Studies on chemical composition of angelica sinensis's V - The Nitrogen-containing compounds

Journal of Chemistry, Vol. 43 (5), P., 2005<br /> <br /> <br /> <br /> studies on chemical composition of angelica<br /> sinensis's<br /> V - The Nitrogen-containing compounds<br /> <br /> Received 2 March 2004<br /> Nguyen Thi Hong Van1, Nguyen Thi Hoang Anh1, Tran Van Sung1<br /> Katrin Franke2 and Ludger Wessjohann2<br /> 1<br /> Institute of Chemistry, Vietnames Academy of Sciences and Technology,<br /> 2<br /> Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle/Saale, Germany<br /> <br /> SUMMARY<br /> Angelica sinensis (OLiv.) Diels ("Danggui") belongs to the family Umbelliferae. Its rhizomes<br /> are reputed to have marked restorative, antianaemic and haemagogic properties. From ethyl<br /> acetate and butanol extracts of rhizomes of this plant 8 nitrogen-containing compounds (1-8)<br /> have been isolated. Their structures were elucidated by means of MS and NMR spectroscopic<br /> methods as 5-oxo-proline, 5-oxo-prolin-ethylester, nicotinamide, tryptophane, perlolidine,<br /> 1,2,3,4-tetrahydro-1-methyl- -carboline-3-carboxylic acide, adenosine and uridine.<br /> Keywords: Angelica sinensis, Umbelliferae, nitrogen-containing compounds.<br /> <br /> I - INTRODUCTION by comparison with the published data these<br /> compounds have been determined as 5-oxo-<br /> Angelica sinensis was already mentioned as proline (1), 5-oxo-prolin-ethylester (2),<br /> a women's medicine in the famous Shen-nung nicotinamide (3), tryptophane (4), perlolidine<br /> Pen-ts'as-ching in China. The rhizomes of this (5), 1,2,3,4-tetrahydro-1-methyl- -carboline-3-<br /> plant has been used in the treatment of pain and carboxylic acide (6), adenosine (7) and uridine<br /> inflammatory diseases such as rheumatism. It (8).<br /> has additionally been used as spasmolytics,<br /> especially against menstrual disorders.<br /> II - experimental<br /> In Europe, Levisticum officinale Koch is a<br /> well known drug and has been called as Plant material<br /> "European Danggui". It was reported that its<br /> The roots of A. sinensis were bought from a<br /> chemical composition is almost identical to that<br /> traditional medicine markt in Hanoi, March<br /> of A. sinensis [1].<br /> 2002. A voucher specimen, No. 06 was<br /> In a previous article we reported the deposited in the Herbarium of Institute of<br /> isolation and structure elucidation of Chemistry, Hanoi.<br /> falcarindiol, its derivate and phenolic<br /> compounds [2]. This work describes the Instruments and chemicals<br /> isolation and structure elucidation of 8 nitrogen- NMR: Varian Unity 300; MS: AMD 402;<br /> containing compounds from rhizomes of this For analytical purposes: Merck TLC aluminium<br /> plant. Using MS, NMR spectroscopic data and sheets silica gel 60 F254 (layer thickness 0.2 mm)<br /> 1<br /> were used. Silica gel Merck 60 (0.040 - 0.063 With 15% MeOH, fractions 353 - 356 were<br /> mm) is used for column chromatography. separated and further purified by using a<br /> sephadex LH-20 column MeOH as eluate<br /> Extraction and isolation<br /> yielded 5 fractions. The first fraction was<br /> The roots of A. sinensis (5 kg) were dried, rechromatographed on sephadex LH-20 column,<br /> powdered and extracted with EtOH : H2O (95: eluting with MeOH, followed by preparative<br /> 5) at room temperature. The organic solvent TLC to furnish compound 1, negative ESI-MS:<br /> was evaporated under vacuum and aq. soln. was m/z 128 [M-H], positive ESI-MS: m/z 152<br /> extracted with EtOAc and n-BuOH, [M+Na]. The third fraction was<br /> successively, giving 160 g EtOAc and 20 g n- chromatographed on RP-18 column with solvent<br /> BuOH extracts. MeOH : H2O = 4 : 1 to afford compound 2, EI-<br /> The EtOAc extract was separated by MS (70eV, rel. int.): m/z 157 [M]+ (6.7%), 84<br /> chromatography on silica gel, eluting with [M-C2H5COO]+ (100%), 56 (10%).