ARECA CATECHU L. EXTRACT 289 3% CC-516, untreated cream as control), and we even prefer the simple left-right control lateral comparison design (36). The cutometer SEM 575 (C+K Electronic GmbH, Germany), which is used for skin elasticity measurements, is based on the suction method. Negative pressure is created in the device, which can be regulated between 20 and 500 mbar. Skin is drawn into the aperture by negative pressure where the skin penetration depth (=ds) is determined by a new non-contact optical measuring system. The optical measuring system consists of a height transmitter and a height recipient. The light intensity will vary due to the penetration depth of the skin. This means the smaller penetration depth, the greater the elasticity. To measure wrinkles by using the image analyzer, we analyzed wrinkles on the monitor by a three-dimensional skin system program, and then measured the number of wrinkle peaks and the depth of the wrinkles. The measuring principle of the skin visiometer SV 400 is based on transmission of a very thin and specially dyed silicon replica. The wrinkles were per- pendicular to the axis of the light beam, and measurements of the number (N) and depth (P) of wrinkles were recorded in this position by the program. Test sites were taken under the eyes and forearm of twelve volunteers. The test sample (cream containing 3% CC-516) was applied to the volunteers twice a day for six weeks on designated test sites (37,38). SEM examination was performed on dyed silicon replicas of the tested areas that had been air-dried, coated with a thin layer of gold-palladium, and viewed in a scanning electron microscope (JSM-840A, Jeol Co.) at 25 kV. RESULTS AND DISCUSSION We previously screened the inhibitory effect on elastase from methanolic extracts of 150 medicinal plants. The Areca catechu extract showed a high inhibitory effect comparable to reference compounds (18). For the inhibitory effect of several solvent extracts on elastase, the ethanolic extract, CC-516, was highest and used as the sample in this study. Table I shows the composition of CC-516, which contains relatively high amounts of protein (26%, w/w) and carbohydrate (37.5%, w/w), and low amounts of lipid (2.8%, w/w). Figure 1 represents the composition and content of amino acids. The presence of alanine, glycine, and proline in CC-516 is worth something, since a basic collagen contains mainly glycine, proline, and alanine. The proline and lysine of amino acids are the biosynthetic precursors to collagen. Table II lists the composition and contents of fatty acids of the hexane fraction. The analysis revealed that the fraction consisted of lauric, myristic, palmitic, stearic, oleic, linoleic, and other acids. Myristic (33.3%) and Table I Composition of CC-516 Protein Carbohydrate Lipid Flavonoid Ash Composition (1) (2) (3) (4) (5) (mg/g) 260 375 34 86 9 (1) Protein concentrations were measured by the Lowry assay (24). (2) The amount of carbohydrate was determined by the phenol-sulfuric assay (25). (3) Total lipid contents were determined by the hexane extraction method. (4) Total flavonoid contents were carried out by using the photometric method of 280-nm absorption detection (26). (5) Ash contents were determined by heating in a muffle furnace (600øC, 6 hr).

290 JOURNAL OF COSMETIC SCIENCE l0 (%) 0 • •: :'' ,9 •.•} 7 •- -. ß _a ' ß ß Figure 1. Percentage of total amino acids in CC-516. Free amino acid content was 0.2% determined by amino acid analyzer (TCX 3100, ACS, UK). Table II Composition Analysis of Fatty Acids in CC-516 by GC/MS Fatty acid Lauric Myristic Palmitic Stearic Oleic Linoleic Composition (12:0) (14:0) (16:0) (18:0) (18:1) (18:2) Others (%) 24.2 33.3 11.5 1.3 14.4 14.3 1.0 Derivated trimethylsilyl was prepared from the dried extracts (1 mg) by dissolution in Bis(trimethylsi- ly)trifiuroacetamide (50 1 fi) and pyridine (50 1 fi) heated at 60øC for 30 min. Aliquots of 0.05 I•1 were injected onto the GC. CC-516 and standards were run alternately on the GC/MS with blanks of BSTFA/ pyridine. A fused HP ultra-2 column (Econocap: 50m capillary: 0.2 mm, thickness: 0.11 l•m) was crosslinked with a PMS. The bonded phase was eluted with He (inlet pressure 6 psi) directly into the ion source ofa HP 5890 GC/HP 5970 MS. The column was temperature programmed from 50øC to 300øC at 10øC/min. The major components and many of the minor components were identified, after trimethylsi- lylation, by comparison of their electron impact mass spectra and GC elution t, mes with standard sample spectra. lauric (24.2%) acids were the major fatty acids of the hexane-soluble fraction. Oleic (14.4%) and linoleic (14.3%) acids appear to be major contents of unsaturated fatty acids. Intercellular lipids, particularly ceramides, play an important role in regulating skin barrier function as well as in maintaining the water-holding capacity of the stratum corneum. Several lipidic substances could inhibit leukocytic elastase, pancreatic elastase,