Oleanane-Type Saponins from the Roots of Ligulariopsis shichuana and Their α-Glucosidase Inhibitory Activities

Abstract

Five new oleanane-type saponins, named ligushicosides A-E, and three known oleanane-type saponins were isolated from the roots of Ligulariopsis shichuana. Their structures were established by a combination of spectroscopic techniques, including 1D and 2D NMR and high resolution electrospray ionization mass spectroscopy (HR-ESI-MS). Furthermore, all isolates were evaluated for their yeast α-glucosidase inhibitory effects and exhibited potent inhibition against α-glucosidase, while compounds 1 and 2 showed excellent inhibitory activities. The 3-O-glycoside moiety in oleanane-type saponin is important for the α-glucosidase inhibitory effects.

1. Introduction

Nowadays, diabetes, especially type II diabetes mellitus, is a significant metabolic disorder that endangers public health, and is regarded as one of the most important public health problems in all nations [1]. One of the effective managements of type II diabetes mellitus is to retard the absorption of glucose by inhibition of carbohydrate hydrolyzing enzymes such as α-glucosidase in the digestive organs, which is the key enzyme catalyzing the final step in the digestive process of carbohydrates. Hence, α-glucosidase inhibitors can retard the liberation of glucose from dietary complex carbohydrates and delay glucose absorption, resulting in reduced postprandial plasma glucose levels and suppression of postprandial hyperglycemia. Among many classes of clinical drugs for diabetes, α-glucosidase inhibitors are useful for preventing the progression of the disease and for treating prediabetic conditions [2]. However, α-glucosidase inhibitors in clinical use usually exhibit decreased efficacy over time, ineffectiveness against some long-term diabetic complications, and low cost-effectiveness [3]. With regard to achieving and maintaining long-term glycemia control, discovering new α-glucosidase inhibitors is an urgent need.

Because of their perceived effectiveness, minimal side effects in clinical experience, and relatively low cost, herbal drugs are recognized as a wonderful source for medicines. Moreover, natural products have always provided new scaffolds for the design and development of new drugs, due to their structural diversity [4,5]. Ligulariopsis shichuana is the only species in genus Ligulariopsis (Compositae) that is endemic to western China. Previous studies on this plant have reported eremophilenolides and triterpenes, showing its chemical similarity with those plants in Ligularia [6,7,8]. Local people currently use the root and rhizome of L. shichuana, as well as some species of Ligularia, as “shanziwan”, a traditional substitute for “ziwan” (root of Aster tataricus L.f.) (Radix Asteris), which is a traditional Chinese medicine used to treat the symptom of xerostomia and frequent drinking caused by diabetes [9].

Aiming to elucidate the antidiabetic constituents from L. shichuana, our team carried out phytochemical investigations on L. shichuana. In this study, five new oleanane-type saponins (1–5) and three known oleanane-type saponins (6–8) were found (Figure 1). All isolated compounds were tested for α-glucosidase inhibitory activity.

2. Results

Phytochemical study of MeOH extract of the roots of L shichuana led to the isolation of five new ligushicosides A-E (1–5) and three known oleanane-type saponins, longispinogenin 3-O-β-d-glucuronopyranoside (6) [10], calenduloside E (7) [11] and calenduloside E 6′-methyl ester (8) [11] (Figure 1). The known structures were identified by comparison of their spectroscopic data with those reported in the literature. Acid hydrolysis of the saponins mixture A (compounds 1–8, each 1.0 mg) only gave one sugar, which was identified as glucuronic acid (GluA) by TLC, and its absolute configuration was determined as D by measuring its optical rotation [12].

Compound 1 was obtained as a pale yellow powder, and was found to have the molecular formula C₃₆H₅₆O₉, as determined through HR-ESI-MS ([M + H]⁺ peak at m/z 633.3998, calc. for C₃₆H₅₇O₉, 633.4003). IR spectrum indicated hydroxyl (3393 cm⁻¹), carbonyl (1715 cm⁻¹) and C=C double bond (1607 cm⁻¹) functions. The ¹H-NMR spectrum (

Table 1. ¹H-NMR (600 MHz) spectroscopic data for compounds 1–5 in CD₃OD (δ_H, J in Hz).

