JPH1067769A - Natural antibacterial agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent having a high antibacterial activity, effective to periodontal bacteria, dental caries bacteria and MRSA and also high in safety by including a hydrophilic organic solvent extract of a bark of mulberry tree as an active ingredient. SOLUTION: This natural antibacterial agent contains a hydrophilic organic solvent extract of a bark of a plant belonging to the genus Morus e. g. a new compound such as a flavonid compound of the formula. As the extracting method, the method of defatting the bark of the plant belonging to the genus Morus with a hydrophobic solvent (preferably a hydrocarbons, an aromatic hydrocarbon, etc.) and then extracting the bark by a hydrophilic organic solvent (preferably a lower fatty acid ester, a lower alcohol, and an ether) is cited. Thus, an antibacterial agent having a high safety to a human body and useful for the prevention and treatment of a periodontal disease and a caries of a tooth, and also the prevention of an intra-hospital infection, is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antibacterial agent containing a mulberry bark hydrophilic organic solvent extract as an active ingredient, a novel compound (flavonoid compound) having an antibacterial action derived from mulberry bark and useful as a medicament, and Antibacterial agent contained as an active ingredient, specifically, periodontal disease bacteria, caries bacteria, and MRS
The present invention relates to an antibacterial agent that suppresses or sterilizes pathogenic bacteria such as A, and is effective for prevention and treatment of periodontal disease and caries, and for prevention of hospital-acquired infection.

[0002]

2. Description of the Related Art It is said that mulberry has various medicinal effects depending on the site to be used. In Chinese medicine, mulberry root bark is called mulberry bark, and it is used as an anti-inflammatory, diuretic, cough, asthma, edema,
Used for treatment of dysuria. The leaves are referred to as mulberry leaves, and are used as antipyretic, antitussive, and anti-inflammatory agents for the treatment of colds, eye diseases, hypertension, and the like. The fruit is called mulberry straw and is used as a tonic, sedative, blood-supplementing and antidiarrheal agent.

On the other hand, in modern pharmacology, many studies have been made on physiologically active substances extracted from mulberry plants, especially mulberry bark, which is the root bark thereof. For example, it has been reported that the methanol extract of mulberry bark has a blood pressure lowering effect and a hyperglycemic lowering effect (JP-A-57-144223, JP-B-53-44530).

[0004] Recently, attention has been paid to the antibacterial activity of the extract component of mulberry bark, and its research has been conducted (Japanese Patent Publication No. Sho 63-2).
4489, JP-A-5-271085). In addition, research on antibacterial active ingredients in mulberry plants was conducted, and Morasin A & B [Tetrahedron Let
t., 797, (1978), Chem. Lett., 1239, (1978)], Quannon L (JP-49152), Quannon J (2-182)
4) Sangenone B (JP-A-57-146718) has been isolated.

Further, studies have been conducted on the use of such an antibacterial active substance of mulberry bark for caries and periodontal disease. For example, the growth inhibitory action of cariogenic bacteria by a hydrophilic organic solvent extract of mulberry bark (Japanese Patent Publication No. 63-24489), Quanone L and J effective against Gram-negative bacteria (Japanese Patent Publication Nos. 1-49152 and 2-82).
1824), periodontal disease preventive agent (JP-A-5-27085)
Have been reported.

However, research on antibacterial substances contained in such mulberry is limited to mulberry bark, which is the root bark of mulberry, and research on other parts has not been carried out much. It is not yet done. In addition, no research has been conducted on an antibacterial agent that is effective against periodontal disease bacteria and tooth decay bacteria contained in parts other than mulberry bark of mulberry. Periodontal disease is one of the two major diseases in the oral cavity that are comparable to caries,
It is a general term for diseases that occur in periodontal tissue such as so-called gingivitis and periodontitis. Porphyromonas gingivalis is a bacterium that causes this periodontal disease.
s ) there.

