1. WOUND HEALINGACCELERATOR
2.
1. (A) A-Z LTD.
(B) Japan
(C) 2-3-19, Kimachidori, Aoba-ku, Sendai-shi, Miyagi 9800801, Japan
The following specification particularly describes the invention and the manner in which it is to
be performed.
2
Technical Field
[0001]
The present invention relates to use of a specific polyphenol for accelerating
wound healing, a wound healing accelerator comprising the same and a pharmaceutical
composition for 5 wound healing comprising the same.
Background Art
[0002]
Periodontal disease is an oral disease spreading across the world and one of major
10 causes of tooth loss. Although someactive ingredients can be mentioned against such a wound as
to include periodontal disease, a demand still exists for compounds capable of accelerating
wound healing.
Examples of the compounds that show the accelerating actionof wound
healinginclude agents, as an active ingredient, such as an extract from aloes or the like, an
15 antibiotic, an anti-inflammatory agent, kallikrein, adenine, nicotinic acid, allantoin, vitamin A,
zinc, a cAMP derivative (Patent Literature 1) and exogenous collagen (Patent Literature 2). In
the conventional technique,the improvement of a formulation,etc., to improve the absorbability
of such active ingredients had been actively practiced, however, recently, as various findings on
the skin histology become clear, attempts to use epidermal growth factor (EGF) for postoperative
20 wound healing have been made (Patent Literature 3).
When an agent is orally administered,a portion of the administered agent acts on
the affected area, thereby making it difficult to obtain a sufficient effect and further leaving
concern over the manifestation of side effects. Therefore,agents such as an external preparation,
which act directly on the skin, are desired for therapy. Few wound healing accelerators,
25 however, are available as an external preparation, and this makes skin wound healing difficult.
At present, as a wound healing acceleratorwhich is commonly used for an
external preparation, solcoseryl ointment containing as a main ingredient a calf blood extract,
Actosin ointment containing as a main ingredientbucladesine sodium which is a cAMP
derivative,and Fiblast spray containing as a main ingredientbFGF (basic fibroblast growth
30 factor), are known. It is, however, requiredfor therapyto apply such an accelerator to the affected
area for a long time, and side effects, such as irritation/aching, redness, itching and the increase
of exudation, have also been reported. Many of specific polyphenolsas disclosed in the present
specification, for example, proanthocyanidin and chlorogenic acid which are abundant in grapes
3
and coffee, respectively,abound in familiarity with eating,and can constitute one characteristic
feature of being highly safe even upon ingestion. In addition, it takes a certain extent of time for
the above drugs to show the effect on fibroblasts, and the action of various growth factors (TGF-
β, IL-1α and PDGF) including bFGF that the present inventors had previously tested became
detectable on a fibroblast wound model (in vitro) after 24 5 hours of treatment (Non Patent
Literature 1). Thus, any drug or the liketo reveal its effect after several minutes of treatment has
not been reported.
On the other hand, it has been known that a polyphenol acts as an antioxidant and
hasa suppressing effect on oxidative damage at inflamed sites in local tissues which are caused
10 by reactive oxygen species produced by inflammatory cells such as neutrophils. A polyphenol is
used foran external dermatologic agent, an agent for use in the oral cavity and the like (Patent
Literatures 4 - 9 and Non Patent Literatures 2 and 3). However, it has not been confirmed that
any specific polyphenol can accelerate wound healing.
15 Citation List
Patent Literature
[0003]
Patent Literature 1
JP 63-107935 A
20 Patent Literature 2
JP 2004-217587 A
Patent Literature 3
JP 03-106823 A
Patent Literature 4
25 International Publication No.WO 2005/092327
Patent Literature 5
JP 06-336422 A
Patent Literature 6
Japanese Patent No. 3917370
30 Patent Literature 7
Japanese Patent Application No. 2005-213234
Patent Literature 8
JP 2009-52185 A
Patent Literature 9
4
JP 2010-511026 A
Non Patent Literature
[0004]
Non Patent Literature 1
O. Ohgoda et al., Journal of Dermatological 5 Science 17 (1998) 123-131.
Non Patent Literature 2
M. A. Soobrattee et al., Mutation Research 579 (2005) 200-213.
Non Patent Literature 3
C. A. RICE-EVANS et al., Free Radical Biology & Medicine, Vol. 20, No. 7
10 (1996) pp. 933-956.
