Three new steroidal saponins from Helleborus thibetanus

Abstract Three new steroidal saponins including two spirostanol glycosides (1–2) and one furostanol glycoside 1-sulphate (3) were isolated from the dried roots and rhizomes of Helleborus thibetanus. Structures of the compounds were determined on the basis of extensive use of 1-D and 2-D NMR experiments, together with HR–ESI–MS and IR measurements, as well as the results of acid hydrolysis. Compounds 1–2 represented steroidal saponins with an unusual substitution pattern, which possessed a double bond at C-25 and were glycosylated at 1-OH.


Introduction
Helleborus, a genus of herbaceous perennials belongs to the family Ranunculaceae. A total of around 25 species are distributed over different parts of South-east Europe and West Asia (Sylla et al. 2014). It is reported that steroids including bufadienolides, phytoecdystones and steroidal saponins (Cheng et al. 2014;Zhang et al. 2014aZhang et al. , 2014b constitute the major components of Helleborus according to previous phytochemical research. Some Helleborus species extracts possess immunostimulatory, anti-inflammatory and different anticancer properties (Čakar et al. 2014). Helleborus thibetanus (FRANCH), a plant endemic to China, is mainly growing wild in Sichuan, Gansu and Shaanxi. The roots and rhizomes of H. thibetanus, commonly known by the local name of 'Xiao-Tao-Er-Qi' , have been used for the treatment of traumatic injury, cystitis and urethritis. Several steroidal saponins, one pregnane, one ABSTRACT Three new steroidal saponins including two spirostanol glycosides (1-2) and one furostanol glycoside 1-sulphate (3) were isolated from the dried roots and rhizomes of Helleborus thibetanus. Structures of the compounds were determined on the basis of extensive use of 1-D and 2-D NMR experiments, together with HR-ESI-MS and IR measurements, as well as the results of acid hydrolysis. Compounds 1-2 represented steroidal saponins with an unusual substitution pattern, which possessed a double bond at C-25 and were glycosylated at 1-OH. spirostanol sulphate, several bufadienolides and phytoecdystones had been isolated from H. thibetanus (Zhang et al. 2014a(Zhang et al. , 2014b. On continuing the study of this plant, we have now isolated two new spirostanol steroidal saponins (1-2) and one new furostanol glycoside 1-sulphate (3) (Figure 1) from the title plant. This paper deals with the isolation and structural elucidation of the three new steroidal saponins by detailed analysis of their NMR spectra and acid hydrolysis.

