Immune Modulating and Antiproliferative Potential of Withania somnifera Crude and Pre-purified Fractions on Selected Cancerous and Normal Cells

Background: Cancer is a major public health problem globally. The burden of cancer is greater in developing countries where the cost of treatment and management is way beyond the reach of many resource challenged individuals. Plants are a good source for anticancer agents. Withania somnifera has been widely used for its great pharmacological value. Previous studies have shown that W. somnifera has potential as an immune modulating agent by up-regulating the Interleukin-7 cytokine gene expression. Interleukin-7 cytokine (IL-7) is required for the development of the immune system. Studies have shown that exogenous IL-7 increases T cell and B cell numbers. Activation of CD4+ and CD8+T cells may be effective agents for the treatment of malignant tumors. The objective of this study was to confirm that Kenyan W. somnifera plant parts extracts have antiproliferative activity and ability to raise IL-7 levels.

Methodology: The MTT assay was used to evaluate the anti-proliferative potential of the extracts. The conventional drug 5-Flourouracil was used as the positive control. For IL-7 analysis, the plant extract with the best antiproliferative activity was used. The cells were recultured in T25 flask then treated with the extract at 50µg/ml for 48hrs. After which the RNA was extracted using Pure Link RNA mini kit (Thermoscientific USA). The extracted RNA was quantified and its quality assessed using a NanoDrop ND-2000 spectrophotometer. Reverse transcription and cDNA amplification was done by a single step reaction technique using Invitrogen Super Script IV Reverse Transcriptase and Thermo scientific real time SYBR green kits according to the manufacturer’s instructions. Quantitative real-time PCR was performed in a reaction volume of 25 μL under different condition and time. A total of 50 cycles using RT- PCR Quant studio V system were performed. The primers were the designed using Expasy and Jusbio after which the ct cycle was used to determine the expression levels of IL-7.

Results: The stem methanol-dichloromethane extracts on HCC (IC50-25.23±1.23), roots water extract on DU 145 (IC50-8.04±1.55), leaves methanol-dichloromethane extracts on 22RVI (IC50-2.21±0.577) had the lowest IC50 values signifying high activity. The methanolic-dichloromethane extract stem bark gave the highest selectivity index on HCC (breast cancer, SI-98.403) compared to the other extracts. The most inhibitive methanol-dichloromethane extracts against cancerous cells did not inhibit the growth of IEC6 (normal cells). However after 48hrs of exposure of the plant’s extract on IEC6 cells expression of IL-7 gene was upregulated 2 times for crude W. somnifera leaves organic extract and up to 5.37 times for the prepurified fraction. Our findings indicate that Kenyan W. somnifera extracts especially in the pre-purified form have potential to treat and manage cancer. Of great significance is the potency of the plants extracts more so the prepurified fraction to up-regulate IL-7 which is an anticancer cytokine important in proliferation and maturation of immune system cells. This cytokine has been implicated in improved cancer prognosis. The potency of the W. somnifera extracts and pre-purified fraction to induce up-regulation of this could infer its probable immune modulation pathway as a possible mechanism of action of the plant’s extracts.