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Open Access Highly Accessed Research article

Screening of Tanzanian medicinal plants for anti-Candida activity

Deborah KB Runyoro1, Mecky IN Matee2, Olipa D Ngassapa1*, Cosam C Joseph3 and Zakaria H Mbwambo4

Author Affiliations

1 Department of Pharmacognosy, School of Pharmacy, Muhimbili University College of Health Sciences P.O Box 65013, Dar Es Salaam, Tanzania

2 Department of Microbiology and Immunology, School of Medicine, Muhimbili University College of Health Sciences, P. O Box 65001, Dar Es Salaam, Tanzania

3 Department of Chemistry, University of Dar Es Salaam, P.O Box 35065, Dar Es Salaam, Tanzania

4 Institute of Traditional Medicine, Muhimbili University College of Health Sciences, P. O Box 65001, Dar Es Salaam, Tanzania

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BMC Complementary and Alternative Medicine 2006, 6:11  doi:10.1186/1472-6882-6-11


The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1472-6882/6/11


Received:3 October 2005
Accepted:30 March 2006
Published:30 March 2006

© 2006 Runyoro et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Candida albicans has become resistant to the already limited, toxic and expensive anti-Candida agents available in the market. These factors necessitate the search for new anti-fungal agents.

Methods

Sixty-three plant extracts, from 56 Tanzanian plant species obtained through the literature and interviews with traditional healers, were evaluated for anti-Candida activity. Aqueous methanolic extracts were screened for anti-Candida activity by bioautography agar overlay method, using a standard strain of Candida albicans (ATCC 90028).

Results

Twenty- seven (48%) out of the 56 plants were found to be active. Extracts of the root barks of Albizia anthelmintica and Balanites aegyptiaca, and roots of Plectranthus barbatus showed strong activity.

Conclusion

The extracts that showed strong anti-Candida activity are worth of further investigation in order to isolate and identify the active compounds.

Background

Numerous studies have shown an association between increased prevalence of HIV infection and the occurrence of opportunistic fungal infections [1,2]. Among the different HIV-associated fungal infections, oral mucosal lesions caused by Candida species are by far the most frequent manifestation [3]. Up to 90% of HIV-infected individuals suffer at least one episode during the course of their disease [3], and the incidence and severity of the episodes increase with decreasing immunity, especially when CD4+ cell counts fall to levels below 200 cells/mm3 [4]. Candida albicansis the most causative agent, accounting for more than 90% of cases [5]. However, other Candida species such as C. glabrata, C. parapsilosis, C. tropicalis, and C. kruseimay also cause symptomatic oral candidiasis in HIV-positive individuals [6]. Other fungal infections seen in HIV-infected individuals include, cryptococcosis due to Cryptococcus neoformans and aspergillosis due to Aspergillus flavus, A. fumigatus and A. niger [7,8].

In sub Saharan Africa, where 70% of the world cases of HIV/AIDS are found [9], oral candidiasis, including oropharyngeal and oesophageal candidiasis, are very common, causing significant morbidity among patients. Oral candidiasis is usually treated by topical antifungal agents, which include nystatin, miconazole, fluconazole, itraconazole and amphotericin B. However, the management of Candida infections faces a number of problems including; limited number of effective antifungal agents [10], toxicity of the available antifungal agents [10-12], resistance of Candida to commonly used antifungals [13-16], relapse of Candida infections [17], and the high cost of antifungal agents [10-12]. When relapses occur, the infections tend to be increasingly refractory to treatment. The difficulties associated with the management of Candida infections necessitate the discovery of new antifungal agents, in order to widen the spectrum of activity against Candida and combat strains expressing resistance to the available antifungal agents.

Plant-derived natural products may offer potential lead to new compounds, which could act on these fungi [18]. This paper reports on the screening of 63 aqueous methanol plant extracts for activity against Candida albicans. The extracts were obtained from 56 plant species, belonging to 29 plant families, collected from four regions of Tanzania including Coast, Dar es Salaam, Morogoro and Tanga. Screening was achieved by using the bioautography agar overlay method, which is very convenient, simple and efficient [19]. The method can also be employed in the target-directed isolation of the active constituents.

