Antifungal activities of six South African Terminalia species (Combretaceae)

Antifungal activities of six South African Terminalia species (Combretaceae)

Journal of Ethnopharmacology 99 (2005) 301–308 Ethnopharmacological Communication Antifungal activities of six South African Terminalia species (Com...

368KB Sizes 0 Downloads 99 Views

Journal of Ethnopharmacology 99 (2005) 301–308

Ethnopharmacological Communication

Antifungal activities of six South African Terminalia species (Combretaceae) P. Masoko a , J. Picard b , J.N. Eloff a,∗ a

b

Phytomedicine Programme, Department of Paraclinical Sciences, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa Department of Veterinary Tropical Diseases, Faculty of Veterinary Sciences, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa Received 10 September 2004; received in revised form 24 January 2005; accepted 25 January 2005 Available online 22 April 2005

Abstract A serial microplate dilution method developed for bacteria was modified slightly and gave good results with several fungi. The antifungal activity of acetone, hexane, dichloromethane and methanol leaf extracts of six Terminalia species (Terminalia prunioides, Terminalia brachystemma, Terminalia sericea, Terminalia gazensis, Terminalia mollis and Terminalia sambesiaca) were tested against five fungal animal pathogens (Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis and Sporothrix schenkii). Methanol extracted the highest quantity, but the acetone extracts had the highest antifungal activity. Some of the extracts had antioxidant activity. Most of the antifungal extracts had MIC values of c. 0.08 mg/ml, some were with MIC values as low as 0.02 mg/ml. Microsporum canis was the most susceptible microorganism and Terminalia sericea extracts were the most active against nearly all microorganisms tested. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Combretaceae; Terminalia species; Antifungal activity; MIC

1. Introduction A large proportion of the South African population use traditional medicine to serve the health needs of humans and animals. Medicinal plants have become the focus of intense study recently in terms of conservation and as to whether their traditional uses are supported by actual pharmacological effects or merely on folklore (Locher et al., 1995). With the increasing acceptance of herbal medicine as an alternative form of health care, the screening of medicinal plants for bioactive compounds is important. More than 80% of the population in developing countries depend on plants for their medical needs (Farnsworth, 1988; Balick et al., 1994). Medicinal and poisonous plants have always played an important role in African society. Traditions of collecting, processing and applying plants and plant∗

Corresponding author. Tel.: +27 12 529 8244; fax: +27 12 529 8252. E-mail address: [email protected] (J.N. Eloff).

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.01.061

based medications have been handed down from generation to generation. In South Africa and also in many other African countries, traditionally used medicinal plants are sold in market places or prescribed by traditional healers in their homes (Fyhrquist et al., 2002). Because of this strong dependence on plants as medicines, it is important to study their safety and efficacy (Farnsworth, 1994). Many medicinal plants produce a variety of compounds of known therapeutic properties. Substances that can either inhibit the growth of pathogens or kill them and have little or no toxicity to host cells are considered good candidates for developing new antimicrobial drugs. In recent years, antimicrobial properties of medicinal plants have been increasingly reported from different parts of the world (Cowan, 1999). Higher plants are still regarded as potential sources of new medicinal compounds. Throughout the world, plants are used traditionally to treat many ailments, particularly infectious diseases, such as diarrhoea, fever and colds (Mitscher et al., 1987).

