A two-month survey for evaluation and documentation of the use of medicinal plants used for the day-to-day treatment of common diseases by traditional healers in Khyang tribe Bangladesh was performed from 5–30 April 2015 according to ethnopharmacological criteria (Cotton 1996). The survey was done on the Khyang tribe residing in villages adjoining Rowangchari bazar and Balaghata village in Bandarban district, Chittagong Hill Tracts, Bangladesh. Information gathered allowed the collection of 18 medicinal plants, which were identified by Professor M. Atique Rahman, University of Chittagong. Voucher herbarium specimens with vernacular names, collecting localities, and dates of collections were deposited at the Medicinal Plants Collection Wing, Department of Pharmacy, University of Development Alternative, Dhaka, Bangladesh. After screening of superfluous matter, the collected leaves, bark, roots, rhizomes, seeds or fruits were separated and air-dried at room temperature for 2 weeks. The dried materials were then finely pulverized by grinding using aluminium collection blender (Philips, Shanghai, China) and the powders obtained were weighted with top loading balance (Sartorius AG, Gottingen, Germany).
Medicinal plants extraction The plant powders (20–60 g) were mixed at room temperature sequentially with organic solvents of increasing polarity starting with hexane (Friendemann Schmidt, Parkwood, Australia), ethyl acetate (Friendemann Schmidt, Parkwood, Australia) and 95% (v/v) ethanol (AR grade, John Kollin Corporation, Midlothian, UK) for differential extraction of non-polar, mid-polar and polar extracts, respectively (Harborne 1998). Each extraction was performed in triplicate by maceration of plant powder-tosolvent ratio of 1:5 (w/v) for three days at room temperature. The respective liquid extracts were subsequently filtered through qualitative filter papers No. 1 (Whatman International Ltd., Maidstone, UK) using aspirator pump (EW-35031-00, 18 L/min, 9.5 L Bath, 115 VAC), and the filtrates were concentrated to dryness under reduced pressure at 40 C using rotary evaporator (Buchi Labortechnik AG, Flawil, Switzerland). The dry extracts obtained were weighed with an analytical balance (Sartorius AG, G€ottingen, Germany) and stored in tightly closed glass scintillation vials (Kimble, Rockwood, TN) at 20 C until further use. For stock solutions, each crude extract was dissolved in 100% dimethyl sulphoxide (DMSO) (R&M Chemicals, Chelmsford, UK) to a concentration of 100 mg/mL. A yield for each extract was calculated.
Tested bacterial strains Stock cultures of bacteria used for this study were kindly provided by the Department of Medical Microbiology, Faculty of Medicine, University of Malaya. The following human pathogenic bacteria were used as tested organisms: Gram positive organisms MRSA (University of Malaya Hospital clinical isolate), Enterococcus faecalis (ATCC 29212) and Gram negative organisms Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 15442), Klebsiella pneumoniae (University of Malaya Hospital clinical isolate) and Acinetobacter baumannii (University of Malaya Hospital clinical isolate).
Phytoconstituents and control antibiotics Cinnamaldehyde, eugenol, gallic acid, rifampicin, vancomycin and cefotaxime were purchased from Sigma-Aldrich (St. Louis, MO, >98% purity).
Broth microdilution assay Determination of minimum inhibitory concentration (MIC) was performed according to the Clinical and Laboratory Standards Institute guidelines (CLSI 2012). Briefly, bacterial strains were grown for 18–24 h at 37 C. Direct suspension of the colonies were made in cationically adjusted Mueller-Hinton broth € (CAMHB) and adjusted to OD625 0.08–0.1 which corresponds to 1–2 108 CFU/mL followed by serial 10-fold dilutions to give 1 106 CFU/mL. Bacterial suspension (50 mL) was added to 96- well round bottom microtiter plates containing an equal volume of extracts or phytoconstituents at different concentrations and the 96-well plates were incubated for 24 h at 370 C. The MIC is defined as the lowest concentration of material tested that completely inhibits the growth of bacteria. Minimum bactericidal concentration (MBC) was determined by sub-culturing the test dilutions on to a sterile agar plate and incubated further for 18–24 h. The highest dilution that yielded 0% bacterial growth on agar plates was taken as MBC. Both MIC and MBC values were calculated as the mean of triplicate experiments. Vancomycin and rifampicin were used as positive control antibiotics.
Time-killing assay Time-killing assay was conducted according to Giacometti et al. (1999). Bacteria (1 106 CFU/mL) were incubated with cinnamaldehyde, eugenol, vancomycin or cefotaxime at 1 MIC in Mueller-Hinton broth (MHB) at 37 € C. Bacterial suspensions (10 lL) were removed at various time intervals (1, 2, 3, 4 and 5 h), serially diluted in PBS, and plated onto Mueller-Hinton agar € with 20–24 h at 37 0C to obtain viable colonies. Bacteria count Log10 values were calculated as the mean of triplicate experiments.