The leaf crude extract of Oroxylum indicum (L.) Kurz induces genomic DNA fragmentation, comet formation, and the inhibition of cell proliferation in the prostate cancer cell line PC3, as assessed by agarose gel electrophoresis, comet assay and MTT assay, respectively. The bioactive compound was purified through bioassay-guided fractionation using preparative HPLC and MTT assay. The light brown and water-soluble compound was characterized using 1H and 13C nuclear magnetic resonance (NMR), Fourier transform infrared (FT-IR), and electrospray ionization (ESI) mass spectrometry. The compound was identified as a glycosylated hydroquinone derivative, 2-[p-(2-Carboxyhydrazino)phenoxy]-6-(hydroxymethyl) tetrahy-dro-2H-pyran-3,4,5-triol (molecular formula, C13H18N2O8; molecular mass = 330). The identified phytocompound has not been reported earlier elsewhere. Therefore, the common name of the novel anticancer phytocompound isolated from Oroxylum indicum in this current study is oroxyquinone. The half-maximal inhibitory concentration (IC₅₀) of oroxyquinone on PC3 cells was 58.9 µM (95% CI = 54.5 to 63.7 µM). Treatment of PC3 cells with oroxyquinone induced genomic DNA fragmentation and chromatin condensation, increased in the annexin-V positive cells, arrested the cell cycle at S phases, and inhibited the cell migration; as assessed by comet assay, DAPI staining, flow cytometry and a wound healing assay, respectively. On the investigation of the molecular mechanism of the induction of apoptosis, the results indicated that oroxyquinone induced caspase-3 and PARP independent apoptosis but through the p38 pathway and the localization of AIF into the nucleus. The present study identifies a novel anticancer molecule and provides scientific evidence supporting the therapeutic potency of Oroxylum indicum for ethnomedicinal uses.

Background: As the number of new cancer cases increases every year, there is a necessity to develop new drugs for the treatment of different types of cancers. Plants' resources are considered to be huge reservoirs for therapeutic agents in nature. Among all the medicinal plants, Oroxylum indicum is one of the most widely used medicinal plants in India, China, and Southeast Asian countries. Combinatorial drug treatment, on the other hand, is favored over single drug treatment in order to target multiple biomolecular moieties that help in the growth and development of cancer. Therefore, combinatorial drug treatment using a co-crystal of multiple drugs gives researchers an idea of the development of a new type of drug for targeting multiple targets. In this study, a new co-crystal of chrysin and oroxylin A was isolated from the leaves of O. indicum, and its anticancer properties were studied in cervical cancer cells HeLa. Aim: This study was conducted with the aim of identifying new anticancer compounds from the leaves of Oroxylum indicum and studying the anticancer properties of the isolated compound. Objective: In this study, we elucidated the structure of a new co-crystal compound, which was isolated from the leaf extract of Oroxylum indicum. The apoptosis induction mechanism of the newly discovered co-crystal in HeLa cells was also studied. Methods: A crystal compound from the chloroform extract of leaves of Oroxylum Indicum was isolated by solvent fractionation and chromatographic methods involving HPLC. The molecular structure of the isolated crystal was elucidated by Single Crystal-XRD, FT-IR analysis, and further determined by LC-MS. The antiproliferative activity was carried out using an MTT assay and fluorescence microscopy, and the mechanism of apoptosis was determined using Western blotting techniques. Results: The novel co-crystal consists of two active pharmaceutical ingredients (APIs) in a 1:1 ratio, i.e., oroxylin A and chrysin. The isolated new co-crystal induced death in HeLa cells with a very low IC50 value of 8.49 μM. It induced caspase-dependent apoptosis in HeLa cells by activation of Caspase-3 through inhibition of ERKs and activation of p38 of MAPK cell signalling pathway. Conclusion: This study presents the first report on the discovery of a naturally occurring co-crystal of chrysin and oroxylin A and the involvement of ERKs and p38 of MAPK pathways in the induction of apoptosis in HeLa cells by the co-crystal. Our study sheds light on the development of a co-crystal of chrysin and oroxylin A in a specific ratio of 1:1 for combination therapy of the two APIs. The purified co-crystal was found to be more efficient compared to the compounds present individually. Further analysis of the physiochemical properties and molecular mechanisms of the isolated co-crystal in different cancer cells is warranted for its application in therapeutics.