α-Glucosidase Inhibitory Constituents from Acanthopanax senticosus Harm Leaves

A new triterpene glycoside, 3-O-[(α-L-rhamnopyranosyl)(1→2)]-[β-D-glucuronopyranosyl-6-O-methyl ester]-olean-12-ene-28-olic acid (1) and a new indole alkaloid, 5-methoxy-2-oxoindolin-3-acetic acid methyl ester (5) were isolated from the leaves of Acanthopanax senticosus Harms along with six known compounds. The structures of the new compounds were determined by means of 2D-NMR experiments and chemical methods. All the isolated compounds were evaluated for their glycosidase inhibition activities and compound 6 showed significant α-glucosidase inhibition activity.


Introduction
Acanthopanax senticosus (Rupr. Maxim) Harms, a member of the Araliaceae family, is a shrub found mainly in the northeast of China, Korea and Japan. It usually grows up to 2 m in height and generally has prickly stems bearing five leaflets (palmate) and umbel-shaped flowers. The young leaves of A. senticosus are cultivated as a vegetable crop in Northeast China. As regards biological active constituents of A. senticosus, many of its triterpenoid saponins [1][2][3][4] were reported to have anti-inflammatory [5], antibacterial [6] and platelet anti-aggregating activities [7]. Our studies indicated that the 30% EtOH fraction of A. senticosus leaves can inhibit plasma glucose levels in alloxan-induced diabetic rats and showed significant α-glucosidase inhibition activity. To further

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investigate the constituents and screen the bioactive compounds from its leaves, a phytochemical study was performed that resulted in the isolation of new compounds 1 and 5 along with six other previously known compounds. In this paper, we describe the structural elucidation of new compounds 1 and 5, together with the α-glucosidase inhibitory activity of all these compounds 1-8 ( Figure 1).

Results and Discussion
Compound 1 was obtained as a white amorphous powder (MeOH  [3,8,9], indicated that the two compounds were very similar, except for two additional sugar units in 1. By comparison of the 13 C-NMR spectral data of 1 with those of oleanolic acid, the signal due to C-3 shifted to downfield by approximately 10 ppm, which indicated the sugar unit was linked to C-3 in 1. On acid hydrolysis, 1 yielded L-rhamnose and D-glucuronic acid as a sugar component by GC analysis using a hydrogen flame detector after treatment with L-cysteine methyl ester hydrochloride in pyridine [9] and oleanolic acid was obtained and identified as the aglycone from NMR data by comparison with authentic samples. The 1 H-NMR spectrum of 1 (Table 1) showed seven methyl singlets δ 0.81 (3H), 0.94 (3H), 0.97 (3H), 1.00 (3H), 1.07 (3H) and 1.29 (6H), one olefinic H-atom (br.s at δ 5.45) and a methoxy group at δ 3.71 (3H). The 13 C-NMR spectrum of 1 showed signals of a pair of olefinic C-atoms at δ 122.5, 144.8, two anomeric C-atoms at δ 102.5, 105.4, and two C=O groups at δ 180.2 and 170.5. The 1 Hand 13 C-NMR signals of 1 were assigned by the use of HMQC and HSQC-TOCSY. The full connectivity of 1 was deduced from the HMBC correlations ( Figure 2). In particular, the long-range correlations between the proton signals at δ 5.64 (H-1′′) and the carbon signals at δ 83.4 (C-2′) suggested that α-L-rhamnose unit was linked to C-2′ of β-D-glucuronic acid. The cross-peak between the proton signals at δ 3.71 (3H) and the carbon signals at δ 170.5 suggested that a methoxy group was linked to C=O of D-glucuronic acid. The anomeric configuration of D-glucuronic acid was determined to be β-form on the basis of the J H-H value (7.6 Hz) in the 1 H-NMR spectrum, and the configuration of   (Table 2) spectra showed the presence of an aromatic ring with an ABX-coupling system at δ 6.64 (1H, dd, J = 8.5, 2.4 Hz), 6.71 (1H, d, J = 8.5 Hz) and 6.73 (1H, d, J = 2.4 Hz). The signals at δ 3.83 (3H, s) and 3.67 (3H, s) were assigned to proton signals of methoxy group. The 13 C-NMR spectrum data (Table 2) showed 12 C-atom signals including an aromatic ring δ (C) 108.4, 110.0, 115.3, 131.6, 135.9 and 154.4 and two C=O groups at δ (C) 181.1 and 173.2.    (7) [13], 3-carboxylperlolyrine (8) [14] by comparing their NMR spectroscopic data with literature values. All these known compounds are reported for the first time in A. senticosus leaves.
We evaluated the isolated compounds for their inhibitory activity against α-glucosidase since some compounds are known α-glucosidase inhibitors [15]. The results are shown in Table 3, with acarbose used as a positive control. Compounds 1, 4 and 6 exhibited α-glucosidase inhibitory activities with IC 50 values of 186.0 μM, 908.5 μM and 819.7 μM, respectively. Thus, this research suggested that the saponins and (+)-afzelechin from 30% EtOH fraction were primary α-glucosidase inhibitors.

