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Cholinesterase Inhibitors from the Aerial Part of Piper hymenophyllum
Cholinesterase Inhibitors from the Aerial Part of Piper hymenophyllum
Bulletin of the Korean Chemical Society. 2014. Feb, 35(2): 655-658
Copyright © 2014, Korea Chemical Society
  • Received : November 14, 2013
  • Accepted : November 27, 2013
  • Published : February 20, 2014
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About the Authors
Hoang Viet Dung
To Dao Cuong
Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
Nguyen Minh Chinh
Do Quyen
Deparment of Pharmacognosy, Hanoi University of Pharmacy, 15 Le Thanh Tong, Hoankiem, Hanoi, Vietnam
Jeong Su Byeon
Faculty of Food Science and Biotechnology, Pukyung National University, Busan 608-737, Korea
Jeong Ah Kim
College of Pharmacy, Kyungpook National University, Daegu 702-701, Korea
Mi Hee Woo
College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712-702, Korea
Jae Sui Choi
Faculty of Food Science and Biotechnology, Pukyung National University, Busan 608-737, Korea
Byung Sun Min
College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712-702, Korea

Abstract
Keywords
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Experimental
General Experimental Procedure. Optical rotations were measured using a JASCO DIP 370 digital polarimeter. UV spectra were recorded in MeOH using a Thermo spectro-meter. IR spectra were obtained using a Fourier Transform infrared spectrometer (Brucker Instrument, Inc., German). 1D- and 2D-NMR spectra were obtained using a Varian Unity Inova 400 MHz spectrometer with tetramethylsilanne (TMS) as the internal standard, and the chemical shifts were recorded in δ values (ppm). HREIMS was recorded using JEOL JMS-700 MStation TM spectrometer (Japan). Silica gel (Merck, 63-200 μm particle size) and RP-18 (Merck, 75 μm particle size) were used for column chromatography. TLC was carried out using Merck silica gel 60 F 254 and RP-18 F 254 plates. HPLC was conducted using a Waters system (515 pump), a UV detector (486 Tunable Absorbance), and an YMC Pak ODS-A column (20 × 250 mm, 5 μm particle size, YMC Co., Ltd., Japan). HPLC solvents were purchased from Burdick Jackson (USA).
Plant Material. The aerial parts of P. hymenophyllum were harvested at the Cuc Phuong National Park, Ninh Binh province, Vietnam in April 2011. Botanical identification was performed by Associate Professor Vu Xuan Phuong, Department of Herbal Specimen, Vietnam Institute of Ecology and Biological resources. A voucher specimen (HVD 004-11) was deposited at the Department of Specimen, Vietnam Institute of Ecology and Biological Resources.
Extraction and Isolation. The aerial parts of P. hymeno-phyllum (15.0 kg) were extracted three times (3 h × 3 L) with MeOH under reflux at 60 ℃. After removing the solvent under reduced pressure, the residue was suspended in H 2 O and then partitioned successively with n -hexane, CHCl 3 , EtOAc, and n -BuOH. An activity-guided fractionation study resulted in the CHCl 3 and EtOAc fractions being chosen for further study.
The CHCl 3 soluble fraction (110.0 g) was chromatographed on a silica gel column using a stepwise gradient of n -hexane: acetone (15:1 to 0:1, each 4 L) to yield ten fractions (Fr.1-Fr.10, determined according to their TLC profiles). Fraction 3 (1.4 g) was subjected to silica gel column chromatography (CC) using n -hexane:acetone (30:1 to 5:1, 2 L for each step) as the eluent, to afford four sub-fractions (Fr.3-1 to Fr.3-4). Further purification of sub-Fr.3-2 (350.0 mg) using a ODS silica gel column eluted with MeOH-H 2 O (4:1 to 1:0, 1 L for each step) resulted in the isolation of compound 6 (27.0 mg). Fraction 6 (43.9 g) was subjected to silica gel CC using CH 2 Cl 2 -acetone (100:1 to 0:1, 2 L for each step) as the eluent, to afford ten fractions (Fr.6-1 to Fr.6-10). Fraction 6-1 (1.1 g) was subjected to ODS silica gel CC, using MeOH-H 2 O (1:2 to 3:1, 1.5 L for each step) gradient, to obtain two sub-fractions (Fr.6-1-1 to Fr.6-1-2). Sub-Fr.6-1-1 (525.0 mg) was purified by silica gel CC using n -hexane-acetone (5:1 to 2:1, 1 L for each step), which resulted in the isolation of compounds 5 (27.0 mg) and 7 (18.0 mg). Fraction 7 (18.3 g) was also subjected to a silica gel CC using n -hexane-acetone (10:1 to 1:1, 1.0 L for each step) gradient, to afford eight sub-fractions (Fr.7-1 to Fr.7-8). Fraction 7-4 (2.1 g) was subjected to silica gel CC using CH 2 Cl 2 -acetone (100:1 to 10:1, 1.0 L for each step), to obtain eight sub-fractions (Fr.7-4-1 to Fr.7-4-8). Further purification of sub-Fr.7-4-2 (86.0 mg) using a semi-preparative Waters HPLC system [using a gradient solvent system of MeOH-H 2 O = 55:45 to 85:15; flow rate 5 mL/min; for 90 min; UV detection at 210 nm; YMC Pak ODS-A column (20 × 250 mm, 5 mm particle size] resulted in the isolation of compound 1 (6.5 mg; t R = 38.2 min).
