Five New Pregnane Glycosides from Gymnema sylvestre and Their α-Glucosidase and α-Amylase Inhibitory Activities

Abstract

Gymnema sylvestre, a medicinal plant, has been used in Indian ayurvedic traditional medicine for the treatment of diabetes. Phytochemical investigation of Gymnema sylvestre led to the isolation of five new pregnane glycosides, gymsylosides A–E (1–5) and four known oleanane saponins, 3β-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (6), gymnemoside-W1 (7), 3β-O-β-D-xylopyranosyl-(1→6)-β-D- glucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (8), and alternoside XIX (9). Their structures were identified based on spectroscopic evidence and comparison with those reported in the literature. All compounds were evaluated for their α-glucosidase and α-amylase inhibitory activities. Compounds 2–4 showed significant α-amylase inhibitory activity, with IC₅₀ values ranging from 113.0 to 176.2 µM.

1. Introduction

Gymnema sylvestre (Retz.) R.Br. ex Sm. (Apocynaceae) is a perennial woody climber native to tropical and subtropical regions, such as India, Africa, and southeast Asia. In folk medicine, G. sylvestre have been used to treat snake bites, arthritis, digestive, and enhancing laxative [1,2]. Moreover, the plant has been explored for its benefits in blocking sugar craving and reducing sugar consumption. The recent studies have indicated that G. sylvestre are potential anti-diabetic plants [3,4]. The bioactive components from this plant include pregnane glycosides [5], triterpene saponins [6,7], and flavonoids [8]. Our previous study reported pregnane glycosides from Gymnema inodorum and their α-glucosidase inhibitory activity [9]. As a part of our ongoing investigation on anti-diabetic compounds from Vietnamese plants [10], a methanol extract of the leaves of G. sylvestre was found to inhibit α-glucosidase and α-amylase activities. Herein, we report the isolation, structural elucidation of pregnane-type saponins and oleanane saponins and the evaluation of α-glucosidase and α-amylase inhibitory activities of these compounds.

2. Results and Discussion

2.1. Isolation of Compounds

The methanol extract of the G. sylvestre leaves was suspended in water and then partitioned with n-hexane, CH₂Cl₂ and EtOAc to obtain four layers. The CH₂Cl₂ and water extracts were chromatographed using combined silica gel and RP-18 columns. The fractions were further purified by HPLC to give five new pregnane glycosides and four known compounds (Figure 1 and Supplementary Materials).

2.2. Compound Identification

Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C₄₇H₇₆O₁₇ by HRESIMS ion at m/z 935.4986 [M + Na]⁺ (calcd for [C₄₇H₇₆O₁₇Na]⁺, 935.4975). The ¹H-NMR spectrum showed proton signals of three methyl groups at δ_H 1.13 (3H, s), 1.53 (3H, s), and 1.05 (3H, d, J = 6.4 Hz), one olefinic proton at δ_H 5.32 (1H, br s), which represented a pregnane aglycone. Two methyl groups at δ_H 1.80 (3H, d, J = 7.2 Hz) and 1.85 (3H, s) and one olefinic proton at δ_H 6.98 (1H, q, J = 7.2 Hz), suggested the presence of a tigloyl moiety. Three anomeric protons [δ_H 4.84 (br d, J = 9.6 Hz), 4.57 (br d, J = 9.2 Hz), and 4.41 (br d, J = 8.0 Hz), three methoxy groups [δ_H 3.39, 3.42, and 3.60 (each 3H, s)], together with three secondary methyl groups [δ_H 1.19 (3H, d, J = 6.0 Hz), 1.25 (d, J = 6.0 Hz), and 1.35 (d, J = 6.0 Hz)], confirmed the presence of three sugar units. The ¹³C NMR and DEPT spectra indicated that 1 contained one carbonyl, seven non-protonated carbons (three oxygenated), nineteen methines (sixteen oxygenateds), nine methylenes, and eleven methyl carbons (three methoxys). The ¹H and ¹³C-NMR spectroscopic data suggested that 1 was a pregnane glycoside [11]. Of these, 21 carbons were assigned to the pregnane skeleton, 5 to one tigloyl moiety, and 21 to a trisaccharide moiety. The HMBC correlations between H-19 (δ_H 1.13) and C-1 (δ_C 39.8)/C-5 (δ_C 140.0)/C-9 (δ_C 44.7)/C-10 (δ_C 38.0) suggested the position of a double bond at C-5/C-6. The HMBC correlations between H-18 (δ_H 1.53) and C-13 (δ_C 57.6)/C-14 (δ_C 89.3)/C-17 (δ_C 89.1); between H-21 (δ_H 1.05) and C-17 (δ_C 89.1)/C-20 (δ_C 71.5); and between H-6 (δ_H 5.32)/H-7 (δ_H 2.11)/H-9 (δ_H 1.49) and C-8 (δ_C 74.9) demonstrated the positions of hydroxyl groups at C-8, C-14, C-17, and C-20 (Figure 2). The constitution of the aglycone of 1 was demonstrated by the analysis of NOESY observations and similar reported-structures [11].

