Journal of Ethnopharmacology 134 (2011) 775–780 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm A comparative study of the antimicrobial and phytochemical properties between outdoor grown and micropropagated Tulbaghia violacea Harv. plants B. Ncube, V.N.P. Ngunge, J.F. Finnie, 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 a r t i c l e i n f o Article history: Received 2 November 2010 Received in revised form 7 January 2011 Accepted 21 January 2011 Available online 1 February 2011 Keywords: Antibacterial Antifungal Micropropagation Phenolic compounds Saponins Tulbaghia violacea a b s t r a c t Aim of the study: The study aimed to compare the antimicrobial and phytochemical properties of in vitro cultured and outdoor grown Tulbaghia violacea plants in the quest to validate the use of micropropagated plants as alternatives to outdoor grown plants in traditional medicine. Tulbaghia violacea is used extensively in South African traditional medicine for HIV/AIDS patients and in the treatment of gastrointestinal ailments, asthma, fever and tuberculosis. Materials and methods: Extracts of micropropagated and outdoor grown Tulbaghia violacea plants were evaluated for their antibacterial and antifungal activities against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and a fungus Candida albicans using microdilution methods. Saponins and phenolic compounds including condensed tannins, gallotannins and flavonoids were quantitatively determined using spectrophotometric methods. A qualitative test for saponins was also carried out. Results: The petroleum ether (PE) extracts of micropropagated plants and dichloromethane (DCM) extracts of outdoor grown plants showed good antibacterial activity, each against two bacterial test strains. PE extracts of micropropagated plants showed the best antibacterial activity with a minimum inhibitory concentration (MIC) of 0.39 mg/ml against Bacillus subtilis. Good MIC (<1 mg/ml) and minimum fungicidal concentration (MFC) values of 0.78 mg/ml were only obtained in DCM extracts of outdoor grown plants. MIC and MFC values for water and ethanol extracts of both micropropagated and outdoor grown plants were similar and in the range 3.125–12.5 mg/ml. Total phenolics, gallotannins, flavonoids and saponins were significantly higher in micropropagated plants than in outdoor grown ones. In all cases, the amounts of phytochemical compounds in micropropagated plants were more than twice that of outdoor grown plants except for condensed tannins. Conclusion: The results form a good basis for the use of Tulbaghia violacea micropropagated plants as a complement to the outdoor grown plants in traditional medicine. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Tulbaghia violacea Harv. (Alliaceae), bulbs and leaves are used traditionally in the southern African region for the treatment of gastrointestinal ailments, asthma, fever, and tuberculosis (Hutchings et al., 1996; Van Wyk and Wink, 2004). The freshly prepared herbal Abbreviations: ATCC, American type culture collection; BA, 6benzylaminopurine; CFU, colony forming unit; CTE, catechin equivalent; DCM, dichloromethane; DE, diosgenin equivalent; EtOH, ethanol; GAE, gallic acid equivalent; INT, p-iodonitrotetrazolium chloride; LC, least concern; MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration; MS, Murashige and Skoog; NAA, a-naphthalene acetic acid; PE, petroleum ether; PGR, plant growth regulators; YM, yeast malt. ∗ Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897. E-mail address: rcpgd@ukzn.ac.za (J. Van Staden). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.01.039 remedies are used as decoctions that are either taken orally or as enemas (Hutchings et al., 1996). Due to the devastating effects and the high prevalence rate of HIV/AIDS in South Africa, the search for anti-HIV agents has stimulated the screening of medicinal plants based on their ethnobotanical data (WHO, 1989; Motsei et al., 2003; Klos et al., 2009). Tulbaghia violacea is one of these plants and has shown promising antimicrobial activity against some medically important pathogenic bacteria and fungi that cause opportunistic infections in HIV/AIDS patients (McGaw et al., 2000; Gaidamashvili and Van Staden, 2002; Motsei et al., 2003). The plant has undoubtedly found its traditional use among HIV/AIDS patients. Young Tulbaghia violacea plants are eaten as vegetables (Hutchings et al., 1996). Apart from the medicinal and nutritive value, the plant is also used extensively as an ornamental in South Africa (Bryan, 2002). As a result of the extensive use and increased demand for medicinal plants, both formal and informal markets for these plants have 776 B. Ncube et al. / Journal of Ethnopharmacology 134 (2011) 775–780 grown tremendously in South Africa. Most of the plant material traded is from wild harvested plant stock (Mander and Mckenzie, 2005) and this acute harvesting is leading to local plant extinction (Mander, 1998). The basic idea behind sustainable harvesting is that a biological