Spirostane-Type Saponins Obtained from Yucca schidigera

Abstract

It is well known that spirostane-type saponins show various bioactivities. In our on-going program of screening these kinds of constituents from natural products, Yucca schidigera was found to be rich in them, and nine new spirostanol saponins, Yucca spirostanosides A₁ (1), A₂ (2), B₁ (3), B₂ (4), B₃ (5), C₁ (6), C₂ (7), C₃ (8), and D₁ (9), together with five known ones (10–14) were isolated from the plant. Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D NMR and MS spectra, and comparing with published data.

1. Introduction

As one of the secondary metabolites, spirostanol saponins have been found to have broad bioactivities, such as antiproliferative, anti-inflammatory [1,2,3,4], anti-HIV [5], anti-bacterial [6], anti-fungi [7], and anti-hyperuricemic [8] activities, which make the phytochemical or bioactive researches for spirostanol saponins meaningful.

In our on-going program of investigating spirostanol saponins [3,4,8] from natural products, we found that Yucca schidigera Roezl (Agavaceae family) is a plant rich in these kinds of constituents. As one of the major industrial sources of steroid saponins, Y. schidigera is native to the desert of the southwestern United States and northern Baja California, Mexico [9]. The commercial extracts of Y. schidigera are approved by the FDA as GRAS (Generally Recognized as Safe) and widely used as animal and human food additives [10].

Then, the isolation of spirostanol saponins from Y. schidigera stems was studied, which led to the separation of nine new spirostane-type saponins, Yucca spirostanosides A₁ (1), A₂ (2), B₁ (3), B₂ (4), B₃ (5), C₁ (6), C₂ (7), C₃ (8), and D₁ (9), along with five known ones, schidigera-saponins A3 (10) and A1 (11) [9], 5β-spirost-25(27)-en-3β-ol-12-one 3-O-{β-d-glucopyranosyl-(1→2)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranoside} (12) [11], schidigera-saponins C2 (13) and C1 (14) [9]. In this paper, their structures were determined by analysis of physical data, spectroscopic analysis, and chemical methods.

2. Results and Discussion

The 70% EtOH extract of Y. schidigera stems were subjected to D101 column chromatography (CC) (H₂O → 95% EtOH), and 95% EtOH eluate was yielded, which was separated by Silica gel, ODS, and finally preparative HPLC to afford compounds 1–14. The structures of them are shown in Figure 1.

Yucca spirostanoside A₁ (1) was obtained as a white powder with negative optical rotation (${\left[\alpha \right]}_D^{25}$ −45.4°, MeOH). The molecular formula, C₃₃H₅₂O₈, of 1 was established by positive-ion HRESI-TOF-MS (m/z 577.3758 [M + H]⁺, calcd for C₃₃H₅₃O₈, 577.3735). The IR spectrum showed absorption bands ascribable to hydroxyl (3370 cm⁻¹), terminal olefinic bond (1651, 1019, 921 cm⁻¹), and O-glycosidic linkage (1077 cm⁻¹). Acid hydrolysis of it yielded d-glucose, which was identified by retention time and optical rotation using chiral detection by HPLC analysis [12]. Thirty-three carbon signals were displayed in the ¹³C NMR (

Table 1. ¹³C NMR data for 1–9 in C₅D₅N.

NO.	1	2	3	4	5	6	7	8	9
1	31.0	30.9	31.0	31.0	31.0	30.6	30.6	30.5	39.6
2	27.0	27.0	26.9	26.9	26.8	26.7	26.6	26.7	66.7
3	74.3	74.4	74.3	74.4	75.7	73.9	74.0	75.0	79.7
4	30.5	30.4	30.5	30.4	31.0	30.2	30.1	30.7	30.5
5	37.0	37.0	36.8	36.8	36.7	36.5	36.5	36.2	35.3
6	27.0	27.0	27.1	27.1	27.2	26.8	26.8	27.0	26.0
7	26.8	26.8	26.7	26.7	26.8	26.4	26.4	26.6	26.3
8	35.6	35.6	34.7	34.7	34.8	34.7	34.7	34.9	34.6
9	40.3	40.3	39.4	39.4	39.6	41.9	41.9	42.2	42.8
10	35.2	35.3	35.3	35.3	35.4	35.7	35.7	35.9	37.3
11	21.2	21.2	31.4	31.5	31.6	37.7	37.7	37.9	37.8
12	40.3	40.3	79.4	79.4	79.5	213.0	213.0	213.2	212.6
13	40.9	41.0	46.7	46.7	46.9	55.6	55.6	55.8	55.4
14	56.5	56.5	55.3	55.3	55.4	56.0	56.0	56.2	55.6
15	32.1	32.1	31.9	31.9	32.0	31.4	31.4	31.6	31.3
16	81.6	81.6	81.7	81.7	81.8	80.1	80.1	80.3	80.0
17	63.2	63.2	63.0	63.0	63.2	54.3	54.3	54.5	54.2
18	16.6	16.6	11.2	11.2	11.3	16.1	16.1	16.2	15.8
19	23.9	23.9	23.8	23.8	24.0	23.0	23.1	23.3	22.8
20	41.9	41.9	42.9	42.9	43.1	42.5	42.5	42.7	42.4
21	15.0	15.0	14.3	14.3	14.5	13.9	13.9	14.0	13.7
22	109.4	109.4	109.7	109.7	109.9	109.5	109.5	109.7	109.3
23	33.3	33.3	33.4	33.4	33.5	33.2	33.2	33.4	33.1
24	29.0	29.0	29.0	29.1	29.2	28.9	28.9	29.1	28.7
25	144.4	144.4	144.6	144.6	144.7	144.2	144.2	144.4	144.1
26	65.0	65.0	65.1	65.1	65.2	65.1	65.1	65.3	64.9
27	108.7	108.7	108.6	108.6	108.8	108.9	108.9	109.0	108.7
1′	103.1	102.5	103.1	102.6	102.1	102.9	102.3	102.0	101.6
2′	75.3	74.2	75.4	74.3	80.1	75.4	74.2	77.8	77.1
3′	78.7	87.8	78.8	87.8	88.4	78.7	87.7	84.3	84.1
4′	71.8	69.6	71.8	69.6	70.1	71.7	69.5	70.0	69.7
5′	78.4	78.1	78.4	78.1	78.0	78.4	78.1	76.6	76.6
6′	62.9	62.5	62.9	62.5	62.6	62.8	62.3	62.5	61.8
1′′		106.3		106.4	104.4		106.3	104.6	104.2
2′′		75.3		75.4	76.6		75.3	76.5	76.2
3′′		78.1		78.2	78.4		78.1	78.5	78.5
4′′		70.9		70.9	72.6		70.9	72.9	72.6
5′′		67.4		67.4	78.4		67.4	78.1	77.8
6′′					63.5			63.6	63.3
1′′′					105.0			105.5	106.0
2′′′					75.5			75.5	75.0
3′′′					78.7			78.7	78.3
4′′′					71.8			71.7	70.9
5′′′					78.8			78.5	67.0
6′′′					62.6			62.7

