3.3. Extraction and Isolation
The whole plants of V. vexillata L. (4.9 kg) were powdered and exhaustively extracted with methanol under reflux (10 L × 5 × 8 h), and the combined extracts were concentrated under reduced pressure to give a dark brown syrup (900 g). The crude extract was partitioned between chloroform and water to afford chloroform (210 g) and water extracts (690 g), respectively.
The chloroform extract was subjected to a silica gel column eluted with n-hexane and a step gradient of acetone (100:1 to 1:1) to afford 13 fractions as monitored by TLC. There were no constituents identified from fractions 1–4. Fraction 5 was subjected to silica gel column chromatography with mixture of n-hexane and acetone (50:1) to yield a mixture of 4 and 5 (3.0 mg), and 6 (1.5 mg). Fraction 6 was further resolved on a silica gel column eluted with n-hexane and a step gradient of ethyl acetate (100:1 to 1:1) to give three subfractions (6.1–6.3). Subfraction 6.1 was recrystallized with chloroform and methanol to afford a mixture of 7 and 8 (900.0 mg). Subfraction 6.2 was purified with silica gel column chromatography eluted with the solvent mixture of n-hexane and ethyl acetate (8:1) to yield 9 (8.2 mg), 10 (10.0 mg), and 11 (4.0 mg). Subfraction 6.3 was separated through silica gel column chromatography eluted with chloroform and methanol (100:1) and further purified with preparative TLC on silica gel to afford 12 (1.0 mg). Column chromatography over silica gel of the seventh fraction by the mixture of benzene and ethyl acetate (8:1) and followed by purification with preparative TLC on silica gel to result in 13 (3.0 mg) and 14 (0.5 mg). Fractions 8 and 9 were combined and further separated by repeated column chromatography over silica gel eluted with chloroform and a step gradient with acetone (300:1 to 1:1) followed by purification with preparative TLC on silica gel to yield 1 (2.6 mg), 15 (1.0 mg), 16 (0.5 mg), and 17 (3.0 mg). The tenth fraction was separated by silica gel column chromatography eluted with benzene and a step gradient with ethyl acetate (100:1 to 1:1) to afford six subfractions (10.1–10.6). Subfraction 10.1 was purified with preparative TLC by chloroform and acetone (300:1) to result in 18 (1.0 mg). Subfraction 10.2 was subjected into preparative TLC eluted by benzene and ethyl acetate (10:1) to afford 2 (2.5 mg). With similar procedures eluted with the solvent mixtures of chloroform and acetone (10:1), subfraction 10.3 was purified and resulted in 3 (6.0 mg). The fourth subfraction 10.4 was purified with preparative TLC by n-hexane and acetone (2:1) to result in 19 (3.0 mg). Subfractions 10.5 and 10.6 were resolved with preparative TLC by n-hexane and ethyl acetate (2:1) to afford 20 (3.0 mg) and 21 (1.5 mg), respectively. Fraction 11 was subjected into silica gel column chromatography with the mixing solvent of chloroform and a step gradient with methanol (100:1 to 1:1), and the resulted subfractions were purified with preparative silica gel TLC by chloroform and acetone (100:1) to yield 22 (2.0 mg), 23 (2.0 mg), and 24 (1.0 mg), respectively. Fraction 12 was separated by silica gel column chromatography eluted with chloroform and a step gradient with methanol (30:1 to 1:1) to afford four subfractions (12.1–12.4). There were no significant spots in subfractions 12.1 and 12.2 as monitored by TLC and thus no principles were identified from these fractions. Subfraction 12.3 was purified with the aid of preparative TLC by n-hexane and ethyl acetate (2:1) to yield 25 (3.0 mg) and 26 (4.0 mg). With the similar procedures, 27 (20.0 mg) was characterized from the subfraction 12.4. The last fraction of chloroform extract was separated by silica gel column chromatography eluted with chloroform and methanol (20:1) to yield 16 (0.5 mg).