[0007] Dental caries is generally called caries, and bacteria that cause the caries include Streptococcus mutans . Conventionally, synthetic antibacterial agents such as cetylpyridinium chloride and antibiotics such as tetracycline have been used as antibacterial agents against these bacteria. However, the use of these antimicrobial agents has major problems. For example, if these antibacterial agents are used in the mouth for a long time and swallowed, there is a possibility that bacteria necessary for the human body such as disturbing the flora of intestinal bacteria may be killed or weakened. Therefore,
An antibacterial agent having a strong antibacterial property against only the above-mentioned periodontal disease bacteria and cariogenic bacteria and a low antibacterial property against other microorganisms, that is, a narrow antibacterial spectrum is desired. Also, considering long-term use in the mouth, it is preferable to use a naturally derived antibacterial agent from the viewpoint of safety. However, to date, naturally-derived antibacterial agents against periodontal disease bacteria and dental caries have not been sufficiently studied.

On the other hand, in recent years, hospital-acquired infections in medical facilities have become a major clinical problem. The microorganisms responsible for this are methicillin-resistant Staphylococcus aureus (Meticillin resistant Staphylococcus aureus; MRS)
A) is a multidrug-resistant bacterium having resistance to antibiotics and disinfectants. For this reason, when infected with MRSA, it becomes difficult for a person with low resistance to remove MRSA from the body, so that treatment becomes difficult and symptoms tend to be serious. Also, once the infection spreads within the hospital, it is difficult to control it.

MRSA has resistance to antibiotics such as β-lactams, macrolides, aminoglycosides, etc., and the multidrug resistance has been broadened year by year, and the degree of resistance has been increasing. To prevent nosocomial infections caused by MRSA,
Although the “SA Guideline” has been formulated, the development of an antibacterial agent with a new mechanism of action, particularly a safe antibacterial agent of natural origin, is desired, but has not been sufficiently studied.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to provide an antibacterial agent which is effective for periodontal disease bacteria, dental decay fungi and MRSA, and which contains a highly safe and natural component as an active ingredient. It is in.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and have studied in detail the antibacterial activity of the extracted components in each part of the mulberry. The extract was found to have a much higher antibacterial activity than the extract of the hydrophilic organic solvent of mulberry bark, and the compound obtained by purifying the extract from the hydrophilic organic solvent by liquid chromatography was converted to a periodontal periodontal. It has been found that it has strong antibacterial activity only against pathogenic fungi, dental caries, and MRSA, and it was confirmed that this compound was a novel flavonoid compound, and the present invention was completed as an antibacterial agent containing these as active ingredients. I came to.

That is, the present invention is an antibacterial agent containing a hydrophilic organic solvent extract of mulberry bark as an active ingredient. Furthermore, the present invention is a method for extracting an antibacterial substance, wherein mulberry plant bark is defatted with a hydrophobic solvent and then extracted with a hydrophilic organic solvent.

Further, the present invention provides a compound of the formula (VI)

[0014]

Embedded image

The flavonoid compound represented by the formula: Formula (VI)

[0016]

Embedded image

An antibacterial agent containing a flavonoid compound represented by the formula (1) as an active ingredient.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The active ingredient of the antibacterial agent of the present invention is a hydrophilic organic solvent extract of mulberry plant bark (hereinafter referred to as the present extract). The mulberry bark used as a raw material can be used as long as it is the bark of a mulberry plant, for example, Yamaguchi (Morus bombycis Koidz), Magwa
(Morus alba L.), bark such as rosou (Morus lhou Koidz) can be used. These mulberry bark can be used alone or in combination of two or more. These mulberry bark is crushed to an appropriate size or powdered for use. This extract can be obtained by extracting mulberry plant bark with a hydrophobic solvent such as toluene, defatting, extracting with lower fatty acid esters, lower alcohols, ethers and ketones, and concentrating under reduced pressure.

The flavonoid compound of the present invention represented by the formula (VI) (hereinafter referred to as the present compound) may be obtained from the bark of a mulberry plant by the method described below, or may be obtained by chemical synthesis. . As an example of the method for producing the present compound, a method for producing from a bark of a mulberry plant will be described.

First, mulberry bark as a raw material can be used as long as it is the bark of a mulberry plant.
Koidz), Magwa ( Morus alba L.), Rosewort ( Morus lhou K
bark such as oidz) can be used. These mulberry bark can be used alone or in combination of two or more. These mulberry bark is crushed to an appropriate size or powdered for use.

