Contribution to results of the chemical constituents of ricinus communis - Trần Thị Hoài Vân

Journal of Science and Technology 54 (2C) (2016) 523-529 CONTRIBUTION TO RESULTS OF THE CHEMICAL CONSTITUENTS OF RICINUS COMMUNIS Tran Thi Hoai Van1, 2, Luan Thi Thu1, Do Tien Lam1, Cam Thi Inh1, Lanh Thi Ngoc2, Nguyen Van Tuyen Anh1, Pham Thi Hong Minh1, *, Pham Quoc Long1 1Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi 2Vietnam University of Traditional Medicine, No.2, Tran Phu, Ha Dong, Hanoi 3Thai Nguyen University of Agriculture and Forestry, Quyet Thang Commune, Thai Nguyen *Email: minhhcsh@gmail.com Received: 15 June 2016; Accepted for publication: 29 October 2016 ABSTRACT Using combined chromatographic methods, four compounds were isolated from the n- hexane and ethyl acetate extracts of the Ricinus communis in Melinh - Hanoi. Their structures were determined to be lupeol (1), epialeuritolic acid (2), ergosterol peroxide (3), and ricinine (4) by 1D-, 2D-NMR spectroscopic methods and in comparison with those reported in literature. Keywords: Ricinus communis, triterpenoids, ricinine. 1. INTRODUCTION Ricinus communis (Euphorbiaceae) is widely distributed in Vietnam and leaves are used in the Vietnamese folk medicine to treat several diseases such as pyoderma, eczema, rashes, ulcers, anti-inflammation, anti-arthritis, to kill worms and mosquito larvae [1, 2, 3]. Seed oil is indicated for treatment of constipation in children, pregnant women, patients after surgery and used to treat rectal prolapse, dysentery, pyoderma, lymphadenitis. The roots are used to treat rheumatism arthralgia, pain swelling kicks, tetanus, epilepsy, schizophrenic [1]. Chemical studies on this plant showed the presence of alkaloids, flavonoids, triterpenoids and sterols [4]. Further study on the chemical constituents of Ricinus communis growing in Melinh - Hanoi province, this paper reports the isolation of lupeol (1), epialeuritolic acid (2), ergosterol peroxide (3), and ricinine (4). Their structures were elucidated by the IR, ESI-MS, 1D and 2D NMR spectral data and by comparison with those of related compounds. 2. MATERIALS AND METHODS 2.1. Plant materials Tran Thị Hoai Van, Luan Thi Thu, Do Tien Lam 524 Ricinus communis (Euphorbiaceae) was collected at Melinh – Hanoi, in Aug. 2015 and was identified by Dr. Nguyen Quoc Binh, Department of Biology, Vietnam National Museum of Nature, VAST. 2.2. General experimental procedures The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer and TMS was used as internal standard. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70 - 230 mesh and 230 - 400 mesh, Merck), and Sephadex LH-20 gel. Thin layer chromatography (TLC) used pre-coated silica gel 60 F254 (1.05554.0001, Merck). Compounds were visualized by spraying with aqueous 5 % H2SO4 and heating for 3 - 5 minutes. MS were obtained using a Varian FT-ICR/HRMS Trap spectrometer. 