Summary of Invention
Technical Problem
[0005]
15 The object of the present invention is to provide a wound healing
acceleratoreffective to accelerate healing of wounds, in particular of intraoral wounds, and
pharmaceutical compositions comprising the same. In particular, the invention provides a wound
healing acceleratorwhich is expected to work within a short duration of contact and which is easy
to handle, and a pharmaceutical composition comprising the same.
20
Solution to Problem
[0006]
After an extensive study to achieve the object, the present inventors have found
that a specific polyphenol with catechol groups is effective in wound healing, thereby completing
25 the present invention.
[0007]
Thus, the present invention is as follows.
[1] A pharmaceutical composition for wound healing, comprising at least one of a polyphenol
represented by the following general formula:
30 [Formula 1]
5
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl 5 group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
10 group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20aromatic hydrocarbon group or a substituted or unsubstituted C3-
20polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
15 salt thereof.
[2] The pharmaceutical composition for wound healing according to [1], wherein both R2 and R3
are a hydrogen atom.
[3] The pharmaceutical composition for wound healing according to [1] or [2], wherein R1 is a
hydrogen atom.
20 [4] The pharmaceutical composition for wound healing according to any one of [1] - [3], wherein
the polyphenol is selected from the group consisting of caffeic acid, (+)-catechinic acid and
chlorogenic acid.
[5] An oral care composition, comprising at least one of a polyphenol represented by the
following general formula:
25 [Formula 2]
6
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl 5 group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
10 group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
15 salt thereof.
[6] The oral care composition according to [5], wherein both R2 and R3 are a hydrogen atom.
[7] The oral care composition according to [5] or [6], wherein R1 is a hydrogen atom.
[8] The oral care composition according to any one of [5] - [7], wherein the polyphenol is
selected from the group consisting of caffeic acid, (+)-catechinic acid and chlorogenic acid.
20 [9] The oral care composition according to claims 5 to 8, wherein the composition is in the form
of a mouthrinse, a dentifrice, a sweet, a medicament or a film.
[10] Amethod for accelerating wound healing, comprising the step of contacting a wound with at
least one of a polyphenol represented by the following general formula:
[Formula 3]
7
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl 5 group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
10 group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
15 salt thereof.
[11] The method according to [10], wherein both R2 and R3 are a hydrogen atom.
[12] The method according to [10] or [11], wherein R1 is a hydrogen atom.
[13] The method according to any one of [10] - [12], wherein the polyphenol is selected from the
group consisting of caffeic acid, (+)-catechinic acid and chlorogenic acid.
20 [14] The method according to any one of [10] - [13], wherein a duration of the contact is 0.01
seconds to 48 hours.
[15] The method according to [14], wherein the duration of the contact is 1 second to 10 minutes.
[16] The method according to any one of [10] - [15], further comprising the step of washing a
contact site after the contact.
25 [17] The method according to any one of claims 10 to 16, wherein the wound is in an oral cavity.
[18] Use of at least one of a polyphenol represented by the following general formula:
[Formula 4]
8
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl 5 group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
10 group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
15 salt thereof for manufacturing a wound healing accelerator.
Advantageous Effects of Invention
[0008]
The wound healing acceleratorof the present invention comprising the
20 specificpolyphenol is easy to handle and can accelerate wound healing even after a short
duration of contact. The acceleratoris also expected to have an inhibitory action on oxidative
damage at inflamed sites in local tissues, which is caused by reactive oxygen species produced
by inflammatory cells such as neutrophils.
25 Brief Description of Drawings
[0009]
[Fig. 1] Fig. 1 shows the results of proanthocyanidin treatment and the effect of laser irradiation
after the proanthocyanidin treatment.
9
[Fig. 2] Fig. 2 shows the results of caffeic acid, (+)-catechin and chlorogenic acid treatments.
[Fig. 3] Fig. 3 shows the results of treating with (+)-catechin for 24 hours.
[Fig. 4] Fig. 4 shows the results of green tea extract treatment.
[Fig. 5] Fig. 5 shows the results of treating human gingival fibroblasts with proanthocyanidin for
5 one minute.
[Fig. 6] Fig. 6 shows the time-course of growth of proanthocyanidin-treated human gingival
fibroblasts.
[Fig. 7] Fig. 7 shows the results of the cytoprotective effect of proanthocyanidin on osmotic
stress.
