Nội dung

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2014, Vol. 59, No. 9, pp. 37-42 This paper is available online at http://stdb.hnue.edu.vn CHEMICAL CONSTITUENTS OF Stemona tuberosa PLANT Pham Thi Huyen1,3 , Pham Van Cong1,2 , Bui Thi Lua1 , Lam Thi Hai Yen1 , Dang Ngoc Quang1 and Pham Huu Dien1 1 Faculty of Chemistry, Hanoi National University of Education Faculty of Bio-Chemistry, Tay Bac University, Son La Province 3 Hai Hau B High School, Hai Phu Commune, Nam Dinh Province 2 Abstract. Two alkaloids, neotuberostemonine and bisdehydrotuberostemonine, and two non-alkaloidal metabolites, methyl trans-4-hydroxycinnamate and 5-(hydroxymethyl)furfural, were isolated from the roots of Stemona tuberosa plant, collected in Buondon Commune (Daklak Province). Their structures were elucidated using various spectroscopic analyses. Keywords: Stemona tuberosa, neotuberostemonine, bisdehydrotuberostemonine, methyl trans-4-hydroxycinnamate, 5-(hydroxymethyl)furfural. 1. Introduction Stemona plants, which are popularly called “Bach Bo” in Vietnamese, have been traditionally used as an anticough remedy and for their antiparasitic properties. Extracts made from the tuberous roots of S. tuberosa Lour., S. saxorum Gagnep. and S. cochinchinensis have been used in Asian traditional medicine in a broad range of applications, including cough and asthma relief and to limit enteric helminths and ectoparasites in humans and cattle [1, 8, 10, 11]. Chemical investigations on Stemona genus have isolated more than 90 different alkaloids, most of which share a common pyrrolo[1, 2-a]azepine basic nucleus while a minority contain a pyrido[1, 2-a]azepine skeleton [2, 4, 6, 9]. However, the non-alkaloids components of this genus have rarely been investigated with fewer than 20 compounds, mostly, dihydrophenanthrenes and dihydrostibenes [3, 12] being identified. Herein, we describe the isolation and structure of four metabolites of Stemona tuberosa Lour. That was growing in Buondon Commune (Daklak Province). Received November 1, 2014. Accepted December 2, 2014. Contact Pham Huu Dien, e-mail address: dienph@hnue.edu.vn 37 Pham Thi Huyen, Pham Van Cong, Bui Thi Lua, Lam Thi Hai Yen, Dang Ngoc Quang and Pham Huu Dien 2. Content 2.1. Experiment * General experimental procedures: IR spectra were recorded on a Shimadzu-FTIR 8101M spectrophotometer, using a KBr disk, and NMR (1 H, 13 C-NMR, DEPT, HSQC, HMBC) spectra were recorded on a Bruker Avance 500 MHz. The chemical shift (δ) values are given in ppm with TMS as the internal standard and the coupling constant J (in Hz). ESI-LC-MS spectra were recorded on an Agilent LC mass spectrometer. Silica gel (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, 302 and 366 nm and visualized by spraying with a vanillin-10% H2 SO4 solution. High-Performance Liquid Chromatography (HPLC) was performed on a Jasco PU-2087 instrument with UV-2070 and RI-2031 detectors. Chromatographic separation was carried out on a Waters 5 SL-II column (10.0 × 250 mm) using a gradient solvent system of n-hexane (A) and ethyl acetate (B) at a flow rate of 1.0 mL/min. * Plant material: The roots of S. tuberosa (1.0 kg) were collected in Buondon Commune (Daklak Province) in October, 2012. The plant material was identified by MSc. Nguyen The Anh, Institute of Chemistry, Vietnamese