The aglycone of 1 was supposed to have the same configurations as those of gymnepregoside F and 12-O-(E)-cinnamoylgymnepregoside F from G. sylvestre [12], biogenetic derivatives of 1 at C-3, C-8, C-12, C-14, C-17, and C-20. In addition, the alkaline hydrolysis of 1 gave sarcostin, ((20S)-3β,8β,12β,14β,17β,20-hexahydroxypregn-5-ene) [13]. The multiplicity of H-12 [δ_H 4.68 (dd, J = 4.0, 11.6 Hz)] suggested that the configuration of H-12 was axial (α-configuration, Figure 2). The NOESY correlations between H-3 (δ_H 3.50) and H_α-1 (δ_H 1.09)/ H_α-4 (δ_H 2.33), and between H-12 (δ_H 4.68) and H-9 (δ_H 1.49)/H_α-15 (δ_H 1.88) suggested the configurations of the oxygenated groups at C-3 and C-12, the hydroxy groups at C-8 and C-14 to be β. The HMBC correlations between Tig H-5 (δ_H 1.85) and Tig C-1 (δ_C 169.1)/Tig C-2 (δ_C 130.0)/Tig C-3 (δ_C 139.5) and between Tig H-4 (δ_H 1.80) and Tig C-2/ Tig C-3 and NOESY correlations between Tig H-4 (δ_H 1.80) and Tig H-5 (δ_H 1.85) suggested the presence of (E)-tigloyl moiety. In addition, the position of this moiety at C-12 was confirmed by HMBC correlation from H-12 (δ_H 4.68) to Tig C-1 (δ_C 169.1). Acid hydrolysis of 1 gave three monosaccharides, which were identified as D-cymarose [14], D-oleandrose [14], and D-thevetose [15], by comparing their specific rotation with those reported [16]. The large coupling constants between H-1 and H-2 of monosaccharide moieties and also HMBC correlations between Thv H-1 (δ_H 4.41) and Ole C-4 (δ_C 84.1), Ole H-1 (δ_H 4.57) and Cym C-4 (δ_C 83.8), and between Cym H-1 (δ_H 4.84) and aglycone C-3 (δ_H 79.2) indicated the sugar linkages as β-D-thevetopyranosyl-(1→4)-β-D-oleandropyranosyl- (1→4)-β-D-cymaropyranoside and at C-3 of aglycone. Based on the above evidence, the structure of 1 was elucidated as (20S)-12β-tigloyloxy-3β,8β,14β,17β,20-pentahydroxypregn-5-ene 3-O-β-D-thevetopyranosyl- (1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside, a new compound named gymsyloside A.

The ¹H and ¹³C-NMR spectra of 2 exhibited a pregnane aglycone, one tigloyl unit, and three sugar units (

Table 1. NMR spectroscopic data for compounds 1–3 in CD₃OD.