resource should be harvested within the limits of its capacity for self-renewal. Importantly, the use of Tulbaghia violacea in traditional medicine involves the destructive harvesting of the whole plant. Despite the plant being listed under the National Red List of South African plants as of least concern (LC) (Raimondo et al., 2009), the emerging interest and research emphasis being placed on the improvement and validation of traditional medicine increases the demand for these plants. This pressure exerts ecological instability on the resource base, hence the need for alternative medicinal sources. Although conventional cultivation of medicinal plants is a logical conservation strategy, it is challenged by a number of factors such as land availability, climate, season, water availability, diseases and pests, and slow growth of plants (Pierik, 1987; Arikat et al., 2004). The development and adoption of tissue culture methods has provided a means for rapid propagation of a large number of uniform plants while maintaining their genotypes (Bryan, 2002). The technology also allows an opportunity for ethnopharmacological exploitation of cells, tissues, organs or entire plants at their early stages of growth. Application of the technology in traditional medicine may offer benefits that include avoidance of collection of endangered wild medicinal plant species and rapid production of pharmacological compounds irrespective of seasonal and climatic conditions (Pierik, 1987; Arikat et al., 2004). The success of this strategy, however, depends on the validation of micropropagated plants through pharmacological screening on their suitability for use in traditional medicine. In view of the medicinal importance of Tulbaghia violacea, the present investigation was undertaken to compare antimicrobial and phytochemical properties of micropropagated and outdoor grown Tulbaghia violacea plants in an effort to validate and promote the use of micropropagated plants in traditional medicine. 2. Materials and methods 2.1. Plant material Tulbaghia violacea Harv. seeds (Silverhill Seeds Nursery, Cape Town) were surface sterilised and germinated for 30 days on onetenth strength of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962). The medium was supplemented with myoinositol (0.1 g/L), without plant growth regulators (PGRs) and sucrose. The pH of the medium was adjusted to 5.8 and solidified with 8 g/L No. 1 bacteriological agar (Oxoid Ltd., England). The medium was sterilised by autoclaving at 121 ◦ C for 20 min. The photoperiod of the growth room in which the cultures were placed was 16/8 h light/dark at 25 ± 2 ◦ C. Hypocotyls derived from in vitro grown seedlings were surface sterilised. These were cultured on full strength MS medium supplemented with sucrose (30 g/L), myo-inositol (0.1 g/L) and various concentrations of PGRs in the range 0–1.5 mM for NAA and 0–12 mM for BA. The pH of the medium was adjusted to 5.8 and solidified with agar (8 g/L). The medium was sterilised by autoclaving at 121 ◦ C for 20 min. Cultures were grown under 24 h light conditions at 25 ± 2 ◦ C for three months. The plantlets were subcultured onto a medium containing 1.5 mM NAA and 12 mM BA for a period of two months before being taken for analysis. Outdoor grown Tulbaghia violacea plants (12 years old) were collected in summer (December) from the University of KwaZuluNatal Botanical Garden, Pietermaritzburg, South Africa and a voucher specimen (NCUBE 04 NU) deposited in the University of KwaZulu-Natal Herbarium (NU), Pietermaritzburg. Whole plant samples (bulbs and leaves), including those of micropropagated plants were then dried at a constant temperature of 50 ◦ C in an oven and ground into fine powders. 2.2. Preparation of plant extracts The ground samples were sequentially extracted with 20 ml/g of petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water in a sonication bath containing ice for 1 h. The crude extracts were then filtered under vacuum through Whatman No. 1 filter paper and the organic extracts concentrated in vacuo at 35 ◦ C using a rotary evaporator. The concentrated extracts were subsequently dried at room temperature under a stream of cold air. Water extracts were freeze dried and kept in airtight containers. 2.3. Antibacterial activity Minimum inhibitory concentrations (MIC) of extracts for antibacterial activity were determined using the microdilution bioassay as described by Eloff (1998). Overnight cultures (incubated at 37 ◦ C in a water bath with an orbital shaker) of two Gram-positive (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600) and two Gram-negative (Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883) bacterial strains were diluted with sterile Mueller–Hinton (MH) broth to give final inoculums of approximately 106 CFU/ml (colony forming units). The dried crude organic plant extracts were resuspended in 70% ethanol to a concentration of 50 mg/ml while water extracts were dissolved in distilled water to the same concentration. One hundred