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Requests concerning with intellectual property rights and others
The catalogues, technical documents, samples and the like materials of our company’s products that you are provided for this time, are supplied in favor
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30 :-;+1.24<' *$' =)0*;4&' >?' @AAB

 CONTENTS

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31
NF-κB and Photo-Aging1) to 7)

Human body has the genetic information of about twenty-two thousand genes and has been
maintained by the information. The gene exhibits its function by expressing the appropriate
amount of information where and when the information is needed. When the information is not
necessary, the gene is locked so as not to exhibit its function. The key to unlock the gene is
called “Transcriptional factor.” One of the representative transcriptional factors is “Nuclear
factor-kappa B <NF-κB>.”
NF-κB is activated in response to various stimulus events to increase the expression of
various genes involved in immunity, proliferation, apotosis, inflammation and so on (Fig. 1). In

@*)C"%OAIH;''*-:%-2%V(H*-/'%);:;'%KG%9@$κD%

9-HQ(R% % % % % % % % M:2R(QQ(.*-:%Z%[\
a normal condition, NF-κB unlocks a
gene only when the gene expression is
necessary. When a cell is led to an
abnormal condition by inflammation
etc., NF-κB is activated enormously to
excessively enhance the gene
expression (Fig. 2). It has been
considered that such excessive gene
expression induces various disorders.
In fact, it has been reported that excess 9;J;''(HG (Q-/:.% -2
9@$κD%2-H%R*2;$'/II-H. OAJ;''*V;%9@$κD
activation of NF- κ B leads to the
@*)C?% M:2R(QQ(.*-:% (:E% OAJ;''*V;% 9@$κD%
occurrence of cancer, rheumatism,
psoriasis and so on. Therefore, NF-κ
B inhibitors are now under development extensively in the world.

1
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NF- κ B exists also in skin epidermal keratinocyte and dermal fibroblasts, and it has
been confirmed that NF- κ B is expressed excessively by stimuli such as ultraviolet
radiation and inflammation to result in an increase in the transcriptional activity (Fig.3).
Excess expression of NF- κ B induces inflammatory cytokines in the cells which
further induces the expression of NF- κ B. That is, uncontrollable spiral production of NF-
κ B occurs. Excess expression of NF- κ B increases the production of bFGF (basic
Fibroblast Growth Factor), a cell growth factor (Fig. 4), which causes epidermal thickening due
to hyperproliferation and abnormal keratinization of epidermal keratinocytes and
pigment deposition due to melanocyte proliferation. It is considered that such

2
changes lead to photo-aging of the skin (Fig. 5).
We confirmed that NF-κB played a major role in photo-aging, verified that various
photo-aging symptoms could be
prevented by NF- κ B inhibitors
(Fig. 6) and published the results *.
We have confirmed at the same
time that the inhibition of the NF-
κB action does not give adverse
effects on normal skin functions 7).
It can be expected that NF-κ
B inhibitors have the capability to
safely protect the skin from
photo-aging.
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*124th Japan Pharmaceutical Society meeting (2004)
104th meeting of Japanese Dermatological Society (2005)
J.Pharmacol. Exp. Ther (2005)

3
Cynaropicrin and Biobenefity7) 8)

Since it can be expected that NF-κB inhibitors have the capability to protect the skin from
photo-aging, a screening test of the candidates was performed on the basis of the inhibitory
activity on the increment of NF-κB gene transcription stimulated by TNF-α (Tumor Necrosis
Factor-α) in a cell. In the screening test, we found that artichoke extract had a powerful
inhibitory activity on the increment of NF-κB transcription. Thus,

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FG:(H-I*JH*:%
F"BP??dN% % ,Se!5N%
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artichoke extract was purified by silica gel
9@$WD%.H(:'JH*I.*-:%(J.*V*.G

column chromatography etc. NMR (H, C) "44

=M:JH;Q;:.(R%V(R/;>

analysis of an isolated component revealed #4

that an active component of artichoke extract
was “cynaropicrin” (Figure shown below). It N4
was cleared that cynaropicrin had very
54
strong inhibitory activity on TNF- α
dependent enhancement of NF- κ B ?4
transcription in a concentration-dependent
4
manner (Fig.7. presented at the 125th
$ a a a a
Annual Meeting of the Pharmaceutical X9@$]
Society of Japan). FG:(H-I*JH*: $ $ ? 5 # =μ,>
Thus we developed “Biobenefity”, highly @*)C6%M:0*K*.-HG%O22;J.%-2%9@$κD%XH(:'JH*I.*-:(R
purified artichoke extract, prepared by &J.*V*.G%-:%FG:(H-I*JH*:%
removing almost all components except
cynaropicrin from artichoke crude extract
using various column chromatography
purification methods. "44
Since Biobenefity contains cynaropicrin
M:0*K*.*-:%Y(.;%-2%9@$WD
PGI;H.H(:'JH*I.*-:%=c>

#4
with powerful inhibitory action on an
increase in NF-κB transcription, it can be
N4
expected to have a powerful capability to
protect the skin from photo-aging. The 54
comparison of a capability to inhibit an D*-K;:;2*.G
increase in NF- κ B transcription between ?4 FG:(H-I*JH*:
Biobenefity and cynaropicrin alone revealed
that both compounds had almost the same 4
capability on the basis of cynaropicrin unit D*-K;:;2*.G 4 " ? ! 5 3 =c>
(Fig. 8). Therefore, it was cleared that the FG:(H-I*JH*: 4 ? 5 N N # =μ,>
powerful effect of cynaropicrin was exhibited
also in Biobenefity. @*)C#% F-QI(H*'-:% K;.b;;:% FG:(H-I*JH*:
(:E%D*-K;:;2*.G%

