Currently, there is a growing interest in screening and quantifying antioxidants from biological samples in the quest for natural and effective antioxidants to combat free radical-related pathological complications. Antioxidant assays play a crucial role in high-throughput and cost-effective assessment of antioxidant capacities of natural products such as medicinal plants and food samples. However, several investigators have expressed concerns about the reliability of existing in vitro assays. Such concerns arise mainly from the poor correlation between in vitro and in vivo results. In addition, in vitro assays have the problem of reproducibility. To date, antioxidant capacities are measured using a panel of assays whereby each assay has its own advantages and limitations. This unparalleled review hotly disputes on in vitro antioxidant assays and elaborates on the chemistry behind each assay with the aim to point out respective principles/concepts. The following critical questions are also addressed: (1) What make antioxidant assays coloured? (2) What is the reason for working at a particular wavelength? (3) What are the advantages and limitations of each assay? and (4) Why is a particular colour observed in antioxidant-oxidant chemical reactions? Furthermore, this review details the chemical mechanism of reactions that occur in each assay together with a colour ribbon to illustrate changes in colour. The review ends with a critical conclusion on existing assays and suggests constructive improvements on how to develop an adequate and universal antioxidant assay.
Keywords: antioxidants; chemical reactions; colorimetry; free radicals; limitations; oxidative stress; spectrophotometer.