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.

JOURNAL OF COSMETIC SCIENCE 420 (p 0.05) in the concentration of hydroxyproline in skin was detected compared to the control group (Figure 5). Also, a signifi cant increase (p 0.05) was seen in the group that received bipolar Gunpatsu pulse stimulation for 10 min with saline (no VC derivative), although treatment with VC derivative without any Gunpatsu pulse stimulation did not have any signifi cant effect compared to the control group. Thus, in order to increase the amount of hydroxyproline in skin, the VC derivative must be administered with bipolar Gunpatsu pulse stimulation through iontophoresis. From the results of Experiments 1 and 2, it is clear that the greatest increase in hydroxy- proline levels occurred in the group treated with unipolar Gunpatsu pulse stimulation for 10 min (164 ± 20% increase), followed by the group treated with bipolar Gunpatsu pulse Figure 4. Changes in rat body weight during unipolar Gunpatsu pulse stimulation. Data show the mean ± SE of 10 animals: ⦁, control group , 0.5-min group , 2-min group , 5-min group ○, 10-min group. Figure 5. Changes in hydroxyproline levels in skin stimulated by iontophoresis using Gunpatsu pulse stim- ulation. Data show the mean ± SE of 10 animals. *Signifi cant difference from the control group. *p 0.05.