microlitres of each extract were serially diluted two-fold with sterile distilled water in a 96-well microtitre plate for each of the four bacterial strains. A similar two-fold serial dilution of neomycin (Sigma–Aldrich, Germany) (0.1 mg/ml) was used as a positive control against each bacterium. One hundred microlitres of each bacterial culture were added to each well. Water and 70% ethanol were included as negative and solvent controls, respectively. The plates were covered with parafilm and incubated at 37 ◦ C for 24 h. Bacterial growth was indicated by adding 50 ml of 0.2 mg/ml p-iodonitrotetrazolium chloride (INT) (Sigma–Aldrich, Germany) and a further incubation at 37 ◦ C for 24 h. Since the colourless tetrazolium salt is biologically reduced to a red product due to the presence of active organisms, the MIC values were recorded as the concentrations in the last wells in which no colour change was observed after adding the INT indicator. Bacterial growth in the wells was indicated by a reddish-pink colour. The assay was repeated twice with two replicates per assay. 2.4. Antifungal activity A microdilution method as described by Eloff (1998) and modified for fungi (Masoko et al., 2007) was used to determine the antifungal activity of the extracts against Candida albicans (ATCC 10231). An overnight fungal culture was prepared in yeast malt (YM) broth. Four hundred microlitres of the overnight culture were added to 4 ml of sterile saline and absorbance was read at 530 nm. The absorbance was adjusted with sterile saline to match that of a 0.5 M McFarland standard solution. From this standardised fungal stock, a 1:1000 dilution with sterile YM broth was prepared giving a final inoculum of approximately 106 CFU/ml. Dried organic extracts were resuspended in 70% ethanol to a concentration of 50 mg/ml and water extracts were dissolved in water to the same concentration. One hundred microlitres of each extract were serially diluted two-fold with sterile water in a 96-well microtitre plate. A similar two-fold dilution of amphotericin B (Sigma–Aldrich, Germany) (2.5 mg/ml) was used as the positive control while water and 70% B. Ncube et al. / Journal of Ethnopharmacology 134 (2011) 775–780 ethanol were used as negative and solvent controls, respectively. One hundred microlitres of the dilute fungal culture were added to each well. The plates were covered with parafilm and incubated at 37 ◦ C for 24 h, after which 50 ml (0.2 mg/ml) INT were added and incubated for a further 24 h at 37 ◦ C. The wells remained clear where there was inhibition of fungal growth. MIC values were recorded as the lowest concentrations that inhibited fungal growth after 48 h. To determine the fungicidal activity, 50 ml of sterile YM broth were added to all the clear wells and further incubated at 37 ◦ C for 24 h after which the minimum fungicidal concentrations (MFC) were recorded as the last clear wells. The assay was repeated twice with two replicates per assay. 2.5. Phenolic content determination 2.5.1. Preparation of extracts Phenolic compounds were extracted from plant material as described by Makkar (1999). Dried plant samples (2 g) were extracted with 10 ml of 50% aqueous methanol by sonication on ice for 20 min. The extracts were then filtered under vacuum through Whatman No. 1 filter paper. 2.5.2. Determination of total phenolic compounds The amounts of total phenolic compounds in plant samples were determined using the Folin Ciocalteu (Folin C) assay for total phenolics as described by Makkar (1999) with slight modification (Ndhlala et al., 2007). Fifty microlitres of each extract from the plant samples were transferred into test tubes into which 950 ml of distilled water were added followed by 1 N Folin C phenol reagent (500 ml) and 2% sodium carbonate (2.5 ml). A blank that contained aqueous methanol instead of plant extracts was also prepared. The test mixtures were incubated for 40 min at room temperature and the absorbance was read at 725 nm using a UV–vis spectrophotometer (Varian Cary 50, Australia). Each extract had three replicates. Total phenolic concentrations were expressed as gallic acid equivalents (GAE). 2.5.3. The butanol–HCl assay for condensed tannins (proanthocyanidins) Three millilitres of butanol–HCl reagent (95:5, v/v) were added to 500 ml of each extract, followed by 100 ml ferric reagent (2% ferric ammonium sulphate in 2 N HCl). The test combination was mixed with a vortex and placed in a boiling water bath for 60 min. Absorbance was then read at 550 nm using a UV–vis spectrophotometer against a blank prepared by mixing the extract (500 ml) with butanol–HCl reagent (3 ml) and ferric reagent (100 ml), but without heating. Each extract had three replicates. Condensed tannins (%) were calculated as leucocyanidin equivalents using the formula developed by Porter et al. (1986). 