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Journal of Ethnopharmacology 115 (2008) 140–146

Antifungal activity of medicinal plant extracts;preliminary screening studies

Duncan Webster a,∗, Pierre Taschereau b, Rene J. Belland c,Crystal Sand d, Robert P. Rennie e

a Department of Medicine, Division of Infectious Diseases, Saint John Regional Hospital,P.O. Box 2100, Saint John, New Brunswick E2L 4L2, Canada

b Department of Biology, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia B3H 4J1, Canadac Devonian Botanic Garden, University of Alberta, Edmonton, Alberta T6G 2E9, Canada

d National Centre for Mycology, Provincial Laboratory for Public Health for Northern Alberta,University of Alberta Hospital, Edmonton, Alberta T6G 2J2, Canada

e Director, National Centre for Mycology, Site Chief, Laboratory Medicine and Pathology,University of Alberta Hospital, Edmonton, Alberta T6G 2B7, Canada

Received 24 August 2007; accepted 19 September 2007Available online 25 September 2007

bstract

thnopharmacological relevance: In the setting of HIV and organ transplantation, opportunistic fungal infections have become a common causef morbidity and mortality. Thus antifungal therapy is playing a greater role in health care. Traditional plants are a valuable source of novelntifungals.im of the study: To assess in vitro antifungal activity of aqueous plant extracts. The minimum inhibitory concentrations were determined for eachxtract in the setting of human pathogenic fungal isolates.aterials and methods: Plants were harvested and identification verified. Aqueous extracts were obtained and antifungal susceptibilities determined

sing serial dilutional extracts with a standardized microdilution broth methodology. Twenty-three fungal isolates were cultured and exposed tohe plant extracts. Five known antifungals were used as positive controls. Results were read at 48 and 72 h.esults: Of the 14 plants analyzed, Fragaria virginiana Duchesne, Epilobium angustifolium L. and Potentilla simplex Michx. demonstrated

trong antifungal potential overall. Fragaria virginiana had some degree of activity against all of the fungal pathogens. Alnus viridis DC., Betulalleghaniensis Britt. and Solidago gigantea Ait. also demonstrated a significant degree of activity against many of the yeast isolates.onclusion: Fragaria virginiana, Epilobium angustifolium and Potentilla simplex demonstrate promising antifungal potential.2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Antifungal agents; Plant extracts

. Introduction

Throughout history and across the globe, the plant kingdomas provided a variety of medicines. In modern times, plantsave been a source of analgesics, anti-inflammatories, antineo-

lastic drugs, medicines for asthma, antiarrhythmic agents, andntihypertensives. Plants with antimicrobial activity are alsonown to be numerous, yet prior to a decade ago, minimal

∗ Corresponding author. Tel.: +1 506 648 6807; fax: +1 506 648 7379.E-mail address: dvwebste@dal.ca (D. Webster).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.09.014

esearch had been conducted in the area of antifungal medicinallants.

Fungi are ubiquitous in the environment, and infection due toungal pathogens has become more frequent (Walsh and Groll,999; Fleming et al., 2002). With the rise of HIV, opportunisticungal pathogens have become a common cause of morbiditynd mortality (Garbino et al., 2001). Furthermore, since the firstuman kidney was transplanted in 1954, the frequency of organ

ransplantation has increased and the number of immunocom-romised hosts has grown steadily with the introduction of novelmmunosuppressive agents and improved rates of survival. Thushe incidence of opportunistic infections, including fungal infec-

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ions, has also increased (Rubin et al., 1981; Winston et al.,995; Fishman and Rubin, 1998). As a result, antifungal ther-py is playing a greater role in health care and the screening ofraditional plants in search of novel antifungals is now more fre-uently performed (McCutcheon et al., 1994; Jones et al., 2000;otsei et al., 2003).The search for novel antifungal agents relies in great

art on ethnobotanical information and ethnopharmacologicxploration. The medicinal knowledge of North American Firstations peoples has been shown to be a valid resource. Studiesave revealed a high degree of correlation between traditionaledicinal uses and laboratory analysis (McCutcheon et al.,

994; Bergeron et al., 1996; Jones et al., 2000). The traditionalreparation of these medicines often involves a broth or tea.owever, much of the antifungal research conducted to dateas assessed ethanol or methanol extracts while few studiesave utilized aqueous extracts, a closer approximation of theraditional medicine.

