Antimicrobial activity of Tanzanian traditional medicinal plants

M.R. KHAN and M.H.H. NKUNYA

Department of Chemistry, University of Dar es Salaam
P.O. Box 35061, Dar es Salaam, Tanzania

ABSTRACT

A large number of plants used in traditional medicine were screened for antimicrobial activity. In the preliminary screening, Staphylococcus aureus (gram positive bacteria) and Escherichia coli (gram negative bacteria) were used to differentiate between active and non-active plant extracts. The extracts which showed activity were then screened for their antigonococcal and also for antifungal activity. A number of active plants were then phytochemically investigated to isolate the active components. A large number of different types of non-active compounds were also isolated and identified. There is some correlation between the activities and the traditional medicinal uses of the plants studied. Some of the compounds isolated could be responsible for the activity and use of the plants. This paper gives only the in-vitro screening and the results should be used with caution when applied to in-vivo effectiveness in humans. Screening needs to be done in-vivo and the toxicity aspect has to be studied very thoroughly before such crude plant extracts could be given as safe treatment with no serious consequences.

Introduction

In African and most developing countries traditional medicine still forms the backbone of rural medical practice. Medicinal herbs are extensively used for various ailments in these countries. This indicates that some of these medicines, if scientifically evaluated and standardized, could make very valuable medicaments. However, although a number of American (Lucas et al., 1951) and Australian (Atkinson et al., 1955) medicinal herbs have been screened for their medicinal properties, up to now there seem to be no serious attempts to evaluate African medicinal plants in a collective form for their biological activities and medicinal usefulness. However, there are scattered reports of such evaluations for individual or small groups of plants, as it will be noted in various presentations in this conference.

In the literature, it can be noted that Nickell (1959) is among the first researchers to compile an extensive review on biological (antibacterial) activity of vascular plants. Nickell's list of plants included only a few of Tanzanian medicinal plants. We therefore considered it worthwhile to investigate the in vitro antibacterial and antifungal activities of some of the Tanzanian medicinal plants, and ultimately to isolate and identify the active constituents (Sawhney et al., 1978a; Sawhney et al., 1978b: Khan et al., 1979).

We chose to screen the medicinal plants for antifungal activity because, of all human microbial infections, fungal diseases are the most difficult to modify in their course, or to prevent (Lucas et al., 1973; Taylor et al., 1961). It is now becoming more evident that the incidence of such diseases is increasingly becoming prominent.

From the literature (Kokwaro, 1976; Watt et al., 1962) and personal communications with Tanzanian traditional medical practitioners, we established that a number of herbs are used for the treatment of skin diseases, and many of them are said to be very effective. Thus the fruits of Solanum incanum, a weed which is widely distributed in East Africa, are extensively used for the treatment of cutaneous mycotic infections and other pathological conditions. The therapeutic action of the fruits has been attributed to solanine and related glycoalkaloids (Beaman-Mbaya et al., 1976). Similarly, the juice of Emilia sagittata is used for ring worms and athletic's foot. Although no chemical work is reported on this plant, a very potent antimicrobial and pharmacological agent, emiline (1), has been obtained from another plant of the same genus, E. flammea (Tomczyk et al., 1971).

Apart from using Staphylococcus aureus and Escherichia coli as test bacteria, we also included the essay of the crude plant extracts for their antigonococcal activity. This is because gonorrhoea is among the most common venereal diseases, both in rural and urban populations in Africa (Becker, 1973). Despite the introduction of sulphonamides and antibiotics, a large proportion of rural populations in developing countries still rely on local herbs for the treatment of gonorrhoea. Thus, in West Africa for example, cottonwood tree (Bombax sp.), Alchronea cordifolia, A. floribunda, Mussaenda elegans, Craterspermum laurinum and Aframomum baumannii are commonly used (Harley, 1970). There are also similar example in East Africa (Kokwaro, 1976).

In this paper we will give an overview of the results on the screening of crude plant extracts for their antibacterial, antigonococcal and antifungal activity and the phyto-chemical investigations on some of the most active plants.