10 [Fig. 8] Fig. 8 shows the results of the cytoprotective effect of proanthocyanidin on starvation
stress.
[Fig. 9] Fig. 9 shows the results of the cytoprotective effect of proanthocyanidin on the
cytotoxicity of bacterial endotoxin (lipopolysaccharide, LPS).
15 Description of Embodiments
[0010]
Details of embodiments according to the present invention are given below.
A wound generally heals through the following steps. First, the wound causes
tissue lesion, followed by hemostasis and the inflammatory phase. Subsequently, granulation
20 tissue is formed with vascularization, epidermis regenerates and extends on the resulting
granulations, and the healing process proceeds through the wound contraction phase and
maturation. During granulation tissue formation (the granulation phase), fibroblasts need to
grow. The growth of fibroblasts is observed if they are brought into contact with a specific
polyphenol which is effective in accelerating wound healing. Thus, such a polyphenol can
25 accelerate wound healing.
[0011]
As used herein, "wound" includes abrasions, lacerations, incised wounds and
bruises, and also ulcers and burns,which occur on the skin (including the intraoral skin) and other
tissues (organs). Wound sites referred to herein are not limited unless explicitly specified, and
30 application can be made on any sites including skin parts of hands, feet, faces, torsos and the
like, and intraoral sites, but is preferably on intraoral wounds since the wound healing according
to the invention are expected to be effective after even a short duration of contact. Wound
healing may also be accelerated in animals other than humans in the same manner.
[0012]
10
A polyphenol effective for wound healing is onewhich has a catechol group
(dihydroxyphenyl group) represented by the following general formula:
[Formula 5]
wherein R1-R3 are each independently selected from the group 5 consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10alkyl group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
10 a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
15 and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group. Hereinafter, the simplifiedexpression "polyphenol(s)
represented by the above general formula" may be used to refer to at least one of the polyphenol
represented by the above general formula, a polymer thereof and a pharmaceutically acceptable
20 salt thereof.
[0013]
Specific examples of the polyphenols represented by the above general formula
include caffeic acid, catechins, chlorogenic acid, quercetin, rosmarinic acid, anthocyanidins such
as cyanidin, delphinidin, aurantinidin, luteolinidin and petunidin, flavonoids such as cinchonine
25 and quercetin, and polymers thereof. The degrees of polymerization of the polymers are not
particularly limited, but polycondensates with the degrees of 2 - 17 (2-17mers) are preferable.
[0014]
Some polyphenols represented by the above general formula are compounds with
11
optical isomerism. All their optical isomers and all the mixtures of the optical isomers are
included in the polyphenols represented by the above general formula, and both the racemic
mixtures and optical isomers can be used. Optical isomers may be obtained by resolving
racemic mixtures through a known method (e.g., preferential crystallization, column
chromatography with an optically active stationary phase, 5 and a method that involves conversion
of the mixture into diastereomers).
[0015]
Among the polyphenols represented by the above general formula, caffeic acid,
(+)-catechin and chlorogenic acid, and proanthocyanidins, which are polymers of catechins, are
10 preferable. Among proanthocyanidins, oligomers of (+)-catechin, (-)-epicatechin and
epicatechingallate are preferable.
[0016]
Examples of pharmaceutically acceptable salts of the polyphenol represented by
the above general formula include Na, K, Li and ammonium salts formed with a phenolic OH;
15 and Na, K, Li and ammonium salts formed with X of the formula (I) in the case where X can
form a salt.
[0017]
Further, crude extracts comprising a compound represented by the above general
formula may also be used for the effect ofwound healing-accelerating. Examples of the crude
20 extracts include green tea extract, grape seed extract, crimson glory vine extract, blueberry
extract and coffee bean extract.
[0018]
To illustrate methods to obtain the crude extracts, an exemplary case of green tea
extract is given below. Green tea extract will be obtained by extracting green tea leaves with
25 water or an organic solvent. In the case of extraction with water, warm or hot water may be
used, the purpose of which is to increase the extraction efficiency. Organic solvents,which can
be used for the extraction, include those acceptable for manufacture of foods or agents, such as
methanol, ethanol and ethyl acetate. Further, supercritical fluid extraction may be performed
using liquefied carbon dioxide.
