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Antimycobacterial activity of some trees used in South African traditional medicine
South African Journal of Botany 73 (2007) 248 – 251 www.elsevier.com/locate/sajb
Short communication
Antimycobacterial activity of some trees used in South African traditional medicine I.M.S. Eldeen, J. Van Staden ⁎ Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Received 8 August 2006; accepted 19 September 2006
Abstract Seventy eight extracts obtained from 10 trees used in South African traditional medicine were investigated for antimycobacterial activity using the broth microdilution method. The extracts (dichloromethane, ethyl acetate and ethanol) were tested against Mycobacterium aurum A+. Of the plant extracts investigated, 30% showed activity against M. aurum at a concentration ranging from 1.5 to 0.195 mg ml− 1. At these concentrations the ethyl acetate and ethanolic extracts of the leaf, bark and roots of Acacia nilotica and Combretum kraussii inhibited growth of the organism. Dichloromethane extracts (with some exceptions) did not show activity against M. aurum at the highest concentration (25 mg ml− 1) tested in this study. © 2006 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Antimycobacterial activity; Tuberculosis; Traditional medicine
Tuberculosis (TB) has reemerged in recent years as a serious public health problem worldwide. The disease spreads more easily in overcrowded settings and in conditions of malnutrition and poverty (Pereira et al., 2005). Despite highly effective drugs, morbidity and mortality due to Mycobacterium tuberculosis are still increasing (Ballell et al., 2005). This has been linked to coinfection with the human immunodeficiency virus (HIV) and to the emergence of strains resistant to available therapies (Chung et al., 1995; Ballell et al., 2005). In South Africa, TB is the most commonly notified disease and the fifth largest cause of death among the black population where one in ten cases of TB is resistant to treatment in some parts of the country (Lall and Meyer, 1999). The search for biologically active agents based on traditionally used plants is still relevant as medicinal plants have the potential to provide pharmacologically active natural products with compounds that act on novel molecular targets. New chemical entities with novel mechanism of action may possess
⁎ Corresponding author. E-mail address: [email protected] (J. Van Staden).
activity against the multi-drug resistant pathogens (Clements et al., 2002; Ballell et al., 2005; Pereira et al., 2005). Southern Africa is one of the richest centers of plant diversity in the world (Arnold and de Wet, 1993). Many of these plants have been used for several centuries in traditional medicine for the prevention and treatment of ailments including microbial diseases (Watt and Breyer-Brandwijk, 1962; Iwu, 1993; Hutchings et al., 1996; Eldeen et al., 2005). Work done in our laboratory on the biological activity of trees used in South African traditional medicine showed that some plant extracts had interesting antibacterial activities against both Gram-positive and Gram-negative bacteria (Eldeen et al., 2005). Based on these results, a further investigation screening specifically for antituberculosis activity was carried out in this study. Due to the slow growth rate and the highly infectious nature of M. tuberculosis, a strain of the rapidly growing, non-pathogenic Mycobacterium aurum A+ was used. Inhibition of M. aurum A+ growth is highly predictive of activity against M. tuberculosis (Chung et al., 1995). Tree species screened in this study were selected based on their uses in South African traditional medicine (Table 1). Plant material (leaves, roots, bark) was collected from the National
0254-6299/$ - see front matter © 2006 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2006.09.004
I.M.S. Eldeen, J. Van Staden / South African Journal of Botany 73 (2007) 248–251
249
Table 1 Minimum inhibitory concentrations (MICs) for antimycobacterial activity of extracts (mg ml− 1) obtained from some trees used in South African traditional medicine as determined by the broth micro-dilution method using Mycobacterium aurum A+ Plant name
Voucher Traditional uses and references specimen no.
