U.S. patent application number 11/886270 was filed with the patent office on 2008-06-26 for anti-inflammatory agent.
Invention is credited to Yasuo Miyake, Ito Yoko.
Application Number | 20080152734 11/886270 |
Document ID | / |
Family ID | 36991366 |
Filed Date | 2008-06-26 |
United States Patent
Application |
20080152734 |
Kind Code |
A1 |
Miyake; Yasuo ; et
al. |
June 26, 2008 |
Anti-Inflammatory Agent
Abstract
An anti-inflammatory agent which comprises an oil-soluble
licorice extract obtained by extraction treatment of at least
either a leguminous plant of the genus Glycyrrhiza or a water
extraction residue of a leguminous plant of the genus Glycyrrhiza
with an organic solvent, and has at least one effect selected from
an inhibitory effect on hyaluronidase activity, an inhibitory
effect on hexosaminidase release (i.e., an inhibitory effect on
histamine release), an inhibitory effect on platelet aggregation
and an inhibitory effect on phospholipase A.sub.2 activity.
Inventors: |
Miyake; Yasuo; (Hiroshima,
JP) ; Yoko; Ito; (Hiroshima, JP) |
Correspondence
Address: |
ARTHUR G. SCHAIER;CARMODY & TORRANCE LLP
50 LEAVENWORTH STREET, P.O. BOX 1110
WATERBURY
CT
06721
US
|
Family ID: |
36991366 |
Appl. No.: |
11/886270 |
Filed: |
March 15, 2005 |
PCT Filed: |
March 15, 2005 |
PCT NO: |
PCT/JP2005/004562 |
371 Date: |
February 26, 2008 |
Current U.S.
Class: |
424/757 |
Current CPC
Class: |
A61K 31/122 20130101;
A61K 36/484 20130101; A61P 43/00 20180101; A61P 17/00 20180101;
A61P 29/00 20180101; A61P 7/02 20180101 |
Class at
Publication: |
424/757 |
International
Class: |
A61K 36/484 20060101
A61K036/484; A61P 29/00 20060101 A61P029/00 |
Claims
1. An anti-inflammatory agent comprising an oil-soluble licorice
extract.
2. The anti-inflammatory agent according to claim 1, wherein the
oil-soluble licorice extract is prepared by subjecting at least one
of a leguminous plant of the genus Glycyrrhiza and a water
extraction residue of a leguminous plant of the genus Glycyrrhiza
to an extraction treatment.
3. The anti-inflammatory agent according to claim 1, wherein the
anti-inflammatory agent is prepared by subjecting at least one of
roots, rhizomes, leaves, and stems of a leguminous plant of the
genus Glycyrrhiza, and water extraction residues thereof to an
extraction treatment with an organic solvent.
4. The anti-inflammatory agent according to claim 3, wherein the
organic solvent is at least one selected from ethanol, hydrous
ethanol, and ethyl acetate.
5. The anti-inflammatory agent according to claim 2, wherein the
leguminous plant of the genus Glycyrrhiza is at least one selected
from Glycyrrhiza glabra, Glycyrrhiza inflata, Glycyrrhiza
araleasis, Glycyrrhiza uralensis, and Glycyrrhiza echinata.
6. The anti-inflammatory agent according to claim 1, wherein the
oil-soluble licorice extract comprises at least one flavonoid of
glabridin and licochalcone A.
7. The anti-inflammatory agent according to claim 6, wherein a
total content of the at least one flavonoid of glabridin and
licochalcone A in the oil-soluble licorice extract is 1% by mass to
80% by mass based on dry solid content.
8. The anti-inflammatory agent according to claim 5, wherein a
content of glabridin in an oil-soluble licorice extract of
Glycyrrhiza glabra is 1% by mass or more based on dry solid
content.
9. The anti-inflammatory agent according to claim 5, wherein a
content of licochalcone A in an oil-soluble licorice extract of
Glycyrrhiza echinata is 1% by mass or more based on dry solid
content.
10. The anti-inflammatory agent according to claim 5, wherein a
content of licochalcone A in an oil-soluble licorice extract of
Glycyrrhiza inflata is 1% by mass or more based on dry solid
content.
11. The anti-inflammatory agent according to claim 1, wherein the
oil-soluble licorice extract has at least one effect selected from
an inhibitory effect on hyaluronidase activity, an inhibitory
effect on hexosaminidase release, an inhibitory effect on platelet
aggregation, and an inhibitory effect on phospholipase A.sub.2
activity.
12. The anti-inflammatory agent according to claim 1, which is used
as an external preparation for skin.
Description
TECHNICAL FIELD
[0001] The present invention relates to an anti-inflammatory agent
which contains an oil-soluble licorice extract, which has at least
one effect selected from an inhibitory effect on hyaluronidase
activity, an inhibitory effect on hexosaminidase release, an
inhibitory effect on platelet aggregation, and an inhibitory effect
on phospholipase A.sub.2 activity, and which is suitably used
especially as an external preparation for skin.
BACKGROUND ART
[0002] Licorice has been known as herbal medicine from long ago,
and currently it is mainly used as raw material of a sweetener for
food, medicines, quasi-drugs, etc. Among these, glycyrrhizin and
glycyrrhetinic acid, a water extract of licorice, have excellent
medicinal effects such as anti-inflammatory effect, antiulcer
effect, and antiallergic effect, and are widely used in e.g. food
and drink, medicines, and cosmetics.