The water extract was applied to a reversed-phase Diaion HP-20 column eluted with water and methanol gradients to afford 14 fractions as monitored by C-18 TLC, however, no constituents were identified from fractions 1–3. Fractions 4 and 5 were combined and subjected into C-18 column chromatography eluted with water and methanol gradients and further recrystallization with chloroform-methanol to yield 28 (6.0 mg), 29 (2.5 mg), and 30 (5.0 mg), respectively. Fractions 6–8 were merged and purified by C-18 column chromatography eluted with water and methanol gradients followed by preparative TLC eluted with chloroform and methanol (20:1) on the resulted subfractions to afford 31 (20.0 mg), 32 (1.5 mg), 33 (2.0 mg), and 34 (15.0 mg), respectively. Fractions 7–9 were merged and purified by silica gel column chromatography eluted with chloroform and methanol (50:1) and further recrystallization of methanol of the subfractions to result in 35 (3.0 mg) and 36 (2.0 mg). Fractions 12 and 13 were combined and separated by silica gel column chromatography eluted with chloroform and methanol (50:1) to afford four subfractions (12.1–12.4). Subfraction 12.1 was recrystallized with chloroform and methanol to yield 35 (2.0 mg). Subfraction 12.2 was purified by reversed-phase HPLC with a Supelco Discovery® HS C-18 (250 × 4.6 mm, 5μm) column eluted with 0.5 mL/min of MeOH-H2O (40:60) to give 37 (5.0 mg) and 38 (4.0 mg). Subfraction 12.3 was recrystallized with chloroform and methanol to yield 39 (6.0 mg). Fraction 14 was resolved by silica gel column chromatography eluted with chloroform and methanol (50:1) and further recrystallization of methanol of the subfraction to result in 40 (30.0 mg).
3.3.1. Vignasterol A (1)
White powder, mp 145–147 °C (CHCl3); [α]D25 −300.0 (c 0.1, CHCl3). UV (MeOH) λmax (log ɛ): 280 (2.29), 229 (2.72, sh) nm. IR (Neat) νmax: 3438, 2956, 2868, 1721, 1642, 1516, 1461, 1377, 1261, 1207, 1172, 1103, 1063, 1024 cm−1. ESI-MS (rel. int. %): m/z 575 ([M−H]−, 47), 559 (100). HR-ESI-MS: m/z 575.4093 [M−H]− (calcd for C38H55O4, 575.4095). 1H-NMR (CDCl3, 500 MHz): δ 6.76 (2H, d, J = 8.3 Hz, H-5′, −9′), 6.57 (2H, d, J = 8.3 Hz, H-6′, −8′), 5.36 (1H, br s, H-6), 4.66 (1H, m, H-3), 4.23 (1H, d, J = 5.9 Hz, H-3′), 3.68 (1H, d, J = 5.9 Hz, H-2′), 2.34 (2H, m, H-4), 2.00 (2H, m, H-7, −12), 1.85–1.87 (2H, m, H-1, −16), 1.70 (1H, m, H-25), 1.57 (3H, m, H-2, −7, −15), 1.46 (2H, m, H-8, −11), 1.36 (2H, m, H-20, −22), 1.26 (2H, m, H-16, −28), 1.18 (2H, m, H-12, −23), 1.07 (1H, m, H-1), 1.01 (1H, m, H-22), 1.01 (3H, s, CH3-19), 0.92 (3H, d, J = 6.4 Hz, CH3-21), 0.85 (3H, t, J = 7.4 Hz, CH3-29), 0.84 (3H, d, J = 6.9 Hz, CH3-27), 0.83 (1H, m, H-28), 0.82 (3H, d, J = 6.9 Hz, CH3-26), 0.68 (3H, s, CH3-18). 13C-NMR (CDCl3, 100 MHz): δ 172.0 (C-1′), 154.0 (C-7′), 139.6 (C-5), 131.1 (C-4′), 129.1 (C-5′, −9′), 122.8 (C-6), 115.0 (C-6′, −8′), 74.7 (C-3), 56.7 (C-14), 56.1 (C-17), 50.0 (C-9), 45.9 (C-24), 44.3 (C-3′), 43.9 (C-2′), 42.3 (C-13), 39.7 (C-12), 38.1 (C-4), 37.0 (C-1), 36.6 (C-10), 36.2 (C-20), 34.0 (C-22), 31.9 (C-7), 31.9 (C-8), 29.2 (C-25), 28.2 (C-16), 27.8 (C-2), 26.2 (C-23), 24.3 (C-15), 23.1 (C-28), 21.0 (C-11), 19.8 (C-26), 19.3 (C-19), 19.0 (C-27), 18.8 (C-21), 12.0 (C-29), 11.9 (C-18).