2.3. Extraction and isolation The powdered sample (5.8 kg) was extracted three times with ethanol (at room temperature). After the solvent was evaporated in vacuum, the combined extracts were then evaporated to give the ethanol residue (225 g). The crude extracts were suspended in water and extracted sequentially with n-hexane, ethyl acetate to afford corresponding extracts: n-hexane (55 g), ethyl acetate (50 g). After that mother solution was evaporated again to give water residue then extracted with ethanol (125.3 g). The n-hexane extract (55 g) was subjected to chromatography on a silica gel column, eluting with n-hexane-ethyl acetate mixtures (gradients for n-hexane/ethyl acetate 0→100, v/v) to afford minor fractions. The fraction H1 (3.9 g) was subjected to chromatography on silica gel column, eluting with n-hexane-EtOAc mixture (5:1, v/v) to obtain compound 1 (13.0 mg). The fraction H3 (5 g) was subjected to chromatography on silica gel column, eluting with n-hexane- ethyl acetate (10:1, v/v) to give two fractions H3a and H3b. Fractions E3a and E3b were subjected to chromatography on silica gel column, eluting with n-hexane-ethyl acetate to obtain compounds 2 (20.5 mg) and 3 (19.2 mg). The ethyl acetate extract (49,5 g) was subjected to chromatography on a silica gel column, eluting with chloroform-methanol mixtures (gradients for chloroform/methanol 0→100, v/v) to give six fractions (E1-E6). The fraction E1 (1.2 g) was subjected to chromatography on silica gel column, eluting with chloroform-methanol mixture (9:1, v/v) to obtain compound 4 (16.2 mg). 2.3.1. Lupeol (1) Colorless needles. Rf = 0.55 (n-hexane : EtOAc = 4 : 1, v/v); mp 213 – 214 oC. IRνmax (cm-1) : 3509, 2937, 1685, 1456. ESI-MS (m/z): 426 [M]+ 1H-NMR (500 MHz, CDCl3), δ (ppm): 4.68 (1H, s, H-29a), 4.56 (1H, s, H-29b), 3.19 (1H, dd, J = 5.0 Hz and 6.5 Hz, H-3), 2.38 (1H, m, J = 4.7 Hz and 1.5 Hz, H-19), 0.68 (1H, d, J = 9.5 Hz, H-5), 0.76 (3H, s, H-24), 0.78 (3H, s, H-28), 0.82 (3H, s, H-25), 0.94 (3H, s, H-27), 0.96 (3H, s, H-23), 1.02 (3H, s, H-26), 1.68 (3H, s, H-30). 13C-NMR (125 MHz, CDCl3), δ (ppm): 38.69 (C-1), 27.44 (C-2), 79.02 (C-3), 38.85 (C-4), 55.28 (C-5), 18.31 (C-6), 34.24 (C-7), 40.82 (C-8), 50.42 (C-9), 37.16 (C-10), 20.92 (C-11), Contribution to results of the chemical constituents of Ricinus communis 525 25.12 (C-12), 38.04 (C-13), 42.82 (C-14), 27.40 (C-15), 35.57 (C-16), 43.00 (C-17), 48.29 (C- 18), 47.98 (d, C-19), 150.99 (C-20), 29.84 (C-21), 40.00 (C-22), 27.99 (C-23), 15.37 (C-24), 16.12 (C-25), 15.97 (C-26), 14.54 (C-27), 18.00 (C-28), 109.32 (C-29), 19.30 (C-30). 2.3.1. Aleuritolic acid (2) White powder. Rf = 0.40 (n-hexane : EtOAc = 4 : 1, v/v). ESI-MS (m/z): 456 [M]+ 1H-NMR (500 MHz, CDCl3), δ (ppm): 5.53 (1H, dd, J = 3.5 and 8.0 Hz, H-15), 3.20 (1H, dd, J = 5.0 and 11.0 Hz, H-3), 2.38 (1H, dd, J = 8.0 and 14.0 Hz, H-16), 2.29 (1H, d, J = 11.0 Hz, H-18), 1.4 (1H, m, H-9), 0.79 (1H, d, J = 12.0 Hz, H-5), 0.80 (3H, s, H-25), 0.91 (3H, s, H- 30), 0.92 (3H, s, H-24), 0.93 (3H, s, H-29), 0.95 (3H, s, H-26), 0.97 (6H, s, H-23, H-27). 13C-NMR (125 MHz, CDCl3), δ (ppm): 37.72 (C-1), 27.13 (C-2), 78.98 (C-3), 38.75 (C-4), 55.48 (C-5), 18.81 (C-6), 40.87 (C-7), 39.02 (C-8), 49.13 (C-9), 38.02 (C-10), 17.31 (C-11), 33.35 (C-12), 37.33 (C-13), 160.69 (C-14), 116.71 (C-15), 31.37 (C-16), 51.38 (C-17), 41.44 (C- 18), 35.35 (C-19), 29.3 (C-20), 33.66 (C-21), 30.72 (C-22), 27.97 (C-23), 15.55 (C-24), 15.45 (C-25), 26.15 (C-26), 22.45 (C-27), 183.31 (C-28), 31.89 (C-29) and 28.67 (C-30). 