2.5.4. Vanillin assay for flavonoids Plant extracts (50 ml), were made up to 1 ml with methanol in test tubes before adding 2.5 ml methanolic-HCl (95:5, v/v) and 2.5 ml vanillin reagent (1 g/100 ml acetic acid). Similar preparations of a blank that contained methanol instead of plant extracts were made. After 20 min at room temperature, absorbance was read at 500 nm using a UV–vis spectrophotometer. The flavonoid levels were expressed as catechin equivalents (CTE) (Hagerman, 2002). 2.5.5. Rhodanine assay for gallotannins Gallotannin contents from plant material were determined as described by Makkar (1999). Plant extracts (50 ml) in test tubes were made up to 1 ml with distilled water. One hundred microlitres of 0.4 N sulphuric acid and 600 ml of rhodanine were added to the diluted extracts. After 5 min, 200 ml of 0.5 N potassium hydroxide was added followed by 4 ml of distilled water after a further 2.5 min. 777 The mixtures were left for a further 15 min at room temperature, after which the absorbance at 520 nm was read using a UV–vis spectrophotometer against a blank that contained methanol instead of sample. Each extract was evaluated in triplicates and gallotannin concentrations were expressed as gallic acid equivalents (GAE). 2.6. Saponin content 2.6.1. Qualitative saponin detection Ten millilitres of distilled water were added to 0.1 g of ground samples in test tubes. The test tubes were corked and vigorously shaken for 2 min. The appearance of stable and persistent foam on the liquid surface for 15 min indicated the presence of saponins (Tadhani and Subhash, 2006). The presence of saponins was confirmed by the formation of an emulsion upon addition of ten drops of olive oil to the 2 ml aqueous extract. 2.6.2. Preparation of extracts for saponin quantification Saponins were extracted from the plant material as described by Makkar et al. (2007). The dried and ground plant samples were defatted with hexane in a Soxhlet apparatus for 3 h. After airdrying, saponins were extracted twice from the defatted samples (10 g) in 100 ml of 50% aqueous methanol by incubating at room temperature overnight with continuous stirring. The extracts were then centrifuged at 3000 × g for 10 min and the supernatant collected. The procedure was repeated with the original residue to obtain a second supernatant. The first and second supernatants were combined and filtered under vacuum through Whatman No. 1 filter paper. Methanol from the filtrate was evaporated from the solution under vacuum at 40 ◦ C to remain with the saponin sample in the aqueous phase. The aqueous phase was then centrifuged at 3000 × g for 10 min to remove water insoluble materials. The aqueous phase was then transferred into a separating funnel and extracted three times with an equal volume of chloroform to remove pigments. The concentrated saponins in the aqueous solution were then extracted twice with an equal volume of n-butanol. The n-butanol was evaporated under vacuum at 45 ◦ C. The dried fractions containing saponins were dissolved in 10 ml of distilled water and freeze-dried. 2.6.3. Quantitative determination of total saponins Total saponin content was determined using a spectrophotometric method as described by Hiai et al. (1976) with modifications. The crude saponin extracts were dissolved in 50% aqueous methanol to a concentration of 10 mg/ml. From this, aliquots of 250 ml (in triplicate) of each sample were transferred into test tubes into which an equal volume of vanillin reagent (8 g/100 ml ethanol) was added followed by 2.5 ml of 72% (v/v) sulphuric acid. The mixture was mixed with a vortex and placed in a water bath adjusted at 60 ◦ C for 10 min. The tubes were cooled in an ice-cold water bath for 3–4 min and absorbance was measured at 544 nm using a UV–vis spectrophotometer against a blank that contained 50% aqueous methanol instead of sample extract. The saponin concentrations were expressed as diosgenin equivalents (DE) calculated from a standard curve. 2.6.4. Quantitative determination of total steroidal saponins Total steroidal saponins were determined following the method by Baccou et al. (1977). Crude saponin extracts were dissolved in 50% aqueous methanol (0.1 mg/ml) from which 300 ml aliquots (corresponding to a sapogenin content of between 1 and 40 mg) were transferred into test tubes and placed in a boiling water bath at 100 ◦ C in order to remove methanol. After cooling, 2 ml of ethyl acetate were added followed by 1 ml of anisaldehyde–ethyl acetate reagent (0.5:95.5, v/v) and 1 ml sulphuric acid–ethyl acetate reagent (50:50, v/v). The test combination was mixed with a vortex 778 B. Ncube et al. / Journal of Ethnopharmacology 134 (2011) 775–780 and incubated in a water bath at 60 ◦ C for 20 min. After cooling for 10 min in a water bath at room temperature, absorbance was measured at 430 nm using a UV–vis spectrophotometer against a blank that contained ethyl acetate instead of sample. Each extract was evaluated in triplicate and steroidal saponin concentrations were expressed as diosgenin equivalents (DE) calculated from a standard curve. 