Separation and Bioactive Assay of 25R/S-Spirostanol Saponin Diastereomers from Yucca schidigera Roezl (Mojave) Stems.

In order to find a simple, generic, efficient separation method for 25R/S-spirostanol saponin diastereomers, the liquid chromatographic retention behaviors of C12 carbonylation and C12 unsubstituted 25R/S-spirostanol saponin diastereomers on different stationary phases (C₈, C18, C30 columns) and different mobile phases (MeOH-1% CH₃COOH and CH₃CN-1% CH₃COOH) were investigated. A C30 column was firstly found to offer the highest efficiency for the separation of this kind of diastereomers than C₈ and C18 columns. Meanwhile, the analysis results indicated that both CH₃CN-1% CH₃COOH and MeOH-1% CH₃COOH eluate systems were selective for C12 unsubstituted 25R/S-spirostanol saponin diastereomers, while MeOH-1% CH₃COOH possessed better selectivity for C12 carbonylation ones. Using the abovementioned analysis method, six pairs of 25R/S-spirostanol saponin diastereomers 1a⁻6a and 1b⁻6b from Yucca schidigera Roezl (Mojave) were isolated successfully by using HPLC on C30 column for the first time. Among them, three pairs were new ones, named as (25R)-Yucca spirostanoside E₁ (1a), (25S)-Yucca spirostanoside E₁ (1b), (25R)-Yucca spirostanoside E₂ (2a), (25S)-Yucca spirostanoside E₂ (2b), (25R)-Yucca spirostanoside E₃ (3a), (25S)-Yucca spirostanoside E₃ (3b), respectively. Moreover, 3a, 5a, 6a, 3b⁻6b showed strong inhibitory activities on the growth of SW620 cell lines with the IC50 values of 12.02⁻69.17 μM.

1. Introduction

Yucca schidigera Roezl (Mojave), belonging to the Yucca genus (Agavaceae family), is mainly distributed in the southwestern United States and the northern desert of Mexico. Steroidal saponins and phenolic acids are reported to be its main constituents. Because of its excellent biological activity and proven safety, it has used worldwide as a kind of additive in foods, beverages, cosmetics and feeds [1]. It is worth mentioning that extracts of Y. schidigera which are enriched in steroidal saponins [2], have already been developed into a commodity for a wide range of applications.

One of the main steroidal saponin aglycone types in Y. schidigera are the C27 spirostanol type steroidal saponins, which can be divided into spirostanol type (25S) and isospiritol type (25R) according to the configuration at C25. The successfully separation of 25R/S-spirostanol saponin diastereomers has scarecely been reported until now, although the different stereo configurations may cause complete different bioactivity and lead to unreliable results. Therefore, the successful separation of 25R/S-spirostanol saponin diastereomers and the determination of their configurations play a crucial role in further pharmacological or molecular biological research on these compounds.

The objective of this study was to establish a simple, generic, efficient separation and analysis method for the 25R/S-spirostanol saponin diastereomers. During the process, the separation ability of three stationary phase (C8, C18 and C30 columns) as well as two kinds of mobile phases (MeOH-1% CH3COOH and CH3CN-1% CH3COOH) were evaluated. As a result, the separation of 25R/S-spirostanol saponin diastereomers was accomplished by a C30 column, and six pairs of 25R/S-spirostanol saponin diastereomers 1a–6a and 1b–6b were thus obtained, the structures of which were identified by spectroscopy and chemical methods. What’s more, the potent in vitro inhibitory effects of these compounds on human colon cancer cells SW620 were assessed by the MTT method.

2. Results and Discussion

2.1. Selection of Separation Conditions for 25R/S-Spirostanol Saponin Diastereomers by HPLC

On the basis of optimizing stationary and mobile phases, 25R/S-spirostanol saponin diastereomer mixtures 1–6 were isolated successfully by using the C30 column which possessed significant advantages for separating diastereomers. As results, six pairs of 25R/S-spirostanol saponin diastereomers were obtained, and their structures were elucidated to be new ones, (25R)-Yucca spirostanoside E1 (1a), (25S)-Yucca spirostanoside E1 (1b), (25R)-Yucca spirostanoside E2 (2a), (25S)-Yucca spirostanoside E2 (2b), (25R)-Yucca spirostanoside E3 (3a), (25S)-Yucca spirostanoside E3 (3b), and known ones, (25R)-5β-spirostan-3β-ol 3-O-β-d-glucopyranoside (4a) [3], asparagoside A (4b) [3], 25(R)-schidigera-saponin D5 (5a) [4], 25(S)-schidigera-saponin D5 (5b) [4], 25(R)-schidigera-saponin D1 (6a) [4], 25(S)-schidigera-saponin D1 (6b) [4] (Figure 1), respectively.

Figure 1

Chemical structures of the spirostanol saponins 1a–6a and 1b–6b.

The 25R/S-spirostanol saponin diastereomers mentioned above could be divided into two classes according to their aglycone types: C12 carbonylated (compounds 1a–3a, 1b–3b) and C12 unsubstituted (compounds 4a–6a, 4b–6b) 25R/S-spirostanol saponins. In the course of comparing separation ability of three stationary phase (C8, C18 and C30 columns) as well as two kinds of mobile phases (MeOH-1% CH3COOH and CH3CN-1% CH3COOH) [using an evaporating light scattering detector (ELSD) detector] for the abovementioned 25R/S-spirostanol saponin diastereomers, we found that the liquid chromatography retention behaviors of two kinds of aglycone type 25R/S-spirostanol saponin diastereomers were different, and the specific rules are summed up in the following subsections.

