Evaluation of phytochemicals from Datura species to induce Granulysin expression in immune cells conferring cytotoxicity towards cancerous cells

Abstract

newline Faculty of Applied Sciences and Biotechnology newlineABSTRACT newlineHimalayan medicinal plants have been used since antiquity to treat a variety of human newlinediseases. The prime purpose of Ayurveda, one of the ancient Indian medicinal systems, is the newlineprevention of different ailment routes. Datura stramonium L. is a very old herb found in the newlineAyurveda and has been used widely due to its pharmacological potential. It has been newlinementioned in Ayurveda that D. stramonium has been used to treat various human diseases newlinesuch as wounds, fever, inflammation, swelling, toothache, asthma, skin disorders, newlineleucoderma, burns etc. whereas D. metel has been used to cure various human problems like newlineskin rashes, ulcers, bronchitis, jaundice and diabetes, skin inflammation and psoriasis. Datura newlinespp. are a rich source of alkaloids but also consisted of other active phytochemicals such as newlineflavonoids, terpenoids, steroids, withanolides, and tannins. In the present study, we focused newlineon the immunomodulatory potential of Datura spp. (D. stramonium and D. metel) on human newlineperipheral blood mononuclear cells (PBMC) assayed by MTT assay. Our data revealed that newlineprepared fractions, non-alkaloid and alkaloid fraction of D. stramonium showed higher newlineimmunostimulatory activity as compared to the non-alkaloid and alkaloid fraction of D. newlinemetel. Rather the non-alkaloid fraction of D. metel showed cytotoxicity towards human newlinePBMC. These results suggest that the phytoconstituents present in extracts and fractions of D. newlinestramonium and D. metel display significant immunoenhancing and immunosuppressing newlinepotential. Furthermore, the bioassay (PBMC proliferation) guided chromatographic newlinefractionation of non-alkaloid fraction of D. stramonium led to the isolation of bioactive newlinefraction (fraction-10) having significant immunostimulatory activity in human PBMC. newlinePhytochemical screening of fraction-10 showed the presence of terpenoids and steroids. newlineMoreover, LC-MS analysis of fraction-10 depicted the presence of a major phytoconstituent newlinenamely daturaolone with corresponding m/z of 440 and the mass spectral data also revealed newlinepresence of daturadiol, stigmasterol and sitosterol with corresponding m/z of 442, 412 and newline414 respectively as fragments of the major compound daturaolone. Additionally, fraction-10 newline(10 and#956;g/ml) treated PBMC exhibited enhanced production of IL-2 (7.4 fold), IFN-and#947; (9.6 fold) newlineand TNF-and#945; (13.4 fold) at RNA level as well as at protein level i.e. IL-2 (8 fold) and IFN-and#947; (10 newlinefold) as compare to the untreated cells. Moreover, fraction-10 enhanced the expression of newlineGranulysin (a cytolytic granule) in peripheral lymphocytes at both RNA and protein level. It newlinehas been reported earlier in the literature that the progression of cancer is directly associated newlinewith decreased Granulysin expression hence it may used as an indicator in the progression of newlinecancer. Therefore, the increased level of Granulysin expression by fraction-10 activated newlineFaculty of Applied Sciences and Biotechnology newlinePBMC would help in the assessment of the cancer cell killing capabilities of the human newlineperipheral lymphocytes. In addition, the cytolytic potential of bioactive fraction-10 treated newlinehuman PBMC was evaluated by MTT microcytotoxicity assay using A549 (lung newlinecarcinomas), MCF-7 (breast cancer) and HepG2 (hepatic carcinoma) cell lines as target cells. newlineFraction-10 (10 and#956;g/ml) enhanced the cytotoxic capability of peripheral blood lymphocytes newlinetowards A549, MCF-7 and HepG2 cancer cells lines after 24 hrs of incubation. Fraction-10 newlinetreated PBMC showed significantly higher cell cytotoxicity (59±2.1%) against A549 at 20:1 newline(effector:target) ratio whereas untreated PBMC showed only 11±3.1% and Con A treated newlinecells (positive control) showed 28±3.3% cytotoxicity against A549 cells. Similarly when newlineMCF-7 and HepG2 cells were taken as target cells, fraction-10 treated PBMC exhibited newline62±2.3% and 53±3.1% cytotoxicity respectively at 20:1 (effector:target) ratio which was newlinesignificantly higher as compare to respective controls. Taken together, the present research newlineprovides a rationale that the bioactive fraction-10 from D. stramonium trigger the immune newlinecells thereby enhancing their anticancer abilities against human lung, breast and liver cancer newlinecells. This research can be a basis for the potential drug design especially in immunotherapy newlineagainst cancer. newlineKeywords: Immunomodulation, PBMC, anticancer, cytokine, cytotoxic, Granulysin, LCMS, newlineDatura stramonium, Datura metel