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Antibacterial activity of Marula (Sclerocarya birrea (A. rich.) Hochst. subsp. caffra (Sond.) Kokwaro) (Anacardiaceae) bark and leaves
Journal of Ethnopharmacology 76 (2001) 305– 308 www.elsevier.com/locate/jethpharm
Short Communication
Antibacterial activity of Marula (Sclerocarya birrea (A. rich.) Hochst. subsp. caffra (Sond.) Kokwaro) (Anacardiaceae) bark and leaves J.N. Eloff * Department of Pharmacology, Uni6ersity of Pretoria, Pretoria, South Africa Received 14 March 2001; received in revised form 10 April 2001; accepted 12 May 2001
Abstract Marula bark is widely used for bacteria-related diseases by indigenous cultures in Africa. This study was undertaken to investigate whether the ethnobotanical use can be validated by laboratory studies. Bark and leaves were extracted with acetone and MIC values were determined using a microplate serial dilution technique with Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as test organisms. All extracts were active with MIC values from 0.15 to 3 mg/ml. Based on minimum inhibitory concentration values, inner bark extracts tended to be the most potent followed by outer bark and leaf extracts, but the differences were not statistically significant. There were two major bioactive components visible after bioautography of leaf extracts: one strongly polar and the other highly non-polar. The bioactive components could be separated from 92% of the non-active dry matter by solvent– solvent fractionation into the carbon tetrachloride, chloroform and n-butanol fractions; these fractions, however, still contained many different compounds. Using bark may be detrimental to the plant, but leaf material can also be used for antibacterial application. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sclerocarya birrea subsp. Caffra; Bark; Leaf; Antibacterial; Bioactivity
1. Introduction The Marula (Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro) is one of the most highly valued indigenous trees in southern Africa. The Tonga people celebrate the Feast of the First Fruits by pouring a drink offering of the fresh juice of the fruit over the tombs of dead chiefs (Palgrave, 1983). The pulp of the fruit is delicious and the large nut is also edible. Some tribes such as the Pedi make a relish from the leaves (Fox and Young, 1982). Some attempts have been made to improve the plant by selection and breeding and it has become a commercial fruit crop recently. Watt and Breyer-Brandwijk (1962), Oliver-Bever (1986) * Tel.: +27-12-319-2139; fax: +27-12-319-2411. E-mail address: [email protected] (J.N. Eloff).
and Hutchings et al. (1996) identified a number of medicinal uses in southern, eastern and tropical West Africa. The presence of antimicrobial constituents may be inferred from the following ethnomedical uses: the Zulu people use bark decoctions administered as enemas for diarrhoea. Traditional Zulu healers wash in bark decoctions before treating patients with gangrenous rectitis and also administer the decoction to the patient (Bryant, 1966). Bark decoctions are taken in 300 ml doses for dysentery and diarrhoea in unspecified parts of southern Africa (Watt and Breyer-Brandwijk, 1962). Bark has also been used in treating proctitis. The Vhavenda use bark for treating fevers, stomach ailments and ulcers (Mobogo, 1990). Roots are used for many purposes including sore eyes in Zimbabwe (Gelfand et al., 1985). In East Africa, roots are an ingredient in an alcoholic medicine taken to treat an
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 6 0 - 4
306
J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308
internal ailment known as kati while bark is used for stomach disorders (Kokwaro, 1976). The Hausas in West Africa use a cold infusion of the bark along with native natron as a remedy for dysentery (Oliver-Bever, 1986). Galves et al. (1993) could show that extracts inhibit diarrhoea in mice. Bark yields 3.5–20.5% tannin, 10.7% tanning matter and traces of alkaloids (Watt and Breyer-Brandwijk, 1962). The fruit is rich in ascorbic acid and juice extracts yield 33 sesquiterpene hydrocarbons (Pretorius et al., 1985). Kernels yield 54– 60% of a non-drying oil and contain as much as 28% protein and some iodine (Watt and Breyer-Brandwijk, 1962). The oil-rich seeds contain 64% oleic acid, myristic, stearic and amino acids with a predominance of glutamic acid and arginine (Busson, 1985). The gum is rich in tannin. Tannins and flavonoids are present in leaves but no alkaloids, steroids or triterpenoids have been detected (Gueye, 1973). Only one paper published on the effect of S. birrea plant extracts on in vitro antimicrobial activity (Hussein and Deeni, 1991) was found. In a preliminary study these authors examined 34 plant