the effect of botanical tinctures and essential oils …...candida albicans is a commensal organism...

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Research Article

1© Under License of Creative Commons Attribution 3.0 License | This article is available in: http://herbal-medicine.imedpub.com/archive.php

DOI: 10.21767/2472-0151.100012

Herbal Medicine: Open AccessISSN 2472-0151

Ferreira V1, Dickerson W1,Farr D1, Barraza M1,Banker R1, Dutton J1,Cervigni M1, Willhauck M1,Tifre L1, Mayman S1,Stone S1, Bushman S1,Hull G1, Morgan R1,Hoover T1, Mittman A1,Sparks L 1, Smithers J1,Miles D1, Calvert D3 andLangland J1,2

1 Southwest College of Naturopathic Medicine, Tempe, AZ 85282, USA

2 BiodesignInstitute,ArizonaStateUniversity, Tempe, AZ 85287, USA

3 Enerpathic Technologies, Tempe, AZ 85282, USA

Corresponding author: Langland J

[email protected]

Southwest College of Naturopathic Medicine, Tempe, AZ 85282, USA.

Citation: Ferreira V, Dickerson W, Farr D, et al. TheEffectofBotanicalTincturesandEssentialOils on the Growth and Morphogenesis of Candida albicans. Herb Med. 2016, 2:1.

The Effect of Botanical Tinctures and Essential Oils on the Growth and Morphogenesis of

Candida albicans

AbstractObjective: Candida albicansisanopportunisticandpolymorphicfungalpathogenthat affectsmucosalmembranes and squamousepithelia aswell asbeingpartof thenormalhumanflora.Historically, therehavebeenmanybotanical-basedremedies used to treat fungal conditions, including C. albicans. This study examinedtheefficacyofbothbotanicaltincturesandessentialoilsonthegrowthandmorphologicaldifferentiationofC. albicans.

Methods: The in vitro growthanddifferentiationofC. albicans was monitored followingtreatmentwithethanol-basedtincturesandessentialoilspreparedfromseveral commonly used botanicals.

Results: Results demonstrated that all ethanol-based botanical tincturestested did not inhibit the growth of C. albicans,butseveraltinctures, includingMarsdenia condurango, Juglans nigra (Black walnut), Anemopsis californica (Yerba mansa) and Piper cubeba (Cubeb berry),significantlyreducedthemorphologicaldifferentiationof theyeast into the invasivehyphae form.Alternatively, severalbotanicalessentialoils,includingthosefromThymus vulgaris (Thyme), Rosmarinus officinalis (Rosemary) and Cymbopogon citratus (Lemon grass) had a dramaticeffectoninhibitingthegrowthofC. albicans.

Conclusions: These results suggest that botanical tinctures commonly used inthe treatment of C. albicansinfectionsmayactbyblockingthedifferentiationoftheyeast intoamorevirulenthyphal formwhilenotaffectingthegrowthrate.Incomparison,therapeuticessentialoilsmaytargetboththedifferentiationandgrowth rate of C. albicans.Theresultssupportthatdifferentactiveconstituentsare present in botanical tinctures as compared to oils thereby contributing toourunderstandingofhowthesebotanicalsmaybeeffectivetherapeuticsinthetreatment of C. albicansinfections.

Keywords: Candida albicans; Fungi; Yeast; Hyphae; Virulence; Botanical; Tincture; Essentialoils

Received: January 02, 2016; Accepted: March 29, 2016; Published: March 31, 2016

Introduction Candida albicans is a commensal organism in the human cutaneous andmucosal flora, but canbecomeamajor humanfungalpathogengiventheproperconditionsallowingforhyphaldifferentiation, biofilm formation, and overgrowth. Some ofthe most common areas for candidiasis include the oral and gastrointestinaltract,vaginalcanalandtopicallyontheskin[1].Inpatientsexperiencingvulvovaginalcandidiasis,commonsignsincludeburning,itchingandredness.Thisconditionaffectsupto

75%ofwomenatleastonceintheirlifetime[2].Skininfectionsarecommoninburnvictimsandneonatesduetotheirdepressedor underdeveloped immune system [2]. In the AIDS patientpopulation,thereisanincreasedlikelihoodofdevelopingoralandesophagealcandidiasis,duetodecreasedimmunefunction[2].Inthegeneralpopulation,Candidaovergrowthinthegastrointestinaltractisexacerbatedbyhigh-sugardietsormedicationsincludingsteroidsandantibiotics.Inthesecases,peoplewillpresentwithdigestivediscomfort,gasorbloating.Moreseriously,C. albicans

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canbecomesystemicandcauseaninfectionofthebloodstreamknownascandidaemia.This infectioncan then further spreadintoorgans,causingconditionssuchashepatospleniccandidiasisor Candida peritonitis [3]. C. albicans has become the fourth-leadingcauseofhospital-acquiredbloodinfectionsintheworld

andmortalityratesforthemoresevereinfectionsremainhighatabout50%[1,4-6].

