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Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus (Ranunculaceae)
Biochemical Systematics and Ecology 38 (2010) 759–763
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus (Ranunculaceae) Feng-Ying Yang a, Yan-Fang Su a, b, *, Ying Wang a, Xin Chai a, Xu Han a, Zhen-Hai Wu c, Xiu-Mei Gao b a
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, PR China Tianjin Key Laboratory of TCM Chemistry and Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China c College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 November 2009 Accepted 10 July 2010
Two new bufadienolide glycosides with an A/B trans ring structure, 14b,16b-dihydroxy-3b(b-D-glucopyranosyloxy)-5a-bufa-20,22-dienolide (1), and 14b,16b-dihydroxy-3b-[b-Dglucopyranosyl-(1/4)-(b-D-glucopyranosyloxy)]-5a-bufa-20,22-dienolide (2), two known ecdysteroids (polypodine B and 20-hydroxyecdysone) (3-4), and six known bufadienolide and its glycosides with 5b-OH (hellebrigenin, 16b-hydroxyhellebrigenin-3-O-a-L-rhamnoside, hellebrigenin 3-O-b-D-glucoside, hellebrin, 16b-hydroxyhellebrigenin-3-O-b-D-glucoside, and deglucohellebrin) (5-10) were isolated from the rhizomes of Helleborus thibetanus. The structures of compounds 1 and 2 were elucidated using various spectroscopic methods. All compounds were reported for the first time from the title plant and their chemotaxonomic significance for the genus Helleborus was discussed. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Ranunculaceae Helleborus Helleborus thibetanus Bufadienolides 5a-Bufadienolides Phytoecdystones
1. Subject and source The genus Helleborus (Ranunculaceae), including about 20 species, are distributed in Southeast Europe and West Asia (Wang, 1979). Helleborus thibetanus Franch is endemic to China and used for treatment of cystitis, urethritis, sores and traumatic injury in Chinese folk medicine (Guo et al., 2003). The rhizomes of H. thibetanus were collected from Mei County, Shaanxi Province in the People’s Republic of China in September 2007 and were authenticated by Prof. Zhen-Hai Wu. A voucher specimen (S200709002) is deposited in School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, People’s Republic of China. 2. Previous work A previous report indicated that the ethanol extract of H. thibetanus presented the digitalis-like cardiotonic activity (Li and Lu, 1981). The polysaccharide extract from H. thibetanus have been also studied on their anticancer and immunomodulating activities (Bao et al., 2003; Liu et al., 2005). To our knowledge, there is no previous report on the phytochemistry of H. thibetanus. 3. Present study In this study, two new 5a-bufadienolide glycosides (1 and 2, Fig. 1) were isolated from the rhizomes of H. thibetanus. The isolation and identification of eight known compounds are reported for the first time from H. thibetanus, including two known ecdysteroids (3 and 4), one known bufadienolide (5) and five known bufadienolide glycosides with 5b-OH (6–10) (Fig. 1). * Corresponding author. School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, PR China. Tel.: þ86 22 27402885; fax: þ86 22 27892025. E-mail address: [email protected] (Y.-F. Su). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.07.002
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F.-Y. Yang et al. / Biochemical Systematics and Ecology 38 (2010) 759–763
O O
24
21
22
18 19 1
9
R
17
16
OH
14 8
5
3
18
20
13
HO 5
3
HO
R
R OGlc OGlc4-Glc
20
27 23
OH 26
14
8
2
OH
OH
17
19
H 1 2
OH
21
OH 6
O 3 4
R OH H
O O 18 17
19
OHC 3
R2
16
14
R1
OH
5
OH 5 6 7 8 9 10
R1 H OH H H OH H
R2 OH ORha OGlc ORha4-Glc OGlc ORha
Fig. 1. Structures of compounds from the rhizomes of Helleborus thibetanus.
The dried rhizomes of H. thibetanus (8.0 kg) were extracted twice using 95% EtOH and then 60% EtOH respectively. The ethanol extract was evaporated to give a residue that was suspended in water and then extracted successively by petroleum ether (60–90 C), CHCl3, EtOAc and n-BuOH. The CHCl3 extract (34.7 g) was applied to silica gel column chromatography with petroleum ether–Me2CO in a gradient manner, and 76 fractions were collected. The further purification of fractions 19–23 proceeded by silica gel column with CHCl3–Me2CO and then by recrystallization with MeOH, giving compound 5 (61 mg). The EtOAc extract (44 g) was subjected to silica gel column eluting with gradient CH2Cl2–MeOH and 38 fractions were obtained. The further isolation of fractions 19–23 proceeded by silica gel column with CH2Cl2–MeOH and then by recrystallization with Me2CO, affording compounds 10 (668 mg) and 3 (115 mg). Fractions 24–28 were fractionated by silica gel column with CH2Cl2–MeOH and EtOAc–MeOH–H2O respectively and further recrystallization with Me2CO to obtain compounds 4 (894 mg) and 1 (70 mg). The n-BuOH extract (934 g) was submitted to D101 macroporous resin eluting with EtOH–H2O (0:100, 30:70, 50:50, 70:30 and 95:5). The 30% EtOH concentration (378 g) was partitioned with silica gel column eluting with EtOAc–MeOH and 126 fractions were obtained. The white power, precipitated from methanol suspension of fractions 10–25, and was further exposed to ODS column twice (40% MeOH and 37.5% MeOH) to yield compound 6 (56 mg). Fractions 26–36 were repeatedly subjected to silica gel column (CHCl3–MeOH and EtOAc–MeOH), Sephadex LH-20 (MeOH) together with ODS column (45% MeOH and 70% MeOH) to obtain compounds 7 (489 mg) and 8 (218 mg). The further purification of fractions 37–46 exposed to silica gel column (CHCl3–MeOH–H2O and EtOAc–MeOH–H2O) and then ODS column (40% MeOH and 50% MeOH), yielded compounds 9 (42 mg) and 2 (36 mg).