Results and discussion
Compound 1 was obtained as a white amorphous solid and its molecular formula was assigned as C 57 H 88 O 29 , deduced from the HR-ESI-MS m/z 1235.5327 ([M − H] − ), as well as its 1 H and 13 C NMR spectroscopic data. The IR spectrum of 1 showed the characteristic absorptions of hydroxyl groups at 3396 cm −1 and carbonyl group at 1729 cm −1 . Its 1 H and 13 C NMR spectra revealed the presence of two angular Me groups at δ H 1.02 (3H, s), 1.36 (3H, s) and δ C 16.7, 14.9, and a characteristic quaternary carbon signal at δ C 111.7, suggesting the occurrence of a spirostanol skeleton in 1. The position of 21-OH was established based on the HMBC correlations between the protons at δ H 1.93 (1H, m, H-17) and δ H 3.31 (1H, m, H-20) and the carbon resonance at δ C 62.2 (C-21). The correlation from the olefinic proton at δ H 5.62 (1H, br d, J = 5.0 Hz) to δ C 124.7 (C-6) was observed in the HSQC spectrum, identifying the double bond at C-5(6), which was also verified by the correlations from olefinic proton at δ H 5.62 (1H, br d, J = 5.0 Hz) to the carbon resonances of δ C 43.8 (C-4), δ C 33.0 (C-8) and δ C 42.8 (C-10), along with correlations between δ H 1.36 (3H, s, Me-19) and δ C 139.4 (C-5) in the HMBC spectrum. Evidence for the presence of the other double bond at C-25(27) came from correlations from the olefinic protons at δ H 5.16 (1H, m) and δ H 5.04 (1H, br s) to δ C 113.7 (C-27) in the HSQC spectrum, which was demonstrated by HMBC correlations from olefinic protons at δ H 5.16 (1H, m) and δ H 5.04 (1H, br s) to the carbon resonances of δ C 82.2 (C-24), δ C 143.7 (C-25) and δ C 61.4 (C-26). The proton signal at δ H 3.79 (1H, m) was assigned as H-1 attached to the oxygenated C-1 by its correlation with C-1 (δ C 83.7) in the HSQC spectrum, which was also confirmed by the HMBC correlation between Me-19 (3H, s, δ H 1.36) and C-1 (δ C 83.7). The signal at δ H 3.87 (1H, m) in the 1 H NMR spectrum showed COSY correlations with H-4ax/H-2ax, giving evidence for its assignment of H-3. In addition, the NOESY cross-peaks between H-1 and H-3, between Me-19 and Me-18/H-2ax/H-4ax, indicated the β-configurations of the oxygenated substituents at C-1 and C-3. Moreover, the configurations of C-23 and C-24 were characterised as S by the NOESY correlations between H-23 and H-20, between H-23 and H 2-21 /H 2-27 , between H-24 and H 2-27 Hayes et al. 2009). Comparison of the 1 H and 13 C NMR spectroscopic data of the aglycone moiety of 1 with those of bethoside A (Hayes et al. 2009), along with the above analysis, the structure of the aglycone of 1 was elucidated as (23S,24S)-1β,3β,21,23,24-pentahydroxy-spirosta-5,25(27)-diene. For the sugar moiety, the five anomeric protons at δ H 6.46 (1H, br s), 5.14 (1H, d, J = 7.0 Hz), 5.13 (1H, d, J = 7.0 Hz), 4.91 (1H, d, J = 7.0 Hz) and 4.67 (1H, d, J = 7.5 Hz) showed correlations with the anomeric carbon resonances at δ C 100.7, 105.9, 106.8, 106.6 and 100.5 in the HSQC spectrum, respectively. And two Me groups were observed at δ H 1.36 (3H, d, J = 5.5 Hz), 1.50 (3H, d, J = 6.0 Hz) in the 1 H NMR spectrum, and δ C 18.3, δ C 17.4 in the 13 C NMR spectrum, respectively, which implied two of them were 6-deoxyhexose units. The Me group at δ H 2.00 (3H, s) and δ C 20.9 and C=O signal at δ C 170.7 were assignable to one acetyl group. Acid hydrolysis of 1 with 1 M HCl in dioxane-H 2 O (1:1) followed by TlC analysis showed the presence of arabinose (Ara), rhamnose (Rha), xylose (Xyl), fucose (Fuc) and glucose (Glc). One glycosyl group attached to C-1 position of the aglycone was established from the HMBC correlations of signals at δ H 4.67 (H-1 of Ara) with δ C 83.7 (C-1 of the aglycone), H-1 (δ H 6.46) of Rha with C-2 (δ C 72.6) of Ara, H-1 (δ H 4.91) of Xyl with C-3 (δ C 85.0) of Ara, which was also supported by the NOESY correlations of signals at H-1 (δ H 3.79) of aglycone with H-1 (δ H 4.67) of Ara, H-2 (δ H 4.59) of Ara with H-1 (δ H 6.46) of Rha, H-3 (δ H 4.07) of Ara with H-1 (δ H 4.91) of Xyl. The other sugar chain located at C-24 (δ C 82.2) of 1 was deduced by a downfield shift of 8.1 ppm of C-24 (δ C 74.1) compared with clintonioside B , and the HMBC correlations between H-24 (δ H 4.75) of the aglycone and C-1 (δ C 105.9) of Fuc, and between C-4 (δ C 83.2) of Fuc to H-1 (δ H 5.13) of Glc proved the linkage of the sugars and the aglycone, which was further supported by the NOESY cross-peaks between H-24 (δ H 4.75) of aglycone and H-1 (δ H 5.14) of Fuc, between H-4 (δ H 4.04) of Fuc and H-1 (δ H 5.13) of Glc. Full assignments of 1 were achieved by a comprehensive analysis of DEPT, COSY, HSQC, NOESY and HMBC spectra. On the basis of the above evidence, the structure of the new spirostanol glycoside 1 was fully determined to be (23S,24S)-21-hydroxymethyl-24- Compound 2, a white amorphous solid, exhibited the ion peak at m/z 1193.5226 [M − H] − in the HRESIMS data, corresponding to the molecular formula C 55 H 85 O 28 , which displayed the lack of C 2 H 3 O compared with 1. The IR absorption at 3424 cm −1 revealed the presence of hydroxyl groups. Complete assignments of the 1 H and 13 C NMR signals of 2 were accomplished by a combined analysis of DEPT, COSY, HSQC, NOESY and HMBC spectra. A detailed comparison of the 1 H, 13 C NMR chemical shifts of 1 and 2, revealed that they shared the same skeleton and same glycosidic positions at C-1 and C-24, except for the disappearance of the carbon signal at δ C 170.7 and δ C 20.9 in the 13 C NMR spectrum and the proton signal of δ H 2.00 (3H, s) in the 1 H NMR spectrum of compound 2. Compound 3 was isolated as a white amorphous solid. Its molecular formula was determined as C 34 H 53 O 13 S, deduced from the HR-ESI-MS (m/z 701.3224 [M] − ), as well as its 13 C NMR spectrum. The 1 H NMR spectrum of 3 displayed signals for two tertiary methyl groups at δ H 0.77 (3H, s) and 1.21 (3H, s), one secondary methyl groups at δ H 1.04 (3H, d, J = 7.5 Hz), one methoxyl group at δ H 3.18 (3H, s), one methine proton signal at 3.84 (1H, m) attributed to secondary alcoholic function, two methylene proton signals at δ H 4.53 (1H, br d, J = 12.5 Hz) and 4.27 (1H, m), indicative of a primary alcoholic function, three olefinic protons at δ H 5.53 (1H, br d, J = 5.5 Hz), δ H 5.27 (1H, br s), δ H 4.98 (1H, br s), along with an anomeric proton at δ H 4.82 (1H, d, J = 7.5 Hz). Besides, the 13 C NMR spectrum for the aglycone moiety exhibited signals ascribable to an acetal carbon at δ C 112.3, one secondary alcoholic function at δ C 67.7, one primary alcoholic function at δ C 71.9 and one methoxyl carbon at δ C 47.2, together with an anomeric carbon at δ C 103.7. The evidence above manifested that 3 had a glycosidic furostanol skeleton with one methoxy. The signal at δ C 85.1 (C-1) of compound 3 was similar to δ C 85.5 (C-1) of ruscogenin 1-sulphate (Asano et al. 1993), δ C 84.1 (C-1) of spirost-5-en-1β,3β-diol 1-sulphate (Oulad-Ali et al. 1996) and δ C 85.1 (C-1) of spirost-5,25(27)-dien-1β,3β-diol 1-sulphate (Yang et al. 2010), indicating a sulphate group at C-1. The existence of the sulphate functional group was further affirmed by a series of characteristic strong absorption bands at 1242, 1067 and 951 cm −1 in its IR (KBr) spectrum. The HMBC spectrum of 3 provided interaction of C-22 at δ C 112.3 with the proton at δ H 3.18 (3H, s), testifying that the methoxyl group connected to C-22. The occurrence of a double bond at C-25(27) was confirmed from a correlation signal between the proton signals at δ H 5.27 (1H, br s, H-27a), δ H 4.98 (1H, br s, H-27b) and the carbon signals of C-24 (δ C 28.0) and C-26 (δ C 71.9) in the HMBC spectrum. The assignments of all the individual protons and carbons of 3 were achieved with the analysis of COSY, HSQC, HMBC and NOESY spectra. In addition, the presence of a glucopyranosyl moiety in 3 was readily recognised by the appearance of an anomeric proton signal at δ H 4.82 (1H, dd, J = 7.5 Hz) in the 1 H NMR spectrum and also by the characteristic six signals at δ C 103.7, 74.9, 78.4, 71.6, 78.3 and 62.7 in the 13 C NMR spectrum. HMBC correlation of anomeric proton signal at δ H 4.82 with C-26 (δ C 71.9) proved the location of the glucopyranosyl moiety at C-26 of aglycone, which is a structural feature in naturally occurring furostanol glycosides (Matsuo et al. 2008). Acid hydrolysis of 3 with 1 M HCl in dioxane-H 2 O (1:1), followed by TlC analysis indicated the presence of Glc. Eventually, the structure of 3 was unequivocally identified to be 26-O-β-d-glucopyranosyl-3β-hydroxy-22α-methoxyfurosta-5,25(27)-diene-1β-yl sulphate.

