PHENOLIC CONSTITUENTS FROM SARCOPYRAMIS NEPALENSIS AND THEIR Α-GLUCOSIDASE INHIBITORY ACTIVITY

. Abstract Background: This study was carried out to fully investigate the phenolic chemical constituents of Sarcopyramis nepalensis and determine their α-glucosidase inhibitory activity. Materials and Methods: S. nepalensis was extracted using the ultrasonic assistant extraction (UAE) method and further fractionated with petroleum ether (PE), chloroform (CHCl 3 ), ethyl acetate (EtOAc) and n -butanol ( n -BuOH), respectively. The active fraction was chromatographed on AB-8 macroporous resin column, silica gel column, Sephadex LH-20 column, RP-ODS column and semi-preparative HPLC column. The isolated phenolic constituents were identified by 1 H-Nuclear Magnetic Resonance (NMR), 13 C-NMR and mass spectral (MS) analyses and detected their α-glucosidase inhibitory activity by micro-plate. Results : Ten phenolic constituents were isolated and identified from the active fraction of S. nepalensis . They were identified as isorhamnetin( 1 ), quercetin( 2 ), isorhamnetin-3-O-β-D-glucopyranoside( 3 ), isoquercetin ( 4 ), astragalin( 5 ),isorhamnetin-3-O-(6"- p -coumaroyl)-β-D-glucopyranoside( 6 ),isorhamnetin-3-O-(6"-caffeoyl)-β-D-glucopyranoside( 7 ), isoferulic acid( 8 ) Caffeic acid( 9 ) and ellagic acid( 10 ). All of the phenolic compounds were assayed for their hypoglycemic activity against α-glucosidase in vitro . Compound 4 , 6 and 7 showed promising α-glucosidase inhibitory activity with the IC 50 values of 0.69 mg/ml, 0.56 mg/ml, 0.45 mg/ml, respectively. Conclusion: Compounds 5 - 8 were isolated for the first time from S. nepalensis . This is the first report on the characterization of phenolic compounds and possible utilization of S. nepalensis for therapeutic intervention in type 2 diabetes.


Introduction
Sarcopyramis nepalensis belongs to the genus of Sarcopyramis, which is comprised of 4 species and 2 varieties in china (Chen 1999).Most species are medicinal plants used in folk medicine to treat liver and other inflammatory diseases.S. nepalensis showed good hepatoprotective activities for lowering aminotransferase and curing choleplania and hepatoma (Guo et al. 2012).Phytochemistry studies of the plant revealed that phenolic acid and flavonoids were (the) major constituents in the plant (Lan 2010;Zhang et al. 2011;Huang et al. 2013;Wei et al. 2014).It is well known that phenolic acid and flavonoids have showed good hypoglycemic activity in vivo and in vitro (Ranilla et al. 2010;Sharma et al. 2008).However, the hypoglycemic activity of S. nepalensis was not fully illuminated.α-glucosidase is a key enzyme hydrolysing dietary carbohydrate into glucose leading to high blood glucose.So the α-glucosidase inhibitory model was frequently used to screen the therapeutic agents for the control of postprandial hyperglycemia arising from use of natural medicinal plants and isolated compounds (Wan et al. 2012ab).The purpose of this study is to fully elaborate the yeast α-glucosidase inhibitory phenolic constituents from S. nepalensis.

Plant material
The specimen of S. nepalensis was collected from Sanming city, Fujian Province, P.R. China, in May 2012.A voucher specimen (2012R06) was deposited at the pharmacy of The First College of Clinical Medical Science, China Three Gorges University.The whole parts of S. nepalensis were dried at 40 °C in an air oven for 48 h and finely powdered.

General experimental procedures
1 H and 13 C-Nuclear Magnetic Resonance (NMR) data were recorded on a Bruker Avance-600 FT NMR spectrometer with Tetramethylsilane (TMS) as internal standard.Mass spectral (ESI-MS) data were acquired on a Q-Star Elite (Applied Biosystems MDS, USA) mass spectrometer.

Extraction and fractionation with organic solvent
The air-dried plant material (4 kg) was ground and extracted exhaustively by UAE with 75% ethanol.The extracts were pooled and concentrated.A total extract (580 g) were fractionated with petroleum ether, CHCl 3 , EtOAc and n-BuOH, respectively.Those solvent extracts were tested for their α-glucosidase inhibitory activity in order to choose the active fraction.The EtOAc fraction showed the best α-glucosidase inhibitory activity and further to isolate the active constituents.

α-glucosidase inhibitory assay
α-glucosidase inhibitory activity was determined by method of literature (Wan et al. 2013).Briefly, a mixture of 50 μL of different concentrations of each isolates and 100 μL of 0.1 M phosphate buffer (pH 6.9) containing yeast α-glucosidase solution (1.0 U/ml) was incubated in 96 well plates at 25 °C for 10 min.Then 50 μL of 5 mM pNPG solution (dissolve in 0.1 M phosphate buffer) was added to each well.
Absorbance was recorded immediately at 405 nm.The reaction mixtures were incubated at 25 °C for 5 min and the Absorbance was recorded immediately at the same wavelength.50 μL buffer solutions instead of test isolates were used as control.α-glucosidase inhibitory activity, expressed as inhibition (%),was calculated as in Eq 1.

Statistical analysis
All experiments were performed in triplicate.Statistical analysis of data was by Microsoft Excel XP and the results were given as mean ± standard deviation (SD).p  0.05 was considered statistically significant difference.

Results and Discussion
Identification of the isolates (Fig. 1) by NMR and ESI-MS

α-glucosidase inhibitory activity
Table 1 shows the α-glucosidase inhibitory activity of the isolates from S. nepalensis.The ethyl acetate soluble fractions showed best α-glucosidase inhibitory activity compared with other solvent extracts and the positive control drug, acarbose.Therefore, further isolation was conducted on the ethyl acetate fraction.The α-glucosidase inhibitory activity of the ten isolates was tested at the original concentration of 2.5 mg/mL.Compound 8 and 9 showed < 50 % inhibition activity (p  0.05), while others showed > 50 %.Hence, all of the compounds except 8 and 9 were further tested and the IC 50 was calculated (Table 1).It was well known that the phenolic constituents maybe responsible for the α-glucosidase inhibitory activity.However, the active constituents were unknown.An activity-guided active compounds isolation method was used to study theα-glucosidase inhibitory active compounds in G.

Table 1 :
Yeast α-glucosidase inhibitory activity (mean ± SD, n = 3) of isolates of S. nepalensis Positive control; b Isolates showed mg/mL as the unit of IC 50 . a