JP2003504402A - Procyanidin oligomers that inhibit matrix metalloproteases and drugs containing them as active ingredients - Google Patents

Abstract

(57) SUMMARY The present invention provides procyanidin oligomers having significant biological activity against matrix metalloproteases (MMPs). The procyanidin oligomer may be extracted from the genus Elm and other plants, and may include trimer to dodecameric flavan-3-ol monomer procyanidin oligomer. The present invention relates to cancer metastasis, rheumatoid arthritis, diabetes, corneal ulcer, epithelial ulcer, gastric ulcer, skin wrinkles and aging, periodontitis, osteoporosis in which uncontrolled high levels of MMPs may play an important role in disease progression Use of procyanidin oligomers to treat trauma, burns and other diseases.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a procyanidin oligomer (Procyanidin Oligomer), and particularly to inhibiting the activity of matrix metalloproteinase (MMP) that decomposes extracellular matrix and basement membrane of connective tissue. It relates to procyanidin oligomers. Such procyanidin oligomers can be obtained from the elm cortex and other plants of the genus Ulmus and range from trimers to 12-mers composed of flavan-3-ol basic units. Such procyanidin oligomers include cancer metastasis, periodontal disease, rheumatoid arthritis, diabetes, corneal ulcer, epithelial ulcer, gastric ulcer, skin wrinkles and aging, periodontitis, osteoporosis, trauma, burns, and MMP.
Can be used as a drug for preventing and treating related diseases that play an important role.

PRIOR ART Matrix metalloproteases (MMPs) are calcium- and zinc-dependent endopeptidases secreted by cells such as polymorphonuclear neutrophils, macrophages, fibroblasts and osteocytes. And acts at neutral pH and utilizes a variety of extracellular matrices as its matrix. Such matrix metalloproteases are not only found in numerous physiological processes such as embryogenesis, tissue formation, salivary gland formation, and tooth development, but also wounds, cancer metastasis, periodontal disease, rheumatoid arthritis, inflammation, diabetes, cornea. Ulcer,
It is also known to be involved in pathological processes and various diseases such as osteoporosis, gastric ulcer, trauma, skin wrinkles and aging, burns and wound healing.

In particular, the type IV collagenase MMPs (MMP2 and 9), 72-kD and 92-kD collagenase, are enzymes that degrade type IV collagen, which is a major component of basement membrane, the first barrier to cancer metastasis. Therefore, it is the most important enzyme in cancer cell invasion and metastasis. In periodontal disease, MMP decomposes collagen, which is a matrix of periodontal tissue, by collagenase secreted by fibroblasts and polymorphonuclear leukocytes, epithelial cells and macrophages, and collagenase secreted by periodontal bacteria. Gum retraction occurs, and if left unattended, it progresses to periodontal disease. Recently, it was also found that MMPs are very closely involved in skin aging, light-induced skin aging, and wrinkle formation (Br. J. Dermatol., 2000; 142, 267-273, Arch. Derm.
atol. Res., 2000: 292, 27-31, Free Radical Biol. Med., 1999; 27, 729-73.
7).

Therefore, collagenase inhibitors are associated with periodontal disease, rheumatoid arthritis, inflammation, skin wrinkles and aging, diabetes, corneal ulcers, skin ulcers caused by cancer invasion and metastasis, and collagenous connective tissue degradation. And functions as a useful drug for the prevention and treatment of diseases such as gastric ulcer, osteoporosis, trauma and burns.

To date, there are tetracycline compounds (tetracycline, minocycline, doxycycline) and peptide derivatives as agents capable of inhibiting the activity of MMP.

Among them, peptide derivatives are similar to collagen, and are capable of chelating zinc ions in the active site of collagenase enzyme, hydroxamic acid, thiol, and carboxylalkyl (ca).
Active research on enzyme inhibitors containing rboxylalkyl) groups (Pharmacol. Ther., 1997
75, 69-75, U.S. Pat. Nos. 4,996,358, 5,183,900, 5,300,674, 5,
861,436 etc.). For example, in US Pat. No. 5,514,677, hydroxamic acid is used to inhibit rheumatoid arthritis, inflammation, skin disorders by collagenase inhibition,
It has been disclosed that osteoporosis, cancer metastasis and wounds can be treated. Also,
U.S. Pat. No. 4,666,897 discloses that tetracycline, minocycline, doxycycline can be used to inhibit excess collagenase activity.

Procyanidins represented by the following chemical formula 1 are catechin (Catechin), epicatechin (Epicatechin), catechin gallate (Catechin gallate), epicatechin gallate (Epicatechin gallate), gallocatechin gallate (Gallocatechin gallate).
ate) or epigallocatechin gallate (Epigallocatechin gallate) as a basic skeleton (Backbone), which is a general name for oligomers and polymers of dimers or higher, and is a non-hydrolyzed substance contained in a wide range of plants. It is sex tannin. It is known that such procyanidins have the ability to bind to proteins, and it has been reported that dimers have an anti-inflammatory effect. In particular, catechin known as green tea tannin is reported to have an excellent antitumor effect.

[0008]

[Chemical 1] In the above formula, when the procyanidin is catechin, R ¹ is OH, R ² and R ³ are H, and when it is epicatechin, R ¹ is H, R ² is OH, and R ³ is H,
In the case of epicatechin-3-O-gallate, R ¹ is H and R ² is O-galloyl (O-ga
lloyl), R ³ is H, R ¹ is H in the case of epigallocatechin, R ² and R ³ are OH, and R ¹ is H and R ² is O in the case of epigallocatechin-3-O-gallate. −
Galloyl, R ³ is OH. Also, the polymer is formed through bonds (4-8 or 4-6) between the monomers.

Techniques relating to the antioxidative effect, antiviral effect, and antibacterial effect of procyanidins have been disclosed (US Pat. Nos. 5,494,661 and 5,877,206).
No. 5, 646, 178, No. 4, 797, 421, International Patent Publication No. 9
3/24106 etc.). In addition, medicinal products using procyanidins (European Patent Publication No. 812592, Japanese Unexamined Patent Publication No. 61-83958, etc.), cosmetics using procyanidins (European Patent Publication No. 694305).
No. JP-A-63-36420), food additives using procyanidin (JP-A-10-004923, JP-A-7-49333, etc.) and procyanidin production method (JP-A-63). -267774, South African Published Patent Publication No. 8205023, etc.) are also disclosed.