302

P. Masoko et al. / Journal of Ethnopharmacology 99 (2005) 301–308

Members of the Combretaceae are used for many medicinal purposes by traditional healers. This includes treating abdominal disorders, abdominal pains, backache, bilharziasis, chest coughs, colds, conjunctivitis, diarrhoea, dysmenorrhoea, earache, fattening babies, fever, headache, hookworm, infertility in women, leprosy, pneumonia, scorpion bite, snake bite, swelling caused by mumps, syphilis, toothache, gastric ulcer, venereal diseases, heart diseases, cleanse the urinary system, dysentery, gallstones, sore throats, nosebleeds and general weakness (Hutchings et al., 1996; Van Wyk et al., 1997). The Combretaceae consists of 18 genera, the largest of which are Combretum with about 370 species, and Terminalia with about 200 species (McGaw et al., 2001). Species from the genus Combretum and to a lesser extent Terminalia are most widely used for medicinal purposes, as they are common and widely distributed throughout western and southern Africa (McGaw et al., 2001). Many members of the Combretaceae are easily characterized by the wing-shaped appendages of the fruits (Carr, 1988). Several investigations into the antimicrobial activity of members of the Combretaceae have been undertaken in recent years. Although the antibacterial properties of various Terminalia species (Silva et al., 1996; Eloff, 1999; Fyhrquist et al., 2002) have been investigated in depth, this is not the case regarding their antifungal properties. Antifungal activity of Terminalia extracts was demonstrated, but no quantitative data was provided (Bhatt and Saxena, 1979; Baba-Moussa et al., 1998). This may be because the adequate methods for determining MIC values are not available. Our aim in this work was to expand a serial microplate technique developed for bacteria (Eloff, 1998c) for fungi in order to determine quantitative data on antifungal activities of Terminalia species occurring in southern Africa. Further motivation was that antifungal agents are becoming more important to combat infections of immune-compromised patients suffering from the acquired immune-deficiency syndrome

(AIDS) and Terminalia species occuring widely in southern Africa.

2. Materials and methods 2.1. Plant collection Leaves were collected in summer from trees in the Lowveld National Botanical Garden in Nelspruit, South Africa. Voucher specimens in the garden herbarium verified the identity of the plants. Leaves collected were from the following five species with the section (Carr, 1988) in brackets: Terminalia prunioides M.A. Lawson (Abbreviatae), Terminalia brachystemma Welw. ex Hiern (Psidiodes), Terminalia sericea Burch ex DC (Psidiodes), Terminalia gazensis Bak.f. (Platycarpae), T. mollis Laws. (Platycarpae) and T. sambesiaca Engl. & Diels. (Platycarpae). 2.2. Plant storage Leaves were separated from stems and dried at room temperature. Most scientists have tended to use dried material because there are a few problems associated with large-scale extraction of dried plants rather than fresh plant material (Eloff, 1998a). The dried plants were milled to a fine powder in a Macsalab mill (Model 200 LAB), Eriez® , Bramley, and stored at room temperature in closed containers in the dark until used. 2.3. Extraction procedure Plant samples from each species were individually extracted by weighing four aliquots of 1 g of finely ground plant material and extracting with 10 ml of acetone, hexane, dichloromethane (DCM) or methanol (technical grade – Merck) in polyester centrifuge tubes. Tubes were vigor-

Fig. 1. Percentage of samples extracted by acetone (), hexane (), dichloromethane , and methanol , from the six Terminalia species: T. pru.: Terminalia prunioides, T. bra.: Terminalia brachystemma, T. ser.: Terminalia sericea, T. gaz.: Terminalia gazensis, T. mol.: Terminalia mollis and T. sam.: Terminalia sambesiaca.

P. Masoko et al. / Journal of Ethnopharmacology 99 (2005) 301–308

303

Fig. 2. Chromatograms sprayed with vanillin to show compounds extracted with acetone (Ac), hexane (Hex), dichloromethane (D) and methanol (Met), in lanes from left to right for each group.

ously shaken for 3–5 min in Labotec model 20.2, shaking machine at a high speed. After centrifuging at 3500 rpm for 10 min, the supernatant was decanted into pre-weighed labeled containers. The process was repeated three times to

exhaustively extract the plant material and the extracts were combined. The solvent was removed under a stream of air in a fume cupboard at room temperature to quantify the extraction.

Fig. 3. Chromatograms of extracts of different Terminalia species separated by EMW and sprayed with 0.2% DPPH, clear zones indicate antioxidant activity. Lanes from left to right in each group are acetone (Ac), hexane (Hex), dichloromethane (D) and methanol (Met).

304

P. Masoko et al. / Journal of Ethnopharmacology 99 (2005) 301–308

0.38 0.54 0.53 0.36 0.43 0.41 0.69 0.37 0.45 0.39 0.41 0.36 0.49 0.46 0.46 0.47 0.54 0.38 0.41 0.45 0.43 0.4 0.38

0.02 0.08

24 48

24 48

Sporothrix schenckii

Microsporum canis

0.4

0.04 0.32

24 48 Aspergillus fumigatus

Average

0.16 2.5

24 48 Cryptococcus neoformans

2.5. Antioxidant activity Positive control: Amphotericin B (Amph.B). a Values in ␮g/ml; originally all were <20 ␮g/ml in combined experiment, data provided here was determined in separate experiments.