The present compound is obtained by extracting mulberry bark with a non-polar solvent such as hexane, defatting, extracting with lower fatty acid esters, lower alcohols, ethers and ketones and concentrating under reduced pressure to obtain a mulberry bark extract. . The extract thus obtained was subjected to adsorption column chromatography such as celite, florisil, silica gel and the like, and reverse phase column chromatography such as ODS to obtain a crude product.
It can be obtained by purification by LC.

The extraction method may be a commonly used method, for example, a method in which the raw material mulberry bark is immersed in an organic solvent for a long time, a method in which the extraction is carried out while heating and stirring at a temperature not higher than the boiling point of the organic solvent, and filtering. To obtain an extract. As the hydrophobic organic solvent used in the degreasing step, pentane, hexane, heptane, hydrocarbons such as cyclohexane, or toluene, aromatic hydrocarbons such as benzene,
Alternatively, halogenated hydrocarbons such as dichloromethane and chloroform may be mentioned, and toluene is preferred from the viewpoint of the yield of the extract.

The hydrophilic organic solvent used in the extraction step includes lower fatty acid esters such as methyl acetate, ethyl acetate and butyl acetate; lower alcohols such as methanol, ethanol and isopropanol; and methyl ether;
Examples thereof include ethers such as ethyl ether, tetrahydrofuran, and dioxane; and ketones such as acetone and methyl ethyl ketone. A mixed solvent of these hydrophilic organic solvents and a mixed solvent of a hydrophilic solvent and a hydrophobic solvent can also be used. From the viewpoint of the antibacterial activity of the obtained extract, dichloromethane / methanol (1: 1), ethyl acetate, acetone and tetrahydrofuran are preferred, and ethyl acetate is more preferred.

It is possible to omit the degreasing step during the extraction / separation operation. However, in order to obtain a highly active mulberry bark extract, mulberry bark is not directly extracted with a hydrophilic organic solvent. An embodiment in which defatting with a hydrophobic organic solvent is followed by extraction with a hydrophilic organic solvent is preferred. When the antibacterial activity and the yield of the mulberry bark extract are comprehensively evaluated together, the method with high antibacterial activity and high extract yield uses toluene as the hydrophobic solvent used during degreasing and ethyl acetate as the extraction solvent. The combination is the best.

The above mulberry bark hydrophilic organic solvent extract is
Further purification by liquid chromatography identified the following six compounds, indicating that these compounds were included as active ingredients in the extract.

[0026]

Embedded image

[0027]

Embedded image

[0028]

Embedded image

[0029]

Embedded image

[0030]

Embedded image

[0031]

Embedded image

As the column chromatography used in the purification step, adsorption chromatography such as celite, florisil, silica gel and the like and reverse phase chromatography such as ODS can be used. In celite column chromatography, an extract is adsorbed using a column packed with celite, developed with a hexane-ethyl acetate mixed solution or the like, and the active compound is eluted and separated. In florisil chromatography, an extract is adsorbed using a column packed with florisil, developed with a hexane-ethyl acetate mixed solution, and the active compound is eluted and separated. In silica gel column chromatography, a column packed with silica gel is used to adsorb the extract, and the active compound is eluted and separated with a dichloromethane-methanol mixture or the like. As the solvent used here, hexane, benzene, toluene, ethyl ether, ethyl acetate, acetone, dichloromethane, chloroform, ethanol, isopropanol, or the like can be used alone or as a mixture.

In the HPLC used in the purification step, ODS,
Reverse-phase chromatographic fillers such as octyl, phenyl, cyanopropyl and the like, and adsorptive chromatographic fillers such as silica gel can be used. In ODS-HPLC, a mixed solution of methanol and distilled water, a mixed solution of acetonitrile and distilled water, and the like are used as eluents. The antimicrobial activity of the present extract and the present compound obtained by the above method can be evaluated by measuring the minimum inhibitory concentration (MIC) using the paper disk method or the liquid medium dilution method.