30 [0019]
When the above polyphenol is used for accelerating wound healing, in the actual
use,a composition comprising the above polyphenol may be used. Such a composition may
comprise additives including solvents,which are not particularly limited as long as they do not
inhibit the wound healing-accelerating effect of the polyphenol.
12
[0020]
Examples of the solvents that can be used for the composition include water, an
acidic solution, an alkaline solution and an alcohol solution, and a suitable one may be chosen
depending on the location of a wound.
5 [0021]
The concentration of the polyphenol in the composition is generally 0.0001 - 5.0
wt % of the total weight of the composition, but not limited to the range.
[0022]
Taking into consideration the wound site, as additives in the composition, for
10 example, polishing agents, foaming agents, wetting agent, sweetening agents, flavoring agents,
preservatives, various medicinal ingredients, water and ethanol may be suitably added to the
composition in therange so as not to interfere with the acceleration of wound healing. The
composition may also contain any nutritional supplement as an ingredient, selected from various
well-known nutritional supplements such as vitamins, minerals, essential and non-essential
15 amino acids, carbohydrates, lipids, foods and dietary supplements.
[0023]
Examples of the foaming agents include anionic surfactants, nonionic surfactants,
cationic surfactants and amphoteric surfactants.
[0024]
20 Examples of the anionic surfactants include salts of acylamino acids such as
sodium acylglutamate and sodium acyl sarcosinate, alkylphosphate salts such as sodium
alkylphosphate, alkyl sulfate salts, higher fatty acid sulfonatedmonoglyceridesalts, salts of fatty
acid esters of isethionic acid, sodium N-methyl-(long-chain acyl)taurates and
polyoxyethylenemonoalkyl phosphate salts.
25 [0025]
Examples of the nonionic surfactants include polyoxyalkylene-adductsurfactants,
amine oxide surfactants, mono- or diethanolamide surfactants, sorbitan esters of fatty acids,
glycerol esters of fatty acids, polyglycerol esters of fatty acids and sucrose esters of fatty acids.
[0026]
30 Examples of the wetting agents include glycerol, sorbitol, polyethylene glycol,
propylene glycol, 1,3-butylene glycol, xylitol, maltitol, lactitol and trehalose, which are suitable
for use. Erythritol is also suitable for use because of its refreshing and flavoring properties.
[0027]
Described below are methods of contacting a wound with a wound healing
13
accelerator comprising the polyphenol represented by the above general formula.
[0028]
In such a contact method, simply contacting the wound site with the wound
healing accelerator achieves a wound healing-accelerating effect. Further, the wound healing
accelerator can be directly injected. Forms of using a 5 polyphenol represented by the above
general formula are not particularly limited, but include solutions, slurries, tablets, powders,
capsules, drinkable preparations, ointments and patches. For an intraoral wound, the wound
healing accelerator can also be formulated in suitable ways for oral administration by adding
them in solutions, drymasses, dentifrices (toothpastes, liquid dentifrices and tooth gels),
10 mouthwashes, mouthrinses, fluid suspensions, topical agents, foodsupplement powders, pastes,
gels, solid foods, sealed foods, wafers, troches, chewing gums, sweets, films and the like.
[0029]
The duration of contact with the wound site is not particularly limited, but can be
short while still achieving a sufficient effect, and is preferably 0.1 seconds - 48 hours, more
15 preferably 0.5 seconds - 30 minutes, and particularly preferably 1 second - 10 minutes.
[0030]
The wound healing accelerator may be washed away with water or the like to stop
its contact with the wound site.
[0031]
20 Irradiation of the wound site with an ultraviolet laser at the wavelength of 200 -
500 nm (preferably 405 nm) during or after the contact still allows the effect of the wound
healing accelerator to maintain. The polyphenol can therefore be combined with laser irradiation
for the purpose of disinfection. Intraoral use of the wound healing acceleratormayalso be
combined with use of a toothbrush whichcan irradiate ultraviolet rays having the above
25 wavelength.
Examples
[0032]
The following examples illustrate the present invention, but do not limit the
30 invention in any way.
