JOURNAL OF COSMETIC SCIENCE 418 His, Arg, Thr, Ala, Tyr, Val, Met, Ile, Leu, Phe, Lys, Pro, and hydroxyproline. All other chemicals were of HPLC grade. Hydrochloric acid (HCl), acetonitrile, potassium dihy- drogenphosphate, sodium tetra borate decahydrate, and boric acid were purchased from Wako Pure Chemical Industries Ltd. Ethylenediamine tetra acetic acid disodium salt dehydrates (EDTA-2Na) was purchased from Pharmacia Biotech AB (Uppsala, Sweden). HYDROLYSIS OF PROTEIN AND DERIVATIVE Tissue samples were hydrolyzed for 22 h at 108°C in evacuated fl ame-sealed Pyrex tubes with 400 μl of 6 N HCl, to which 1% phenol was added. The hydrolysates were dried in vacuo. For the HPLC analysis, the dry residue of each hydrolysate was re-suspended in 500 μl of 50 mM borate buffer containing 20 mM EDTA-2Na (pH 8.0). The resuspended samples were diluted to 1/2000 with a 50-mM borate buffer containing 20 mM EDTA- 2Na and 7.5 μM of L-homoserine (pH 8.0). Next, 30 μl of either the hydrolyzed sample or a mixed amino acid standard solution diluted to 1/100 with a 50-mM borate buffer containing 20 mM EDTA-2Na (pH 8.0) was poured into a 500-μl conical tube. Then 10 μl of NBD-F (10 mM in acetonitrile, freshly prepared) was added to this solution and the tube was capped and covered with aluminum foil. The vessel was heated to 60°C for 1 min, and after it had cooled in ice water, 40 μl of 0.05 M HCl was added to the reaction mixture. Finally, 10 μl of the solution was injected into the column. HPLC CONDITIONS Briefl y, an EP-300 (Eicom Corp., Kyoto, Japan) pump equipped with an L-7200 auto sampler (Hitachi, Tokyo, Japan) was employed. A guard column (200 mm × 3.9 mm) and a main column of SC-5ODS (200 mm × 4 mm, 10 μm Eicom Corp., Kyoto, Japan) were used. All solvents were fi ltered and degassed prior to use. The fl ow rate was 0.8 ml/min, and the column temperature was kept at 30°C using a column oven. A Hitachi L-7485 spectrofl uorometer equipped with a 12-μl fl ow cell was used with an excitation wave- length of 470 nm and emission at 540 nm. A phosphate buffer (pH 5.0) containing 8% acetonitrile was used as the mobile phase. STATISTICAL ANALYSES Results are presented as the mean ± SE. All results were analyzed with Dunnett’s multi- ple comparison test of the Pearson correlation after an analysis of variance. RESULTS During this experiment, there was no signifi cant difference between the groups in body weight or quantity of water ingested. No adverse effects were observed in any of the rats (data not shown). All results, particularly for the analysis of hydroxyproline content, ob- tained by HPLC are clearly documented in Figure 2. The retention times of hydroxypro- line and Pro were about 8 and 34 min, respectively. L-homoserine, as an internal standard, was eluted for about 12 min. Experiment 1: Changes in hydroxyproline levels in the skin and increase in body weight. In the control group (Figure 3 control: the electrode for Gunpatsu pulse was tied around the

EFFECTS OF PULSE STIMULATION ON COLLAGEN INCREASE 419 abdominal and dorsal skin but with no Gunpatsu pulse stimulation), hydroxyproline lev- els in the skin were 102.6 ± 9.3 nmol/mg (10 rats/group). These levels were increased depending on the length of stimulation with a unipolar Gunpatsu pulse, with signifi cant increases when the stimulation lasted longer than 5 min (Figure 3). In addition, the in- creases in body weight seen in the Gunpatsu pulse groups were likely to be less than those seen in the control group. More than 10 min of Gunpatsu pulse stimulation seems to prevent a gain in body weight (Figure 4). Experiment 2: Changes in hydroxyproline levels in skin stimulated by iontophoresis using Gunpatsu pulse. In the control group (Figure 5 saline + non-Gunpatsu pulse: the electrode for Gun- patsu pulse was tied around the abdominal and dorsal skin but with no Gunpatsu pulse stimulation), the hydroxyproline levels in rat skin were 102.8 ± 11.9 nmol/mg (10 rats/ group). Signifi cant increases (p 0.05) were found in two Gunpatsu pulse-stimulated groups (Figure 5). In the bipolar Gunpatsu pulse 5-min + VC group, a signifi cant increase Figure 2. Chromatogram of derivatized rat skin sample (control group) analyzed by isocratic HPLC with the NBD-F method. Homoserine is the internal standard amino acid. Hydroxyproline was eluted at about 8 min. Figure 3. Changes in hydroxyproline levels in skin stimulated by unipolar Gunpatsu pulse stimulation. Each column shows the mean ± SE of 10 animals. The dotted line expresses the value of 100% of the control group. ∗Signifi cant difference from the control group. ∗p 0.05.