Note: Descriptions are shown in the official language in which they were submitted.
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
1
COSMETIC COMPOSITION HAVING WHITENING EFFECT COMPRISING
EXTRACT OF PULSATILLA RADIX AS MAIN INGREDIENT
Technical Field
The present invention relates to a cosmetic composition comprising extract of
Pulsatillae Radix as main ingredient.
The skin of human being consisted of epidermis and dermis. Nails and hairs are
formed in the epidermis and sweat glands exist there too. In dermis there
exist nerve nets,
blood vessels and sweat glands. Melanin-producing- melanocytes exist in
epidermis. The
starting material for producing melanin is tyrosine, an essential amino acid
and the tyrosine
is oxidized by tyrosine hydroxylase(TH) into 3,4-dihydroxyphenylalanine(DOPA)
and the
DOPA is experienced through several reaction steps to be changed into melanin,
a black
polymer. If the TH-function is congenitally deficient, albinism is appeared.
Accordingly, The most important target of development of whitening material in
biosynthetic mechanism of melanin is to find any material which prevents the
activity of ~. .
tyrosine hydroxylase.
Intensive studies have been being focused to find tyrosine hydroxylase
inhibitor
from natural products. Korean Patent Laid-open Publication No. 2002-0023168
discloses
that the extract of Gardenias fructus has inhibitory effect against tyrosine
hydroxylase by 3
times than arbutin in comparative test. However, as the said patent
specification did not
disclose which material in the extract of Gardenias fructus has such
inhibitory action against
tyrosine hydroxylase, it could not evaluate whether the extract of Gardenias
fructus has toxic
effect on the skin of human being or not. Until now, materials such as
dihydroxybenzene
derivatives, retinoid series and steroid hormones had been being used as
tyrosine
hydroxylase inhibitor. However, the said materials showed to have side effects
on the skin
and therefore the use of the said materials is restricted.
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
2
Background Art
The present invention relates to a whitening cosmetic composition comprising
extract of Pulsatillae Radix as main ingredient.
The present invention relates to a whitening cosmetic composition comprising
extract of Pulsatillae Radix and one or more ingredients) selected from the
group consisting
ranunculin, deoxypodophyllotoxin and 3-O-a-L-ramnopyranosyl(1-~2)-[(3-D-
glucopyranosyl(1-->4)]-a-L-arabinopyranoside(SB365) extracted and isolated
from the
extract of Pulsatillae Radix as main ingredients.
The present invention relates to a whitening cosmetic composition comprising
extract of Pulsatillae Radix and extract of bark of Ulmus macrocarpa as main
ingredients.
Disclosure of Invention
One object of the present invention is to provide a whitening cosmetic
composition
comprising the extract of Pulsatillae Radix as main ingredient.
The other object of the present invention is to provide a whitening cosmetic
composition comprising the extract of Pulsatillae Radix and the extract of the
bark of the
Ulmus macrocarpa as main ingredients.
Another object of the present invention is to provide a whitening cosmetic
composition comprising the extract of Pulsatillae Radix and one or more
ingredients)
selected from the group consisting ranunculin, deoxypodophyllotoxin and 3-O-a-
L-
ramnopyranosyl( 1-->2)-[(3-D-glucopyranosyl( 1--~4)]-a-L-arabinopyranoside(SB3
65)
extracted and isolated from the extract of Pulsatillae Radix as main
ingredients.
Still another object of the present invention is to provide a whitening
cosmetic
composition comprising the extract of Pulsatillae Radix as main ingredient and
more
comprising one or more extracts selected from the group consisting the extract
of the bark of
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
3
the Ulmus macrocarpa, extract of Ginseng Radix and extract of Glycyrrhizae
Radix as
auxiliary ingredients.
Still another object of the present invention is to privide a whitening
cosmetic
composition comprising the extract of Pulsatillae Radix and one or more
ingredients)
selected from the group consisting ranunculin, deoxypodophyllotoxin and 3-O-a-
L-
ramnopyranosyl(1-~2)-[(3-D-glucopyranosyl(1--~4)]-a-L-arabinopyranoside(SB365)
extracted and isolated from the extract of Pulsatillae Radix as main
ingredients and more
comprising one or more extracts selected from the group consisting the extract
of the bark of
the Ulmus macrocarpa, the extract of Ginseng Radix and the extract of
Glycyrrhizae Radix
as auxiliary ingredients.
