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

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Title Evaluation of phytochemicals from Datura species to induce Granulysin expression in immune cells conferring cytotoxicity towards cancerous cells
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Contributor Saini Reena Vohra
 
Subject anticancer
cytokine
cytotoxic
Immunomodulation
PBMC
 
Description 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
Summary and Conclusion p.,76-79;Recommendation and Future Direction p.,80; References p., 81-98; Appendices p.,99-106;
 
Date 2018-10-01T10:37:32Z
2018-10-01T10:37:32Z
27/08/2012
2017
08/09/2017
 
Type Ph.D.
 
Identifier http://hdl.handle.net/10603/217888
 
Language English
 
Relation 218
 
Rights university
 
Format 106p.
29cm
DVD
 
Coverage Biotechnology
 
Publisher Solan
Shoolini University of Biotechnology and Management Sciences
Faculty Of Biotechnology
 
Source University