Mycobiology 31(2): 99-104 (2003)Copyright © 2003 by The Korean Society of Mycology

d-

ee

mto

1.3the-st

andn

ashatts.ned

e

-rA,t-cy,

ta

at

ted

In vitro Evaluation of Antidermatophytic Activity of Egyptian Bee Propolis inCombination with Plant Essential Oils in Sheep Hoof Plate: An ExperimentalModel

Yehia A.-G. Mahmoud�

Tanta University, Faculty of Science, Botany Department, Mycology research lab. Tanta 31527, Egypt

(Received January 3, 2003)

Bee propolis ethanolic extract with some plant essential oils was investigated for its antidermatophytic properties. The testeplant essential oils included jasmine, clove, lemon, Arabian jasmine, mint, rosa, olive and basil. The antidermatophytic activity has been compared to Naftifine-HCl and Clotrimazole used for dermatophyte treatment. Experimental model has beentested using sheep hoof plate for the in vitro tests to stimulate human nails. Mint, clove and basil with 4 mg/ml of bee pro-polis have a comparable efficacy to those of Naftifine-HCl and Clotrimazole. There is a great necessity for new effectivlow price and safe antidermatophyte agents to avoid recurrent infection. Propolis synergistic could be of great importancwith essential oils of plants in dermatophyte therapy.

KEYWORDS: Antifungal drugs, Bee propolis, Dermatophytes, Plant essential oils

The available antifungal agents have a limited effective-ness and lack specificity against the dermatophytes. Der-matophytes are attacking skin, nails, hair and foot. It iswell known that there are three fungal genera responsiblefor the dermatophytic infection namely Trichophyton, Mi-crosporum and Epidermophyton. Foot and nail mycosesare resistant to antifungal agents and there are recurrentinfections. As Williams (1993) indicated that fungal infec-tion of the nails include primary nail pathogens, whichinvade healthy nail plate, and those, which invade second-arily in subjects with pre-existing nail disease. Fungal nailinfections onychomycoses are caused by three groups ofpathogens: dermatophytic fungi such as T. rubrum, yeastssuch as Candida albicans and non-dermatophytes such asScopulariopsis brevicaulis, Aspergillus species or Hender-sonula toruloidea and by various combinations of thesethree groups (Moharrem, 1999). In Egypt, onychomycosisrepresented 2% of human dermatophytic disease (Mah-moud, 1991).

Recently, the number of the antidermatophytic medica-tions for systemic and topical treatment has been multi-plied. The available antifungal agents include allylamine,azoles and others have severe side effects and inefficient.Dermatologists often attribute treatment failures to shorttherapies and lacking the drug specificity. Actually thepenetration of antifungal drugs is very poor in respect tonails. Many researchs used different keratinous substratesin order to evaluate the efficiency of antidermatophyticagents, Ceshin-Roques et al. (1991) used pigskin andMalecky and McClausland (1982) used ovine hoof. How-

ever, Hemidy et al. (1994) found that sheep hooves seeto possess quite similar physico-chemical properties those of human nails, although they are thicker about mm and harder. Benfields and Clissold (1988) studied antifungal efficiency of 1% (w/v) solution of Caryophyllene oxide, sulconazole, and cicloprioxolamine againdermatophytes strains and found that sulconazole ciclopiroxolamine have the ability to penetrate humanails in onychomycosis model. The aim of this work wto develop new and safe antidermatophytic agents tmight be more effective for treatment with no side effecTherefore, some plant essential oils have been screefor its antidermatophytic activity with and without bepropolis.

Material and Methods

Essential oils and antifungal agents. Essential oilswere obtained from industrial oil company in Egypt, however Naftifine-HCl (Exoderil) was obtained from OctobePharma S.A.E., Cairo under license of Pfizer Inc. USand Clotrimazole (Lotrimin) was obtained from Deparment of Pharmaceutical Chemistry, Faculty of PharmaTanta University, Egypt.

Propolis. Propolis (bee glue) was collected from Tanarea, Egypt during March 2002.

