Pharmaceutical Biology 2004, Vol. 42, No. 7, pp.

491498

Antifungal activity of Allium, Aloe, and Solanum species

Sumbul Shamim1, S. Waseemuddin Ahmed2, and Iqbal Azhar2 Faculty of Pharmacy, Hamdard University, Karachi, Pakistan; 2Department of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Karachi, Pakistan
1

Abstract
The current study evaluated and established the activity of Allium sativum Linn., Aloe barbadensis Mill., and Solanum nigrum Linn. against some common fungal species associated with superficial mycoses. The ethanol and aqueous extracts of these plants were tested to establish the antimycological effects against dermatophytes, saprophytes, and Candida species isolated from infected hospitalized patients. The in vitro antifungal activity was established by observing and measuring the zones of inhibition formed on selective nutrient media. Zones of inhibition were categorized as very high (4150 mm), high (3140 mm), medium (2130 mm), and low (1120 mm). High zones of inhibition were noted with ethanol extracts of Allium sativum, Aloe barbadensis, and Solanum nigrum. Keywords: Allium sativum Linn., Aloe barbadensis Mill., antimycological evaluation, Candida species, dermatophytes, saprophytes, Solanum nigrum Linn. ringworm of the scalp and may give rise to ringworm in other parts of the body. Trichophyton causes ringworm of the scalp, beard, and other areas of the skin and nails. Epidermophyton is largely responsible for ringworm of the skin, hands, and feet and appears as interlacing threads in the skin but does not invade the hair (Rippon, 1982). Candida spp. have been reported to be commensal fungi commonly found in the gastrointestinal tract, mouth, and vagina; they become pathogenic only when natural defense mechanism fails. Candida albicans has been observed most commonly associated with infection, although infections with other species, notably Candida glabrata, Candida krusei, Candida parapsiolosis, and Candida tropicalis, also occur (Grabue, 1994). Until recently, comparatively little research for new antifungal agents has been carried out. However, with the upsurge in the number of immunocompromised and immunosuppressed patients succumbing to fungal infections, the demand for new antifungal compounds has been raised dramatically. Volatile oil of Allium sativum Linn. has antimicrobial activity against bacterial and fungal organisms (Petricic et al., 1977). Candida albicans have been reported to be more sensitive to Allium sativum juice than Staphylococcus aureus and Escherichia coli. Small amounts of Allium sativum did not simulate endogenous respiration in C. albicans not show inhibitory effects on growth of bacteria and fungi (Tynecka Gos, 1973). Mustard=coconut oil, in which garlic is fried, is an excellent antiseptic application for sores and scabies when rubbed over ringworm for relief (Behl et al., 1993). Polysaccharides from juice of Aloe vera are useful in treating burns and wounds, show resistance to depolymn by airborne fungi and enzymes, and hasten healing of wounds from thermal burns and radiation injury. This material is also used in the treatment of dry and moist epidermis, second- and third- degree burns, prophylactic

Introduction
Since prehistoric times, people have used natural resources for medicinal purposes. Folk medicines employ many plants to counteract diverse diseases including skin infections. A large number of plants have been reported by researchers and practitioners of traditional medicine to be useful in the treatment of skin diseases (Miranda, 1976; Berlin et al., 1990). Agents to prevent growth of fungi are important in medicine. Fungi that infect the skin, nails, and hair, generally called ringworm or tinea, are classified as dermatophytes. The three important genera that are closely related botanically are Microsporum, Trichophyton, and Epidermophyton. The genus Microsporum is the most frequent cause of

Accepted: August 16, 2004 Address correspondence to: Sumbul Shamim, Faculty of Pharmacy, Hamdard University, Karachi 74600, Pakistan. E-mail: hfpsumbul@hotmail.com DOI: 10.1080/13880200490891845 # 2004 Taylor & Francis Ltd.

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action, prevention of kraurosis, dermatitis, eczema, psoriasis, neurodermatitis, herpes, and is suitable for subcutaneous infections. The application of fresh Aloe pith relieves pain, burning, and itching and has antiseptic action. Aloe vera gel is used in the treatment of seborrhea, acne vulgaris, and alopacia (Hakim Muhammad Said et al., 1986; Behl et al., 1993). Solanum nigrum Linn. extract is used for the treatment of chronic skin diseases, like psoriasis (Duke, 1987; Dymock, 1972). Among the plants reported to be used for the treatment of diverse skin infections, the genus Solanum shows a high index of citation. Considering the epidemiological importance of skin infections in tropical countries such as Pakistan and searching for new remedies for the treatment of this type of ailment, a specific study was performed to inquire deeper into the ethnomedical botany of three different plants, Allium sativum, Aloe barbadensis Mill., and Solanum nigrum.