0.4

0.2

Chemical constituents of the extracts were analyzed by thin layer chromatography (TLC) using aluminium-backed TLC plates (Merck, silica gel 60 F254 ). The TLC plates were developed under saturated conditions with one of the three eluent systems developed in our laboratory that separates components of Combretaceae extracts well, i.e., ethyl acetate/methanol/water (40:5.4:5): [EMW] (polar/neutral); chloroform/ethyl acetate/formic acid (5:4:1): [CEF] (intermediate polarity/acidic); benzene/ethanol/ammonia hydroxide (90:10:1): [BEA] (non-polar/basic) (Kotze and Eloff, 2002). To detect the separated compounds, vanillin-sulphuric acid (0.1 g vanillin (Sigma® ): 28 methanol: 1 ml sulphuric acid) was sprayed on the chromatograms and heated at 110 ◦ C to optimal colour development.

0.03 0.1 0.02 0.02 0.08 0.08 0.08 0.08 0.02 0.02 0.02 0.08 0.02 0.16 0.02 0.08 0.02 0.08 0.02 0.16 0.02 0.16

0.02 0.16

0.02 0.16

0.02 0.16

0.02 0.32

0.02 0.04

0.02 0.08

0.02 0.08

0.02 0.08

0.02 0.04

0.02 0.08

0.02 0.02

0.02 0.08

0.02 0.04

0.05 0.27 0.04 0.02 0.04 0.16 0.04 0.04 0.02 0.04 0.02 0.04 0.08 0.16 0.08 0.16 0.08 0.32 0.04 0.64 0.04 0.32

0.04 0.32

0.02 0.64

0.02 0.32

0.04 0.64

0.08 0.32

0.02 0.64

0.02 0.64

0.02 0.32

0.08 0.08

0.04 0.16

0.08 0.08

0.04 0.16

0.08 0.02

0.3

0.4

0.2 0.45 2.5 0.16 2.5 1.25 2.5 1.25 2.5 0.16 2.5 0.32 2.5 0.16 2.5 0.64 2.5 0.32 2.5 0.16 2.5 0.16 2.5

0.32 2.5

0.16 2.5

0.08 2.5

0.16 2.5

0.16 2.5

0.32 2.5

0.16 2.5

0.64 2.5

0.16 2.5

0.32 2.5

0.32 2.5

2.5 2.5

0.64 2.5

0.12 0.15 0.02 0.02 0.16 0.16 0.16 0.16 0.02 0.02 0.08 0.08 0.08 0.08 0.16 0.16 0.02 0.02 0.04 0.04 0.16 0.16 0.08 0.08 0.04 0.04 0.16 0.16 0.16 0.32 0.08 0.16 0.16 0.16 0.32 0.16 0.08 0.32 0.16 0.16 0.16 0.32 0.16 0.16 0.04 0.16 0.08 0.16

24 48 Candida albicans

Terminalia sambesiaca Terminalia mollis Terminalia gazensis Terminalia sericea Terminalia brachystemma

Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol 0.16 0.16 0.16 0.32 0.32 0.16 0.16 0.64 0.64 0.16 0.08 0.32 0.32 0.16 0.16 0.64 0.32 0.64 0.32 0.16 0.16 0.32 0.32 0.32 0.3 0.16 0.16 0.16 0.32 0.32 0.16 0.16 0.64 0.64 0.64 0.64 0.64 0.32 0.32 0.32 0.64 0.32 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.46

Microorganisms Time MIC values (mg/ml) (h) Terminalia prunioides

Table 1 Minimum Inhibitory Concentration (MIC) of six Terminalia species after 24- and 48-h incubation at 37 ◦ C

