African Health Sciences, Vol. 1, No. 1, December, 2001, pp. 66-72
Antibacterial and antifungal activities of extracts of Zanthoxylum chalybeum and Warburgia ugandensis, Ugandan medicinal plants
*Olila, D.1*, Olwa-Odyek2, and Opuda-Asibo,J3
1Departments of Veterinary Physiological Sciences, Makerere University, P.O. Box 7062, Kampala, Uganda.
*Corresponding Author: Dr. D. Olila, E-mail:email@example.com, Tel: 256-077-593926
Code Number: hs01021ABSTRACT
Measles is a killer disease of children in Uganda. The treatment of the disease is mainly directed at the secondary microbial infections. A large proportion of the population in Uganda still relies on the use of herbal remedies, which have been claimed to produce beneficial responses. In this study, the efficacy of Warburgia ugandensis and Zanthoxylum chalybeum against common bacteria and fungi was investigated. Bactericidal and antifungal assays were done using extracts derived from Z. chalybeum and W. ugandensis (agar well diffusion, disc diffusion and colony count assays). All extracts (ethanolic, petroleum ether and aqueous) of Z. chalybeum did not show antimicrobial activity. Phytochemical investigations of Zanthoxylum chalybeum (seed) yielded a pure crystalline alkaloid (27-135D) which was characterized as skimianine based on 1H-NMR spectroscopy and comparison with spectra of authentic samples. Skimmianine did not have antimicrobial activity in this test system. W. ugandensis water extracts elicited antibacterial activity against both Escherischia coli and Staphylococcus aureus in the agar well assay but not in the disc diffusion assay. Warburgia ugandensis water extracts and fraction 27-163D also showed antifungal activity against Candida albicans. Chromatography of extracts of Warburgia ugandensis stem bark afforded compound 49-169K, which was characterized as the sesquiterpine muzigadial (by 1H-NMR spectroscopy), which did not show antibacterial activity but had antifungal activity against C. albicans. Therefore, the claimed efficacy of W. ugandensis could be attributable to antibacterial and antifungal activity of its components. Since Z. chalybeum extracts had neither antifungal nor antibacterial activities, its mode of action is unclear from these results.INTRODUCTION
Despite the wide availability of clinically useful antibiotics and semisynthetic analoques, a continuing search for new anti-infective agents remains indispensable because some of the major antibacterial agents have considerable drawbacks in terms of limited antimicrobial spectrum or serious side effects. Moreover, the combination of genetic versatility of microbes and widespread overuse of antibiotics has led to increasing clinical resistance of previously sensitive microorganisms and the emergence of previously uncommon infections. Studies aimed at discovery and characterization of the substances that exhibit activity against infectious micro-organisms, yet showing no cross resistance with existing antibiotics, are highly called for.
Human and veterinary health services in Uganda are still very poor and are compounded by many people living in rural areas several kilometers from health centers. This has resulted in a large proportion of the population relying on traditional methods of treatment, using herbal extracts, which have been claimed to produce beneficial responses. This is in consonance with past experience of many useful drugs being derived from plants (such as atropine, ergometrine and the antibiotics).
Measles is one of the major killer diseases of children in Uganda1. There is currently no treatment for this primarily viral disease. Intervention treatment is usually directed at controlling secondary bacterial infections using antibacterial compounds. In Uganda, plants may represent a potential source of antibacterial drugs since extracts from some plants have been used in treatment of measles. The claimed efficacy of W. ugandensis and Z. chalybeum extracts in the treatment of measles was studied and here we report the evaluation of the antimicrobial properties of these plant extracts.
MATERIALS AND METHODS
i) Phytochemical studies
Z. chalybeum stem bark was chopped into small pieces and dried under the sun for three weeks. The seedswere already dry when collected, but they were also further sun dried for one week.
W. ugandensis stem bark was likewise chopped and sundried for three weeks. The dried specimens were then crushed by pounding in a wooden mortar into fine powder, ready for extraction.