Propolis extraction and sample preparation. One gramof propolis was cut into small pieces and extracted room temperature with 50 ml of 70% ethanol (twice after24 hours). Then the alcoholic extract was evapora*Corresponding author <E-mail: yehiam@decl.tanta.edu.eg>

99

100 Yehia A.-G. Mahmoud

infteraltoe4).etted

ithd for

nt-het,cts

ced

e

under vacuum at 50oC until dryness.

Fungal strains. Epidermatophyton floccosum, Trichophy-ton rubrum, Microsporum canis and Candida albicanswere isolated from about 10 patients with dermatophyto-sis of Tinea cruris, Tinea unguium, and Tinea corporis asdiagnosed by dermatologist of Tanta University hospitals,Tanta, Egypt. These organisms were isolated on Sab-ouraud dextrose agar amended with 0.05% of each of cy-cloheximide and chloramphenicol (Al-Doory, 1980). Theisolated cultures were incubated up to 3 weeks at 28oCduring which the growing fungi were identified and purified.

Antifungal assay on agar medium. To evaluate theeffect of essential oil and bee propolis on dermatophytegrowth, these were sterilized by Millipore filter (0.22µm)and mixed with cooled autoclaved Sabouraud dextrosemedium at concentration at 0.5, 1.0, and 5.0%. Tween 80was added to the medium at concentration of 1% foremulsification. Also, bee propolis was added to the me-dium containing the essential oil to give final concentra-tion of 4 mg/ml. The Petri dishes with control plates con-taining media without and with oils were inoculated withmycelial discs (10 mm diameter) of each of the testedorganisms and incubated at 28oC for the specified periodof time. The inhibition percentage was calculated usingthe formula of Vincent (1927):

Where I = percent of inhibition of fungal growth, C = fun-gal growth of check and T = fungal growth of treatment.

In vitro determination of penetration and absorptionof essential oils with bee propolis into sheep hoof (anexperimental model). Sheep hoofs were cut into platesof 2.5 cm length and 1.3 cm width and 0.5 cm (thickness)and then sterilized by 75% ethanol and washed in steriledist.water. Then, in sterile Petri-dish, culture agar blocksof the dermatophyte fungus under investigation of 1.6 cmwere cut and placeded above sterile filter paper and thehoof plates were placed on the culture block, where theexternal hoof surface is in contact with the dermatophyte.The whole plates were transferred into a larger petri-dishcontaining sterile water to prevent dehydration (Fig. 5).Daily adding of a Sabouraud broth at the side and thebase of culture block kept the dermatophyte moist. Theessential oils with bee propolis and the commercial anti-fungal drugs were added with a micropipette directly ontothe hoof internal surface on daily basis for 14 days.

Strategy of dermatophyte treatments. Two protocolswere tried, simultaneous treatment and post-treatment. Insimultaneous treatment an essential oils with bee propolis

were added into the hoof at the same time. Howeverthe post-treatment the antifungal agents were applied a7 days of fungal agar block implantation on the externface of hoof plate in order to get a similar environment clinical infection. Post treatment is more similar to thtopical therapy in practice (Cohen and Richard, 199Growth controls without antifungal treatments were sup. The percentage of growth inhibition exerted by tesagents was evaluated.

Results

Antifungal activity of essential oils with or without beepropolis in agar medium. In search for new antifungalagents with a lower toxicity redundant, essential oils wbee propolis as a natural components have been testeantifungal activity against dermatophytic fungi and C.albicans. Current antifungal treatment has had disappoiingly low success rate and high prices. Evaluation of tinhibitory effect of jasmine, clove, Arabian jasmine, minrosa, olive at 0.5, 1.0 and 5.0% showed inhibitory effeto the tested dermatophytic fungi (T. rubrum, M. canis, E.floccosum) and C. albicans. Largely incorporating 4 mg/ml of bee propolis to the all tested essential oils enhanthe inhibitory effect of the tested essential oils.