Antibacterial activity

In all, 134 plant extracts were tested for their activity against S. aureus and E. coli in vitro. An extract which failed to inhibit the growth of the test bacteria was regarded as being inactive. Results are summarized in Table 1, in which the inactive extracts are not shown.

Phytochemical investigations on some of the most active extracts have revealed the active constituents of the plants. Thus the activity of Euclea natalensis can be attributed to 7- methyljuglone (2), mamegakinone (3) and diospyrin (4). These compounds, which were isolated from the plant, have been found to be active against S. aureus and a few other bacteria (Table 2).

The antibacterial activity of Harrisonia abyssinica root bark, which showed an activity against S. aureus, comparable to 5 units of penicillin G, has been traced to be due to the limonoid harrisonin (5) (Kubo et al., 1976). The latter compound, which was the only active component of this plant, showed a minimum inhibitory concentration of 5 mg/ml (Mosile, 1980).

Another most active plant is Acacia nilotica. This plant is known to contain phenylethyl alkaloids and flavonoids. Although these compounds have not been tested, we found the activity to be concentrated in the acidic fraction of the extract, which contains the flavonoids.

Active compounds which have been isolated from some of the most active plant extracts are shown in Chart 1.

Antigonococcal activity

In this category of assay, extracts from 88 Tanzanian medicinal plants were tested for their in vitro activity against Neisseria gonorrhoea isolates from clinical cases, which were isolated and maintained at the Department of Microbiology and Immunology, Faculty of Medicine, University of Dar es Salaam (Sawhney et al, 1978a). Results are shown in Table 3. It is interesting to note that some of the plants used locally for the treatment of gonorrhoea are very active against the pathogenic bacteria. Furthermore, 82% of the plants listed in Table 4 were also active against S. aureus. More than 40% of the plant extracts without antigonococcal activity showed various levels of inhibition of S. aureus. This, in a way, ruled out the effect of nonspecific factors, such as acidity, on the observed activity.

Antifungal activity

In all, 124 plants were screened for activity against the common dermatophyte, Trichophyton mentagrophytes, as well as Candida albicans. Results are summarized in Table 4.

As it can be noted in Table 4, the highest level of antifungal activity was exhibited by extracts of Emillia sagittata, Securrinega virosa (pulp) and Sida serratifolia (roots) (Sawhney et al., 1978b). Apparently, none of these plants is used to treat dermatomycoses in East Africa. Instead these plants are used for miscellaneous ailments, such as eye inflammation, topical dressing for wounds and contusions, diarrhoea, gonorrhoea, pneumonia, pulmonary tuberculosis and dysentery, most of which are bacterial diseases (Kokwaro, 1976; Watt et al, 1962). Incidentally, among the above plants only S. serratifolia showed antibacterial activity in vitro (Table 1). Such results may suggest that either the antibacterial activity is exhibited only in vivo, in patients, or the plants are used just as a matter of tradition. Again, the observed antifungal activity, despite the plants not being used traditionally for fungal related diseases, gives us a good indication that a lot is yet to be discovered regarding the diverse usefulness of medicinal plants.

Phytochemical investigations

We have carried out extensive phytochemical investigations on some of the most active plants shown in Tables 1, 4 and 5, with the aim of isolating the active constituents. Thus from Euclea natalensis we isolated several naphthaquinones, among which the active ones are listed in Table 2 (Khan et al., 1979).

Several triterpenoids and naphthaquinones have been isolated from various Diospyros species (Ebenaceae), but only 7-methyljuglone, diospyrin and mamegakenone were the active compounds in this series. Eleven Cassia species have been analysed for their constituents, and in addition to emodine (6), aloe-emodine (7) and barakol (8), several other anthraquinones have been isolated, some of which were obtained for the first time (Mutasa, et al., 1990).

Maerua angolensis (Capparidaceae) is among the plants which exhibited a high antifungal activity. We have isolated several C₁₂, C₁₄ and C₁₈ fatty acids and esters from this plant, and most of these compounds showed antifungal activity (Nkunya, 1985).

