EFFECTS OF PULSE STIMULATION ON COLLAGEN INCREASE 421 stimulation for 5 min with VC (144 ± 21% increase), and then the group treated with bipolar Gunpatsu pulse stimulation for 10 min without VC treatment (129 ± 11%), al- though the values did not differ signifi cantly between the three groups. Gunpatsu pulse stimulation with either a unipolar or bipolar pulse is effective at increasing skin hydroxy- proline levels and can also be useful for enhancing the iontophoresis of VC. In general, however, a longer electric stimulation can have adverse effects, such as a suppression of body weight gain, as shown in Figure 4. The decrease in body weight can be seen in the 10-min Gunpatsu pulse group alone, and the correlation between the duration of treat- ment and the changes in body weight cannot be seen in other groups. Additionally, a bipolar Gunpatsu pulse for 5 min showed no disturbance of growth and a more effective increase in hydroxyproline levels compared to those of a 10-min Gunpatsu pulse. There- fore, the iontophoresis of VC using a bipolar Gunpatsu pulse (5 min) is the best way to produce skin collagen. DISCUSSION In this paper, we evaluated the usefulness of the iontophoresis of a VC derivative for regen- erating collagen in skin by using a newly developed method of electrical stimulation, Gun- patsu pulse, with either unipolar or bipolar pulses. To measure levels of collagen in skin, we used a newly developed, highly sensitive HPLC with NBD-F for detecting the quantity of hydroxyproline. This method clearly showed the changes in hydroxyproline levels in rat skin following Gunpatsu pulse stimulation and/or treatment with the VC derivative. Two approaches to the regeneration of collagen, a laser/far-infrared ray technique and the iontophoresis of VC derivatives, are widely used in the fi eld of cosmetic dermatology and esthetic training, although they have weak effects and can cause skin damage (10–15). Thus, the techniques of producing collagen in skin require some improvements (5,15). As shown in Figure 3, daily treatment for 5 or 10 min with unipolar Gunpatsu pulse stimulation promoted the production of collagen, although stimulation for longer than 10 min may suppress body weight gain. Furthermore, as shown in Figure 5, the iontophoresis of the VC derivative was completely achieved with 5 min of bipolar Gunpatsu pulse stim- ulation without any severe damage (e.g., burn and body weight gain) to rat skin. These results suggest that collagen can be regenerated by the iontophoresis of a VC derivative potentiated by a short bipolar Gunpatsu pulse stimulation without any adverse effects. Gunpatsu pulse stimulation (4800 Hz pulse) is patented, since it differs from ordinary pulse stimulation, and also can be reversed as unipolar or bipolar (see Figure 1). As our preliminary data, between 1200 and 4800 Hz, the increase in the VC-derivative ionto- phoresis was seen in a frequency-dependent manner. Between 4800 Hz and 50,000 Hz, such increases were not shown to be signifi cant. Furthermore, Gunpatsu pulse stimula- tion did not induce any signifi cant damage in the skin. Attempts to increase the amount of collagen in skin have mostly used pulses for stimula- tion. For instance, pulses of UV rays and infrared rays are more than 10,000 Hz, whereas pulses widely used in iontophoresis are between 1 and 500 Hz (3–8). Gunpatsu pulse stimulation uses moderate pulses of 4800 Hz. In this experiment, we demonstrated that Gunpatsu pulse is neither weak nor strong, requires less stimulus to regenerate the collagen in skin, and can be used for the ionto- phoresis of VC derivatives. Additionally, bipolar Gunpatsu pulse stimulation is useful for