species for antimicrobial activity. Methanolic extracts from dried powdered stembark had strong antibacterial activity (zone of inhibition\8 mm) against Corynebacterium diphtheriae, Pseudomonas aeruginosa and a Streptococcus spp. and less activity against unidentified Neisseria, Streptobacillus and Salmonella species. The extracts were inactive against Staphylococcus aureus and Escherichia coli. The authors indicated that their results differed from results obtained by earlier authors without specifying the differences or authors. Unfortunately no voucher specimens of the plants were retained. Subsequently, subspecies of Sclerocarya were recognised. It is therefore not clear which taxon Hussein and Deeni (1991) investigated. With a widely distributed plant that is valued highly for edible purposes and for treating many other ailments, Marula could be considered a ‘power plant’ (Balick, 1990). Power plants have been defined as widely used plants without any apparent or demonstrable pharmacological basis for their use. Some workers have eliminated ‘power plants’ from their list of plants to investigate. A disadvantage of using bark as source for the medicinal component is that injudicial removal of bark can lead to the death of the plant. If bark can be sold for a high price, valuable plant populations may be collected to extinction in nature. The aim of this paper is to determine if the use of extracts of Marula for ailments that may be related to bacterial pathogens can be substantiated in in vitro experiments and to compare the antibacterial activity of leaf and bark extracts.
2. Materials and methods Plant material was collected after fruiting in late May from a single tree growing in Pretoria. A voucher specimen was deposited at the National Herbarium (J N Eloff 503, PRE). Stem bark was collected by making two longitudinal cuts ca. 1 cm apart and ca. 25 cm long and removing the bark from the stem. The inner bark had a light buff colour and was separated from the dark brown flaky outer bark. Bark and leaves were dried indoors at ambient temperature and then ground to a fine powder in a Jankel and Ku¨ nkel Model A10 mill. The finely ground plant material (0.5 g) was extracted (5 ml) in a centrifuge tube and the marc collected by centrifuging at 300× g for 5 min. The extraction was repeated two times on the marc. The acetone was removed by a stream of cold air and redissolved in acetone to yield a concentration of 50 mg/ml. The plant extracts were analysed by TLC (5 ml of 100 mg extract/ml solution) on Merck TLC F254 plates with chloroform/ethyl acetate/formic acid (5:4:1) as eluent. Plates were sprayed with 0.5 g vanillin dissolved in 100 ml sulphuric acid/ethanol (40:10). Minimum inhibitory concentrations (MIC) were determined by two-fold serial dilution of extracts beyond the concentration where no inhibition of growth of Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 25922 was observed. The wells in the dilution series were inoculated with the relevant cultures, incubated overnight at 37 °C and 0.2 mg/ml p-iodonitrotetrazolium violet [INT] (Sigma) was added. After further incubation bacterial growth was indicated by the red colour of the INT formazan produced (Eloff, 1998). For bioautography, developed TLC plates were dried overnight and sprayed with a concentrated suspension of actively growing S. aureus cells, before incubating at 38 °C in a chamber at 100% relative humidity. Plates were sprayed with a 2 mg/ml solution of p-iodonitrotetrazolium violet (Sigma). Clear zones on the chromatogram indicated inhibition of growth (Begue and Kline, 1972). The acetone extract was taken to dryness in a rotary evaporator under reduced pressure and this extract was dissolved in a 1:1 mixture of chloroform/water. The water fraction was extracted with an equal volume of n-butanol to yield the water (W) and butanol (B) fractions. The chloroform fraction was taken to dryness in a rotary evaporator under reduced pressure and extracted with a 1:1 mixture of hexane and 10% water in methanol. The 10% water in methanol extract was diluted to 20% water in methanol and extracted with carbon tetrachloride to yield the carbon tetrachloride (CT) fraction. The 20% water in methanol extract was
J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308
diluted to 35% methanol in water and extracted with chloroform to yield the chloroform (CHL) fraction and the 35% water in methanol (MW) fractions (Suffness and Douros, 1979). In all cases equal volumes of the solvents were used and the extraction was repeated with a small volume ca. three more times or until all the colour was extracted. In some instances centrifugation was used to separate the fractions and a small quantity of an insoluble pellicle was formed that was discarded.