Acacia nilotica (L.) Willd. ex Del. subsp. kraussiana (BENTH.) Brenan Acacia sieberiana Dc. var.woodii (Burtt Davy) Keay & Brenan
Eldeen8
Plant MIC values (mg ml− 1) part Plant extracts tested analyzed Dichloromethane Ethyl acetate Ethanol
Dry coughs, respiratory ailments including tuberculosis (Sawhney et al., 1978; Khan et al., 1980)
Leaf Bark Roots Eldeen1 Fever, infectious diseases, inflammation and pain Leaf in the back (Gelfand et al., 1985; Hutchings et al., 1996) Bark Roots Albizia adianthifolia (Schumach.) Eldeen6 Fever, headaches, stomach problems, internal Bark W. F. Wight. parasites and dysentery (Jenkins, 1987; Pujol, 1990) Roots Combretum kraussii Hochst. Eldeen9 Various stomach complaints, fever, wounds and Leaf inflammation (Neuwinger, 1996; Hutchings et al., 1996) Bark Roots Faidherbia albida (Del.) A. Chev. Eldeen2 Stomach disorders, colds, ophthalmia and infections Leaf (Watt and Breyer-Brandwijk, 1962) Bark Ficus sur Forssk. Eldeen4 Tuberculosis, influenza and colic (Watt and Leaf Breyer-Brandwijk, 1962; Hutchings et al., 1996) Bark Roots Prunus africana (Hook.f.) Kalkm. Eldeen10 Chest pain, diarrhoea, fever, allergies, stomachache, prostrate Leaf gland disease and kidney disease (Pujol, 1990; Iwu, 1993) Bark Salix mucronata Thunb. Eldeen7 Rheumatism, fever, rheumatic fever, wound dressing, Leaf pain relief and anti-infective (Iwu, 1993; Rood, 1994) Bark Roots Terminalia sericea Burchel ex Dc. Eldeen3 Stomach disorders, wounds, diarrhoea and swollen Bark painful eyes (Neuwinger, 1996; Hutchings et al., 1996) Roots Trichilia dregeana Sond. Eldeen5 Gonorrhea, fever, diarrhoea, pain in the back and as a Leaf purgative (Watt and Breyer-Brandwijk, 1962; Iwu, 1993; Bark Hutchings et al., 1996) Roots
na na na na na 6.25 na na na na na na na 3.2 na na na 6.25 6.25 na 6.25 3.12 na na na 3.12
0.78 0.78 0.78 3.12 na 3.12 na na 1.56 1.56 0.78 6.25 12.5 0.195 na 0.78 0.78 1.56 na na 6.25 3.12 1.56 6.25 na na
0.195 1.56 0.195 3.12 3.12 0.78 na 6.25 0.195 0.195 0.195 3.12 na 3.12 3.12 3.12 na na 1.56 1.56 1.56 1.56 3.12 0.78 1.56 6.25
Voucher specimens and traditional uses are also included. Minimum inhibitory concentration (MIC) of ciprofloxacin (standard) against M. aurum A+ was 1.8 μg ml− 1. na = not active at the highest concentration tested (25 mg ml− 1).
Botanical Garden Pietermaritzburg and the Botanical Gardens of the University of KwaZulu-Natal Pietermaritzburg. Voucher specimens were deposited in the Natal Herbarium, University of KwaZulu-Natal (Table 1). The collected materials were dried in an oven at 50 °C, powdered and extracted using dichloromethane, ethyl acetate and ethanol (10 ml/g) sequantially by sonication for 1 h. The extracts were filtered using Whatman No.1 filter paper and dried under a fan at room temperature. Middlebrook 7H10 agar base (ref 453982) and Middlebrook 7H9 broth base (ref 454012) were used. The supplement OADC (oleic acid + albumin + dextrose + catalase) (Remel, USA) was added (10%) to both agar and broth media. A stock culture of M. aurum A+ was obtained from the Microbiology Laboratory, Division of Pharmacology, University of Cape Town. The bacteria was first isolated from human sputum in France in 1965 (Tsukamura, 1966). The organism (stock number CIP 104482) was prepared as described in the Standard Operating Procedures (SOP) for determination of minimum inhibitory concentration (MIC) of M. aurum (SOP no. GL 2005/01). Two (5 ml) supplemented 7H9 broths (Middlebrook 7H9 + 10% OADC) were inoculated by the stock bacterial culture and grown for 72 h at 37 °C. Gram and Ziel-Neelson stains were performed on these cultures to ensure culture purity (SOP no. GL 2005/01). Twenty percent sterile glycerol was added to each culture and 500 μl aliquots were made into sterile 1.5 ml
Eppendorf tubes. These stocks represent G0 stocks and were stored at − 70 °C. A single stock was used to inoculate two further broth cultures. Further glycerol stocks were prepared from these cultures as described above and stored at − 70 °C, representing G1 stocks. A single G1 stock was used to inoculate two supplemented Middlebrook 7H10 agar (7H10 + 10% OADC) plates and incubated at 37 °C for 4 days. From this culture a single colony was used to inoculate 5 ml supplemented 7H9 broth. This was grown at 37 °C for 72 h and used for the experiment. The broth microdilution method (Swenson et al., 1982) was used to determine the minimum inhibitory concentration (MIC) of M. aurum A+ of the investigated plant extracts using 96-well microtiter plates. The extracts were dissolved in water: ethanol (1:1 v/v). One hundred microliter of the supplemented 7H9 broth was added to each well using a multi-channel pipette. A further 100 μl of supplemented 7H9 broth was added to all the wells of the first column to serve as a medium control and blank. For the drug and solvent controls, as well as the test samples, 100 μl of the respective sample was added to the first well of the column. A multi-channel pipette was used to make a two fold serial dilution starting with a concentration of 25 mg ml− 1 of the extracts and 62.5 μg ml− 1 of the ciprofloxacin (standard drug control). The optical density (OD) of the 72 h liquid culture was adjusted to 0.125 at 550 nm. One hundred microliter of the
