Isolation and structural determination of some chemical constituents from Hedyotis diffusa Willd. (Rubiaceae) growing in Tuyen Quang province

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From the methanol extract of dried powders of Hedyotis diffusa Willd. (Rubiaceae) plant growing in Tuyen Quang Province, three compounds were isolated and identified: B-sitosterol, daucosterol and ursolic acid. Their structures were confirmed by various spectral methods (IR, NMR, MS, 1H-NMR, 13C-NMR, etc.) and by comparison with those of references.

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Nội dung Text: Isolation and structural determination of some chemical constituents from Hedyotis diffusa Willd. (Rubiaceae) growing in Tuyen Quang province

JOURNAL OF SCIENCE OF HNUE Natural Sci., 2010, Vol. 55, No. 6, pp. 57-61 ISOLATION AND STRUCTURAL DETERMINATION OF SOME CHEMICAL CONSTITUENTS FROM Hedyotis diffusa Willd. (RUBIACEAE) GROWING IN TUYEN QUANG PROVINCE Pham Huu Dien(∗) and Tuong Hong Nhung Hanoi National University of Education (∗) E-mail: dienhp@gmail.com Abstract. From the methanol extract of dried powders of Hedyotis diffusa Willd. (Rubiaceae) plant growing in Tuyen Quang Province, three com- pounds were isolated and identified: β-sitosterol (1), daucosterol (2) and ursolic acid (3). Their structures were confirmed by various spectral meth- ods (IR, NMR, MS, 1 H-NMR, 13 C-NMR, etc.) and by comparison with those of references. Keywords: Hedyotis diffusa Willd., β-sitosterol, daucosterol, ursolic acid, isolation. 1. Introduction Bach Hoa Xa Thiet Thao is the Vietnamese name of the Hedyotis diffusa Willd. (Rubicaceae) plant, which belongs to the family of Rubiaceae and is one of the most popular traditional Vietnamese medicines [1]. Extensive pharmacological investigations performed so far on Hedyotis diffusa have shown that this herbal medicine has several biological activities such as human neutrophil elastase inhibitor [2], neuroprotection [3], antitumor activity [4]. Hedyotis diffusa contains a majority of anthraquinone [5], irridoid glucoside, flavonoids [2, 3], glucans [6]. In this paper we report our investigation on the chemical constituents of Hedyotis diffusa growing in Tuyen Quang Province. 2. Content 2.1. Experiments ∗ General experimental procedures: IR spectra were recorded on SHIMADZU-FTIR 8101M spectrophotometer using KBr disks. NMR spectra (1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC) were recorded on Brucker Advance 500MHz. The chemical shift values (δ) are given in ppm with TMS as internal standard, coupling constant J (by Hz). EIMS, LC- MSD spectra were recorded on HP 5989B, Agilent mass spectrometer. Silica gel 57
Pham Huu Dien and Tuong Hong Nhung (Merck Co., Germany) was used for flash chromatography. TLC was carried out on precoated Si gel GF254 (Merck Co., Germany) and TLC spots were viewed at 254 nm and visualized by spraying with vanillin-10% H2 SO4 solution. ∗ Plant material: Bach hoa xa thiet thao plants collected in Tuyen Quang Province (in July, 2009) were identified as Hedyotis diffusa Willd. by Dr. Ninh Khac Ban (Institute of Ecology & Biological Resources, Vietnam Academy of Science and Technology, VAST). A voucher specimen (No.DHP20090601) is deposited in the Herbarium of the Dept. of Organic Chemistry, Hanoi National University of Education. ∗ Extraction and Isolation: The air-dried aerial parts of Hedyotis diffusa Willd. (Rubiaceae) (1.5 kg) were ground into powder and extracted with 80% methanol (3L x 7 days x 3 times). After evaporation of collected percolation, the crude extract (125 g) was extracted in n-hexane:H2 O (1:1) biphasic solvent system. Evaporating the organic phase gave 45 g of condensate. The crude condensate was subjected to column chromatography over