Methods

Plant collection

Plants were collected from four regions, which are in the eastern part of Tanzania, namely, Coast, Dar es Salaam, Morogoro and Tanga (Figure 1). The plants included those reported in the literature [20,21] to be active against Candida albicans, but which have not been investigated further to identify the active compounds. The second category comprised of plants obtained through interviews with traditional healers.

thumbnailFigure 1. Map of Tanzania showing the regions where plants were collected.

Interviews with traditional healers

Traditional healers in the four regions of Tanzania (Figure 1) were interviewed on plants they used to treat Candida infections. Symptoms of the various forms of Candida infections associated with HIV/AIDS were described to the traditional healers so as to enable them give the appropriate plants they used in the management of these conditions. The symptoms which have been described in the literature [22] include; oral thrush, mouth ulcers and lesions of epithelial cells of the lips, erythematous lesions on the dorsum of the tongue and angular cheilitis. Those for Candida oesophagitis were painful swallowing, a feeling of obstruction on swallowing, substernal chest pain and discrete ulceration of the oesophagus [23]. The symptom for vaginal candidiasis was a cuddle milk discharge [24].

Prior to the interview each traditional healer was asked to sign an agreement/consent form if she/he agreed with the terms given in the forms. The form, in short, explained the importance of the information they were providing and the type of research that was to be done on the plants they provided. They were also informed that the results and any profitable outcome would be communicated to them. This was done in order to safeguard the interests of both the parties.

Identification of the plants

Preliminary identification of the plants was done in the field by a botanist. Herbarium specimens were prepared and photographs were taken to aid in the confirmation of the identity of the plants. Voucher specimens were deposited in the Herbarium of the Botany Department, University of Dar es Salaam, Tanzania, where identity of the plants was confirmed by comparison with available voucher specimens.

Processing of the plant materials

The plant materials were sorted, washed and chopped into smaller pieces, where necessary, before drying. The materials were dried outdoors; however, leaves were dried in the sun for one day, then in the shade. The dried plant materials were ground to various degrees of fineness depending on their botanical structures. This was done using a Fitz mill Type 6 (Manesty machines Ltd. Liverpool, England).

Screening of the extracts for anti-Candida activity

Preparation of extracts and standards

Ground plant materials (100 to 200 g) were macerated with 80% methanol at room temperature (28°C). In each case the amount of solvent used was just enough to cover the plant materials. Maceration was carried out for two days and the procedure was repeated three times. Extracts were decanted then filtered using filter funnels fitted with Whatman No 1 filter papers. The extracts were pooled and concentrated using a Büchi rotary evaporator (Büchi Labortechnik, Flawil, Switzerland) set at 40–50°C, followed by freeze-drying using the Edwards freeze drier (Edwards High Vacuum International Crawley, Sussex, England). For each extract 1 g was weighed and dissolved in 5 ml of methanol to produce a solution with a concentration of 200 μg/μl.

Amphotericin B (Calbiochem, USA and Canada) was used as the standard anti-fungal agent.

Inoculum

Sabouraud dextrose agar (SDA; Biotec Laboratories Ltd. UK) was used to prepare the culture medium according to the manufacturer's directions. Candida albicans ATCC 90028 (provided by Prof. Paul Verweij, Department of Medical Microbiology, University Medical Center, St. Radboud Hospital, Nijmegen, The Nertherlands) was aseptically inoculated on petridishes containing autoclaved, cooled and settled medium. The petridishes were incubated at 31°C for 48 h to give white round colonies against a yellowish background. These were aseptically sub-cultured on SDA slants.

Candida albicans colonies from SDA slants were suspended in sterilised 0.9% sodium chloride solution (normal saline), which was compared with a 5 McFarland solution. The microbial suspension (1 ml) in normal saline was added to 74 ml of sterile medium, kept at 45°C, to give concentration of 2 × 107 cells/ml.

