African Journal of Traditional, Complementary and Alternative Medicines African Ethnomedicines Network ISSN: 0189-6016 Vol. 4, Num. 4, 2007, pp. 510 – 523

African Journal of Traditional, Complimentary and Alternative Medicines, Vol.4, No. 4, 2007, pg. 510 – 523

Research Paper

IN VITRO ANTIMICROBIAL ASSAY OF PLANTS USED IN TRADITIONAL MEDICINE IN BUKOBA RURAL DISTRICT, TANZANIA.

¹Department of Botany, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania.
²Department of Molecular Biology and Biotechnology, P.O. Box 35060, University of Dar es Salaam, Dar es Salaam, Tanzania.,³Department of Chemistry, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania. *E-mail: kisangau@yahoo.com

Code Number: tc07068

Plants used in traditional medicine in Bukoba Rural district in Tanzania were evaluated for their in vitro antimicrobial activities. Plant materials from eight plant species (Harungana madagascariensis (Lam) Poir., Jatropha curcas L., Lantana trifolia L., Plectranthus barbatus Andr., Pseudospondias microcarpa Engl., Psorospermum febrifugum Spach, Teclea nobilis Del. and Vernonia adoensis [Warp.] SL) were collected based on ethnomedical information provided by traditional herbal practitioners. Results of the study indicate that extracts from the eight plant species were active against at least one or more of the test organisms (Bacillus subtilis, Staphylococcus aureus [gram positive], Escherichia coli, Pseudomonas aeruginosa [gram negative] and Candida albicans [Yeast]). A profile of secondary metabolites (alkaloids, terpenoids, triterpenes, phenolics, tannins, flavonoids, anthraquinones, flavonols/flavones and /or chalcones, sterols and saponins) was obtained for three plant species (Jatropha curcas L., Plectranthus barbatus Andr., and Pseudospondias microcarpa Engl.). The paper discusses the probable therapeutic basis of these traditional plants based on their secondary metabolite profiles and for the first time draws research attention to Bukoba Rural district as a source for plants with potential pharmaceutical applications.

Key words: Antimicrobial activity, Secondary metabolites, Traditional medicine, Tanzania.

Traditional medicine continues to provide health coverage for over 80% of the world population, especially in the developing world (WHO, 2002). In Africa, traditional healers have for centuries been the main providers of primary health care (Scheinman, 2002; Kala et al., 2004). In Tanzania, the use of traditional medicine for the treatment of various diseases has been practiced for generations and a large number of the population in the country use traditional medicines for their day to day healthcare needs (Moshi et al., 2006; Hamza et al., - In press).

Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as micro organisms, animals and plants (Janovka et al., 2003). However, the increasing prevalence of multidrug resistant bacteria and fungi, the recent appearance of strains with reduced susceptibility to antibiotics, the side effects associated with antibiotics, the high costs of antimicrobial drugs and the re-emergence of diseases like tuberculosis are the key factors that obstruct resonant management of bacterial and fungal infections in many developing countries including Tanzania (Janovka et al., 2003; Runyoro et al., 2006; Hamza et al., -In Press). The increased prevalence of HIV/AIDS virus has also in the recent past augmented the magnitude of many bacterial and fungal opportunistic infections with frequent episodes resulting from the immune suppression of the affected persons, (Bii, 2001; Runyoro et al., 2006). Consequently, this has raised the spectre of untreatable microbial infections and adds the urgency to the search for new infection-fighting strategies. (Janovka et al., 2003).

Plants that are traditionally used in the treatment of bacterial and fungal infections or related ailments could be a good source for new, safe and biodegradable antimicrobial drugs (Hamza et al., - In press), and could offer potential lead towards development of novel compounds that are active against pathogenic microbes (Runyoro et al., 2006). Thus, chemical studies of Tanzanian medicinal plants could provide a valuable material base for the discovery and development of new drugs of natural origin (Moshi et al., 2006).

