262 JOURNAL OF COSMETIC SCIENCE research group has demonstrated that high protection against oxidation and degradation of ascorbic acid occurs when it is dissolved in the inner aqueous phase of the W/O/W multiple emulsion (4,5). We also demonstrated that in an W/O emulsion, ascorbic acid is more stable than in an O/W emulsion, and that the stability of ascorbic acid is increased by reducing the pH value of the inner aqueous phase (4,5). Retinoids are a large class of compounds that are important in modern therapy for the dermatological treatment of wrinkled skin (6). Retinal and its congeners are present in all living organisms, either as vitamin A or as carotenoids, some of which are provita­ mins A (7). Among the retinoids, retinal and retinyl palmitate are thought to induce thickening of the epidermis and to be effective for treatment of skin diseases (8). These functional substances, however, are known to be unstable if exposed to light or heat. A characteristic feature of retinoids is their sensitivity to ultraviolet radiation in fact, UVB and UV A radiation reduces the vitamin A content of the human epidermis (9). Although retinal and retinyl palmitate are less dangerous than retinoic acid (which, due to its irritative properties, is acceptable only for therapeutic treatment), the effect of ultraviolet rays on these compounds makes their use in dermatology more difficult. In a previous paper (10) we studied the photostability of vitamin A and of its palmitic ester in ethanol and in octyl octanoate solutions. The vitamins were irradiated alone and upon addition of sunscreens and butylated hydroxytoluene. The photostability of vita­ mins was also studied in an O/W fluid emulsion with and without butylated hydroxy­ toluene (BHT). Among the tested sunscreens, butyl methoxy dibenzoyl methane (Eu­ solex®9020) showed the strongest protective action toward vitamin A and vitamin A palmitate. Butylated hydroxytoluene inhibited the photodegradation of both vitamins, suggesting that oxygen may be involved in their degradation. In two other studies (11,12) we evaluated the protective effect of SLN (solid lipid nanoparticles) encapsula­ tion on the photodegradation and thermal degradation of retinyl palmitate introduced in different media. The aim of this work was to study the stability and effectiveness of two commercial products containing retinal in their formulations. In particular, we were interested in investigating the anti-aging effectiveness of the two products by studying the physico­ chemical stability of retinal. It is clear that the aluminum tubes and the appropriate steps adopted by the factory in packaging the RETI C and RETI C concentrate emul­ sions assure their stability during the storage period, as they minimize the contact of the functional ingredients with oxygen and light, but we were primarily interested in studying the behavior of these two products once applied to the skin. On the other hand, everybody knows that, especially during the summer season, the tubes, during storage, could be subjected to a sudden change in temperature, reaching even higher than 30°C, especially after purchase. Moreover, once applied to the skin, the thin layer of product remains there for several hours, coming in contact with lots of damaging factors like air (oxygen), UV-IR irradiation, and high temperature. In this context, our tests could be very important because they are accelerated tests of stability-hence they allow us to predict what could happen to the products in the worst conditions. MATERIALS AND METHODS Ethanol absolute and methyl alcohol were purchased from Carlo Erba, aluminum sulfate was from Schiapparelli, octyl octanoate was a gift from Dragoco, and all-trans-retinal

ANTI-AGING EFFECTIVENESS OF RETINOL EMULSIONS 263 was from Sigma. RETI C emulsion (water, glycerin, hydrogenated polysobutene, octo­ crylene, prunus armeniaca, zea maize, ascorbic acid, butyl methoxydibenzoylmethane, isohexadecane, retinal, BHT, glycine soja, triethanolamine, dimethicone, methylpara­ ben, sodium hydroxide, arginine PCA, phenoxyethanol, magnesium sulfate, ammonium polyacryloyldimethyl taurate, disodium EDTA, tocopherol, propylparaben, tetrasodium EDTA, acrylates/C10�3o alkyl acrylate crosspolymer, polyglyceryl-4 isostearate, cetyl dimethicone copolyol, hexyl laurate, butylparaben, ethylparaben, and perfume) was a gift from Vichy® (Milan, Italy). RETI C concentrate emulsion (water, hydrogenated polysobutene, squalane, glycerin, ascorbic acid, cetyl alcohol, PEG-40 stearate, glyceril stearate, prunus armeniaca/apricot kernel oil, sodium hydroxide, zea maize/corn starch, petrolatum, retinal, PEG-10, sor­ bitan tristearate, glycine soja/soybean oil, methylparaben, arginine PCA, phenoxyetha­ nol, tocopherol, disodium EDTA, chlorhexidine digluconate, propylparaben, hydroge­ nated lecithin, phosphoric acid, polycaprolactone, CI 14700, FD&C red N°4, CI 47005/ D&C yellow N°10, and perfume) was a gift from Vichy® (Milan, Italy). RETINOL CONCENTRATION The determination of retinal concentration in two commercial products (RETI C and RETI C concentrate emulsions) was performed by HPLC at the opening and after three months. The emulsions studied were supplied by the factory (Vichy), kept in their original aluminum tubes, and stored protected from direct sunlight and sources of heat. HPLC METHOD ANALYSIS The concentration of retinal was determined by HPLC analysis using a LC-10 AD pump unit control, a SPD-10 AV UV-Vis spectrophotometric detector set at 325 nm, and a C-R6A Chromatopac integrator (Shimadzu Corporation, Kyoto, Japan). An RP Cl8 column (80 mm x 4.6 mm) was used. The mobile phase consisted of methanol/ethanol/ water (66.6/16.7 /16.7, v/v). The flow rate was 0.8 ml/min. Samples were prepared as follows: 0.5 grams of emulsion were diluted with 2.5 ml of octyl octanoate, and 0.5 grams of aluminum sulfate were added. The mixture was stirred on vortex and centrifuged at 3000 rpm for ten minutes. After centrifugation, the oil phase was diluted in a 1 :20 ratio with ethanol and analyzed by HPLC. The determi­ nations were repeated three times. RADIATION STABILITY STUDY RETI C and RETI C concentrate emulsions were irradiated under UVB and UV A lamps in a solarbox. The radiation intensity under the lamps was measured, under the same conditions adopted for sample irradiation, using a multimeter (CO.FO.ME.GRA., Mi­ lan, Italy), at 10 cm from the lamps, equipped with a probe sensitive to radiation in the wavelength interval of 290-400 nm. The radiation power per unit area emitted by the UVA lamp was 8.9 x 10- 4 W cm- 2 and that emitted by the UVB lamp was 2.6 x 10- 4 W cm - 2 . It must be considered that the skin, all the year round, receives a high amount of UVA radiation and, especially during summertime, a large amount of UVB radiation.