Academy of Science and Technology, Vietnam. * Extraction and isolation: Air-dried roots of S. tuberosa (1.0 kg) were ground into a powder and extracted with 80% MeOH (5L × 3) at room temperature to give a crude extract (125.5 g) which was subjected to column chromatography over Silica gel and eluted gradient with petroleum ether-acetone from 9:1 to 1:1 and CH2 Cl2 -methanol from 10:1 to 0:10. Eight fractions were successively obtained. From fraction 2 (1.05g) were precipitated white crystals, recrystallized to afford 1 (125 mg). Fraction 5 (610 mg) was separated by column chromatography (SiO2 ; CH2 Cl2 ) to afford 3 (20 mg). Fraction 4 (230 mg) was separated by HPLC prep. HPLC A/B 33 - 50% over 28.74 min to afford 2 (9 mg) and over 30.87 min to afford 4 (12 mg). Compound 1: Colorless crystals; mp. 160.5 - 162 ◦ C. ESI-LC-MS (m/z): 374.2 [M-H]-; IR (KBr, v cm−1 ): 2942, 1759 (C=O), 1456, 1384, 1167. 1 H-NMR (500MHz, CDCl3 , TMS) and 13 C-NMR (125 MHz, CDCl3 , TMS) (see Table 1). Compound 2: Colorless crystals; mp. 172 - 173.5 ◦ C. ESI-LC-MS (m/z): 370.4 [M-H]-. 1 H-NMR (500 MHz, CDCl3 , TMS) and 13 C-NMR (125 MHz, CDCl3 , TMS) (see Table 1). Compound 3: Colorless viscous oil. IR (KBr, v cm−1 ): 3358 (OH), 2942, 1703 (C=O), 1602, 1513, 1446, 1270, 1172. 1 H-NMR (500MHz, CDCl3 , TMS): 7.61 (1H, d, J = 16.0Hz, H-3), 7.47 (2H, d, J = 8.5Hz, H-2’, 6’), 6.83 (2H, d, J = 8.5Hz, H-3’, 5’), 6.34 (1H, d, J = 16.0Hz, H-2), 3.92 (3H, s, OCH3 ). 13 C-NMR (125 MHz, CDCl3 , TMS): 169.7 38 Chemical constituents of Stemona tuberosa plant (C-1), 161.3 (C-4’), 146.5 (C-3), 131.1 (C-2’, 6’), 127.1 (C-1’), 116.8 (C-3’, 5’), 114.9 (C-2), 52.0 (OCH3 ). Compound 4: Yellowish oil. 1 H-NMR (500MHz, CDCl3 , TMS): 9.61 (1H, s, 2-CHO), 7.22 (1H, d, J = 3.5Hz, H-3), 6.52 (1H, d, J = 3.5Hz, H-4), 4.73 (2H, s, 5-CH2 OH). 13 C-NMR (125 MHz, CDCl3 , TMS): 177.6 (2-CHO), 160.4 (C-5), 152.5 (C-2), 122.4 (C-3), 110 (C-4), 57.7 (5-CH2 OH). 2.2. Results and discussion Compound 1 was obtained as colorless crystals, mp. 160.5 - 162◦ C. Its molecular formula was deduced to be C22 H33 NO4 on the basis of the quasi-molecular ion [M-H]peak at m/z 374.2 in the LC-MS spectrum, with seven degrees of unsaturation. Its IR spectrum (KBr) showed the presence of a γ-lactone ring (1759 cm−1 ). The 1 H-NMR of 1 in CDCl3 showed a triplet (3H) at δH 0.96 for the C-17 methyl group, two doublets (3H each) at δH 1.08 and 1.10 for a secondary C-15 and C-22 methyl group. These signals indicated that 1 was a tuberostemonine type of alkaloid. The 13 C-NMR and HSQC spectra of 1 presented 22 carbon signals: three methyl carbons, seven methylene carbons, ten methine carbons and two quaternary carbons (Table 1). Of these, two quarternary carbons at δC 178.9 and 179.3 were assigned to two γ-lactone ring carboxyl carbons. From the above analyses of the NMR, MS spectra and melting point of 1, and making a comparison with those in [7], we concluded that 1 is neotuberostemonine, one of the most commonly found alkaloids in Stemona plants. Compound 2 was obtained as colorless crystals, mp.172 - 173.5 ◦ C. Its molecular formula was deduced to be C22 H29 NO4 on the basic of the quasi-molecular ion [M-H]peak at m/z 370.4 in the LC-MS spectrum, with nine degrees of unsaturation. The 1 H-NMR of 2 in CDCl3 was similar to that of 1 with