stilbene, Ferulic acid and its derivatives from the roots of Angelica sinensis

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1 T¹p chý Hãa häc, T. (), Tr , 00 stilbene, Ferulic acid and its derivatives from the roots of Angelica sinensis Received Nguyen Thi Hong Van, Nguyen Thi Hoang Anh, Tran Van Sung, Katrin Franke and Ludger Wessjohann Institute of Chemistry, National Center for Natural Science and Technology, Institute of Plant Biochemistry, Halle/S., Germany SUMMARY The rhizome of Angelica sinensis (liv.) Diels (Umbelliferae) is one of the most commonly used traditional drugs in China, indicated against anemia, menstrual disorders, amenorroehea and rheumatism. From the ethyl acetate extract of the roots of A. sinensis a stilbene, ferulic acid, coniferylferulate and decorticate have been isolated. Their structures were identified by MS and NMR spectroscopic methods. I - INTRDUCTIN The roots of Angelica sinensis are also known as "Danggui" in China. In China it serves as remedy against anemia, menstrual disorders, amenorroehea and rheumatism []. In the Vietnamese folk medicine it is used for the treatment of constipation and pain []. In Japan, Angelica acutiloba is used as a substitute for A. sinensis. Research reports on the analysis its ingredients are appeared in the literature since 9. Recently, many advances have been made in chemical and pharmacological studies of this drug. A series of components have been found in the roots of A. sinensis by GC, HPLC-MS analysis. 5 constituents in its essential oil and compounds in non-volatile fraction have been reported by Chen Yaozu []. Z-ligustilide is the main component and coniferyl ferulate, butylphthalide etc. are the major components. The present communication deals with the isolation and structural elucidation of four compounds ( - ). Their structures have been determined by MS, H and C-NMR spectroscopy and by comparison with the data published in the literature to be,'-dihydroxy,,'-dimethoxystilben (), ferulic acid (), coniferylferulate () and decorticate (). II - Experimental. Plant material The roots of A. sinensis were bought in the traditional medicine mark in Hanoi, March 00. A voucher specimen was deposited in the Herbarium of Institute of Chemistry.. Instruments and Chemicals NMR: Varian Unity 00; MS: AMD 0; for analytical purposes: Merck TLC aluminum sheets silica gel 60 F 5 (layer thickness 0. mm) were used. Silica gel Merck 60 ( mm) is used for column chromatography.. Extractions and Isolation Air dried and powdered tubers of A. sinensis were extracted with Et : H (95 : 5) at room temperature. The ethanol extract was 508

2 concentrated under vacuum and aq. soln. was extracted with EtAc and n-bu, successively. The solvent was evaporated in vacuum to afford 60g EtAc and 0g n-bu extracts. The EtAc extract was separated by chromatography on silica gel, eluting gradient with n-hexan, EtAc and Me to furnish 5 fractions, which were combined according to TLC monitoring. The fraction containing compound was chromatographed on Sephadex LH-0, washing with Me, and further purified by preparative TLC eluted with CHCl : Me (9:) to afford compound, an amorphous powder, EI-MS m/z (rel. int.): 7 [M] + (0), [M-xCH ] + (), [M-CH -C-H +H] + (9), 08 (8). The fraction containing compound was subjected on a Sephadex LH-0 column, eluting with Me and following by preparative TLC, developing with CHCl : Me (9 : ) to yield compound, an amorphous powder, negative ESI-MS: 9 [M-H] +, HRESIMS m/z 7.07 [M+Na] +, C 0 H 0 cal Chromatography of fraction 5 on Sephadex LH-0 with Me as solvent afforded compounds and, compounds and were isolated. Compound : colourless oil, EIMS: 56 [M] + (.8), 0 [M - C 8 H 9 + H] + (.5), 9 [M - C 0 H + H] + (ferulic acid) (6), 76 [M - C 0 H - H + H] + (00). Compound : yellow powder, positive ESI-MS: 5 [M+Na] +. III - RESULTS AND DISCUSSIN The roots of Angelica sinensis were extracted with ethanol at room temperature. The aqueous ethanol extract has been treated further by extraction with ethyl acetate and n-butanol, successively. Chromatographic separation of the ethyl acetate extract yielded compounds -. Compound was obtained as an amorphous powder. Its EI-MS spectrum indicated the molecular ion peak at m/z 7 [M] +. The H- NMR of contained protons at 6.89 d (8.06), 6.99 dd (.95, 8.06) and 7.0 d (.95) corresponding to an ABX aromatic ring. Additionally, three singlets at 6.88, 5.6 and.95 with the integrals of, and protons, respectively, were also observed. The signal at 5.6 could be assigned to a hydroxyl group. The last signal (.95) exhibited the presence of a methoxy group. The C-NMR and APT spectra revealed only 8 carbons, including quaternary signals, four protonated and one methoxyl signals. The analysis of spectroscopic data suggested that, compound consisted of two equivalent moieties. The singlet at 6.88 and one protonated carbon more in the aromatic range led to conclude that two moieties of were connected with each other by a double bond. Its structure was confirmed by comparison with the data described in the literature [] to be,'-dihydroxy-,'- dimethoxy-stilben. This compound was isolated for the first time from the roots of Leuzea carthamoides []. Compound was isolated as an amorphous powder and gave the molecular ion peak at m/z 9 [M] + in the EI-MS spectrum. Its molecular formula was obtained from the HRESIMS as C 0 H 0. The H-NMR spectrum indicated a trisubstituted aromatic ring [7.7 d (.0), 7.05 dd (.0, 8.) and 6.0 d (8.)], two trans ethenyl protons with coupling constant of 5.9 Hz. Furthermore, only methoxyl group was observed. C-NMR spectrum revealed 0 carbons. Among them, 8 signals could be assigned for aromatic ring and one double bond ( ppm). Besides these carbons, a carbonyl (7. ppm) and a methoxyl groups (56. ppm) have been seen. Comparison of the spectroscopic data of with the data described in the literature [5] could conclude that compound is ferulic acid. But it seems that compound existed as salt because of the large difference of chemical shifts of carbonyl signal (.), when compared with that of ferulic acid. Some studies [6, 7] reported that ferulic acid showed anti-inflammatory and inhibitory effects on uterine movement. The EI-MS spectrum of compound, an amorphous powder, showed the molecular ion peak at m/z 56 [M] +. H-NMR spectrum indicated the signals of aromatic rings [7.0 d 509

