Steroidal and pregnane glycosides from Ypsilandra thibetica

Introduction

In a continuation of our study on saponin constituents of medicinal plants, we have examined the saponin riched fraction prepared from the EtOH extract of the air-dried whole plants of Ypsilandra thibetica (Liliaceae). This perennial plant distributes in southwestern China and has been used as hemostatic agent in Chinese folk medicine.^{1, 2} In our recent study, we found that this species was a rich source of steroidal saponins. Two sapogenin, 22 spirostanol saponins, and two C-22 steroidal lactone glycosides were obtained from the title plants.^3-6 Further phytochemical investigation has been carried out on this species, with particular attention to the steroidal glycoside constituents, and has resulted in the isolation of two new bisdesmosidic furostanol saponins (1 and 2) and one new pregnane glycoside (3), together with nine known steroidal glycosides: protoprogenin Ⅱ (4), ⁷ proto-Pb (5), ⁸ saponin Th (6), ⁹ pseudoproto Pb (7), ¹⁰pregnane 5, 16-dien-3β-ol-20-oxo 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (8), ¹¹ smilaxchinoside B (9), ¹² parispseudoside C (10), ¹³ parispseudoside A (11), ¹³ and 26-O-β-D-glucopyranosyl-3β, 26-dihydroxy-20, 22-seco-25(R)-furost-5-en-20, 22-dione 3-O-α-Lrhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-Lrhamnopyranosyl-(1→2)]-β-D-glucopyranoside (12).¹⁴ This paper reports the isolation, structural determination, and cytotoxic activity of these glycosides.

Results and Discussion

Compound 1, obtained as a white amorphous powder, gave a pseudo-molecular ion peak [M – H]^– at m/z 1207.5736 (calcd. 1207.5747) in its HRESIMS. Combined with ¹³C NMR spectroscopic data (Table 2), its molecular formula was determined as C₅₇H₉₂O₂₇. The ¹H NMR spectrum of 1 (Table 1) showed signals of four steroid methyl groups at δ_H 0.97 (3H, d, J = 6.5 Hz, Me-27), 1.06 (3H, s, Me-19), 1.15 (3H, s, Me-18), and 1.53 (3H, d, J = 6.7 Hz, Me-21), an olefinic proton at δ_H 5.22 (1H, br. s), as well as signals for five anomeric proton signals at δ_H 4.80 (1H, d, J = 7.7 Hz, H-1'''''), 4.93 (1H, d, J = 7.2 Hz, H-1'), 5.84 (1H, br. s, H-1'''), 6.29 (1H, br. s, H-1''''), and 6.41 (1H, br. s, H-1''). The three methyl carbon signals at δ_C 18.7, 18.9, and 18.5 and their corresponding proton signals at δ_H 1.58 (3H, d, J = 5.2 Hz, H-6''), 1.58 (3H, d, J = 5.2 Hz, H-6'''), and 1.75 (3H, d, J = 6.1 Hz, H-6'''') indicated that 1 had three deoxy sugars. The monosaccharides of the acidic hydrolysate of 1 were identified as D-glucose and L-rhamnose on the basis of GC analysis and comparison with authentic standards. The above ¹H NMR and chemical data, together with an acetalic carbon signal at δ_C 110.9 in the ¹³C NMR spectrum¹⁵ and a positive coloration with Ehrlich reagent, ^{16, 17} indicated 1 to be a furostanol saponin with up to five monosaccharides. A comparison of the 1H and ¹³C spectroscopic signals of the aglycone moiety of 1 with ProtoPb (5)⁸indicated that the signals were similar except for the presence of a carbonyl group (δ_C 213.1). In the HMBC spectrum (Figure 1), the long-range correlations from δ_H 1.15 (Me-18, s) to δ_C 213.1 (C-12, s), 55.4 (C-13, s), 56.0 (C-14, d), and 54.9 (C-17, d) indicated that the carbonyl group was attached at C-12 of the aglycone of 1. The configuration of the methyl group at C-25 is R on the basis of the proton signals of C-26 at δ_H 3.94 (1H, 26-H_a) and 3.60 (1H, 26-H_b), and the difference (∆_ab) of the proton signals at C-26 was 0.34.^{18, 19} From the above evidence, the aglycone of 1 was identified as (25R)-furost-5-en-3β, 22α, 26-triol-12-one.

