2.3. Extraction and isolation
The dried mature fruits powder (47.9 kg) was extracted with 70% aqueous ethanol under reflux. After extracted for three times (first 479 kg for 2 h, and then 384 kg for 2 h two times), the crude extract (8.2 kg, yield 17.12%) was obtained. And then, part of the residue (6.0 kg) was suspended in H2O (8 L) and extracted with cHE (8 L × 8), EtOAc (8 L × 8) and n-butanol (BuOH, 8 L × 8) successively and afforded corresponding extract for 1.2 kg, 2.2 kg and 0.7 kg. The cHE extract (1.0 kg) was fractionated by silica gel column (SGC, 140 mm i.d. × 800 mm) with gradient eluent (PE-EtOAc, 5:1, 3:1, 1:1, 2:3, 1:3, 0:1, v/v) to give 26 fractions (Fr. A–Z). The Fr. B (27.2 g) was separated by SGC (55 mm i.d. × 650 mm) with a step gradient eluent of PE-EtOAc (100: 1, 50: 1, 25: 1, 7: 1, 5: 1, 3:1, 1: 1, 1:3, 0:1, v/v) to afford 15 subfractions (Fr. B-1–Fr. B-15). Fr. B-8 (2.1 g) was separated by reversed-phase column (RPC), eluted with MeCN-H2O (40:60 to 100:0, v/v), to give 13 subfractions. Fr. B-8-5 was purified by SP-RP-HPLC (MeCN-H2O, 95:5, v/v), to yield compound 10 (5 mg, tR = 85 min). The Fr. C (136 g) was separated by SGC (120 mm i.d. × 600 mm) with a step gradient eluent of PE-EtOAc (100: 1, 50: 1, 20: 1, 15: 1, 10: 1, 5:1, 5: 2, 1:1, 0:1, v/v) to afford 16 subfractions (Fr. C-1–Fr. C-16). Fr. C-4-7 was separated by Sephadex LH-20 column (SC) and purified by SP-RP-HPLC (MeCN-H2O, 93:7, v/v), to yield compound 1 (230 mg, tR = 45 min) and 11 (60 mg, tR = 80 min). Fr. C-5 (20 g) was separated by RPC, eluted with MeOH-H2O (80:20 to 100:0, v/v), to give 9 subfractions. Fr.C-5-9 was separated by SC and purified by SP-RP-HPLC (MeCN-H2O, 93:7, v/v), to yield compound 12 (34 mg, tR = 71 min). By SP-RP-HPLC (MeCN-H2O, 90:10, v/v) and preparative TLC (PE-CHCl3, 10:1, v/v), compound 2 (104 mg, tR = 252 min) was obtained from Fr. C-11 (2.1 g). The Fr. D (335 g) was separated by SGC (140 mm i.d. × 800 mm) with a step gradient eluent of PE-CHCl3 (100: 1, 50: 1, 20: 1, 10: 1, 5: 1, 4:1, 3: 1, 2: 1, 1: 1, 1:3, 0:100, v/v) to afford 14 subfractions (Fr. D-1–Fr. D-14). Fr. D-4 (20.7 g) was separated by RPC, eluted with MeOH-H2O (45:55 to 100:0, v/v), to give 13 subfractions. Fr. D-4-12 was purified by SP-RP-HPLC (MeOH-H2O, 93:7, v/v), to yield compounds 8 (15 mg, tR = 131 min) and 9 (28 mg, tR = 136 min). By SP-RP-HPLC (MeCN-H2O, 90:10, v/v), Fr. D-7(1.7 g)was separated to yield compounds 3 (5 mg, tR = 95 min) and 4 (2 mg, tR = 87 min). The Fr. D-9 (37 g) was separated by SGC (55 mm i.d. × 650 mm) with a step gradient eluent of PE-CHCl3 (100: 1, 50: 1, 20: 1, 10: 1, 9: 1, 8:1, 7: 1, 6: 1, 5: 1, 4:1, 3:1, 1:1, 1:3, 0:1, v/v) to afford 28 subfractions. By SP-RP-HPLC (MeOH-H2O, 92:8, v/v), Fr. D-9-13 was separated to yield compounds 5 (11 mg, tR = 77 min), 6 (9 mg, tR = 100 min) and 7 (19 mg, tR = 110 min). Fr. D-9-25 was purified by SP-RP-HPLC (MeOH-H2O, 92:8, v/v), to give compounds 13 (18 mg, tR = 86 min) and 14 (25 mg, tR = 92 min). Fr. D-9-26 was purified by SP-RP-HPLC (MeOH-H2O, 92:8, v/v), to give compound 15 (15 mg, tR = 76 min). Fr. D-11 (19.2 g) was separated by RPC, eluted with MeOH-H2O (45:55 to 100:0, v/v), to give 27 subfractions. The Fr. D-11-25 (5.9 g) was separated by SGC (35 mm i.d. × 500 mm) with a step gradient eluent of PE-CHCl3 (8:1, 7: 1, 6: 1, 5: 1, 4:1, 3:1, 1:1, 1:3, 0:100, v/v) to afford 18 subfractions. By SP-RP-HPLC (MeOH-H2O, 75:25, v/v), Fr. D-11-25-12 was separated to yield compounds 16 (14 mg, tR = 286 min) and 17 (15 mg, tR = 328 min).
