BSc - MSc (Int.)

The research work entitled “Antiproliferative and anticancer properties of butterfly ginger lily (Hedychium coronarium Koenig ex Retz)” was carried out at the Department of Plant Biotechnology, College of Agriculture Vellayani, Thiruvananthapuram during the year 2021-2022 and the objective was in vitro analysis of cytotoxic, antiproliferative, and anticancer properties of methanolic extract of Hedychium coronarium in lung and colon cancer cell lines.
The rhizome samples of Hedychium coronarium were collected from the Aromatic and Medicinal Plants Research Station, Odakkali, Ernakulam. The samples were washed, dried, powdered, and the methanolic extract of the rhizome was prepared via soxhlet extraction. The crude extract obtained (205 mg/5g of powdered rhizome) was dissolved in absolute ethanol (10 mg/mL w/v) for treatment on cell lines.
All the assays were performed on the lung cancer cell line (A549), colon cancer cell line (HCT 116), and normal cell line (HEK-293). The cell lines were treated with varying concentrations of the extract and MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)] assay was carried out to determine the cytotoxicity. Treatments with absolute ethanol and the standard chemotherapeutic drug, 5-Fluorouracil served as vehicle control and positive control respectively. The crude methanolic rhizome extract exhibited dose-dependent cytotoxicity in cancer cell lines with IC50 values of 44 μg/mL and 74.60 μg/mL in A549 and HCT 116 respectively. No cytotoxicity was observed in the normal cell line, HEK-293 on treatment with the extract up to a concentration of 125μg/mL.
Clonogenic assay was performed to identify the reproductive death of cancer cells by analyzing the ability of a single cell to form a colony. The results indicated that the inhibition rate in colony formation was 38.7% and 50% in A549 and HCT 116 cell lines respectively on treatment with the respective IC50 concentrations of the extract in the cell lines.
DNA was isolated from the cell lines treated with varying concentrations of the extract and subjected to agarose gel electrophoresis and observed for DNA fragmentation, which is an indicator of apoptosis. Intact bands were observed in the gel profile of DNA isolated from the treated A549 cell line while slight shearing was observed in the gel profile of DNA isolated from the treated HCT 116 cell line indicating the onset of apoptosis. Microscopic observations of the cell lines after treatment with the extract revealed marked morphological changes in the cells including shrinkage of the cell, loss of intact morphology, detachment from the surface, and decreased cell population compared to the untreated cell lines.

Expression analysis of key apoptotic genes viz., BAX, BCL-2, CASPASE 3, CASPASE 9, and PARP-1 were carried out in the treated cell lines using Real-time qPCR with ACTIN as the reference gene. In A549 cell line, downregulation of BAX (0.10 fold), BCL-2 (0.033 fold), CASPASE 3 (0.015 fold), CASPASE 9 (0.029 fold) and upregulation of PARP-1(1.14 fold) was recorded. Whereas in HCT 116 cell line, upregulation of BAX (1.34 fold), CASPASE 3 (1.10 fold) CASPASE 9 (1.06 fold) and downregulation of BCL-2 (0.02 fold) and PARP-1(0.90) was recorded. The BAX: BCL-2 ratio was 2.97 fold in the A549 cell line and 58.96 fold in the HCT 116 cell line. The results indicate that the mechanism of action of the extract in the two cancer cell lines may be different and is dependent on the nature of the cancer.
The study revealed that methanolic rhizome extract of H. coronarium possess cytotoxic, antiproliferative, and anticancer activities in the lung (IC50 = 44 μg/mL) and colon cancer cell lines (IC50 = 74.60 μg/mL) without affecting the normal cell line at these concentrations. The mechanism of action of the extract may be by induction of apoptosis in colon cancer cell line and by cell cycle arrest in lung cancer cell line. To conclude, the results indicate that methanolic rhizome extract of Hedychium coronarium contains constituents with potential for drug development against cancer.