Pulsatillae Radix is the root of Pulsatilla koreana, P. cerna and P.
chinensis, etc. The
Pulsatillae Radix has been being used as oriental medicine for treating child
bed fever,
detoxication, antidiarheal, bactericide, amebicide, fungicide, etc. Recently,
the Pulsatillae
Radix is reported to have antitumor activity and is now under clinical study.
The bark of Ulmus marocarpa has being been used as oriental medicine for
treating
various parasites, various kinds of epidermophytid(Oriental Materia Medica, H-
Y Hsu et. al.,
Oriental Healing Arts Institute, pp 749). Therefore, the bark of UImus
marocarpa is regarded
to have protecting effect for the skin.
Deoxypodophyllotoxin isolated from the extract of Pulsatillae Radix is known
compound and has the following structural formula.
1
~'/
~Q
Deoxypodophyllotoxin
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
4
Ranunculin isolated from the extract of Pulsatillae Radix is known compound
and
has the following structural formula.
10 Ranunculin
3-O-a-L-ramnopyranosyl( 1-~2)-[(3-D-glucopyranosyl( 1--~4)]-a-L-
arabinopyranoside(SB365) of which generic name is hederagenin, which isolated
from the
extract of Pulsatillae Radix is known compound and has the following
structural formula.
20
SB365
The said compounds are confirmed to have excellent whitening effects by the
present invention.
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
The present whitening composition can be formulated by adding conventional
vehicles to cream, injection, capsule, tablet, etc., by conventional
preparation methods.
Extracting solvent such as water; lower alkanol e.g. methanol, ethanol,
propanol or
butanol; methylenechloride; acetone; or mixture thereof can be used in the
present invention.
5
Brief Description of Drawings
Fig. 1 indicates the results of chromatogram of PT fraction through Sepadex
LH20
column chromatography.
Best Mode for Carrying Out the Invention
The present invention is explained in more detail by the following examples
and
experimental examples.
Example 1
General preparation of the extract of Pulsatilla Radix
The Pulsatillae Radix were powdered to obtain powder of 40-100 mesh. A certain
volume of the powder was put in an extractor and extraction was carried out
with lower
alcohol-water mixed solvent. Lower alcohol is selected from the group
consisting methanol,
ethanol, propanol and butanol. Etanol-water mixed solvent among them is the
best
selectivity in extraction. In the case of the use of ethanol-water mixed
solvent of 50°!o(v/v),
materials having very big polarity and materials of large molecular weight and
polymers, etc.
were not extracted. Extraction were carried out under the temperature of 15 C
~ 35 C, most
preferably 20 C ~ 25 °C . Extraction time carried out from 1 hour unit
and gradually
increased. Extraction time of 3hours is preferable. The extract which
Pulsatillae Radix was
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
6
extracted by using 50% aqueous ethanol solvent under the temperature of 25 C
for 3 hours is
expressed as ET fraction and the ET fraction was used for measuring the
whitening effect.
Example 2
1. Isolation of materials having whitening effect.
The whitening effect was confirmed by clinical trial with a group of human
beings
of the following experimental examples. The isolation of materials having
whitening effect
was carried out by the method of measurement for tyrosinase-inhibition effect
which is
generally used.
Low molecular, polar materials were extracted from acetone and the acetone-
extract was expressed as PA fraction and the remaining part was expressed as
PT fraction.
PT and PA fractions showed respectably 10% and 73% inhibition effects against
tyrosinase.
2. Active material 1 of the PT fraction
The fraction 3 which was obtained by re-fractionation of the PT fraction with
Sephadex was refined to obtain an active ingredient which was ascertained as 3-
O-a-L-
ramnopyranosyl(1-~2)-[[3-D-glucopyranosyl(1-~4)]-a-L-arabinopyranoside(SB365)
of
which generic name is hederagenin. This compound is ascertained to have
antitumor
activity(Now patent pending). This material showed no effect against
tyrosinase but showed
an excellent whitening effect in clinical trials. So, this material is sure to
have whitening
effect through another mechanism. It is to be assumed that when relation
between saponin
derivative and the surface of skin cell is to be thought, the compound hinders
the migration
of melanosome and prevents melanin to be accumulated on keratinocyte.