Bioautography agar overlay

Preamble

The bioautography agar overlay method is an improved version of a disc diffusion method. Unlike the ordinary diffusion method in which the drug to be evaluated is adsorbed on a paper disc and placed onto the inoculated petridish, in the bioautography agar overlay method the drug to be evaluated is adsorbed onto the Thin Layer Chromatography (TLC) plate and the inoculum is laid onto the plate as a very thin layer of about 1 mm in thickness. In both cases the drug diffuses from the adsorbent into the inoculum where it exerts its effects. The bioautography agar overlay method is advantageous in that, firstly it uses very little amount of sample when compared to the normal disc diffusion method and hence it can be used for bioassay- guided isolation of compounds, and secondly, since the crude extract is resolved into its different components it simplifies the process of identification and isolation of the active compounds [19].

Bioautography agar overlay process

Five microliters (5 μl) of the solution of each extract was applied on a glass backed Silica gel G60 F254 TLC plates (20 × 20 cm, 250 μm thickness, Merck, Darmstadt, Germany) and reference plate was similarly prepared. Both plates were developed to a distance of about 10 cm in the same tank using the pre-determined mobile phases. The mobile phase was removed from the plate by drying with a stream of cool air from a heating gun. The reference plate was observed in UV light to see if the separated spots were UV active after which it was sprayed with vanillin sulphuric acid followed by heating using a heating gun.

The positive control, amphotericin B (0.002 μg/μl) equivalent to 0.01 μg per spot was applied on the test plate in triplicate followed by removal of solvent using a stream of cold air. About 22 ml of the freshly prepared inoculum described above was uniformly spread using a sterile Pasteur pipette. After solidification the plates were placed in a polythene container lined at the bottom, with moist cotton wool. The plates were incubated at 31°C for 18 to 24 h, after which they were removed and sprayed with an aqueous solution (2.5 mg/ml) of thiazolyl blue (3-(4,5 dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide) (MTT) (BDH, Poole England). They were incubated for a further 4 h, after which the inhibition zones appeared colourless against a purple background. Spots showing any inhibition were noted and their hRf values and inhibition zones measured. The test was performed in duplicate.

Results and discussion

The plants studied gave aqueous methanolic extracts with yields ranging from 2 to 26.5% (Table 1). Twenty-eight out of the sixty-three extracts, belonging to 27 plant species and constituting 48% of all the plants collected, could be resolved into TLC spots exhibiting activity against C. albicans (Table 1). The number of active resolved spots for each extract ranged from 1 to 4. Table 1 shows the TLC bioautography results for the various extracts developed using various solvent systems and Figures 2, 3, 4 show the bioautograms of some of the most active extracts.

Table 1. TLC Bioautography results of various extracts.

thumbnailFigure 2. Chromatograms (i) and bioautograms (ii) for Holarrhena febrifuga leaves (a), Balanites aegyptiaca root bark (b) and Albizia anthelmintica root bark (c). Mobile phase: Chloroform:Methanol:Ethylacetate:water 28:30:35:5; Amphotericin B (A)

thumbnailFigure 3. Chromatograms (i) and bioautograms (ii) for Combretum zeyheri leaves (d), Plectranthus barbatus roots (e) and Vitex fischeri root bark (f) mobile phase: Chloroform: Methanol 4:1 3; Amphotericin B (A).

thumbnailFigure 4. Chromatograms (i) and bioautograms (ii) for Margaritaria discoidea roots (g), Cajanus cajan leaves (h) and Zanha africana roots (j) mobile phase: Chloroform: Methanol 4:1 3; Amphotericin B (A).

Bioautography agar overlay method is considered as one of the most efficient methods for the detection of antimicrobial compounds [19]. It involves the transfer of the active compounds from the stationary phase into the agar layer through a diffusion process. From Table 1 it can be seen that a total of 27 plants, screened by using this method, exhibited anti-Candida activity. However, only 8 out of the 18 plants previously reported to be active by Sawhney et al. [20], were found to be active in this bioassay. Ziziphus abyssinica and Asparagus africanus which were collected as substitutes for Ziziphus pubescens and Asparagus falcatus respectively (reported active by Sawhney et al. [20] were also found to be active. This proves the fact that allied species may contain similar or related active compounds [25]. Eriosema psoraleoides, a plant, reported by Khan et al. [21] to be active was found to be inactive in this bioassay.

