Journal of Ethnopharmacology 108 (2006) 124–132

Antifungal activity of some Tanzanian used traditionally for the treatment of fungal infections Omar J.M. Hamza a,b,c,∗,1, Carolien J.P. van den Bout-van den Beukel b,c,1, Mecky I.N. Matee d, Mainen J. Moshi e, Frans H.M. Mikx f, Haji O. Selemani g, Zakaria H. Mbwambo e, Andre´ J.A.M. Van der Ven c,h, Paul E. Verweij b,c a Department of Oral Surgery and Oral Pathology, Muhimbili University College of Health Sciences (MUCHS), P.O. Box 65014, Dar es Salaam, Tanzania b Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands c Nijmegen University Center for Infectious diseases, Nijmegen, The Netherlands d Department of Microbiology and Immunology, MUCHS, Dar es Salaam, Tanzania e Institute of Traditional Medicine, MUCHS, Dar es Salaam, Tanzania f WHO Collaborating Centre, Dentistry, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands g Department of Botany, University of Dar es Salaam, Dar es Salaam, Tanzania h Department of General Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Received 25 April 2005; received in revised form 5 April 2006; accepted 26 April 2006 Available online 22 May 2006

Abstract Using the ethnobotanical approach, some Tanzanian plants reported to be used by traditional healers for the treatment of and fungal infections of the skin were collected and screened for their antifungal activity against , , , , and . A total of 65 crude methanol extracts belonging to 56 species and 38 families were screened using the broth microdilution method, according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI) (formerly, National Committee for Clinical and Laboratory Standards) [National Committee for Clinical Laboratory Standards, 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-2nd Edition M27-A2, National Committee for Clinical Laboratory Standards, Wayne, PA, USA]. Among the tested plant species, 45% (25 species) showed antifungal activity against one or more of the test fungi. The most susceptible yeasts were Cryptococcus neoformans, followed by Candida krusei, Candida tropicalis, and Candida parapsilosis. The least susceptible were Candida albicans and Candida glabrata. Strong antifungal activity was exhibited by extracts of anisata Oliv., Sclerocariya birrea Sond, Turraea holstii Gurk, Sterculia africana (Lour) Fiori, Acacia robusta subsp. Usambarensis (Taub) Brenan, Cyphosterma hildebrandti (Gilg), Desc, Elaeodendron buchannanii (Lows), Acacia nilotica (L.) Wild ex Del, Jatropha multifida L., and Pteridium aquilinum (L.) Kuhn. © 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Antifungal activity; Tanzanian plants; Traditional treatment; Fungal infections

1. Introduction Oral candidiasis is the earliest and most frequent fungal infection in the HIV-infected patients (Schiodt et al., 1990; Matee et al., It has been shown that up to 90% of all HIV/AIDS patients 2000; Reichart, 2003). This may extend to the oesophagus and contract fungal infections at some point during the course of prevent intake of adequate oral nutrition, resulting in increased the disease (Diamond, 1991) and that 10–20% die as a direct morbidity and mortality, and reduction of the length and qual- consequence of fungal infection (Drouhent and Dupont, 1989). ity of life (Klein et al., 1984). Candida albicans is the most common causative agent of oral candidiasis, but non-albicans Candida spp., such as Candida tropicalis, Candida glabrata, ∗ Candida parapsilosis Candida krusei Candida guillier- Corresponding author. Tel.: +255 741 769769; fax: +255 22 2150465. , , and E-mail address: [email protected] (O.J.M. Hamza). mondii, have also emerged as significant pathogens (Powderly, 1 The authors contributed equally to this work. 1992; Barchiesi et al., 1993). Infection with the pathogenic

