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.

147 Repairing Bleach-Damaged Hair
The specimens for the optical microscope were prepared in the following manner: the
natural hair, the bleached hair and chicoric acid, and the copper (II) chlorophyllin treated
bleached hairs, were cut to about 10mm in length. About 30 strands of the shortened
sample were then immersed in aqueous 5 M urea and warmed at 60°C for 1 hour. After
cooling to ambient temperature, 10 strands of the swollen sample were mounted parallel
to a microscope slide glass, where the medium was aqueous 5 M urea. No weight was
placed on the micro cover glass (22 mm × 22 mm thickness, 0.13–0.17 mm) (Matsunami
Glass, Osaka, Japan) while sealing the edges with UV Curable Urethane Acrylate
Oligomer (Mitsubishi Chemical, Tokyo, Japan) and irradiating with a 365 nm LED-light
for 3 minutes at ambient temperature.
MEASUREMENT OF SWELLING DEGREE OF HAIR FIBER
Various hair fibers (about 15mm in length) were warmed in aqueous 5 M urea solution at
60°C for 1 hour. The swollen fiber in the solution was kept at ambient temperature for
1 day, then gently placed on the slide glass with a cover glass using 5 M urea solution.
The cross-sectional length (Ls) was measured by means of the optical microscope using
the Plan ×20 objective lenses. The swelling degree (length−%) was calculated by 100×
(Ls − Ld) ÷ Ld, where Ld is the cross-sectional length of the fiber before swelling. 7
strands of the same hair sample were tested to average the swelling degree.
TENSILE TESTING OF HAIR SAMPLES
The polyphenol treated fibers (20–100 strands) were immersed in purified water for
12 hours, and then set in an ALS1500 +MTT690 automatic tensile tester (Dia-Stron,
Hampshire, UK). Tensile measurement was performed at pulling speed of 3mm/min while
immersing the hair sample (30mm in length) in water. Under the testing condition, which
was similar to ones used previously,18,19 we experienced that the tensile data was obtainable
with small dispersion. 60 fibers per test cell (N =60, 3 persons, 20 fiber samples each)
were individually applied to the tests for estimating the mechanical properties (the breaking
stress, the breaking extension, and the elastic modulus in the initial pilling stage). The
breaking stress (J/m3) applied to breaking total work (J) per the volume of sample hair ((d
÷ 2)2 × π × L
0 ).In the denominator, d is an average value of a hair diameter at 5 points as
measured with a laser contour measuring device (FDAS770, Dia-Stron Hampshire, UK).
L
0 is the initial hair length.The breaking extension (%)is given by 100 × (L – L
0 )÷ L
0 ,
where L is the breaking length of hair specimen. The initial elastic modulus (J/m3) was the
Hookean slope of at the 3% extension.
RESULTS AND DISCUSSION
POLYMERIZATION OF THE POLYPHENOL TREATED PROTEINS
This study was first conducted to examine an action of various kinds of polyphenols
(0.9 mM) on lysozyme (0.28 mM, MW: 14,300), a model protein, in the presence of a
catalytic concentration (4 ppm) of copper (II) chlorophyllin (Figure 1). Figure 2 shows
the SDS-PAGE pattern of the reaction mixtures. When the model protein was treated