Alcohol extracts provide a more complete extraction, includ-ng less polar compounds, and many of these extracts haveeen found to possess antifungal properties. Yet success withqueous extracts has also been observed (Ali-Shtayeh and Abuhdeib, 1999). In this study we analyse aqueous plant extracts

hus more accurately replicating the preparation of the tradi-ional medicines. Furthermore, in cases where alcohol extractsave previously shown antifungal potential, should the aqueousxtract of the same plant also show activity it would suggesthat the active components may be the more polar compounds.ikewise, should the aqueous extract not demonstrate activityhere the alcohol extract had, this would suggest that the lessolar compounds are the active components.

In this study, aqueous extracts of 14 plants were ana-yzed. These plants were chosen based on either traditionalsage suggestive of antifungal activity or previous studies that

ave demonstrated antifungal properties using alcohol extractsMcCutcheon et al., 1994; Jones et al., 2000; Filipowicz et al.,003; Thirach et al., 2003). The antifungal activity of a com-ercial preparation of Melaleuca alternifolia (tea tree) oil was

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able 1lant extract sources

lant species Common name Part

eracleum maximum Cow Parsnip Roolnus viridis Green alder Catkotentilla simplex Old-field cinquefoil Stemralia nudicaulis Wild sarsparilla Rooragaria virginiana Wild strawberry Leavetula alleghaniensis Yellow birch Branuniperus communis Juniper Berrolidago gigantea Giant goldenrod Flowlycyrrhiza lepidota Licorice Roopilobium angustifolium Fireweed Roocorus calamus Sweetflag Roortemisia campestris Field sagewort Branrtemisia frigida Prairie sagewort Aeriopulus tremuloides Trembling aspen Catk

B, New Brunswick, Canada; Alberta, Alberta, Canada.

rmacology 115 (2008) 140–146 141

lso analyzed. The essential oil of this bush has been used byustralian Aborigines for centuries and has known antifungalroperties (Hammer et al., 2002, 2003).

The plant extracts were assessed for antifungal activitygainst 23 human fungal pathogens. A broad range of pathogensas chosen, and in many cases a single strain of a given speciesas used, thus providing a preliminary screening assessment.

. Materials and methods

.1. Plant collection

Ethnobotanical literature was reviewed, information was pro-ided by elders and plant species were selected for analysis.lants were obtained from the province of New Brunswick

n eastern Canada and the province of Alberta in westernanada. Plants from eastern Canada included: Heracleum max-

mum Bartr., Alnus viridis DC., Potentilla simplex Michx.,ralia nudicaulis L., Fragaria virginiana Duchesne, andetula alleghaniensis Britt. Plants harvested in western Canada

ncluded: Artemisia frigida Willd., Artemisia campestris L.,opulus tremuloides Michx., Juniperus communis L., Solidagoigantea Ait., Glycyrrhiza lepidota Pursch., Epilobium angusti-olium L., and Acorus calamus L. The plants with their commonames are listed in Table 1. Table 1 also provides a description ofhe parts of the plant used, the month the plants were gatherednd a geographic and geologic description of the region fromhich the plants were harvested.Once plants from eastern Canada were harvested, they were

leaned, air-dried and stored under dark refrigerated conditions.oucher specimens were prepared and submitted to the Dal-ousie Herbarium in Halifax, Nova Scotia. Plant identificationas verified by Dr. P. Taschereau. The western Canadian plants

ere also cleaned air dried and stored under dark cool conditions.oucher specimens were submitted to the Devonian Botanicarden Herbarium, University of Alberta. Plant identificationas verified by Dr. R. Belland.