0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.04.026 O.J.M. Hamza et al. / Journal of Ethnopharmacology 108 (2006) 124–132 125 fungus Cryptococcus neoformans is a more serious infection 2.2. Interviews with traditional healers that often produces pneumonia and meningitis in HIV-infected patients. Cryptococcus neoformans is estimated to have a preva- Information on the plants used for treatment of oral candidi- lence of between 8% and 10% among the AIDS patients group asis and fungal infections of the skin, parts used, preparations, (Brol et al., 2002). other uses and adverse effects were obtained through interviews Management of candidiasis and other fungal infections, with traditional healers using a questionnaire, after obtaining among HIV/AIDS patients, is facing a number of problems their informed consent. Interviews were conducted by the main including resistance to antifungal agents, drug toxicity, high author in Swahili language. Two traditional healers could only costs of antifungal agents and relapse of infections (Sangeorzan understand Sukuma and Shambaa language, respectively in et al., 1994; Debruyne, 1997). Literature reports show that HIV- which case close relatives assisted with translation during the infected patients often develop resistance to treatment with flu- interview. Fourteen traditional healers were interviewed, 11 men conazole, the most currently used antifungal agent (Redding et and 3 women. al., 1994; Ruhnke et al., 1994; Goldman et al., 2005). Sometimes, resistance to fluconazole triggers cross-resistance to other azoles 2.3. Collection and identification of plants or pathogen shift from Candida albicans to less sensitive species such as Candida glabrata and Candida krusei (Powderly, 1992; Various plant parts such as leaves, whole plant, fruits, stem, Bastert et al., 2001). This reduces the number of available, effec- stem barks, roots and root barks reported by interviewed tradi- tive antifungal agents, thus necessitating the development of new tional healers to be used for treatment of oral candidiasis and antifungal remedies. fungal infections of the skin were collected from the forest by Plants that are traditionally used in the treatment of fun- the main and second author with the help of a traditional healer. gal infections or related ailments could be a good source for The plants were identified at the Department of Botany, Uni- new, safe, biodegradable and renewable antifungal drugs. This versity of Dar es Salaam where also voucher specimens were is an important possibility for Tanzania, where medicinal plants deposited in the herbarium. have been used for centuries. Both rural and urban populations still depend on traditional healers for provision of health care. 2.4. Extraction of plant materials One report of a survey performed in Dar es Salaam showed that 21% of the people that went to seek care from public All plant samples were air-dried and ground. Approximately services had first consulted a traditional healer (Kilima et al., 400 g of the plant materials were macerated with 80% methanol 1993). Also according to Medicine du Monde, a French non- at room temperature and after 24 h filtered through Whatman governmental organisation, in Kagera region five out of every six number 1 filter paper. The procedure was repeated three times HIV-infected patients receive their medical attention from a tra- to ensure exhaustive extraction of the plant material. The extracts ditional healer rather than from a hospital or primary health care were pooled together, concentrated, and the solvent removed by facility (Anonymous, 1996). Despite the broad use of medicinal evaporation under reduced pressure in a rotavapor at 40 ◦C. The plants there is very limited scientific support for their use in the extracts were further dried by freeze-drying and kept in a freezer, treatment of fungal infection. at −20 ◦C, until used for antifungal testing. The purpose of this study was to identify and obtain infor- mation on the plants that are used by traditional healers in the 2.5. Assay for antifungal activity management of fungal infections and to determine their in vitro antifungal activities against Candida spp. and Cryptococcus The activity of the plant extracts were tested against six neoformans. American Type Culture Collection (ATCC) yeast strains includ- ing; Candida albicans ATCC 90028, Candida glabrata ATCC 2. Materials and methods 90030, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, and Cryptococcus 2.1. Study areas neoformans ATCC 90112. Candida spp. and Cryptococcus neo- formans were subcultured onto Sabouraud dextrose agar (Oxoid The fieldwork was carried out in Tanga, Singida, Coast, and Ltd., Hampshire England) and incubated at 35 ◦C for 24 and 48 h, Morogoro regions located on northeast, central and eastern Tan- respectively. zania in February–March 2004. Earlier studies reported that Testing of the plant extracts for antifungal activity was done some plants used traditionally in these regions possess antifun- by the broth microdilution method, according to the guidelines gal activity (Khan and Nkunya, 1991; Khan et al., 2000). The of the Clinical and Laboratory Standard Institute (CLSI) (for- villages that were visited included Nyasa, (Mlalo ward, Lushoto merly, National Committee for Clinical and Laboratory Stan- district), on the western Usambara highlands with an altitude of dards) (NCCLS, 2002). All the testing was done in duplicate. 1341–1829 m. Mwaru, Nduamghangha and Mgori villages in Stock solutions of dried methanol extracts were prepared by dis- Singida rural district bordering Mgori forest reserve, approxi- solving 100 mg of each extract in 1 ml 100% dimethylsulfoxide mately 120 km from Singida town. Bwawani, and Mikese village (DMSO). The resulting 100 mg/ml extract solutions were diluted (Morogoro region), Chamakwesa, Chalinze, Mabwepande, and 12.5 times in milli-Q water to obtain a concentration of 8 mg/ml. Bagamoyo (Coast region). To avoid bacterial contamination each solution was sterilized 126 O.J.M. Hamza et al. / Journal of Ethnopharmacology 108 (2006) 124–132

Table 1 Herbal plants parts reported to be used by traditional healers for treatment of fungal infections and other diseases in Tanzania Family Species (voucher specimen no.) Local name Part useda Life form Preparation Other uses

Aloaceae Aloe lateritia Engl. (OH 10) Mapunisinyamviri WP Shrub Topical Snake poison Anacardiaceae Lannea stuhlmanii Engl. (OH 7) Muhungilo L Tree Topical b Sclerocariya birrea Sond (OH 8) Muongozi L, R Tree Topical Snake poison Annonaceae Annona senegalensis Purs. (OH Mnene kanda L, R Shrub Topical (L), Hernia, 11) Oral (R) asthma Apocynaceae Strophanthus eminii Asch and Muhunguti RB Shrub Oral b Pax (OH 25) Araceae Gonatopus boivinii Hook. F. (OH Kunzulu T Herb Topical Swellings 1) Balanitaceae Balanites aegyptiaca (L.) Del Mudughuyu RB Tree Topical b (OH 17) Bignoniaceae Kigelia africana L. (OH 49) Mungungu RB, F Tree Oral b Boraginaceae Cordia africana Lam (OH 9) Mgwengweni R Shrub Topical b Bursaraceae Commiphora pteleifolia Engl. Twini ndedemu R Shrub Topical b (OH 34) Campanulaceae Lobelia giberroa Neumeleg (OH Gongoa L Herb Topical b 35) Caesaepinaceae Cassia abbreviata Oliv. (OH 20) Mufafati R, SB Tree Oral b Cassia singuena Del (OH 12) Muhufia R Shrub Topical/Oral Hernia, skin rashes Caryophyllaceae Drymaria cordata (L.) A.Schult Ugurashishi WP Herb Topical b (OH 46) Celastraceae Elaeodendron buchananii (Loes) Muhorachwi SB Tree Oral Pneumonia (OH 19) Elaeodendron schlechteranum Mkandekande SB Tree Oral Herpes zoster (Loes) (OH 50) Compositae Tagetes minuta L. (OH 43) Mbangi L Climber Topical b Cruciferae Coronopus didymus (L.) (OH 47) Kissango WP Herb Oral Haemorrhoids Cucurbitaceae Cucumis aculeatus Cogn. (OH Ingang´ aa´ F Climber Topical b 32) Senecio deltoidea Less (OH 33) Ulenge WP Climber Oral b Zehneria scabra (L.F.) Sond (OH Foiza WP Climber Topical Dermatitis 42) Dennsstraediaceae Pteridium aquilinum (L.) Kuhn Shilu L Herb Topical b (OH 41) Ebenaceae Diospyros usambarensis F. (OH Muriorio R Shrub Topical b 26) Ericaceae Agauria salicifolia Oliv. (OH 45) Mwomboa L Tree Topical b Euphorbiaceae Acalypha fruticosa Forsk. (OH Siaiti L, R Shrub Topical (L), b 56) Oral (R) Croton scheffleri Pax (OH 24) Muhalange R Shrub Oral b Euphorbia tirucali L. (OH 57) Injokii L Tree Topical b Euphorbia heterophylla L. (OH Loo WP Herb Oral b 31) Jatropha multifida L. (OH 53) Maugwamwipoli L, S, R Shrub Topical b Spirostachys africana Sonder Ormotanga S Tree Topical b (OH 54) Gutteferae Hypericum roeperanum A. Rich Mwambaziwa L Shrub Topical b (OH 44) Labiatae Ocimum basilicum L. (OH 29) Irumbasi WP Herb Oral b Ocimum suave Oliv. (OH 13) Suameno L Herb Topical Roots used for hernia Loganiaceae Strychnos potatorum Gilg. (OH Mumpande L Tree Oral Pain, asthma, 21) lack of appetite Meliaceae Turraea holstii Gurk (OH 37) Muhenga L Shrub Oral Convulsions Khaya anthotheca C.Dc (OH 52) Mgolaminzi SB Tree Topical b O.J.M. Hamza et al. / Journal of Ethnopharmacology 108 (2006) 124–132 127

