Four phthalides from the roots of Angelica sinensis

Journal of Chemistry, Vol. 43 (2), P. 228 - 231, 2005<br /> <br /> <br /> Four phthalides from the roots of Angelica sinensis<br /> Received 22nd-Dec-2004<br /> Nguyen Thi Hong Van1, Nguyen Thi Hoang Anh1, Tran Van Sung1<br /> Katrin Franke2, Ludger Wessjohann2<br /> 1<br /> Institute of Chemistry, Vietnam Academy of Science and Technology<br /> 2<br /> Institute of Plant Biochemistry, Halle/S., Germany<br /> <br /> <br /> SUMMARY<br /> The rhizome of Angelica sinensis (Oliv.) Diels (Umbelliferae) is known as "Danggui" in<br /> China, has been used widely in traditional Chinese medicine to treat irregular menstruation,<br /> anaemia, headache, hypertension, chronic asthma and rheumatism. From the ethyl acetate<br /> extract of its roots four phthalides have been isolated. Their structures were identified by MS and<br /> NMR spectroscopic methods to be (Z)-3-butylidenphthalide, senkyunolide H, senkyunolide I and<br /> dihydroxyligustilide.<br /> <br /> <br /> I - INTRODUCTION phthalides from the ethyl acetate extract of this<br /> plant. Their structures were elucidated by<br /> The Umbelliferae plants were known to be spectroscopic methods and by comparison with<br /> source of alkylphthalide derivatives. The first the published data in the literatures as (Z)-3-<br /> study to use high-performance liquid chroma- butylidenphthalide (1), senkyunolide H (2),<br /> tography electrospray ionization-mass spectro- senkyunolide I (3) and dihydroxyligustilide (4).<br /> scopy method (HPLC-ESP-MS) for identifi-<br /> cation of the phthalides in Danggui's extracts II - experimental<br /> was reported by Long Ze-Lin in 1998 [1]. In this<br /> report the existence of more than 30 phthalides 1. Plant material<br /> have been demonstrated by using the above<br /> mentioned method. Twelve of them were The roots of A. sinensis were bought in the<br /> unambiguously identified as senkyunolide I, H, traditional medicine market in Hanoi in March<br /> sedanenolide,... The main essential component, 2002. A voucher specimen, No. 06 was<br /> Z-ligustilide, other phthalides and ferulic acid deposited in the Herbarium of Institute of<br /> are though to be the biologically active Chemistry.<br /> components of Danggui [2]. Various phthalide 2. Instruments and chemicals<br /> derivatives, named senkyunolides B-L have<br /> been also isolated from the rhizomes of NMR: Varian Unity 300; MS: AMD 402 in<br /> Ligusticum chuangxiong [3]. A variety of the Institute of Plant Biochemistry, Halle/S.,<br /> volatile non-polar alkylphthalide derivatives Germany; For analytical purposes: Merck TLC<br /> showed to have antifungal and smooth muscle aluminium sheets silica gel 60 F254 (layer<br /> relaxing activities [4]. thickness 0.2 mm) were used. Silica gel Merck<br /> We describe in this communication the 60 (0.040 - 0.063 mm), Sephadex LH 20 are<br /> isolation and structure determination of four used for column chromatography.<br /> <br /> 228<br /> 3. Extraction and Isolation positive ESI-MS spectrum showed the molecular<br /> ion peak at m/z 189 [M+H]+. The 1H-NMR<br /> The roots of A. sinensis (5 kg) were dried,<br /> spectrum revealed 4 protons of an aromatic ring<br /> powdered and extracted with EtOH : H2O (95:<br /> 5) at room temperature. The organic solvent in the range of from 7.5 to 7.9 ppm and the<br /> was evaporated under vacuum and aq. soln. was signals due to the butylidene side chain [ 0.99<br /> extracted with EtOAc and n-BuOH, succes- (3H, t, J = 7.4 Hz), 1.56 (2H, sext, J = 7.4 Hz),<br /> sively giving 160 