used Habitat location Month gathered

t NB flood plains Octoberins NB woods Junes, leaves NB woods June

t NB woods Junees NB woods Junech bark NB woods Juneies Alberta canyon Augusters Alberta canyon August

t Alberta riverbank Augustt Alberta woods Septembert Alberta marsh Septemberches Alberta prairie Augustals Alberta prairie Augustins Alberta riverbank August

1 nopharmacology 115 (2008) 140–146

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Table 2Fungal pathogen clinical isolates, control strains and stock strains

Fungal species Isolate source

Yeast isolatesCandida krusei Control strain ATCC 6258Candida krusei Catheter tip culture October 2001Candida albicans Blood culture August 2001Candida albicans Mouth isolate November 2001Candida albicans Control strain ATCC 24433Candida albicans Control strain ATCC 90028Candida parapsilosis Control strain ATCC 90018Candida parapsilosis Control strain ATCC 22019Candida glabrata Urine culture October 2001Candida tropicalis Control strain ATCC 750Candida lusitaniae Subphrenic abscess November 2001Saccharomyces cerevisiae High vaginal swab culture November 2001Cryptococcus neoformans Cerebrospinal fluid culture July 2002

DermatophytesTrichophyton tonsurans NCM stock strain 334Trichophyton rubrum Toenail March 2002Trichophyton mentagrophytes Skin scraping August 2002Microsporum canis NCM stock strain 336Epidermophyton floccosum NCM stock strain 335

Hyaline hyphomycetesAspergillus fumigatus Bronchial washing August 2002Aspergillus fumigatus NCM stock strain 338Aspergillus flavus Bronchial washing August 2002Fusarium solani Pig pasture soil June 1956

ZygomycetesRhizopus species NCM stock strain 337

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.2. Aqueous extract and broth preparation

Extracts of Heracleum maximum, Aralia nudicaulis, Gly-yrrhiza lepidota and Fragaria virginiana were prepared bydding ground compound to distilled H2O at a ratio of 1 g:10 ml.n each case the mixture was brought to a boil and allowed toimmer for 3 h. Extracts of Populus tremuloides, Artemisiarigida and Artemisia campestris were also prepared using a ratiof 1 g of ground plant for every 10 ml distilled H2O, however,he former simmered for 1.5 h and the latter 2 for 4.5 h.

Solidago gigantea, Alnus viridis, Potentilla simplex, Junipe-us communis, Epilobium angustifolium, Betula alleghaniensisnd Acorus calamus extracts were prepared by adding 1 g ofround compound to 20 ml of distilled H2O. Each was broughto a boil and allowed to simmer for 3 h. Differing boiling timesnd ratios of ground compound to distilled H2O were used inrder to obtain a sufficient volume of extract solution for furthernalysis.

Subsequently, each extract was sterile filtered using either0.22 �m or 0.45 �m sterile filter. Each sterile filtered extractas stored at −60 ◦C. The extracts were later thawed and serialilutions were prepared using RPMI media. Eleven serial dilu-ions were prepared, ranging from neat to a 1:2 dilution up to a:2048 dilution. One hundred microliters aliquots of each extractilution were added to microtitre tray wells and 100 �l aliquotsf each fungal isolate were subsequently added to each of thextract aliquots.

RPMI was used as a negative control. Voriconazole, flu-onazole, itraconazole, amphotericin B and the essential oilf Melaleuca alternifolia were used as positive controls. Thentifungal agents were prepared for minimum inhibitory concen-ration (MIC) testing as per Clinical and Laboratory Standardsnstitute (CLSI – formerly NCCLS) (NCCLS M27-A2, 2002;CCLS M38-A, 2002). A commercial preparation of 100%elaleuca alternifolia oil (DIN 02012081: Australian Bodyare; Vancouver, British Columbia, Canada) was diluted bydding 1.6 ml of essential oil to 8.4 ml of a 200:1 mixturef RPMI and 0.5% Tween 80. Subsequent serial 1:2 dilu-ions were prepared with RPMI for MIC testing using broth

icrodilution.

