WO2010119638A1

WO2010119638A1 - Biofilm formation inhibitor

Info

Publication number: WO2010119638A1
Application number: PCT/JP2010/002477
Authority: WO
Inventors: 土屋友房; 塩田澄子
Original assignee: 国立大学法人岡山大学; 学校法人就実学園
Priority date: 2009-04-13
Filing date: 2010-04-05
Publication date: 2010-10-21
Also published as: JPWO2010119638A1

Abstract

A biofilm formation inhibitor which effectively acts on the formation of a biofilm without disrupting the resident bacterial flora or developing drug resistant bacteria is provided. More specifically, a biofilm formation inhibitor which effectively acts on the formation of a biofilm derived from a Gram-positive coccus belonging to the genus Staphylococcus is provided. Further, a method for inhibiting the formation of a biofilm in which a treatment is performed using the biofilm formation inhibitor is provided. A diterpenoid compound contained in a plant belonging to the family Pinaceae effectively has a strong inhibitory effect on the formation of a biofilm without disrupting the resident bacterial flora or developing drug resistant bacteria, and therefore can be used as an effective biofilm formation inhibitor.

Description

Biofilm formation inhibitor

The present invention relates to a biofilm formation inhibitor containing a diterpenoid compound as an active ingredient. Furthermore, it is related with the biofilm formation inhibition method processed using this biofilm formation inhibitor.

This application claims the priority of Japanese Patent Application No. 2009-97207, which is incorporated herein by reference.

A biofilm is a biological group formed by being covered with an extracellular matrix and adhering to each other or from cells to the surface / interface. Biofilm formation is a problem in the medical field, wastewater treatment facilities, general households, and the like, and particularly in the medical field.

In the medical field, since biofilms by oral bacteria are related to periodontal disease, many inhibitors for biofilms have been reported in the dental field. For example, epigallocatechin-3-gallate (EGGG), a kind of catechin, has been patent-patented and disclosed as a biofilm inhibitor by Eikenella corrodens , a periodontal disease-related bacterium (Patent Document) 1). Cranberry extract has been patented and disclosed as a biofilm inhibitor by Porphyromonas gingivalis and Treponema denticola , which are one of the causative agents of periodontal disease ( Patent Document 2).

Of the bacteria, many Gram-positive bacteria are attenuated bacteria that are often present in humans and the environment and are not a problem for healthy people. However, these attenuated bacteria are also serious in immunocompromised and susceptible patients. May cause infection. Staphylococci are Gram-positive cocci and are resident bacteria such as human skin, nasal cavity and gastrointestinal tract, but in medical settings, surgical site infection, bloodstream infection, respiratory infection, urinary tract infection mainly in easily infected patients. May cause endocarditis, meningitis, septicemia, etc., and is one of the most important hospital infection-causing bacteria.

In the medical field, catheter-related bloodstream infection (CR-BSI) and urethral indwelling catheter-related urinary tract infection (UA-UTI) involving biofilms have become problems. Infection is caused by bacteria, such as staphylococci, colonizing medical devices such as vascular and urinary catheters to form a biofilm. Once a biofilm is formed, it becomes difficult to treat because bacteria escape from attack by the host's immune system or the biofilm prevents the penetration of antimicrobial agents. Staphylococcus aureus is a causative agent for surgical site infections, bloodstream infections, respiratory infections, urinary tract infections, etc. in highly infected patients mainly in medical institutions. It may become. In particular, the invasion route of methicillin-resistant Staphylococcus aureus (MRSA), which is the causative agent of nosocomial infection, is said to be 47.8% by blood indwelling catheter and 13.9% by urinary catheter indwelling. It becomes a problem because it becomes intractable and becomes severe at the same time.

Examples of substances that suppress staphylococcal biofilms include farnesol, a kind of linear sesquiterpene (Non-patent Document 1), and berberine, a kind of benzylisoquinoline alkaloid (Non-patent Document 2). It has been reported.

