CHEVALIERINOSIDE A: A NEW ISOFLAVONOID GLYCOSIDE FROM THE STEM BARK OF ANTIDESMA

. Chevalierinoside A ( 1 ), a new isoflavonoid glycoside determined as biochanin A 7-O -[ α -L- rhamnopyranosyl-1 → 6-apiofuranosyl-1 → 2-β -D-glucopyranoside], together with the known friedelin ( 2 ), friedelan-3 β -ol ( 3 ) and betulinic acid ( 4 ), were isolated from the stem bark of Antidesma chevalieri Beille. Their structures were established by direct interpretation of their spectral data, mainly TOF-HRESIMS, 1D-NMR ( 1 H, 13 C and DEPT) and 2D-NMR (COSY, ROESY, TOCSY, HSQC and HMBC), and by comparison with the literature.


INTRODUCTION
The genus Antidesma (Euphorbiaceae) contains about 170 species, forest shrubs and trees, distributed in the world [1,2].Antidesma chevalieri Beille (syn.Antidesma laciniatum Muell.Arg.var.laciniatum) is a tree of about 9 m high encountered in Africa, particularly in Cameroon, Democratic Republic of Congo and Equatorial Guinea [3], and occupies a prominent position in traditional African medicine [4].Previous studies on the genus afforded terpenoids, steroids, flavonoids [2] and alkaloids [5], some of which possessed various biological activities [5][6][7].Particularly, squalene, (E)-phyt-2-en-1-ol, sitosterol and amentoflavone are the only compounds previously isolated from the leaves of Antidesma laciniatum Muell.Arg.[2].Continuing with the phytochemical investigation of plants of the genus Antidesma, we herein report the isolation and structure elucidation of a novel isoflavonoid glycoside, chevalierinoside A (1) from the methanol extract of the stem bark of A. chevalieri Beille.

EXPERIMENTAL
Melting points were recorded with a Reichert microscope and are uncorrected.Optical rotations were measured on a Belligham and Stanley ADP 220 polarimeter. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 600 spectrometer equipped with a cryoplatform ( 1 H at 600 MHz and 13 C at 150 MHz).2D-NMR experiments were performed using standard Bruker microprograms (Xwin-NMR version 2.1 software).Chemical shifts (δ) are reported in parts per million (ppm) with the solvent signals as reference relative to TMS (δ = 0) as internal standard, while the coupling constants (J values) are given in Hertz (Hz).The IR spectra were recorded with a Shimadzu FT-IR-8400S spectrophotometer.UV spectra were determined as methanol solution with a Cary 50 UV/VIS Spectrophotometer.TOF-ESIMS and HR-TOF-ESI experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK) with an electrospray source.The samples were introduced by direct infusion in a solution of MeOH at a rate of 5 µL min -1 .Column chromatography was run on Merck silica gel 60 (70-230 mesh) and gel permeation on Sephadex LH-20 while TLC was carried out on silica gel GF 254 pre-coated plates with detection accomplished by spraying with 50% H 2 SO 4 followed by heating at 100 o C, or by visualizing with an UV lamp at 254 and 365 nm.
Plant material.The stem bark of Antidesma chevalieri Beille was collected at Bansoa, Menoua Division, West Region of Cameroon, in July 2011.Authentication was done by Mr Victor Nana, a botanist of the Cameroon National Herbarium, Yaoundé, where a voucher specimen (No. 9667/SRF/Cam) has been deposited.
Chevalierinoside A (1) Acid hydrolysis and GC-MS analysis.Compound 1 (10 mg) was dissolved in MeOH-2 N HCl (1:4) (10 mL) and refluxed at 80 °C for 3 h.After removal of MeOH under reduced pressure, the aqueous layer was extracted with CH 2 Cl 2 (3 x 5 mL).The combined CH 2 Cl 2 extracts were washed with H 2 O and evaporated to dryness to afford the aglycon (2 mg) identified as 5,7dihydroxy-4'-methoxyisoflavone by comparison of its 1D-NMR data with those of literature [8].The aqueous layer was neutralized by dilute NaOH.The sugar components were analyzed by co-TLC with the mixture CHCl 3 /MeOH/H 2 O (7:3:0.2).After spraying, apiose gave a weak yellow spot at Rf 0.78, rhamnose gave a green spot at R f 0.75 and glucose gave a blue spot at Rf 0.70.
The previous aqueous layer was concentrated to dryness.The residue obtained was dissolved in pyridine (1 mL), then (CH 3 ) 3 SiNHSi(CH 3 ) 3 (1 mL) was added.After 10 min at room temperature, the solution was blown to dryness with a stream of nitrogen.The residue was dissolved in diethyl ether then subjected to GC-MS analysis.
GC-MS experiments were carried out on an MD 800 instrument.Trimethylsilyl ether derivatives were separated using an HP Ac-5 capillary column (0.25 x 30 m).Nitrogen was used as the carrier gas.The initial column oven temperature was 180 °C, then increased at 5 °C min -1 to a final value of 240 °C.The sugars were determined by comparison of retention times (t R ) with standard sugars: t R (min) Glc 6.87, Rha 4.32, Api 2.80.