Among the plants of the family Annonaceae, which were included in the screening tests, were those belonging to the genus Uvaria. In the literature some of these plants are reported to possess a wide range of biological activities. Furthermore, these plants have been found to contain compounds with interesting chemical structures, some of which are also the active components of the plant extracts. These findings prompted us to carry out extensive phytochemical investigations of these plants. In the course of these investigations, we have isolated more than forty compounds from nine Uvaria species found in Tanzania. An account of these compounds, regarding their biological activities, has been given by Nkunya (1990, this conference), in a paper on the antimalarial activity of the compounds. Apart from this, the compounds have shown activity against some bacteria and tumour cells. Among the active compounds are (+)-b-senepoxide (9), (+)-pandoxide (10) and (-)-pipoxide (11) (Nkunya et al, 1986). Results on the antibacterial activity are shown in Table 5.

Conclusion

The results discussed in this paper do not claim that the plants we have investigated and the pure compounds therefrom are safe medicines. Their efficacy and safety can only be established by very careful toxicity and pharmacological studies, followed by clinical trials using usual protocols. Our results definitely have provided a basis for further investigations on similar lines, as well as on the toxicity and pharmacological aspects of the extracts, and pure compounds. We hope that the leads presented here will be pursued exhaustively by the scientific community.

References

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Beaman-Mbaya, V. and Muhammed, S. I. (1976). Antimicrob. Agents Chemother. 9: 920 - 924.

Becker, N. L. (1973). In Clinical Medicine in Africans in Southern Africa. Campbell, G.D., Seedat, Y.K. and Daynes, G. (Eds). Churchill/Livingstone, London: 465.

Harley, G.W. (1970). Native African Medicine, Frank Cass, London.

Khan, M.R., Mutasa, S.L., Ndaalio, G., Wevers, H. and Sawhney, A.N. (1978): Pakistan J. Sci. Ind. Res. 21: 197 - 199.

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Kokwaro, J. O. (1976). Medicinal Plants of East Africa, East African Literature Bureau, Nairobi.

Kubo, I., Tanis, S. P., Lee, Y., Miuva, F., Nakanishi, K. and Chapya, A. (1976). Heterocycles 5: 485.

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Lucas, E. H., Lickfield, A., Gottshall, F. and Jennings, J. C. (1951). Bull. Torrey Bot. Club 78: 310 - 321.

Mosile, F. W. (1980). Chemical studies and antimicrobial activity of some Tanzanian medicinal plants: M.Sc, Thesis, University of Dar es Salaam.

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Nickell, L. G. (1959). Econ. Bot. 13: 281 - 318.

Nkunya, M. H. H. (1985). A search for potentially useful compounds from some Tanzanian plants: In Proc. Sci. Symp. Univ. Dar es Salaam, Publisher: Tanzania Commission for Science and Technology: 73-75.

Nkunya, M. H. H. and Weenen, H. (1986). Chemical investigations of a Tanzanian medicinal plant: Uvaria pandensis Verdc (Annonacese). In: Proc. 3rd Internat. Chem. Conf. Africa, Lome (Togo): 313 - 317.

Nkunya, M. H. H. (1990). Chemical evaluation of Tanzania Medicinal Plants for active constituents as a basis for the medicinal usefulness of the plants. In Proc., this conference.

Sawhney, A. N., Khan, M.R., Ndaalio, G., Nkunya, M.H.H. and Wevers, H. (1978a). Pakistan J. Sci. Ind. Res. 21: 189 - 192.

Sawhney, A. N., Khan, M.R., Ndaalio, G., Nkunya, M. H. H. and Wevers, H. (1978b). Pakistan J. Sci. Ind. Res. 81: 193 - 196.

Taylor, E. P. and D'Arcy, P. F. (1961). Progress in Medicinal Chemistry, Plenum Press, New York: 220.