3. Whitening material 2 of the PT fraction
The SPX2 fraction which was obtained by fractionation of the PT fraction by
Sephadex showed inhibition of 48% against tyrosinase. A material which was
obtained by
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
7
passing the SPX2 fraction through silicagel column (Eluting solvent
methylenechloride/methanol=4:1) and showed R~0.3 was confirmed. The Rf value
was
identified as those of ranunculin. The NMR data of the material which was
'isolated by a
known method was the same with those of the ranunculin. Ranunculin is broadly
spread in
plants which belonged to Ranunculaceae and is known to have mitotoxicity
(Vonderbank,
Pharmazie 5, 21(1950)). Pure ranunculin showed inhibition of 51% against
tyrosinase.
4. Active material of the PA fraction
Deoxypodophyllotoxin is spread in various plants including Anthriscus
sylvestris
Hoffm and shows to have mitotoxicity (Byung-Zun Ahn, Song-Bae Kim, Yong Kim,
et al.
Use of Deoxypodophyllotoxin as antitumor agent, Korean Patent No. 315,200). It
is reported
that this material inhibits the formation of blood vessel of endotheliocyte of
umbilicus in
human being(Yong Kim, Song-Bae Kim, Byung-Zun Ahn, et al.,
Deoxypodophyllotoxin;
the cytotoxic and antiangiogenic component from Pulsatilla koreana Nakai,
Planta Medica,
68, 271-274(2002)). This material exhibits inhibition of 38% at 0.03p,g/ml
against tyrosinase.
It was difficult to experiment at higher concentration because this material
has low water
solubility.
Example 3
SOg of dried finely powdered Pulsatilla Radix and SOOmI of 50% ethanol were
added to an extractor. The mixture was stirred at room temperature for 3 hours
and filtered.
The filtrate was stored and the procedure was carried out with the remaining
residue for 2
times. The combined filtrate was concentrated under reduced pressure and dried
to obtain
23g of extract.
Example 4
PT fraction having inhibition against tyrosinase and preparation of the PA
fraction
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
8
20g of the extract obtained in Example 3 was added to 200m1 of acetone. The
mixture was stirred for 10 minutes to obtain a suspension. The suspension was
filtered to
obtain solution and residue. The residue was suspended in 200m1 of acetone and
stirred for
minutes and filtered. The residue was dried to obtain PT fraction (17.4g). The
combined
5 solution was concentrated and dried to obtain PA fraction (3.Sg).
Example 5
Isolation of PTpur, an inhibitor material against tyrosinase from PT fraction
560mg of the PT fraction was eluted and fractionated through Sephadex LH20
10 column (200g, 60x4cm) (velocity of elution: lml/lmin.). Fractionation was
carried out and
filled O.SmI of volume of the fractionation per 1 test tube by use of test
tubes. These
fractions were in turn spotted on thin layers of silica gel and developed for
fractions to be
divided(Developing solvent: butanol : acetic acid : water = 4:1:1, coloration
: spraying of
sulfuric acid and heated). The results were showed in the Figure 1.
In the Figure 1, the PT1 fraction (139mg, 24.8%) was collected from the test
tube
Nos. 26 ~ 66 and main spots are 4. The lower parts were reacted with sulfuric
acid to appear
yellow color.
The PT2 fraction (344mg, 61.4%) was collected from the test tube Nos. 66 ~ 91
and
main sports are 2.
The PT3 fraction (6lmg, 10.9%) was collected from the test tube Nos. 91 ~ 111.
When spraying of sulfuric acid and heating, firstly red color was appeared and
the time being,
blue color appeared. The fraction is the main material of which spots existed
between Rf
value is in mean 0.48 ~ 0.50.
The PT4 fraction (15.7mg, 2.8%) was collected from the test tube Nos. 111 ~
138.
The SPX3 fraction and the SPX4 fraction were appeared each one spot on the
thin
layers and were fractions which were relatively pure. The fractions were shown
on the
Figure 1.
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
9
The SPXl, SPX2, SPX3 and SPX4 fractions were obtained through Sephadex
LH20 column chromatography.
The SPX3 fraction was relatively pure fraction and was obtained from white
precipitation which the fraction was dissolved in O.SmI of water and stored to
precipitate
(PTpur).
Example 6
Identification of the PTpur structure
As the above isolated material PTpur has the identifying data, that is, m.p.