250
I.M.S. Eldeen, J. Van Staden / South African Journal of Botany 73 (2007) 248–251
diluted culture was added to every well of the microtitre plate excluding the wells of the first column that served as the medium control. The plates were loosely sealed in plastic bags to prevent dryness and incubated at 37 °C for 72 h. After incubation, 40 μ1 of 0.4 mg ml− 1 solution of p-iodonitrotetrazolim salt (INT) was added to each well of the plate. The plates were left sealed in plastic packets overnight 37 °C. The lowest concentration containing no indication of red colour as a result of INT was deemed to be the MIC. Plant materials were extracted sequentially in order to ensure extraction of a wide range of polar and non-polar compounds using dichloromethane, ethyl acetate and ethanol. The minimum inhibitory concentrations (MIC) of the plant extracts against M. aurum A+ as determined using the broth micro-dilution method are shown in Table 1. Of the 78 plant extracts tested, 30% inhibited growth of M. aurum A+ at a concentration ranging from 1.5 to 0.195 mg ml− 1 (Table 1). All ethyl acetate and ethanolic extracts (leaf, bark and roots) of Acacia nilotica and Combretum kraussii showed strong activity against the organism. These findings are in agreement with previous reports of antimycobacterium activity of extracts from Acacia and Combretum species (Lall and Meyer, 1999). Extracts from different plant parts of both Combretum and Acacia species also possessed broad spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria (Iwu, 1993; Eldeen et al., 2005). Several bioactive compounds are present in Combretum and Acacia species. The isomeric flavonoids vitexin and saponaretin, catechins and the alkaloids combretacin occur in Combretum species (Iwu, 1993). Hydroxyproline, serine, dimethyl-triptamine, β-amyrin and betulin amongst many others were isolated from Acacia species (Ayoub, 1982; Anderson and McDougall, 1987; Hutchings et al., 1996). Inhibition of M. aurum A+ observed in this study may in part be due to the presence of similar or related compounds. With the exception of Trichilia dregeana (roots), Terminalia sericea (bark), Salix mucronata (leaf and roots), Ficus sur (leaf) and Acacia sieberiana (roots), dichloromethane extracts in general did not show any activity against the test organism at the highest concentration tested (25 mg ml− 1). Best activities were obtained from the ethanolic extracts (with some exceptions). This is very interesting as traditional healers normally use polar solvents such as water and alcohol. The use of alcohol in this study may be more relevant to the traditional extraction methods. However, traditional healers in most cases use mixtures in which whole plants or plant parts are prepared and consumed in combination or in sequences. Pharmacological evaluation of such preparations therefore remains more complicated (Taylor et al., 2001; Elgorashi and Van Staden, 2004). Plant species investigated in this study are used by South African traditional healers for treatment of tuberculosis or related symptoms such as cough and fever, amongst other diseases (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). Although some of these plant extracts were previously reported to have strong antibacterial activities, they showed weak or no activity against M. aurum A+ in this study. A key target for antimycobacterial chemotherapy is cell wall biosynthesis. Due to the complex lipoglycan calyx on the cell surface