, C₅D₅N) spectrum. In addition to the carbon signals represented by d-glucose, the other 27 indicated 1 was a spirostane-type steroid saponin. Its ¹H NMR spectrum showed signals for two tertiary methyl groups at δ 0.84, 0.85 (3H each, both s, H₃-18, 19), a secondary methyl group at δ 1.11 (3H, d, J = 7.0 Hz, H₃-21), one oxygenated methylene group at δ 4.04, 4.48 (1H each, both d, J = 12.5 Hz, H₂-26), two oxygenated methine protons at δ [4.37 (1H, m, H-3), 4.61 (1H, q like, ca. J = 8 Hz, H-16)], one terminal olefinic moiety at δ 4.79, 4.82 (1H each, both br. s, H₂-27), together with the sugar portion signal of one anomeric proton at δ 4.93 (1H, d, J = 8.0 Hz, H-1′). The ¹H–¹H COSY spectrum of 1 suggested the presence of three partial structures written in bold lines as shown in Figure 2. The planar structure of the aglycon was determined based on the key HMBC correlations from H₃-18 to C-12-14, C-17; H₃-19 to C-1, C-5, C-9, C-10; H₃-21 to C-17, C-20, C-22; H₂-26 to C-22; H₂-27 to C-24-26, which was very close to that of 5β-spirost-25(27)-en-3β-ol 3-O-β-d-glucopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside [9]. Thus, the aglycon of 1 was determined to be 5β-spirost-25(27)-en-3β-ol. Meanwhile, the long-range correlation from H-1′ to C-3 observed in the HMBC spectrum suggested d-glucose was attached to C-3 of the aglycon. On the basis of above mentioned evidence, the structure of Yucca spirostanoside A₁ (1) was identified as 5β-spirost-25(27)-en-3β-ol 3-O-β-d-glucopyranoside.

The molecular formula of Yucca spirostanoside A₂ (2) was assigned as C₃₈H₆₀O₁₂ on the basis of ¹³C NMR data and positive-ion HRESI-TOF-MS (m/z 709.4178 [M + H]⁺, calcd for C₃₈H₆₁O₁₂, 709.4158). A detailed comparison between compounds 2 and 1 indicated that they have the consistent ¹H and ¹³C NMR spectroscopic data from their aglycon moieties (Table 1), except for the signals due to the sugar moieties. Meanwhile, its ¹H NMR spectrum suggested the presence of two anomeric proton signals at δ 4.91 (1H, d, J = 7.5 Hz, H-1′) and 5.26 (1H, d, J = 8.0 Hz, H-1′′), which correlated to the corresponding anomeric carbon signals at δ_C 102.5 (C-1′) and 106.3 (C-1′′), respectively. With the help of ¹H–¹H COSY, HSQC, and HMBC NMR analysis, the ¹H and ¹³C NMR chemical shifts for the sugar moiety were assignable. On acid hydrolysis, 2 yielded d-glucose and d-xylose [12]. Furthermore, the sugar sequence was consolidated by key HMBC correlations from δ_H 4.91 (H-1′) to δ_C 74.4 (C-3); δ_H 5.26 (H-1′′) to δ_C 87.8 (C-3′). Consequently, the structure of 2 was elucidated to be 5β-spirost-25(27)-en-3β-ol 3-O-β-d-xylopyranosyl(1→3)-β-d-glucopyranoside.

Yucca spirostanoside B₁ (3) was determined to possess the molecular formula, C₃₃H₅₂O₉ by its quasi-molecular ion peak at m/z 593.3700 [M + H]⁺ (calcd for C₃₃H₅₃O₉, 593.3684) in the positive HRESI-TOF-MS experiment, which was 16 amu greater than that of 1. Moreover, the ¹³C NMR signals of 3 were coincident with those of 1 except for the C ring carbons. Furthermore, comparing the DEPT spectrum of compound 3 with that of 1, showed that 3 had one oxygenated methine more and one methylene less than 1. According to the HMBC correlations from δ_H 1.09 (H₃-18) to δ_C 79.4 (C-12), δ_H 3.54 (H-12) to δ_C 31.4 (C-11), 46.7 (C-13), the position of the oxygenated methine was determined. Meanwhile, the configuration of C-12 hydroxyl group in 3 was deduced to be β by comparing carbon signals of C-11–14, 17, and 18 (δ_C 11.2 (C-18), 31.4 (C-11), 46.7 (C-13), 55.3 (C-14), 63.0 (C-17), 79.4 (C-12)) with those of its similar compounds, (25R)-26-O-β-d-glucopyranosyl-5β-furostane-3β,12β,22,26-tetraol-3-O-β-d-glucopyranosyl(1→2)-β-d-galactopyranoside (δ_C 11.3 (C-18), 31.4 (C-11), 47.0 (C-13), 55.2 (C-14), 63.8 (C-17), 79.6 (C-12)) [13] and (25R)-26-O-β-d-glucopyranosyl-5β-furostane-3β,12α,22,26-tetraol-3-O-β-d-glucopyranosyl(1→2)-β-d-galactopyranoside (δ_C 17.5 (C-18), 29.6 (C-11), 45.8 (C-13), 48.6 (C-14), 54.6 (C-17), 71.7 (C-12)) [14]. Finally, the β-d-glucopyranosyl was proved to link at C-3 of aglycon by the observed HMBC correlation from δ_H 4.93 (H-1′) to δ_C 74.3 (C-3). Thus, the structure of 3 was determined to be 5β-spirost-25(27)-en-3β,12β-diol 3-O-β-d-glucopyranoside.

Yucca spirostanoside B₂ (4) was isolated as white powder with negative optical rotation (${\left[\alpha \right]}_D^{25}$ –36.2°, MeOH). The molecular formula, C₃₈H₆₀O₁₃ of 4 was deduced by the positive-ion HRESI-TOF-MS signal at m/z 725.4121 [M + H]⁺ (calcd for C₃₈H₆₁O₁₃, 725.4107). The ¹H and ¹³C NMR (Table 1) spectroscopic data analysis indicated that 4 had the same aglycon, 5β-spirost-25(27)-en-3β,12β-diol as 3, and the same sugar moiety, β-d-xylopyranosyl(1→3)-β-d-glucopyranosyl as 2, which was supported by ¹H–¹H COSY, HSQC, and HMBC experiments (Figure 2). Moreover, the HMBC correlation from δ_H 4.91 (H-1′) to δ_C 74.4 (C-3) suggested β-d-xylopyranosyl(1→3)-β-d-glucopyranosyl was attached to C-3 of the aglycon. Therefore, the structure of 4 was established as 5β-spirost-25(27)-en-3β, 12β-diol 3-O-β-d-xylopyranosyl(1→3)-β-d-glucopyranoside.