The present extract and the present compound have a unique spectrum of antibacterial activity. That is, the present extract is much better than the mulberry bark extract in antibacterial activity against the periodontal disease bacteria Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, and against the decay bacteria Streptococcus mutans and Streptococcus sobrinas. ing. In particular, the antibacterial activity against the periodontal disease bacterium Porphyromonas gingivalis has an antibacterial activity comparable to that of cetylpyridinium chloride, which is a typical synthetic antibacterial agent, and is superior to that of hinokitiol, which is a naturally occurring antibacterial agent.
On the other hand, this extract is different from cetylpyridinium chloride,
The antibacterial activity against Escherichia coli, a typical intestinal bacterium, is very weak, and the antibacterial spectrum is narrow.

Accordingly, the present extract can be widely used as an antibacterial component derived from natural products having a mild effect, such as pharmaceuticals, quasi-drugs, food additives, animal drugs, animal feed additives, etc. It is useful as an agent for preventing and treating periodontal disease and dental caries. Specifically, it can be used by blending it in toothpaste, mouthwash, troche, chewing gum, candy and the like.

The present extract is also extremely excellent in antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), which is a causative agent of hospital-acquired infection, and is used as an anti-MRSA agent. Comparable to the antibiotic antibiotic vancomycin. Therefore, the present extract and the present compound can be used as an anti-MRSA agent in antibacterial processing of fibers, cloths, nonwoven fabrics, papers such as gauze, bed sheets, white coats, clothes, curtains and the like used in hospitals.

The present extract can be used as it is, or in any state, such as a concentrated one or a dried one. In addition, since the present extract is derived from natural products, there is no danger to the human body and there is no problem in terms of safety. The use mode and dosage form of the antimicrobial agent containing the present extract and the present compound are not particularly limited, and include, for example, solid, powder, liquid, paste, powder,
Sprays, mousses, tablets and the like are widely selected depending on the application, and preparation into these is carried out by a conventional method.

The content of the active ingredient in the antibacterial agent of the present invention can be appropriately changed depending on the use mode and dosage form. For example, when the present compound is used as the active ingredient, the content is 0.000005 to 10%.
% By weight, preferably about 0.00005 to 5% by weight.
For example, it is contained in an amount of about 0001 to 20% by weight, preferably about 0.00001 to 10% by weight. Exceeding the upper limit of this range is undesirable because it affects the flavor of the periodontal disease preventive agent,
On the other hand, when the value is below the lower limit, the effect is hardly obtained, which is not desirable.

The microorganism whose growth can be inhibited and sterilized by the antibacterial agent of the present invention is Porphyromonas gingivaris, a periodontal disease bacterium.
lis381), Actinobacillus actinomycetemcomitans Y4,
Streptococcus mutans (Str.
eptococcus mutans GS-5), Streptococcus sobrinus OMZ 176, MRSA (Methicillin Resistant Staphylo), a nosocomial infection-causing bacterium
coccus aureus RIM 0310925) and Staphylococcus aureus 209P IFO 1273
2) can be mentioned.

[0040]

EXAMPLES The present invention will now be described specifically with reference to examples, comparative examples and test examples, but the present invention is not limited to these examples. Example 1 The bark of Yamagusu (Morus bombycis Koidz) was dried (800 g dry weight) and extracted with 3 L of a mixed solvent of dichloromethane-methanol (1: 1) at room temperature for 3 days. The solvent was distilled off from the extract under reduced pressure to obtain about 40 g of extract I.

[Embodiment 2] In the same manner as in Embodiment 1, Magwa (M
orus alb a L.) was dried (800 g dry weight) and extracted with 3 L of a dichloromethane-methanol (1: 1) mixed solvent at room temperature for 3 days. The solvent was distilled off from the extract under reduced pressure to obtain about 40 g of Extract II.

Example 3 Yamaguswa (Morus bombycis Ko)
Dry the bark of idz) (dry weight 750 g) 3 L n-hexane
After defatting at room temperature for 7 days, the mixture was extracted with 3 L of ethyl acetate at room temperature for 3 days. The solvent of the extract was distilled off under reduced pressure, and the extract III
About 18 g were obtained.

[Embodiment 4] In the same manner as in Embodiment 3, Yamaguchi (M
orus bombycis Koidz) dried bark (dry weight 750)
g) After defatting with 3 L of n-hexane at room temperature for 7 days, the mixture was extracted with 3 L of a mixed solvent of dichloromethane-methanol (1: 1) at room temperature for 3 days. The solvent of the extract was distilled off under reduced pressure.
About 22 g of IV was obtained.

