Unit 5: Antioxidant Activity

I. Orientation

Antioxidant activity describes the ability of a substance or food extract to delay, prevent, or reduce oxidation by reactive oxygen species (ROS), reactive nitrogen species, or other free radicals. In laboratory analysis, activity is measured by allowing antioxidants in a sample to react with a defined oxidant or radical and then determining the change in colour or absorbance, usually with a spectrophotometer. Thin layer chromatography (TLC) provides a complementary separation technique for examining individual antioxidant compounds in a mixture.

• Oxidation: A chemical process involving electron loss; in food, it can cause rancidity, pigment loss, nutrient destruction, and off-flavour formation.
• Antioxidant: A molecule that inhibits oxidation by donating an electron or hydrogen atom, chelating pro-oxidant metals, or decomposing reactive intermediates.
• Free radical: A species containing an unpaired electron, such as the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•).
• Spectrophotometric principle: The concentration of a coloured species is related to absorbance according to Beer–Lambert law:

  A = εbc

Here, A is absorbance, ε is molar absorptivity, b is cuvette path length in centimetres, and c is concentration.

• Control and comparison: A reagent blank measures background absorbance, while a standard such as Trolox or ascorbic acid permits results to be expressed quantitatively.
• Important convention: Antioxidant capacity is assay-dependent; a value from a DPPH assay is not automatically equivalent to a value from a ferric-reducing assay.

II. Determination of Total Antioxidant Activity of Food Sample — Spectrophotometric estimation

A. Purpose and principle

The determination measures the combined radical-scavenging or reducing effect of compounds extracted from a food sample rather than identifying each compound individually. A commonly demonstrated method uses DPPH•, a stable purple radical that becomes pale yellow after accepting an electron or hydrogen atom from an antioxidant.

• Reaction basis: Antioxidants reduce DPPH• to its non-radical form, DPPH-H, producing a fall in absorbance near 517 nm.
• General reaction:

  DPPH• + AH → DPPH-H + A•

AH represents an antioxidant molecule and A• is the antioxidant-derived radical.

• Measured response: Greater absorbance decrease indicates greater radical-scavenging activity under the selected conditions.
• Meaning of “total”: The result represents the combined response of extractable antioxidants, including possible contributions from phenolics, flavonoids, ascorbate, tocopherols, and other reducing substances.

B. Determination of total antioxidant activity of food sample

This subsection describes the preparation, reaction, measurement, and calculation used to estimate antioxidant activity in a food extract.

• Sample preparation: Homogenize the food to obtain a representative material; weigh a known mass, such as 1.00 g, and extract it with a measured volume of solvent.
  • Solvent choice: Methanol, ethanol, acetone–water, or aqueous buffer may be selected according to the expected polarity and stability of the antioxidants.
  • Extraction condition: Mix or shake for a fixed time, protect light-sensitive compounds from strong light, and filter or centrifuge to remove particles.
• Reagent preparation: Prepare a DPPH solution at a known concentration, commonly in methanol, and keep it in an amber container because light promotes radical degradation.
• Blank and control: Prepare a reagent control containing DPPH solution and solvent but no food extract; its absorbance is represented by A_control.
• Sample reaction: Mix a measured volume of extract with DPPH reagent, incubate for a fixed period such as 20–30 minutes in darkness, and read absorbance at approximately 517 nm.
  • Sample absorbance: Record the absorbance after reaction as A_sample.
  • Sample blank: If the food extract is strongly coloured, measure extract plus solvent without DPPH and correct for its intrinsic absorbance.
• Percentage inhibition: Calculate the decrease in DPPH absorbance using:

  % inhibition = [(A_control − A_sample) / A_control] × 100

A_control is the absorbance of DPPH without extract, and A_sample is the absorbance after reaction with the extract.

• Standard-curve expression: Prepare several Trolox or ascorbic-acid concentrations, measure their responses, and plot antioxidant response against concentration.
  • Possible units: Results may be expressed as mg Trolox equivalents per gram of food (mg TE/g) or ascorbic-acid equivalents per gram.
  • Interpretation: A result of 2.5 mg TE/g means the extract response is equivalent, under the assay conditions, to 2.5 mg of Trolox per gram of sample.
• Worked example: If A_control = 0.800 and A_sample = 0.320, then:

  % inhibition = [(0.800 − 0.320) / 0.800] × 100
               = 60%

This indicates that the tested extract removed or reduced 60% of the measurable DPPH response under the specified conditions.

C. Experimental variables, controls, and interpretation

Reliable antioxidant measurements depend on controlling factors that affect reaction rate, extraction yield, and absorbance independently of antioxidant concentration.

