|Year : 2009 | Volume
| Issue : 6 | Page : 387-391
Antifungal activity of Tabernaemontana stapfiana Britten (Apocynaceae) organic extracts
EK Ruttoh1, C Bii2, PK Tarus3, A Machocho4, LK Karimi5, P Okemo6
1 Kenya National Public Health Laboratory Services, P.O. Box 20176, 00100 GPO, Nairobi, Kenya
2 Center for Microbiology, Research Kenya Medical Research Institute, P.O. Box 2632, 00202, Nairobi, Kenya
3 Department of Physical Science, Masinde Muliro University of Science and Technology, P.O. Box 190, 50100 Kakamega, Kenya
4 Chemistry Department, School of Pure and Applied Sciences, Kenyatta University, P.O. Box 43844, 00100 GPO, Nairobi, Kenya
5 Department of Pharmacy and Complementary Medicine, School of Pure and Applied Sciences, Kenyatta University, P.O. Box 43844, 00100 GPO, Nairobi, Kenya
6 Department of Plant and Microbial Sciences, School of Pure and Applied Sciences, Kenyatta University, P.O. Box 43844, 00100 GPO, Nairobi, Kenya
|Date of Web Publication||2-Jan-2010|
Department of Plant and Microbial Sciences, School of Pure and Applied Sciences, Kenyatta University, P.O. Box 43844, 00100 GPO, Nairobi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The methanol and successive (hexane, dichlroromethane, ethyl acetate and methanol) extracts of leaves, fruits, root and stem barks of Tabernaemontana stapfiana Britten were investigated for antifungal activity against four fungi; Candida albicans, Cryptococcus neoformans, Microsporum gypseum and Trichophyton mentagrophytes. The extracts of the different plant parts showed varied activities against the tested fungi with the stem and root barks showing significant antifungal activities. The minimum inhibitory concentrations (MICs) of methanol and sequential extracts ranged between 31.25 and 8000 μg/ml while the minimum fungicidal concentrations (MFCs) ranged between 125 and 8000 μg/ml. Among the test microorganisms, Candida albicans was the most susceptible with the lowest MIC of 31.25 μg/ml followed by Cryptococcus neoformans with an MIC of 62.25 μg/ml. These results show that it is possible to find antifungal compounds in the extracts of T. stapfiana.
Keywords: Tabernaemontana stapfiana Britten; Apocynaceae; Antifungal activity; Minimum inhibitory concentration; Minimum fungicidal concentration.
|How to cite this article:|
Ruttoh E K, Bii C, Tarus P K, Machocho A, Karimi L K, Okemo P. Antifungal activity of Tabernaemontana stapfiana Britten (Apocynaceae) organic extracts. Phcog Res 2009;1:387-91
|How to cite this URL:|
Ruttoh E K, Bii C, Tarus P K, Machocho A, Karimi L K, Okemo P. Antifungal activity of Tabernaemontana stapfiana Britten (Apocynaceae) organic extracts. Phcog Res [serial online] 2009 [cited 2018 Feb 21];1:387-91. Available from: http://www.phcogres.com/text.asp?2009/1/6/387/58022
| Introduction|| |
Emergence of HIV/AIDS has led to increasing incidences of fungal infections. Although Candida albicans infections remain the major species isolated from clinical samples in HIV patients, other infections such as those caused by Cryptococcus neoformans, Microsporum gypseum and Trichophyton menthagrophytes are on the rise  . Some of these fungal infections are showing resistance to common antifungals such as fluconazole and amphotericin B , . Others like griseofulvin and most of the azoles have adverse side effects such as gastrointestinal disturbances, hepatotoxicity and leucopeania  . It is therefore important to search for novel, more effective and less toxic agents for the management of fungal infections, especially the HIV/AIDS related opportunists.