C		1		2		3
	δC	δH (mult., J, in Hz)	δC	δH (mult., J, in Hz)	δC	δH (mult., J, in Hz)
1	39.8	1.09, m, α/1.80, m, β	39.8	1.09, m, α/1.79, m, β	39.8	1.09, m, α/1.80, m
2	30.2	1.57, m, β/1.84, m, α	30.2	1.56, m, β/1.84, m, α	30.1	1.57, m, β/1.83, m, α
3	79.2	3.50, m	79.3	3.50, m	79.3	3.49, m
4	39.8	2.20, m, β/2.33, m, α	39.8	2.20, m, β/2.33, m, α	39.8	2.19, m, β/2.33, m, α
5	140.0	-	140.0	-	140.0	-
6	120.0	5.32, br s	120.0	5.32, br s	119.9	5.33, br s
7	35.2	2.11, m	35.2	2.12, m	35.2	2.12, m
8	74.9	-	75.0	-	75.0	-
9	44.7	1.49, m	44.7	1.48, m	44.7	1.49, m
10	38.0	-	38.0	-	38.0	-
11	26.0	1.63, m, α/2.00, m, β	26.0	1.62, m, α/2.00, m, β	25.9	1.62, m, α/2.00, m, β
12	75.1	4.68, dd, 4.0, 11.6)	75.2	4.68, m	75.2	4.70, dd, 4.0, 9.6)
13	57.6	-	57.6	-	57.6	-
14	89.3	-	89.3	-	89.2	-
15	34.3	1.83, m, β/1.88, m, α	34.3	1.82, m, β/1.91, m, α	34.3	1.81, m, β/1.89, m, α
16	33.5	1.74, m	33.5	1.75, m	33.5	1.74, m
17	89.1	-	89.2	-	89.3	-
18	11.2	1.53, s	11.2	1.53, s	11.2	1.52, s
19	18.5	1.13, s	18.5	1.14, s	18.5	1.14, s
20	71.5	3.45, m	71.5	3.44, m	71.6	3.46, m
21	18.9	1.05, d (6.4)	18.9	1.04, d (6.4)	18.9	1.06, d (6.4)
	Tig		Tig		Tig
1	169.1	-	169.2	-	169.1	-
2	130.0	-	130.1	-	130.1	-
3	139.5	6.98, q (7.2)	139.6	6.98, q (7.2)	139.6	7.00, q (6.8)
4	14.5	1.80, d (7.2)	14.5	1.80, d (7.2)	14.5	1.81, d (6.8)
5	12.2	1.85, s	12.1	1.85, s	12.1	1.87, s
	Cym		Cym		Cym I
1	97.2	4.84, br d (9.6)	97.2	4.85, br d (9.6)	97.2	4.84, br d, 9.6)
2	36.6	1.52, m, a/2.05, m, e	36.7	1.53, m, a/2.04, m, e	36.6	1.55, m, a/2.05, m, e
3	78.5	3.82, m	78.5	3.83, m	78.5	3.82, m
4	83.8	3.24, m	83.8	3.24, m	83.8	3.23, m
5	69.9	3.79, m	70.0	3.79, m	70.1	3.79, m
6	18.5	1.19, d (6.0)	18.5	1.19, d (6.0)	18.5	1.18, d (6.4)
3-OMe	58.5	3.42, s	58.5	3.42, s	58.5	3.42, s
	Ole		Ole		Cym II
1	102.6	4.57, br d (9.2)	102.6	4.56, br d (6.8)	101.2	4.78, br d (9.6)
2	37.6	1.40, m, a/2.30, m, e	37.5	1.39, m, a/2.30, m, e	36.4	1.55, m, a/2.10, m, e
3	80.2	3.36, m	80.4	3.36, m	78.6	3.82, m
4	84.1	3.18, m	84.0	3.17, m	83.9	3.23, m
5	72.5	3.36, m	72.6	3.35, m	69.9	3.79, m
6	18.9	1.35, d (6.0)	19.0	1.34, d (6.0)	18.6	1.21, d (6.0)
3-OMe	57.6	3.39, s	57.5	3.39, s	58.4	3.42, s
	Thv		All		Ole
1	104.3	4.41, br d (8.0)	102.2	4.70, br d (7.6)	102.6	4.58, br d (8.4)
2	75.6	3.18, m	73.6	3.29, m	37.6	1.40, m, a)/2.30, m, e)
3	87.7	3.00, t (6.8)	83.8	3.60, m	80.2	3.36, m
4	76.6	3.00, t (6.8)	75.0	3.16, m	84.1	3.18, m
5	73.2	3.25, m	71.2	3.64, m	72.5	3.35, m
6	18.1	1.25, d (6.0)	18.5	1.21, d (6.0)	18.9	1.36, d (5.6)
3-OMe	61.0	3.60, s	62.5	3.58, s	57.5	3.41, s
					Thv
1	-	-	-	-	104.3	4.42, d (8.0)
2	-	-	-	-	75.6	3.18, m
3	-	-	-	-	87.7	3.00, m
4	-	-	-	-	76.6	3.00, m
5	-	-	-	-	73.2	3.25, m
6	-	-	-	-	18.1	1.27, d (6.0)
3-OMe	-	-	-	-	61.1	3.60, s

Assignments were done by Heteronuclear Single Quantum Coherence (HSQC), Heteronuclear Multiple Bond Correlation (HMBC), Correlation Spectroscopy (COSY), and Rotating frame Overhauser Effect Spectroscopy (ROESY) experiments. Tig, Tigloyl; Cym, β-d-cymaropyranosyl; Ole, β-d-oleandropyranosyl; Thv, β-d-thevetopyranosyl; All, 6-deoxy-3-O-methyl-β-d-allopyranosyl; a, axial; e, equatorial; α, atoms or groups laying below the plane of structure; β, atoms or groups laying above the plane of structure.