0.32 0.32

Average Amph.Ba

2.4. Phytochemical analysis

TLC was used to separate extracts as above. To detect antioxidant activity, chromatograms were sprayed with 0.2% 1,1-diphenyl-2-picryl-hydrazyl (Sigma® )(DPPH) in methanol as an indicator (Deby and Margotteaux, 1970). 2.6. Fungal test organisms Five microorganisms namely, Candida albicans, Cryptococcus neoformans var. gattii, Aspergillus fumigatus, Sporothrix schenckii and Microsporum canis cultured from clinical cases of disease in animals were used in the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science. Candida albicans was isolated from a Goldian finch, Cryptococcus neoformans from a cheetah, and Aspergillus fumigatus from a chicken, all of which suffered from a systemic mycosis. Microsporum canis was isolated from a cat suffering from dermatophytosis and Sporothrix schenckii from a horse with cutaneous lymphangitis. None of the animals had been treated prior to sampling. These fungi represent the different morphological forms of fungi, namely yeasts (Candida albicans and Cryptococcus neoformans), thermally dimorphic fungi (Sporothrix schenckii) and moulds (Aspergillus fumigatus) and are the most common and important diseasecausing fungi of animals. All fungal strains were maintained on Sabouraud dextrose agar (Oxoid, Basingstoke, UK). 2.7. Antifungal assays 2.7.1. Microdilution assay A serial microdilution assay with tetrazolium violet added as growth indicator (Eloff, 1998c) was used to determine the minimum inhibitory concentration (MIC) values for plant extracts. This method had previously been used only for antibacterial activities (Eloff, 1998c; McGaw et al., 2001). Motsei et al. (2003) also used a serial microplate dilution assay in determining the antifungal activity of 24 South African medicinal

Average

859

269

453

261

776

321

617

128

621

461

663

27

1303

118

186

1173

1102

121

307

2085

3242

187

157

830

2036 799 1450 1450 263 263 250 250 5600 5600 6050 3025 900 225 550 138 2350 127 3900 975 900 113 500 125 4000 2000 100 25 2300 575 1600 400 2750 1375 650 41 2050 256 1150 144 3750 469 1300 325 1700 213 3700 925 24 48 Microsporum canis

1100 138

1150 692 725 1450 525 131 500 500 5600 2800 1513 6050 900 450 275 69 588 2350 1950 488 225 113 125 63 1000 1000 100 6 2300 72 1600 50 688 172 325 20 2050 128 1150 36 1875 234 325 81 850 53 1850 231

550 69

194 41 181 12 17 8 16 8 700 45 189 48 56 7 4 4 19 588 244 31 113 7 16 4 500 32 3 1 288 18 100 13 344 22 81 5 513 16 144 9 234 30 81 10 213 14 138 9

807 645 1450 1450 131 131 125 125 5600 5600 1513 1513 225 225 69 69 2350 147 1950 1950 113 113 125 125 2000 2000 13 13 288 144 400 200

Terminalia sericea

344 344 41 81 513 128 144 144 469 234 163 163 850 213 275 138

24 48

0.68 0.75 0.80 0.64 0.62 0.66

Sporothrix schenckii

48 h

0.124 0.146 0.163 0.162 0.293 0.232

463 30

24 h Terminalia prunioides Terminalia brachystemma Terminalia sericea Terminalia gazensis Terminalia mollis Terminalia sambesiaca

24 48

Average MIC values (mg/ml)

Aspergillus fumigatus

Terminalia ssp.

231 231

Table 3 Average MIC values of different Terminalia species

24 48

0.259 0.147 0.146 0.195

Cryptococcus neoformans

Hexane DCM Acetone Methanol

24 48

Average MIC values (mg/ml)

Terminalia brachystemma

Extractants

Time Total activity (ml/g) (h) Terminalia prunioides

Table 2 Average MIC values for different extractants on all test organisms

Microorganisms

Six Terminalia species were selected for antifungalactivity screening based on their use in traditional medicinal treatments for both domestic animals and humans in southern Africa and availability. The majority of traditional healers use water to isolate active compounds from these plants, because water is not harmful to domestic animals and humans and is generally the only extractant available. Using only water

Table 4 Total activity in ml/g of six Terminalia species after 24- and 48-h incubation at 37 ◦ C

