66 a) 100 80 60 % 40 20 0 __ t JOURNAL OF COSMETIC SCIENCE b) 100 � 80 53' = I = % Q. 40 i � ') ai ') ') Jl1.L1H�_L. 20 JL 0 200 400 m/z 200 Figure 4. Mass spectra for (a) GHK and (6) GSH from emulsion in acceptor cells. Table I Ionized Species Observed in ESI-MS Spectra of GHK-Cu and GSH-Cu Solutions Positive ion Negative ion mlz Proposed ion mlz Proposed ion 341 [GHK + H} + 339 [GHK + H}- 402 [CuGHK + H} + 400 [CuGHK - H} 306 [GSH - Hr 304 [GSH - Hr 369 [GSH - Hr 367 [GSH - Hr 400 m/z GSH-Cu (Figure 56). The mass spectrum for these studies consists of two signals at mlz 306 for GSH and 369 for GSH-Cu. The participation of the GSH ligand form in the penetration ability of copper shows the important role of these species in the transport process. The ligand influence can be confirmed by the fact that the K P for copper from these complexes is lower than for copper from the GHK-Cu complex. All signals were compared to the theoretical profiles. This result supports the thesis that copper com­ plexes with bioligands can be formed and penetrate through the model membrane from the emulsion. CONCLUSIONS The biological activities of the copper tripeptide complex play an important role in the protection and regeneration of skin tissue, and GHK-Cu and GSH-Cu are a very good copper ion source. The research on copper transport through membrane modeling stra­ tum corneum proved that the tripeptide-copper complex may permeate through a horny layer of epidermis. GHK significantly participates in the wound-healing process: due to its properties it influences the elasticity and strength of the skin. What's more, GSH play a very

a) 100 80 60 % 40 20 a .LJ SKIN PENETRATION BY COPPER-PEPTIDE COMPLEXES 67 b) 100 80 200 400mlZ Figure 5. Mass spectra for (a) GHK-Cu and (b) GSH-Cu from emulsion in acceptor cells. important role in skin lightening. The research proves that the copper complexes might be a good source not only for copper ions but also for peptides. The investigations of the influence of complexing agents on the skin migration rate of copper ions have evidenced their hampering role in this process. At the present time, the incorporation of copper ions in cosmetics formulations is still very popular in Europe. The beneficial effect of the active substances depends strictly on their skin penetration ability. Our investigations show the possibility of the effective penetration of copper complexes with tripeptide into the stratum corneum, which allows one to exploit the biological activity of these complexes in cosmetics. ACKNOWLEDGMENTS The authors are thankful to Lipoid GmBH (Germany) for the kind gift of Cerasome 9005 and to ProCyte (USA) for the sample of Prezatitde copper acetate. The authors are grateful to Rafal Ruzik for valuable help with the ESI-MS study. REFERENCES (1) R.H. Guy, J. J. Hostynek, R. S. Hinz, and C.R. Lorence, Metals and the Skin (Marcel Dekker, New York, 1999), pp. 179-189. (2) M. C. Linder, Copper and genomic stability in mammals, Mut. Res., 475, 141-152 (2001). (3) K. Lintner and 0. Peschard, Biologically active peptides: From a laboratory bench curiosity to a functional skin care product, Int.]. Cosmet. Sci., 22, 207-218 (2000). (4) L. Pickart and M. M. Thaler, Tripeptide in human serum which prolongs survival of normal liver cells and stimulates growth in neoplastic liver, Nature New Biol., 243, 85-87 (1973). (5) S. J. Lau and B. Sarkar, The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth­ modulating tripeptide from plasma, Biochemj., 199, 649-656 (1981). (6) C. Conato, R. Gavioli, R. Guerrini, H. Kozlowski, P. Mlynarz, C. Pasti, F. Pulidori, and M. Remelli, Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: Chemical behaviour and biological activity, Biochim. Biophys. Acta, 1526, 199-210 (2001). (7) F. X. Maquart, L. Pickart, M. Laurent, P. Gillery, J. C. Monboisse, and J. P. Borel, Stimulation of