4
Cynara scolymus L. (Compositae)9) to 12)

Biobenefity is extract obtained from the above ground

portion of Cynara scolymus L. (Compositae), genus Cynara
in Composite family. Artichoke is called artichoke in
English, Artichaut in French and Chosenazami in
Japanese. It has been said that the original home of
Cynara scolymus L. is European Mediterranean coastal
region and African Northern region. It has been, however,
considered that Cynara scolymus L. was produced by
improving Cynara cardunculus originally grown wild in
Mediterranean Midwest region and used the leafstalk for
food. Cynara scolymus L. was cultivated in Italy in the 15th century and it was enthusiastically
improved much better for food vegetable in France from the 16th century and afterwards. It
came to Japan from the Meiji era and the majority of
the plant commercially available now has been
imported from EU or American continent.
Cynara scolymus L. is 60 to 200 cm in height, the
leaves have long deeply serrated bases and thick white
tomenta on the back. A large purple head-like
inflorescence with about 15 cm in diameter blooms
early summer to summer. The pulpy portion of
involucral bracts of buds just before bloom is used as
herbal medicine and as food after boiling. The flower is
enjoyed as cut flowers.
The tori and involucres contain cynarin, chrologenic
acid, caffeic acid, protein, carotene, vitamin C etc. and it
has been known that the leaves, tori and involures have
various pharmacological actions such as diuresis, making
the body strong, acceleration of bile secretion, a decrease in
blood cholesterol concentration and liver protection.
Therefore, they have been used for the treatment of
arteriosclerosis, jaundice and dyspepsia, and appetite
enhancement.
Biobenefity is highly purified extract of artichoke prepared by removing almost all
components except cynaropicrin, an active component, from crude extract of artichoke with
various column chromatographies. By such high purification, the product has very good
stability and compatibility.
Biobenefity was named using bio and benefit, which means that it is a raw material for
cosmetics having benefit of bio (plant) cultivated on the earth.

5
 Introduction

Biobenefity is a 1,3-butylene glycol water solution to dissolve an ethanol extract of an aerial

part of Cynara scolymus L. (Compositae).

"##$%&%'!
Biobenefity inhibits NF-κB Hypertranscription in skin and below mentioned prevention of
photo-ageing was observed.

Photo-Aging $(!)$*+,!
PGI;HU;H(.*:*i(.*-: ●% M:0*K*.-HG%O22;J.%-2%K@1@%8H-E/J.*-:%
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"-$./+0$1
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2/+0$1 TU*:

Visible Pore Also, prominent skin’s pores was observed by

Inhibitory Effect of NF-κB Hypertranscription
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6
Inhibitory Effect of NF-κB Hypertranscription

Transcription of NF-κB gene is enhanced by stimulus such as inflammation and ultraviolet

irradiation in the skin cells to lead to epidermal hyperkeratinization and disrupture of
extracellular matrix such as collagen, which causes photo-aging of the skin.
The effect of Biobenefity on TNF-α dependent NF-κB transcription was examined in
human cells.

X;'.%T(QIR;%
Biobenefity is applied 1 to 3% as final concentration. As control, 50% 1,3-Butylene Glycol
was applied.

X;'.%,;.0-E%
293 cells (human embryonic kidney cells, Riken) were seeded on a 12-well dish using DMEM
(Sigma) containing 10% fetal bovine serum (Thermo Trace). At 24 hours after seeding, firely
luciferase reporter vector, pGL-3-BasicVector with a base sequence of NF-κB binding site and
an internal standard, pRL-TK Vector expressing renilla luciferase were transfected into the cells
with Fugene-6 transfection reagent (Roche Diagnostics). Twenty-four hours later, a test sample
was added and 2 hours later, TNF-α was added to make the final concentration 0.5 ng/mL.
Twenty-two hours later, cell contents were extracted to assay luciferase activity in the extract
with Luciferase Assay System (Promega) by a luminometer (Promega TD-20/20 Luminometer).
The amount of NF-κB expressed was obtained by dividing the assay value of firefly luciferase
(amount of NF-κB transcribed) by the assay value of renilla luciferase (amount of cells). The
amount of NF-κB expressed in the addition of TNF-α was defined as 100 to calculate the
relative value in the treatment of each test sample.

Y;'/R.%(:E%_*'J/''*-:%
The effect of Biobenefity on TNF-α dependent NF-κB transcription was shown in Fig.9.
Although amount of NF-κB Hypertranscription was caused by stimulation of NF-α, NF-κB
Hypertranscription was strongly inhibited by concentration of Biobenefity.
According to this result, Biobenefity is expected to inhibit photo-aging by inhibitory effect of
NF-κB hypertranscription.

X9@]=$>

F-:.H-R

D*-K;:;2*.G%"c

D*-K;:;2*.G%?c

D*-K;:;2*.G%!c

4 ?4 54 N4 #4 "44 "?4
9@$WD%.H(:'JH*I.*-:%(J.*V*.G%=M:JH;Q;:.(R%V(R/;>

@*)CB%M:0*K*.-HG%O22;J.%-2%9@$κD%PGI;H.H(:'JH*I.*-:%
7
Inhibitory Effect of bFGF Production (NF-κB)

bFGF (basic Fibroblast Growth Factor) is produced excessively at the inflammatory sites
and the skin exposed ultraviolet rays to enhance abnormal proliferation (hyperkeratinization) of
the hyperkeratinization, which leads to dry skin due to the thickness of the horny layer and
abnormal differentiation of epidermal keratinocyte, and to pigment deposition. It has been
known that NF-κB is expressed excessively at the occurrence of inflammation to increase bFGF
production. Therefore, a substance that inhibits NF-κB dependent bFGF production can be
expected to prevent horny layer thickness, dry skin and pigment deposition due to inflammation
and ultraviolet irradiation (photo-aging).
Using epidermal keratinocyte expressing NF- κ B excessively, the inhibitory effect of
Biobenefity on bFGF production was examined.

X;'.%T(QIR;%
Biobenefity is applied 3% as final concentration. Also, cynaropicrin of active component
was applied as same concentration as Biobenefity. As control, 50% 1,3-Butylene Glycol was
applied.