The present study was carried out in Bukoba Rural district which is faced by severe inadequacy of health facilities, with a population-doctor ratio of 1:95,000, the lowest of all the six districts forming Kagera region in the Northern part of Tanzania (TUROT, 2003; TACAIDS et al., 2005). Majority of the population in the district are poor and cannot afford the usually expensive conventional drugs which are hardly available in many of the local health centers. Besides, many people are forced to walk for long distances to reach a health facility (TUROT, 2003). This has forced the local population to turn to traditional plant remedies which are readily available, more affordable, less toxic, and have a wide acceptance around the world (WHO, 2002). Therefore, the current study forms a strong basis for identifying plants with potential pharmaceutical applications not only in the study area, but also in the world.

Different parts of eight plant species were collected from Bukoba Rural district and prepared for bioactivity and phytochemical screening. The plant species collected for analysis were Harungana madagascariensis (Lam) Poir., Jatropha curcas L., Lantana trifolia L., Plectranthus barbatus Andr., Pseudospondias microcarpa (A. Rich) Engl., Psorospermum febrifugum Spach, Teclea nobilis Del. and Vernonia adoensis (Warp.) SL. (Table 1 ). With a Prior Informed Consent (PIC), ethnomedical information about the eight plant species was sourced from three authentic herbal practitioners (One man and two women), who were identified with the help of local administration officers and interviewed using an openended semi-structured questionnaire according to Martin (1995). Voucher specimens were collected and identified by Mr. Frank Mbago and Mr. Suleiman Haji of the University of Dar es Salaam Botany Department Herbarium, where they were coded and deposited (Table 1 ). Collection of the plant materials from the eight species for in vitro bioactivity assays was pegged on informant consensus on their ethnomedical uses enumerated in Table 1 . Only the parts of the plants used by the herbal practitioners were collected for analysis. Leaves were collected for seven of the eight plant species while stem/bark was collected for only one plant species. No roots were collected for analysis (Tables 1 , 2 and 3 ).

Fresh plant materials were chopped into smaller pieces before drying and then air dried under shade for a period of ten days. The dried plant materials were then pulverized using a NATIONAL SUPER^® mixer grinder [MX-119] (Emerging planet India Ltd., Coimbatore 641011, India). About 300 g of each of the pulverized material was extracted using petroleum ether, dichloromethane and water in that order as successfully done in other studies (Janovka et al., 2003; Moshi et al., 2006). These crude extracts were concentrated in vacuo using a rotary evaporator (HEIDOLPH^®, Essex Scientific laboratory supplies Ltd. Crucible house, Endway, Hadleigh, Benfleet, essex, SS7 2AN) with the bath temperature maintained at 40⁰C to prevent thermal decomposition of labile compounds. The aqueous extracts were obtained by using either cold percolation or hot water to mimic the mode of extraction by the herbal practitioners. The organic solvent extracts were dissolved in DMSO while the aqueous extracts were dissolved in water at varying concentrations as done in recently reported procedure (Motsei et al., 2003).

Five test microorganisms were used in antimicrobial sensitivity tests. They were the gram positive bacteria Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (DSM 347) and gram negative bacteria Escherichia coli (DSM 1103) and Pseudomonas aeruginosa (DSM 1117) and the yeast Candida albicans (ATCC 90028). These were obtained from the Department of Molecular Biology and Biotechnology (MBB), University of Dar es Salaam.

Nutrient Broth (NB), (OXOID^®, Unipath Ltd. Basingstoke, England) and Malt Extract Broth (MEB), (PRONADISA^®, Conda Ltd., Madrid, Spain) were used to prepare broth cultures of bacteria and yeast test organisms respectively. The broth media were prepared according to the manufacturers' instructions. The autoclaved media was asceptically transferred to sterile capped test tubes of about 6ml each. Pure isolates of subcultured bacteria and yeast colonies were asceptically transferred to the respective broth media. The approximate cell concentration in the broth was 10⁵ CFU/ml. These were incubated in DEPEX MEMMERT^® incubator (Bellevue WA 98005, US) for an overnight at 37⁰C and for 48 hours at 30⁰C for bacteria and Candida respectively.

Antimicrobial activity of the crude extracts was determined by Agar well and Disc diffusion assay methods as previously described by Rojas et al. (2006) and Moshi et al. (2006).