Figure 1(A) shows that clove and olive oils have thhighest percentage of growth inhibition for T. rubrum at

I = C T–

C------------- 100×

Fig. 1. Effect of plant essential oils on Trichophyton rubrumgrowth at different concentrations for two weeks at 28oCwithout bee propolis (A) and with bee propolis (B).

In vitro Evaluation of Antidermatophytic Activity of Egyptian Bee Propolis 101

e

nger-

edof

stlusnst

r-

er-

inpro-ilf

On

different tested oil concentrations; clove oils performed70, 85 and 90% of growth inhibition for T. rubrum at 0.5,1 and 5% of oils respectively.

Adding the bee propolis to the different oils greatlyenhance the antidermatophytic activity, where clove oil per-formed 90% growth inhibition for T. rubrum at 1% concen-tration which was produced at 5% concentration of clove oilonly (Fig. 1B). In case of M. canis arabian jasmine oilproved to be the most effective one a mong eight tested oils,where it produced nearly 100% inhibition at 5% (Fig. 2A).

The addition of bee propolis enhanced the antifungalactivity of Arabian jasmine oil against M. canis; the anti-fungal activity of 5% of Arabian jasmine oil alone wascomparable with that of 1% Arabian jasmine oil in com-bination with bee propolis (Fig. 2B).

Epidermatophyton flocossum was largely inhibited withmint essential oil followed by clove oil (Fig. 3A, B). Mintas an essential oil produced 80% E. floccosum growthinhibition at 5%. The addition of 4 mg/ml of bee propolisto that oil produced nearly the same inhibition percentageat 1%. The antimycotic activity of the essential oils withbee propolis was also screened against C. albicans a clini-

cally important infectant. Clove oil followed by mint andjasmine performed the strongest growth inhibition of C.albicans (Fig. 4A, B). Also, bee propolis enhanced thantifungal efficacy of plant essential oils tested.

Sheep hoof onychomycosis experimental model.Resultsobtained from onychomycosis experimental model usisheep hoof as testing material infected with the tested dmatophytes indicated a treatment ability for the testessential oils with bee propolis (Table 1). The mixture essential oil (1%) and bee propolis (4 mg/ml) proved to beeffective in eliminating the dermatophyte infection. In potreatment of infected sheep hoof with essential oils pbee propolis, rosa, clove showed highest activity agaiC. albicans, E. floccosum, T. Rubrum and M. canis,respectively. Essential oil (mint, clove and basil) at diffeent concentrations (0.5, 1.0, 5 and 10% with 4 mg/ml ofbee propolis) was tested for antifungal activity against dmatophytic fungi and C. albicans (Tables 2, 3 and 4).Mint, clove and basil have been chosen to be testedsheep hoof experimental model because they gave a nounced inhibition effect on petri dishes testing. Mint o(10%) with bee propolis performed a good inhibition oC. albicans and a considerable inhibition for T. rubrumand M. canis. However, E. floccosum is the least affectedone during the dermatophyte post treatment (Table 2).

Fig. 2. Effect of essential oils on Microsporum canis growthwithout bee propolis (A), and with bee propolis (B)for two weeks at 28oC.

Fig. 3. Effect of essential oils on Epidermatophyton floccosumgrowth without bee propolis (A), and with bee pro-polis (B) for two weeks at 28oC.

102 Yehia A.-G. Mahmoud

stntsth

refi-eeic

eem-).

tle,es

4 mg/m

nis

the other hand, clove oil (10%) with bee propolis in pofungal treatment proved to be excellent antifungal agefor against the four tested fungi where it gave 80% growinhibition (Table 3). Additionally, basil oil (10%) with beepropolis exhibited 65~78% of growth inhibition of thefour fungi in case of the post treatment which is the moreal to the fungal infection (Table 4). Comparing the efciency of the essential oils (clove, mint and basil with bpropolis) with commercially available antidermatophytdrugs like Naftifine-HCl (Table 5) and Clotrimazole(Table 6) indicated that 10% of the essential oil with bpropolis more or less has the same efficiency of the comercial antidermatophytic agents (with 5% concentration

Discussion

Plant essential oils are not real oils. They are the subaromatic and volatile liquids extracted from the leav

Fig. 4. Effect of essential oils on Candida albicans growthwithout bee propolis (A), and with bee propolis (B)for two weeks at 28oC.

