| dc.description.abstract | Growth factors used as pharmaceuticals and cosmetics are one of the interesting and major applications in biotechnology. Epidermal growth factor, EGF, is one of growth factors, and is a potent mitogenic factor for a variety of cultured cells originated from ectodermal and mesodermal layers. EGF and FGF have been utilized as one of specific components in cosmetics recently. However, low stability of growth factors is considered to limit pharmaceutical and cosmetic applications of growth factors, generating problems in formation, administration and storage. Therefore, the effect of stabilizers in EGF solution was investigated on the stability of human EGF (hEGF) from conformation of heat-treated hEGF using circular dichroism (CD) spectroscopy and from bioactivity of heat-treated hEGF from cell cycle analysis using flow cytometry. The following stabilizers were selected as the candidates of optimal stabilizers for hEGF: mannitol, polyacrylic acid (PAA), heparin, polyvinylalcohol (PVA), fullerene-OH, hydroxypropyl-cellulose (HPMC) and polymethacrylic acid (PMAA). The goal of this research is to develop physical evaluation method of hEGF stability and activity, and to find the relationship between physical evaluation method and bioassay of hEGF activity. The unfolding fraction of hEGF measured by CD spectroscopy at 232nm increased with increasing heat-treated temperature of hEGF with and without stabilizers. The temperature of hEGF solution, which showed the temperature where the unfolding fraction of hEGF was 80% was also analyzed in hEGF solution containing no or 0.05 wt% of stabilizers, and shifted to higher temperature when hEGF solution contained heparin, PVA or mannitol. These results indicates heparin, PVA and mannitol are one of good candidates of hEGF stabilizers as single component stabilizers. PMAA, heparin and PVA are found to be good candidates of hEGF containing multi-component stabilizers to stabilize the conformation of PARS-hEGF (hEGF containing 0.31 wt% mannitol) compared to PARS-hEGF containing no or other stabilizers from unfolding fraction analysis of PARS-hEGF.
DNA synthesis of fibroblast cells was also analyzed from cell cycle measurements using flow cytometry, and was used as the bioassay of hEGF activity. We demonstrated physical evaluation method of hEGF stability and activity, and found the relationship between physical evaluation method and bioassay of hEGF activity. In the summary of combination between physical evaluation and bioassay for hEGF, mannitol, heparin and HPMC were found to be optimal stabilizers to stabilize hEGF.
The effect of cosmetics containing hEGF on human skin conditions was also evaluated from monitoring test for ten volunteers using cosmetics containing with and without hEGF as one of the application of hEGF in bioengineering. The elasticity, melanin amount, erythem amount and surface sebum amount of skin on the human volunteers were measured from cutometer MPA580 instrument. Cosmetic A containing hEGF was found to be effective to improve the elasticity, erythem amount and control of sebum amount on the human skin in this study.
| en_US |