[0033]
Test 1: Effect of Treating with Proanthocyanidin for One Minute
Cells (mouse-derived 3T3-L1 fibroblasts purchased from DS Pharma Biomedical
Co., Ltd. and assayed within five passages), cultured in a 25 cm2 flask under conditions of 37°C
14
and 5% CO2, were treated with 0.25% Trypsin-EDTA solution (from Life Technologies Corp.),
and prepared in medium by the conventional technique to obtain a cell suspension at a
concentration of 2x104 cells/ml. For the culture, 2 ml of glutamine-containing Dulbecco's
Modified Eagle Medium (DMEM, Life Technologies Corp., Frederick, MD USA) supplemented
with bovine serum (from Life Technologies Corp.) and penicillin/5 streptomycin solution (10000
units/ml penicillin and 10 mg/ml streptomycin, fromWako Pure Chemical Industries, Ltd.) at
10% (v/v) and 1% (v/v), respectively, of the total medium was used as the growth medium.
Each 100 μl aliquot of the cell suspension was plated in each well of a 96-well
microplate (from ASAHI GLASS CO., LTD.), which had been coated with type I collagen. After
10 28 - 30 hours of culture under conditions of 37°C and 5% CO2, the medium was removed and
100 μl of proanthocyanidin solution (Leucoselect (TM), from Indeda Co. Ltd.) was added. One
1 minute later, the proanthocyanidin solution was removed, and the cells were washed with fresh
medium. Fresh medium was added again and the cells were further cultured under conditions of
37°C and 5% CO2. In order to examine the effect of light, another group was prepared from the
15 cells, to which the proanthocyanidin solution had been added, by 405 nm laser irradiation with
an output of 300 mW at an irradiation intensity of 930 mW/cm2 for 1 minute (an InGaN
semiconductor laser apparatus for experimental use, from RICOH OPTICAL INDUSTRIES
CO., LTD.). After the irradiation of this group, the medium was replaced with fresh medium,
and the cells were further cultured under conditions of 37°C and 5% CO2, in the same way as for
20 the other group. After 24 hours of culture, the extent of cell growth was determined by MTT
assay. Specifically, after 20 hours of culture, 10 μl of MTT reagent (from Trevigen Inc.) was
added to each well and the cells were further cultured for 4 hours under conditions of 37°C and
5% CO2. After the culture, 100 μl of the Detergent Reagent (Trevigen Inc.) was each added and
the cells were left in the dark overnight at room temperature. The absorbance at 595nm was then
25 determined using a microplate reader (FilterMax (TM) F5 Multi-Mode Microplate Reader,
Molecular Devices, LLC.). Another group, treated with sterile physiological saline, served as the
control group, and its absorbance was set at 100% so that the absorbance of each treatment group
was calculated as a proportion (%). The results are shown in Fig. 1. The absorbance of the
control group in Test 1 was 0.289±0.016.
30 [0034]
Test 2: Effect of Treating with Different Polyphenols for One Minute
Gallic acid (from Tokyo Chemical Industry Co., Ltd.), caffeic acid (from Tokyo
Chemical Industry Co., Ltd.), (+)-catechin (from Tokyo Chemical Industry Co., Ltd.) and
chlorogenic acid (from Sigma-Aldrich Co. LLC.) were assayed. Similarly to the above Test 1,
15
each 100 μl aliquot of the cell suspension was plated in each well of a 96-well microplate, which
had been coated with type I collagen. After 27 - 28 hours of culture under conditions of 37°C
and 5% CO2, the medium was removed and 100 μl of a solution of each test substance was
added. One minute later, the solution of the test substance was removed, and the cells were
washed with fresh medium. Fresh medium was added again and the 5 cells were further cultured
under conditions of 37°C and 5% CO2. After 24 hours of culture, the extent of cell growth was
determined by MTT assay. The results are shown in Fig. 2. The absorbance of the control group
in Test 2 was 0.196±0.011.
[0035]
10 Test 3: Effect of Treating with Catechin for 24 Hours
(+)-Catechin was assayed. As in Test 1, each 100 μl aliquot of the cell suspension
was plated in each well of a 96-well microplate, which had been coated with type I collagen.
After 24 hours of culture under conditions of 37°C and 5% CO2, the medium was replaced with
medium containing the test substance, and the cells were cultured for 24 hours under conditions
15 of 37°C and 5% CO2. The medium containing the test substance was replaced with fresh
medium. 10 μl of MTT reagent (Trevigen Inc.) was added to each well and the cells were further
cultured for 4 hours under conditions of 37°C and 5% CO2. An aliquot (100 μl) of the Detergent
Reagent (Trevigen Inc.) was then added and the extent of cell growth was determined as in other
Tests. The results are shown in Fig. 3. The absorbance of the control group in Test 3 was
20 0.163±0.014.