[0003] Oil-soluble licorice extract, which is obtained by
extracting licorice with an organic solvent such as ethanol and
ethyl acetate, It is known to contain a lot of flavonoids other
than the above-mentioned glycyrrhizin and glycyrrhetinic acid. It
is disclosed that this oil-soluble licorice extract has useful
effects such as an antioxidant effect (See, Patent Literature 1),
an oxidation preventing effect (See, Patent Literature 2), a
whitening effect (See, Patent Literatures 3 and 4), and an
ultraviolet absorbing effect (See, Patent Literature 5).
[0004] However, it has not been known that the oil-soluble licorice
extract has at least one effect selected from an inhibitory effect
on hyaluronidase activity, an inhibitory effect on hexosaminidase
release, an inhibitory effect on platelet aggregation, and an
inhibitory effect on phospholipase A.sub.2 activity, and is useful
for, for example, contact dermatitis (rash), psoriasis, pemphigus
vulgaris, and other various skin diseases associated with chapped
skin. [0005] Patent Literature 1: Japanese Patent Application
Laid-Open JP-A) No. 58-217583 [0006] Patent Literature 2: JP-A No.
5946210 [0007] Patent Literature 3: JP-A No. 01-149706 [0008]
Patent Literature 4: JP-A No. 01-311011 [0009] Patent Literature 5:
JP-A No. 01-157909
DISCLOSURE OF INVENTION
[0010] Under such circumstances, the present invention has been
achieved. An object of the present invention is to solve
conventional problems mentioned above and to achieve the following
objects. Specifically, an object of the present invention is to
provide an anti-inflammatory agent that can prevent and improve
inflammatory diseases through at least one effect selected from an
inhibitory effect on hyaluronidase activity, an inhibitory effect
on hexosaminidase release, an inhibitory effect on platelet
aggregation, and an inhibitory effect on phospholipase A.sub.2
activity.
[0011] Means for solving the above-mentioned problems are as
follows.
[0012] <1> An anti-inflammatory agent including an
oil-soluble licorice extract.
[0013] <2> The anti-inflammatory agent according to
<1>, wherein the oil-soluble licorice extract is prepared by
subjecting at least one of a leguminous plant of the genus
Glycyrrhiza and a water extraction residue of a leguminous plant of
the genus Glycyrrhiza to an extraction treatment.
[0014] <3> The anti-inflammatory agent according to one of
<1> and <2>, wherein the anti-inflammatory agent is
prepared by subjecting at least one of roots, rhizomes, leaves, and
stems of a leguminous plant of the genus Glycyrrhiza, and water
extraction residues thereof to an extraction treatment with an
organic solvent.
[0015] <4> The anti-inflammatory agent according to
<3>, wherein the organic solvent is at least one selected
from ethanol, hydrous ethanol, and ethyl acetate.
[0016] <5> The anti-inflammatory agent according to any one
of <2> to <4>, wherein the leguminous plant of the
genus Glycyrrhiza is at lease one selected from Glycyrrhiza glabra,
Glycyrrhiza inflata, Glycyrrhiza araleasis, Glycyrrhiza uralensis,
and Glycyrrhiza echinata.
[0017] <6> The anti-inflammatory agent according to any one
of <1> to <5>, wherein the oil-soluble licorice extract
includes at least one flavonoid of glabridin and licochalcone
A.
[0018] <7> The anti-inflammatory agent according to
<6>, wherein a total content of the at least one flavonoid of
glabridin and licochalcone A in the oil-soluble licorice extract is
1% by mass to 80% by mass based on dry solid content.
[0019] <8> The anti-inflammatory agent according to any one
of <5> to <7>, wherein a content of glabridin in an
oil-soluble licorice extract of Glycyrrhiza glabra is 1% by mass or
more based on dry solid content.
[0020] <9> The anti-inflammatory agent according to any one
of <5> to <8>, wherein a content of licochalcone A in
an oil-soluble licorice extract of Glycyrrhiza echinata is 1% by
mass or more based on dry solid content.
[0021] <10> The anti-inflammatory agent according to any one
of <5> to <9>, wherein a content of licochalcone A in
an oil-soluble licorice extract of Glycyrrhiza inflata is 1% by
mass or more based on dry solid content.
[0022] <11> The anti-inflammatory agent according to any one
of <1> to <10>, wherein the oil-soluble licorice
extract has at least one effect selected from an inhibitory effect
on hyaluronidase activity, an inhibitory effect on hexosaminidase
release, an inhibitory effect on platelet aggregation, and an
inhibitory effect on phospholipase A.sub.2 activity.
[0023] <12> The anti-inflammatory agent according to any one
of <1> to <11>, which is used as an external
preparation for skin.
[0024] The anti-inflammatory agent of the present invention
includes an oil-soluble licorice extract, is prepared, particularly
by subjecting at least one of a leguminous plant of the genus
Glycyrrhiza and a water extraction residue of a leguminous plant of
the genus Glycyrrhiza to an extraction treatment with an organic
solvent, and, through an inhibitory effect on hyaluronidase
activity, an inhibitory effect on hexosaminidase release (i.e.,
inhibitory effect on histamine release), an inhibitory effect on
platelet aggregation, and an inhibitory effect on phospholipase
A.sub.2 activity, can attain efficient prevention and improvement
of inflammations related to these.