Extraction, purification and spectroscopy
Extraction and isolation of compounds from Z. chalybeum.
Extraction and isolation of compounds from W. ugandensis
To obtain muzigadial, ground stem bark (200g) was macerated first with petrol and then with Ethyl acetate (EtOAC). The petrol extract was concentrated to yield 10g of crude extract which was fractionated over silica gel column eluting with petrol and then petrol containing increasing amounts of EtOAC.In order of elution, the following fractions were obtained: 49-169b (1.17g), 49-169c (500 mg), 49-169d (500mg), 49-169e (1.15g), 49-169f (1.15g), 49-169g (1.16g), 49-169h (150mg), 49-169i (651mg), 49-169j (1.56mg), 49-169k (850mg), 49-181a (665mg). Fraction 49-169k was pure muzigadial.
ii) Antibacterial and antifungal studies
Test organisms and media
Extracts tested and the standard bactericidal agent
Bactericidal and antifungal tests
Filter paper disc assay
Tube incubation and colony plate assays
The analysis of all the differences between means was performed using a paired t-test5. The confidence interval was set at 95% (p<0.05). In all cases means are shown with ± standard error.
Bactericidal and antifungal studies
Warburgia ugandensis extracts were bactericidal. It was not possible to demonstrate this effect in the paper disc assay, probably because the paper disc retains the active component and does not allow it to diffuse into the Muller Hinton Agar. In the agar well assay, the water extract showed a slightly higher activity as compared to the ethanolic extract. This was to be expected since the aqueous phase of the Muller Hinton agar would be expected to more easily dissolve water-soluble extracts and hence enable easier diffusion. This emphasizes the fact that agar well diffusion assay is ideal for water extracts. The antibacterial activity of W. ugandensis is only detectable at very high concentration of the extract (i.e. upto 50 mg/ml of crude extract). This highlights the problem of deciding on the starting point of assaying crude extracts. Most assays of extracts start at 50-100ug/ml, at which level it is not possible to detect activity in W. ugandensis extracts. This may explain why there is as yet no report of antibacterial activity of W. ugandensis. It is probably not proper to start assaying crude extracts at concentration levels comparable to tetracycline since tetracycline is a known pure compound. It is quite likely that the active ingredient is present in W. ugandensis in very low quantities, requiring the use of large amounts of crude extracts.
Warburgia ugandensis is known to have potent antifeedant and antifungal activity 8. Warburganal and Muzigadial have been characterized as the active compounds. This study has also demonstrated antifungal activity in W. ugandensis (Table 1). To the best of our knowledge this is the first report of antibacterial activity in extracts derived from W. ugandensis. The extracts inhibited S. aureus more than E. coli.
In the colony count assay, both the alcohol and the water extracts exhibited bactericidal activity against both S. aureus and E. coli. There was, however, a marked difference in the activity of the two. This was rather paradoxical since both ethanol and water are highly polar solvents, which would be expected to extract similar components from the plants. It was also noted that the agar well diffusion assay revealed a low activity of ethanolic extracts. In the tube incubation test, however, the bactericidal activity of the crude extracts against both S. aureus and E. coli was quite high. Staphylococcus aureus was more sensitive than E. coli to W. ugandensis extracts (agar well assay). Since S. aureus is more often associated with secondary bacterial infections in measles than E. coli 10, it may explain the value of the plant in measles therapy.
Zanthoxylum chalybeum had no antibacterial activity against E. coli and S. aureus. This was true of the crude as well as the purified fractions. The extracts also had no antifungal activity against Candida albicans. In a study carried out by Odebiyi and Sofowora 11, antibacterial activity was demonstrated in extracts of Zanthoxylum zanthoxyloides, a closely related plant to the one in this study. There are a number of possible explanations for the differences in biological activities. Firstly, it is known that similar plants growing in different geographical locations may be phytochemically very different 12. Secondly, the active constituents of various Zanthoxylum species vary greatly 16,4,3,9 and the taxonomy in certain cases is somewhat obscure (both scientifically and by local momenclature). This makes it usually necessary to specify not only the botanical sources used but also give some indication of the chemical composition. This is because, apart from the interspecific variation in chemistry, the extent of the variations of active constituents within a species (particularly from different geographical locations) is still not clear 13.
