JOURNAL OF COSMETIC SCIENCE 390 Besides its biological and physiological functions, skin is a predominant factor in terms of physical appearance, like a “social interface,” it is an essential beauty criterion (3). Skin structure and appearance alterations caused, for instance, by aging or by sun exposure are often not well accepted. Hence, use of cosmetic treatments formulated to slow down skin damages is extremely widespread. Furthermore, progresses made in the fi eld of medicine increase life expectancy (4) we can easily assume that demand of this type of topical treat- ment will constantly increase (5). By contrast to most internal organs that mainly age through intrinsic mechanisms (chronological aging), aging of the skin is also subjected to extrinsic factors (environmen- tal stresses) (6). Together, these two types of alterations often result in well-known char- acteristics: skin undergoes pigmentation changes and becomes wrinkled, saggy, and dry. Actually, most physical damages have their origin in dermis thickness reduction induced by fi broblast number and activity decreases. Hence, preservation or restoration of dermal fi broblast function is a goal that many anti-aging cosmetics claim to reach. It is usually considered that free radicals are largely responsible for skin aging. Thus, most cosmetic treatments contain natural (i.e., retinol, ascorbic acid, tocopherol, etc.) or modifi ed (i.e., tetra-isopalmitoyl ascorbate, tocopherol acetate, etc.) antioxidant com- pounds (7,8). It is also generally known that extracts from yeast S. cerevisiae may also be benefi cial with regard to skin texture and appearance (8). To date, mainly two types of bioactive yeast extracts have emerged: chromium- and β-glucan-rich extracts (9–15). Meanwhile, another type of yeast extract (MYE for Methanol Yeast Extract), has been described in a different context which is that of the alcoholic fermentation. This extract differs from traditional yeast extracts by its mode of production. Our group and others have shown that it had a positive effect on S. cerevisiae (16–19). Actually, MYE has been shown to accelerate the metabolism of carbohydrates via an increase in glucose transport, glycolytic fl ux, and intracellular content of fructose-2,6-bisphosphate and via the activa- tion of the pyruvate decarboxylase. It was also shown that this extract was able to increase S. cerevisiae resistance to various stresses such as oxidative, alcoholic, osmotic, and UV- induced. In addition, MYE increases longevity of S. cerevisiae, a reference model for study- ing genetic and physiological modulations regulating longevity in higher organisms (20). Thus, all these observations led us to study the effect of this extract on higher eu- karyotic cells and particularly on fi broblasts that play a major role in dermis structure. MATERIAL AND METHODS YEAST EXTRACTION METHOD Two kilograms of pressed yeast S. cerevisiae (Algist Bruggeman, Gent, Belgium) were ex- tracted with 2 l of pure methanol at room temperature and stirred for 24 h. After cen- trifugation and fi ltration, the bioactive fraction contained in the supernatant was precipitated by addition of 4 volumes of acetone. The precipitate was then recovered in 500 ml of distilled water and the bioactive fraction was isolated by differential precipita- tion with ethanol. Briefl y, after addition of 35% v/v ethanol, precipitated fraction was discarded and after adding 50% ethanol, precipitated fraction was, this time, recovered (viscous precipitate). Consecutive additions of ethanol at 70% and 100% induced the precipitation of inactive compounds. Note that all fractions were tested with regard to

EFFECT OF METHANOL YEAST EXTRACT ON 3T3 FIBROBLAST CELLS 391 cell proliferation (data not shown). As a result, we obtained a viscous brownish and water soluble bioactive complex (MYE for Methanol Yeast Extract). EVALUATION OF THE ANTIOXIDANT PROPERTY OF MYE The antioxidant capacity of MYE was evaluated by the DPPH method (1,1 diphenyl pycril 2 hydrazil) following a protocol adapted from Szabo et al. (21). Briefl y, a methanol solution of 1 mM DPPH was incubated in the presence or absence (control) of MYE at 0.001%, 0.01%, 0.1%, and 1% v/v. After 15 min incubation at room temperature, ab- sorbance at 517 nm was measured using an Oasis UVM340 spectrophotometer. Antioxi- dant effect of the yeast extract was compared to the one of ascorbic acid at concentrations ranging from 0.001 to 1mM. MEASUREMENT OF YEAST PROLIFERATION About 5 × 106 cells of S. cerevisiae previously grown on rich medium were used to inoculate 50 ml of glycerol minimal medium (4% glycerol/0.17% yeast nitrogen base/0.5% ammo- nium sulphate Difco, Detroit, MI) (22) supplemented or not (control) with MYE at 0.01%, 0.1%, and 1%. After 24, 48, and 96 h of incubation at 30°C with shaking, growth was assessed by measuring the absorbance at 660 nm using a Unicam UV1 spectrophotometer. ANIMAL CELL CULTURE 3T3 cells (mouse embryonic fi broblasts) were from DSMZ bank (Braunschweig, Ger- many). Cells were cultured in DMEM medium supplemented with 4 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal bovine se- rum (FBS) (all reagents from Invitrogen/Gibco, Carlsbad, CA). Cells were maintained at 37°C in a humid atmosphere containing 5% CO2. MTT CYTOTOXICITY ASSAY 3T3 cells were seeded in 96-well plates (3000 cells/well). Twenty four hours after seed- ing, cells were treated or not (control) with MYE at 0.01%, 0.1%, and 1% for a period of 48 h. Cell viability was then assayed by exposure to 0.03% MTT w/v. After removal of medium, produced formazan was dissolved in DMSO (1 h, room temperature, under agitation) for measurement by spectrometry at 550 nm using an Oasis UVM340 spectrophotometer. 3T3 CELL GROWTH MEASUREMENT 3T3 cells were seeded in 96-well plates (3000 cells/well). Twenty four hours after seeding, cells were treated or not (control) with MYE at 0.01% and 0.1% for a period