2.3.3. Ergosterol peroxide (3) Colorless needles, Rf = 0,69 (n-hexane : EtOAc = 2 : 1, v/v); mp: 180 - 181 oC; ESI-MS m/z: 428 [M]+ 1H-NMR (500MHz, CDCl3) (δ ppm): 6,50 (1H, d, J = 8.5 Hz, H-7); 6,23 (1H, d, J = 8.5 Hz, H-6); 5,24 (1H, dd, J = 8.5 and 15.0 Hz, H-22); 5,18 (1H, dd, J = 8.5 and 15.0 Hz, H-23); 3,97 (1H, m, H-3); 1.00 (3H, d, J = 6.5 Hz, H-21); 0,91 (3H, d, J = 6.5 Hz, H-28); 0,88 (3H, s, H-19); 0.84 (3H, d, J = 6.5 Hz, H-27); 0.82 (3H, d, J = 6.5 Hz, H-26); 0,81 (3H, s, H-18). 13C-NMR (125MHz, CDCl3) (δ ppm): 34,2 (C-1); 30.15 (C-2); 64,49 (C-3); 36.97 (C-4); 82.16 (C-5); 135.43 (C-6); 130.77 (C-7); 79.44 (C-8); 51.15 (C-9); 37.00 (C-10); 23.42 (C-11); 39.38 (C-12); 44.59 (C-13); 51.72 (C-14); 20.65 (C-15); 28.64 (C-16); 56.25 (C-17); 12.89 (C- 18), 18.18 (C-19); 39.71 (C-20); 20.89 (C-21); 135.22 (C-22); 132.35 (C-23); 42.86 (C-24); 33.09 (C-25); 19.65 (C-26); 19.95 (C-27); 17.57 (C-28). 2.3.4. Ricinine (4) Colorless needles, Rf = 0,61 (chloroform : methanol = 12 : 1, v/v); mp: 192 – 193 oC; ESI-MS (m/z): [M]+ 164. 1H-NMR (500 MHz, MeOD-d6) δH (ppm): 7.98 (1H, d, J = 7.5 Hz, H-6), 6.46 (1H, d, J = 7.5 Hz, H-5), 4.06 (3H, s, 4-OCH3), 3.63 (3H, s, N-CH3). 13C-NMR (125 MHz, MeOD-d6) δC (ppm): 174.72 (C-4), 163.76 (C-2), 146.77 (d, C-6), 114.74 (3-CN), 95.69 (C-5), 88.26 (C-3), 58.19 (4-OCH3), 37.76 (N-CH3). Tran Thị Hoai Van, Luan Thi Thu, Do Tien Lam 526 3. RESULTS AND DISCUSSION Compound 1 was isolated as colorless needles. The ESI-MS of 1 provided an molecular ion peak at m/z: 426 [M]+, corresponding to the formula C30H48O. The 13C-NMR and DEPT spectra of 1 exhibited 30 carbon signals, including 7 methyl, 11 methylene, 6 methine, and 6 quartenary carbons. The 13C-NMR spectrum revealed the signals of an exocyclic methylene carbon at δC 109.32 (C-29), an quaternary carbon at δC 150.99 (C-20) that were characteristic of a vinylic carbon signals for triterpenoid of the lupane (skeleton) series [5], and a methine carbon signal which directly attached to OH group at δC 79.02 (C-3) is also displayed. The 1H-NMR of 1 showed 7 methyl protons singlets at δH 0.76 (3H, s, H-24), 0.78 (3H, s, H-28), 0.82 (3H, s, H-25), 0.94 (3H, s, H-27), 0.96 (3H, s, H-23), 1.02 (3H, s, H-26), 1.68 (3H, s, H-30) corresponding to δC 15.37, 18.00, 16.12, 14.54, 27.99, 115.97 and 19.3 in the 13C-NMR spectrum, and two exomethylene protons at δH 4.68 (1H, s, H-29a) and 4.56 (1H, s, H-29b) which are corresponding with carbon at δC 109.32. The proton signal at δH 3.19 (H-3) was taken to be the oxymethine proton at C-3 corresponded to a carbon resonating at δC 79.1. Further, two methine protons signals at δH 2.38 (H-19), and 0.68 (H-5) corresponding with carbons at δC 47.98 (C-19) and 55.28 (C-5). These data revealed that compound 1 was a lupane-type triterpene. The HMBC spectrum indicated the correlations between the proton H-3 (δH 3.19) with C- 23, C-24, and between the proton H-19 (δH 2.38) with C-20, C-29, C-30, C-18, C-13 and C-21. According to the 1H, 13C-NMR, DEPT, HSQC, and HMBC spectra and comparison these data with those published in references [6, 7], compound 1 was therefore identified as lupeol and its structure was shown in Figure 1. Compound 2 was isolated as white powder. The ESI-MS of 2 provided an molecular ion peak at m/z: 456 [M]+, corresponding to the formula C30H48O3. The 13C-NMR and DEPT spectra of 2 exhibited 30 carbon signals, including 7 methyl, 10 methylene, 5 methine, and 8 quartenary carbons. The 13C-NMR spectrum revealed the signals