Table 1 (Continued) Family Species (voucher specimen no.) Local name Part useda Life form Preparation Other uses

Mimosaceae Albizia anthelmintica (A. Rich) Mfuleta R Tree Oral Worms, Brogn (OH 3) asthma Acacia robusta subsp. Mkame L Tree Topical Convulsions Usambarensis (Taub) Brenan (OH 38) Acacia nilotica (L.) Wild ex Del Kloriti S Shrub Topical b (OH 58) Moraceae Ficus sur. Benth (OH 51) Mkuyu SB Tree Oral/topical b Myrsinaceae Rapanea melanophloeus (L.) Mpaja L, SB Tree Oral b Mez (OH 5) Papilionaceae Indigofera rhynchocarpa Bak. Igangula R Shrub Topical Syphilis Var (OH 16) Erythrina abyssinica Lam (OH Mkalalwanhuwa R Tree Topical Worms 18) Polygonaceae Securidaca longipedunculata Musatu R Shrub Oral b Fres (OH 28) Plumbaginaceae Plymbago zeylannica L. (OH 36) Chambula R Herb Oral b Rhamnaceae Ziziphus pubercens Oliv. (OH 55) Indigrishi L Shrub Topical b Clausena anisata Oliv. (OH 6) Mjavikali L, SB, R Shrub Oral Convulsions, gonorrhea Zanthoxylum chalybeum L. (OH Mulungu RB Tree Topical/oral Cough 22) Rubiaceae Hymenidictyon parvifolium Brig Pekawake R Shrub Topical b (OH 2) Salvadoraceae Salvadora persica L. (OH 30) Mukunkuni R Tree Topical Gonorrhea Sterculiaceae Sterculia africana (Lour) Fiori Muhoza L Tree Oral Convulsions (OH 39) Sapotaceae Chrysophyllum bangweolense Mseweye RB Tree Topical b RE Fris (OH 15) Solanaceae Solanum incanum L. (OH 23) Mtula ndulele WP Herb Oral b Vitaceae Rhoicissus tridentata (Fresm) Iforiyo T Climber Oral b Placium (OH 27) Cissus petiolata Hook. F. (OH Mswilaswila R Climber Topical Edema of the 48) legs Cyphostemma hildebrandtii Damanyamwili L Herb Topical b (Gilg) Desc. (OH 14) a F, fruit; L, leaves; R, roots; RB, root bark; S, stem; SB, stem bark; T, tubor; WP, whole plant. b No other uses reported. by filtration through a 0.22 ␮m millipore-filter. Serial dilutions neoformans, after which readings were performed visually by were made in RPMI 1640 medium with l-glutamine but with- comparing with growth in control wells and the extract blank, out bicarbonate (GIBCO BRL, Life Technologies, Woerden,The which consisted of uninoculated plates (the score of 0–4 was Netherlands). Aliquots of 100 ␮l of the diluted extracts were dis- given) and spectrophotometrically with a microplate reader pensed into the wells of flat-bottomed 96-well microtitre plates (Anthos htIII; Anthos Labtec Instruments, Salzburg, Austria) (Costar, Corning, NY). at 405 nm. The optical densities (ODs) of the blanks were sub- The inocula were prepared spectrophotometrically (NCCLS, tracted from the ODs of the inoculated plates and the percentage 2002) to give a final concentration of 0.5–2.5 × 103 CFU/ml in growth for each well was calculated. MICs of the extracts were RPMI 1640 medium buffered to pH 7 with 0.165 M morpholine- defined as the lowest concentration of plant extract that visually propanesulfonic acid (MOPS). A constant volume (100 ␮l) of showed no growth and spectrophotometrically percentage the inoculum was added to each microdilution well containing growth was less than 5% in comparison to that of growth con- 100 ␮l of the serial dilution of the methanol extract to reach final trol (MIC0). Minimum fungicidal concentrations (MFCs) were concentrations of 4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016 and determined by subculturing each optically clear wells (no growth 0.008 mg/ml, respectively. The microtiter plates were incubated seen). Using a micropipette 50 ␮l was removed from clear wells at 35 ◦C for 48 h for Candida spp. and 72 h for Cryptococcus and inoculated onto SDA plates. The plates were then streaked neoformans. DMSO 4–0.008% was used as growth control and with a sterile loop and incubated for 24–48 h at 35 ◦C. The MFC amphotericin B was used as control against tested fungi. was defined as the concentration of plant extract at which the Minimum inhibitory concentrations (MICs) were deter- number of colony forming units was zero (Hawser and Islam, mined after 48 h for Candida species and 72 h for Cryptococcus 1999). 128 O.J.M. Hamza et al. / Journal of Ethnopharmacology 108 (2006) 124–132

