Antifungal activity of Allium, Aloe, and Solanum species

Sumbul Shamim1, S. Waseemuddin Ahmed2, and Iqbal Azhar2

1Faculty of Pharmacy, Hamdard University, Karachi, Pakistan; 2Department of Pharmacognosy,Faculty of Pharmacy, University of Karachi, Karachi, Pakistan

Abstract

The current study evaluated and established the activityof Allium sativum Linn., Aloe barbadensis Mill., andSolanum nigrum Linn. against some common fungalspecies associated with superficial mycoses. The ethanoland aqueous extracts of these plants were tested toestablish the antimycological effects against dermato-phytes, saprophytes, and Candida species isolated frominfected hospitalized patients. The in vitro antifungalactivity was established by observing and measuringthe zones of inhibition formed on selective nutrientmedia. Zones of inhibition were categorized as very high(41–50mm), high (31–40mm), medium (21–30mm), andlow (11–20mm). High zones of inhibition were notedwith ethanol extracts of Allium sativum, Aloe barbadensis,and Solanum nigrum.

Keywords: Allium sativum Linn., Aloe barbadensis Mill.,antimycological evaluation, Candida species, dermato-phytes, saprophytes, Solanum nigrum Linn.

Introduction

Since prehistoric times, people have used naturalresources for medicinal purposes. Folk medicines employmany plants to counteract diverse diseases including skininfections. A large number of plants have been reportedby researchers and practitioners of traditional medicineto be useful in the treatment of skin diseases (Miranda,1976; Berlin et al., 1990). Agents to prevent growth offungi are important in medicine. Fungi that infect theskin, nails, and hair, generally called ‘‘ringworm’’ or‘‘tinea,’’ are classified as dermatophytes. The threeimportant genera that are closely related botanicallyare Microsporum, Trichophyton, and Epidermophyton.The genus Microsporum is the most frequent cause of

ringworm of the scalp and may give rise to ringwormin other parts of the body. Trichophyton causes ringwormof the scalp, beard, and other areas of the skin and nails.Epidermophyton is largely responsible for ringworm ofthe skin, hands, and feet and appears as interlacingthreads in the skin but does not invade the hair (Rippon,1982). Candida spp. have been reported to be commensalfungi commonly found in the gastrointestinal tract,mouth, and vagina; they become pathogenic only whennatural defense mechanism fails. Candida albicans hasbeen observed most commonly associated with infection,although infections with other species, notably Candidaglabrata, Candida krusei, Candida parapsiolosis, and Can-dida tropicalis, also occur (Grabue, 1994).

Until recently, comparatively little research for newantifungal agents has been carried out. However, withthe upsurge in the number of immunocompromisedand immunosuppressed patients succumbing to fungalinfections, the demand for new antifungal compoundshas been raised dramatically. Volatile oil of Allium sati-vum Linn. has antimicrobial activity against bacterialand fungal organisms (Petricic et al., 1977). Candida albi-cans have been reported to be more sensitive to Alliumsativum juice than Staphylococcus aureus and Escherichiacoli. Small amounts of Allium sativum did not simulateendogenous respiration in C. albicans not show inhibi-tory effects on growth of bacteria and fungi (TyneckaGos, 1973). Mustard=coconut oil, in which garlic is fried,is an excellent antiseptic application for sores and scabieswhen rubbed over ringworm for relief (Behl et al., 1993).Polysaccharides from juice of Aloe vera are useful intreating burns and wounds, show resistance to depolymnby airborne fungi and enzymes, and hasten healing ofwounds from thermal burns and radiation injury. Thismaterial is also used in the treatment of dry and moistepidermis, second- and third- degree burns, prophylactic

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.