3. Results and discussion

Candida albicans

Average Terminalia gazensis

Terminalia mollis

Terminalia sambesiaca

plants to three Candida albicans isolates. They determined growth with an ELISA reader. In our experience, measuring growth by turbidity measurement has several complications (Eloff, 1998c). By applying the tetrazolium violet assay for measuring the antifungal activities, a slight modification was made to suit fungal growth conditions. Residues of the different extracts were dissolved in acetone to a concentration of 10 mg/ml. The plant extracts (100 ␮l) were serially diluted up to 50% with water in 96 well microtitre plates (Eloff, 1998c). Fungal cultures were transferred into fresh Sabouraud dextrose broth, and 100 ␮l of this was added to each well. Amphotericin B was used as the reference antibiotic and positive control, and appropriate solvent blanks were included. As an indicator of growth, 40 ␮l of 0.2 mg/ml of p-iodonitrotetrazolium violet (Sigma® ) (INT) dissolved in water was added to each of the microplate wells. The covered microplates were incubated for 2–3 days at 35 ◦ C and 100% relative humidity. The MIC was recorded as the lowest concentration of the extract that inhibited antifungal growth after 24 and 48 h. One is tempted to consider the 48-h value as a minimal lethal concentration, especially since no growth was apparent in the particular well after 120 h. When the cells from wells showing no growth after 48 h were incubated in fresh growth medium, fungal growth however resumed. We are following up on this matter.

305

Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol Acetone Hexane DCM Methanol 463 138 213 81 234 144 256 20 86 200 575 6 250 63 113 122 147 17 56 756 700 63 66 91 202 463 138 213 81 234 144 256 20 86 50 72 3 250 31 56 122 2350 17 28 189 175 31 33 45 212

P. Masoko et al. / Journal of Ethnopharmacology 99 (2005) 301–308

306

P. Masoko et al. / Journal of Ethnopharmacology 99 (2005) 301–308

leads to difficulties in extracting non-polar active compounds. Success in isolating compounds from plant material is largely dependent on the type of the solvent used in the extraction procedure. The total percentages extracted by using different solvents (acetone, hexane, DCM and methanol) are shown in Fig. 1. Methanol was the best extractant, extracting a greater quantity of plant material than any of the other solvents. There was a major difference in the methanol extractability of Terminalia gazensis leaves compared with all the other species. This difference is not related to the sectional division of the species (Carr, 1988). The extract probably contained more polar compounds (Fig. 2) that may not be that interesting for clinical application. Hexane, dichloromethane and methanol extracts were redissolved in acetone because this solvent was not found to be harmful towards bacteria (Eloff, 1998b). We found that acetone was also not harmful towards fungi at the concentrations used in the plant extracts. Because fungi grow slower than bacteria, we modified Eloff’s (1998c) method by adding the tetrazolium violet before incubating the fungi. This made it possible to see when sufficient growth had taken place to determine MIC value. There was similarity in the chemical composition of the non-polar components of extracts using extractants of varying polarity. This may indicate the presence of saponin-like compounds in the leaves. The acetone and methanol extracts had antioxidant activity after spraying chromatogram with 0.2% DPPH (Fig. 3). Hexane and dichloromethane extracts apparently did not have any antioxidant activity. To determine MIC values, growth was checked after 24 and 48 h (Table 1) to determine the end point. The MIC values of most of the extracts were in the order of 0.08 mg/ml and some had values as low as 0.02–0.04 mg/ml after 24-h incubation. Amphotericin B was used as a positive control and there was no growth in any of the wells containing antibiotic indicating an MIC < 0.02 mg/ml. In subsequent experiments, the MIC value for Candida albicans, Cryptococcus neofor-

