Rapid identification of the main constituents of an antimalarial plant extract using LC-SPE-NMR

Previous phytochemical investigation of Ormocarpum kirkii S.Moore (Papilionaceae) has led to the isolation and identification of (I-3,II-3)-biflavonoids, some of them with antiplasmodial activity (Dhooghe et al., 2010). The major compound of the total extract (80% MeOH) was (+)-chamaejasmin. However, a series of minor constituents remained unidentified. The aim of this work was to further characterise the 80% MeOH ectract from O. kirkii and to identify the minor constituents using an integrated platform of LC-MS and LC-SPE-NMR.

The crude 80% MeOH extract of O. kirkii was partitioned into four fractions by means of liquid-liquid extraction: n-hexane, chloroform, ethyl acetate and water. The chloroform and ethyl acetate fractions were used for the LC-SPE-NMR study. By using the multiple trapping technique several components were enriched on the SPE cartridges. After drying and eluting with deuterated methanol into 3mm NMR tubes, high-resolution NMR spectra were recorded. The structure elucidation of these compounds was based on 1D and 2D NMR, and MS data.

A total of sixteen compounds were identified and assigned in the HPLC chromatogram: sikokianin B (1), sikokianin C (2), glabroisoflavanone A (3), diphysolone (4), (+)-chamaejasmin (5), isochamaejasmin (6), apigeninyl-(I-3,II-3)-naringenin (7), liquiritigeninyl-(I-3,II-3)-naringenin (8), (I-3,II-3)-biliquiritigenin (9), 5,5“-dimethoxydiphysin (10), 4“-hydroxydiphysolone (11), 7-O-glucosylchamaejasmin (12), diphysin (13), ormocarpin (14), 7-O-glucosyldiphysin (15), and isovitexin (16). In addition to the twelve constituents (5-16) that had been isolated before using semi-preparative HPLC, four more compounds were obtained (1-4).

References: Dhooghe L et al. (2010) Phytochemistry 71: 785–791