Journal of Ethnopharmacology 143 (2012) 372–376 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep Antidiabetic, anti-oxidant and antimicrobial activities of Fadogia ancylantha extracts from Malawi K.K. Nyirenda a,b,n, J.D.K. Saka a, D. Naidoo b, V.J. Maharaj b, C.J.F. Muller c a Chemistry Department, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi Biosciences, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, South Africa c Diabetes Discovery Platform, Medical Research Council, Tygerberg 7505, South Africa b a r t i c l e i n f o a b s t r a c t Article history: Received 16 April 2012 Received in revised form 29 June 2012 Accepted 5 July 2012 Available online 16 July 2012 Ethnopharmacological relevance: Communities in Chilumba, Malawi use herbal tea prepared from Fadogia ancylantha Schweinf (Rubiaceae) leaves for the management of diabetes, hypertension and alleviation of symptoms of gastrointestinal disorders and pneumonia. The objective of the study was to evaluate the in vitro antidiabetic, anti-oxidant and antimicrobial activities of the crude extracts of the leaves prepared by using three different extraction methods. Materials and methods: Each of the organic, cold and hot aqueous extracts of the herbal tea was evaluated for its effect on glucose uptake in C2C12 muscle and Chang cell lines. Metformin and insulin were used as positive controls. The anti-oxidant activity, based on neutralisation of DPPH free radicals, was determined spectrophotometrically. The Agar serial dilution method was utilised to determine the minimum inhibitory concentration (MIC) of the extracts for the selected fungal and bacterial strains. Results and discussion: The organic extract (12.5 mg/ml) exhibited the highest in vitro glucose uptake increases in Chang cells (181.247 0.29%) and C2C12 muscle cells (172.29 7 0.32%) while the hot and cold aqueous extracts gave lower uptakes, 145.947 0.37% and 138.70 7 0.52% in Chang cells respectively. At 100 mg/ml, aqueous extracts gave significantly higher (p o 0.01) anti-oxidant activity (range 85.78–86.29%) than their organic counterpart (68.16%). The minimum inhibitory concentration (156 mg/ml) was obtained in the organic extract against the fungus Aspergillus fumigatus and moderate growth inhibition was observed with other test micro-organisms. The hot aqueous extract inhibited the growth of all test organisms except Pseudomonas aeruginosa. The cold aqueous extract was inactive against Pseudomonas aeruginosa and Candida albicans. The differences in the MIC values between the aqueous extracts seem to suggest that raised temperatures, as traditionally practised, facilitate the extraction of secondary bioactive metabolites. Conclusion: These results show that Fadogia ancylantha extracts have high antidiabetic and anti-oxidant properties. & 2012 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-oxidant Antimicrobial Diabetes Fadogia ancylantha Malawi 1. Introduction Plants play an important role in maintenance of human health and contribute greatly to the management of various ailments and nutrition of the African communities. Plants also form a valuable source of medicines and despite remarkable progress in synthetic organic chemistry, over 25% of the prescribed drugs in industrialised countries are derived directly or indirectly from plants (Newman et al., 2000). In developing countries, the World Health Organization (WHO) estimates that about three quarters of the population relies on plant based preparations in their n Corresponding author at: Chemistry Department, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi. E-mail address: kk_nyirenda2004@yahoo.com (K.K. Nyirenda). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.07.002 traditional medicinal system and over 80% of Malawians rely fully or partly on plants for human primary health care (WHO, 2003). Systematic indigenous knowledge gathering on the utilisation of known plants among communities may result in the discovery of novel and effective compounds and plant-derived commercial food products (Cortella and Pocheltino, 1999; Roja and Rao, 2000; Tomoko et al., 2002). Indigenous knowledge gathered from Chilumba indicates that the majority of the elderly population prefers tea prepared from a local herbal plant, Fadogia ancylatha Scheinf over the commercial product derived from Camellia sinensis (Saka, Personal communication). Several studies have