However, farnesol and berberine, which have already been reported, are known to have antibacterial activity against Staphylococcus aureus, and at the reported concentrations, they suppress biofilm formation while inhibiting the growth of Staphylococcus aureus. There is a possibility. Berberine is a substrate for NorA. NorA is a major multidrug efflux pump for Staphylococcus aureus and is said to contribute to the multidrug resistance of Staphylococcus aureus. By using berberine regularly, the expression of this NorA is enhanced, and a multidrug-resistant mutant strain may be induced. Those with antibacterial activity, such as farnesol and berberine, still have problems in disturbing the resident bacterial flora and eventually inducing resistance, and medical devices such as pharmaceuticals and medical instruments There is nothing in practical use.

Also, in general households and other facilities, the formation of biofilms is a problem, for example, around water and wastewater treatment. Even in such a case, development of a biofilm formation inhibitor that is capable of effectively inhibiting the formation of a biofilm while being safe for a living body such as a human is desired.

Japanese Patent Laid-Open No. 2008-13458 JP 2007-91703 A

An object of the present invention is to provide a biofilm formation inhibitor that effectively acts on biofilm formation without disturbing the resident bacterial flora or inducing drug-resistant bacteria. More specifically, an object of the present invention is to provide a biofilm formation inhibitor that effectively acts on biofilm formation derived from gram-positive cocci belonging to the genus Staphylococcus. Furthermore, it aims at providing the biofilm formation inhibition method processed using this biofilm formation inhibitor.

As a result of intensive studies to solve the above problems, the present inventors have found that diterpenoid compounds contained in Pinaceae plants do not disturb the resident bacterial flora or induce drug-resistant bacteria, and thus biofilms. The present invention was completed by finding that it has a strong inhibitory effect on formation.

That is, this invention consists of the following.
1. A biofilm formation inhibitor comprising a diterpenoid compound as an active ingredient.
2. 2. The biofilm formation inhibitor according to 1 above, wherein the diterpenoid compound is a cyclic diterpenoid compound.
3. 3. The biofilm formation inhibitor according to 2 above, wherein the cyclic diterpenoid compound is an abietane diterpenoid compound.
4). Item 4. The abietane diterpenoid compound is any one or more of abietic acid, dehydroabietic acid, neoabietic acid, pimaric acid, parastrinic acid, levopimaric acid, and pharmacologically acceptable derivatives thereof. Biofilm formation inhibitor.
5). 5. The biofilm formation inhibitor according to any one of items 1 to 4, wherein the biofilm formation inhibitor comprises rosin.
6). 8. The biofilm formation inhibitor according to any one of 1 to 7 above, wherein the biofilm is derived from gram-positive cocci belonging to the genus Staphylococcus.
7). 7. The biofilm formation inhibitor according to 6 above, wherein the gram-positive cocci belonging to the genus Staphylococcus is any one selected from Staphylococcus aureus, S. epidermidis and rot staphylococci.
8). 8. The biofilm formation inhibitor according to 7 above, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Staphylococcus aureus (VRSA).
9. 9. A medical device treated with the biofilm formation inhibitor according to any one of 1 to 8 above.
10. The medical device treated with the biofilm formation inhibitor is coated with the biofilm formation inhibitor on the surface of the medical device, or the biofilm formation inhibitor penetrates the medical device 10. The medical device according to 9 above.
11. 9. A biofilm formation inhibition method comprising a step of treating with the biofilm formation inhibitor according to any one of 1 to 8 above.
12 12. The biofilm formation inhibition method according to 11 above, wherein the object to be treated using the biofilm formation inhibitor is a medical device.
13. 12. The biofilm formation inhibition method according to 11 above, wherein the object to be treated using the biofilm formation inhibitor is wastewater treatment equipment.

One or more Gram-positive cocci belonging to the staphylococcus that are clinically problematic, for example, Staphylococcus aureus (including MRSA), Staphylococcus epidermidis and rot staphylococci, which are clinically problematic by the biofilm formation inhibitor of the present invention Biofilm formation from certain staphylococci can be inhibited. Unlike an antibacterial agent, the biofilm formation inhibitor inhibits only biofilm formation without killing bacteria. This property is 1. Does not disturb the resident bacterial flora at each site including the skin. It has two advantages of not inducing drug-resistant bacteria. Catheters that have been soaked with antibacterial substances (antibacterial drugs and silver) have already been clinically applied, but none of them are intended for antibacterial properties, and have not been designed to inhibit biofilm formation. A method of using the biofilm formation inhibitor containing the diterpenoid compound of the present invention as an active ingredient by coating or immersing a medical device, for example, a medical device such as a catheter, a medical dressing material or a medical tape, It can be a new prevention method for infectious diseases related to medical devices.