[0036]
Test 4: Effect of Treating with Green Tea Extract for One Minute
Green tea extract (from MARUZEN PHARMACEUTICALS CO., LTD.) was
assayed. Similarly to the above Test 1, each 100 μl aliquot of the cell suspension was plated in
25 each well of a 96-well microplate, which had been coated with type I collagen. After 28 hours of
culture under conditions of 37°C and 5% CO2, the medium was removed and 100 μl of a solution
of the test substance was added. One minute later, the solution of the test substance was
removed, and the cells were washed with fresh medium. Fresh medium was added again and the
cells were further cultured under conditions of 37°C and 5% CO2. After 24 hours of culture, the
30 extent of cell growth was determined by MTT assay. The results are shown in Fig. 4. The
absorbance of the control group in Test 4 was 0.276±0.010.
[0037]
Proanthocyanidin, caffeic acid, (+)-catechin, chlorogenic acid and green tea
extract used in the tests had been dissolved in physiological saline and sterilized by filtration
16
through a membrane filter (diameter: 0.22 μm).
[0038]
As can alsobe seen from the above results, the growth of fibroblasts was observed
when the catechol group-containing polyphenols represented by the above general formula were
used, and their wound healing-accelerating effect was confirmed. 5 Further, it was also observed
that the effect was retained when combined with the use of the laser. The effect on the growth of
granulation was also confirmed whenthe duration of contact was 1 minute - 24 hours. On the
other hand, no growth of fibroblasts was observed for gallic acid, which is a polyphenol but has a
pyrogallol group.
10 [0039]
Test 5: Effect of Treating Human Gingival Fibroblasts with Proanthocyanidin for One Minute
Cells (human gingival fibroblasts purchased from Primary Cell Co., Ltd. and
assayed within five passages), cultured in a 25 cm2 flask under conditions of 37°C and 5% CO2,
were treated with 0.25% Trypsin-EDTA solution (from Life Technologies Corp.), and prepared in
15 medium by conventional technique to obtain a cell suspension at a concentration of 2x104
cells/ml. For the culture, 2 ml of glutamine-containing Dulbecco's Modified Eagle Medium
(DMEM, Life Technologies Corp., Frederick, MD USA) supplemented with bovine serum (from
Life Technologies Corp.) and penicillin/streptomycin solution (10000 units/ml penicillin and 10
mg/ml streptomycin, fromWako Pure Chemical Industries, Ltd.) at 10% (v/v) and 1% (v/v),
20 respectively, of the total medium was used as the growth medium.
Each 100 μl aliquot of the cell suspension was plated in each well of a 96-well
microplate (Corning Corp., NY, USA). After 24 hours of culture under conditions of 37°C and
5% CO2, the medium was removed and 100 μl of a solution of proanthocyanidin (Leucoselect
(TM), from Indeda Co. Ltd.) in sterile physiological saline was added. One minute later, the
25 proanthocyanidin solution was removed, and the cells were washed with fresh medium. Fresh
medium was added again and the cells were further cultured under conditions of 37°C and 5%
CO2. After 24 hours of culture, the extent of cell growth was determined by MTT assay.
Another group, treated with sterile physiological saline, served as the control group, and its
absorbance was set to 100% so that the absorbance of each treatment group was calculated as a
30 proportion (%). The results are shown in Fig. 5. The absorbance of the control group in Test 5
was 0.073±0.007.
[0040]
Test 6: Time-Course of Growth of Proanthocyanidin-Treated Human Gingival Fibroblasts
Similarly tothe above Test 5, each 100 μl aliquot of the cell suspension was plated
17
in each well of a 96-well microplate. After 24 hours of culture under conditions of 37°C and 5%
CO2, the medium was removed and 100 μl of a solution of 0.25 mg/ml or 1
mg/mlproanthocyanidin in sterile physiological saline was added. One minute later, the
proanthocyanidin solution was removed, and the cells were washed with fresh medium. Fresh
medium was added again and the cells were further 5 cultured under conditions of 37°C and 5%
CO2. The extent of cell growth was determined by MTT assay over time. The control group was
treated with sterile physiological saline in the same way. The results are shown in Fig. 6. The
absorbance of the control group at time 0 in Test 6 was 0.088±0.013.