Position	1	2	3	4	5
1a	1.62, m	1.60, m	1.55, m	1.55, m	1.58, m
1b	1.00, m	0.98, m	0.95, m	0.93, m	0.95, m
2a	1.98, m	1.96, m	1.78, m	1.80, m	1.78, m
2b	1.72, m	1.70, m	1.70, m	1.76, m	1.68, m
3	3.19, dd (11.7, 4.3)	3.13, dd (11.8, 4.4)	3.07, dd (9.9, 5.7)	3.07, dd (9.9, 5.7)	3.14, dd (11.2, 4.6)
5	0.81, m	0.77, m	0.75, m	0.75, m	0.76, m
6a	1.59, m	1.57, m	4.51, br s	4.51, br s	1.57, m
6b	1.45, m	1.43, m			1.43, m
7a	1.43, m	1.41, m	1.72, m	1.73, m	1.55, m
7b	1.27, m	1.25, m	1.55, m	1.58, m	1.36, m
9	1.58, m	1.56, m	1.58, m	1.58, m	1.54, m
11a	1.93, m	1.92, m	2.03, m	2.02, m	1.92, m
11b	1.92, m	1.91, m	1.91, m	1.90, m	1.85, m
12	5.34, t-like (1.5)	5.31, t-like (1.5)	5.27, br s	5.27, br s	5.34, br s
15a	1.82, m	1.79, m	1.80, m	1.40, m	1.40, m
15b	1.53, m	1.51, m	1.25, m	1.20, m	1.21, m
16	4.32, dd (11.9, 4.5)	4.29, dd (12.0, 4.6)	4.13, dd (11.4, 4.5)	4.24, dd (11.7, 4.8)	4.24, dd (12.0, 4.8)
18	2.78, dd (14.0, 4.2)	2.76, dd (14.0, 4.5)	2.18, m	2.21, m	2.18, m
19a	1.70, m	1.67, m	1.73, m	1.72, m	1.71, m
19b	1.19, m	1.16, m	1.03, m	1.06, m	1.02, m
21a	1.58, m	1.56, m	1.90, m	1.41, m	1.41, m
21b	1.37, m	1.34, m	1.15, m	1.21, m	1.21, m
22a	2.09, m	2.06, m	1.39, m	2.21, m	2.10, m
22b	1.26, m	1.23, m	1.11, m	1.41, m	1.40, m
23	1.08, s	1.05, s	1.13, s	1.13, s	1.06, s
24	0.88, s	0.85, s	1.23, s	1.23, s	0.86, s
25	0.98, s	0.95, s	1.33, s	1.33, s	0.97, s
26	0.83, s	0.81, s	1.28, s	1.30, s	1.03, s
27	1.25, s	1.23, s	1.19, s	1.20, s	1.24, s
28a	9.76, s	9.73, s	0.79, s	3.80, d (9.7)	3.81, d (9.7)
28b				3.26, m	3.27, m
29	0.96, s	0.93, s	0.89, s	0.90, s	0.90, s
30	0.96, s	0.94, s	0.92, s	0.93, s	0.92, s
GluA-1′	4.38, d (8.2)	4.37, d (7.8)	4.37, d (7.7)	4.37, d (7.8)	4.40, d (7.5)
2′	3.24, t (8.3)	3.22, t (8.5)	3.24, t (8.4)	3.24, t (8.4)	3.26, t (8.4)
3′	3.39, t (9.2)	3.34, t (9.1)	3.35, t (9.1)	3.35, t (9.1)	3.37, t (9.0)
4′	3.47, t (8.8)	3.49, t (9.4)	3.50, t (9.4)	3.49, t (9.4)	3.53, t (9.2)
5′	3.62, d (10.0)	3.81, d (9.8)	3.80, d (9.8)	3.80, d (9.7)	3.83, br d (10.0)
6′-OCH3		3.77, s	3.76, s	3.76, s	3.79, s

