COM

MPOSITION F
FOR PREVENT
TING GRAY HAIR
H
(WO2009066938)
(USA
A).
Expe
erimental proccedure is as fo
ollows. Hairs at
a the back of 12-week-old
1
m
mice
are removed by depilattion. The area
a of depilation is
cons
stant for all ind
dividual mice. From the nextt day, hair graying inhibiting
g substances are
a applied on
n the depilated
d area, twice a day.
As th
he vehicle for the hair grayin
ng inhibiting substances, a mixture
m
of EtO
OH, 1,3-BG an
nd DW (3:2:5, v/v/v) is used. The vehicle itself
is us
sed as negativve control subsstance, and 50
0 mM IBMX ad
dded to the ve
ehicle is used as positive co
ontrol substancce.
2.5%
% arrowroot ad
dded to the ve
ehicle is used as test substa
ance. About 3 weeks later, when
w
the effecct of inhibiting gray hair of th
he
subs
stances are ap
ppreciable with
h eyes, newly grown hairs are
a collected and
a the melanin content in the hairs is me
easured. The
prote
ease Esperase
e (Novozymess) is used to measure
m
the melanin
m
conten
nt in the hairs. A reaction bu
uffer is prepare
ed by dissolvin
ng
Espe
erase in a bufffer (50 mM Triis-HCl, 5 mM DTT, pH 9.3) to a concentra
ation of 1 NPU
U/mL. 5 mg off mouse hair iss added to 1 mL
m of
the reaction
r
bufferr, and reaction
n is carried outt at 37C for 13 hours, while
e shaking at 1,000 rpm. The
ereafter, the hair is instantlyy
sepa
arated from the
e reaction solu
ution by centriifugation. Thus obtained rea
action solution
n is placed on a 96-well platte and absorba
ance
is me
easured at 405 nm to meassure the melan
nin content in the
t reaction so
olution.

Fig. 3 shows the re

esult of macro
oscopic observvation and me
elanin assay in
n the hair afterr treating the hair
h graying-in
nduced leukop
plakic
mice
e with the nega
ative control substance, the
e positive contrrol substance,, and the test substance. Fig
g. 3a shows th
he photograph
hs of
the hairs
h
grown on
n the back of the
t mice after 3 weeks of ap
pplication. Fig. 3b is a graph
h showing the melanin conte
ent in grown hair.
h
As seen in Fig. 3, arrowroot prevents gray ha
air and increasses the melanin content in hair
h in vivo in hair
h graying-induced mice.
The composition d
disclosed here
ein may be pre
epared as follo
ows. Howeverr, the following
g preparation examples
e
are not intended to
t be
limiting of this discclosure.
Prep
paration Example 1: Hair shampoo

Prep
paration Example 2: Hair conditioner

Prep
paration Example 5: Ointment

While the exempla

ary embodime
ents have been
n shown and described,
d
it will
w be understo
ood by those skilled
s
in the art
a that variouss
chan
nges in form and details mayy be made the
ereto without departing
d
from
m the spirit and
d scope of this
s disclosure ass defined by th
he
appe
ended claims.

In ad
ddition, many modifications can be made to adapt a particular situatio
on or materiall to the teachin
ngs of this disclosure withou
ut
depa
arting from the
e essential sco
ope thereof. Therefore, it is intended that this disclosure
e not be limite
ed to the particcular exemplary
embodiments discclosed as the best
b
mode con
ntemplated forr carrying out this disclosure
e, but that thiss disclosure will include all
embodiments fallin
ng within the scope
s
of the appended
a
claim
ms.