g EtOAc- and 20 g n-BuOH- 2.46 (2H, dd, J = 7.5 and 7.7 Hz)]. In the 13C-<br /> extracts. NMR spectrum of this compound, 12 carbon<br /> signals have been observed. Four of them could<br /> The EtOAc extract was chromatographed on be assigned for signals of butylidene side chain<br /> silica gel, eluting with increasing amounts of<br /> ( 13.9, 22.6, 27.8 and 109.5). Besides 6<br /> EtOAc (0 - 30%) in n-hexane and then with<br /> EtOAc : MeOH (7 : 3) to yield 345 fractions, aromatic carbons ( 119.5, 124.3, 125.1, 129.2,<br /> which were combined according to TLC 134.1, 139.4), one carbonyl ( 167.0) and an<br /> monitoring. oxygenated quartenary carbon ( 145.6) have<br /> been also seen. This spectroscopic analysis<br /> The fraction containing compound 1 was<br /> suggested that compound 1 was butylidene-<br /> rechromatographed over flash silica gel column<br /> phthalide. This suggestion was confirmed by<br /> eluting with n-hexane : CHCl3 (1 : 1) and then<br /> comparison with the published reference data<br /> with n-hexane : CHCl3 : MeOH (1 : 1 : 0.1)<br /> [5]. Butylidenephthalide was isolated previously<br /> yielded 1, positive ESI-MS: m/z 211 [M + Na]+.<br /> from Angelica sinensis and Cnidium officinale<br /> The fraction containing compound 2, 3 and [4].<br /> 4 was further purified by using sephadex LH-20<br /> column, eluted with MeOH to furnish pure The spectroscopic data of compounds 2 and<br /> compound 2 and 10 other fractions. Compound 3 were very similar. Their EI-MS spectra<br /> 2 was obtained as an oil, positive ESI-MS: m/z indicated the molecular ion peaks at m/z 224<br /> 247 [M+Na]+. The second fraction of this [M]+ for both compounds. They showed the<br /> sephadex column was subjected over a flash signals due to the butylidene side chain in 1H-<br /> column on silica gel, eluting with CHCl3 : NMR spectra [ 0.95 (3H, t, J = 7.3 Hz), 1.50<br /> MeOH (9 : 1), followed by preparative TLC (2H, sext, J = 7.3 Hz), 2.36 dd (2H, J = 7.5 and<br /> washing with CHCl3 : MeOH (98 : 2) furnished 7.5 Hz)]. They have a glycol group in the<br /> compound 3, EI-MS (rel. int.): m/z 224 [M]+ cyclohexene ring and their vicinal hydroxy-<br /> (25.7), 206 (2.5), 180 (100), 164 (20), 95 (21.4). methine protons appeared at 4.07 (ddd, J =<br /> Compound 4 was obtained from the fourth 2.4, 3.7, 7.7 Hz) and 4.61 (d, J = 3.7 Hz) in 2<br /> fraction by using a silica gel column, eluted and at 3.94 (ddd, J = 3.0, 6.2, 9.7 Hz) and 4.49<br /> with CHCl3 : MeOH (9 : 1), and followed by (d, J = 6.2 Hz) in 3. The 13C-NMR of them<br /> preparative TLC with CHCl3 : MeOH as eluted showed almost identical chemical shifts, except<br /> solvent system. Its ESI-MS: m/z 224 [M]+. the signals of C-6 and C-7. These carbons<br /> resonanced at 67.3, 63.4 in compound 2 and at<br /> III - RESULTS AND DISCUSSION 71.8, 68.0 in compound 3 for C-6 and C-7,<br /> respectively. The base peaks at m/z 180<br /> The roots of Angelica sinensis were (C12H12O3) in their EI-MS spectra, due to retro-<br /> extracted with ethanol at room temparature. The Diels-Alder cleavage at the cyclohexene ring<br /> aqueous ethanol extract has been treated further and established the position of the glycol group<br /> by extraction with ethyl acetate and n-butanol, at C-6 and C-7. These spectroscopic data<br /> successively. Four phthalides (1 - 4) have been confirmed that these compounds were an<br /> obtained after chromatographic separation of the isomeric pair of dihydroxyphthalides by<br /> ethyl acetate extract. comparison with the published data [8]. The<br /> Compound 1 was isolated as