, Figure S1) displayed signals for seven methyl groups at δ_H 0.83, 0.88, 0.96, 0.96, 0.98, 1.08 and 1.25 (each 3H, s, H₃-26, 24, 29, 30, 25, 23, 27). Further features included signals observed at δ_H 3.19 (1H, dd, J = 11.7, 4.3 Hz, typical for an axial proton attached to a hydroxylate carbon), δ_H 5.34 (1H, br t, J = 1.5 Hz, assignable to a vinylic proton), and at δ_H 9.76 (1H, s, an aldehyde proton signal). The ¹³C NMR spectrum (

Table 2. ¹³C-NMR (150 MHz) spectroscopic data for compounds 1–5 in CD₃OD (δ_C, type).

Position	1	2	3	4	5
1	38.4, CH2	38.3, CH2	42.1, CH2	42.1, CH2	39.9, CH2
2	25.5, CH2	25.6, CH2	27.3, CH2	27.3, CH2	27.0, CH2
3	89.4, CH	89.6, CH	91.3, CH	91.3, CH	91.1, CH
4	38.8, C	38.8, C	41.2, C	41.2, C	41.6, C
5	55.6, CH	55.5, CH	57.3, CH	57.3, CH	57.0, CH
6	17.9, CH2	17.8, CH2	68.6, CH	68.6, CH	19.3, CH2
7	32.6, CH2	32.6, CH2	41.6, CH2	41.7, CH2	33.7, CH2
8	39.5, C	39.4, C	40.4, C	40.4, C	40.9, C
9	46.8, CH	46.7, CH	48.7, CH	48.6, CH	48.3, CH
10	36.4, C	36.4, C	37.4, C	36.7, C	36.7, C
11	23.1, CH2	23.1, CH2	24.7, CH2	24.7, CH2	24.1, CH2
12	123.1, CH	123.0, CH	123.8, CH	124.3, CH	124.0, CH
13	142.0, C	141.9, C	144.5, C	143.6, C	144.3, C
14	43.4, C	43.4, C	45.4, C	45.1, C	44.7, C
15	36.5, CH2	36.5, CH2	36.4, CH2	34.9, CH2	35.0, CH2
16	63.8, CH	63.8, CH	66.4, CH	68.0, CH	67.8, CH
17	52.8, C	52.7, C	38.6, C	41.5, C	40.2, C
18	41.8, CH	41.7, CH	50.8, CH	45.1, CH	45.1, CH
19	45.7, CH2	45.8, CH2	48.1, CH2	47.9, CH2	47.9, CH2
20	30.0, C	30.0, C	31.9, C	30.8, C	31.7, C
21	32.6, CH2	32.6, CH2	35.4, CH2	34.9, CH2	34.8, CH2
22	22.3, CH2	22.3, CH2	31.7, CH2	26.0, CH2	26.0, CH2
23	27.1, CH3	27.0, CH3	28.3, CH3	28.2, CH3	28.5, CH3
24	15.6, CH3	15.5, CH3	18.5, CH3	18.5, CH3	17.0, CH3
25	14.6, CH3	14.6, CH3	17.6, CH3	17.5, CH3	16.2, CH3
26	16.3, CH3	16.2, CH3	18.9, CH3	18.6, CH3	17.7, CH3
27	25.6, CH3	25.5, CH3	27.8, CH3	27.5, CH3	27.0, CH3
28	208.0, CH	208.0, CH	22.4, CH3	69.0, CH2	69.0, CH2
29	32.0, CH3	32.0, CH3	33.9, CH3	31.8, CH3	33.7, CH3
30	22.6, CH3	22.6, CH3	24.5, CH3	24.3, CH3	24.3, CH3
GluA-1′	105.3, CH	105.6, CH	107.1, CH	107.1, CH	107.1, CH
2′	75.3, CH	73.9, CH	75.4, CH	75.4, CH	75.4, CH
3′	78.1, CH	78.1, CH	77.6, CH	77.6, CH	77.6, CH
4′	74.1, CH	71.8, CH	73.3, CH	73.3, CH	73.3, CH
5′	76.6, CH	76.1, CH	76.7, CH	76.7, CH	76.7, CH
6′	175.5, C	170.0, C	171.5, C	171.5, C	171.5, C
6′-OCH3		51.4, CH3	52.9, CH3	52.8, CH3	52.8, CH3