reported the health benefits of the Camellia sinensis tea in humans including protection against degenerative diseases as well as antiproliferative activity on hepatoma cells (Vanessa and Gary, 2004. and antitumorigenic properties (Roomi et al., 2007). Furthermore, its extracts and isolated constituents are effective in K.K. Nyirenda et al. / Journal of Ethnopharmacology 143 (2012) 372–376 preventing oxidative stress (Babu et al., 2006) and neurological problems (Unno et al., 2007). Fadogia ancylantha Scheinf (Rubiaceae), locally known as ‘Masamba gha Muthondo’ in Karonga, Malawi, is a wild perennial shrub, 0.5–1.8 m in height (Petit, 2002). The plant has been used in cosmetic compositions to improve skin tone and also to improve anti-aging, antibacterial, and anti-free-radical effects on aged, photoaged, stressed, and tired skin, showing simultaneously protective and restorative properties (Mencherini et al., 2010). In the Eastern Highlands of Zimbabwe, the herbal tea, known as the Makoni tea, has been used traditionally to treat a variety of ailments including abdominal pain, asthma, loss of appetite and constipation (Muchuweti et al., 2008). Until recently, the use of Fadogia ancylantha as a herbal tea and thus as an important substitute of Camellia sinensis tea in Malawi was unknown. The tea is used to manage diabetes, hypertension and to alleviate symptoms of gastrointestinal disorders and pneumonia. The antidiabetic and antimicrobial properties of the local plant species have not been reported. Therefore, the objective of the present study was to determine the bioactivity of preparations based on three different extraction methods: (i) traditional procedure of brewing tea, (ii) cold aqueous extraction and (iii) organic solvent extraction. 2. Materials and methods 2.1. Plant material Plants used in this study were harvested and processed based on the indigenous knowledge of the community in Chilumba, Malawi. The plant was identified at the National Herbarium and Botanical Gaderns of Malawi (NHBGM), Zomba, where a voucher specimen, HB 720410, was deposited. The plants were harvested between March and April, 2010, after flowers changed colour from yellow to brown. Leaves of Fadogia ancylantha were harvested and dried under shed for three days. Fermentation, under local conditions, was complete after two days of drying when the green leaves turned brown. The dried leaves were ground using a mortar and pestle, sieved and the resultant powdery product (1.5 kg) was collected and stored in plastic bags until analysis. 2.2. Preparation of extracts 2.2.1. Cold aqueous extraction Approximately 50 g of plant material was weighed into 5 l conical flask and de-ionised water (2 l) was added. The mixture was left for overnight agitation, after which it was filtered through Whatman (no. 2) filter paper into a bottle and left in a cold room at  20 1C for 24 h. The filtrate was freeze dried to give 12.13 g of crude extract, A. 2.2.2. Hot water extraction (traditional procedure) A traditional method of preparing tea was mimicked in the laboratory in order to obtain the extract, B. De-ionised water (1 l) was heated in a beaker to boiling point. Five tablespoonfuls (24.6 g) of the plant material was added to the boiled water and this was allowed to boil for a further 5 min. The brewed herbal tea was cooled to room temperature, filtered, placed in a cold room at 20 1C for 24 h and freeze dried. Crude extract, B (3.17 g), was obtained and stored at 4 1C until analysis. 2.2.3. Organic extraction Approximately 50 g of the plant material was weighed, added to 2 l of methanol/dichloromethane (1:1), stirred and stored overnight at room temperature. The mixture was filtered; the 373 filtrate was evaporated under vacuum using a rotary evaporator and 12.1 g of a maroon syrup organic extract, labelled as C, was obtained. The extract was stored at 4 1C in a refrigerator until analysis. 2.3. In vitro antidiabetic activity To determine the effect of the three crude extracts on glucose uptake, an antidiabetic assay, described previously by Van de Venter et al. (2008) with slight modifications, was performed using two in vitro cell culture models. C2C12 muscle and Chang liver cells originating from the American Type Culture Collection (ATCC) (Manassas, USA) were obtained from Highveld Biological (Johannesburg, South Africa). 