**3. In vitro analysis of biological activity of substances.**

In search of bioactive substances, researchers have directed their interest towards substances found in plants. Parts of plants which have been used in natural medicine have proved to be a rich source of bioactive compounds; however, to make use of them, they have to be isolated and their properties determined. Using selective techniques of extraction has resulted in obtaining concentrated preparations of bioactive substances. To achieve comprehensive knowledge of their properties, it was necessary to develop methods of isolation of individual components and testing these methods. This could be done with chromatographic techniques. Isolated compounds were tested in order to show which of them (and to what extent) are responsible for bioactivity of plant preparations from which they were obtained. Due to the fact that many of the substances have the opposite effect, it is frequently impossible to use extracts without isolating individual compounds.

*In vitro* tests, used in evaluation of antioxidant properties make use of the ability of antioxidants to quench free radicals. Based on this mechanism, the methods are divided into two groups: SET – single electron transfer, and HAT – hydrogen atom transfer. Reactions with antioxidants in assays with the DPPH radical, ABTS and the Folin-Ciocalteu reagent both operate according to the SET and HAT mechanism. Due to the kinetics of the reaction, they are included in the group of SET assays. The HAT mechanism is of lesser importance in those assays [2]. SET assays include: DPPH, TEAC, FRAP, CUPRAC, DMPD, Folin-Ciocalteu; HAT assays include: ORAC, TRAP, CBA, β-carotene – linoleic model system. Those classified as "other" in literature include: cellular antioxidant activity (CAA), chemiluminescence, electrochemiluminescence, Total Oxyradical Scavenging Capacity Assay (TOSCA) and others [3].