3. Results and discussion Acetone extracted 6.4% of the leaf, 9.4% of the outer bark and 9.8% of the inner bark. Extracts from leaves, inner bark and outer bark inhibited the growth of all the test organisms (Table 1). The average values for leaf, inner bark and outer bark for all the test organisms were 1.4 (SD=1.33), 0.49 (SD= 0.51) and 0.81 (SD = 1.23) mg/ml, respectively. The extremely high standard deviations are caused by combining MIC values for the different organisms. The standard deviation of MIC values with the same test organisms were much lower (Table 1). The MIC values for the positive control, gentamycin, ranged from 0.02 to 0.6 mg/ml. One microwell where the bacterial growth inhibition was not noted would lead to a doubling of the MIC value. Although the differences were not statistically significant, the inner bark extract tended to be more active (i.e. have a lower MIC) than the outer bark and the leaf extracts. Based only on MIC values, one could speculate that leaves would also be a useful source for treating ailments caused by bacteria. It should be kept in mind that extracts were dried and made up to a known concentration before bioassay and the MIC values found does not relate to the total quantity present in the different plant parts. The total antibacterial activity present in different parts of the plant can be calculated
307
by dividing the quantity extracted from one gram of plant material with the MIC value in mg/ml (Eloff, 2000) (Table 1). The average total activity for the four test organisms, with leaf extracts is 56, with outer bark is 189 and with inner bark is 385 ml/g. Staphylococcus aureus was the most sensitive with a total activity of 653 ml/g. This means that the antibacterial compounds present in 1 g of inner bark material diluted to 653 ml would still inhibit the growth of the test organisms. When the total activity is compared it explains why Marula leaves are not traditionally used for ailments related to bacteria. The total activity values obtained here are in the same order of magnitude as that found for leaves of members of the Combretaceae and Celastraceae (Eloff, 1999, 2000). To obtain some information on the active component(s) a larger quantity of dried leaves was extracted in acetone and fractionated by solvent–solvent extraction. After solvent–solvent extraction, most of the extract (85%) was in hexane, the least polar fraction. In increasing order of polarity the carbon tetrachloride fraction contained 2%, chloroform 1%, n-butanol 5%, 35% water in methanol 3% and water 3% of the dry weight of the original extract. Most of the antibacterial activity was in highly non-polar compounds in the chloroform and carbon tetrachloride fractions (Rf = 0.93). There was also some activity in the n-butanol fraction (Rf = 0.04) with chloroform/ethylacetate/formic acid (5:4:1) as eluent. According to the bioautograms there are only two major inhibiting compounds present in the leaf extracts. The solvent–solvent fractionation was effective in concentrating more than 95% of the activity of the non-polar compound in 3% of the original dry mass extracted (2% in carbon tetrachloride and 1% in the chloroform fraction). More than 95% of the activity of the polar compound was concentrated in 5% of the original dry weight extracted in the n-butanol fraction. According to the TLC separation, the components present in the bark and leaves differed (results not
Table 1 The MIC values in mg/ml of acetone extracts of the inner bark, outer bark and leaves of Marula using Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as test organisms and total activity present in different plant parts Organism and extract tested S. S. S. P. P. P. E. E. E. E. E. E.
aureus leaves aureus inner bark aureus outer bark aeruginosa leaves aeruginosa inner bark aeruginosa outer bark coli leaves coli inner bark coli outer bark faecalis leaves faecalis inner bark faecalis outer bark
Average
SD
Quantity (mg extracted from 1 g)
Total activity (mg/ml)
1.15 0.15 0.50 1.27 0.37 0.47 3.00 1.33 2.43 1.58 0.60 0.67
0.38 0.05 0.24 0.53 0.08 0.16 1.64 0.41 1.89 1.55 0.22 0.21
64 98 94 64 98 94 64 98 94 64 98 94
56 653 188 50 265 200 21 74 39 41 163 140
308
J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308
Fig. 1. Left bioautogram of Marula acetone leaf extract separated into different fractions by solvent – solvent extraction. TLC plate developed in chloroform/ethylacetate/formic acid (5:4:1) and sprayed with E. faecalis culture, incubated overnight then sprayed with INT. Growth inhibition indicated by colourless areas on TLC. Right TLC of fractions sprayed with vanillin – sulphuric acid. Lanes from left to right 35% MeOH in water, hexane, water, butanol, chloroform and carbon tetrachloride fractions.
shown), but in both cases there were compounds present with Rf values similar to the antibacterial compounds visible on bioautograms of leaf extracts (Fig. 1). This may mean that the same compounds are responsible for the antibacterial activity in leaves and stem bark. Attempts are under way to isolate and characterise these compounds. The results substantiate the ethnobotanical use of Marula bark for bacteria-related diseases. The results also show that leaf material is also useful for antibacterial uses. Because leaves are a more sustainable resource than bark it could be used without any detrimental effect on the plant. Bark is probably preferred because it contains a larger total quantity of antibacterial activity and because it is easier to transport and store for trading purposes.