.3. Microorganisms

Fungal isolates were subcultured and prepared for assess-ent of plant extract activity. Thirteen yeast isolates and 10onomorphic mold isolates were selected. The mold isolates

ncluded five dermatophytes, four hyaline hyphomycetes andne zygomycete. The clinical isolates and American Type Cul-ure Collection (ATCC) control strains used in this study areisted in Table 2. All isolates had been stored in skim milk at

70 ◦C with the exception of the Fusarium solani isolate whichad been stored in sterile H2O at room temperature.

The 13 yeasts were each diluted in sterile H2O to a concen-

ration of 104 yeast cells/l. This was further diluted with RPMIo ∼103 yeast cells/l and 100 �l were then added to each of theerial dilutions of the plant extracts. The 10 molds were eachiluted in sterile H2O in order to obtain ∼103 CFU/l. One hun-

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TCC, American Type Culture Collection; NCM, National Centre for Mycologyn Edmonton, Canada.

red microliters of each dilution were then added to the serialilutions of the plant extracts.

.4. Antifungal assays

The plant extract/fungal isolate microtitre trays were storedt 35 ◦C and assessed for growth at 48 and 72 h. Using the brothicrodilution technique, MICs were determined for each extract

gainst the fungal isolates at 48 and 72 h. An 80% inhibition ofrowth as compared to the RPMI negative control well was con-idered evidence of fungistatic activity. Where a prozone-likeffect or “eagle” phenomenon was observed in the dilutionaleries, the MIC was taken at the lower concentration wherenhibition ceased.

. Results

Table 3 demonstrates the MIC ranges of the positive controlsith reference to the yeasts and Table 4 with reference to theolds. Table 5 demonstrates the antifungal activity of the plant

xtracts with reference to the yeast isolates. Fragaria virgini-na, Epilobium angustifolium and Potentilla simplex showed

trong activity overall and demonstrated at least some degreef activity against all of the yeast isolates. The Fragaria vir-iniana extract inhibited both Candida krusei strains as wells Candida glabrata and Candida lusitaniae at concentrations

D. Webster et al. / Journal of Ethnopharmacology 115 (2008) 140–146 143

Table 3Antifungal MIC (mg/l) against yeast isolates at 48 and 72 h

Pathogen Voriconazole Fluconazole Itraconazole Amphotericin B Melaleuca alternifolia (%, v/v)

Candida krusei (48 h) 0.5 32 0.25 0.5 16258 (72 h) 1 64 0.5 0.5 2Candida krusei 0.5 64 0.25 0.5 19/65 2 64 0.5 0.5 2Candida albicans > > > 0.25 0.58/67 > > > 0.25 1Candida albicans 0.12 8 0.03 0.25 0.1210/3 0.12 8 0.06 0.25 0.25Candida albicans 0.12 0.25 0.03 0.25 124433 0.12 0.5 0.06 0.25 2Candida albicans 0.06 0.25 0.03 0.25 190028 0.25 0.5 0.06 0.25 2Candida parapsilosis 0.06 0.5 0.03 0.25 0.2590018 0.03 0.5 0.06 0.5 0.25Candida parapsilosis 0.03 2 0.06 0.25 0.2522019 0.06 2 0.12 0.5 0.5Candida glabrata 0.5 16 0.5 0.25 19/54 1 16 0.5 0.5 1Candida tropicalis 0.12 1 0.12 0.25 0.5750 0.12 1 0.12 0.25 0.5Candida lusitaniae 0.016 2 0.12 0.12 0.510/8 0.016 2 0.12 0.12 1Saccharomyces cerevisiae 0.25 16 1 0.25 0.59/67 0.5 16 1 0.25 0.5Cryptococcus neoformans 0.06 2 0.25 0.06 0.251 0.

