Rat Intestinal Sucrase and α-Glucosidase Inhibitory Activities of Isocoumarin and Flavonoids from the Zanthoxylum schinifolium Stems

Experimental

General Experimental Procedures. Optical rotations were determined using a Jasco DIP-370 automatic polarimeter. CD spectra were recorded with a Jasco J-810 spectropolarimeter. The FT-IR spectra were measured using a Jasco Report-100 infrared spectrometer; The NMR spectra were recorded using a Jeol ECA 600 spectrometer (1H, 600 MHz; 13C, 150 MHz), High-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained using an Agilent 6530 Accurate-Mass Q-TOF LC/MS system. Column chromatography was performed using a silica gel (Kieselgel 60, 70-230, and 230-400 mesh, Merck, Darmstadt, Germany), YMC RP-18 resins, and thin layer chromatography (TLC) was performed using pre-coated silica-gel 60 F254 and RP-18 F254S plates (both 0.25 mm, Merck, Darmstadt, Germany).

Plant Material. Dried stems of Z. schinifolium were collected from Daejeon, Korea in September 2012 and identified by one of the authors (Prof. Young Ho Kim). A voucher specimen (CNU 12102) was deposited at the Herbarium of College of Pharmacy, Chungnam National University.

Extraction and Isolation. Dried stems (2.3 kg) were extracted with MeOH (5 L × 3) under reflux. The MeOH extract (102.0 g) was suspended in water and partitioned with n-hexane, EtOAc and n-BuOH. The EtOAc fraction (12.0 g) was subjected to silica gel (5 × 30 cm) column chromatography with a gradient of hexane-EtOAc-MeOH (10:1:0, 6:1:0, 3:1:0, 1.5:1:0.1, 1:1:0.2; 1.5 L for each step) to give 6 fractions (Fr. 1A-1F). The fraction 1C was separated using a silica gel (1.5 × 80 cm) column chromatography with a gradient of hexane-acetone-MeOH (7:1:0.1, 6:1:0.1, 5:1:0.1; 1.0 L for each step) elution solvent to give compound 1 (11.0 mg). The n-BuOH fraction (28.0 g) was subjected to silica gel (3.0 × 30 cm) column chromatography with a gradient of CH2Cl2-MeOH-H2O (16:1:0, 10:1:0, 7.5:1:0.1, 3:1:0.15, 1.5:1:0.2; 2.5 L for each step) to give 6 fractions (Fr. 2A-2F). The fraction 2C was subjected to silica gel (1.0 × 70 cm) column chromatography with a gradient of CH2Cl2-MeOH-H2O (12:1:0, 10:1:0, 8.5:1:0.1, 6.5:1:0.1, 4:1:0.1; 1.5 L for each step) to give four subfractions (Fr. 2C-1-2C-4). The fraction 2C-3 was separated using an YMC (1.0 × 80 cm) column chromatography with a MeOH-H2O (0.2:1, 0.3:1, 0.6:1, 1:1; 750 mL for each step) elution solvent to give compounds 2 (43.0 mg) and 3 (21.0 mg). The fraction 2C-4 was separated using an YMC (1.0 × 80 cm) column chromatography with a MeOH-H2O (0.2:1, 0.67:1; 1.0 L for each step) elution solvent to give compound 4 (12.0 mg).

Schinifolisatin (1): Yellowish oil; C12H12O5; : +56.8 (c 0.3, MeOH); CD (MeOH): Δε 240+0.16, Δε 265+0.21, Δε 285—0.18 (c 0.71 × 107 M); IR (KBr): νmax 3458, 1761, 1705, 1610, 1471 cm-1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR data (methanol-d4, 150 MHz), see Table 1; HR-ESI-MS: m/z 237.0765 [M+H]+ (calcd. 237.0763).

Hesperidin (2): Amorphous powder, mp 268-271 ℃. : -36.7 (c 0.3, pyridine); IR (KBr): νmax 3474, 2918, 1649, 1609, 1096, 1069 cm-1; FAB-MS m/z: 611 [M+H]+. This compound exhibited comparable spectroscopic data (1H- and 13C-NMR) to published values.17

Homoesperetin-7-O-rutinoside (3): Amorphous powder; : -42.7 (c 0.3, pyridine); IR (KBr): νmax 3470, 2921, 1655, 1612, 1073 cm-1; FAB-MS m/z: 625 [M+H]+. This compound exhibited comparable spectroscopic data (1H-and 13C-NMR) to published values.18

Hyperin (4): Yellow needles. IR νmax(KBr) cm-1: 3316, 2900, 1655, 1607, 1060. FAB-MS m/z: 465 [M+H]+. This compound exhibited comparable spectroscopic data (1H-and 13C-NMR) to published values.19

Table 1.1H (600 MHz) and 13C NMR (150 MHz) spectroscopic data of compound 1 (methanol-d4, δ , ppm, J/Hz)

Table 2.aPercentage of enzyme inhibition at the concentration of 1 mM. bAcarbose (0.05 mM) was used as positive control. cQuercetin (1 mM) was used as positive control. Data presented is the mean ± SD of samples runs in triplicate.

Inhibition Assay for Sucrase Inhibitory Activity. The crude enzyme solution prepared from rat intestinal acetone powder Sigma-Aldrich Co. (St. Louis, MO, USA) was used as the small intestinal sucrase, showing specific activities of 0.34 units/mL. Rat-intestinal acetone powder (1.0 g) was suspended in 3 mL of 0.9% saline, and the suspension was sonicated twelve times for 30 sec at 4 ℃. After centrifugation (10000 × g, 30 min, 4 ℃), the resulting supernatant was used for the assay. Sucrase inhibitory activities were assayed by modifying a method developed by Kwon et al.24 The inhibitory activity was determined by incubating a solution of an enzyme (50 μL), 0.1 M phosphate buffer (pH 7.0, 100 μL) containing 0.4 mg/mL sucrose and a solution (50 μL) with a concentration of sample (1.0 mM) at 37 ℃ for 30 min. Acarbose (0.05 mM) was used as positive control. The reaction mixture was heated in a boiling water bath to stop the reaction for 10 min, and then the amount of liberated glucose was measured by the glucose oxidase method.25 The inhibitory activity was calculated from the formula as follows. Inhibition (%) = (C-T)/C × 100, where C is the enzyme activity without inhibitor and T is the enzyme activity with inhibitor.

Inhibition Assay for α-Glucosidase Activity. The α-glucosidase inhibition assay was performed according to (Li et al., 2009) with some modification. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Briefly, a mixture of 50 μL samples and 50 μL of 0.1 M phosphate buffer (pH 7.0) containing α-glucosidase solution (0.3 U/mL) and 50 μL distilled water was incubated in 96 well plates at 37 ℃ for 15 min. After pre-incubation, 100 μL of 3 mM p-NPG solution in 0.1 M phosphate buffer (pH 7.0) was added to each well at timed intervals. Quercetin (1 mM) was used as positive control. The reaction mixtures were incubated at 37 ℃ for 10 min and stopped by adding 750 μL of 0.1 M Na2CO3. The absorbance was recorded at 405 nm by FLUO star Optima (BMG Labtech, Offenburg, Germany). The results were expressed as a percent of μ-glucosidase inhibition and calculated according to the following equation: % inhibition = [(A control - A compound) / A control] × 100

Statistical Analysis. All experiments were performed in triplicate. Data is presented as the means ± SD. The results were statistically analyzed by ANOVA and Duncan’s multiple range tests. Statistical significance was accepted at a level of p ＜ 0.05.