DOI 10.1007/s10600-015-1236-5 Chemistry of Natural Compounds, Vol. 51, No. 1, January, 2015
TRITERPENOIDS AND STEROIDS FROM THE LEAVES OF Forsythia suspensa
Qing Zhang,1,2 Zhen Lu,2 Xing Li,3 Yuan Zheng,1 Donggui Yao,1 Yucheng Gu,1,4 Changhong Huo,1* and Bin Cong1*
Forsythia suspensa (Thunb.) Vahl is a deciduous shrub of the Oleaceae family, which is widely distributed in China, North and South Korea, and Japan. Its fruits, “Lian qiao”, have been used for treating conditions such as gonorrhea, erysipelas, inflammation, pharyngitis, pyrexia, tonsillitis, and ulcers in Chinese and Japanese medicine [1]. The immature leaves of this plant have been used as a health tea by residents of longevity villages in Wuan, Hebei Province and Pingding, Shanxi Province in China [2]. In recent years, studies have shown that the F. suspensa leaf extract has a variety of pharmacological activities, such as antioxidant, hepatoprotective, anti-aging, and cardioprotective [3]. However, its chemical constituents have not been thoroughly investigated, and only (+)-pinoresinol, (–)-pinoresinol, and phillygenin (lignans), forsythiaside (phenylpropanoid), quercetin and rutin (flavonoids), and ursolic acid (triterpenoid) have been reported [4–6]. The leaves of F. suspensa were collected in October 2009 from the Dongyangpo production base, Jingxing County, Hebei Province, and identified by Prof. Jianhua Wang, Pharmacognosy Laboratory, School of Pharmaceutical Sciences, Hebei Medical University. Chemical investigation of the ethanol extract of Forsythia suspensa led to the isolation of ten compounds (1–10). Dried F. suspensa leaves (4.4 kg) were extracted with 95% ethanol at room temperature. After evaporation of the solvent under reduced pressure, the residue was suspended in water and extracted with petroleum ether, CH2Cl2, EtOAc, and n-BuOH, successively. The CH2Cl2 fraction (288.6 g) was subjected to silica gel column chromatography, eluting with petroleum ether–CH3COCH3 (10:1o1:1). Fraction 1 afforded 1 (33.3 mg). Fraction 9 was subjected to repeated chromatography to obtain 5 (20.0 mg). Fraction 10 was chromatographed on silica gel, eluting with CH2Cl2–CH3COCH3 (80:1o1:1) to yield 6 (30.1 mg), 3 (8.9 mg), and 8 (306.5 mg). The petroleum ether fraction (194.1 g) was subjected to silica gel column chromatography, eluting with CH2Cl2–EtOAc (50:1o1:1). Fraction 1 was subjected to a silica gel column, eluting with petroleum ether–CH3 COCH3 (10:1o2:1) to afford 4 (15.5 mg), 9 (3.1 mg), and 10 (231.6 mg). Fraction 2 afforded 7 (10.3 mg) and fraction 5 afforded 2 (7.8 mg). Their chemical structures were identified by NMR spectroscopy and were determined to be 3E-acetoxy-11-en-olean28,13-olide (1) [7], 3E -acetoxyolean-12-en-28-oic acid (2) [8], 3 E-hydroxy-11-oxo-olean-12-en-28-oic acid (3) [9], 3 E-acetoxyurs-12-en-28-oic acid (4) [10], 2D,3D-hydroxyursolic acid (5) [11], ursolic acid (6) [12], betulin (7) [13], alphitolic acid (8) [7], stigmast-4-en-3-one (9) [14], and E-sitosterol (10) [15] by comparing the data with those in the literature. Compounds 8 and 10 are reported in the leaves of this plant for the first time; the other compounds except 6 were isolated from the Forsythia genus for the first time. As some of the 1H and 13C NMR data had been incorrectly assigned for 3E-acetoxyolean12-en-28-oic acid (2) and alphitolic acid (8) due to NMR limitations, in particular no access to 2D NMR, in the original reported papers, all the 1H and 13C NMR spectral data of these two compounds were reassigned after interpretation of their 2D NMR spectroscopic data.