2.1.1. General Rules and Characteristics HPLC Analysis for C12 Carbonylation 25R/S-Spirostanol Saponin Diastereomers 1a–3a, 1b–3b

When using a C30 column as stationary phase, better separation effect could be obtained on C12 carbonylated 25R/S-spirostanol saponin diastereomers (Figure 2A, Figure 3A and Figure 4A). During the process of optimizing the separation conditions on the C30 column, MeOH-1% CH3COOH was found to possess better selectivity. Moreover, the longer for the retention time (tR), the better resolution (Figure 2B, Figure 3B and Figure 4B) was obtained. On the other hand, we found that the tR of 25R-spirostanol saponins 1a–3a was always shorter than that of 25S ones 1b–3b with the MeOH-1% CH3COOH eluate system, which was not related to the type or number of substituted sugars.

Figure 2

(A) Chromatograms of 1a and 1b separated on C8, C18, C30 columns; (B) Chromatograms of 1a and 1b separated on the C30 column in different solvent system.

Figure 3

(A) Chromatograms of 2a and 2b separated on C8, C18, C30 columns; (B) Chromatograms of 2a and 2b separated on the C30 column in different solvent systems.

Figure 4

(A) Chromatograms of 3a and 3b separated on C8, C18, C30 columns; (B) Chromatograms of 3a and 3b separated on the C30 column in different solvent systems.

2.1.2. General Rules and Characteristics HPLC Analysis for C12 Unsubstituted 25R/S-Spirostanol Saponin Diastereomers 4a–6a, 4b–6b

The C30 column was also found to be more suitable for the separation of C12 unsubstituted 25R/S-spirostanol saponin diastereomers (Figure 5A, Figure 6A and Figure 7A) than the C8 and C18 columns. The difference from the C12 carbonylated 25R/S-spirostanol saponin diastereomers was that both the CH3CN-1% CH3COOH and MeOH-1% CH3COOH eluate systems were selective to this type of compounds, while CH3CN-1% CH3COOH system could guarantee the resolution in shorter tR (Figure 5B, Figure 6B and Figure 7B).

Figure 5

(A) Chromatograms of 4a and 4b separated on C8, C18, C30 column; (B) Chromatograms of 4a and 4b separated on the C30 column in different solvent systems.

Figure 6

(A) Chromatograms of 5a and 5b separated on C8, C18, C30 column; (B) Chromatograms of 5a and 5b separated on the C30 column in different solvent systems.

Figure 7

(A) Chromatograms of 6a and 6b separated on C8, C18, C30 column; (B) Chromatograms of 6a and 6b separated on the C30 column in different solvent systems.

In addition, the tR of monosaccharide substituted 25R-spirostanol saponin 4a was shorter than that of 25S-spirostanol saponin 4b whether in MeOH-1% CH3COOH or in CH3CN-1% CH3COOH system. As the number of glycosyl groups increase (compounds 5a, 5b, 6a, 6b), the tR of 25R-spirostanol saponin was always shorter than that of 25S-spirostanol saponin (5a vs. 5b, 6a vs. 6b) in MeOH-1% CH3COOH eluate system, a phenomenon in contrast in the CH3CN-1% CH3COOH eluate system case.

2.2. Structure Identification for 25R/S-Spirostanol Saponin Diastereomers

Both (25R)-Yucca spirostanoside E1 (1a) and (25S)-Yucca spirostanoside E1 (1b) were isolated as white powders with negative optical rotation [([α] − 11.2°, MeOH) for 1a, ([α] − 6.6°, MeOH) for 1b]. Their molecular formulae were deduced to be C33H52O9 by the positive-ion HRESI-MS analysis (m/z 593.3705 [M + H]+ for 1a, and 593.3703 [M + H]+ for 1b, both calcd. for C33H53O9, 593.3684). Treatment with 1 M hydrochloric acid (HCl) liberated d-glucose, which was identified by HPLC analysis using an optical rotation detector [5]. Thirty-three carbon signals were displayed in their 13C-NMR (Table 1, C5D5N) spectrum. Besides the carbon signals represented by d-glucose, the other twenty-seven ones, especially the quaternary carbon signal at δC 109.3 (1a)/109.8 (1b) indicated that they were spirostane-type steroid saponins. The 1H-NMR spectrum suggested the presence of four methyls [δ 0.70 (3H, d, J = 6.0 Hz, H3-27), 0.85, 1.09 (3H each, both s, H3-19 and 18), 1.37 (3H, d, J = 6.5 Hz, H3-21) for 1a; δ 1.07 (3H, d, J = 6.5 Hz, H3-27), 0.84, 1.08 (3H each, both s, H3-19 and 18), 1.37 (3H, d, J = 7.0 Hz, H3-21) for 1b], two methines bearing an oxygen function [δ 4.32 (1H, m, H-3), 4.55 (1H, q like, ca. J = 8 Hz, H-16) for 1a; δ 4.32 (1H, m, H-3), 4.52 (1H, q like, ca. J = 7 Hz, H-16) for 1b], one oxygenated methene {[δ 3.50 (1H, dd, J = 10.5, 10.5 Hz), 3.60 (1H, dd, J = 4.0, 10.5 Hz), H2-26] for 1a; [δ 3.38 (1H, br. d, ca. J = 11 Hz), 4.06 (1H, dd, J = 2.5, 11.0 Hz), H2-26] for 1b} and one β-d-glucopyranosyl [δ 4.93 (1H, d, J = 7.5 Hz, H-1′) for 1a; δ 4.92 (1H, d, J = 7.5 Hz, H-1′) for 1b] in their aglycones. The existence of carbonyl was clarified by the 13C-NMR signal at δC 213.0 (C-12) (1a/1b). The 1H-1H COSY spectra of 1a and 1b suggested the presence of the three partial structures written in bold lines in Figure 8. The planar structure of their aglycons were determined to be spirostan-3-ol-12-one based on the key HMBC correlations from H2-11, H-14, 17 to C-12; H3-18 to C-12–14, 17; H3-19 to C-1, 5, 9, 10; H3-21 to C-17, 20, 22; H2-23, 26 to C-22; H3-27 to C-24–26. Moreover, the β-d-glucopyranosyl was determined to link at C-3 position of aglycone by the long-range correlation from H-1′ to C-3 observed in the HMBC experiment. Their 1H- and 13C-NMR data for the protons and carbons in A–E ring were identical to those of Yucca spirostanoside C1 [5], and the configuration of A–E ring was determined. Comparing the proton chemical shifts, we found CH3-27 of 1a (δ 0.70) displayed signal at the higher field than that of 1b (δ 1.07); what’s more, there was a smaller difference between the two protons of CH2-26 of 1a (∆δa,b = 0.10 ppm) than that of 1b (∆δa,b = 0.68 ppm). According to the rules summarized by Boll et al. [6] and Schreiber et al. [7], the absolute configuration of C-25 was elucidated to be R and S for 1a and 1b, respectively. On the other hand, the comparision resultsof their 13C-NMR data for F ring (C-22–26) and C-27 [δ 17.3 (C-27), 29.2 (C-24), 30.5 (C-25), 31.8 (C-23), 66.9 (C-26), 109.3 (C-22) for 1a; δ 16.3 (C-27), 26.1 (C-24), 26.4 (C-23), 27.5 (C-25), 65.2 (C-26), 109.8 (C-2) for 1b] with those of (25R)-5β-spirostan [δ 17.1 (C-27), 28.8 (C-24), 30.3 (C-25), 31.4 (C-23), 66.8 (C-26), 109.2 (C-22)] and (25S)-5β-spirostan [δ 16.1 (C-27), 25.8 (C-24), 26.0 (C-25), 27.1 (C-23), 65.2 (C-26), 109.7 (C-22)] [8], clarified the absolute configuration of C-25 furtherly. On the basis of above mentioned evidence, the structure of 1a and 1b was elucidated to be (25R)-5β-spirostan-3β-ol-12-one 3-O-β-d-gluco- pyranoside and (25S)-5β-spirostan-3β-ol-12-one 3-O-β-d-glucopyranoside, respectively.