Nutrient Agar (LAB M^TM, Lancashire, UK) and Malt Extract Agar (PRONADISA^®, Conda Ltd. Madrid, Spain) were prepared for bacteria and Candida respectively according to the manufacturers' instructions. Immediately after autoclaving, the media was allowed to cool in a 45 to 50⁰C water bath. The freshly prepared and cooled media was poured into glass, flat-bottomed petri dishes (90mm in diameter) placed on a level, horizontal surface (FASTER^® Laminar flow, Cornaredo via Merendi, 22 20010, Italy) to give a uniform depth of approximately 4 mm. The agar media was allowed to cool and solidify at room temperature and the plates were incubated at 35⁰C for 18-20 hours before use to ensure sterility. About 0.2ml of the test inoculum was evenly spread on the surface of the solidified agar media using a sterile grigalsky spatula. Four equidistant wells of 5mm in diameter and 4mm in depth were then made on the agar using a sterile cork borer. Two more wells for positive and negative controls were made at the middle of the agar. About 25 µl of the plant extracts and controls were filled into the wells (Rojas et al., 2006). Concentrations for the crude extracts ranged from 25-200mg/ml. The positive controls were Ampicillin (SIGMA^®, Sigma chemical Co., st. Louis, USA.), (0.5mg/ml) for gram positive bacteria, Gentamicin (IVEE Aqua EPZ Ltd. Nairobi, Kenya), (0.5mg/ml) for gram negative bacteria and Fluconazole (ZOCON^®, FDC Ltd. India), (1mg/ml) for Candida. The negative controls were DMSO for organic solvent extracts and distilled water for aqueous extracts. The wells were then labeled to correspond with the code numbers of the test crude extracts and controls. The treated plates were stored in a refrigerator (DAEWOO^®, Daewoo Electronics, Europe GmbH, Germany) at 4 ⁰C for atleast six hours to allow diffusion of the extracts into the agar while arresting the growth of the test microbes. The plates were then incubated for 24 hours at 37 ⁰ C for bacteria and for 48 hours at 30⁰C for Candida. The test was carried out in duplicates. Antimicrobial activity was determined by measuring the diameters of zones of inhibition in mm. The means and standard deviations (±SD) of the diameters of zones of growth inhibitions for the treatments are shown in Table 2 .

Preparation of the media and inoculation of the test microbes were performed as described in the agar well method. However, instead of punching out wells on the agar, sterile 5 mm-Whatman No. 1 filter paper discs (WHATMAN^®, Springfield Mill, Maidstone, Kent, England) were used in the disc diffusion method (Moshi et al., 2006). The discs were soaked into the dissolved crude extracts for a minimum of two hours. Blank discs were impregnated with Ampicillin (10µg/disc) for gram positive bacteria, Gentamicin (15µg/disc) for gram negative bacteria and Fluconazole (0.4mg/disc) for Candida and used as positive controls. Negative control discs were soaked in DMSO and distilled water for organic solvent and water extracts respectively. By use of a sterile forcep, four seeded discs of the plant extracts were placed equidistantly onto each of the inoculated plates. Two extra discs for positive and negative controls were placed at the middle of plate. The treated plates were stored in a refregerator (DAEWOO^®, Daewoo Electronics, Europe GmbH, Germany) at 4⁰C for atleast six hours then transferred to DEPEX MEMMERT^® incubator (Bellevue WA 98005, US) for 24 hours at 37⁰ C for bacteria and for 48 hours at 30⁰C for Candida. The test was carried out in duplicates. Antimicrobial activities were determined by measuring the diameters of zones of inhibition in mm. The means and standard deviations (±SD) of the diameters of zones of growth inhibitions for the treatments are shown in Table 3 .

Screening for secondary metabolites was done for three plants, Jatropha curcas, Plectranthus barbatus and Pseudospondias microcarpa whose information on their bioactivity and phytochemistry is scanty with regard to their medicinal uses. Chromatograms were developed on commercial Thin Layer Chromatography (TLC) plates using different solvent systems of varying polarities (Houghton and Raman, 1998; Kinge, 2002; Orech et al., 2005, Edeoga et al., 2005). The chromatograms were observed under normal light and UV (254nm, 365nm) before being sprayed with the respective test reagents. Secondary metabolites were identified by color establishment using standard spray reagents on the TLC Chromatograms as exemplified in figures 2, 3 and 4. The secondary metabolites tested in all the extracts of the three plant species were alkaloids, terpenoids, triterpenes, phenolics, tannins, flavonoids, anthraquinones, flavonols/flavones and/or chalcones, sterols and saponins.