F.-Y. Yang et al. / Biochemical Systematics and Ecology 38 (2010) 759–763
761
On the basis of 1H, 13C, and 2D NMR, HRMS and IR spectroscopic analysis, ten compounds were elucidated respectively as two new 5a-bufadienolide glycosides including 14b,16b-dihydroxy-3b-(b-D-glucopyranosyloxy)-5a-bufa20,22-dienolide (1) (Agrawal et al., 1985; Kamano et al., 2001; Watanabe et al., 2003; Su et al., 2009) and 14b,16bdihydroxy-3b-[b-D-glucopyranosyl-(1/4)-(b-D-glucopyranosyloxy)]-5a-bufa-20,22-dienolide (2), two known ecdysteroids named polypodine B (3) (Lafont et al., 2002) and 20-hydroxyecdysone (4) (Lafont et al., 2002), one known bufadienolide as hellebrigenin (5) (Kamano et al., 2001) and five known bufadienolide glycosides as 16b-hydroxyhellebrigenin-3-O-a-Lrhamnoside (6) (Watanabe et al., 2003), hellebrigenin-3-O-b-D-glucoside (7) (Watanabe et al., 2003), hellebrin (8) (Meng et al., 2001; Su et al., 2003), 16b-hydroxyhellebrigenin-3-O-b-D-glucoside (9) (Watanabe et al., 2003) and deglucohellebrin (10) (Watanabe et al., 2003) (Fig. 1). Compound 1 was isolated as a white amorphous powder. Its 1H and 13C NMR spectroscopic data (Table 1) were similar to those of 9, which has an A/B cis, B/C trans and C/D cis ring structure with an a-pyrone ring at C-17 position (Nogawa et al., 2001; Watanabe et al., 2003). Comparison of the 1H and 13C NMR spectra of compounds 1 and 9 allowed us to observe that dH 10.45 (s) of CHO-19 in 9 was displaced by dH 0.60 (s) of CH3-19 in 1, and only one oxygenated carbon signal at d 77.1 (C-3) in the A ring of 1 instead of two oxygenated carbon signals at d 72.5 (C-3) and d 73.3 (C-5) in that of 9, suggesting one hydrogen atom linked at C-5. The angular methyl carbon signal at dC 12.1 (CH3-19) in 1 was very similar to the signal at dC 12.5 (CH3-19) in both 5a-furostan and 5a-spirostan with 5a-H (Agrawal et al., 1985; Su et al., 2009), indicating an a-configuration of H-5. Full assignments of the proton and carbon resonances of 1 were accomplished by analysis of COSY, HSQC and HMBC spectra. In the HMBC spectrum (Fig. 2), a three-bond correlation between H-10 of glucosyl (dH 5.01, d, J ¼ 7.5 Hz) and C-3 (dC 77.1) confirmed that the glucosyl group was connected to aglycone at C-3; the signal of dC 44.2, as the tertiary carbon, was assigned to C-5 by its three-bond correlation with the proton signal of CH3-19 (dH 0.60) together with comparison to the literature data (Agrawal et al., 1985; Su et al., 2009). In the NOESY spectrum (Fig. 2), the NOE correlations between CH3-19 (dH 0.60) and Hax-2 (dH 1.60)/Hax-4 (dH 1.33)/ Hax-6 (dH 1.08)/H-8 (dH 1.63)/Hax-11 (dH 1.12), and between H-3 (dH 3.97) and Hax-1 (dH 0.81)/H-5 (dH 0.82) verified the configuration of 5a-H and the A/B trans ring structure. Additionally, the molecular formula of 1 was confirmed as C30H44O10 by HRMS (m/z 563.2864 [M H]). Therefore, the structure of 1 was unambiguously identified as 14b,16b-dihydroxy-3b-(b-Dglucopyranosyloxy)-5a-bufa-20,22-dienolide. Compound 2 was obtained as a white amorphous powder. Its 1H and 13C NMR spectroscopic data (Table 1) were similar to those of 1, suggesting that 2 is a bufadienolide glycoside structurally related to 1. However, one more b-D-glucosyl was illustrated in 2 by six more carbons signals at dC 101.7, 74.6, 