Screening, isolation and evaluation of antioxidative compounds from Geranium macrorrhizum, Potentilla fruticosa and Rhaponticum carthamoides

Food molecules (lipids, proteins, carbohydrates) can be widely involved in oxidation reactions. These reactions, caused by so called reactive oxygen species (ROS) are a major cause of food deterioration. In the case of lipid containing foods this process is defined as rancidity. Significant changes can occur in product colour, texture and nutritive value. Eventually the oxidation can result in complete spoilage. Oxidation reactions caused by ROS also play an important role in the ageing processes of living organisms and are associated with numerous diseases, like coronary heart disease, cancer, cataracts, ageing. Although protective mechanisms exist both in living cells and in foods, in many cases there is a need to strengthen this mechanism. A daily intake of antioxidants can prevent or delay problems caused by ROS.The research described in this study is aimed at the evaluation of several plant extracts as a possible source of food antioxidants.An initial screening for radical scavenging activity of extracts from medicinal and aromatic plants namely Salvia sclarea , Salvia glutinosa , Salvia pratensis , Salvia officinalis, Lavandula angustifolia , Calendula officinalis , Matricaria recutita , Echinacea purpurea , Rhaponticum carthamoides , Juglans regia , Melilotus officinalis , Geranium macrorrhizum and Potentilla fruticosa has been performed using DPPH and ABTS radical scavenging assays. Of these extracts Geranium macrorrhizum and Potentilla fruticosa possessed the highest radical scavenging activity in both assays, higher than that of the Salvia officinalis extract.In further steps of this study the structures of the radical scavenging compounds present in leaf extracts ofGeranium macrorrhizum, in blossoms extracts ofPotentilla fruticosaand leaf extracts of Rhaponticum carthamoides were elucidated.The radical scavenging activity of the isolated compounds from Geranium macrorrhizum and Potentilla fruticosa was measured using DPPH^·and ABTS^·+scavenging assays and compared with the activity of rosmarinic acid and Trolox.A quantitative comparison of the major antioxidant compounds in different botanical parts of Geranium macrorrhizum and Potentilla fruticosa was also made.A hyphenated LC-DAD-SPE-NMR set-up in combination with on-line radical scavenging detection was used for the identification of radical scavenging compounds from Rhaponticum carthamoidesextracts. The technique enabled selective detection and identification of individual radical scavenging compounds without any prior off-line chromatographic steps. One of identified a new natural compound. Its radical scavenging activity was tested against DPPH radical and was found to be weaker than that of the reference antioxidants rosmarinic acid and Trolox.Antioxidant activity of all plant fractions from Geranium macrorrhizum and Potentilla fruticosa was assessed by model system assays and in real food systems consisting of edible oil or fermented sausages.A preliminary safety evaluation of some plant extracts has been conducted (enzymatic oxidation assay;effects against singlet oxygen caused erythrocyte hemolysis;mutagenicity evaluation) andindicated the plant extracts not to be genotoxic or mutagenic.Research on natural products, like plant antioxidants, demands the application of various analytical techniques. Apart from state-of-the-art analytical instruments, a lot of specific techniques needed to be applied at certain stages, e.g. on-line radical scavenging detection of individual plant constituents in complex plant mixtures. These DPPH/ABTS assays coupled on-line to liquid chromatography-diode array detection set-ups have been developed earlier at the Laboratory of Organic Chemistry of Wageningen University. Expansion of these assays by coupling a mass spectrometer to the system was one part of the methodological research.The developed system enabled a quick analysis (simultaneous separation, isolation-identification and detection of radical scavenging activity) of complex Ginkgo and rosemary extracts.The hyphenated LC-DAD-SPE-NMR system is another powerful technique for compound isolation and structure elucidation in complex mixtures. The investigation of the application of LC-DAD-SPE-NMR with a semi-preparative HPLC column and comparison of the obtained results with those of an analytical column was described in the thesis.It is concluded that extracts from Geranium and Potentilla are unlikely to be a new source of antioxidants for lipophilic food products. Extracts proved to be effective in assays with polarmedia,however their application in such products is also unlikely. Reasons are problems, like odour and colour of the extracts, and the complex extraction procedure.