To test for alkaloids, chromatograms were sprayed with dragedorff's reagent and over sprayed with aqueous iron (III) chloride. Presence of terpenoids was detected by spraying the chromatograms with anisaldehyde and then heating them with a hot air stream from a heating gun (BLACK & DECKER^®, Black & Decker, Hunt valley, MD 21031, USA). Triterpenes were detected by treating the chromatograms with 10% ammonium hydroxide. Phenolic compounds were detected by spraying the plates with 5% aqueous iron (III) chloride, then heating them with a hot air stream. Test for presence of flavonoids was done by passing the chromatograms on Ammonium hydroxide vapour. Anthraquinones were tested by spraying the chromatograms with 10% ammonium hydroxide. Presence of flavonols, flavones and/or chalcones was detected by treating the chromatograms with aluminium chloride followed by heating with hot air stream from the heating gun. Detection of tannins was done by treating the chromatograms with ferric chloride. Sterols were detected by treating chromatograms with 50% acetic anhydride in sulphuric acid followed by heating from a hot air stream. Presence of saponins was tested by shaking about 2ml of the plant extracts in distilled water.

All plant species exhibited activity against at least one or more of the microorganisms tested (Tables 2 and 3 ). Amongst the eight plants studied, J. curcas and P. barbatus had the broadest range of activity inhibiting the growth of the gram positive, gram negative and the yeast fungus tested. L. trifolia had the least range of antimicrobial activity inhibiting the growth of only one of the five microbes tested. E. coli was the most susceptible of all the microbes tested followed by B. subtilis. The least susceptible microbes were P. aeruginosa and C. albicans. Generally, bacteria were more susceptible to the plant extracts than the yeast Candida, with Activity Index (AI) ranging between 0.22-2.96 and 0.23-0.32 for bacteria and Candida respectively. Moreover, activity against C. albicans was observed only in the disc diffusion assays but none in the agar well assays.

The highest activity was observed in the crude extracts of V. adoensis against E. coli with an Activity index (AI) of 1.76 (Table 3 ). The lowest activity was observed in the crude extracts of P. microcarpa with an Activity index of 0.22 (Table 3 ). However, the highest Activity index was realized in the J. curcas extracts at 2.96 against S. aureus (Table 3 ). The Activity Index (AI) is an expression of the degree of potency of the plant extract in relation to the standard drug (Moshi et al. 2006). Based on the Activity index, water extracts portrayed the highest potency of the herbal drugs screened compared to Petroleum ether or Dichloromethane extracts. There were 37 treatments which showed activity against the test organisms in the disc diffusion assays as opposed to 24 in the agar well assays (Tables 2 and 3 ). In some cases, the three extracts of the same plant (petroleum ether, dichloromethane and water) had antimicrobial activity against the same microorganism. For instance, the three extracts of H. madascariensis were active against B. subtilis, the three extracts of P. febrifugum were active against E. coli while those of P. barbatus were active against C. albicans. Different kinds of secondary metabolites were detected in all the plant extracts tested (Table 5 ). Appearance of colour establishments shown in Table 4 was positive indication of the secondary metabolites tested. P. barbatus had the highest number of secondary metabolites, with eight out the ten metabolites screened. The plant with the least number of secondary metabolites was P. microcarpa with only three of the ten metabolites screened. Phenolics, tannins and sterols were present in all the extracts, while alkaloids and saponins were absent in all the extracts screened.

Table 1: Traditional uses of the plants recorded in the field.