239 ~ 241 C, [a]D = +23.6°(c, 0.2, MeOH), white amorphous, and positive
in Riberman-
Bucart Reaction is identified as a glycoside. In addition, as the material has
IR (cm ~) data,
3400(br, -OH), 2940(br, C-H), 1695(C=O), 1455, 1040(C-O) and adsorption band
of 1000-
1100, 3000-3400 or more, the material was largely regarded as a possibility of
glycoside.
'H-NMR of the material follows the typical pattern of those of saponin and 6-
CH3
groups were observed at 0.91, 0.92, 0.98, 1.00, 1.07 and 1.21 ppm and another -
CH3 group
was observed at 1.64 ppm as doublet. From these, one saccharide among
consisting
saccharides is assumed as rhamnose. Anomeric proton was observed at 6.25(br.),
5.11(1H,
J=7.80Hz) and 4.97ppm (1H, J=6.66 Hz). Therefore, PTpur was identified as a
glycoside
bonded 3 saccharides.
From '3C-NMR, hydroxymethyl group was observed at 65.4ppm(C-23); 3 anomeric
carbon signals were observed respectively at 140.2(C-1'), 106.7(C-1 "') and
101.7ppm(C-13);
2 olefin carbons were respectively at 122.Sppm(C-12) and 144.8ppm(C-13); a
carboxyl
carbon was observed at 180.2ppm(C-28). Conventionally, when a saccharide is
bonded at C-
28, glycosylation upheld shift of about 4Hz is observed. But, such shift was
not observed on
this compound. Therefore, It could be ascertained that this compound is not
glycoside which
saccharide was not bonded at C-28.
And then, for identifying the structure of aglycone, this compound was
hydrolyzed
by ethanol/sulfuric acid. When physical and chemical data, '3C-NMR and 1H-NMR
data of
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
the hydrolyzed product were compared, it was ascertained that PTpur was
hederagenin. The
hydrolyzed saccharides were ascertained as rhamnose, arabinose and glucose by
comparative
TLC.
When generally analyzing the above results and data published in arts, PTpur
is
5 ascertained to be 3-O-a-L-ramnopyranosyl(1-j2)-[[i-D-glucopyranosyl(1-~4)]-a-
L-
arabinopyranoside(SB365) of which generic name is hederagenin, a saponin
already isolated
from the Pulsatilla Radix.
'H-NMR and 13H-NMR data of the PTpur showed in the following table.
15
25
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
11
Table 1
position'H "'C position~H
(ppm) (ppm) (NPm) J ~ (PPm)
J (Hz) (Hz)
G1. 98,9 Arabinoss
G2 ' 28.1 G1' 4.97 6.88 140.2
~ d
C-3 3.28 d 10.9 81.0 G2' 80.4
C-4 43.5 C.3' 76,4
G6 48.1 C-4' 78.2
C-8 18.1 C-6' 83.9
C-7 32.8 ptta~
GB 39.7 C-1" 6.26 101.T
br
G9 4T.8 G2' 72.3
C-10 98.9 C-3" 724
G11 23.9 C-4' 74.1
C-i2 5.45 s 1226 C-6" ~.g
C-13 144.8 C8' 1.84 6.94 18.8
C14 42.1 (~llluopse
C15 28.8 C-1' 5.11 7.80 108.7
d
C18 23.8 G2" 75.0
C17 48.2 C-3" 78.5
G18 41.9 C-4"' 71.2
C19 48.4 C-5" 78.8
CW 30.9 C-8" 82.5
C-21 34,2
C-22 93.2
C-23 4.38, 3.87 85.4
overlap
C-24 1.07 a 14.0
C25 0.91 s 18,0
C-28 0.98 s 17.4
C-27 1.21 s 28.3
C-28 - 1802
C-29 0.92 s 32.8
C-30 1.00 s 23.7
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
12
Example 7
Isolation of PTculin, an inhibitor material against tyrosinase from the PT
fraction
When the PT fraction and pure ranunculin were developed on thin layer of
silica gel,
the same spots were observed at Rf = 0.3.
(Solvent: methylenechloride/methanol=4:1)
Example 8
Isolation of PApur, an inhibitor material against tyrosinase from the PA
fraction
30mg of PA fraction was dissolved in 2m1 of methanol and 3m1 of hexane was
added thereto and the mixture was stirred. After standing a while, hexane
layer was decanted.