3.3.2. Vigvexin A (2)
White powder, mp > 300 °C (MeOH); [α]D25 −100.0 (c 0.2, MeOH). UV (MeOH) λmax (log ɛ): 340 (1.86), 296 (sh, 2.28), 265 (2.84), 222 (sh, 2.52), 213 (2.53) nm. IR (Neat) νmax: 3418, 2928, 1656, 1614, 1515, 1480, 1433, 1402, 1320, 1286, 1251, 1205, 1169, 1131, 1065, 1027 cm−1. ESI-MS (rel. int. %): m/z 335 ([M−H]−, 100), 321 (76), 305 (88). HR-ESI-MS: m/z 335.0905 [M−H]− (calcd for C20H15O5, 335.0914). 1H-NMR (Acetone-d6, 400 MHz): δ 13.26 (1H, s, OH), 8.77 (1H, s, OH), 8.18 (1H, s, H-2), 7.43 (2H, d, J = 8.3 Hz, H-2′, −6′), 6.90 (2H, d, J = 8.3 Hz, H-3′, −5′), 6.27 (1H, s, H-6), 5.46 (1H, t, J = 8.6 Hz, H-2″), 5.12 (1H, s, H-4″), 4.95 (1H, s, H-4″), 3.51 (1H, dd, J = 15.2, 8.6 Hz, H-1″), 3.09 (1H, dd, J = 15.2, 8.6 Hz, H-1″), 1.78 (3H, s, CH3-5″). 13C-NMR (Acetone-d6, 100 MHz): δ 181.8 (C-4), 167.4 (C-7), 164.7 (C-5), 158.6 (C-4′), 154.0 (C-2), 153.8 (C-9), 144.6 (C-3″), 131.3 (C-2′, −6′), 124.2 (C-3), 122.9 (C-1′), 116.0 (C-3′, −5′), 112.9 (C-4″), 106.5 (C-10), 104.0 (C-8), 94.5 (C-6), 88.8 (C-2″), 31.3 (C-1″), 17.1 (C-5″).
3.3.3. Vigvexin B (3)
White plates, mp 227–229 °C (MeOH); [α]D25 −80.0 (c 0.2, MeOH). UV (MeOH) λmax (log ɛ): 307 (2.25), 250 (2.71), 243 (sh, 2.67), 216 (2.38) nm. IR (Neat) νmax: 3292, 2928, 2858, 1627, 1594, 1515, 1449, 1389, 1318, 1270, 1208, 1175, 1105, 1066, 1021 cm-1. ESI-MS (rel. int. %): m/z 343 ([M+Na]+, 28), 321 ([M+H]+, 100). HR-ESI-MS: m/z 343.0944 [M+Na]+ (calcd for C20H16O4Na, 343.0946), 321.1128 [M+H]+ (calcd for C20H17O4, 321.1127). 1H-NMR (Acetone-d6, 400 MHz): δ 8.17 (1H, s, H-2), 8.05 (1H, d, J = 8.6 Hz, H-5), 7.46 (2H, d, J = 8.6 Hz, H-2′, −6′), 6.95 (1H, d, J = 8.6 Hz, H-6), 6.88 (2H, d, J = 8.6 Hz, H-3′, −5′), 5.52 (1H, t, J = 7.8 Hz, H-2″), 5.15 (1H, s, H-4″), 4.97 (1H, s, H-4″), 3.64 (1H, dd, J = 16.0, 7.8 Hz, H-1″), 3.23 (1H, dd, J = 16.0, 7.8 Hz, H-1″), 1.81 (3H, s, CH3-5″). 13C-NMR (Acetone-d6, 100 MHz): δ 175.8 (C-4), 165.7 (C-7), 158.3 (C-4′), 154.5 (C-9), 153.0 (C-2), 144.6 (C-3″), 131.2 (C-2′, −6′), 128.4 (C-5), 125.3 (C-3), 124.3 (C-1′), 119.8 (C-10), 115.9 (C-3′, −5′), 114.3 (C-8), 112.8 (C-4″), 109.0 (C-6), 88.7 (C-2″), 32.0 (C-1″), 17.1 (C-5″).