No obvious reasons can be given as to why some of the plants reported previously to be active were found inactive in this study. The plant parts used were more or less similar, with very few peculiar exceptions, such as Xerroderris stuhlmanii, which Sawhney et al. [20] reported that the whole plant was active. However this plant is a tree, and, in this study only the stem barks was evaluated. The bioassay method employed in this study is quite different from the one used by Sawhney et al. [20]. In the present study the bioautography method was used on partially resolved extracts, whereas Sawhney et al. [20] used the agar dilution method for unresolved crude extracts. Also it was not possible to compare concentrations because in the bioautography method absolute amounts of extracts were chromatographed while in the agar dilution method concentrations of the extracts were used. Such differences could have contributed to the observed differences in the bioassay results.

Eighteen (49%) out of 37 plants, obtained through interviewing traditional healers, were found active in this bioassay. Ten of these have also been reported to be active in previous studies; these include; Ziziphus mucronata [26], Cajanus cajan [27], Acacia nilotica [28], Harrisonia abyssinica and Zanthoxylum chalybeum [20]. Others were Securidaca longepedunculata, [29,30]Salvadora persica [31], Balanites aegyptiaca [32], Zanha africana [33], Combretum molle [21]. Harrisonia abyssinica and Z. chalybeum were selected through the literature and were also mentioned by the interviewed traditional healers. Physalis peruviana, was reported previously as inactive [34], but was found to be active in this study. The remaining eight active plants, including Margaritaria discoidea, Suregada zanzibariensis, Plectranthus barbatus, Albizia anthelmintica, Securidaca longepedunculata, Catunaregum nilotica, Pseudovigna argentea, Agathisanthemum bojeri and Chassalia umbraticola, were screened for anti- Candida activity for the first time. Other plants obtained from the traditional healers, which were reported active previously were found to be inactive in this study and they include Ozoroa insignis [35], Carica papaya [26] and Dichrostachys cinerea [28]. The discrepancy in the results could be due to a number of factors, including differences in the plant parts used, extraction solvents, time of collection of plant materials, geographical location of the plants and bioassay methods.

Eleven plants were resolved into several spots showing large inhibition zones (≥ 10 mm). The plants included Agathisanthemum bojeri, Albizia anthelmintica, Balanites aegyptiaca, Cajanus cajan, Cassia auriculata, Harrisonia abyssinica,Holarrhena febrifuga, Plectranthus barbatus, Salvadora persica, Sida serratifolia and Suregada zanzibariensis. Eight among these were obtained through interviewing traditional healers including A. anthelmintica, B. aegyptiaca and P. barbatus the most active of the screened plants. This observation shows that ethnomedical information is important in drug discovery.

Albizia anthelmintica and Balanites aegyptiaca have been reported to contain several triterpene saponins and steroidal saponins, respectively [36-38]. Also the extracts of these plants formed strong foam, with water indicating that they contain saponins. The presence of saponins in these plants is also supported by the chromatogram obtained by visualizing with vanillin-sulphuric acid (Fig 2) in which the probable active compounds gave purple or blue colour with this reagent, a normal positive test for triterpenoidal and steroidal saponins respectively [39]. The roots of B. aegyptiaca have, also been reported to contain flavonoids [40]. The root bark of A. anthelmintica was reported to contain a histamine alkaloid and proteins [41]. A. anthelmintica was screened for anti-Candida activity for the first time and, although B. aegyptiaca was reported to be active in the previous study [32] no identification of compounds responsible for the activity was done.

Plectranthus, barbatus another promising plant has been reported to contain diterpenes [42] and an essential oil containing α-pinene, myrcene and caryophylene as the major constituents [43]. The plant has never been evaluated for anti-Candida activity before, and thus, it has been shown for the first time in this study to have anti-Candida activity.

