218 JOURNAL OF COSMETIC SCIENCE moisturization depends on lipid presence in the stratum corneum, and it guarantees a softer and healthy skin with a more pleasing appearance (4). When the barrier function is damaged by a surfactant, organic solvent, or tape stripping, a series of homeostatic systems is accelerated and the barrier function recovers its original level (5). First, exocytosis of lipid-containing granules, lamellar bodies, is accelerated and the inside lipids are secreted into the intercellular domain between the stratum granu­ losum and the stratum corneum and form a water-impermeable membrane (5). Then, lipid synthesis and processing are speeded up. Finally, the barrier function recovers to its original level. In order to increase the rate of the homeostatic response in the epidermis and the return of the barrier function to normal, different strategies have been studied. Many research­ ers (6-8) demonstrated that a topically applied mixture of stratum corneum lipids, i.e, ceramide, cholesterol, and free fatty acids, accelerated repair of the barrier function after its damage. Furthermore, Yang et al. demonstrated the efficacy of an optimal non­ physiological lipid mixture in increasing the recovery rate of the disrupted skin barrier (9). Soybean-germ oil deserves an important place among nutraceuticals because of its very high content of tocopherols, phytosterols, polyunsaturated fatty acids, carotenoids, and other vitamins (10). This pool of substances represents a natural defense against oxidative damage caused by free radicals derived from cellular aging (10). Particularly, various scientific works have demonstrated the efficacy of soy phytosterols, such as �-sitosterol, in the treatment of erythema, chilblains, and chapping, and as ingredients in topic formulations in the treatment of burns (11-13). As previously observed, the stratum corneum represents a valid shield against the penetration of exogenous and toxic substances and its removal is often followed by an amplification of skin irritant phenomena compared to normal conditions. Therefore, the aim of the present work was to study the effect of the topical application of soybean phytosterols on skin barrier recovery in human volunteers using the extent of methyl nicotinate (MN)-induced erythema in damaged skin as a parameter to evaluate the rate of stratum corneum recovery. MN was chosen as an erythematogenous substance for its capability to cause an erythema whose intensity and duration are proportional to the quantity of the substance that has entered the living epidermis over time (14). MN­ induced erythema was monitored using reflectance spectrophotometry as a noninvasive instrumental technique. MATERIALS AND METHODS MATERIALS Soy phytosterol mixture was obtained from Sochim International (Milan, Italy) and its composition is reported in Table I methyl nicotinate (MN) was obtained from Fluka (Switzerland) Cetiol B (d-n-butyl adipate) was obtained from Henkel (Dusseldorf, Ger­ many). All other chemicals were of reagent grade. INSTRUMENTATION MN-induced skin erythema was monitored by using a reflectance visible spectropho-

SOYBEAN PHYTOSTEROLS AND SKIN BARRIER RECOVERY 219 Table I Composition of Soy Phytosterol Extract Compound 13-Si tosterol Campesterol Stigmasterol Brassicasterol Stigmastanol Ergostanol Total tocopherols % (w/w) 47 25 22 2-3 l l l tometer, X-Rite model 968 (X-Rite Inc. Grandville, MI), calibrated and controlled as previously reported (15). From the reflectance spectra, obtained over a wavelength range of 400-700 nm, the erythema index (E.I.) was calculated using equation 1: E.I. = lOO[log - 1 -+ 1.5 ( log - 1 -+ log- 1 -)- 2(1og - 1 -+ log - 1 -)] (1) Rs6o \ Rs4o Rsso Rs10 R610 where 1/R is the inverse reflectance at a specific wavelength (560, 540, 580, 510, and 610). PROTOCOL In vivo experiments were performed on eight volunteers of both sexes in the age range of 25-35 years. The volunteers followed a "sterol-free" diet with a duration of one month, to exclude the phytosterol dietary contribution. They were recruited after medi­ cal screening, including a health questionnaire followed by physical examination of the application sites. After they were fully informed of the nature of the study and of the procedures involved, they gave their written consent. The participants did not suffer from any ailment and were not on any medication at the time of the study. They rested for 15 min prior to the experiments, and room conditions were set at 22° ± 2°C and 40-50% relative humidity. In the first part of the experiment, six sites of the volar aspect of the forearm of each volunteer were defined using a circular template (1 cm2) and demarcated with perma­ nent ink. In each site 50 µl of MN aqueous solution (0.5% w/v) was applied for 15 min using Hill Top chambers (Hill Top Research, Cincinnati, OH) (1 cm2). After MN­ occlusive treatment, the chambers were removed and the skin surface was washed, to remove the formulations tested, and allowed to dry for 15 min. The induced erythema was monitored for 10 h. E.I. baseline values were taken before application of the formulations tested, and they were subtracted from the E.I. values obtained after MN application at each time point, to obtain the LiE.I. values. After plotting the LiE.I. values versus time, area-under-curve (AUC) values were determined for each subject by calcu­ lating the areas between the response curve and the x-axis. The day after (24 h), twenty individual 1-cm2 squares of adhesive tape (Scotch® Book Tape 845, 3M) were utilized to sequentially tape-strip the stratum corneum on the application sites. Again the application of MN aqueous solution (0.5% w/v) for 15 min was repeated and, for each site, LiE.I. values were obtained and plotted as reported before.