Acknowledgements Maryna Steinmann and Nataly Martini gave valuable technical assistance. The Research Committee of the Faculty of Medicine, University of Pretoria, provided financial support.
References Balick, M.J., 1990. Ethnobotany and the identification of therapeutic agents from the rainforest. In: Chadwick, D.J., Marsh, J. (Eds.), Bioactive Compounds from Plants. Wiley, Chichester, pp. 22 – 39. Begue, W.J., Kline, R.M., 1972. The use of tetrazolium salts in bioautographic procedures. Journal of Chromatography 64, 182 – 184. Bryant, A.T., 1966. Zulu medicine and medicine men. C. Struik, Cape Town. Busson, F., 1985. Plantes Alimentaires de l’Quest Africain. Leconte, Marseille as cited by Oliver-Bever, 1986.
Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64, 711 – 713. Eloff, J.N., 1999. The antibacterial activity of 27 southern African members of the Combretaceae. South African Journal of Science 95, 148 – 152. Eloff, J.N., 2000. A proposal on expressing the antibacterial activity of plant extracts — a small first step in applying scientific knowledge to rural primary health care in South Africa. South African Journal of Science 96, 116 – 118. Fox, F.W., Young, M.E.N., 1982. Food from the veld. Edible wild plants of southern Africa. Delta Books, Johannesburg. Galves, J., Crespo, M.E., Zarzuelo, A., 1993. Pharmacological activity of a procyanidin isolated from Sclerocarya birrea bark: antidiarrhoeal activity and effects on isolated guinea-pig ileum. Phytotherapy Research 7, 25 – 28. Gelfand, M., Mavi, S., Drummond, R.B., Ndemera, B., 1985. The Traditional Medical Practitioner in Zimbabwe. Mambo Press, Gweru, Zimbabwe. Gueye, M.S., 1973. Contribution a` l’e´ tude pharmacodynamique d’une plante antidiabe´ tique (Sclerocarya birrea). Thesis in Pharmacy, Dakar cited by Oliver-Bever, 1986. Hussein, H.S.N., Deeni, Y.Y., 1991. Plants in Kano ethnomedicine: screening for antimicrobial activity and alkaloids. International Journal of Pharmacognosy 29, 51 – 56. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu medicinal plants — an inventory. University of Natal Press, Pietermaritzburg, South Africa. Kokwaro, J.O., 1976. Medicinal Plants of East Africa. East African Literature Bureau, Nairobi, Dar es Salaam. Mobogo, D.E.N., 1990. Unpublished MSc Thesis, University of Pretoria. Oliver-Bever, B., 1986. Medicinal Plants in Tropical West Africa. Cambridge University Press, Cambridge. Palgrave, K.C., 1983. Trees of Southern Africa. C. Struik, Cape Town. Pretorius, V., Rohwer, E., Rapp, A., Holtzhausen, L.C., Mandery, H., 1985. Volatile flavour components of marula Sclerocarya birrea subsp. caffra juice. Zeitschrift fu¨ r Lebensmittel Untersuchung und Forschung 181, 458 – 461. Suffness, M., Douros, J., 1979. Drugs of plant origin. Methods in Cancer Research 26, 73 – 126. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The medicinal and poisonous plants of southern and eastern Africa. E.S. Livingstone, Edinburgh.