76.7, 81.3, 76.4 and 62.1 together with one more anomeric proton at dH 5.22 (d, J ¼ 8.0 Hz) than 1. The assignments of 2 were established by analysis of COSY, HSQC and HMBC spectra. HMBC correlation from H-10 (dH 5.01) to C-3 (dC 77.2) provided the linkage of inner b-D-glucosyl to C-3. The correlation between H-100 (dH 5.22) and C-40 (dC 81.3) illustrated the linkage of terminal b-D-glucosyl (Watanabe et al., 2003; Su et al., 2009) to the inner b-D-glucosyl at C-40 (dC 81.3) (Koto et al., 2004), which also could be deduced by a downfield shift of 9.5 ppm from dC 71.8 (C-40 of 1) to dC 81.3 (C-40 of 2). The molecular formula of 2 was unequivocally established to be C36H54O15 by HRMS (m/z 725.3387 [M H]). Thus, the structure of 2 was elucidated eventually as 14b,16b-dihydroxy-3b-[b-D-glucopyranosyl-(1/4)-(b-Dglucopyranosyloxy)]-5a-bufa-20,22-dienolide. 14b,16b-Dihydroxy-3b-(b-D-glucopyranosyloxy)-5a-bufa-20,22-dienolide (1): White amorphous power; [a]26 D 27.0 (c 1.00, C5H5N); IR (KBr) vmax: 3430 (OH), 2936 and 2857 (CH),1699 (C]O),1630,1535,1440, 1366,1220,1161,1118,1078,1022 cm1; 1H and 13C NMR data see Table 1; HRMS: m/z 563.2864 [M H], Calc. for [C30H43O10] 563.2862. 14b,16b-Dihydroxy-3b-[b-D-glucopyranosyl-(1/4)-(b-D-glucopyranosyloxy)]-5a-bufa-20,22-dienolide (2): White amor phous power; [a]19 D 31.0 (c 0.42, C5H5N); IR (KBr) vmax: 3402 (OH), 2931 and 2862 (CH), 1700 (C]O), 1629, 1536, 1445, 1372, 1318, 1221, 1155, 1077, 1033 cm1; 1H and 13C NMR data see Table 1; HRMS: m/z 725.3387 [M H], Calc. for [C30H43O10] 725.3379. 4. Chemotaxonomic significance The genus Helleborus consists of six sections: Griphopus, Chenopus, Helleborus, Syncarpus, Dicarpon and Helleborastrum. Chemotaxonomically, Helleborus genus is characterized by the presence of steroids, including bufadienolides, phytoecdystones and steroidal saponins (Kissmer and Wichtl, 1987; Colombo et al., 1990; Meng et al., 2001; Watanabe et al., 2003; Braca et al., 2004). H. thibetanus, the single species of the section of Dicarpon, had been predicted to contain high levels of phytoecdysteroids (Dinan et al., 2002). In our ongoing study, two known ecdysteroids (3 and 4) isolated from the rhizomes of H. thibetanus for the first time. 20-Hydroxyecdysone (4) is a phytoecdysteroid that is found in other species within Hellerborus (Dinan et al., 2002). It was obtained at a high level. All the isolated bufadienolides from species of Helleborus have had an A/B cis ring structure (Kissmer and Wichtl, 1987; Colombo et al., 1990; Meng et al., 2001; Watanabe et al., 2003) except for 14-hydroxy-3-oxo-5a-bufa-20,22-dienolide from H. odorus, which has an A/B trans ring structure together with 5a-H and a carbonyl group at C-3 (Hauser et al.,1972). The present work not only reported two new bufadienolide glycosides (1 and 2) with features of an A/B trans ring structure, 5a-H and glycosidation of the hydroxyl group at C-3, but also identified six known bufadienolide and its glycosides with 5b-OH (5–10). This is the first report of the occurrence of 5a-bufadienolide glycosides (1 and 2) not only in the section of Dicarpon but also in the genus of Helleborus. The contribution of this work may open opportunities to explore 5a-bufadienolide glycosides as chemotaxonomic marker for Dicarpon section.