It is a figure which shows biofilm formation ability when it culture | cultivates about the various S. aureus strains in the culture medium containing glucose of each concentration. (Reference Example 1) It is a figure which shows the biofilm formation inhibitory ability by a Staphylococcus aureus (N315 strain | stump | stock) of abietic acid and another naturally-derived substance. (Reference Example 2) It is a figure which shows the biofilm formation inhibitory ability by a Staphylococcus aureus (N315 strain | stump | stock) of abietic acid and six types of naturally-derived substances. (Reference Example 3) It is a figure which shows the biofilm formation inhibitory ability by Staphylococcus aureus (N315 strain | stump | stock, Newman strain | stump | stock) of abietic acid. Example 1 It is a figure which shows the influence which abietic acid has on the biofilm formation inhibitory ability by S. aureus (N315 strain | stump | stock), and the viable count of free bacteria. (Example 2) It is a figure which shows the influence which abietic acid and its analog have on the biofilm formation inhibitory ability by Staphylococcus aureus (N315 strain | stump | stock), and the viable count of free bacteria. (Example 3) It is a figure which shows the biofilm formation inhibitory ability by a Staphylococcus aureus (N315 strain | stump | stock) of abietic acid, dehydroabietic acid, and rosin. (Example 4) It is a figure which shows the biofilm formation inhibitory ability by Staphylococcus epidermidis (clinical isolate) of abietic acid, dehydroabietic acid, and rosin. (Example 5)

The present invention relates to a biofilm formation inhibitor containing a diterpenoid compound as an active ingredient. In the present invention, the diterpenoid compound contained in the biofilm formation inhibitor is preferably a cyclic diterpenoid compound. As the diterpenoid compound, a labdan type, a pyramant type, an abietane type, and the like are known, and as the biofilm formation inhibitor of the present invention, an abietan diterpenoid compound is particularly suitable. Abietane diterpenoid compounds are compounds that are extracted from Pinaceae plants such as pine and cedar, for example, and are compounds that can also be extracted from plants of the family Lamiaceae. Specific examples of such compounds include abietic acid, dehydroabietic acid, neoabietic acid, pimaric acid, parastolic acid, levopimaric acid, and the like. In the present invention, the abietane diterpenoid compound may be any of these pharmacologically acceptable derivatives in addition to the above compound. In the present invention, the pharmacologically acceptable derivative is a diterpenoid compound, preferably an abietane diterpenoid compound, more preferably a derivative having a basic skeleton in common with the above-mentioned compound and having a biofilm formation inhibitory ability Say. The diterpenoid compound of the present invention may be derived from a natural product extracted from the above plant, or may be a synthesized compound. Moreover, all the stereoisomers (diastereomers, epimers, enantiomers, etc.) or racemates are included.

Examples of abietic acid, which is a typical abietane diterpenoid compound, and similar compounds are shown below.

The above-mentioned compound used as an active ingredient in the biofilm formation inhibitor of the present invention may be obtained commercially or extracted from a natural product. When extracting from a natural product, this extract can be manufactured by a well-known method or all the methods developed in the future. For example, an extract can be obtained by extracting by an appropriate method using water, an organic solvent, or a mixture thereof. The organic solvent is not particularly limited, and examples thereof include alcohols such as methanol, ethanol, isopropanol, and butanol, acetone, ethyl acetate, ethyl ether, and the like, and these may be used alone. Two or more kinds can be used in combination. These compounds are preferably extracted by immersion using, for example, 70% acetone aqueous solution and 100% methanol as solvents. The extracted compound may be further purified. For example, it may be purified by column chromatography or the like. Further, rosin (a sap obtained by scratching a pine trunk by steam distillation to remove a volatile substance) can be obtained alone or by adding an acid and heating.