[0025] In addition, the anti-inflammatory agent of the present
invention is particularly suitably used for an external preparation
for skin due to its excellent feel and high safety. Here, in the
present invention, the "external preparation for skin" means
various drugs that are applied for skin and is a concept that
include, for example, cosmetics, quasi-drugs, and medicines.
BEST MODE FOR CARRYING OUT THE INVENTION
[0026] The anti-inflammatory agent of the present invention
contains an oil-soluble licorice extract and further contains
additional components on as-needed basis.
[0027] The oil-soluble licorice extract is not particularly limited
and can be appropriately selected depending on the application, but
oil-soluble licorice extracts are suitable that are prepared by
subjecting at least one of a leguminous plant of the genus
Glycyrrhiza and a water extraction residue of a leguminous plant of
the genus Glycyrrhiza to an extraction treatment with an organic
solvent.
[0028] The leguminous plant of the genus Glycyrrhiza is not
particularly limited and can be appropriately selected depending on
the intended active ingredient. Examples thereof include
Glycyrrhiza glabra, Glycyrrhiza inflata, Glycyrrhiza araleasis,
Glycyrrhiza uralensis, Glycyrrhiza echinata, and the like. Among
these, Glycyrrhiza glabra, Glycyrrhiza echinata, and Glycyrrhiza
inflata are particularly preferable.
[0029] Licorice is often called with the name of the place of
production, for example, licorice of northeast and northwest China
(Glycyrrhiza uralensis), Sinkiang licorice or Shinkyo kanzoh in
Japanese (Glycyrrhiza echinata), Russian licorice (Glycyrrhiza
glabra), Spanish licorice (Glycyrrhiza echinata), Mongolian
licorice, and Afghan licorice.
[0030] Any site of roots, rhizomes, leaves, and stems of the
above-mentioned licorice can be used as a raw material for the
extraction of the oil-soluble licorice extract. However, at least
one of roots and rhizomes are particularly preferable in that the
content of flavonoid such as glabridin and licochalcone A is high.
Although the raw materials may be used for extraction as it is or
may be used for extraction after drying, but dried roots and dried
rhizomes are particularly preferable.
[0031] A water extraction residue of the raw material for
extraction, i.e., a water extraction residue of the above-mentioned
licorice, can also be used as a raw material for the extraction of
the oil-soluble licorice extract.
[0032] The water extraction residue of licorice means a solid
residue after extraction of licorice with at least one of water,
warm water, and hot water, and neutral or weak alkaline cold water,
warm water, and hot water in order to obtain glycyrrhizin and the
like from the licorice. The residue after water extraction may
contain water or may be dried.
[0033] Use of water extraction residue of licorice as a raw
material for extraction as mentioned above has an advantage that
water extraction residue of licorice can be utilized efficiently,
improving productivity.
[0034] The organic solvent is not particularly limited and can be
appropriately selected depending on the application. Examples
thereof include benzene, toluene, xylene, ethyl ether, methyl ethyl
ketone, methyl isobutyl ketone, dichloromethane, dichloroethane,
chloroform, ethyl acetate, propyl acetate, butyl acetate, acetone,
methanol, ethanol, propanol, hydrous methanol, hydrous ethanol,
hydrous propanol, and the like. Further, carbon dioxide can also be
used as a supercritical fluid. Among these organic solvents,
ethanol, hydrous ethanol, and ethyl acetate are preferably used in
terms of safety. For the hydrous ethanol, those with an ethanol
concentration of 30% by mass to 99% by mass are suitable.
[0035] The condition of extraction for obtaining oily extracts of
licorice from the licorice or water extraction residues of licorice
with use of the organic solvent is not particularly limited and can
be appropriately selected depending on the application. For
example, 2- to 15-fold amount of organic solvent based on raw
material for extraction is added, and extracted with stirring at
room temperature or extracted by heating and dry distillation. In
addition, repeated operation of these methods singly or in
combination is more preferable since extraction efficiency is
improved.
[0036] The obtained extract is subjected to centrifugation and
filtration, by which insoluble matter is removed. The resulting
solution can be used as an oil-soluble licorice extract without
further treatment, or after further treatment where the resulting
solution is concentrated in the usual manner. In addition,
deodorization, decoloration, and the like may be performed
appropriately unless the intended physiological effect is impaired.
For this deodorization and decoloration, an activated carbon,
synthetic adsorbent resin, ion-exchange resin, and the like are
typically used. By drying extract by an appropriate method,
yellowish brown extract powder can be obtained as an oil-soluble
licorice extract.
[0037] The obtained liquid extracts without further treatment, or
those obtained after concentration of liquid extracts, and dry
powder of or dry solid of liquid extracts are utilized as an
oil-soluble licorice extract.
[0038] Flavonoid can be purified from the oil-soluble licorice
extract by any method without limitation, and the purification
method can be appropriately selected from known methods for
purifying an organic compound depending on the application. For
example, purification can be performed by treating the oil-soluble
licorice extract by means of one of normal phase silica gel
chromatography and reverse phase chromatography, and then
crystallizing from acetone. By this method, pure product of active
ingredient can be obtained relatively easily. Other purification
methods include column chromatography using a synthetic adsorbent
such as Diaion HP-20 (manufactured by Mitsubishi Chemical
Corporation), liquid-liquid countercurrent extraction, and the
like.
[0039] The flavonoid that is contained in the oil-soluble licorice
extract is different depending on the species of licorice, which is
a raw material for extraction, so that it is impossible to define
definitely. However, examples of the flavonoid include glabridin,
glabrene, licochalcone A, licochalcone B, glycycoumarin,
glisoflavone and the like. Among these, glabridin, glabrene,
licochalcone A, and licochalcone B are particularly preferable, and
glabridin and licochalcone A are most preferable since they have a
high anti-inflammatory effect.
