Myristica fragrans subsp. seeds

Kruakaew, Sudarat, Singha, Suriphon, Sangvichien, Ek, Yahuafai, Jantana & Sutthivaiyakit, Somyote, 2022, Neolignans from Myristica fragrans seeds, revision of their absolute configurations, reduction products and biological activities, Phytochemistry (113174) 199, pp. 1-9 : 5

4.3. Extraction of M. fragrans seeds and isolation

The fresh seeds (weight 4.5 kg) were crushed to small pieces and extracted by soaking in methanol (10 L) at ambient temperature for seven days. The extract was filtered and concentrated to remove solvent under reduced pressure on a rotary evaporator to obtain green residue of MeOH extract. The methanol extract after addition 15% of water was partitioned successively with hexanes (3 × 700 mL), CH 2 Cl 2 (3 × 700 mL) and EtOAc (3 × 700 mL) to afford hexanes (40 g), CH 2 Cl 2 (33.7 g), EtOAc (10.4 g) and MeOH (38.5 g) extracts. The crude CH 2 Cl 2 extract of seeds (33.7g) was subjected to column chromatography (CC) using gradient of hexanes-CH 2 Cl 2 85:15 to CH 2 Cl 2 –MeOH 50:50 as eluting solvent to obtain 7 fractions. Fraction 2 (274.2) was fractionated by CC (Sephadex LH20, MeOH) to give four subfractions (2.1–2.4), subfraction 2.4 (5.4 mg) gave 3. Fraction 3 (8.89 g) was purified by CC (hexanesCH 2 Cl 2 50:50 to 0:100) to provide seven subfractions (3.1–3.7), subfraction 3.2 provided additional quantity of 3 (288.6 mg). Subfraction 3.3 (208.1 mg) was purified by column chromatography (hexanesCH 2 Cl 2 60:40 to 50:50) to give four subfractions (3.3.1–3.3.4), subfraction 3.3.2 (178.5 mg) after purification (CC, Sephadex LH20, CH 2 Cl 2 –MeOH 5:95 then silica gel, hexanes-EtOAc 98:2) provided 9 (5.6 mg) and 4 (83.3 mg). Subfraction 3.4 (4.97 g) after CC (silica gel, hexanes-CH 2 Cl 2 50:50 to 40:60) gave three subfractions (3.4.1-3.4.3), subfraction 3.4.2 (974 mg) provided additional 4 (103.7 mg) after CC (hexanes- CH 2 Cl 2 50:50 to 40:60), and 1 (365 mg) was obtained from subfraction 3.4.3 after recrystallization in MeOH. Subfraction 3.6 (1.07 g) was purified by CC (hexanes-EtOAc 90:10 to 80:20) to give 2 (47.5 mg). Subfraction 3.5 (663.0 mg) was purified by CC (silica gel, hexanesCH 2 Cl 2 30:70 to 80:20) to give five subfractions. Subfraction 3.5.4 provided 10 (21.1 mg). Subfraction 3.6 (1.07 g) was fractionated by CC (silica gel, hexanes-EtOAc 90:10 to 80:20) and gave subfractions 3.6.1- 3.6.4. Subfraction 3.6.1 (201.3 mg) after CC (RP-18, MeOH–H 2 O 70:30 then silica gel, hexanes-CH 2 Cl 2 10:90) provided 9 (4.4 mg). Subfraction 3.6.2 (250 mg) was purified by CC (silica gel, CH 2 Cl 2 –MeOH 99.5:0.5 to 98:2) and gave five subfractions (3.6.2.1–3.6.2.5). Subfraction 3.6.2.2 (21.9 mg) after fractionation (RP-18, MeOH–H 2 O 70:30) gave 14 (3.4 mg). Subfraction 3.6.2.3 (146.9 mg) was purified by CC (RP-18, MeOH–H 2 O 70:30 to 80:20) to give 18 (58.1 mg). Subfraction 3.6.2.4 gave additional 18 (8.7 mg). Subfraction 3.6.3 (111.0 mg) was purified by CC (silica gel, hexanes-CH 2 Cl 2 20:80 to CH 2 Cl 2 –MeOH 99:1 then hexanes-CH 2 Cl 2 10:90) to give of guaiacin (7) (16.6 mg). Subfraction 3.7 (1.151 g) was fractionated by CC (hexanes-EtOAc 80:20 to 60:40) to give three subfractions (3.7.1-3.7.3), subfraction 3.7.2 (282.7 mg) after CC (Sephadex LH20, MeOH) provided 2 (26.0 mg). Fraction 4 (6.92 g) was fractionated (silica gel, hexanes-EtOAc 80:20 to 50:50) to give four subfractions (4.1–4.4), subfraction 4.2 (581.6 mg) was purified (RP-18, MeOH–H 2 O 80:20) to give three subfractions (4.2.1–4.2.3). Subfraction 4.2.2 (182.4 mg) after further purification (silica gel, hexanes-EtOAc 70:30) provided 13 (10.3 mg). Subfraction 4.3 (391.5 mg) was purified by CC (silica gel, CH 2 Cl 2) and gave 12 (9.3 mg) and 8 (50.9 mg). Fraction 5 (1.59 g) was fractionated by CC (silica gel, hexanes-EtOAc 90:10 to 40:60) to give four subfractions (5.1–5.4), subfraction 5.2 (186.4 mg) after purification (silica gel, CH 2 Cl 2 –MeOH 99.5:0.5 to 96:4) gave three subfractions, subfraction 5.2.2 after reversed phase CC (RP-18, MeOH–H 2 O 70:30 to 100:0) gave 5 (70.5 mg). Subfraction 5.4 (719.3 mg) was purified by CC (silica gel, CH 2 Cl 2 –MeOH 99.5:0.5 to 96:4) to give five subfractions (5.4.1–5.4.5), subfraction 5.4.1 (64.7 mg) after purification (Sephadex LH20, MeOH, then RP-18, MeOH–H 2 O 60:40 to 100:0) gave 17 (13.3 mg). Subfraction 5.4.2 (433.4 mg) was fractionated by CC (silica gel, CH 2 Cl 2 –MeOH 98:2 then reversed phase RP-18, MeOH–H 2 O 70:30) to give two subfractions (5.4.2.1–5.4.2.2), subfraction 5.4.2.1 gave 8.8 mg of 16. Subfraction 5.4.3 (99.0 mg) was purified by CC (silica gel, hexanes-EtOAC then RP-18, MeOH–H 2 O 70:30) and gave 15 (9.4 mg). Fraction 6 (3.24 g) was fractionated by CC (silica gel, hexanes-EtOAc 80:20 to 72:28) to obtain four subfractions (6.1–6.4). Subfraction 6.3 (139.4 mg) after purification (RP-18, MeOH–H 2 O 60:40 to 80:20) gave 11 (5.7 mg). Subfraction 6.4 (182.7 mg) was purified by CC (RP-18, MeOH–H 2 O 80:20) to obtain 6 (11.3 mg). Fraction 7 (981 mg) was fractioned by CC (silica gel, hexanes-EtOAc 88:12 to 60:40) to obtain four subfractions (7.1–7.4). Subfraction 7.3 (32.5 mg) after fractionation (Sephadex LH20, MeOH) gave additional quantity of 6 (4.5 mg).