JOURNAL OF COSMETIC SCIENCE 422 the iontophoresis of VC derivatives and some medicines directed to not only the skin but other tissues, since the different polarities between skin/target organs and VC derivatives/ medicines can be regulated by bipolar stimulation. CONCLUSION Gunpatsu pulse was effective at increasing the amount of collagen in skin with either unipolar or bipolar stimulation for less than 10 min. Furthermore, in terms of the ionto- phoresis of VC derivatives, bipolar Gunpatsu pulse stimulation for 5 min was most effec- tive. These results suggest that our methods can be used to increase the amount of collagen in skin, particularly in the case of iontophoresis of substances to the target organ, where bipolar stimulation was more effective. Furthermore, Gunpatsu pulse stimulation has much less of an adverse effect compared to ordinary pulse stimulation. This method can be developed as a novel and safe treatment for the application of medicines. This is the fi rst report that substantiates that the amount of collagen in skin is increased by elec- tric stimulation, Gunpatsu pulse. REFERENCES (1) W. D. Tian, R. Gillies, L. Brancaleon, and N. Kollias, Aging and effects of ultraviolet A exposure may be quantifi ed by fl uorescence excitation spectroscopy in vivo, J. Invest. Dermatol., 116(6), 840–845 (2001). (2) T. Quan, T. He, S. Kang, J. J. Voorhees, and G. J. Fisher, Solar ultraviolet irradiation reduces collagen in photoaged human skin by blocking transforming growth factor-beta type II receptor/Smad signaling, Am.J. Pathol., 165(3), 741–751 (2004). (3) L. Reinisch, J. A. Muccini, Jr., and T. Fuller, Quantitative and qualitative evaluation of skin laser, Lasers Surg. Med., 167(Suppl. 11), 41 (1999). (4) J. A. Muccini, Jr., F. E. O’Donnell, Jr., T. Fuller, and L. Reinisch, Laser treatment of solar elastosis with epithelial preservation, Lasers Surg. Med., 23, 121–127 (1998). (5) K. M. Kelly, J. S. Nelson, G. P. Lask, R. G. Geronemus, and L. J. Bernstein, Cryogen sprays cooling in combination with nonabrasive laser treatment of facial rhytides, Arch Dermatol., 135, 691–694 (1999). (6) L. Zhang, S. Lerner, W. V. Rustrum, and G. A. Hofmann, Electroporation mediated topical delivery of vitamin C for cosmetic applications, Bioelectrochem. Bioenerg., 48(2), 453–461 (1999). (7) P. Batheja, R. Thakur, and B. Michniak, Transdermal iontophoresis, Expert Opin. Drug Deliv., 3(1), 127–138 (2006). (8) M. Ebihara, M. Akiyama, Y. Ohnishi, S. Tajima, K. Komata, and Y. Mitsui, Iontophoresis promotes percutaneous absorption of L-ascorbic acid in rat skin, J. Dermatol. Sci., 32(3), 217–222 (2003). (9) M. Kakinuma, Y. Watanabe, Y. Hori, T. Oh-I, and R. Tsuboi, Quantifi cation of hydroxyproline in small amounts of skin tissue using isocratic high performance liquid chromatography with NBD-F as fl uoro- genic reagent, J. Chromatogr. B, 824(1–2), 161–165 (2005). (10) G. Menaker, Treatment of facial rhytides with a nonabrasive laser: A clinical and histologic study, Der- matol. Surg., 25, 440–444 (1999). (11) S. N. Doshi and T. S. Alster, 1,450 nm long-pulsed diode laser for nonablative skin rejuvenation, Der- matol, Surg., 31, 1223–1226 (2005). (12) M. A. Trelles, I. Allones, J. L. Levy, R. G. Calderhead, and G. A. Moreno-Arias, Combined nonablative skin rejuvenation with the 595- and 1450-nm lasers, Dermatol. Surg., 30(10), 1292–1298 (2004). (13) E. F. Rostan, Laser treatment of photodamaged skin, Facial Plast. Surg., 21(2), 99–109 (2005). (14) N. N. Byl, A. L. McKenzie, J. M. West, J. D. Whitney, T. K. Hunt, H. W. Hopf, and H. Scheuenstuhl, Pulsed microamperage stimulation: A controlled study of healing of surgically induced wounds in Yucatan pigs, Phys. Ther., 74(3), 201–218 (1994). (15) L. J. Bernstein, A. N. Kauvar, M. C. Grossman, and R. G. Geronemus, The short- and long-term side effects of carbon dioxide laser resurfacing, Dermatol. Surg., 23(7), 519–525 (1997).