silica gel and eluted with n-hexane:ethyl acetate (100:0, 50:1, 10:1, 1:1, 1:10) and ethyl acetate:methanol (4:1, 1:1, 1:2, 1:4), giving fractions (Fr.) A (0.52 g), B (0.87 g), C (0.8 g), D (1.50 g), E (0.65 g), F (0.43 g), G (0.85 g), H (0.45 g) and I (0.98 g). By repeating column chromatography, 45 mg of compound (1) (from Fr. B), 22 mg of compound (2) (from Fr. C) and 31 mg of compound (3) (from Fr. E) were isolated. β-Sitosterol (1): white needles, m.p.: 139.5 - 140.5◦C; Rf = 0.34 (n-hexane: EtOAC = 4:1), well dissolved in n-hexane, ethyl acetate, chloroform; IR (KBr, cm−1 ): 3434 (wide, νOH ), 2936, 2856, 1642, 1463 (νC=C ), 1376, 1059, 963, 803, 588; 1 H-NMR (δ, ppm): 5.35 (1H, m, H-6), 3.52 (1H, m, H-3), 2.28 and 2.23 (2H, m, H-4), 0.68 (3H, s, H-18), 1.10 (3H, s, H-17), 0.917 (3H, d, J = 5.5Hz, H-21), 0.82 (3H, d, J = 7.1Hz, H-26), 0.811 (3H, d), 0.85 (3H, d, H-29); EIMS (m/z, %): 414 [M]+ (8), 396 (4), 381 (3), 314 (5), 255 (18), 213 (12), 159 (18), 145 (20), 133 (19), 105 (33), 81 (50), 69 (62), 55 (100). Daucosterol (2): white amorphous powders, m.p.: 280◦ C; dissolved in DMSO, methanol, CHCl3 , Rf = 0.28 (in n-hexane:EtOAc = 1:1); IR (KBr, cm−1 ): 3440 (OH), 2948, 1638, 1463, 1377, 1020, 600; LC-MSD (m/z): 577 [M+H]+ ; 1 H-NMR (δ, ppm): 5.37 (1H, brd, H-6), 3.24 (1H, t 7.5, H-3), 1.03 (3H, s, H-19), 0.92 (3H, d 5.5, H-21), 0.85 (3H, t 6.5, H-29), 0.84 (1H, d 7.5, H-25), 0.83 (3H, d 6.5, H-27), 0.68 (3H, s, H-18). Ursolic acid (3): white powder, m.p.: 261 - 262◦ C; Rf = 0.7 (n-hexane:EtOAC = 1:2), well dissolved in methanol, ethanol; IR (KBr, cm−1 ): 3451 (OH), 2931, 2867, 1693, 1462, 1382, 1279, 1185, 1036, 999, 734; LC-MSD (m/z): 455 [M+H]+ ; 1 H-NMR and 13 C-NMR (δ, ppm) (see Table 1). 58
Isolation and structural determination of some chemical constituents... 2.2. Results and discussion ∗ Compound 1: The EI-MS data of (1) afforded m/z [M]+ 414, corresponding to molecular formula of C29 H50 O. The strong, wide IR absorption at 3450 cm−1 (assigned for O-H band absorption), at 1646 cm−1 (assigned for olefinic C=C band absorption) together with 1 H-NMR signals at 3.49 ppm (1H, m, H-3), 5.35 ppm (1H, brd m, H-6) suggested the presence of OH (at C-3) and C=C groups at C5-C6. Especially six methyl signals at 0.683 (3H, s, H-18), 1.00 (3H, s, H-19), 0.918 (3H, d, J = 5.5Hz, H-21), 0.824 (3H, d, J = 6.5 Hz, H-26), 0.811 (3H, d, J = 6.5 Hz, H-27), 0.849 (3H, d, J = 7.5, H-29) are typical for sterols. From above analysis of IR, 1 H-NMR, MS spectra and melting point of (1) and in comparison with those of stigmast-5-en-3β-ol (or β-sitosterol) [7], it shows that (1) must be the β-sitosterol, one of the most popular sterols in plants. ∗ Compound 2: The LC-MS data of (2) afforded m/z [M+H]+ 577, so m/s of [M]+ was 576, corresponding to molecular formula of C35 H60 O6 . The IR of (2) has a strong, wide absorption peak at 3440 cm−1 (assigned for free OH band absorption), a middle peak at 1638 cm−1 (assigned for olefinic C=C band absorption). The 1 H-NMR data of (2) are similar to those of (1), except a group of six multiplet proton signals with δ from 3.24 ppm to 4.41 ppm, assigned for seven protons of glucose. From above analysis of IR, NMR, MS spectra, melting point of (2) and in comparison with those of (1) and stigmast-5-en-3β-ol-3-O-β-D-glucoside (or dau- costerol) [8], it shows that (2) must be the daucosterol, one of the most popular glycoside in plants. ∗ Compound 3: The LC-MSD data of (3) afforded m/z [M]+ 456. The strong, wide IR absorp- tion at 3451 cm−1 (assigned for free O-H band absorption), at 1693 cm−1 (assigned for carboxylic C=O band absorption) together with 13 C-NMR signal at 180.4 ppm suggested the presence of a carboxylic group COOH. One middle peak at 1444 cm−1 is assigned for cyclic C=C band absorption. The 