General
Open column chromatography was carried out using silica gel (200-300 mesh, Qingdao Marine Chemical Co., Qingdao, China) or octadecyl silica gel (ODS, 25-40 μm, Fuji, Tokyo, Japan) as stationary phases. TLC employed precoated silica gel plates (5-7 μm, Qingdao Marine Chemical Co., Qingdao, China). Preparative HPLC was carried out on a Waters 600 instument equipped with a Waters RID-2414 detector. A Waters Sunfire prep C 18 OBD (19 × 250 mm i.d.; Milford, PA, USA) column was used for preparative purposes. The IR spectra were recorded as KBr pellets on a Jasco 302-A spectrometer. Optical rotation was recorded on a Jasco P-2000 polarimeter. HRESIMS were measured on a FTMS-7 instrument (Bruker Daltonics, Karlsruhe, Germany). Melting points were determined on a Gallenkemp apparatus and are uncorrected. The 1 H-, 13

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The

Acid Hydrolysis of 1
Compound 1 (3 mg) was hydrolyzed in 1 M HCl (1.0 mL) for 2 h at 85°. The reaction mixture was cooled and partitioned between CHCl 3 (2.0 mL) and H 2 O (2.0 mL). The aqueous layer was washed with CHCl 3 (3 × 3.0 mL), neutralized with Ba(OH) 2 , filtered, and evaporated under reduced pressure. The residue was dissolved in pyridine (1.0 mL) and L-cysteine methyl ester hydrochloride in pyridine (0.1 M, 2.0 mL) was added. The mixture was heated at 60° for 1 h. An equal volume of Ac 2 O was added with heating and incubation was continued for 1 h. The acetylated thiazolidine derivatives were analyzed by GC with a DM-5 column (30 m × 0.25 mm, 0.25 μm) using authentic samples as standards. Temperatures of both injector and detector were 280°. A temperature gradient system was used for the oven, starting at 160° and increasing up to 195° at a rate of 5°/min. The determination of the sugar units in compound 1 was achieved by comparison of retention times of peaks between the hydrolysate and authentic samples of D-glucuronic acid (10.08 min) and L-rhamnose (7.05 min).

α-Glucosidase Inhibition Assay
The α-glucosidase (EC.3.2.1.20) enzyme inhibition assay has been performed according to the literature [16]. α-Glucosidase (25 μL, 0.2 U/mL), various concentrations of samples (25 μL), and 67 mM phosphate buffer (pH 6.8, 175 μL) were mixed at room temperature for 10 min. Reactions were initiated by the addition of 23.2 mM p-nitrophenyl-α-D-glucopyranoside (25 μL). The reaction mixtures were incubated at 37 °C for 15 min in a final volume of 250 μL, and then 1 M Na 2 CO 3 (50 μL) was added to the incubation solution to stop the reaction. The activities of glucosidase were detected in a 96-well plate, and the absorbance was read at 405 nm by a microplate spectrophotometer (Spectra Max, Molecular Devices, Sunnyvale, CA, USA). The negative control was prepared by adding phosphate buffer instead of the sample in the same way as the test. Acarbose was utilized as the positive control. The blank was prepared by adding phosphate buffer instead of α-glucosidase using the same method. The inhibition rates (%) were calculated from the formula: [(OD negative control − OD blank ) − (OD test − OD test blank )]/(OD negative blank − OD blank ) × 100%.