which provide a significant physical barrier to intracellularacting compounds (Ballell et al., 2005). This may explain the lack of activities showed by the plant extracts against M. aurum A+ in this study. These plants may also be effective against other microorganisms that cause related TB diseases (Lall and Meyer, 1999). In general antimycobacterium activities observed in this study indicate the presence of promising compounds that can be further investigated as antimycobacterials. Results obtained verified in part the traditional use of some of the tested plants as traditional remedies against TB. Acknowledgements P. Smith and T. Seaman from the Division of Pharmacology, University of Cape Town are thanked for providing a strain of Mycobacterium auarum A+. This project was financially supported by the National Research Foundation (NRF), Pretoria. References Anderson, D.M.W., McDougall, F.J., 1987. The composition of the proteinaceous gums exuded by Acacia gerrardii and Acacia goetzii subsp. goetzii. Food Hydrocolloids 1, 327–331. Arnold, T.H., de Wet, B.C., 1993. Plants of Southern Africa: Names and Distribution. Memoirs of the Botanical Survey of South Africa, vol. 62. National Botanical Institute, Pretoria. Ayoub, H.S.M., 1982. Mulluscicidal properties of Acacia nilotica. Planta Medica 46, 181–183. Ballell, L., Field, R.A., Duncan, K., Young, R.J., 2005. New small molecule synthetic antimycrobacterials. Antimicrobial Agents and Chemotherapy 49, 2153–2163. Chung, G.A.C., Aktar, Z., Jackson, S., Duncan, K., 1995. High-throughput screen for detecting antimycobacterial agents. Antimicrobial Agents and Chemotherapy 39, 2235–2238. Clements, J.M., Coignard, F., Johnson, I., Chandler, S., Palan, S., Waller, A., Wijkmans, J., Hunter, M.G., 2002. Antibacterial activities and characterization of novel inhibitors of LpxC. Antimicrobial agents and Chemotherapy 46, 1793–1799. Eldeen, I.M.S., Elgorashi, E.E., Van Staden, J., 2005. Antibacterial, antiinflammatory, anti-cholinesterase and mutagenic effects of extracts obtained from some trees used in South African traditional medicine. Journal of Ethnopharmacology 102, 457–464. Elgorashi, E.E., Van Staden, J., 2004. Pharmacological screening of six Amaryllidaceae species. Journal of Ethnopharmacology 90, 27–32. Gelfand, M., Mavis, S., Drummond, R.B., Ndemera, B., 1985. The Traditional Medical Practitioner in Zimbabwe. Mambo Press, Gweru, Zimbabwe. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants. An Inventory. University of Natal Press, Pietermaritzburg. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, Boca Raton, Florida, USA. Jenkins, M.D., 1987. Madagascar: an Environmental Profile. IUCN, Gland, Switzerland. Khan, M.R., Ndaalio, G., Nkunya, M.H.H., Wevers, H., Sawney, A.N., 1980. Studies on African medicinal plants. Part 1, preliminary screening of medicinal plants for antibacterial activity. Planta Medica Supplement, pp. 91–97. Lall, N., Meyer, J.J.M., 1999. In vitro inhibition of drug-resistant and drugsensitive strains of Mycobacterium tuberculosis by ethnobotanically selected South African plants. Journal of Ethnopharmacology 66, 347–354. Neuwinger, H., 1996. African Ethnobotany Poisons and Drugs Chemistry. (Germany). Chapman and Hall Gm bH, D-69469 Weinheim (Bundes Republik Deutschland). Pereira, M., Tripathy, S., Indamdar, V., Ramesh, K., Bhavsar, M., Date, A., Iyyer, R., Acchammachary, A., Mehendale, S., Risbud, A., 2005. Drug
I.M.S. Eldeen, J. Van Staden / South African Journal of Botany 73 (2007) 248–251 resistance pattern of Mycobacterium tuberculosis in seropositive and seronegative HIV-TB patients in Pune, India. Indian Journal for Medical Research 121, 235–239. Pujol, J., 1990. Naturafrica — the Herbalist Handbook. Jean Pujol Natural Healers, Foundation, Durban, South Africa. Rood, B., 1994. Uit die Veldapteek. Tafelberg, Uitgewers, Cape Town, South Africa. Sawhney, A.N., Khan, M.R., Ndaalio, G., Nkunya, M.H.H., Wevers, H., 1978. Studies on the rationale of African traditional medicine 2. Preliminary screening of medicinal plants for antigonococcal activity. Pakistanian Journal of Scientific and Industrial Research 21, 189–192. Standard Operating Procedures (SOP), 2005. A Protocol for Determination of Minimum Inhibitory Concentrations (MICs) Involving Mycobacterium aurum A+. SOP no. GL2005/01.
Edited by JJM Meyer
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Swenson, J.M., Thornsberry, C., Silcox, V.A., 1982. Rapidly growing mycobacteria: testing of susceptibility to 34 antimicrobial agents by microdilution. Antimicrobial Agent and Chemotherapy 22, 186–192. Taylor, J.L.S., Rabe, T., McGaw, L.J., Jäger, A.K., Van Staden, J., 2001. Towards the scientific validation of traditional medicinal plants. Plant Growth Regulation 34, 23–37. Tsukamura, M., 1966. Adansonian classification of mycobacteria. Journal of General Microbiololgy 45, 253–273. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and East Africa. E & S Livingstone Ltd., London.