[Embodiment 5] In the same manner as in Embodiment 3, Yamagwa (M
orus bombycis Koidz) dried bark (dry weight 750)
g) After degreasing with 3 L of n-hexane for 7 days at room temperature, acetone 3
L was extracted at room temperature for 3 days. The solvent was distilled off from the extract under reduced pressure to obtain about 19 g of extract V.

[Embodiment 6] In the same manner as in Embodiment 3, Yamaguchi (M
orus bombycis Koidz) dried bark (dry weight 750)
g) After defatting with 3 L of n-hexane at room temperature for 7 days, the mixture was extracted with 3 L of tetrahydrofuran at room temperature for 3 days. The solvent was distilled off from the extract under reduced pressure to obtain about 18 g of Extract VI.

Example 7: Yamaguswa (Morus bombycis Ko)
Dry the bark of (idz) (dry weight 900g) n-hexane 3L
At room temperature for 7 days, then use 3 L of toluene at room temperature for 3 days.
Extracted for days. Further, use 3 L of ethyl acetate at room temperature for 3 hours.
Extracted for days. The solvent is distilled off from the toluene extract under reduced pressure,
About 5 g of a toluene extract was obtained. Similarly, the solvent was distilled off from the ethyl acetate extract under reduced pressure to obtain about 18 g of extract VII.

Example 8 Yamagusu (Morus bombycis Ko)
Dry the bark of idz) (750 g dry weight) with 3 L of toluene.
After defatting at room temperature for 3 days, the mixture was extracted with 3 L of ethyl acetate at room temperature for 3 days. The extract was evaporated under reduced pressure to remove the solvent and extract VIII about 15
g was obtained.

Example 9 Yamagusu (Morus bombycis Ko)
Dry the bark of idz) (750 g dry weight) and add dichloromethane 3
After defatting at room temperature for 3 days, use 3 L of ethyl acetate at room temperature for 3 days.
Extracted for days. The solvent of the extract was distilled off under reduced pressure, and the extract IX
About 12 g were obtained.

COMPARATIVE EXAMPLE In the same manner as in Example 1, commercially available mulberry bark was dried (dry weight: 500 g) and extracted with 3 L of a mixed solvent of dichloromethane-methanol (1: 1) at room temperature for 3 days. The solvent was distilled off from the extract under reduced pressure to obtain about 26 g of extract X.

[Reference Example 1] The 12 microorganisms used in the antibacterial activity test for each extract obtained in the examples and the culture conditions thereof are as shown in Table 1.

[0051]

[Table 1]

Test Example 1 Measurement of Antibacterial Activity (Paper Disk Method) (1) Test Microorganisms The following four microorganisms were used in this test. Microbial name Porphyromonas gingi
valis) Streptococcus mutans (Streptococcus mut
ans) Streptococcus sobrinus (Streptococcus sobri
nus ) Staphylococcus aureus
us )

(2) Test Method As a method for measuring the antibacterial activity, the antibacterial activity against various microorganisms including periodontal disease bacteria was evaluated by a paper disk method.
That is, three round pieces of circular filter paper (diameter 8 mm) were prepared for each sample after impregnating a methanol solution containing three levels of 1000 μg, 100 μg, and 10 μg, and placed on an agar plate uniformly inoculated with the above microorganisms. And incubated at 37 ° C for 24 hours.
After the cultivation, the smallest sample filter paper in which an inhibition circle where no microorganisms proliferate was formed around the filter paper was examined. The antibacterial effect is +++ when the inhibition circle is formed with 10 μg, ++, 1000 μg when 100 μg
Was evaluated as +, and the case where the inhibition circle could not be formed was evaluated as-.

Table 2 shows the results of the antibacterial activity of the yamagwa bark extract I and magwa bark extract II obtained in Examples 1 and 2. In addition, mulberry bark extract X obtained in Comparative Example, and synthetic antibacterial agents cetylpyridinium chloride (CPC), antibiotic tetracycline (TC), and natural antibacterial agent hinokitiol (H) as positive controls
The antibacterial activity was measured in the same manner, and the results are shown in Table 2.

