Induction of cell-specific apoptosis and protection of mice from cancer challenge by a steroid positive compound from Zornia diphylla (L.) Pers

Objectives: Zornia diphylla (L.) Pers is an ethnomedical herb. The aim of the study is to scientifically verify the traditional use of Z. diphylla as an anticancer medicine. Materials and Methods: Different extracts, fractions, and chemical isolates of the whole plant were screened for cytotoxicity to Dalton’s lymphoma ascites (DLA) cells by the Trypan blue exclusion method and MTT assay. Column chromatographic and preparative TLC techniques were used for the isolation of active fraction (AF) and active principle. Cytotoxicity of AF to different cell types was tested. The apoptotic activity of AF was evaluated by morphological observations, nuclear condensation, and comet assay. In vivo antitumor activity of AF was determined in DLA-challenged mice. Short-term (29 days) preliminary toxicity evaluation of AF was done in mice. Results: n-Hexane extract (but not water and ethanol extracts) showed significant cytotoxicity. AF, isolated from n-hexane extract, induced apoptotic cell death (in vitro) to DLA cells, but not to normal thymocytes and macrophages. A steroid positive active principle was isolated which showed 100% cytotoxicity at 5 µg/mL level. Interestingly, AF (50 mg/kg) protected all the mice challenged with one million DLA cells/mouse. AF (up to 10 times higher than the therapeutic dose) did not exhibit any conspicuous adverse toxic symptoms in the toxicity evaluation. Conclusion: Z. diphylla (AF) showed promising in vitro and in vivo anticancer activity against DLA cells, and it was devoid of any toxicity to mice in short-term toxicity evaluation. The herb is promising for the development of a valuable anticancer medicine.