Short communication
Antimycobacterial activity of some trees used in South African traditional medicine I.M.S. Eldeen, J. Van Staden ⁎ Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Received 8 August 2006; accepted 19 September 2006
Abstract Seventy eight extracts obtained from 10 trees used in South African traditional medicine were investigated for antimycobacterial activity using the broth microdilution method. The extracts (dichloromethane, ethyl acetate and ethanol) were tested against Mycobacterium aurum A+. Of the plant extracts investigated, 30% showed activity against M. aurum at a concentration ranging from 1.5 to 0.195 mg ml− 1. At these concentrations the ethyl acetate and ethanolic extracts of the leaf, bark and roots of Acacia nilotica and Combretum kraussii inhibited growth of the organism. Dichloromethane extracts (with some exceptions) did not show activity against M. aurum at the highest concentration (25 mg ml− 1) tested in this study. © 2006 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Antimycobacterial activity; Tuberculosis; Traditional medicine
Tuberculosis (TB) has reemerged in recent years as a serious public health problem worldwide. The disease spreads more easily in overcrowded settings and in conditions of malnutrition and poverty (Pereira et al., 2005). Despite highly effective drugs, morbidity and mortality due to Mycobacterium tuberculosis are still increasing (Ballell et al., 2005). This has been linked to coinfection with the human immunodeficiency virus (HIV) and to the emergence of strains resistant to available therapies (Chung et al., 1995; Ballell et al., 2005). In South Africa, TB is the most commonly notified disease and the fifth largest cause of death among the black population where one in ten cases of TB is resistant to treatment in some parts of the country (Lall and Meyer, 1999). The search for biologically active agents based on traditionally used plants is still relevant as medicinal plants have the potential to provide pharmacologically active natural products with compounds that act on novel molecular targets. New chemical entities with novel mechanism of action may possess
⁎ Corresponding author. E-mail address: [email protected] (J. Van Staden).
activity against the multi-drug resistant pathogens (Clements et al., 2002; Ballell et al., 2005; Pereira et al., 2005). Southern Africa is one of the richest centers of plant diversity in the world (Arnold and de Wet, 1993). Many of these plants have been used for several centuries in traditional medicine for the prevention and treatment of ailments including microbial diseases (Watt and Breyer-Brandwijk, 1962; Iwu, 1993; Hutchings et al., 1996; Eldeen et al., 2005). Work done in our laboratory on the biological activity of trees used in South African traditional medicine showed that some plant extracts had interesting antibacterial activities against both Gram-positive and Gram-negative bacteria (Eldeen et al., 2005). Based on these results, a further investigation screening specifically for antituberculosis activity was carried out in this study. Due to the slow growth rate and the highly infectious nature of M. tuberculosis, a strain of the rapidly growing, non-pathogenic Mycobacterium aurum A+ was used. Inhibition of M. aurum A+ growth is highly predictive of activity against M. tuberculosis (Chung et al., 1995). Tree species screened in this study were selected based on their uses in South African traditional medicine (Table 1). Plant material (leaves, roots, bark) was collected from the National
0254-6299/$ - see front matter © 2006 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2006.09.004
I.M.S. Eldeen, J. Van Staden / South African Journal of Botany 73 (2007) 248–251
249
Table 1 Minimum inhibitory concentrations (MICs) for antimycobacterial activity of extracts (mg ml− 1) obtained from some trees used in South African traditional medicine as determined by the broth micro-dilution method using Mycobacterium aurum A+ Plant name
Voucher Traditional uses and references specimen no.
