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

Feb 1, 2010 | Contact Author | By: Hongbo Zhai, MD, and Howard I. Maibach, MD, University of California San Francisco
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Title: A Rapid and Sensitive In vitro Method to Ascertain Antioxidative Capacity*
antioxidative capacityx skinx UV radiationx photoagingx photochemiluminescencex
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Keywords: antioxidative capacity | skin | UV radiation | photoaging | photochemiluminescence

Abstract: 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 formulations using photochemiluminescence to provide rapid, accurate and sensitive measurements.

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H Zhai and HI Maibach, A dermatological view—A rapid and sensitive in vitro method to ascertain antioxidative capacity, Cosm & Toil 125(2) 20-24 (Feb 2010); 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, J Cosmetic Derm 7(2) 96-100 (2008)

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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.

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. Supplying exogenous antioxidants may therefore play a key role in preventing or minimizing UVR-induced photoaging.

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 formulations 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) of plant extracts suggested by the manufacturer to possess a powerful antioxidative capacity. In addition, idebenone—a lower molecular weight antioxidant analogue of coenzyme Q10—was tested for comparison purposes. The antioxidant capacities of 3% NAOC and 1% idebenone were determined using a photochemiluminometer system. These concentrations were chosen since they typically are used in popular non-prescription antioxidant skin formulations.

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Figure 1. Standard calibration curve of synthetic vitamin E

Figure 1. Standard calibration curve of synthetic vitamin E

As far as the standard calibration curve for vitamin E, 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.

Footnotes

a NAOC (INCI: Water (aqua) (and) Hibiscus Sabdariffa Flower Extract (and) Ferula Assa Foetida Root Extract (and) Pyrus Communis (Pear) Fruit Extract (and) Camellia Sinensis Leaf Extract) is a product from Basic Research Inc.
b The idebenone used for this study was obtained from Basic Research Inc.
c The Photochem system used for this study is manufactured by Analytik Jena AG and Analytik Jena USA, Inc.
d Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble derivative of vitamin E, is a product of Hoffman-LaRoche.
e Antioxidative Capacity Water-soluble (ACW) Reagent 1 is a product of Analytik Jena AG and Analytik Jena USA, Inc.
f Antioxidative Capacity of Lipid-soluble (ACL) Reagent 1 is a product of Analytik Jena AG and Analytik Jena USA, Inc.
g The HPLC grade methanol used is a product of Fisher Scientific, Fair Lawn, NJ.
h The HPLC grade hexanes used are products of Fisher Scientific, Fair Lawn, NJ.
j The heptane used is a product of Mallinckrodt Chemical Works, St. Louis, MO.
k The butanol used is a product of Mallinckrodt Chemical Works, St. Louis, MO.

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