Polyanxanthone A, B and C, three xanthones from the wood trunk of Garcinia polyantha Oliv.

Three xanthones, polyanxanthone A ( 1 ), B ( 2 ) and C ( 3 ) have been isolated from the methanol extract of the wood trunk of Garcinia polyantha , along with ﬁve known xanthones: 1,3,5-trihydroxyxanthone ( 4 ); 1,5-dihydroxyxanthone ( 5 ); 1,3,6,7-tetrahydroxyxanthone ( 6 ); 1,6-dihydroxy-5-methoxyxanthone ( 7 ) and 1,3,5,6-tetrahydroxyxanthone ( 8 ). Their structures were determined by means of 1D-and 2D-NMR techniques. Some of the above compounds were screened for their anticholinesterase activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes.


Introduction
The genus Garcinia of the Guttiferae family is well known to be a rich source of bioactive isoprenylated xanthones (Kanda et al., 2006;Deachathai et al., 2006;Komguem et al., 2005) and benzophenones (Nilar et al., 2005;Williams et al., 2003). In continuation of our search for bioactive substances from Garcinia species, we have investigated the methanol extract of the wood trunk of Garcinia Polyantha Oliv, a tree distributed in the lowland tropical rainforest of west, east and central Africa (Ampofo and Waterman, 1986;Brehaut, 1975). This investigation led to the isolation of three new xanthones (1-3) and five known xanthones (4-8). We report herein, the structure elucidation of the above compounds along with their anticholinesterase activities.
The following spectral data showed that all the new xanthones have a prenyloxy groups at C-1 and C-5. UV absorption of these compounds showed two strong bands at k max 240-248 and 254-266 nm, a medium bands at k max 302-312 nm, and weak broad bands at k max 352-390 nm indicated the conjugated chromophore systems (Ito et al., 2003). The IR spectra showed evenly bands at m max 1656-1665 and 1560-1601 cm À1  suggesting a carbonyl and an aromatic double bonds, respectively.
Polyanxanthone C (3) was isolated as yellow oil. This compound was showed to have the molecular formula C 28 H 32 O 4 by HREIMS which showed the [M+] ion peak at m/z 432.1683 (Calc. 432.1679). The 1 H NMR features were similar to those of (2), except for the appearance of prenyl group at d H 3.45 (2H, d, J = 7.2 Hz), 5.30 (1H, t, J = 7.2 Hz), 1.71 (3H, s) and 1.74 (3 H, s) instead of an aromatic proton appearing at d H 6.77 in ring C of (2). The lack of ABX system aforenamed on (2) and the presence of two aromatic protons ortho-coupled firmly indicated that H-2 was substituted by a new prenyl moiety. This hypothesis was confirmed after recording of HMBC and NOESY experiments showing on one side the expected long range correlation between H-1 00 of prenyl group and C-1, C-2 and C-3 (Table 1) together with the strong nuclear overhauser effects between H-1 0 /H-1 00 ,and in the other side the strong nuclear overhauser between H-3/H-4, H-3/H-2 00 and H-1 000 /H-6 ( Fig. 1). These results indicated the structure of polyanxanthone C (3), identified as 1,5-prenyloxy-2-(3methylbut-2-enyl)xanthone.

General procedure
The melting points were determined on a micro melting point apparatus (Yanaco MP-S3 apparatus) and are uncorrected. Infrared spectra were obtained on a JASCO A-302 spectrophotometer using KBr pellets. Ultraviolet spectra were recorded on a Shimadzu UV 240 spectrophotometer in methanol. Mass spectra (EI and HREIMS) were measured in an electron impact mode on Varian MAT 312 spectrometers. The 1 H NMR spectra were registered on a Bruker Avance AMX 500 NMR spectrometer with tetramethylsilane (TMS) as an internal standard; while 13 C NMR spectra were recorded on a Bruker Avance AMX 500 NMR spectrometer operating at 125 MHz using CDCl 3 as solvent. Methyl, methylene and methine carbons were distinguished by DEPT experiments. Homonuclear 1 H connectivities were determined by using the COSY experiment. One-bond 1 H-13 C connectivities were determined with HMQC gradient pulse factor selection. Two and three bond 1 H-13 C connectivities were determined by HMBC experiment. Chemical shifts were reported in d(ppm) and coupling constants (J) were measured in Hz. TLC was performed on precoated silica gel cards (E. Merck), spots were viewed under ultraviolet light at 254 nm for fluorescence quenching spots and at 366 nm for fluorescent spots and stained by spraying with a solution of ceric sulphate (0.2%) in H 2 SO 4 (5%). For column chromatography, silica gel (E. Merck, 230-400 mesh) were used. All reagents used were of analytical grades.

Plant material
The wood trunk of G. polyantha was collected from Mt Kala, central-province Cameroon in August 2003, and identified by Dr. Tchiengue Bathelemy of the Cameroon Nation Herbarium (Yaoundé), where a voucher specimen (21337/SRF/Cam/Mt Kala) was deposited.

Anticholinesterase assays
Electric-eel AChE (EC 3.1.1.7), horse-serum BChE (EC 3.1.1.8), acetylthiocholine iodide, butyrylthiocholine chloride, 5,5 0 -dithio-bis-nitrobenzoic acid (DTNB) and galanthamine were purchased from the Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. AChE and BChE inhibiting activities were measured by the spectrophotometric method developed by Ellman (Ellman et al., 1961). Acetylthiocholine iodide and butyrylthiocholine chloride were used as substrates to assay AChE and BChE, respectively. The reaction mixture contained 130 ll (100 mM) sodium phosphate buffer (pH 8.0), 20 ll of DTNB, 10 ll of test compound solution and 20 ll of AChE or BChE solution, which were mixed and incubated for 15 min at 25°C. The reaction was then initiated by the addition of 20 ll acetylthiocholine or butyrythiocholine, respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine were monitored by the formation of yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine, respectively at a wavelength of 412 nm (15 min). Test compounds and the positive control (galanthamine) were dissolved in EtOH. All the reactions were performed in triplicate in 96-well micro-plates in SpectraMax 340 (molecular Devices, USA). The concentrations of test compounds that inhibited the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) by 50% (IC 50 ) were determined by monitoring the effect of increasing concentrations of these compounds in the assays on the inhibition values. The IC 50 values were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA.).