Plants and their extractives have been used all over the world since antiquity in folk medicine  . This use has been supported by the isolation of active antifungal compounds from plants  . Medicinal plants are widely used in Kenya in controlling various ailments including fungal infections and Tabernaemontana stapfiana Britten (Apocinaceae) is one of the commonly used medicinal plants  . This species grows up to 21 m high and is usually found in disturbed forests  . The roots and stem barks are used by the Keiyo community in Rift Valley for management of abdominal problems, skin infections, sexually transmitted infections and upper respiratory tract infections. There has been no antifungal study on T. stapfiana. In this study, T. stapfiana methanol and sequential (hexane, dichloromethane, ethyl acetate and methanol) extracts were assayed for antifungal activity.
| Materials and Methods|| |
Plant samples were collected from Kaptagat Forest in Keiyo district about 40 Km East of Eldoret town, in Kenya. The samples were authenticated by Mr. Karimi, a taxonomist at Kenyatta University, Kenya. The leaves, fruit, root and stem barks from mature plants were collected in March 2006 and floral reference parts (Reference number ER/001/06) deposited at the Kenyatta University herbarium. The plant samples were dried in the shade, and ground into powdered material using a grinding mill (Christy and Norris Ltd, England). Powdered plant material were hermetically sealed in polythene bags and stored in a refrigerator at 4°C in the dark until the time of extraction.
Powdered plant parts leaves, stem bark, root bark and fruits', (100 g), were extracted using cold extraction method by soaking in methanol for 72 h in the dark in an Erlenmeyer flask, covered with aluminium foil with frequent swirling. The extracts were then filtered using Whatman filter paper No 1, and the filtrate evaporated under reduced pressure. The final extracts were stored in labeled screw-capped bottles in a deep freezer at -20° C.
Powdered root and stem barks (100 g) of T. stapfiana were extracted using cold extraction method by successively extracting three times with solvents of increasing polarity starting with hexane, dichloromethane, ethyl acetate and methanol for 48 h each, with occasional swirling to ensure thorough extraction. The filtrates were concentrated at a temperature of 45°C under reduced pressure to obtain gummy extracts.
Bioassay culture strains
The microorganisms used in the bioassay tests were obtained from Kenya Medical Research Institute (KEMRI). These included reference strains and local clinical isolates. The microorganisms that were used included: two yeasts; Candida albicans (ATCC 90028) and Cryptococcus neoformans (ATCC 66031) and two clinical isolates of dermatophytes; Trichophyton mentagrophytes (KMR 100) and Microsporum gypseum (KMR 101). The test strains were maintained on Potato Dextrose Agar (PDA) (Becton Dickinson, BBL, Heidelberg, Germany) at 4°C. These fungi were subjected to two successive transfers in broth and tested for purity and viability using biochemical and morphological characteristics before use as described by Elgayyar et al.,  . The 1.0 McFarland standard was prepared by mixing 0.1 ml of 1.175% barium chloride dihydrate (BaCl2·2H2O), with 9.9 ml of 1% sulfuric acid (H2SO4) .
Antifungal disk assay
Antifungal activities of plant extracts were investigated using the Baur and Kerby disk diffusion method as described in Clinical and Laboratory Standards Institute (CLSI), 2002  . The PDA broth containing 24 h broth culture diluted to match 1.0 MacFarlands standard, 108 Colony Forming Units (CFU)/ml for yeasts and 108 spores/ml for filamentous fungi, were spread aseptically on the solid PDA plates using sterile L-shaped glass rod . Disks (6.00 mm diameter) impregnated with 10 μl of each extracts at a concentration of 200 mg/ml of solvent used in extraction and air dried, were then placed on the inoculated plates. The controls had 10 μl of each extract solvent on the disc without the extract but evaporated to dryness. Discs containing fluconazole 25 μg (Pfizer, New York, N.Y.), were used as standard controls. Both test and control discs were run parallel. All of the plates were incubated at 37 °C for 48 h for yeast and at 25 °C for 5 days for filamentous fungi. The zones of growth inhibition around the disks were measured after incubation.
The sensitivity of the microorganisms to the plant extracts was determined by measuring the diameters of inhibition zones. Values < 9 mm were considered as not active against microorganisms. All of the experiments were performed in triplicate. The results are reported as the average of the 3 experiments.
Minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC)
The MICs were determined by broth dilution methods . Two-fold serial dilutions (1.95 to 8000.00 μg/ml) of the extracts were prepared in PDA broth. The diluted extracts were then inoculated with 0.1 ml of approximately 108 CFU/ml of fungal suspension. The inoculated tubes were then incubated at either 37 °C for 48 h for yeast or 25 °C for 5 days for moulds. The MICs were taken as the average of the first tubes showing no visible growth of the micro-organisms  . Each assay was performed in triplicate.
The MICs to fluconazole powder (Pfizer, New Tork)was used as a standard and quality control as the range of MIC is known. Sterility of the media was controlled by use of broth only in a negative control tube, and growth of the organism was checked by broth plus microoganism in question in a positive control tube. All the controls were run parallel to the tests.
The MFC were determined by sub-culturing on PDA plates 0.1 ml of all the tubes showing no growth in PDA broth. They were incubated as already described previously for MIC cultures. After incubation, the first plates showing no growth were recorded as the MFCs  .
| Results|| |
Activity of Methanolic extracts of T. stapfiana using disk diffusion method
The root and stem bark extracts showed activity against Candida albicans (10 mm), Cryptococcus neoformans (10 mm) and Trichophyton mentagrophytes (10 mm) [Table 1]. There was no antifungal activity exhibited by the leaf and fruit methanol extracts and there was no further studies done on them. The controls using solvents exhibited no activity.
Disk diffucsion test for Sequential extracts
The successive extracts of the root and stem barks against the test fungi showed an increased activity compared to methanol extracts. The ethyl acetate extracts of the root exhibited inhibition zones of 19 and 18 mm against C. albicans and C. neoformans, respectively [Table 2]. The ethyl acetate stem bark extract exhibited activity with an inhibition zone size of 18 mm against both test yeasts [Table 3]. There was a 20 mm zone of inhibition for methanol sequential extract of the root and stem barks against C. albicans and also 17 mm (stem bark) and 18 mm (root bark) zones of inhibition against C. neoformans. The zone of inhibition of ethyl acetate sequential extracts of the root bark against T. mentagrophytes was 14 mm. The M. gypseum was sensitive to ethyl acetate extract with the root bark giving 12 mm zone of inhibition while the stem bark did not show any activity. The stem bark methanol portions had activity against M. gypseum with a 9 mm zone diameter but the root bark showed no activity. The hexane and dichloromethane portions of both the root and stem bark exhibited no antifungal activity. The controls using solvents exhibited no activity.
Minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC)
The methanol extracts of both root and stem barks showed strong MICs against C. albicans and C. neoformans with MICs of 62.5 and 125 μg/ml, respectively [Table 1]. There was also strong fungicidal activity against the test yeasts with both parts showing MFCs of 250 μg/ml. Weak activities were demonstrated against the dermatophytes tested, with the root and stem bark methanol extracts showing an MIC of 1000 μg/ml against T. mentagrophytes. The MFCs were 4000 μg/ml (root) and 8000 μg/ml (stem) against T. mentagrophytes.
The MICs and MFCs of the methanolic sequential extracts of the root bark against C. albicans were 31.25 and 125 μg/ml, respectively while that for the stem bark were 31.25 and 250 μg/ml, respectively [Table 2],[Table 3]. The MICs of ethyl acetate extracts of both the stem and root barks were 31.25 μg/ml and MFCs of 500 μg/ml against C. albicans. The MICs of methanol extracts of both the root and stem barks against C. neoformans was 62.5 μg/ml while the MFCs were 125 μg/ml for the root and 250 μg/ml for the stem. The MICs of ethyl acetate extracts of both the root and the stem barks against T. mentagrophytes were 500 μg/ml, while the MFCs were 1000 μg/ml. For the methanolic extract of the stem bark, the MIC was 1000 μg/ml and MFC, 4000 μg/ml against M. gypseum.
| Discussion|| |
Medicinal plants constitute an effective source of both traditional and modern medicine. World Health Organization (WHO) encourages countries to examine traditional medicine with a view to identifying and exploiting aspects that provide safe and effective remedies for ailments  . The results obtained from this study support the WHO recommendation as it provides scientific evidence that the root and stem bark extracts of T. stapfiana have antifungal properties. The sequential extracts showed a better antifungal activity as compared to methanol extracts. The root and stem bark methanol extracts that had MIC of 62.5 μg/ml and MFC of 250 μg/ml against C. albicans got lowered to MIC of 31.25 μg/ml and MFC of 125 μg/ml (root bark) and MIC of 31.25 μg/ml and MFC of 250 μg/ml (stem bark) in the methanolic extract of the sequential extracts. This activity is fungicidal as the ratios of MFCs to MICs are less than 4  .
