J. Cosmet. Sci., 60, 415–422 (July/August 2009) 415 Skin collagen reproduction increased by ascorbic acid derivative iontophoresis by frequent-reversal bipolar electric stimulation YUSUKE HORI, RYUJI AKIMOTO, AKIKO HORI, KATSUHIKO KATO, DAISUKE CHINO, SHOHEI MATSUMOTO, SHOHEI KAMIYA, and YASUO WATANABE, Tes Holdings Co., Ltd, Tokyo 111-0032 (Y.H) Homer Ion Laboratory Co., Ltd, Tokyo 150- 0045 (R.A., S.K.) Gene Expression and Regulation, Institute of Medical Science, University of Tokyo 108-8639 (A.H.) Department of Genetical Education, School of Veterinary Medicine, University of Azabu, Kanagawa 229-8501 (K.K.) Department of Pharmacology and Pharmacotherapy, School of Medicinal Pharmacy, Nihon Pharmaceutical University, Saitama 362-0806 (D.C.,Y.W.) and Department of Anesthesiology, Tokyo Medical University, Tokyo 160-0023 (S.M.), Japan. Accepted for Publication February 11, 2009. Synopsis The effect of the iontophoresis of ascorbic acid (Vitamin C VC) derivative with frequent-reversal bipolar electric stimulation on the production of collagen in rat skin was evaluated in terms of hydroxyproline con- tent through high-performance liquid chromatography. First, a control group was not given electrical stimu- lation and four groups were stimulated with a unipolar pulse for 0.5–10 min every day for one week. The hydroxyproline level in the skin was increased depending on the length of the stimulation. Second, a control group was not given any electrical stimulation, and three groups were treated with (a) VC solution without any stimulation, (b) a bipolar pulse for 10 min with saline, or (c) a bipolar pulse for 5 min with the VC solu- tion. Signifi cant increases were found in all the stimulation groups, although these treated with the VC solution without any stimulation did not have any effects compared to the control. Thus, in order to increase the hy- droxyproline levels in skin, a VC must be delivered with bipolar stimulation as a method of iontophoresis. These results suggest that our newly developed electric stimulation is effective at increasing skin collagen content, and that bipolar stimulation is more effective on the iontophoresis of not only VC but also some medicines such as low- and high-molecular drugs directed to the target organ (7). INTRODUCTION Collagen is a major constituent of the dermis, and is therefore involved in many facets of skin disease as well as the recovery process. Collagen also plays an important role in main- taining the moisture content and the elasticity of skin, although collagen is altered and

JOURNAL OF COSMETIC SCIENCE 416 damaged by aging and/or ultraviolet rays (1,2). Generally, in order to avoid such damage and changes, attempts have been made to regenerate collagen in skin by stimulation with either a laser or far-infrared rays (3). However, these treatments induce erythema or red- ness of the skin for a few weeks to several months (4,5). Meanwhile, it has been already reported that ascorbic acid (vitamin C VC) promotes the synthesis of collagen. Furthermore, in the fi eld of esthetics, VC derivatives, which have a high affi nity for skin cells, have been used to regenerate skin collagen because they are highly effective and safe, as shown in previously reported papers (6,7). However, treat- ment with VC takes a long time to obtain effects because of its weak activity. Recently, a method of treatment using VC derivatives with a weak electric current has been devel- oped for improving the absorption of VC derivatives by the skin, so-called iontophoresis, although satisfying effects have yet to be obtained (7,8). Recently, we developed a fre- quent-reversal electric stimulation machine, and in preliminary human studies, the ef- fects of the VC derivative iontophoresis with this machine were much more satisfying than those of the VC derivative alone, although the actual increased amount of skin col- lagen could not be detected (Watanabe, Hori, Oh-I, Urushibata, Akiyama, and Kamiya unpublished data, 2008). In addition, frequent-reversal electric stimulation is much more harmless and can be used as sequential stimulation for a much longer period than fre- quent unipolar stimulation. The main purpose of this study is to prove the effects of a newly developed electric stim- ulation, “Gunpatsu pulse®” (Application number 2006-048423 fi ling date, May 26, 2006 registration number 5026198, Japanese Patent Offi ce) on the regeneration of col- lagen by measuring the hydroxyproline content of the skin using high-performance liquid chromatography (HPLC) with a fl uorogenic agent, 7-fl uoro-4-nitrobenzo-2-oxa-1,3-di- azole (NBD-F) (9). Furthermore, we compared the effects of unipolar Gunpatsu pulse stimulation and bipolar Gunpatsu pulse stimulation either with or without VC deriva- tive (aminopropy1 ascorbyl phosphate) on the increase of rat skin hydroxyproline content within the most effective and shortest duration (one week). MATERIALS AND METHODS ANIMALS AND GUNPATSU PULSE STIMULATION Healthy seven-week-old male Sprague-Dawley rats (SPF) (Charles River Japan Inc., Yokohama, Japan) were used in this experiment. These rats were housed in wire- mesh-bottom cages in an animal room with controlled temperature, humidity, and light- ing, with food and water available ad libitum. At breeding, plain tap water was supplied. All rats were anesthetized with pentobarbital-Na (45 mg/kg, i.p., Abbot Laboratories, IL, U.S.A) until unconscious and removed of hair on the abdominal and dorsal skin before the experiment, since the electrode for the Gunpatsu pulse was tied around the abdomi- nal and dorsal skin. Before any stimulation, circular dorsal skin samples 8 mm in diam- eter from the right of the midline as a control were stored at −80°C until analyzed. Electric stimulation (4800 Hz, 5 V duty: 50% and 1.2 mA) was then applied for one week, and more than one week later, dorsal skin samples from the left of the midline were stored as above (9). This paper is focused on the evaluation of the several effects of Gunpatsu pulse stimulation on the increases in rat skin collagen using the following two experiments: