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

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.


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], [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 αamylase inhibitory activities of these compounds.

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).

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

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.

Acid Hydrolysis
Each compound (1-5, 3.0 mg) was separately dissolved in 1.0 N HCl (dioxane-H 2 O, 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 N 2 overnight. After extraction with CHCl 3 , the aqueous layer was dried using N 2 to give aqueous residue (A). The aqueous residue (A) was separated by silica gel CC eluting with CH 2 Cl 2 -MeOH (10:1, v/v) and then further fractionated by RP-18 CC using a solvent gradient of MeOH-H 2 O (6:4, 7:3, and 8:2, v/v), to give the monosaccharides (50% yield). The specific rotations of these sugars were determined.

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 CHCl 3 to give CHCl 3 layer. CHCl 3 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.

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.