*3.5. Identification of Compounds* **6***–***8** *and* **10***–***12** *by LC–MS*

LC–MS analysis was carried out on an Agilent system using Luna 5 μ C18 (2), 150 × 4.6 mm, λ 254, flow 1 mL/min, gradient 0–2 min [95% H2O; 5% MeCN], 2–30 min, 5% MeCN→ 100% MeCN, 30–35 min 100% MeCN, 35–45 min [95% H2O; 5% MeCN]. The retention times (*Rt*) of the compounds **6** (m/z 349.2 [M + H]<sup>+</sup>; C24H29O2), **8** (365.2 [M + H]<sup>+</sup>; C24H29O3), **7** (363.2 [M + H]<sup>+</sup>; C24H27O3), **10** (349.2 [M + H]<sup>+</sup>; C24H29O2); **11** (363.2 [M + H]<sup>+</sup>; C24H27O3), and **12** (365.2 [M + H]<sup>+</sup>; C24H29O2) were found to be 4.4, 4.5, 9.9, 10.0, 9.4, and 9.7 min<sup>−</sup>1, respectively. Compounds **6**–**9** and **10**–**12** were identified from leaves, stem bark, and root extracts through HPLC and LC–MS.

#### *3.6. Antimicrobial Assays*

All organisms were obtained from the American Type Culture Collection (Manassas, VA, USA) or the National Collection of Type Cultures (Colindale, UK), unless specified otherwise. These included the yeasts *Candida albicans* ATCC 90028, *C. glabrata* ATCC 90030, and *C. krusei* ATCC 6258; the fungi *Cryptococcus neoformans* ATCC 90113 and *Aspergillus fumigatus* ATCC 204305; and the bacteria *Escherichia coli* ATCC 35218, NCTC 12923, *Klebsiella pneumoniae* NCTC 13368, M6 (Colindale, UK), *Acinetobacter baumannii* AYE (ATCC BAA-1710), ATCC 17978, *Pseudomonas aeruginosa* ATCC 27853, PAO1 (Manoil collection, University of Washington, Washington, DC, USA), NCTC 13437, *Mycobacterium intracellulare* ATCC 23068, methicillin-resistant *Staphylococcus aureus* ATCC 33591 (MRSa), USA-300 MRSa (ATCC BAA-1717), USA-400 MRSa (ATCC BAA-1696), Mupirocin-resistant *S. aureus* (ATCC BAA-1708), *Enterococcus faecium* ATCC 700221 (VRE), *E. faecalis* ATCC 29212 (Vancomycin-sensitive) and *Enterococcus faecium* ATCC 51299 (Vancomycin-intermediate). Drug controls ciprofloxacin, methicillin and vancomycin (ICN Biomedicals, Aurora, OH, USA) for bacteria and amphotericin B (ICN Biomedicals) for yeasts and fungi were included in each assay. Susceptibility testing was performed using a modified version of the CLSI (formerly NCCLS) method [16–18]. *M. intracellulare* was tested using a modified Franzblau method [18]. Samples were serially diluted in 20% DMSO/saline and transferred in duplicates to 96-well flat-bottomed microplates. Microbial inocula were prepared by correcting the OD630 of microbe suspensions in incubation broth to give final target inocula. All organisms were read at either 530 nm, using the Biotek Powerwave XS plate reader (Bio-Tek Instruments, Winooski, VT, USA) or 544ex/590em, (*M. intracellulare, A. fumigatus*) using the Polarstar Galaxy Plate Reader (BMG Lab Technologies, Ortenburg, Germany), prior to and after incubation. Minimum fungicidal or bactericidal concentrations were determined by removing 5 μL from each clear well, followed by transferring to agar, and incubating. The MFC/MBC was defined as the lowest test concentration that kills the organism (allows no growth on agar).

Gram-negative MICs were determined using the CLSI microbroth dilution method, modified as described previously [19]. Bacteria were added at a starting concentration of 5 <sup>×</sup> 10<sup>5</sup> cfu/mL and incubated for 20 h at 37 ◦C in the dark. Absorbance at OD600 was then read using the CLARIOstar plate reader (BMG Lab Technologies, Germany). The MIC was defined as the lowest concentration where visible growth could not be detected, equivalent to an OD600 of 0.1. MICs were also determined in the presence of the membrane permeabilizer, polymyxin-B-nonapeptide (PMBN) following the same method, with an additional step; after the 2-fold dilution of compound was prepared and before the bacteria were added, PMBN was added to all wells at a final concentration of 30 μg/mL. This concentration was shown to not significantly inhibit growth of the test panel.

#### *3.7. Antimicrobial Combination Study by Checkerboard Method*

The combination study of the compounds was carried out using a standard Checkerboard method [13,14]. Strains were grown on Eugon agar at 35 ◦C, prior to assays. Test samples were dissolved in DMSO (2 mg/mL) to the desired concentrations, and serially-diluted with 20% DMSO/saline. Samples were transferred to 96 well assay plates (10 μL) in a checkerboard layout. Inocula were prepared by suspending growth from agar in 0.9% saline, determining the OD630, and correcting in incubation broth (cation-adjusted Mueller-Hinton, Difco) to afford 5 <sup>×</sup> <sup>10</sup><sup>5</sup> colony forming units per mL, after addition to samples (180 μL) using standard inocula calculations. Final sample test concentrations were 1/100th the DMSO stock concentrations. The assay plates were read at 530 nm prior to and after incubation at 35 ◦C for 18–20 h. IC50s of each test compound were calculated using the XLfit 4.2 software (IDBS, Alameda, CA, USA) using the fit model 201. After incubation, all 96 wells were also pinned to Eugon Agar and incubated at 35 ◦C overnight to determine bactericidal activity. Fractional inhibitory concentrations (FICs) were calculated to evaluate possible synergy with FICS < 0.5 synergistic.