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Comparison of the carbon chemical shift thus assigned with those of the reference methyl glycosides, ^{15, 19} taking into account the known effects of O-glycosylation, indicated that 1 contained a terminal β-D-glucopyranosyl units, two terminal α-L-rhamnopyranosyl unit, a C-4 substituted α-L-rhamnopyranosyl unit, and a C-2 and C-4 disubstituted β-Dglucopyranosyl unit. The β-configuration of the anomeric protons of the glucopyranosyl residue were assigned based on its J_1H-2H value (J = 7.2~7.7 Hz), while the anomeric configuration of the three rhamnopyranosyls were determined as α-oriented on the ground the chemical shift values of the C-3", C-5", C-3''', C-5''', C-3'''', and C-5'''' with those of the corresponding carbons of methyl α- and β-rhamnopyranoside.^{20, 21} In the HMBC spectrum, a correlation peak between the signals at δ_H 4.80 (H-1 of terminal glucosyl) and δ_C 75.4 (C-26 of aglycon) implied that one glucose unit was attached at C-26 of the aglycon, which is a structural feature most frequently encountered in the plant furostanol saponins.¹⁵ Consequently, a tetraglycoside was assumed to be located at C-3 of the aglycon. The sequence of the tetrasaccharide, which was the same as the known compounds 5–12, was established from the further HMBC correlations: H-1'(δ_H 4.93) of Glc with C-3 (δ_C 77.8) of the aglycone, H-1''(δ_H 6.41) of 2'-Rha with C-2'(δ_C 78.0) of Glc, H-1'''(δ_H 5.84) of 4'-Rha with C-4' (δ_C 77.8) of Glc, and H-1''''(δ_H 6.29) of 4''-Rha with C-4'''(δ_C 80.5) of 4'-Rha. Therefore, 1 was determined to be 26-O-β-D-glucopyranosyl-(25R)-3β, 22α, 26-trihydroxyfurost-5-en-12-one 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside, and named ypsilandroside P.

Compound 2 displayed a [M – H]^– ion at m/z 1189.5645 (calcd. for C₅₇H₈₉O₂₆, 1189.5642) in the HRESIMS and gave a red color with Ehrlich's reagent. The NMR spectral data suggested 2 is a furostanol saponin closely related to 1. It differed from 1 in the presence of one more olefinic functionality [δ_C 153.2 (s) and 103.1 (s)] in addition to the 5(6)-en group. Furthermore, the Me-21 methyl doublet signal observed at δH 1.53 (J = 6.7 Hz) in the ¹H NMR spectrum of 1 was absent from 2, but was replaced by a methyl singlet at δ_H 1.74. These data were suggestive of 2 being the corresponding Δ²⁰⁽²²⁾-furostanol saponin of 1, which was confirmed by the mass difference of m/z = 18 and HMBC correlations. In the HMBC spectrum of 2, the correlations of Me-21 (δ_H 1.74) with C-17 (δ_C 56.1), C-20 (δ_C 103.1), and C-22 (δ_C 153.2) were observed. Thus, the structure of 2 was established as 26-O-β-D-β-D-glucopyranosyl-(25R)-3β, 26-dihydroxyfurost-5, 20 (22)-diene-12-one 3-O-α-L-rhamnopyranosyl-(1→4)-α-Lrhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-Dglucopyranoside, and named ypsilandroside Q.