During growth, C. albicans may undergo a reversible morphological transition from single-celled yeast; round budding spores (alsoknown as blastospores), to filamentous pseudohyphae andfinally, multicellular, elongated tubal cells attached end-to-endknownas truehyphae [1,2,6].This transition,which is inducedupon exposure of C. albicans to a number of host conditions,is required for increased virulence. The most common hostconditions required for differentiation include the presence ofserum, the environmental temperature, and pH levels [2]. Instudyingmucosal infections,thehyphal formofC. albicans has been found embedded andovergrown in tissue leading to theconclusionthatinitsmulticellularformiswhenC. albicans is at itsmostinvasiveandinfectiousstage[7].

Currently, there are a large variety of anti-Candida treatments dependentontheanatomicallocationoftheyeastovergrowth.PharmacologicaloralagentssuchasfluconazoleandamphotericinB have been used in cases of internal or invasive Candida overgrowth[8].Topicalagentssuchasnystatinandclotrimazolehave been used to treat cutaneous candidiasis as well as certain oralcandidaovergrowths.Miconazoleandtioconazolearemorecommonly used topically in the vaginal canal for treatment of vaginalyeastinfections[3,8].DuetoCandidabeingaeukaryoticpathogen, many of the antifungal agents available interactnegatively with human cells. These anti-fungal treatmentshavemanyadverseeffectsincludinghepatotoxicityasthemostcommon and concerning [2]. Invasive and systemic forms ofcandidiasis aremore difficult to treat using thesemedicationsbecauseoftheirhighsideeffectprofile,particularly inpatientswhoarealreadyimmunocompromised[3].

In this study, we examined the efficacy of botanical tincturesandessentialoilstoinhibitboththegrowthandmorphologicaltransformationofC. albicans.

Materials and MethodsBotanical tincture and essential oil preparationPlant material was obtained from reputable sources with documentation of authenticity. All plant material wassubsequently verified by qualified botanical specialists usingherbal pharmacopoeia monographs and reference keys. A voucher specimen of all plant material was deposited in a repository. Fortincturepreparation,thedriedbotanicalsweregroundtoafinepowder, resuspended in extraction solutionand incubatedfor 3 days at room temperature. The extract was centrifuged at3000×gfor10mintoremovecelldebrisandtheextractionsolutionfilteredthrougha0.2µMfilter.Forstandardizationandcomparison, all botanical extracts had an average non-volatileconstituent concentration averaging 32.1mg/ml (ranging from22.4-37.2mg/ml).Thisconcentration(mg/ml)isbasedonthe

weight of non-volatile constituents present in the extract perml of aqueous liquid. The source, extraction solution (percentethanol:water), and plant dry to extraction solution ratio foreach of the botanicals used in this study is shown in Table 1. The essentialoilsusedinthisstudywerepreparedbysteamdistillationorcoldpressedextraction.ThesourceandpreparationmethodforeachessentialoilusedinthisstudyisshowninTable 2.

Yeast growthMedia and the yeast culture Candida albicans ATCC 10231 were obtainedfromHardyDiagnostics(SantaMonica,CA).Forgrowthstudies,18-hourcultures(rangingfrom3-8×107 colony forming units (CFU)/ml) grown at 30°C in YPD broth were diluted intomedia (1:1,000 dilution; yeast peptone dextrose broth (YPD))followed by the addition of indicated concentrations of eachbotanical extract, essential oil, or control solution. For single-celled yeast growth, the cultures were incubated in YPD broth at30°Cwithaeration(bycontinuousrotation)foramaximumof24hours.At0,4,8,and12hrs,sampleswereremovedandthecellconcentrationdeterminedbyserialdilutionsonSabdexagar(CFU/mlmedia).Forhyphalgrowth,thecultureswereincubatedinYPDbrothwith5%fetalbovineserumat37°Cwithaeration(bycontinuousrotation)foramaximumof24hours.DeterminationofCFU/mlwasdoneasabove.Forhyphal reversal studies, the18-hourstarterculturesweregrownat37°CinYPDbrothplus5%fetal bovine serum.