In addition, various research results have been reported regarding the inhibition of matrix metalloproteinases by the green tea component. For example, epicatechin gallate and epigallocatechin gallate, which are green tea components, inhibit the activity of microorganisms and collagenase in gingival crevicular fluid (J. Periodontal., 1993; 64, 630-636), and epicatechin gallate, epicatechin gallate Garocatechin gallate and theaflavin inhibit the penetration of cancer cell lines into the gelatin membrane (J. Agric. Food. Chem. 1999; 47, 2350-2354), and green tea epicatechin gallate and epigallocatechin The effect of gallate on MMP-2, 9 and 12 was shown (Biochem. Biophys. Acta, 2000; 1478, 51-60), and catechins and theaflavins derived from green tea and black tea were found to inhibit the lung cancer cell line MMP2, 9. Inhibitory activity was shown (Biosci. Biotech Biochem., 1997; 61, 1504-1506). However, these publications are all related to catechins and catechin gallate derivatives having a small molecular weight, and inhibit the MMP enzyme inhibition of procyanidin oligomer mixture in which 3 to 12 flavan-3-ol monomers of the present invention are polymerically linked. Not known about.

On the other hand, the elm skin layer (Ulmus cortex) is Ulmus macrocarp of the genus Elm.
a, Pumila, Davidiana, Americana roots and trunk skins, which have been traditionally used for inflammation and gastric ulcers. Recently, it was reported in U.S. Pat. No. 6,045,800 that the elm skin layer has excellent inhibitory activity against periodontitis-related collagenase and other results of evaluation of inflammation inhibition (J. Ethanopharm., 1998;
62, 129-135).

As a result of research to clarify which component of elm skin layer component is excellent in collagenase inhibitory ability, the present inventors have revealed that procyanidin oligomer is the main component.

Therefore, the present invention aims to provide procyanidin oligomers that inhibit the activity of matrix metalloproteases (MMPs).

Another object of the present invention is to provide a pharmaceutical composition containing procyanidin oligomer, which is a safe and natural ingredient, which is superior to conventional synthetic MMP enzyme inhibitors such as doxycycline and natural epigallocatechin gallate. is there.

The invention and its advantages will be more apparent from the following detailed description of the invention and the accompanying drawings.

In order to achieve the above object, the present invention provides matrix metalloprotease (MM).
A procyanidin oligomer that inhibits the activity of P) is provided.

The present invention also provides a drug for preventing and / or treating a disease caused by the activity of matrix metalloprotease (MMP), which comprises the procyanidin oligomer as an active ingredient.

[0018]   Hereinafter, the present invention will be described in detail.

The present inventors sought to search for an MMP activity inhibitor that is safe for the human body but is highly effective, in order to prevent injury, cancer metastasis, periodontal disease, rheumatoid arthritis, inflammation, corneal ulcer,
The active ingredients of the elm skin layer (US Pat. No. 6,045,800) traditionally known to be effective against osteoporosis, gastric ulcer, trauma, wrinkles, acne, burns, etc. were investigated. As a result, a procyanidin oligomer mixture in which 3 to 12 flavan-3-ol basic units are polymerized and linked is the main active ingredient of the elm skin layer, and its inhibitory activity against matrix metalloprotease is conventional doxycycline and epigallocatechin gallate. After confirming that it excels, the present invention has been completed.

The procyanidin oligomer is preferably used as an MMP activity inhibitor. The MMPs are collagen-degrading proteases (derived from Kamchatka crab, manufactured by Sigma) MMP-1, MMP-8, and MMPs that are type IV collagenases.
-2 and MMP-9.

The product form of the procyanidin oligomer is preferably tablets, capsules, powders, ointments, solutions, gels, pastes, patches, granules and the like. The content of the procyanidin oligomer in the preparation is preferably 0.0001 to 5% by weight.

The procyanidin is separated from the n-butanol fraction obtained when the primary extract extracted from the elm skin layer of Elm genus with a polar solvent is subjected to solvent fractionation with n-hexane, dichloromethane, ethyl acetate and n-butanol. . When this Sephadex (registered trademark) LH-20 (Sephadex LH-20) column chromatography is performed on this n-butanol fraction, the procyanidin oligomer is separated so as to be concentrated. For example, when water-methanol is used as a mixed solvent to elute in an increasing order of methanol, the eluent of methanol is 80% to 100%, and the other method is to elute various fractions with 100% methanol successively, and thin layer chromatography is performed. Through confirmation, the procyanidin oligomer is recombined so as to be concentrated and fractionated.

The procyanidin oligomer is a mixture of trimer and dodecer in which 3 to 12 flavan-3-ol basic units are linked, and has an average molecular weight of 1,
518, an average degree of polymerization of 5.3, and in addition to the elm skin layer, the grape seeds that contain a lot of the same components as the elm skin layer, Daihoku, Heisei crow, Kusu, cinnamon, side Kashiwa, camellia fruit, It can be extracted more than the group consisting of Koryan, buckwheat, and Kashiwa.

The present invention will be described in more detail through the following experimental examples, examples and comparative examples. However, the examples are for the purpose of illustrating the present invention and are not limited to these.

(Example) Experimental Example 1 (Separation of procyanidin oligomer as an active ingredient and identification of its structure) A procyanidin oligomer was separated from an elm skin layer extract and its structure was identified. However, procyanidins are generally known to be abundant in plants, so they are also extracted from plants such as grape seeds, rhubarb, Heishu, Kusu, cinnamon, side oysters, camellia seeds, kollyan, buckwheat, and kashiwa. , Can be used after purification.

Experimental Example 1-1 (Preparation of crude extract and inhibition of collagenase activity) The elm skin layer was crushed to a size of 10 to 200 mesh, the extraction solvent was added to the crushed plant powder, and cold soaked at room temperature for 72 hours. After filtering, the filtered stock solution was concentrated to obtain a primary extract. The extraction solvent usable at this time was selected from the group consisting of purified water, methanol, ethanol, propanol, butanol, glycerol, ethylene glycol, propylene glycol, 1,3-butylene glycol, ethyl acetate and acetone. It is a mixed solvent of one kind or two or more kinds.