3. Results and discussion stem topically applied) for 31 plant species; infusions taken orally/water decoctions for 20 species; or both topical and water 3.1. Ethnomedical information decoctions for 5 species. The commonest plant parts mentioned to be used were leaves (30%), followed by roots (29%) and the The results of the field study are presented in Table 1. A total least used were tubers and fruits (3% each). Analysis of the data of 65 plant parts belonging to 56 plant species and 38 families based on their habits showed that 21 species (37%) were trees, were reported to be used for treatment of oral candidiasis and 18 species (32%) were shrubs, 11 species (20%) were herbs fungal infections of the skin. The most represented family and 6 species (11%) were climbers. Adverse effects were only was Euphorbiaceae (six species), followed by Cucurbitaceae, reported for three species; Euphorbia tirucali L. juice from Mimosaceae and Vitaceae (three species each). Out of 56 plant leaves may cause blindness when in contact with eyes, and species, three species were mentioned by two or more tradi- bitter and sour taste for Rhoicissus tridentata (Fresm) Placium tional healers. These included Clausena anisata Oliv. (three and Cucumis aculeatus Cogn, respectively. Literature reports times), Albizia anthelmintica (A. Rich) Brogn, and Rapanea (Table 2) showed that 15 (27%) plant species have reports of melanophloeos (L.) Mez (two times each). The methods of similar ethnomedical uses or proven in vitro antifungal activity. preparation and administration were generally topical (plant Two plants Euphorbia heterophylla (Rocha e Silva, 1943) parts were ground into powder, mixed with water or sesame oil and Jatropha multifida were reported to be toxic (Levin et al., and used as mouthwash or ointment, and juice from leaves and 2000).

Table 2 Reports from the literature showing similar ethnomedical claims and proven laboratory results showing antifungal activity for some of the plants reported by Tanzanian traditional healers Plant name Literature reports of related ethnomedical uses and proven antifungal activity

