Antibacterial activities of fourteen medicinal plants from the endemic plant diversity of Madagascar

Methanolic extracts of 14 plant species used in traditional medicine in Madagascar were investigated for their antibacterial activities. Several extracts showed high inhibitory activities ( Maytenus polyacantha , Mystroxylon aethiopicum , Psychotria bridsoniae, Dombeya tsaratananensis, Psychotria oreotrephes , Raza ﬁ mandimbisonia sambiranensis ) against Listeria monocytogenes , Staphylococcus aureus and Streptococcus pyogenes strains, some of which reached a MIC value lower than 0.1 mg/ml. The extract of P. oreotrephes was the most active against Clostridium perfringens, Proteus mirabilis and Yersinia enterocolitica . Escherichia coli and Salmonella Enteritidis were resistant to all tested extracts. This study demonstrates that plant extracts from species belonging to the Lauraceae, Proteaceae,


Introduction
Plant use in traditional medicine remains a major practice of the health care system in Madagascar based on a rich know-how from the population and that the country represents a hot spot for biodiversity in the world. Some studies have already been reported on the activities of some plants used in the traditional medicine in Madagascar. A methanol extract of Tetradenia riparia was active against Staphylococcus aureus, Escherichia coli, Bacillus cereus, Shigella flexneri, and Klebsiella pneumoniae with MIC values ranging between 1 and 10 mg/ml (Ndamane et al., 2013). Active molecules have been elucidated from the ethyl acetate leaf extract of Dilobeia thouarsii Roem and Schult, which is used as decoctions for diarrhea and skin infections (Rabesa, 1986;Razafintsalama et al., 2013a). Fourteen species endemic to Madagascar and which have been reported in the literature for their use in traditional medicine for treatment of gastroenteritis, diarrhea, skin infections and fatigue were selected for this study (Bost, 1961;Debray et al., 1971;Schmitt, 1971;Boiteau, 1975;Boiteau, 1986;Rabesa, 1986;Rakotobe et al., 1993;Boiteau et al., 1999). These include Beilschmiedia microphylla Kosterm (Lauraceae); Cryptocarya dealbata . The aim of this study was to assess the antibacterial activities of their methanolic extracts in order to provide a better characterization of the biological activity of the plants endemic to Madagascar and scientific validation for their traditional use.

Preparation of extracts
Voucher specimens of collected plant samples from different regions of Madagascar (around Tsaratanana Mountain, Mandraka, Arivonimamo) ( Fig. 1) were deposited at the Botany Department of the "Centre National d'Application des Recherches Pharmaceutiques" (CNARP, Antananarivo, Madagascar). Once shade-dried, plant samples were ground into powder and stored at room temperature before performing individual methanol extraction procedure. The powder (500 mg) was extracted successively through maceration using 500 ml × 4 of methanol. Methanol is among the best solvents used for extraction of antimicrobial substances when compared to other solvents, such as water, ethanol and hexane (Eloff, 1998). The solvent was evaporated under reduced pressure to yield methanol extracts. Extracts were stored in glass vials at room temperature until use.

Antibacterial activity
Four Gram-positive and five Gram-negative strains (Table 1) were used for susceptibility-screening tests using the disc diffusion method (Kil et al., 2009). Sterilized filter paper discs of 6 mm (Biomérieux, Marcy l'Etoile, France) were saturated with 10 μl of the methanol extract (1 mg/disc). Kanamycin 30 μg and Streptomycin 10 μg (Bio-Rad, Marnes-la-Coquette, France) were used as positive controls. Tests were performed in triplicate. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were evaluated using the microdilution method (Razafintsalama et al., 2013b). Each extract concentration (100 μl), ranging from 0.001 to 5 mg/ml, was distributed in a well (96-well microplate) containing 95 μl of Mueller-Hinton broth and 5 μl of the inoculum (10 8 CFU/ml by adjusting the optical density to 0.125 at 600 nm corresponding to 0.5 McFarland) and incubated for 24 h at 37°C. The MIC of each extract was the lowest concentration that inhibited the bacterial growth which was visually 3.52 h 3.52 g 10 3.500 d 3.500 c 8 0.310 ab 2.250 c 12 3.500 e 3.500 d Cryptocarya dealbata (fruit) -  evaluated based on the degree of turbidity and the measure of absorbance at 600 nm. Thereafter, 5 μl from each well was deposited on to Mueller-Hinton agar. The MBC was determined as the lowest concentration at which no colony growth occurred on the plates after incubation for 24 h at 37°C.

Statistical analysis
For comparison of MIC and MBC values, analysis of variance was performed using ANOVA. Significant differences between mean values were determined by Fisher LSD test at the threshold of P b 0.05.

Results and discussion
Inhibition zones of extracts were lower than those of the antibiotics for all strains (Table 1). Gram-positive strains were more sensitive than Gram-negative strains with MIC values lower than 0.1 mg/ml, which can be considered as significant activity according to Kuete (2010) and reaching in some cases values lower than 0.01 mg/ml which are noteworthy (Maytenus spp., Mystroxylon spp., Psychotria spp., R. sambiranensis bark). Plants displaying moderate activity (0.1 mg/ml b MIC b 0.625 mg/ml) were more frequent for Grampositive bacteria as well. Streptococcus pyogenes was the most sensitive (MIC range of 0.002-0.197 mg/ml) for the most active group (R. sambiranensis 1-2, R. affinis, D. thouarsii, P. oreotrephes, P. bridsoniae, Hyperacanthus sp.1-sp.2, C. floribunda) (P b 0.001). R. sambiranensis bark, M. aethiopicum 1-2, P. bridsoniae and D. tsaratananensis showed the highest activity (P b 0.001) against S. aureus (MICs from 0.027 mg/ml to 0.167 mg/ml). Both M. aethiopicum extracts were the most active against Listeria spp. (P b 0.001), showing the lowest MIC values of 0.001-0.004 mg/ml, which are noteworthy. E. coli and Salmonella spp. were resistant to all extracts, as was observed by Khan et al. (2009) for the crude ethanolic extracts of Terminalia arjuna and Eucalyptus globulus. Higher resistance of Gram-negative bacteria has been attributed to the greater complexity of their double membrane-containing cell wall in contrast with the single membraneglycoprotein/teichoic acid of Gram-positive bacteria (Ndamane et al., 2013). Considering the ratio of MBC/MIC b 4 (Oussou et al., 2008), several extracts (D. thouarsii, P. bridsoniae, P. oreotrephes, R. sambiranensis) were bactericidal against Listeria spp., Clostridium spp. and Streptococcus spp. Different parts of the plant may possess varying antimicrobial properties including the flowers, bark, stems, leaves, twigs or roots (Okigbo et al., 2009). For instance, the MICs of the bark extract from R. sambiranensis (0.002 mg/ml) or Mystroxylon spp. (0.75 mg/ml) were, respectively, lower than those from the leaf extracts (0.173 mg/ml) or the roots (0.9 mg/ml) of both plants against Streptococcus spp.
To the best of our knowledge, this is the first study that demonstrates the antibacterial activity of the selected parts of the plants belonging to the Lauraceae, Proteaceae, Celastraceae, Malvaceae and Rubiaceae families, some of which are noteworthy, and may contribute to support the traditional use against bacterial infections.