• Reaction time: DPPH reduction is not always instantaneous; compare samples only after the same incubation period, because 10 and 30 minutes can produce different inhibition values.
• Wavelength: Use the method’s selected wavelength, typically 517 nm for DPPH; measuring at another wavelength changes the relationship between colour and concentration.
• Extract concentration: Test a dilution range when possible. An excessively concentrated extract can produce complete or near-complete quenching, reducing the ability to distinguish samples.
• Replicates: Perform at least duplicate or triplicate measurements and report the mean with variation, such as standard deviation.
• Blank correction: Pigments in berries, spices, or leafy vegetables can absorb near the analytical wavelength; a sample blank prevents colour from being incorrectly counted as radical scavenging.
• Calibration quality: A standard curve should be approximately linear over the working range; dilute samples that fall outside that range rather than extrapolating substantially.
• Interpretation limit: Antioxidant activity is not identical to health benefit. Bioavailability, metabolism, digestion, and concentration in tissues are not measured by this in-vitro assay.

D. Applications and limitations

The method is useful for comparing foods or processing treatments, but the result must be reported with its assay conditions and sample basis.

• Applications: Compare fresh and stored food, evaluate cooking effects, screen plant extracts, or monitor antioxidant loss during processing.
• Processing interpretation: Heat may destroy ascorbic acid but release phenolic compounds from plant cell walls, so total activity may increase or decrease depending on the food.
• Chemical interference: Sugars, organic acids, pigments, and reducing agents may affect the reagent response without all acting as biologically important antioxidants.
• Extraction limitation: A single solvent does not extract every antioxidant; an ethanol extract and an aqueous extract may give different values from the same food.
• Safety and technique: Wear gloves and eye protection, handle organic solvents in a fume hood, label all extracts, and dispose of DPPH and solvent waste according to laboratory rules.

III. Demonstration of Thin Layer Chromatography — Separation and detection of antioxidants

A. Purpose and principle

Thin layer chromatography separates compounds according to their differing interactions with a stationary phase and a mobile solvent phase. It can demonstrate whether a food extract contains several components and can help locate antioxidant-active spots after development.

• Stationary phase: A thin coating of silica gel, commonly silica gel 60, is fixed to a plate; polar compounds interact strongly with its surface.
• Mobile phase: A solvent or solvent mixture rises through the plate by capillary action and carries dissolved compounds at different rates.
• Separation mechanism: A compound that interacts strongly with silica moves less, whereas a compound more soluble in the mobile phase travels farther.
• Chromatographic measure: The retention factor is:

  Rf = distance travelled by solute spot / distance travelled by solvent front

Rf has no unit and normally lies between 0 and 1 when measured from the origin to the solvent front.

B. Demonstration of thin layer chromatography

This procedure demonstrates the separation of antioxidant-containing compounds from a food extract and their visual or chemical detection on the plate.

• Plate preparation: Use a silica TLC plate without touching the silica surface; draw a light pencil origin line approximately 1 cm from the lower edge.
• Sample application: Apply a small, concentrated spot of food extract to the origin using a capillary tube.
  • Spot size: Allow each application to dry before adding more extract so the final spot remains narrow rather than forming a broad streak.
  • Comparison: A standard such as ascorbic acid, quercetin, or another known compound may be spotted beside the food extract.
• Developing chamber: Add a shallow layer of mobile phase to a closed chamber, ensuring that the solvent level remains below the origin line.
• Development: Place the plate upright in the chamber and close it. The solvent rises by capillary action until it approaches the upper boundary.
  • Atmosphere: Chamber saturation with solvent vapour improves reproducibility.
  • Stopping development: Remove the plate, immediately mark the solvent front in pencil, and dry the plate.
• Visualization: Observe spots under ultraviolet light when compounds absorb UV, or spray with a suitable reagent.
  • Antioxidant-specific demonstration: Spray a developed plate with DPPH solution; antioxidant compounds can appear as yellow or pale spots against a purple background because they reduce local DPPH•.
  • Alternative detection: Ferric chloride may reveal some phenolic compounds through coloured complexes, but colour response is not a universal test for all antioxidants.
• Rf calculation: Measure from the origin to the centre of each spot and from the origin to the solvent front:

  Rf = d_spot / d_front

If a spot travels 3.2 cm and the solvent front travels 6.4 cm, its Rf is 0.50.

• Interpretation: Several spots indicate several separated components; a spot with antioxidant bleaching suggests radical-reducing activity at that position.

C. Applications and limitations

TLC links separation with activity detection, but it is mainly qualitative or semi-quantitative unless carefully calibrated.

• Applications: Compare antioxidant profiles among fruits, teas, spices, or oils; monitor extraction; and distinguish a simple extract from a chemically complex mixture.
• Identification: Matching Rf values with a standard under the same solvent and plate conditions supports identification but does not prove chemical identity.
• Solvent dependence: Rf changes with solvent composition, humidity, silica type, sample loading, and development distance; values must therefore be compared only under comparable conditions.
• Resolution limitation: Compounds with similar polarity may overlap in one solvent system; changing the mobile phase may improve separation.
• Detection limitation: A compound may be present but invisible under UV or unreactive toward DPPH spray, while a strongly coloured or highly reducing compound may produce a misleadingly intense signal.
• Quantification caution: Spot intensity is affected by loading and reagent distribution; accurate concentration requires calibration or an instrumental method such as HPLC.
• Method relationship: Spectrophotometry measures the combined response of an extract, whereas TLC separates components and shows where activity occurs; using both methods gives complementary rather than identical information.