The primary extract obtained here was suspended in water and then subjected to sequential solvent fractionation using n-hexane, dichloromethane, ethyl acetate and n-butanol to obtain respective fractions. The filtrate remaining after the solvent fractionation was used as the water fraction. Using the five solvent fractions obtained by the solvent fractionation, an activity inhibition experiment of collagenase, which is a type of MMP, was carried out.

As a result, no enzyme activity inhibitory effect appeared in the n-hexane fraction and the dichloromethane fraction, but an activity inhibitory effect appeared in the ethyl acetate fraction and n-butanol fraction.

The following experiment was conducted in order to clarify the active ingredients of the ethyl acetate fraction and the n-butanol fraction in which the enzyme activity-inhibiting effect appeared. Silica gel column chromatography was performed using the obtained ethyl acetate fraction as a sample.

Chloroform, ethyl acetate, acetone and methanol were sequentially used as elution solvents to obtain silica chloroform fraction, silica ethyl acetate fraction, silica acetone fraction and silica methanol fraction, respectively. As a result of conducting an activity inhibitory effect experiment on collagenase of this fraction, an activity inhibitory effect appeared in the silica ethyl acetate (hereinafter referred to as silica ethyl acetate) fraction, but it was not superior to elm skin primary extract and doxycycline. (See Experimental Example 2 below).

The silica ethyl acetate fraction was analyzed by high performance liquid chromatography / electrospray ionization mass spectrometry (Finnigan LCQ) and thin layer chromatography to obtain the results shown in FIG. 1, and the procyanidin monomer having a small molecular weight and two kinds were analyzed. It was found that there are unidentified substances of procyanidin dimer.

As a result of conducting a collagenase activity inhibition experiment with respect to each component, similar to the result of the silica ethyl acetate fraction, the effect is not so good as compared with doxycycline and elm skin primary extract conventionally used as drugs. Not shown.

Meanwhile, Sephadex (registered trademark) LH-20 column chromatography was performed using the n-butanol fraction obtained through the solvent fractionation of the primary extract.
They were named fractions 1, 2, 3, 4 after recombining by sequential elution with 100% by weight methanol, fractionation and thin layer chromatography.

In another method, 20% by weight methanol, 50% by weight methanol, 80% by weight methanol and 100% by weight methanol were used as an elution solvent to obtain 20% methanol fraction, 50% methanol fraction, 80% methanol fraction, respectively. And a 100% methanol fraction were obtained.

The fractions 1, 2, 3, 4 and the methanol fractions obtained here were subjected to a collagenase activity inhibition experiment. As a result, the fractions 1, 2, 3 and 20 wt% and 50 wt% methanol fractions were analyzed. No effect was seen in the above, but fraction 4, 80% by weight methanol fraction and 100
The effect appeared in the wt% methanol fraction. In particular, the effects were outstanding in the fraction 4 and the 100% by weight methanol fraction, and the enzyme inhibitory activity was higher than that of the conventional drugs doxycycline and epigallocatechin gallate.

When a 10% FeCl ₃ solution was added to the fraction 4 and the 100% by weight methanol fraction obtained here, it was confirmed that procyanidins were present in view of the change in dark blue color. In the following, reference will be made to procyanidin oligomers.

Experimental Example 1-2 (Structural analysis of fraction 4: matrix-assisted laser desorption / ionization-time of flight (M
ALDI-TOF mass spectrometer) PE Biosystems Voyager System 4
(95% (Perkin Elmer)) was used to measure the mass ([M + sodium] ⁺ ) of sodium added to the procyanidin oligomer molecular weight as cyano-4-hydroxycinnamic acid (cyan
o-4hydroxycinnamic acid) was used as a matrix for detection.

FIG. 2 shows the results of the MALDI-TOF mass spectrometer of Fraction 4, which was 889 (m / m).
z) From the fact that the mass increases from the ion peak by about 288,
It can be inferred that fraction 4 is a procyanidin oligomer in which catechin or epicatechin monomers are linked by a single bond. Ion 889m / z here
The peak is a trimer in which a sodium mass of 23 is added to a molecular weight of 866. The remaining peaks are also calculated in this way and are found to correspond to various oligomers polymerized from 3 to 12 procyanidin monomers. If it is a dimer,
From the fact that the expected peak of 601 m / z could not be detected, it can be seen that the trimer to the 12-mer are distributed in the fraction 4 having an excellent effect.

This result is very similar to the procyanidin distribution contained in cocoa and chocolate (J. Agric. Food Chem., 1999, 47, 490-496, US Pat. No. 5,877,20.
6). This mass spectrometric method does not generate procyanidin oligomer molecules as fragments and does not generate multiple charges, so it can be used to calculate the degree of molecular distribution and degree of polymerization (Phytochemist
ry 2000; 54: 173-181, and Rapid Comm. Mass Spectrometry 1997; 11: 31-36.
). Fraction 4 was analyzed at peak height ratios and respective molecular weights and the results are set forth in Table 1 below, with an average molecular weight of 1,518 and an average degree of polymerization of 5.3. Do you get it.

[0040]

[Table 1] Experimental Example 1-3 (Structural analysis of fraction 4: high performance liquid chromatography / electrospray ionization mass spectrometry, Finnigan LCQ) Obtaining structural information complementary to the matrix-assisted laser desorption / ionization-time of flight mass spectrometer analysis results Therefore, it is used to measure the molecular weight of biological materials such as procyanidin oligomers, which have poor ionization (Phytochemistry 1997.
44: 351-357, and J. Agric. Food Chem., 1999: 47: 3693-3701), high performance liquid chromatography / electrospray ionization mass spectrometry capable of generating multiple charges was performed.

