Discovery of a Novel Co-crystal of Chrysin and Oroxylin A with Anticancer Properties from Leaves of Oroxylum indicum

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.