Microscopy differentiation analysisAt 24 hours post-treatment, light microscopy was done toevaluate the morphological state of the C. albicans. Each broth culturewasstainedwithLactophenolCottonBlueandvisualizedby light microscopy. A minimum of 100 cells were counted and the percent of single cells verses hyphal/pseudohyphal cellsdetermined.

ResultsIn this study, several botanical tinctures and oils were utilizedbasedon their historical and/or suggested therapeutic efficacyin the treatment of C. albicans infections.UpontestingtincturesforgrowthinhibitoryeffectsonC. albicans,littleornoeffectwasobserved. Figure 1 illustrates the lack of inhibitory growth with Olea europea, Origanum vulgare, Thymus vulgaris and Cymbopogon citratus on C. albicans. Eachtincturewastestedatconcentrationsof0μl/ml(noherbaltreatment),10μl/mland30μl/ml.Atboth37°Cwithserumand30°Cwithoutserum,allofthetinctureshadnosignificanteffectonthegrowthofC. albicans. Similar results wereobservedwithallotherethanoltincturestested includingMarsdenia condurango, Cinnamomum zeylanicum, Juglans nigra, Chilopsis linearis, Anemopsis californica, Arctium lappa, Tabebuia avellanedae, Alpinia officinarum, Carum carvi, Arcostaphylos uva-ursi, Nepeta cataria, Allium sativum, Cuminum cyminum and Artemisia absinthium (data not shown). Vehicle control samples treatedwith 60% ethanol also showed no inhibitory effect ongrowth (data not shown).

Since morphological transformation of C. albicans to hyphal form isakeyvirulence factor, theherbaltinctureswere testedfor their ability to inhibit this transformationprocess.Figure 2

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Botanical name Common name Part Origin Company source Extraction Soln (Ethanol:Water)

Extract ratio (Dry:liquid)

Marsdenia condurango Condurango Vine Equador Raintree Health 45%:55% 1:08

Cinnamomum zeylanicum Cinnamon Bark United States Starwest Botanicals 65%:35% 1:08

Juglans nigra Black Walnut Hull United States Starwest Botanicals 40%:60% 1:08

Chilopsis linearis Desert Willow Leaf Sri Lanka Starwest Botanicals 45%:55% 1:08

Anemopsis californica Yerba Mansa Root United States Starwest Botanicals 60%:40% 1:08

Arctium lappa Burdock Root China Starwest Botanicals 60%:40% 1:08

Tabebuia avellanedae Pau D'Arco Bark Brazil Starwest Botanicals 60%:40% 1:08

Alpinia officinarum Lesser Galangal Root India Starwest Botanicals 25%:75% 1:08

Carum carvi Caraway Seed Egypt Starwest Botanicals 60%:40% 1:08

Arctostaphylos uva ursi Uva Ursi Leaf Croatia Starwest Botanicals 60%:40% 1:08

Nepeta cataria Catnip Leaf Canada Starwest Botanicals 50%:50% 1:08

Allium sativum Garlic Bulb China Starwest Botanicals 0%:100% 1:08

Cuminum cyminum Cumin Seed India Starwest Botanicals 60%:40% 1:08

Artemisia absinthium Wormwood Aerial Croatia Starwest Botanicals 60%:40% 1:08

Origanum vulgare Oregano Leaf Turkey Starwest Botanicals 40%:60% 1:08

Piper cubeba Cubeb Berries India Starwest Botanicals 60%:40% 1:08

Olea europaea Olive Leaf Egypt Starwest Botanicals 60%:40% 1:08

Thymus vulgaris Thyme Leaf Egypt Starwest Botanicals 0%:100% 1:08

Cymbopogon citratus Lemon Grass Aerial Egypt Starwest Botanicals 0%:100% 1:08

Table 1 Thesource,extractionsolution(percentethanol:water),andplantdrytoextractionsolutionratioforeachofthebotanicals.