an oil. Its observed coupling constant (J = 3.7 Hz)<br /> <br /> 229<br /> between H-6 and H-7 in compound 2 suggested Contrast to compound 2 the J value of the vicinal<br /> that the configuration of the two hydroxyl coupling between H-6 and H-7 in compound 3<br /> groups was cis. Thus, compound 2 was was 6.2 Hz. This value indicated that it has a 6,7-<br /> established to be senkyunolide H. The X-ray trans glycol. The spectral data of 3 were in good<br /> diffraction analysis of senkyunolide H was agreement with those of senkyunolide as<br /> carried out by Takashi Naito [3] resulting that described in the literature [3]. Both isomers,<br /> two hydroxyl groups in the cyclohexene ring senkyunolide H and I, were previously isolated<br /> have axial (C-7) and equatorial (C-6) orientation. from Cnidium officinale [4].<br /> <br /> 9<br /> 8<br /> 4 11<br /> 3a 3 10 O O<br /> 5 O<br /> O HO HO<br /> 6 1 HO<br /> 7a O OH O<br /> 7 OH OH O<br /> O<br /> 1 2 3 4<br /> <br /> Table 1: 1H-NMR data of compounds 1 - 4 (300 MHz, CDCl3)<br /> H 1 2 3 4<br /> +<br /> 4 7.9 d (7.7) 1.80 m, 2.16++ m<br /> ++<br /> 1.90* m, 2.10* m 1.84 m, 2.02# m<br /> #<br /> <br /> 5 7.5 - 7.7+ m 2.61++ m 2.55* m 2.50# m<br /> 4.07 ddd 3.94 ddd 3.96 ddd<br /> 6 7.5 - 7.7 m<br /> (2.4, 3.7, 7.7) (3.0, 6.2, 9.7) (2.9, 5.2, 8.4)<br /> 7 7.5 - 7.7 m 4.61 d (3.7) 4.49 d (6.2) 4.46 d (5.2)<br /> 8 5.64 t (7.7) 5.31 t (7.7) 5.29 t (7.9) 5.28 t (7.9)<br /> 9 2.46 dt (7.4, 7.7) 2.36 dt (7.4, 7.7) 2.36 dt (7.3, 7.9) 2.32 dt (7.3, 7.9)<br /> 10 1.56 sext (7.4) 1.50 sext (7.4) 1.50 sext (7.3) 1.48 sext (7.3)<br /> 11-CH3 0.99 t (7.4) 0.95 t (7.4) 0.95 t (7.3) 0.94 t (7.3)<br /> + ++<br /> , , *, #: may be exchanged in each columns.<br /> <br /> Table 2: 13C-NMR data of compounds 1 - 4 (75 MHz, CDCl3)<br /> C 1 2 3 4<br /> 1 167.0 169.2 168.9 169.1<br /> 3 145.6 153.1 152.6 153.3<br /> 3a 139.4 148.0 148.0 147.8<br /> 4 119.5 18.4 19.2 18.7<br /> 5 134.1 25.7 26.6 26.1<br /> 6 125.1 67.5 71.8 71.2<br /> 7 129.2 63.4 68.0 66.7<br /> 7a 124.3 125.2 125.9 125.5<br /> 8 109.4 114.5 114.3 114.0<br /> 9 27.8 28.2 28.1 28.0<br /> 10 22.6 22.3 22.2 22.2<br /> 11 13.9 13.9 13.7 13.7<br /> 230<br />  Compound 4 was obtained as an oil. Its references<br /> EIMS exhibited the same molecular peak at m/z<br /> 224 [M]+ as of compound 2 and 3. Like 2 and 3, 1. Long-Ze Lin, Xian-Guo He, Li-Zhi Lian,<br /> compound 4 was also an isomer of dihydroxy- Wayne King, Jerry Elliott. J. Chromato., A.<br /> phthalide by NMR spectroscopic analysis. The 810, P. 71 - 79 (1998).<br /> carbons of 4 were different from those of 3 only 2. P. -M. Hou, C. -M. Lee, T. F. Choang, J.-<br /> by chemical shifts of C-6 and C-7. They YChui, H. N. C. Wong. Phytochemistry 29,<br /> appeared in the 13C-NMR spectra of 4 at 71.2 P. 1189 (1990).<br /> and 66.7, while of 3 at 71.8 and 68.0. On the 3. Takashi Naito, Takao Katsuhara, Kazuaki<br /> other hand, the coupling constant of the vicinal Naitsu, Yukinobu Ikeya, Minoru Okada and<br /> protons between H-6 and H-7 in compound 4 Hiroshi Mitsuhashi. Phytochem., 31, P. 639<br /> was 5.1 Hz. This value indicated that the - 642 (1992).<br /> configuration of its two hydroxyl groups was 4. Masaru Kobayashi, Miyuki Fujita and<br /> trans by comparison with the value of Hiroshi Mitsuhasshi. Chem. Pharm. Bull.<br /> senkyunolide I. Thus, compound 4 was 35, No. 4, P. 1427 - 1433 (1987).<br /> elucidated as depicted structure for 4 with the 5. T. Yamagishi and H. Kaneshima. Yakugaku<br /> opposite configuration to those of 3. Zasshi, 97, 237 (1977).<br /> <br /> <br /> <br /> <br /> 231<br />