The HRESI-MS of Yucca spirostanoside B₃ (5) showed the [M + Na]⁺ ion at m/z 939.4565 (calcd for C₄₅H₇₂O₁₉Na, 939.4560), consistent with the molecular formula of C₄₅H₇₂O₁₉. The ¹H and ¹³C NMR spectroscopic data comparison of 5, 4 and 3 revealed that all the three compounds have the same aglycon pattern and the difference was only in the signals due to the sugar moieties. Compound 5 was subjected to acid hydrolysis and give d-glucose only. The ¹H NMR spectrum of 5 indicated there were three β-d-glucopyranosyl moieties [δ 4.87 (1H, d, J = 7.5 Hz, H-1′), 5.35 (1H, d, J = 7.5 Hz, H-1′′′), 5.65 (1H, d, J = 7.5 Hz, H-1′′)]. A combination of HSQC, HSQC-TOCSY, and ¹H–¹H COSY spectra analysis led to the assignment of three β-d-glucopyranosyl units. In the HSQC-TOCSY spectrum, the correlations between the following proton and carbon pairs were observed: δ_H 4.87 (H-1′) and δ_C 70.1 (C-4′), 78.0 (C-5′), 80.1 (C-2′), 88.4 (C-3′), 102.1 (C-1′); δ_H 3.79 (H-5′) and δ_C 62.6 (C-6′); δ_H 5.65 (H-1′′) and δ_C 72.6 (C-4′′), 76.6 (C-2′′), 78.4 (C-3′′), 104.4 (C-1′′); δ_H 3.97 (H-5′′) and δ_C 63.5 (C-6′′), 72.6 (C-4′′), 78.4 (C-3′′ and 5′′); δ_C 104.4 (C-1′′) and δ_H 4.28 (H-3′′); δ_H 5.35 (H-1′′′) and δ_C 71.8 (C-4′′′), 75.5 (C-2′′′), 78.7 (C-3′′′), 105.0 (C-1′′′); δ_C 105.0 (C-1′′′) and δ_H 4.22 (H-3′′′). Once again, direct evidence of the sugar sequence and the linkage sites was derived from HSQC-TOCSY and HMBC experiments. The glycosidation shifts on C-3 (δ 75.7), C-2′ (δ 80.1), and C-3′ (δ 88.4) suggested the linkage sites. Long-range correlations from δ_H 4.87 (H-1′) to δ_C 75.7 (C-3); δ_H 5.65 (H-1′′) to δ_C 80.1 (H-2′); δ_H 5.35 (H-1′′′) to δ_C 88.4 (H-3′) were observed in the HMBC spectrum of it. Therefore, compound 5 was established as 5β-spirost-25(27)-en-3β, 12β-diol 3-O-β-d-glucopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranoside.

Yucca spirostanoside C₁ (6) presented as a white powder with negative optical rotation (${\left[\alpha \right]}_D^{25}$ −8.9°, MeOH). The positive-ion HRESI-TOF-MS spectrum of 6 (m/z 591.3557 [M + H]⁺, calcd for C₃₃H₅₁O₉, 591.3528) supported a molecular formula of C₃₃H₅₀O₉, two proton less than that of 3. Meanwhile, the ¹³C NMR spectrum of 6 was similar to that of 3, except for the signals due to the C-ring carbons of aglycon moiety. The above-mentioned evidence suggested the hydroxyl at C-12 in 3 changed into carboxyl in 6, which was identified by the presence of carbon signal at δ_C 213.0 (C-12) and the long-range correlations from δ_H 1.10 (H₃-18), 1.49 (H-14), 1.76 (H-9), 2.21, 2.38 (H₂-11), 2.83 (H-17) to δ_C 213.0 (C-12) showed in HMBC spectrum of 6. On the other hand, the ¹H, ¹³C NMR (Table 1, C₅D₅N) and 2D NMR (¹H–¹H COSY, HSQC, HMBC) experiments suggested the aglycon moiety of 6 was identical with that of 5β-spirost-25(27)-en-3β-ol-12-one 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl (1→2)]-β-d-glucopyranoside [9], which indicated the aglycon of 6 was 5β-spirost-25(27)-en-3β-ol-12-one. On acid hydrolysis, 6 afforded glucose [12]. Finally, the linkage position of β-d-glucopyranosyl was identified by the HMBC correlation from δ_H 4.94 (H-1′) to δ_C 73.9 (C-3). Then, 6 was formulated as 5β-spirost-25(27)-en-3β-ol-12-one 3-O-β-d-glucopyranoside.

The molecular formula of Yucca spirostanoside C₂ (7) was established as C₃₈H₅₈O₁₃ by positive-ion HRESI-TOF-MS analysis (m/z 723.3959 [M + H]⁺, calcd for C₃₈H₅₉O₁₃, 723.3950). The ¹H and ¹³C NMR (Table 1) spectroscopic data for aglycon of 7 were identical to those of 6. The remaining eleven carbon signals were assigned to the sugar moiety, which was determined to be β-d-xylopyranosyl(1→3)-β-d-glucopyranosyl group by comparing the NMR data of it with those of compounds 2 and 4. Finally, according to the ¹H–¹H COSY, HSQC, and HMBC experiments, the structure of 7 was identified as 5β-spirost-25(27)-en-3β-ol-12-one 3-O-β-d-xylopyranosyl(1→3)-β-d-glucopyranoside.

Yucca spirostanoside C₃ (8) was obtained as white powder, and its molecular formula was deduced as C₄₅H₇₀O₁₉ from [M + Na]⁺ quasi-molecular ion at m/z 937.4412 (calcd for C₄₅H₇₀O₁₉Na, 937.4404 ) in the positive-ion HRESI-TOF-MS spectrum. On acid hydrolysis, 8 gave d-glucose and d-galactose as sugar components. The ¹H and ¹³C NMR (Table 1) spectra indicated that 8 possessed the same aglycon, 5β-spirost-25(27)-en-3β-ol-12-one as that of 6 and 7. The presence of one β-d-galactopyranosyl (δ 4.83 (1H, d, J = 7.5 Hz, H-1′)) and two β-d-glucopyranosyl (δ 5.37 (1H, d, J = 7.5 Hz, H-1′′′), 5.57 (1H, d, J = 7.5 Hz, H-1′′)) were suggested by its ¹H and ¹³C NMR spectra, too. The linkage positions between sugar and sugar, as well as aglycon and sugar were elucidated by HMBC correlations from δ_H 4.83 (H-1′) to δ_C 75.0 (C-3); δ_H 5.57 (H-1′′) to δ_C 77.8 (C-2′); δ_H 5.37 (H-1′′′) to δ_C 84.3 (C-3′′). The combined use of ¹H–¹H COSY, HSQC, and HSQC-TOCSY experiments allowed the sequential assignments of all resonances for each monosaccharide. In the HSQC-TOCSY spectrum of 8, the correlations between δ_H 4.83 (H-1′) and δ_C 70.0 (C-4′), 77.8 (C-2′), 84.3 (C-3′), 102.0 (C-1′); δ_H 3.98 (H-5′) and δ_C 62.5 (C-6′), 70.0 (C-4′), 76.6 (C-5′); δ_H 5.57 (H-1′′) and δ_C 72.9 (C-4′′), 76.5 (C-2′′), 78.5 (C-3′′), 104.6 (C-1′′); δ_H 3.81 (H-5′′) and δ_C 63.6 (C-6′′), 72.9 (C-4′′), 76.5 (C-2′′), 78.1 (C-5′′), 78.5 (C-3′′); δ_H 5.37 (H-1′′′) and δ_C 71.7 (C-4′′′), 75.5 (C-2′′′), 78.7 (C-3′′′), 105.5 (C-1′′′); δ_H 3.92 (H-5′′′) and δ_C 62.7 (C-6′′′), 71.7 (C-4′′′), 78.5 (C-5′′′), 78.7 (C-3′′′) were found. On the basis of above mentioned evidence, the structure of Yucca spirostanoside C₃ (8) was formulated as 5β-spirost-25(27)-en-3β-ol-12-one 3-O-β-d-glucopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-galactopyranoside.