). In addition, the NMR data of 2 were similar to those of gymsyloside A (1), except for the difference of sugar unit at Ole C-4: D-thevetose replaced by 6-deoxy-3-O-methyl-D-allose. Acid hydrolysis of 2 confirmed the presence of D-cymarose, D-oleandrose, and 6-deoxy-3-O-methyl-D-allose as sugar components. Furthermore, the ¹H and ¹³C-NMR data of 2 showed the sugar units as β-D-cymaropyranosyl, β-D-oleandropyranosyl, and 6-deoxy-3-O-methyl-β-D-allopyranose. The HMBC correlations between All H-1 (δ_H 4.70) and Ole C-4 (δ_C 84.0), Ole H-1 (δ_H 4.56) and Cym C-4 (δ_C 83.8), and between Cym H-1 (δ_H 4.85) and aglycone C-3 (δ_C 79.3) confirmed the sugar linkages to be 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside. Consequently, compound 2 was elucidated to be (20S)-12β-tigloyloxy-3β,8β,14β,17β,20-pentahydroxypregn-5-ene 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside, a new compound named gymsyloside B.

The HRESIMS of 3 gave a pseudo-molecular ion peak at m/z 1079.5769 [M + Na]⁺, corresponding to the molecular formula of C₅₄H₈₈O₂₀. The ¹H and ¹³C-NMR spectra of 3 showed the presence of one pregnane aglycone, four sugar units, and one tigloyl unit (Table 1). The NMR data of 3 were compared to gymsyloside A (1) and found the addition of one sugar unit in the sugar linkages. Acid hydrolysis of 3 gave three monosaccharides, which were identified as D-cymarose, D-oleandrose, and D-thevetose. The tetrasaccharide was determined to be β-D-thevetopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, by the analysis of HMBC and COSY correlations. The location of sugar linkages at C-3 was confirmed by the HMBC correlation between Cym I H-1 (δ_H 4.84) and C-3 (δ_C 79.3). Consequently, the structure of 3 was determined to be (20S)-12β-tigloyloxy-3β,8β,14β,17β, 20-pentahydroxypregn-5-ene 3-O-β-D-thevetopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, a new compound named gymsyloside C.

The molecular formula of 4 was determined as C₅₄H₈₈O₂₀ by the HRESIMS. The ¹H and ¹³C-NMR data (

Table 2. NMR spectroscopic data for compounds 4 and 5 in CD₃OD.

