Antioxidant Capacity: DPPH Radical Scavenging Assay

Antioxidant claims are among the most common in skin care and among the least often substantiated with a measurement. The DPPH assay is the measurement: a chemical test of how effectively a product, extract or raw material neutralises a stable free radical, expressed as a number that can be compared between formulations, between batches and over time.

Oxford Biosciences runs the assay in its own laboratory under the Head of Laboratory Sciences. The report states the result in the three conventional forms, states what was done to the sample to obtain it, and states what the result can and cannot be used to claim.

The chemistry

DPPH is 2,2-diphenyl-1-picrylhydrazyl. It is a nitrogen-centred free radical that is unusual in being stable at room temperature in solution: the unpaired electron on the hydrazyl nitrogen is delocalised across the two phenyl rings and the electron-withdrawing picryl group, which prevents the molecule from dimerising as most radicals do. The delocalisation also gives the radical a deep violet colour, with an absorbance maximum at approximately 517 nm in methanol or ethanol.

When DPPH• accepts a hydrogen atom, or an electron followed by a proton, the unpaired electron is paired, the radical becomes the hydrazine DPPH-H, the conjugation is broken, and the violet colour is lost in favour of pale yellow. The loss of absorbance at 517 nm is stoichiometric with the number of radicals reduced. An antioxidant is any substance that donates the hydrogen or electron; the more of them it can donate, and the faster, the greater the fall in absorbance.

Two mechanisms operate. In hydrogen atom transfer (HAT), the antioxidant donates a hydrogen atom directly from a phenolic hydroxyl or a similar bond, as vitamin E, ferulic acid and most polyphenols do. In single electron transfer (SET), the antioxidant donates an electron to form the DPPH anion, which then takes a proton from the solvent, a route favoured in alcohol solvents and by ascorbic acid and readily ionised phenols. The assay does not distinguish the two, which is one reason results depend on solvent and pH and must be compared only between samples run under the same conditions.

Applications

• Formulation development. Comparing the antioxidant capacity of candidate formulations, or of one formulation at different concentrations of an active, before committing to a final recipe.
• Ingredient screening. Ranking botanical extracts, oils and antioxidant additives from different suppliers or batches. Extracts vary considerably with origin, extraction solvent and age, and a supplier’s typical value is not evidence for the batch in hand.
• Stability programmes. Measuring the antioxidant capacity of a product at intervals through accelerated and real-time storage, which shows whether the active claimed on the label is still active at the end of shelf life. This is the use with the most direct bearing on the CPSR.
• Claims substantiation. Generating the quantitative data behind “contains antioxidants”, “with antioxidant [ingredient]” and comparative statements between the brand’s own products.
• Benchmarking. Positioning a product against a reference antioxidant or against alternative formulations.

Regulatory basis

In the EU and UK, the DPPH assay is not a mandatory test under Annex I of Regulation (EC) No 1223/2009 in the way that microbiological quality and stability are. Its regulatory relevance is through claims. Regulation (EU) No 655/2013 lays down the Common Criteria that every cosmetic claim must meet, of which evidential support is the one the assay addresses: a claim must be supported by adequate and verifiable evidence, and the evidence must be appropriate to the claim. The Commission’s Technical Document on Cosmetic Claims (2017) adds that in vitro data can support a claim about a property of the product, and that a claim about an effect on the consumer needs evidence of that effect.

That distinction governs what a DPPH result can do. It is direct evidence that the product scavenges free radicals in a chemical system, and therefore substantiates a claim that the product contains antioxidants or has antioxidant activity. It is not, on its own, evidence that the product protects the skin from free radical damage, oxidative stress, pollution or ageing, because those are effects on the consumer that a chemical assay in a cuvette does not demonstrate. The report says which claims the result supports and which would need further work, such as an ex vivo or in vivo study, before they could be made.

Quantitative antioxidant data on a product also belong in the Product Information File as part of the analytical characterisation, and where an antioxidant is claimed, the stability data on that antioxidant are what the safety assessor and any inspector will look for.

How the assay is done

1. Sample preparation

The result depends heavily on how the sample is prepared, so the method is chosen for the product and recorded in the report.

Extracts, tinctures and hydrophilic actives are diluted directly in methanol or in a methanol-water mixture to a series of concentrations spanning the expected active range.

Oils and lipophilic actives are diluted in methanol where they are soluble, or in ethanol or a methanol-chloroform mixture where they are not, with the solvent recorded because it affects the result.

Emulsions and finished products cannot be measured directly: the oil phase, emulsifiers and pigments scatter light and the water phase is immiscible with the reagent. A weighed portion is extracted with methanol, with vortexing and sonication, centrifuged to remove the insoluble fraction, and the supernatant is used as the sample. Where the antioxidant is lipophilic, a second extraction with a non-polar solvent recovers the oil-phase antioxidants, and both extracts are assayed. The extraction efficiency is checked by spiking a portion of the product with a known amount of Trolox and confirming its recovery.

Coloured samples, particularly those containing anthocyanins, carotenoids, chlorophyll or a synthetic colourant, absorb at or near 517 nm and would read as a false loss of DPPH absorbance. A sample blank containing the sample and solvent but no DPPH is measured at the same concentration and subtracted.

