ARECA CATECHU L. EXTRACT 287 lyophilization. The dried extract was designated the "CC-516," and used as the sample in this study. Areca catechu seed was extracted with other solvents (methanol, ethanol, ether, hexane, chloroform, ethyl acetate, and butanol) by the same extraction process as for the CC-516, and the inhibitory effect on elastase was assayed. Especially, the etha- nolic extract showed a high inhibitory effect on elastase. Thus CC-516 was used as the sample in this study. DETERMINATION OF ELASTIC PROPERTIES Assay for elastase activity. Porcine pancreatic elastase (PPE Sigma, Type IV) was assayed spectrophotometrically by the method of James et al. (27), using N-Succ-(Ala) 3- nitroanilide (S.A.N.A.) as the substrate, and by monitoring the release ofp-nitroaniline for 20 min at 25øC. The amount of p-nitroaniline was determined by measuring the absorbance at 410 nm. The reaction mixture contained 0.2 M Tris-HCl buffer (pH 8.0), ! lag/ml elastase, 0.8 mM Succinyl-Ala-Ala-Pro-p-nitroanilide (ESIV elastase substrate IV, Calbiochem) as substrate, and CC-516 dissolved in methanol. The CC-516 was preincubated for 20 min at 25øC. The reaction was started by adding the substrate. Blanks contained all the components except the enzyme. Human leukocyte elastase (HLE, Sigma) activity was spectrophotometrically determined by measuring the amount ofp-nitroaniline at 410 nm for 20 min at 25øC. The reaction mixture contained 0.! M HEPES buffer (pH 7.5) ! lng/ml elastase 0.5 M NaC1 9.8% DMSO, 1% (v/v) 10 mg/ml BSA (Sigma, fraction V) 1. ! 2 mM Meo-Succinyl-Ala-Ala-Pro-Val-p-nitroaniline (ESI, elastase substrate I, Calbiochem) as a substrate and CC-516. The CC-516 dissolved in methanol was preincubated for 20 min at 25øC, and the reaction was started by adding the substrate. The rate of the reaction was determined by the slope of the line recorded and was proportional to elastase activity. A control curve was prepared with elastase in the absence of CC-516. One unit of elastolytic activity is defined as the activity releasing 1 uM of p-nitroaniline/min. The percentage of inhibition was calcu- lated as: Inhibition (%) = (! - B/A) x 100, where A is the enzyme activity without CC-516 and B is the activity in the presence of CC-516. Assay for elastase activity on dermal sections. The elastase activity on dermal sections was assayed by the modified method of Donald et aL (28). In this study, CC-516 was diluted in 0.! M HEPES buffer (pH 7.5) containing 0.! M NaCI. Skin sections were obtained from plastic surgery on the mammary gland of a woman. The plastic surgery cut the mammary gland into fragments of 1 cm on each side. Fragments were placed on a cork support and frozen in liquid nitrogen. Cross sections carried out with a freezing micro- tome were placed on glass plates and maintained hydrated with the HEPES buffer. The CC-516 dilutions were deposited on skin sections, at the rate of 100 ul per section, and incubated for 30 min at 37øC. After 30 min, an enzyme-impregnated thread (HLE, ! lag/ml) was deposited on the section. After three hours of incubation, sections were rinsed with HEPES and colored with orcein. The effect of the CC-516 on the enzyme activity was assayed by observing sections by a semi-automated image analysis (section photos were taken), and the sections were graded. ASSAY FOR ANTI-AGING EFFECTS WITH FIBROBLAST IN VITRO Fibroblast cultures were initiated from biopsies of normal human skin. Tissue was minced and plated onto 75-T plastic tissue flasks. Cells were maintained in Dulbecco's

288 JOURNAL OF COSMETIC SCIENCE modified Eagle's medium (DMEM) containing 0.48 mg/ml glutamine, 100 IU/ml penicillin, 50 mg/ml streptomycin, and 10% fetal bovine serum (FBS, Gibco BRL) at 37øC in a 5% CO 2 humidified atmosphere. The cells were obtained after the dermis was separated by trypsine. They were then employed upon the fourth passage and mixed for the purpose of testing. Preparation of three-dimensional dermal equivalents system. Three-dimensional dermal equivalents were prepared with a 24-well multichamber plate in the Coulomb B method (29) by mixing type I collagen gel, reconstituted buffer (2.2 g NaHCO3, 4.77 g HEPES made up to 1000 ml 0.06N NaOH), and fibroblasts. The collagen matrix contracted during one day due to an active organization of collagen fibrils by the fibroblasts. After one day, three-dimensional dermal equivalents were maintained as described monolayer cell cultures. Cellprolij•ration. CC-516 was added into the culture system containing DMEM supple- mented with 10% FBS, and the cell proliferation was measured by an MTT assay (30). Collagen synthesis. The activity of CC-516 on the monolayer and three-dimensional cul- ture was measured by the [H3]-proline incorporation method (31). Cells cultured onto 24-well multichamber plates were assayed for confluence. The culture medium was changed to Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS containing 20 mCi/ml L[2,3,4,5-H3]-proline (NEN Chemicals) and incubated at 37øC in 5 % CO2 incubator for 24 hr. The protein concentrations per well were assayed with the protein assay kit (Bio-Rad). After labeling, protease inhibitors were added to the cultures. The media and the cells were mixed and then sonicated. Collagen synthesis was assayed by measuring the radioactivity of the media and cells together after limited degradation with purified bacterial collagenase, according to the method of Peterkofsky et al. (32). The relative rate of collagen synthesis to total protein synthesis was calculated with the assumption that collagen had an amino acid content 5.4 times higher than that of other proteins (33). Collagen synthesis of three-dimensional dermal equivalents was measured in the same manner as the above procedures. ASSAY OF IMPROVEMENTS OF SKIN CONDITION The improvements in skin condition were evaluated for three aspects: moisture content of the skin, skin elastic properties, and wrinkles. The instruments used were the cor- neometer CM 820 (Courage + Khazaka, C+K, Germany) for moisture content of the skin, the cutometer SEM 474 (C+K, Germany) for skin elasticity, and the skin visiom- eter SV 400 (C+K, Germany) for wrinkles. All the percentage values were calculated as: (%) = (value at measuring point - value at initial point)/value at initial point x 100), and the statistical significance of the two test groups was verified by a paired t-test at the significant level, p 0.05. Evaluation of skin moisturizing efficiency was performed by measuring stratum corneum hydration by the capacitance method (34,35). The long-term (six weeks) effects of CC-516 were topically assessed by twenty healthy volunteers with dry to very-dry skin. After application twice daily to cheek and eye regions, measurements were taken before the first treatment and one week, two weeks, four weeks and six weeks after treatment. In order to minimize the risk of treatment errors we routinely limit the number of treatment areas to two groups (cream containing