Figure 1. Hyphae of a dermatophytic fungus in skin (KOH preparation). Obtained from the neck of female patient no. 59.

brown in color, and that of Solanum nigrum was dark green (Ahmad, 1992). Preparation of extracts of testing Ethanol and aqueous extract were prepared in three different concentrations. The stock solutions were prepared by dissolving 100 mg of dry extract in 1 ml of ethanol and water separately to obtain a concentration of 100 mg=ml Dilutions (1:10, 1:100, 1:500) of these stock solutions were used in phosphate buffer at pH 6.0 to evaluate the antifungal activity (Champion et al., 1992). Collection of test organisms

Materials and Methods

Plant material Bulbs of Allium sativum Linn. (GH no. 67686), leaves of Aloe vera Mill. (GH no. 67687), and air-dried berries of Solanum nigrum Linn. (GH no. 67685) were purchased from a local market in Karachi. Voucher specimens are deposited at the Department of Pharmacognosy, University of Karachi (Karachi, Pakistan).

Extraction of plant material Allium sativum bulb cloves (5 kg after removing the sheath) and Aloe vera leaf latex (4.5 kg after removing the epidermis with the help of a sharp knife) were cut into small pieces, and dried berries of Solanum nigrum (3.5 kg) were crushed in a homogenizer separately. All these plant materials were soaked in ethanol (95%) and in distilled water in separate jars for 3 weeks. The extracts (aqueous and ethanol) obtained were evaporated at reduced pressure (45C) to a syrupy residue. The dried ethanol and aqueous extracts of Allium sativum were slightly gummy in appearance and reddish brown in color. Similarly, dried extract of Aloe vera was dark

For the isolation and identification of fungal species, 315 samples were collected from the Hospital Institute of Skin Diseases, Karachi, Sindh. Out of the 315 samples, 250 indicated positive results under a microscope for the presence of fluorescent hyphae (Fig. 1). Identification of all fungal species was primarily based on the characteristic morphological character seen on the mycobiotic agar media and on the bases of microscopical characteristics. Out of 250 isolates, 204 were identified as dermatophytes, 30 as saprophytes, and 16 as Candida species. The prevalence of these dermatophytes is summarized in Table 1. For the isolation of dermatophytes, mycobiotic agar medium was used, whereas Sabourauds dextrose agar

Table 1. Data obtained during the study of patients infected with superficial mycoses. T. T. T. T. T. E. A. A. A. A. Candida rubrum verrucosum violaceum tonsurans mentagrophytes floccosum flavus fumigatus glaucus terreus spp. Males Females Children Total 47 35 2 84 15 19 14 48 5 1 18 24 9 8 1 18 2 3 13 18 12 0 0 12 2 4 0 6 2 4 0 6 3 9 0 12 1 4 1 6 5 5 6 16

Table 2. Effect of the ethanol extract and aqueous extract obtained from Solanum nigrum, Aloe barbadensis, and Allium sativum on different fungal species isolated from infected patients suffering from superficial mycosis. Plants Solanum nigrum Aqueous extract Ethanolic extract Aqueous extract Ethanolic extract Aloe barbadensis Allium sativum Aqueous extract

Ethanolic extract

Fungal species

No. of hosts 1:500 1:10 1:100 1:500 1:10 1:100 1:500 1:10 1:100 1:500 1:10

1:10

1:100

1:100

1:500

1:10

1:100 1:500

6 6

12 6 10 3 3 12

18

84

18

48

Aspergillus flavus Aspergillus fumigatus Aspergillus glaucus Aspergillus tereus Candida albicans Candida glabrata Candida tropicalis Epidermophyton floccosum Trichophyton mentagrophytes Trichophyton rubrum Trichophyton tonsurans Trichophyton verrucosum Trichophyton zviolaceum

24

Key: , negative antifungal activity: , positive antifungal activity (in the following combinations): , low inhibition; , medium inhibition; , high inhibition; , very high inhibition.

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was used to isolate saprophytes and Candida, as well as for the maintenance of all isolates (Champion et al., 1992). Test organisms The test organisms isolated from different hosts were Trichophyton rubrum (84 hosts), T. verrucosum (48 hosts), T. violaceum (24 hosts), T. tonsurans (18 hosts), T. mentagrophytes (18 hosts), Epidermophyton floecosum (12 hosts), Aspergillus glaucus (12 hosts), A. flavus (6 hosts),

Antifungal assay The antifungal activity of ethanol and aqueous extracts of plants were assayed by the hole-plate method (Sakharkar Patil, 1998). Sabourauds dextrose agar was employed as medium. In vitro screening of antifungal activity was

Figure 2. A comparison of in vitro antifungal activity of aqueous and ethanol extracts of plants in three different concentrations. A. f Aspergillus flavus, A. fu A. fumigatus, A. g A. glaucus, A. te A. terreus, C. al Candida albicans, C. gl C. glabrata, C. tr C. tropicalis, T. m Trichophyton mentagrophytes, T. r T. rubrum, T. to T. tonsurans, T. v T. verrucosum, T. vi T. violaceum.