<br /> increasing amounts of EtOAc (0 - 30%) in n- With 20% MeOH, fractions 364 and 366<br /> hexan and then with EtOAc : MeOH (7 : 3) to have been separated. The fraction 364 was<br /> yield 345 fractions, which were combined purified by using a silica gel column, eluting<br /> according to TLC monitoring. with CHCl3 : MeOH = 75 : 25 to yield<br /> Fr. 182 - 188 of EtOAc extract was chroma- compound 4, positive ESI-MS: m/z 227 [M+Na],<br /> tographed over a sephadex LH-20 column, negative ESI-MS: m/z 203 [M-H]. The fraction<br /> eluting with MeOH to give 4 fractions. The 366 was rechromatographed over sephadex LH-<br /> second fraction was further purified by 20, eluting with MeOH, followed on a RP-18<br /> preparative TLC (CHCl3 : MeOH = 8 : 2) column using MeOH : H2O = 3 : 5 as eluate<br /> furnishing compound 7, 1H-NMR (Pyridin-d5, furnishing compound 6, positive ESI-MS: m/z<br /> 300MHz): 8.73s (H-2), 8.63s (H-8), 8.47s 253 [M+Na]+, negative ESI-MS: m/z 459 [M-H].<br /> (NH2), 6.75d (5.9, H-1'), 5.08d (4.68), 4.79d<br /> (2.73), 4.3dd (2.34, 12.2, H-5'), 4.16dd (2.34, III - RESULTS and discussion<br /> 12.2, H-5'). The third fraction was rechroma-<br /> tographed over a sephadex LH-20 column The roots of Angelica sinensis were<br /> eluted with MeOH to give compound 8, positive extracted with ethanol at room temparature. The<br /> ESI-MS: m/z 267.0 [M+Na]+, 1H-NMR aquoues ethanol extract has been treated further<br /> (CD3OD, 300MHz): 8.0 d (8.0, H-6), 5.69 d by extraction with ethyl acetate and n-butanol,<br /> (8.0, H-5), 5.9 d (4.4, H-1'), 3.4 - 4.3 m (H-2', successively. Compounds 1 - 8 have been<br /> 3', 4' and 5'); 13C-NMR (CD3OD, 75 MHz): obtained after further chromatographic<br /> 165.9 (C-2), 152.2 (C-4), 142.5 (C-6), 102.5 (C- separation of both ethyl acetate and butanol<br /> 5), 90.5 (C-1'), 86.3 (C-4'), 75.6 (C-3'), 71.2 (C- extracts.<br /> 2') and 62.2 (C-5'). Compound 1 showed the molecular ion peak<br /> The BuOH extract was chromatographed on at m/z 128 [M-H] in the negative ESI-MS<br /> silica gel column with CHCl3 and increasing spectrum, indicating the presence of one<br /> amounts of MeOH afforded 380 fractions. nitrogen atom in its molecule. The 1H-NMR<br /> With 10% MeOH, fractions 124 - 223 were spectrum contained 5 proton signals, including a<br /> obtained. These fractions were rechromato- proton at H 4.22 dd (4.8, 8.8) and 4 protons<br /> graphed over a sephadex LH-20 column eluted resonanced in the range from 2.1 to 2.6 ppm.<br /> with MeOH to give compounds 3 and 5. There are only 5 carbons in the 13C-NMR<br /> Compound 3: positive ESI-MS: m/z 145 spectrum. Two of them are carbonyl groups ( C<br /> [M+Na], negative ESI-MS: m/z 121 [M-H]. 180.9, 176.3). In addition, the signals of one<br /> Compound 5: positive ESI-MS: m/z 287 methine and two methylene groups appeared at<br /> [M+Na], negative ESI-MS: m/z 263 [M-H]. C 57.3 and 30.6, 26.2, respectively. These<br /> <br /> <br /> <br /> <br /> 2<br /> spectral data are in good agreement with those of 5-oxo-proline published in [3].