In the present invention, the biofilm formation inhibitor is a diterpenoid compound having an ability to inhibit biofilm formation, preferably an abiethane diterpenoid compound, more preferably one of the above-mentioned compounds alone or as a mixture of two or more. can do. In addition, the biofilm formation inhibitor of the present invention may contain a purified compound or a natural product as long as it contains the above-mentioned compound having biofilm formation inhibiting ability. The extract may be contained as it is. An example of such an extract is rosin. The biofilm formation inhibitor of the present invention may contain rosin or rosin itself. Rosin contains about 90% of diterpene resin acid, and its main component is abietic acid. In other words, it is a non-volatile component of pine resin that is contained in a large amount in Pinaceae plants, and is technically composed mainly of various isomers called resin acids. Rosin can be produced by removing essential oils and volatile substances from the secretions of Pinaceae plants. The rosin may be natural rosin or various synthetic rosins.

In the present invention, the diterpenoid compound contained in the biofilm formation inhibitor may be used at any concentration as long as it can inhibit biofilm formation. The minimum inhibitory concentration: MIC (hereinafter also referred to simply as “MIC”) is characterized by being able to be used at a lower concentration. For example, it can be used at a concentration of 1/2 or less, preferably 1/4 or less, more preferably 1/8 or less of MIC. By inhibiting the formation of biofilm at such a low concentration, it is possible to prevent disturbance of the resident bacterial flora and induction of drug-resistant bacteria. Specifically, the concentration of abietic acid can be 8 to 64 ppm, preferably 16 to 32 ppm. In other diterpenoid compounds, the concentration of dehydroabietic acid, neoabietic acid, and pimaric acid can be 2 to 8 ppm, preferably 2 to 4 ppm.

The biofilm formation inhibitor of the present invention is not particularly limited as long as it contains the diterpenoid compound as the active ingredient in the above concentration. Examples of the solvent for the diterpenoid compound include dimethyl sulfoxide (DMSO), alcohols, acetone and the like.

The origin of the biofilm on which the biofilm formation inhibitor of the present invention acts is not particularly limited, but the biofilm formation inhibitor of the present invention is against that formed by gram-positive cocci belonging to Staphylococcus. In particular, it has inhibitory activity. The biofilm formation inhibitor of the present invention is preferably a staphylococcus genus, preferably a biofilm formation derived from S. aureus , S. epidermidis or S. saprophyticus. Is effectively inhibited. Some Staphylococcus aureus strains have capsular polysaccharides, and these strains form biofilms, which can reduce the effectiveness of antimicrobial agents and can reduce their effectiveness. The biofilm formation inhibitor of the present invention effectively acts on inhibition of biofilm formation from Staphylococcus aureus, MRSA and vancomycin-resistant Staphylococcus aureus (VRSA).

The present invention also extends to medical devices processed with the biofilm formation inhibitor, such as medical devices, medical dressings and medical tapes. A medical device treated with a biofilm formation inhibitor means that a part or the whole of the surface of the medical device is coated with a biofilm formation inhibitor, or a part or the whole of the medical device is a biomaterial. This refers to the penetration of a film formation inhibitor.

In the present invention, medical tools include, but are not particularly limited to, apparatuses, devices, and devices used for medical purposes. For example, it is feared that a medical device that can be placed in a living body, that is, an implant, will lead to serious infection by the formation of a biofilm. Examples of implants include, but are not limited to, for example, catheters, artificial teeth (dental implants), bolts for fixing bones in the treatment of fractures, rheumatism, pacemakers, artificial heart valves, artificial joints, voice prostheses, contact lenses, etc. Is mentioned. Examples of the catheter include a urinary catheter, an abdominal catheter, and a central venous catheter. In this way, by treating a medical device that can be placed in a living body with the biofilm formation inhibitor of the present invention, the resident bacterial flora of the placed living body is disturbed or drug-resistant bacteria are induced. Without effectively inhibiting biofilm formation in medical devices. In addition, even medical devices that are not intended to be placed in a living body, but are feared to have some undesirable effects due to the formation of a biofilm, such as medical devices, medical dressing materials and medical tapes, It is included in the scope of the present invention.