Phytotchemical screening of T stapfiana documented before, indicate presence of plant metabolites well known for antimicrobial activity such as alkaloids, flavonoids, coumarins and saponins  . The alkaloids and saponins have been demonstrated to have a powerful anti-fungal effects and are some of a number of natural substances tested against pathogens by functional medicine labs , . The coumarins have been shown to inhibit Candida albicans in vitro  while flavonoids on the other hand have been found in vitro to be effective antimicrobial substance against a wide range of microorganisms including fungi  . Therefore, the antifungal activity of T stapfiana could be associated with any of these classes of compounds present. The activity could also be due to synergism or additive activity of two or more of the compounds present in the extracts .
The antifungal demonstrated in vitro, supports ethnobotanic uses of T. stapfiana among the Keiyo community. Extracts from T. stapfiana is traditionally used for dermatological problems of fungal nature  and this is confirmed in the present study. These results correlate fairly well with the literature on other Tabernaemontana species as shown by previous studies that indicated extracts from T. cymosa and T. pachysiphon have significant antifungal activities , . This shows the potential of members of the genus Tabernaemontana in antifungal therapy.
| Conclusion|| |
These antifungal studies suggest that T. stapfiana could be a rich source of antifungal agents. The present results further show that there is some merit in the use of this plant in traditional medicine. However, further studies with multiple strains of test fungi are recommended to confirm the utility of this plant extracts as alternative medicine in the war against fungal infections.
The fact that current dermatophytosis therapy is associated with considerable adverse effects in some patients, make these plant-derived compounds the subject of future research. This calls for further investigations on the isolation of the active principle(s) which is currently underway in our research laboratories and will be reported at a later stage.
| Acknowledgement|| |
The authors are greateful to the traditional medical practitioners in Keiyo District especially Kimoi Kiplagat and Arap Alwela who gave the ethnobotanical uses of the plant, Kenyatta University Chemistry laboratory, Kenya Medical Research Institute and Kenya National Public Health Laboratory Services' members of staff for their support during the study.
| References|| |
|1.||Irobi O.N. and aramola S.O.. Antifungal activities of crude extracts of Mitracarpus villosus (Rubiaceae). J Ethnopharmacol. 40 : 137-140 (1993). |
|2.||Zhanel G.G., Karlowsky J.A., Zelenitsky S.A., Turik M.A. and Hoban D.J.. Susceptibilities of Candida species isolated from the lower gastrointestinal tracts of the high-risk patients to the new semisynthetic echinocandin LY303366 and other antifungal agents. Antimicrob Agents Chemother. 42 : 2446-8 (1998). |
|3.||Reichart P.A., Samaranayake L.P., Samaranayake Y.H., Grote M., Pow E. and Cheung B.. High oral prevalence of Candida krusei in leprosy patients in northern Thailand. J Clin Microbiol. 40 : 479-85 (2002). |
|4.||Gupta A.K., Lynde C.W., Lauzon G.J.. Cutaneous adverse effects associated with terbinafine therapy: 10 case reports and a review of the literature. Br J Dermatol. 138 : 529-32 (1998). |
|5.||Thangadurai D., Murthy K.S.R., Prasad P.J.N. and Pullaian T.. Antimicrobial screening of Decapelis hamiltonii WIGHT and ARN. (Asclepiadaceae) root extracts against food-related micro-organisms. Journal of Food Safety. 24 : 239-245 (2004). |