, Figure S2) displayed a methine carbon signal at δ_C 208.0 (supporting the presence of a CHO group), a quaternary carbon signal at δ_C 175.5 (ester carbon), and two olefinic carbon signals (one quaternary at δ_C 142.0 and one methine at δ_C 123.1, suggesting the presence of a double bond). Furthermore, the spectrum also showed a signal at δ_C 105.3 (CH) assignable to an anomeric carbon in a sugar unit. On the basis of these data, compound 1 was determined to be a triterpene glycoside. Its aglycone was determined to be gummosogenin [13], due to their similar NMR data, except for the presence of one set of resonances attributable to a β-d-glucuronopyranosyl moiety [H-1′ (δ_H 4.38, 1H, d, J = 8.2 Hz), δ_C 74.1, 75.3, 76.6, 78.1, 105.3 and 175.5] in 1 [14]. Comparison of the ¹³C NMR data of 1 with that of gummosogenin showed the downfield shift of C-3 (+11.4 ppm) and upfield shift of C-2 (−2.6 ppm) in 1, indicating glycosylation at C-3. This assignment was further confirmed by the HMBC correlations (Figure 2, Figure S5) between H-3 (δ_H 3.19, 1H, dd, J = 11.7, 4.3 Hz) in the aglycon and C-1′ (δ_C 105.3) in β-d-glucuronopyranosyl moiety, and between H-1′ and C-3. The relative configuration of 1 was confirmed by nuclear overhauser enhancement spectroscopy (NOESY) (Figure 3, Figure S6). The α-orientation of H-3 was deduced from the correlations of H-3 with H-5 (δ_H 0.81, 1H, m) and H₃-23 respectively. The α-orientation of H-16 (δ_H 4.32, 1H, dd, J = 11.9, 4.5 Hz) was deduced from the correlations of H-16 with H₃-27. The structure of 1 was confirmed by a complete acid hydrolysis of the saponin mixture A, which afforded D-glucuronic acid, as identified by co-TLC with an authentic sample [12]. On the basis of these findings, the structure of 1 was elucidated and named ligushicoside A.

The structure determination of compound 2 was performed in a similar manner to that described for 1. Its molecular formula was determined as C₃₇H₅₈O₉ on the basis of HR-ESI-MS ([M + Na]⁺ peak at m/z 669.3972, calc. for C₃₇H₅₈O₉Na, 669.3979) and NMR experiments. Compared with the NMR data of 1, the ¹H NMR spectrum of 2 (Table 1, Figure S7) displayed a signal for an additional methoxy group at 6′-OCH₃ (δ_H 3.77, 3H, s), and the sugar carbon data revealed a noticeable difference in the δ value of C-6′ (−5.5 ppm). This evidence, together with the observed correlation between 6′-OCH₃ and C-6′ (δ_C 170.0) in its HMBC (Figure S12), suggested that there was a 6′-O-methyl-β-glucuronopyranoside in compound 2 [15]. The relative configuration of 2 was confirmed by the NOESY spectrum, which was identical to that of 1, thus permitting assignment of the molecular structure of 2, and its naming as ligushicoside B.