The molecular formula of Yucca spirostanoside D₁ (9) was elucidated as C₄₄H₆₈O₁₉ from [M + Na]⁺ quasi-molecular ion at m/z 923.4293 (calcd for C₄₄H₆₈O₁₉Na, 923.4247) in the positive-ion HRESI-TOF-MS spectrum. Acid hydrolysis of it yielded d-galactose, d-glucose, and d-xylose [12]. The ¹H and ¹³C NMR (Table 1) suggested the presence of one β-d-galactopyranosyl [δ 4.98 (1H, d, J = 7.5 Hz, H-1′)], one β-d-glucopyranosyl (δ 5.57 (1H, d, J = 8.0 Hz, H-1′′)], together with one β-d-xylopyranosyl (δ 5.22 (1H, d, J = 7.5 Hz, H-1′′′)). The ¹³C NMR signals due to the sugar moieties of 9 were in good agreement with those of schidigera-saponin A₂, which was 5β-spirost-25(27)-en-3β-ol 3-O-β-d-xylopyranosyl(1→3)[β-d-glucopyranosyl(1→2)]-β-d-galactopyranoside [9]. To assign the badly overlapped protons in sugar chemical shift range, HSQC-TOCSY and ¹H−¹H COSY experiments were determined. In the HSQC-TOCSY spectrum, the correlations between the following proton and carbon pairs were observed: δ_H 4.98 (H-1′) and δ_C 69.7 (C-4′), 77.1 (C-2′), 84.1 (C-3′), 101.6 (C-1′); δ_H 5.57 (H-1′′) and δ_C 72.6 (C-4′′), 76.2 (C-2′′), 78.5 (C-3′′), 104.2 (C-1′′); δ_H 3.77 (H-5′′) and δ_C 63.3 (C-6′′), 72.6 (C-4′′), 77.8 (C-5′′); δ_H 5.22 (H-1′′′) and δ_C 67.0 (C-5′′′), 70.9 (C-4′′′), 75.0 (C-2′′′), 78.3 (C-3′′′), 106.0 (C-1′′′); δ_H 4.38 (H-5′) and δ_C 61.8 (C-6′), 76.6 (C-5′). Meanwhile, the correlations between δ_H 4.11 (H-5′) and δ_H 4.39 (H₂-6′), 4.74 (H-4′) were found in the ¹H–¹H COSY spectrum. Meanwhile, the ¹³C NMR data for aglycon of 9 were almost superimposable on those of 6 except for the signals due to the A-ring carbons. Moreover, comparing the ¹³C NMR data of C-1–6 and C-10 of 9 [δ_C 26.0 (C-6), 30.5 (C-4), 35.3 (C-5), 37.3 (C-10), 39.6 (C-1), 66.7 (C-2), 79.7 (C-3)] with those of 3-O-β-d-xylopyranosyl(1→3)[β-d-glucopyranosyl(1→2)]-β-d-galactopyranosyl-5β-spirost-25(27)-ene-2β,3β-diol (schidigera-saponin C1) [δ_C 26.4 (C-6), 31.8 (C-4), 36.1 (C-5), 37.1 (C-10), 40.3 (C-1), 67.1 (C-2), 81.6 (C-3)], which obtained Y. schidigera [9], the aglycon of 9 was clarified as 5β-spirost-25(27)-en-2β,3β-diol-12-one. Finally, in the HMBC experiment (Figure 2), long-range correlations were observed from δ_H 4.98 (H-1′) to δ_C 79.7 (C-3); δ_H 5.57 (H-1′′) to δ_C 77.1 (C-2′); δ_H 5.22 (H-1′′′) to δ_C 84.1 (C-3′), then the connectivities between oligoglycoside moieties and aglycon were characterized. Thus, the structure of Yucca spirostanoside D₁ (9) was elucidated to be 5β-spirost-25(27)-en-2β,3β-diol-12-one 3-O-β-d-xylopyranosyl(1→3)-[β-d-glucopyranosyl(1→2)]-β-d-galactopyranoside.

The structures of known compounds 10–14 were identified by comparing their ¹H, ¹³C NMR data with references.

3. Experimental

3.1. General

Optical rotations were measured on a Rudolph Autopol^® IV automatic polarimeter (l = 50 mm) (Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectra were recorded on a Varian 640-IR FT-IR spectrophotometer (Varian Australia Pty Ltd., Mulgrave, Australia). NMR spectra were determined on a Bruker 500 MHz NMR spectrometer (Bruker BioSpin AG Industriestrasse 26 CH-8117, Fällanden, Switzerland) at 500 MHz for ¹H and 125 MHz for ¹³C NMR (internal standard: TMS). Positive-ion mode HRESI-TOF-MS were obtained on an Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer (Agilent Corp., Santa Clara, CA, USA).

Column chromatographies (CC) were performed on macroporous resin D101 (Haiguang Chemical Co., Ltd., Tianjin, China), Silica gel (48–75 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and ODS (40–63 μm, YMC Co., Ltd., Tokyo, Japan). Preparative high-performance liquid chromatography (PHPLC) columns, Cosmosil 5C₁₈-MS-II (20 mm i.d. × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan), Wacopak Navi C₃₀-5 (7.5 mm i.d. × 250 mm, Wako Pure Chemical Industries, Ltd., Osaka, Japan), and Cosmosil PBr (20 mm i.d. × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) were used to separate the constituents.

3.2. Plant Material

The stems of Y. schidigera were collected from the State of Florida, the United States of America, and identified by Dr. Li Tianxiang (The Hall of TCM Specimens, Tianjin University of TCM, China). The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20160301).

3.3. Extraction and Isolation

The dried stems of Y. schidigera (5.0 kg) were refluxed with 70% ethanol-water for three times. Evaporation of the solvent under pressure provided a 70% ethanol-water (800.0 g). The residue (700.0 g) was dissolved in H₂O, and subjected to D101 CC (H₂O → 95% EtOH) to afford H₂O (380.4 g) and 95% EtOH (310.1 g) eluates, respectively.

