Some triterpenoids and steroids from Bruguiera cylindrica leaves collected from Can Gio mangrove forest - Nguyen Thi Huong Que

Vietnam Journal of Chemistry, International Edition, 55(5): 569-572, 2017 DOI: 10.15625/2525-2321.2017-00509 569 Some triterpenoids and steroids from Bruguiera cylindrica leaves collected from Can Gio mangrove forest Nguyen Thi Huong Que 1 , Tran Minh Chi 2 , Pham Nguyen Kim Tuyen 2 , Nguyen Kim Phi Phung 1* 1 University of Science, National University Ho Chi Minh City, Vietnam 2 Sai Gon University, Ho Chi Minh City, Vietnam Received 3 March 2017; Accepted for publication 20 October 2017 Abstract Bruguiera cylindrica (Rhizophoracea), distributed in the Southeast Asia, has been used as a traditional medicine in the treatment of diarrhea and healing of wounds. Some triterpenoids and steroids were isolated from the dried leaves of Bruguiera cylindrica, collected at Can Gio mangrove forest, Ho Chi Minh City, including lupeol (1), 3β- hydroxyoleana-9(11),12-diene (2), a mixture of stigmasterol (3a) and -sitosterol (3b), cholesta-4-ene-3-one (4) and 3β-hydroxycholesta-5-ene-7-one (5). Their chemical structures were elucidated by spectroscopic analysis as well as comparing their data with the ones in the literature. Although these compounds were known in other plants this is the first time they are reported in this species. Keywords. Bruguiera cylindrica, triterpenoids, steroids. 1. INTRODUCTION Bruguiera cylindrica Blume (Rhizophoraceae), a mangrove plant, is distributed widely in Southeast Asia [6]. This plant has been used in Vietnamese traditional medicine for the treatment of diarrhea and healing of wounds [6, 7]. Four species of this genus are found in Vietnam including B. cylindrica, B. gymnorhiza, B. parviflora and B. sexangula [1]. In the study on Vietnamese mangrove plants, we investigated the chemical constituents of B. cylindrica leaves collected in Can Gio mangrove forest, Ho Chi Minh City, Viet Nam. 2. EXPERIMENTAL Leaves of Bruguiera cylindrica were collected at Can Gio mangrove forest in Ho Chi Minh city, Vietnam in September 2015. The scientific name of the plant was authenticated by Dr. Pham Van Ngot, Faculty of Biology, Ho Chi Minh city Pedagogical University. A voucher specimen (No US-B020) was deposited in the herbarium of the Department of Organic Chemistry, University of Science, National University - Ho Chi Minh City. Column chromatography was carried out on silica gel 60 F254 (Merck) and gel Sephadex LH-20. Extraction of Bruguiera cylindrica The air-dried leaves of Bruguiera cylindrica (6 kg) were dried, ground then extracted with n-hexane, ethyl acetate, and methanol (3 x 20 L for each solvent), successively. The n-hexane extract (25 g) was subjected to column chromatography, eluted with a gradient of n-hexane/acetone to afford nine fractions (A1–A9). Fraction A3 (1.5 g) was purified by silica gel column chromatography to afford compound 1 (10 mg). Compound 3 (5 mg) was isolated from the fraction A5. Fraction A7 (2.49 g) was applied to silica gel column chromatography to give compound 4 (5 mg) and 5 (4 mg) The ethyl acetate extract (100 g) was subjected to silica gel column chromatography, eluted with a gradient of n-hexane/ethyl acetate to afford nine fractions (B1–B9). Fraction B4 was subjected to Sephadex LH-20 column to give five fractions (B4.1– B4.5). Fraction B4.4 was further purified by silica gel column chromatography (chloroform/methanol, 95.5/0.5) to give compound 2 (5 mg). 3. RESULTS AND DISCUSSION Compound 1 obtained as a white crystal is a triterpene due to the positive sulfuric acid test. The 1 H-NMR spectral data of compound 1 gave signals of an isopropenyl group at δ 4.72 (1H, d, J = 2.0 Hz, H- 29a), 4.61 (1H, dd, J = 2.0, 1.5 Hz, H-29b) and 1.72 (3H, s, H-30), a methine proton at  3.16 (1H, dd, J = 11.5, 4.5 Hz, H-3), six singlets belonging to six VJC, 55(5), 2017 Nguyen Kim Phi Phung et al. 570 methyl groups, including  0.79 (3H, s, H-24), 0.86 (3H, s, H-28), 0.91 (3H, s, H-25), 0.99 (3H, s, H-23), 1.02 (3H, s, H-27) and 1.11 (3H, s, H-26). The 13 C- NMR spectral data of compound 1 are shown in table 1. All the