Anti-proliferative and apoptotic activities of constituents of chloroform extract of Juglans regia leaves.

OBJECTIVES: To evaluate anti-proliferative as well as apoptotic activities of compounds identified in chloroform extract of Juglans regia leaves, on human breast and oral cancer cell lines (MCF-7 and BHY).
MATERIALS AND METHODS: Column chromatography, MTT assay, flowcytometry and western blotting have all been used in the study.
RESULTS: Bioassay-guided fractionation of chloroform extract of J. regia afforded isolation of 5-hydroxy-3,7,4'-trimethoxyflavone [1], lupeol [2], daucosterol [3], 4-hydroxy-α-tetralone [4], β-sitosterol [5], 5,7- dihydroxy-3,4'-dimethoxyflavone [6] and regiolone [7]. Structures of the compounds were established on the basis of spectroscopic analyses [Nuclear magnetic resonance (NMR) and mass]. All compounds inhibited proliferation of MCF-7 (human breast adenocarcinoma) and BHY (human oral squamous carcinoma) cells in a concentration-dependent manner. Compounds 6 and 7 had potent cytotoxic effects on both MCF-7 and BHY cells (IC50 21-51 μm), yet were not toxic to normal cells. MCF-7 growth inhibition was attributed to apoptosis; population of apoptotic cells increased from 1.12% in controls to 5.64 and 8.1% after 48-h treatment with compounds 6 and 7, indicating their potential at inducing early and late apoptosis. The caspase cascade was not activated, as indicated by only insignificant cleavage of caspase-3.
CONCLUSIONS: Our results suggest that compounds 6 and 7 can induce apoptosis in MCF-7 cells through the caspase-3 independent pathway.