Such extraction was carried out 2 times. The combined hexane solution was
dried and
carried out a comparative chromatography with deoxypodophyllotoxin on thin
layer of silica
gel. It was ascertained that the same spot with the deoxypodophyllotoxin was
observed from
the hexane solution. The spot was isolated from fractionating silica gel thin
layer to obtain
3mg of deoxypodophyllotoxin which was the same with the standard of the
deoxypodophyllotoxin when NMR was measured.
The present invention is described in more detail with the following
preparative
examples.
Preparative Example 1
Emulsion
Composition of emulsion base (g)
Cetyl octanoate 4.0
Cyclomethicone 3.0
Fluid paraffin 4.0
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
13
Polysorbate 60 5.0
Sorbitan stearate 3.0
To the each bases(each 19g), each 100mg, 400mg, 600mg, 800mg and 1000mg of
the ET fraction were added and mixed uniformly to obtain each preparation
P100, P400,
P600, P800, P1000mg.
Preparative Example 2
Injection
Pulsatilla Radix as main ingredient, bark of Ulmus macrocarpa, Ginseng Radix,
Glycyrrhizae Radix as auxiliary ingredients were extracted with 50% ethanol.
The extract
was prepared, filled in vials and lyophilized to obtain injections by
conventional preparation
method of injections.
Preparative Example 3
Composition of Example 3 100mg
Starch 1 OOmg
Lactose SOmg
Magnesium stearate q.s.
The above ingredients were prepared to obtain a capsule by a conventional
preparation method of capsules.
Preparative Example 4
Composition of Example 3 100mg
Lactose SOmg
Magnesium stearate q.s.
Talc q.s.
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
14
The above ingredients were prepared to obtain a tablet by a conventional
preparation method of tablets.
Preparative Example 5
Composition of Example 3.Og
3
Isomerized saccharide SO.Og
Sodium alginate SO.Omg
Sodium benzoate q.s.
Purified water q.s. to
100m1
The above ingredients were prepared and filled in brown bottle of 100m1 to
obtain
solution by a conventional preparation method of solution.
Preparative Example 6
Injection
Composition of Example 3 100mg
Ranunculin l Omg
Water for injection q.s. to lml
The above ingredients were prepared and filled in ampoule of lml to obtain
injection by a conventional preparation method of injection.
Preparative Example 7
Composition of Example 3 100mg
Deoxypodophyllotoxin l Omg
SB 365 lOmg
Starch 100mg
Lactose SOmg
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
Magnesium stearate q.s.
The above ingredients were prepared to obtain a capsule by a conventional
preparation method of capsules.
5
Preparative Example 8
Composition of Example 3 100mg
. Deoxypodophyllotoxin lOmg
Lactose SOmg
10 Magnesium stearate q.s.
Talc q.s.
The above ingredients were prepared to obtain tablet by a conventional
preparation
method of tablets.
Preparative Example 9
Composition of Example 3 3.Og
SB 365 100mg
Isomerized saccharide SO.Og
Sodium alginate SO.Omg
Sodium benzoate q.s.
Purified water q.s. to
100m1
The above ingredients were prepared and filled in the brown bottle of 100m1 to
obtain solution by a conventional preparation method of solution.
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
16
Experimental Example 1
Whitening effect of the extract of Pulsatilla Radix on clinical trial
Whitening effect was carried out with 20 volunteer women. Among them 10 of the
women had freckles on their faces and remaining 10 women had liver spots on
their faces.
women of freckles were divided into A, A'(A group), B B'(B group), C, C'(C
group), D, D'(D group), E and E' (E group). P100 was administered to A group;
P400 to B
group; P800 to C group; P1000 to D group, respectively and only the base was
administered
10 to E group. The methods of administrations were to rub on the places of
freckles of the faces.
The same methods were applied to the women of liver spots. The groups were
divided into G, G'(G group), H, H'(H group), I, f (I group), J, J'(J group),
K, L'(K group) and
L, L'(L group).
The volumes of the composition and the base were each SOOmg per SxScmZ. The
numbers of administrations were 3 times a day. Before administrations, faces
were washed.
The administrations were continued for 3 weeks.
Three persons comparatively observed from the beginning of the clinical
trials,
discussed and judged effects on the base of criteria of cured, effective,
slightly effective and
non-effective of the whitening effects.
The results were shown on the Table 2.
Table 2.