X;'.%,;.0-E%
Normal human epidermal keratinocyte (Kurabo NHEK) was seeded on a 12-well plate
(Corning) with serum-free medium EpiLife-KG2 (Kurabo) and preincubated for 24 hours at 37°C
under 5% CO2 condition. Thereafter, pCMV-p65 Vector expressing p65, a subunit of NF-κB,
was transfected into the cells with Fugene-6 transfection reagent (Roche Diagnostics) so that the
cells are changed to those that can express active NF-κB strongly. After further 24-hour
incubation, the medium was changed to fresh one. Then a test sample was added and the cells
were further incubated for 48 hours. After the incubation, the supernatant of the culture
medium was isolated to determine bFGF with bFGF EIA kit (Cytimmune). At the same time,
the number of cells was counted with Cell Counting Kit-8 (Dojindo) to calculate the amount of
bFGF per cell, which was used for the evaluation.

8
Y;'/R.%(:E%_*'J/''*-:%
The inhibitory effect of Biobenefity on bFGF production is shown in Fig. 10. In the
modified cells with strong expression of NF-κB, the amount of bFGF produced was significantly
increased. Biobenefity inhibited bFGF production significantly in the cells. The addition of
cynaropicrin also inhibited bFGF production like the addition of Biobenefity, confirming that
cynaropicrin played a major role in the inhibitory effect of Biobenefity.
It was confirmed by above-mentioned results that Biobenefity inhibited bFGF production
caused by excess NF-κB. Therefore, it can be expected that Biobenefity improves the aging
skin symptoms such as abnormal keratinization and pigment deposition due to inflammation
and ultraviolet irradiation.

**
9-HQ(R%J;RR
=L-b%9@$WD>

F-:.H-R

D*-K;:;2*.G%!c
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FG:(H-I*JH*:
=F-HH;'I-:E%.- **
%%%%%!c%D*-K;:;2*.G>
4 4C3 " "C3 ?
5
8H-E/J.*-:%(Q-/:.%-2%K@1@%=:)Z"4 %J;RR'>%jj%I^443

@*)C"4%M:0*K*.-HG%O22;J.%-2%K@1@%8H-E/J.*-:%

9
Inhibitory Effect of Epidermal Melanocyte Proliferation (NF-κB)

Epidermal melanocyte producing melanin pigment in the skin is increased by ultraviolet

irradiation, chronic inflammation, etc. and it has been said that the increased epidermal
melanocyte leads to an increase in the amount of melanin production in the skin. NF-κB is
expressed excessively in epidermal keratinocyte at the occurrence of inflammation and increases
the production of bFGF with proliferating activity on melanin cells in epidermal keratinocyte.
Therefore, it can be expected that a substance inhibiting NF- κ B activity prevents the
occurrence of chloasma and freckle caused by inflammation and ultraviolet irradiation, that is,
pigment deposition.
Using human epidermal keratinocyte modified to express NF-κB excessively and human
epidermal melanocyte, the inhibitory effect of Biobenefity on epidermal melanocyte proliferation
was examined.

X;'.%T(QIR;%
Biobenefity is applied 3% as final concentration. Also, cynaropicrin of active component
was applied as same concentration as Biobenefity. As control, 50% 1,3-Butylene Glycol was
applied.

X;'.%,;.0-E%
Normal human epidermal OI*E;HQ(R%Q;R(:-JG.; OI*E;HQ(R%J;RR%
keratinocyte (Kurabo NHEK)
was seeded on a 12-well plate
(Corning) with serum-free
medium EpiLife-KG2 (Kurabo)
F0(:);%9@$κD% %
and preincubated for 24 hours at
PGI;H$;AIH;''*-:%J;RR'%
37°C under 5% CO2 condition.
Thereafter, pCMV-p65 Vector =M:E/J;%IN3%);:;>%
&EE%.;'.%'(QIR;
expressing p65, a subunit of NF-
κ B, was transfected into the
cells with Fugene-6 transfection F/R./H;%'/I;H:(.(:.%
F/R./H;%
reagent (Roche Diagnostics) so &EE
that the cells are changed to
those that can express active F-/:.%:/QK;H%-2%J;RR'
NF-κB strongly. After further @*)C%""%F;RR%F/R.*V(.*-:%,;.0-E%
24-hour incubation, the medium
was changed to fresh one. Then a test sample was added and the cells were further incubated
for 48 hours and the supernatant of the culture medium was isolated.
Human epidermal melanocyte (Kurabo NHEM) was seeded on a 24-well plate (Corning)
with low-serum medium Medium 154S (Kurabo) and incubated for 24 hours at 37°C under 5%
CO2 condition. Thereafter, the medium was changed to a medium prepared by mixing Medium
154S without a proliferation promoter and the supernatant of epidermal keratinocyte culture
medium collected in the previous test at the volumetric ratio of 1 to 4 and the cells were
incubated for a further 72 hours (Fig. 11). After the completion of incubation, the cells were
isolated and the number of cells was counted with a blood cell counter.

10
Y;'/R.%(:E%_*'J/''*-:%
The inhibitory effect of Biobenefity on epidermal melanocyte proliferation is shown in Figs.
12 and 13. The addition of the culture medium supernatant of epidermal keratinocyte with
excessive expression of NF-κB to the medium for the cultivation of epidermal melanocyte
significantly increased epidermal melanocyte proliferation. On the other hand, the culture
medium supernatant obtained from epidermal keratinocyte incubated in the addition of
Biobenefity did not increase epidermal melanocyte proliferation. In the addition of cynaropicrin,
the inhibition of epidermal melanocyte proliferation was also observed like in the addition of
Biobenefity. Thus it could be confirmed that cynaropicrin also played a major role in the effect
of Biobenefity.
It was confirmed by above-mentioned results that Biobenefity inhibited epidermal
melanocyte proliferation caused by excess NF- κ B. Therefore, it can be expected that
Biobenefity improves the pigmentation such as black spot and freckle to inflammation and
ultraviolet irradiation.