|6.||Fabry W., Okemo P. and Ansorg R.. Fungistatic and fungicidal of East Africa medicinal plants. Mycoses. 39 : 67-70 (1996). |
|7.||Omino E.A. and Kokwaro J.O.. Ethnobotany of Apocynaceae species in Kenya. J Ethnopharmaco. 40 : 167-180 (1993). |
|8.||Beenje H.J.. Kenya trees, shrubs and lianas. National Museums of Kenya, Nairobi, (1994). |
|9.||Elgayyar M., Draughon F.A., Golden D.A. and Mount J.N.. Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. J Food Prot. 64 : 1019-1024 (2000). |
|10.||National Committee for Clinical Laboratory Standards (NCCLS). Performance standards for antimicrobial disk susceptibility test: Approved Standard M2-A7. National Committee for Clinical Standards, Wayne, PA, USA. |
|11.||Clinical and Laboratory Standards Institute (CLSI). Reference methods for disk diffusion antifungal susceptibility testing; Approved standard. Clinical and Laboratory Standards Institute, Villanova, PA (2002). |
|12.||Chattopadhyay D., Maiti K., Kundu A.P., Chakrabarty M.S., Bhadra R., Mandal S.C. and Mandal A.B.. Antimicrobial activity of Alstonia macrophylla: folklore of Bay Islands. J Ethnopharmacol. 77 : 49-55 (2001). |
|13.||Burrowes O.J., Hadjicharalambous C., Diamond G. and Lee T.C.. Evaluation of antimicrobial spectrum and cytotoxic activity of pleurocidin for food applications. Journal of Food Science. 69 : 66-71 (2004). |
|14.||Micheal J.P., Chan E.C.S. and Krieg N.R., Microbiology, 5th Edition, Tata McGraw-Hill Publishing Company Limited, New Delhi, 510-539 (2003). |
|15.||Akinyemi K.O., Oladapo O., Okwara C.E., Ibe C.C. and Fasure K.A.. Screening of crude extracts of six medicinal plants used in South-West Nigeria unorthodox medicine for anti-methicillin resistant Staphylococci aureus activity. Complementary and Alternative Medicine. 5 : 6 (2005). |
|16.||Meletiadis J., Antachopoulos C., Stergiopoulou T., Pournaras S., Roilides E. and Walsh T.J.. Differential antifungal activities of amphotericin B and vericonazole against Aspergilus species determined by microbroth methodology. Antimicrob Agents Chemother 51 : 3329-3337 (2007). |
|17.||Ruttoh E. K., Tarus P. K., Bii C. C., Machocho A. K., Karimi L. K. and Okemo P. O.. Antibacterial activity of Tabernaemontana stapfiana Britten (Apocynaceae) extracts. Afr J Trad CAM. 6 : 186-194 (2009). |
|18.||Agarwal A. K., Xu T., Jacob M. R., Feng Q., Lorenz M. C., Walker L. A. and Clark A. M.. Role of Heme in the Antifungal Activity of the Azaoxoaporphine Alkaloid Sampangine. Eukaryot Cell. 7 : 387-400 (2008). |
|19.||Zehavi U., Polacheck I.. Saponins as antimycotic agents: glycosides of medicagenic acid. Adv Exp Med Biol. 404 : 535-46 (1996). |
|20.||Cowan M.M.. Plant products as antimicrobial agents. Clin Microbiol Rev. 12 : 564-582 (1999). |
|21.||Afolayan A.J. and Meyer J.J.M.. The antimicrobial activity of 3,5,7-trihydroxyflavone isolated from the shoots of Helichrysum aureonitens. J Ethnopharmacol. 57 : 177-181 (1997). |
|22.||Ruttoh E.K.. Antimicrobial activity of selected plants used by traditional medical practitioners in Keiyo District, Kenya. Msc Thesis, 75-76 (2007). |
|23.||Achenbach H., Benirschke M. and Torrenegra R.. Alkaloids and other compounds from seeds of Tabernaemontana cymosa. Phytochemistry. 45 : 325-335 (1997). |
|24.||Uwumaronngie H., Onwukaeme N.D. and Obasuyi O.. Antimicrobial activity of the methanolic leaf extract of Tabernaemontana pachysiphon stapf. (Apocynaceae). Nigerian Journal of Natural Products and Medicine. 11 : 23-25 (2007). |
[Table 1], [Table 2], [Table 3]