Conclusion

Almost 50% of the collected plants were found to be active against Candida albicans (ATCC 90028) with varied degrees of activity. About half (49%) of the plants, obtained by interviewing traditional healers, demonstrated activity against C. albicans. This study gives an indication of the efficacy of the plants obtained from the traditional healers. The results from this study form a basis for further studies on the active plants so as to isolate the compounds responsible for the observed anti-Candida activity.

Abbreviations

1. ATCC: American Type Culture Collection

2. BDH: British Drug House

3. CD4: Cluster Designation 4

4. HIV/AIDS: Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome

5. ITM: Institute of Traditional Medicine

6. MTT: Methylthiazolyltetrazolium Chloride

7. MUCHS: Muhimbili University College of Health Sciences

8. SDA: Sabouraud dextrose agar

9. TLC: Thin layer Chromatography

10. UDSM: University of Dar es Salaam

Authors' contributions

Deborah KB Runyoro : Conception and designing of the research, acquisition of data, drafting the manuscript and revising it critically for important intellectual content.

Mecky IN. Matee: Conception and designing of the research and revising the manuscript critically for important intellectual content and gave final approval for its publication.

Olipa D Ngassapa: Conception and designing of the research and revising it critically for important intellectual content and gave final approval for its publication.

Cosam C Joseph: Conception and designing of the research and revising it critically for important intellectual content.

Zakaria H Mbwambo: Acquisition of data and revising the manuscript critically for important intellectual content.

Acknowledgements

This work has been financially supported by, Sida/SAREC through the Muhimbili, University College of Health Sciences (MUCHS). The authors would like to thank the following; botanists of the Department of Botany, University of Dar es Salaam and those of the Institute of Traditional Medicine (ITM), MUCHS, technicians of the School of Pharmacy, ITM and Department of Microbiology and Immunology, School of Medicine all of MUCHS, who assisted in this study in their various capacities. Authors are also grateful to Prof. Paul Verweij of the Department of Medical Microbiology, University Medical Center, St. Radboud, Nijmegen, Holland for providing the standard strains of Candida albicans ATCC 90028 which was used in the study. They are also indebted to the Director and researchers at ITM, MUCHS, where part of this study was done. They also thank all the traditional healers who participated in this study.

References

  1. Ghannoum MA: Future of antimycotic therapy.

    Dermatologic Therapy 1997, 3:104-11. OpenURL

  2. Vazquez JA: Therapeutic options for the management of oropharyngeal and esophageal candidiasis in HIV/AIDS patients.

    HIV Clin Trials 2000, 1:47-59. PubMed Abstract | Publisher Full Text OpenURL

  3. Vazquez JA: Options for the management of mucosal candidiasis in patients with AIDS and HIV infection.

    Pharmacotherapy 1999, 19:76-87. PubMed Abstract | Publisher Full Text OpenURL

  4. Anteyi KO, Thacher TD, Yohanna S, Idoko JI: Oral manifestations of HIV-AIDS in Nigerian patients.

    Int J STD AIDS 2003, 14:395-8. PubMed Abstract | Publisher Full Text OpenURL

  5. Edwards JE Jr: Candida species. In Mandell, Douglas and Bennett's principles and practice of infectious diseases. Volume 2. 4th edition. Edited by Mandell GL, Bennett JE, Dolin R. New York: Churchill Livingstone; 1995::2289-2301. OpenURL

  6. Krcmery V, Barnes AJ: Non-albicans Candida species causing fungaemia: Pathogenicity and antifungal resistance.

    J Hosp Infect 2002, 50:243-60. PubMed Abstract | Publisher Full Text OpenURL

  7. Musial CE, Cockenii FR, Roberts GD: Fungal infections of the immunocompromised host. Clinical laboratory aspects.

    Clin Microbiol Rev 1988, 1:349-364. PubMed Abstract | PubMed Central Full Text OpenURL

  8. Marty F, Mylonakis E: Antifungal use in HIV infection.

    Expert Opin Pharmacother 2002, 3:91-102. PubMed Abstract | Publisher Full Text OpenURL