of an carbonyl carbon at δC 183.31 (C-28), an quaternary carbon at δC 160.69 and methine carbon at δC 116.71 indicating the presence of double bond, which is a typical 14-15 double bond of taraxerane skeletone [5], and methine carbon signal which attached directly to OH group at δC 78.98 (C-3) was also displayed. The 1H-NMR of 2 showed 7 methyl protons singlets at δH 0.80 (3H, s, H-25), 0.91 (3H, s, H-30), 0.92 (3H, s, H-24), 0.93 (3H, s, H-29), 0.95 (3H, s, H-26), 0.97 (6H, s, H-23, H-27) representing seven methyl groups instead of the expected eight methyl groups of a triterpene. Furthermore, an olefinic proton doublet at δH 5.53 (dd, J = 3.0, 8.0 Hz, H-15) and an oxymethine proton at δH 3.20 (dd, J = 5.0, 11.0 Hz, H-3) corresponding with carbons at δC 116.71 (C-15) and δC 78.98 (C-3) were also observed. The HMBC spectrum indicated the correlations between the proton H-15 (5.53) with C-16, C-13, C-8, between the proton H-16 (2.38) with C-28, C-14, C-15, C-17, and C-18. The above data of 2 were consistent with those published in references [8, 9], compound 2 was therefore identified as epialeuritolic acid or 3-hydroxy-14-taraxeren-28-oic acid and its structure was shown in figure 1. Compound 3 was obtained as optically active white powder. The ESI-MS of 3 provided an molecular ion peak at m/z: 428 [M]+, corresponding to the formula C28H44O3. The 13C-NMR and Contribution to results of the chemical constituents of Ricinus communis 527 DEPT spectra of 3 exhibited 28 carbon signals, including 6 methyl, 7 methylene, 11 methine, and 4 quartenary carbons. The 13C-NMR showed carbon signals of two double bonds in the downfield region at δC 130.77 (C-7), 132.35 (C-22), 135.22 (C-23), 135.43 (C-6). In addition, the 13C-NMR spectrum of compound 3 exhibited signals consistent with the presence of three oxygenated carbons at δC 82.16 (C-5); 79.44 (C-8); 64.49 (C-3), in which there are two quaternary carbons attached directly to oxy (C-5, C-8). Notably, these spectroscopic data of 3 were consistent with those reported in the literature for a known compound ergosterol peroxide (5α,8α-epidioxyergosta-6,22-dien-3β-ol) [10, 11]. The 1H-NMR displayed signals of six methyl groups at δH 0.81 (s, H-18), 0.88 (s, H-19), 0.82 (d, J = 6.5 Hz, H-26), 0.84 (d, J = 6.5 Hz, H-27), 0.91 (d, J = 7,0 Hz, H-28), 1,00 (d, J = 6,5 Hz, H-21). Downfield signals at δH 6.51 (d, J = 8.5 Hz, H-7), and 6.24 (d, J = 8.5 Hz, H-6) H-7) were also observed. The 1H-NMR spectrum showed other characteristic signals of double bond at δH 5.24 (dd, J = 7.5 and 15.0 Hz, H-22), and 5.17 (dd, J = 7.5 and 15.0 Hz, H-23) with corresponding to carbons at δC 132.35 (C-22), 135.22 (C-23). In addition, the 1H-NMR spectrum confirmed the presence of methine protons at 0.99 (d, J = 6.5 Hz, H-21), 2.01 (m, H-20), and one oxygenated methine proton at δH 3.97 (m, H-3). These data suggested that 3 was a ergosterol-type with two double bonds in the structure. The HMBC spectrum showed the correlations between the proton H-7 (δH 6.51) with carbons C-5 at δC (82.16), C-8 (79.44), and C-9 (51.15), between the proton H-6 (δH 6.24) with C-5 (δC 82.16), H-8 (δC 79.44), and C-4 (δC 36.97), between the proton H-22 (δH 5.24) with carbons C-20 (δC 39.71), C-24 (δC 42.86) and between the proton H-23 (δH 5.17) with carbons C- 20 (δC 