Short Communication
Antibacterial activity of Marula (Sclerocarya birrea (A. rich.) Hochst. subsp. caffra (Sond.) Kokwaro) (Anacardiaceae) bark and leaves J.N. Eloff * Department of Pharmacology, Uni6ersity of Pretoria, Pretoria, South Africa Received 14 March 2001; received in revised form 10 April 2001; accepted 12 May 2001
Abstract Marula bark is widely used for bacteria-related diseases by indigenous cultures in Africa. This study was undertaken to investigate whether the ethnobotanical use can be validated by laboratory studies. Bark and leaves were extracted with acetone and MIC values were determined using a microplate serial dilution technique with Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as test organisms. All extracts were active with MIC values from 0.15 to 3 mg/ml. Based on minimum inhibitory concentration values, inner bark extracts tended to be the most potent followed by outer bark and leaf extracts, but the differences were not statistically significant. There were two major bioactive components visible after bioautography of leaf extracts: one strongly polar and the other highly non-polar. The bioactive components could be separated from 92% of the non-active dry matter by solvent– solvent fractionation into the carbon tetrachloride, chloroform and n-butanol fractions; these fractions, however, still contained many different compounds. Using bark may be detrimental to the plant, but leaf material can also be used for antibacterial application. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sclerocarya birrea subsp. Caffra; Bark; Leaf; Antibacterial; Bioactivity
1. Introduction The Marula (Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro) is one of the most highly valued indigenous trees in southern Africa. The Tonga people celebrate the Feast of the First Fruits by pouring a drink offering of the fresh juice of the fruit over the tombs of dead chiefs (Palgrave, 1983). The pulp of the fruit is delicious and the large nut is also edible. Some tribes such as the Pedi make a relish from the leaves (Fox and Young, 1982). Some attempts have been made to improve the plant by selection and breeding and it has become a commercial fruit crop recently. Watt and Breyer-Brandwijk (1962), Oliver-Bever (1986) * Tel.: +27-12-319-2139; fax: +27-12-319-2411. E-mail address: [email protected] (J.N. Eloff).
and Hutchings et al. (1996) identified a number of medicinal uses in southern, eastern and tropical West Africa. The presence of antimicrobial constituents may be inferred from the following ethnomedical uses: the Zulu people use bark decoctions administered as enemas for diarrhoea. Traditional Zulu healers wash in bark decoctions before treating patients with gangrenous rectitis and also administer the decoction to the patient (Bryant, 1966). Bark decoctions are taken in 300 ml doses for dysentery and diarrhoea in unspecified parts of southern Africa (Watt and Breyer-Brandwijk, 1962). Bark has also been used in treating proctitis. The Vhavenda use bark for treating fevers, stomach ailments and ulcers (Mobogo, 1990). Roots are used for many purposes including sore eyes in Zimbabwe (Gelfand et al., 1985). In East Africa, roots are an ingredient in an alcoholic medicine taken to treat an
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 6 0 - 4
306
J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308
internal ailment known as kati while bark is used for stomach disorders (Kokwaro, 1976). The Hausas in West Africa use a cold infusion of the bark along with native natron as a remedy for dysentery (Oliver-Bever, 1986). Galves et al. (1993) could show that extracts inhibit diarrhoea in mice. Bark yields 3.5–20.5% tannin, 10.7% tanning matter and traces of alkaloids (Watt and Breyer-Brandwijk, 1962). The fruit is rich in ascorbic acid and juice extracts yield 33 sesquiterpene hydrocarbons (Pretorius et al., 1985). Kernels yield 54– 60% of a non-drying oil and contain as much as 28% protein and some iodine (Watt and Breyer-Brandwijk, 1962). The oil-rich seeds contain 64% oleic acid, myristic, stearic and amino acids with a predominance of glutamic acid and arginine (Busson, 1985). The gum is rich in tannin. Tannins and flavonoids are present in leaves but no alkaloids, steroids or triterpenoids have been detected (Gueye, 1973). Only one paper published on the effect of S. birrea plant extracts on in vitro antimicrobial activity (Hussein and Deeni, 1991) was found. In a preliminary study these authors examined 34 plant species for antimicrobial activity. Methanolic extracts from dried powdered stembark had strong antibacterial activity (zone of inhibition\8 mm) against Corynebacterium diphtheriae, Pseudomonas aeruginosa and a Streptococcus spp. and less activity against unidentified Neisseria, Streptobacillus and Salmonella species. The extracts were inactive against Staphylococcus aureus and Escherichia coli. The authors indicated that their results differed from results obtained by earlier authors without specifying the differences or authors. Unfortunately no voucher specimens of the plants were retained. Subsequently, subspecies of Sclerocarya were recognised. It is therefore not clear which taxon Hussein and Deeni (1991) investigated. With a widely distributed plant that is valued highly for edible purposes and for treating many other ailments, Marula could be considered a ‘power plant’ (Balick, 1990). Power plants have been defined as widely used plants without any apparent or demonstrable pharmacological basis for their use. Some workers have eliminated ‘power plants’ from their list of plants to investigate. A disadvantage of using bark as source for the medicinal component is that injudicial removal of bark can lead to the death of the plant. If bark can be sold for a high price, valuable plant populations may be collected to extinction in nature. The aim of this paper is to determine if the use of extracts of Marula for ailments that may be related to bacterial pathogens can be substantiated in in vitro experiments and to compare the antibacterial activity of leaf and bark extracts.
