*3.3. Extraction and Isolation of Compounds from Stem Bark and Leaves*

The powdered stem bark (0.5 kg) was extracted by percolation with 95% EtOH (3 × 2 L) and the combined extracts were evaporated under reduced pressure (yield 17.7 g). A portion of the dried EtOH extract (15 g) was percolated with *n*-hexane, followed by DCM, and finally the residual extract was washed with MeOH (each 200 mL × 3). The *n*-hexane, DCM, and MeOH fractions were separately filtered and dried, which afforded 3.8, 8.9, and 4.5 g, respectively. The antimicrobial activity was detected in the DCM fraction (IC50 < 20 μg/mL against *S. aureus* and MRSA). A portion of the dried DCM fraction (1.65 g) was fractionated by CPTLC with a Chromatotron®instrument, using a 4 mm custom-made C18 RP silica gel ChromatoRotorTM [15], eluting with a gradient of 60% to 100% MeCN-H2O to afford 30 fractions. The fractions were pooled by TLC analyses.

Fractions 1–8 (475 mg) were combined and further subjected to CPTLC, using a 4 mm silica gel P254 disc, and gradient elution with MeCN:DCM. Elution with 2% MeCN:DCM afforded medicarpin (**4**; 4.5 mg), followed by elution with 4%MeCN:DCM, which gave 3,8-dihydroxy-9-methoxy-pterocarpan (**5**; 7.5 mg), and finally elution with 5% MeCN:DCM yielded vestitol (**2**; 9.8 mg). The combined fractions 10–15 (70 mg) was subjected to preparative C18 RP-HPLC, using 90% MeCN:H2O as solvent, which afforded machaeridiol B (**12**), followed by machaeridiol A (**10**) and machaeridiol C (**11**). Similarly, combined fractions 25–32 (100 mg) was also separated by preparative C18 RP-HPLC, which afforded additional quantities of **10**–**12** [total yields: **10** (10 mg), **11**; (18 mg), **12** (21 mg)] and machaeriol C (**8**; 34.6), however, the minor compound machaeriol D (**9**) could not be re-isolated due to a paucity of material. Further elution with 75% MeCN:H2O afforded 13 fractions, which contained the mixture of two compounds **6** + **7**, (50 mg). The mixture was then separated by preparative C18 RP-HPLC

(column: ODS prodigy 10μ, 250 × 10 mm; detector: UV-254 nm), using 95% MeCN:H2O as solvent, which afforded **6** (16 mg), followed by **7** (16 mg). Finally, the dried *n*-hexane fraction (77 mg) was subjected to CPTLC, using a 2 mm C18 RP rotor, and eluted with 65% MeCN:H2O, which afforded 7-*O*-methylvestitol (**3**; 8 mg). A sub-fraction of DCM (15 mg) was subjected to prep-HPLC (Waters LC module I plus, using Phenomenex C18, 2 mm), which afforded compound **1a**+**1b** (5 mg). The structures of (+)-vestitol (**2**), 7-*O*-methylvestitol (**3**), (+)-medicarpin (**4**) and 3,8-dihydroxy-9-methoxypterocarpan (**5**) were determined by physical and spectroscopic data (1H and 13C NMR, see SI 1), and also by comparison with those reported [11,12]. The structures of the re-isolated compounds **6**–**8** and **10**–**12** were identified by NMR data [5,6] and by direct comparison (TLC, HPLC/LC–MS) with their respective authentic samples available in our laboratories. Finally, powdered leaves (560 g) and root bark (50 g) of *Machaerium* sp. were extracted using the method described previously [5,6], and compounds 6–8 and 10–12 were isolated from leaves as describe below.

The powdered leaf was percolated with *n*-hexane, followed by DCM and EtOH (each 3 × 2 L) to yield 5, 14, and 9 g of extracts, respectively. A portion of the DCM extract (10 g) was subjected to reversed phase (RP) cartridge (10 G, 60 mL Giga tube), and eluted with MeCN-H2O to afford 30 fractions. The combined fractions 20–21 (102 mg; eluted by 60–65% MeCN-H2O) were subjected to centrifugal preparative thin layer chromatograph (CPTLC, 1 mm Si gel P254 disc), eluting with 0.5–1% MeCN-DCM to yield **12** (11.4 mg). Fraction 22 (60 mg; eluted with 75% MeCN-H2O) was further subjected to CPTLC (1 mm RP-C18 ChromatoRotor), eluted with 50–100% H2O-CH3CN to afford 115 fractions, of which fractions 42–45 and 90–115 eluting with 80% and 90% MeCN-H2O yielded **10** (2.7 mg) and **11** (4.5 mg), respectively. Combined fractions 46–89 (28.4 mg) were enriched with **12** (+ traces of **10** + **11**). Similarly, RP cartridge purified fractions 23 and 24 (60 and 70 mg; eluted with 70 and 80% MeCN-H2O, respectively) were further purified (1 mm RP-C18 ChromatoRotor) by eluting separately with 50–100% H2O-MeCN to yield a mixture of **8** + **10** + **11** (32 mg and 31 mg respectively). The above enriched mixtures were further purified preparative RP-HPLC, using 90% MeCN-H2O as solvent to afford compounds **12**, **11** + **12**, **11**, and **8** (5, 32, 4 and 31.7 mg, respectively).

A portion of *n*-hexane extract (2.5 g) was fractionated with CPTLC (6 mm, Si gel P254 disc) eluting with 5% DCM in hexane to yield 10 fractions. The fractions 3–7 (840 mg) that enriched with compounds **6** and **7** were combined and further attempted to purify with an additional CPTLC (4 mm, Si gel P254 disc) eluting with 5% DCM in hexane to yield semi-pure **6** (40 mg), **6**+**7** (50 mg), and semi-pure **7** (6 mg). In addition, the presence of these compounds in leaves, stem bark, and root extracts were confirmed by HPLC and LC–MS (vide infra).