2. Reagent

A fresh DPPH solution in methanol is prepared for each run at a concentration, typically 60 to 100 µmol/L, that gives an initial absorbance of approximately 1.0 at 517 nm. DPPH is light-sensitive and degrades slowly in solution, so the reagent is prepared in amber glassware, kept in the dark, and its absorbance verified immediately before use. A reagent that has drifted is discarded.

3. Reaction

A fixed volume of sample dilution is mixed with a fixed volume of DPPH solution in a cuvette or a microplate well, in triplicate at each concentration. The reaction is allowed to proceed in the dark at 25 °C. The original Brand-Williams method (1995) reads at 30 minutes; because slow-acting antioxidants continue to reduce DPPH for longer, the absorbance is followed kinetically and the reading taken at the plateau, and the time to plateau is recorded. Fast antioxidants such as ascorbic acid reach it within minutes; sterically hindered phenols and some botanical extracts take an hour or more, and reading them at 30 minutes underestimates their capacity.

Each run includes a control of DPPH with solvent only, which gives the initial absorbance A₀; the sample blanks described above; a Trolox calibration series; and an ascorbic acid or quercetin positive control whose result must fall within its established range for the run to be accepted.

4. Measurement

Absorbance is read at 517 nm on a UV-Vis spectrophotometer, blanked against methanol. The instrument’s wavelength and photometric accuracy are verified against certified reference filters on a routine schedule, and a spectrum of the DPPH reagent is recorded to confirm that the absorbance maximum is where the method expects it in the solvent used.

5. Calculation

Percentage inhibition, also called radical scavenging activity, at each sample concentration is calculated as (A₀ minus A_sample, corrected for the sample blank) divided by A₀, multiplied by 100.

IC50 is the concentration of sample at which 50 percent of the DPPH radicals are scavenged. It is obtained by plotting percentage inhibition against sample concentration and interpolating, or by non-linear regression where the response is sigmoidal. A lower IC50 means a more potent antioxidant. Because IC50 depends on the DPPH concentration used, the report states the DPPH concentration and, where comparison with published values is intended, also gives the EC50 as the ratio of antioxidant to DPPH at 50 percent inhibition.

Trolox equivalents express the sample’s capacity relative to Trolox, a water-soluble analogue of vitamin E, from a calibration curve of percentage inhibition against Trolox concentration run alongside the samples. The result is reported as micromoles of Trolox equivalents per gram of sample (µmol TE/g), sometimes called TEAC. This is the figure most useful for comparing products and batches, because it is normalised to a reference and less sensitive to the assay conditions than IC50.

All three are reported with the mean and standard deviation of the triplicates and the concentration range over which the response was linear.

Interpreting the result

A high Trolox equivalent value in an extract does not guarantee a high value in the finished product. Dilution into the formulation, partitioning between phases, interaction with the emulsifier, and losses during manufacture and storage all reduce the capacity actually present, which is why the assay is run on the finished product where a claim is made on it. A product whose antioxidant capacity falls by half over 12 weeks at 40 °C has an antioxidant claim with a shelf-life problem, and the stability report says so.

Comparisons are valid only between samples prepared and assayed under the same conditions. A Trolox equivalent value from one laboratory using a 30-minute read in ethanol is not comparable with one from another using a plateau read in methanol, and published values for botanical extracts should be treated as indicative at best.

Limitations

DPPH is a synthetic radical that does not occur in skin; it is soluble only in organic solvents; and its response is influenced by solvent, pH, steric hindrance around the antioxidant’s active site, and the presence of coloured compounds. It measures a property of the product, not an effect on the consumer.

Those limits are managed in the method rather than removed by it. Reading to the plateau rather than at a fixed time captures slow-acting antioxidants that a 30-minute read underestimates. Running the assay on the finished product, and on aged samples through a stability programme, answers the question that matters for a claim: whether the antioxidant is present and active in the product the consumer buys, for as long as they use it. Where the brand’s claim is about an effect on the skin, such as protection from free radical damage, oxidative stress or pollution, the DPPH result is the starting point and the substantiation comes from ex vivo or in vivo work under our clinical efficacy studies.

What the report contains

• Sample identification, form, batch and date of receipt
• Sample preparation: solvent, extraction, centrifugation and any correction applied
• Assay conditions: DPPH concentration, solvent, temperature, read time and time to plateau
• Percentage inhibition at each concentration, IC50 with the DPPH concentration used, and Trolox equivalents in µmol TE/g, each with mean, standard deviation and replicate count
• Trolox calibration curve and positive control result
• For comparative or time-series work, a table and plot across formulations or time points
• Interpretation: what the result substantiates under Regulation (EU) No 655/2013 and what it does not

What to send

10 g or mL of finished product, or 5 g or mL of extract, oil or raw material, from a single batch, in amber glass or opaque packaging protected from light. Send the formulation with percentages so that the antioxidant of interest can be identified and the extraction designed around it. For a stability series, send the aged samples with their storage history, or arrange for the assay to be run as part of a stability programme in our laboratory. Results are reported within 5 working days.

How it fits with the CPSR

Where an antioxidant claim is made on the label, the DPPH result and its stability series are the substantiation filed in the Product Information File under the claims section, and the CPSR records that the claimed active was present and active at the end of the tested shelf life. Where we prepare both, the assay is scheduled into the stability programme so that the data exist when the claim is written rather than being sought afterwards.