2.7. Statistical analysis Data on phytochemical evaluation was subjected to one way analysis of variance (ANOVA) using SPSS software for Windows (SPSS® , version 15.0, Chicago, USA). Where there was statistical significance (P ≤ 0.05), the means were further separated using least significant difference (LSD). 3. Results and discussion 3.1. Antimicrobial activity The antibacterial MIC values and antifungal MIC and MFC values of Tulbaghia violacea extracts are presented in Table 1. Only MIC and MFC values less than 1 mg/ml were considered sufficiently active for crude extracts (Aligiannis et al., 2001). The best antibacterial activity was shown by the PE extracts of micropropagated plants with an MIC value of 0.39 mg/ml compared to 0.78 mg/ml from the DCM extracts of the outdoor grown plants against Bacillus subtilis. Petroleum ether extracts of micropropagated plants showed good activity (MIC < 1 mg/ml) against Bacillus subtilis and Klebsiella pneumoniae while DCM extracts were active from the outdoor grown plants against Bacillus subtilis and Staphylococcus aureus. Although HIV/AIDS has relatively minimal ethnobotanical treatments, the good MIC values shown by Tulbaghia violacea against these bacterial strains provides prospects for the treatment of such ailments since the plant is used traditionally among HIV/AIDS patients. None of the extracts showed good activity against Escherichia coli. This is surprising due to the plant’s widespread use in traditional medicine in the treatment of gastrointestinal ailments (Hutchings et al., 1996) which may be caused by Escherichia coli. Petroleum ether extracts of micropropagated plants, however, showed promising activity (1.56 mg/ml) although slightly higher than 1 mg/ml. Manipulation of the culture environment may possibly result in the accumulation of high enough levels of bioactive compound(s) in these plants. Compared to DCM, PE is a non-polar solvent and accordingly extracts less polar compounds than DCM. The observed activity from the two extracts in different plants indicates that the activity in micropropagated plants is due to non-polar compound(s) compared to the less polar compound(s) in outdoor grown plants. Most of these phytochemical compounds are produced in response to external stimuli (Derita et al., 2009) such as light, moisture stress and temperature among other factors. It is possible therefore, that the consistent exposure to constant temperature and photoperiod, and media compositional balance in micropropagated plants, might have favoured the production of particular compound(s), and hence the increase in the bioactivity. In terms of the antifungal activity, greater than 1 mg/ml MIC and MFC values were recorded in all extracts of micropropagated plants as compared to the outdoor grown plants (Table 1). Only DCM extracts of outdoor grown plants showed noteworthy fungicidal activity (0.78 mg/ml). The results are, however, consistent with those found in previous studies using outdoor grown plants against the same fungus (Motsei et al., 2003). Candida albicans is more resistant to plant extracts (Heisey and Gorham, 1992; Buwa and Van Staden, 2006), and the good fungicidal activity demonstrated by Tulbaghia violacea offers promising prospects for the treatment of candidiasis. Particularly interesting were the stable MIC and MFC values (1.56 mg/ml) shown by the PE extracts of micropropagated plants, although they were slightly higher than 1 mg/ml. The concentration of the bioactive compound(s) in these extracts, if identified, can possibly be further increased to effective levels by manipulating the plants’ culture environment. However, antifungal activities of EtOH and water extracts were the same for both outdoor grown and micropropagated plants. The extract yields (%) and total activity of the plants from different extracting solvents are shown in Table 2. Although the yields of micropropagated plants were lower than the outdoor grown ones in most of the extracts, their antimicrobial activities were fairly comparable. A comparison of the total activity of the corresponding extracts between the outdoor grown and micropropagated plants reveals that micropropagated plants had more concentrated active compounds in the PE extracts than the outdoor grown ones. For example, the yield (1.52%: equivalent to 15.2 mg/g) and MIC value (0.39 mg/ml) of the PE extracts of micropropagated plants against Bacillus subtilis gives a total activity of 39 ml/g (15.2 mg/0.39 mg/ml) (Eloff, 2004) compared to the PE extracts of the outdoor grown plants with a total activity of 11.4 ml/g (17.8 mg/1.56 mg/ml) against the same bacterium. The same PE extracts of the micropropagated plants showed good activity against Klebsiella pneumoniae with a total activity of 19.5 ml/g compared to 11.4 ml/g from the outdoor grown plants, despite the fact that the outdoor grown plants had higher extract yield than the micropropagated ones. Total activity (ml/g) indicates the degree to which the active compound(s) in one gram of plant material can be diluted and still inhibit the growth of the tested microorganism (Eloff, 2000). The DCM extracts of the outdoor