2. Materials and methods Plant material was collected after fruiting in late May from a single tree growing in Pretoria. A voucher specimen was deposited at the National Herbarium (J N Eloff 503, PRE). Stem bark was collected by making two longitudinal cuts ca. 1 cm apart and ca. 25 cm long and removing the bark from the stem. The inner bark had a light buff colour and was separated from the dark brown flaky outer bark. Bark and leaves were dried indoors at ambient temperature and then ground to a fine powder in a Jankel and Ku¨ nkel Model A10 mill. The finely ground plant material (0.5 g) was extracted (5 ml) in a centrifuge tube and the marc collected by centrifuging at 300× g for 5 min. The extraction was repeated two times on the marc. The acetone was removed by a stream of cold air and redissolved in acetone to yield a concentration of 50 mg/ml. The plant extracts were analysed by TLC (5 ml of 100 mg extract/ml solution) on Merck TLC F254 plates with chloroform/ethyl acetate/formic acid (5:4:1) as eluent. Plates were sprayed with 0.5 g vanillin dissolved in 100 ml sulphuric acid/ethanol (40:10). Minimum inhibitory concentrations (MIC) were determined by two-fold serial dilution of extracts beyond the concentration where no inhibition of growth of Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 25922 was observed. The wells in the dilution series were inoculated with the relevant cultures, incubated overnight at 37 °C and 0.2 mg/ml p-iodonitrotetrazolium violet [INT] (Sigma) was added. After further incubation bacterial growth was indicated by the red colour of the INT formazan produced (Eloff, 1998). For bioautography, developed TLC plates were dried overnight and sprayed with a concentrated suspension of actively growing S. aureus cells, before incubating at 38 °C in a chamber at 100% relative humidity. Plates were sprayed with a 2 mg/ml solution of p-iodonitrotetrazolium violet (Sigma). Clear zones on the chromatogram indicated inhibition of growth (Begue and Kline, 1972). The acetone extract was taken to dryness in a rotary evaporator under reduced pressure and this extract was dissolved in a 1:1 mixture of chloroform/water. The water fraction was extracted with an equal volume of n-butanol to yield the water (W) and butanol (B) fractions. The chloroform fraction was taken to dryness in a rotary evaporator under reduced pressure and extracted with a 1:1 mixture of hexane and 10% water in methanol. The 10% water in methanol extract was diluted to 20% water in methanol and extracted with carbon tetrachloride to yield the carbon tetrachloride (CT) fraction. The 20% water in methanol extract was
J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308
diluted to 35% methanol in water and extracted with chloroform to yield the chloroform (CHL) fraction and the 35% water in methanol (MW) fractions (Suffness and Douros, 1979). In all cases equal volumes of the solvents were used and the extraction was repeated with a small volume ca. three more times or until all the colour was extracted. In some instances centrifugation was used to separate the fractions and a small quantity of an insoluble pellicle was formed that was discarded.