9-HQ(R%J;RR%=R-b%9@$κD> F-:.H-R%=9@$κD%;AJ;''*V;%J;RR%>

!c%D*-K;:;2*.G% FG:(H-I*JH*:%
=9@$κD%;AJ;''*V;%J;RR%> =9@$κD%;AJ;''*V;%J;RR%>%
@*)C"?%M:JH;(';%;22;J.%-2%OI*E;HQ(R%Q;R(:*:%J;RR'%"%

9-HQ(R%J;RR **
=L-b%9@$WD>

F-:.H-R

D*-K;:;2*.G%!c
**

FG:(H-I*JH*:
=F-HH;'I-:E%.-
**
%%%%%!c%D*-K;:;2*.G>
4 " ? ! 5 3
3
X0;%:/QK;H%-2%J;RR'%=k%"4 %J;RR'ZQL>%%%jjI^4C43
@*)C"!%M:JH;(';%;22;J.%-2%OI*E;HQ(R%Q;R(:*:%J;RR'%?%
11
Whitening Effect on Human Skin

NF-κB is hyper-released by inflammation in epidermal cell and production of bFGF; which

has efficacy of melanin production from epidermal keratinocyte causes hyperkeratinization. We
investigate the whitening effect of Biobenefity on human skin.

X;'.%T(QIR;%
Biobenefity is diluted 20 times by 50% 1,3-Butylene Glycol, adjusted to 5% solution and use
as test sample. 50% 1,3-Butylene Glycol is used as control.

X;'.%,;.0-E%
The test detail is explained to volunteers in advance, and after confirming consent, they
cooperated in these tests. Test sample and control was applied on both cheeks twice a day for 3
months. Volunteers were allowed to use their general cosmetic items during this test. The test
was conducted from December 20, 2004 to March 22, 2006
Black (dark) value was measured before application, after application and one to three
months later by MEXAMETER MXI8 (Courage + Khazka electronic GmbH, Germany).

Y;'/R.%(:E%_*'J/''*-:%
Transition of melanin index of 5 volunteers is shown in Fig. 14. Melanin index was down on
skin applied with Biobenefity improved.
According to this result, Biobenefity has a whitening effect on human.

@;Q(R;%&%=?4'> ,(R;%&%=54'> ,(R;%D%=!4'>

?44 ?34 !44

,;R(:*:%M:E;A

?44 ?34

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4 " ? !%Q-:.0 4 " ? !%Q-:.0 4 " ? !%Q-:.0

,(R;%F%=!4'> ,(R;%_%=54'>

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D*-K;:;2*.G
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4 " ? !%Q-:.0 4 " ? !%Q-:.0

@*)C"5%S0*.;:*:)%O22;J.%-:%P/Q(:%TU*:%

12
Inhibitory Effect of B16 Melanoma Cell

We investigated melanin production inhibition test on B16 melanoma cells for Biobenefity

X;'.%T(QIR;%
Biobenefity is applied 1% and 2% as final concentration. As control, 50% 1,3-Butylene Glycol
was applied.

X;'.%,;.0-E%
B16 melanoma cells were used. MEM culture medium (GIBCO BRL) including 5% fetal
bovine serum (ThermoTrace) were used and cultured at 37℃ and 5% CO2. 2 x 105 B16
melanoma cells were planted in a 60 mm plastic culture dish and were pre-cultured 24 hours.
Then they were transferred to a fresh culture medium and test materials were added. The cells
were collected by processing with trypsin after culturing for three days. Cells were dissolved in
1N NaOH and 10% DMSO and then absorbance was measured at 420nm. At the same time, the
number of the cells were measured, and amount of melanin per cell were measured and
analyzed.

Y;'/R.%(:E%_*'J/''*-:%
Amount of melanin in medium was shown in Fig. 15. Biobenefity was observed to have
significant inhibition effect of melanin production.
According to results, Biobenefity is expected to have strong whitening effect.

F-:.H-R

D*-K;:;2*.G%"c
**

D*-K;:;2*.G%?c
**

4 4C3 " "C3

&Q-/:.%-2%Q;R(:*:%I;H%J;RR%=d_5?4:Q>%%%jjI^4C43

@*)C"3%M:0*K*.-HG%O22;J.%-2%,;R(:*:%

13
Improvement Effect of Skin Elasticity on Human Skin

A decrease in skin elasticity finally leads to aging symptoms such as wrinkles and pouches.
It has been considered that such decrease in skin elasticity is caused by the degradation and
denaturation of dermic components such as collagen due to the action of active oxygen and due to
inflammation. A decrease in elasticity due to epidermal hardness can be also considered to be
one of the causes. Since bFGF produced by the action of NF-κB increases epidermal thickness,
there is a possibility that bFGF decreases skin elasticity by hardening the skin. Thus an
improving effect of Biobenefity with an inhibitory action on NF-κB on skin elasticity was
examined.

X;'.%T(QIR;%
Biobenefity is diluted 20 times by 50% 1,3-Butylene Glycol, adjusted to 5% solution and use
as test sample. 50% 1,3-Butylene Glycol is used as control.

X;'.%,;.0-E"!>%
5 volunteers at age 20s to 40s that gave us written informed consent were enrolled in this
study. Each test sample was applied around the left and right eyes of each subject twice a day
for 3 months. Volunteers were allowed to use their general cosmetic items during this test. The
test was conducted from December 20, 2004 to March 22, 2006.
Before and at 3 months after the commencement of the application, the elasticity of the skin
was measured by a skin viscosity and elasticity meter ! Cutometer (CUTOMETER SEM474,
COURAGE + KHAZAKA Electronic GmbH). The elasticity was calculated by the change of the
skin condition when the skin was sucked for 5 sec by instantly reducing the pressure to 500 mb
and thereafter the negative pressure was instantly released, which were done twice.
The test was performed several times each in right and left application sites, and the
percentage of the change of the mean values between the value before the commencement of the
application and that at 3 months after the commencement of the application was calculated to
obtain the rate of the change (Ur/Uf). The measurement was performed 20 min after
acclimation in an air conditioning room (room temperature: 20"C and humidity: 50%) after
washing the face.

