Rapid detection of bioactive compounds plays an important role in the phytochemical investigation of natural plant extracts.The hyphenated techniques, which couple on-line chromatographic separation and biochemical detection, are called high-resolution screening methods. In this system, high-performance liquid chromatography separates complex mixtures, and a post-column biochemical assay determines the activity of the individual compounds present in the mixtures. At the same time, parallel chemical detection techniques (e.g., diode-array detection, mass spectrometry, and nuclear magnetic resonance) identify and quantify the active compounds. In recent years, bioassays for radical scavenging (antioxidant) activity and immunoassays for antibodies have particularly been developed and applied. Assays for enzymes and receptors are limited. In the literature section of this thesis the development of on-line, post-column biochemical detection systems for the screening of bioactive compounds from complex mixtures was investigated. The interaction of drugs with proteins has gained significant importance in various areas of analytical chemistry. It can also be expected that more drugs will certainly be discovered with the development of biotechnology in the future.
In the experimental section of this thesis comprehensive two-dimensional gas chromatography- time-of-flight mass spectrometry was used for screening for chemical composition of birch bark (Betula pendula). The exploitation of the mass spectra and retention index information allowed the identification of more than 600 organic compounds. Altogether, 59 phenolic compounds were identified in the inner layer of birch bark. To the best of our knowledge some of these compounds (e.g., raspberry ketone and tyrosol) have not been reported as extractives of Betula species before. The results achieved by gas chromatography mass spectrometry showed that several phenols with biological activity were present at relatively high concentrations in the sample. It was noticed that content of the compounds were dependent on the solvents with different polarities and volatilities. Phenols were extracted from birch bark using an environmental friendly pressurized hot water extraction technique. It provided good extraction efficiencies for phenolic compounds compared to those achieved with Soxhlet extraction. With pressurized hot water extraction the amount of extractable phenolic compounds approached for up to 23% of the dry weight whereas the amount was 2-5% (w/w) by Soxhlet extraction. Typical extraction time varied from 20 to 40 minutes. Most of the phenolic compounds were extracted at 180 °C for 40 minutes. Increase in the extraction temperature from 150 to 180°C resulted in an increase in the number of phenols extracted. However, enhanced temperature can accelerate hydrolysis and oxidation, so that unwanted or thermo-labile compounds can compose. Pressurized hot water extraction using water as a solvent proved to be a very promising extraction technique, and it surely has a great potential for the extraction of phenols from birch bark in the future.