The present invention also extends to a biofilm formation inhibition method including a step of treating with a biofilm formation inhibitor. Here, the processing object to be processed using the biofilm formation inhibitor can be any object that can cause the formation of a biofilm in addition to the medical device described above. For example, pollution caused by biofilm such as slime becomes a problem in wastewater treatment facilities in ordinary households and other business establishments. Specifically, facilities related to water such as a kitchen, a washroom, and a bathroom, other water supply facilities, and drainage facilities such as a drain pipe and a drain ditch are included. Moreover, it can be applied to uses such as tackifiers for adhesive bandages, base materials for ointments, antiskid, and mixed with soap.

In the above, the treatment includes a step of bringing a treatment object into contact with the biofilm formation inhibitor of the present invention. Contacting with a biofilm formation inhibitor refers to operations such as coating, spraying, dipping, and washing. If the object to be treated is a small-scale object such as a medical device, a biofilm formation inhibitor is applied to or sprayed on a part of the surface of the medical device to perform a coating process or immerse it. It can be soaked or washed. In the case where the treatment object is a large scale such as a water supply facility or a wastewater treatment facility, the biofilm formation inhibitor of the present invention can be poured into a desired site, applied, or washed. In such a case, the compound itself contained in the biofilm formation inhibitor may contain an extract from a natural product as it is.

In the above case, if necessary, an antiseptic effective against bacteria capable of forming a biofilm, such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, amphoteric surfactant, alcohol, sodium hypochlorite, povidone iodine , Glutaral or the like may be used in combination, or may be processed before or after the above processing. In addition, it is said that chlorhexidine is excellent in terms of a sustained effect against staphylococci.

Hereinafter, in order to make the understanding of the present invention more reliable, details of the present invention will be described in a reference example, and the contents of the present invention will be described in detail in a specific example. However, it is clear that the present invention is not limited to the scope of the following examples.

(Reference Example 1) Production of Biofilm In this reference example, biofilm production conditions used for screening an active ingredient of a biofilm formation inhibitor were examined. Each cell of various strains of S. aureus is diluted with BHI (Brain Heart Infusion) medium, and then 200 μl is dispensed into a 96-well plate, so that each glucose is 0.1-2.0%. Was added. This was incubated at 37 ° C. for 48 hours, and the formed biofilm was stained with crystal violet. After washing each well with distilled water, the absorbance at 570 nm was measured for crystal violet eluted with 200 μl of DMSO to determine the biofilm forming ability.

The above examination results are shown in FIG. As a result, among various S. aureus strains, S. aureus N315 was considered suitable for biofilm formation and used in the following examples. The N315 strain is a standard strain of MRSA, the entire genome sequence has already been determined, and it is easy to perform genetic analysis. In order to further enhance the biofilm forming ability of this strain, it was cultured using a medium containing 1% glucose.

Reference Example 2 Screening for Biofilm Formation Inhibiting Substance In this reference example, screening was performed to select a biofilm formation inhibiting substance that is an active ingredient of a biofilm formation inhibitor. For screening, 35 kinds of naturally derived substances were used. First, the minimum growth inhibitory concentration (MIC) of these naturally-derived substances against S. aureus N315 strain was determined, and the biofilm when the concentration of 1/8 or less of MIC that does not affect the growth was added to the medium. The ability to form was confirmed. Each candidate compound was diluted in a medium containing 1% DMSO. The MIC for S. aureus N315 strain is shown in Table 1.

S. aureus N315 strain cells were diluted 100-fold with BHI (1% glucose added) medium, then dispensed 200 μl each into a 96-well plate, and 2 μl of each sample solution was further added. Here, among the candidate compounds, eugenol and quercetin were adjusted so that the final concentration was 1/16 MIC, and the others were 1/8 MIC. The control was BHI medium containing neither sample nor solvent. These were cultured at 37 ° C. for 48 hours.
The formed biofilm was dyed using crystal violet, and the biofilm forming ability was measured by the same method as in Reference Example 1. The number after the substance name represents the concentration, and the unit is μg / ml.