[0040] The total content of at least one flavonoid of the glabridin
and licochalcone A in the oil-soluble licorice extract is
preferably 1% by mass to 80% by mass, and more preferably 5% by
mass to 60% by mass, based on dry solid content.
[0041] The content of glabridin in the oil-soluble licorice extract
of the Glycyrrhiza glabra is preferably 1% by mass or more, more
preferably 10% by mass or more, and still more preferably 20% by
mass to 50% by mass, based on dry solid content.
[0042] The content of licochalcone A in the oil-soluble licorice
extract of the Glycyrrhiza echinata is preferably 1% by mass or
more, more preferably 5% by mass or more, and still more preferably
10% by mass to 40% by mass, based on dry solid content.
[0043] The content of licochalcone A in the oil-soluble licorice
extract of the Glycyrrhiza inflata is preferably 1% by mass or
more, more preferably 5% by mass or more, and still more preferably
10% by mass to 40% by mass, based on dry solid content.
[0044] The anti-inflammatory agent of the present invention can
prevent and improve various inflammatory diseases including skin
diseases such as contact dermatitis (rash), psoriasis and pemphigus
vulgaris through at least one effect selected from an inhibitory
effect on hyaluronidase activity, an inhibitory effect on
hexosaminidase release, (i.e., inhibitory effect on histamine
release), an inhibitory effect on platelet aggregation, and an
inhibitory effect on phospholipase A.sub.2 activity.
[0045] Here, the hyaluronidase is a hyaluronan hydrolase and exists
in a mast cell, and it is said that the hyaluronidase is involved
in degranulation of mast cell through its activation. Thus, by
inhibiting the activation of hyaluronidase, stabilization of
hyaluronan can be attained, release of various chemical mediators
from a mast cell can be prevented, and enhancement of moisture
retention or anti-inflammatory effect can be expected.
[0046] In addition, since hexosaminidase is also released
simultaneously upon release of histamine inside a cell, the
inhibitory effect on histamine release can be evaluated using
hexosaminidase release as an indicator of histamine release. The
histamine is present in a mast cell, and when the mast cell is
stimulated, it is released by degranulation reaction and acts as
prophlogistic or inflammation-causing substance and as an
allergenic substance. The histamine released from activated mast
cell causes increased vascular permeability, smooth muscle
contraction, increased mucus secretion, etc., resulting in allergic
diseases such as bronchial asthma, allergic rhinitis, and
urticaria. Therefore, inhibition of release of hexosaminidase,
i.e., inhibition of release of histamine enables prevention and
treatment of allergic diseases and inflammatory diseases.
[0047] The platelet aggregation invites activation of phospholipase
A.sub.2 in the arachidonate cascade, thereby leukotriene B,
prostaglandin E.sub.2, lo and the like are released, and these
substances cause inflammation. Thus, allergic diseases and
inflammatory diseases can be prevented and treated by substances
that inhibit aggregation of platelet.
[0048] The phospholipase A.sub.2 is an important enzyme in the
arachidonate cascade, which is a metabolic pathway of arachidonic
acid, and excessive activation of phospholipase A.sub.2 leads to
abnormal metabolism of arachidonic acid, causing inflammation,
allergy, asthma, ischemia, myocardial infarction, etc. Therefore,
by inhibiting the activation of phospholipase A.sub.2, allergic
diseases and inflammatory diseases can be prevented and
treated.
[0049] The anti-inflammatory agent of the present invention is not
particularly limited and can be appropriately selected depending on
the application, but it is suitably used as an external preparation
for skin. The external preparation for skin means various drugs
that are applied for skin, including, for example, cosmetics,
quasi-drugs, and medicines. Examples of the external preparation
for skin include ointment, cream, milky lotion, lotion, pack,
jelly, lip cream, lipstick, bath agent, tonic, rinse, shampoo,
astringent, and the like.
[0050] The amount of the anti-inflammatory agent to be mixed or
compounded in the external preparation for skin can be
appropriately adjusted depending on the type of the external
preparation for skin, bioactivity of extract, etc., but is
preferably 0.001% by mass to 10% by mass and more preferably 0.01%
by mass to 5% by mass.
[0051] The anti-inflammatory agent of the present invention,
described above, is suitably applied to humans, but is also applied
to animals other than humans as long as each effect is
achieved.
[0052] Hereafter, the present invention will be described in detail
by means of examples, but it will be understood that the present
invention should not be construed as being limited thereby.
PRODUCTION EXAMPLE 1
Preparation of Oil-Soluble Licorice Extract
[0053] 10 L of dehydrated ethanol was added to 1 kg of licorice
(Glycyrrhiza glabra Linne var. glandulifera Regel et Herder) root
and extraction was performed for 5 hours under reflux. The obtained
extract was subjected to vacuum concentration. To this concentrated
extract, was added 1 L of ethyl acetate, extracted for 5 hours
under reflux, subjected to vacuum drying, and then grinded to
prepare 10 g of oil-soluble licorice extract.
[0054] The content of flavonoid in the prepared oil-soluble
licorice extract of Production Example 1 was determined
quantitatively using high-performance liquid chromatography
(manufactured by JASCO Corporation). The prepared oil-soluble
licorice extract of Production Example 1 contained 20% by mass of
glabridin.
