14‑Deoxy‑11,12‑Didehydroandrographolide: A Novel Compound Isolated from Andrographis paniculata Nees. Induces Robust Apoptosis in Leukemic Cells

Objective: Andrographis paniculata is widely cultivated in South and Southeast Asian countries and popularly used in “Ayurveda” medicine. We attempted to investigate antileukemic activity of the biomolecules extracted from this plant and a probable mechanism of action. Materials and Methods: Biomolecules from methanolic extract were isolated using silica gel column chromatography and high‑performance liquid chromatography. The structures were determined by liquid chromatography‑mass spectrometry (LC‑MS), 1H nuclear magnetic resonance (NMR), and 13C NMR. In vitro antiproliferative activity was measured by 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assay. The apoptotic efficacy of the most potent molecule was investigated by annexin V and propidium iodide (V/PI) staining and flow cytometry. Caspase activation, cell cycle distribution pattern, and nuclear morphology of the treated leukemic cells were also investigated. Results: From the methanolic extract, four biomolecules were isolated, namely 14‑deoxy‑11,12‑didehydroandrographolide (1), andrographolide (2), neoandrographolide (3), and 14‑deoxy‑11,12‑didehydroandrographiside (4). Results of MTT assay exhibited that out of four compounds, compound (1) showed the most potent activity against all the cell lines tested with the lowest IC50 values of 13 μM on U937 cells. Annexin V/PI staining revealed that the compound was able to induce apoptosis in concentration‑dependent manner with IC50 value being 17.66 µM. Apoptotic induction was mediated through elevated activation of caspase‑3 and caspase‑9. Cell cycle analysis revealed that the compound (1) effectively increased the sub‑G0‑G1 population in the treated U937 cells (73.25% at 50 µM) in comparison to control set (3.12%). DAPI nuclear staining indicated that compound (1) increased the number of deformed nuclei and an increased level of apoptotic body formation in the treated cells.