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F.-Y. Yang et al. / Biochemical Systematics and Ecology 38 (2010) 759–763
Table 1 1 H and 13C NMR Data for Compounds 1 and 2a (C5D5N). Position
1
2
dH (ppm) 1axb 1eqc 2ax 2eq 3 4ax 4eq 5 6ax 6eq 7ax 7eq 8 9 10 11ax 11eq 12ax 12eq 13 14 15a 15b 16 17 18 19 20 21 22 23 24 Glc 10 20 30 40 50 60 a 60 b Glc 100 200 300 400 500 600 a 600 b
dC (ppm)
0.81 1.57 1.60 2.02 (m) 3.97 1.33 1.83 (m) 0.82 1.08 1.20 1.10 2.35 (m) 1.63 0.78
37.3
77.1 34.6 44.2 29.1 28.1 41.8 49.8 35.9 21.5
49.4 84.5 43.1
(dd, 7.0, 14.0) (br d, 14.0) (t-like, 7.5) (d, 8.0) (s) (s)
2.51 2.18 4.80 2.79 0.99 0.64
72.5 59.0 17.2 12.1 119.3 150.6 151.3 112.6 162.2
(d, 7.5)
(dd, 2.0, 11.5) (dd, 5.5, 11.5)
37.2 29.7 (m) 77.2 34.5 (m) 44.0 28.9 28.0 (m) (m)
41.7 49.7 35.8 21.4 40.9
(dd, 7.5, 14.5) (br d, 14.5) (t-like, 7.5) (d, 7.5) (s) (s)
7.51 (br s) 8.56 (br d, 9.5) 6.32 (d, 10.0)
102.1 75.3 78.6 71.8 78.5 62.9
(dd, 8.5, 9.0) (dd, 8.5, 9.0)
dC (ppm)
1.16 1.39 1.24 1.49 (m)
41.1
7.46 (d, 2.0) 8.46 (dd, 2.5, 10.0) 6.27 (d, 10.0)
5.01 3.99 4.25 4.19 3.99 4.56 4.36
0.85 1.60 1.62 2.04 3.98 1.37 1.85 0.87 1.13 1.21 1.08 2.41 1.68 0.83
29.9
1.12 1.33 1.22 1.45 (m)
2.47 2.13 4.75 2.74 0.95 0.60
dH (ppm)
49.3 84.3 43.0 72.3 58.8 17.1 12.0 119.2 150.4 151.3 112.4 162.1
5.01 (d, 8.0) 4.06 4.34 4.37 4.00 4.58 4.55
101.7 74.6 76.7 81.3 76.4 62.1
5.22 (d, 8.0) 4.14 (m) 4.23 4.22 4.03 4.57 4.32
104.8 74.7 78.3 71.3 78.1 62.3
a Full assignments of the proton and carbon were accomplished by analysis of COSY, HSQC and HMBC spectra, and coupling pattern and coupling constants (J in Hz) are in parentheses. Overlapped signals were given without designating multiplicity. b ax ¼ axial. c eq ¼ equatorial.
H
OH HO HO
O OH 1' H
O 3
H
19CH3
H
2
10
H
1 4
H
H
9 5
H
CH3
H 8
14
OH
6
24
21 20
13
11
O
O
18
22 17 16
NOE HMBC
Fig. 2. Selected HMBC and NOE correlations for compound 1.
OH
F.-Y. Yang et al. / Biochemical Systematics and Ecology 38 (2010) 759–763
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Acknowledgements Financial support from Tianjin Municipal Science and Technology Commission (Grant No. 09JCYBJC13700) and Program for New Century Excellent Talents in University (NECT-09-0589) is gratefully acknowledged. References Agrawal, P.K., Jain, D.C., Gupta, R.K., Thakur, R.S., 1985. Phytochemistry 24, 2479. Bao, G.S., Deng, Y., Shi, D.Z., 2003. J. Trop. Med. 3, 260. Braca, A., Prieto, J.M., Tommasi, N.D., Tomè, F., Morelli, I., 2004. Phytochemistry 65, 2921. Colombo, M.L., Tome’, F., Servettaz, O., Bugatti, C., 1990. Int. J. Crude Drug Res. 28, 219. Dinan, L., Savchenko, T., Whiting, P., 2002. Biochem. Syst. Ecol. 30, 171. Guo, Z.J., Wang, J.X., Su, Y.F., Wei, Y.X., Zhu, Y.H., 2003. Shaanxi Qiyao. Shaanxi Science and Technology Press, Shaanxi, China, 35 pp. Hauser, E., Linde, H.H.A., Zivanov, D., 1972. Helv. Chim. Acta 55, 2625. Kamano, Y., Nogawa, T., Yamashita, A., Pettit, G.R., 2001. Collect. Czech. Chem. Commun. 66, 1841. Kissmer, B., Wichtl, M., 1987. Arch. Pharm. 320, 541. Koto, S., Hirooka, M., Tashiro, T., Sakashita, M., Hatachi, M., Kono, T., Shimizu, M., Yoshida, N., Kurasawa, S., Sakuma, N., Sawazaki, S., Takeuchi, A., Shoya, N., Nakamura, E., 2004. Carbohydr. Res. 339, 2415. Lafont, R., Harmatha, J., Marion-Poll, F., Dinan, L., Wilson, I.D., 2002. The Ecdysone Handbook, third ed. on-line. http://ecdybase.org. Li, G.R., Lu, F.H., 1981. Acta Acad. Med. Wuhan 4, 58. Liu, W.Y., Pan, Y.B., Liu, X., 2005. Pharm. Clin. Chin. Mat. Med. 21, 29. Meng, Y.H., Whiting, P., Sik, V., Rees, H.H., Dinan, L., 2001. Phytochemistry 57, 401. Nogawa, T., Kamano, Y., Yamashita, A., Pettit, G.R., 2001. J. Nat. Prod. 64, 1148. Su, L., Chen, G., Feng, S.G., Wang, W., Li, Z.F., Chen, H., Liu, Y.X., Pei, Y.H., 2009. Steroids 74, 399. Su, Y.F., Koike, K., Nikaido, T., Liu, J.S., Zheng, J.H., Guo, D.A., 2003. J. Nat. Prod. 66, 1593. Wang, W.C., 1979. Flora Republicae Popularis Sinicae, vol. 27. Science Press, Beijing, China, 106 pp. Watanabe, K., Mimaki, Y., Sakagami, H., Sashida, Y., 2003. J. Nat. Prod. 66, 236.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus (Ranunculaceae) Feng-Ying Yang a, Yan-Fang Su a, b, *, Ying Wang a, Xin Chai a, Xu Han a, Zhen-Hai Wu c, Xiu-Mei Gao b a
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, PR China Tianjin Key Laboratory of TCM Chemistry and Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China c College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 November 2009 Accepted 10 July 2010