Table 1

13C-NMR data for 1a–3a and 1b–3b in C5D5N.

No.	1a	1b	2a	2b	3a	3b	No.	1a	1b	2a	2b	3a	3b
1	30.6	30.6	30.6	30.6	30.6	30.6	21	14.0	13.8	14.0	13.8	13.9	13.8
2	26.7	26.7	26.6	26.7	26.6	26.6	22	109.3	109.8	109.3	109.8	109.3	109.8
3	73.9	73.9	74.0	74.0	74.9	74.9	23	31.8	26.4	31.8	26.4	31.9	26.4
4	30.2	30.2	30.1	30.1	30.7	30.7	24	29.2	26.1	29.2	26.2	29.3	26.2
5	36.5	36.5	36.5	36.5	36.4	36.4	25	30.5	27.5	30.5	27.5	30.6	27.5
6	26.8	26.8	26.8	26.8	26.8	26.8	26	66.9	65.2	66.9	65.2	67.0	65.2
7	26.4	26.4	26.4	26.4	26.4	26.4	27	17.3	16.3	17.3	16.3	17.3	16.3
8	34.7	34.7	34.7	34.7	34.7	34.7	1′	102.9	102.9	102.3	102.4	102.0	101.8
9	41.9	41.9	41.9	41.9	42.0	42.0	2′	75.3	75.3	74.2	74.3	83.1	83.1
10	35.7	35.7	35.7	35.7	35.8	35.8	3′	78.7	78.7	87.7	87.8	78.2	78.2
11	37.7	37.7	37.7	37.8	37.8	37.8	4′	71.7	71.7	69.5	69.5	71.6	71.6
12	213.0	213.0	213.0	213.0	213.0	213.0	5′	78.4	78.4	78.1	78.2	78.3	78.3
13	55.6	55.6	55.6	55.6	55.7	55.6	6′	62.8	62.8	62.3	62.4	62.7	62.7
14	56.0	56.0	56.0	56.0	56.1	56.1	1″			106.3	106.4	106.0	106.0
15	31.5	31.4	31.5	31.4	31.5	31.5	2″			75.3	75.4	77.1	77.1
16	79.8	79.9	79.8	79.9	79.8	79.9	3″			78.2	78.2	78.0	78.0
17	54.3	54.2	54.3	54.2	54.4	54.2	4″			70.9	70.9	71.9	71.9
18	16.1	16.1	16.1	16.1	16.1	16.1	5″			67.4	67.4	78.6	78.6
19	23.0	23.0	23.1	23.1	23.2	23.2	6″					63.0	63.0
20	42.6	43.1	42.6	43.1	42.7	43.2

Figure 8

The main 1H-1H COSY and HMBC correlations of 1a–3a and 1b–3b.

The Q-TOF-ESI-MS analysis results indicated that (25R)-Yucca spirostanoside E2 (2a) and (25S)-Yucca spirostanoside E2 (2b), (25R)-Yucca spirostanoside E3 (3a) and (25S)-Yucca spirostanoside E3 (3b) had the same molecular formula, C38H60O13 and C39H62O14, respectively. Acid hydrolysis reaction experiments proved that 2a and 2b contained d-glucose and d-xylose [5], while only d-glucose existed in 3a and 3b. Their 1H-, 13C- (Table 1, C5D5N) and 2D- (1H-1H COSY, HSQC, HMBC) NMR spectra suggested that the aglycons of compounds 2a and 3a, 2b and 3b were (25R)-5β-spirostan-3β-ol-12-one, (25S)-5β-spirostan-3β-ol-12-one, respectively [6,7,8]. Meanwhile, the long-rang correlations from H-1′ to C-3; H-1″ to C-3′ could be observed in the HMBC spectra of compounds 2a and 2b; and in the HMBC spectra of 3a and 3b, the correlations from H-1′ to C-3; H-1″ to C-2′ could be observed. Consequently, the structures of 2a, 2b, 3a, and 3b were elucidated.

The structures of known compounds 4a–b [3], 5a–b [4] and 6a–b [4] were identified by comparing their 1H-, 13C-NMR data with references.