The 95% EtOH eluate (200.0 g) was subjected to silica gel CC (CH₂Cl₂ → CH₂Cl₂–MeOH (100:1 → 100:3 → 100:7 → 5:1 → 3:1 → 2:1, v/v) → MeOH) to afford 13 fractions (Fr. 1–Fr. 13). Fraction 6 (12.0 g) was separated by ODS CC [MeOH–H₂O (30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)], and fourteen fractions (Fr. 6-1–Fr. 6-14) were obtained. Fraction 6-11 (596.5 mg) was purified by PHPLC (CH₃CN–1% CH₃COOH (40:60, v/v), Cosmosil 5C₁₈-MS-II column) to give Yucca spirostanosides B₁ (3, 12.5 mg, t_R 38.76′) and B₂ (4, 15.6 mg, t_R 29.24′). Fraction 6-12 (800.9 mg) was isolated by PHPLC (MeOH–1% CH₃COOH (75:25, v/v), Cosmosil 5C₁₈-MS-II column) to provide eight fractions (Fr. 6-12-1–Fr. 6-12-8). Fraction 6-12-4 (143.4 mg) was purified by PHPLC (CH₃CN–1% CH₃COOH (48:52, v/v), Cosmosil 5C₁₈-MS-II column) to gain Yucca spirostanoside C₂ (7, 49.4 mg, t_R 23.06′). Fraction 6-12-5 (165.4 mg) was separated by PHPLC (CH₃CN–1% CH₃COOH (45:55, v/v), Cosmosil 5C₁₈-MS-II column) to yield Yucca spirostanoside C₁ (6, 32.2 mg, t_R 21.02′). Fraction 6-13 (1.2 g) was subjected to silica gel CC (CH₂Cl₂–MeOH (100:3 → 100:5 → 100:7) → MeOH, v/v) to produce nine fractions (Fr. 6-13-1–Fr. 6-13-9). Fraction 6-13-3 (446.3 mg) was isolated by PHPLC [MeOH–1% CH₃COOH (90:10, v/v), Cosmosil 5C₁₈-MS-II column] to provide Yucca spirostanoside A₁ (1, 42.1 mg, t_R 33.03′). Fraction 6-13-5 (740.6 mg) was further purified by PHPLC (MeOH–1% CH₃COOH (85:15, v/v), Cosmosil 5C₁₈-MS-II column) to give Yucca spirostanoside A₂ (2, 56.6 mg, t_R 31.04′). Fraction 7 (10.0 g) was separated by PHPLC (MeOH–1% CH₃COOH (80:20, v/v), Cosmosil 5C₁₈-MS-II column), and 13 fractions (Fr. 7-1–Fr. 7-13) were obtained. Fraction 7-11 (984.6 mg) was isolated by PHPLC (MeOH–1% CH₃COOH (95:5, v/v), Cosmosil PBr column) to gain four fractions (Fr. 7-11-1–Fr. 7-11-4). Fraction 7-11-4 (60.8 mg) was purified by PHPLC (CH₃CN–1% CH₃COOH (55:45, v/v), Wacopak Navi C₃₀-5 column) to provide schidigera-saponin A1 (11, 10.3 mg, t_R 42.24′). Fraction 8 (10.0 g) was separated by PHPLC (MeOH–1% CH₃COOH (80:20, v/v), Cosmosil 5C₁₈-MS-II column], and 16 fractions (Fr. 8-1–Fr. 8-16) were given. Fraction 8-15 (80.4 mg) was purified by PHPLC (MeOH–1% CH₃COOH (75:25, v/v), Cosmosil 5C₁₈-MS-II column) to yield schidigera-saponin C2 (13, 40.2 mg, t_R 26.17′). Fraction 9 (12.4 g) was isolated by PHPLC (MeOH–H₂O (80:20, v/v) + 1% CH₃COOH, Cosmosil 5C₁₈-MS-II column) to afford 16 fractions (Fr. 9-1–Fr. 9-16). Fraction 9-5 (120.1 mg) was purified by PHPLC (CH₃CN–1% CH₃COOH (32:68, v/v), Cosmosil PBr column) to yield Yucca spirostanosides B₃ (5, 10.0 mg, t_R 65.87′) and D₁ (9, 33.5 mg, t_R 64.75′). Fraction 9-7 (220.2 mg) was separated by PHPLC (MeOH–1% CH₃COOH (85:15, v/v), Cosmosil 5C₁₈-MS-II column] to gain Yucca spirostanoside C₃ (8, 11.7 mg, t_R 42.85′). Fraction 9-8 (549.4 mg) was purified by PHPLC (CH₃CN–1% CH₃COOH (38:62, v/v), Cosmosil 5C₁₈-MS-II column] to produce 5β-spirost-25(27)-en-3β-ol-12-one 3-O-{β-d-glucopyranosyl-(1→2)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranoside} (12, 253.8 mg, t_R 46.73′). Fraction 9-15 (151.9 mg) was subjected to PHPLC (CH₃CN–1% CH₃COOH (45:55, v/v), Cosmosil 5C₁₈-MS-II column] to obtain schidigera-saponin C1 (14, 97.8 mg, t_R 28.06′). Fraction 9-16 (400.0 mg) was separated by PHPLC (MeOH–1% CH₃COOH (85:15, v/v), Cosmosil 5C₁₈-MS-II column) to provide six fractions (Fr. 9-16-1–Fr. 9-16-6). Fraction 9-16-5 (189.1 mg) was further purified by PHPLC [MeOH–1% CH₃COOH (80:20, v/v), Wacopak Navi C₃₀-5 column] to afford schidigera-saponin A3 (10, 68.0 mg, t_R 23.04′).

Yucca spirostanoside A₁ (1): White powder; ${\left[\alpha \right]}_D^{25}$ −45.4° (c = 0.41, MeOH); IR ν_max (KBr) cm⁻¹: 3370, 2928, 1651, 1451, 1375, 1231, 1169, 1077, 1043, 1019, 921; ¹H NMR (C₅D₅N, 500 MHz) data: δ 1.47, 1.72 (1H each, both m, H₂-1), [1.54 (1H, m, overlapped), 1.90 (1H, m), H₂-2], 4.37 (1H, m, H-3), [1.75 (1H, m, overlapped), 1.82 (1H, m), H₂-4], 2.02 (1H, m, H-5), [1.09 (1H, m), 1.76 (1H, m, overlapped), H₂-6], [0.99 (1H, m), 1.30 (1H, m, overlapped), H₂-7], 1.51 (1H, m, H-8), 1.31 (1H, m, overlapped, H-9), 1.21, 1.33 (1H each, both m, H₂-11), [1.10 (1H, m, overlapped), 1.69 (1H, m), H₂-12], 1.11 (1H, m, overlapped, H-14), 1.42, 2.04 (1H each, both m, H₂-15), 4.61 (1H, q like, ca. J = 8 Hz, H-16), 1.86 (1H, dd, J = 6.5, 8.5 Hz, H-17), 0.84 (3H, s, H₃-18), 0.85 (3H, s, H₃-19), 1.98 (1H, m, H-20), 1.11 (3H, d, J = 7.0 Hz, H₃-21), 1.79 (2H, m, H₂-23), [2.25 (1H, m), 2.72 (1H, dt, J = 5.0, 13.0 Hz), H₂-24], [4.04 (1H, d, J = 12.5 Hz), 4.48 (1H, d, J = 12.5 Hz), H₂-26], 4.79, 4.82 (1H each, both br. s, H₂-27), 4.93 (1H, d, J = 8.0 Hz, H-1′), 4.03 (1H, dd, J = 8.0, 9.0 Hz, H-2′), 4.25 (2H, m, H-3′, 4′), 3.94 (1H, m, H-5′), [4.40 (1H, dd, J = 4.5, 12.0 Hz), 4.53 (1H, dd, J = 2.5, 12.0 Hz), H₂-6′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z - 577.3758 [M + H]⁺ (calcd for C₃₃H₅₃O₈, 577.3735).

Yucca spirostanoside A₁ (2): White powder; ${\left[\alpha \right]}_D^{25}$ –53.5° (c = 0.40, MeOH); IR ν_max (KBr) cm⁻¹: 3373, 2928, 1651, 1451, 1374, 1233, 1161, 1080, 1042, 922; ¹H NMR (C₅D₅N, 500 MHz) data: δ [1.47 (1H, m), 1.71 (1H, m, overlapped), H₂-1], [1.54 (1H, m, overlapped), 1.91 (1H, m), H₂-2], 4.34 (1H, m, H-3), [1.75 (1H, m, overlapped), 1.83 (1H, m), H₂-4], 2.02 (1H, m, H-5), 1.14, 1.80 (1H each, both m, H₂-6), [1.01 (1H, m), 1.31 (1H, m, overlapped), H₂-7], 1.53 (1H, m, overlapped, H-8), 1.32 (1H, m, overlapped, H-9), [1.23 (1H, m), 1.33 (1H, m, overlapped), H₂-11], 1.10,1.70 (1H each, both m, overlapped, H₂-12), 1.11 (1H, m, overlapped, H-14), 1.42, 2.05 (1H each, both m, H₂-15), 4.62 (1H, q like, ca. J = 8 Hz, H-16), 1.86 (1H, dd, J = 6.5, 8.5 Hz, H-17), 0.84 (3H, s, H₃-18), 0.87 (3H, s, H₃-19), 1.98 (1H, m, H-20), 1.10 (3H, d, J = 6.5 Hz, H₃-21), 1.78 (2H, m, H₂-23), [2.26 (1H, m), 2.72 (1H, dt, J = 5.0, 12.5 Hz), H₂-24], [4.04 (1H, d, J = 11.0 Hz), 4.48 (1H, d, J = 11.0 Hz), H₂-26], 4.79, 4.82 (1H each, both br. s, H₂-27), 4.91 (1H, d, J = 7.5 Hz, H-1′), 4.05 (1H, dd, J = 7.5, 8.0 Hz, H-2′), 4.22 (1H, dd, J = 8.0, 9.0 Hz, H-3′), 4.15 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.89 (1H, m, H-5′), [4.32 (1H, dd, J = 5.0, 12.0 Hz), 4.47 (1H, dd, J = 2.5, 12.0 Hz), H₂-6′], 5.26 (1H, d, J = 8.0 Hz, H-1′′), 4.00 (1H, dd, J = 8.0, 8.5 Hz, H-2′′), 4.11 (1H, dd, J = 8.5, 8.5 Hz, H-3′′), 4.13 (1H, m, H-4′′), [3.67 (1H, dd, J = 11.0, 11.0 Hz), 4.29 (1H, dd, J = 4.5, 11.0 Hz), H₂-5′′]; ¹³C NMR (C₅D₅N, 125 MHz) data: see Table 1; HRESI-TOF-MS Positive-ion mode m/z 709.4178 [M + H]⁺ (calcd for C₃₈H₆₁O₁₂, 709.4158).

