Spirostane-Type Saponins Obtained from Yucca schidigera.

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.

Figure 1

The compounds 1–14 obtained from the stems of Y. schidigera.

Yucca spirostanoside A1 (1) was obtained as a white powder with negative optical rotation ( −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, 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.

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

Figure 2

The main 1H–1H COSY and HMBC correlations of 1–9.

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 δC 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 δH 4.91 (H-1′) to δC 74.4 (C-3); δH 5.26 (H-1′′) to δC 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 δH 1.09 (H3-18) to δC 79.4 (C-12), δH 3.54 (H-12) to δC 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 (δC 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 (δC 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 (δC 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 δH 4.93 (H-1′) to δC 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 ( –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 δH 4.91 (H-1′) to δC 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: δH 4.87 (H-1′) and δC 70.1 (C-4′), 78.0 (C-5′), 80.1 (C-2′), 88.4 (C-3′), 102.1 (C-1′); δH 3.79 (H-5′) and δC 62.6 (C-6′); δH 5.65 (H-1′′) and δC 72.6 (C-4′′), 76.6 (C-2′′), 78.4 (C-3′′), 104.4 (C-1′′); δH 3.97 (H-5′′) and δC 63.5 (C-6′′), 72.6 (C-4′′), 78.4 (C-3′′ and 5′′); δC 104.4 (C-1′′) and δH 4.28 (H-3′′); δH 5.35 (H-1′′′) and δC 71.8 (C-4′′′), 75.5 (C-2′′′), 78.7 (C-3′′′), 105.0 (C-1′′′); δC 105.0 (C-1′′′) and δH 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 δH 4.87 (H-1′) to δC 75.7 (C-3); δH 5.65 (H-1′′) to δC 80.1 (H-2′); δH 5.35 (H-1′′′) to δC 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 ( −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 δC 213.0 (C-12) and the long-range correlations from δH 1.10 (H3-18), 1.49 (H-14), 1.76 (H-9), 2.21, 2.38 (H2-11), 2.83 (H-17) to δC 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 δH 4.94 (H-1′) to δC 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 δH 4.83 (H-1′) to δC 75.0 (C-3); δH 5.57 (H-1′′) to δC 77.8 (C-2′); δH 5.37 (H-1′′′) to δC 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 δH 4.83 (H-1′) and δC 70.0 (C-4′), 77.8 (C-2′), 84.3 (C-3′), 102.0 (C-1′); δH 3.98 (H-5′) and δC 62.5 (C-6′), 70.0 (C-4′), 76.6 (C-5′); δH 5.57 (H-1′′) and δC 72.9 (C-4′′), 76.5 (C-2′′), 78.5 (C-3′′), 104.6 (C-1′′); δH 3.81 (H-5′′) and δC 63.6 (C-6′′), 72.9 (C-4′′), 76.5 (C-2′′), 78.1 (C-5′′), 78.5 (C-3′′); δH 5.37 (H-1′′′) and δC 71.7 (C-4′′′), 75.5 (C-2′′′), 78.7 (C-3′′′), 105.5 (C-1′′′); δH 3.92 (H-5′′′) and δC 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: δH 4.98 (H-1′) and δC 69.7 (C-4′), 77.1 (C-2′), 84.1 (C-3′), 101.6 (C-1′); δH 5.57 (H-1′′) and δC 72.6 (C-4′′), 76.2 (C-2′′), 78.5 (C-3′′), 104.2 (C-1′′); δH 3.77 (H-5′′) and δC 63.3 (C-6′′), 72.6 (C-4′′), 77.8 (C-5′′); δH 5.22 (H-1′′′) and δC 67.0 (C-5′′′), 70.9 (C-4′′′), 75.0 (C-2′′′), 78.3 (C-3′′′), 106.0 (C-1′′′); δH 4.38 (H-5′) and δC 61.8 (C-6′), 76.6 (C-5′). Meanwhile, the correlations between δH 4.11 (H-5′) and δH 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 [δC 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) [δC 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 δH 4.98 (H-1′) to δC 79.7 (C-3); δH 5.57 (H-1′′) to δC 77.1 (C-2′); δH 5.22 (H-1′′′) to δC 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, tR 38.76′) and B2 (4, 15.6 mg, tR 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, tR 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, tR 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, tR 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, tR 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, tR 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, tR 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, tR 65.87′) and D1 (9, 33.5 mg, tR 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, tR 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, tR 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, tR 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, tR 23.04′).

Yucca spirostanoside A1 (1): White powder; −45.4° (c = 0.41, MeOH); IR νmax (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; –53.5° (c = 0.40, MeOH); IR νmax (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; −45.2° (c = 0.42, MeOH); IR νmax (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; –36.2° (c = 0.37, MeOH); IR νmax (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; –33.0° (c = 0.23, MeOH); IR νmax (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 –8.9° (c = 0.09, MeOH); IR νmax (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; –8.5° (c = 0.77, MeOH); IR νmax (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; –5.0° (c = 0.16, MeOH); IR νmax (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; –26.2° (c = 0.45, MeOH); IR νmax (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.