Botanical Common name Origin Company source Method

Copaifera officinalis Copaifera Brazil Mountain Rose Herbs Steamdistillationofthebalsam

Oenothera biennis Evening Primrose N/A Starwest Botanicals Coldpressedextractionoftheseed

Cannabis sativa Hemp N/A Starwest Botanicals Coldpressedextractionoftheseed

Thymus vulgaris Thyme Spain Starwest Botanicals Steamdistillationoftheleaf

Cymbopogon citratus Lemon Grass India Esoteric Oils Steamdistillationoftheleaf

Rosmarinus officinalis Rosemary N/A Starwest Botanicals Coldpressedextractionoftheleaf

Melaleuca alternifolia Tea Tree Australia Starwest Botanicals Steamdistillationoftheleafandaerials

Mentha balsamea Peppermint United States Starwest Botanicals Steamdistillationofthefloweringherb

Origanum vulgare Oregano Turkey Starwest Botanicals Steamdistillationoftheleaf

Lavandula officinalis Lavender India Starwest Botanicals Steamdistillationofthefloweringtop

Cymbopogon martini Palmarosa India Starwest Botanicals Steamdistillationofthegrass

Table 2 Thesourceandpreparationmethodforeachessentialoil.

representsthepercenthyphaeformationofC. albicans cultures treatedwith herbal tinctures for 24 hours. As shown,whenC. albicans culturesweregrownat37°Cwiththeadditionofserum,80-100% of the culture formed hyphae, whereas when grownat30°Cwithoutserum,typicallyonly10%ofthecultureformedhyphae with 90% as single celled form. Upon the addition ofMarsdenia condurangotinctureat10μl/mlat37°Cwithserum,decreased hyphae formation was observed to about 15% ofthepopulation and further decreased to 5%of thepopulationat30μl/ml.ThiswasthestrongestactingtinctureuponhyphaeinhibitionwithPiper cubeba, Juglans nigra, Anemopsis californica and Cinnamomum zeylanicum also being significantly potent

withonly10-25%hyphaegrowthat30μl/ml.At10μl/mlthesebotanicalsprovidedbetween50-75%hyphalgrowth.

Visually, Figure 3A shows microscopic images of the C. albicansgrownat0μl/ml,10μl/mland30μl/mlofMarsdenia condurango and Piper cubebatinctures.At30°Cwithoutserumateachconcentration,therewasmostlyonlysingle-celledyeastpresentforbothtinctures.At37°Cplusserumwithoutanyherbaltreatment large, hyphal structures were observed throughout theculture.At10μl/ml forbothherbs, largehyphaewerenotdetected,butpseudohyphaewerepresent.At30μl/ml,theM. condurango treatment resulted with nearly only single-celledyeastbeingpresentuponobservationof thebrothculture.For


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Figure 1 EffectofbotanicaltincturesonC. albicans growth rate. A starter culture of C. albicanswasgrownat30°CinYPD.Diluted(1,000-fold)C. albicanscultureswereleftuntreated(solidline),ortreatedwiththeindicatedbotanicalextractsintheamountof10or30µl/mlat0hours(dashedlines).Thecultureswereincubatedat37°CinYPDplusserumor30°CinYPDwithoutserumwithcontinuousaeration(byrotation)andCFU/mldeterminedevery4hoursoveraperiodof12hours.Assayswererepeatedintriplicate.

P. cubeba at 30 μl/ml, pseudohyphaewere still prevalent, butas shorter structures. These results agree with Figure 1 where the growth rate of C. albicanswasnotaffectedbytheherbs,butratherthemorphologicalstateofthecellswasaffected.

The effect of these tinctures onC. albicans differentiation canalso be observed at a gross level with the broth cultures. Figure 3B shows M. condurango treatment of C. albicans cultures at 0 μl/ml,10μl/mland30μl/mlat37°Cplusserumand30°Cwithoutserum.At 30°Cwithout serum, the broth cultureswere turbidateveryherbconcentrationduetotheC. albicans growing in a single-celledform.Whengrownat37°Cwithserumandnoherb,thecultureappearedflocculentduetolarge,hyphaeformation(Figure 3B). This thendecreased to a lessflocculent andmoreturbid appearance at 10 μl herb/ml and a completely turbidappearance(similarto30°Cwithoutserum)at30μlherb/ml.

For other herbs tested, intermediate acting herbs showed asignificanteffectonthemorphologicalgrowthofC. albicans only atthehigherdosesof30μl/mlandwereabletoinhibithyphalgrowthto40-60%ofthepopulation(Figure 2). These herbs included Chilopsis linearis, Alpinia officinarum and Carum carvi. More minor effectsongrowth,with75-80%oftheculturestillforminghyphaewas seen with Olea europea, Thymus vulgaris and Cymbopogon citratus at thehighest (30μl/ml) concentrationsofeachtincture.