14
Y;'/R.%(:E%_*'J/''*-:%
The change of elasticity before application and 3 months later of 5 volunteers is shown in
Fig.16. According to application of Biobenefity, elasticity were improved for 5 volunteers
compared with control.
According to this result, Biobenefity has improvement effect of elasticity.

@;Q(R;%&%=?4'> ,(R;%&%=54'> Male B (30s)

4C6 0.5
Elasticity (Ur/Uf)

4C5

4CN3 4C!3 0.45

4CN 4C! 0.4

4C33 4C?3 0.35

4C3 4C? 0.3

4C53 4C"3 0.25

4C5 4C" 0.2

4 !%Q-:.0 4 !%Q-:.0 0 3 month

,(R;%F%=!4'> ,(R;%_%=54'>
4C3 4C3

4C53 4C53

4C5 4C5
D*-K;:;2*.G
4C!3 4C!3
F-:.H-R%
4C! 4C!

4C?3 4C?3

4C? 4C?
4 !%Q-:.0 4 !%Q-:.0

@*)C"N%MQIH-V;Q;:.%O22;J.%-2%TU*:%OR('.*J*.G%

15
NF-κB and Action of Skin Pore14 to 17)

One of the major recent skin trouble topics

is “Noticease skin pores.” Until now, so much Spread-Open
attention has not been paid to such topic. The
recent beauty skin boom and increased
consciousness about the skin appearance
because of the increased chance of seeing the
skin condition much more closely due to the
advent of high resolution digital cameras and @*HQ%I-H;% dI;:;E% I-H;% KG% KR-JU*:)
high vision are one of the reasons why the ;AJ;''*V;% R*I*E+% 0-H:G
concern for skin’s pores was heightened. '/K'.(:J;%;.JC%

It is considered that there are roughly two

factors for the noticease skin pores (right figure).
One is opening of the skin’s pores. It has been
said that excessive sebaceous secretion makes
the skin’s pores around the nose open.
However, it has been recently reported that the @*HQ;E%I-H;% DG%(K:-HQ(R%U;H(.*:*i(.*-:
-2% I-H;% -/.'U*H.'+% (% I-H;
extend of the skin’s pores by conically sinking of
-I;:'%J-:;$'0(I;EC%
the pores caused by abnormal keratinization,
enhances parakeratosis, in epidermal cells Blackhead
around the skin’s pores led to the prominence of
the skin’s pores.
Another one is darkening of skin’s pores.
The pores apparently darken due to the dirty
staining of sebum of keratotic plug packed in
the skin’s pores and of keratin. Furthermore, [II;H ,;R(Q-JG.;%
pigment deposition due to ultraviolet
irradiation is listed as one of the causes.
Epidermal melanocyte exists also in the inside
of the skin’s pores so that melanin is made in M:V*'*KR;%I-H; 8*)Q;:.%J;RR%(:E%Q;R(:*:%(H;
the pores. Epidermis in the pores appears *:JH;(';EC% _;;I% I-H;+
;'I;J*(RRG+%R--U'%KR(JUC%
dense from the outside because of the deep
positioning. By ultraviolet irradiation, not
Visible Pore only dirtiness but also the skin’s pores themselves
apparently darken.
/;$*&,%+ It is expected that excess expression of NF-#B
D4&%01$1E%01*$
$
may be involved in these two causes for the
1; 1 01* κ H ;FGF (M&4%<J=M4$ prominence of the skin’s pores (following table).
N $2 IFJκH !"*&$J.2%M4<':*&4.5
Abnormal keratinization considered as a cause for
opening of the skin’s pores and an increase in the
N$21 κH melanin production causing darkening of pores
;101* :&*+174&%01*$'*7'
$ K4+%$*)#04. are phenomenon caused by the increased NF-κB
H+%)324%< action.

L1.1;+4':*&4.

16
Improvement Effect of Pore on Human Skin

We investigated improvement effect of pore on Human Skin for Biobenefity.

X;'.%T(QIR;%
Biobenefity is diluted by purified water, adjusted to 5% solution and use as test sample. 50%
1,3-Butylene Glycol is used as control.

X;'.%,;.0-E%
Fifteen male and female healthy volunteers in their
twenties to forties that gave us written informed consent were
employed as subjects in the present study. Each test
substance was applied overall on left and right face twice a day
for 2 months after face washing. There was no limitation on
the usage of cosmetics other than the test substance. The test
was performed during June 9, 2005 and August 16, 2005.
Before the commencement of application, and 1 and 2
months after the commencement application, the number of
the prominent skin’s pores around the nose and the cheek was
counted by a Roboskin Analyzer RSA-50 (Inforward, right
photo).
The measurement was performed after 20-min acclimation in a constant temperature and
humidity controlled room (temperature: 20"C and humidity: 50%) after face washing.

17
Y;'/R.%(:E%_*'J/''*-:%
The images of the skin’s pores before the commencement of application and the
representative improvement images of the skin’s pores 2 months after the commencement of
application at each application site of 15 subjects are shown in Fig. 17 and Fig. 19, respectively,
and the change of the number of the skin’s pores opened is shown in Fig. 20. Nine of 15 subjects
showed a decrease in the number of the skin’s pores opened at the Biobenefity application site
and three other subjects showed a tendency towards improvement. It was only three subjects
that showed no improvement (Fig. 18). There was no subject whose symptom was aggravated
compared with the pre-application condition.
From these results, it can be expected that Biobenefity shows an improving effect on the
prominent skin’s pores.