C		4		5
	δC	δH (mult., J, in Hz)	δC	δH (mult., J, in Hz)
1	39.8	1.07, m, α/1.79, m, β	39.8	1.08, m, α/1.80, m, β
2	30.2	1.57, m, β/1.83, m, α	30.2	1.58, m, β/1.84, m, α
3	79.3	3.50, m	79.3	3.50, m
4	39.8	2.18, m, β/2.33, m, α	39.8	2.20, m, β/2.33, m, α
5	140.0	-	140.0	-
6	120.0	5.32, br s	120.0	5.32, br s
7	35.2	2.11, m	35.2	2.11, m
8	74.9	-	74.9	-
9	44.7	1.49, m	44.7	1.49, m
10	38.0	-	38.0	-
11	26.0	1.63, m, α/2.00, m, β	25.9	1.63, m, α/2.00, m, β
12	75.1	4.67, m	75.1	4.68, m
13	57.6	-	57.6	-
14	89.3	-	89.3	-
15	34.3	1.80, m, β/1.88, m, α	34.3	1.83, m, β/1.90, m, α
16	33.5	1.74, m	33.5	1.75, m
17	89.1	-	89.1	-
18	11.2	1.53, s	11.2	1.53, s
19	18.6	1.13, s	18.6	1.12, s
20	71.5	3.46, m	71.5	3.45, m
21	19.0	1.05, d (6.0)	18.9	1.05, d (6.4)
	Tig		Tig
1	169.1	-	169.1	-
2	130.1	-	130.1	-
3	139.5	6.98, q (7.2)	139.5	6.98, q (7.2)
4	14.6	1.81, d (7.2)	14.5	1.80, d (7.2)
5	12.2	1.85, s	12.2	1.85, s
	Cym I		Cym
1	97.2	4.83, br d (9.6)	97.2	4.84, br d (9.6)
2	36.6	1.54, m, a/2.04, m, e	36.7	1.53, m, a/2.05, m, e
3	78.5	3.82, m	78.5	3.81, m
4	83.8	3.24, m	83.8	3.24, m
5	69.8	3.78, m	69.9	3.80, m
6	18.3	1.17, d (6.4)	18.4	1.19, d (6.4)
3-OMe	58.4	3.41, s	58.5	3.42, s
	Cym II		Ole
1	101.2	4.77, br d (9.6)	102.6	4.57, br d (9.2)
2	36.4	1.54, m, a/2.04, m, e	37.6	1.40, m, a/2.30, m, e
3	78.4	3.82, m	80.3	3.35, m
4	83.8	3.24, m	84.1	3.18, m
5	69.9	3.78, m	72.6	3.38, m
6	18.6	1.19, d (5.6)	18.8	1.36, d (6.0)
3-OMe	58.5	3.40, s	57.6	3.39, s
	Ole		Thv
1	102.6	4.56, br d (9.2)	104.2	4.43 (d (7.6)
2	37.5	1.40, m/2.30, m	75.2	3.23, m
3	80.3	3.35, m	86.3	3.17, m
4	83.7	3.18, m	82.9	3.30, m
5	72.5	3.35, m	72.5	3.38, m
6	18.9	1.35, d (6.0)	18.5	1.36, d (7.2)
3-OMe	57.4	3.39, s	61.2	3.60, s
	All		Glc
1	102.2	4.70, d (8.0)	104.3	4.41, d (7.6)
2	73.5	3.29, m	75.6	3.15, m
3	83.9	3.60, m	78.3	3.23, m
4	75.0	3.16, m	71.8	3.20, m
5	71.2	3.63, m	77.9	3.31, m
6	18.6	1.21, d (6.0)	63.2	3.62, m/3.84, m
3-OMe	62.5	3.58, s

Tig, Tigloyl; Cym, β-d-cymaropyranosyl; Ole, β-d-oleandropyranosyl; Thv, β-d-thevetopyranosyl; Glc, β-d-glucopyranosyl; All, 6-deoxy-3-O-methyl-β-d-allopyranosyl; a, axial; e, equatorial; α, atoms or groups laying below the plane of structure; β, atoms or groups laying above the plane of structure.

) indicated that the structure of 4 was similar to those of 3, except for the difference of monosaccharide at Ole C-4. The sugar components were found to be similar to those of 2 (D-cymarose, D-oleandrose, and 6-deoxy-3-O-methyl-D-allose) [17]. Moreover, the sugar linkages, 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside was confirmed by the HMBC correlations from All H-1 (δ_H 4.70) to Ole C-4 (δ_C 83.7), Ole H-1 (δ_H 4.56) to Cym II C-4 (δ_C 83.8), and from Cym II H-1 (δ_H 4.77) to Cym I C-4 (δ_C 83.8). Similar to those of 1–3, the aglycone was found as (20S)-12β-tigloyloxy-3β,8β,14β,17β, 20-pentahydroxypregn-5-ene. Consequently, the structure of 4 was determined as (20S)- 12β-tigloyloxy-3β,8β,14β,17β,20-pentahydroxypregn-5-ene 3-O-6-deoxy-3-O-methyl-β-D- allopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside and named gymsyloside D.

The molecular formula of 5, C₅₃H₈₆O₂₂ was determined by the HRESIMS pseudo-ion at m/z 1097.5530 [M + Na]⁺. The ¹H and ¹³C NMR data of 5 were similar to 2, except for an additional sugar unit at Thv C-4 (Table 2). The sugar moieties were determined as D-cymarose [14], D-oleandrose [14], D-thevetose [15], and D-glucose [17] by acid hydrolysis. The HMBC correlations between Glc H-1 (δ_H 4.41) and Thv C-4 (δ_C 82.9), Thv H-1 (δ_H 4.43) and Ole C-4 (δ_C 84.1), Ole H-1 (δ_H 4.57) and Cym C-4 (δ_C 83.8), and between Cym H-1 (δ_H 4.84) and aglycone C-3 (δ_C 79.3) confirmed the sequence of sugar linkages, previously reproted from G. sylvestre [5]. Thus, compound 5 was characterized as (20S)-12β-tigloyloxy-3β,8β,14β,17β,20-pentahydroxypregn-5-ene 3-O-β-D-glucopyranosyl-(1→4)-β-D-thevetopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside and named gymsyloside E.