As a result, abietic acid (C ₂₀ H ₃₀ O ₂ ), eugenol (eugenol, C ₁₀ H ₁₂ O ₂ ), capsaicin (capsaicin, C ₁₇ H ₂₇ NO ₃ ), nerol (nerol, C ₁₀ H ₁₈ O ), naringenin _{_{(naringenin, C 15 H 12 O}} 5), citronellol _{_{(citronellol, C 10 H 20 O}} ), dihydrocapsaicin _{_{(dihydrocapzaicin, C 18 H 29 NO}} 3), 8 of geraniol _{_{(geraniol, C 10 H 18 O}} ) Inhibition of biofilm formation was observed in several types of substances. In particular, it was confirmed that abietic acid strongly inhibits biofilm formation by Staphylococcus aureus N315 strain. Citronellol, an essential oil component, was also confirmed to inhibit biofilm formation to some extent. Here, geraniol is a kind of linear monoterpenoid, and nerol is a kind of monoterpene. Capsaicin is a kind of compound called capsaicinoid among alkaloids. Eugenol is a kind of phenylpropanoid having a structure in which an allyl group is substituted for guaiacol. Naringenin is a flavanone and belongs to the flavonoids.

(Reference Example 3) Screening for biofilm formation inhibitors (2)
In the present Reference Example, as a result of screening in Reference Example 2, in the N315 strain of Staphylococcus aureus, biofilm production was relatively strongly suppressed, and abietic acid, eugenol, capsaicin, nerol that did not significantly affect the growth of the bacteria , Citronellol, and geraniol were examined for concentration dependence of biofilm formation inhibitory activity using samples having a concentration lower than 1/8 of MIC.

As a result, it was found that abietic acid and citronellol suppressed biofilm formation in a concentration-dependent manner. Abietic acid having an inhibitory action at a low concentration was used as an effective biofilm formation inhibitor.

(Example 1) Confirmation of biofilm formation inhibitory action of abietic acid Based on the results of Reference Examples 2 and 3, this example was confirmed with respect to abietic acid that was particularly excellent in biofilm formation inhibitory action. Effect of abietic acid on biofilm formation of Newman strain used in experiments related to the pathogenicity of methicillin-sensitive Staphylococcus aureus to confirm whether the biofilm formation inhibitory action is unique to S. aureus N315 I also investigated. S. aureus N315 cell line or Newman cell line is diluted 100 times with BHI (1% glucose added) medium, then 200 μl is dispensed into 96-well plates, and final concentrations are 0, 4, 8, 16, 32 μg. 2 μl of abietic acid solution (final concentration of DMSO as a solvent is 1%) was added so as to be / ml. These were cultured at 37 ° C. for 48 hours. The formed biofilm was dyed using crystal violet, and the biofilm forming ability was measured by the same method as in Reference Example 1.

As a result, as shown in FIG. 4, it was confirmed that biofilm formation was inhibited by abietic acid in both the N315 strain and the Newman strain.

(Example 2) Confirmation of biofilm formation inhibitory action of abietic acid In this example, the relationship between the biofilm formation inhibitory action of abietic acid and the growth inhibitory action of bacteria was examined. After the S. aureus N315 strain cells were diluted 100-fold with BHI (1% glucose added) medium by the same method as in Example 1, 200 μl each was dispensed into a 96-well plate, and 0, 4, 8, 16, 32 μg. 2 μl / ml of abietic acid solution (final concentration of DMSO as a solvent is 1%) was added. The control was BHI medium containing neither sample nor solvent. These were cultured at 37 ° C. for 48 hours. The formed biofilm was dyed using crystal violet, and the biofilm forming ability was measured by the same method as in Reference Example 1. In addition, the number of bacteria in the medium in the well (floating bacteria) was also measured. Culture supernatants of 96 well plates each well was applied to nutrient agar bacterial suspension was diluted with sterile saline to 1 of 10 ⁴ minutes, and counted the number of colonies after static culture at 37 ° C., the number of bacteria Was measured. Viable counts were expressed as log ₁₀ CFU / ml.