[0055]

[Table 2]

As shown in Table 2, as shown in Table 2,
I and Magwa bark extract II have much stronger antibacterial activity than mulberry bark extract X against four microorganisms. Especially,
The antibacterial property against periodontal disease bacteria (Porphyromonas gingivalis) is excellent, and it can be seen that it has the same antibacterial activity as a commercially available antibacterial agent.

Test Example 2 Measurement of Antibacterial Activity (Liquid Medium Dilution Method) (1) Test Microorganisms The following 12 microorganisms were used in this test. Microbial name Porphyromonas gingi
valis ) Actinobacillus actinomycetemcomitance (Act
inobacillus actinomycetemcomitans) Streptococcus mutans (Streptococcus mut
ans) Streptococcus sobrinus (Staphylococcus aureus)
us ) Methicillin-resistant Staphylococcus aureus (Meth
icillin Resistant St aphylococcus aureus = MRSA) E. coli (Esherichia coli) Pseudomonas putida (Pseudomonas putida) Bacillus subtilis (Bacillus subtilis) Arthrobacter globiformis (Arthrobacter gl
obiformis) Aspergillus niger (Aspergillus niger) Penicillium citrinum (Penicillium citrinum)

(2) Test method As a method for measuring the antibacterial activity, the minimum inhibitory concentration (MIC) for various microorganisms including periodontal disease bacteria was determined by a liquid medium dilution method. That is, the above 12 microorganisms were cultured, and the number of bacteria was adjusted to about 1 × 10 ⁸ cells / mL, and the number of spores of fungi was adjusted to about 1 × 10 ⁶ cells / mL. Next, sample concentration 2500
50 μL each of a 0.2-μg / mL methanol solution was added to 1 mL of the above bacterial suspension to give a final sample concentration of 125-0.01 μg / m
L. After the bacterial suspension was cultured under the culture conditions shown in Table 1, the presence or absence of bacterial growth was visually observed. The minimum concentration (μg / mL) at which no growth was observed was defined as the MIC.

Mulberry bark extract obtained in Examples 1, 2 and 3
Table 3 shows the MIC results for I, II, and III. Also,
Mulberry bark extract obtained in Comparative Example, and a synthetic antibacterial agent cetylpyridinium chloride (CP
The MICs of C), the antibiotic tetracycline (TC), and the natural antibacterial agent hinokitiol (H) were also measured in the same manner, and the results are shown in Table 3.

[0060]

[Table 3]

As shown in Table 3, Yamagwa bark extract I,
III and Magwa bark extract II show a unique spectrum of antibacterial activity against 12 microorganisms. That is, each of the above-mentioned extracts is much more antibacterial than the mulberry bark extract against the periodontal disease bacteria Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, and the cariogenic bacteria Streptococcus mutans and Streptococcus sorbinus. It turns out that it is excellent. In particular, it can be seen that the antibacterial activity against the periodontal disease bacterium Porphyromonas gingivalis has an antibacterial activity comparable to that of a synthetic antibacterial agent. On the other hand, the antibacterial activity against Escherichia coli, which is a typical intestinal bacterium, is very weak, and the antibacterial spectrum is narrow.

[Test Example 3] Examination of extraction method Mulberry bark extract obtained by each extraction method of Examples 1 to 9
The antibacterial activities of I to IX were measured. In addition, the yield of mulberry bark extract by hydrophilic organic solvent extraction was also examined together with the antibacterial activity, and the optimal extraction method was evaluated. In the same manner as in Test Example 2, the antibacterial activity of the extract was determined by using a liquid medium dilution method to determine the minimum inhibitory concentration (MIC) against the periodontal disease bacterium Porphyromonas gingivalis.

Mulberry bark extract I obtained in Examples 1 to 9
Table 4 shows the results of the MIC for to IX. Table 4 also shows the results of MIC of the mulberry bark extract obtained in Comparative Example and the synthetic antibacterial agents cetylpyridinium chloride (CPC) and vancomycin (VCM) as positive controls.

[0064]

[Table 4]

As shown in Table 4, Yamaguchi bark extract I
VII = VIII>IX> III
> IV = V = VI> I, so that mulberry bark extract is highly active by degreasing with a hydrophobic organic solvent and then extracting with a hydrophilic organic solvent, rather than extracting mulberry bark directly with a hydrophilic organic solvent. Is obtained. When the antibacterial activities of the extracts obtained by the difference in the hydrophobic solvent for degreasing are compared, the order is as follows: toluene = dichloromethane> hexane. Considering the yield of the extract, toluene is the best.

