Analyzing Antioxidant Properties of Plants

Jul 3, 2024

Analyzing Antioxidant Properties of Plants

Introduction

  • Objective: How to analyze antioxidant properties in plants.

Key Concepts

Antioxidants

  • Definition: Molecules that donate electrons to free radicals to neutralize them.
  • Function: Stabilize unstable free radicals to prevent cell damage and disorders.
  • Sources: Green tea, citrus fruits, tocopherols, etc.

Free Radicals

  • Definition: Unstable molecules with an unpaired electron in the outermost orbit.
  • Causes: Exposure to toxic chemicals, radiation, smoking, adulterated food, cosmetics, and alcohol.
  • Consequences: Can lead to arthritis, inflammation, cell toxicity, cancer, etc.
  • Chain Reaction: One free radical leads to the formation of many, causing extensive damage.

Free Radical Scavenging

  • Process: Neutralizing free radicals by donating electrons to stabilize them.
  • Significance: Reduces damage and disorders caused by free radicals.

Antioxidant Assay by DPPH Method

Chemicals and Reagents

  • DPPH (2,2-Diphenyl-1-picrylhydrazyl): An organic, stable free radical, crystalline, purple compound, insoluble in water, soluble in methanol/ethanol.
  • Solvents: Prefer methanol over ethanol (cost-effective, less volatile).
  • Concentration: 0.004% (4 mg DPPH in 100 ml methanol).
  • Preparation: Light-sensitive; work in dark conditions; cover with aluminum foil.

Experiment Principle

  • Baseline: DPPH in methanol forms a purple solution.
  • Reaction: Plant extracts reduce DPPH (purple to colorless or yellow) if antioxidants are present.
  • Measurement: Absorbance taken at 517 nm; higher reduction indicates higher antioxidant properties.

Experimental Procedure

Preparing Solutions

  1. DPPH Solution: 0.004% in methanol.
  2. Plant Extracts: Methanolic extracts in varying concentrations (20-200 µL).

Conducting the Assay

  1. Setup: Take plant extract in test tubes in varying concentrations, make up to 3 ml with methanol.
  2. Reaction: Add 1 ml of DPPH solution to each test tube. Incubate in the dark for 30 minutes.
  3. Control Sample: 3 ml methanol + 1 ml DPPH (no plant extract).
  4. Blank: Only methanol (no DPPH).

Calculating Antioxidant Activity

  • Formula: (Abs. of Control - Abs. of Sample) / Abs. of Control * 100 to get the percentage.

Comparative Analysis

  • Use standard antioxidants (e.g., ascorbic acid, BHT, BHA) as benchmarks.
  • Prepare standards similarly and compare absorbance.

Observations and Results

  • Color Change: Higher concentrations of plant extracts lead to a yellow or colorless solution if antioxidants are present.

  • Measurement: Absorbance at 517 nm; calculate antioxidant activity percentage.

  • Example Calculation:

    • Absorbance control: 0.118
    • Absorbance sample: 0.082
    • [(0.118 - 0.082) / 0.118] * 100 = 30.5%

Applications and Industrial Uses

  • Antioxidants are used in food preservation, cosmetics, and pharmaceuticals.
  • Common preservatives: BHT, BHA, tocopherols.

Conclusion

  • Antioxidant assay helps to determine the effectiveness of plant extracts compared to standard antioxidants.
  • Important for developing pharmaceuticals and preservatives.

Additional Notes

  • Experiment Maintenance: Keep all reagents and samples in dark conditions, handle with care.
  • Protocols: Follow the method that gives reproducible and accurate results.

End of Lecture.