Compound 3 had a molecular formula of C₄₅H₆₈O₂₀, established by analysis of HRESIMS (m/z 963.3953 [M + Cl]^–, calcd. 963.3992) and ¹³C NMR spectrum (45 signals). The ¹H NMR spectrum of 3 displayed two three-proton singlet signals at δ_H 1.07 (s) and 1.33 (s), indicating the presence of two angular methyl groups, and a methyl singlet at δ_H 2.32 (s) attached to a deshielding moiety, as well as four anomeric proton signals at δ_H 4.93 (1H, d, J = 7.4 Hz), 5.83 (1H, br. s), 6.29 (1H, br. s), and 6.41 (1H, br. s). The existence of an α, β-unsaturated carbonyl group was verified by the IR (1657 cm⁻¹), UV [227 nm (logε 2.8)], and ¹³C NMR [δ_C 196.0 (C=O), 150.7 (C), and 142.8 (CH)] spectra. These spectral data and comparison with those of the known compound 8¹¹ indicated that 3 differed from 8 by the presence of a carbonyl group (δ_C 209.2) instead of a methylene moiety at C-12 in the latter. The HMBC correlations of δ_H 1.33 (Me-18) with δ_C 209.2 (C-12, s), 61.3 (C-13, s), 56.2 (C-14, d), 150.7 (C-17, s) indicated that the location of the carbonyl group at C-12. Thus, the aglycone of 3 was identified as 3α-hydroxypregna-5, 16-dien-12, 20-dione. The ¹H and ¹³C NMR shifts of the tetraglycoside moiety linked to C-3 of the pregnane were superimposable on those of 1, 2, and 5–12. On the basis of all the information above, the structure of 3 was characterized as pregnane 5, 16-dien-3β-ol-12, 20-dione 3-O-α-L-rhamnopyranosyl-(1→4)-α-Lrhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-Dglucopyranoside, and named ypsilandroside R.

The cytotoxic activities of saponins 1, 2, and 12 against the growth of human tumor cell lines (A549 and HL-60) were evaluated. The results indicated that only compound 1 showed 86.4% inhibition to A549 cell lines and 75.9% inhibition to HL-60 cell lines at the tested concentration (10 μM).

Experimental Section

General Experimental Procedures. Optical rotations were measured on a Jasco P-1020 automatic digital polarimeter. UV spectra were measured using a Shimadzu UV-2401PC spectrophotometer. IR spectra were measured on a Bio-Rad FTS-135 spectrometer with KBr pellets. NMR spectra were run on Bruker AM-400 and DRX-500 instruments with TMS as internal standard. FAB-MS spectra were recorded on a VG Auto Spec-300 spectrometer, HRESIMS spectra were recorded on an API Qstar Pulsar instrument. Column chromatography (CC) was performed over silica gel (200–300 mesh, 10–40 μm, Qingdao Marine Chemical Co., China), Rp-18 (40–63 μm, Merck), and Sephadex LH-20 (GE Healthcare, Sweden). TLC was performed on HSGF254 (0.2 mm, Qingdao Marine Chemical Co., China) or Rp-18 F₂₅₄ (0.25 mm, Merck). Semipreparative HPLC was run on Agilent 1100 liquid chromatograph with diode array detector (DAD) setting at 200nm and 254 nm, ZORBAX SB-C18 (5 μm) column (25 cm × 9.4 mm i.d.). GC analysis was performed on a Shimadzu GC-2010 gas chromatograph equipped with an H₂ flame ionization detector.

Plant Material. The plant material of Y. thibetica was collected in November 2006 from Luding County, Sichuan Province, China, and identified by Prof. Xin-Qi Chen, Institute of Botany, Chinese Academy of Sciences, Beijing. A voucher specimen (No. HY0002) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China.

Extraction and Isolation. The air-dried whole plants of Y. thibetica (10 kg) were extracted three times with 70% EtOH (50 L × 3) under reflux for a total of 6 h and the combined extract was concentrated under reduced pressure. Then the concentrated extract was loaded onto a macroporous resin column (YWD-3F) and eluted successively with H₂O, 40% EtOH (F1 fraction), 70% EtOH (F2 fraction), and 95% EtOH (F3 fraction), respectively. The 40% EtOH elutes were evaporated to dryness. Fraction F1 (33 g) was fractioned by silica gel column and eluted with a gradient of CHCl₃-MeOH-H₂O (8:2:0.2→7:3:0.5, v/v) to get two subfractions (F11 and F12). Fraction F11 was subjected to column chromatography on Rp-18 gel (MPLC, MeOH-H₂O 4:6→6.5:3.5) and semipreparative HPLC (MeOH-H₂O 38:62 v/v; flow rate: 3 mL.min⁻¹) to obtain 3 (14 mg), 4 (28 mg), and 8 (19 mg). Fraction F12 was chromatographed over Rp-18 gel (MPLC, MeOH-H₂O 3:7→7:3) and semi-preparative HPLC (MeCNH₂O 20:80→35:65 v/v; flow rate: 3 mL.min⁻¹) to yield 1 (17 mg), 2 (8 mg), 5 (43 mg), 6 (80 mg), 7 (23 mg), 9 (20 mg), 10 (17 mg), 11 (18 mg), and 12 (24 mg).