39.71), C-24 (δC 42.86). Based on the above analysis and comparison with published data [11,12], compound 3 was thus identified as ergosterol peroxide (5α,8α-epidioxyergosta-6,22- dien-3β-ol). HO OH O 1 3 4 5 810 12 13 15 16 17 18 20 21 22 23 24 25 26 28 lupeol (1) Epialeuritolic acid (2) Ergosterol peroxide (3) ricinine (4) Figure 1. Chemical structures of compounds 1 - 4. Compound 4 was isolated as colorless needles with melting point 193 oC. The ESI-MS of 4 provided an molecular ion peak at m/z: 164 [M]+, corresponding to the formula C8H8N2O2. The 13C-NMR and DEPT spectra of 4 exhibited 8 carbon signals, including 2 methyl, 2 methine, and 4 quartenary carbons. In the 13C-NMR spectrum, two tertiary carbon signals belong to carbonyl group at δC 174.72 and 163.76, in addition to the two methine carbon signals at δC 146.77 and 95.69. The 1H-NMR spectrum showed aromatic protons signals at δH 7.98 (1H, d, J = 7.5 Hz, H- 6) and 6.46 (1H, d, J = 7.5 Hz, H-5) with corresponding to carbons at δC 146.77 (C-6) and 95.69 (C-5). In addition, the 1H-NMR spectrum confirmed the presence of methoxy protons singlet at δH 4.06 (3H, s) with carbon at δC 58.19 in the HSQC spectra. Analysis of the NMR data of 4 and comparing with reference led to the confirmed structure of compound 4 to be ricinine [13]. Tran Thị Hoai Van, Luan Thi Thu, Do Tien Lam 528 4. CONCLUSION Repeated column chromatographies of an ethyl acetate extract of the Ricinus communis, collected in Melinh - Hanoi, four compounds were isolated. Their chemical structures were elucidated as lupeol (1), epialeuritolic acid (2), ergosterol peroxide (3), and ricinine (4) by combined analyses of their NMR spectroscopic data (including 1H, 13C, DEPT, 2D, and ESI-MS) and the comparison with literature data was well. This is the first time that these compounds have been isolated from the Ricinus communis plant, excepted ricinine. Acknowlegments. This research was supported by research project granted by Ministry of Agriculture & Rural Development (Code: 04/HĐ-KHCN). REFERENCES 1. Vo Văn Chi. - Vietnamese Medical Plants Dictionary, Medicine Publishing House – HCM, Vol II, 2012, tr. 855. 2. Pham Hoang Ho. - Viet Nam plants, HCM Publishing House, II, 2003, tr. 267. 3. 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Contribution to results of the chemical constituents of Ricinus communis 529 TÓM TẮT ĐÓNG GÓP VÀO KẾT QUẢ NGHIÊN CỨU THÀNH PHẦN HÓA HỌC CÂY THẦU DẦU (RICINUS COMMUNIS) Trần Thị Hoài Vân1, 2, Luân Thị Thu1, Đỗ Tiến Lâm1, Cầm Thị Ính1, Lành Thị Ngọc2, Nguyễn Văn Tuyến Anh1, Phạm Thị Hồng Minh1, *, Phạm Quốc Long1 1Viện Hóa học các Hợp chất thiên nhiên, VAST, 18 Hoàng Quốc Việt, Cầu Giấy, Hà Nội 2Học viện Y dược học cổ truyền Việt Nam, Số 2, Trần Phú, Hà Đông, Hà Nội 3Trường Đại học Nông lâm Thái Nguyên, Xã Quyết Thắng, Thành phố Thái Nguyên *Email: minhhcsh@gmail.com Từ các dịch chiết n-hexane và ethyl acetate cây Thầu dầu (Ricinus communis) ở Mê Linh- Hà Nội, kết hợp sử dụng các phương pháp sắc kí đã phân lập được bốn hợp chất lupeol (1), axit epialeuritolic (2), ergosterol peroxid (3), và ricinine (4). Cấu trúc của các hợp chất này được xác định bằng các phương pháp phổ NMR với kỹ thuật một chiều, hai chiều và kết hợp so sánh với những tài liệu đã công bố. Từ khóa: thầu dầu, Ricinus communis, tritecpen, ricinine.