[0041]   For chromatography, HP1100 series HPLC (Hewlett-Packard,
 Palo Alto, USA) was used. Column is silica (ZoRBOX Sil, 4.6 × 250mm, 5
μm) and solvent conditions are solvent 1 (CH₂Cl₂: CH₃OH: H₂O: CH₃COOH = 82: 14: 2: 2) and solvent 2
(CH₃OH: H₂O: CH₃COOH = 96: 2: 2) and the ratio of 12:88 after 50 minutes from the initial 100: 0.
The flow rate was 1 m / min, determined to be the rate. Procyanidin oligomer
280 nm absorption value was detected with a DAD detector. Connected to the DAD detector
Matrix-assisted laser desorption / ionization results using a mass spectrometer-in flight
The results were compared with the results obtained by the intermass mass spectrometer. In selective mass tracking mode, procyan
Hydrogen excluded from gin oligomer molecular weight ([M-H])^- ) Mass and trifluoro
One molecule of acetic acid (TFA) was added to one molecule of procyanidin oligomer ([M +
TFA^- ]^- ) Mass and charge number of 2 are expected [M-H + TFA^- ]^-2And [M + 2TFA] ^-2 The mass peak of was used. For example, as shown in Fig. 3, the pentamer is ([M-H]^- )mass
1441 and ([M + TFA^- ]^- ) Specify 1555, which is the mass, and track it.
In the same manner as above, the masses were traced from the trimer to the hexamer. Twelve to seven
The number of charges up to 1 is not 1 but 2 (J. Agric. Food Chem., 1999, 47, 490-496)
Therefore, the corresponding mass, for example, the heptamer is [M-H + TFA^- ]^-2Is 1
065 peak and [M + 2TFA]^-2And trace the 1122 peak, which is
Tracked from body to dodecamer.

As shown in FIG. 4, in the active fraction 4, predicted procyanidin oligomer peaks were detected from the trimer to the dimer, and as the degree of polymerization increased from 3, the distribution of the ions having the predicted mass was determined. It was found that the chromatogram of screen 4 moved from the initial part to the latter half.

Table 2 below shows the expected procyanidin oligomer ion mass of fraction 4 and the type of ions detected by the electrospray ionization mass spectrometer, blank means that none were detected.

[0044]

[Table 2] FIG. 5 shows the results of detection of peaks in scan mode in the retention time zone on the chromatogram near the designated mass peak using the HPLC / ESI mass spectrometer, and shows the mass peaks estimated to be procyanidin oligomers, for example, tetramer. If there is 1153 (
[MH] ^- ) and 1267 ([M + TFA ^- ] ^- ), 1441 ([M-
H] ^- ) and 1555 ([M + TFA ^- ] ^- ) are the main mass peaks, indicating that the main component of Fraction 4 is procyanidin oligomer.

When the results of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and high-performance liquid chromatography / electrospray ionization mass spectrometry of Fraction 4 are combined, the components of Fraction 4 having excellent MMP inhibitory activity are Flavan-3-ol monomer 3 to 1
It is a procyanidin oligomer mixture in which up to 2 are linked by a single bond, and has a molecular weight of 1,518 and an average degree of polymerization of 5.3.

Experimental Example 1-4 (Analysis of constituent monomers of procyanidin oligomer) In order to analyze constituent monomers of procyanidin oligomer of fraction 4, phloroglucinol acidolysis (J. Chromatogr., J. Chromatogr. 1992; 59
4, 117-123).

Phloroglucinol acidolysis means that when a polymer and phloroglucinol are reacted under acidic conditions, phloroglucinol causes an addition reaction while cutting bonds between monomers in the polymer. , A reaction producing a monomer-phloroglucinol compound as the reaction product. After reacting phloroglucinol with fraction 4 under acidic conditions, the resulting reaction products were separated and compared using high performance liquid chromatography, and as a result, epicatechin was present outside the peaks presumed to be catechin-phloroglucinol. , Catechin gallate, epicatechin gallate, gallocatechin gallate, or phloroglucinol derivative of epigallocatechin gallate was not detected. The putative catechin-phloroglucinol purified here was analyzed by an ESI mass spectrometer and found to have a molecular weight of 414 from the predicted mass peak.

Carbon and hydrogen nuclear magnetic resonance spectroscopy ( ¹³ C, ¹ H NMR) was used to distinguish between catechin and epicatechin. Chemical shift, that is, the chemical shift of carbon number 2 is different between catechin and epicatechin depending on the orientation of phloroglucinol linked to carbon number 4 in the heterocycle (J. Chem. Soc. Perkin, Trans., L 1980: 2278
-2286, J. Chem. Soc. Perkin, Trans., L 1983; 1535 to 1543, and JCSPerk.
in 1982, 1217-1221), the chemical shift of carbon number 2 of the phloroglucinol derivative was 83.4 ppm, and it was found to be catechin-4-phloroglucinol.

Therefore, it was found that the first monomer of the procyanidin oligomer of the present invention was catechin.

Comparing the results of the MALDI-TOF mass spectrometer, HPLC / ESI mass spectrometry and nuclear magnetic resonance spectroscopy, the component of Fraction 4, which is excellent in MMP inhibitory power, is the silica ethyl acetate fraction, which has a weak effect. Unlike the above, it is a procyanidin oligomer in which flavan-3-ol monomers are linked by 3 to 12 single-bonds, and its average molecular weight is 1,518 and average polymerization degree is 5.3.

Experimental Example 2 (Evaluation of inhibitory effect of procyanidin oligomer fraction on MMP activity using azochol, a red collagen matrix (Anal. Biochem., 1984, 136;
446-450) Collagen-degrading proteolytic enzyme (from Kamchatka crab, Sigma), MM
P-1 (fibroblast collagenase), MMP-8 (polymorphonuclear leukocyte collagenase)
The inhibitory effect of the procyanidin oligomer fraction was measured by the following method.

To a 1.5 ml Eppendorf tube, 100 μl of an azochol solution, which is a 2% red collagen matrix, was added. One Eppendorf tube was used as a blank, and three tubes were each filled with 10 collagen standard degrading enzyme standard solutions purchased from Sigma in order to prepare a standard activity curve of the enzyme.
, 100, 200 ppm. In each tube, 100 μl of collagen degrading enzyme standard solution, elm skin primary extract, silica ethyl acetate fraction, procyanidin oligomer fraction, tetracycline, minocycline and doxycycline, epigallocatechin gallate 0.0001, 0.001, 0. ．
It was made to have four kinds of concentrations of 005 and 0.01% by weight.

A buffer solution (0.05 M Tris-HCl, 1 nM CaCl ₂ , pH 7.8) was added so that the total reaction solution was 500 μl, and the mixture was reacted in a thermostat at 37 ° C. for 18 hours, and then the Eppendorf tube was placed in the tube. Undegraded collagen was precipitated by centrifugation at 10,000 g for 5 minutes. The supernatant containing the degraded collagen is then removed and
The absorbance was measured at 0 nm to prepare a standard activity curve, and the enzyme activity concentration was converted from the standard curve to compare and evaluate the enzyme activity of the experimental group and the control group. MMP-1 and MMP
The activity inhibitory effect experiment on −8 was also performed in the same manner as described above. Each enzyme standard solution was purchased from Calbiochem. The results are as follows.