As a result, as shown in FIG. 5, it was confirmed that as the concentration of abietic acid increased, the biofilm forming ability decreased, while the number of floating bacteria in each well increased. This indicates that abietic acid only inhibited the ability to form biofilm without killing S. aureus.

(Example 3) Confirmation of biofilm formation inhibitory action of abietic acid and its similar substances Since abietic acid was found to have a biofilm formation inhibitory action, similar compounds of abietic acid were also confirmed in this example. The S. aureus N315 strain cells were diluted 100-fold with BHI (1% glucose added) medium by the same method as in Example 1, and then 200 μl each was dispensed into a 96-well plate, and each concentration of abietic acid and dehydroabietic acid 2 μl each of (dehydroabietic acid), neoabietic acid, pimaric acid or tanshinones (final concentration of DMSO as solvent is 1%) was added. The control was BHI medium containing neither sample nor solvent. A BHI medium containing 1% DMSO was also used as a comparative control. These were cultured at 37 ° C. for 48 hours. The formed biofilm was dyed using crystal violet, and the biofilm forming ability was measured by the same method as in Reference Example 1. The structural formulas of abietic acid and its similar substances are as follows.

As a result, as shown in FIG. 6, a strong biofilm formation inhibitory action was observed for dehydroabietic acid, neoabietic acid, and pimaric acid, which are diterpenoids contained in Pinaceae plants, as in the case of abietic acid. Further, the concentration of each abietic acid-like compound added to the medium was 2-4 μg / ml, which was lower than that of abietic acid, compared to 32 μg / ml for the concentration of abietic acid.

(Example 4) Confirmation of biofilm formation inhibitory action of rosin (1)
Based on the fact that abietic acid and dehydroabietic acid had a biofilm formation inhibitory effect, it was examined whether rosin containing abietic acid as a main component also had a biofilm inhibitory effect against Staphylococcus aureus. In this example, N315 strain cells were used for Staphylococcus aureus, and rosin was used from Wako Pure Chemical Industries, Ltd. The composition of rosin used is resin acid (80-97%), non-fighting and other small amounts. Resin acids mainly include abietic acid, neoabietic acid, and parastrinic acid, and also include levopimaric acid, dehydroabietic acid, pimaric acid, and the like. Moreover, the biofilm inhibitory effect was confirmed also about abietic acid and dehydroabietic acid. The final concentrations of abietic acid and dehydroabietic acid were 1/16 and 1/8 of MIC, and the final concentrations of rosin were 1/32, 1/16 and 1/8 of MIC.

S. aureus N315 strain cells were diluted 100-fold with BHI (1% glucose added) medium, then 200 μl each was dispensed into a 96-well plate, and further 2 μl of abietic acid solution (16 μl / 32 μg / ml) was added. The final concentration of DMSO as a solvent was 1%). For dehydroabietic acid, the solution was added to 2 μl medium to 4 or 8 μg / ml and rosin to a final concentration of 8, 16 or 32 μg / ml. These were then statically cultured at 37 ° C. for 48 hours. After the formed biofilm was stained with crystal violet, each well was washed with distilled water, and then the absorbance at 570 nm was measured for crystal violet eluted with DMSO 200 μl to determine the biofilm formation ability. .

As a result, in staphylococcus aureus, the same biofilm inhibitory effect as that of abietic acid was observed for rosin containing abietic acid as a main component. Rosin is a resin obtained by removing essential oils and volatile substances from the secretions of Pinaceae plants. It contains 90% diterpene resin acid and its main component is abietic acid. It has a wide range of uses such as adhesives for adhesive bandages and base materials for ointments, anti-slip materials, and soap.

(Example 5) Confirmation of biofilm formation inhibitory action of rosin (2)
In the same manner as in Example 4 except that Staphylococcus epidermidis (clinical isolate) was used instead of Staphylococcus aureus, the biofilm formation inhibitory action of abietic acid, dehydroabietic acid and rosin on Staphylococcus epidermidis was confirmed. did.

As a result of the above, rosin was found to inhibit biofilm formation in Staphylococcus epidermidis as well as Staphylococcus aureus.