<br /> O<br /> 3 1<br /> 8 OH<br /> O 1' NH2 7 4<br /> 5 NH 2<br /> H O H O 10<br /> 3 6 NH<br /> O 5 N 2 O 5 N 2' 2 4 11<br /> 1 OH 1 O 1'<br /> N 5<br /> 4 3<br /> 6<br /> 1: 5-oxo-proline 2: 5-oxo-prolin-ethylester 3: Nicotinamide 4: Tryptophane<br /> <br /> NH2 O<br /> 5 4<br /> 4b 4a O 6 4<br /> 6 3 5 N<br /> 5 4 N HN<br /> N 4b 4a 3 8<br /> 7 8a N 8b 1' OH 2 6<br /> 1 2<br /> 8 H N 4 N O N<br /> 7 NH HO<br /> 3' 8a N 8b HO 5'<br /> 8 1 O 5' O<br /> O H 1'<br /> 1'<br /> 5' 3'<br /> 3'<br /> HO OH OH OH OH<br /> 5: Perlolidine 6: 1,2,3,4-tetrahydro-1-methyl-<br /> -carboline-3-carboxylic acid 7: Adenosine 8: Uridine<br /> <br /> <br /> The molecular ion peak of compound 2 at Compound 4 indicated the molecular ion<br /> m/z 157 [M]+ has been indicated in the EI-MS peak at m/z 203 [M-H] in its negative ESI-MS<br /> spectrum. The base peak at m/z 84 [M- spectrum. The 1H-NMR spectrum contained<br /> C2H5COO]+ suggested that its molecule possess signals of 5 protons in the aromatic region and 3<br /> an ethylester group. The NMR data of compound protons in the range from 3.1 to 3.9 ppm. Four<br /> 2 are very similar to those of compound 1. of aromatic protons [ H 7.69 d (7.5), 7.36d<br /> Besides the signals of 5-oxo-proline moiety as in (7.5), 7.11 t (7.5) and 7.03 t (7.5)] corresponded<br /> 1, the signals at H 1.28 t (3H, J = 7.1), 4.2 dd to an ortho-disubstituted aromatic ring and an<br /> (2H, J = 7.1, 14.3) and C 14.5, 62.5 in the NMR another proton appeared as a singlet at 7.2ppm.<br /> spectra of compound 2 reveal the presence of The 13C-NMR spectrum revealed 11 carbons,<br /> an ethoxyl group. This spectral analysis led to including one carbonyl ( C 174.3), 5 methine<br /> conclusion that compound 2 is ethylester of 5- together with 3 quartenary carbons in the<br /> oxo-proline [4]. aromatic range ( C : 109.4 - 138.2), a methine<br /> and a methylene signals in the aliphatic region.<br /> Compound 3 showed a molecular ion peak The spectroscopic data of this compound were<br /> at m/z 121 [M-H] in the negative ESI-MS completely identical with those of tryptophane<br /> spectrum. The 1H-NMR spectrum exhibited in the literature [6].<br /> signals of 4 aromatic protons in the range from<br /> 7.54 to 9.02 ppm. Six carbon signals are present Compound 5 was isolated as yellow powder,<br /> in the 13C-NMR spectrum. One of them is having a molecular ion peak at m/z 263 [M-H]<br /> carbonyl group. The others are 4 methine and in its negative ESI-MS spectrum. The 1H-NMR<br /> one quartenary carbons. The structure of this spectrum showed four protons of an ortho-<br /> compound has been elucidated as nicotinamide disubstituted aromatic ring [8.18 d (7.9), 7.7 d<br /> by comparison with the published data [5]. (7.9), 7.59 t (7.9), 7.29 t (7.9)] and two olefinic<br /> Nicotinamide, a well known water soluble protons at H 8.29 d (5.3), 8.01 d (5.3) of an<br /> vitamin, was first isolated from liver as an anti- unsaturated heterocyclic ring. Eight carbon<br /> pellagra factor. Nishimura reported that signals including 6 protonated and two<br /> nicotinamide exhibits shell-opening activity quartenary carbons corresponded to above<br /> against common bivalve molluscs [5]. mentioned protons have been observed in the<br /> <br /> <br /> 3<br /> 13<br /> C-NMR. This analysis suggested that the at m/z 229 [M-H] in the negative ESI-MS<br /> structure 5 contained an -carboline skeleton. spectrum. In comparison its NMR spectral data<br /> Furthermore, the typical signals of an 5- with those of compound 5 suggested that<br /> hydroxymethyl-2-furyl at H 4.89 (2H, s), 6.59 d compound 6 has the structure of 1,2,3,4-<br /> (1H, 3.2) and 7.22 d (1H, 3.2); C 57.5, 110.9 tetrahydro- -carboline. Besides the signals of<br /> and 111.0 have been seen in its 1H- and 13C- 1,2,3,4-tetrahydro- -carboline moiety, a methyl<br /> NMR spectra. Consequently, the structure of group at H 1.47 d (6.59), C 20.1 and a carboxyl<br /> compound 5 was concluded to be 1-(5- group at C 181.1 are present. Finally, the<br /> hydroxymethyl-2-furyl)- -carboline. The struc- structure of 6 was elucidated to be 1,2,3,4-<br /> ture 5 is identical with perlolidin isolated from tetrahydro-1-methyl- -carboline-3-carboxylic<br /> perenial rye-grass (Lolium perenn L.) and from acide, when compared with the published data<br /> Lolium chuanxiong a medicinal plant for [8].