Two new bufadienolide glycosides with an A/B trans ring structure, 14b,16b-dihydroxy-3b(b-D-glucopyranosyloxy)-5a-bufa-20,22-dienolide (1), and 14b,16b-dihydroxy-3b-[b-Dglucopyranosyl-(1/4)-(b-D-glucopyranosyloxy)]-5a-bufa-20,22-dienolide (2), two known ecdysteroids (polypodine B and 20-hydroxyecdysone) (3-4), and six known bufadienolide and its glycosides with 5b-OH (hellebrigenin, 16b-hydroxyhellebrigenin-3-O-a-L-rhamnoside, hellebrigenin 3-O-b-D-glucoside, hellebrin, 16b-hydroxyhellebrigenin-3-O-b-D-glucoside, and deglucohellebrin) (5-10) were isolated from the rhizomes of Helleborus thibetanus. The structures of compounds 1 and 2 were elucidated using various spectroscopic methods. All compounds were reported for the first time from the title plant and their chemotaxonomic significance for the genus Helleborus was discussed. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Ranunculaceae Helleborus Helleborus thibetanus Bufadienolides 5a-Bufadienolides Phytoecdystones
1. Subject and source The genus Helleborus (Ranunculaceae), including about 20 species, are distributed in Southeast Europe and West Asia (Wang, 1979). Helleborus thibetanus Franch is endemic to China and used for treatment of cystitis, urethritis, sores and traumatic injury in Chinese folk medicine (Guo et al., 2003). The rhizomes of H. thibetanus were collected from Mei County, Shaanxi Province in the People’s Republic of China in September 2007 and were authenticated by Prof. Zhen-Hai Wu. A voucher specimen (S200709002) is deposited in School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, People’s Republic of China. 2. Previous work A previous report indicated that the ethanol extract of H. thibetanus presented the digitalis-like cardiotonic activity (Li and Lu, 1981). The polysaccharide extract from H. thibetanus have been also studied on their anticancer and immunomodulating activities (Bao et al., 2003; Liu et al., 2005). To our knowledge, there is no previous report on the phytochemistry of H. thibetanus. 3. Present study In this study, two new 5a-bufadienolide glycosides (1 and 2, Fig. 1) were isolated from the rhizomes of H. thibetanus. The isolation and identification of eight known compounds are reported for the first time from H. thibetanus, including two known ecdysteroids (3 and 4), one known bufadienolide (5) and five known bufadienolide glycosides with 5b-OH (6–10) (Fig. 1). * Corresponding author. School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, PR China. Tel.: þ86 22 27402885; fax: þ86 22 27892025. E-mail address: [email protected] (Y.-F. Su). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.07.002
760
F.-Y. Yang et al. / Biochemical Systematics and Ecology 38 (2010) 759–763
O O
24
21
22
18 19 1
9
R
17
16
OH
14 8
5
3
18
20
13
HO 5
3
HO
R
R OGlc OGlc4-Glc
20
27 23
OH 26
14
8
2
OH
OH
17
19
H 1 2
OH
21
OH 6
O 3 4
R OH H
O O 18 17
19
OHC 3
R2
16
14
R1
OH
5
OH 5 6 7 8 9 10
R1 H OH H H OH H
R2 OH ORha OGlc ORha4-Glc OGlc ORha
Fig. 1. Structures of compounds from the rhizomes of Helleborus thibetanus.
The dried rhizomes of H. thibetanus (8.0 kg) were extracted twice using 95% EtOH and then 60% EtOH respectively. The ethanol extract was evaporated to give a residue that was suspended in water and then extracted successively by petroleum ether (60–90 C), CHCl3, EtOAc and n-BuOH. The CHCl3 extract (34.7 g) was applied to silica gel column chromatography with petroleum ether–Me2CO in a gradient manner, and 76 fractions were collected. The further purification of fractions 19–23 proceeded by silica gel column with CHCl3–Me2CO and then by recrystallization with MeOH, giving compound 5 (61 mg). The EtOAc extract (44 g) was subjected to silica gel column eluting with gradient CH2Cl2–MeOH and 38 fractions were obtained. The further isolation of fractions 19–23 proceeded by silica gel column with CH2Cl2–MeOH and then by recrystallization with Me2CO, affording compounds 10 (668 mg) and 3 (115 mg). Fractions 24–28 were fractionated by silica gel column with CH2Cl2–MeOH and EtOAc–MeOH–H2O respectively and further recrystallization with Me2CO to obtain compounds 4 (894 mg) and 1 (70 mg). The n-BuOH extract (934 g) was submitted to D101 macroporous resin eluting with EtOH–H2O (0:100, 30:70, 50:50, 70:30 and 95:5). The 30% EtOH concentration (378 g) was partitioned with silica gel column eluting with EtOAc–MeOH and 126 fractions were obtained. The white power, precipitated from methanol suspension of fractions 10–25, and was further exposed to ODS column twice (40% MeOH and 37.5% MeOH) to yield compound 6 (56 mg). Fractions 26–36 were repeatedly subjected to silica gel column (CHCl3–MeOH and EtOAc–MeOH), Sephadex LH-20 (MeOH) together with ODS column (45% MeOH and 70% MeOH) to obtain compounds 7 (489 mg) and 8 (218 mg). The further purification of fractions 37–46 exposed to silica gel column (CHCl3–MeOH–H2O and EtOAc–MeOH–H2O) and then ODS column (40% MeOH and 50% MeOH), yielded compounds 9 (42 mg) and 2 (36 mg).