  9. UNAIDS: Report on the Global AIDS epidemic.

    UNAIDS 2004. OpenURL

  10. Mehta DK, Martin J, Jordan B, Macfarlane CR, Hashimi FT, Kouimtzi M, Ryan RSM, Shing T, Wagle SMS, Gallagher GP, (eds): British National Formulary. 43rd edition. London, Pharmaceutical Press; 2002:294-298. OpenURL

  11. Feldmesser M: New and emerging antifungal agents: Impact on respiratory infections.

    Am J Respir Med 2003, 2:371-83. PubMed Abstract OpenURL

  12. Wong-Beringer A, Kriengkauykiat J: Systemic antifungal therapy: New options, new challenges.

    Pharmacotherapy 2003, 23:1441-62. PubMed Abstract | Publisher Full Text OpenURL

  13. Perea S, López-Ribot JL, Kirkpatrick WR, McAtee RK, Santillán RA, Martínez M, Calabrese D, Sanglard D, Patterson TF: Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains, displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients.

    Antimicrob Agents Chemother 2001, 45:2676-2684. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  14. Klepser ME: Antifungal resistance among Candida species.

    Pharmacotherapy 2001, 21:124S-132S. PubMed Abstract | Publisher Full Text OpenURL

  15. Pelletier R, Loranger L, Marcote H, De Carolis E: Variconazole and fluconazole susceptibility of Candida isolates.

    J Med Microbiol 2002, 51:479-83. PubMed Abstract | Publisher Full Text OpenURL

  16. Khan ZU, Chandy R, Metwali KE: Candida albicans strain carriage in patients and nursing staff of an intensive care unit: A study of morphotypes and resistotypes.

    Mycoses 2003, 46:476-86. Publisher Full Text OpenURL

  17. Debruyne D: Clinical pharmacokinetics of fluconazole in superficial and systemic mycoses.

    Clin Pharmacokinet 1997, 33:52-77. PubMed Abstract OpenURL

  18. Lachoria L, Jain PC, Agrawal SC: Activity of some plant extracts against dermatophytes.

    Hindustan Antibiot Bull 1999, 41:17-21. PubMed Abstract OpenURL

  19. Rahalison L, Hamburger M, Hostettmann K, Monod M, Frenk E: A bioautographic agar overlay method for the detection of anti-fungal compounds from higher plants.

    Phytochem Anal 1991, 2:199-203. OpenURL

  20. Sawhney AN, Khan MR, Ndaalio G, Nkunya MHH, Wever SH: Studies on the rationale of African Traditional medicine. Part III. Preliminary screening of medicinal plants for antifungal activity.

    Pak J Sci Ind Res 1978, 21:193-196. OpenURL

  21. Khan MN, Ngassapa O, Matee MIN: Antimicrobial activity of Tanzanian chewing sticks against oral pathogenic microbes.

    Pharmaceutical Biol 2000, 38:235-240. OpenURL

  22. Laskaris G, Hadjivassiliou M, Stratigos J: Oral signs and symptoms in 160 Greek HIV-infected patients.

    J Oral Path Med 1992, 21:120-3. OpenURL

  23. Connolly GM, Hawkins D, Harcourt-Webster JN, Parsons PA, Husain OA, Gazzard BG: Oesophageal symptoms, their causes, treatment, and prognosis in patients with the acquired immunodeficiency syndrome.

    BMJ 1989, 30:1033-1039. OpenURL

  24. Namkinga LA, Matee MIN, Kivaisi AK, Moshiro C: Prevalence and risk factors for vaginal candidiasis among women seeking primary care for genital infections in Dar es Salaam, Tanzania.

    E Afr Med J 2005, 82:138-143. OpenURL

  25. Evans WC: Trease and Evans' Pharmacognosy. Thirteenth edition. Bailliere Tindall, London, Philadelphia, Toronto, Marrickville, Tokyo; 1989. OpenURL

  26. Gundidza M: Screening of extracts from Zimbabwean higher plants II: Antifungal properties.

    Fitoterapia 1986, 57:111-113. OpenURL

  27. Boily Y, Van Puyvelde L: Screening of medicinal plants of Rwanda (Central Africa) for antimicrobial activity.

    J Ethnopharmacol 1986, 16:1-13. PubMed Abstract | Publisher Full Text OpenURL

  28. Almagboul AZ, Bashir AK, Karim A, Salih M, Farouk A, Khalid SA: Antimicrobial activity of certain Sudanese plants used in folkloric medicine. Screening for antifungal activity (VI).