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f 100 mg/l and less. The Epilobium angustifolium root extractlso had activity against Candida glabrata and Candida lusita-

iae at low concentrations, and it also revealed activity againstaccharomyces cerivisiae at concentrations as low as 50 mg/l.he extract of Potentilla simplex was also found to inhibit therowth of Candida glabrata at concentrations as low as 25 mg/l

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athogen Voriconazole Fluconazole I

richophyton tonsurans (48 h) ng ng nontrol (72 h) 0.12 16 0richophyton rubrum ng ng n8/23 0.016 0.5 0richophyton mentagrophytes 0.25 4 00/71 0.03 8 0icrosporum canis ng ng n

#2 ng ng npidermophyton floccosum 0.03 2 0#3 0.03 2 0spergillus fumigatus 0.25 > 00/76 0.5 > 0spergillus fumigatus 0.5 > 0#4 0.5 > 0spergillus flavus 0.5 64 00/55 0.5 64 0usarium solani 2 > >-96 2 > >hizopus 4 > 0#8 8 > 1

>” implies no inhibition; “ng” implies no growth.

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t 48 h and 50 mg/l at 72 h. Alnus viridis, Betula alleghaniensisnd Solidago gigantea also demonstrated a significant degree of

ntifungal potential in the setting of the yeast isolates.

The plant extract activity against the monomorphic mold iso-ates is shown in Table 6. Fragaria virginiana was the onlyxtract that demonstrated complete inhibition of all dermato-

traconazole Amphotericin B Melaleuca alternifolia (%, v/v)

g ng ng.12 0.03 0.03g ng ng.06 0.12 0.12.06 0.5 0.12.5 1 0.12g ng ngg 0.25 ng.03 0.25 0.03.06 0.25 0.03.5 1 0.12.5 1 0.25.5 2 0.06.5 1 0.25.5 2 0.25.5 2 0.5

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144 D. Webster et al. / Journal of Ethnopharmacology 115 (2008) 140–146

Table 5Plant extract antifungal activity (mg/l)a against yeast isolates at 48 and 72 h

Pathogen Hm Av Ps An Fv Ba Jc Sg Gl Ea Acc Arc Af Pt

Candida krusei (48 h) > 200 400 > 100 400 > 1600 > 200 12500 50000 6000 >6258 (72 h) > 200 400 > 100 400 > 1600 > 200 25000 50000 6000 >Candida krusei > 100 200 > 50 400 > 800 > 200 12500 50000 6000 >9/65 > 200 200 > 100 400 > 800 > 200 25000 50000 6000 >Candida albicans > 12500 1600 > 800 1600 > 1600 > 200 12500 > > >8/67 > 12500 1600 > 800 800 > > > 200 25000 > > >Candida albicans > > 3000 > 800 > > > > 400 12500 > > >10/3 > > 3000 > 1600 > > > > 1600 12500 > > >Candida albicans > > 1600 > 1600 > > > > 400 12500 > > >24433 > > 1600 > 1600 > > > > 800 12500 > > >Candida albicans > > 1600 > 800 > > > > 400 12500 > > >90028 > > 1600 > 800 > > > > 400 12500 > > >Candida parapsilosis > 25000 1600 > 800 3000 > > > 400 12500 50000 50000 5000090018 > > 1600 > 800 3000 > > > 800 12500 50000 50000 50000Candida parapsilosis > 25000 400 > 200 800 > > > 100 12500 > > >22019 > 25000 400 > 400 50 > > > 100 12500 > > >Candida glabrata > 25000 25 > 50 50 > 100e > 25 > 50000 400 >9/54 > > 50 > 50 100e > 400e > 50 > 25000 1600 >Candida tropicalis > 3000 1600 > 800 6000 > 12500 > 1600 > 25000 6000 50000750 > 6000 1600 > 800 6000 > 12500 > 1600 > 25000 50000 >Candida lusitaniae > 6000 200 > 100 400 > 1600 > 50 12500 25000 12500 >10/8 > 6000 200 > 100 800 > 1600 > 200 25000 25000 25000 >Saccharomyces cerevisiae > 25000 12500 > 1600 100e > 200 > 50e 25000 200e 200e >9/67 > 25000 25000 > 12500 200e > 400 > 50e > 25000 400 >Cryptococcus neoformans > 25000 400 > 200 25000 > 6000 > 400 6000 50000 50000 >13/47 > 25000 800 > 200 25000 > 12500 > 400 25000 50000 50000 >