Whitening effects of the extract of Pulsatilla Radix on frecke and liver spot
of women:
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
17
Freckes Liverspots
,A'(100mg)A: non effective G,G'(100mg) G: non effective
A': non effective G': non effective
,B'(400mg)B: effective H,H'(400mg) H: slightly effective
B': slightly effectiveH': slightly effective
C,C'(600mg)C: cured I,f(600mg) I: cured
C': effective f : cured
D,D'(80mg)D: cured ,J'(800mg) J: cured
D': cured J': cured
E,E'(1000mg)E: cured K,K'(1000mg) IC: cured
E': cured K': cured
F,F'(base) ,L'(base) L: non effective
F: non
effective
F' non L': non effective
effective
Remarks:
Cured : Freckles or liver spots were disappeared.
Effective : Great parts of freckles or liver spots were disappeared.
Slightly effective:Some of freckles or liver spots were decreased.
As shown on the Table 2, P100 did not show effects on freckle group
A,A'(100mg)
or liver spot group G,G'(100mg); P400 showed slightly effective on freckle
group
B,B'(400mg) or liver spot group H,H'(400mg); More dosage group than 400mg
showed
effective. More dosage group than 600mg showed effective on all the groups in
case of liver
spots. More dosage group than 800mg showed effective on all the groups in case
of freckles.
Experimental Example 2
Whitening effect of injection on clinical trial
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
18
Five women having liver spots were selected and marked as A, B, C, D and E.
Each
S.OmI of the injection were injected to the women respectively for a day for 3
days
successively and after that the same procedures carried out and ointment
administered at the
same time. Three women, A, B, C were cured 5 days after the administrations of
injection
and ointment and women D and E were effective.
A combined treatment of the administration of injection and ointment was more
immediate effective than ointment treatment on skin without injection.
Clinical trials confirmed that the present composition has effectiveness. The
effectiveness was carried out by the measuring method of inhibition of
tyrosinase generally
used in this field at the same time.
The PT and PA fractions inhibited 10% and 73% of activity of tyrosinaseat at
Smg/ml respectively.
IS
Pure ranunculin inhibited 51 % of activity of tyrosinase
Deoxypodophyllotoxin inhibited 38% of activity of tyrosinase at 0.03pg/ml. It
was
difficult that experiment at higher concentration could not be carried out
because the
material has very low solubility in water.
Experimental Example 3
Whitening effect of preparation composed of ranunculin, deoxypodophylotoxin
and SB365 on clinical trial
Pharmaceutical composition: lOmg of ranunculin, SOmg of deoxypodophyllotoxin,
and l5mg of SB365 were mixed well together with the base (19g) to obtain
pharmaceutical
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
19
composition and the composition Was rubbed on the area of liver spots three
times a day till
the time of curing.
Among five women (A,B,C,D and E), A and B were cured after lOdays; C was
cured after l7days; D was cured after 27days and E was not cured, though the
composition
was slightly effective.
A composition from which SB365 which was not effective on tyrosinase was
deleted was used. The composition were administered to five women (F,G,H,I,K)
having
liver spots. F and G were cured after 26days; I was cured after 35days; and J
and K were the
level of effectiveness.
From the result of the clinical trial, SB365 did not inhibit the activity of
tyrosinase
but have whitening effect. There is a possibility that SB 365 acts on
melanocyte and
interrupts the procedure of the migration of melanin to karatinocyte. An exact
mechanism
may be disclosed scientifically.
By adding the extract of the bark of the Ulmus macrocarpa, the extract of
Ginseng
Radix and/or the extract of Glycyrrhizae Radix as auxiliary ingredients to the
preparation of
Pulsatillae Radix having whitening effect, the combined composition can be
anticipated to
have merits of beauty arts, such as synergic effect of whitening action, skin-
protection
through bacteriocidal effect of saponin, skin-elutriation and skin-penetration
effect, etc.
Experimental Example 4
Measurement of inhibition action against tyrosinase
Tyrosinase was purchased from Sigma Company. The enzyme was dissolved in 3,
4-dihydrophenylalanine. Sodium phosphate buffer(O.1M, pH 6.0), a substrate of
enzyme and
adjusted l.6mg/ml of concentration to obtain an enzyme solution. Sample was
dissolved in
sodium phosphate buffer and the undissolved residue was filtered out. 0.7m1 of
the buffer,
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
0.2m1 of sample and O.lml (15.7unit/ml) of the enzyme solution were mixed and
after
60seconds, absorbance was measured at 475nm by spectrophotometer.
