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

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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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 diffusaWilld. (Rubicaceae) plant, which belongs to the family of Rubiaceae and is one ofthe most popular traditional Vietnamese medicines [1]. Extensive pharmacologicalinvestigations performed so far on Hedyotis diffusa have shown that this herbalmedicine has several biological activities such as human neutrophil elastase inhibitor[2], neuroprotection [3], antitumor activity [4]. Hedyotis diffusa contains a majorityof anthraquinone [5], irridoid glucoside, flavonoids [2, 3], glucans [6]. In this paperwe report our investigation on the chemical constituents of Hedyotis diffusa growingin Tuyen Quang Province.2. Content2.1. Experiments ∗ General experimental procedures: IR spectra were recorded on SHIMADZU-FTIR 8101M spectrophotometerusing KBr disks. NMR spectra (1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC) wererecorded on Brucker Advance 500MHz. The chemical shift values (δ) are givenin 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 onprecoated Si gel GF254 (Merck Co., Germany) and TLC spots were viewed at 254nm 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 (Instituteof Ecology & Biological Resources, Vietnam Academy of Science and Technology,VAST). A voucher specimen (No.DHP20090601) is deposited in the Herbarium ofthe 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) wereground 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 extractedin n-hexane:H2 O (1:1) biphasic solvent system. Evaporating the organic phase gave45 g of condensate. The crude condensate was subjected to column chromatographyover 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-NMRand 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 molecularformula of C29 H50 O. The strong, wide IR absorption at 3450 cm−1 (assigned forO-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. Especiallysix 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 ...