Supercritical CO2 fluid extraction of croton crassifolius Geisel root: Chemical composition and anti-proliferative, autophagic, apoptosis-inducing, and related molecular effects on A549 tumour cells.

The use of plant essential oils as pharmaceuticals is a fast-growing market especially in China. Throughout the 20th century, a rapid increase took place in the use of many essential oil-derived products in the medicinal industry as nutraceuticals, medicinal supplements, and pharmaceuticals.The objective of this study was to explore the chemical composition of Croton crassifolius essential oil as well as its potential anti-tumour properties and related anti-proliferative, autophagic, and apoptosis-inducing effects.

Supercritical CO₂ fluid extraction technology was used to extract CCEO and the chemical constituents of the essential oil were identified by comparing the retention indices and mass spectra data taken from the NIST library with those calculated based on the C7-C40 n-alkanes standard. The cytotoxic activity and anti-proliferative effects of CCEO were evaluated against five cancer cell lines and one normal human cell line via CCK-8 assays. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of CCEO treatments in controlling cancer cell proliferation was assessed by cell cycle analysis, clonal formation assays, RT-qPCR, and western blot analysis. Autophagic and apoptosis-inducing effects of oils and the associated molecular mechanisms were assessed by flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR, and western blot analysis.

Forty compounds representing 92.90% of the total oil were identified in CCEO. The results showed that CCEO exerted a measurable selectivity for cancer cell lines, especially for A549 with the lowest IC₅₀ value of 25.00 ± 1.62 μg/mL. Assessment of the anti-proliferative effects of CCEO on A549 cells showed that the oil inhibited cell proliferation and colony formation in a dose- and time-dependent manner. Investigation of the molecular mechanisms of cell cycle regulation confirmed that the oil arrested A549 cells in G2/M phase by decreasing the expression of cyclin B1-CDK1 and cyclin A-CDK1 and increasing the expression of cyclin-dependent kinase inhibitor (CKI) P21 at both the transcriptional and translational levels. Autophagy staining assays and western blot analysis revealed that CCEO promoted the formation of autophagic vacuoles in A549 cells and increased the expression of autophagy-related proteins beclin-1 and LC3-II in a dose-dependent manner. A series of apoptosis analyses indicated that CCEO induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that CCEO is a promising candidate for development into an anti-tumour drug of the future.