Acacia nilotica Different extracts of the plant have been reported to have antifungal activity against yeasts and other fungi (Gupta and Bilgrami, 1970; Abd el Nabi et al., 1992; Sinha and Anjana, 1984; Srinivasan et al., 2001; Ahmad et al., 1998; Almagboul et al., 1988; Srivastava et al., 1984) Acalypha fruticosa Cold-water extract of fresh leaves is used for skin disease; patient is bathed with cold-water extract, or smoke from burning dry leaves is inhaled. It is used in combination with Zanthoxylum chalybeum and Suregada zanzibariensis (Hedberg et al., 1983a). Acetone, chloroform and hexane extracts of exhibited antifungal activity against Candida albicans (Alasbahi et al., 1999) Balanites aegyptiaca Ethanol extract exhibited antifungal activity against Penicillium crustosum, Saccharomyces cerevisiae (Taniguchi et al., 1978; Liu and Nakanishi, 1982), and the saponin fraction showed antifungal activity against , Aspergillus niger and Candida albicans (Saeed et al., 1995) Clausena anisata Decoction is used orally to treat mouth infections (Adesina and Adewunmi, 1985). A 50% aqueous ethanol extract exhibited antifungal activity against Aspergillus niger, Microsporum canis, and Trichophyton mentagrophytes (Aswal et al., 1984). Clausenol, carbazole alkaloid isolated from alcoholic extracts exhibited antifungal activity against Candida albicans (Chakraborty et al., 1995) Diospyros usambarensis Used as chewing stick (Khan et al., 2000) Drymaria cordata Plant juice of the leaves is used externally for skin diseases (Rao, 1981; Saklani and Jain, 1989) Erythrina abbyssinica Infusions of the bark three times a day per oral reported for treatment of candidiasis. (Hamill et al., 2000). Saccharomyces cerevisiae, Penicillium crustosum (Taniguchi et al., 1978; Kamat et al., 1981; Taniguchi and Kubo, 1993) Euphorbia heterophylla Reported to be toxic (Rocha e Silva, 1943). Hymenodictyon parvifolium A methanol extract exhibited antifungal activity against Candida albicans, Trichophyton mentagrophytes and Microsporium gypseum (Kariba, 2002). Jatropha multifida Reported to cause toxicity to two children (Levin et al., 2000) Ocimum basilicum Essential oil reported to have antifungal activity against Trichophyton mentagrophytes, Microsporium gypseum, Trichophyton rubrum (Dikshit and Husain, 1984), Yeast (Ela et al., 1996), Penicillium digitatum (Arora and Pandey, 1984) and Aspergillus niger (Meena and Sethi, 1994) Rhoicissus tridentata Methanol extract exhibited antifungal activity against Candida albicans and Saccharomyces cerevisiae (Lin et al., 1999) Salvadora persica It is used as toothbrush (Johns et al., 1996; Al-Said, 1993); dentrifice (Farooqi and Srivastava, 1968); chewing stick (Hattab, 1997); has anti-ulcer activity (Islam et al., 2000; Monforte et al., 2001). The plant exhibited weak anti yeast activity (Chhabra et al., 1991) Sclerocarya birrea Stem bark ethanol extracts exhibited strong activity against Candida albicans (Adoum et al., 1997) Solanum incanum Used for skin diseases and treatment of ringworm (Hedberg et al., 1983b; Sawhney et al., 1978); Methanol extract exhibited antifungal activity against Candida albicans (Sawhney et al., 1978; Al-Yahya et al., 1983) and Trichophyton mentagrophytes (Sawhney et al., 1978) Securidaca longipedunculata Root bark extracts exhibited antifungal activity against Saccharomyces cerevisiae (Taniguchi et al., 1978). Tagetes minuta Used for treatment of ringworm infections (Bii et al., 2000); oil from flowers and leaves exhibited antifungal activity against Candida albicans, Cryptococcus neoformans, T. metangrophytes and Microsporum gypseum (Bii et al., 2000); ethanol extracts of leaves exhibited activity against Candida albicans (Motsei and Lindsey, 2003) Zanthoxylum chalybeum Used for skin diseases. Leaves of Acalypha fruticosa, Zanthoxylum chalybeum and Suregada zanzibariensis are pounded together and juice is applied to affected area (Hedberg et al., 1983a; Chhabra et al., 1991); exhibited antifungal activity against Penicillium crustosum and Saccharomyces cerevisiae (Taniguchi et al., 1978) O.J.M. Hamza et al. / Journal of Ethnopharmacology 108 (2006) 124–132 129

