Bioteksynergy4 multimode microplate reader

Deoxyribose assay is a practical application of studying free radical reactions in biological systems induced by fenton reaction and has been utilized to determine antioxidant activity.[20 ] Reaction mixture contained 50 μL each of vegetable juice, 2-deoxy-D-ribose (2.8mM), FeCl₃ (25mM) premixed with EDTA (100 μM) in 10mM potassium phosphate buffer (KH₂PO₄/KOH) of pH 7.4, H₂O₂ (2.8mM). Ascorbic acid (100 μM) was added as promoter of the reaction reducing Fe (III) to Fe (II). Tubes containing samples were incubated in water bath at 37⁰ C for 1 h. 1 mL each of 2.8% (w/v) TCA and 1% (w/v) TBA in 50mM NaOH were added thereafter and reaction mixtures were heated in a water bath at 80ºC for 20 min. Pink colored TBA-MDA adduct generated due to oxidative degradation of 2-deoxy-D-ribose was measured spectrophotometrically (BioTek^synergy4 multimode microplate reader, BioTek Instruments Inc, Winooski, VT, USA) at 532 nm.[20 ]
The percentage prevention (%P) of 2-deoxy-D-ribose degradation by test material was calculated from the absorbance (A) with respect to control absorbance as follows:
where A_Control and A_Juice represent absorbance recorded for control and samples incubated with vegetables’ juice.

Total polyphenol content was measured using Folin-Coicalteu reagent. In brief, fresh juice (25 μL) was reconstituted in 2.5mL distilled de-ionized water, followed by addition of Folin-Coicalteu reagent (1 N, 250 μL) and Sodium carbonate (20% w/v Na₂CO₃, 250 μL). Mixture was incubated at room temperature (60 min). Absorbance (765 nm) was recorded spectrophotometrically on microplate reader (BioTek^synergy4 multimode microplate reader, BioTek Instruments Inc, Winooski, VT, USA). Total polyphenolic content was expressed as micrograms of Gallic Acid Equivalent per milliliter of the juice (μg GAE/mL).[12 ]

The antioxidative stress potentials of vegetables juice was determined by measuring H₂O₂ induced hemolysis of erythrocytes.[19 ] 2.8% suspension of washed and packed erythrocytes was prepared in sodium phosphate buffer saline (150 mM NaCl, 8.1 mM Na₂HPO₄, 19 mM NaH₂PO₄) of pH 7.4. In 96 well micro plate 100 μL of vegetable juice was incubated with 50 μL of erythrocyte suspension for 10 min at room temperature with shaking (Eppendorf centrifuge 5430R, Eppendorf AG, 22331 Hamburg, Germany) at 1200rpm. 100 μL of 2.5mM H₂O₂ was added and shaking was continued for another 20 min at 37°C. The increase in absorbance (A) due to lysis of erythrocytes was measured spectrophotometrically (BioTek^synergy4 multimode microplate reader, BioTek Instruments Inc, Winooski, VT, USA) at 660nm. Percentage inhibition of erythrocytes hemolysis (I_Hemolysis) by vegetables’ juice was calculated using the following formula:
where A_Control and A_Juice represent absorbance recorded for control and samples incubated with vegetables’ juice.