**4. ROS Measuring Tools**

ROS levels' evaluation and measurement are important, practical steps to improve these reactive species' effects. By measuring these species, a more accurate view of them can be achieved, and the appropriate treatment method can be used for each of them. Of course, direct measurement of ROS has its problems and difficulties [40]. This is important because some ROS, such as O2 •− and •OH, while having very short half-lives of 5−<sup>10</sup> and 9−<sup>10</sup> s, respectively, also have very high reactivity. Over the years, countless indicators have been discovered to measure these species, but many of them did not provide consistent and reliable results and were easily discarded. However, in the following years, valid indicators were calculated to measure the oxidation of various tissues and cells of lipids, proteins, and DNA [41].

In general, the grouping of OS indicators is very important. In one group, the biochemical nature of molecules such as proteins, lipids, carbohydrates, and DNA is considered. While in the second group, products of oxidation of cellular compounds are formed to balance cellular mechanisms (oxidation-reduction). This group itself is divided into several subgroups [42]. The first group to free radicals leads to the change of various biomolecules such as malondialdehyde (MDA) from lipid oxidation, 4-hydroxy-2-nonenal (4-HNE), and reactive carbonyls from protein oxidation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) of nucleic acid oxidation have been noted. In the second group, the relationship between free radical metabolism and physiological antioxidant defense molecules such as reduced glutathione (GSH) and CAT is considered. Furthermore, in the third group, modulation of free radicals with transcription factors such as c-Myc and Nuclear factor-κB (NF-κB) is important [16]. Here are the most common and commonly used indicators of OS:

#### *4.1. MDA*

When lipid molecules are exposed to OS, various products are produced in lowdensity lipoproteins (LDL) or cell membranes. One of its end products is MDA. The reactive substance, thiobarbituric acid reactive substances (TBARS), is responsible for measuring MDA levels [13].