3. Results and discussion Acetone extracted 6.4% of the leaf, 9.4% of the outer bark and 9.8% of the inner bark. Extracts from leaves, inner bark and outer bark inhibited the growth of all the test organisms (Table 1). The average values for leaf, inner bark and outer bark for all the test organisms were 1.4 (SD=1.33), 0.49 (SD= 0.51) and 0.81 (SD = 1.23) mg/ml, respectively. The extremely high standard deviations are caused by combining MIC values for the different organisms. The standard deviation of MIC values with the same test organisms were much lower (Table 1). The MIC values for the positive control, gentamycin, ranged from 0.02 to 0.6 mg/ml. One microwell where the bacterial growth inhibition was not noted would lead to a doubling of the MIC value. Although the differences were not statistically significant, the inner bark extract tended to be more active (i.e. have a lower MIC) than the outer bark and the leaf extracts. Based only on MIC values, one could speculate that leaves would also be a useful source for treating ailments caused by bacteria. It should be kept in mind that extracts were dried and made up to a known concentration before bioassay and the MIC values found does not relate to the total quantity present in the different plant parts. The total antibacterial activity present in different parts of the plant can be calculated
307
by dividing the quantity extracted from one gram of plant material with the MIC value in mg/ml (Eloff, 2000) (Table 1). The average total activity for the four test organisms, with leaf extracts is 56, with outer bark is 189 and with inner bark is 385 ml/g. Staphylococcus aureus was the most sensitive with a total activity of 653 ml/g. This means that the antibacterial compounds present in 1 g of inner bark material diluted to 653 ml would still inhibit the growth of the test organisms. When the total activity is compared it explains why Marula leaves are not traditionally used for ailments related to bacteria. The total activity values obtained here are in the same order of magnitude as that found for leaves of members of the Combretaceae and Celastraceae (Eloff, 1999, 2000). To obtain some information on the active component(s) a larger quantity of dried leaves was extracted in acetone and fractionated by solvent–solvent extraction. After solvent–solvent extraction, most of the extract (85%) was in hexane, the least polar fraction. In increasing order of polarity the carbon tetrachloride fraction contained 2%, chloroform 1%, n-butanol 5%, 35% water in methanol 3% and water 3% of the dry weight of the original extract. Most of the antibacterial activity was in highly non-polar compounds in the chloroform and carbon tetrachloride fractions (Rf = 0.93). There was also some activity in the n-butanol fraction (Rf = 0.04) with chloroform/ethylacetate/formic acid (5:4:1) as eluent. According to the bioautograms there are only two major inhibiting compounds present in the leaf extracts. The solvent–solvent fractionation was effective in concentrating more than 95% of the activity of the non-polar compound in 3% of the original dry mass extracted (2% in carbon tetrachloride and 1% in the chloroform fraction). More than 95% of the activity of the polar compound was concentrated in 5% of the original dry weight extracted in the n-butanol fraction. According to the TLC separation, the components present in the bark and leaves differed (results not
Table 1 The MIC values in mg/ml of acetone extracts of the inner bark, outer bark and leaves of Marula using Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as test organisms and total activity present in different plant parts Organism and extract tested S. S. S. P. P. P. E. E. E. E. E. E.
aureus leaves aureus inner bark aureus outer bark aeruginosa leaves aeruginosa inner bark aeruginosa outer bark coli leaves coli inner bark coli outer bark faecalis leaves faecalis inner bark faecalis outer bark
Average
SD
Quantity (mg extracted from 1 g)
Total activity (mg/ml)
1.15 0.15 0.50 1.27 0.37 0.47 3.00 1.33 2.43 1.58 0.60 0.67
0.38 0.05 0.24 0.53 0.08 0.16 1.64 0.41 1.89 1.55 0.22 0.21
64 98 94 64 98 94 64 98 94 64 98 94
56 653 188 50 265 200 21 74 39 41 163 140
308
J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308
Fig. 1. Left bioautogram of Marula acetone leaf extract separated into different fractions by solvent – solvent extraction. TLC plate developed in chloroform/ethylacetate/formic acid (5:4:1) and sprayed with E. faecalis culture, incubated overnight then sprayed with INT. Growth inhibition indicated by colourless areas on TLC. Right TLC of fractions sprayed with vanillin – sulphuric acid. Lanes from left to right 35% MeOH in water, hexane, water, butanol, chloroform and carbon tetrachloride fractions.
shown), but in both cases there were compounds present with Rf values similar to the antibacterial compounds visible on bioautograms of leaf extracts (Fig. 1). This may mean that the same compounds are responsible for the antibacterial activity in leaves and stem bark. Attempts are under way to isolate and characterise these compounds. The results substantiate the ethnobotanical use of Marula bark for bacteria-related diseases. The results also show that leaf material is also useful for antibacterial uses. Because leaves are a more sustainable resource than bark it could be used without any detrimental effect on the plant. Bark is probably preferred because it contains a larger total quantity of antibacterial activity and because it is easier to transport and store for trading purposes.
Acknowledgements Maryna Steinmann and Nataly Martini gave valuable technical assistance. The Research Committee of the Faculty of Medicine, University of Pretoria, provided financial support.
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