1) School of Pharmaceutical Sciences, Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, 361 Zhongshan East Road, 050011, Shijiazhuang, Hebei Province, P. R. China, e-mail:
[email protected]; 2) Department of Pharmacy, Huangshi Central Hospital, 435000, Huangshi, Hubei Province, P. R. China; 3) CSPC-NBP Pharmaceutical Co., Ltd, 88 Yangzi Road, Shijiazhuang Economic and Technological Development Zone, 052160, Hebei Province, P. R. China; 4) Syngenta Jealottcs Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom. Published in Khimiya Prirodnykh Soedinenii, No. 1, January–February, 2015, pp. 154–155. Original article submitted March 25, 2013. 178
0009-3130/15/5101-0178 2015 Springer Science+Business Media New York
3E-Acetoxyolean-11-en-28,13-olide (1). White crystals (CH2Cl2–EtOAc). 1H NMR (500 MHz, CDCl3, G, ppm, J/Hz): 6.03 (1H, d, J = 10.5, H-12), 5.42 (1H, dd, J = 10.5, 3.0, H-11), 4.49 (1H, dd, J = 11.0, 5.5, H-3), 2.14 (1H, dd, J = 13.0, 6.0, H-18), 2.06 (3H, s, 3-COCH3), 1.91 (1H, d, J = 3.0, H-9), 1.06 (6H, s, 2 u CH 3), 0.98, 0.94, 0.88, 0.87, 0.86 (each 3H, s, 5 u CH3), 0.83 (1H, dd, J = 12.0, 3.0, H-5). 13C NMR (125 MHz, CDCl3, G, ppm): 180.0 (C-28), 171.0 (COCH3), 135.7 (C-11), 127.0 (C-12), 89.8 (C-13), 80.6 (C-3), 54.8 (C-5), 53.1 (C-9), 50.5 (C-18), 44.0 (C-17), 41.6 (C-14), 41.4 (C-8), 37.9 (C-1), 37.8 (C-4), 37.3 (C-19), 36.2 (C-10), 34.3 (C-7), 33.3 (C-29), 31.4 (C-20), 31.1 (C-21), 27.7 (C-23), 27.1 (C-22), 25.4 (C-15), 23.6 (C-30), 23.3 (C-2), 21.3 (C-16), 21.3 (COCH3), 19.0 (C-27), 18.3 (C-26), 18.0 (C-25), 17.5 (C-6), 16.0 (C-24). 3E-Acetoxyolean-12-en-28-oic Acid (2). White crystals (petroleum ether–EtOAc). 1H NMR (500 MHz, CDCl3, G, ppm, J/Hz): 5.28 (1H, t, J = 3.5, H-12), 4.50 (1H, t, J = 8.0, H-3), 2.82 (1H, dd, J = 14.0, 4.0, H-18), 2.05 (3H, s, 3-COCH3), 1.99 (1H, m, H-16a), 1.91 (2H, m, H-11a, b), 1.78 (1H, m, H-22a), 1.70 (1H, m, H-15a), 1.62 (3H, m, H-2a, 16b, 19a), 1.61 (1H, m, H-1a), 1.58 (1H, m, H-22b), 1.55 (1H, m, H-9), 1.53 (1H, m, H-6a), 1.42 (1H, m, H-7a), 1.37 (2H, m, H-21a, 6b), 1.29 (1H, m, H-7b), 1.22 (1H, m, H-21b), 1.17 (1H, m, H-19b), 1.13 (3H, s, 27-CH3), 1.06 (2H, m, H-2b, 15b), 1.04 (1H, m, H-1b), 0.94 (3H, s, 25-CH3), 0.93 (3H, s, 30-CH3), 0.91 (3H, s, 29-CH3), 0.87 (3H, s, 24-CH3 ), 0.85 (3H, s, 23-CH3), 0.84 (1H, m, H-5), 0.75 (3H, s, 26-CH3). 