Screening of Irish Fruit and Vegetable Germplasm for Novel Anti-Tumour and Pesticidal Compounds.
Document Type Theses, Ph.D
Thesis submitted to the Dublin Institute of Technology in fulfilment of requirements for the Degree of Doctor of Philosophy, July 2013.
Abstract
Phytochemicals are a source of potential novel therapeutic and insecticidal agents. This project is focused on screening Irish grown fruit and vegetables for novel bioactive compounds that may have applications, for use in anti-tumour and biopesticides applications. The cytotoxicity using aqueous extracts of 173 plant samples has been screened using the brine shrimp bioassay (BSB). Six crops showed positive lethality to brine shrimp from the aqueous extracts (LC50<1/50 (v/v)), beetroot (root), cranberry (fruit), garlic (bulb), ginger (rhizome), red onion (bulb) and rhubarb (petiole). These samples also had significant inhibition of crown gall tumours in the potato disc bioassay (PDB) (>20% inhibition). Methanolic extracts of cranberry (fruit), ginger (rhizome) and rhubarb (petiole) had positive results in the BSB LC50<1000>μg/ml. Methanolic extracts of cranberry (fruit) and rhubarb (petiole) had significant inhibition of crown gall tumours in the PDB (>20%). The polyphenolic compounds in aqueous extracts of the six positive crop extracts were separated and analysed using thin layer chromatography (TLC) plates sprayed with Folin-Denis’ reagent. Three crops, beetroot Beta vulgaris, red onion Allium cepa and rhubarb Rheum rhabarbarum, which can be grown in Ireland, were then chosen for further investigation. Different tissues from beetroot (‘Pablo’ and ‘Rocket’) and red onion (‘Red Spark’, ‘Red Baron’ and ‘Lilia’) had negative results in the BSB for both aqueous (LC50>1/50 (v/v)) and methanolic extracts (LC50>1000 μg/ml). Petiole and leaf tissues of rhubarb varieties (‘Red Early Superb’, ‘Sutton’ and ‘Victoria’) had positive results in the BSB for both aqueous (LC50<1/50 (v/v)) and methanolic extracts (LC5020%) apart from ‘Sutton’ (petiole) which gave -11.5% inhibition. All extracts of rhubarb varieties had antibacterial activity against Agrobacterium tumefaciens, and measured by minimum inhibition concentration (MIC). Petiole methanolic extracts of all rhubarb varieties inhibited the spore germination of Penicillium digitatum spores for 72 hours. All rhubarb varieties had high radical scavenging capacity (DPPH) and total phenolic content (TPC). TLC was carried out for all rhubarb varieties for both aqueous and methanolic extracts and the polyphenolic compounds were detected using Folin-Denis’ spray reagent. Three fractions out of eleven fractions from petiole and leaf tissues of ‘Victoria’ variety had positive lethality to BSB (LC50>1000 μg/ml). The polyphenolic compounds were identified in the three positive fractions using high performance liquid chromatography (HPLC-DAD) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The three positive fractions were purified and sub-fractioned to 23 sub-fractions using preparative high performance liquid chromatography (PREP-HPLC). Two polyphenolic aglycone compounds had positive cytotoxicity to BSB were purified and characterised using LC-ESI-MS/MS.