Pharmaceutical Biology2004, Vol. 42, No. 7, pp. 491–498

action, prevention of kraurosis, dermatitis, eczema,psoriasis, neurodermatitis, herpes, and is suitable forsubcutaneous infections. The application of fresh Aloepith relieves pain, burning, and itching and has antisepticaction. Aloe vera gel is used in the treatment of sebor-rhea, acne vulgaris, and alopacia (Hakim MuhammadSaid 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 treat-ment of diverse skin infections, the genus Solanum showsa high index of citation. Considering the epidemiologicalimportance of skin infections in tropical countries suchas Pakistan and searching for new remedies for the treat-ment of this type of ailment, a specific study was per-formed to inquire deeper into the ethnomedical botanyof three different plants, Allium sativum, Aloe barbadensisMill., and Solanum nigrum.

Materials and Methods

Plant material

Bulbs of Allium sativum Linn. (GH no. 67686), leaves ofAloe vera Mill. (GH no. 67687), and air-dried berries ofSolanum nigrum Linn. (GH no. 67685) were purchasedfrom a local market in Karachi. Voucher specimens aredeposited at the Department of Pharmacognosy, Univer-sity of Karachi (Karachi, Pakistan).

Extraction of plant material

Allium sativum bulb cloves (5 kg after removing thesheath) and Aloe vera leaf latex (4.5 kg after removingthe epidermis with the help of a sharp knife) were cutinto small pieces, and dried berries of Solanum nigrum(3.5 kg) were crushed in a homogenizer separately. Allthese plant materials were soaked in ethanol (95%) andin distilled water in separate jars for 3 weeks. Theextracts (aqueous and ethanol) obtained were evaporatedat reduced pressure (45�C) to a syrupy residue. The driedethanol and aqueous extracts of Allium sativum wereslightly gummy in appearance and reddish brown incolor. Similarly, dried extract of Aloe vera was dark

brown in color, and that of Solanum nigrum was darkgreen (Ahmad, 1992).

Preparation of extracts of testing

Ethanol and aqueous extract were prepared in three dif-ferent concentrations. The stock solutions were preparedby dissolving 100mg of dry extract in 1ml of ethanol andwater separately to obtain a concentration of 100mg=mlDilutions (1:10, 1:100, 1:500) of these stock solutionswere used in phosphate buffer at pH 6.0 to evaluatethe antifungal activity (Champion et al., 1992).

Collection of test organisms

For the isolation and identification of fungal species, 315samples were collected from the Hospital Institute ofSkin Diseases, Karachi, Sindh. Out of the 315 samples,250 indicated positive results under a microscope forthe presence of fluorescent hyphae (Fig. 1). Identificationof all fungal species was primarily based on the charac-teristic morphological character seen on the mycobioticagar media and on the bases of microscopical character-istics. Out of 250 isolates, 204 were identified as derma-tophytes, 30 as saprophytes, and 16 as Candida species.The prevalence of these dermatophytes is summarizedin Table 1.

For the isolation of dermatophytes, mycobiotic agarmedium was used, whereas Sabouraud’s dextrose agar

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

Table 1. Data obtained during the study of patients infected with superficial mycoses.

T.rubrum

T.verrucosum

T.violaceum

T.tonsurans

T.mentagrophytes

E.floccosum

A.flavus

A.fumigatus

A.glaucus

A.terreus

Candidaspp.

Males 47 15 5 9 2 12 2 2 3 1 5Females 35 19 1 8 3 0 4 4 9 4 5Children 2 14 18 1 13 0 0 0 0 1 6Total 84 48 24 18 18 12 6 6 12 6 16

492 S. Shamim et al.

Table

2.

Effectoftheethanolextract

andaqueousextract

obtained

from

Solanum

nigrum,Aloebarbadensis,andAllium

sativum

ondifferent

fungalspeciesisolatedfrom

infected

patients

sufferingfrom

superficialmycosis.