[0041]
10 Test 7: Cytoprotective Effect of Proanthocyanidin on Osmotic Stress
Osmotic stress was applied to cells by exposing them to pure water for defined
durations of time, and the effect of proanthocyanidin on the cell were examined. Similarly to the
above Test 5, each 100 μl aliquot of the cell suspension was plated in each well of a 96-well
microplate. After 15 - 16 hours of culture under conditions of 37°C and 5% CO2, the medium
15 was removed and 100 μl of sterile pure water or of a solution of 1 mg/ml proanthocyanidin in
sterile pure water was added. One, four or sixteen minutes later, the pure water or the solution of
proanthocyanidin in pure water was removed, and the cells were washed with fresh medium.
Fresh medium was added again and the cells were further cultured under conditions of 37°C and
5% CO2. After 24 hours of culture, the extent of cell growth was determined by MTT assay.
20 Another group, cultured for 40 hours without treatment (treated only with medium), served as
the control group. The results are shown in Fig. 7. The absorbance of the control group (without
treatment) in Test 7 was 0.228±0.030.
[0042]
Test 8: Cytoprotective Effect of Proanthocyanidin on Starvation Stress
25 Starvation stress was applied to cells by exposing them to sterile physiological
saline for 1, 2 and 3 hours, and the effect of proanthocyanidin on the cell were examined.
Similarly to the above Test 5, each 100 μl aliquot of the cell suspension was plated in each well
of a 96-well microplate. After 19 hours of culture under conditions of 37°C and 5% CO2, the
medium was removed and 100 μl of sterile physiological saline or of a solution of 1 mg/ml
30 proanthocyanidin in sterile physiological saline was added. One, two or three hourslater, the
sterile physiological saline or the solution of proanthocyanidin in sterile physiological saline was
removed, and the cells were washed with fresh medium. Fresh medium was added again and the
cells were further cultured under conditions of 37°C and 5% CO2. After 22 - 24 hours of culture,
the extent of cell growth was determined by MTT assay. Another group, cultured for 44 hours
18
without treatment (treated only with medium), served as the control group. The absorbance of
the control group (without treatment) in Test 8 was 0.137±0.004. The results are shown in Fig.
8.
[0043]
Test 9: Cytoprotective Effect of Proanthocyanidin on Cytotoxicity 5 of Bacterial Endotoxin
(Lipopolysaccharide, LPS)
Similarly to the above Test 5, each 100 μl aliquot of the cell suspension was
plated in each well of a 96-well microplate. After 20 hours of culture under conditions of 37°C
and 5% CO2, the medium was removed and 100 μl of a solution of 1 mg/ml proanthocyanidin in
10 sterile physiological saline was added. The proanthocyanidin solution was removed after 1
minute and the cells were washed with fresh medium. Medium containing Escherichia coliderived
lipopolysaccharide (LPS,Wako Pure Chemical Industries, Ltd.) at a concentration of 2.5
μg/ml was added and the cells were further cultured under conditions of 37°C and 5% CO2.
After 24 hours of culture, the extent of cell growth was determined by MTT assay. Another
15 group, cultured for 44 hours without treatment (treated only with medium), served as the control
group. The absorbance of the control group in Test 9 was 0.109±0.006. Each sample was
analyzed by evaluating the significance of its difference from the mean value of the control
group, using Dunnett's multiple comparison test. The results are shown in Fig. 9.
[0044]
20 As can be seen from the above results, similarly to mouse fibroblasts, only
treating human gingival fibroblasts with proanthocyanidin for one minute also accelerated cell
growth in a concentration-dependent manner. Further, the time-course of cell growth shows that
the growth was significantly accelerated when compared to the control group at 6 hours after the
treatment. Proanthocyanidin, in the testof dissolving it in pure waterto evaluate the effect of
25 treatment time,suppressively acted against both the reduction of cell growth by the pure water
treatment and of the cells treated by the saline substitution. It was therefore suggested that the
polyphenols represented by the above general formula, including proanthocyanidin, also have the
cytoprotective effect for fibroblasts, in addition to the growth-accelerating effect therefor.
Furthermore, proanthocyanidin not only exerted the protective effect on the cytotoxicity of
30 bacterial endotoxin, but also accelerated cell growth. Thus, it was suggested that
proanthocyanidin exerts a wound healing-accelerating effect through the protective and growthaccelerating
actions on cells also at the inflammatory damage due to bacterial infection.