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On the basis of 1H, 13C, and 2D NMR, HRMS and IR spectroscopic analysis, ten compounds were elucidated respectively as two new 5a-bufadienolide glycosides including 14b,16b-dihydroxy-3b-(b-D-glucopyranosyloxy)-5a-bufa20,22-dienolide (1) (Agrawal et al., 1985; Kamano et al., 2001; Watanabe et al., 2003; Su et al., 2009) and 14b,16bdihydroxy-3b-[b-D-glucopyranosyl-(1/4)-(b-D-glucopyranosyloxy)]-5a-bufa-20,22-dienolide (2), two known ecdysteroids named polypodine B (3) (Lafont et al., 2002) and 20-hydroxyecdysone (4) (Lafont et al., 2002), one known bufadienolide as hellebrigenin (5) (Kamano et al., 2001) and five known bufadienolide glycosides as 16b-hydroxyhellebrigenin-3-O-a-Lrhamnoside (6) (Watanabe et al., 2003), hellebrigenin-3-O-b-D-glucoside (7) (Watanabe et al., 2003), hellebrin (8) (Meng et al., 2001; Su et al., 2003), 16b-hydroxyhellebrigenin-3-O-b-D-glucoside (9) (Watanabe et al., 2003) and deglucohellebrin (10) (Watanabe et al., 2003) (Fig. 1). Compound 1 was isolated as a white amorphous powder. Its 1H and 13C NMR spectroscopic data (Table 1) were similar to those of 9, which has an A/B cis, B/C trans and C/D cis ring structure with an a-pyrone ring at C-17 position (Nogawa et al., 2001; Watanabe et al., 2003). Comparison of the 1H and 13C NMR spectra of compounds 1 and 9 allowed us to observe that dH 10.45 (s) of CHO-19 in 9 was displaced by dH 0.60 (s) of CH3-19 in 1, and only one oxygenated carbon signal at d 77.1 (C-3) in the A ring of 1 instead of two oxygenated carbon signals at d 72.5 (C-3) and d 73.3 (C-5) in that of 9, suggesting one hydrogen atom linked at C-5. The angular methyl carbon signal at dC 12.1 (CH3-19) in 1 was very similar to the signal at dC 12.5 (CH3-19) in both 5a-furostan and 5a-spirostan with 5a-H (Agrawal et al., 1985; Su et al., 2009), indicating an a-configuration of H-5. Full assignments of the proton and carbon resonances of 1 were accomplished by analysis of COSY, HSQC and HMBC spectra. In the HMBC spectrum (Fig. 2), a three-bond correlation between H-10 of glucosyl (dH 5.01, d, J ¼ 7.5 Hz) and C-3 (dC 77.1) confirmed that the glucosyl group was connected to aglycone at C-3; the signal of dC 44.2, as the tertiary carbon, was assigned to C-5 by its three-bond correlation with the proton signal of CH3-19 (dH 0.60) together with comparison to the literature data (Agrawal et al., 1985; Su et al., 2009). In the NOESY spectrum (Fig. 2), the NOE correlations between CH3-19 (dH 0.60) and Hax-2 (dH 1.60)/Hax-4 (dH 1.33)/ Hax-6 (dH 1.08)/H-8 (dH 1.63)/Hax-11 (dH 1.12), and between H-3 (dH 3.97) and Hax-1 (dH 0.81)/H-5 (dH 0.82) verified the configuration of 5a-H and the A/B trans ring structure. Additionally, the molecular formula of 1 was confirmed as C30H44O10 by HRMS (m/z 563.2864 [M H]). Therefore, the structure of 1 was unambiguously identified as 14b,16b-dihydroxy-3b-(b-Dglucopyranosyloxy)-5a-bufa-20,22-dienolide. Compound 2 was obtained as a white amorphous powder. Its 1H and 13C NMR spectroscopic data (Table 1) were similar to those of 1, suggesting that 2 is a bufadienolide glycoside structurally related to 1. However, one more b-D-glucosyl was illustrated in 2 by six more carbons signals at dC 101.7, 74.6, 76.7, 81.3, 76.4 