grown plants on the other hand, were the only active ones compared to those of the micropropagated plants, with total activities of 11.9 ml/g against Bacillus subtilis and Staphylococcus aureus, respectively, compared to 4.2 ml/g from the micropropagated plants against the same bacterial strains. The DCM extracts of the outdoor grown plants were the only extracts that showed good fungicidal activity and had a total activity of 11.9 ml/g compared to 2.1 mg/g from the DCM extracts of micropropagated plants. This indicates that gram for gram; micropropagated plants had more concentrated active compound(s) of the less polar class (PE extractable) while the outdoor grown plants have more concentrated active compound(s) extractable by DCM. This, however, suggests some differences in the chemical profile composition of the two plant samples. Results of the antimicrobial and total activity of the two plant samples indicate that the PE extracts of micropropagated plants can supplement outdoor grown ones in the traditional medicinal use of Tulbaghia violacea, particularly in the treatment of Bacillus subtilis and Klebsiella pneumoniae related ailments. 3.2. Phenolic composition The total phenolic compounds, flavonoid, gallotannin and condensed tannin contents of both outdoor grown and micropropagated plants are presented in Table 3. Micropropagated plants showed higher total phenolic, flavonoid and gallotannin concentrations compared to the outdoor grown plants. Particularly interesting to note was the markedly high levels of the flavonoids in micropropagated plants (15.3 ± 1.5 mg CTE/g) compared to those that are outdoor grown (0.44 ± 0.03 mg CTE/g dry matter). Flavonoids are reported to have multiple biological effects including antioxidant (Rice-Evans et al., 1997; Tapas et al., 2008), hepatoprotective (Tapas et al., 2008), and antimicrobial activities (Tapiero et al., 2002; Makkar et al., 2007). In medicinal plants, the plant’s ability to concentrate bioactive compounds in high amounts within a limited period of time, makes it more preferable to use. The high levels of phenolic compounds in micropropa- B. Ncube et al. / Journal of Ethnopharmacology 134 (2011) 775–780 779 Table 1 Antimicrobial activity of Tulbaghia violacea extracts expressed as MIC (mg/ml) against bacteria and MIC (mg/ml) and MFC (mg/ml) against Candida albicans. Sample Extract Yield (%) Antibacterial MIC (mg/ml) Antifungal Bacteria Candida albicans Bs Ec Kp Sa MIC (mg/ml) MFC (mg/ml) 3.125 0.78 12.5 12.5 0.39 0.78 3.125 12.5 6.25 0.78 6.25 12.5 1.56 3.125 3.125 3.125 1.56 3.125 3.125 12.5 1.56 6.25 6.25 12.5 Tulbaghia violacea (outdoor grown) PE DCM EtOH Water 1.78 0.93 26.1 17.95 1.56 0.78a 6.25 >12.5 3.125 3.125 3.125 12.5 1.56 1.56 12.5 12.5 Tulbaghia violacea (micropropagated) PE DCM EtOH Water 1.52 1.31 19.98 14.05 0.39 3.125 12.5 >12.5 1.56 3.125 3.125 12.5 0.78 3.125 12.5 3.125 0.098 3.13 1.56 1.56 – – – – – – 0.15 9.80 Neomycin (mg/ml)a b Amphotericin B (mg/ml) MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration; Bs., Bacillus subtilis; Sa., Staphylococcus aureus; Ec., Escherichia coli; Kp., Klebsiella pneumoniae; PE, petroleum ether; DCM, dichloromethane; EtOH, 80% ethanol. Values boldly written are considered very active (1 ≤ mg/ml). a Positive control for the antibacterial assay. b Positive control for the antifungal assay. Table 2 Extract yields (%) and total antibacterial and antifungal activity (ml/g) of Tulbaghia violacea extracts. Sample Extract Yield (%) Total activity (ml/g) Total activity Bacteria Candida albicans Bs Ec Kp Sa (ml/g) (ml/g) Tulbaghia violacea (outdoor grown) PE DCM EtOH Water 1.78 0.93 26.1 17.95 11.4 11.9 41.76 >14.4 5.7 3.0 83.5 14.4 11.4 6.0 20.9 14.4 5.7 11.9 20.9 14.4 45.6 11.9 83.5 14.4 2.8 11.9 41.76 14.4 Tulbaghia violacea (micropropagated) PE DCM EtOH Water 1.52 1.31 19.98 14.05 39.0 4.2 16.0 >11.2 9.7 4.2 63.9 11.2 19.5 4.2 16.0 45.0 9.7 4.2 63.9 45.0 9.7 4.2 63.9 11.2 9.7 2.1 32.0 11.2 Bs., Bacillus subtilis; Sa., Staphylococcus aureus; Ec., Escherichia coli; Kp., Klebsiella pneumoniae; PE, petroleum ether; DCM, dichloromethane; EtOH, 80% ethanol. Values boldly written represent total activity of extracts that were active. gated plants observed in this study, demonstrate that these plants have the capacity to produce large quantities of these secondary metabolites and thus, have potential to be exploited commercially to accumulate these valuable compounds for their medicinal benefits. The biosynthesis and accumulation of plant secondary metabolites depend on exogenous factors, but the plant’s intrinsic factors, developmental stage and tissue differentiation determine the site of synthesis (Treutter, 2001; Mirdehghan and Rahemi, 2007). Accordingly, based on the results obtained in this study, it therefore, follows that the physiological and/or environmental conditions are more favourable for phenolic compound production in micropropagated than in outdoor grown plants. Nevertheless, both types of plants accumulate sufficient quantities of various