The results of evaluating the inhibitory effects of various drugs on collagen degrading enzyme activity are shown in Table 3 below.

[0055]   Here, the enzyme inhibition rate was calculated by the following mathematical formula, Formula 1.

[0056]

[Equation 1] Enzyme inhibition rate (%) = 100- (enzyme activity of experimental group x 100 / enzyme activity of control group)

[Table 3] The inhibitory effect evaluation of the procyanidin oligomer fraction on MMP-1 (fibroblast collagenase) activity is shown in Table 4 below. Here, the enzyme inhibition rate was calculated by the above-mentioned numerical formula 1.

[0057]

[Table 4] In addition, the inhibitory effect evaluation of the procyanidin oligomer fraction on MMP-8 (polymorphonuclear leukocyte collagenase) activity is shown in Table 5 below. Here, the enzyme inhibition rate was calculated by the above formula 1.

[0058]

[Table 5] Based on the above test results, the procyanidin oligomer fraction was
llagenolytic) enzyme, MMP-1 and MMP-8 inhibitory effect on enzyme activity is superior to epigallocatechin gallate, which is a known green tea component, and doxycycline, and elm skin primary extract and silica ethyl acetate fraction It turns out to be better. Therefore, the procyanidin oligomer fraction is M in the elm skin layer.
It is considered to be the main active component of MP enzyme inhibitory activity.

Experimental Example 3 (Evaluation of inhibitory effect of procyanidin oligomer fraction on MMP activity using Collageno Kit (CLN-100, Japan Cosmo Bio)) This test method was carried out using a fluorescent substance, fluorescein isothiocyanate (FITC).
This is based on the fact that when collagen type I covalently bound to) is treated with collagenase, only the degraded collagen is denatured at 35 ° C. and extracted with ethanol. After centrifuging the reaction solution, the supernatant was collected at 520 nm (EM) / 49
5 (EX), the fluorescence intensity (FI) was measured with a spectrofluorometer (Inflammation,
1994, 18; 613-623). Add 0.5% F to a 1.5 ml Eppendorf tube.
200 μl of ITC-collagen was added respectively. One Eppendorf tube was used as a blank and used to generate a standard enzyme activity curve.
Each tube contains 1 MMP-1 standard enzyme solution purchased from Calbiochem.
The amount added was 0, 100, and 200 ppm. In each of the other tubes, 100 μl of the enzyme standard solution, procyanidin oligomer fraction, silica ethyl acetate fraction, tetracycline, minocycline, and doxycycline were added to 0.00 each.
It was added in an amount of 1, 0.01, 0.03, 0.05% by weight. Buffer solution (0.
05M Tris-HCl, 1 mM CaCl ₂ , pH 7.8) was added to each tube so that each reaction solution had a volume of 500 μl, and the reaction was allowed to proceed for 18 hours in a thermostat at 37 ° C. 10 μl of 80 mM o-phenanthroline dissolved in 50% by weight ethanol as a reaction stop solution
Was added, and the mixture was maintained in a thermostat at 37 ° C. for 18 hours and then cooled. 0.5 ml of 70% by weight ethanol was added to each cooled Eppendorf tube, and the mixture was centrifuged at 3000 rpm for 10 minutes. 5 from each tube after centrifugation
The supernatant (00 μl) was taken and the fluorescence intensity (F) was measured at 520 nm (EM) / 495 (EX).
I) was measured with a spectrofluorimeter to first create a standard curve for enzyme activity. The enzyme activity concentration was converted from the prepared standard curve, and the enzyme activities of the experimental group and the control group were compared. The activity inhibition experiment on MMP-8 was also performed by the same method as described above. The above results are as follows.

Evaluation of the inhibitory effect of procyanidin oligomer fraction on MMP-1 activity was performed by
The results are shown in Table 6 below. Here, the enzyme inhibition rate was calculated by the above formula 1.

[0061]

[Table 6] The evaluation of the inhibitory effect of procyanidin oligomer fraction on MMP-8 activity is shown in Table 7 below. Here, the enzyme inhibition rate was calculated by the above formula 1.

[0062]

[Table 7] The above test results also revealed that the procyanidin oligomer fraction had a more excellent inhibitory effect on the activity of MMP than the tetracycline system.
Almost 100% of the activity-inhibiting effect was exhibited for the two enzymes (MMP-1, MMP-8) at the concentration of wt%. The inhibitory effect of the silica ethyl acetate fraction composed of flavan-3-ol monomer, two dimers, some trimers and unidentified substances,
It can be seen that it is significantly inferior to the procyanidin oligomer of the present invention. The effect of doxycycline is the best among the tetracycline series, and shows a 90% activity inhibitory effect on MMP-1 and MMP-8 at a concentration of 0.05% by weight,
The inhibitory effect of tetracycline and minocycline on the enzyme activity was lower than this.

Experimental Example 4 (Type IV collagenase (MMP-using Zymography)
2 and MMP-9) Evaluation of inhibitory effect of procyanidin oligomer fraction on activity) MMP-2 and 9 enzyme standard solution purchased from Calbiochem was buffered (2.
5% SDS (sodium dodecyl sulfate), 50 mM Tris-HCl, pH
6.8, 10% glycerol, 0.005% bromphenol blue, 3%
Sucrose) was mixed and then electrophoresed on a gel plate (8% SDS-polyacrylamide gel) containing 0.2% gelatin as a matrix of type IV collagenase. After electrophoresis, the gel was washed with 50 mM Tris-HCl (pH 7.5) buffer solution containing 2.5% Tritox X-100 twice for 30 minutes to remove SDS. Then, the gel was cut into several pieces in the longitudinal direction, and the enzyme standard solution and procyanidin oligomer fraction, tetracycline, minocycline and doxycycline were added to 0.0001%, 0.001%, 0.005% and 0.01%, respectively. The mixture was placed in a reaction buffer solution prepared to each concentration and reacted at 37 ° C. for 18 hours.