Plants

Solanum

nigrum

Aloebarbadensis

Allium

sativum

Ethanolic

extract

Aqueous

extract

Ethanolic

extract

Aqueous

extract

Ethanolic

extract

Aqueous

extract

Fungalspecies

No.of

hosts

1:10

1:100

1:500

1:10

1:1001:500

1:10

1:100

1:500

1:10

1:100

1:500

1:10

1:100

1:500

1:10

1:100

1:500

Aspergillusflavus

6þþþ

þþ

þþþ

þ�

þþþ

þþþþ

þþ

þþþþ

þþþ

þþ

þþþþ

þþþ

þþ

þþ

þþþþ

þþ

Aspergillus

fumigatus

6þþþþ

þþþ

þþ

þþ

þþþ

þþþ

þþ

þþ

þþþ

þþ

þþ

þþ

þþþ

þþ

þþ

þþþ

þþ

þþ

þþþþ

þþ

Aspergillusglaucus

12

þþþ

þþ

þþ

þþ

þ�

þþþþ

þþ

þþþþ

þþþ

þþ

þþþ

þþþ

þþ

þþ

þþ

þþ

Aspergillustereus

6þþþ

þþ

þþþ

þ�

þþ

þþþ

þþþþ

þþ

þþþ

þþþ

þþþ

þþ

þCandidaalbicans

10

þþþ

þþ

þþþ

þþþ

þþþ

þ�

þþ

þ�

þþþþ

þþþ

þþ

þþþþ

þþþ

þþ

Candidaglabrata

3þþ

þ�

þþ

�þþ

þ�

þþ

þ�

þþ

þþþ

þþ

þþ

þþ

þCandidatropicalis

3þþþþ

þþþ

þþ

þþþ

þþþ

þþ

þþþ

þþ

þþ

þþþ

þþ

þþþ

þþ

þþ

þþþþ

þþþ

þþþ

þþ

Epidermophyton

floccosum

12

��

��

��

��

��

��

��

��

��

Trichophyton

mentagrophytes

18

þþ

þ�

þ�

�þþ

þþþ

þþ

þþ

þþ

þþþ

þþþ

þþ

þþ

þþ

Trichophyton

rubrum

84

þþ

þ�

þ�

�þþ

þþ

þþþ

þþ

þþ

þþþ

þþþ

þ�

Trichophyton

tonsurans

18

þþþ

þþ

þþ

þþ

þ�

þþþ

þþþ

þþ

þþ

þþþ

þþ

þþ

þþ

þþþ

þ�

Trichophyton

verrucosum

48

þþ

þ�

þ�

�þþ

þþþ

þþ

þþ

þþþ

þþ

þ�

þ�

�

Trichophyton

zviolaceum

24

þþ

þþ

þþ

�þþ

þþ

þþþ

þþ

þþ

þþ

þþ

þ�

Key:�,negativeantifungalactivity:þ

,positiveantifungalactivity(inthefollowingcombinations):þ,low

inhibition;þ

þ,¼

medium

inhibition;þ

þþ,highinhibition;

þþþþ,veryhighinhibition.

493

was used to isolate saprophytes and Candida, as well asfor the maintenance of all isolates (Champion et al.,1992).

Test organisms

The test organisms isolated from different hosts were Tri-chophyton rubrum (84 hosts), T. verrucosum (48 hosts),T. violaceum (24 hosts), T. tonsurans (18 hosts), T. menta-grophytes (18 hosts), Epidermophyton floecosum (12hosts), Aspergillus glaucus (12 hosts), A. flavus (6 hosts),

A. fumigatus (6 hosts), A. terreus (6 hosts), Candida albi-cas (10 hosts), C. glabrata (3 hosts), and C. tropicalis (3hosts).

Antifungal assay

The antifungal activity of ethanol and aqueous extracts ofplants were assayed by the hole-plate method (SakharkarPatil, 1998). Sabouraud’s dextrose agar was employed asmedium. 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 ¼ Aspergillus flavus, A. fu ¼ A. fumigatus, A. g ¼ A. glaucus, A. te ¼ A. terreus, C. al ¼ Candida albicans, C. gl ¼ C. glabrata,C. tr ¼ C. tropicalis, ¼ T. m ¼ Trichophyton mentagrophytes, T. r ¼ T. rubrum, T. to ¼ T. tonsurans, T. v ¼ T. verrucosum, T. vi ¼T. violaceum.

