In-vitro Antioxidant Assay-An Insight

Antioxidants are chemicals that protect cells by neutralizing free radicals generated by external factors (such as damage from the sun, pollution, wind, temperature, etc.) and internal factors (for example, stress emotions, metabolism, contaminants, toxic substances, etc.). Free radicals are byproducts that are formed inside our body during various metabolic processes when oxygen is used by the body cells. Antioxidants resist that action of free radicals which facilitate the development of degenerative diseases. They are also responsible for the pathogenesis of diabetes, liver damage, nephrotoxicity, inflammation, cancer, cardiovascular disorders, neurological disorders, aging, and many other diseases.

Different fruits, vegetables, medicinal herbs, oil, etc are known to be antioxidants due to the presence of vitamins and various phytochemicals. These foods inhibit/stop the generation of radicals, stop the propagation of the radicals, scavenge free radicals, minimize their impact and eliminate them from the system or donate hydrogen to radicals to reduce them to inactive molecules.

In vitro Antioxidant Assay

Numerous methods can be applied for determining the in vitro antioxidant potential (AOP) of single compounds or their mixtures. Due to their simplicity, spectrophotometric methods based on the reaction of the antioxidants with chromomeric radicals such as 2,2′-azino-bis-3-ethylbenzotiazolin-6-sulfonic acid (ABTS^⋅⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPH^⋅) and Ferric Reducing Antioxidant Power (FRAP) assay are widely applied.

Sample Preparation

Sample preparation for different commodities to carry out in vitro antioxidant assays differs greatly. While simpler extraction may be sufficient for fruits, more complex sample preparation is needed for edible oils and fats.

ABTS cation radical decolourisation assay

ABTS-, the oxidant, is generated by persulfate oxidation of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS^2-). This solution is diluted with phosphate buffer (pH 7.4) until the absorbance reaches 0.7 to 0.8 at λ 734 nm. One ml of the resulting solution was mixed with the sample. The absorbance is read at 30 °C, 4 min after mixing at 30 °C. The difference of the absorbance reading is plotted versus the antioxidant concentrations to give a straight line. The concentration that caused a decrease in the initial ABTS concentration by 50% is defined as IC₅₀.

2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) scavenging capacity assay

DPPH is one of a few stable and commercially available organic nitrogen radicals and has a UV-vis absorption maximum at λ 517 nm. Upon reduction, the solution color fades. The DPPH assay is typically run according to the following procedure. DPPH solution is mixed with a sample solution. The absorbance of the mixture during the reaction progress is monitored at λ 517 nm for 20 min or until the absorbance is stable. Upon reduction, the colour of the solution fades. The concentration that causes a decrease in the initial DPPH concentration by 50% is defined as IC₅₀.

Ferric ion reducing antioxidant power (FRAP) assay

The oxidant in the FRAP assay is prepared by mixing TPTZ, acetate buffer, and FeCl₃.H₂O. The conglomerate is referred to as “FRAP reagent”. To measure FRAP value, freshly prepared FRAP reagent is warmed to 37 °C and a reagent blank reading is taken at λ 593 nm, then sample and 30 μL of water are added. Absorbance readings are taken λ 593 nm. The concentration that causes an increase in the initial FRAP concentration by 50% is defined as IC₅₀.

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In-vitro Antioxidant Assay-An Insight

Testimonial

Mitra S.K. have added value to our business process by effective inspection, strict quality control and timely response. It is a pleasant experience to work with “Team Mitra S.K.”. We hope the same service level will continue in the future too....

Sirsendu Mukherjee

Mitra SK Inspection Gulf LLC has been appointed by us and the services provided by them has been up to the mark and as per our expectations....

Ajay Mathur

Amar Singh Gohil

The Inspection services provided by Mitra S. K. Inspection Gulf has been prompt and accurate and has been in the line with our expectations....

Naser Bustami

We would like to express our high appreciation of the business association our company had with Mitra S. K. as a testing and inspection house for their quality of service, good documentation and reporting during the job ....

R. Jeyakumar

Tulus Situmeang

Their job was excellent and showed Mitra S.K. differential from others....

Geraldo Pimenta

Konstantin Gertsen

We’ve been pleased with the improvement we have seen in CIQ results, and Mitra S.K’s reports have certainly helped us to resolve any disputes with our customers in a more timely manner….....

Brian Mcdonald

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