A new dihydrobenzofuran lignan and potential α-glucosidase inhibitory activity of isolated compounds from Mitrephora teysmannii

Abstract A new dihydrobenzofuran lignan, (2R,3S)-2-(3′,4′-dimethoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-2,3-dihydrobenzofuran-3-methyl acetate, named as mitredrusin (1), was isolated from the leaves of Mitrephora teysmannii (Annonaceae) together with 12 known compounds including a related dihydrobenzofuran lignan: (−)-3′,4-di-O-methylcedrusin (2), four polyacetylenic acids: 13(E)-octadecene-9,11-diynoic acid (3), 13(E),17-octadecadiene-9,11-diynoic acid (4), octadeca-9,11,13-triynoic acid (5) and octadeca-17-en-9,11,13-triynoic acid (6), five lignans: (−)-eudesmin (7), (−)-epieudesmin (8), (−)-phillygenin (9), magnone A (10) and forsythialan B (11) and two megastigmans: (3S,5R,6S,7E,9R)-7-megastigmene-3,6,9-triol (12) and annoionol A (13). The chemical structures of these compounds were established on the basis of their 1-D and 2-D NMR spectroscopic data. All compounds were evaluated for their α-glucosidase inhibitory activity. Among these isolates, polyacetylenic acids 3 and 4 showed more than 20-fold much higher activity compared with that of the antidiabetic drug acarbose.

been used as a Thai folk medicine for tonic purposes (Smitinand 2001). Previous phytochemical investigations of the bark and twigs of M. teysmannii have resulted in the isolation of alkaloids, polyacetylenes, lignans and terpenoids (lee et al. 1999;yu et al. 2005;Deepralard et al. 2007;Zhang et al. 2010). However, there have not been any previous studies of extracts from the leaves of this plant. As part of our search for new natural products and bioactive compounds from Thai Mitrephora plants (Rayanil et al. 2013), extracts from the leaves of M. teysmannii became of interest due to their inhibitory activity against α-glucosidase in our preliminary screening. Therefore, a chemical investigation of the α-glucosidase inhibition guided fractions was undertaken, aiming to discover new potent antidiabetic drugs. We describe herein the isolation, characterisation and α-glucosidase inhibitory assay of all isolated compounds.

General experimental procedures
Melting points were measured on a Kofler hot stage apparatus and are uncorrected. Optical rotations were obtained using a Jasco P1010 digital polarimeter. iR spectra were obtained on a Perkin elmer GX FT-iR spectrophotometer. uV spectra were recorded on a Hewlett Packard 8453 uV-vis spectrometer. 1-D and 2-D NMR experiments were recorded on a Brüker AVANCe 300 MHz spectrometer operating at 300 MHz for proton and 75 MHz for carbon, respectively. Mass spectra were acquired on a Micromass lCT mass spectrometer and the lock mass calibration was applied for the determination of accurate masses. Column chromatography (CC) was carried out on silica gel (Merck, 70-230 mesh or 230-400 mesh) or RP-18 (Merck, 40-63 mesh). TlC was performed on Merck precoated silica gel 60 F 254 plates and spots were visualised under uV light (254 and 365 nm) or by spraying with 1% CeSO 4 in 10% aqueous H 2 SO 4 followed by heating.

Plant material
The leaves of M. teysmannii were collected in February 2011 from Ton Pariwat Wildlife Conservation Area, Phang-nga Province, Thailand and were identified by Dr. Piya Chalermglin of the Thailand institute of Scientific and Technological Research. A voucher specimen (SS614/272) was deposited at the Herbarium of the Faculty of Science and Technology, Phuket Rajabhat university, Phuket, Thailand.

Basic hydrolysis of mitredrusin (1)
To a solution of compound 1 (4.3 mg) in MeOH (0.5 ml), 1.0 ml of 0.5 M NaOH/H 2 O solution was added and the mixture was stirred for 5 h at room temperature. The mixture was acidified with 1.0 ml of 1.0 M HCl and extracted with CH 2 Cl 2 (1.0 ml × 3). The combined organic layer was concentrated and purified by preperative TlC to afford (−)-3′,

α-Glucosidase inhibitory assay
The α-glucosidase (from Saccharomyces cerevisiae; Sigma-Aldrich, St. louis, MO, uSA) inhibitory activity was determined by the method described by Worawalai et al. (2012). Briefly, 20 μl of the test compound was mixed with 20 μl of the enzyme solution (1 u/ml) in 0.01 M phosphate buffer (pH 6.8). The mixture was pre-incubated at 25 °C for 15 min and then a p-nitrophenyl-α-D-glycopyranoside (PNPG) solution (20 μl) was added. After 15 min of continuous incubation, a 0.5 M Na 2 CO 3 solution (40 μl) was added to quench the reaction. The enzymatic activity was evaluated by monitoring the absorbance at 405 nm of released p-nitrophenol. The per cent inhibition was calculated according to the equation: % inhibition = (A blank − A sample )/A blank × 100; where A sample and A blank are the absorbances of the solutions containing PNPG and α-glucosidase with and without the sample, respectively. The iC 50 values of the samples possessing an inhibition rate >50% were then determined. Acarbose was used as the positive control.

Conclusion
The chemical investigation of the leaves of M. teysmannii resulted in the isolation of 13 compounds (1-13). Compound 1 was a new natural product and the known compounds 2-5, 8, 9 and 11-13 were isolated for the first time from M. teysmannii. in addition, the strong α-glucosidase inhibitory activity of polyacetylenic acids (3-6) presented here encouraged us to further develop this type of compounds as potent α-glucosidase inhibitors.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Faculty of Science, Silpakorn university[grant number SRF-JRG-2557-02].