j. Cosmet. sci., 49, 285-297 (September/October 1998) Areca catechu L. extract. I. Effects on elastase and aging KUN KOOK LEE and JUNG DO CHOI, R&D Center, Corearia Cosmetics Co. Ltd., 204-1 Jeongchon-ri, Cheonan-si, Chungnam 330-830, South Korea (K.K.L.), and Department of Biochemistry, College of Natural Science, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea (J.D.C.). Accepted for publication August 31, 1998. Synopsis The anti-aging effects ofAreca catechu L. extract (CC-516) on skin were investigated both in vitro and in vivo. The CC-516 is composed of a relatively high amount of protein (26%) and carbohydrate (37.5 %), and it has a high proportion of proline (13%) of free amino acid content. The inhibitory effect of CC-516 on the elastase exhibited a 37% to 98% inhibition for a 10-500 tng/ml concentration, and IC5o values of 40.8 tng/ml for porcine pancreatic elastase (PPE) and 48.1 lag/ml for human leukocyte elastase (HLE), respectively. One of the effects of elastase is that it is known to reduce the number of elastin fibers at the level of enzyme deposition. The number of elastin fibers was increased when we drifted from the deposit point of elastase with 100 tng/rnl of CC-516. CC-516 showed protection of elastic fiber against degradation by the enzyme in an ex vivo assay. The CC-516 increased proliferation of human fibroblast cells by 85% at 10-4% concentration, compared with control, whereas the increase with ascorbic acid was 50%. The monolayer and three-dimensional collagen synthesis was increased by 50% at 10-4% and 20% at 0.1% concentrations of CC-516, respectively. The treatment with CC-516 improved skin hydration, skin elasticity, and wrinkle reduction. From this study, we suggest that CC-516 can be used as a new anti-aging component in cosmetics. INTRODUCTION With aging, especially of people over 40 years, the elasticity of skin is significantly decreased by elastase activity, which results in sagging (1). Histological examination reveals a thickened epidermis and dermis, an increased number of mast cells and hy- pertropic cysts, the infiltration of inflammatory cells, the partial absence and aggrega- tion of elastin fibers, and a decrease in collagen fibers. Biologically, elastase activity significantly increases with age (2). The increase results in reduced skin elasticity and in the appearance of wrinkles or stretch marks (3). A number of studies have been interested in interactions between elastase and its inhibitors, which include unsaturated fatty acids, peptides, fiavonoids, and terpenoids (4-10). It has been proposed but not yet fully demonstrated (1 l) that the beneficial effects of these inhibitors on the typical signs of cellulitis are obtained by inhibition of elastase, the lysosomal enzyme which regulates the turnover of the structural constituents of the extravascular matrix that surrounds the capillary walls (12-15). Plant sources have been evaluated for developing natural active 285

286 JOURNAL OF COSMETIC SCIENCE agents that may be involved in anti-aging and anti-wrinkle care (16-17). To develop active agents for skin anti-aging, we previously screened the inhibitory effects of 150 medicinal plants on elastase activity. The Areca catechu metanolic extract showed high inhibitory effect on elastase compared with reference compounds (18), and we selected the Areca catechu extract as a new candidate for anti-aging agent in cosmetics. Areca catechu L. is widely cultivated, especially in southern Asia, and its seed is used as a chewing material, as an anthelmintic, and also as a kompo-traditional medicine. Prepa- rations containing Areca are used also as digestive medicine since Areca promotes the secretion of saliva (19-20). Areca catechu L. contains a number of chemical components, such as alkaloids, tannins, flavonoids, and fatty acids (21,22). These compounds have been tested for their individual mutagenicity in bacterial and some rodent test systems (23). To clarify the mechanism of Areca catechu L. extract against anti-aging, we have studied the inhibitory capacity of elastase in vitro and also performed ex vivo assessments using human skin tissue and automated image analysis. Furthermore, we investigate here the effects of the Areca catechu extract on collagen synthesis, extracellular matrix deposition of human dermal fibroblasts in culture, wrinkle reduction, skin moisturizing, and skin elasticity in vivo, MATERIALS AND METHODS CHEMICALS All solvents were of analytical grade. Human leukocyte and porcine pancreatic elastase, ascorbic acid, reference substances, and the basic fibroblast growth factors were pur- chased from Sigma Chemical Co. (St. Louis, MO). [N-Succ-(Ala)3-p-nitroanilide and Meo-Succ-(Ala)2-Pro-Val-p-nitroanilide were purchased from Calbiochem. Other re- agents were analytical grade from commercial sources. EQUIPMENT Spectrophotometric measurements were performed with a UV-visible spectrophotometer (Beckman). The composition analyses of CC-516 were performed with a GC/MS (HP 5890 GC/HP 5970 MS), an amino acid analyzer (TCX 3100, ACS, UK) a TLC (Kiesel gel 60, F254 glass plate, Merck), and an HPLC (Shimatzu LC-9A, Japan). The anti-aging effects of the cream containing CC-516 wer• measured by a corneometer CM 820 (Courage + Khazak, C+K, Germany), a cutometer SEM 474 (C+K Electronic GmbH, Germany), a skin visiometer SV 400 (C+K Electronic GmbH, Germany), an automated image analyzer (BIOCOM 200, France), and a scanning electron microscope (JSM-840 A, Jeol Co.). PREPARATION OF CC-516 Areca catechu seed was purchased from the oriental medicinal market, Seoul, South Korea, and it was sliced and weighed. 100 g of Areca catechu was soaked in 500 ml of 90% ethanolic aqueous solution at room temperature for seven days. After filtration, the ethanolic flitrate was evaporated to dryness under vacuum, and then completely dried by