J. Cosmet. Sci., 57, 369-376 (September/October 2006) A horse chestnut extract, which induces contraction forces in fibroblasts, is a potent anti-aging ingredient TSUTOMU FUJIMURA, KAZUE TSUKAHARA, SHIGERU MORIWAKI, MITSUYUKI HOTTA, TAKASHI KITAHARA, and YOSHINORI TAKEMA, Biological Science Laboratories, Kao Corporation, 2606, Akabane, Ichikai-rnachi, Haga-gun, Tochigi, 321-3497, Japan. Accepted for publication May 4, 2006. Synopsis Contraction forces generated by non-muscle cells, such as fibroblasts, play important roles in determining cell morphology, vasoconstriction, and/or wound healing. We have searched among various plant extracts for ingredients that generate cell contraction forces using fibroblast-populated collagen gels. Using that model, we found that an extract of horse chestnuts (Aesculus hippocastanum) is able to generate such con­ traction forces in fibroblasts. The involvement of stress fiber formation in that response is suggested by the inhibition of such force generation by cytochalasin D and rhodamine phalloidin stain. Clinical testing of the extract was carried out using 40 healthy female volunteers. A gel formulation that included 3% of the extract was applied topically to the skin around the eye three times daily for nine weeks. The efficacy of the extract to diminish wrinkles was evaluated by visual scoring based on photo scales. After six weeks, significant decreases in the wrinkle scores at the corners of the eye or in the lower eyelid skin were observed compared with controls. After nine weeks, similar resLilts were obtained. Taken together, our results suggest that an extract of horse chestnuts can generate contraction forces in fibroblasts and is a potent anti-aging ingredient. INTRODUCTION Contraction forces generated by non-muscle cells, such as fibroblasts, play important roles in determining cell morphology, vasoconstriction, and/or wound healing (1-3). In general, non-muscle cells are thought to generate from 1/10 to 1/100 the force of smooth muscle cells, which specialize in generating such forces (1 ). Therefore, active contraction forces generated by fibroblasts appear to influence the morphology and/or mechanical properties of the skin. However, ingredients that generate cell contraction forces are hardly known except for lysophosphatidic acid (LP A), thrombin, or fetal bovine serum and the like (1,3). Address all correspondence to Tsutomu Fujimura.

370 JOURNAL OF COSMETIC SCIENCE We have searched among various plant extracts for ingredients that can generate cell contraction forces, using fibroblast-populated collagen gels, the so-called dermal equiva­ lent model ( 4). We now report that an extract of horse chestnuts (Aesculus hippocastanurn) is able to induce such contraction forces in fibroblasts. The horse chestnut extract is safe and suitable for cosmetic formulation. Therefore, the wrinkle smoothing efficacy of this extract was evaluated for its efficacy as an anti-aging agent. MATERIALS AND METHODS PLANT EXTRACT AND REAGENTS The horse chestnut (Aesculus hippocastanurn) extract was purchased from Maruzen Phar­ maceutical Co., Ltd. (Hiroshima, Japan). The residue of this extract was about 1.0 weight/volume % (residue weight %). Other extracts, sage (Salvia officinalis), Baikal skullcup (Scutellaria baicalensis), and Amur corktree (Phellodendron arnurense), were pur­ chased from Maruzen. Ginger (Zingiber officinale) and milk vetch (Astragalus sinicus) were purchased from Ichimaru Pharcos Co. (Gifu, Japan). Thrombin, lysophosphatidic acid (LPA), and cytochalasin D were purchased from Sigma Chemical Co. (St. Louis, MO). CELL CULTURE Human fibroblasts were purchased from Dainihon Pharmaceutical Co., Ltd. (Osaka, Japan). They were cultured in Dulbecco's Modified Eagle's Medium (DMEM), supple­ mented with 5 % fetal calf serum (FCS) and an antibiotic-antimycotic (Gibco), at 3 7 degrees in humidified 5 % CO 2 , 95 % air. The cells were removed from the dishes by treatment with trypsin/EDT A and were subcultivated at a 1 :4 split ratio. Fibroblasts from the third to the eighth passage were used in this study. PREPARATION OF FIBROBLAST-POPULATED COLLAGEN GELS Collagen gels containing fibroblasts were prepared as previously described (1). Briefly, the required amount of collagen solution (cell matrix Type I, Nitta Gelatin Inc, Tokyo, Japan), concentrated DMEM, HEPES-NaCO 3 solution, FCS, distilled water, and fibro­ blasts were gently mixed at 4°C, giving a suspension at a final density of 3.0 x 10 5 cells/ml and 1.5 mg/ml collagen. The collagen/cell suspension was poured into a silicon elastomer mold (25 mm x 45 mm, 5 mm depth), and incubated at 3 7°C to gel, as described previously (1). Twenty-four hours later, serum-free medium was added, the mold was removed, and the gel was allowed to float in the serum-free medium. MEASUREMENT OF CONTRACTION FORCE GENERATION Contraction forces generated by fibroblasts cultured in the collagen gel were measured as described previously (1). Briefly, each collagen gel containing fibroblasts was mounted in an isometric transducer at its original length and bathed in serum-free DMEM kept at 3 7°C. Each gel was allowed to equilibrate for one hour prior to each experiment to ensure a stable baseline. Serum-free DMEM, including the plant extract, was added