By the following equation, inhibition ratio(%) of tyrosinase was calculated.
5 Inhibition ratio(%) of tyrosinase = [1-(S-B)/C]x100
wherein, S: Test tube having enzyme and sample,
B: Test tube having sample,
C: Test tube having enzyme.
Experimental Example 5
To each EAGLE MEM culture mediums containing 10% of foetus serum of
cow were added each concentrations of the composition of Example 3 listed in
Table 3
below and B-16 cells originated from mouse melanoma were inoculated to each
culture
mediums and were cultured under the condition of 37 C, 5% C02 for 5 days. The
cells were
dispersed with trypsin; centrifuged at 1,OOOrpm for Sminutes; collected and
the melanoid
degrees were measured macroscopically.
The criteria of judgement were as follows:
- : About same degree of not adding of the composition of Example 3,
+ : Slightly whitening,
++ : fairly whitening.
+++ : mostly whitening.
Table 3
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
21
Amount of the composition of Example
3 Results
(concentratiion, weight %)
control
0.01 +-
0.1 ++
0.3 ++
0.5 ++
0.7 +++
1.0 +~-~-
As ascertained from the above results, the composition of the present
invention has
excellent whitening effect on melanin originated from the mouse melanoma.
Experimental Example 6
On each inside parts of right brachiums of volunteers(heathy mem and women,
each
15, total 30persons), each 2x2 cm2 parts were chosen ; the chosen parts were
washed well
with warm water; other parts than the chosen parts were masked with aluminum
foil in order
for ultraviolet ray to be irradiated only on the chosen parts; and with two
FL20SBLB
lamp(Toshiba Co., Ltd.) and FL20SE-30 lamp(Toshiba Co., Ltd.) at the same
time,
ultraviolet ray was irradiated the volume of 0.8x10erg/cm3/time/day for
successively 3 times.
After irradiation, to the irradiated parts there applied the sample of below
Table 4 three times
a day(morning, noon and night). Evaluations were measured, after 3 weeks, the
melanoid
degrees macroscopically. The improvement degrees were evaluated as three
degrees as
excellent effective, effective and not effective.
Table 4
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
22
Ingredients concentration(weight part)
Composition of Example 3 0.5
Polyoxyethylene(40)monostearate2.0
Glycerolmonostearate 5.0
Stearic acid 5.0
Behenyl alcohol 1.0
Fluid paraffin 1.0
Glyceryltrioctanoate 10.0
1,3-butylenglycol 5.0
Purified water q.s.
Other ingredients exempt 1,3-butylenglycol and purified water were dissolved
by
warming.(Oily state). Separately, 1,3-butyleneglycol and purified water were
mixed and
warmed to obtain a solution. The solution was added to the oil and the mixture
was stirred;
emulsified; cooled to obtain a vanishing cream.(Vanishing cream of the extract
of Pulsatilla
Radix).
Separately, A vanishing cream exempt the extract of Pulsatilla Radix was
prepared
and used as control.
The results are as follows:
Table 5
ingredient excellent effectiveeffective not effective
ext.of P.Radix17(persons) 10(persons) 3(persons)
control 0(persons) 3(persons) 27(persons)
CA 02499603 2005-03-21
WO 2004/026275 PCT/KR2003/001905
23
As ascertained from the above experimental results, the cosmetic composition
comprising extract of Pulsatilla Radix of the present invention has excellent
inhibiting effect
against melanin formation.
Industrial Applicability
The present invention relates to a whitening cosmetic composition comprising
the
extract of Pulsatillae Radix and if necessary, one or more ingredients)
selected from the
group consisting ranunculin, deoxypodophyllotoxin and 3-O-a-L-ramnopyranosyl(1-
j2)-[[i-
D-glucopyranosyl(1~4)]-a-L-arabinopyranoside(SB365) extracted and isolated
from the
extract of Pulsatillae Radix as main ingredients and if necessary, more
comprising one or
more extracts selected from the group consisting the extract of the bark of
the Ulmus
macrocarpa, the extract of Ginseng Radix and the extract of Glycyrrhizae Radix
as auxiliary
ingredients. The present composition has an excellent whitening effect.
20