2.3. Inhibitory Activities on the Growth of SW620 Cell Lines Study of Extract, Fractions, and Compounds Obtained from Y. schidigera

The inhibitory effects of Y. schidigera 70% EtOH extract, Y. schidigera 95% EtOH eluate, Y. schidigera H2O eluate, as well as 25R/S-spirostanol saponin diastereomers 1a–6a, 1b–6b on the growth of SW620 cell lines were measured by the MTT method. As the results in Table 2 show, Y. schidigera 70% EtOH extract and Y. schidigera 95% EtOH eluate displayed IC50 values as 85.20 and 93.04 μg/mL, respectively. Meanwhile, compound 6b exhibited strong activity with IC50 value of 12.02 μM comparable with that of the positive control 5-fluorouracil (5-FU, IC50 10.00 μM), and 3a, 5a, 6a, 3b–5b showed the IC50 values of 29.81–69.17 μM. Moreover, through the summary of structure-activity relationships of 25R/S-spirostanol saponin diastereomers 1a–6a, 1b–6b, it could be found that the configuration of C25 had significant influence of the inhibitory activities towards SW620 cells. For C12 unsubstituted 25R/S-spirostanol saponin diastereomers, the bioactivity of 25S-spirostanol saponins was stronger than that of 25R-ones (4b vs. 4a; 5b vs. 5a; 6b vs. 6a); however, it was exactly opposite for C12 carbonylation 25R/S-spirostanol saponin diastereomers (3b vs. 3a). What’s more, the numbers of substituted glycosyls also affected their activities. For example, as the substituted glycosyls increased, C12 unsubstituted 25R/S-spirostanol saponin diastereomers showed stronger inhibitory effects on SW620 cells (6b vs. 5b vs. 4b; 6a vs. 5a vs. 3a).

Table 2

The inhibitory effects of Y. schidigera extract, fractions, and 25R/S-spirostanol saponin diastereomers on the growth of SW620 cell.

Sample	IC50	Sample	IC50
Positive control	10.00 ± 0.15	3a	29.81 ± 0.21
Y. schidigera 70% EtOH extract	85.20 ± 0.95	3b	55.90 ± 0.78
Y. schidigera 95% EtOH eluate	93.04 ± 1.21	4a	>100
Y. schidigera H2O eluate	>100	4b	60.26 ± 4.53
1a	>100	5a	63.37 ± 0.70
1b	>100	5b	33.91 ± 1.27
2a	>100	6a	69.17 ± 1.24
2b	>100	6b	12.02 ± 1.43

n = 4; Positive control: 5-FU; IC50: μg/mL for Y. schidigera 70% EtOH extract, Y. schidigera 95% EtOH eluate, and Y. schidigera H2O eluate; μM for positive control and compounds1a–6a and 1b–6b.

3. Materials and Methods

3.1. General Information

The following instruments were used to measure physical data: IR spectra were determined on a 640-IR FT-IR spectrophotometer (Varian Australia Pty Ltd., Mulgrave, Australia). Optical rotations were run on an Autopol® IV automatic polarimeter (l = 50 mm, Rudolph Research Analytical, Hackettstown, NJ, USA). NMR spectra were obtained 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 HRESI-TOF-MS were recorded on an Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer (Agilent Corp., Santa Clara, CA, USA). High performance liquid chromatography (HPLC) analyses were performed on an Agilent 1260 Infinity system (Agilent Technologies Inc.) equipped with ELSD (Alltech 2000 ES, Chengdu, China).

Column chromatographies (CC) were performed on macroporous resin D101 (Haiguang Chemical Co., Ltd., Tianjin, China), Silica gel (74–149 µm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and ODS (40–63 μm, YMC Co., Ltd., Tokyo, Japan). High performance liquid chromatography (HPLC) columns: Cosmosil 5C18-MS-II (4.6 mm i.d. × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan), Cosmosil C8-MS (4.6 mm i.d. × 250 mm, Nacalai Tesque, Inc.), Cosmosil PBr (4.6 mm i.d. × 250 mm, Nacalai Tesque, Inc.), Wacopak Navi C30-5 (4.6 mm i.d. × 250 mm, Wako Pure Chemical Industries, Ltd., Osaka, Japan) were used to analyze the mixture. Preparative high performance liquid chromatography (PHPLC) columns: Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, Nacalai Tesque, Inc.), Wacopak Navi C30-5 (7.5 mm i.d. × 250 mm, Wako Pure Chemical Industries, Ltd.), and Cosmosil PBr (20 mm i.d. × 250 mm, Nacalai Tesque, Inc.) were used to separate the constituents.

3.2. Plant Material

The stems of Y. schidigera were collected from National City, in southwest Califonia, USA, and identified by Dr. Li Tianxiang (The Hall of TCM Specimens, Tianjin University of TCM, Tianjin, 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

3.3.1. Extraction and Isolation of 25R/S-Spirostanol Saponin Diastereomer Mixtures 1–6

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 thirteen 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 6-12 (800.9 mg) was isolated by PHPLC [MeOH-1% CH3COOH (75:25, v/v), Cosmosil 5C18-MS-II column] to yield mixtures 1 and 2. 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 mixture 4. Fraction 7 (10.0 g) was subjected to PHPLC [MeOH-1% CH3COOH (80:20, v/v), Cosmosil 5C18-MS-II column] to produce thirteen fractions (Fr. 7-1–Fr. 7-13). Fraction 7-5 (712.6 mg) was separated by PHPLC [CH3CN-1% CH3COOH (40:60, v/v), Cosmosil 5C18-MS-II column] and PHPLC [MeOH-1% CH3COOH (70:30, v/v), Cosmosil 5C18-MS-II column] to obtain mixture 3. Fraction 7-12 (984.6 mg) was separated by PHPLC [MeOH-1% CH3COOH (95:5, v/v), Cosmosil PBr column] to give mixtures 5 and 6.

