1. Introduction

Antioxidants play a crucial role in protecting our cells and tissues from oxidative damage caused by free radicals. These highly reactive molecules can lead to various health issues, including aging and chronic diseases. To assess the antioxidant potential of various substances, researchers rely on a range of in vitro methods. This review delves into the intricacies of these in vitro antioxidant assays, highlighting the importance of understanding their variations and limitations ^[1].

Antioxidant activity should not be gauged based solely on a single in vitro antioxidant test model. In practice, researchers employ several in vitro test procedures to evaluate the antioxidant activity of samples under investigation. However, it's essential to recognize that antioxidant test models can differ significantly in various respects. Therefore, comparing one method directly to another is often challenging ^[1][2].

Badarinath et al. (2010) did attempt some comparisons among different in vitro methods, but even their findings demonstrated that no single method is absolute in nature. Thus, it is crucial for researchers to critically verify the methods of analysis before selecting one for their specific research objectives. In this article, we explore various in vitro antioxidant methods, emphasizing the need to optimize the chosen method to suit the experimental goals effectively ^[3].

2. DPPH Scavenging Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stable free radical widely used to assess the antioxidant capacity of a substance. This method measures the ability of a substance to scavenge and neutralize DPPH radicals, providing insights into its radical scavenging potential ^[4].

3. Hydrogen Peroxide Scavenging (H₂O₂) Assay

The hydrogen peroxide scavenging assay evaluates a substance's capacity to neutralize hydrogen peroxide, a reactive oxygen species involved in oxidative stress. This method assesses the substance's ability to mitigate oxidative damage caused by hydrogen peroxide ^[5].

4. Nitric Oxide Scavenging Activity

Nitric oxide (NO) is a signaling molecule, but excess NO can lead to oxidative stress and cell damage. The nitric oxide scavenging assay measures a substance's ability to neutralize excess NO, indicating its potential to regulate nitric oxide levels and reduce oxidative stress ^[5].

5. Peroxynitrite Radical Scavenging Activity

Peroxynitrite is a highly reactive and damaging molecule formed by the reaction of nitric oxide (NO) and superoxide (O2•−). The peroxynitrite radical scavenging assay evaluates a substance's ability to quench peroxynitrite radicals, demonstrating its protective role against oxidative and nitrosative stress ^[6].

6. Phosphomolybdenum Method

The phosphomolybdenum method quantifies the total antioxidant capacity of a substance by measuring its ability to reduce molybdenum(VI) to molybdenum(V). This assay provides an overall assessment of an antioxidant's capacity to neutralize free radicals and reduce oxidative stress ^[7].

7. Ferric Thiocyanate (FTC) Method

The FTC method assesses the antioxidant activity of a substance by measuring its ability to inhibit the oxidation of lipids (fats). It evaluates how well an antioxidant can prevent lipid peroxidation, a process associated with cell damage and aging ^[8].

8. Ferric Reducing-Antioxidant Power (FRAP) Assay

The FRAP assay measures the reducing power of a substance by evaluating its capacity to reduce ferric (Fe³⁺) ions to ferrous (Fe²⁺) ions in a chemical reaction. This method quantifies the substance's ability to donate electrons and act as a reducing agent, reflecting its antioxidant potential ^[9].

Antioxidants are added to the FRAP reagent, and the absorbance is recorded after incubation at 37 °C. FRAP values are calculated and expressed as mM of Fe2

9. Superoxide Radical Scavenging Activity (SOD)

Superoxide radicals are highly reactive molecules produced in the body during various metabolic processes. SOD is a technique used to measure the ability of a substance (usually an antioxidant) to scavenge and neutralize these superoxide radicals. It's a crucial indicator of an antioxidant's ability to combat oxidative stress ^[10].

10. Hydroxyl Radical Scavenging Activity

Hydroxyl radicals are potent and highly reactive oxygen species involved in oxidative damage to cells and biomolecules. The hydroxyl radical scavenging activity assay assesses a substance's capacity to neutralize these radicals, which is essential for evaluating its antioxidant potential ^[11].

11. Hydroxyl Radical Averting Capacity (HORAC) Method

HORAC is a technique used to measure the ability of antioxidants to prevent the formation of hydroxyl radicals. It quantifies the capacity of a substance to inhibit the generation of these damaging radicals, thus evaluating its protective role against oxidative stress ^[12].

12. Oxygen Radical Absorbance Capacity (ORAC) Method

ORAC is a widely used method to assess the antioxidant capacity of a substance. It measures the substance's ability to neutralize peroxyl radicals, which are one of the most common and damaging forms of free radicals. A higher ORAC value indicates stronger antioxidant activity ^[13].

13. Reducing Power Method (RP)

The reducing power method evaluates the ability of a substance to donate electrons and reduce other compounds. This technique measures the reducing capacity of antioxidants, indicating their potential to counteract oxidative reactions and protect cells from damage ^[14].

14. Thiobarbituric Acid (TBA) Method

The TBA method is used to quantify the extent of lipid peroxidation in a sample. It measures the concentration of malondialdehyde (MDA), a byproduct of lipid peroxidation. This method helps assess the protective effect of antioxidants against lipid damage ^[15].

15. DMPD (N,N-dimethyl-p-phenylene diamine dihydrochloride) Method

The DMPD method measures the antioxidant capacity of a substance by assessing its ability to scavenge free radicals, particularly the stable radical DMPD. This technique helps evaluate the substance's overall antioxidant potential ^[16].

16. Trolox Equivalent Antioxidant Capacity (TEAC) Method/ABTS Radical Cation Decolorization Assay

The TEAC method, based on the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation decolorization assay, quantifies the ability of a substance to reduce ABTS radicals. This method measures the substance's antioxidant capacity by comparing it to a known antioxidant standard, Trolox, providing a relative antioxidant value ^[17].

17. Total Radical-Trapping Antioxidant Parameter (TRAP) Method

The TRAP method assesses the overall antioxidant potential of a substance by measuring its ability to trap and neutralize various radicals, such as peroxyl radicals. It provides a comprehensive evaluation of the substance's radical-scavenging capacity ^[18].

18. β-carotene Linoleic Acid Method

The β-carotene linoleic acid method evaluates the ability of a substance to protect β-carotene from oxidation when exposed to oxygen and linoleic acid. It measures the substance's capacity to inhibit the formation of conjugated dienes, which are indicative of oxidative damage ^[19].

19. Xanthine Oxidase Method

Xanthine oxidase is an enzyme involved in the production of uric acid and reactive oxygen species. This method assesses the ability of a substance to inhibit xanthine oxidase activity, thereby reducing the generation of free radicals and oxidative stress ^[20].

20. Cupric Ion Reducing Antioxidant Capacity (CUPRAC) Method

The CUPRAC method evaluates the antioxidant capacity of a substance by measuring its ability to reduce copper ions (Cu²⁺) to copper ions (Cu⁺). It provides a quantitative assessment of the substance's reducing power and antioxidant potential ^[21].

21. Conclusion

These various antioxidant assays and methods provide insights into the ability of substances to combat oxidative stress and protect cells from damage caused by free radicals and reactive oxygen species. Researchers use these techniques to assess the antioxidant potential of natural compounds, pharmaceuticals, and dietary supplements, aiding in the development of therapies and interventions to promote health and well-being.