“>” Implies no inhibition; “ng” implies no growth; “e” implies eagle. Plant abbreviations: Hm, Heracleum maximum; Av, Alnus viridis; Ps, Potentilla simplex;An, Aralia nudicaulis; Fv, Fragaria virginiana; Ba, Betula alleghaniensis; Jc, Juniperus communis; Sg, Solidago gigantea; Gl, Glycyrrhiza lepidota; Ea, Epilobiumangustifolium; Acc, Acorus calamus; Arc, Artemisia campestris; Af, Artemisia frigida; Pt, Populus tremuloides.

a Expressed as the concentration at which 80% inhibition of fungal growth occurs.

Table 6Plant extract antifungal activity (mg/l)a against monomorphic mold isolates at 48 and 72 h

Pathogen Hm Av Ps An Fv Ba Jc Sg Gl Ea Acc Arc Af Pt

Trichophyton tonsurans (48 h) ng ng ng ng ng ng ng ng ng ng ng ng ng ngControl (72 h) > 1600 1600 50000 800 3000 25000 6000 > 3000 3000 12500 12500 25000Trichophyton rubrum ng ng ng ng ng ng ng ng ng ng ng ng ng ng18/23 > 1600 1600 50000 800 3000 25000 6000 > 3000 12500 12500 12500 12500Trichophyton mentagrophytes > 1600 1600 50000 400 6000 25000 12500 > 6000 6000 25000 25000 2500020/71 > 1600 1600 50000 400 6000 25000 12500 > 3000 12500 25000 25000 25000Microsporum canis ng ng ng ng ng ng ng ng ng ng ng ng ng ngS#2 ng 1600 800 > 400 3000 12500 6000 > 1000 12500 ng ng ngEpidermophyton floccosum > 800 800 12500 400 3000 6000 3000 50000 3000 6000 3000 6000 6000S#3 > 800 800 12500 400 3000 12500 6000 > 3000 12500 6000 6000 12500Aspergillus fumigatus > > > > 25000 > > > > > > > > >20/76 > > > > 50000 > > > > > > > > >Aspergillus fumigatus > > > > 25000 > > > > > > > > >S#4 > > > > 50000 > > > > > > > > >Aspergillus flavus > 25000 > > 25000 > > > > > 12500 > > >20/55 > 25000 > > 50000 > > > > > 25000 > > >Fusarium solani > 25000 > > 50000 > > > > > > > > >H-96 > > > > 50000 > > > > > > > > >Rhizopus > 25000 12500 > 6000 > > 12500 > 25000 25000 > > >S#8 > 25000 12500 > 3000 > > 25000 > 200 25000 > > >

“>” Implies no inhibition; “ng” implies no growth. Plant abbreviations: Hm, Heracleum maximum; Av, Alnus viridis; Ps, Potentilla simplex; An, Aralia nudicaulis;Fv, Fragaria virginiana; Ba, Betula alleghaniensis; Jc, Juniperus communis; Sg, Solidago gigantea; Gl, Glycyrrhiza lepidota; Ea, Epilobium angustifolium; Acc,Acorus calamus; Arc, Artemisia campestris; Af, Artemisia frigida; Pt, Populus tremuloides.

a Expressed as the concentration at which 80% inhibition of fungal growth occurs.

