Process for Isolating the Active Ingredients from Different Parts of the Unripe Fruit of Diospyros peregrina and their Characterization to Define the Cancer Preventive Functions

Abstract

Diospyros peregrina is a plant indigenous to India, belonging to the family Ebenaceae. It is a medium-sized evergreen tree having ethnomedicinal significance as an aphrodisiac, astringent, bactericide and is used for the treatment of several diseases. Herbal practitioners have indicated that all parts of this plant specially bark, leaves, flowers and fruits are used in different medicinal preparations. Diverse bioactive compounds have been isolated from various parts of this plant. Every year, there are 500,000 cases of cancer, and compounds in this fruit could be an answer to its treatment, saving millions of lives. The Milli-Q water extracts of the epicarp, mesocarp and endocarp of the unripe fruit of Diospyros peregrina will be prepared and lyophilized separately. In vitro cytotoxicity assay will be performed on different kinds of cancer cell lines using the lyophilized fractions through MTT assay. In vitro lymphocytic proliferation will be also tested on the lymphocytes isolated from normal and cancerous subjects using the lyophilized fractions through MTT assay. Cytotoxic T lymphocyte (CTL) assay will be carried out coculturing lyophilized fractions treated lymphocytes (effector cells) and cancerous cells (target cells) and cellular cytotoxicity will be determined by measuring lactate dehydrogenase (LDH) released by target cancerous cells using a commercially available kit (Roche Diagnostics, Mannheim, Germany). Further, methanolic extract of the unripe fruit of Diospyros peregrina will be carried out to identify the secondary metabolites which might possess cancer preventive properties. Further the cancer preventive function of the extracts through immunostimulation was confirmed by using the effector cells (Gab extract treated lymphocytes) against COLO 205 (Colorectal cancer cell lines) by CTL reaction through LDH release assay. Thus, the fractions having the principle active ingredient responsible for the cancer preventive activity will be further confirmed through HPLC and silica gel column purification. This aims to be a significant milestone in finding a non-invasive cure for cancer. The cancer preventive active ingredient from the unripe fruit of Diospyros peregrina could revolutionize cancer treatment.