3.3.2. Extraction and Isolation of 25R/S-Spirostanol Saponin Diastereomers 1a–6a, 1b–6b by Using C30 Column

Mixture 1 (190.3 mg) was purified by PHPLC [MeOH-1% CH3COOH (85:15, v/v)] to give (25R)-Yucca spirostanoside E1 (1a, 37.8 mg) and (25S)-Yucca spirostanoside E1 (1b, 23.0 mg). Mixture 2 (160.7 mg) was separated by using the same method as that for mixture 1 to gain (25R)-Yucca spirostanoside E2 (2a, 47.5 mg) and (25S)-Yucca spirostanoside E2 (2b, 32.0 mg). Mixture 3 (15.0 mg) was separated by HPLC [MeOH-1% CH3COOH (70:30, v/v)] to yield (25R)-Yucca spirostanoside E3 (3a, 3.8 mg) and (25S)-Yucca spirostanoside E3 (3b, 7.2 mg). Mixture 4 was (180.0 mg) isolated by PHPLC [CH3CN-1% CH3COOH (80:20, v/v)] to provide (25R)-5β-spirostan-3β-ol 3-O-β-d-gluco- pyranoside (4a, 10.0 mg) and asparagoside A (4b, 36.5 mg). Mixture 5 (10.0 mg) was purified by HPLC [CH3CN-1% CH3COOH (40:60, v/v)] to give 25(R)-schidigera-saponin D5 (5a, 2.0 mg) and 25(S)-schidigera-saponin D5 (5b, 4.2 mg). Using the same HPLC condition, 25(R)-schidigera-saponin D1 (6a, 5.5 mg) and 25(S)-schidigera-saponin D1 (6b, 10.0 mg) were obtained.

(25R)-Yucca spirostanoside E (1a): White powder; [α] − 11.2° (c = 0.97, MeOH); IR νmax (KBr) cm−1: 3395, 2929, 2871, 1705, 1454, 1379, 1345, 1244, 1161, 1074, 1027, 985, 921, 897, 867. 1H-NMR (C5D5N, 500 MHz): δ 0.70 (3H, d, J = 6.0 Hz, H3-27), 0.85 (3H, s, H3-19), 0.97, 1.33 (1H each, both m, H2-7), 1.09 (3H, s, H3-18), 1.11, 1.76 (1H each, both m, H2-6), [1.28 (1H, m), 1.73 (1H, m, overlapped), H2-1], 1.37 (3H, d, J = 6.5 Hz, H3-21), 1.42, 1.86 (1H each, both m, H2-2), 1.47 (1H, m, H-14), 1.57 (2H, m, overlapped, H2-24), 1.58 (1H, m, overlapped, H-25), [1.61 (1H, m, overlapped), 2.14 (1H, ddd, J = 5.5, 8.0, 11.5 Hz), H2-15], 1.63, 1.71 (1H each, both m, H2-23), 1.73 (2H, m, overlapped, H2-4), 1.75 (1H, m, overlapped, H-9), 1.83 (1H, m, H-8), 1.94 (1H, quin, J = 6.5 Hz, H-20), 2.08 (1H, m, H-5), [2.21 (1H, dd, J = 4.5, 14.0 Hz), 2.37 (1H, dd, J = 14.0, 14.0 Hz), H2-11], 2.82 (1H, dd, J = 6.5, 8.5 Hz, H-17), [3.50 (1H, dd, J = 10.5, 10.5 Hz), 3.60 (1H, dd, J = 4.0, 10.5 Hz), H2-26], 3.95 (1H, m, H-5′), 4.05 (1H, dd, J = 7.5, 8.5 Hz, H-2′), 4.27 (1H, m, overlapped, H-3′), 4.27 (1H, m, overlapped, H-4′), 4.32 (1H, m, H-3), [4.40 (1H, dd, J = 5.0, 11.5 Hz), 4.54 (1H, m, overlapped), H2-6′], 4.55 (1H, q like, ca. J = 8 Hz, H-16), 4.93 (1H, d, J = 7.5 Hz, H-1′); 13C-NMR (C5D5N, 125 MHz) spectroscopy data: see Table 1. HRESI-TOF-MS: Positive-ion mode m/z 593.3705 [M + H]+ (calcd. for C33H53O9, 593.3684).

(25S)-Yucca spirostanoside E (1b): White powder; [α] − 6.6° (c = 1.06, MeOH); IR νmax (KBr) cm−1: 3391, 2930, 2874, 1704, 1454, 1378, 1345, 1269, 1170, 1070, 1025, 988, 920, 897, 849. 1H-NMR (C5D5N, 500 MHz): δ 0.84 (3H, s, H3-19), [0.96 (1H, m), 1.35 (1H, m, overlapped), H2-7] 1.08 (3H, s, H3-18), 1.07 (3H, d, J = 6.5 Hz, H3-27), [1.11 (1H, m), 1.76 (m, overlapped), H2-6], [1.27 (1H, m), 1.72 (1H, m, overlapped), H2-1], [1.33 (1H, m, overlapped), 1.91 (1H, m, overlapped), H2-23], [1.33 (1H, m, overlapped), 2.13 (1H, m, overlapped), H2-24], 1.37 (3H, d, J = 7.0 Hz, H3-21), [1.41 (1H, m, overlapped), 1.86 (1H, m), H2-2], 1.45 (1H, m, H-14), 1.59 (1H, m, overlapped, H-25), [1.59 (1H, m, overlapped), 2.12 (1H, m, overlapped), H2-15], 1.72 (m, overlapped, H2-4), 1.75 (1H, m, overlapped, H-9), 1.82 (1H, m, H-8), 1.88 (1H, m, overlapped, H-20), 2.08 (1H, m, H-5), [2.20 (1H, dd, J = 4.5, 14.5 Hz), 2.36 (1H, dd, J = 14.5, 14.5 Hz), H2-11], 2.79 (1H, dd, J = 6.5, 8.5 Hz, H-17), [3.38 (1H, br. d, ca. J = 11 Hz), 4.06 (1H, dd, J = 2.5, 11.0 Hz), H2-26], 3.94 (1H, m, H-5′), 4.05 (1H, dd, J = 7.5, 8.0 Hz, H-2′), 4.25 (1H, m, overlapped, H-3′), 4.25 (1H, m, overlapped, H-4′), 4.32 (1H, m, H-3), [4.40 (1H, dd, J = 5.0, 11.5 Hz), 4.54 (1H, dd, J = 2.5, 11.5), H2-6′], 4.52 (1H, q like, ca. J = 7 Hz, H-16), 4.92 (1H, d, J = 7.5 Hz, H-1′); 13C-NMR (C5D5N, 125 MHz) spectroscopy data: see Table 1. HRESI-TOF-MS: Positive-ion mode m/z 593.3703 [M + H]+ (calcd. for C33H53O9, 593.3684).