,(R;%&%=!4'>%

=,!"##/%*
<676!8

9$:(!,#
;0-+,)/0/(*
<676!8

3/(/#$%$&4!"##/%*
5676!8

,(R;%O%=54'>%

@*)C"#%MQIH-V;Q;:.%-2%'U*:%I-H;

@*)C"6%OA(QIR;%-2%*QIH-V;Q;:.%;22;J.%-2%I-H;%

@;Q(R;%&%=?4'>%

D;2-H;%&IIR*J(.*-:% ?%,-:.0%L(.;H%

18
@;Q(R;%D%=?4'>%

D;2-H;%&IIR*J(.*-:% ?%,-:.0%L(.;H%

@;Q(R;%F%=?4'>%

D;2-H;%&IIR*J(.*-:% ?%,-:.0%L(.;H%

,(R;%D%=54'>%

D;2-H;%&IIR*J(.*-:% ?%,-:.0%L(.;H%

@*)C"B%OA(QIR;%-2%*QIH-V;Q;:.%;22;J.%-2%I-H;

19
 MQIH-V;Q;:. X;:E;:JG%.-b(HE'%*QIH-V;Q;:. 9-%MQIH-V;Q;:.

@;Q(R;%&%=?4'> ,(R;%D%=54'> @;Q(R;%_%=?4'> ,(R;%M%=?4'>

#44 544
Visiable pore (Number)

?44
644
"N4 !N4
644 N44
"?4 !?4
344
#4 ?#4
N44
54 544 ?54

4 344 !44 ?44

4 " ?%,-:.0 4 " ?%,-:.0 4 " ?%Q-:.0 "ヶ月 ?ヶ月 !ヶ月

@;Q(R;%D%=?4'> ,(R;%F%=!4'> ,(R;%@%=54'> @;Q(R;%O%=?4'>

!44 B44 644 "#4

?#4 #44 N44 "N4

?N4
644 344 "54
?54
N44 544 "?4
??4

?44 344 !44 "44

4 " ?%Q-:.0 4 " ?%,-:.0 4 " ?%Q-:.0 4 " ?%%,-:.0

@;Q(R;%F%=?4'> ,(R;%_%=!4'> ,(R;%P%=?4'> @;Q(R;%@%=?4'>

"?44
!44
!44 B44
"444
?44
?44 #44
#44

"44 644 "44

N44

4 544 N44 4
4 " ?%Q-:.0 4 " ?%Q-:.0 4 " ?%Q-:.0 4 " ?%,-:.0

,(R;%&%=!4'> ,(R;%O%=54'> ,(R;%1%=!4'>

""44 "?44

344 "444
"444 D*-K;:;2*.G
B44
544
#44 F-:.H-R%
#44

!44 644 N44

4 " ?%Q-:.0 4 " ?%Q-:.0 4 " ?%Q-:.0

@*)C%?4%MQIH-V;Q;:.%-2%'U*:%I-H;%
=T-Q;%2H(Q;%J-R-H%*'%2-Hb(HE%.-%I();%"#%(:E%"BC>%

20
Stability Test

Stability of Biobenefity was investigated.

1.Long Term Stability

Store Biobenefity in a cool dark place(4℃), room temperature, window side and at 50℃.
Absorbance value and concentration of cynaropicrin at 410nm were determined.

Y;'/R.%(:E%_*'J/''*-:%
Change of Absorbance value at 410 is shown in Fig. 21 and concentration of cynaropicrin is
shown in Fig.22. Biobenefity is stable at any condition.
According to this result, Biobenefity does not have time dependent change and is
significantly stable product.

" "?4
F-:J;:.H(.*-:%-2%FG:(H-I*JH*:

4C# "44
=T.(H.*:)%.*Q;%('%"44>
&K'-HK(:J;%=5"4:Q>

#4
4CN Y--Q%X;QC
Y--Q%X;QIC
F--R%8R(J; N4
F--R%8R(J;
4C5 S*:E-b%T*E;
S*:E-b:%T*E;
34℃ 54
34℃
4C?
?4

4 4
4 " ? ! 4 " ? !
,-:.0' ,-:.0'
@*)C?"%L-:)%.;HQ%'.(K*R*.G%"% % % @*)C??%L-:)%.;HQ%'.(K*R*.G%?%

2. Thermal stability
Biobenefity was heated in a water bath at 90℃. After cooling down, absorbance value was
measured at 410 nm.

Y;'/R.%(:E%E*'J/''*-:%
"
Thermal stability of Biobenefity is shown in Fig.
4C#
23. The increase of absorbance of Biobenefity and
&K'-HK(:J;%=5"4:Q>

precipitate were not observed. According to the 4CN

result, Biobenefity is significantly stable for short 4C5

heating.
4C?

4
4 " ? ! 5 3 N
P-/H'

@*)C?!%X0;HQ(R%T.(K*R*.G

21
3.pH Stability
pH of Biobenefity is adjusted from 3 to 10 by HCl and NaOH. Absorbance value at 410nm
were determined.

Y;'/R.%(:E%_*'J/''*-:%
Absorbance value of Biobenefity is shown in "

Fig.24 and color tone is shown in Fig.25. pH 3 to 8,

4C#

&K'-HK(:J;%=5"4:Q>
it was almost stable on color, but over pH8 the
4CN
color was darkened. Color tone was also changed a
little by little. Precipitation was not observed at 4C5

any pH range. 4C?

According to the result, Biobenefity is

4
necessary to take care at alkali range. ! 5 3 N 6 # B "4
IP

@*)C?5%IP%T.(K*R*.G

22
Compatibility Test

Compatibility of Biobenefity with various ingredients was evaluated.

X;'.%,;.0-E%
Biobenefity was diluted to 5 %, and the test samples were adjusted by purified water as
shown on the each table. After 24 hours later, mixed solution was evaluated.

Y;'/R.%(:E%_*'J/''*-:%
Results are shown on table 1 to 3.

