In vitro antioxidant assays: Are they relevant?

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.

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Bioassay guided fractionation of Cyclea peltata using in vitro RAW 264.7 cell culture, antioxidant assays and isolation of bioactive compound tetrandrine

Journal of Ayurveda and Integrative Medicine ◽

10.1016/j.jaim.2018.05.009 ◽

2020 ◽

Vol 11 (3) ◽

pp. 281-286 ◽

Cited By ~ 1

Author(s):

V.J. Shine ◽

G.I. Anuja ◽

S.R. Suja ◽

Gopan Raj ◽

P.G. Latha

Keyword(s):

Cell Culture ◽

Bioactive Compound ◽

Raw 264.7 ◽

Antioxidant Assays ◽

Raw 264.7 Cell ◽

Bioassay Guided Fractionation

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In-vitro and in-vivo antioxidant assays of chicory plants (Cichorium intybus L.) as influenced by organic and conventional fertilisers

BMC Plant Biology ◽

10.1186/s12870-020-2256-2 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 2

Author(s):

Lovro Sinkovič ◽

Polona Jamnik ◽

Mojca Korošec ◽

Rajko Vidrih ◽

Vladimir Meglič

Keyword(s):

Cichorium Intybus ◽

Antioxidant Assays

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Polyphenol Characterization and Skin-Preserving Properties of Hydroalcoholic Flower Extract from Himantoglossum robertianum (Orchidaceae)

Plants ◽

10.3390/plants8110502 ◽

2019 ◽

Vol 8 (11) ◽

pp. 502 ◽

Cited By ~ 4

Author(s):

Miriam Bazzicalupo ◽

Bruno Burlando ◽

Marcella Denaro ◽

Davide Barreca ◽

Domenico Trombetta ◽

...

Keyword(s):

Iron Chelation ◽

Bioactive Substances ◽

Protective Properties ◽

Biological Assays ◽

Antioxidant Assays ◽

Flower Extract ◽

Polyphenol Profile ◽

Collagenase Inhibition ◽

Β Carotene

Himantoglossum robertianum (Loisel.) P. Delforge is a Mediterranean orchid whose propagation in vitro has been achieved, making it eligible as a source of bioactive substances. Flowers were analyzed by light and SEM microscopy and used to obtain a polyphenol-rich, hydroalcoholic flower extract (HFE). HFE was characterized for total phenols, flavonoids and proanthocyanidins, and for polyphenol profile by RP-LC-DAD. Antioxidant assays, in vitro collagenase and elastase inhibition, and MTT and cell motility assays on HaCaT keratinocytes were done. Microscopy showed epidermal cells containing anthocyanins in the flower labellum. Flavonoids (flavones and flavan-3-ols) represented the most abundant compounds (42.91%), followed by scopoletin (33.79%), and phenolic acids (23.3%). Antioxidant assays showed strong activities, rating ORAC > FRAP > TEAC > β-carotene bleaching > DPPH > iron-chelation. Biological assays showed elastase and collagenase inhibition (up to 42% and 78%, respectively), improvement of HaCaT cell viability after treatment with 500 μM H2O2 (from 30% to 84% of control), and stimulation of cell migration rate up to 210% of control. In summary, HFE counteracted different free radicals, while protective properties were shown by cell-free and cell-based bioassays, suggesting the possible use of H. robertianum flowers for skin-preserving, repair, and anti-aging applications.

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A Concise Review of Current In Vitro Chemical and Cell-Based Antioxidant Assay Methods

Molecules ◽

10.3390/molecules26164865 ◽

2021 ◽

Vol 26 (16) ◽

pp. 4865

Author(s):

Ifeanyi D. Nwachukwu ◽

Roghayeh Amini Sarteshnizi ◽

Chibuike C. Udenigwe ◽

Rotimi E. Aluko

Keyword(s):

Free Radicals ◽

Negative Impact ◽

Ex Vivo ◽

Scientific Information ◽

Antioxidant Properties ◽

Antioxidant Assays ◽

Antioxidant Agents ◽

Synthetic Molecules ◽

Negative Side Effects

Antioxidants remain interesting molecules of choice for suppression of the toxic effects of free radicals in foods and human systems. The current practice involves the use of mainly synthetic molecules as potent antioxidant agents. However, due to the potential negative impact on human health, there is an intensive effort within the research community to develop natural alternatives with similar antioxidant efficacy but without the negative side effects of synthetic molecules. Still, the successful development of new molecules depends on the use of reliable chemical or cell culture assays to screen antioxidant properties. Chemical antioxidant assays include the determination of scavenging ability against free radicals such as DPPH, superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, and nitric oxide. Other antioxidant tests include the ability of compounds to bind and sequester prooxidant metal cations, reduce ferric iron, and attenuate the rate of lipid oxidation. Ex vivo tests utilize cell cultures to confirm entry of the molecules into cells and the ability to quench synthetic intracellular free radicals or to stimulate the increased biosynthesis of endogenous antioxidants. In order to assist researchers in their choice of antioxidant evaluation methods, this review presents background scientific information on some of the most commonly used antioxidant assays with a comparative discussion of the relevance of published literature data to food science and human nutrition applications.