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hyte and mold isolates. Fragaria virginiana strongly inhibitedhe growth of the dermatophyte isolates at concentrations of00 mg/l and less at 72 h. Notably, this extract was the onlyxtract to demonstrate any antifungal activity against Fusariumolani at 72 h. Also of note, at 72 h Epilobium angustifoliumemonstrated 80% growth inhibition of the Rhizopus sp. isolatet a concentration of 200 mg/l.

. Discussion and conclusions

The body of literature identifying the antimicrobial poten-ial of many traditional plants is growing. The increasing dataegarding the in vitro and clinical efficacy of the essential oilf Melaleuca alternifolia serves as an excellent example. Thisraditional Australian Aborigine medicine has demonstratedungicidal activity against a range of fungal pathogens (Hammert al., 2002). As a positive control in our study, the essential oilf this traditional plant demonstrated impressive activity againstach of the fungal isolates. In particular, it had very strong activ-ty against the dermatophytes and other monomorphic molds,onsistent with previous findings.

The oil of Melaleuca alternifolia, as well as the aque-us extracts obtained in this study, are mixtures of multipleomponents. Thus the range of MICs identified must be inter-reted in the appropriate context. While the positive controlsn this study demonstrate MICs in the range of 1 mg/l, thelant extract MICs are rarely found to be less than 100 mg/l.he plant extracts are crude fusions and therefore higher MICsre expected. For example, in an analysis by Thirach et al,he antifungal activity of an ethanol extract of Acorus calamusas assessed against 28 clinical isolates of Candida albicanssing a broth microdilution technique. The MIC was identifieds 28800 ± 16320 mg/l and in this setting Acorus calamus wasnterpreted as being a potential fungistatic agent (Thirach et al.,003).

Quite simply, it must be recognized that the extraction pro-ess employed is not specific. Crude plant extracts are generallymixture of active and non-active compounds. With more

efined and solitary compounds, MICs of less than 100 mg/l maye suggestive of good anti-microbial activity. As an example,miganan, a cationic peptide with MICs in this range againstandida spp., has shown promise as an antimicrobial for therevention of catheter-related bloodstream infections (Sader etl., 2004). Yet, crude mixtures will have higher MICs and forhis reason, an MIC of less than 1000 mg/l is interpreted in thistudy as showing strong antifungal potential.

Of the 14 plants analyzed, 3 – Fragaria virginiana, Epilobiumngustifolium and Potentilla simplex – demonstrated strong anti-ungal potential overall. Three others – Alnus viridis, Betulalleghaniensis and Solidago gigantean – showed a significantegree of activity against many of the yeast isolates. Amonghe positive controls, amphotericin B and the oil of Melaleucalternifolia also had some degree of activity against all of the

east and mold isolates.

Fragaria virginiana was also noted to have some degree ofntifungal activity against all of the yeast and mold isolatesmployed in this study. Though we are not aware of previous

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rmacology 115 (2008) 140–146 145

tudies that have reported fungal growth inhibition by Fragariairginiana, other Fragaria spp. have been reported as demon-trating significant levels of antifungal activity. McCutcheont al found that methanol extracts of the leaves of Fragariaesca and Fragaria chiloensis demonstrated strong antifungalctivity against a number of yeasts and molds (McCutcheon etl., 1994). These included Candida albicans, Saccharomyceserevisiae, Aspergillus fumigatus, Aspergillus flavus, and Tri-hophyton mentagrophytes – pathogens also used in our studyin addition to Fusarium tricuictum, Microsporum cookerii,icrosporum gypseum and Trichoderma viridae. Specifically,

he Fragaria spp. extracts tested by McCutcheon et al. as wells the extract used in our study showed relatively high levels ofntidermatophyte activity against Trichophyton mentagrophytesnd Microsporum spp.

