Carica papaya is widely cultivated in tropical and subtropical countries and is used as a traditional medicine to treat a range of diseases as well as a foodstuff. Increasing anecdotal reports of the use of papaya leaf in successful cancer treatment and prevention have warranted the scientific authentication of the pharmacological properties of papaya leaf. An extensive review that was conducted at the beginning of this study further confirmed that more research is needed to explore the bioactive compounds in C. papaya for their anticancer activities.
Fresh C. papaya leaves that were not sprayed with any chemicals were collected from Tropical Fruit World, a plantation orchard farm and research park in northern New South Wales, Australia. Chapter 2 of this thesis focuses on an investigation of the in vitro cytotoxicity on human oral squamous cell carcinoma SCC25 cells and non-cancerous human keratinocyte HaCaT cells, of eight different papaya leaf extracts, which were obtained by two extraction schemes – serial and parallel with a medium polar solvent (ethanol) and a polar solvent (water) at acidic and basic pH values. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method utilised for cytotoxicity assays has been thoroughly optimised for this study. The serial extraction scheme showed better discrimination of cytotoxic effects in comparison to the parallel scheme. Two acidic extracts, of the four serial extractions, demonstrated significantly selective cytotoxicity towards the cancer cells relative to non-cancerous cells. Total phenolic and flavonoid contents were measured and found to be elevated in these two acidic serial extracts.
In Chapter 3, extractions of papaya leaf were carried out using traditional methods: leaf decoction/tea (Australian Aboriginal remedy for cancer treatment) prepared by boiling and concentrating papaya leaves in water, and papaya leaf juice prepared by mechanical crushing followed by squeezing of the juice from fresh leaves. In comparison with the leaf decoction, the leaf juice not only exhibited a more potent cytotoxic effect on SCC25 cancer cells, but also produced much greater selective effects on cancer cells as shown by tests on HaCaT cells. In this chapter, leaf juice and leaf decoction were also tested on colon cancer cells and the viability of neither H29 cells nor HCT116 cells was decreased by papaya leaf extracts; this indicates that the effect of papaya extracts on the survival of cancer cells is likely to be cancer-type specific.
The chemical compositions of the extracts were then explored using Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by chemometric analysis. Comparative analysis in Chapter 2 and multivariate data analysis in Chapter 3 were used to analyse the metabolomic profiles of papaya leaf extracts to identify tentatively the compounds responsible for the significantly selective anti-cancer effects towards the cancer cells. A customised personal compound database library consisting of 1,574 entries of plant-derived compounds with reported anticancer activities was assembled and curated to facilitate the putative identification of compounds. Comparative analysis of LC-MS profiles of serial extracts in Chapter 2 revealed that the principal compounds identified were flavonoids or flavonoid glycosides particularly compounds from the kaempferol and quercetin families, of which several have previously been reported to possess anticancer activities. Multivariate data analysis of chemical profiles of leaf juice and leaf decoction in Chapter 3 revealed 90 and 104 peaks in positive and negative ionisation mode respectively as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound principally contributing to the selective activity of leaf juice.
Subsequently, the chemical composition of papaya leaf juice, which has the greatest selectivity in cytotoxic effects, was further explored via bioassay-guided fractionation using preparative chromatography. Thirteen fractions were isolated using preparative reversed phase HPLC and tested on SCC25 and HaCaT cells. The results suggested the possibility of multi-component additive effects among the fractions with different polarities and C. papaya probably belongs to the category of plants with a complement of active compounds. In conclusion, the results from this thesis provide further scientific evidence of the anticancer activities of C. papaya leaves. To the best of our knowledge, this is the first study to report the selective cytotoxic effect of papaya leaf juice on squamous carcinoma cells and the effects imparted by the long heating process of the Australian Aboriginal remedy preparation. Other key contributions of this study are the investigation of bioactive compounds in C. papaya leaf extracts by LC-MS based-metabolomics and detail bioactivity-guided fractionation of papaya leaf juice with the highlight of potential additive interactions among its bioactive components.