Antioxidant Activity Test

WHY CONDUCT AN ANTIOXIDANT TEST?

The OxiSelect™ method predicts the antioxidant potential of ingredients or formulations against all major types of Reactive Oxygen Species (ROS), using human epidermal keratinocyte cultures as an in vitro system with a high level of physiological relevance for human skin. The test can be used to provide supporting data for a number of claims including antioxidant, anti-ageing, anti-pollution and anti-inflammatory.

OVERVIEW

Oxygen is an essential requirement for life, but it is also a very reactive molecule and can be harmful when converted to reactive oxygen species (ROS) within the body. ROS are unstable and react with most biological molecules, causing damage to vital cellular components such as DNA, proteins and lipids, contributing to inflammation and ageing. Stress and exposure to air pollutants can dramatically increase ROS levels in the skin and lead to an acceleration of the inflammatory and ageing pathways. Our cells are equipped with natural antioxidant enzymes to prevent ROS from forming, or to remove them before they cause damage. Providing our skin with an antioxidant boost through active ingredients in cosmetic products can help to enhance the skin’s own natural protective mechanisms. Antioxidant ingredients may therefore support a variety of claims including anti-pollution and anti-ageing.

The OxiSelect™ test method exposes human skin cells (epidermal keratinocytes) to a range of concentrations of the active ingredient(s), either alone or in combination. Unlike antioxidant tests that utilise simple biochemical reactions in a test tube, the use of whole cells provides a more complete environment with higher physiological relevance, accounting for important aspects such as pH, cellular uptake and metabolism, as well as demonstrating the efficacy of the test item. OxiSelect™ captures anti-oxidant contributions from all major types of ROS, including peroxides, superoxides, hydroxyl radicals and singlet oxygen.

The test is based on the intracellular conversion of DCFH to its fluorescentmetabolite (DCF). The reaction is inhibited by antioxidants, reducing the amount of fluorescence observed. OxiSelect™ is suitable for ingredients or formulations with sufficient aqueous solubility in cell culture medium.

TEST SYSTEM: HUMAN KERATINOCYTES IN ANIMAL-FREE CONDITIONS

A OxiSelect™ uses human skin cells (epidermal keratinocytes) in animal product-free culture, providing a physiologically relevant environment. Cells are obtained from a commercial source with full donor consent and QC screening.

B The method uses a specialised marker called DCFH-DA, which diffuses into cells and is metabolised by cellular esterases. The metabolite (DCFH) is rapidly oxidised to highly fluorescent 2’,7’-DCF by reactive oxygen species (ROS) in the presence of the proprietary “Free Radical Initiator”. The fluorescence intensity is proportional to the levels of ROS within the cell, and is therefore reduced in the presence of antioxidant active ingredients. The plant flavonoid, quercetin, is used as a positive control.

SUMMARY OF THE TEST METHOD

• Preliminary steps establish the solubility of the active ingredient or product and a cytotoxicity screen determines the highest concentration that the cells will tolerate without causing cell damage.
• Human epidermal keratinocytes are cultured until 80-90% confluent in 96-well plates, in animal product-free culture conditions.
• DCFH-DA probe solution is added to the cells along with Quercetin (positive control), or the active ingredient(s) or product, at the specified concentrations.
• After incubation for 60 minutes at 37°C, cells are rinsed and Free Radical Initiator solution is added to the wells.
• Fluorescence intensity (to quantify DCF) is read immediately and monitored over a 60-minute period, in increments of 5 minutes.
• Results for each of the test conditions are plotted in graph format, to demonstrate any dose-dependent antioxidant potential of the active ingredients or product, either alone or in combination.

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ANTIOXIDANT TESTING CASE STUDY

One of our clients wanted help to determine the antioxidant potential of five different active ingredients for a new anti-ageing skincare formulation. Read the case study to discover how our in vitro test allowed the active ingredients to be ranked based on different concentrations and combinations.