Subsequently, the gel was stained with 0.1% Coomassie Brilliant Blue and then decolorized, and the gelatin resolution was measured with a densitometer to prepare a standard activity curve. Then, the enzyme activity concentrations were converted from the standard curve, and the enzyme activities of the experimental group and the control group were compared. Further, an activity inhibition experiment on MMP-9, which is another type IV collagenase, was also conducted by the same method as described above. The results are as follows.

The results of evaluation of the inhibitory effect of procyanidin oligomer fraction on MMP-2 activity are shown in Table 8 below. Here, the enzyme inhibition rate was calculated by the above formula 1.

[0066]

[Table 8] The evaluation results of the inhibitory effect of the procyanidin oligomer fraction on MMP-9 activity are shown in Table 9 below. Here, the enzyme inhibition rate was calculated by the above formula 1.

[0067]

[Table 9] From the above experimental results, it was revealed that the procyanidin oligomer fraction was superior to the tetracycline system in inhibiting type IV collagenase enzyme activity such as MMP-2 and MMP-9, and at a concentration of 0.01%. MMP-2, MMP
Both showed 100% inhibitory effect on the activity of -9. Among the tetracyclines, doxycycline was the most effective, while minocycline and tetracycline were less effective. The inhibitory effect of the silica ethyl acetate fraction composed of flavan-3-ol monomer, two dimers, a part of trimer and an unidentified substance is significantly inferior to that of the procyanidin oligomer of the present invention. From the above, it can be seen that the titer of elm skin primary extract is mainly due to the procyanidin oligomer specified in the present invention.

Experimental Example 5 (Inhibition experiment of MMP secreted by periodontal ligament cells) The periodontal ligament tissue attached to the surface of the extracted tooth was scraped with a scalpel and cultured in a medium. 6th to 8th generation cells grown outward from the tissue were used.

The supernatant of the periodontal ligament tissue was used as an enzyme source in Experimental Example 3 described above. The MMP secreted from the periodontal ligament has a molecular weight, complete inhibition of titer by EDTA, and PM
It is considered to be a type IV collagenase because it is not inhibited by SF (serine-based protease inhibitor) and other protease inhibitors such as pepstatin.

As shown in FIG. 6, it was found that the procyanidin oligomer effect was about 10 times better than that of doxycycline.

[0071]Experimental example 6 (Effect of periodontitis bacteria on collagenase)   Two bacteria that are periodontitis (Porphyromonas gingivalis, Treponema dentico)
for the inhibition experiment of the collagenase activity from
Collagen matrix (³H-collagen type 4 (N-propionate-2, 3- ³ H- Propionated, mCi / ml: NEN Life Science Products, Boston, MA)
) Was mixed with various concentrations of drugs and a bacterial culture solution, and cultured for 18 hours. Disassembled
After precipitating the missing collagen with 0.05% tannic acid and trichloroacetic acid,
The radioactivity remaining in the supernatant was measured to compare the inhibitory activity of the drugs.

Here again, it was revealed that the procyanidin oligomer of the present invention was 10 times or more superior to the silica ethyl acetate fraction and doxycycline.

Experimental Example 7 (Selective Enzyme Inhibitory Property of Procyanidin Oligomer) In order to examine whether procyanidin is a non-specific enzyme inhibitor by general protein binding, azocol (azocoll) in Experimental Example 2 was tested. The collagenase inhibitory effect of the procyanidins used and its elastase inhibitory effect were compared with that of doxycycline.

First, elastin-congo red (Sigma) to which congo red is bound
The reaction solution (10 mM TES buffer, pH 7.0) prepared to various drug concentrations was mixed with the above. Elastase (Sigma E-150) was added to the mixture and cultured for 18 hours. Then, the undegraded elastin was precipitated, and the absorbance of the supernatant was adjusted to 495n.
It was measured in m. As for the titer, only the enzyme and no drug were used, that is, the enzyme activity of the control group in which the degradation was sufficiently advanced was 100 (that is, the inhibition rate was 0%), and the degree of relative reaction inhibition is shown in Table 10 below. It was Here, the enzyme inhibition rate was calculated by the above formula 1.

[0075]

[Table 10] From the above results, the inhibition of the collagenolytic enzyme of procyanidin oligomers is superior to that of doxycycline, but the inhibitory activity of elastase is inferior to that of doxycycline, showing that procyanidins are nonspecific due to general protein binding. It turns out that it is not an enzyme inhibitor.

The following examples [0076] Examples of the present invention to collagenolytic activity ointment (Example 1
The evaluation of the effects of (8) to (8) was performed.

The procyanidin oligomer fraction according to the present invention can be prepared in any form for inhibiting the activity of MMP such as collagenase. As such a form, tablets, capsules, powders, ointments, solutions, gels, pastes, patches, granules and the like are possible, but in the present invention, experiments were conducted with ointments.

The content of the procyanidin oligomer fraction used at this time is from 0.0001 to
It is 5% by weight, preferably 0.01 to 1% by weight. If the content is less than 0.0001% by weight, the enzyme activity inhibitory effect or efficacy cannot be expected, and if it exceeds 5% by weight, the procyanidin oligomer fraction is discolored to brown, which makes it difficult to use. .

The ointment compositions used in the following Examples and Comparative Examples were prepared by the following methods.

With procyanidin oligomer fraction, pluronic, lower alcohol, glycerin, gelatin, pectin, carboxymethylcellulose, poloxamer (poloxamer 407), monoglyceride (Myverol 18-99), propylene glycol or polyethylene glycol, menthol and preservatives. The composed mixture was prepared by dissolving in a buffer solution and gelling. In Comparative Example, doxycycline was used in place of the procyanidin oligomer fraction, and the same procedure as described above was performed except for the above.

As the lower alcohol used in the present invention, 0 to 20% by weight of ethanol or isopropyl alcohol is used individually or in combination, and as the pluronic derivative used for the state stability of the ointment composition, Pluronic F-127
Alternatively, 5 to 30% by weight of Pluronic F-108 can be used. A wetting agent is used for maintaining the state of the ointment composition and preventing drying, and examples of such a wetting agent include glycerin, sorbitol, polyethylene glycol and propylene glycol, poloxamer (Plolxamer 407), monoglyceride (Myverol 18
-99) and mixtures thereof, used in an amount of 5 to 30% by weight. Particularly, as the polyethylene glycol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, or polyethylene glycol 1000 can be used.

