General Papers ARKIVOC 2007 (xiii) 116-123
Experimental Section
Plant material
The twigs of D. barteri var. subtriangularis were collected at Tombel in the South-western province of Cameroon in November 2001. The plant was identified by Mr. Victor NANA from the National Herbarium in Yaoundé, where a voucher specimen (No 19534/SRF Cam.) was deposited.
Extraction and compound isolation
Air-dried and powdered twigs (450 g) were successively macerated with a mixture of CH2Cl2- MeOH (1:1) and MeOH for 24 and 2 hours, respectively, at room temperature. These two crude extracts were combined (70 g) based on their thin layer chromatography (TLC) patterns. 65 g of this organic extract was subjected to column chromatography (CC) on silica gel 60 (200 g) and eluted with petroleum ether (60/80) followed by pet. Ether-EtOAc (3:1, 1:1, 1:3) mixtures and then EtOAc to give fractions A to E of 500 ml each. The fraction A (5 g) eluted with pet. Ether contained mainly mixtures of oils and was not investigated further. Fraction B (30.2 g) was passed through Sephadex LH-20 column and eluted with CHCl3-MeOH (2:1). The post chlorophyll fraction (10.5 g) was subjected to silica gel 60 (150 g) CC separations and eluted with hexane followed by hexane-EtOAc gradient. 25 fractions of 250 ml each were collected and combined on the basis of TLC patterns. Fractions 1-5 eluted with hexane-EtOAc (95:5) yielded 4-hydroxylonchocarpin 4 (25 mg). Fractions 6-12 obtained with hexane-EtOAc (9:1, 4:1) gave isobavachalcone 5 (15 mg). Fractions 13-25 obtained with hexane-EtOAc (7:3) were combined to give 3 g of a mixture of three compounds from the TLC patterns; part (1g) of this mixture was purified by preparative TLC using CHCl3-MeOH (97:3) to give stipulin 3 (40 mg); bartericin A 1 (35 mg) and B 2 (40 mg). Combined fractions C - E (14.5 g) were also passed through Sephadex LH-20 and eluted with CHCl3-MeOH (2:1); the post chlorophyll fractions (2 g) was subjected to CC (silica gel, 50 g) using CH2Cl2-MeOH (95:5) to afford kanzonol B 6 (20 mg) and bartericin A 1 (5 mg). The structures of the purified compounds were elucidated by spectroscopic methods, mainly 1D and 2D-NMR spectroscopy.14
Evaluation of erythrocyte susceptibility to compounds 1-6 in vitro
A preliminary toxicological assessment was carried out to determine the highest drug concentrations that can be incubated with erythrocytes without any significant damage. This was done according to the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide/phenazine methosulfate (MTT/PMS) colorimetric assay described by Cedillo-Rivera et al. (1992),15 with some modifications. The drugs were serially diluted in 96 well culture plates, and each concentration incubated in triplicate with erythrocytes (2% hematocrit) in a final 100ml culture volume (at 37 °C, in a 3% O2, 5% CO2 and 91% N2 atmosphere, in the presence of RPMI 1640, 25mM HEPES, pH7.4 for 48 h). At the end of the incubation period, the cultures were
ISSN 1424-6376 Page 121 ©ARKAT USA, Inc.