TinctureswhichprovedtohavenoeffectonhyphaegrowthwereArtemisia absinthium, Arcostaphylos uva-ursi, Nepeta cataria, Allium sativum, Cuminum cyminum, Arctium lappa, Tabebuia avellanedae and Origanum vulgare (datanotshown).Inaddition,vehiclecontrolsamplestreatedwith60%ethanolalsoshowednoeffectonhyphaegrowthordifferentiation(datanotshown).

Since essential oils are also historically used in treating C. albicans infections, the effect of various essential oils on thegrowth and differentiation of C. albicans was also examined. Figure 4 shows that certain essential oils were able to inhibitthe replication ofC. albicans at both 30°Cwithout serum and37°C with serum. Although Thymus vulgaris and Cymbopogon citratusastincturesshowednoeffectonthereplicationrateofC. albicans,theessentialoilsinhibitedgrowthapproximately1000-foldat1µl/mlandover10,000-foldatdosesof3µl/mlorhigher.This appeared to be a fungicidal effect since levels of viablecellsdecreasedover2-logsbelow the initial concentration.Forotheressentialoils,includingthosefromRosmarinus officinalis, Melaleuca alternifolia, Mentha balsamea, Organum vulgare, Lavandula officinalis, and Cymbopogon martini, a more dose dependent inhibition ingrowthwasobserved(Figure 4). These herbsgenerally inhibitedgrowthapproximately10-foldat1µl/ml,100-1,000-foldat3µl/ml,andgreaterthan10,000-foldat10µl/mlandhigher.NotallessentialoilstestedinhibitedC. albicans

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Figure 2 EffectofbotanicaltincturesonC. albicansmorphologicaldifferentiation,AstartercultureofC. albicanswasgrownat30°CinYPD.Diluted(1,000-fold)C. albicanscultureswereleftuntreated(solidbar),ortreatedwiththeindicatedbotanicalextractsintheamountof10or30µl/mlat0hours(dottedbars).Thecultureswereincubatedat37°CinYPDplusserumor30°CinYPDwithoutserumwithcontinuousaeration(byrotation)for24hoursfollowedbyobservationbylightmicroscopy.Thepercentofsinglecellsversushyphal/pseudohyphalcellswasdetermined.Assayswererepeatedintriplicate.

growth. PalmarosaoilshowednoactivityonthegrowthoftheC. albicansevenat thehigherconcentrationstested(Figure 4). Similarnon-inhibitoryeffectswereobservedwithessentialoilsfrom Copaifera officinalis, Oenothera biennis and Cannabis sativa (data not shown).

Since the herbal tinctures had an effect on themorphologicaldifferentiation of C. albicans, similar assays were done using the essential oils. As shown in Figure 5, Lavandula officinalis, Rosmarinus officinalis, Mentha balsamea and Melaleuca alternifolia oils were able to inhibit hyphal formation atconcentrations below that which inhibited replication. Forexample, L. officinalis reduced hyphal formation to 5-15% at10and1µl/ml, respectively,butdidnotsignificantlyaffectthereplicationrateofC. albicansuntil30µl/ml.ForThymus vulgaris, Cymbopogon citratus and Origanum vulgare oils there was no significanteffectonhyphal formationatdoses thatdidnotlead togrowth inhibition (Figure 5). These results may suggest

that herbs like R. officinalus, may have separate constituentswhichaffectbothgrowthandmorphologicaldifferentiationofC. albicans, whereas herbs like T. vulgaris,onlyaconstituentwhichaffects only the growth rate.Cymbopogon martini essential oilshowednoinhibitionofhyphaldifferentiationateventhehighestconcentration of the essential oil (Figure 5). Other essentialoils tested with no effect on hyphal growth include Copaifera officinalis, Oenothera biennis and Cannabis sativa (data not shown).