Table 3 In vitro activity of plant extracts against selected yeast

Botanical name: species Plant C.ab, C.g, C.p, C.t, C.k, Cr.n, a c part MIC0/MFC MIC0/MFC MIC0/MFC MIC0/MFC MIC0/MFC MIC0/MFC Acacia robusta subsp. Usambarensis L −/− 1000/− 63/− 500/1000 31/− 4000/− (Taub) Brenan Acacia nilotica (L.) Wild ex Del. SB −/−−/− 31/− 63/− 1000/1000 4000/− Agauria salicifolia Oliv. L −/− 500/4000 1000/− 4000/− 2000/− 1000/− Clausena anisata Oliv. SB 1000/1000 4000/− 63/125 125/250 250/500 250/250 Cyphostemma hildebrandtii (Gilg) Desc L −/− 1000/− 1000/− 250/500 1000/− 500/− Drymaria cordata (L.) A. Schult WP −/−−/−−/−−/− 4000/4000 −/− Elaeodendron schlechteranum (Loes) SB −/−−/−−/−−/− 2000/− 250/− Elaeodendron buchannanii (Loes) SB −/−−/−−/− 250/− 63/− 31/− Euphorbia heterophylla L. WP −/−−/−−/− 4000/4000 −/− 4000/− Hymenidictyon parvifolium Brig. R −/−−/−−/−−/−−/− 125/− Jatropha multifida L. S −/−−/− 1000/− 250/250 −/− 1000/− Jatropha multifida L. R −/−−/−−/−−/−−/− 4000/− Khaya anthotheca C.Dc. SB −/−−/−−/−−/− 200/−−/− Kigelia africana L. F −/−−/−−/−−/−−/− 1000/− Pteridium aquilinum (L.) Kuhn L −/−−/−−/− 500/− 500/−−/− Rapanea melanophloeus (L.) Mez. SB −/−−/−−/−−/− 1000/− 500/− Rhoicissus tridentata (Fresm) Placium. T −/−−/−−/−−/− 125/−−/− Sclerocarya birrea Sond R 250/−−/− 125/− 63/− 63/− 250/− Senecio deltoidea Less WP −/−−/−−/− 250/500 −/−−/− Solanum incanum L. L −/−−/− 4000/− 4000/−−/−−/− Sterculia africana (Lour) Fiori. L −/−−/− 1000/− 63/500 250/− 1000/1000 Spirostachys africana Sonder S 2000/2000 1000/2000 −/−−/−−/− 1000/− Tagetes minuta L. L −/−−/−−/− 500/− 1000/− 500/− Turraea holstii Gurk L 500/−−/− 1000/− 63/500 500/− 125/− Zanthoxylum chalybeum L. RB −/−−/− 2000/− 2000/4000 2000/2000 2000/− Ziziphus pubercens Oliv. L −/−−/−−/−−/− 1000/− 4000/− Amphotericin B 0.25/0.25 0.5/0.5 0.25/0.25 0.5/0.5 0.5/0.5 0.25/0.25

MFC, minimum fungicidal concentration (␮g/ml); −,noin vitro activity at concentration equal or less than 4000 ␮g/ml. a F, fruits; L, leaves; R, root; RB, root bark; S, stem; SB, stem bark; T, tuber; WP, whole plant. b C.a, Candida albicans; C.g, Candida glabrata; C.p, Candida parapsilosis; C.t, Candida tropicalis; C.k, Candida krusei; Cr.n, Cryptococcus neoformans. c MIC0, concentration (␮g/ml) that visually showed no growth and percentage growth less than 5% spectrophotometrically.