mans var. gattii, Sporothrix schenckii and Microsporum canis were 0.4, 0.3, 0.4, and 0.2 ␮g/ml, respectively, after 48-h incubation and for Aspergillus fumigatus, it was 0.2 ␮g/ml after 24 h. The average value for all the extracts and Terminalia species after 24 h was 0.3 mg/ml (Table 1). Motsei et al. (2003) found much less activity with different plants and extractants. Of the 273 extracts evaluated, 211 (77%) had MIC values as high or even higher than the highest concentration tested (8.35 mg/ml). This indicates that the tested plants were much less active than the Terminalia species, or that the technique they used was not as sensitive as the tetrazolium violet technique we used. Acetone and DCM extracts had the lowest average MIC values on the tested organisms (Table 2) which were 0.146 and 0.147 mg/ml, respectively, after 24 h. Acetone and DCM are followed by methanol which had 0.195 mg/ml after 24 h, and hexane had the highest average MIC value after 24 h which was 0.259 mg/ml. Terminalia prunioides, Terminalia brachystemma, Terminalia sericea and Terminalia gazensis had the lowest average values after 24 h (Table 3), which were 0.124, 0.146, 0.163 and 0.162 mg/ml, respectively. All the Terminalia species had almost the same average MIC value after 48 h ranging between 0.62 and 0.80 mg/ml. The quantity of antifungal compounds present was also determined in Table 4. To determine which plants can be used for further testing and isolation, not only the MIC value is important, but also the total activity. Because the MIC value is inversely related to the quantity of antifungal compounds present, an arbitrary measure of the quantity of antifungal compounds present was calculated by dividing the quantity extracted in milligrams from 1 g leaves by the MIC value in mg/ml. This value indicates the volume to which the biologically active compound present in 1 g of the dried plant material can be diluted and still kill the fungi (Eloff, 1999). Extracts with higher values were considered the best to work with. From Table 4, all 96 extracts had substantial total

Fig. 4. The average antifungal activity of different leaf extracts of six Terminalia species towards Candida albicans , Cryptococcus neoformans (), Aspergillus fumigatus () Sporothrix schenckii , Microsporum canis . (Antifungal activity expressed as the inverse of MIC in mg/ml indicating the volume to which 1 mg of extract can be diluted and still inhibit the growth of the fungus).

P. Masoko et al. / Journal of Ethnopharmacology 99 (2005) 301–308

Fig. 5. The sensitivity of different fungal pathogens to different acetone (), hexane (), dichloromethane (Antifungal activity expressed as inverse of MIC in mg/ml).

activity against Microsporum canis followed by Sporothrix schenckii, especially after 24 h. Cryptococcus neoformans and Candida albicans were also reasonably sensitive, but Aspergillus fumigatus was relatively resistant. All of the six tested Terminalia species (Fig. 4) had antifungal properties against the tested organisms. The values are expressed as the reciprocal of MIC, which means that 1 mg of the acetone extract diluted to 50 ml would still inhibit the growth of Microsporum canis. Microsporum canis and Sporothrix schenckii were highly sensitive and Aspergillus fumigatus was less sensitive (Fig. 5). The acetone extract of Terminalia mollis was the only extract which was not active against Aspergillus fumigatus after 24 h at the highest level tested. After 48-h incubation, there were changes in Aspergillus fumigatus wells; all showed fungal growth, even at the highest extract concentrations. Aspergillus fumigatus is a filamentous fungus and has hyphae which grows over surfaces and inside nearly all wells. Although there was inhibition of the Aspergillus fumigatus after 24 h of incubation, this inhibition was overcome by 48 h of incubation. The active antifungal compounds may have broken down allowing the inhibited fungus to grow, or the fungus was able to overcome the initial inhibitory effects of the antifungal compounds, and that the minimal lethal concentration was higher than highest level tested (final concentration 2.5 mg/ml).

4. Conclusion The serial dilution microplate method worked well with fungi after slight modifications. The antifungal activity of some of the extracts were at levels that would probably already therapeutically useful, leading to the distinct possibility that some of the extracts may be applied clinically for dermatophyte infections, e.g., Microsporum canis. If one extrapolates from in vitro to in vivo activity, especially in topical applications, it means that an acetone leaf extract from 1 g of Terminalia sericea leaves diluted to 2.7 l would still inhibit the growth of Microsporum canis. Extracts from other

, and methanol

307

extracts of six Terminalia

species had values as high as 6.05 l/g. We intend to follow the in vitro experiments up with in vivo work. An important question is, whether the same compound inhibits different fungi and also whether the same compound is present in different Terminalia species. We hope to address these aspects by bioautography, but there are still difficulties in the bioautography using fungi as test organisms. The results of the present work indicate that the Terminalia species assayed possess substantial antifungal properties. This explains the use of these plants in folk medicine for the treatment of various diseases related to fungal infections. We have started isolating the active compounds responsible for the antifungal effects from Terminalia sericea.

Acknowledgement The National Research Foundation (NRF) and the Research Committee, Faculty of Veterinary Science, University of Pretoria provided financial assistance.

