusarium solani Fusarium dermatitidisExophiala dermatitidisExophiala Aspergillus niger Aspergillus niger Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus flavus Aspergillus flavus Yarrowia lipolytica Malassezia yamatoensis Malassezia sympodialis Malassezia slooffiae Malassezia restricta Malassezia pachydermatis Malassezia nana Malassezia globosa Malassezia furfur Malassezia dermatis sero D CR neoformans 96Cryptococcus sero C neoformans Cryptococcus sero AD neoformans Cryptococcus sero AD neoformans Cryptococcus sero A neoformans Cryptococcus neoformans Cryptococcus neoformans Cryptococcus laurentiiCryptococcus gattiiCryptococcus albidus Cryptococcus tropicalisCandida tropicalisCandida parapsilosis Candida krusei Candida glabrata Candida albicans Candida albicans Candida TC2884 8 ATCC6258 UOA/HCFP 2213 UOA/HCPF 14744 O/CF54 4 8 8 8 8 4 4 8 UOA/HCPF15749 CS52 .2 0.5 4 4 0.125 CBS 562 CBS9559 O/CF58 8 4 8 4 8 UOA/HCPF15489 UOA/HCPF 3908 8 8 8 8 ATCC90028 UOA/HCPF15587 UOA/HCPF 12726 CBS 7983 8 8 CBS 7904 B9 8 8 8 8 8 4 8 0.032 2148-93IP CBS94 B62- 8 8 CBS6124-1 CR86 O/CF14382 8 UOA/HCPF 15443 UOA/HCPF15428 B75 2 4 CBS7956 CBS9145 TC01 8 8 8 8 ATCC90018 CR 127 UOA/HCFP 12739 O/CF162 8 UOA/HCPF15831 UOA/HCPF 7431 UOA/HCPF 14662 CR 125 UOA/HCPF3801 UOA/HCPF 3801 CBS7222 CBS 9725 UOA/HCPF 9235 CR192 CBS1880 CR 81 CR 12 CR 49 CR 48 aasznIZPtrairnIdrbnFICZ Indirubin Pityriacitrin ICZ Malassezin 23 21 23 216 32 32 32 32 32 32 16 16 32 32 32 32 32 32 23 23 23 232 32 32 32 32 32 16 32 32 32 32 32 32 32 32 32 32 32 32 32 16 32 32 32 32 32 16 32 32 32 32 32 16 32 32 32 32 32 32 32 32 32 32 16 32 16 32 32 32 32 32 32 16 32 32 32 32 32 32 32 16 32 32 32 32 16 16 32 32 16 32 16 32 16 32 16 32 16 16 32 32 32 16 16 16 32 32 32 32 32 32 23 61 63 632 16 32 16 16 16 32 32 23 21 23 232 32 32 32 32 32 32 32 32 32 16 32 16 32 32 16 16 32 32 16 32 16 32 16 32 16 32 32 32 32 32 32 232 32 63 632 16 32 16 8 8 8 8 8 8 8 4 8 4 2 4 8 4 16 16 16 16 16 16 32 16 16 16 8 4 8 61 61 16 16 16 16 16 4 1 8 8 4 2 8 1 8 4 2 2 8 8 8 8 4 8 8 8 4 4 32 16 32 616 16 16 16 16 16 32 216 32 61 616 16 16 16 32 216 32 16 6-hydroxy-methyl- b]carbazole indolo[3,2- .152 0.0125 .528 2 0.25 16 32 16 16 8 8 8 8 8 8 8 8 4 8 4 y (MIC) of 4 the Concentrations Tryptanthrin 16 616 16 32 8 8 8 4 8 8 8 8 8 8 8 4 6-methyl-indolo 6-methyl-indolo [3,2-b]carbazole 16 16 16 16 16 16 16 16 16 32 8 8 8 8

Malassezia Malassezia Table 2. be Concentrations skin. human ondiseased inhibited are from adapted –carbazole [3,2b] Accepted articleThis protected is rights by All copyright. reserved. Article SD2 SD1 PV3 PV2 PV1 SD6 SD4 SD3 Malassezia Malassezia associated indole concentration (g/ml) on skin extracts and respective and ge extracts fungal onskin (g/ml) associated indole concentration 0. 0. 0. 0. Malassezin 33 76 06 06

5. 2. ICZ 97 84

6. 1. 34 Pityriacitrin 84 87 . 24

9 . PV: Pityriasis versicolor; SD: Seborrheic dermatitis; ICZ: . PV: indolo- Pityriasis Seborrheic SD: versicolor; dermatitis; 3. 75 1. 43 23 Indirubin 78 22 . . . 68 66 57

Candida, Malassezia Malassezia Fusarium,Candida, Cryptococcus, Aspergillus, Candida, Malassezia Candida Candida Malassezia Fusarium,Candida Cryptococcus, Aspergillus, Malassezia Fusarium,Candida, Cryptococcus, Aspergillus, Malassezia Candida,Cryptococcus, Species inhibited nera least at tested would strain one that

This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article in thein study i.e. B.genera all strains testedthe Antifungal ofthe indoles against of activity incl P Susceptibilitystudy genera fungal included againstof the in the each indole. Figure 1 indolo – [3,2 b] –carbazole– [3,2 indolo for FICZ: 4 generastudy indolesarray employed. the in against of the included one active most the PanelactiveB.FICZ and substance less Susceptibi the statistical significance. . Susceptibility of the 33 fungal strains included in the study. Panel A. ofPanel 33fungal the . Susceptibility study. the in strains included Malassezia , ; ; Candida ICZ: -carbazole. asterisk The [3,2b] denotes indolo- , Cryptococcus and Aspergillus . Indirubin. was lity ofthe anel uded uded myl- This article is protected by copyright. All rights reserved. articleThis protected is rights by All copyright. reserved. Accepted Article