494 S. Shamim et al.

carried out against 13 stock cultures of fungi. Threecups were made aseptically with a cork borer having6-mm diameter. The plates were inoculated withrespective fungi, under aseptic conditions, and 0.2mlof test solution in 3 dilutions (i.e., 1:10, 1:100, 1:500)

was poured in the holes using a dropping pipette underaseptic conditions. The plates were incubated at 35�Cfor 24–48 h for the observation of zones of inhibition.Each experiment was carried out in triplicate (Sakhar-kar Patil, 1998).

Figure 2. Continued.

Antifungal activity of Allium, Aloe, and Solanum species 495

Results and Discussion

The results of antifungal screening are given in Table 2.The antifungal activity of the ethanol extracts of allplants was found to be quite impressive as compared toaqueous extracts. However, none of the plant extractswere found active against Epidermophyton floccosum.Growth inhibition (zone of inhibition) was recorded asvery high (þþþþ ), high (þþþ ), medium (þþ ),and low (þ ), which ultimately indicated zones of

inhibition between 41–50, 31–40, 21–30, and 11–20mm,respectively. Some very interesting outcomes were notedin this study. The ethanol extracts of all the plants werenoted to possess more antimycological effects as com-pared to the aqueous extracts (Fig. 2).

The high zones of inhibition noted in the ethanolextracts of Solanum nigrum, Aloe barbadensis, and inAllium sativum (using a 1:10 concentration) suggestfurther explanation of the possibility of using these

Figure 2. Continued.

496 S. Shamim et al.

Table3.

Minim

um

inhibitory

concentrationsobserved

indifferentconcentrations,preparedfrom

stock

solutionsof100mg=ml,ofaqueousandethanolextractsoftheplants.

Solanum

nigrum

Aloebarbadensis

Allium

sativum

Fungalspecies

No.of

hosts

Ethanolicextract

Aqueousextract

Ethanolicextract

Aqueousextract

Ethanolicextract

Aqueousextract

Aspergillusfumigatus

61:500

1:500

1:500

1:500

1:500

1:500

Aspergillusglaucus

12

1:500

1:100

1:500

1:500

1:500

1:500

Aspergillustereus

61:500

1:100

1:500

1:500

1:500

1:500

Candidaalbicans

10

1:500

1:500

1:100

1:100

1:500

1:500

Candidaglabrata

31:100

1:100

1:100

1:100

1:500

1:500

Candidatropicalis

31:500

1:500

1:500

1:500

1:500

1:500

Epidermophytonfloccosum

12

��

��

��

Trichophytonmentagrophytes

18

1:100

1:10

1:500

1:500

1:500

1:500

Trichophytonrubrum

84

1:100

1:10

1:500

1:500

1:500

1:100

Trichophytontonsurans

18

1:500

1:100

1:500

1:500

1:500

1:100

Trichophytonverrucosum

48

1:100

1:10

1:500

1:500

1:100

1:10

Trichophytonviolaceum

24

1:500

1:100

1:500

1:500

1:500

1:100

Key:�,noinhibitionwasseen.

497

plants against certain skin infections caused by the abovefungal organisms. The minimum inhibitions observed aregiven in Table 3.

For work on antifungal activity, the test organismsused are of considerable importance because dermato-phyte species 9 and 7 other fungal species were isolatedfrom patients. As the number of organisms increases,the results become more credible.

Further, these findings could be used to develop suit-able dosage forms such as cream, ointment, and lotion asper the requirement of the treatment.

Acknowledgments

The authors wish to express their appreciation toDr. Ehtishamuddin and Dr. Rubina Dawar for the identi-fication of specimens. Thanks also to the University ofKarachi for financial support.

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