*3.3.1. Cellular Antioxidant Activity (CAA) assay*

A novel method of determination of antioxidant activity was proposed in 2007 by Wolfe and Liu of Cornell University [49]. They devised a method which is based on reactions running inside cells. According to them, the method better reflects reality than in-vitro methods due to the intake, metabolism and distribution of antioxidants in cells and, consequently, in a live organism. In a CAA assay, a solution of DCFH-DA (2',7'-Dichlorofluorescein diacetate) and a solution of the substances under analysis in PBS (phosphate buffered saline) at pH 7.4 is added to human hepatocarcinoma (HepG2) cells. Cells are incubated at 37o C and, during the incubation, DCFH-DA and components of the sample diffuse through the cell membrane into the cell. After that, the unabsorbed remainder is washed out with PBS and a solution of AAPH is added which – after its infiltration into a cell – generates free radicals oxidising DCFH-DA to DCF. Antioxidants quench the radicals, which reduces the amount of DCF, whose fluores‐ cence is measured at 485 nm (ex.) and 520 nm (em.) during the analysis. The area under the fluorescence curve can be compared to the calibration curve prepared for a standard antioxi‐ dant, e.g. Trolox, and expressed as its equivalent. There are also modifications of the method, in which blood erythrocytes are used instead of HepG2 cells.

### *3.3.2. Total Oxyradical Scavenging Capacity (TOSCA) assay*

Total Oxyradical Scavenging Capacity assay was proposed by Winston and co-workers as a rapid gas chromatographic method. They used this assay as a method of quantifiable meas‐ urement of the ability of sample antioxidants to quench free radicals [3]. The assay is based on the reaction between free radicals (peroxyl, hydroxyl, alkoxyl) and α-keto-γ-methiolbutyric acid (KMBA). The reaction yields ethylene, which can be simply analysed by gas chromatog‐ raphy. The assay involves incubation of solutions of AAPH, KMBA and the analysed sample at 39o C, with resulting ethylene production. Its content is determined every 12 minutes for the 96–120 minute period of the assay. The values obtained in the measurement form the basis for plotting the curve illustrating the changes in ethylene content. Quantitative determination of TOSC is possible only by comparison of the area under the curve for the analysed sample (∫SA) and the control sample (∫CA). The value of TOSC was calculated by Winston and co-workers as the difference between 100 and the ratio of the area for the analysed sample and the control sample, multiplied by 100.

$$TOSC = 100 - 100 \text{ \* } \left\{ \int\_{SA} \Big/ \int\_{CA} \right\}$$

acid. In the method proposed by Miller [44], a decrease in the absorbance of aqueous emulsion of "β-carotene – linoleic acid – analysed sample" depends on the antioxidant activity of the sample components. The antioxidant under study reacts with the radicals generated by linoleic acid in an incubated sample. As the ability of the analyte to scavenge radicals decreases, the oxidative effect on β-carotene increases. Measurement results of absorbance at 470 nm are read out every 15 minutes until the plateau is reached. The oxidative strength of the analyte is

Crocin is one of carotenoids present in saffron. It is present there as several isomers, differing by biological activity. The method of determination of antioxidant activity using crocin as an indicator was proposed in 1984 by Bors and co-workers [45]. In order to determine the antioxidant activity of the components of an analysed sample, it is put into a reaction tube together with solution of crocin diluted with phosphate buffer at pH 7.4. Thus obtained, the mixture is treated with radicals generated by solution of AAPH. The reaction runs at the

at the wavelength of 443 nm and recorded for 10 minutes relative to the blank sample. The method has been modified many times [46, 47, 48]. Considering the problem of the unrepeat‐ ability of the composition of the saffron dye extract and, consequently, the differences in biological activity of the mixture of crocin isomers, Bathaie and co-workers [48] used α-crocin in their modification of the method. The results are expressed as "percent of inhibition of crocin degradation" (% Inh) and refer to the calibration curve prepared with Trolox and expressed

A novel method of determination of antioxidant activity was proposed in 2007 by Wolfe and Liu of Cornell University [49]. They devised a method which is based on reactions running inside cells. According to them, the method better reflects reality than in-vitro methods due to the intake, metabolism and distribution of antioxidants in cells and, consequently, in a live organism. In a CAA assay, a solution of DCFH-DA (2',7'-Dichlorofluorescein diacetate) and a solution of the substances under analysis in PBS (phosphate buffered saline) at pH 7.4 is added

incubation, DCFH-DA and components of the sample diffuse through the cell membrane into the cell. After that, the unabsorbed remainder is washed out with PBS and a solution of AAPH is added which – after its infiltration into a cell – generates free radicals oxidising DCFH-DA to DCF. Antioxidants quench the radicals, which reduces the amount of DCF, whose fluores‐ cence is measured at 485 nm (ex.) and 520 nm (em.) during the analysis. The area under the fluorescence curve can be compared to the calibration curve prepared for a standard antioxi‐ dant, e.g. Trolox, and expressed as its equivalent. There are also modifications of the method,

to human hepatocarcinoma (HepG2) cells. Cells are incubated at 37o

in which blood erythrocytes are used instead of HepG2 cells.

C. Decrease in the absorbance of the solution is measured colorimetrically

C and, during the

presented as the amount of β-carotene which was protected against oxidation.

*3.2.4. Crocin Bleaching Assay (CBA)*

temperature of 40o

108 Column Chromatography

as its equivalent (% InhTrolox).

*3.3.1. Cellular Antioxidant Activity (CAA) assay*

**3.3. Other methods**

When the radical inhibition reaches its theoretical maximum, ethylene is not produced and the value of TOSC is equal to 100 [3].
