In vitro anti-cancer properties of a commercially available polyherbal nutraceutical (Vernolac) capsule on cancer stem cell-like (NTERA-2 cl.D1) cells.

Rathnayake, P. C.; Senavirathna, D. C.; Milhan, M. M.; Gunawaradane, S. D. V.; Seneviratne, N. N.; Perera, D. N.; Rajagopalan, U. -; Senathilake, K. S.; Samarakoon, S. R.

doi:10.1101/2025.07.15.665027

bioRxiv · 10.1101/2025.07.15.665027

In vitro anti-cancer properties of a commercially available polyherbal nutraceutical (Vernolac) capsule on cancer stem cell-like (NTERA-2 cl.D1) cells.

Rathnayake, P. C.·Senavirathna, D. C.·Milhan, M. M.·Gunawaradane, S. D. V.·Seneviratne, N. N.·Perera, D. N.·Rajagopalan, U. -·Senathilake, K. S.·Samarakoon, S. R.

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Abstract

Vernolac is a commercially available polyherbal nutraceutical formulation comprising Vernonia zeylanica aerial parts, Nigella sativa seeds, Hemidesmus indica roots, Smilax glabra rhizome, and Leucas zeylanica aerial parts. Previous studies have demonstrated anti-cancer activities of phytochemicals derived from these individual plant components. However, the anti-cancer properties of the supercritical CO2 extract of Vernolac remain unexplored against cancer stem-like cell populations. The current study is focused on the anti-cancer potential of Vernolac extract on NTERA-2 cl.D1 cancer stem-like model, a human embryonal carcinoma-derived pluripotent cell line. Gas Chromatography-Mass Spectrometry (GC-MS) was performed for phytochemical analysis. Several in vitro assays evaluated the anti-cancer properties of the Vernolac extract. Cytotoxicity was assessed using the Sulforhodamine B assay, and apoptosis induction was determined by Acridine Orange/Ethidium Bromide staining and the caspase 3/7 activity assay. Scratch assay was used to evaluate cell migration, and Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) was performed to analyze the expression levels of apoptosis-related genes (TP53, BIRC5) and the autophagy-related gene mTOR. Further, the free-radical scavenging activity of Vernolac extract was assessed using 2,2-diphenyl-1-picrylhydrazyl and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay. The reactive oxygen species levels in NTERA-2 cl.D1 cells were quantified using the nitroblue tetrazolium assay. GC-MS analysis revealed 20 phytochemical constituents in the supercritical CO2 extract. In vitro assay results demonstrated that Vernolac extract exhibits significant anti-proliferative activity in NTERA-2 cl.D1 cells, with an IC50 of 41.12 {micro}g/mL at 48 h, while exerting minimal effects on non-cancerous MCF-10A cells (IC50 > 1000 g/mL). Fluorescence microscopy and caspase 3/7 assay showed that Vernolac extract leads to early apoptosis in NTERA-2 cl.D1 cells. RT-qPCR revealed the upregulation of tumor suppressor protein P53 while downregulating BIRC5 and mTOR. Additionally, Vernolac extract inhibited the migration rate of NTERA-2 cl.D1 cells and elevated intracellular reactive oxygen species levels. These findings suggest that the supercritical CO2 extract of Vernolac exerts potent anticancer properties against NTERA-2 cl.D1 cancer stem-like model, highlighting its therapeutic potential for targeting cancer stem cells.

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Rathnayake, P. C., Senavirathna, D. C., Milhan, M. M., Gunawaradane, S. D. V., Seneviratne, N. N., Perera, D. N., Rajagopalan, U. -, Senathilake, K. S., Samarakoon, S. R.. 2025-07-19. In vitro anti-cancer properties of a commercially available polyherbal nutraceutical (Vernolac) capsule on cancer stem cell-like (NTERA-2 cl.D1) cells.. https://doi.org/10.1101/2025.07.15.665027

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