Ypsilandroside P (1): white amorphous powder; [α]_D²⁴ −65.0 (c 0.26, MeOH); IR (KBr) ν_max 3431, 2934, 1706, 1640, 1453, 1381, 1130, 1044, 985, 911, 839, 804 cm⁻¹ (intensity: 839 > 911); ¹H and ¹³C NMR data see Tables 1 and 2; negative FABMS m/z 1208 M⁻, 1062 [M − 146]⁻, 915 [M − H − 2 × 146]⁻, 769 [M⁻ H − 3 × 146]⁻; negative ion HRESIMS m/z 1207.5736 (calcd. for C₅₇H₉₁O₂₇ [M − H]⁻, 1207.5747).

Ypsilandroside Q (2): white amorphous powder; α [α]_D²⁴ −66.8 (c 0.47, MeOH); IR (KBr) ν_max 3426, 2933, 1707, 1640, 1453, 1382, 1131, 1043, 984, 911, 841, 804 cm⁻¹ (intensity: 841 > 911); ¹H and ¹³C NMR data see Tables 1 and 2; negative FABMS m/z 1190 [M]⁻, 1043 [M − H − 146]⁻, 897 [M − H − 2 × 146]⁻; negative ion HRESIMS m/z 1189.5645 (calcd. for C₅₇H₈₉O₂₆ [M − H]⁻, 1189.5642).

Ypsilandroside R (3): white amorphous powder; α_D²² −58.3 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) 227 (2.8) nm; IR (KBr) ν_max 3418, 2934, 1713, 1657, 1376, 1132, 1053, 983 cm⁻¹; ¹H and ¹³C NMR data see Tables 1 and 2; negative FABMS m/z 927 [M − H]⁻, 781 [M − H − 146]⁻, 635 [M − H − 2 × 146]⁻; negative ion HRESIMS m/z 963.3953 (calcd. for C₄₅H₆₈O₂₀Cl [[M + Cl]]⁻, 963.3992).

Acid Hydrolysis of Compounds 1–3 and GC Analysis. Compounds 1–3 (4 mg each) were refluxed with 4 M TFAdioxane (1:1 v/v, 2 mL) on water bath for 4h. The reaction mixture was neutralized with 1 M NaOH and filtered. The filtrate was extracted with CHCl₃ and H₂O. The H₂O-soluble fraction was evaporated to dryness. The dried sugar residues was diluted in 1 mL pyridine without water and treated with 0.5 mL trimethyl-chlorsilan (TMCS) and stirred at 60℃ for 5 min. After drying the solution with a stream of N₂, the residue was extracted with ether (1 mL). The ether layer was analyzed by GC under the following conditions: column, SGE AC-10 quartz capillary column (30 m × 0.32 mm × 0.25 μm); column temperature 180–280℃; programmed increase, 3 ℃/min; carrier gas, N₂ (2 ml/min); injector and detector temperature, 250℃; injection volume, 2 μL; split ratio, 1/50. Peaks of the hydrolysate were detected by comparison with retention times of authentic samples of glucose and rhamnose after treatment with trimethyl-chlorsilan (TMCS) in pyridine. The absolute configurations of the sugar residues were determined to be Lrhamnose (t_R 7.67 min) and D-glucose (t_R 14.22 min).

Cell-Growth Inhibition Assay. Growth inhibition of compounds on tumor cells was determined by microculture 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium hydrobromide (MTT) assay.²²

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-011-0039-z and is accessible for authorized users.

Notes

Acknowledgments

This work was financially supported by National Natural Science Funding of China (No. 31170333), the Natural Science Foundation of Yunnan Province (No. 2009CC019), the West Light Foundation of the Chinese Academy of Sciences (No. 2908025712W1), and a fund (No.540806321211) of State Key Laboratory of Phytochemistry and Plant Resources in West China, Germplasm Bank of Wild Species and CAS Innovation Program of Kunming Institute of Botany.