3 (.0), 7.07 dd (.0, 8.), 6.88 d (8.) and 6.9 d (.0), 6.9 d (8.), 6.9 dd (.0, 8.)], each corresponding to an ABX system, two double bonds with coupling constant of 5.9 and 5.7 Hz, two methoxyl signals (.90 s and.9 s), two hydroxyl groups (5.7 s and 5.9 s) and one oxygenated methylene (.8 dd). Besides the carbons, which according to above mentioned proton signals, a carbonyl signal at 66.9 and 6 quaternary aromatic carbons have been seen in the C-NMR. These spectroscopic data suggested that, could be an ester of ferulic acid. This suggestion was confirmed by comparison with the spectroscopic data of coniferylferulate, published in the literature []. Thus, the structure of has been determined to be coniferylferulate. It was described for the first time as a constituent of A. sinensis, Ligusticum porteri and Levisticum officinale. Compound, an amorphous powder, has the molecular ion peak at m/z 0 [M] + in the ESI-MS spectrum. The NMR spectra of were similar to those of. Compound consisted also trans-feruloyl moiety [ 7.7 d (.8), 7.06 dd (.8, 8.) 6.78 d (8.)] and a,-disubstituted aromatic ring [7. d (8.0), 6.80 d (8.0)]. Additionally, the signals resonanced at.86 (H, dd, J =.8 and 7.) and. m (H) showed the existence of a -CH()CH - subunit. Consequently, the structure was elucidated to be -['- hydroxyphenyl]-glycol mono trans-ferulate by detailed comparison its spectroscopic data with published literature [8]. This compound has been isolated for the first time from Peucedanum decursivum. Its trivial name was decorticated. The assignments of all protons and carbons of compound - were listed in the tables and. 50 Table : H-NMR data of compounds - (00 MHz, measured in CDCl and,, in CD D) H 7.0 d (.95) 7.7 d (.0) 7.0 d (.0) 7.7 d (.8) d (8.06) 6.0 d (8.) 6.88 d (8.) 6.78 d (8.) dd (.95, 8.06) 7.05 dd (.0, 8.) 7.07 dd (.0, 8.) 7.06 dd (.8, 8.) s 7.57 d (5.9) 7.65 d (5.9) 7.6 d (5.9) d (5.9) 6. d (5.9) 6.7 d (5.9) -Me.95 s.89 s.90* s.88 s s s ' 6.9 d (.0) 7. d (8.0) ' 6.80 d (8.0) '-Me '-.9* s s 5' 6.9 d (8.) 6.80 d (8.0) 6' 6.9 dd (8.,.0) 7. d (8.0) 6.6 d (5.7).86 dd (.76, 7.) 8' 6. dt (6.59, 5.7). m 9'.8 dd (.8, 6.59) +, * : Could be exchanged in the column.

4 Me 7 6' 5' ' ' ' Me Me H H 5 6 : Ferulic acid H :,'-dihydroxy,,'-dimethoxystilben ' 8' 6' Me : Coniferylferulate ' ' Me H Me : Decursidate ' Table : C-NMR chemical shift values of compounds - (75 MHz, measured in CDCl and,, in CD D) C * * Me ' ' '-Me 55.9 ' ' ' ' ' 65. *: Could be exchanged in the column. Acknowledgements: We thank the Bundesministerium fuer Bildung und Forschung (BMBF), Bonn, Germany for financial support, Dr. A. Porzel and Dr. J. Schmidt, Institute for Plant Biochemistry Halle/ S., Germany for their discussion on NMR and MS-spectra. REFERENCES. Sibylle Zschocke, Jiang-Hua Liu, Hermann Stuppner and Rudolf Bauer. Phytochemical Analysis, 9, P (998).. Do Tat Loi. Glossary of Vietnamese Medicinal Plants. Medicinal Publication, Hanoi, Vietnam, P. 55 (999).. Chen Yaozu, Zhang Huidi, Chen Nengyu, Zhao Tianzeng and Wang Maotian, Hexue Tongbao, 9 (), P (98).. Zsuzsanna Hajdu, Erzsebet Varga, Judit Hohmann, Alajos Kalman, Gyula Argay and Gabor Guenther. J. Nat. Prod., 6, P (998). 5. Ralph et al. NMR Database of Lignin and Cell Wall Model Compounds, Yukihiro zaki. Chem. Pharm. Bull. 0 (), P (99). 7. Yukihiro zaki and Jian-Ping Ma. Chem. Pharm. Bull. 8 (6), P (990). 8. Ling Yi Kong, Nian Huan Yao. Chinese Chemical Letters, (), P. 5-8 (000). 5

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