Discussion

The nominal susceptibility of C. albicans to the plant extracts could be associated with the presence of supramolecular complexes of its cell wall including chitin, which may be difficult to digest, thus imposing resistance to prospective drugs (Marcilla et al., 1991). In the cases where the three kinds of extracts of the same plant depicted activity against the same microorganism, would possibly mean that the compound responsible for the antimicrobial activity was present in each extract at a different concentration as similarly observed by Rojas et al. (2006).An antimicrobial assay on the root extracts of J. curcas by Atindehou et al. (2002) reported a negative activity against C. albicans. The anti Candida activity of the leaf extracts recorded in the present study (Table 3 ) may be an indication that the active principles against the fungus are present in the leaves of the plant. It is however important that further studies on toxicity potential on leaf extracts of J. curcas are carried out as the oil from the seeds of the plant have been reported to be rich in toxalbumins which are poisonous proteins and usually irritant in nature (Kokwaro, 1993).

An antimicrobial flavonoid umuhengerin isolated from L. trifolia (Rwangabo, 1988) showed activity against S. aureus as opposed to the present data where there was no activity against the microbe. However, it was not clear from which part of the plant the active compound was isolated. The effects of extracts from H. madagascariensis against S. aureus and B. subtilis (Atindehou et al., 2002; Moulari et al., 2006) and the anti Candida activity of P. barbatus (Runyoro et al., 2006) are confirmed in this work Most of the medicinal applications of the secondary metabolites reported in literature are in harmony with the traditional medicinal uses of the plants recorded here (Table 6 ), thus confirming the rationalization for the traditional use of the herbal remedies in treating various disease conditions.

The presence of terpenoids in J. curcas and P. barbatus could be responsible for the medicinal properties associated with these plants as they were the major group of compounds expressed on the chromatograms after treatment with Anisaldehyde spray reagent (Figure 2). The presence of tannins in P. barbatus could also contribute to its medicinal use for treatment of diarrhea as reported by the traditional healers. Tannins are glycosides of gallic or protocatechuic acids and have the property of precipitating proteins and mucus and also constricting blood vessels. Such stringent action of tannins gives them the medicinal value of preventing diarrhea, controlling haemorrhages and treating wounds (Kokwaro, 1993; Kisangau, 1999).

Moreover, the skin healing properties of P. barbatus could be associated with the presence of anthraquinones detected in the plant, which could be responsible for the plant's inhibitory activities against S. aureus (Tables 2 and 3 ). Previous reports indicate that anthraquinones are useful in healing wounds and have been used as dressing for burns and other skin lesions (Kokwaro, 1993). They are also a remedy for general skin eruptions and have the ability to inhibit the growth of S. aureus, a major cause of many skin infections (Mona, 1997). They could also be responsible for its use as purgative, treatment of colics, diarrhea, peptic ulcers, typhoid and stomach aches as they have the ability to stimulate bile flow and promote activity of the entire digestive process (Mona, 1997).

Phenolic compounds have been recorded to have diuretic and choleretic effects decreasing the viscosity of the blood, reducing blood pressure and stimulating intestinal peristalsis (Farombi, 2003). This means that the presence of phenolics in the three traditional plants could be possible candidates for use in the management of hypertensive cases. However, it is imperative to note that the healing effects of the reported plants could be due to synergistic action of the various compounds found in the plants. For example, the traditional use of P. barbatus for the treatment of diarrhea, colics or as purgative could be an effect of the presence of triterpenes, tannins, anthraquinones, flavonols/flavones or chalcones detected in the plant extracts (Table 5 ).

The fact that water extracts portrayed the highest potency of the herbal extracts against the test microbes justifies the rationale for use of water as medium for extracting herbal medicines from plants. The study unveils rich resources of bioactive plants in Bukoba rural district and lays crucial needs for isolation and development of pharmaceuticals from the extracts reported here. In this study, we are starting efforts to incorporate the traditional healers in all stages of our research by signing Material Transfer Agreement (MTA) with them. This will ensure that they stand to benefit in the event of pharmaceutical production of their knowledge. Any new studies on ethnobotany and ethnomedicine are encouraged to do the same.

DPK is highly indebted to DAAD/NAPRECA for a scholarship on this study. We also thank the Inter-University Council of East Africa Research initiative-VicRes for supplementary financial support. We thank the respondents and the general community in Bukoba Rural district for their cooperation during the field data collection period. Messrs F.M. Mbago and S. Haji of the Herbarium, Botany department are thanked for identifying the plants.