3E-Hydroxy-11-oxo-olean-12-en-28-oic Acid (3). White crystals (CH2Cl2–CH3COCH3). 1H NMR (500 MHz, CDCl3, G, ppm, J/Hz): 5.63 (1H, s, H-12), 3.22 (1H, dd, J = 11.0, 5.5, H-3), 2.98 (1H, dd, J = 14.0, 4.0, H-18), 1.36 (3H, s, 27-CH3), 1.01 (3H, s, 23-CH3), 0.95 (3H, s, 30-CH3), 0.94 (3H, s, 29-CH3), 0.93 (3H, s, 26-CH3 ), 0.92 (3H, s, 25-CH3), 0.78 (3H, s, 24-CH3). 13C NMR (125 MHz, CDCl3, G, ppm): 200.4 (C-11), 181.7 (C-28), 168.4 (C-13), 128.1 (C-12), 78.8 (C-3), 61.8 (C-9), 55.0 (C-5), 46.0 (C-17), 45.0 (C-14), 44.1 (C-19), 43.4 (C-8), 41.4 (C-18), 39.13 (C-4), 39.12 (C-1), 37.3 (C-10), 33.6 (C-21), 32.88 (C-29), 32.85 (C-7), 31.6 (C-22), 30.7 (C-20), 28.1 (C-23), 27.8 (C-15), 27.3 (C-2), 23.6 (C-27), 23.4 (C-30), 22.7 (C-16), 19.2 (C-26), 17.4 (C-6), 16.3 (C-25), 15.6 (C-24). 3E-Acetoxyurs-12-en-28-oic Acid (4). White crystals (petroleum ether–CH3COCH3). 1H NMR (500 MHz, CDCl3, G, ppm, J/Hz): 5.23 (1H, t, J = 3.5, H-12), 4.50 (1H, dd, J = 10.0, 6.0, H-3), 2.18 (1H, d, J = 11.0, H-18), 2.05 (3H, s, COCH3), 1.07 (3H, s, 25-CH3), 0.96 (3H, s, 27-CH3), 0.95 (3H, d, J = 6.0, 30-CH3), 0.87 (3H, s, 23-CH3), 0.86 (3H, d, J = 3.0, 29-CH3), 0.85 (3H, s, 24-CH3), 0.77 (3H, s, 26-CH3). 2D ,3D-Dihydroxyursolic Acid (5). White crystals (CH2 Cl2 –CH3COCH3). 1 H NMR (500 MHz, CDCl3 , G, ppm, J/Hz): 5.26 (1H, t, J = 4.0, H-12), 4.00 (1H, dt, J = 11.5, 4.0, H-2), 3.43 (1H, d, J = 3.0, H-3), 2.19 (1H, d, J = 11.0, H-18), 1.92 (1H, dd, J = 9.0, 3.5, H-11), 1.09, 1.02, 0.97, 0.85, 0.78 (each 3H, s, 23, 24, 25, 26, 27-CH3), 0.94 (3H, d, J = 6.5, 30-CH3), 0.86 (3H, d, J = 3.0, 29-CH3). Ursolic Acid (6). White crystals (CH2Cl2–CH3COCH3) >12@. Betulin (7). White crystals (petroleum ether–EtOAc) >13@. Alphitolic Acid (8). White crystals (CH3OH). 1H NMR (500 MHz, DMSO-d6, G, ppm, J/Hz): 4.69 (1H, s, H-29a), 4.57 (1H, s, H-29b), 3.40 (1H, m, H-2), 2.95 (1H, m, H-19), 2.72 (1H, d, J = 9.5, H-3), 2.22 (1H, m, H-13), 2.12 (1H, br.d, J = 9.5, H-16a), 1.81 (3H, m, H-1a, 21a, 22a), 1.65 (3H, s, H-30), 1.63 (1H, m, H-12a), 1.52 (1H, t, J = 11.5, H-18), 1.42 (2H, m, H-6a, 22b), 1.40 (1H, m, 15a), 1.37 (2H, m, H-11a, 16b), 1.32 (4H, m, H-6b, 7a, b, 21b), 1.28 (1H, m, H-9), 1.18 (1H, m, H-11b), 1.09 (1H, m, H-15b), 0.98 (1H, m, H-12b), 0.93 (3H, s, 27-CH3), 0.90 (3H, s, 23-CH3), 0.86 (3H, s, 26-CH3), 0.82 (3H, s, 25-CH3), 0.71 (2H, m, H-1b, H-5), 0.68 (3H, s, 24-CH3). 