*3.2.2. Oxygen Radical Absorbance Capacity (ORAC) assay*

TheORACmethodwas firstproposedbyCaoandco-workers in1993.Like intheTRAPmethod, they used a fluorescent indicator. Determination of antioxidant activity by this method is based on measurement of decreasing fluorescence of the indicator caused by the radicals generated in the system. The reaction mixture in their proposal consisted of a fluorescent indicator βphycoerythrin (β-PE), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator and the analysed sample [42]. Attributing the low purity of β-phycoerythrin (approx. 30%) to the low reproducibility of fluorescence and the occurrence of different forms of phycoerythrin, Ou and co-workers [43] modified the method by replacing the indicator with fluorescein (3',6'-dihydroxyspiro[isobenzofuran-1[3H],9'[9H]-xanthen]-3-one).

The reaction of antioxidants in a sample with the radicals generated by AAPH and fluorescein is conducted in a phosphate buffer at pH 7.4 and at the temperature of 37o C. As the reaction progresses, antioxidants in the analysed sample react with the radicals. With an excess of radicals, the ability of antioxidants to reduce them becomes exhausted and radicals react with fluorescein, oxidising it to a non-fluorescing form. Observation of the fluorescence of the reaction mixture is conducted at the excitation wavelength of 485 nm and emission wavelength of 525 nm. Measurements are conducted every 60-90 seconds until the resulting curve reaches a plateau.

**Figure 3.** ORAC antioxidant activity determination of *Echium vulgare* defatted seeds methanolic extract expressed as net area under the curve (net AUC)

Surface area under the curve (AUC) was calculated by Ou and co-workers [43] from the following formula:

$$\text{ALIC} = 1 + f\_1 / f\_o + f\_2 / f\_o + f\_3 / f\_o + f\_4 / f\_o + \dots + f\_{34} / f\_o + f\_{35} / f\_o$$

where *fo* denotes fluorescence read out at the beginning of the assay and *fi* denoted the value of fluorescence read after the time i.

The area under curve for the sample (AUCsample), reduced by the area under curve plotted for the blank sample (AUCblanc) is referred to as "net AUC". Moreover, the net AUC is calculated for a series of dilutions of Trolox and a calibration curve is plotted, showing the relationship between net AUC and the concentration of Trolox. The results of the assay which refer the net AUC of the sample to the calibration curve are expressed as Trolox equivalent.

### *3.2.3. β-carotene bleaching test*

compounds on the assay results. One such method is to remove the solvent from the sample and to dissolve phenolic compounds in alcohol, which eliminates the compounds insoluble in

Performing the assay is reduced to putting an alcoholic solution of the analysed sample, Folin-Ciocalteu reagent and solution of sodium carbonate into a reaction tube, which brings the pH of the reaction environment to approx. 10. According to various literature reports, the reaction runs in the darkness for 10 to 120 minutes. After that time, the blue colour of the solution is observed colorimetrically at 725 nm – 760 nm [34, 35, 36, 37, 38]. The results are expressed

This method is based on the measurement of the fluorescence of a molecular sample. Canadian researchers proposed this method to determine total peroxyl radical-trapping antioxidant capability of plasma [39]. They used a water-soluble azo compound, such as AAPH [2,2'-azobis-(2-amidinopropane)]. They measured the induction time electrochemically by measuring the time in which the antioxidants contained in the analysed sample prevent the capture of oxygen atoms. Four years later, DeLange and Glazer [40] proposed R-phycoerythrin in an induction measurement as a fluorescence indicator. They observed the time in which antiox‐ idants in the sample protect R-phycoerythrin from oxidation and compared it with the time by which Trolox, added at a known amount, prevented a decrease in fluorescence. The reaction kinetics curve was monitored at the excitation wavelength of 495 and emission wavelength of 575, and antioxidant properties were expressed as Trolox equivalent. However, the method is time-consuming and rather complicated, which makes it susceptible to considerable errors, especially in inter-laboratory studies. Another modification of the method was developed by Valkonen and Kuusi [41], who used dichlorofluorescein diacetate (CDFH-DA) as an indicator of oxidation progress. DCFH-DA is hydrolysed in the presence of AAPH to highly fluorescent dichlorofluoresceine (DCF). An increase in fluorescence is a sign that the antioxidant activity

TheORACmethodwas firstproposedbyCaoandco-workers in1993.Like intheTRAPmethod, they used a fluorescent indicator. Determination of antioxidant activity by this method is based on measurement of decreasing fluorescence of the indicator caused by the radicals generated in the system. The reaction mixture in their proposal consisted of a fluorescent indicator βphycoerythrin (β-PE), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator and the analysed sample [42]. Attributing the low purity of β-phycoerythrin (approx. 30%) to the low reproducibility of fluorescence and the occurrence of different forms of phycoerythrin, Ou and co-workers [43] modified the method by replacing the indicator with

fluorescein (3',6'-dihydroxyspiro[isobenzofuran-1[3H],9'[9H]-xanthen]-3-one).

that environment or ones which become denatured.

106 Column Chromatography

**3.2. Hydrogen Atom Transfer (HAT) methods**

of the analysed sample is exhausted.

*3.2.2. Oxygen Radical Absorbance Capacity (ORAC) assay*

based on calibration curves prepared for catechol and gallic acid.

*3.2.1. Total Radical Trapping Antioxidant Parameter (TRAP) assay*

Determination of the antioxidant activity in the system comprising β-carotene and linoleic acid is based on competitive oxidation of β-carotene during heat-induced auto-oxidation of linoleic 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 presented as the amount of β-carotene which was protected against oxidation.

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

at 39o

sample, multiplied by 100.

*SA* / *∫ CA* )

the value of TOSC is equal to 100 [3].

reactions are presented below.

*TOSC* = 100 – 100 \* (*∫*

**compounds**

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

Chromatography in Bioactivity Analysis of Compounds

http://dx.doi.org/10.5772/55620

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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

When the radical inhibition reaches its theoretical maximum, ethylene is not produced and

The methods of determination of antioxidant activity are popular, which does not mean that they are only used in scientific research. A number of modifications of those methods along with methods which are not presented here are still used in analytical procedures applied in examination of bioactive substances. Owing to constantly broadening knowledge on the mechanism of oxidation and action of antioxidants, the choice and development of analytical methods is also changing. Increasing awareness of biological activity and the availability of analytical methods has changed the way substances are analysed. Regarding the different transport mechanisms of substances in organisms, observations have been conducted with different test cells. An analysis of different substances in mixtures has revealed differences in their biological activity. Due to such differences within a sample, it may contain both strong antioxidants and biologically inactive compounds as well as pro-oxidants. Their separation may obtain individual substances, or their mixtures, with beneficial biological properties. In search of rich sources of bioactive substances, screening studies are conducted in which isolated components of mixtures are analysed for their activity, e.g. as antioxidants. Time and money which must be spent on such analyses, as well as new testing capabilities, combined with chromatographic methods have made looking for such sources much cheaper and easier. Examples of using different chromatographic methods to inhibit or promote oxidation

**4. On-line liquid chromatography in bioactivity determination of**

### *3.2.4. Crocin Bleaching Assay (CBA)*

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 temperature of 40o C. Decrease in the absorbance of the solution is measured colorimetrically 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 as its equivalent (% InhTrolox).