1. Amonkar, A; Chang, C-J. and Cassady, J.M. (1981). 6-Geranyloxy-3-methyl-1,8-dihydroxyanthrone, a novel antileukemic agent from Psorospermum febrifugum Sprach var.ferrugineum (Hook. fil). J. Cell molecular life sci.. 37(11):1138-1139.
2. Atindehou, K.K; Kone, M; Traore, D; Hostettman, K. and Dosso, M. (2002). Evaluation of the antimicrobial potential of medicinal plants from the Ivory coast. Phytother. Res. 16:497-502.
3. Beentje, H.J. (1994). Kenya trees, shrubs and lianas. National Museums of Kenya, Nairobi, Kenya.
4. Berger, A; Jones, P.J.h and Abumweis, S.S. (2004). Plant sterols: factors affecting their efficacy and safety as functional food ingredients. Lipids Health Dis. 3:5
5. Bii, C.C. (2001). The Potential use of Allium sativum (Garlic) for management of HIV-AIDS Oral Candidiasis. Abstracts of the 9^th Symposium of the Natural Product Research Network for Eastern and Central Africa (NAPRECA) held at Kenyatta International Conference centre (KICC), Nairobi, Kenya, Pg.24.
6. Craig W.J. (1999). Health promoting properties of common herbs. Am. J. Clin. Nutr. 70(3):491S-499
7. S.Edeoga, H.O; Okwu, D.E. and Mbaebie, B.O. (2005). Phytochemical constituents of some Nigerian medicinal plants. Afric.J. Biotech. 4 (7): 685-688.
8. Farombi E.O.(2003). African indigenous plants with chemotherapeutic potentials and biotechnological approach to the production of bioactive prophylactic agents. Afric. J. Biotech. 2(12):662-671.
9. Hamza, O.J. M; Beukel, C; Matee, M.I; Moshi, M.J; Ajam, V.V; Mikx, F. and Verweij, P.E. (2006). Antifungal activity of some Tanzanian plants used traditionally for the treatment of fungal infections J. Ethnopharmacol. 108(1): 124-132.
10. Houghton, P.J. and Raman, A. (1998). Laboratory handbook for the fractionation of Natural extracts.
11. Chapman & Hall, London. Janovka, D; Kubikova, K. and Kokoska, L. (2003). Screening for antimicrobial activity of some medicinal plant species of traditional Chinese medicine. Czech J. Food Sci. 21:107-110.
12. Kala, C.P. Farooquee, N.A. and Dhar, U. (2004). Prioritization of medicinal plants on the basis of available knowledge, existing practices and use value status in Uttaranchal, India. J. Biodiver. and Conserv. 13: 453-469.
13. Kinge, R.W. (2002). Investigations on the Bioactivities of Aloe secundiflora (Aloeaceae) on Newcastle disease and fowl typhoid in local chickens (Gallus domesticus) in Tanzania. Unpublished PhD thesis, University of Dar es Salaam.
14. Kisangau D.P. (1999). An ethnobotanical and Pytochemical study of medicinal plants of Makueni district. Unpublished MSc. Thesis, University of Nairobi, Kenya.
15. Kokwaro J.O. (1993). Medicinal plants of East Africa (2^nd Ed.). Kenya Literature Bureau, Nairobi, Kenya.
16. Marcilla, A; Elorza, M.V, Mormeneo, S; Rico, H. and Sentandreu, R. (1991). Candida albicans mycelial wall structure: supramolecular complexes released by zymolyase, chitinase and beta-mercaptoethanol. Arch Microbiol. 155(4):312-9.
17. Martin, G.J. (1995). Ethnobotany: A people and plants conservation manual. Chapman & Hall,London. Mona, H. (1997). Health benefits of Morinda citrifolia: consumer health. 20:12.
18. Moshi, M.J; Mbwambo, Z.H; Kapingu, M.C; Mhozya, V.H. and Marwa, C. (2006). Antimicrobial and Brine Shrimp Lethality of extracts of Terminalia mollis Laws. Afri J. Trad. CAM. 3(3):1-10.