a triplet (3H) at δH 1.06 for the C-17 methyl group, two doublets (3H each) at δH 1.30 and 1.35 for a secondary C-15 and C-22 methyl group. One singlet for H-2 at δH 5.93 and the absence of an H-1 and H-9a proton in comparison with those of 1 reveals the presence of two double bonds in ring A (small ring) of the perhydroazaazulene-core ring. The 13 C-NMR and HSQC spectra of 2 presented 22 carbon signals: three methyl carbons, six methylene carbons, eight methine carbons and five quaternary carbons (Table 1). Among them, two quarternary carbons at δC 178.7 and 178.8 were assigned to two γ-lactone ring carboxyl carbons; three quarternary carbons at δC 107.1, 127.5, 137.5 and one methine carbon at δC 1108.6 were assigned to ring A of the perhydroazaazulene-core ring. From the above analyses of the NMR, MS spectra and melting point of 2, and making a comparison with those in [13], we concluded that 2 is bisdehydroneotuberostemonine, the second most common alkaloid found in Stemona plants. 39 Pham Thi Huyen, Pham Van Cong, Bui Thi Lua, Lam Thi Hai Yen, Dang Ngoc Quang and Pham Huu Dien No 1 2 3 5 6 7 8 9 9a 10 11 12 13 14 15 16 17 18 19 20 21 22 Table 1. 1 H-NMR and 13 C-NMR spectral data of 1 and 2 Compound 1 Compound 2 1 13 1 13 H-NMR C-NMR H-NMR C-NMR (CDCl3 , J Hz) (CDCl3 ) (CDCl3 , J Hz) (CDCl3 ) 1.72 m 36.8 107.1 1.58 m (2H) 32.3 5.93 s 108.6 3.11 dd 7.0 14.0 66.3 137.5 4.21 dd 6.0 3.05 m; 2.81 m 49.6 44.8 14.5;3.70 dd 14.0 11.0 1.63 m (2H) 29.9 2.00 m ; 1.30 m 28.6 1.45 m; 1.57 m 22.8 2.03 m; 1.52 m 29.1 1.54 m; 1.80 m 28.9 1.18 m; 1.30 m 35.0 1.62 m; 1.53m 35.8 2.70 m 36.0 3.23 q 8.0 65.8 127.5 1.65 m 34.6 1.99 m 34.8 4.40 m 80.4 4.70 dd 2.0 4.5 80.8 2.07 m 40.8 3.45 brt 5.0 39.5 2.32 dd 8.0 13.5 41.6 2.97 m 41.9 178.8 178.7 1.08 d 5.0 9.9 1.30 d 7.0 15.0 1.18 m; 1.60 m 20.9 1.75-1.85 m 23.2 0.96 t 7.0 11.0 1.06 t 6.5 11.4 4.56 t 3.0 78.1 5.53 dd 5.0 8.0 71.7 2.32 m; 1.45 m 33.9 2.70 m; 2.15 m 30.9 2.66 m 34.1 2.81 m 44.8 179.2 178.8 1.10 d 5.0 14.5 1.35 d 7.0 11.8 Neotuberostemonine (1) 40 Bisdehydrotuberostemonine (2) Chemical constituents of Stemona tuberosa plant Compound 3 was obtained as colorless viscous oil. Its molecular formula was deduced to be C10 H10 O3 on the basic of the 1 H- NMR and 13 C-NMR spectral data. Its IR spectrum (KBr) showed the presence of a hydroxyl (3358 cm−1 ), carboxylate (1703 cm−1 ) groups and a double C=C bond (1513, 1446 cm−1 ). The 13 C-NMR and HSQC spectra of 3 presented 10 carbon signals: one methoxy carbon, six methine carbons and three quaternary carbons, including a carboxyl carbon at δC 169.7. The 1 H-NMR spectrum of 3 displayed the characteristic signals for four symmetric aromatic protons at δH 7.13 (2H, d, J = 16.0 Hz), 7.47 (2H, d, J = 8.5 Hz), for three methoxy protons at δH 3.92 (3H, s) and one phenolic hydroxyl proton at δH 4.87 (1H, s). From the above analyses of the IR and NMR data, we concluded that 3 is methyl trans-4-hydroxycinnamate, a substance isolated from Stemona cochinchinensis in 2012 [7]. Compound 4 was obtained as a yellowish oil. Its molecular formula was deduced to be C6 H6 O3 on the basic of the 1 H-NMR and 13 C-NMR spectral data. The 13 C-NMR and HSQC spectra of 4 presented 6 carbon signals: one hydroxymethylene carbon (δC 57.7), four aromatic carbons (δC 110.0 122.4 152.5 and 160.4) and one