Table 1. Dermatophyte development on implanted sheep hoof treated with some essential plant oils and bee propolis at (l)after two weeks

Treatments at (1%)Dermatophyte development (%)

Candida albicans Epidermatophyton floccosum Trichophyton rubrum Microsporum ca

Simultaneous treatmentJasmineCloveLemonArabian JasmineMintRosaOliveBasilPost treatmentJasmineCloveLemonArabian JasmineMintRosaOliveBasil

a23±2.0

a

42±3.517±1.019±1.628±2.042±4.038±4.133±2.0

26±2.045±4.020±2.023±1.630±3.050±4.242±3.241±3.2

40±3.237±3.025±2.138±3.246±4.528±1.618±2.145±3.5

47±3.535±3.429±2.045±4.087±7.625±2.023±2.051±4.2

39±3.053±4.542±3.440±3.235±3.043±4.128±2.548±4.0

42±3.570±6.546±4.145±3.242±3.246±4.532±3.151±4.6

30±2.242±3.428±1.228±2.152±4.638±2.817±1.061±5.3

34±3.140±4.031±2.532±2.663±5.041±3.223±2.062±5.2

aMean±SD (n = 3).

Fig. 5. Experimental Model for in vitro evaluation of antider-matophytic activity of bee propolis and essential oils.

In vitro Evaluation of Antidermatophytic Activity of Egyptian Bee Propolis 103

eyint

on-ne.

),hatyp-been bepel-re

uye,

stems, seeds bark, flowers and roots of various plantsthrough distillation. Essential oils are soluble in the lipidsin the skin and in most cases easily penetrate it and areabsorbed into the bloodstream. They have advantages overthe antidermatophytic agents, because essential oils haveno side effects. Ahmed and Agnihotri (1977) reportedantifungal activity of volatile oils obtained from severalplants including, Mentha piperata against Alternaria bras-sicae, Colletotrichum papaya and Helminthosporium spp.Deshnukh et al. (1986) also studied the mycotoxicity ofsome essential oils against Trichophyton equinum, T. men-tagrophytes, T. rubrum, T. terrestre, M. gypseum andKeratinomyces ayelloi. El-Naghy et al. (1992) tested theinhibitory effects of some natural oils and fatty acids on

the growth of some of the dermatophytes and threvealed high fungistatic effects of clove and peppermoils. Eucalyptus melliodora (yellow box) leaves werefound to have a cineole level of 71.2% the remainder csisting mainly of terpenes such as pinene and limoneAnalysis of the red gum E. blakelyi revealed a wide rangeof constituents, the highest levels were for cymene (30%cryptone (11.7%) and pinene. Investigation revealed tcymene is used in perfumery and as a solvent, while crtone possesses marked germicidal properties and had used in the manufacture of disinfectants. Plant oils canapplied and used as liquid, sprays, crystals, gels and lets, and by impregnating material. Other variations aalso used, which means the safe use of those oils. Ino

Table 2. Percentage of dermatophyte development on implanted sheep hoof treated with bee propolis at (4 mg/ml) and with differentconcentrations of Mint, after two weeks

Treatments MicroorganismsMint (%)

0.5 1.0 5.0 10

Simultaneous treatment

Post treatment

C. albicansE. floccosumT. rubrumM. canisC. albicansE. floccosumT. rubrumM. canis

a36±3.0a

50±4.138±3.261±4.543±3.093±7.854±4.279±6.8

30±3.045±4.030±3.256±4.834±3.285±7.946±4.570±6.4

28±2.047±3.135±2.154±4.230±3.089±7.044±3.565±5.2

18±1.032±3.028±2.145±3.627±1.678±6.338±2.645±3.2

aMean±SD (n = 3).