19. Moshi, M.J; Mbwambo, Z.H; Nondo, R.S.O; Masimba, P.J; Kamuhabwa, A; Kapingu, M.C; Thomas, P. and Richard. M. (2006). Evaluation of ethnomedical claims and Brine shrimp toxicity of some plants used in Tanzania as traditional medicines. Afri. J. Trad. CAM, 3(3):48-58.
20. Motsei, M.L; Lindsay, K.L; Staden J.V and Jager, A.K. (2003). Screening of traditionally used South African plants for antifungal activity against Candida albicans. J Ethnopharmacol. 86:235-241.
21. Moulari, B; Pellequer, Y; Lboutounne, H; Girad, C; Chaumont, J.P; Millet, J and Muyard, F. (2006). Isolation and in vitro antibacerial activity of astilbin, the bioactive flavanone from the leaves of Harungana madagascariensis Lam.ex Poir. (Hypericaceae). J Ethnopharmacol. 106(2):272-8.
22. Njoroge, N.G ; Rainer, B.W ; Barbara, G ; Newton, L.E. and Ngumi, V.W.(2004).Utilisation of weed species as sources of traditional medicines in Central Kenya. Lyonia jJ. Ecol. Application. 7(2):71-87.
23. Okoli, A.S; Okeke, M.I; Iroegbu, C.U. and Ebo, P.U. (2002). Antibacterial activity of Harungana madagascariensis leaf extracts. Phytother. Res. 16(2):174-9.
24. Orech, F.O; Akenga, T; Ochora, J; Friis, H. and Aagaard-Hansen, J. (2005). Potential toxicity of some leafy vegetables consumed in Nyang'oma Division, Western Kenya. Afri. J. Food and Nutrit. Sci..5 (1):1-13.
25. Repetto, M.G and Llesuy, S.F. (2002). Antioxidant properties of natural compounds used in popular medicine for gastric ulcers. Braz. J. Med.Biol. Res. 35(5):523-534.
26. Richelle, M; Enslen, M; Hager, C; Groux, M; Tavazzi, I; Godin, J.P; Berger, A; Métairon, S; Quaile, S; Piguet-Welsch, C; Sagalowicz, L; Green, H. and Fay, L.B. (2004). Both free and esterified plant sterols reduce cholesterol absorption and the bioavailability of ß-carotene and -tocopherol in normocholesterolemic humans. Am.J.Clin. Nutr, 80(1):171-177.
27. Rojas, J.J; Ochoa, V.J ; Ocampo, S.A. and Munoz, J.F. (2006). Screening for antimicrobial activity of ten medicinal plants used in Colombian folkloric medicine: A possible alternative in the treatment of non-nosocomial infections. BMC Complement Altern Med. 6: 2.
28. Runyoro D.K.B; Matee M.I.N; Ngassapa O.D; Joseph C.C and Mbwambo Z.H. (2006). Screening of Tanzanian medicinal plants for anti-Candida activity. BMC Complement Altern Med. 6: 11.
29. Rwagabo, P.C. (1988). Umuhengerin, a new antimicrobially active flavonoid from Lantana trifolia. J.Natural Products. 51(5):966-968.
30. Scheinman, D. (2002). The ancient and modern worlds unite to fight HIV/AIDS in Tanga, Tanzania. Merck; Science in Africa on-line magazine http://www.scienceinafrica.co.za/2002/september/tanga.htm
31. Tanzania Commission for AIDS (TACAIDS); National Bureau of Statistics (NBS) and ORC Macro. (2005). Tanzania HIV/AIDS indicator survey 2003-04.Calverton, Maryland, USA: TACAIDS, NBS and ORC Macro
32. The United Republic of Tanzania (TUROT). (2003). Kagera Region Socio-economic profile (2^nd.). National Bureau of Statistics (NBS) and Kagera Regional Commissioner's office, Tanzania.
33. Wagner, K.H. and Emadfa, I. (2003). Biological relevance of terpenoids: Overview focusing on mono-, di and tetraterpenes. Annals Nutrition metabolism. 47(3-4):95-106.
34. WHO. (2002). Traditional Medicine: Growing Needs and Potential. WHO Policy Perspectives on Medicines. World Health Organization, Geneva pp. 1-6.

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