<br /> treatment of angina pectoris [7, 8]. The NMR assignment of compounds 1 - 6<br /> Compound 6 indicated a molecular ion peak were listed in the tables 1 and 2.<br /> <br /> Table 1: 1H-NMR data of compounds 1 - 6 (1-5 measured in CD3OD and 6 in D2O + CD3OD, 300 MHz)<br /> H 1 2 3 4 5 6<br /> 1 - - - - - 4.16 d (6.59)<br /> 2 4.22 dd 4.28 dd 9.02 s 3.87 dd - -<br /> (4.8, 8.8) (4.5, 8.8) (3.7, 9.5)<br /> 3 2.15*m, 2.17* m, - 3.15 dd 8.29 d 3.51 dd<br /> 2.34* m 2.36* m (9.5, 15.2) (5.3) (4.3, 11.4)<br /> 3.51 dd<br /> (3.7, 15.2)<br /> 4 2.34* m, 2.36* m, 8.68* d - 8.01 d 2.7 ddd (2.7, 11.4, 15.4)<br /> 2.48* m 2.45* m (4.2) (5.3) 3.07 dd (2.7, 15.4)<br /> 5 - - 7.54 dd 7.2 s 8.18 d (8.0) 7.52 d (7.5)<br /> (4.2, 8.0)<br /> 6 - - 8.28* d - 7.29 d (8.0) 7.08 t (7.5)<br /> (8.0)<br /> 7 - - - - 7.59 t (8.0) 7.15 t (7.5)<br /> 8 - - - 7.69 d (7.5) 7.7 t (8.0) 7.4 d (7.5)<br /> 9 - - - 7.03 t (7.5) - -<br /> 10 - - - 7.11 t (7.5) - -<br /> 11 - - - 7.36 d (7.5) - -<br /> 1' 1.28 t<br /> (7.1)<br /> 2' 4.2 dd<br /> (7.1, 14.3)<br /> 3' - 4.2 dd - - 7.22 d -<br /> (7.1, 14.3) (3.2)<br /> 4' - 1.28 t - - 6.59 d (3.2) -<br /> (7.1)<br /> CH2-OH - - - - 4.89 s -<br /> 1-CH3 - - - - - 1.47 d (6.59)<br /> *: may be exchanged in each column.<br /> <br /> <br /> 4<br />  Table 2. 13C-NMR data of compounds 1 - 6 (1-5 measured in CD3OD and 6 in D2O + CD3OD, 75 MHz)<br /> C 1 2 3 4 5 6<br /> 1 176.3 173.8 - 174.3 143.0 59.7<br /> 2 57.3 57.0 125.0 56.6 - -<br /> 3 30.6 30.3 131.3 28.5 138.5 49.2<br /> 4 26.2 25.9 137.2 109.4 114.9 26.7<br /> 4a - - - - 122.1 107.9*<br /> 4b - - - - 132.1 127.6*<br /> 5 180.9 180.7 149.2 125.0 122.5 119.9<br /> 6 - - 152.6 128.3 121.1 118.7<br /> 7 - - - 138.2 129.9 122.1<br /> 8 - - - 122.6 113.2 112.1<br /> 8a - - - - 132.5 137.1*<br /> 8b - - - - 134.3 138.0*<br /> 9 - - - 119.2 - -<br /> 10 - - - 119.9 - -<br /> 11 - - - 112.3 - -<br /> 1' 14.5 169.6 - - 181.1<br /> 2' - 62.5 - - 157.1 -<br /> 3' - - - - 110.9 -<br /> 4' - - - - 111.0 -<br /> 5' - - - - 154.2 -<br /> 1-CH3 - - - - - 20.1<br /> 5'-CH2OH - - - - 57.5<br /> *: may be exchanged in the column<br /> <br /> The structures of compound 7 and 8 were 1. S. Zschocke, J-H Liu, H. Stuppner and R.<br /> determined as two common nucleosides: adenosine Bauer. Phytochemical analysis, 9, 283-290,<br /> [9-( -D-ribofuranosyl)adenine] and uridine [1-( - (1998).<br /> D-ribofuranosyl]uracil, respectively. The spectro- 2. Nguyen Thi Hong Van, Nguyen Thi Hoang<br /> scopic data of both were completely identical with Anh, Tran Van Sung, Katrin Franke and<br /> those reported previously [6, 10]. It was reported Ludger Wessjohann. J. of Chem. (accepted<br /> that adenosine and uridine have been isolated from for publication).<br /> fennel [a drug prepared from the fruit of<br /> Foeniculum vulgare Miller (Umbelliferae)]. The 3. F. Benz, Helv. Chim. Acta, 57, 2459,<br /> spectral data of compounds 7 and 8 were given in (1974).<br /> the experimental. 4. B. Rigo. J. Het. Chem. 25, 49 (1988).<br /> Acknowledgements: We thank the 5. N. Yamashita, K. Sakata, H. Ina and K. Ina,<br /> Bundesministerium fuer Bildung und Forschung Agric. Biol. 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