BSC -MSc

The study entitled “Anticancer activity of nanocurcuminoids prepared from black turmeric (Curcuma caesia Roxb.)” was carried out at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, in the year 2022-2023 to evaluate the cytotoxic and antiproliferative potential of nanocurcuminoids prepared from methanolic extract of Curcuma caesia rhizome in cancer cell lines and to elucidate its action on the apoptotic pathway.

The methanolic extract prepared from 30g of dried rhizome of Curcuma caesia using cold extraction method yielded 500mg of crude extract. To prepare the nano form of curcuminoids, the crude extract was dissolved in DMSO (Dimethyl sulfoxide) and ultrasonication was done for 10 minutes with an amplitude of 40µm and a pulse of 10 seconds. The colour change from yellow to orange indicated the formation of nanocucuminoids. The particles showed a spherical shape with a 63.4nm size by transmission electron microscopy and an average hydrodynamic size (Z-value) of 63.1nm by the dynamic light scattering (DLS) particle size analyzer. The zeta potential of nanocurcuminoids was -25.5mV and the maximum peak was observed at 380nm.

The evaluation of cytotoxic activity and antiproliferative effect of nanocurcuminoids were performed in the liver (HepG2), breast (MDA-MB-231), colon (HCT-116) cancer cell lines, and normal (HEK-293) cell lines cultured in DMEM medium. 3-[4,5- dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was performed in the cell lines treated with different concentrations of the nanocurcuminoids and crude extract for 48h and 72 h, by keeping DMSO as vehicle control and the standard chemotherapeutic drug, 5 Fluorouracil as the positive control. The nanocurcuminoids showed cytotoxicity to the cancer cells with IC50 values of 92.97µg/mL and 75.67µg/mL in the breast and colon cancer cell lines respectively without cytotoxicity to the normal cell lines at concentration up to 100 µg/ml in 48h of incubation. The crude methanolic extract showed IC50 values of 144.16µg/ml and 111.49µg/ml in the breast and colon cancer cell lines respectively. Treatment of HepG2 cells with 200µg/ml of the nanocurcuminoids and crude extract showed inhibition of 45.42% and 42.19% at 72 hours of incubation and at 48 hours it was 39.21% and 36.05% respectively. Treatment



of HEK-293 cells with 100µg/ml of nanocurcuminoids showed cytotoxicity up to 18.27% and 73.91% at 48h and 72h of incubation respectively.

In the clonogenic assay, the nanocurcuminoids at IC50 showed 92.68% inhibition of colony formation in HCT-116 cells and 83.3% in MDA-MB-231 cells. Gene expression profiles of five apoptotic genes with Β-ACTIN as a reference gene were analysed using real-time PCR. The ratio of expression of the apoptotic marker genes, BAX and BCL-2 were 7.45 and 1.13 in the colon and breast cancer cell lines respectively, indicating the possibility of activation of the apoptotic pathway. The BAX gene was found upregulated and the BCL-2 gene downregulated in HCT-116 but, BAX and BCL-2 were upregulated in MDA-MB-231 treated with the nanocurcuminoids compared to the control. While CASPASE 9 was upregulated, CASPASE 3 was downregulated in both the HCT-116 and the MDA-MB-231 cell lines. PARP1 gene was downregulated in HCT-116 but upregulated in MDA-MB-231. The results suggest activation of apoptosis as a mechanism of action of nanocurcuminoids from C. caesia in HCT-116, but it could not be confirmed in MDA-MB-231. The study suggests that the nanocurcuminoids of C. caesia possesses greater cytotoxic effects and antiproliferative effect in the breast and colon cancer cell lines than the crude extract without affecting normal cell lines for 48 hours of incubation.