Industrial Applicability
19
[0045]
The polyphenols represented by the above general formula can, due to their effect,
be used as a medical agent, and are therefore useful in medicine, particularly in the fields of skin
therapy and therapy in the oral cavity.
5
20
We Claim:
[Claim 1]
A pharmaceutical composition for wound healing, comprising at least one of a
polyphenol represented by the following general formula:
[Formula 1]
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
salt thereof.
[Claim 2]
The pharmaceutical composition for wound healing according to claim 1, wherein
both R2 and R3 are a hydrogen atom.
[Claim 3]
The pharmaceutical composition for wound healing according to claim 1 or 2,
wherein R1 is a hydrogen atom.
[Claim 4]
The pharmaceutical composition for wound healing according to any one of
21
claims 1 to 3, wherein the polyphenol is selected from the group consisting of caffeic acid, (+)-
catechinic acid and chlorogenic acid.
[Claim 5]
An oral care composition, comprising at least one of a polyphenol represented by
the following general formula:
[Formula 2]
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
salt thereof.
[Claim 6]
The oral care composition according to claim 5, wherein both R2 and R3 are a
hydrogen atom.
[Claim 7]
The oral care composition according to claim 5 or 6, wherein R1 is a hydrogen
atom.
22
[Claim 8]
The oral care composition according to any one of claims 5 to 7, wherein the
polyphenol is selected from the group consisting of caffeic acid, (+)-catechinic acid and
chlorogenic acid.
[Claim 9]
The oral care composition according to claims 5 to 8, wherein the composition is
in the form of a mouthrinse, a dentifrice, a sweet, a medicament or a film.
[Claim 10]
Amethod for accelerating wound healing, comprising the step of contacting a
wound with at least one of a polyphenol represented by the following general formula:
[Formula 3]
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
salt thereof.
[Claim 11]
The method according to claim 10, wherein both R2 and R3 are a hydrogen atom.
23
[Claim 12]
The method according to claim 10 or 11, wherein R1 is a hydrogen atom.
[Claim 13]
The method according to any one of claims 10 to 12, wherein the polyphenol is
selected from the group consisting of caffeic acid, (+)-catechinic acid and chlorogenic acid.
[Claim 14]
The method according to any one of claims 10 to 13, wherein a duration of the
contact is 0.01 seconds to 48 hours.
[Claim 15]
The method according to claim 14, wherein the duration of the contact is 1 second
to 10 minutes.
[Claim 16]
The method according to any one of claims 10 to 15, further comprising the step
of washing a contact site after the contact.
[Claim 17]
The method according to any one of claims 10 to 16, wherein the wound is in an
oral cavity.
[Claim 18]
Use of at least one of a polyphenol represented by the following general formula:
[Formula 4]
wherein R1-R3 are each independently selected from the group consisting of a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a
heteroaryloxy group, a carbonyl group, an amino group, a C1-10alkylamino group, a sulfonamide
group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl group, a C3-12cycloalkyl
group, a C3-12heterocycloalkyl group, a C9-12bicycloalkyl group, a C3-12heterobicycloalkyl group,
a C1-10arylalkyl group, a C1-5heteroarylalkyl group, a C1-10perhaloalkyl group, a C1-
3carbonylalkyl group, a C1-3thiocarbonylalkyl group, a C1-3sulfonylalkyl group, a C1-
24
3sulfinylalkyl group, a C1-10aminoalkyl group, a C1-3iminoalkyl group, an aryl group, a heteroaryl
group, a C9-12bicycloaryl group and a C4-12heterobicycloaryl group, each of which is substituted
or unsubstituted, or any two of R1, R2 and R3 together form a substituted or unsubstituted ring:
and X represents a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a substituted
or unsubstituted C3-20 aromatic hydrocarbon group or a substituted or unsubstituted C3-20
polycyclic aromatic hydrocarbon group, a polymer thereof and a pharmaceutically acceptable
salt thereof for manufacturing a wound healing accelerator.
Dated this 22nd day of June, 2015.
25
ABSTRACT
WOUND HEALINGACCELERATOR
The purpose of the present invention is to provide a specific wound healing accelerator effective
for accelerating healing of wounds, particularly of wounds in the oral cavity, or a composition
which includes the wound healing accelerator. This wound healing accelerator including a
specific polyphenol is easy to handle and is capable of accelerating would healing even by
contact for a short time.