Bmc Complementary and Alternative Medicine Antimicrobial Activity and Brine Shrimp Toxicity of Extracts of Terminalia Brownii Roots and Stem

Background: Ternimalia brownii Fresen (Combretaceae) is widely used in traditional medicine to treat bacterial, fungal and viral infections. There is a need to evaluate extracts of this plant in order to provide scientific proof for it's wide application in traditional medicine system.


Background
Terminalia brownii Fries (Combretaceae) is found in many parts of Africa and it has different uses. It is found in the Democratic Republic of Congo, Ethiopia, Kenya, Tanza-nia [1,2]. In Tanzania the plant grows in Morogoro, Coast Region, Tanga and Arusha. It has different vernacular names in different places such as kuuku, muvuku (Kamba, Kenya), koloswa (northern region, Kenya), weba (Ethiopia), lbukoi (Samburu, Kenya), orbukoi (Maasai, Tanzania), and mbarao or mwalambe, in Kiswahili. The leaves are used by traditional healers in Tanzania to treat diarrhoea and stomach ache, gastric ulcers, colic, and heartburn [2,3]. In the Democratic Republic of Congo barks from the stems, branches, and trunks are used to treat urogenital infections, urethral pain, endometritis, cystitis, leucorrhoea, syphilis, and gonorrhoea [4]. It is also used by traditional healers in Kenya to treat malaria [5]. The decoction of the stem bark, trunk and branches is taken orally to treat dysmenorrhoea, nervosity, hysteria, epilepsy, beriberi, dyspepsia, stomachache, gastric ulcers, and colitis [2,6]. Stem barks are chewed to treat cough and as emetic, infusion of barks and leaves are mixed with meat to treat hepatitis [7]. Traditional healers in Ethiopia use the stem and barks to treat jaundice, hepatitis, liver cirrhosis, and yellow fever [8][9][10].

Collection of Plant material
Ternimalia brownii Fresen (Combretaceae) roots and stem were collected in Mombo, Tanga Region, Tanzania. The plant was identified by Haji, Selemani of Department of Botany, University Dar es salaam, and the voucher specimen no. RKR 222 is kept in the Herbarium of the Institute of Traditional Medicine, Muhimbili University College of Health Sciences.

Antimicrobial tests
Antibacterial and antifungal activities were tested by the disc-diffusion method [11]. Eight standard bacteria, Staphylococcus aureus (NCTC 6571), Escherichia coli (NCTC 10418), Pseudomonas aeruginosa (NCTC 10662), Klebsiella pneumoniae (NCTC 9633), Salmonella typhi (NCTC 8385), Bacillus anthracis (NCTC 10073), Bacillus cereus (NCTC 7464), and Proteus mirabilis (NCTC 10975) and the fungi, Candida albicans (Strain HG 392), and local strains of Cryptococcus neoformans extracted from hospitalized patient were used. Filter paper discs (Whatman No. 1; 5 mm diameter) were impregnated with crude extracts (5 mg/disc) or standard drugs (10 μg/disc gentamicin; for bacteria) and (20 μg/disc clotrimazole; for fungi). The discs were overlayed on tryptone soya agar plates (for bacteria) and Saborauld's dextrose agar plates (for fungi) and incubated at 37°C, for 24 h. The discs were tested in triplicate, including one with a solvent blank and 3 for the standard drugs. Inhibition zones were calculated as the difference between disc diameter (5 mm) and the diameters of inhibition [12]. The mean inhibition zones were used to calculate the activity index. Activity index (AI) was calculated as the mean inhibition zone for test sample divided by the mean inhibition zone for the standard drug [11].

Brine shrimp lethality test
The brine shrimp lethality test (BST) was used to predict the presence, in the extracts, of cytotoxic activity [13]. Assay procedures and analysis of results was done as reported earlier [14]. Cyclophosphamide was used a standard anticancer test drug.

Anti-microbial activity
With the exception of Petroleum ether extracts, all the other extracts of stem bark were active against one or more of the tested organisms (Tables 1 and 2). The best activity of the stem bark extracts was exhibited by methanolic extract against S. aureus and B. anthracis; aqueous extract against S. aureus, B. anthracis, K. pneumoniae and both C. albicans and C. neoformans. Similarly, in the case of stem wood extracts, the Petroleum ether extract was inactive against all tested organisms whereas all other extracts exhibited activity against a number of tested organisms with the best activity being recorded for against B. anthracis, B. cereus, S. aureus, C. albicans and C. neofaormans. All the root extracts tested (

Brine shrimp lethality test
Brine shrimp results presented (Table 4) show that the root extracts were virtually non-toxic on the shrimps. They exhibited very low toxicity, giving LC 50 values greater than 100 μg/ml. The root aqueous extract was the most toxic. On the other hand the stem wood extracts were all very toxic to brine shrimps with LC 50 values ranging from 2.6-14.9 μg/ml. The LC 50 values for stem bark extracts ranged between 68.4-88.0 μg/ml for dichloromethane, 1:1 dichloromethane:methanol, methanol, and aqueous extracts. The petroleum ether extract (LC 50 1458.81 μg/ ml) exhibited very low toxicity.

Discussion
Terminalia brownii is used in traditional medicine to treat bacterial and viral infections [2][3][4]7,8,10]. The claims for treatment of gastrointestinal conditions, such as diarrhea [3] are similar to previous claims on two other plants of the genus Terminalia [14,15]. These claims have been supported by the current bioassay results, which have shown activity against bacterial and fungal pathogens. The root extracts were particularly more active on bacteria and fungi than the stem extracts. It is worth noting that, much as the root extracts were more active against bacteria and fungi, they exhibited very low toxicity on brine shrimps. This may suggest inherent selectivity of the extracts, thus making the root preparations more suitable for treatment of bacterial and fungal infections. It is also notable that  The results are presented as inhibition zone diameters in mm. Each value represents mean ± SD (n = 3). Key: PE = petroleum ether extract; DM = dicloromethane extract; 1:1 D:M = 1:1 dicloromethane:methanol extract; M = methanol extract; Aqua = aqueous extract; Gent = gentamicin; Clotr = Clotrimazole.
The results are presented as inhibition zone diameters in mm. Each value represents mean ± SD (n the aqueous extracts, which in most cases are the ones used by traditional healers, were active on bacteria and fungi, and slightly less toxic on brine shrimps. The stem extracts also had antimicrobial activity, but high brine shrimp toxicity. This indicates less selectivity of the stem extracts, and hence possibility of toxicity or other biological activities such as anticancer activity [13].
One of popular use of Terminalia species in Tanzania is for the treatment of diarrhea, particularly in HIV patients [14,15]. The leaves of this plant are used by traditional healers in Tanzania to treat diarrhoea, stomach ache, gastric ulcers, colic, and heartburn [2,3]. Since almost all the extracts that were tested showed activity against either Candida albicans or Cryptococcus neoformans, or both, it may be speculated here that the extracts would be useful for treatment of diarrhoea caused by gastrointestinal Candida infection, the most frequently encountered fungal infection in HIV patients.

Conclusion
Both organic and aqueous extracts of the stem and roots of Terminalia brownii have indicated varied levels of antibacterial and antifungal activity. Some of these extracts also exhibited cytotoxic activity on brine shrimps, comparable to that of a standard anticancer drug, cyclophosphamide. The traditional use of Terminalia brownii extracts to treat diarrhoea, cut wounds, gonnorrhea, and other infec-   tions has been supported by laboratory results from this study, suggesting a need to isolate and evaluate active constituents responsible for the exhibited biological activities.