and 62.1 together with one more anomeric proton at dH 5.22 (d, J ¼ 8.0 Hz) than 1. The assignments of 2 were established by analysis of COSY, HSQC and HMBC spectra. HMBC correlation from H-10 (dH 5.01) to C-3 (dC 77.2) provided the linkage of inner b-D-glucosyl to C-3. The correlation between H-100 (dH 5.22) and C-40 (dC 81.3) illustrated the linkage of terminal b-D-glucosyl (Watanabe et al., 2003; Su et al., 2009) to the inner b-D-glucosyl at C-40 (dC 81.3) (Koto et al., 2004), which also could be deduced by a downfield shift of 9.5 ppm from dC 71.8 (C-40 of 1) to dC 81.3 (C-40 of 2). The molecular formula of 2 was unequivocally established to be C36H54O15 by HRMS (m/z 725.3387 [M H]). Thus, the structure of 2 was elucidated eventually as 14b,16b-dihydroxy-3b-[b-D-glucopyranosyl-(1/4)-(b-Dglucopyranosyloxy)]-5a-bufa-20,22-dienolide. 14b,16b-Dihydroxy-3b-(b-D-glucopyranosyloxy)-5a-bufa-20,22-dienolide (1): White amorphous power; [a]26 D 27.0 (c 1.00, C5H5N); IR (KBr) vmax: 3430 (OH), 2936 and 2857 (CH),1699 (C]O),1630,1535,1440, 1366,1220,1161,1118,1078,1022 cm1; 1H and 13C NMR data see Table 1; HRMS: m/z 563.2864 [M H], Calc. for [C30H43O10] 563.2862. 14b,16b-Dihydroxy-3b-[b-D-glucopyranosyl-(1/4)-(b-D-glucopyranosyloxy)]-5a-bufa-20,22-dienolide (2): White amor phous power; [a]19 D 31.0 (c 0.42, C5H5N); IR (KBr) vmax: 3402 (OH), 2931 and 2862 (CH), 1700 (C]O), 1629, 1536, 1445, 1372, 1318, 1221, 1155, 1077, 1033 cm1; 1H and 13C NMR data see Table 1; HRMS: m/z 725.3387 [M H], Calc. for [C30H43O10] 725.3379. 4. Chemotaxonomic significance The genus Helleborus consists of six sections: Griphopus, Chenopus, Helleborus, Syncarpus, Dicarpon and Helleborastrum. Chemotaxonomically, Helleborus genus is characterized by the presence of steroids, including bufadienolides, phytoecdystones and steroidal saponins (Kissmer and Wichtl, 1987; Colombo et al., 1990; Meng et al., 2001; Watanabe et al., 2003; Braca et al., 2004). H. thibetanus, the single species of the section of Dicarpon, had been predicted to contain high levels of phytoecdysteroids (Dinan et al., 2002). In our ongoing study, two known ecdysteroids (3 and 4) isolated from the rhizomes of H. thibetanus for the first time. 20-Hydroxyecdysone (4) is a phytoecdysteroid that is found in other species within Hellerborus (Dinan et al., 2002). It was obtained at a high level. All the isolated bufadienolides from species of Helleborus have had an A/B cis ring structure (Kissmer and Wichtl, 1987; Colombo et al., 1990; Meng et al., 2001; Watanabe et al., 2003) except for 14-hydroxy-3-oxo-5a-bufa-20,22-dienolide from H. odorus, which has an A/B trans ring structure together with 5a-H and a carbonyl group at C-3 (Hauser et al.,1972). The present work not only reported two new bufadienolide glycosides (1 and 2) with features of an A/B trans ring structure, 5a-H and glycosidation of the hydroxyl group at C-3, but also identified six known bufadienolide and its glycosides with 5b-OH (5–10). This is the first report of the occurrence of 5a-bufadienolide glycosides (1 and 2) not only in the section of Dicarpon but also in the genus of Helleborus. The contribution of this work may open opportunities to explore 5a-bufadienolide glycosides as chemotaxonomic marker for Dicarpon section.
