Modified Rancimat Method for Evaluation of Antioxidative Effect against Skin Lipids

Abstract

Skin is a highly complicated tissue which possesses the largest surface area in the body and serves as the protective layer for internal organs. It is designed to give both physical and biochemical protection, and is equipped with a large number of defense mechanisms. 1 However, skin is very susceptible to oxidative reaction, because there are rich contents of lipids, proteins and DNA in the skin cell, all of which are very sensitive to the oxidation process. 2 Lipid peroxidation could be induced from excessive exposure to UV light, and has been considered as a major factor for skin aging processes. 3 In particular, squalene, main component of skin surface polyunsaturated lipids, is easily peroxidized. 4 The initial product of peroxidized squalene, squalene-monohydroperoxide (Sq-OOH), is produced at the human skin by natural exposure to sun light. Recently, the damages of hairless mice skin by the topical application of Sq-OOH 5 have been reported. Repeated application of Sq-OOH to the hairless mice induced clear skin wrinkles. Thus, in order to prevent or retard skin aging process, prevention of skin lipid peroxidation by natural or chemical antioxidants is desirable. To identify efficient antioxidants in cosmetic applications, a convenient assay method suitable for the estimation of skin lipid peroxidation is necessary. Rancimat 6 is a convenient method which can evaluate the lipid peroxidation by observing the change of electrical conductivity due to the formation of volatile dicarboxylic acids. It has been used in food industry to determine the peroxidation of unsaturated fatty acid components. However, the rancimat method is not suited for skin research, because the composition of oils used as a lipid source in this method is very different from that of skin lipid. 6c In this report, we demonstrate that modified rancimat method is a powerful tool for screening natural antioxidants for skin lipid peroxidation. We changed composition of lipid source to what is very close to skin lipid. At first, we tested antioxidative activities of several natural extracts by using general rancimat method. As shown in Table 1, extracts from Korean medicinal plants presented different induction time on retarding the peroxidation of corn oil. Their activities were compared with BHT as a positive control. Among the three natural extracts, Scutellaria baicalensis Georgi (SBG) extract was found to have the most powerful antioxidative activity. Portulaca oleracea L. (POL) extracts appeared to be the compound with the next activity and Rheum coreanum Nakai (RCN) extract was less active than other extracts. The order of activity is BHT > SBG > POL > RCN. However, ranges of Pf values of tested natural product are too small (from 1.2 to 1.6). Thus, sensitivity is not enough for primary screening of antioxidative activity. To establish the modified rancimat system suitable for skin lipid peroxidation studies, we prepared the artificial skin lipid and used it instead of corn oil. The prepared artificial skin lipid 7 was consisted of pork lard (33%), stearic acid (24%), lanolin (22%), squalene (12%) and cholesterol (4%). This composition is similar to that of human skin. According to the Table 2, the results of new rancimat method were different from previous general rancimat data. Surprisingly, SBG extract shows the strongest antioxidative activity among the tested samples. Induction period of SBG was 11.4 times longer than that of control with this new method. The sensitivity for evaluation of antioxidative activity was dramatically increased due to the wide ranges of Pf value (from 2.2 to 11.4). It is interesting to observe that the order of activity between RCN and ROL was reversed. In contrast to previous rancimat data, RCN is more active than POL. These interesting and meaningful differences between general rancimat and modified method may be due to changing of lipid composition which is suited to skin research. In search of new antioxidant applicable to prevent skin lipid peroxidation, we selected meso-dihydroguaiaretic acid (MDGA) 8 as a candidate. MDGA, a naturally occurring polyphenol,