The previous results demonstrated that several tinctures andoilscouldinhibitthedifferentiationofC. albicans fromasingle-celled yeast to a hyphal form. In order to test if these herbs couldalsoreversedifferentiationfromahyphalformtoayeastform, pre-existing hyphal C. albicans was maintained under growthconditionsof37°Cwithserumandsubsequentlytreatedwith various herbs and examined for morphological changes. These studies had the goal of indirectly assessing if an already


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established invasive C. albicans infection could be reversed tothe less invasive andmore benign state of single-celled yeast.Basedonearlierresults,selecttincturesandoilswerechosenforthese assays including Marsdenia condurango and Piper cubeba tincturesandMelaleuca alternifolia, Rosmarinus officinalis, and Lavandula officinalis oils. As shown in Figure 6A and 6B, with M. condurango, theinitialculturestartedoutwithapproximately90%hyphaepopulation.At24hourswithno treatment, thehyphalgrowthremainedunchanged.At10μltincture/ml,asignificantreductiontopseudohyphaeandsinglecellswasobservedat24hours post treatment. At 30 μl tincture/ml, hyphae formationwas decreased to 95% single-celled yeast following 24 hourspost-treatment. Figure 6B shows similar results for the other tincturesandoils testedwithPiper cubebatincture,Melaleuca alternifolia oil, Rosmarinus officinalis oil and Lavandula officinalis oilallsignificantlyreversinghyphalgrowthtoasingle-celledyeastform.

DiscussionHistorically, many botanical treatments have been used as therapeuticagents for fungal infections. Inthisstudy,botanicaltinctures and essential oils were tested individually for theirefficacy towards both inhibiting the growth and preventingdifferentiationofC. albicans. Juglans nigra has been used to treat oralinfectionsandhasbeenshowntohaveinhibitorycompoundstowards C. albicans [9,10].Piper cubebahasreportedconstituentswith bacteriostatic, fungistatic and fungicidal properties thathavebeenfoundusefulhistoricallyintreatingoralpathogens[11-14].Anemopsis californica has reported very strong antifungalpropertiesmakingitapotentiallyeffectivetopicaltreatmentforfungalinfections[15].Someherbs,suchasMarsdenia condurango are commonly used by practitioners without laboratory-basedstudiesorevaluations.Similarly,essentialoilshavealonghistoryof therapeutic use for fungal infections. Thymus vulgaris, as an essential oil, has been suggested to inhibit the growth ofC. albicans intracellularly [16-18]; Rosmarinus officinalis was demonstratedtoinhibitgermtubeformationasanessentialoil[19,20];andCymbopogon citratus has been suggested to inhibite mycelialformationaswellasyeastbudformation[21,22].

Thebotanicaltincturesusedinthisstudywerepreparedasethanol-based herbal extractions. Unexpectedly, based on historicaluse,noherbaltincturewasabletoinhibitthegrowthrateofC. albicans.However,severaltincturesinhibitedthemorphologicaldifferentiationofC. albicans to a hyphal form. These results may suggest that therapeuticherbal tincturesmay act by inhibitingthe progression of single-celled yeast into the more invasivehyphaeform.Thestrongestherbswiththismechanismofactionincluded Marsdenia condurango, Piper cubeba, Juglans nigra, Anemopsis californica and Cinnamomum zeylanicum. C. albicans is typicallya seriousproblemonlywhen itdifferentiates intoahyphal form, therefore, it may be therapeutically valuable tolimitdifferentiationofC. albicansinsteadofcompletelyinhibitinggrowthandmaintainingtheyeastformaspartofthestablefloraof the microbiome in the human body.

There have been very limited previous studies done on herbs and their constituents that effectively act on inhibiting hyphalgrowth. Gymnemic acid is found in the herb Gymnema sylvestre and has been traditionally used in the treatment of diabetes.Similar to the herbs tested in this study, gymnemic acid did nothaveaneffectonthegrowthofC. albicans, but did inhibit differentiationofthesingle-celledyeasttohyphae.Inthiscase,the effective constituent was isolated and tested in hyphae-inducing conditions, which supports the future possibility ofisolating novel compounds from various botanicalswithwhicheffecthyphalgrowthandvirulenceofC. albicans [5].

To further study the original goal of the experiment, which was toevaluatethefungicidaleffectsofherbs,essentialoilswerealsoevaluated.Essentialoilsarethesteam-extracted,volatilearomacompoundsofplants.Theytypicallyhavedifferentconstituentsandahigherconcentrationofactiveconstituentsrequiringasmalldoseforatherapeuticeffect.Inthisstudy,severalessentialoil-basedbotanicalswereabletocompletelyblockhyphaeformationaswellasinhibitingthereplicationofC. albicans. Thymus vulgaris and Cymbopogon citratuswerethemosteffectiveessentialoils,although they had no fungicidal effect as botanical tinctures.These two herbs as tinctures only had an effect on inhibitingdifferentiationofC. albicans. This suggests that the same herb extractedintoeitheroilortincturemaycontaindifferentactiveconstituents.