Yucca spirostanoside B₁ (3): White powder; ${\left[\alpha \right]}_D^{25}$ −45.2° (c = 0.42, MeOH); IR ν_max (KBr) cm⁻¹: 3385, 2928, 2857, 1649, 1451, 1372, 1234, 1158, 1078, 1040, 919; ¹H NMR (C₅D₅N, 500 MHz) δ [1.46 (1H, m), 1.77 (1H, m, overlapped), H₂-1], 1.53, 1.90 (1H each, both m, H₂-2), 4.35 (1H, m, H-3), [1.75 (1H, m, overlapped), 1.81 (1H, m), H₂-4], 2.04 (1H, m, H-5), [1.12 (1H, m), 1.76 (1H, m, overlapped), H₂-6], 0.98, 1.34 (1H each, both m, H₂-7), 1.55 (1H, m, H-8), 1.49 (1H, m, overlapped, H-9), 1.50, 1.78 (1H each, both m, overlapped, H₂-11), 3.54 (1H, dd, J = 5.5, 10.0 Hz, H-12), 1.16 (1H, m, H-14), 1.61, 2.11 (1H each, both m, H₂-15), 4.70 (1H, q like, ca. J = 8 Hz, H-16), 2.23 (1H, m, overlapped, H-17), 1.09 (3H, s, H₃-18), 0.87 (3H, s, H₃-19), 2.23 (1H, m, overlapped, H-20), 1.39 (3H, d, J = 6.5 Hz, H₃-21), 1.84 (2H, m, H₂-23), [2.26 (1H, m), 2.75 (1H, dt, J = 6.0, 13.0 Hz), H₂-24], [4.06 (1H, d, J = 12.0 Hz), 4.54 (1H, d, J = 12.0 Hz), H₂-26], 4.79, 4.82 (1H each, both br. s, H₂-27), 4.94 (1H, d, J = 8.0 Hz, H-1′), 4.04 (1H, dd, J = 8.0, 9.0 Hz, H-2′), 4.25 (2H, m, H-3′, 4′), 3.96 (1H, m, H-5′), [4.39 (1H, dd, J = 5.5, 12.0 Hz), 4.55 (1H, dd, J = 2.5, 12.0 Hz), H₂-6′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z 593.3700 [M + H]⁺ (calcd for C₃₃H₅₃O₉, 593.3684).

Yucca spirostanoside B₂ (4): White powder; ${\left[\alpha \right]}_D^{25}$ –36.2° (c = 0.37, MeOH); IR ν_max (KBr) cm⁻¹: 3389, 2928, 2872, 1650, 1452, 1373, 1264, 1161, 1079, 1041, 921; ¹H NMR (C₅D₅N, 500 MHz) δ [1.48 (1H, m), 1.77 (1H, m, overlapped), H₂-1], 1.51, 1.86 (1H each, both m, H₂-2), 4.32 (1H, m, H-3), 1.75, 1.81 (1H each, both m, overlapped, H₂-4), 2.04 (1H, m, H-5), 1.16, 1.80 (1H each, both m, overlapped, H₂-6), 1.00, 1.34 (1H each, both m, H₂-7), 1.56 (1H, m, H-8), 1.49 (1H, m, overlapped, H-9), 1.50, 1.79 (1H each, both m, overlapped, H₂-11), 3.55 (1H, dd, J = 5.0, 10.0 Hz, H-12), 1.16 (1H, m, overlapped, H-14), 1.61, 2.11 (1H each, both m, H₂-15), 4.70 (1H, q like, ca. J = 8 Hz, H-16), 2.23 (1H, m, overlapped, H-17), 1.09 (3H, s, H₃-18), 0.89 (3H, s, H₃-19), 2.23 (1H, m, overlapped, H-20), 1.40 (3H, d, J = 6.0 Hz, H₃-21), 1.84 (2H, m, H₂-23), [2.26 (1H, m), 2.76 (1H, dt, J = 5.5, 13.0 Hz), H₂-24], [4.06 (1H, m, overlapped), 4.53 (1H, d, J = 12.0 Hz), H₂-26], 4.79, 4.82 (1H each, both br. s, H₂-27), 4.91 (1H, d, J = 8.0 Hz, H-1′), 4.06 (1H, m, overlapped, H-2′), 4.24 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 4.18 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.89 (1H, m, H-5′), [4.30 (1H, dd, J = 5.0, 12.0 Hz), 4.48 (1H, br. d, ca. J = 12 Hz), H₂-6′], 5.28 (1H, d, J = 7.5 Hz, H-1′′), 4.02 (1H, dd, J = 7.5, 8.0 Hz, H-2′′), 4.13 (1H, dd, J = 8.0, 9.0 Hz, H-3′′), 4.15 (1H, m, H-4′′), [3.69 (1H, dd, J = 11.5, 11.5 Hz), 4.29 (1H, dd, J = 4.5, 11.5 Hz), H₂-5′′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z 725.4121 [M + H]⁺ (calcd for C₃₈H₆₁O₁₃, 725.4107).