Table 3. Percentage of dermatophyte development on implanted sheep hoof treated with bee propolis at (4 mg/ml) and differentconcentrations of Clove, after two weeks

Treatments MicroorganismsClove oil (%)

0.5 1.0 5.0 10

Simultaneous treatment

Post treatment

C. albicansE. floccosumT. rubrumM. canisC. albicansE. floccosumT. rubrumM. canis

a58±4.5a

52±4.969±5.255±3.857±4.253±3.888±6.867±4.

47±4.245±3.960±5.743±4.148±4.040±3.875±6.856±4.1

25±2.130±2.735±3.520±2.835±3.227±2.728±2.135±3.1

18±2.023±2.018±1.014±1.220±2.218±1.918±1.722±2.8

aMean±SD (n = 3).

Table 4. Percentage of dermatophyte development on implanted sheep hoof treated with bee propolis at (4 mg/ml) and differentconcentrations of Basil, after two weeks

Treatments MicroorganismsBasil (%)

0.5 1.0 5.0 10

Simultaneous treatment

Post treatment

C. albicansE. floccosumT. rubrumM. canisC. albicansE. floccosumT. rubrumM. canis

a46±3.0a

52±4.168±3.271±4.553±3.063±7.853±4.279±6.8

34±3.045±4.050±3.261±4.841±3.252±7.949±4.560±6.4

25±2.037±3.145±2.144±4.232±3.041±3.034±3.545±5.2

18±1.022±3.028±2.135±3.622±1.628±6.328±2.635±3.2

aMean±SD (n = 3).

104 Yehia A.-G. Mahmoud

5%itus a

f

ry

itst-

,Case

J.,er

eat-

n, of

on

-tty

J.ails

O.,f

alis,

e

he

of

in

et al. (2000) studied the inhibitory effect of essential oilson apical growth of Aspergillus fumigatus by vapour con-tact and they concluded that suppression of the fungusapical growth by vapour contact was ascribed to the directdisposition of essential oils on fungal mycelia, to getherwith an indirect effect via the agar medium absorption. Ithas been reported that the most of plant essential oils con-tains phenols mostly, particularly thymol (Sainsbury andSafowora, 1971) and that these are probably responsiblefor its reported antimicrobial action. In addition to thephenolic nature of some essential oils there are some oth-ers contain terpenoid and aldehyde class of compounds(Cowan et al., 1999). So, the degree of antidermatophyticactivity of mint, clove and basil might return to its con-tents of aldehyde or phenols and/or terpenoid class present.Studying the mode of antimicrobial action of the essen-tial oil of Melaleuca alternifolia (tea tree oil) revealed thatminimum bactericidal/fungicidal concentrations of tea treeoil inhibited respiration and increased the permeability oforganisms plasma membranes as indicated by uptake ofpropidium iodide. It is worth to be mentioned out thatadding bee propolis to essential oils greatly enhanced theiractivity towards dermatophytes. The actual composition ofpropolis varies according to the area of collection. In gen-eral terms, it is composed of 50% resins and vegetables

balsams, 30% wax, 10% essential and aromatic oils, pollen and 5% other substances including organic detr(Callejo et al., 2001). Propolis has been shown to benon-specific immunostimulators.

References

Ahmed, S. K. and Agnihotri, J. P. 1977. Antifungal activity osome plant extracts. Indian. J. Mycology and Plant Pathol. 7:180-181

Al-Doory, Y. 1980. Media in Lea and Febiger (eds.). LaboratoMedical Mycology. Henry Kimpton London, pp. 357-375.

Benfields, P. and Clissold, S. P. 1988. Sulconazole: a review ofantimicrobial activity and therapeutic use in superficial dermaomycosis. Drugs Auckland. 35: 143-153.

Callejo, A., Armentia, A., Lombardero, M., Martinez, C., RebolloS., Sedano, E., de la Fuente, R. and Fernandez, A. 2001. report, hypersensitivity to propolis. Alergol Immunol Clin. 16:113-117.

