Modified 2,2-Azinobis(3-Ethylbenzo Thiazoline)-6- Sulphonic Acid Radical Cation Decolorization Assay for Antioxidant Activity of Human Plasma and Extracts of Traditional Medicinal Plants

Muhammad Nadeem Asghar, Islam Ullah Khan,* Iram Zia, Mukhtar Ahmad# and Fahim Ashraf Qureshi

Department of Chemistry, Government College University, Lahore-54000, Pakistan
#Department of Biochemistry, Allama Iqbal Medical College, Canal Road, Lahore, Pakistan
* Corresponding author: E-mail: iukhan@gcu.edu.pk
Office Tel: 92-042-111-000-010

Abstract
2,2-Azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation decolorization assay has been applied under different pH conditions for the determination of trolox equivalent antioxidant capacity (TEAC) of biological and food samples. Due to innate nature of ABTS radical cation, it is unstable at physiological pH. Further, its sensitivity is also lost at acidic pH. No single method has yet claimed to cover both drawbacks of the ABTS procedure. The present investigation was aimed at developing an improved ABTS decolorization method for the measurement of antioxidant activity of human plasma and herbal extracts with greater stability and sufficient sensitivity. A relatively stable ABTS radical cation was generated through a reaction between ABTS and potassium persulfate in the presence of sodium acetate buffer at pH 6.5 (20 mM). The electron or hydrogen atom transfer from antioxidative components of plasma or herbal extracts reduced the radical cation. Standard antioxidants like trolox, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT) and ascorbic acid demonstrated stable end-point readings showing a promising aspect of determination of total antioxidant activity (TAA) of all the other samples with reference to these indicators. The modified assay proved to be highly reproducible. TEAC values for plasma samples ranged between 1.4102.025 mM with mean percent relative standard deviation (% RSD) 1.64 and between 1.721.98 with mean % RSD 1.27 for older and modified ABTS assays, respectively. The efficacy of the assay was ascertained further by determining antioxidant activity of organic and aqueous extracts of Cuscuta reflexa and Terminalia arjuna.

Keywords: ABTS radical cation, antioxidant activity, TEAC, plasma, potassium persulfate.