Antifungal activity of Allium, Aloe, and Solanum species

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Figure 2. Continued.

carried out against 13 stock cultures of fungi. Three cups were made aseptically with a cork borer having 6-mm diameter. The plates were inoculated with respective fungi, under aseptic conditions, and 0.2 ml of test solution in 3 dilutions (i.e., 1:10, 1:100, 1:500)

was poured in the holes using a dropping pipette under aseptic conditions. The plates were incubated at 35C for 2448 h for the observation of zones of inhibition. Each experiment was carried out in triplicate (Sakharkar Patil, 1998).

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Figure 2. Continued.

Results and Discussion

The results of antifungal screening are given in Table 2. The antifungal activity of the ethanol extracts of all plants was found to be quite impressive as compared to aqueous extracts. However, none of the plant extracts were found active against Epidermophyton floccosum. Growth inhibition (zone of inhibition) was recorded as very high ( ), high ( ), medium ( ), and low ( ), which ultimately indicated zones of

inhibition between 4150, 3140, 2130, and 1120 mm, respectively. Some very interesting outcomes were noted in this study. The ethanol extracts of all the plants were noted to possess more antimycological effects as compared to the aqueous extracts (Fig. 2). The high zones of inhibition noted in the ethanol extracts of Solanum nigrum, Aloe barbadensis, and in Allium sativum (using a 1:10 concentration) suggest further explanation of the possibility of using these

Table 3. Minimum inhibitory concentrations observed in different concentrations, prepared from stock solutions of 100 mg=ml, of aqueous and ethanol extracts of the plants. Solanum nigrum Aloe barbadensis Allium sativum

Fungal species 1:500 1:500 1:500 1:500 1:100 1:500 1:100 1:100 1:500 1:100 1:500 1:500 1:100 1:100 1:500 1:100 1:500 1:10 1:10 1:100 1:10 1:100 1:500 1:500 1:500 1:100 1:100 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:100 1:100 1:500 1:500 1:500 1:500 1:500 1:500

No. of hosts Ethanolic extract Aqueous extract Ethanolic extract Aqueous extract

Ethanolic extract 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:100 1:500

Aqueous extract 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:100 1:100 1:10 1:100

Aspergillus fumigatus Aspergillus glaucus Aspergillus tereus Candida albicans Candida glabrata Candida tropicalis Epidermophyton floccosum Trichophyton mentagrophytes Trichophyton rubrum Trichophyton tonsurans Trichophyton verrucosum Trichophyton violaceum

6 12 6 10 3 3 12 18 84 18 48 24

Key: , no inhibition was seen.

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Behl PN, Arora RB, Srivastana G, Malhotra SC (1993): Herbs Useful in Dermatological Theraphy. BS Publishers and Distributors, Delhi, pp. 2024. Champion RH, Burton JL, Ebling FJG (1992): Textbook of Dermatology, 5th ed, Vol. 3. London, Blackwell Scientific Publications, pp. 11301175. Duke JA (1987): Handbook of Medicinal Herbs, Boca Raton, CRC Press, Inc. Dymock W (1972): Pharmagraphia Indica: A History of Principal Drugs of Vegetable Origin, not within British India, Vol. II. pp. 431433. Reprinted by Hamdard Foundation, Pakistan. Grabue GE (1994): Treatment of oral Candida mucositis infections. Drugs 47: 734740. Said HM, Ahmed VU, Rahman A (1986): Pakistan Encyclopedia, Planta Medica, Vol. 1. Nazimabad, Karachi, Hamdard Foundation Press, Hamdard Centre. Miranda (1976): Vegetacion Chipas, Vol. 2. Chiapas, Mexico, Gobiernodel Estado, pp. 4750. Petricic J, Lulic B, Kupinic M (1977): Antimicrobial efficiency and stabilities of active components of garlic (Allium sativum). Acta Pharm Jugosl 27: 3541. Rippon JW (1982): Medical Mycology: The Pathogenic Fungi and the Pathogen Antimycetes, 2nd ed. London, W.B. Saunders, pp. 154248. Sakharkar PR, Patil AT (1998): Antifungal activity of Cassia alata. Hamdard Medicus, XLI (3), 2021. Tynecka Z, Gos Z (1973): Inhibitory action of garlic (Allium sativum) on growth and respiration of some microorganisms. Acta Microbial Pol. Ser. B 5: 2227.

plants against certain skin infections caused by the above fungal organisms. The minimum inhibitions observed are given in Table 3. For work on antifungal activity, the test organisms used are of considerable importance because dermatophyte species 9 and 7 other fungal species were isolated from patients. As the number of organisms increases, the results become more credible. Further, these findings could be used to develop suitable dosage forms such as cream, ointment, and lotion as per the requirement of the treatment.

Acknowledgments
The authors wish to express their appreciation to Dr. Ehtishamuddin and Dr. Rubina Dawar for the identification of specimens. Thanks also to the University of Karachi for financial support.

References
Ahmad SW (1992): Ph.D. Thesis, Department of Pharmacognosy, University of Karachi, p. 79. Berlin B, Berlin EA, Breedlove DE, Duncan T, et al. (1993): La Herbolaria Medica Tzeltal-Tzotzil en los altos de Chiapas. Chicagas, Mexico, Gobiernodel Estado, p. 154.