Yucca spirostanoside B₃ (5): White powder; ${\left[\alpha \right]}_D^{25}$ –33.0° (c = 0.23, MeOH); IR ν_max (KBr) cm⁻¹: 3364, 2928, 2861, 1648, 1452, 1369, 1264, 1159, 1078, 1039, 920; ¹H NMR (C₅D₅N, 500 MHz) δ 1.46, 1.84 (1H each, both m, overlapped, H₂-1), [1.47 (1H, m, overlapped), 1.88 (1H, m), H₂-2], 4.30 (1H, m, overlapped, H-3), 1.84 (2H, m, H₂-4), 2.19 (1H, m, H-5), [1.20 (1H, m), 1.82 (1H, m, overlapped), H₂-6], 0.96, 1.31 (1H each, both m, H₂-7), 1.55 (1H, m, H-8), 1.47 (1H, m, overelapped, H-9), 1.50, 1.77 (1H each, both m, H₂-11), 3.55 (1H, dd, J = 5.5, 10.0 Hz, H-12), 1.13 (1H, m, H-14), 1.60, 2.11 (1H each, both m, H₂-15), 4.71 (1H, q like, ca. J = 8 Hz, H-16), 2.23 (1H, m, overlapped, H₃-17), 1.09 (3H, s, H₃-18), 0.97 (3H, s, H₃-19), 2.23 (1H, m, overlapped, H-20), 1.40 (3H, d, J = 6.0 Hz, H₃-21), 1.84 (2H, m, overlapped, H₂-23), [2.27 (1H, m), 2.76 (1H, dt, J = 6.0, 13.0 Hz), H₂-24], [4.08 (1H, d, J = 11.5 Hz), 4.53 (1H, d, J = 11.5 Hz), H₂-26], 4.79, 4.83 (1H each, both br. s, H₂-27), 4.87 (1H, d, J = 7.5 Hz, H-1′), 4.37 (1H, dd, J = 7.5, 9.0 Hz, H-2′), 4.27 (1H, m, overlapped, H-3′), 4.04 (1H, m, overlapped, H-4′), 3.79 (1H, m, H-5′), [4.23 (1H, m, overlapped), 4.41 (1H, m, overlapped), H₂-6′], 5.65 (1H, d, J = 7.5 Hz, H-1′′), 4.07 (1H, m, overlapped, H-2′′), 4.28 (1H, m, overlapped, H-3′′), 4.20 (1H, dd, J = 9.0, 9.0 Hz, H-4′′), 3.97 (1H, m, H-5′′), [4.43 (1H, m, overlapped), 4.56 (1H, m, overlapped), H₂-6′′], 5.35 (1H, d, J = 7.5 Hz, H-1′′′), 4.06 (1H, m, overlapped, H-2′′′), 4.22 (1H, dd, J = 8.0, 9.0 Hz, H-3′′′), 4.15 (1H, dd, J = 9.0, 9.0 Hz, H-4′′′), 4.04 (1H, m, H-5′′′), [4.28 (1H, m, overlapped), 4.54 (1H, m, overlapped), H₂-6′′′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z 939.4565 [M + Na]⁺ (calcd for C₄₅H₇₂O₁₉Na, 939.4560).

Yucca spirostanoside C₁ (6): White powder; [α${]}_D^{25}$ –8.9° (c = 0.09, MeOH); IR ν_max (KBr) cm⁻¹: 3388, 2928, 2872, 1703, 1650, 1452, 1375, 1266, 1164, 1078, 1042, 922; ¹H NMR (C₅D₅N, 500 MHz) δ [1.28 (1H, m), 1.75 (1H, m, overlapped), H₂-1], [1.42 (1H, m), 1.85 (1H, m, overlapped), H₂-2], 4.33 (1H, m, H-3), 1.74 (2H, m, overlapped, H₂-4), 2.09 (1H, m, H-5), [1.12 (1H, m), 1.76 (1H, m, overlapped), H₂-6], 0.98, 1.34 (1H each, both m, H₂-7), 1.84 (1H, m, overlapped, H-8), 1.76 (1H, m, overlapped, H-9), [2.21 (1H, dd, J = 4.5, 14.0 Hz), 2.38 (1H, dd, J = 14.0, 14.0 Hz), H₂-11], 1.49 (1H, m, H-14), [1.63 (1H, m), 2.15 (1H, m), H₂-15], 4.56 (1H, m, overlapped, H-16), 2.83 (1H, dd, J = 6.5, 8.5 Hz, H-17), 1.10 (3H, s, H₃-18), 0.86 (3H, s, H₃-19), 1.96 (1H, m, H-20), 1.34 (3H, d, J = 6.5 Hz, H₃-21), 1.77 (2H, m, overlapped, H₂-23), [2.26 (1H, m), 2.72 (1H, dt, J = 6.0, 13.0 Hz), H₂-24], [4.06 (1H, d, J = 12.5 Hz), 4.47 (1H, d, J = 12.5 Hz), H₂-26], 4.80, 4.84 (1H each, both br. s, H₂-27), 4.94 (1H, d, J = 8.0 Hz, H-1′), 4.07 (1H, m, overlapped, H-2′), 4.28 (2H, m, H-3′, 4′), 3.96 (1H, m, H-5′), [4.41 (1H, dd, J = 5.5, 12.0 Hz), 4.56 (1H, m, overlapped), H₂-6′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z 591.3557 [M + H]⁺ (calcd for C₃₃H₅₁O₉, 591.3528).

Yucca spirostanoside C₂ (7): White powder; ${\left[\alpha \right]}_D^{25}$ –8.5° (c = 0.77, MeOH); IR ν_max (KBr) cm⁻¹: 3371, 2928, 1705, 1651, 1452, 1375, 1265, 1163, 1082, 1041, 922; ¹H NMR (C₅D₅N, 500 MHz) δ [1.28 (1H, m), 1.70 (1H, m, overlapped), H₂-1], [1.42 (1H, m), 1.85 (1H, m, overlapped), H₂-2], 4.29 (1H, m, overlapped, H-3), 1.74 (2H, m, overlapped, H₂-4), 2.08 (1H, m, H-5), [1.15 (1H, m), 1.80 (1H, m, overlapped), H₂-6], 1.00, 1.36 (1H each, both m, H₂-7), 1.85 (1H, m, overlapped, H-8), 1.76 (1H, m, overlapped, H-9), [2.20 (1H, dd, J = 5.0, 13.5 Hz), 2.38 (1H, dd, J = 13.5, 13.5 Hz), H₂-11], 1.49 (1H, m, H-14), 1.63, 2.15 (1H each, both m, H₂-15), 4.55 (1H, q like, ca. J = 9 Hz H-16), 2.82 (1H, dd, J = 7.0, 9.0 Hz, H-17), 1.10 (3H, s, H₃-18), 0.87 (3H, s, H₃-19), 1.95 (1H, m, H-20), 1.33 (3H, d, J = 7.0 Hz, H₃-21), [1.77 (1H, m, overlapped), 1.82 (1H, m, overlapped), H₂-23], [2.26 (1H, m), 2.72 (1H, dt, J = 5.0, 13.0 Hz), H₂-24], [4.06 (1H, d, J = 12.5 Hz), 4.46 (1H, d, J = 12.5 Hz), H₂-26], 4.80, 4.84 (1H each, both br. s, H₂-27), 4.90 (1H, d, J = 8.0 Hz, H-1′), 4.06 (1H, dd, J = 8.0, 9.0 Hz, H-2′), 4.24 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 4.17 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.89 (1H, m, H-5′), [4.33 (1H, dd, J = 5.0, 12.0 Hz), 4.49 (1H, dd, J = 2.0, 12.0 Hz), H₂-6′], 5.28 (1H, d, J = 7.0 Hz, H-1′′), 4.02 (1H, dd, J = 7.0, 8.5 Hz, H-2′′), 4.14 (1H, dd, J = 8.5, 8.5 Hz, H-3′′), 4.15 (1H, m, H-4′′), [3.67 (1H, dd, J = 11.0, 11.0 Hz), 4.31 (1H, m, overlapped), H₂-5′′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z 723.3959 [M + H]⁺ (calcd for C₃₈H₅₉O₁₃, 723.3950).

