A Rapid and Sensitive In vitro Method to Ascertain Antioxidative Capacity*

By: Hongbo Zhai, MD, and Howard I. Maibach, MD, University of California San Francisco

Posted: January 29, 2010, from the February 2010 issue of Cosmetics & Toiletries.

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Photochemiluminescence Analysis
Briefly, the principle of photochemiluminescence involves generating defined free radicals—superoxide anion radicals, in this case—by exposing a photosensitizer to a UV light source. The free radicals produced are detected by the system via their reaction with a chemiluminogenic substance and the light they subsequently emit; these light flashes are detected in the device by a photomultiplier.

The radicals generated are then partially scavenged by a reaction with the sample antioxidants and the remaining radicals are quantified by the same detection principle described. The intensity of the photochemiluminescence measured is attenuated as a function of the antioxidant concentration within a sample, and the results are presented in equivalent concentration units of synthetic vitamin E^d for lipid-soluble substances or ascorbic acid for water-soluble substances.

Vitamin E is a collective term used to describe eight related tocopherols and tocotrienols; these fat-soluble antioxidant vitamins are considered to be key indicators of the skin’s response to oxidative stress.¹² Concentrations ranging from 10 μg to 100 μg of standard vitamin E were used to establish a calibration curve, and a detector signal for each run was monitored for 180 sec. Analyses were performed according to the standard method described by Popov and Lewin.¹³

A light emission curve was recorded for a duration of 180 sec and the amounts of antioxidative substances tested were calculated and expressed as nmol equivalents in antioxidant activity of synthetic vitamin E; this establishes a standard calibration curve prior to testing the samples. Details of measuring method and principles of photochemiluminescence analysis can be found elsewhere.^{13, 14}

Sample Preparations
Solubility samples: To determine the compatibility of the NAOC and idenenone with commonly used test compounds, 10 mg of each was mixed with 5 mL of the six following common solvents: antioxidative capacity of water-soluble (ACW) reagent 1^e, antioxidative capacity of lipid-soluble (ACL) reagent 1^f, methanol (HPLC grade)^g, hexanes (HPLC grade)^h, heptane^j and butanol^k.