Chemical constituents from the stems of Michelia alba

We have continued the study of constituents from Michelia alba [1]. To further understand the chemotaxonomy and to continue searching for novel agents from Magnoliaceous plants, the stems of M. alba were chosen for the first time for phytochemical investigation. In this paper, we report the isolation of 20 pure substances. The compounds, including six aporphines, (–)-anonaine (1), (–)-norushinsunine (2), (–)-ushinsunine (3), (–)-N-formylanonaine (4) [2], (–)-romerine (5) [3], and (–)-asimilobine (6) [4]; two oxoaporphines, liriodenine (7) and oxoxylopine (8); one lignan, (+)-syringaresinol (9); one amide, N-trans-feruloyltyramine (10); seven benzenoids, 4-hydroxybenzaldehyde (11), p-anisaldehyde (12) [5], veratraldehyde (13) [5], 3,4,5-trimethoxybenzoic acid (14) [6], 3,4-dimethoxybenzoic acid (15) [7], eugenol (16) [8], and methyl isoeugenol (17) [8]; one triterpenoid, ficaprenol (18) [9]; two steroids, β-sitosterol (19) [10] and stigmasterol (20) [10], are isolated from the stems of Michelia alba. Compounds 4, 5, and 6 and 12–18 were isolated for the first time from this species [2].

The MeOH extract (156.7 g) was obtained from M. alba according to the literature method [1] (89.2 g) and H₂O (60.8 g). The CHCl₃-soluble fraction was chromatographed over silica gel (800 g, 70–230 mesh) using n-hexane–EtOAc– CHCl₃–MeOH mixtures as eluents to produce five fractions. Part of fraction 1 (12.86 g) was subjected to silica gel chromatography by eluting with n-hexane–EtOAc (40:1) and enriched gradually with EtOAc to furnish three fractions (1-1–1-3). Fraction 1-1 (5.32 g) was further purified on a silica gel column using n-hexane–EtOAc mixtures to obtain eugenol (16) (23 mg), methylisoeugenol (17) (18 mg), and ficaprenol (18) (20 mg). Fraction 1-2 (4.63 g) was further purified on a silica gel column using n-hexane–EtOAc mixtures to obtain p-anisaldehyde (12) (19 mg), veratraldehyde (13) (22 mg), 3,4,5-trimethoxybenzoic acid (14) (26 mg), and 3,4-dimethoxybenzoic acid (15) (25 mg). Fraction 1-3 (3.28 g) was further purified on a silica gel column using n-hexane–EtOAc mixtures to obtain {-sitosterol (19) (45 mg) and stigmasterol (20) (38 mg). Part of fraction 2 (9.33 g) was subjected to silica gel chromatography by eluting with n-hexane–EtOAc (30:1) and enriched with EtOAc to furnish two further fractions (2-1–2-2). Fraction 2-1 (5.22 g) was further purified on a silica gel column using n-hexane–EtOAc mixtures to obtain liriodenine (7) (62 mg). Part of fraction 2-2 (3.62 g) was further purified on a silica gel column using n-hexane–EtOAc mixtures to obtain (+)-syringaresinol (9) (30 mg). Part of fraction 3 (11.36 g) was subjected to silica gel chromatography by eluting with n-hexane–EtOAc (10:1) and enriched with EtOAc to furnish three further fractions (3-1–3-3). Fraction 3-1 (5.59 g) was further purified on a silica gel column using n-hexane–EtOAc mixtures to obtain oxoxylopine (8) (18 mg). Fraction 3-2 (5.45 g) was further purified on a silica gel column using n-hexane–EtOAc mixtures to obtain 4hydroxybenzaldehyde (11) (28 mg). Part of fraction 4 (25.63 g) was subjected to silica gel chromatography by eluting with CHCl₃–MeOH (100:1) and enriched with MeOH to furnish three fractions (4-1-4-3). Fraction 4-1 (11.62 g) was further purified on a silica gel column using CHCl₃–MeOH mixtures to obtain (–)-anonaine (1) (68 mg) and (–)-ushinsunine (3) (32 mg). Fraction 4-2 (5.32 g), eluted with CHCl₃–MeOH (60:1), was further separated using silica gel column chromatography and preparative TLC (CHCl₃–MeOH (100:1) and gave (–)-N-formylanonaine (4) (18 mg) and (–)-romerine (5) (15 mg). Fraction 4-3 (4.58 g), eluted with CHCl₃–MeOH (50:1), was further separated using silica gel column chromatography and preparative TLC (CHCl₃–MeOH (80:1) and gave N-trans-feruloyltyramine (10) (22 mg). Part of fraction 5 (10.82 g) was subjected to silica gel chromatography by eluting with CHCl₃–MeOH (30:1) and enriched with MeOH to furnish two fractions (5-1–5-2). Fraction 5-1 (6.32 g) was further purified on a silica gel column using CHCl₃–MeOH mixtures to obtain (–)-norushinsunine (2) (33 mg). Fraction 5-2 (2.88 g), eluted with CHCl₃–MeOH (20:1), was further separated using silica gel column chromatography and preparative TLC (CHCl₃–MeOH (60:1) and gave (–)-asimilobine (6) (16 mg).

(–)-N-Formylanonaine (4) as in [5], brownish needles (MeOH), UV (λ_max, nm): 211, 270, 316, IR (ν_max, cm^–1): 1655 (C=O), 1044, 936 (OCH₂O). ¹H NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 2.73 (2H, m, H-4), 2.85 (1H, m, H-7α), 3.26 (1H, dd, J = 14.0, 4.4, H-7β), 3.16 and 3.42 (total 1H, m and td, J = 11.8, 2.8, H-5β), 3.85 and 4.50 (total 1H, each ddd, J = 12.8, 4.8, 3.6, H-5α), 4.65 and 5.07 (total 1H, each dd, J = 14.4, 4.4, H-6a), 5.99 and 6.12 (each 1H, d, J = 1.4, OCH₂O), 6.59 and 6.62 (total 1H, s, H-3), 7.20–7.30 (3H, m, H-8, 9, 10), 8.11 (1H, d, J = 7.6, H-11), 8.27 and 8.40 (total 1H, s, CHO), EI-MS m/z: 293 [M]⁺.

(–)-Romerine (5) as in [6], colorless needles (CHCl₃), UV (λ_max nm): 234, 272, 312. IR (ν_max, cm^–1): 3420, 1045, 942. ¹H NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 2.60 (3H, s, N-CH₃), 5.94 and 6.09 (each 1H, d, J = 1.2, OCH₂O), 6.56 (1H, s, H-3), 7.23–7.32 (3H, m, H-8, 9, 10), 8.08 (1H, d, J = 6.0, H-11), EI-MS m/z: 279 [M]⁺.

(–)-Asimilobine (6) as in [6], brown powder (CHCl₃), UV (λ_max, nm): 216, 246, 275, 308. IR (ν_max, cm^–1): 3500, 1560, 1452, 1059, 960. ¹H NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 3.60 (3H, s, 1-OCH₃), 6.73 (1H, s, H-3), 7.25–7.27 (3H, m, H-8, 9, 10), 8.30 (1H, d, J = 8.8, H-11), EI-MS m/z: 267 [M]⁺.