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In Vitro Antioxidant Assays

Progress in Drug Research - Pharmacological Assays of Plant-Based Natural Products ◽

10.1007/978-3-319-26811-8_9 ◽

2015 ◽

pp. 57-72 ◽

Cited By ~ 1

Author(s):

Parimelazhagan Thangaraj

Keyword(s):

Antioxidant Assays

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Natural Methoxyphenol Compounds: Antimicrobial Activity against Foodborne Pathogens and Food Spoilage Bacteria, and Role in Antioxidant Processes

Foods ◽

10.3390/foods10081807 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1807

Author(s):

Elena Orlo ◽

Chiara Russo ◽

Roberta Nugnes ◽

Margherita Lavorgna ◽

Marina Isidori

Keyword(s):

Foodborne Pathogens ◽

Antioxidant Activities ◽

Food Additives ◽

Radical Scavenging ◽

Food Spoilage ◽

Antioxidant Assays ◽

Scavenging Capacity ◽

Spoilage Bacteria ◽

Brochothrix Thermosphacta

The antibacterial and antioxidant activities of three methoxyphenol phytometabolites, eugenol, capsaicin, and vanillin, were determined. The in vitro antimicrobial potential was tested on three common foodborne pathogens (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) and three food spoilage bacteria (Shewanella putrefaciens, Brochothrix thermosphacta, and Lactobacillus plantarum). The antioxidant assays were carried out for studying the free radical scavenging capacity and the anti-lipoperoxidant activity. The results showed that eugenol and capsaicin were the most active against both pathogens and spoilage bacteria. S. aureus was one of the most affected strains (median concentration of growth inhibition: IC50 eugenol = 0.75 mM; IC50 capsaicin = 0.68 mM; IC50 vanillin = 1.38 mM). All phytochemicals slightly inhibited the growth of L. plantarum. Eugenol was the most active molecule in the antioxidant assays. Only in the oxygen radical absorbing capacity (ORAC) test did vanillin show an antioxidant activity comparable to eugenol (eugenol ORAC value = 2.12 ± 0.08; vanillin ORAC value = 1.81 ± 0.19). This study, comparing the antimicrobial and antioxidant activities of three guaiacol derivatives, enhances their use in future applications as food additives for contrasting both common pathogens and spoilage bacteria and for improving the shelf life of preserved food.

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In Vitro and In Vivo Antioxidative Activity against Radiation-Induced Damage and the Systematic Chemical Components of Different Extracts of Lagotis brevituba Maxim

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/9726431 ◽

2020 ◽

Vol 2020 ◽

pp. 1-14

Author(s):

Dan Zhang ◽

Lihong Tan ◽

Ling Yao ◽

Wei Tao ◽

Ruixue Gong ◽

...

Keyword(s):

Antioxidant Activity ◽

Antioxidant Activities ◽

Whole Body ◽

Chemical Components ◽

Control Group ◽

Dependent Manner ◽

Antioxidant Assays ◽

Radiation Induced

Lagotis brevituba Maxim is a perennial species distributed in the highlands of China, which has been used for more than 2000 years as a traditional Tibetan medicinal plant. However, no attention has been paid to the antioxidant activities of Lagotis brevituba Maxim in vitro or in vivo. Thus, this study aimed to evaluate the in vitro and in vivo antioxidant activity of Lagotis brevituba Maxim against radiation-induced damage as well as the systematic chemical components. To explore the relationship between the antioxidant activity and extraction solvent, Lagotis brevituba Maxim was extracted with three different solvents: methanol, water, and acetone. In antioxidant assays in vitro, the water extract had the strongest reducing power, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity compared with the methanol and acetone extracts. However, the methanol extract was more potent in the β-carotene/linoleic acid cooxidation assay. In antioxidant assays in vivo, mice that were exposed to 6.0 Gy60Co γ-ray whole-body radiation on day 15 after administration of Lagotis brevituba Maxim decreased their level of malondialdehyde (MDA) in a dose-dependent manner compared with the control group, indicating that Lagotis brevituba Maxim had favorable antioxidant activities in vivo. In addition, a total of 44 compounds were tentatively identified by liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS), including 19 flavonoids, 14 phenols, 8 phenylethanoid glycosides, 2 iridoid glycosides, and 1 carbohydrate. We obtained 25 compounds from plants in the genus Lagotis for the first time. These results suggested that Lagotis brevituba Maxim had potent antioxidant activity and could be explored as a novel natural antioxidant.

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Characterization of biochemical compounds and antioxidant activity of “dedo-de-moça” chili pepper accessions

Horticultura Brasileira ◽

10.1590/s0102-053620190411 ◽

2019 ◽

Vol 37 (4) ◽

pp. 429-436

Author(s):

Gisely Paula Gomes ◽

Leonel Vinicius Constantino ◽

Ligia Erpen-Dalla Corte ◽

Cristiano Jorge Riger ◽

Douglas S de A Chaves ◽

...

Keyword(s):

Antioxidant Activity ◽

Vitamin C ◽

Fruit Quality ◽

Chili Pepper ◽

Total Phenolic ◽

Cell Protection ◽

Antioxidant Assays ◽

Horticultural Crops

ABSTRACT Capsicum baccatum is one of the main chili pepper species grown and consumed in South America. In Brazil, C. baccatum var. pendulum is widely cultivated, especially “dedo-de-moça” type. For most horticultural crops, including Capsicum species their diversity remains under-explored for traits related to fruit quality, since over the past half century breeding efforts have focused mainly on good agronomic performances. The investment in screening traits such as flavor, nutritional compounds and antioxidant content in traditional varieties is very important to support fruit quality breeding efforts. Thus, the objective of this study was accessing the variations into content of soluble solid, acidity, capsaicinoid, vitamin C, total phenolic compounds and antioxidant activity (in vitro and in vivo) of five “dedo-de-moça” peppers accessions. The results evidenced that UEL111 and UEL112 accessions showed highest TSS and ratio. Accessions UEL110 and UEL112 showed highest concentration of vitamin C and total phenolic content and UEL113 accession showed highest concentration of capsaicinoids. In vitro antioxidant assays suggest the UEL114 with highest antioxidant activity. In vivo antioxidant assays have not identified differences among the accessions and proved the antioxidant effect of chili pepper extracts on cell protection against stress agent. The results highlighted accessions that can be exploited in chili pepper breeding programs.

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