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Table 1: Susceptibility of Staphylococcus aureus and Escherichia coli to various plant extracts

Table 1a: Sensitivity of test organisms against a number of standard antibiotics

Table 2: Susceptibility of some microorganisms to some naphthoquinones

The 10 - 15mm zone of inhibition is comparable to the one caused by 25 mg of tetracycline

Table 3: In vitro antigonococcal activity of some Tanzanian medicinal plants

The following plants did not show any antigonococcal activity:

Acanthaceae: Barleria prionitis L. (roots, leaves and bark); Amaranthes aspera L. (plant);

Anacardiaceae: Rhus natalensis Bernh. (leaves), Lannea stuhlmannii Engl. (leaves);

Annonaceae: Anona senegalensis Pers (bark), Uvaria acuminata Oliv. (roots);

Apocynaceae: Calotropis gigantea Ait. f. (leaves), Dictyophleba lucida Pierre. (leaves, trunk), Nerium oleander L. (leaves), Plumeria rubra L. (bark);

Araceae: Stylochiton hennigii Engl. (roots and leaves);

Boraginaceae: Ehretia amoena Klotzch. (root bark);

Capparidaceae: Boscia salifolia Oliv. (bark, leaves), Maerua angolensis DC. (bark); Carica papaya L. (leaves, roots, (bark);

Celastraceae: Elaeodendron schlechteranum Loes. (roots);

Combretaceae: Combretum zeyheri Sond. (fruits, plant), Terminalia catappa L. (leaves); Compositae: Aspilia natalensis Willd. (roots), Emilia sagittata D.C. (plant);

Convolvulaceae: Bonamia mossambicensis Hall. f. (roots);

Cyperaceae: Cyperus rotundus L. (tuber);

Ebenaceae: Diospyros mespiliformis Hochst ex DC (leaves);

Euphorbiaceae: Acalypha fruticosa Forsk, (roots), Fluggea virosa Baill. (bark), Phylanthus niruri L. (plant), Pseudolachmaestylis maprouncaefolia Pax. (bark), Securinega virosa Baill, (bark, pulp);

Icacinaceae: Pyrenacantha kaurabassana Baill (tuber, green fruits); Labiatae: Hoslundia opposita Vahl. (leaves), Leonotis nepetaefolia R. Br. (plant);

Lauraceae: Cassytha filiformis L. (plant);

Leguminosae: Acacia robusta Burch (rootbark), A. Senegal Wild. (roots), Adenanthera pavonina L. (seeds), Caesalpinia pulcherrina Swartz (bark), Cassia fistula L. (bark), C. amiculata L. (seeds and bark), Desmodium sp. (plant), Dichrostachys cinerea Wight. Am. (roots), Peltophorum petocarpum K. (roots, bark), Pongania pinnata L. (leaves, rootbark, seeds), Pterocarpus angolensis DC (bark), Stylosanthes fruticosa Alston. (plant), Xeroderris stuhlmannii Taub. (plant);

Liliaceae: Asparagus falcatus L. (plant);

Malvaceae: Sida spinosa L. (leaves);

Rhamnaceae: Ziziphus pubescens Oliv (leaves);

Rubiaceae: Lamprathamnus zanguebaricus Hiern. (leaves);

Rutaceae: Citrus aurantifolia Swingle. (roots);

Sapindaceae: Allophylus rubifolius Engl. (stem);

Solanaceae: Withania somnifera Dun (plant);

Sterculiaceae: Dombeya shupangae K. Schum (leaves), Melhania velutina Forsk. (leaves), Waltheria indica L. (flowers, leaves);

Tiliaceae: Corchorus olitorius L. (fruits and seeds), Grewia forbesii Hary ex Mast. (bark and roots), G. Stuhlmannii K. Schum (roots), Trimimfetta rhomboidea Jacq. (bark and roots);

Verbenaceae: Lantania camara L. (leaves), Vitex fischeri Guerke. (leaves), Vitex sp. (roots);

Vitaceae: Cissus integrifolia Manch. (stem), Rhoicissus rovoilii Planch (roots).