With reference to Candida albicans, the aqueous extractf Epilobium angustifolium demonstrated the most antifungalctivity among the plant extracts used in our study. Epilobiumngustifolium had strong antifungal activity against each of theour Candida albicans strains with concentrations of 200, 1600,00 and 400 mg/l, respectively, at 72 h. Fragaria virginianaemonstrated modest to strong activity and Potentilla simplexad modest activity against all four strains. Betula alleghanien-is had strong activity against a single strain at a concentrationf 800 mg/l, however, it had no activity against the other 3trains. Among the positive controls, only amphotericin B andelaleuca alternifolia had activity against this single strain.sing ethanol extracts with a disc diffusion assay to assess anti-

ungal activity, Jones et al. also found the root of Epilobiumngustifolium had significant activity against Candida albicans.he stembark of Betula alleghaniensis, however, had no activity

Jones et al., 2000).The three extracts with strong activity against the Cryp-

ococcus neoformans isolate were again Fragaria virginiana,pilobium angustifolium and Potentilla simplex. Jones et al.lso identified that an ethanol extract of the root of Epilobiumngustifolium had significant activity against Cryptococcus neo-ormans (Jones et al., 2000). This suggests that the active com-onents of the extract may be among the more polar compoundss they are found in both the aqueous and alcohol extracts.

Betula alleghaniensis had the strongest activity against Sac-haromyces cerevisiae, with activity observed at 200 mg/l at2 h. Extracts of Epilobium angustifolium, Solidago giganteand Artemisia frigida also had activity at concentrations less than000 mg/l. Jones et al. have also previously noted that ethanolxtracts of Betula allegheniensis and Epilobium angustifoliumhow activity against Saccharomyces cerevisiae, again suggest-ng activity involving polar molecules (Jones et al., 2000).

Though our study observes antifungal trends similar to othertudies, some differences are also noted. Discrepancy in find-ngs between studies may be the result of the pathogen strainstilized. With multiple fungal strains, differences are notedven within studies. A single extract is seen to have vary-

ng degrees of activity against differing strains of a commonpecies.

Differences between studies may also be due to the extractionrocess used. Alcohol is a general solvent and tends to provide a

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46 D. Webster et al. / Journal of Eth

ore complete extraction of compounds with a variety of polari-ies (Evans, 1996). Thus, aqueous extracts may not contain somef the less polar compounds. It is also possible that climate vari-tion and seasonal discrepancy regarding the harvesting of thelants may account for some of the differences observed. Theuantity and quality of active compounds will vary depending onhe growing conditions and compounds will concentrate in cer-ain parts of the plant during different growth cycles and duringpecific seasons.

Storage is also an issue. In a study by Filipowicz et al., thessential oil of Juniperus communis was assessed for antimicro-ial activity using a modified disc diffusion method (Filipowiczt al., 2003). Three different oils obtained via steam distillationf the berries of Juniperus communis were analyzed. One of theils demonstrated an extremely high degree of antifungal activ-ty in the setting of two Candida albicans isolates. The secondil also demonstrated significant zones of inhibition while thehird oil had no activity at all. It was determined that the first oilad the highest levels of �-pinene, �-pinene and p-cymene andhat the increased antifungal activity was likely a result of syn-rgy among these compounds. Furthermore, it was concludedhat the high levels of these compounds resulted from storageonditions and ‘ageing’.

In conclusion, the body of literature identifying the antimi-robial potential of many traditional plants is growing. Of the4 plants analyzed in this study, Fragaria virginiana, Epilo-ium angustifolium and Potentilla simplex demonstrated strongntifungal potential overall and Fragaria virginiana showedctivity against all of the fungal pathogens. Alnus viridis, Betulalleghaniensis and Solidago gigantea also demonstrated a sig-ificant degree of activity against many of the yeast isolates.ragaria virginiana, Epilobium angustifolium and Potentillaimplex demonstrate promising antifungal potential.

cknowledgements

Thanks to Anne Hill, Russell Webster, Sandy Cardinal andob Cardinal for assistance with the gathering of plant materials.hank you to Tannis Jurgens for information provided regarding

he extraction process.

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