aldehyde carbon (δC 177.6). The 1 H-NMR spectrum of 4 displayed the characteristic signals for four two aromatic protons in ortho-composition at δH 6.52 (1H, d, J = 3.5Hz), 7.22 (1H, d, J = 3.5 Hz), two methylene protons at δH 4.73 (2H, s) and one aldehyde proton at δH 9.61 (1H, s). From above analyses of the NMR data and in making comparison with those of [5], we concluded that 4 is 5-(hydroxymethyl)furfural. Methyl trans-4-hydroxycinnamate (3) 5-(hydroxymethyl)furfural (4) 3. Conclusion From roots of Stemona tuberosa plants that were collected in Buondon Commune (Daklak Province) in October 2012, we isolated four compounds. By various spectral methods, these compounds were elucidated as neotuberostemonine, bisdehydrotuberostemonine, methyl trans-4-hydroxycinnamate and 5-(hydroxymethyl)furfural. Acknowledgement: This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED), Grant No. 104.01-2012.14. 41 Pham Thi Huyen, Pham Van Cong, Bui Thi Lua, Lam Thi Hai Yen, Dang Ngoc Quang and Pham Huu Dien REFERENCES [1] Vo Van Chi, 2001. A dictionary of Traditional Vietnamese Medicinal plants. Bio-pharmaceutical Publishing House, Hanoi, p. 162. [2] Pham Huu Dien, Phan Van Kiem, Luu Van Chinh, Chau Van Minh, 2000. Alkaloid was isolated from roots of "Bach Bo" Stemona tuberosa Lour. (Stemonaceae). Part I. Journal of Chemistry, No. 38, No. 1, pp. 64-67 (in Vietnamese). [3] P. H. Dien, L. G. Lin, C. P. Tang, C. Q. Ke, Y. Ye, 2008. Bisbenzopyrans and alkaloids from the roots of Stemona cochinchinensis. Nat. Prod. Res., 22, No. 10, pp. 915-920. [4] H. Greger, 2006. Structural relationships, distribution and biological activities of Stemona alkaloids. Planta medica, 72, pp. 99-113. [5] Suqin Hu, Zhaofu Zhang, Jinliang Song, Yinxi Zhou and Buxing Han, 2009. Efficient conversion of glucose into 5-(hydroxymethyl)furfural catalyzed by a common Lewis acid SnCl4 in an ionic liquid. Green Chem., No. 11, pp. 1746-1749. [6] E. Kaltenegger, B. Brem, K. Mereiter, H. Kalchhauser, H. Kahlig, O. Hofer, S. Vajirodaya, H. Greger, 2003. Insecticidal pyrido[1,2-a]azepine alkaloids and related derivatives from Stemona specie. Phytochemistry, 63, No. 7, pp. 803-816. [7] Vonganatha Khamko, Dang Ngoc Quang, Pham Huu Dien, 2012. A cytotoxic stilbenoid and 4-hydroxycinnamates from Stemona cochinchinensis plants, growing in Savannakhet province, Laos. J. of Sciences of HNUE, Vol. 57, No. 7, pp. 3-9. [8] Do Tat Loi, 2001. Vietnamese Traditional Medicinal Plants and Drugs, 3rd. Ed. Publishing House of Medicine, Hanoi, p. 180. [9] P. Mungkornasawakul, S. G. Pyne, A. Jatisatienr, D. Supyen, C. Jatisatienr, W. Lie, A. T. Ung, B. W. Skelton, A. H. White, 2004. Phytochemical and larvicidal studies on Stemona curtisii:Structure of a new pyrido[1,2-a]azepine Stemona alkaloid. J. Nat. Prod., 67, pp. 675- 679. [10] R. A. Pili, M. C. Oliveira, 2000. Recent progress in the chemmistry of Stemona alkaloids. Nat. Prod. Rep., 17, No. 1, pp. 117-127. [11] R. S. Xu, 2000. Studies in Nat. Products Chemistry. Elsevier Press, Amsterdam, 21, pp. 729-742. [12] X. Z. Yang, C. P. Tang, C. Q. Ke, Y. Ye, 2007. Stibenoids from Stemona sessilifolia. J. of Asian Nat. Prod. Res., Vol. 9, No. 3, pp. 261- 266. [13] Yang Ye, Guo-Wei Qin, Ren-Sheng Xu, 1994. Alkaloids from Stemona tuberosa. Phytochemistry, Vol. 37, No. 4, pp. 1201-1203. 42