Table 4: Susceptibility of fungi to various plant extracts

Plant extracts which did not show any in vitro antifungal activity:

Acanthaceae: Barleria prionitis L. (roots, leaves and bark);

Amaranthaceae: Achyranthes aspera L. (plant);

Anacardiaceae: Rhus natalensis Bernh. (leaves), Lannea stuhlmannii Engl. (leaves);

Annonaceae: Anona senegalensis Pers. (bark), Uvaria acuminata Oliv. (leaves, roots);

Apocynaceae: Calotropis gigantea Ait. F. (leaves), Nerium oleander L. (leaves); Stylochiton hennigii. (roots and leaves);

Bignoniaceae: Kigelia africana Benth. (bark), Tecomaria capensis Spach. (leaves);

Boraginaceae: Ehretia amoena Klotzch. (root bark);

Capparidaceae: Boscia salicifolia Oliv. (leaves), Maerua angolensis DC. (bark, leaves);

Caricaceae: Carica papaya L. (green fruits, bark);

Celastraceae: Elaeodendron schlechteranum Loes. (roots, leaves);

Combretaceae: Combretum zeyheri Sond: (fruits), Terminalia catappa L. (leaves); Compositae: Vernonia hildebranditii Vatke, (leaves and stem), V. cinerea Less. (plant);

Connaraceae: Byrsocarpus orientalis Bak. (plant);

Dilleniceae: Tetracera boiviniana Baill. (rootbark);

Ebenaceae: Diospyros mespiliformis Hochst. ex. DC (leaves);

Euphorbiaceae: Acalypha fruticosa Forsh (leaves, roots), Antidesma venosum E. May. (root bark), Bridelia cathartica Bertol. f. (leaves), Euphorbia hirta L. (plant), Fluggea virosa Baill, (bark), Phyllanthus reticulatus Poir. (leaves), Securinega virusa Baill (roots);

Icacinaceae: Pyrenacantha caurabassana Baill (tuber, green fruits); Labiatae: Hoslundia opposita Vahl (leaves), Leonotis nepetaefolia R. Br. (plant);

Lauraceae: Cassytha piliformis L. (plant);

Leguminosae: Acacia mellifera Vehl. (bark), A. robusta Burch. (root bark), A. senegal Willd. (roots), Adenanthera pavonina L. (seeds, leaves), Bauhinia reticulata DC. (plant), Caesalpinia pulcherrina Swartz. (flowers, rootbark), Cassia fistula L. (bark), C. obtusifolia L. (plant), C. occidentalis L. (plant), Desmodium sp. (plant), Dichrostachys cenerea Wight. Arn.(stem), Peltophorum petocarpum K. (roots, bark), Pongamia pinnata P. (leaves and rootbark), Pterocarpus angolensis DC. (bark), Stylosanthes fruticosa Alston. (plant);

Liliaceae: Asparagus sp. (plant);

Loganiaceae: Strychnos madagascarensis Poir. (root bark);

Malvaceae: Malvastrum coromandelianum Garcke. (plant), Sida cordifolia L. (roots), S. serratifolia L. (plant), S. spinosa L. (roots, leaves);

Ochnaceae: Brackenridgea Zanguebarica Oliv. (root bark);

Rhamnaceae: Zizyphus pubescens Oliv. (stem);

Solanaceae: Withania somnifera Dun. (plant),

Sterculiaceae: Dombeya shupangae K. Schum. (bark, leaves), Melhania velutina Forsk. (leaves), Waltheria indica L. (leaves);

Tiliaceae: Corchorus olitorius L. (fruits and seeds), Grewia stuhlmannii K. Schum. (roots), Triumfetia rhomboidea jacq. (bark and roots);

Verbenaceae: Lantana camara L. (leaves), Vitex sp. (plant, roots);

Vitaceae: Cissus rotundifolia Vahl. (leaves),

Table 5: Zones of inhibition of bacterial growth (nun) by (+)-b-senepoxide and (+)-pandoxide

Both the compounds showed bacteristatic activity and no bactericidal properties. (+)-b-Senepoxide showed a minimum inhibitory concentration (MIC) of 62.5 mg/ml.

Chart 1: Some antibacterial compounds from Tanzanian medicinal plants.

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