PRODUCTION EXAMPLE 2
Preparation of Oil-Soluble Licorice Extract
[0055] 10 L of dehydrated ethanol was added to 1 kg of licorice
(Glycyrrhiza glabra Linne var. glandulifera Regel et Herder) root
and extraction was performed for 5 hours under reflux. The obtained
extract was subjected to vacuum concentration. To this concentrated
extract, was added 1 L of ethyl acetate and extraction was
performed for 5 hours under reflux. This was roughly purified with
a synthetic adsorbent (manufactured by Mitsubishi Chemical
Corporation), subjected to vacuum drying, and then grinded to
prepare 5 g of oil-soluble licorice extract.
[0056] The content of flavonoid in the prepared oil-soluble
licorice extract of Production Example 2 was determined
quantitatively using high-performance liquid chromatography
(manufactured by JASCO Corporation). The prepared oil-soluble
licorice extract of Production Example 2 contained 40% by mass of
glabridin.
PRODUCTION EXAMPLE 3
Preparation of Oil-Soluble Licorice Extract
[0057] 10 L of dehydrated ethanol was added to 1 kg of licorice
(Glycyrrhiza inflata Batalin) root and extraction was performed for
5 hours under reflux. The obtained extract was subjected to vacuum
concentration. To this concentrated extract, was added 1 L of ethyl
acetate and extraction was performed for 5 hours under reflux. The
obtained extract was subjected to vacuum concentration. To this
concentrated extract, was added 1 L of ethyl acetate, extracted for
5 hours under reflux, subjected to vacuum drying, and then grinded
to prepare 30 g of oil-soluble licorice extract.
[0058] The content of flavonoid in the prepared oil-soluble
licorice extract of Production Example 3 was determined
quantitatively using high-performance liquid chromatography
(manufactured by JASCO Corporation). The prepared oil-soluble
licorice extract of Production Example 3 contained 20% by mass of
licochalcone A.
PRODUCTION EXAMPLE 4
Purification of Glabridin
[0059] 20 g of oil-soluble licorice extract prepared in Production
Example 2 was dissolved in chloroform, mixed with silica gel
(silica gel 60, manufactured by Merck Ltd.), and then dried. This
dried material was deposited or loaded on a column packed with 1 kg
of silica gel the same as that mentioned above and eluted with a
chloroform/methanol mixture (30:1). The fraction containing
glabridin was collected. The solvent of this fraction was distilled
away under reduced pressure to obtain 5.8 g of solid. Next, the
obtained solid was dissolved in a small amount of methanol, mixed
with reversed-phase silica gel (ODS DM1020T, manufactured by Fuji
Silysia Chemical Ltd.), dried, and deposited or loaded on a column
which was packed with 800 g of reversed-phase silica gel
previously. A methanol/water mixture (60:40) was passed through
this column as an elution solvent, and the fraction containing
glabridin was collected. The solvent was distilled away from this
fraction under reduced pressure. The obtained solid (4.3 g) was
dissolved in 40 ml of acetone and left at rest at 5.degree. C. for
3 days to obtain 3.8 g of crystalline glabridin.
PRODUCTION EXAMPLE 5
Purification of Licochalcone A
[0060] 120 g of oil-soluble licorice extract prepared in Production
Example 3 was dissolved in chloroform, mixed with silica gel
(silica gel 60, manufactured by Merck Ltd.), and then dried. This
dried material was deposited or loaded on a column packed with 3 kg
of silica gel the same as that mentioned above and eluted with an
n-hexane/ethyl acetate mixture (2:1). The fraction containing
licochalcone A was collected. The solvent of this fraction was
distilled away under reduced pressure to obtain 50 g of solid.
Then, the obtained solid was dissolved in a small amount of
methanol, mixed with reversed-phase silica gel (ODS DM1020T,
manufactured by Fuji Silysia Chemical Ltd.), dried, and deposited
or loaded on a column which was packed with reversed-phase silica
gel previously. A methanol/water mixture (60:40) was passed through
this column as an elution solvent, and the fraction containing
licochalcone A was collected. The solvent was distilled away from
this fraction under reduced pressure. The obtained solid (25 g) was
dissolved in a methanol/water mixture (70:30) and left at rest at
room temperature for 1 day to obtain 15.2 g of crystalline
licochalcone A.
EXAMPLE 1
Hyaluronidase Activity Inhibition Assay
[0061] For the extracts and purified materials prepared in
Production Examples 1 to 5 (hereinafter, may be referred to as
"sample"), inhibitory effect on hyaluronidase activity was tested
as follows.
[0062] First, 0.1 mL of hyaluronidase solution (Type IV-S(from
bovine testis; SIGMA 400 NF units/mL) was added to 0.1 mol/L acetic
acid buffer (pH 3.5) in which each sample was dissolved, and
allowed to react at 37.degree. C. for 20 minutes.
[0063] Next, 0.2 mL of 2.5 mmol/L calcium chloride was added as an
activating agent and allowed to react at 37.degree. C. for 20
minutes. To this solution, 0.5 mL of 0.4 mg/mL potassium
hyaluronate solution (from robster comb) was added and allowed to
react at 37.degree. C. for 40 minutes. After that, reaction was
stopped by adding 0.2 mL of 0.4 mol/L sodium hydroxide and cooled.