References Baba-Moussa, F., Akpagana, K., Bouchet, P., 1998. Antifungal activities of seven West African Combretaceae used in traditional medicine. Journal of Ethnopharmacology 66, 335–338. Balick, M.J., Arvigo, R., Romero, L., 1994. The development of an enthnobiomedical forest reserve in Belixe: its role in the preservation of biological and cultural diversity. Conservation Biology 8, 316–317. Bhatt, S.K., Saxena, V.K., 1979. Efficacy of successive extracts of seeds of Anogeissus leiocarpus against some human pathogenic fungi. Indian drugs 16, 263–264. Carr, J.D., 1988. Combretaceae in southern Africa. Tree Society of southern Africa, Johannesburg. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12, 564–582. Deby, C., Margotteaux, G., 1970. Relationship between essential fatty acids and tissue antioxidant levels in mice. C R Seances Society Biology Fil. 165, 2675–2681. Eloff J.N., 1998a. Conservation of Medicinal Plants: Selecting Medicinal Plants for Research and Gene Banking. Monographs in systematic

308

P. Masoko et al. / Journal of Ethnopharmacology 99 (2005) 301–308

botany from the Missouri Garden 71, 209–222 In: Conservation of plants Genes III: Conservation, utilisation of African plants., Robert, P., Adams, Janice, E., Adams, eds., Missouri Botanical Garden Press, St. Louis, USA. Eloff, J.N., 1998b. Which extractant should be used for the screening and isolation of antimicrobial components from plants? Journal of Ethnopharmacology 60, 1–8. Eloff, J.N., 1998c. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plants extracts for bacteria. Planta Medica 64, 711–713. Eloff, J.N., 1999. The antibacterial activity of 27 sourthern African members of the Combretaceae. South African Journal of Science 95, 148–152. Farnsworth, N.R., 1988. Screening plants for new medicines. In: Wilson, E.O. (Ed.), Biodiversity. National Academic Press, Washington, DC, pp. 83–97. Farnsworth N.R., 1994. The ethnobotanical approach to drug discovery: strengths and limitations. In: Prance G.T. (Ed), Ethnobotany and the search for new drugs. In: Ciba Foundation Symposium, 185. Chicherster, 42–59. Fyhrquist, P., Mwasumbi, L., Haeggstrom, C.A., Vuorela, H., Hiltunen, R., Vuorela, P., 2002. Ethnobotanical and antimicrobial investigation of some species of Terminalia and Combretum (Combretaceae) growing in Tanzania. Journal of Ethnopharmacology 79, 169– 177.

Hutchings, A., Scott, A.H., Lewis, G., Cunninghan, A., 1996. Zulu Medicinal Plants, An Inventory. University of Natal Press, Pietermarizburg, South Africa. Kotze, M., Eloff, J.N., 2002. Extraction of antibacterial compounds from Combretum microphyllum (Combretaceae). South African Journal of Botany 68, 62–67. Locher, C.P., Burch, M.T., Mower, H.F., Berestecky, J., Davis, H., van Poel, B., Lasure, A., Vanden Berghe, D.A., Vlietinck, A.J., 1995. Antimicrobial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants. Journal of Ethnopharmacology 49, 23–32. McGaw, L.J., Rabe, T., Sparg, S.G., J¨ager, A.K., Eloff, J.N., van Staden, J., 2001. An investigation of the biological activity of Combretum species. Journal of Ethnopharmacology 75, 45–50. Mitscher, L.A., Drake, S., Goliapudi, S.R., Okwute, S.K., 1987. A modern look at folkloric use of anti-infective agents. Journal of Natural Products 50, 1025–1040. Motsei, M.L., Lindsey, K.L., van Staden, J., Jager, A.K., 2003. Screening of traditionally used South African plants for antifungal activity against Candida albicans. Journal of Ethnopharmacology 86, 235–241. Silva, O., Duarte, A., Cabrita, J., Pimentel, M., Diniz, A., Gomes, E., 1996. Antimicribial activity of Guinea-Bissau traditional remedies. Journal of Ethnopharmacology 50, 55–59. Van Wyk, B.-E., van Oudtshoorn, B., Gericke, N., 1997. Medicinal plants of South Africa. Briza Publications, Pretoria, South Africa.