The known compounds were identified as 3β-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (6) [7], gymnemoside-W1 (7) [18], 3β-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (8) [7], and alternoside XIX (9) [18]. These compounds were already reported from G. sylvestre. Thus, for oleanane saponins, the main components could be hightly species-specific of G. sylvestre [7,18]. In addition, new pregnane glycosides were also found in G. alternifolium [19], G. sylvestre [5], and G. griffithii [15]. Five new pregnane glycosides in this report will contribute specific compounds in Gymnema genus.

2.3. α-Glucosidase and α-Amylase Inhibitory Activities

All compounds were evaluated for the α-glucosidase and α-amylase inhibitory assays. Compound 4 showed the weak α-glucosidase inhibitory activity (16.4 ± 2.3%) at the concentration of 200 µM, compared tos the positive control, acarbose (inhibition percentage of 57.8 ± 3.2% at the concentration of 155 µM) (Figure 3). Compounds 2–4 showed α-amylase inhibitory activity with inhibition percent ranging from 57.9% to 66.8% at the concentration of 200 µM (Figure 4). In the subsequent concentration-dependent assay, compounds 2, 3, and 4 showed significant α-amylase inhibitory activity, with IC₅₀ values of 175.8 ± 2.3, 162.2 ± 2.7, and 113.0 ± 0.7 µM, respectively, compared to positive control, acarbose (IC₅₀ value of 72.4 ±0.8 µM). This is the first report of α-glucosidase and α-amylase inhibitory activities of compounds from G. sylvestre. Recent reports have shown insulin secretion stimulation of G. sylvestre extract [4], antihyperglycemic effects of gmynemic acids [20], α-glucosidase and α-amylase inhibitory activities of pregnane glycosides from G. latifolium [11]. Previous studies have indicated that pregnane glycosides from G. griffithii showed moderate α-glucosidase inhibitory activity [15]. Russelioside B, a pregnane glycoside, possessed antidiabetic and antihyperlipidemic effect in streptozotocin induced diabetic rats [21]. Therefore, the results suggest that the discovery of pregane glycosides may increase the possibility of finding antidiabetic agents.

3. Materials and Methods

3.1. General

All NMR spectra were recorded on an Agilent 400-MR-NMR (Agilent technologies, Santa Clara, CA, USA) spectrometer operated at 400 and 100 MHz for hydrogen and carbon, respectively. Data processing was carried out with the MestReNova ver.6.0.2 program. HRESIMS spectra were obtained using an AGILENT 6550 iFunnel Q-TOF LC/MS system (Agilent technologies, Santa Clara, CA, USA). Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. Preparative HPLC was carried out using an AGILENT 1200 HPLC system. Column chromatography was performed on silica-gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) or YMC RP-18 resins (30–50 µm, Fuji Silysia Chemical Ltd., Aichi, Japan). For thin layer chromatography (TLC), a pre-coated silica-gel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) and RP-18 F254S plates (0.25 mm, Merck, Darmstadt, Germany) were used.

3.2. Plant Material

The leaves of Gymnema sylvestre (Retz.) R.Br. ex Sm. were collected in Hai Loc, Hai Hau, Nam Dinh in November, 2015, and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources. A voucher specimen (NCCT-P20) was deposited at the Herbarium Institute of Marine Biochemistry, VAST.

