Isolation, cytotoxicity evaluation and HPLC-quantification of the chemical constituents from Artemisia amygdalina Decne

Abstract

The hexane extracts of both shoot and root parts of Artemisia amygdalina Decne displayed potent cytotoxic effects. Phytochemical analysis of these active extracts led to the isolation of six cytotoxic constituents, viz., Ergostadien-3β-ol (1), ludartin (2), 5-hydroxy-6,7,3',4'-tetramethoxyflavone (3) (from shoot) and trans-matricaria ester (4), diacetylenic spiroenol ether (5) and cis-matricaria ester (6) (from root) for the first time from this plant. The constituents were identified using spectral techniques in the light of literature. Sulphorhodamine B cytotoxicity screening of the isolated constituents was carried out against four human cancer cell lines including Lung (A-549), Leukaemia (THP-1), Prostate (PC-3) and Colon (HCT-116) cell lines. Ludartin (2) exhibited the highest cytotoxicity with IC50 values of 7.4μM, 3.1μM, 7.5μM and 6.9μM against Lung (A-549), Leukaemia (THP-1), Prostate (PC-3), Colon (HCT-116) cancer cell lines respectively. To test against in vitro skin cancer models [human dermal fibroblasts (CRL-1635)] all the isolates were further subjected to 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) cytotoxicity screening. Ludartin (2) being highly cytotoxic was again evaluated against mouse melanoma (B16F10) and human epidermoid carcinoma (A-431) cells by MTT assay displaying IC50 values of 6.6μM and 19.0μM respectively. Finally a simple and reliable HPLC method was developed (RP-HPLC-DAD) and validated for the simultaneous quantification of these cytotoxic constituents in A. amygdalina Decne. Excellent specificity and high linearity for all the standard calibration curves having regression coefficients of the respective linear equations in the range of 0.9962-0.9999 was observed. Relative recovery rates varied between 98.37±0.90 and 105.15±1.74 with relative standard deviation less than 4%. Based on our results, the developed method features good quantification parameters, accuracy, precision and can serve as effective quality control method for standardisation of A. amygdalina Decne.