non polar metabolites to effect some good antimicrobial activities. Table 3 Total phenolic, flavonoid, gallotannin, condensed tannin, total saponin and steroidal saponin contents in micropropagated and outdoor grown Tulbaghia violacea extracts. Values represent the means ± standard error (n = 3). Total phenolics (mg GAE/ml) Flavonoids (mg CTE/ml) Gallotannins (mg GAE/ml) Condensed tannins (% LCE/g) Total saponins (mg DE/ml) Total steroidal saponins (mg DE/ml) Micropropagated Outdoor grown 3.56 15.3 4.03 0.72 25.14 10.03 0.45 0.44 2.32 0.84 8.94 3.77 ± ± ± ± ± ± 0.06a 1.5a 0.24a 0.22b 0.74a 0.58a ± ± ± ± ± ± 0.021b 0.03b 0.02b 0.078a 0.11b 0.43b Values in a row with different letters are significantly different at P ≤ 0.05. GAE, gallic acid equivalents; CTE, catechin equivalents; LCE, leucocyanidin equivalents; DE, diosgenin equivalent. 3.3. Saponin composition The qualitative froth test for the presence of saponins was positive for both types of plant material. Table 3 represents total saponins and total steroidal saponins in micropropagated and outdoor grown Tulbaghia violacea plants. As was the trend with the phenolic content, saponins and steroidal saponins were significantly higher in micropropagated plants than in the outdoor grown ones. Saponins, particularly steroidal saponins have been reported in Tulbaghia violacea (Burton, 1990). The results obtained in this study agree with these previous findings. However, the markedly high saponin content obtained in micropropagated plants compared to the outdoor grown plants is an indication that in vitro culture conditions are favourable for the production of plant secondary metabolites. Saponins are reported to have antimicrobial activity (Bader et al., 2000), anti-inflammatory (Navarro et al., 2001) and haemolytic (Oda et al., 2000) properties. This markedly high saponin composition recorded in micropropagated plants shows their promising pharmacological potential as sources of bioactive molecules. Manipulation of the culture environment may be effective in increasing accumulation of these compounds. 4. Conclusions The antibacterial results exhibited by micropropagated Tulbaghia violacea plants in comparison with the outdoor grown ones were fairly comparable. Although micropropagated plants did not 780 B. Ncube et al. / Journal of Ethnopharmacology 134 (2011) 775–780 show good antifungal activity compared to the outdoor grown plants, their PE extracts appeared to contain some active antifungal compound(s). However, the concentration(s) of the compound(s) might have been lower than those required to effect good activity. Micropropagated plants of Tulbaghia violacea may be used as alternative sources of treatments of some ailments caused by bacteria in traditional medicine and have a promising potential to produce better activity. The generally high total phenolics, gallotannin, flavonoid and saponin contents recorded in micropropagated plants in contrast to those grown outdoors, makes them potential sources for the production of secondary metabolites for medicinal purposes. The fact that micropropagated plants produce secondary metabolites at an early stage of growth provides an opportunity for rapid production of pharmacological compounds that can be utilised for medicinal purposes. Although the yields of polar extracts (water and EtOH) of the outdoor grown plants were higher than those from the micropropagated ones, the later yielded high levels of the screened polar compounds (phenolics and saponins) in this study. This may, however, indicate the presence of other polar compounds in these extracts, such as carbohydrates. Further synergistic studies to determine the biological activity of all the combined extracts are required to give an overall picture of the whole extracts. Acknowledgements The National Research Foundation (NRF), Pretoria and the University of KwaZulu-Natal are gratefully acknowledged for financial assistance. References Aligiannis, N., Kalpoutzakis, E., Mitaku, S., Chinou, I.B., 2001. Composition and antimicrobial activity of the essential oils of two Origanum species. Journal of Agricultural and Food Chemistry 49, 4168–4170. Arikat, N.A., Jawad, F.M., Karam, N.S., Shibli, R.A., 2004. Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa Mill.). Scientia Horticulturae 100, 193–202. Baccou, J.C., Lambert, F., Sauvaire, Y., 1977. Spectrophotometric method for the determination of total steroidal sapogenin. Analyst 102, 458–465. Bader, G., Seibold, M., Tintelnot, K., Hiller, K., 2000. Cytotoxicity of triterpenoid saponins. Part 2. Relationships between the structures of glycosides of polygalacic acid and their activities against pathogenic Candida species. Pharmazie 55, 72–74. Bryan, J.E., 2002. Bulbs. Timber Press Inc., Oregon. Burton, J.S., 1990. A chemical investigation of Tulbaghia violacea. MSc Thesis, Department of Chemistry, Rhodes University. Buwa, L.V., Van Staden, J., 2006. Antibacterial and antifungal activity of traditional medicinal plants used against venereal diseases in South Africa. Journal of Ethnopharmacology 103, 139–142. Derita, M.G., Leiva, M.L., Zacchino, S.A., 2009. Influence of plant part, season of collection and content of the main active constituent, on the antifungal properties of Polygonum acuminatum Kunth. Journal of Ethnopharmacology 124, 377–383. 