When comparing hydrophilic solvents for extraction, there was little difference between dichloromethane / methanol (1: 1), ethyl acetate, acetone and tetrahydrofuran.
The antibacterial activity of the ethyl acetate extract was high. Therefore, when the antibacterial activity and the yield of the mulberry bark extract are comprehensively evaluated together, the method with high antibacterial activity and high extract yield shows that the hydrophobic solvent used at the time of degreasing is toluene, and the extracting solvent is acetic acid. It turns out that ethyl is the best.

EXAMPLE 10 Morus bombycis
The bark of Koidz) was dried (800 g dry weight), defatted with n-hexane at room temperature for 7 days, and extracted with ethyl acetate at room temperature for 3 days. Extract was evaporated under reduced pressure to remove the solvent, acetic acid extract from about 1
8 g were obtained. Next, the ethyl acetate extract was dissolved again in ethyl acetate and then adsorbed on Celite. Further, heptane was added and concentration under reduced pressure was repeated, and ethyl acetate was removed by azeotropic distillation. Celite on which the above extract was adsorbed was added to the column containing Celite. The components were fractionated by first flowing n-hexane and then ethyl acetate through the celite column. The fraction eluted with ethyl acetate was concentrated under reduced pressure to obtain 12.7 g of an ethyl acetate fraction.

Next, the ethyl acetate fraction was subjected to column chromatography using silica gel to give dichloromethane-
Elution was performed with a methanol system. Dichloromethane / methanol
= 85/15 was subjected to ODS-HPLC,
Separation was performed with methanol-distilled water = 85/15 to obtain 2.5 g of compound (VI) (content in bark: 0.31%). The physicochemical properties of this compound are as follows.

Optical rotation: [α] _D ²⁵ = −288,7 ° Molecular weight: m / z 286 (EI-MS), Molecular formula: C ₁₇ H ₁₈ O ₄ UV (λ _max MeOH): 288 nm (ε = 30300) IR (ν _max KBr): 3296, 2944, 2924, 1649, 1595, 151
6, 1428, 1374, 1344, 1305, 1270, 1246, 1156, 1089,
^{974, 834, 796cm -1 1 H} -NMR (δdmso-d6, 70 ℃): 1.57 (3H, s), 1.58 (3H,
s), 1.64 (3H, s), 1.67 (3H, s), 2.00 (2H), 2.0-2.1 (3
H), 2.41 (1H, m), 2.54 (1H, brd, J = 15.4), 3.02 (1H, d
d, J = 20.2, 13.2), 3.09 (2H), 3.48 (1H, m), 4.19 (1H,
brd, J = 10.0), 4.75 (1H, m), 5.03 (1H, brt, J = 7.1),
5.17 (1H, m), 5.18 (1H, brs), 5.43 (1H, m), 5.75 (1H,
m), 5.95 (1H, dd, J = 8.3, 2.4), 6.10 (1H, brd, J = 10.
5), 6.11 (1H, d, J = 2.2), 6.25 (1H, dd, J = 8.2, 2.2),
6.32 (1H, d, J = 2.2), 6.73 (1H, d, J = 8.3), 7.11 (1H, d,
J = 8.5), 7.43 (1H, d, J = 9.0), 8.56 (1H, s), 8.76 (1H,
s), 9.11 (1H, s), 9.31 (1H, s), 9.91 (1H, s), 13.16
(1H, s) ppm ¹³ C-NMR (δdmso-d6, 70 ° C): 17.20, 17.21, 20.85,
24.9, 25.08, 25.70, 35.3, 36.49, 36.57, 39.0, 41.0
8, 45.0, 73.0, 94.0, 102.37, 102.64, 105.77,105.9
3, 106.25, 108.45, 113.07, 113.98, 115.40, 120.46,
122.2, 123.69,123.74, 124.0, 127.6, 129.05, 129.1
3, 129.65, 130.06, 135.01, 155.22, 155.25, 155.67,
158.18, 160.89, 160.89, 161.08, 161.51, 161.53, 1
96.3, 208.28 ppm

[Reference Example 2] The 12 microorganisms used in the antibacterial activity test for each compound obtained in Example 10 and the culture method thereof are as shown in Table 5.