Thirdly, the difference in extraction procedures could also explain the differences in the findings. For instance, previous studies 14 indicated that chelerythrine, berberine and canthin-6-one are antibacterial components of Zanthoxylum spp. There has been also a report of antibacterial activity of two groups of compounds (phenolic acids and alkaloids), which occur in the root of Z. zanthoxyloides 11. In other studies 11, in which antibacterial activity was reported, the Zanthoxylum species was Soxhlet-extracted and the extract concentrated to a low volume before defatting with light petroleum. The precipitate observed was passed through a column of alumina. In all cases of this study 11, the alcoholic extracts produced the highest zones of inhibition. The antibacterial compound was characterized as canthin-6-one, which has also been isolated from Zanthoxylum elephantiasis. The observed differences in activity of extracts may, therefore, be attributable to the differences in extraction procedures.
Finally, the absence of antibacterial activity may suggest that the extracts of Z. chalybeum may act in an indirect way; the active ingredient may exist as a precursor, which requires activation in the body by some as yet unknown mechanism. It is known that skimmianine (isolated in this study from the seed of Z. chalybeum) has ephedrine-like action 15. and antihistaminic effect. It is possible that the extract may achieve effect via an immunopharmacological mechanism. This, however, requires further investigation.ACKNOWLEDGEMENTS
This study was funded by the GTZ veterinary project based at the Faculty of Veterinary Medicine, Makerere University. The phytochemical analyses were partly done at the Department of Chemistry, Addis Ababa University (AAU). We are grateful to Prof. E. Dagne and NAPRECA for part-support to enable D. Olila to do part of the study in Ethiopia. We thank Ms. Tenaye Asrat (AAU) for excellent technical assistance.REFERENCES
The antibacterial effect on S. aureus was higher than that on E. coli in the agar well assay. Both the alcohol and the water extracts showed antibacterial activity against both E. coli and S. aureus in the colony count assay (Tables 2 and 3).
Table 2: Cidal studies for Staph aureus and E. coli in tetracycline and 1% Gelatin as positive and negative controls respectively.
ND = Not done; TNTC = too numerous to count; GNS = % gelatin in normal saline; S.E = Standard error of the mean; CFUs = Colony forming units, S.A = S. aureus; E.C. = E. coli.
Table 3: Cidal studies for Staph. aureus and E. coli in Warburgia ugandensis extracts.
W = W. ugandensis; S.E = Standard error of the mean; CFUs = Colony forming units, S.A = S. aureus; E.C. = E. coli.
Table 4: Percentage bacterial kill or growth following incubation with W. ugandensis extracts (1% gelatin in normal saline and tetracycline used as negative and positive controls respectively).
TNTC = too numerous to count; GNS = % gelatin in normal saline; S.E = Standard error of the mean; S.A = S. aureus; E.C. = E. coli.; W. = Warburgia ugandensis; K = Kill; G = Growth
The alcohol extracts inhibited E. coli more; so that by 15 minutes of antibacterial effect on S. aureus was more pronounced than on E. coli in % CFU’s from 100.0 ± 24.0% (at zero time) to 0.0% CFU’s (Table 3). W. ugandensis showed antibacterial activity against S. aureus and E. coli in the agar well diffusion assay. This effect was not demonstrable in the paper disc assay (Table 1). The antibacterial effect on S. aureus was more pronounced than on E. coli in the agar well assay. Both the alcohol and the water extracts showed antibacterial activity against both E. coli and S. aureus in the colony count assay. The alcohol and water extracts also had antifungal activity against C. albicans (paper disc assay, Table 1).
Copyright 2001 - Makerere Medical School, Uganda
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