2.3.1. C2C12 in vitro glucose uptake model Following differentiation, C2C12 cells were acutely exposed for 1 h at 37 1C in humidified air with 5% CO2 to the relevant extracts and controls. The respective plant extracts at a concentration of 12.5 mg/ml, insulin (1 mM), metformin (1 mM) or vehicle control consisting of Dimethylsulphoxide (DMSO) solvent diluted in media were added to pyruvate and serum free Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 8 mM glucose. After incubation, an aliquot (10 ml) of the media was collected and mixed with glucose oxidase reagent (200 ml). The mixture was incubated for 15 min at 37 1C and the absorbance was read at 492 nm on a multiplate reader (Multiscan MSsversion 4.0 Labsystems type 352). Glucose concentrations were calculated from a glucose standard curve using 0, 2, 4, 6, 7 and 8 mM glucose. Vehicle controls (diluted DMSO) were included to establish basal glucose uptake levels. Insulin was included as a positive control and metformin as a diabetic drug control. The assay was performed in triplicate. 2.3.2. Chang liver cell in vitro glucose uptake model Plant extracts (12.5 mg/ml) were added to Chang cells in culture three days prior to performing the glucose uptake experiments. Following chronic exposure, the cells were acutely exposed for 3 h at 37 1C in humidified air with 5% CO2 to either 12.5 mg/ml of the respective plant extracts, insulin (1 mM), metformin (1 mM) or solvent control in pyruvate and serum free DMEM supplemented with 8 mM glucose. After incubation the glucose concentration of the media was determined as described for C2C12 cells. 2.4. Determination of anti-oxidant activity The radical scavenging activity was determined using the procedure described by Brand-Williams et al. (1995) that involves quenching free radicals of DPPH (2, 2-diphenyl-1-picrylhydrazyl hydrate) by the anti-oxidant. The stable free radical DPPH was dissolved in methanol to give a stock solution (100 mM). Each of the three plant extracts was previously dissolved in DMSO and diluted to produce 7 concentrations (100, 50, 25, 12.5, 6.25, 3.13 and 1.56 mg/ml) in a 96-well plate. To each mixture DPPH (100 ml) was added, after which it was shaken vigorously and kept in the dark at room temperature for 30 min. The decrease in absorption was measured at 540 nm using a Shimadzu UV-1601 UV–vis spectrophotometer. One plate filled by DPPHþ DMSO was read immediately and designated T0. Negative controls consisted of DPPHþDMSO and methanolþDMSO without drug, while gallic acid served as a standard. Each experiment was done in triplicates. The radical scavenging activity was calculated using the following formula (Brand-Williams et al., 1995): 374 K.K. Nyirenda et al. / Journal of Ethnopharmacology 143 (2012) 372–376 The Agar dilution method (Eloff, 1998) was used to determine the antimicrobial activity of the extracts. All microorganisms were obtained from the American Type Culture Collection (ATCC) through Highveld Biological (Johannesburg, South Africa). Three fungal strains: Candida albicans (ATCC 10231), Aspergillus fumigatus (ATCC 29214) and Cryptococcus neoformans (ATCC 10239); two Gram-positive bacterial strains: Staphylococcus aureus (ATCC 13709), and Enterococcus faecalis (ATCC 29212); and two Gramnegative bacteria: Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 9637) were used in this study. 2.5.1. Minimum inhibitory concentration All test organisms with densities ranging from approximately 5  107 to 9  107 cfu/ml were incubated in nutrient broth at 37 1C for 24 h before inoculation. Exactly 100 ml of 10 mg/ml extract was added in the first row of a 96-well microtitre plate to produce an initial concentration of 2.0 mg/ml. Using the serial dilution method, a total of eight different concentrations (20–2000 mg/ml) of the plant extracts were obtained. Two columns were used as sterility control (no cultures were added) and growth control (extracts replaced by blank solvent). The 96-well microtitre plates were sealed in plastic bags and incubated for 18 h in a 100% humidified incubator. After incubation, 40 ml of 0.2 mg/ml r-iodonitrotetrazolium violet (INT) (Sigma, Germany) was added to each well and the plates were incubated for a further 2 h. The development of red colour due to the formation of the red/purple formazan, indicated microbial growth. The lowest concentration of extracts that inhibited the growth of test organisms was recorded as MIC. Amphotericin B and gentamicin were used as positive controls in antifungal and antibacterial activity assays, respectively. Three replicate analyses were done. 2.6. Statistical analysis All assays were done in triplicate, and the antidiabetic and antioxidant values were expressed as mean7standard errors. Data were analysed by one-way analysis of variance (ANOVA) followed by post-hoc Dunnett’s multiple comparison test (Tallarida and Murray, 1984). Differences were considered significant if po0.05. 