1.Compatibility of 5% of Biobenefity with Surfactant

% Ingredients Result
2.8 Stearyl Trimethyl Ammonium Chloride ○
Cation 3.0 Cetyltrimehylammonium Chloride ○
2.7 Lauryltrimethylammonium Chloride ○
10.0 Triethanolamine Lauryl Sulfate ○
25.0 Sodium Laureth Sulfate ○
25.0 Triethanolamine Laureth Sulfate ○
6.25 Laureth-6 Carboxylic Acid ○
Anion
10.0 Sodium N-Cocoyl-N-methyl Taurate ○
10.0 Potassium N-Cocoyl Glycinate ○
7.5 Sodium Lauroyl Methylaminopropionate ○
25.0 Sodium Tetradecenesulfonate ○
10.0 Polyethylene Glycol (50) Oleyl Ether ○
10.0 Coconut Tatty Acid Diethanolamide ○
Nonion 10.0 Sorbeth-60 Tetraoleate ○
10.0 Polyoxyethylene Sorbitan Monooleate (20E.O.) ○
10.0 Polyoxyethylene Hydrogenated Castor Oil (60E.O.) ○
Silicone 10.0 Polyoxyethylene・Methylpolysiloxane Copolymer ○
3.5 Lauryl Dimethylaminoacetic Acid Betaine ○
Ampholytic 4.0 Sodium N-Cocoyl-N-Carboxymethyl-N-Hydroxyethyl Ethylenediamide ○
2.9 Lauroyl Amide Propylhydroxysulfobetaine ○
○<%1--E+% % △<%TR*)0.%X/HK*E*.G+% % ×<%8H;J*I*.(.;%

23
2.Compatibility of 5% of Biobenefity with other ingredients

% Ingredients Result
50 Glycerin ○
50 1,3-Butylene Glycol ○
Solvent 50 Propylene Glycol ○
50 Isopropyl Alcohol ○
50 Ethanol ○
0.1 Carboxyvinyl polymer ○
Synthetic 1 Polyvinylpyrrolidone ○
polymer 1 Polyvinyl Alcohol ○
1 Polyethylene glycol (6000) ○
1 Sodium alginate ○
1 Carboxymethyl cellulose ○
Natural
1 Cationic cellulose ○
polymer
0.1 Bio Sodium Hyaluronate HA12 ○
1 Hydroxypropyl cellulose ○
Phospholipid 1 Lipidure-PMB ○
Vitamin-C 2 Ascorbyl Glucoside ○
derivative 2 Pacificos VAP ○
○<%1--E+% % △<%TR*)0.%X/HK*E*.G+% % ×<%8H;J*I*.(.;%

24
3.Compatibility of 5% Biobenefity with other our products

% Product name INCI Name Result

5 FM Extract LA-B Lactobacillus / Milk Ferment Filtrate ○
5 Absorage Plantago Major Seed Extract ○
5 OUGON Liquid B Scutellaria Baicalensis Root Extract ○
5 Caffenoage Coffea Arabica (Coffee) Seed Extract ○
5 CHITIN Liquid (N) Carboxymethyl Chitin ○
5 HPCH Liquid Hydroxypropyl Chitosan ○
5 CureBerry Vaccinium Myrtillus Leaf Extract
5 Clairju Hydrolyzed Prunus Domestica ○
5 KOTHALAHIMBUTU Liquid B Salacia Reticulata Wood Extract ○
5 SAKURA Extract B Prunus Yedoensis Leaf Extract ○
5 MARINWORT IPC-14 SBW Algae Extract ○
5 SILKGEN G Soluble Hydrolyzed Silk ○
5 SILKGEN G Soluble-S Hydrolyzed Silk ○
5 TREHALOSE 30 Trehalose ○
5 NEEM Leaf Liquid B Melia Azadirachta Leaf Extract ○
0.1 Bio-PHA Na Powder Polyglutamic Acid ○
5 Bio-PGA Solution HB Polyglutamic Acid ○
5 Bio-PGA Solution LB Polyglutamic Acid ○
Pueraria Lobata Root Extract
5 Bio antiage B Chlorella Vulgaris Extract ○
Aloe Barbadensis Leaf Extract
5 PEACH Leaf Liquid B Prunus Persica (Peach) Leaf Extract ○
5 Biocellact ALOE VERA B Aloe Barbadensis Leaf Extract ○
5 Fermentage Chardonnary B Lactobacillus/Grape Juice Ferment ○
5 Fermentage Pear B Lactobacillus/Pyrus Communis (Pear) Fruit Juice Ferment ○
5 Pharconix CTP-F (BG) Hydrolyzed Collagen ○
5 JIOU Liquid Rehmannia Chinensis Root Extract ○
5 SOUHAKUHI Liquid (BG) Morus Alba Root Extract ○
5 HIOUGI Liquid Belamcanda Chinensis Root Extract ○
5 BOTANPI Liquid E Paeonia Suffruticosa Root Extract ○
5 LEMONGRASS Liquid B Cymbopogon Schoenanthus Extract ○
5 Phyto COLLAGE (N) Natto Gum ○
5 Phyto HYALURON B Hibiscus Esculentus Fruit Extract ○
5 FLAVOSTERONE SB Glycine Soja (Soybean) Protein ○
5 PrinessCare Geranium Robertianum Extract ○
5 YUZU Ceramide B Citrus Junos Fruit Extract ○
5 LACTOSACCHARIDES B Yogurt Filtrate ○
5 RYOKUCHA Liquid Camellia Sinensis Leaf Extract ○
5 LUNAWHITE B Oenothera Biennis (Evening Primrose) Seed Extract ○
○<%1--E+% % △<%TR*)0.%X/HK*E*.G+% % ×<%8H;J*I*.(.;%

25
Specification

Subject Specification
Appearance Light brown to yellowish brown liquid, having
characteristic odor.
Identification
Terpenoid Positive
Purity
Heavy metals 20 ppm max.
Arsenic 2 ppm max.
Residue on Evaporation 0.18 w/v% min.
Butylene Glycol
INCI Name Water
Cynara Scolymus (Artichoke) Leaf Extract
CAS Number 84012-14-6
EINECS Number 281-659-3

26
&EE*.*-:(R%E-J/Q;:.%

Mechanism of Cynaropicrin (Biobenefity)18)

The action mechanism of the inhibitory N$21;101*$'IFJκH

QIFJα
N$%)01O%01*$
effect of cynaropicrin, an active component IFJ3H IFJκH NJκH /)01O%01*$
:2*.M2*+#+%01*$
/)01O%01*$'(04M
of Biobenefity, on NF- κ B actions was :
:
NDD'"*,M*-$<.