Then, 0.2 mL of boric acid solution was added to each reaction
solution and boiled for 3 minutes. After cooling on ice, 6 mL of
p-dimethyl aminobenzaldehyde (p-DABA) reagent was added and allowed
to react at 37.degree. C. for 20 minutes. After that, absorbance at
the wavelength of 585 nm was determined.
[0064] Next, similar operations and absorbance determination were
performed without addition of enzyme. Further, as a control, using
distillated water instead of sample solution, similar operations
and absorbance determination were performed.
[0065] From the results of the measurements mentioned above, the
percentage inhibition of hyaluronidase activity was calculated
according to the following formula 1:
% Inhibition of hyaluronidase
activity=[1-(St-Sb)/(Ct-Cb)].times.100 <Formula 1>
[0066] where St represents the absorbance at the wavelength of 585
nm of the sample solution; Sb represents the absorbance at the
wavelength of 585 nm of the sample solution blank; Ct represents
the absorbance at the wavelength of 585 nm of the control solution;
and Cb represents the absorbance at the wavelength of 585 nm of the
control solution blank.
[0067] Next, the sample concentration was reduced in a stepwise
manner, and the above-mentioned percentage inhibition of
hyaluronidase activity was measured. The sample concentration, at
which 50% of hyaluronidase activity is inhibited, was determined by
interpolation. The results are shown in Table 1. The smaller this
value is, the stronger the inhibitory effect on hyaluronidase
activity is.
TABLE-US-00001 TABLE 1 Sample concentration for 50% inhibition of
hyaluronidase activity Oil-Soluble Licorice Extract 189.4 .mu.g/ml
of Production Example 1 Oil-Soluble Licorice Extract 54.5 .mu.g/ml
of Production Example 2 Oil-Soluble Licorice Extract 14.6% (when
400 .mu.g/ml of Production Example 3 was added) Glabridin of
Production 18.3 .mu.g/ml Example 4 Licochalcone A of 17.2 .mu.g/ml
Production Example 5
[0068] From the results of Table 1, it was confirmed that
oil-soluble licorice extracts of Production Examples 1 to 3,
glabridin of Production Example 4, and licochalcone A of Production
Example 5 have an inhibitory effect on hyaluronidase activity.
EXAMPLE 2
Assay of Inhibitory Effect on Hexosaminidase Release
[0069] For the extracts and purified materials prepared in
Production Examples 1 to 5 (hereinafter, may be referred to as
"sample"), inhibitory effect on hexosaminidase release was tested
as follows. Since hexosaminidase is also released simultaneously
upon release of histamine inside a cell, the inhibitory effect on
histamine release can be evaluated using hexosaminidase release as
an indicator of histamine release.
[0070] First, rat basophilic leukemia cells (RBL-2H3) were cultured
using a medium (S-MEM medium with 15% FBS; hereinafter the same)
placed in 25 mL culture flasks, and then cells were collected by
trypsin treatment. The collected cells were diluted with S-MEM
medium to a concentration of 4.0.times.10.sup.5 cells/mL, and mouse
monoclonal anti-dinitrophenyl group IgE (DNP-specific-IgE) was
added to a final concentration of 0.5 .mu.g/mL. Then, 100 .mu.l of
this cell suspension was seeded in a well of a 96-well plate and
cultured overnight. After cultivation, S-MEM medium was removed,
and washed twice with 500 .mu.L of Siraganian buffer.
[0071] Next, 30 .mu.L of Siraganian buffer and 10 .mu.L of each
sample, prepared using Siraganian buffer, were added and left at
rest at 37.degree. C. for 10 minutes. After that, 10 .mu.L of 100
ng/mL dinitrophenylated bovine serum albumin (DNP-BSA) was added
and left at rest at 37.degree. C. for 15 minutes to release
hexosaminidase.
[0072] Then, release was stopped by leaving the 96 well plate at
rest on ice. 110 .mu.L of cell supernatant of each well and 10
.mu.L of 1 mmol/L p-nitrophenyl-N-acetyl-.beta.-D-glucosaminide
(p-NAG) solution were added to a fresh 96 well plate and allowed to
react at 37.degree. C. for 1 hour. After completion of the
reaction, 250 .mu.L of 0.1 mol/L Na.sub.2CO.sub.3/NaHCO.sub.3 was
added to each well, and the absorbance at the wavelength of 415 nm
was determined.
[0073] Next, as a blank test, the absorbance of a mixture of 10
.mu.L of cell supernatant and 250 .mu.L of 0.1 mol/L
Na.sub.2CO.sub.3/NaHCO.sub.3 at the wavelength of 415 nm was
determined and was used for correction.
[0074] From the results of the measurements mentioned above, the
percentage inhibition of hexosaminidase release was determined
according to the following formula 2:
% Inhibition of hexosaminidase release=[1-(B-C)/A].times.100
<Formula 2>
[0075] where A represents the absorbance at the wavelength of 415
nm without the addition of sample; B represents the absorbance at
the wavelength of 415 nm with the addition of sample; and C
represents the absorbance at the wavelength of 415 nm with the
addition of sample and without the addition of p-NAG.
[0076] Next, the sample concentration was reduced in a stepwise
manner, and the above-mentioned percentage inhibition of
hexosaminidase release was measured. The sample concentration, at
which 50% of release of hexosaminidase is inhibited, was determined
by interpolation. The results are shown in Table 2. The smaller
this value is, the stronger the inhibitory effect on hexosaminidase
release is.