(25R)-Yucca spirostanoside E (2a): White powder; [α] − 0.29° (c = 0.67, MeOH); IR νmax (KBr) cm−1: 3427, 2928, 2869, 1707, 1454, 1375, 1240, 1162, 1074, 1040, 984, 922, 897, 866. 1H-NMR (C5D5N, 500 MHz): δ 0.70 (3H, d, J = 6.0 Hz, H3-27), 0.87 (3H, s, H3-19), 1.10 (3H, s, H3-18), 1.16, 1.81 (1H each, both m, H2-6), [1.28 (1H, m), 1.71 (1H, m, overlapped), H2-1], 1.37 (3H, d, J = 7.0 Hz, H3-21), [1.37 (1H, m, overlapped), 1.81 (1H, m), H2-2)], 1.48 (1H, m, H-14), 1.57 (2H, m, overlapped, H2-24), 1.58 (1H, m, overlapped, H-25), [1.60 (1H, m, overlapped), 2.15 (1H, ddd, J = 6.5, 7.5, 13.5 Hz), H2-15], [1.64 (1H, m), 1.71 (1H, m, overlapped), H2-23], 1.73 (2H, m, H2-4), 1.76 (1H, m, H-9), 1.84 (1H, m, H-8), 1.94 (1H, quin, J = 7.0 Hz, H-20), 2.08 (1H, m, H-5), [2.21 (1H, dd, J = 5.0, 14.5 Hz), 2.38 (1H, dd, J = 14.5, 14.5 Hz), H2-11], 2.82 (1H, dd, J = 6.5, 8.5 Hz, H-17), [3.50 (1H, dd, J = 10.5, 10.5 Hz), 3.60 (1H, dd, J = 3.5, 10.5 Hz), H2-26], [3.69 (1H, dd, J = 11.0, 11.0 Hz), 4.31 (1H, m, overlapped), H2-5″], 3.90 (1H, m, H-5′), 4.02 (1H, dd, J = 7.5, 8.0 Hz, H-2″), 4.07 (1H, dd, J = 8.0, 8.5 Hz, H-2′), 4.14 (1H, dd, J = 8.0, 9.0 Hz, H-3″), 4.16 (1H, m, H-4″), 4.18 (1H, dd, J = 9.0, 9.5 Hz, H-4′), 4.20 (1H, m, overlapped, H-3), 4.25 (1H, dd, J = 8.5, 9.0 Hz, H-3′), [4.34 (1H, dd, J = 5.0, 12.0 Hz), 4.48 (1H, dd, J = 2.0, 12.0 Hz), H2-6′], 4.55 (1H, q like, ca. J = 7 Hz, H-16), 4.91 (1H, d, J = 8.0 Hz, H-1′), 5.29 (1H, d, J = 7.5 Hz, H-1″); 13C-NMR (C5D5N, 125 MHz) spectroscopy data: see Table 1. HRESI-TOF-MS: Positive-ion mode m/z 725.4117 [M + H]+ (calcd. for C38H61O13, 725.4107).

(25S)-Yucca spirostanoside E (2b): White powder; [α] − 0.45° (c = 0.45, MeOH); IR νmax (KBr) cm−1: 3398, 2930, 2869, 1707, 1454, 1375, 1246, 1162, 1074, 1041, 986, 919, 896. 1H-NMR (C5D5N, 500 MHz): δ 0.87 (3H, s, H3-19), [0.97 (1H, m), 1.35 (1H, m, overlapped), H2-7], 1.08 (3H, d, J = 8.0 Hz, H3-27), 1.09 (3H, s, H3-18), 1.16, 1.80 (1H each, both m, H2-6), 1.28, 1.71 (1H each, both m, H2-1), 1.34, 2.13 (1H each, both m, overlapped, H2-24), 1.38 (3H, d, J = 7.0 Hz, H3-21), 1.40, 1.85 (1H each, both m, overlapped, H2-2), 1.41, 1.83 (1H each, both m, overlapped, H2-23), 1.46 (1H, m, H-14), 1.59 (1H, m, H-25), 1.61, 2.13 (1H each, both m, H2-15), 1.74 (2H, m, overlapped, H2-4), 1.75 (1H, m, overlapped, H-9), 1.83 (1H, m, overlapped, H-8), 1.89 (1H, m, H-20), 2.09 (1H, m, H-5), [2.20 (1H, dd, J = 5.0, 14.5 Hz), 2.37 (1H, dd, J = 14.5, 14.5 Hz), H2-11], 2.80 (1H, dd, J = 7.0, 8.5 Hz, H-17), [3.38 (1H, br. d, ca. J = 12 Hz), 4.05 (1H, dd, J = 3.5, 11.5 Hz), H2-26], [3.70 (1H, dd, J = 11.0, 11.0 Hz), 4.31 (1H, m, overlapped), H2-5″], 3.91 (1H, m, H-5′), 4.04 (1H, dd, J = 7.5, 8.0 Hz, H-2″), 4.08 (1H, dd, J = 7.5, 8.5 Hz, H-2′), 4.15 (1H, dd, J = 8.0, 9.0 Hz, H-3″), 4.17 (1H, m, H-4″), 4.19 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 4.26 (1H, dd, J = 8.5, 9.0 Hz, H-3′), 4.30 (1H, m, overlapped, H-3), [4.34 (1H, dd, J = 5.0, 11.5 Hz), 4.49 (1H, dd, J = 2.0, 11.5 Hz), H2-6′], 4.52 (1H, m, H-16), 4.92 (1H, d, J = 7.5 Hz, H-1′), 5.31 (1H, d, J = 7.5 Hz, H-1″); 13C-NMR (C5D5N, 125 MHz) spectroscopy data: see Table 1. HRESI-TOF-MS: Positive-ion mode m/z 725.4117 [M + H]+ (calcd. for C38H61O13, 725.4107).