Yucca spirostanoside C₃ (8): White powder; ${\left[\alpha \right]}_D^{25}$ –5.0° (c = 0.16, MeOH); IR ν_max (KBr) cm⁻¹: 3398, 2929, 2879, 1701, 1650, 1451, 1375, 1223, 1166, 1158, 1077, 1036, 921; ¹H NMR (C₅D₅N, 500 MHz) δ [1.24 (1H, m), 1.78 (1H, m, overlapped), H₂-1], [1.26 (1H, m), 1.81 (1H, m, overlapped), H₂-2], 4.24 (1H, m, overlapped, H-3), 1.78 (2H, m, overlapped, H₂-4), 2.28 (1H, m, H-5), [1.21 (1H, m), 1.81 (1H, m, overlapped), H₂-6], [0.95 (1H, m), 1.33 (1H, m, overlapped), H₂-7], 1.82 (1H, m, overlapped, H-8), 1.73 (1H, m, H-9), [2.18 (1H, dd, J = 4.5, 14.0 Hz), 2.36 (1H, dd, J = 14.0, 14.0 Hz), H₂-11], 1.46 (1H, m, H-14), [1.62 (1H, m), 2.12 (1H, m), H₂-15], 4.57 (1H, q like, ca. J = 7 Hz, H-16), 2.83 (1H, dd, J = 6.5, 8.5 Hz, H-17), 1.10 (3H, s, H₃-18), 0.98 (3H, s, H₃-19), 1.96 (1H, m, H-20), 1.33 (3H, d, J = 7.0 Hz, H₃-21), 1.82 (2H, m, overlapped, H₂-23), [2.27 (1H, m), 2.73 (1H, dt, J = 6.0, 13.0 Hz), H₂-24], [4.06 (1H, d, J = 12.5 Hz), 4.47 (1H, d, J = 12.5 Hz), H₂-26], 4.80, 4.83 (1H each, both br. s, H₂-27), 4.83 (1H, m, overlapped, H-1′), 4.80 (1H, m, overlapped, H-2′), 4.36 (1H, m, overlapped, H-3′), 4.80 (1H, m, overlapped, H-4′), 3.98 (1H, m, H-5′), 4.35 (2H, m, overlapped, H₂-6′), 5.57 (1H, d, J = 7.5 Hz, H-1′′), 4.03 (1H, dd, J = 7.5, 8.5 Hz, H-2′′), 4.23 (1H, m, overlapped, H-3′′), 4.16 (1H, dd, J = 9.0, 9.0 Hz, H-4′′), 3.81 (1H, m, H-5′′), [4.35 (1H, m, overlapped), 4.50 (1H, dd, J = 3.0, 11.5 Hz), H₂-6′′], 5.37 (1H, d, J = 7.5 Hz, H-1′′′), 4.00 (1H, dd, J = 7.5, 8.0 Hz, H-2′′′), 4.23 (1H, m, overlapped, H-3′′′), 4.22 (1H, m, overlapped, H-4′′′), 3.92 (1H, m, H-5′′′), [4.29 (1H, dd, J = 5.5, 11.5 Hz), 4.43 (1H, dd, J = 2.0, 11.5 Hz), H₂-6′′′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z 937.4412 [M + Na]⁺ (calcd for C₄₅H₇₀O₁₉Na, 937.4404).

Yucca spirostanoside D₁ (9): White powder; ${\left[\alpha \right]}_D^{25}$ –26.2° (c = 0.45, MeOH); IR ν_max (KBr) cm⁻¹: 3461, 3389, 2930, 2877, 1696, 1654, 1450, 1376, 1266, 1231, 1159, 1071, 1042, 916; ¹H NMR (C₅D₅N, 500 MHz) δ 1.72, 1.96 (1H each, both m, overlapped, H₂-1), 3.75 (1H, m, H-2), 4.41 (1H, m, overlapped, H-3), 1.82, 1.97 (1H each, both m, overlapped, H₂-4), 2.26 (1H, m, overlapped, H-5), [1.32 (1H, m, overlapped), 1.78 (1H, m), H₂-6], [0.94 (1H, m), 1.32 (1H, m, overlapped), H₂-7], 1.81 (1H, m, overlapped, H-8), 1.72 (1H, m, overlapped, H-9), [2.37 (1H, dd, J = 5.0, 14.0 Hz), 2.41 (1H, dd, J = 14.0, 14.0 Hz), H₂-11], 1.46 (1H, m, H-14), 1.62, 2.13 (1H each, both m, H₂-15), 4.56 (1H, q like, ca. J = 8 Hz, H-16), 2.82 (1H, dd, J = 6.5, 8.5 Hz, H-17), 1.09 (3H, s, H₃-18), 0.99 (3H, s, H₃-19), 1.95 (1H, m, overlapped, H-20), 1.31 (3H, d, J = 6.5 Hz, H₃-21), 1.79 (2H, m, overlapped, H₂-23), [2.27 (1H, m, overlapped), 2.73 (1H, dt, J = 6.0, 13.0 Hz), H₂-24], 4.06, 4.47 (1H each, both m, overlapped, H₂-26), 4.80, 4.84 (1H each, both br. s, H₂-27), 4.98 (1H, d, J = 7.5 Hz, H-1′), 4.87 (1H, dd, J = 7.5, 9.5 Hz, H-2′), 4.31 (1H, dd, J = 2.5, 9.5 Hz, H-3′), 4.75 (1H, m, overlapped, H-4′), 4.11 (1H, m, overlapped, H-5′), 4.39 (2H, m, H₂-6′), 5.57 (1H, d, J = 8.0 Hz, H-1′′), 4.03 (1H, dd, J = 8.0, 8.0 Hz, H-2′′), 4.23 (1H, dd, J = 8.0, 9.0 Hz, H-3′′), 4.09 (1H, m, overlapped, H-4′′), 3.77 (1H, m, H-5′′), [4.28 (1H, dd, J = 4.5, 11.5 Hz), 4.48 (1H, m, overlapped), H₂-6′′], 5.22 (1H, d, J = 7.5 Hz, H-1′′′), 3.95 (1H, dd, J = 7.5, 9.5 Hz, H-2′′′), 4.12 (1H, m, overlapped, H-3′′′), 4.13 (1H, m, overlapped, H-4′′′), [3.60 (1H, dd, J = 10.0, 10.0 Hz), 4.19 (1H, dd, J = 4.5, 10.0 Hz), H₂-5′′′]; ¹³C NMR (C₅D₅N, 125 MHz) data, see Table 1; HRESI-TOF-MS Positive-ion mode m/z 923.4293 [M + Na]⁺ (calcd for C₄₄H₆₈O₁₉Na, 923.4247).

Acid Hydrolysis of 1–9: A solution of each saponin (about 3.0 mg) in 1 M HCl (1 mL) was heated under reflux for 3 h, respectively. Then each reaction mixture was neutralized with Amberlite IRA-400 (OH⁻ form) and removed by filtration. The aqueous layer was subjected to HPLC analysis: HPLC column, Kaseisorb LC NH₂-60-5, 4.6 mm i.d. × 250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation (Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan)]; mobile phase, CH₃CN-H₂O (75:25, v/v; flow rate 1.0 mL/min). As a result, d-xylose (6.0 min, positive optical rotation) for 2, 4, 7, 9; d-galactose (7.2 min, positive optical rotation) for 8, 9; and d-glucose (12.6 min, positive optical rotation) for 1–9 were identified by comparison of their retention times and optical rotations with those of authentic samples.

4. Conclusions

In summary, during the investigation of spirostanol saponins from natural products, fourteen spirostane-type saponins, including nine new ones, Yucca spirostanosides A₁ (1), A₂ (2), B₁ (3), B₂ (4), B₃ (5), C₁ (6), C₂ (7), C₃ (8), and D₁ (9), along with five known ones (10–14) were obtained from the stems of Y. schidigera. Their structures were determined by means of chemical and spectroscopic methods.

In accordance to the increasing applications of yucca extracts, further analytical, biological and physicochemical studies are still required. The presented study will make people understand the phytochemical constituents of Y. schidigera more fully and will lay a solid foundation for further pharmacodynamics research.