Characterization and in vitro antioxidation of papain hydrolysate from black-bone silky fowl (Gallus gallus domesticus Brisson) muscle and its fractions

Black-bone silky fowl (Gallus gallus domesticus Brisson) (BSF) muscle was hydrolyzed by papain, and the hydrolysate was separated by preparative high performance liquid chromatography (HPLC). The amino acid composition of the BSF hydrolysate (BSFH) and its fractions was determined by HPLC precolumn derivation with 2,4-dinitrofluorobenzene. The molecular weight (MW) distribution of the BSFH and its fractions was measured by a peptide column on an HPLC system. Antioxidant activities of the BSFH and its fractions were studied by testing the reducing power and four radical scavenging systems: superoxide anion (O₂⁻), hydroxyl (·OH), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) radicals. The results demonstrated that the BSFH had strong antioxidant capacity to scavenge O₂⁻, DPPH and ABTS⁺, and displayed strong reducing power, but revealed less powerful ability to scavenge ·OH. Fraction II of the BSFH exhibited the highest activity in scavenging O₂⁻ and DPPH, and reducing power, whereas fraction I displayed the strongest ·OH scavenging ability. Besides Glu, Asp and Gly, the rich amino acids of Ala and Leu played an important role in antioxidant activity. The small-size peptides with MW ranging from approximately 200-6000 Da probably contributed to higher antioxidant activity. Results from this study indicated that BSFH and its fractions could be used as food additives and diet nutrients.     

On practical problems in estimation of antioxidant activity of compounds by DPPH method (Problems in estimation of antioxidant activity)

2,2′-Diphenyl-1-picrylhydrazyl (DPPH) method is commonly applied for the estimation of antioxidant activity of single compounds and their mixtures. The method is based on the spectrophotometric measurement of DPPH concentration changes resulting from the DPPH reaction with an antioxidant. The amount of remaining DPPH in the examined system is a measure of the antioxidative activity of compounds. The study shows that the type and amount of solvent used for the antioxidant dissolution, water content, and hydrogen or metal ion concentration in the measuring system significantly influence the differences in the amount of unreacted DPPH. The presence of ethyl acetate or dioxane or metal ions in the system decreases the kinetics of the reaction between DPPH and butylohydroxytoluene in relation to its kinetics in the system containing only methanolic solutions. The opposite effect is observed for water, low concentration of chloroform or hydrogen ions. The presented relationships confirm the necessity of standardising the DPPH method and show the complexity of the problem, even in very simple DPPH/antioxidant systems.     

Rapid antioxidant capacity screening in herbal extracts using a simple flow injection-spectrophotometric system

A simple flow injection (FI)-spectrophotometric system for the screening of antioxidant capacity in herbal extracts was developed. The analysis was based on the color disappearance due to the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical by antioxidant compounds. DPPH and ascorbic acid were used as reagent and antioxidant standard, respectively. Effects of the DPPH concentration, DPPH flow rate, and reaction coil length on sensitivity were studied. The optimized condition provided the linear range of 0.010-0.300 mM ascorbic acid with less than 5%RSD_(n = 10). Detection limit and quantitation limit were 0.004 and 0.013 mM, respectively. Comparison of antioxidant capacity in some herbal extracts determined by the FI system and a standard method was carried out and no significant difference was obtained.