The HR-ESI-MS ([M + Na]⁺ peak at m/z 671.4128, calc. for C₃₇H₆₀O₉Na, 671.4135) of compound 3 established a molecular formula of C₃₇H₆₀O₉. The ¹H (Table 1, Figure S14) and the ¹³C-NMR (Table 2, Figure S15) spectra of 3 were very similar to those of 2, except for the disappearance of the aldehyde group and the appearance of a methyl group and a hydroxy group. The HMBC correlations (Figure S18) between H₃-28 (δ_H 0.79, 3H, s) and C-16 (δ_C 66.4), C-17 (δ_C 38.6), C-18 (δ_C 50.8), and C-22 (δ_C 31.7) indicated that the C-28 aldehyde group in 2 had been changed into a methyl group in 3. The second hydroxy group in 3 was assigned to C-6 (δ_C 68.6), due to the COSY correlation (Figure S16) between the protons at H-6 (δ_H 4.51, 1H, br s) and H-5 (δ_H 0.75, 1H, m), and between those at H-6 and H₂-7 (δ_H 1.55, 1H, m; 1.72, 1H, m), as well as the HMBC correlation. The relative configuration of 3 was confirmed by its NOESY spectrum (Figure S19), which was identical to that of 1. Furthermore, the β-orientation of OH-6 was deduced from the correlations of H-6 and H-5, H₃-23 (δ_H 1.13, 3H, s), and the α-orientation of H-16 (δ_H 4.13, 1H, dd, J = 11.4, 4.5 Hz) was deduced from the correlations of H-16 and H₃-27 (δ_H 1.19, 3H, s) in the NOSEY spectrum. Thus, the structure of ligushicoside C was assigned to 3.

The molecular formula of compound 4 was established as C₃₇H₆₀O₁₀ ([M + Na]⁺ peak at m/z 687.4076, calc. for C₃₇H₆₀O₁₀Na, 687.4084) by the HR-ESI-MS and NMR data. The ¹H (Table 1, Figure S21) and ¹³C NMR (Table 2, Figure S22) spectra of 4 were similar to those of olean-12-en-3β,6β,16β,28-tetraol, previously obtained from this plant [8]. The presence of a 6′-O-methyl-β-glucuronopyranoside moiety [H-1′ (δ_H 4.37, 1H, d, J = 7.8 Hz) and 6′-OCH₃ (δ_H 3.76, 3H, s); δ_C 52.8, 73.3, 75.4, 76.7, 77.6, 107.1 and 171.5] in 4, which was attached to the C-3 (δ_C 91.3) supported by the glycosylation shift at the C-3 position (+11.5 ppm) in its ¹³C NMR spectra. On the basis of these findings, the structure of 4 was elucidated and named ligushicoside D.

The molecular formula of compound 5 was determined as C₃₇H₆₀O₉ ([M + Na]⁺ peak at m/z 671.4128, calc. for C₃₇H₆₀O₉Na, 671.4135) by its HR-ESI-MS and NMR data. The NMR signals (Table 1 and Table 2, Figures S28 and S29) of 5 were similar to those of longispinogenin 3-O-β-d-glucuronopyranoside [10], except for an additional methoxy group at 6′-OCH₃ (δ_H 3.79, 3H, s). The HMBC correlation (Figure S32) between 6′-OCH₃ and C-6′ (δ_C 171.5) suggested that there was a 6′-O-methyl-β-glucuronopyranoside in compound 5. Thus, 5 was named ligushicoside E.

All isolated compounds 1–8 were tested for α-glucosidase inhibitory activity. The IC₅₀ values, defined as the compound concentration that inhibits α-glucosidase activity by 50%, are summarized in

Table 3. Inhibitory effects of compounds 1–8 and acarbose against α-glucosidase.

Compounds	IC50 (μM)	Compounds	IC50 (μM)
1	18.7 ± 1.7	5	154.3 ± 11.2
2	37.9 ± 3.6	6	42.6 ± 5.9
3	104.9 ± 4.3	7	57.6 ± 6.8
4	89.7 ± 5.1	8	133.7 ± 3.3
Acarbose	190.5 ± 3.1