Further, as a system for transmitting the drug effect of the active ingredient into the skin tissue, gelatin or pectin is used alone or in a mixture of 1 to 20% by weight. Alkali metal salts of o-phosphoric acid as buffering agents for adjusting the pH of the ointment composition, especially the first, second and third sodium phosphates, and citric acid and sodium citrate,
Examples include phosphoric acid, hydrochloric acid, sodium hydroxide or sodium pyrophosphate or pyrophosphate. In the ointment composition of this example, two of the first, second and third sodium phosphates were appropriately mixed and the pH was adjusted to 5 to 8.0 before use.

Furthermore, sodium metabisulfite is used to prevent discoloration of the procyanidin oligomer fraction, but 0.05 to 1% by weight is used in the ointment composition. As the fragrance, peppermint and spearmint oil, which are natural fragrances, are mainly used, and 0.1 to 1% by weight is used in the ointment composition. Further, in order to prevent contamination of microorganisms that may occur during the production and use of the ointment composition, methyl paraoxybenzoate, propyl paraoxybenzoate, benzoic acid, and benzoic acid that are generally licensed for use in foods and pharmaceuticals are used. Sodium, salicylic acid, etc. may be used alone or in a mixture of 0.01 to 0.5% by weight.

Examples 1 to 8 and Comparative Examples 1 to 8 The ointment compositions of Examples and Comparative Examples are shown in Tables 11 to 14 below.

[0085]

[Table 11]

[Table 12]

[Table 13]

[Table 14] The experimental process and results of the inhibitory effect on collagen degrading enzyme with each of the ointments are as follows.

100 μl of a 2% Azocoll solution, which is a red collagen matrix, was added to each of 20 1.5 ml Eppendorf tubes. One Eppendorf tube was used as a blank, and collagen-degrading enzyme standard solution purchased from Sigma Co. was added to each of the three tubes so as to have a concentration of 10, 100 and 200 ppm in order to prepare a standard activity curve of the enzyme. . Each of the experimental group ointment (Examples 1 to 8) and the comparative group ointment (Comparative Examples 1 to 8) was mixed with distilled water at a ratio of 1: 2 and homogenized, and then centrifuged at 5,000 g for 10 minutes. 10 μl of the resulting supernatant and 100 μl of enzyme standard solution were added to each of the 16 remaining tubes. Buffer solution (0.
05MTris-HCl, 1 mM CaCl ₂ , pH 7.8) was added to the total reaction solution at 500
After adding to each tube so that it becomes μl and reacting in a 37 ° C. incubator for 18 hours,
Undegraded collagen was precipitated by centrifuging the Eppendorf tube at 10,000 g for 5 minutes. Take the supernatant containing the degraded collagen and
The absorbance was measured at 0 nm to prepare a standard activity curve, and the enzyme activity concentration was converted from the standard curve to compare and evaluate the enzyme activity of the experimental group and the control group. This yielded the following results. Here, the enzyme inhibition rate was calculated by the above formula 1.

[0087]

[Table 15] From the above experimental results, the inhibitory effect of procyanidins on the activity of collagen-degrading enzymes at a concentration of 0.05% or less was found that the compositions containing the procyanidin oligomer fraction of Examples of the present invention contained doxycycline. It turns out that it was superior to the comparative example.

Therefore, the drug containing the procyanidin oligomer of the present invention as an active ingredient includes cancer metastasis, rheumatoid arthritis, diabetes, inflammation, corneal ulcer, epithelial ulcer, gastric ulcer, skin wrinkles and aging, periodontitis, osteoporosis, It can be used as a drug for the prevention and treatment of diseases associated with the activity of MMPs such as acne, trauma and burns, hyperparathyroidism and the like.

[Brief description of drawings]

FIG. 1 is a result of analyzing a silica ethyl acetate fraction by a high performance liquid chromatography / electrospray ionization mass spectrometer (HPLC / ESI Massspectrometer, Finnigan LCQ), showing a procyanidin monomer having a molecular weight of 290 (9.1 minutes). 2 kinds of molecular weight 5
It can be seen that there are 78 procyanidin dimers (17.08 min, 18.33 min) and unidentified substances of molecular weight 422, 13.34 min, 13.75 min).

FIG. 2 MALDI-TOF (Matrix-assisted laser desorption / ionization-time of flight type)
Using a mass spectrometer, the mass of [M + sodium] ⁺ in which sodium was added to the molecular weight of procyanidin oligomer, which is the main component of Fraction 4, was detected using cyano-4-hydroxycinnamic acid as a matrix. is there.

FIG. 3 shows the results of tracing the pentamer ([MH] ⁻ ) mass 1441 and ([M + TFA ⁻ ] ⁻ ) mass 1555 using a high performance liquid chromatography / electrospray ionization mass spectrometer. It is shown.

FIG. 4 shows the results of high performance liquid chromatography / electrospray ionization mass spectrometry,
In fraction 4, procyanidin oligomer peaks were detected from trimer to dodecer, and as the degree of polymerization increased from 3, the distribution of ions having the predicted mass was obtained from the beginning to the latter half of the chromatogram of fraction 4. You can see that it will move to the club.

FIG. 5 shows the results of detection of mass peaks in scan mode in the retention time zone on the chromatogram near the designated mass peaks using the high performance liquid chromatography / electrospray ionization mass spectrometer, showing procyanidin oligomer mass peaks. For example, if the retention time range is set to tetramer, 1153 ([MH] ^- ) and 12
67 ([M + TFA ^- ] ^- ), 144 when the retention time range is set to pentamer
Since it can be seen that 1 ([MH] ⁻ ) and 1555 ([M + TFA ⁻ ] ⁻ ) are the main peaks, it can be seen that the main component of Fraction 4 is procyanidin oligomer.