Ceshin-Roques, C. G., Hanel, H., Pruja-Bougaret, S. M., Luc,Vandermander, J. and Michel, G. 1991. Ciclopirox nail lacqu80%: in vivo penetration into and through nails and in vitroeffect on pig skin. Skin Pharmacol. 4: 89-94.

Cohen, P. R. and Richard, K. S. 1994. Topical and surgical trment of onchomycosis. J.Amer.Acad.Dermatol. 3: 68-74.

Cowan, M. M. 1999. Plant products as antimicrobial agents. Clin-ical Microbiol. Rev.12: 564-582.

Cox, S. D., Mann, C. M., Markham, J. L., Bell, H. C., GustafsoJ. E., Warmington, J. R. and Wyllie, S. G. 2000. The modeantimicrobial action of the essential oil of Melaleuca alternifo-lia (tea tree oil). J. Appl. Microbiol. 88: 170-175.

Deshnukh, S. K., Jain, P. C. and Agrawal, S. C. 1986. A notemycotoxicity of some essential oils. Fitoterapia 57: 295-297.

El-Naghy, M. A., Maghazy, S. N., Fadl-Allah, E. M. and ElGendy, Z. K. 1992. Fungi static action of natural oils and faacids on dermatophytic and saprophytic fungi. Zentralbl. Mik-robiol. 147: 214-220.

Hemidy, P. Y., Makki, S., Muret, P., Chaumont, J. P. and Millet,1994. The use of sheep hoof plates for substituting human nin transungual absorption studies. J. Appl. Cosmetol. 12: 73-84.

Inouye, S., Tsuruoka, A., Watanabe, S., Takeo, K., Akao, Nishiyama, Y. and Yamaguchi, M. 2000. Inhibitory effects oessential oils on apical growth of Aspergillus fumigatus byvapour contact. Mycoses 43: 17-23.

Mahmoud, A. L. E. 1991. Some physiological and biochemicstudies on fungi isolated from skin of mammals. Ph.D. ThesBot. dept., Fac. Sci. Assiut Univ., Egypt.

Malecky, J. C. and McClausland, J. P. 1982. In vitro penetrationand absorption of chemicals into the ovine hoof. Res. Veterin.Sci. 33: 192-197.

Moharram, A. M. 1999. Comparison of the effect of allylaminand azole derivatives on fungi isolated from human nails. Bull.Fac. Sci. Assiut univ. 28(2-D): 21-33.

Sainsbury, M. and Sofowora, E. A. 1971. Essential oil from tleaves and inflorescence of Ocimum gratissimum. Phytochemis-try 10: 3309-3310.

Vincent, J. M. 1927. Distortion of fungal hypha in the presencecertain inhibitors. Nature 159: 850.

Williams, H. C. 1993. The epidemiology of onychomycosis Britain. Br. J. Dermat. 129: 101-109.

Table 5. Percentage of dermatophyte development on implantedsheep hoof treated with different concentrations ofNaftifine-HCl, after two weeks

Treatments MicroorganismsNaftifine-HCl (%)

0.1 1.0 5.0

Simultaneoustreatment

Post treatment

C. albicansE. floccosumT. rubrumM. canisC. albicansE. floccosumT. rubrumM. canis

45±3.235±2.648±3.461±5.150±4.328±1.937±3.245±4.0

50±5.024±2.131±3.042±3.562±5.118±1.020±2.030±3.0

85±7.410±1.012±1.321±2.075±6.211±1.014±1.322±2.0

aMean±SD (n = 3).

Table 6. Percentage of dermatophyte development on implantedsheep hoof treated with different concentrations ofClotrimazole, after two weeks

MicroorganismsClotrimazole (%)

0.1 1.0 5.0

Simultaneoustreatment

Post treatment

C. albicansE. floccosumT. rubrumM. canisC. albicansE. floccosumT. rubrumM. canis

a45±3.2a

42±3.638±3.251±5.040±3.838±2.839±3.247±4.3

30±3.022±2.027±2.532±3.032±3.128±2.620±2.130±3.0

18±1.110±1.012±0.921±1.820±2.011±0.914±1.322±2.0

aMean±SD (n = 3).