. The results showed that all of the oleanane-type saponins isolated from the roots of L. shichuana displayed inhibitory activity against α-glucosidase, with IC₅₀ values in the range of 18.7–154.3 μM, which was obviously stronger than the positive control of acarbose (IC₅₀ = 190.5 μM). This result was in agreement with a report suggesting that the 3-O-glycoside moiety was essential to the inhibition activity of oleanane-type glycosides [16]. Furthermore, compounds 1 (IC₅₀ = 18.7 μM), 6 (IC₅₀ = 42.6 μM) and 7 (IC₅₀ = 57.6 μM) showed stronger activities than compounds 2 (IC₅₀ = 37.9 μM), 5 (IC₅₀ = 154.3 μM) and 8 (IC₅₀ = 133.7 μM), respectively, suggesting that the 6′-methyl ester of the glucuronic acid moiety reduced the α-glucosidase inhibitory activities [17]. Compared to 6 and 5, respectively, both 1 and 2 were more potent inhibitors, and the formyl group at C-28 may be responsible for their significant inhibitory properties. Compound 4, containing the 6-OH group, showed more potent α-glucosidase inhibitory activity (IC₅₀ = 89.7 μM) than compound 5 (IC₅₀ = 154.3 μM), indicating that 6-OH may increase the activity. Interestingly, according to previous literature, compounds 7 and 8 have also shown significant effects on the incensement of serum glucose levels in glucose-loaded rats, especially compound 7, which strongly inhibited the incensement in serum glucose levels [18].

3. Materials and Methods

3.1. General Experimental Material

Optical rotations were measured on a Perkin-Elmer 241 polarimeter (Perkin-Elmer, Waltham, MA, USA). IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer (Nicolet Instrument Company, Madison, WI, USA). 1D and 2D NMR spectra were recorded on a Bruker AV-600 spectrometer (600 MHz for ¹H and 150 MHz for ¹³C, respectively, Bruker Biospin, Fallanden, Switzerland), using tetramethylsilane (TMS) as an internal standard. HR-ESI-MS were measured by an Agilent 6520 Q-TOF LC-MS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Analytical TLC were run on silica gel plates (GF₂₅₄, Yantai Institute of Chemical Technology, Yantai, China). Spots were observed under UV light and visualized by spraying with 10% H₂SO₄ in ethanol, followed by heating. Column chromatography (CC) was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden). Acarbose was purchased from Sigma-Aldrich (St. Louis, MO, USA).

3.2. Plant Material

The roots of L. shichuana were collected in Baoji, Shaanxi Province, People’s Republic of China, in August 2011, and identified by one of the authors (W.-S.W.). A voucher specimen (No. 20110802) was deposited in the herbarium of the College of Life and Environmental Sciences, Minzu University of China, Beijing, People’s Republic of China.

3.3. Extraction and Isolation

The chopped, dried roots of L. shichuana (1.5 kg) were pulverized and extracted three times with MeOH (each for 7 days) at room temperature. After filtration, the filtrate was concentrated under reduced pressure to yield a residue (135.0 g), and then was suspended in water and partitioned successively with petroleum ether, CHCl₃, EtOAc, and n-butanol, to afford five fractions. The n-butanol fraction (15.5 g) was subjected to silica gel CC (500.0 g) eluting with CHCl₃–MeOH (15:1–0:1, gradient system). On the basis of TLC analysis, seven fractions A-G were obtained. Fraction B (1.1 g) was eluted with CHCl₃–MeOH (8:1) on CC, further purified by Sephadex LH-20 (MeOH) to afford 2 (16.3 mg). Fraction C (0.9 g) was subjected to CC eluting with CHCl₃–MeOH (5:1) to afford 3 (2.6 mg) and 5 (9.1 mg). Fraction D (0.5 g) was subjected to CC eluting with CHCl₃–MeOH (4:1) to afford 4 (20.6 mg). Fraction E (1.3 g) was subjected to CC eluting with CHCl₃–MeOH (3:1) to afford 1 (9.7 mg) and 7 (7.3 mg). Fraction F (0.7 g) was subjected to Sephadex LH-20 (MeOH) and then was subjected to CC eluting with EtOAc–MeOH (1:1) to afford 6 (5.1 mg). Fraction G (2.4 g) was eluted with CHCl₃–MeOH (1:1) on CC, further purified by Sephadex LH-20 (MeOH) to afford 8 (7.2 mg).

3.4. Spectroscopic Data

Ligushicoside A (1): Pale yellow, amorphous powder; [α]${}_D^{20}$ + 5.1 (c = 0.023, MeOH); IR (KBr) ν_max 3393, 2952, 1715, 1607, 1459, 1033 cm⁻¹; ¹H-NMR (CD₃OD, 600 MHz) data see Table 1, Figure S1; ¹³C-NMR (CD₃OD, 150 MHz) data see Table 2, Figure S2; HR-ESI-MS m/z 633.3998 (calcd. for C₃₆H₅₇O₉, 633.4003, Figure S7).