Plant material
The roots and rhizomes of H. thibetanus were collected in September of 2007 from Mei County, Shaanxi Province in the People's Republic of China and were authenticated by Prof. Zhen-Hai Wu, College of life Sciences, Northwest A&F university, China. A voucher specimen (S200609002) has been deposited in School of Pharmaceutical Science and Technology, Tianjin university, Tianjin.

Acid hydrolysis of 1-3
A solution of compound 1 (10 mg) in 1 M HCl with (dioxane-H 2 O, 1:1, 6 ml) was heated at 80 °C for 2 h. After cooling, the reaction mixture was evaporated under reduced pressure to remove dioxane and diluted to 8 ml with H 2 O, and then extracted with EtOAc (4 ml × 6 ml). The aqueous layer was neutralised by Ag 2 CO 3 to pH 6-7 and filtered with the microporous membrane and further concentrated to an appropriate volume. Five sugars were identified as Glc, Xyl, Ara, Fuc and Rha by comparison with authentic samples (d-glucose, d-xylose, l-arabinose, d-fucose and l-rhamnose) through TlC (silica gel) detection with the solvent system EtOAc: MeOH: HAc: H 2 O (20:5:3:3) and CHCl 3 : MeOH: HAc: H 2 O (16:10:3:3). In the same way, compound 2 (6 mg) was subjected to acid hydrolysis to give a sugar fraction. TlC analysis of the sugar fraction under the same condition as in the case of that of 1 showed the presence of Glc, Xyl, Ara, Fuc and Rha. Compound 3 (6 mg) was subjected to acid hydrolysis as described for 1 to give a sugar fraction. TlC analysis of the sugar fraction indicated the presence of Glc.

Conclusion
In summary, the present study describes the isolation and characterisation of three new steroidal saponins including two spirostanol glycosides (1-2) and one furostanol glycoside 1-sulphate (3) from H. thibetanus. According to previous investigations, steroidal saponins from plants are common with a double bond at C-5(6) or C-9(11), furthermore, glycosidation position is usually at 3-OH in most cases. It's worthy to note that the presence of a C-25(27) double bond and meanwhile glycosyl at 1-OH found in spirostanol glycosides 1-2 is unusual.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1-S27 and Tables S1-S3.

Disclosure statement
No potential conflict of interest was reported by the authors.