    Fitoterapia 1988, 59:393-396. OpenURL

  29. Taniguchi M, Chapya A, Kubo I, Nakanishi K: Screening of East African plants for antimicrobial activity.

    Chem Pharm Bull 1978, 26:2910-2913. OpenURL

  30. Desta B: Ethiopian traditional herbal drugs. Part II: Antimicrobial activity of 63 medicinal plants.

    J of Ethnopharmacol 1993, 39:129-139. Publisher Full Text OpenURL

  31. Al-Bagieh NH, Idowu A, Salako NO: Effect of aqueous extract of miswak on the in vitro growth of Candida albicans.

    Microbios 1994, 80:107-113. PubMed Abstract OpenURL

  32. Saeed A, Ibrahim N, Bashand YS, El-Gengaihi S: Saponins of Balanites aegyptiaca Del fruits and biological evaluation.

    Bull Fac Pharm Cairo Univ 1995, 33:105-109. OpenURL

  33. Fabry W, Okemo P, Ansorg R: Fungistatic and fungicidal activity of East African medicinal plants.

    Mycoses 1996, 39:67-70. PubMed Abstract OpenURL

  34. Dhawan BN, Patnaik GK, Rastogi RP, Singh KK, Tandon JS: Screening of Indian plants for biological activity. VI.

    Indian J Exp Biol 1977, 15:208-219. PubMed Abstract OpenURL

  35. Abreu PM, Martins ES, Kayser O, Bindseil KU, Siems K, Seemann A, Frevert J: Antimicrobial, antitumor and antileishmania screening of medicinal plants from Guinea-Bissau.

    Phytomedicine 1999, 6:187-195. PubMed Abstract OpenURL

  36. Carpani G, Orsini F, Sisti M, Verrota L: Soponins from Albizia anthelmintica.

    Phytochemistry 1989, 28:863-866. Publisher Full Text OpenURL

  37. Tschesche R, Kammerer FJ: On triterpenes XXVII. The structure of musennin and deglucomusennin. (Über Triterpene XXVII Die strukturvon musennin und desglucomusennin).

    Justus Liebigs Ann Chem 1969, 724:183-193. OpenURL

  38. Farid H, Haslinger E, Kunert O, Wegner C, Hamburger M: New steroidal glycosides from Balanites aegyptiaca.

    Helv Chim Acta 2002, 85:1019-1026. Publisher Full Text OpenURL

  39. Wagner H, Bladt S, Zygainski EM: Plant Drug Analysis. A thin Layer Chromatography Atlas. Springer Verlag, Berlin, Heidelberg, New York, Tokyo; 1984. OpenURL

  40. Gaudin O, Vacherat R: Rotenone and the ichthyotoxic power in some plants of the French Sudan.

    Bull Sci Pharmacol 1938, 45:385-394. OpenURL

  41. Mazzanti G, Erspamer GF, Mugne Y, Piccinelli D: Occurrence of Histamine and related imidazole compounds in extracts from root and trunk bark of Albizia adianthifolia, Albizia anthelmintica and Albizia julibrissin.

    Fitoterapia 1983, 54:275-280. OpenURL

  42. Abdel-Mogib M, Albar HA, Battrjee SM: Review. Chemistry of the Genus Plectranthus.

    Molecules 2002, 7:271-301. OpenURL

  43. Camara CC, Nascimento NR, Macedo-Filho CL, Almeida FB, Fonteles MC: Antispasmodic effect of the essential oil of Plectranthus barbatus and some major constituents on the guinea pig ileum.

    Planta Medica 2003, 69:1080-1085. PubMed Abstract | Publisher Full Text OpenURL

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