145 Repairing Bleach-Damaged Hair
G, Japan Chlorophyll, Saitama, Japan), Fluorescein-4-isothiocyanate (FITC-I) (Dojindo,
Kumamoto, Japan), 2,2-Dithiobis(5-nitropyridine) (DTNP) (Sigma-Aldrich, Tokyo, Japan)
and 10–20 wt% polyacrylamide gradient gel plates (Kishida Chemical, Osaka, Japan)
were also commercially available. The bleaching was conducted using a mixture of Milbon
Powder Bleach® (Milbon, Tokyo, Japan) and Ordeve Oxidane® 6.0 (Milbon, Tokyo,
Japan) the former consisted of alkaline components and persulfate salt, and the latter
contained 6 wt% hydrogen peroxide as an essential ingredient.
PREPARATION OF HAIR SAMPLES
Bleaching treatment. The previously mentioned bleaching and oxidizing agents were mixed
in a ratio of 1:2 (w/w). After immersing the black hair in the aqueous mixture at a bath
ratio of 1:50 (w/w) at 25°C for 30 minutes, the fibers were washed well with water and dried
at 60°C with a hairdryer. These operations were repeated three times. The resulting hair
will be from now on be referred to as bleached hair.
Cross-linking treatment of hair (a general procedure). The bleached hair fibers were immersed in
pure water or a mixture of an aqueous solution of the polyphenol (8.4 mM) and copper (II)
chlorophyllin (4 ppm) at a bath ratio of 1:50 (w/w) at 25°C for 12 hours. The resulting hair
samples were rinsed with water and dried by using a hairdryer.
Cross-linking treatment of keratin proteins (a general procedure). The water-soluble keratin was
diluted with the phosphate buffer (PBS) (0.1 M, pH 7.4) to 5 wt%. Similarly, the PBS
solution of polyphenol (hydroquinone, catechol, resorcinol and chicoric acid) was prepared
at the appropriate concentration. The reaction was started by adding an aqueous solution
of copper (II) chlorophyllin (0.060mL) and the polyphenol solution (0.24mL) to the protein
solution (2.7mL). After standing still at 40°C for 12 hours, the reaction mixture was
obtained. In control experiments of the reaction, the keratin was replaced with an equal
volume of lysozyme (0.8 wt%).
SODIUM DODECYL SULFATE POLY(ACRYLAMIDE) GEL ELECTROPHORESIS (SDS-PAGE)
The SDS-PAGE was carried out in a manner similar to a previous method.13 The
polyphenol treated keratin solution (10 µL) was diluted with the X2 Laemmli buffer (Bio-
Rad Laboratories, Hercules, California, USA) (10 µL). Each of the mixtures was heated at
100°C for 5 minutes, cooled to ambient temperature and applied to the polyacrylamide
gradient gel plate (10-20 wt%). The resulting gel plate was subjected to electrophoresis
at 20 mA for about 90 minutes. The protein molecular weight maker II (TEFCO, Tokyo,
Japan) was also electrophoresed at the same time. The protein bands were visualized by
staining with Coomassie brilliant blue (CBB).
AMINO ACID ANALYSIS
The amino acid analyses of the polyphenol treated keratin and hair were performed
according to a high-speed RP-HPLC/FL method.14,15 Polyphenol treated product (1mg) was
hydrolyzed in the vapor phase with 6 M HCl at 110°C for 24 hours according to the PicoTag
protocol (Waters Corporation, Milford, Massachusetts, USA). The resulting hydrolysate

146 JOURNAL OF COSMETIC SCIENCE
was assayed for an amino acid distribution by means of a fully automatic NEXERA XR
system amino analyzer (Shimadzu, Kyoto, Japan) equipped with the separation column
(150mm x 3.0mm) packed with 3.0 µm diameter YMC-Triart C18 beads (YMC Co., LTD,
Kyoto, Japan). Solution A (10 mM Na
2 HPO
4 ,10 mM Na
2 B
4 O
7 ,pH 8.2) and B (acetonitrile
:MeOH :H
2 O =45 :45 :10 v/v) were used as a mobile phase.
QUANTIFICATION OF THIOL RESIDUE OF KERATIN
The thiol residue was quantified according to the method adapted by Obata et al.16 A
mixture of the keratin (1 wt%), chicoric acid (4.0 mM) and copper (II) chlorophyllin (4 ppm)
was dissolved in phosphate buffer (0.1 M, pH 7.4) and warmed at 40°C for 12 hours. The
resulting reaction mixture was diluted by a factor of 10× with PBS and dialyzed against
the same buffer at 4°C for 12 hours using a membrane tubing (molecular weight cut-off of
14.0 kDa). Subsequently, the dialyzed solution (0.4mL) was mixed with an ethanol solution
of DTNP (0.5 mM, 0.1mL) and warmed at 37°C for 30 minutes, then was subjected to the
absorption measurement at 386 nm using a U-3310 UV-spectrometer (Hitachi, Ibaraki,
Japan). The number of thiol residue in keratin protein was calculated with the calibration
curve, which was obtained using L-cysteine as a reference compound viz. the cysteine
concentration was well correlated to the absorbance at 386 nm by equation (absorbance at
386 nm) =cysteine concentration (μM) × 0.0092 +0.3144.
FLUORESCENCE MICROSCOPIC OBSERVATIONS OF FITC-I DYEING HAIR SAMPLE
The fluorescence measurement was carried out according to the procedure reported by
Nakamura et al.17 Several strands of hairs were added into carbonate buffer (0.1 M, pH 5.0)
containing FITC-I (180 ppm) and left for 12 hours. The resulting hair fibers were washed
three times with 0.5mL of carbonate buffer (0.1 M, pH 9.5) and sliced cross-sectionally by
means of a microtome to obtain the specimens (about 20 µm in thickness), then subjected
to the fluorescent measurement at excitation wavelength of 470–495 nm and fluorescence
wavelength of 510–550 nm using a Olympus BX51 fluorescence microscope (Olympus
Corporation, Tokyo, Japan) equipped with a U-MNIBA3 mirror unit (Olympus Corporation,
Tokyo, Japan). The shutter speed was 2.5 seconds and the ISO sensitivity was 800.
OPTICAL MICROSCOPIC OBSERVATIONS FOR EXAMINATION OF POLYPHENOL-TREATMENT OF
BLEACHED HAIR
Biological microscopes (Olympus Photomax with the proper attachments) were used for
bright field observations (Olympus Corporation, Tokyo, Japan). The Olympus objective
lenses were: (bright field) Plan ×10, PlanApo ×20 and PlanApo ×40 (Olympus Corporation,
Tokyo, Japan). Original photo tube accessories were modified to adapt the digital camera
that was automatically controlled by a desktop computer to optimize for lighting, ISO
levels and focusing measures. The images in JPEG and RAW formats were developed by
means of Lightroom ver.3 and Photoshop Elements ver. 9 software (Adobe Inc., San Jose,
CA). Image enhancement included color level correction, noise reduction, and contrast and
brightness adjustments the purpose of the enhancement was to make the image appear
nearly identical to that seen actually by the observer.