148 JOURNAL OF COSMETIC SCIENCE
with catechol, hydroquinone, chicoric acid, and resorcinol, the former three polyphenols
showed ladder-like lanes which ranged from about 25–70 kDa as seen in lanes 2, 4, and 5,
respectively. By contrast, resorcinol was not active for increasing the molecular weight
distribution of the protein see lane 3.
It was also found that a combination of the polyphenols with the copper (II) chlorophyllin
(often referred to hereafter as copper salt) was highly effective in increasing the molecular
weight-range of the lysozyme see Figure 2 and lanes 7, 9, and 10. In other words, the
molecular weight-range of the protein was significantly increased when the protein was
treated with catechol, hydroquinone, and chicoric acid. Although the reaction using
hydroquinone and the copper salt just showed only faintly stained medium molecular
weight substances (25–120 kDa) (lanes 7 and 9), the product was considered as the
highly polymerized proteinous substances separated from the buffer medium did not
run through the gel disk. A similar electrophoresis phenomenon was observed in other
systems using gelatin proteins.12 Although the detailed analysis of the catalytic role
of the copper salt is not described here, the optimal concentration of the copper salt
was around about 4 ppm. The salt concentration was therefore employed throughout
this study.
Figure 2. SDS-PAGE patterns of the lysozyme (0.4 wt.-%) which was treated with various polyphenols
(0.9 mM) in the presence or absence of copper (II) chlorophyllin (Cu, 4 ppm) at 40°C for 24h. CBB was used
as a staining agent. It is considered that the very large proteins products, if any, were retained in the starting
wells without running through the gel.

149 Repairing Bleach-Damaged Hair
Figure 3 is the SDS-PAGE band pattern of the reaction products which were obtained by
treating lysozyme (0.4 wt.-%) with various concentrations of the polyphenols (hydroquinone
and chicoric acid) in the presence of the copper salt (4 ppm). At the low hydroquinone
concentration (0.9 mM), a well-defined ladder-like lane was clearly observed. In the higher
concentration (4.5–36 mM), however, not only lysozyme (ca. 14 kDa) but also the protein
ladder (25–210 kDa) disappeared. Like the previous results, it is likely that high molecular
weight and insoluble products were not run through the SDS-PAGE gel. However, chicoric
acid was a relatively milder reagent to provide the polymerized protein bands in a wide
concentration range (0.3–12 mM), showing well-defined ladder-like patterns in the
medium molecular weight-range.
Figure 4 shows the SDS-PAGE pattern observed by the keratin (5 wt.-%) with the polyphenol
(hydroquinone and chicoric acid) in the presence of the copper salt (4 ppm) at 40°C for 24 h.
With hydroquinone, the broad band (about 6–120 kDa) of the starting keratin was disappeared
while the new band (about 80–350 kDa) appeared, particularly in the low concentration (9.0-
0.9 mM). It seemed, similar to the lysozyme, that very large molecular weight-substances were
insoluble and retained in the electrophoresis-wells without flowing through the gel. In the
case of chicoric acid, the product bands were clearly observed and steadily increased with the
increasing concentration of the polyphenol. It appears that the activity of chicoric acid at 6.0–
12 mM concentration was comparable to that of 0.9–4.5 mM hydroquinone. The previously
mentioned electrophoresis data suggested that the polyphenols act on the proteins to polymerize
to the higher molecular weight substances and that the hydroquinone and chicoric acid were
especially effective in promoting the polymerization reaction.
Figure 3. SDS-PAGE patterns of the lysozyme (0.4 wt.-%) which was treated with various concentrations
(mM) of hydroquinone or chicoric acid in the presence of copper (II) chlorophyllin (4 ppm). CBB was used as
a staining agent.