3.3. Extraction and Isolation

The dried powders of G. sylvestre leaves (4.0 kg) were sonicated with hot methanol (3 times × 10 L, each 3 h) to give MeOH extract (450 g), after evaporation of the solvent. The MeOH extract was suspended in water and successively partitioned with n-hexane, CH₂Cl₂ and EtOAc to obtain the n-hexane (GS1, 47.0 g), CH₂Cl₂ (GS2, 60.0 g), EtOAc extracts (GS3, 27.0 g) and H₂O layer (GS4). GS2 was chromatographed on a silica gel column (180.0 g, silica gel) eluting with gradient solvent of n-hexane:acetone (40:1, 20:1, 10:1, 5:1, 1:1, and 0:1, v/v, each 2 L), to give seven fractions, GS2A-GS2G. The GS2F fraction was chromatographed on a silica gel column eluting with CHCl₃:MeOH (11:1, v/v) to give four fractions, GS2F1-GS2F4. GS2F1 was chromatographed on a RP-18 column using MeOH:H₂O (4:1, v/v) as a solvent, to give five fractions, GS2F1A-GS2F1E. Compounds 1 (17.0 mg, t_R 38.5 min) and 2 (9.0 mg, t_R 42.1 min) were yielded from GS2F1B fraction using HPLC system: J’sphere H-80 column (150 × 20 mm), flow rate of 3 mL/min, and solvent condition of 40% acetonitrile in water. Compounds 3 (5.0 mg, t_R 44.7 min) and 4 (5.0 mg, t_R 49.4 min) were yielded from GS2F1D fraction on J’sphere H-80 column (150 × 20 mm), flow rate of 3 mL/min, and solvent condition of 40% acetonitrile in water. GS2G was chromatographed on RP-18 column eluting with MeOH:H₂O (4:1, v/v) to give four smaller fractions, GS2G1-GS2G4. Finally, GS2G3 was chromatographed on J’sphere H-80 column (150 × 20 mm), flow rate of 3 mL/min, and solvent condition of 35% acetonitrile in water to yield compound 5 (14.0 mg, t_R 39.7 min). GS4 was chromatographed on a Diaion column and eluted with H₂O then increased concentrations of MeOH in H₂O, to obtain sub-fractions, GS4A-GS4C. GS4C was chromatographed on a silica gel column eluting with a gradient of CHCl₃:MeOH (20:1, 10:1, 5:1, 1:1, v/v) to give smaller fractions, GS4C1-GS4C4. GS4C4 was chromatographed on an RP-18 CC eluting with MeOH:water (2:1, v/v) to give smaller fractions, GS4C4A-GS4C4E. GS4C4B was chromatographed on an RP-18 column eluting with acetone:H₂O (0.8:1, v/v) to yield 7 (5.0 g) and 9 (40.0 mg). GS4C4E was chromatographed on an RP-18 column eluting with acetone:H₂O (1:1, v/v), to yield 6 (100.0 mg) and 8 (5.0 mg).

3.3.1. Gymsyloside A (1)

White amorphous powder; ${\left[\alpha \right]}_D^{25}$ −20.0 (c 0.1, MeOH); C₄₇H₇₆O₁₇, HRESIMS m/z: 935.4986 [M + Na]⁺ (calcd for [C₄₇H₇₆O₁₇Na]⁺, 935.4975); ¹H (CD₃OD, 400 MHz) and ¹³C-NMR (CD₃OD, 100 MHz) data, see Table 1.

3.3.2. Gymsyloside B (2)

White amorphous powder; ${\left[\alpha \right]}_D^{25}$ + 35.0 (c 0.1, MeOH); C₄₇H₇₆O₁₇, HRESIMS m/z: 935.4996 [M + Na]⁺ (calcd for [C₄₇H₇₆O₁₇Na]⁺, 935.4975); ¹H (CD₃OD, 400 MHz) and ¹³C-NMR (CD₃OD, 100 MHz) data, see Table 1.

3.3.3. Gymsyloside C (3)

White amorphous powder; ${\left[\alpha \right]}_D^{25}$ + 80.0 (c 0.1, MeOH); C₅₄H₈₈O₂₀, HRESIMS m/z: 1079.5769 [M + Na]⁺ (calcd for [C₅₄H₈₈O₂₀Na]⁺, 1079.5769); ¹H (CD₃OD, 400 MHz) and ¹³C-NMR (CD₃OD, 100 MHz) data, see Table 1.

3.3.4. Gymsyloside D (4)

White amorphous powder; ${\left[\alpha \right]}_D^{25}$+ 58.7 (c 0.1, MeOH); C₅₄H₈₈O₂₀, HRESIMS m/z: 1079.5778 [M + Na]⁺ (calcd for [C₅₄H₈₈O₂₀Na]⁺, 1079.5769); ¹H (CD₃OD, 400 MHz) and ¹³C-NMR (CD₃OD, 100 MHz) data, see Table 2.

3.3.5. Gymsyloside E (5)

White, amorphous powder; ${\left[\alpha \right]}_D^{25}$+ 54.0 (c 0.1, MeOH); C₅₃H₈₆O₂₂, HRESIMS m/z: 1097.5530 [M + Na]⁺ (calcd for [C₅₃H₈₆O₂₂Na]⁺, 1097.5503); ¹H (CD₃OD, 400 MHz) and ¹³C NMR (CD₃OD, 100 MHz) data, see Table 2.