1 H-NMR of (3) showed triterpene- type protons signals in high field with seven methyl (mainly single) groups, from 0.77 ppm to 1.04 ppm. One CH signal at 3.2 ppm are signed for a proton linked to C3 -OH groups; other at 5.23 ppm assigned for a cyclic C=C group. The 13 C-NMR and DEPT spectra indicated that (3) had thirty carbons, including seven CH3 (δC from 16.6 to 30.2 ppm), nine CH2 (δC from 18.2 to 39.3 ppm), seven cyclic CH (δC from 39.4 to 77.3 ppm) and six cyclic C (δC from 36.8 to 47.7 ppm, not including COOH). From above analysis of IR, NMR, MS spectra, melting point of (3) and in comparison with those of ursolic acid, C30 H48 O3 [9], it shows that (3) must be ursolic acid. 59
Pham Huu Dien and Tuong Hong Nhung Table 1. The 1 H-NMR and 13 C-NMR data of (3) in comparison with those of ursolic acid in [9] (3) and ursolic acid (U.A) (3) and ursolic acid (U.A) No. 1 H-NMR 13 C-NMR No. 1 H-NMR 13 C-NMR U.A U.A U.A U.A (3) (3) (3) (3) [9] [9] [9] [9] 1 39.3 39.2 t 16 24.3 24.7 t 2 27.8 27.8 t 17 47.7 47.7 s 1H 1H 1H 1H 2.10 3 767.3 77.7 18 52.7 53.2 d 3.20 m 3.00 m 2.18 m d 11.25 4 39.3 39.2 19 39.4 39.4 d 5 55.2 55.6 d 20 39.3 39.3 d 6 18.2 18.9 21 30.6 31.1 t 7 32.9 33.6 t 22 37.0 37.2 t 3H 3H 8 39.7 40.0 s 23 30.2 29.1 q 0.67 s 0.68 s 3H 3H 9 47.7 47.9 d 24 16.6 16.1 q 0.89 s 0.89 s 3H 3H 10 36.8 37.4 s 25 16.8 16.9 q 0.87 s 0.87 s 3H 3H 11 24.0 23.7 t 26 18.1 17.8 q 0.75 s 0.75 s 1H 1H 3H 3H 12 125.4 125.4 d 27 24.1 24.1 q 5.23 m 5.13 m 1.04 s 1.04 s 13 138.0 139.0 s 28 180.4 179.1 s 3H 0.81 3H 0.81 14 41.9 42.5 s 29 18.1 17.9 q d 6.25 d 6.25 3H 3H 0.92 15 27.9 28.4 t 30 20.9 21.9 q 0.91 m d 6.5 1H 1H 4.31 OH - - 4.27 q brd Structure of compounds from the plant of Bach Hoa Xa Thiet Thao Hedyotis diffusa Willd. (Rubiaceae) is below: β-Sitosterol (1) Daucosterol (2) 60
Isolation and structural determination of some chemical constituents... Ursolic acid (3) 3. Conclusion In our first investigation, we isolated and structurally identified three com- pounds from Bach Hoa Xa Thiet Thao Hedyotis diffusa Willd. (Rubiaceae) plant by various spectral methods: IR, NMR, MS, 1 H-NMR, 13 C-NMR, etc. Three com- pounds were β-sitosterol (1), daucosterol (2) and ursolic acid (3). Acknowledgement This work is supported by the Ministry of Education and Training (project number: B-2010-17-275TD). REFERENCES [1] Vo Van Chi, 1999. Dictionary of Vietnamese Trad. Medicinal Plants. Medicine Publishing House. [2] Xu G.H., Kim Y.H., Chi S.W., Choo S.J., Ryoo I.J., Ahn J.S. and Yoo I.D., 2009. Evaluation of human neutrophil elastase inhibitory effect of iridoid glycosides from Hedyotis diffusa. Bioorg. Med. Chem. Lett., Vol. 20, No. 2, pp. 513-515. [3] Kim Y., Park E.J., Kim J., Kim Y., Kim S.R. and Kim Y.Y, 2001. Neuroprotec- tive constituents from Hedyotis diffusa. J. Nat. Prod., Vol. 61, No. 1, pp. 75-78. [4] Liao L.L., Chen C.H. and Chen G.C., 1979. Formosan medicinal herb, Hedyotis diffusa Willd, as an antitumor agent. Taiwan Yi Xue Hui Za Zhi, Vol. 78, No. 7, pp.658-660. [5] Huang W.H., Yu S.H., Li Y.B. and Jiang J.Q., 2008. Four anthraquinones from Hedyotis diffusa. J. Asian Nat. Prod. Res., No. 10, pp. 887-889. [6] Cui J., Wang S.C., Shi S.S. and Wang Z.T., 2006. Structural characterization of a glucan isolated from Hedyotis diffusa Willd. Zhong Yao Cai, Vol. 29, No. 9, pp. 912-915. [7] John G.L. and Toshihiro A., 1997. Analysis of sterols. Blackie Acad. & Profes- sional, Chapman & Hall, p. 378. [8] Laurence V. and et al., 1999. Cytotoxic polyisoprenes and glycosides of long-chain fatty alcohols from Dimocarpus fumatus. Phytochem., Vol. 50, pp. 63-69. [9] Maria Paula P.Ramos and et al., 2003. Computer-aided identification of chemical constituents isolated from Cybistax antisyphilitica. J. of Molecular Design, No. 2, pp. 1232-1237. 61