FIG. 6 is a result of comparing procyanidin oligomer inhibitory effects on MMP, which is a type 4 collagenase secreted from periodontal ligament cells, by zymography as compared with doxycycline, which was about 10 times better than doxycycline. I understand. Reference numeral 1 in the drawing is procyanidin oligomer, and reference numeral 2 is doxycycline.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 9/16 A61K 9/16 9/20 9/20 9/48 9/48 9/70 401 9/70 401 31/353 31/353 35/78 35/78 C XY A61P 1/02 A61P 1/02 1/04 1/04 3/10 3/10 17/02 17/02 17/10 17/10 17 / 16 17/16 19/02 19/02 19/10 19/10 27/02 27/02 29/00 29/00 101 101 35/04 35/04 43/00 105 43/00 105 105 111 111 (81) designation Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, S) D, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU. , ID, IL, IN, IS, JP, KE, KG, KP, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ahn, Hojeong, Taejeong-City, 305-728, U.S.A. Seongguk, Jeongmin-Dong 460-5, Sejong Apartment 107-1106 (72) Inventor Park, Sang-ki Taejeong-city 305-340, Youseon-guk, Drong-dong 386-4, Ergie Sataek 2- 404 (72) Inventor Kim, Moon-Mu Taejeong-city, Republic of Korea 305-340, Youseon Guuk, Dorong-dong 386-4, Lucky Apartment 1-202 (72) Inventor Kim, Jun-Hoon Republic of Korea Taeyeon-city 305-340, Yousseonguk, Doriangsong, Elsey Apartments 1-103 (72) Inventor Lee, Hak-mo Taejeong City, Republic of Korea 305-340, Yousseonguk, Drsong Dong, Elsie Apartments 2-404 (72) Inventor Park, Hyung-Cook Taejeong-City, Republic of Korea 305-75 2, Yuseonguk, Songgang-Dong 8-2, Cheonggol Apart 512-705 (72) Inventor Cho, Hi-Sang Taejeong-City 305-755, Youseonguk, Eo Eun-Dong 99, Hanbit Apartment 110 − 1401 (72) Inventor Jeong, Seung-young Republic of Korea Tae Jeong City 302 −727, Seok, Nae Dong 151, Coron Apartment 6−502 F Term (reference) 4C062 FF56 4C076 AA06 AA09 AA11 AA16 AA29 AA31 AA36 AA53 AA72 CC04 CC09 CC10 CC16 CC18 CC19 CC21 CC27 CC29 DD09 DD24 DD26 DD37 DD38 DD41 DD46 EE23 EE30 EE32. BA10 BA11 BA21 BA22 BA32 CA06 CA14 MA07 MA17 MA21 MA28 MA32 MA35 MA37 MA41 MA43 NA14 ZA33 ZA67 ZA68 ZA89 ZA96 ZA97 ZB11 ZB15 ZB26 ZC20 ZC35

Claims (15)

[Claims] 1. A procyanidin oligomer which inhibits the activity of matrix metalloprotease (MMP). 2. The matrix metalloprotease, MMP-2, which is a collagenolytic protease, MMP-1, MMP-8, and type IV collagenase.
And MMP-9, and a procyanidin oligomer according to claim 1, selected from the group consisting of bacterial secretory collagenase. 3. The procyanidin oligomer is an n-butanol fraction produced by solvent-fractionating a primary extract extracted from the elm skin layer, which is the root or skin of an elm plant, with a polar solvent. The procyanidin oligomer according to claim 1. 4. The n-butanol fraction produced by solvent partitioning an extract obtained by extracting the procyanidin oligomer from the elm skin layer, which is the root or skin of a plant of the genus Elm, with a polar solvent is sephadex. The n-butanol fractions are sequentially eluted in the order of increasing methanol when the chromatography is further carried out with a (registered trademark) LH-20 column and a water-methanol mixed solution, and the eluates are recombined based on thin layer chromatography. The procyanidin oligomer according to claim 1, which is a formulation that is thereby concentrated. 5. An n-butanol fraction produced by solvent partitioning an extract of the procyanidin oligomer extracted from the elm skin layer, which is the root or skin of an elm plant, with a polar solvent, is sephadex. (Registered trademark) LH-20 column and 1
A formulation which is concentrated by sequentially eluting the n-butanol fractions with 100% methanol when further chromatographed with 00% methanol and recombining the eluates based on thin layer chromatography. The described procyanidin oligomer. 6. The procyanidin oligomer is an oligomer in which 3 to 12 monomers each having a basic skeleton of flavan-3-ol are linked by a single bond.
The procyanidin oligomer according to claim 1. 7. A pharmaceutical composition for preventing and / or treating a disease caused by the activity of matrix metalloprotease (MMP), which comprises a procyanidin oligomer as an active ingredient. 8. The disease is cancer metastasis, rheumatoid arthritis, diabetes, inflammation,
The pharmaceutical composition according to claim 7, which is selected from the group consisting of corneal ulcer, epithelial ulcer, gastric ulcer, skin wrinkles and aging, periodontitis, osteoporosis, acne, trauma and burns, or hyperparathyroidism. object. 9. The matrix metalloprotease, MMP-2, which is a collagenolytic protease, MMP-1, MMP-8, and type IV collagenase.
And MMP-9, and a pharmaceutical composition according to claim 7, selected from the group consisting of bacterial secretory collagenase. 10. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is in the form of tablets, capsules, powders, ointments, solutions, gels, pastes, patches or granules. 11. The content of the procyanidin oligomer is 0.0 in the composition.
The pharmaceutical composition according to claim 7, which is 001 to 5% by weight. 12. The procyanidin oligomer is an n-butanol fraction produced by solvent fractionating an extract extracted from a elm skin layer, which is the root or skin of an elm plant, with a polar solvent. The pharmaceutical composition according to claim 7. 13. An n-butanol fraction produced by solvent partitioning an extract of the procyanidin oligomer extracted from a elm skin layer, which is a root or a skin of an elm plant, with a polar solvent, is sephadex. The n-butanol fractions are sequentially eluted in the order of increasing methanol when the chromatography is further carried out with a (registered trademark) LH-20 column and a water-methanol mixed solvent, and the eluates are recombined based on thin layer chromatography. The pharmaceutical composition according to claim 7, which is a formulation that is thereby concentrated. 14. Sephadex is an n-butanol fraction produced by solvent partitioning an extract of the procyanidin oligomer extracted from the elm skin layer, which is the root or skin of an elm plant, with a polar solvent. Concentrated by sequentially eluting the n-butanol fractions with 100% methanol when chromatographed with a (registered trademark) LH-20 column and 100% methanol and recombining the eluates based on thin layer chromatography. The pharmaceutical composition according to claim 7, which is a formulation. 15. The pharmaceutical composition according to claim 7, wherein the procyanidin oligomer is an oligomer in which 3 to 12 monomers each having a basic skeleton of flavan-3-ol are linked by a single bond.