Bisbakuchiol M (1). Brown-red needle crystals; mp 114–116 ºC; [α]25 D + 50.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 204 (4.85), 258 (4.80), 386 (4.28) nm; IR (KBr) νmax 3315, 2967, 2930, 1692, 1609, 1582, 1504, 1385, 1358, 1255, 1035 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 537.3004 [M + H]+ (calcd for C36H41O4, 537.3005).
Bisbakuchiol N (2). Yellow oils; [α]25 D + 20.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (4.41), 253 (4.44) nm; IR (KBr) νmax 3319, 2966, 2924, 1703, 1633, 1603, 1496, 1409, 1373, 1231, 969 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 511.3573 [M + H]+ (calcd for C36H47O2, 511.3576).
Bisbakuchiol O (3). Yellowish oils; [α]25 D + 30.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (4.57), 267 (4.33) nm; IR (KBr) νmax 3373, 2962, 2926, 1704, 1604, 1507, 1454, 1372, 1238, 1172, 1007 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 555.3468 [M + HCOO]− (calcd for C37H47O4, 555.3474).
Bisbakuchiol P (4). Yellowish oils; [α]25 D − 26.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (4.61), 267 (4.32) nm; IR (KBr) νmax 3381, 2968, 2927, 1703, 1604, 1507, 1452, 1375, 1240, 1172, 1000 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 509.3417 [M – H]− (calcd for C36H45O2, 509.3420).
Bisbakuchiol Q (5). Yellowish oils; [α]25 D + 70.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (4.76), 264 (4.55) nm; IR (KBr) νmax 3370, 2964, 2921, 1704, 1607, 1507, 1459, 1370, 1238, 1171, 1099 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 525.3364 [M – H]− (calcd for C36H45O3, 525.3369).
Bisbakuchiol R (6). White amorphous powder; [α]25 D + 70.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (4.57), 260 (4.35) nm; IR (KBr) νmax 3372, 2967, 2924, 1704, 1613, 1506, 1451, 1365, 1253, 1143, 1094 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 603.3676 [M + HCOO]− (calcd for C38H51O6, 603.3686).
Bisbakuchiol S (7). White amorphous powders; [α]25 D + 60.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 204 (4.42), 260 (4.32) nm; IR (KBr) νmax 3373, 2968, 2925, 1705, 1614, 1506, 1450, 1364, 1235, 1143, 1082 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 557.3635 [M − H]− (calcd for C37H49O4, 557.3631).
Bisbakuchiol T (8). Yellowish oils; [α]25 D − 20.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (4.68), 222 (4.57), 262 (4.33) nm; IR (KBr) νmax 3395, 2967, 2921, 1702, 1588, 1507, 1450, 1375, 1267, 1171, 1010 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 525.3367 [M − H]− (calcd for C36H45O3, 525.3369).
Bisbakuchiol U (9). Yellowish oils; [α]25 D + 20.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (4.63), 265 (4.21) nm; IR (KBr) νmax 3387, 2966, 2922, 1703, 1587, 1507, 1451, 1374, 1267, 1171, 1009 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 525.3371 [M − H]− (calcd for C36H45O3, 525.3369).
Bakuchiol ether A (10). Yellowish oils; [α]25 D + 10.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 204(4.26),260(4.15) nm; IR (KBr) νmax 3424, 2969, 2929, 1712, 1603, 1505, 1453, 1369, 1224, 1136, 913 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 3; 13C NMR (CDCl3, 100 MHz), see Table 3; HRESIMS m/z 445.3080 [M + Na]+ (calcd for C29H42O2Na, 445.3083).
Bakuchiol ether B (11). Yellowish oils; [α]25 D + 20.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 206(4.50), 265(4.45) nm; IR (KBr) νmax 3420, 2926, 2864, 1715, 1606, 1507, 1463, 1364, 1245, 1173, 969 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 3; 13C NMR (CDCl3, 100 MHz), see Table 3; HRESIMS m/z 477.3713 [M + H]+ (calcd for C33H49O2, 477.3733).
Bakuchiol ether C (12). Yellowish oils; [α]25 D + 30.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 206(4.46), 263(4.42) nm; IR (KBr) νmax 3420, 2952, 2927, 1710, 1604, 1505, 1452, 1375, 1241, 1171, 967 cm− 1; 1H NMR (CDCl3, 400 MHz), see Table 3; 13C NMR (CDCl3, 100 MHz), see Table 3; HRESIMS m/z 475.3532 [M − H]− (calcd for C33H47O2, 475.3576).