IFJκH NJκH P P P
analyzed. Cynaropicrin showed the
/)01O%01*$ :
:
inhibitory effect on an increase in TNF-$ /)01O%01*$ IFJκH
NJκH P P P
IFJ3H
Q&%$.+*)%01*$'(04M R4S&%<%01*$
dependent NF- κ B transcription. (*2) Q&%$.+*)%01*$

3$,>/(/#$*' I-)+4-.
Since TNF- α dependent NF- κ B ?@'(&+,-$%+$(A
IFJκH ()*
!"#$%$&$'" "#0*M+%.,
transcription is increased by an increase in
phosphorylation of I-κB (*1) (Fig. 26), the @*)C?N% &J.*V;% IH-J;''% -2% 9@$ κ D% (:E% M:0*K*.-HG
IH-J;''%-2%D*-K;:;2*.G%
action site of cynaropicrin was presumed to
be the downstream of this step. Thus using the cells transfected with p65 gene (*3) to express

active NF-κB, the action of cynaropicrin on the steps after activation of NF-κB was examined.
As the result, cynaropicrin inhibited the increase in NF-#B transcription even in the cells
strongly expressing active NF-κB. The inhibitory degree was almost the same as that in TNF-
α dependent NF- κ B transcription (Fig. 27), suggesting that the inhibitory action of

cynaropicrin on the increase in NF-κB transcription occurs mainly at the steps after activation
of NF-κB.
Using an immune staining method, the intracellular localization of NF-κB at the increase

in TNF-α dependent NF-κB transcription was examined. The histological image revealed
that the transfer of cytoplasmic NF-κB to the nucleus by stimulation of TNF-α was blocked by
cynaropicrin (Fig. 28). Thus it was cleared that the inhibitory action of cynaropicrin, namely
Biobenefity on the increase in NF-κB transcription was produced by blocking the transfer of
active NF-κB into the nucleus (Fig. 26).

27
 "?4

"44

9@$WD%.H(:'JH*I.*-:%(J.*V*.G
#4

=M:JH;Q;:.(R%V(R/;>
N4

54

?4

4
9@$WD%=IN3> $ a a a a
FG:(H-I*JH*: $ $ ! 5 #
@*)C?6%M:0*K*.*-:%-2%9@$WD%0GI;H.H(:'JH*I.*-: -2%JG:(H-I*JH*:
KG%;AIH;''%J;RR%-2%(J.*V;%9@$κD%

T.(*:;E%&:.*K-EG%()(*:'.%9@$κD%

T.(*:;E%1;:;%

X9@$α% $% a a
FG:(H-I*JH*: $% $ a

@*)C?#%XH(:'2;H%9@$κD%*:.-%:/JR;(H%(:E%*:0*K*.*-:%KG%JG:(H-I*JH*:%

*1: It has been said that 1-#B blocks the transfer of NF-κB into the nucleus by binding with NF-
κB.
*2: Degradation of I-κB is induced by signal phosphorylation by IKK complex.
*3: NF-κB is a dimer of p65 and a cells transfected with p65 gene expresses a large amount of
active NF-κB.

28
Reference

1) Kiyotaka TANAKA et al, The Pharmaceutical Society of Japan 124 Year’s lecture edition, 3,
163 (2004)
2) Kiyotaka TANAKA et al., The Journal of Biochemistry, 76 (8) 1113 (2004)
3) Kiyotaka TANAKA et al., The Japanese Journal of Dermatology, 115 (3) 466 (2005)
4) Msamistu ICHIHASHI TAMATA et al., FOOD Style 21, 9 (9) 31-39 (2005)
5) Sewon Kang et al.,, Am J.Pathol., 166 (6) 1691-1699 (2005)
6) Kiyotaka TANAKA et al., J.Pharmacol. Exp. Ther., 315 (2) 624-630 (2005)
7) Kiyotaka TANAKA et, al, The Pharmaceutical Society of Japan 125 Year’s lecture edition, 4,
180 (2005)
8) Kaori ASAMITSU et al., Inflammation and Regeration, 26 (4) 1691-1699 (2005)
9) Minoru OKADA, Newly Revised llustrated Medicinal Plants of The World in color Hokuryu
Co.,Ltd., 554 (2002)
10) Mitsuru HOTTA., Useful Plants of the World , Heibonsya, Ltd., 351-352 (1989)
11) Kazuo IZAWA, Color Encyclopedia of Medical Hearb, SHUFUNOTOMO Co.Ltd. Tokyo
699-700 (1998)
12) Hidealo OHBA , Asahi Encyclopedia The World Plant 1, Asahishinbunsya, 26-27 (1977)
13) Tomoko SUGAWARA et al., Comprehensive Medical Examination, 20 (3) 141-148 (2001)
14) Takashi NISHIJIMA et al., SCCJ., 35 (2) 141-158 (2001)
15) Keiko TAKADA, Fragrance Journal, 33 (9) 15-19 (2005)
16) Takashi NISHIJIMA et al., Fragrance Journal, 33 (9) 27-32 (2005)
17) Satsuki KURIKI, Fragrance Journal, 33 (9) 33-38 (2005)
18) Takaki TAMURA et al., Chemical Biology Super Schematic Note, YODOSHA CO.,Ltd., 74-75
(2006)

29