Compound Identification and In Vitro Cytotoxicity of the Supercritical Carbon Dioxide Extract of Papaya Freeze-Dried Leaf Juice

Abstract

Carica papaya leaves are used as a remedy for the management of cancer. Freeze-dried C. papaya leaf juice was extracted using a supercritical fluid extraction system. Compound identification was carried out using analytical techniques including liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry (LC–QToF-MS) and gas chromatography–mass spectrometry (GC–MS). The cytotoxic activities of the scCO2 extract and its chemical constituents were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on squamous cell carcinoma (SCC25) and human keratinocyte (HaCaT) cell lines. The chemical constituents were quantified by QToF-MS. The supercritical carbon dioxide (scCO2) extract of papaya freeze-dried leaf juice showed cytotoxic activity against SCC25. Three phytosterols, namely, β-sitosterol, campesterol, and stigmasterol, together with α-tocopherol, were confirmed to be present in the scCO2 extract. Quantitative analysis showed that β-sitosterol was the major phytosterol present followed by α-tocopherol, campesterol, and stigmasterol. β-Sitosterol and campesterol were active against SCC25 (half maximal inhibitory concentration (IC50) ≈ 1 µM), while stigmasterol was less active (~33 µM) but was biologically more selective against SCC25. Interestingly, an equimolar mixture of phytosterols was not more effective (no synergistic effect was observed) but was more selective than the individual compounds. The compounds identified are likely accountable for at least part of the cytotoxicity and selectivity effects of C. papaya.