12 JOURNAL OF COSMETIC SCIENCE
Therefore, it is considered to be an important pathway for the penetration of substances
into the inner parts of the hair (3 ).Observations of cuticles by transmission electron
microscopy have shown evidence of a penetration pathway through the CMC (4,5)
however, that observation method requires the use of heavy metals and thin-sliced hair
samples. We considered that a less invasive technique is needed to understand the
penetration properties of the cuticle.
In the present study, we utilized microbeam X-ray diffraction for structural analysis of
the CMC in the hair cuticle. With this method, we were able to elucidate the structure
of the CMC in whole hair samples under ambient conditions without pretreatment, such
as staining with heavy metals. The pioneering experiment on the structure of the cuticle
using a microbeam X-ray technique was performed by Kreplak et al. (6), in which they
observed small-angle X-ray scattering (SAXS) patterns of cuticles, and determined the
average thickness values of the r,3- and 8-layers in the CMC. Thereafter, a mathematical
model for precise estimation of the thickness of those layers was proposed by Ohta et al.
(7). Using that model, we determined the thickness of the r,3- and o-layers in hair fibers
treated with various solvents. Our results showed a relationship between the CMC
structure and the extent of hair dyeing as a possible indicator of penetration.
MATERIALS AND METHODS
HAIR SAMPLES
For SAXS-pattern experiments, hair strands were obtained from Japanese women who
had not used any chemical treatments and were then washed with a 0.2% sodium laureth
sulfate solution. After being rinsed with deionized water and drying on a paper towel,
the strands were subjected to extraction with four different solvents, methanol, acetone,
hexane, and a chloroform/methanol mixture (2: 1 v/v) at 3 7°C for six hours. After cutting
to a length of 8 cm, each hair fiber was fixed on a hair holder and subjected to SAXS
analysis.
DYEING EXPERIMENTS
For the dyeing experiments, strands of gray hair (1 gram, 10 cm BM-W, Beaulax,
Tokyo, Japan) were used and subjected to extraction with the four solvents, using the
same method noted above. The hair tresses were then soaked in a solution containing 3.0
g of acid orange 7, 9.0 g of citric acid, and 1.0 g of sodium citrate per liter (pH 2.7) at
35°C for five minutes. After rinsing the tresses with deionized water and drying, the
color index [light (1), red (a), and yellow-blue (b)} of each hair strand was determined
using a chromometer (CR-400, Konica Minolta Sensing, Osaka, Japan). The extent of
dyeing was then determined by calculating the differences in the indexes between
untreated and dyed hair samples (i.e., Lil, Lia, db) using the following formula: dyeing
extent (dE) =SQRT{(d1)2 +(Lia)2 +(Lib)2 }.
SAXS EXPERIMENT
SAXS experiments were performed with a 5-µm high-flux beam (BL40XU High Flux
Beamline) at the SPring-8 facility (Hyogo, Japan) using human hair fibers under an

CMC STRUCTURE IN HAIR CUTICLE 13
atmosphere of 30 ° C and 50% relative humidity. A high-flux beam emitted from a
helical undulator (A. =0.083 nm) was focused with two mirrors laid horizontally and
vertically (8). In an experimental hutch, an X-ray beam 5 µm in diameter was produced
behind two pinholes, the first 5 µm in diameter and the second 100 µm in diameter. The
sample-to-detector distance was set at approximately 2.3 m. The reciprocal spacing (S)
was calibrated by a spacing of 4.894 nm for lead stearate. The X-ray diffraction profile
was recorded by a two-dimensional detector with an exposure time of 1 second, using an
X-ray image intensifier coupled to a cooled CCD camera (1024 x 1024 pixels). The
thickness of the �-layer and 3-layer was estimated using the method of Ohta et al. (7).
RESULTS
EFFECTS OF SOL VENT EXTRACTION ON HAIR DYEING
The effects of solvent extraction on the properties of hair dyeing are shown in Figure 1.
All of the solvents used in the present study elevated the extent of dyeing as compared
with that of non-extracted hair fibers. The differences seemed slight but have statistical
significance. There were differences in the extent of hair dyeing among the solvents used,
as hair subjected to extraction with hexane tended to become smaller.
DIFFRACTION FROM CUTICLE
An illustration of a SAXS diffraction pattern from a portion of the cuticle is shown in
Figure 2, in which signals vertical to the hair axis are tilted approximately 3° with an
46 ***
45
44
***
]..__,,,
+'43
C
Q.) .µ 42 X
Q)
b.O 41 -�
Q) 40 �
0
39
38
None MeOH Ace Hex Cl/Me
Solvent extraction
Figure 1. Effect of solvent extraction on hair dyeing properties. None: non-extracted. MeOH: extracted
with methanol. Ace: extracted with acetone. Hex: extracted with hexane. Cl/Me: extracted with a mixture
of chloroform and methanol (2:1). Mean ± standard derivation (n =8). :-itat1st1cal significance was analyzed
using a Dunnett test. *p 0.05, ***p 0.001.