13C NMR (125 MHz, DMSO-d6, G, ppm): 177.2 (C-28), 150.3 (C-20), 109.6 (C-29), 82.2 (C-3), 67.3 (C-2), 55.4 (C-17), 54.8 (C-5), 49.8 (C-9), 48.5 (C-18), 47.1 (C-1), 46.6 (C-19), 42.0 (C-14), 40.3 (C-8), 38.9 (C-4), 37.8 (C-10), 37.6 (C-13), 36.3 (C-22), 33.8 (C-7), 31.7 (C-16), 30.1 (C-21), 29.2 (C-15), 28.6 (C-23), 25.0 (C-12), 20.6 (C-11), 19.0 (C-30), 18.0 (C-6), 17.1 (C-25), 16.9 (C-24), 15.7 (C-26), 14.4 (C-27). Stigmast-4-en-3-one (9). White crystals (petroleum ether–EtOAc) >14@. E-Sitosterol (10). White crystals (petroleum ether–CH3COCH3) >15@.
ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (81072551, 81241101). We also wish to extend our sincere thanks for the financial support of Syngenta Ltd. (2011-Hebei Medical University-Syngenta-03).
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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Q. Zhang, C. H. Jia, H. Y. Xu, Y. F. Wang, M. L. Zhang, C. H. Huo, Q. W. Shi, and S. H. Yu, Mini-Rev. Org. Chem., 9, 303 (2012). Y. Wang, R. B. Wang, L. Sun, B. H Wang, Y. X. Ding, L. Q. Xu, and S. Tian, Zhongguo Shiyan Fangjixue Zazhi, 17, 109 (2011). H. L. Xue and W. J. Wang, Shizhen Guoyiguoyao, 20, 1149 (2009). S. Kitagawa, S. Nishibe, R. Benecke, and H. Thieme, Chem. Pharm. Bull., 36, 3667 (1988). W. Y. Kang, J. M. Wang, and L. Zhang, Zhongguo Zhongyao Zazhi, 35, 1156 (2010). H. Jing, J. S. Li, and Y. J. Yang, Zhongyaocai, 33, 1487 (2010). M. Ma, S. J. Wang, S. Li, Y. C. Yang, J. G. Shi, F. Ye, and J. Y. Tian, Zhongcaoyao, 37, 1128 (2006). A. Ikuta and H. Itokawa, J. Nat. Prod., 52, 623 (1989). S. Werner, S. Nebojsa, W. Robert, S. Robert, and K. Olaf, Magn. Reson. Chem., 41, 636 (2003). Y. P. Jin, G. G. Zhang, H. T. Zheng, Q. Shang, J. Oo-yang, and S. S. Du, Zhongnan Yaoxue, 6, 43 (2008). M. Katai, T. Terai and H. Meguri, Chem. Pharm. Bull., 31, 1567 (1983). G. S. Zhou, X. Yao, Y. P. Tang, S. Guo, N. Y. Yang, C. Jin, Y. Qin, D. W. Qian, and J. A. Duan, Zhongguo Yaoxue Zazhi, 47, 1362 (2012). J. M. Sun and J. S. Yang, Chin Pharm J, 42, 489 (2007). Y. X. Guan, X. S. Yang, L. H. Tong, B. Yang, and X. J. Hao, Zhongcaoyao, 38, 342 (2007). H. Kojima, N. Sato, A. Hatano, and H. Ogura, Phytochemistry, 29, 2351 (1990).