TABLE-US-00002 TABLE 2 Sample concentration for 50% inhibition of
hexosaminidase release Oil-Soluble Licorice Extract 19.1 .mu.g/ml
of Production Example 1 Oil-Soluble Licorice Extract 18.6% (when 10
ppm was of Production Example 2 added) Oil-Soluble Licorice Extract
17.0 .mu.g/ml of Production Example 3 Glabridin of Production --
Example 4 Licochalcone A of 23.5 .mu.g/ml Production Example 5
[0077] From the results of Table 2, it was confirmed that
oil-soluble licorice extracts of Production Examples 1 to 3, and
licochalcone A of Production Example 5 have an inhibitory effect on
hexosaminidase lo release (i.e., inhibitory effect on histamine
release).
EXAMPLE 3
Platelet Aggregation Inhibitory Effect
[0078] For the extracts and purified materials prepared in
Production Examples 1 to 5 (hereinafter, may be referred to as
"sample"), inhibitory effect on platelet aggregation was tested as
follows.
[0079] First, rabbit blood, which was collected with the addition
of 1/10 amount of 77 mmol/L EDTA (pH 7.4), was centrifuged
(180.times.g, 10 minutes, room temperature) to obtain platelet
suspension. Next, the platelet suspension was centrifuged
(810.times.g, 10 minutes, 4.degree. C.), and the supernatant was
removed to obtain platelet. This was suspended in platelet washing
solution (0.15 mol/L sodium chloride: 0.15 mol/L Tris-HCL buffer
(pH 7.4): 77 mmol/L EDTA (pH7.4)=90:8:2), centrifuged in the same
way as mentioned above. The obtained platelet was suspended in
platelet suspension (10 mmol/L HEPES buffer (pH 7.4) containing 145
mmol/L sodium chloride, 5 mmol/L potassium chloride and 5.5 mmol/L
glucose), and thereby the number of platelet was adjusted
(3.0.times.10.sup.5 cells/.mu.L) to prepare washed platelet
suspension.
[0080] Next, to 222 .mu.L of the prepared washed platelet
suspension, 1 .mu.L of 200 mmol/L calcium chloride solution was
added and allowed to react at 37.degree. C. for 1 minute. To this
solution, 2 .mu.L of each sample was added and further allowed to
react for 2 minutes. A stirring bar was placed, the solution was
stirred for 1 minute, and then 25 .mu.L of 10 ppm collagen solution
was added as an aggregation inducing agent. Rate of platelet
aggregation at 37.degree. C. for 10 minutes was measured with a
platelet aggregation measuring device (PAM12CL, manufactured by
Mebanics Inc.), and the percentage inhibition of platelet
aggregation, A, was determined according to the following formula
3. Separately, rate of platelet aggregation, B, was determined in a
similar operation to that mentioned above except that the solvent
of sample solution was not added instead of the sample
solution.
% Inhibition of platelet aggregation=[(B-A)/B].times.100
<Formula 3>
[0081] where A represents the rate of platelet aggregation when an
aggregation inducing agent and a sample solution were added; and B
represents the rate of platelet aggregation when an aggregation
inducing agent was added and a sample solution was not added.
[0082] Next, the sample concentration was reduced in a stepwise
manner, and the above-mentioned percentage inhibition of platelet
aggregation was measured. The sample concentration, at which 50% of
platelet aggregation is inhibited, was determined by interpolation.
The results are shown in Table 3. The smaller this value is, the
stronger the inhibitory effect on platelet aggregation is.
TABLE-US-00003 TABLE 3 Sample concentration for 50% inhibition of
platelet aggregation Oil-Soluble Licorice Extract 2.0 .mu.g/ml of
Production Example 1 Oil-Soluble Licorice Extract 4.3 .mu.g/ml of
Production Example 2 Oil-Soluble Licorice Extract 0.38 .mu.g/ml of
Production Example 3 Glabridin of Production 400 .mu.g/ml Example 4
Licochalcone A of 79.7 .mu.g/ml Production Example 5
[0083] From the results of Table 3, it was confirmed that
oil-soluble licorice extracts of Production Examples 1 to 3,
glabridin of Production Example 4, and licochalcone A of Production
Example 5 have an inhibitory effect on platelet aggregation.
EXAMPLE 4
Phospholipase A.sub.2 Activity Inhibitory Effect
[0084] For the extracts and purified materials prepared in
Production Examples 1 to 5 (hereinafter, may be referred to as
"sample"), inhibitory effect on phospholipase A.sub.2 activity was
tested as follows.
[0085] First, rat leukemia cells, RBL-2H3 cells, were cultured in a
MEM medium containing 15 v/v % FBS in 75 cm.sup.2 flasks at
37.degree. C. under 5% C0.sub.2-95% air, and cells were collected
in the usual manner. The collected cells were adjusted using a MEM
medium containing 15 v/v % FBS to 5.times.10.sup.5 cells/mL.
Further, [3H] arachidonic acid (50 .mu.Ci/500 .mu.L) was added to a
concentration of 3 .mu.L/10 mL. 1 mL of the prepared solution was
seeded in a 24-well plate and cultured overnight at 37.degree. C.
under 5% CO.sub.2 95% air. The medium in each well was discarded,
washed with PBS(-), followed by the addition of serum free MEM
medium, and incubated at 37.degree. C. for 30 minutes.