spectral data of compound 1 were similar to the one of lupeol in the literature [2, 5]. Figure 1: Chemical structure of isolated compounds Compound 2 obtained as a white crystal. The molecular formula was established as C30H48O from the APCI-MS data at m/z 425.44 [M+H] + (calc. 425.37). The 1 H-NMR spectral data of compound 2 gave one oxymethine doublet of doublet at δ 3.24 (1H, dd, J = 12.0, 5.0 Hz, H-3), two signals of olefin protons at δ 5.57 (1H, d, J = 5.5 Hz, H-11) and 5.50 (1H, d, J = 5.5 Hz, H-12), eight singlets belonging to six methyl groups, including δ 0.81 (3H, s, H-24), 0.87 (3H, s, H-23), 0.89 (3H, s, H-29), 0.90 (3H, s, H-30), 0.99 (3H, s, H-28), 1.03 (3H, s, H-27), 1.14 (3H, s, H-25) and 1.19 (3H, s, H-26). The 13 C-NMR spectral data of compound 2 are shown in table 1. All the spectral data of compound 2 were similar to the ones of 3-hydroxyoleana-9(11),12-diene in the literature [4, 5]. Compound 3 (3a and 3b) was isolated as a white amorphous powder. The APCI-MS gave a molecular ion peaks at m/z 397.36 [M+H-H2O] + (calc. 397.38) of -sitosterol and 395.42 [M+H- H2O] + (calc. 395.38) of stigmasterol, the molecular ion peak of -sitosterol is taller. The 1 H-NMR spectrum of 3 showed signals of olefin protons at δ 5.35 (3H, d, J = 5.5 Hz, H-6, H-6'), 5.15 (1H, dd, J = 15.0, 8.5 Hz, H-22) and 5.01 (1H, dd, J = 15.0, 8.5 Hz, H-23), along with one oxymethine multiplet at δ 3.52 (3H, m, H-3, H-3') and many other signals in the high field from δ 2.5-1.0. The 13 C-NMR spectrum showed four olefin carbon signals at  140.9 (C-5, C-5'), three olefin methines VJC, 55(5), 2017 Some triterpenoids and steroids from 571 at δ 138.5 (C-22), 129.5 (C-23), 121.9 (C-6, C-6') and one oxygenated methine at C 71.9 (C-3, C-3') of a mixture -sitosterol and stigmasterol [3]. All signals showed that 3 was a mixture of -sitosterol and stigmasterol with the ratio of 1:2 based on their protons integrations. Table 1: NMR data of compounds 1, 2, 4 and 5 Po s. Compound 1 (CD3OD) Compound 2 (CDCl3) Compound 4 (CDCl3) Compound 5 (CDCl3) δC δH, J (Hz) δC δH, J (Hz) δC δH, J (Hz) δC δH, J (Hz) 1 38.8 38.7 35.9 36.5 2 27.4 27.9 34.1 31.4 3 79.0 3.16 (1H, dd, 11.5, 4.5) 78.7 3.24 (1H, dd, 12.0, 5.0) 199.7 70.7 3.67 (1H, m) 4 38.7 38.9 123.8 5.72 (1H, s) 38.9 5 55.4 51.2 171.8 165.2 6 18.3 18.4 33.1 126.3 5.69 (1H, s) 7 34.3 32.1 32.2 202.5 8 40.9 37.1 35.8 45.6 9 50.5 154.3 54.0 50.1 10 37.2 40.7 38.8 38.4 11 21.0 115.7 5.57 (1H, d, 5.5) 21.2 21.4 12 25.2 120.7 5.50 (1H, d, 5.5) 39.8 42.9 13 38.1 147.1 42.6 43.3 14 42.9 42.8 56.0 50.2 15 27.5 25.7 24.3 26.3 16 35.6 27.2 28.3 28.7 17 43.0 31.9 56.2 54.9 18 48.4 45.6 12.1 0.71 (3H, s) 12.1 0.68 (3H, s) 19 48.0 2.44 (1H, m) 46.9 17.5 1.17 (3H, s) 17.5 1.20 (3H, s) 20 151.0 31.1 35.8 35.9 21 29.9 34.6 18.9 0.91 (3H, d, 6.5) 19.1 0.93 (3H, d, 6.5) 22 40.0 37.2 36.3 36.2 23 27.9 0.99 (3H, s) 28.2 0.87 (3H, s) 23.2 23.2 24 15.4 0.79 (3H, s) 15.4 0.81 (3H, s) 39.7 40.0 25 16.1 0.91 (3H, s) 20.1 1.14 (3H, s) 28.3 29.3 26 16.0 1.11 (3H, s) 21.0 1.19 (3H, s) 22.8 0.84 (3H, d, 7.0) 23.2 0.84 (3H, d, 7.0) 27 14.6 1.02 (3H, s) 25.3 1.03 (3H, s) 22.9 0.81 (3H, d, 7.0) 21.6 0.82 (3H, d, 7.0) 28 18.0 0.86 (3H, s) 28.7 0.99 (3H, s) 29 109.3 4.72 (1H, d, 2.0) 4.61 (1H, dd, 2.0, 1.5) 23.7 0.89 (3H, s) 30 19.3 1.72 (3H, s) 33.2 0.90 (3H, s) Compound 4 was isolated as a white amorphous powder. Based on the HR-ESI-MS spectrum, the molecular formula of 4 was determined as C27H44O with a pseudomolecular ion peak at m/z 385.3448 [M+H] + (calc. for C27H44O+H, 385.3470). The 1 H-NMR spectrum of 4 displayed a singlet at δ 5.72 (1H, s, H-4), three methyl doublets at δ 0.91 (3H, d, J = 6.5 Hz, H-21), 0.84 (3H, d, J = 7.0 Hz, H-26), 0.81 (3H, d, J = 7.0 Hz, H-27), and two methyl singlets at δ 0.71 (3H, s, H-18), and 1.17 (3H, s, H-19). The 13 C-NMR spectral data of compound 4 were shown in table 1. All signals showed that 4 was cholesta-4-ene-3-one due to the compatibility of its spectroscopic data with the VJC, 55(5), 2017 Nguyen Kim Phi Phung et al. 572 one in the literature [8]. Compound 5 was isolated as a white amorphous powder. 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