Phytochemical studies on Lusitanian heath species and their effects on angiogenesis and melanogenesis

Abstract

Overview: The genus Erica belongs to the Ericaceae family, which includes approximately 4250 species and 124 genera. In Ireland, there are 14 species and 7 genera. Of these, the Lusitanian heaths, Erica erigena, Erica mackayana, Daboecia cantabrica and Erica � stuartii, represent an underexplored groups of plants in terms of their phytochemical composition and biological properties.

Aims of the work: The overall aim of the work described in this thesis was to carry out a phytochemical analysis of the triterpenoid constituents in the Lusitanian heaths and secondly to determine their potential in in vitro and ex vivo angiogenesis and anticancer assays. An additional aim was to profile the flavonoid glycoside constituents in the Lusitanian heaths and determine their effects on melanogenesis using a melanoma cells line (FM55) and cells from a donor patient (NHEM).

Materials and methods: The plants used in the study were collected from different areas in counties Mayo and Galway. The identity of each plant was confirmed with specimens deposited in the Trinity herbarium. Phytochemical analysis on the triterpenoid constituents in the Lusitanian heaths was conducted using GCMS with N,O-Bis (trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% of trimethylchlorosilane (TCMS) used as the derivatizing reagent. HPTLC, HPLC and NMR analysis was carried out on the phenolic rich fractions. Human umbilical vein endothelial cells (HUVEC) and prostate cancer cells (PC-3) were used to test compounds/quantified extracts in cellular viability, migration and invasion assays. The ex vivo aortic ring assay was used to evaluate the antiangiogenic and vasculature disruption effects of test compounds/extracts. The anti- melanogenic activity of the flavonoid rich extracts on cellular melanogenesis was conducted using both the melanoma cell line, FM55 and NHEM cells from a donor patient.

Results and discussion: In E. erigena the triterpenoid composition varied by season, geographical location and plant part used, with the leaves collected in January exhibiting the highest overall content (59,204.62 ± 1021.03 μg/g). Lupeol levels in the leaves significantly decreased throughout the year dropping from 14,590.47 ± 267.64 μg/g in January to 5,048.03 ± 229.32 μg/g in September. Notably, the presence of micromeric acid was confirmed by NMR and GC-MS for the first time in Erica species. The analysis of the coumaroyl triterpenes in E. erigena posed challenges as these compounds rapidly isomerized between their cis and trans forms and co-eluted from the flash column with ursolic aldehyde and other minor constituents. Separation of the coumaroyl triterpenes from the other co-eluting triterpenes was achieved through a methylation reaction, followed by their isolation using flash column chromatography. Their identity as conjugates of lupeol, α-amyrin, and β-amyrin was confirmed by NMR spectroscopy and synthesis of each individual isomer. An important step in their synthesis involved using UV light at 366 nm to convert the trans coumaroyl triterpenes into their cis counterparts. Scanning electron microscopy was used to distinguish botanically E. mackayana, E. tetralix, and E. � stuartii from each other. Phytochemical differences between all three plants were determined by GC-MS with notably lower levels of triterpenoids in E. mackayana. The highest triterpenoid content was found in the leaves of E. tetralix, 38,229.83 ± 367.16 μg/g, while the lowest level was recorded in the flowers of E. mackayana, 24,936.54 ± 565.59 μg/g. Samples of D. cantabrica were collected from nine locations across counties Mayo and Galway. Analysis of D. cantabrica collections and cultivated D. cantabrica alba by GC-MS revealed distinct differences in their triterpenoid profiles depending on their area of collection. A notable difference between D. cantabrica and the other Lusitanian heaths studied was the absence of micromeric acid in D. cantabrica. In the cell-based assays, using HUVEC and PC-3 cells, the quantified fractionated extracts rich in lupeol, uvaol/erythrodiol, and ursolic acid were particularly effective as inhibitors of cellular proliferation, migration, and invasion of these cells, as well as in the ex vivo aortic ring assay that determined their effects on angiogenesis and as vasculature-disrupting agents. In melanogenesis assays, the most promising extract identified was the EEF extract which reduced melanin content in FM55 cells. Among the eight extracts tested, EEF and the compounds EEL-MR, EEL-MG, EEL-QR, and DCF-MR- 5-ME demonstrated significant depigmentation of NHEM cells compared to the control group.

Conclusion: The Lusitanian heaths represent an interesting subclass of plants with their secondary metabolite constituents demonstrating potential as angiogenesis and melanogenesis inhibitors. Further studies are required at the phytochemical level to fully characterize the minor constituents present in the Lusitanian heaths. This will necessitate their isolation with follow-on studies required to assess their true potential in angiogenesis and melanogenesis assays. While the pentacyclic triterpenes were evaluated against the viability of PC-3 cells, a more extensive panel of cancer cell lines should be used to determine the true potential of these constituents in the Lusitanian heaths against a wider panel of tumour cell lines.