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Table 1 1 H and 13C NMR Data for Compounds 1 and 2a (C5D5N). Position
1
2
dH (ppm) 1axb 1eqc 2ax 2eq 3 4ax 4eq 5 6ax 6eq 7ax 7eq 8 9 10 11ax 11eq 12ax 12eq 13 14 15a 15b 16 17 18 19 20 21 22 23 24 Glc 10 20 30 40 50 60 a 60 b Glc 100 200 300 400 500 600 a 600 b
dC (ppm)
0.81 1.57 1.60 2.02 (m) 3.97 1.33 1.83 (m) 0.82 1.08 1.20 1.10 2.35 (m) 1.63 0.78
37.3
77.1 34.6 44.2 29.1 28.1 41.8 49.8 35.9 21.5
49.4 84.5 43.1
(dd, 7.0, 14.0) (br d, 14.0) (t-like, 7.5) (d, 8.0) (s) (s)
2.51 2.18 4.80 2.79 0.99 0.64
72.5 59.0 17.2 12.1 119.3 150.6 151.3 112.6 162.2
(d, 7.5)
(dd, 2.0, 11.5) (dd, 5.5, 11.5)
37.2 29.7 (m) 77.2 34.5 (m) 44.0 28.9 28.0 (m) (m)
41.7 49.7 35.8 21.4 40.9
(dd, 7.5, 14.5) (br d, 14.5) (t-like, 7.5) (d, 7.5) (s) (s)
7.51 (br s) 8.56 (br d, 9.5) 6.32 (d, 10.0)
102.1 75.3 78.6 71.8 78.5 62.9
(dd, 8.5, 9.0) (dd, 8.5, 9.0)
dC (ppm)
1.16 1.39 1.24 1.49 (m)
41.1
7.46 (d, 2.0) 8.46 (dd, 2.5, 10.0) 6.27 (d, 10.0)
5.01 3.99 4.25 4.19 3.99 4.56 4.36
0.85 1.60 1.62 2.04 3.98 1.37 1.85 0.87 1.13 1.21 1.08 2.41 1.68 0.83
29.9
1.12 1.33 1.22 1.45 (m)
2.47 2.13 4.75 2.74 0.95 0.60
dH (ppm)
49.3 84.3 43.0 72.3 58.8 17.1 12.0 119.2 150.4 151.3 112.4 162.1
5.01 (d, 8.0) 4.06 4.34 4.37 4.00 4.58 4.55
101.7 74.6 76.7 81.3 76.4 62.1
5.22 (d, 8.0) 4.14 (m) 4.23 4.22 4.03 4.57 4.32
104.8 74.7 78.3 71.3 78.1 62.3
a Full assignments of the proton and carbon were accomplished by analysis of COSY, HSQC and HMBC spectra, and coupling pattern and coupling constants (J in Hz) are in parentheses. Overlapped signals were given without designating multiplicity. b ax ¼ axial. c eq ¼ equatorial.
H
OH HO HO
O OH 1' H
O 3
H
19CH3
H
2
10
H
1 4
H
H
9 5
H
CH3
H 8
14
OH
6
24
21 20
13
11
O
O
18
22 17 16
NOE HMBC
Fig. 2. Selected HMBC and NOE correlations for compound 1.
OH
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Acknowledgements Financial support from Tianjin Municipal Science and Technology Commission (Grant No. 09JCYBJC13700) and Program for New Century Excellent Talents in University (NECT-09-0589) is gratefully acknowledged. References Agrawal, P.K., Jain, D.C., Gupta, R.K., Thakur, R.S., 1985. Phytochemistry 24, 2479. Bao, G.S., Deng, Y., Shi, D.Z., 2003. J. Trop. Med. 3, 260. Braca, A., Prieto, J.M., Tommasi, N.D., Tomè, F., Morelli, I., 2004. Phytochemistry 65, 2921. Colombo, M.L., Tome’, F., Servettaz, O., Bugatti, C., 1990. Int. J. Crude Drug Res. 28, 219. Dinan, L., Savchenko, T., Whiting, P., 2002. Biochem. Syst. Ecol. 30, 171. Guo, Z.J., Wang, J.X., Su, Y.F., Wei, Y.X., Zhu, Y.H., 2003. Shaanxi Qiyao. Shaanxi Science and Technology Press, Shaanxi, China, 35 pp. Hauser, E., Linde, H.H.A., Zivanov, D., 1972. Helv. Chim. Acta 55, 2625. Kamano, Y., Nogawa, T., Yamashita, A., Pettit, G.R., 2001. Collect. Czech. Chem. Commun. 66, 1841. Kissmer, B., Wichtl, M., 1987. Arch. Pharm. 320, 541. Koto, S., Hirooka, M., Tashiro, T., Sakashita, M., Hatachi, M., Kono, T., Shimizu, M., Yoshida, N., Kurasawa, S., Sakuma, N., Sawazaki, S., Takeuchi, A., Shoya, N., Nakamura, E., 2004. Carbohydr. Res. 339, 2415. Lafont, R., Harmatha, J., Marion-Poll, F., Dinan, L., Wilson, I.D., 2002. The Ecdysone Handbook, third ed. on-line. http://ecdybase.org. Li, G.R., Lu, F.H., 1981. Acta Acad. Med. Wuhan 4, 58. Liu, W.Y., Pan, Y.B., Liu, X., 2005. Pharm. Clin. Chin. Mat. Med. 21, 29. Meng, Y.H., Whiting, P., Sik, V., Rees, H.H., Dinan, L., 2001. Phytochemistry 57, 401. Nogawa, T., Kamano, Y., Yamashita, A., Pettit, G.R., 2001. J. Nat. Prod. 64, 1148. Su, L., Chen, G., Feng, S.G., Wang, W., Li, Z.F., Chen, H., Liu, Y.X., Pei, Y.H., 2009. Steroids 74, 399. Su, Y.F., Koike, K., Nikaido, T., Liu, J.S., Zheng, J.H., Guo, D.A., 2003. J. Nat. Prod. 66, 1593. Wang, W.C., 1979. Flora Republicae Popularis Sinicae, vol. 27. Science Press, Beijing, China, 106 pp. Watanabe, K., Mimaki, Y., Sakagami, H., Sashida, Y., 2003. J. Nat. Prod. 66, 236.