The normal biochemical reactions in our body, increased exposure to the environment, and higher levels of dietary xenobiotic's result in the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The ROS and RNS create oxidative stress in different pathophysiological conditions. The reported chemical evidence suggests that dietary antioxidants help in disease prevention. The antioxidant compounds react in one-electron reactions with free radicals in vivo/in vitro and prevent oxidative damage. Therefore, it is very important to understand the reaction mechanism of antioxidants with the free radicals. This review elaborates the mechanism of action of the natural antioxidant compounds and assays for the evaluation of their antioxidant activities. The reaction mechanisms of the antioxidant assays are briefly discussed (165 references). Practical applications: understanding the reaction mechanisms can help in evaluating the antioxidant activity of various antioxidant compounds as well as in the development of novel antioxidants.

https://pubs.rsc.org/en/content/articlepdf/2015/RA/C4RA13315C

Free radicals, natural antioxidants, and their reaction mechanisms

Oxidative stress has been identified as the root cause of the development and progression of several diseases. Supplementation of exogenous antioxidants or boosting endogenous antioxidant defenses of the body is a promising way of combating the undesirable effects of reactive oxygen species (ROS) induced oxidative damage. Plants have an innate ability to biosynthesize a wide range of non-enzymatic antioxidants capable of attenuating ROS- induced oxidative damage. Several in vitro methods have been used to screen plants for their antioxidant potential, and in most of these assays they revealed potent antioxidant activity. However, prior to confirming their in vivo therapeutic efficacy, plant antioxidants have to pass through several physiopharmacological processes. Consequently, the findings of in vitro and in vivo antioxidant potential assessment studies are not always the same. Nevertheless, the results of in vitro assays have been irrelevantly extrapolated to the therapeutic application of plant antioxidants without undertaking sufficient in vivo studies. Therefore, we have briefly reviewed the physiology and redox biology of both plants and humans to improve our understanding of plant antioxidants as therapeutic entities. The applications and limitations of antioxidant activity measurement assays were also highlighted to identify the precise path to be followed for future research in the area of plant antioxidants.

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Significance of antioxidant potential of plants and its relevance to therapeutic applications.

Antioxidants had a growing interest owing to their protective roles in food and pharmaceutical products against oxidative deterioration and in the body and against oxidative stress-mediated pathological processes. Screening of antioxidant properties of plants and plant-derived compounds requires appropriate methods, which address the mechanism of antioxidant activity and focus on the kinetics of the reactions including the antioxidants. Many studies evaluating the antioxidant activity of various samples of research interest using different methods in food and human health have been conducted. These methods are classified, described, and discussed in this review. Methods based on inhibited autoxidation are the most suited for termination-enhancing antioxidants and for chain-breaking antioxidants, while different specific studies are needed for preventive antioxidants. For this purpose, the most common methods used in vitro determination of antioxidant capacity of food constituents were examined. Also, a selection of chemical testing methods was critically reviewed and highlighted. In addition, their advantages, disadvantages, limitations and usefulness were discussed and investigated for pure molecules and raw extracts. The effect and influence of the reaction medium on the performance of antioxidants are also addressed. Hence, this overview provides a basis and rationale for developing standardized antioxidant methods for the food, nutraceuticals, and dietary supplement industries. In addition, the most important advantages and shortcomings of each method were detected and highlighted. The chemical principles of these methods are outlined and critically discussed. The chemical principles of methods of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical (ABTS·+) scavenging, 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radical scavenging, Fe3+-Fe2+ transformation assay, ferric reducing antioxidant power (FRAP) assay, cupric ions (Cu2+) reducing power assay (Cuprac), Folin-Ciocalteu reducing capacity (FCR assay), peroxyl radical (ROO·), superoxide radical anion (O2·-), hydrogen peroxide (H2O2) scavenging assay, hydroxyl radical (OH·) scavenging assay, singlet oxygen (1O2) quenching assay, nitric oxide radical (NO·) scavenging assay and chemiluminescence assay are outlined and critically discussed. Also, the general antioxidant aspects of main food components were discussed by a number of methods, which are currently used for the detection of antioxidant properties of food components. This review consists of two main sections. The first section is devoted to the main components in the food and pharmaceutical applications. The second general section comprises some definitions of the main antioxidant methods commonly used for the determination of the antioxidant activity of components. In addition, some chemical, mechanistic and kinetic basis, and technical details of the used methods are given.

https://link.springer.com/content/pdf/10.1007/s00204-020-02689-3.pdf

Antioxidants and antioxidant methods: an updated overview

Phenolic compounds are the most abundant antioxidants in the human diet. They have a considerable structural diversity, characterized by the hydroxyl groups on aromatic rings. According to the number of phenol rings and the structural elements that bind rings to one another, such compounds are grouped and classified as simple phenols, phenolic acids, flavonoids, xanthones, stilbenes, and lignans. Each group has different mechanisms of action correlated to a structural specificity, which confer the antioxidant properties to the compounds. They act mainly in scavenging reactive species of oxygen, nitrogen, and chlorine or can also chelate metal ions, acting in both the initiation stage and in the propagation of the oxidative process. The most common in vitro methods used to evaluate antioxidant activity of phenolic compounds are in vitro methods which are based on single electron transfer reaction or single hydrogen atom transfer reaction.

Phenolic Compounds: Structure, Classification, and Antioxidant Power

Maize is a staple food source in the world, whose ancient varieties or landraces are receiving a growing attention. In this work, two Italian maize cultivars with pigmented kernels and one inbred line were investigated for untargeted phenolic profile, in vitro antioxidant capacity and resistance to Fusarium verticillioides infection. “Rostrato Rosso” was the richest in anthocyanins whilst phenolic acids were the second class in abundance, with comparable values detected between cultivars. Tyrosol equivalents were also the highest in “Rostrato Rosso” (822.4 mg kg-1). Coherently, “Rostrato Rosso” was highly resistant to fungal penetration and diffusion. These preliminary finding might help in breeding programs, aiming to develop maize lines being more resistant to infections and with improved nutraceutical value.

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Phenolic Profile and Susceptibility to Fusarium Infection of Pigmented Maize Cultivars.

A good number of abstracts and research articles (in total 74) published, so far, for evaluating antioxidant activity of various samples of research interest were gone through where 407 methods were come across, which were repeated from 29 different methods. These were classified as in vitro and in vivo methods. And those are described and discussed below in this review article. In the later part of this review article, frequency of in vitro as well as in vivo methods is analyzed with a bar diagram. Solvents are important for extracting antioxidants from natural sources. Frequency of solvents used for extraction is also portrayed and the results are discussed in this article. As per this review there are 19 in vitro methods and 10 in vivo methods that are being used for the evaluation of antioxidant activity of the sample of interest. DPPH method was found to be used mostly for the in vitro antioxidant activity evaluation purpose while LPO was found as mostly used in vivo antioxidant assay. Ethanol was with the highest frequency as solvent for extraction purpose.

Nusrat Jahan Bristi

Papers

Review on in vivo and in vitro methods evaluation of antioxidant activity.