3.4. Acid Hydrolysis

Each compound (1–5, 3.0 mg) was separately dissolved in 1.0 N HCl (dioxane—H₂O, 1:1, v/v, 1.0 mL) and heated to 80 °C in a water bath for 3 h. The acidic solution was dried under N₂ overnight. After extraction with CHCl₃, the aqueous layer was dried using N₂ to give aqueous residue (A). The aqueous residue (A) was separated by silica gel CC eluting with CH₂Cl₂–MeOH (10:1, v/v) and then further fractionated by RP-18 CC using a solvent gradient of MeOH–H₂O (6:4, 7:3, and 8:2, v/v), to give the monosaccharides (50% yield). The specific rotations of these sugars were determined. The specific rotations (${\left[\alpha \right]}_D^{25}$) of sugars was determined after dissolving in H₂O for 24 h and compared to the literature (lit):D-cymarose: found +50.1 (c 0.4, H₂O), lit +51.8 [14]; D-oleandrose: found −12.1 (c 0.4, H₂O), lit +11.7 [14]; D-thevetose: found +40.3 (c 0.4, H₂O); lit +42.3 [15]; 6-deoxy-3-O-methyl-D-allose: found +10.9 (c 0.4, H₂O); lit +10.0 [17]; D-glucose: found + 49.2 (c 0.4, H₂O); lit +48.0 [17]. Based on the above evident, sugar components were found in: compounds 1 and 3: D-cymarose, D-oleandrose, and D-thevetose; compounds 2 and 4: D-cymarose, D-oleandrose, and 6-deoxy-3-O-methyl-D-allose; compound 5: D-cymarose, D-oleandrose, D-thevetose, and D-glucose.

3.5. Alkaline Hydrolysis

A solution of compound 1 (8 mg) in 1.0 mL of 5% KOH/MeOH was heated at 40 °C four 4 h and then neutralized with HCl 0.1 M. After that, the solution was partitioned with CHCl₃ to give CHCl₃ layer. CHCl₃ layer was separated on HPLC system: J’sphere H-80 column (150 × 20 mm), solvent condition of 55% acetonitrile, to give sarcostin (54% yield). In a similar way, sarcostin was found as aglycone of compounds 2–5.

3.6. α-Glucosidase Inhibitory Assay

The α-glucosidase (G0660-750UN, Sigma-Aldrich, St. Louis, MO) enzyme inhibition assay was performed according to the previously described method [11]. The sample solution (2 mL dissolved in dimethyl sulfoxide (DMSO)) and 0.5 U/mL α-glucosidase (40 mL) were mixed in 120 mL of 0.1 M phosphate buffer (pH 7.0). After 5 min pre-incubation, 5 mM p-nitrophenyl-α-D-glucopyranoside solution (40 mL) was added, and the solution was incubated at 37 °C for 30 min. The absorbance of released 4-nitrophenol was measured at 405 nm by using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

3.7. α-Amylase Inhibitory Assay

The α-amylase (A8220, Sigma-Aldrich, St. Louis, MO, USA) enzyme inhibitory activity was measured using the reported method [11]. Substrate was prepared by boiling 100 mg potato starch in 5 mL phosphate buffer (pH 7.0) for 5 min, then cooling to room temperature. The samples (2 mL dissolved in DMSO) and substrate (50 mL) were mixed in 30 mL of 0.1 M phosphate buffer (pH 7.0). After 5 min pre-incubation, 5 mg/mL α-amylase solution (20 mL) was added, and the solution was incubated at 37 °C for 15 min. The reaction was stopped by adding 50 mL 1 M HCl and then 50 mL iodine solution was added. The absorbances were measured at 650 nm by a microplate reader.

4. Conclusions

In summary, five new pregnane glycosides and four known oleanane saponins were isolated and identified from G. sylvestre. Besides oleanane saponins-hightly species-specific of G. sylvestre, new pregnane glycosides in this report will provide secondary metabolisms as specific compounds in Gymnema genus. Compound 4 showed the weak α-glucosidase inhibitory activity. Compounds 2, 3, and 4 showed significant α-amylase inhibitory activity. This is the first report of α-amylase and α-glycosidase inhibitory activities of compounds from G. sylvestre.