(25R)-Yucca spirostanoside E (3a): White powder; [α] − 12.0° (c = 0.50, MeOH); IR νmax (KBr) cm−1: 3426, 2927, 2870, 1707, 1456, 1376, 1238, 1161, 1072, 1043, 981, 920, 898, 865. 1H-NMR (C5D5N, 500 MHz): δ 0.70 (3H, d, J = 6.0 Hz, H3-27), 0.93, 1.31 (1H each, both m, H2-7), 1.01 (3H, s, H3-19), 1.09 (3H, s, H3-18), [1.21 (1H, m), 1.84 (1H each, both m), H2-6], [1.28 (1H, m), 1.72 (1H, m, overlapped), H2-1], 1.37 (3H, d, J = 7.0 Hz, H3-21), 1.37, 1.82 (1H each, both m, overlapped, H2-2), 1.44 (1H, m, H-14), 1.58 (2H, m, overlapped, H2-24), 1.58 (1H, m, overlapped, H-25), [1.64 (1H, m), 1.72 (1H, m, overlapped), H2-23], [1.64 (1H, m), 2.12 (1H, ddd, J = 6.0, 7.0, 12.5 Hz), H2-15], 1.73 (2H, m, overlapped, H2-4), 1.74 (1H, m, H-9), 1.84 (1H, m, overlapped, H-8), 1.94 (1H, quin, J = 7.0 Hz, H-20), [2.20 (1H, dd, J = 4.5, 14.0 Hz), 2.38 (1H, dd, J = 14.0, 14.0 Hz), H2-11], 2.25 (1H, m, H-5), 2.82 (1H, dd, J = 6.5, 8.5 Hz, H-17), [3.50 (1H, dd, J = 10.5, 10.5 Hz), 3.59 (1H, dd, J = 4.0, 10.5 Hz), H2-26], 3.86 (1H, m, H-5′), 3.98 (1H, m, H-5″), 4.09 (1H, dd, J = 8.0, 9.0 Hz, H-2″), 4.18 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 4.23 (1H, dd, J = 7.5, 8.5 Hz, H-2′), 4.26 (1H, dd, J = 9.0, 9.0 Hz, H-3″), 4.28 (1H, m, H-3), 4.32 (1H, m, overlapped, H-3′), 4.32 (1H, m, overlapped, H-4″), [4.34 (1H, dd, J = 4.5, 11.5 Hz), 4.51 (1H, m, overlapped), H2-6′], [4.50 (1H, m, overlapped), 4.57 (1H, dd, J = 2.0, 12.0 Hz), H2-6″], 4.55 (1H, m, H-16), 4.93 (1H, d, J = 7.5 Hz, H-1′), 5.40 (1H, d, J = 8.0 Hz, H-1″); 13C-NMR (C5D5N, 125 MHz) spectroscopy data: see Table 1. HRESI-TOF-MS: Positive-ion mode m/z 777.4037 [M + Na]+ (calcd. for C39H62O14Na, 777.4032).

(25S)-Yucca spirostanoside E (3b): White powder; [α] − 8.2° (c = 2.7, MeOH); IR νmax (KBr) cm−1: 3408, 2929, 2987, 1704, 1451, 1373, 1171, 1075, 1032, 990, 919, 896, 850. 1H-NMR (C5D5N, 500 MHz): δ 0.92 (3H, s, H3-19), [0.94 (1H, m), 1.31 (1H, m, overlapped), H2-7], 1.07 (3H, d, J = 7.5 Hz, H3-27), 1.08 (3H, s, H3-18), 1.22, 1.84 (1H each, both m, H2-6), 1.29, 1.72 (1H each, both m, H2-1), 1.31, 1.86 (1H each, both m, overlapped, H2-2), 1.37 (3H, d, J = 7.0 Hz, H3-21), 1.38, 2.13 (1H each, both m, overlapped, H2-24), 1.43 (1H, m, H-14), 1.43, 1.91 (1H each, both m, H2-23), [1.59 (1H, m, overlapped), 2.10 (1H, m), H2-15], 1.60 (1H, m, overlapped, H-25), 1.74 (1H, m, overlapped, H-9), 1.83 (2H, m, overlapped, H2-4), 1.84 (1H, m, overlapped, H-8), 1.88 (1H, m, overlapped, H-20), 2.26 (1H, m, H-5), [2.19 (1H, dd, J = 5.0, 14.0 Hz), 2.37 (1H, dd, J = 14.0, 14.0 Hz), H2-11], 2.79 (1H, dd, J = 6.5, 8.5 Hz, H-17), [3.37 (1H, br. d, ca. J = 12 Hz), 4.06 (1H, dd, J = 2.5, 11.5 Hz), H2-26], 3.87 (1H, m, H-5′), 3.97 (1H, m, H-5″), 4.09 (1H, dd, J = 8.0, 8.0 Hz, H-2″), 4.18 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 4.23 (1H, dd, J = 7.5, 8.5 Hz, H-2′), 4.24 (1H, m, H-3), 4.26 (1H, dd, J = 8.0, 9.0 Hz, H-3″), 4.31 (1H, m, overlapped, H-3′), 4.31 (1H, m, overlapped, H-4″), [4.34 (1H, dd, J = 5.0, 11.5 Hz), 4.50 (1H, m, overlapped), H2-6′], [4.49 (1H, m, overlapped), 4.56 (1H, dd, J = 3.0, 12.0 Hz), H2-6″], 4.51 (1H, m, H-16), 4.93 (1H, d, J = 7.5 Hz, H-1′), 5.40 (1H, d, J = 8.0 Hz, H-1″); 13C-NMR (C5D5N, 125 MHz) spectroscopy data: see Table 1. HRESI-TOF-MS: Positive-ion mode m/z 777.4035 [M + Na]+ (calcd. for C39H62O14Na, 777.4035).

