# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 6 Bioassay
### Microbiochemical Detection  
Thin-layer chromatography can also be a good tool for microbiological detection. As in microbiological detection, the first step involves separating the components of the mixture by thin-layer chromatography (TLC or HPTLC), followed by the determination of the effect of individual substances on the biochemical reactions under investigation, which is usually achieved by a direct bioassay. Another reagent used to determine the antioxidant properties of substances is 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid (ABTS). It has the ability to react with potassium persulphate. Antioxidants inhibit this reaction and are visible as colourless or pink spots on a green background. The antioxidant properties of a substance can also be tested with \(\upbeta\)-carotene using two methods. In the first method, a chloroform solution of \(\upbeta\)-carotene is used. As a result of the photochemical reaction of the antioxidant substance with \(\upbeta\)-carotene under visible light, orange-coloured spots on a white or light-yellow background become visible on the chromatographic plate. A modification of this method involves the addition of a  
\begin{table}
\begin{tabular}{l l l} \hline \hline
**Microorganism** & **Derivatization** & **Ref. ** \\ \hline \multicolumn{3}{c}{Bacteria} \\ \hline _Aeromonas hydrophulia_ & MTT & [127] \\ \hline _Aliivibrio fischeri_ & Luminescence & [128; 129; 130; 124; 131; 132; 133; 134; 135] \\ \hline _Bacillus subtilis_ & MTT, INT & [131; 132; 133; 134; 135] \\ \hline _Clostridium perfringens_ & MTT & [136] \\ \hline Drug-resistant _Staphylococcus aureus_ & Natural & [137] \\ \hline _Enterococcus faecalis_, & MTT & [127] \\ \hline _Escherichia coli_ & MTT & [138; 139; 140] \\ \hline _Listeria monocytogenes_ & MTT & [127] \\ \hline _Pseudomonas syringae_ pv. _maculicola_ & Luminescence & [141; 142] \\ \hline Luminescence genetagged & Luminescence & [128; 124] \\ _Pseudomonas syringae_ pv. _maculicola_ & & [128; 124] \\ \hline methicillin resistant _S. aureus_ & MTT & [128] \\ \hline _Micrococcus leteus_ & MTT & [128; 143] \\ \hline _Morganella morganii_ & Natural & [144] \\ \hline _Mycobacterium tuberculosis_ & Luminescence & [145] \\ \hline _Pseudomonas savastanoi_ pv. _maculicola_ & MTT & [146; 147; 148; 149; 150; 151; 152; 153; 154; 155; 156; 157; 158; 159; 160; 161; 162; 163; 164; 165; 166; 167; 168; 169; 170; 171; 172; 173; 174;  
linoleic acid solution to the \(\beta\)-carotene solution. Substances with antioxidant properties become visible on the chromatographic plate in the form of orange-coloured spots on a white or light-yellow background. As a result of this modification, substances that stabilise lipid oxidation can also be seen on the plate in addition to the substances with antioxidant properties. The type of stationary phase influences the determination of the antioxidant properties of a substance. Non-polar stationary phases, such as C-18, have no influence on the reaction between radicals and antioxidants. If the investigation of antioxidant properties is carried out on a silica gel layer and the plate is sprayed with a methanolic DPPH solution, the results of the antioxidant activity of substances may be overestimated. Detection of Enzyme Inhibitors by Bioassay  
Enzyme reactions are very important in every living organism. They should be specifically catalysed by enzymes that provide optimal amounts of reaction products. Too many products of enzymatic reactions in the body lead to various diseases. In such cases, inhibition of reactions catalysed by a specific enzyme eliminates the causes of disease. The detection of enzyme inhibitors (i.e., the determination of which component of the analysed samples has inhibitory properties against a specific enzyme) can be performed directly on the chromatographic plate on which the components of the test mixtures are separated. The chromatographic plate is sprayed with a solution of the enzyme in question and the substance whose reactions the enzyme catalyses.