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actual increased amount of skin col- lagen could not be detected (Watanabe, Hori, Oh-I, Urushibata, Akiyama, and Kamiya unpublished data, 2008).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:

EFFECTS OF PULSE STIMULATION ON COLLAGEN INCREASE 417 Experiment 1: Electric stimulation with unipolar Gunpatsu pulse (Figure 1) (time course). A total of 50 rats were randomly divided into fi ve groups and housed individually. The control group (10 rats/group) did not receive any electric stimulation. The other four groups (10 rats/group) received the unipolar Gunpatsu pulse stimulation (see Figure 1) for 0.5, 2, 5, and 10 min every day for one week. Experiment 2: Electric stimulation with bipolar Gunpatsu pulse (combined with a VC solution). A total of 40 rats were randomly classifi ed into four groups and were housed individually. The control group (10 rats/group) was treated with a saline solution without any electric stimulation. The other three groups (10 rats/group) were treated with (a) the VC deriva- tive (aminopropyl ascorbyl phosphate, pH 7.4) solution without any Gunpatsu pulse stimulation, (b) bipolar Gunpatsu pulse stimulation (see Figure 1) for 10 min with saline, or (c) bipolar Gunpatsu pulse stimulation for 5 min with the VC solution. Gunpatsu pulse stimulation was performed at 4800 Hz and 5 V every day for one week. MEASUREMENT OF THE AMOUNT OF COLLAGEN IN SKIN Most methods for determining collagen levels are based on measurements of the quanti- ties of proline and/or hydroxyproline, the principal amino acid components of collagen. Recently, we developed a simple and highly sensitive method of measuring the amount of hydroxyproline in skin by using isocratic HPLC with NBD-F (9). REAGENTS NBD-F was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The standards for HPLC were hydroxyproline (Wako Pure Chemical Industries Ltd.) and a standard amino acid mixture, Type AN-2 (Wako Pure Chemical Industries Ltd.). The Wako standard amino acid solution is an equimolar mixture of Asp, Cys, Glu, Ser, Gly, Figure 1. Typical pulse pattern of either unipolar or bipolar stimulation of Gunpatsu pulse. Unipolar stim- ulation shows the only positive site of 4800 Hz, and the bipolar stimulation shows the both positive and negative sites at each 1.5 sec. Electric stimulation (4800 Hz, 5 V, duty: 50% and 1.2 mA) was obtained by the new device (see photograph).