Simultaneous HPTLC analysis and in vitro antileishmanic activity of various secondary metabolites in extract of the traditional medicinal herb Artabotrys hexapetalus (L.f.)

BACKGROUND: Artabotrys hexapetalus [(L.F) Bhandari] a medicinal plant is commonly known as ‘Hari Champa’ and its roots and fruits are used for treating malaria and scrofula, respectively. OBJECTIVE: The aim of this work was to develop a sensitive, fast and reproducible high-performance thin-layer chromatographic (HPTLC) method for simultaneous analysis of quercetin and apigenin in various extracts of Artabotrys hexapetalus (L. f.) Bhandari (Family Annonaceae) and further to assess antileishmanic effects of different extracts of A. hexapetalus against Leishmania donovani. MATERIALS AND METHODS: Metabolic fingerprinting was developed using HPTLC with quantification of markers (quercetin and apigenin). The method was validated for linearity, specificity, precision, accuracy and robustness. Among the different combinations of mobile phases used, best separation was achieved in toluene:ethyl acetate:formic acid (6.5:3:0.5, v/v/v). Densitometric scanning of the plates directly at 254 nm was used for analysis of quercetin as well as apigenin. The concentration-response curve was plotted and IC(50) values were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS: Compact bands for quercetin and apigenin were obtained at R(f) 0.52 ± 0.001 and 0.73 ± 0.002, linearity were found satisfactory for quercetin and apigenin. Linearity range for quercetin and apigenin were 100–1000 ng/spot and 100–2000 ng/spot, respectively, with r(2) = 0.996 ± 0.002 and 0.993 ± 0.003, limit of detection (15.56 and 13.78 ng/spot), limit of quantification (51.8 and 45.94 ng/spot), recovery (98.7%–99.7% and 96.8%–98.8%) and precision with %RSD <2%. Various dried extracts were found to contain quercetin in the range of 0.35%–4.26% (w/w) and apigenin in the range of 0.64%–8.46% (w/w). Cytotoxicity assay of extracts over promastigotes showed that petroleum ether extract was found to be most cytotoxic (IC(50) 30.28 ± 1.06 μg/mL) after 96 h in comparison to other extracts. The finding of this study indicates that this plant is effective against L. donovani in vitro. CONCLUSION: The present HPTLC method is being reported for the first time and can be used for routine quality control. The petroleum ether extract of A. hexapetalus displayed potent antileishmanial activity and can be further explored for the development of antileishmanial treatment regimen.