[0071]

[Table 5]

Test Example 4 Measurement of Antibacterial Activity (Liquid Medium Dilution Method) (1) Test Microorganisms The following 12 microorganisms were used in this test. Microorganisms name Porphyromonas gingivalis (Porphyromonas ging
ivalis ) Actinobacillus actinomycetemcomitance ( Act
inobacillusactinomycetemcomitans) Streptococcus mutans (Streptococcus mu
tans ) Streptococcus sobrina
inus ) Staphylococcus aureus
us ) MRSA (Methicillin Resistant Staphylococcusaure
us) E. coli (Esherichiacoli) Pseudomonas putida (Pseudomonas putida) Bacillus subtilis (Bacillussubtilis) Asuroha Bu restrictor-Gurohi Bu Forumisu (Arthrobacterglobiformis) Aspergillus niger (Aspergillus niger) Penicillium citrinum (Penicillium citrinum)

(2) Test Method As a method for measuring the antibacterial activity, the minimum inhibitory concentration (MIC) for various microorganisms including periodontal disease bacteria was determined by a liquid medium dilution method. That is, the above 12 microorganisms were cultured, and the number of bacteria was adjusted to about 1 × 10 ⁸ cells / mL, and the number of spores of fungi was adjusted to about 1 × 10 ⁶ cells / mL. Next, sample concentration 2500
50 μL each of a 0.2-μg / mL methanol solution was added to 1 mL of the above bacterial suspension to give a final sample concentration of 125-0.01 μg / m
L. After the bacterial suspension was cultured under the culture conditions shown in Table 5, the presence or absence of bacterial growth was visually observed. The minimum concentration (μg / mL) at which no growth was observed was defined as the MIC.

The results of MIC of the antibacterial compound VI obtained in Example 10 are shown in Table 2 Example. At the same time, as a positive control, a synthetic antibacterial agent cetylpyridinium chloride (CPC) and an antibiotic tetracycline (T
C), the natural antibacterial agent Hinokitiol (H)
Was measured and the results are shown in Table 6.

[0075]

[Table 6]

As shown in Table 6, the present compound VI, which is an example, has a stronger antibacterial activity than a commercially available antibacterial agent as a comparative example, has a specific antibacterial activity spectrum, and exhibits periodontal disease bacteria and dental caries bacteria. (Streptococcus mutans) has high antibacterial activity, but has weak antibacterial activity against other microorganisms. This indicates that the antibacterial agent has a narrow antibacterial activity spectrum and is highly safe for the human body.

[0077]

According to the present invention, there is provided an antibacterial agent which is effective for prevention and treatment of periodontal disease and dental caries and for prevention of hospital-acquired infection and which is highly safe for the human body.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location // C07D 307/80 C07D 307/80 493/04 106 493/04 106A

Claims (8)

[Claims] 1. An antibacterial agent containing a hydrophilic organic solvent extract of mulberry bark as an active ingredient. 2. The antibacterial agent according to claim 1, which is effective against periodontal disease or tooth decay fungi selected from any of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Streptococcus mutans, Streptococcus sobrinas. Agent. 3. The antibacterial agent according to claim 1, which is effective against methicillin resistant Staphylococcus aureus (MRSA). 4. After defatting mulberry bark with a hydrophobic solvent,
A method for extracting an antibacterial substance with a hydrophilic organic solvent. 5. The method according to claim 4, wherein the hydrophobic solvent used for degreasing is any of hydrocarbons, aromatic hydrocarbons, and halogenated hydrocarbons. 6. The method according to claim 1, wherein the hydrophilic organic solvent used for the extraction is a lower fatty acid ester, a lower alcohol, an ether, a ketone, a hydrophilic organic solvent thereof, or a mixture of the hydrophilic organic solvent and the hydrophobic solvent. The method according to claim 4, which is any one of a mixed solvent. 7. A compound of the formula (VI) A flavonoid compound represented by the formula: 8. A compound of the formula (VI) An antibacterial agent containing a flavonoid compound represented by the formula (1) as an active ingredient.