3.4. Acid Hydrolysis of –, –

The solution of 1a–3a, 1b–3b (each 2.0 mg) in 1 M HCl (1.0 mL) was treated by using the same method as described in reference [5]: They were heated under reflux for 3 h. Then each reaction mixture was detected by CH3CN–H2O (75:25, v/v; flow rate 1.0 mL/min). As a result, d-glucose was found from the aqueous phase of 1a–3a, 1b–3b, and d-xylose was detected from 2a and 3b by comparison of their tR and optical rotation with those of the authentic sample, d-glucose (tR 12.4 min (positive)), d-xylose (tR 6.1 min (positive)).

3.5. Bioassay

3.5.1. Materials

SW620 cell line was obtained from Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College (Beijing, China). Fetal Bovine Serum (FBS) was purchased from Biological Industries (Beit-Haemek, Israel). Roswell Park Memorial Institute (RPMI) 1640 medium was from Corning (Shanghai, China). Penicillin, streptomycin, and methyl thiazolyl tetrazolium (MTT) were ordered from Thermo Fisher Scientific (Shanghai, China). Dimethyl sulfoxide (DMSO) and 5-fluorouracil were purchased from Sigma-Aldrich (St. Louis, MO, USA).

3.5.2. MTT Assay

The inhibitory effects of Y. schidigera 70% EtOH extract, Y. schidigera 95% EtOH eluate, Y. schidigera H2O eluate, as well as 25R/S-spirostanol saponin diastereomers 1a–6a, 1b–6b were tested for their individual inhibitory activities on the growth of SW620 cell lines. Cell viability in the presence or absence of tested samples (with the positive control, 5-fluorouracil) was determined using the MTT method [9].

The SW620 cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin and kept in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. For cell viability determination, exponentially growing cells were harvested and plated in 96-well plates (5 × 104/mL) in RPMI 1640 medium for 24 h. After the cells had been washed with PBS, the medium was changed to serially diluted test samples in RPMI. After 48 h of incubation, the cells were washed twice with PBS, and MTT solution was added and incubated for 4 h at 37 °C. Then, MTT was removed. After 100 μL DMSO was added, the 96-well plate was shaken (90 R/S) for 5 min at room temperature under avoiding light, then the absorbance was determined at 490 nm by microplate reader. The tested compounds were independently performed four times. Values are expressed as mean ± S.D. The IC50 values were statistically determined using the SPSS 11.0 software (International business machines corporation (IBM Co.), Armonk, NY, USA).

4. Conclusions

Analysis of spirostanol saponins is usually performed by ultra-high performance supercritical fluid chromatography (UHPSFC) and ultra-high performance liquid chromatography (UHPLC) [10], which are not suitable for mass preparation. Until now, successful preparation examples for (25R/S)-spiromeric epimers were accomplished by SFC isolation [11,12] usually. Meanwhile, it is very rare still for preparing this type of R/S mixture using HPLC methods [13,14,15,16]. On the other hand, among the rare isolation examples, all of them are only for the isolation of C12 unsubstituted 25R/S-spirostanol saponin diastereomers, but no reference was found to separate C12 carbonylated 25R/S-ones. Though the emergence of SFC technology provides a new idea for the qualitative and quantitative analysis of drugs, there are still some difficulties for the further promotion and application of this technology [17]: (1) the types of stationary phases are rare, compounds with strong polarity show poor separation; (2) stationary phase, mobile phase and compounds often do not have good compatibility; (3) not all samples have good solubility in CO2; (4) when combined with mass spectrometry due to the delayed effect of the porous structure of the stationary phase, a large flow rate is often required, so desirable environmental protection objectives (less mobile phase usage) cannot be achieved. Therofore, it is very important to establish a simple, universal, fast and mass separation analysis method for 25R/S-spirostanol saponin diastereomers.

In this paper, the liquid chromatographic retention behaviors of C12 carbonylated and C12 unsubstituted 25R/S-spirostanol saponin diastereomers on three kinds of stationary phases (C8, C18, C30 columns) and with two kinds of mobile phases (MeOH-1% CH3COOH and CH3CN-1% CH3COOH) were investigated. A C30 column was firstly found to be more suitable for the separation of this kind of diastereomers than C8 and C18 column. Meanwhile, the analysis results indicated that both CH3CN-1% CH3COOH and MeOH-1% CH3COOH eluate systems were selective to C12 unsubstituted 25R/S-spirostanol saponin diastereomers, while MeOH-1% CH3COOH possessed better selectivity for C12 carbonylated ones. Compared with SFC, this method is more simple and versatile. Since 25R/S-spirostanol saponin diastereomers are widely distributed in natural herbs, this research provides a rapid and reliable method for the isolation of similar compounds from plant materials.

On the basis of the abovementioned analysis method, six pairs of 25R/S-spirostanol saponin diastereomers 1a–6a, 1b–6b were isolated from Y. Schidigera succefully. Among them, 1a–3a, 1b–3b were new compounds and 4a and 4b were isolated from Y. schidigera for the first time. The NMR spectrum of compounds: 1a–3a, 1b–3b are in the Supplementary Material.

On the other hand, the study of inhibitory activity and structure-activity relationships of 25R/S-spirostanol saponin diastereomers from Y. schidigera on the growth of SW620 cells will lay a foundation for the further mechanism studies, and it will supply an example for searching for anti-colon cancer drugs in natural products. What’s more, the summary of the structure-activity relationships affords a basis for structural modification, and semi- or total synthesis of new anti-cancer drugs.