Since C. albicans inpatientswithsevereinfectionsiscommonlydifferentiated into the hyphal form, several tinctures and oilswere also tested for their ability to reverse the hyphal form to a single-celledyeastform.M. condurango wasthemosteffectivetinctureagainstpreventingdifferentiationandit,alongwiththeotheractivetincturesandoilswereshowntobehighlyeffectivein the reversal trial. These results support the possibility for these tincturesandoilstobeusedinactiveC. albicansinfections.

Tincturesareappropriatetotreatbothoralandtopicalinfections,whereasessentialoilsaremoresuitabletotreattopicalinfectionsdue to their highly concentrated and caustic nature. Most ofthe botanical tinctures and oils used in this study have a lowside-effect profile when taken internally or applied topically,respectively, when compared to conventional anti-fungaltreatments [3]. There is a potential for both the tinctures andtheoilstobecombinedinasynergisticblendthatcouldbemoreefficaciousintheoveralltreatmentofaC. albicans overgrowth by providingdifferentmechanismsofaction.

Botanicalsofferatherapeuticeffectonmanyaspectsofafungalormicrobialimbalance.Futureresearchcanexplorethepotentialriskofantifungalresistancetotheseherbs,whichmaysupporttheuseofcombinedbotanicalpreparationsasopposedtoasingle-herb therapy and provide support for which herbs may work synergisticallyinordertobestcontrolC. albicans overgrowth in the human body.

Financial SupportSupport for this project was provided by internal funding from the Southwest College of Naturopathic Medicine.

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Figure 3a VisualrepresentationoftheeffectofbotanicaltincturesonC. albicansmorphologicaldifferentiationMicroscopicimagesofC. albicans cultures treated with M. condurango or P. cubebatincturesunderconditionsdescribedinFigure2.Examplesofhyphae, pseudohyphae and single celled yeast cells are shown.

Figure 3b Visual representation of the effect of botanical tinctures on C. albicans morphological differentiation Image of culturecharacteristicsofC. albicans cultures treated with M. condurango tincture.


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Figure 4 EffectofbotanicalessentialoilsonC. albicans growth rate, A starter culture of C. albicanswasgrownat30°CinYPD.Diluted(1,000-fold)C. albicanscultureswereleftuntreated(solidline),ortreatedwiththeindicatedbotanicaloilsintheamountof1.0,3.0,10or30µl/mlat0hours(dashedlines).Thecultureswereincubatedat37°CinYPDplusserumor30°CinYPDwithoutserumwithcontinuousaeration(byrotation)andCFU/mldeterminedevery4hoursoveraperiodof12hours.Assayswererepeated in triplicate.

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Figure 5 EffectofbotanicalessentialoilsonC. albicansmorphologicaldifferentiation;AstartercultureofC. albicanswasgrownat30°CinYPD.Diluted(1,000-fold)C. albicanscultureswereleftuntreated(solidbar),ortreatedwiththeindicatedbotanicaloilsintheamountof1.0,3.0,10or30µl/mlat0hours(dotted/dashedbars).Thecultureswereincubatedat37°CinYPDplusserumor30°CinYPDwithoutserumwithcontinuousaeration(byrotation)for24hoursfollowedbyobservationbylightmicroscopy.Thepercentofsinglecellsversushyphal/pseudohyphalcellswasdetermined.“ND” indicatessampleswhichcouldnotbeevaluated due to a lack of growth. Assays were repeated in triplicate.


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Figure 6 Abilityofbotanicaltincturesandoilstoreverse C. albicans hyphalformation,AstartercultureofC. albicans was grown at 37°CinYPDplusserum.Diluted(1,000-fold)C. albicanscultureswereleftuntreated(solidbar),ortreatedwiththeindicatedbotanicalextracts/oilsintheamountof1.0,10or30µl/mlat0hours(dottedbars).Thecultureswereincubatedat37°CinYPDplusserumor30°CinYPDwithoutserumwithcontinuousaeration(byrotation)for24hoursfollowedbyobservationbylightmicroscopy (microscopic image shown for M. condurangoinPartA).Thepercentofsinglecellsversushyphal/pseudohyphalcells was determined and graphed in Part B. Assays were repeated in triplicate.

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the effect of botanical tinctures and essential oils …...candida albicans is a commensal organism...

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