Acacia nilotica (L.) Willd. ex Del. subsp. kraussiana (BENTH.) Brenan Acacia sieberiana Dc. var.woodii (Burtt Davy) Keay & Brenan
Eldeen8
Plant MIC values (mg ml− 1) part Plant extracts tested analyzed Dichloromethane Ethyl acetate Ethanol
Dry coughs, respiratory ailments including tuberculosis (Sawhney et al., 1978; Khan et al., 1980)
Leaf Bark Roots Eldeen1 Fever, infectious diseases, inflammation and pain Leaf in the back (Gelfand et al., 1985; Hutchings et al., 1996) Bark Roots Albizia adianthifolia (Schumach.) Eldeen6 Fever, headaches, stomach problems, internal Bark W. F. Wight. parasites and dysentery (Jenkins, 1987; Pujol, 1990) Roots Combretum kraussii Hochst. Eldeen9 Various stomach complaints, fever, wounds and Leaf inflammation (Neuwinger, 1996; Hutchings et al., 1996) Bark Roots Faidherbia albida (Del.) A. Chev. Eldeen2 Stomach disorders, colds, ophthalmia and infections Leaf (Watt and Breyer-Brandwijk, 1962) Bark Ficus sur Forssk. Eldeen4 Tuberculosis, influenza and colic (Watt and Leaf Breyer-Brandwijk, 1962; Hutchings et al., 1996) Bark Roots Prunus africana (Hook.f.) Kalkm. Eldeen10 Chest pain, diarrhoea, fever, allergies, stomachache, prostrate Leaf gland disease and kidney disease (Pujol, 1990; Iwu, 1993) Bark Salix mucronata Thunb. Eldeen7 Rheumatism, fever, rheumatic fever, wound dressing, Leaf pain relief and anti-infective (Iwu, 1993; Rood, 1994) Bark Roots Terminalia sericea Burchel ex Dc. Eldeen3 Stomach disorders, wounds, diarrhoea and swollen Bark painful eyes (Neuwinger, 1996; Hutchings et al., 1996) Roots Trichilia dregeana Sond. Eldeen5 Gonorrhea, fever, diarrhoea, pain in the back and as a Leaf purgative (Watt and Breyer-Brandwijk, 1962; Iwu, 1993; Bark Hutchings et al., 1996) Roots
na na na na na 6.25 na na na na na na na 3.2 na na na 6.25 6.25 na 6.25 3.12 na na na 3.12
0.78 0.78 0.78 3.12 na 3.12 na na 1.56 1.56 0.78 6.25 12.5 0.195 na 0.78 0.78 1.56 na na 6.25 3.12 1.56 6.25 na na
0.195 1.56 0.195 3.12 3.12 0.78 na 6.25 0.195 0.195 0.195 3.12 na 3.12 3.12 3.12 na na 1.56 1.56 1.56 1.56 3.12 0.78 1.56 6.25
Voucher specimens and traditional uses are also included. Minimum inhibitory concentration (MIC) of ciprofloxacin (standard) against M. aurum A+ was 1.8 μg ml− 1. na = not active at the highest concentration tested (25 mg ml− 1).
Botanical Garden Pietermaritzburg and the Botanical Gardens of the University of KwaZulu-Natal Pietermaritzburg. Voucher specimens were deposited in the Natal Herbarium, University of KwaZulu-Natal (Table 1). The collected materials were dried in an oven at 50 °C, powdered and extracted using dichloromethane, ethyl acetate and ethanol (10 ml/g) sequantially by sonication for 1 h. The extracts were filtered using Whatman No.1 filter paper and dried under a fan at room temperature. Middlebrook 7H10 agar base (ref 453982) and Middlebrook 7H9 broth base (ref 454012) were used. The supplement OADC (oleic acid + albumin + dextrose + catalase) (Remel, USA) was added (10%) to both agar and broth media. A stock culture of M. aurum A+ was obtained from the Microbiology Laboratory, Division of Pharmacology, University of Cape Town. The bacteria was first isolated from human sputum in France in 1965 (Tsukamura, 1966). The organism (stock number CIP 104482) was prepared as described in the Standard Operating Procedures (SOP) for determination of minimum inhibitory concentration (MIC) of M. aurum (SOP no. GL 2005/01). Two (5 ml) supplemented 7H9 broths (Middlebrook 7H9 + 10% OADC) were inoculated by the stock bacterial culture and grown for 72 h at 37 °C. Gram and Ziel-Neelson stains were performed on these cultures to ensure culture purity (SOP no. GL 2005/01). Twenty percent sterile glycerol was added to each culture and 500 μl aliquots were made into sterile 1.5 ml
Eppendorf tubes. These stocks represent G0 stocks and were stored at − 70 °C. A single stock was used to inoculate two further broth cultures. Further glycerol stocks were prepared from these cultures as described above and stored at − 70 °C, representing G1 stocks. A single G1 stock was used to inoculate two supplemented Middlebrook 7H10 agar (7H10 + 10% OADC) plates and incubated at 37 °C for 4 days. From this culture a single colony was used to inoculate 5 ml supplemented 7H9 broth. This was grown at 37 °C for 72 h and used for the experiment. The broth microdilution method (Swenson et al., 1982) was used to determine the minimum inhibitory concentration (MIC) of M. aurum A+ of the investigated plant extracts using 96-well microtiter plates. The extracts were dissolved in water: ethanol (1:1 v/v). One hundred microliter of the supplemented 7H9 broth was added to each well using a multi-channel pipette. A further 100 μl of supplemented 7H9 broth was added to all the wells of the first column to serve as a medium control and blank. For the drug and solvent controls, as well as the test samples, 100 μl of the respective sample was added to the first well of the column. A multi-channel pipette was used to make a two fold serial dilution starting with a concentration of 25 mg ml− 1 of the extracts and 62.5 μg ml− 1 of the ciprofloxacin (standard drug control). The optical density (OD) of the 72 h liquid culture was adjusted to 0.125 at 550 nm. One hundred microliter of the
250
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diluted culture was added to every well of the microtitre plate excluding the wells of the first column that served as the medium control. The plates were loosely sealed in plastic bags to prevent dryness and incubated at 37 °C for 72 h. After incubation, 40 μ1 of 0.4 mg ml− 1 solution of p-iodonitrotetrazolim salt (INT) was added to each well of the plate. The plates were left sealed in plastic packets overnight 37 °C. The lowest concentration containing no indication of red colour as a result of INT was deemed to be the MIC. Plant materials were extracted sequentially in order to ensure extraction of a wide range of polar and non-polar compounds using dichloromethane, ethyl acetate and ethanol. The minimum inhibitory concentrations (MIC) of the plant extracts against M. aurum A+ as determined using the broth micro-dilution method are shown in Table 1. Of the 78 plant extracts tested, 30% inhibited growth of M. aurum A+ at a concentration ranging from 1.5 to 0.195 mg ml− 1 (Table 1). All ethyl acetate and ethanolic extracts (leaf, bark and roots) of Acacia nilotica and Combretum kraussii showed strong activity against the organism. These findings are in agreement with previous reports of antimycobacterium activity of extracts from Acacia and Combretum species (Lall and Meyer, 1999). Extracts from different plant parts of both Combretum and Acacia species also possessed broad spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria (Iwu, 1993; Eldeen et al., 2005). Several bioactive compounds are present in Combretum and Acacia species. The isomeric flavonoids vitexin and saponaretin, catechins and the alkaloids combretacin occur in Combretum species (Iwu, 1993). Hydroxyproline, serine, dimethyl-triptamine, β-amyrin and betulin amongst many others were isolated from Acacia species (Ayoub, 1982; Anderson and McDougall, 1987; Hutchings et al., 1996). Inhibition of M. aurum A+ observed in this study may in part be due to the presence of similar or related compounds. With the exception of Trichilia dregeana (roots), Terminalia sericea (bark), Salix mucronata (leaf and roots), Ficus sur (leaf) and Acacia sieberiana (roots), dichloromethane extracts in general did not show any activity against the test organism at the highest concentration tested (25 mg ml− 1). Best activities were obtained from the ethanolic extracts (with some exceptions). This is very interesting as traditional healers normally use polar solvents such as water and alcohol. The use of alcohol in this study may be more relevant to the traditional extraction methods. However, traditional healers in most cases use mixtures in which whole plants or plant parts are prepared and consumed in combination or in sequences. Pharmacological evaluation of such preparations therefore remains more complicated (Taylor et al., 2001; Elgorashi and Van Staden, 2004). Plant species investigated in this study are used by South African traditional healers for treatment of tuberculosis or related symptoms such as cough and fever, amongst other diseases (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). Although some of these plant extracts were previously reported to have strong antibacterial activities, they showed weak or no activity against M. aurum A+ in this study. A key target for antimycobacterial chemotherapy is cell wall biosynthesis. Due to the complex lipoglycan calyx on the cell surface
