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Antioxidant Assay

The document outlines a method for extracting antioxidants from Moringa oleifera leaves using Soxhlet extraction with various solvents. It describes three assays to evaluate antioxidant activity: DPPH radical scavenging, total phenolic content using the Folin-Ciocalteu method, and FRAP for ferric reducing antioxidant power. Statistical analysis was performed with three duplicates for each experiment to determine mean values and IC₅₀ concentrations.

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19 views2 pages

Antioxidant Assay

The document outlines a method for extracting antioxidants from Moringa oleifera leaves using Soxhlet extraction with various solvents. It describes three assays to evaluate antioxidant activity: DPPH radical scavenging, total phenolic content using the Folin-Ciocalteu method, and FRAP for ferric reducing antioxidant power. Statistical analysis was performed with three duplicates for each experiment to determine mean values and IC₅₀ concentrations.

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palwashasahar0001
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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antioxidant assay

1. Plant Extraction and Material

Moringa oleifera fresh leaves were collected, cleaned, dried in the shade, and ground into a powder.

Using Soxhlet extraction, the powdered material was successively extracted using solvents of increasing
polarity (hexane, ethyl acetate, methanol, and water).

Reduced pressure was used to concentrate the extracts, which were then kept at 4°C until they were
needed.

2. Assays for Antioxidants (a) DPPH (1,1-Diphenyl-2-picrylhydrazyl) Radical Scavenging Assay Principle:
evaluates an extract's capacity to scavenge stable DPPH free radicals.

Method:

In methanol, a 0.1 mM DPPH solution was made.

DPPH solution was combined with extracts at varying concentrations (50–500 µg/mL).

The mixture was allowed to sit at room temperature for 30 minutes in the dark.

A UV-Vis spectrophotometer was used to measure absorbance at 517 nm.

As a common antioxidant, ascorbic acid was utilized.

The percentage of scavenging activity was was calculated as:


Scavenging %=(AcontrolAcontrol−Asample)×100
b) Total Phenolic Content (TPC) by Folin-Ciocalteu Method Principle: Determines the amount
of phenolic compounds by calculating how much of the Folin-Ciocalteu reagent they reduce.

Method:

Folin-Ciocalteu reagent and Na2CO3 solution were combined with the extracts.

Absorbance was measured at 765 nm following a 2-hour dark incubation period.

Results were reported as mg GAE/g extract, with gallic acid serving as a standard.

(c) FRAP, or ferric reducing antioxidant power The assay principle quantifies the extracts'
capacity to convert Fe³⁺ to Fe²⁺.

Method:

Acetate buffer, TPTZ, and FeCl₃ were combined to create the FRAP reagent.

After mixing the extracts with the FRAP reagent, they were incubated for 10 minutes at 37°C.

At 593 nm, absorbance was measured.

The results were expressed as µM Fe²⁺ equivalent/g extract, with ferrous sulfate (FeSO₄)
serving as the standard.

3. Analysis of Statistics
Three duplicates of each experiment were conducted, and the results were reported as mean ±
SD.

Regression analysis was used to determine the IC₅₀ values (concentration needed for 50%
inhibition).

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