3.2. Antifungal activity There is no validated criteria for the MIC end points for in vitro testing of plant extracts, however, Aligiannis et al. Among the 65 plant extracts tested 26 (40%) extracts, belong- (2001) proposed classification for plant materials, based on MIC ing to 25 (45%) plant species, completely inhibited the growth results as follows: strong inhibitors—MIC up to 0.5 mg/ml; of one or more of the tested fungi. Table 3 shows the MICs and moderate inhibitors—MIC between 0.6 and 1.5 mg/ml and weak MFCs of the active methanol plant extracts. The most suscep- inhibitors—MIC above 1.6 mg/ml. In our study we tested up to tible yeasts based on the high number of active plant extracts maximum concentration of 4 mg/ml. Based on the above classi- inhibiting its growth were Cryptococcus neoformans followed fication Clausena anisata Oliv. stem bark, Sclerocariya birrea by Candida krusei, Candida tropicalis and Candida parapsilo- Sond, Turraea holstii Gurk leaves, Sterculia africana (Lour) sis. Out of 26 active plant extracts, 21 (80%) showed activity Fiori, Acacia robusta subsp. Usambarensis (Taub) Brenan, against Cryptococcus neoformans, 17 (65%), 16 (61.5%), 11 Cyphosterma hildebrandti (Gilg) Desc Elaeodendron buchan- (42%) and 5 (19%) against Candida krusei, Candida tropi- nanii (Lows) leaves, Acacia nilotica L. Wild ex Del stem, calis, Candida parapsilosis and Candida glabrata, respectively. Agauria salicifolia, Jatropha multifida L., Pteridyum aquillinum Candida albicans was the least susceptible of all the yeasts. showed strong activity to one or more tested microorganisms. Only 4 (15%) of the plant extracts, including Clausena anisata This study shows that information obtained from traditional Oliv., Sclerocariya birrea Sond, Turraea holstii Gurk, and Spiro- healers can lead to discovery of therapeutically useful agents. tachys africana Sonder inhibited its growth. The extracts that From the 56 plant species which were reported by the healers exhibited the broadest spectra of antifungal activity (activity only 2 (3.6%) plants, Euphorbia heterophylla (Rocha e Silva, against at least four yeasts) were Clausena anisata Oliv., Agau- 1943) and Jatropha multifida (Levin et al., 2000) had previous ria salicifolia Oliv., Sclerocariya birrea Sond, Acacia robusta literature reports of toxicity, while 14 plant species had previ- subsp. Usambarensis (Taub) Brenan, Cyphosterma hildebrandtii ous reports of confirmed in vitro antifungal activity. Among the (Gilg) Desc, Turraea holstii Gurk, Sterculia africana (Lour) 14 plant species previously reported to have in vitro antifungal Fiori, Zanthoxylum chalybeum L. and Acacia nilotica (L.) Wild activity the results were confirmed in the present study for 8 ex Del. species; Acacia nilotica, Hymenodictyon parvifolium, Clausena 130 O.J.M. Hamza et al. / Journal of Ethnopharmacology 108 (2006) 124–132 anisata, Rhoicissus tridentate, Solanum incanum, Sclerocariya activity. These results corroborate the importance of ethnob- birrea, Tagetes minuta and Zanthoxylum chalybeum (Gupta and otanical surveys for screening plants as a potential source for Bilgrami, 1970; Sawhney et al., 1978; Chhabra et al., 1982, bioactive compounds. Hence these could result in discovery of 1991; Aswal et al., 1984; Sinha and Anjana, 1984; Srivastava novel antifungal agents. Further bio-guided fractionation will et al., 1984; Almagboul et al., 1988; Abd el Nabi et al., 1992; be conducted for roots of Sclerocarya birrea Sond, leaves of Chakraborty et al., 1995; Ahmad et al., 1998; Srinivasan et al., Acacia robusta subsp. Usambarensis (Taub) Brenan, Turraea 2001; Motsei and Lindsey, 2003). The antifungal activity for holstii Gurk, Agauria salicifolia, Sterculia africana (Lour) Fiori, the above plants were previously determined by other methods Cyphosterma hildebrandtii (Gilg), Elaeodendron buchannanii except for Tagetes minuta (Motsei and Lindsey, 2003), which (Lows) and stem of Spirostachys africana Sonder to identify the similar to our study used a broth microdilution method (NCCLS, active compounds. 2002). The detection of antifungal activity in nearly 50% of the Acknowledgements plant species gives the claims by traditional healers a signifi- cantly high credibility. However, 31 plant species (39 extracts) The authors wish to thank all interviewed traditional healers did not show activity against any of the test microorganisms. for their help and willingness to share knowledge on traditional In contrast to previous reports several plant extracts did not medicine. We also thank the NAPRALERT database of the Uni- exhibit in vitro antifungal activity including Salvadora persica versity of Illinois, at Chicago, for allowing us access to literature. L., Securidaca longipedunculata, Acalypha fruticosa, Balan- This study was funded by a grant from WOTRO and WHO ites aegyptiaca, Erythrina abbyssinica and Ocimum basilicum Collaborating Centre for Oral Health Care Planning and Future (Taniguchi et al., 1978; Taniguchi and Kubo, 1993; Al-Bagieh Scenarios, Radboud University Nijmegen Medical Center, The et al., 1994; Meena and Sethi, 1994; Saeed et al., 1995; Ela Netherlands. et al., 1996; Alasbahi et al., 1999). 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