which provide a significant physical barrier to intracellularacting compounds (Ballell et al., 2005). This may explain the lack of activities showed by the plant extracts against M. aurum A+ in this study. These plants may also be effective against other microorganisms that cause related TB diseases (Lall and Meyer, 1999). In general antimycobacterium activities observed in this study indicate the presence of promising compounds that can be further investigated as antimycobacterials. Results obtained verified in part the traditional use of some of the tested plants as traditional remedies against TB. Acknowledgements P. Smith and T. Seaman from the Division of Pharmacology, University of Cape Town are thanked for providing a strain of Mycobacterium auarum A+. This project was financially supported by the National Research Foundation (NRF), Pretoria. References Anderson, D.M.W., McDougall, F.J., 1987. The composition of the proteinaceous gums exuded by Acacia gerrardii and Acacia goetzii subsp. goetzii. Food Hydrocolloids 1, 327–331. Arnold, T.H., de Wet, B.C., 1993. Plants of Southern Africa: Names and Distribution. Memoirs of the Botanical Survey of South Africa, vol. 62. National Botanical Institute, Pretoria. Ayoub, H.S.M., 1982. Mulluscicidal properties of Acacia nilotica. Planta Medica 46, 181–183. Ballell, L., Field, R.A., Duncan, K., Young, R.J., 2005. New small molecule synthetic antimycrobacterials. Antimicrobial Agents and Chemotherapy 49, 2153–2163. Chung, G.A.C., Aktar, Z., Jackson, S., Duncan, K., 1995. High-throughput screen for detecting antimycobacterial agents. Antimicrobial Agents and Chemotherapy 39, 2235–2238. Clements, J.M., Coignard, F., Johnson, I., Chandler, S., Palan, S., Waller, A., Wijkmans, J., Hunter, M.G., 2002. Antibacterial activities and characterization of novel inhibitors of LpxC. Antimicrobial agents and Chemotherapy 46, 1793–1799. Eldeen, I.M.S., Elgorashi, E.E., Van Staden, J., 2005. Antibacterial, antiinflammatory, anti-cholinesterase and mutagenic effects of extracts obtained from some trees used in South African traditional medicine. Journal of Ethnopharmacology 102, 457–464. Elgorashi, E.E., Van Staden, J., 2004. Pharmacological screening of six Amaryllidaceae species. Journal of Ethnopharmacology 90, 27–32. Gelfand, M., Mavis, S., Drummond, R.B., Ndemera, B., 1985. The Traditional Medical Practitioner in Zimbabwe. Mambo Press, Gweru, Zimbabwe. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants. An Inventory. University of Natal Press, Pietermaritzburg. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, Boca Raton, Florida, USA. Jenkins, M.D., 1987. Madagascar: an Environmental Profile. IUCN, Gland, Switzerland. Khan, M.R., Ndaalio, G., Nkunya, M.H.H., Wevers, H., Sawney, A.N., 1980. Studies on African medicinal plants. Part 1, preliminary screening of medicinal plants for antibacterial activity. Planta Medica Supplement, pp. 91–97. Lall, N., Meyer, J.J.M., 1999. In vitro inhibition of drug-resistant and drugsensitive strains of Mycobacterium tuberculosis by ethnobotanically selected South African plants. Journal of Ethnopharmacology 66, 347–354. Neuwinger, H., 1996. African Ethnobotany Poisons and Drugs Chemistry. (Germany). Chapman and Hall Gm bH, D-69469 Weinheim (Bundes Republik Deutschland). Pereira, M., Tripathy, S., Indamdar, V., Ramesh, K., Bhavsar, M., Date, A., Iyyer, R., Acchammachary, A., Mehendale, S., Risbud, A., 2005. Drug
I.M.S. Eldeen, J. Van Staden / South African Journal of Botany 73 (2007) 248–251 resistance pattern of Mycobacterium tuberculosis in seropositive and seronegative HIV-TB patients in Pune, India. Indian Journal for Medical Research 121, 235–239. Pujol, J., 1990. Naturafrica — the Herbalist Handbook. Jean Pujol Natural Healers, Foundation, Durban, South Africa. Rood, B., 1994. Uit die Veldapteek. Tafelberg, Uitgewers, Cape Town, South Africa. Sawhney, A.N., Khan, M.R., Ndaalio, G., Nkunya, M.H.H., Wevers, H., 1978. Studies on the rationale of African traditional medicine 2. Preliminary screening of medicinal plants for antigonococcal activity. Pakistanian Journal of Scientific and Industrial Research 21, 189–192. Standard Operating Procedures (SOP), 2005. A Protocol for Determination of Minimum Inhibitory Concentrations (MICs) Involving Mycobacterium aurum A+. SOP no. GL2005/01.
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Swenson, J.M., Thornsberry, C., Silcox, V.A., 1982. Rapidly growing mycobacteria: testing of susceptibility to 34 antimicrobial agents by microdilution. Antimicrobial Agent and Chemotherapy 22, 186–192. Taylor, J.L.S., Rabe, T., McGaw, L.J., Jäger, A.K., Van Staden, J., 2001. Towards the scientific validation of traditional medicinal plants. Plant Growth Regulation 34, 23–37. Tsukamura, M., 1966. Adansonian classification of mycobacteria. Journal of General Microbiololgy 45, 253–273. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and East Africa. E & S Livingstone Ltd., London.