The EtOAc (19.3 g) soluble fraction was also subjected to silica gel CC using CHCl 3 -MeOH (80:1 to 0:1, 2.0 L for each step) gradient, to obtain seven fractions (Fr.E-1 to Fr.E-7). Fraction E-2 (0.43 g) was subjected to silica gel CC using CHCl 3 -MeOH (30:1 to 5:1, 0.5 L for each step) gradient, to obtain two sub-fractions (Fr.E-2-1 to Fr.E-2-2). Further puri-fication of sub-Fr.E-2-1 (65.0 mg) using a semi-preparative Waters HPLC system [using a gradient solvent system con-sisting of MeOH-H 2 O = 40:60 to 70:30; flow rate 5 mL/min; for 90 min; UV detection at 210 nm; YMC Pak ODS-A column (20 × 250 mm, 5 mm particle size] resulted in the isolation of compound 4 (11.0 mg; t R = 40.2 min). Fraction E-4 (0. 87 g) was subjected to a silica gel CC using CHCl 3 -MeOH (20:1 to 10:1, 1.0 L for each step) gradient, to afford three sub-fractions (Fr.E-4-1 to Fr.E-4-3). Sub-Fr.E-4-2 (565.0 mg) was further subjected to ODS CC using MeOH-H 2 O (1:3 to 1:1, 1.0 L for each step) gradient, to obtain two sub-fractions (Fr.E-4-2-1 to Fr.E-4-2-2). Compound 2 (24.0 mg; t R = 35.6 min) was obtained from sub-Fr.E-4-2-1 (154.0 mg) by preparative HPLC [using MeOH-H 2 O = 50:50 to 85:15 gradient; flow rate 5 mL/min; for 90 min; UV detec-tion at 210 nm; YMC Pak ODS-A column (20 × 250 mm, 5 mm particle size]. Sub-Fr.E-4-2-2 (258.0 mg) was purified by silica gel CC using with CHCl 3 -MeOH (15:1 to 5:1, 0.5 L for each step) to afford compound 3 (42.0 mg).
N-3,5-Dimethoxy-4-hydroxycinnamoylpyrrole (1): yellow amorphous solid; UV λ max (MeOH): 252, 353 nm; IR (KBr) v max 3490, 3124, 1710, 1620, 1615, 1519, 1460 cm; −1 HREIMS m/z 273.1003 [M] + (calcd for C 15 H 15 NO 4 , 273.1001). 1 H NMR (400 MHz, acetone- d 6 ) and 13 C NMR (100 MHz, acetone- d 6 ) spectroscopic data, see Table 1 .
In vitro AChE Inhibitory Activity Assay. The inhibitory activities of AChE and BChE were measured using the spectrophotometric method developed by Ellman with a slight modification. 6 Essentially, ACh and BCh were used as substrates to detect the inhibitions of AChE and BChE, respectively. The reaction mixture contained: 140 μL of sodium phosphate buffer (pH 8.0); 20 μL of tested sample solution [final concentration (f.c.) 100 μM for either compound]; and 20 μL of AChE or BChE solution, which were mixed and incubated for 15 min at room temperature. All tested samples and the positive control (berberine) were dissolved in 10% analytical grade dimethyl sulfoxide (DMSO). Reactions were started by adding 10 μL of 5,5'-dithio- bis -(2-nitrobenzoic acid) (DTNB) and 10 μL of ACh or BCh. The hydrolysis of ACh or BCh was monitored by following the formation of the yellow 5-thio-2-nitrobenzoate anion (formed by the reaction between DTNB and thiocho-line, released by the enzymatic hydrolysis of ACh or BCh) at 412 nm for 15 min. All reactions were performed in tripli-cate and recorded in 96-well microplates using a VERSA max ELISA Microplate Reader (Molecular Devices, Sunny-vale, CA, U.S.A.). Percent inhibition was calculated using (1−S/E) × 100, where E and S are enzyme activities with or without the tested sample, respectively. The ChEs inhibitory activity of each sample was expressed as IC 50 values (μM, defined as the concentration required inhibiting the hydrolysis of substrate by ACh or BCh by 50%, as calculated using log-dose inhibition curves).
Acknowledgements
This study was financially supported by the National Foundation for Science and Technology Development, Vietnam (Project No: 104.01-2010-16), and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (BK21 Plus program).
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