Staphylococcus epidermidis is a bacterium that is resident in most human epidermis and nasal cavity and has almost no pathogenicity. However, severe infection may occur if Staphylococcus epidermidis resident on the skin infects an easily infected patient when wearing a foreign body. Staphylococcus epidermidis is highly adherent to a catheter or the like placed in the body like S. aureus, and easily forms a biofilm. As a causative bacterium of catheter-related bloodstream infection, Staphylococcus epidermidis has a higher ratio than Staphylococcus aureus.

As described above in detail, the biofilm formation inhibitor of the present invention can inhibit biofilm formation derived from Staphylococcus aureus (including MRSA), which is a clinical problem. Unlike an antibacterial agent, the biofilm formation inhibitor can inhibit only biofilm formation without killing bacteria. This property is 1. Does not disturb the normal bacterial flora at each site including the skin. It has two advantages of not inducing drug-resistant bacteria. Catheters that have been impregnated with antibacterial substances (antibacterial drugs and silver) have already been clinically applied, but none of them are intended for antibacterial properties, and none have been designed to inhibit biofilm formation.

The method of using the biofilm formation inhibitor containing the diterpenoid compound of the present invention as an active ingredient by coating or infiltrating various medical devices such as catheters is used for infectious diseases related to medical devices such as catheters. It can be a new prevention method. Furthermore, the formation of biofilms can be effectively inhibited by treating with the biofilm formation inhibitor of the present invention even around water in each facility including general households.

In addition, the rosin containing abietic acid as a main component has the same biofilm inhibitory effect as abietic acid against Staphylococcus aureus and Staphylococcus epidermidis, so the biofilm formation inhibitor of the present invention containing rosin is It can be used in a wide range of applications, including adhesives for adhesive bandages and base materials for ointments, anti-slip materials, and soaps. Staphylococcus epidermidis is highly adherent to a catheter or the like placed in the body like S. aureus, and easily forms a biofilm. As a causative bacterium of catheter-related bloodstream infection, Staphylococcus epidermidis has a higher ratio than Staphylococcus aureus. From these things, it is possible to provide a highly safe medical device in which the formation of biofilm is suppressed by coating or infiltrating various medical devices with the biofilm formation inhibitor of the present invention. it can.

Claims

A biofilm formation inhibitor comprising a diterpenoid compound as an active ingredient.
The biofilm formation inhibitor according to claim 1, wherein the diterpenoid compound is a cyclic diterpenoid compound.
The biofilm formation inhibitor according to claim 2, wherein the cyclic diterpenoid compound is an abietane diterpenoid compound.
4. The abietane diterpenoid compound is any one or more of abietic acid, dehydroabietic acid, neoabietic acid, pimaric acid, parastrinic acid, levopimaric acid and pharmacologically acceptable derivatives thereof. The biofilm formation inhibitor as described.
The biofilm formation inhibitor according to any one of claims 1 to 4, wherein the biofilm formation inhibitor comprises rosin.
The biofilm formation inhibitor according to any one of claims 1 to 7, wherein the biofilm is derived from gram-positive cocci belonging to the genus Staphylococcus.
The biofilm formation inhibitor according to claim 6, wherein the Gram-positive cocci belonging to the genus Staphylococcus is any one selected from Staphylococcus aureus, S. epidermidis and rot staphylococci.
The biofilm formation inhibitor according to claim 7, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Staphylococcus aureus (VRSA).
A medical device treated with the biofilm formation inhibitor according to any one of claims 1 to 8.
The medical device processed with the biofilm formation inhibitor is coated with the biofilm formation inhibitor on the surface of the medical device, or the biofilm formation inhibitor penetrates the medical device. The medical device according to claim 9.
A biofilm formation inhibiting method comprising a step of treating with the biofilm formation inhibitor according to any one of claims 1 to 8.
The biofilm formation inhibition method of Claim 11 whose target object processed using a biofilm formation inhibitor is a medical device.
The biofilm formation inhibition method of Claim 11 whose target object processed using a biofilm formation inhibitor is a wastewater treatment facility.