Anti-tumor and pro-apoptotic activity of ethanolic extract and its various fractions from Polytrichum commune L.ex Hedw in L1210 cells

Ethnopharmacological relevance: Polytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for lymphocytic leukemia, but the antitumor effect and its potential mechanism remains unclear. Aims of the study: The present study was to investigate the possible anti-proliferative activity of ethanolic extract and the organic fractions from P. commune on murine leukemia L1210 cells. Materials and methods: The content of ethanolic extract and its fractions was performed on HPLC analysis with gradient elution. L1210 cells were treated with different concentrations of ethanolic extract and its fractions at different time intervals. Cell viability was evaluated using MU assay. Apoptotic cell death was monitored by nuclear condensation and confirmed by exposure of phosphatidylserine to outer leaflet of plasma membrane. Loss of mitochondrial membrane potential was analyzed by flow cytometry using rhodamine 123 staining. Results: The obtained results showed that the cell viability of L1210 cells was reduced by ethanolic extract of P. commune in a concentration-dependent manner, and the IC50 value was about 77.22 mu g/ml at 24 h post treatment. The ethylacetate fraction displayed higher anti-tumor effect than that of chloroform and butanol fractions with 32.29 mu g/ml (IC50 value, 48 h). Microscopy studies revealed that ethanolic extract and ethylacetate fraction treated cells showed morphological characteristics of apoptosis such as chromatin condensation and DNA aggregation. Further, Annexin V-PE/ 7-AAD double staining showing the out leaflet of phosphatidylserine and the decline of mitochondrial membrane potential by flow cytometry confirmed that the extracts do, in fact, induce apoptosis in L1210 cells. Conclusion: This is the first report on anti-tumor and pro-apoptotic effect of P. commune in cultured leukemia cells, which provides scientific basis for its usefulness as traditional medicine. Further studies are needed to confirm the precise mechanism not only the crude extract but as well the monomeric chemical substances of Polytrichum commune L.ex Hedw. (c) 2012 Elsevier Ireland Ltd. All rights reserved.