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

Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that can damage cells. Fortunately, plants and animals maintain complex systems of antioxidants such as glutathione and vitamins C and E, as well as enzymes including catalase, superoxide dismutase and various peroxidases, to defend against oxidative stress. Antioxidants may terminate the chain reactions that damage cells either by removing radical intermediates or by inhibiting other oxidation reactions by being oxidized themselves.1-3

Skin is directly and frequently exposed to oxidative stress such as UV radiation (UVR), which is recognized as the most oxidative exogenous factor behind skin problems. While healthy skin possesses an antioxidant defense system against oxidative stress, excessive free radical attack (for example, overexposure to UVR) can overwhelm the cutaneous antioxidant capacity and lead to oxidative damage, which can ultimately cause skin cancer, immunosuppression and premature skin aging.2-4 Supplying exogenous antioxidants may therefore play a key role in preventing or minimizing UVR-induced photoaging.4-8

New methodologies have recently been developed to determine antioxidant effects but they often require extensive training and are time-consuming to conduct. In the present article, however, the authors describe an in vitro method to detect the effects of antioxidant-containing formulations8, 9 using photochemiluminescence to provide rapid, accurate and sensitive measurements.

Antioxidant Selections
Many plants have developed naturally protective substances to enable their continuous survival under direct and intense UVR. One advantage of natural products is their high structural diversity and variety of biological activity; and while they often are chemically complex structures, they also can be obtained through simple extractions and in high quantities and at a low cost. Therefore, antioxidants extracted from plants are of great interest.

The present study examined a novel antioxidant complex (NAOC)a of plant extracts suggested by the manufacturer to possess a powerful antioxidative capacity. In addition, idebenoneb—a lower molecular weight antioxidant analogue of coenzyme Q1010, 11—was tested for comparison purposes. The antioxidant capacities of 3% NAOC and 1% idebenone were determined using a photochemiluminometer systemc. These concentrations were chosen since they typically are used in popular non-prescription antioxidant skin formulations.

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 Ed 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 1e, antioxidative capacity of lipid-soluble (ACL) reagent 1f, methanol (HPLC grade)g, hexanes (HPLC grade)h, heptanej and butanolk.

Antioxidant samples: A solution of 3% NAOC in water was prepared (v/v). In addition, 1 g of idebenone was mixed with 100 mL of methanol (w/v). As noted, a standard curve was established with a series concentrations using these samples with synthetic vitamin E.

Results of the solubility test indicated that the ACL reagent 1 was suitable for both test materials, hence it was used for this study. The NAOC retained a wide solubility range: from the hydrophilic solvent and ACW reagent 1, to the five liphophilic solvents tested. The solubility range of idebenone, however, was relatively narrow.

As far as the standard calibration curve for vitamin E (see Figure 1), the X and Y axis reciprocal and linear regression followed: 1/Y(X) = 94.69251 * and (1/X)2 + 53.56752 * (1/X) + 0.57010; R2 = 0.9938.

Antioxidant capacity: The quantity of antioxidant capacity measured for the 3% NAOC and 1% idebenone were 525 ± 23 (nmol) and 213 ± 14 (nmol), respectively; these are equivalent to the antioxidant activity of synthetic vitamin E. The 3% NAOC, however, showed significant (p < 0.0001) antioxidative capacity and measured nearly 2.5 times stronger than the 1% idebenone.

This study demonstrates that the antioxidant capability of 3% NAOC as a superoxide anion radical quencher, as measured by photochemiluminescence, exceeded idebenone by a factor of 2.5. One possible explanation for this result could be a synergistic effect between the NAOC plant extracts, which include known antioxidants such as anthocyanins, ferulic acid, caffeic acid and other polyphenols. This combination could generate benefits beyond that of a single antioxidant.

Additionally, NAOC retained a wide solubility range, from a hydrophilic solvent to the five liphophilic solvents tested, whereas the solubility of idebenone was relatively narrow. This compatibility would give NAOC an added advantage when considering its combination with other cosmetic ingredients.

Providing a reliable analytical method to detect the antioxidative capacities of even low antioxidant concentrations in a sample is of major interest, and photochemiluminescence analysis has advantages over other methodologies.4-11 Taken together, it is a simple, sensitive, economical, convenient, and reliable method to provide a practical preclinical screening of antioxidant materials—prior to animal/human trials.

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1. KJ Davies, An overview of oxidative stress, IUBMB life 50 241-244 (2000)
2. JJ Thiele, F Dreher and L Packer, Antioxidant defense systems in skin, in Cosmeceuticals, Drugs vs. Cosmetics, P Elsner and HI Maibach, eds, New York: Marcel Dekker (2000) pp 145-187
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*Revised with the permission of Blackwell Publishing from: H Zhai, M Cordoba-Diaz, C Wa, X Hui and HI Maibach, Determination of the antioxidative capacity of an antioxidant complex and idebenone: An in vitro rapid and sensitive method, Journal of Cosmetic Derm 7(2) 96-100 (2008).

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