[0086] Next, a solution, in which sample was dissolved, was added
to each well and incubated for 10 minutes in the same way. Further,
10 .mu.L of 1 mmol/L A23187 (manufactured by Sigma-Aldrich) was
added and incubated at 37.degree. C. for 5 minutes. After the
reaction, 500 .mu.L of supernatant was taken under water cooling, 6
mL of scintillation cocktail was added, and radioactivity was
measured in a liquid scintillation counter.
[0087] Next, in the same manner, radioactivity was measured for a
blank test (without A23187 stimulation) and for a control (solvent
of sample solution).
[0088] From the results of the measurements obtained, the
percentage inhibition of phospholipase A.sub.2 activity was
determined according to the following formula 4:
% Inhibition of phospholipase A.sub.2 activity=[(B-A)/(
B-C)].times.100 <Formula 4>
[0089] where A represents the radioactivity when sample was added;
B represents the radioactivity of control; and C represents the
radioactivity of blank test.
[0090] Next, the sample concentration was reduced in a stepwise
manner, and the above-mentioned percentage inhibition of
phospholipase A.sub.2 activity was measured. The sample
concentration, at which 50% of phospholipase A.sub.2 activity is
inhibited, was determined by interpolation. The results are shown
in Table 4. The smaller this value is, the stronger the inhibitory
effect on phospholipase A.sub.2 activity.
TABLE-US-00004 TABLE 4 Sample concentration for 50% inhibition of
phospholipase A.sub.2 activity Oil-Soluble Licorice Extract 3.7
.mu.g/ml of Production Example 1 Oil-Soluble Licorice Extract 1.5
.mu.g/ml of Production Example 2 Oil-Soluble Licorice Extract 0.4
.mu.g/ml of Production Example 3 Glabridin of Production -- Example
4 Licochalcone A of -- Production Example 5
[0091] From the results of Table 4, it was confirmed that
oil-soluble licorice extracts of Production Examples 1 to 3 have an
inhibitory effect on phospholipase A.sub.2 activity.
COMPOUNDING EXAMPLE 1
[0092] A cream with anti-inflammatory effect that has the following
composition was produced by a conventional method.
TABLE-US-00005 Liquid paraffin 5.0 g White beeswax 4.0 g Cetanol
3.0 g Squalane 10.0 g Lanolin 2.0 g Stearic acid 1.0 g
Polyoxyethylene sorbitan oleate (20 E. O) 1.5 g Glyceryl
monostearate 3.0 g 1,3-Butylene glycol 6.0 g Methyl
parahydroxybenzoate 1.5 g Perfume 0.1 g Oil-soluble licorice
extract of Production Example 1 0.01 g Purified water balance Total
100 g
COMPOUNDING EXAMPLE 2
[0093] A milky lotion with anti-inflammatory effect that has the
following composition was produced by a conventional method.
TABLE-US-00006 Jojoba oil 4.0 g Placenta extract 0.1 g Olive oil
2.0 g Squalane 2.0 g Cetanol 2.0 g Glyceryl monostearate 2.0 g
Polyoxyethylene cetyl ether (20 E. O) 2.5 g Polyoxyethylene
sorbitan oleate (20 E. O) 2.0 g 1,3-Butylene glycol 3.0 g
Hinokitiol 0.15 g Perfume 0.05 g Oil-soluble licorice extract of
Production Example 2 0.01 g Purified water balance Total 100 g
COMPOUNDING EXAMPLE 3
[0094] A pack with anti-inflammatory effect that has the following
composition was produced by a conventional method.
TABLE-US-00007 Polyvinyl alcohol 15 g Polyethylene glycol 3 g
Propylene glycol 7 g Ethanol 10 g Ethyl parahydroxybenzoate 0.05 g
Perfume 0.05 g Oil-soluble licorice extract of Production Example 3
0.05 g Purified water balance Total 100 g
COMPOUNDING EXAMPLE 4
[0095] A cream with anti-inflammatory effect that has the following
composition was produced by a conventional method.
TABLE-US-00008 Liquid paraffin 5.0 g White beeswax 4.0 g Cetanol
3.0 g Squalane 10.0 g Lanolin 2.0 g Stearic acid 1.0 g
Polyoxyethylene sorbitan oleate (20 E. O) 1.5 g Glyceryl
monostearate 3.0 g 1,3-Butylene glycol 6.0 g Methyl
Parahydroxybenzoate 1.5 g Perfume 0.1 g Glabridin of Production
Example 4 0.01 g Purified water balance Total 100 g
COMPOUNDING EXAMPLE 5
[0096] A pack with anti-inflammatory effect that has the following
composition was produced by a conventional method.
TABLE-US-00009 Polyvinyl alcohol 15 g Polyethylene glycol 3 g
Propylene Glycol 7 g Ethanol 10 g Ethyl parahydroxybenzoate 0.05 g
Perfume 0.05 g Licochalcone A of Production Example 5 0.05 g
Purified water balance Total 100 g
INDUSTRIAL APPLICABILITY
[0097] The anti-inflammatory agent of the present invention is
prepared by subjecting at least one of a leguminous plant of the
genus Glycyrrhiza and a water extraction residue of a leguminous
plant of the genus Glycyrrhiza to an extraction treatment with an
organic solvent, has at least one effect selected from an
inhibitory effect on hyaluronidase activity, an inhibitory effect
on hexosaminidase release, an inhibitory effect on platelet
aggregation, and an inhibitory effect on phospholipase A.sub.2
activity, and is suitably used especially as a skin cosmetic such
as skin toner, cream, milky lotion, lotion, and pack.
* * * * *