Ligushicoside B (2): Pale yellow, amorphous powder; [α]${}_D^{20}$ + 10.0 (c = 0.032, MeOH); IR (KBr) ν_max 3404, 2943, 1601, 1456, 1027 cm⁻¹; ¹H-NMR (CD₃OD, 600 MHz) data see Table 1, Figure S8; ¹³C-NMR (CD₃OD, 150 MHz) data see Table 2, Figure S9; HR-ESI-MS m/z 669.3972 (calcd. for C₃₇H₅₈O₉Na, 669.3979, Figure S13).

Ligushicoside C (3): Pale yellow, amorphous powder; [α]${}_D^{20}$ + 7.8 (c = 0.026, MeOH); IR (KBr) ν_max 3413, 2952, 1604, 1459, 1024 cm⁻¹; ¹H-NMR (CD₃OD, 600 MHz) data see Table 1, Figure S14; ¹³C-NMR (CD₃OD, 150 MHz) data see Table 2, Figure S15; HR-ESI-MS m/z 671.4128 (calcd. for C₃₇H₆₀O₉Na, 671.4135, Figure S20).

Ligushicoside D (4): Pale yellow, amorphous powder; [α]${}_D^{20}$ + 8.1 (c = 0.031, MeOH); IR (KBr) ν_max 3403, 2942, 1608, 1461, 1029 cm⁻¹; ¹H-NMR (CD₃OD, 600 MHz) data see Table 1, Figure S21; ¹³C-NMR (CD₃OD, 150 MHz) data see Table 2, Figure S22; HR-ESI-MS m/z 687.4076 (calcd. for C₃₇H₆₀O₁₀Na, 687.4084, Figure S27).

Ligushicoside E (5): Pale yellow, amorphous powder; [α]${}_D^{20}$ + 7.3 (c = 0.017, MeOH); IR (KBr) ν_max 3433, 2952, 1605, 1464, 1032 cm⁻¹; ¹H-NMR (CD₃OD, 600 MHz) data see Table 1, Figure S28; ¹³C-NMR (CD₃OD, 150 MHz) data see Table 2, Figure S29; HR-ESI-MS m/z 671.4128 (calcd. for C₃₇H₆₀O₉Na, 671.4135, Figure S33).

3.5. Acid Hydrolysis of Saponins

The crude saponin mixture A (compounds 1–8, each 1.0 mg) were treated with 5 N TFA (trifluoroacetic acid, aqueous solution, 3 mL) at 90 °C for 6 h. After extraction with CHCl₃ (3 × 3 mL), the aqueous layer was neutralized with 0.1 M NaOH and freeze-dried. The sugar was analyzed by TLC (silica gel, CHCl₃–MeOH–AcOH–H₂O, 60:32:12:8) for glucuronic acid (R_f 0.30) in comparison with standard sugar. Moreover, the optical rotations of the purified sugar were measured as for d-glucuronic acid, [α]${}_D^{20}$ + 8.1 (c = 0.26, H₂O) [12].

3.6. α-Glucosidase Inhibitory Activity Assay

α-Glucosidase inhibitory activity was examined by the method described by Omar et al. [19]. Acarbose, a definite α-glucosidase inhibition, was used as the positive drug.

4. Conclusions

In this study, eight oleanane-type saponins, including five new ones, were isolated from the roots of L. shichuana. All the structures were established by extensive spectroscopic analysis. The isolates were evaluated on the basis of their α-glucosidase inhibitory activity assays. All of the oleanane-type saponins exhibited significant inhibitory activities, with IC₅₀ values in the range of 18.7–154.3 μM, which were obviously stronger than the positive control of acarbose (IC₅₀ = 190.5 μM). Compounds 1 and 2 showed excellent inhibitory activity against α-glucosidase, which might be useful for further developing α-glucosidase inhibitors.