New methods for the screening of antioxidants in three Sideritis species

This thesis describes the rapid and robust evaluation of antioxidant activity of complex mixtures such as plant extracts. The study was directed to establish the best screening and isolation procedures for components with antioxidative properties to ensure the development of an “algorithm” for studying natural antioxidants. Three different screening methods (the b-carotene bleaching test (BCBT), the headspace GC method (HS-GC) and the off-line radical scavenging assay with the DPPH radical (DPPH method) and various extraction schemes were used. Three plants from the genus Sideritis grown in Bulgaria (S. scardica, S. syriaca and S. montana) served as test species. The strengths and limitations of each method were illustrated by testing a number of extracts of different polarity. A novel HPLC-DPPH method to rapidly and sensitively pinpoint individual antioxidants in complex mixtures with as little as possible fractionation procedures was developed. The use of the alternative ABTS·+ radical cation made the method in most cases even more sensitive. The instrumental set-up and physico-chemical parameters of both methods were studied. The methods were successful for qualitative and semi-quantitative measurements of pure antioxidants and extracts. A review is under preparation on the use of on-line methods. As a result, a range of flavonoid-, phenylpropanoid- and iridoid glycosides were isolated. The radical scavenging activity of the isolated compounds by off- and on-line DPPH methods was studied. Three novel compounds were isolated: flavonoid glycosides luteolin-4'-methylether-7-O-[6'''-O-acetyl-β-D-allopyranosyl-(1-2)-β-D-glucopyranoside and hypolaetin-4'-methylether-7-O-[β-D-allopyranosyl-(1-2)-6''-O-acetyl-β-D-glucopyranoside, and iridoid glycoside 3''-O-p-coumaroyl-6''-O-acetyl melittoside from Sideritis species. Preliminary studies by the HS-GC method were applied to investigate how Sideritis extracts and pure components would behave in real systems: bulk oils and oil-in-water emulsions. Conjugated dienes and hexanal formation were monitored using rosmarinic acid and BHT as standards. They showed good inhibitory effects with regard to dienes and hexanal formation in both test systems.