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., 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. Eloff, J.N., 2004. Quantification the bioactivity of plant extracts during screening and bioassay guided fractionation. Phytomedicine 11, 370–371. Gaidamashvili, M., Van Staden, J., 2002. Interaction of lectin-like proteins of South African medicinal plants with Staphylococcus aureus and Bacillus subtilis. Journal of Ethnopharmacology 80, 131–135. Hagerman, A.E., 2002. Tannin Chemistry. Miami University. Heisey, R.M., Gorham, B.K., 1992. Antimicrobial elects of plant extracts on Streptococcus mutans, Candida albicans, Trichophyton rubrum and other microorganisms. Letters in Applied Microbiology 14, 136–139. Hiai, S., Oura, H., Nakajima, T., 1976. Color reaction of some sapogenins and saponins with vanillin and sulphuric acid. Planta Medica 29, 116–122. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A., 1996. Zulu Medicinal Plants. An Inventory. University of Natal Press, Pietermaritzburg. Klos, M., Van de Venter, M., Milne, P.J., Traore, H.N., Meyer, D., Oosthuizen, V., 2009. In vitro anti-HIV activity of five selected South African medicinal plant extracts. Journal of Ethnopharmacology 124, 182–188. Makkar, H.P.S., 1999. Quantification of tannins in tree foliage: a laboratory manual for the FAO/IAEA co-ordinated research project on “Use nuclear and related techniques to develop simple tannin assay for predicting and improving the safety and efficiency of feeding ruminants on the tanniniferous tree foliage”. In: Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture , Vienna. Makkar, H.P.S., Sidhuraju, P., Becker, K., 2007. Plant Secondary Metabolites. Humana Press Inc., New Jersey. Mander, M., 1998. The Marketing of Indigenous Medicinal Plants in Southern Africa: A Case Study in KwaZulu-Natal. FAO, Rome. Mander, M., Mckenzie, M., 2005. Southern African Trade Directory of Indigenous Natural Products. Commercial Products from the Wild Group, Matieland. Masoko, P., Picard, J., Eloff, J.N., 2007. The antifungal activity of twenty-four southern African Combretum species. South African Journal of Botany 73, 173– 183. McGaw, L.J., Jäger, A.K., Van Staden, J., 2000. Antibacterial, anthelmintic and anti-amoebic activity in South African medicinal plants. Journal of Ethnopharmacology 72, 247–263. Mirdehghan, S.H, Rahemi, M., 2007. Seasonal changes of mineral nutrients and phenolics in pomegranate (Punica granatum L.) fruit. Scientia Horticulturae 111, 120–127. Motsei, M.L., Lindsey, K.L., Van Staden, J., Jäger, A.K., 2003. Screening of traditionally used South African plants for antifungal activity against Candida albicans. Journal of Ethnopharmacology 86, 235–241. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497. Navarro, P., Giner, R.M., Recio, M.C., Máñez, S., Cerdá-Nicolás, M., Ríos, J.L., 2001. In vivo anti-inflammatory activity of saponins from Bupleurum rotundifolium. Life Sciences 68, 1199–1206. Ndhlala, A.R., Kasiyamhuru, A., Mupure, C., Chitindingu, K., Benhura, M.A., Muchuweti, M., 2007. Phenolic composition of Flacourtia indica, Opuntia megacantha and Sclerocarya birrea. Food Chemistry 103, 82–87. Oda, K., Matsuda, H., Murakami, T., Katayama, S., Ohgitani, T., Yoshikawa, M., 2000. Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biological Chemistry 381, 67–74. Pierik, R.L.M., 1987. In Vitro Culture of Higher Plants. Martinus Nijhoff, Dordrecht. Porter, L.J., Hrstich, L.N., Chan, B.G., 1986. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25, 223–230. Raimondo, D., Agenbag, L., Foden, W., Victor, J., Helme, N., Turner, R., Kamundi, D.A., Manyama, P.A., 2009. Red List of South African Plants. South African National Botanical Institute, Pretoria. Rice-Evans, C.A., Miller, N.J., Paganga, G., 1997. Antioxidant properties of phenolic compounds. Trends in Plant Science 2, 152–159. Tadhani, M., Subhash, R., 2006. Preliminary studies on Stevia rebaudiana leaves: proximal composition, mineral analysis and phytochemical screening. Journal of Medical Science 6, 321–326. Tapas, A.R, Sakarkar, D.M., Kakde, R.B., 2008. Flavonoids as nutraceuticals: a review. Tropical Journal of Pharmaceutical Research 7, 1089–1099. Tapiero, H., Tew, K.D., Matheı̌, G., 2002. Polyphenols: do they play a role in the prevention of human pathologies? Biomedicine and Pharmacotherapy 56, 200–207. Treutter, D., 2001. Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regulation 34, 71–89. Van Wyk, B.-E., Wink, M., 2004. Medicinal Plants of the World. Briza Publications, Pretoria. World Health Organisation (WHO), 1989. In vitro screening of traditional medicines for anti-HIV activity: memorandum from a WHO meeting. Bulletin of the World Health Organization 87, 613–618.