3. Results and discussion 3.1. Effect of extracts on glucose uptake The effect of the three different crude extracts of Fadogia ancylantha on glucose uptake in the two cell lines is presented in Fig. 1. The respective antidiabetic activities of the plant extracts were determined relative to the glucose uptake obtained in the cells exposed to the vehicle control, the value of which was 100%. The in vitro assay in C2C12 myocytes indicated that at concentration of 12.5 mg/ml, the organic extract registered the highest relative increased glucose uptake (172.29 70.32%) compared to the cold aqueous extract (144.84 70.67%). The Fadogia ancylantha organic extract gave about 10% more potency than the positive drug control, metformin. Metformin is a hypoglycaemic drug effective in the treatment of type II diabetes mellitus (Klip and Leiter, 1990; Morioka et al., 2005). Relative glucose uptake (%) 2.5. Antimicrobial screening 300 C2C12 muscle cells Chang cells 250 200 150 100 50 0 Insulin Metformin Vehicle Extract A Extract B Extract C control Sample identity Fig. 1. In vitro antidiabetic activity of various Fadogia ancylantha extracts. Key: A ¼ Cold aqueous extract, B ¼ Hot aqueous extract, C ¼Organic extract. 100 90 80 Radical scavenging (%) If Aa rAb % Radical scavenging¼[(Ab Aa)/Ab]  100 If Aa4Ab % Radical scavenging¼[(T0 Aa)/T0]  100, where Ab¼DPPH solution absorption after 30 min Aa ¼DPPHþanti-oxidant solution absorption T0 ¼DPPH solution absorption before experiment. 70 60 50 40 30 Extract A Exract B Extract C Gallic Acid 20 10 0 0 10 20 30 40 50 60 70 80 Concentration (µg/ml) 90 100 110 Fig. 2. DPPH free radical scavenging activity of different concentrations of three Fadogia ancylantha extracts and the reference anti-oxidant, gallic acid. Using Chang cells, the organic and hot aqueous extracts significantly (po0.05) increased glucose uptake by 181.247 0.29% and 145.9470.37%, respectively. In contrast, the cold aqueous extract showed significant (p o0.05), albeit slightly lower, increased glucose uptake of 138.7070.52%. This probably means boiling of the tea extracted more active compounds and contributes to the greater antidiabetic activity in Chang cells. In the Chang cells, the plant extracts exhibited lower glucose uptake response than the positive controls (insulin and metformin). However, the organic and hot aqueous extracts still showed higher glucose uptake than that previously reported from Kankerbos (  130%) in Chang cells (Wilson, 2006). 3.2. Radical scavenging activity In Fig. 2, the antiradical activities of various extracts at different concentrations using the free radical, 2,2-diphenyl-1picrylhydrazyl (DPPH) are provided. 375 K.K. Nyirenda et al. / Journal of Ethnopharmacology 143 (2012) 372–376 Table 1 Antimicrobial activity of different extracts. Strain MIC values (mg/ml) Fadogia ancylantha extracts Escherichia coli (ATCC 9637) Pseudomonas aeruginosa (ATCC 27853) Staphylococcus aureus (ATCC 13709) Enterococcus faecalis (ATCC 29212) Aspergillus fumigatus (ATCC 29214) Candida albicans (ATCC 10231) Cryptococcus neoformans (ATCC 10239) Positive controls A B C Gentamicin Amphotericin B 1250 2000 1250 321 1250 2000 625 1250 2000 321 625 1250 1250 625 321 625 321 321 156 625 321 19 39 321 1250        78 321 39 Key: A ¼Cold aqueous extract, B ¼ Hot aqueous extract, C¼ Organic extract. The IC50 anti-oxidant activities obtained in the organic, hot and cold aqueous extracts were 63.8171.72, 46.2371.09, and 44.3171.42 mg/ml, respectively. The organic extract showed a significantly (po0.05) lower inhibition than aqueous extracts. Fig. 2 also indicated that at 100 mg/ml, aqueous extracts A and B gave significantly higher (po0.05) radical scavenging rates (range 85.78–86.29%) than their organic counterpart, C (68.16%). The hot aqueous extract showed DPPH percentage inhibition values ranging from 12.2871.89% (1.56 mg/ml) to 85.7871.22% (100 mg/ml). The organic extract gave significantly lower activity (po0.01), ranging from 7.7171.84% (1.56 mg/ml) to 68.1672.46% (100 mg/ml). Thus, the active compounds are less soluble in organic solvents, hence the reduced activity. Cuvelier and co-workers (1992) attributed the antioxidative activity in plants to the presence of some acid-phenols. Therefore, such polar compounds which are more soluble in aqueous solvents than organic solvents account for higher antiradical activities in aqueous extracts than in the organic extract. Similar findings for Sesbania sesban by Mani et al. (2011) showed that increasing dosage resulted in higher activity. The anti-oxidant activities of Fadogia ancylantha aqueous extracts were comparable to DPPH radical scavenging capacity of 76.3% (100 mg/ml) and 87.8% (100 mg/ml) obtained for selected known medicinal plants, Sesbania sesban (Mani et al., 2011) and Andrographis paniculata (Cuvelier et al., 1992), respectively. Thus, the herbal tea contains bioactive compounds that may have potential applications in managing chronic and degenerative diseases caused by oxidative stress such as diabetes mellitus, ischaemic heart diseases and immunosuppression. 3.3. Antimicrobial activity of plant extracts The antimicrobial activities of the three crude extracts of the herbal preparation are shown in Table 1. The organic extract showed the highest activity against the tested micro-organisms (MIC range 156–625 mg/ml), while the hot aqueous extract gave moderate activity (321 1250 mg/ml). The cold aqueous extract was the least active (321 2000 mg/ml). It is interesting to note that the crude organic extract also displayed broad spectrum activity; with the fungal strains exhibiting greater susceptibility than the bacterial microorganisms. The MIC values for fungal microbes Aspergillus fumigatus, Cryptococcus neoformans and Candida albicans were 156, 321 and 625 mg/ml, respectively. Thus, the Aspergillus fumigatus fungus was the most inhibited by the organic extract of the Fadogia ancylantha herbal preparation. The results for antimicrobial activity revealed that the hot aqueous extract exhibited higher potency than the cold aqueous extraction method. The hot aqueous based extract inhibited the growth of all test organisms except Pseudomonas aeruginosa, and the cold aqueous extract was inactive against Pseudomonas aeruginosa and Candida albicans. The differences confirm that boiling of the tea results in a better extraction of the active constituents. Consequently, continuing study is directed at unravelling the chemistry of the hot tea extracts and the efficiency of the other extraction methods. The results obtained indicated that the organic and the aqueous extracts of Fadogia ancylantha contain compounds that largely inhibit the growth of Aspergillus fumigatus, Escherichia coli and Cryptococcus neoformans. 4. Conclusions The present study has shown that extracts obtained by the cold aqueous method gave the highest anti-oxidant activity, followed by the hot aqueous extract and the organic extract was the least active. The anti-oxidant activities of Fadogia ancylantha extracts were dose dependent; and at 100 mg/ml, the hot and cold aqueous extracts gave 85.78–86.29% DPPH decolourisation, which was significantly (p o0.01) higher than that obtained in the organic extract (68.16%). Using Chang cells, the relative mean glucose uptake of the organic and hot aqueous extracts was increased by 181.2470.29% and 145.9470.37% respectively. Therefore, the herbal tea processed from Fadogia ancylantha has significant potential in the treatment of diabetes and other degenerative diseases caused by oxidative stress. Continuing study is underway to identify and characterise the active constituents responsible for the observed anti-oxidant and antidiabetic effects. Acknowledgements We wish to acknowledge and appreciate the financial support from the Carnegie Corporation of New York through the Regional Initiative in Science and Education (RISE) scholarship. We would like to thank communities, especially Mr. Andrew Zulu, in Chilumba for providing the study plant material and indigenous knowledge, Natasha Kolesnikova for the technical assistance in anti-oxidant assay and Mrs. Linda Saka for giving us information about the tea. We also thank Professor Andrew Marston for the useful comments on the manuscript. References Babu, P.V., Sabitha, K.E., Shyamaladevi, C.S., 2006. 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