KR20060075868A - New quorum sensing inhibitor - Google Patents

Abstract

The present invention relates to a novel quorum sensing inhibitor, and more particularly, to a new antimicrobial agent using the quorum detection inhibitory effect of vanillin derived from vanilla planifolia . Vanilla Planifolia extract and vanillin of the present invention efficiently inhibit quorum detection of Gram-negative bacteria such as Chromobacterium violaceum CV026, Pseudomonas aeruginosa , etc. Thereby providing antimicrobial activity without side effects.

Quorum Detection, Vanilla Planifolia, Vanillin, Antibacterial

Description

New Quorum Sensing Inhibitor}

1 is a graph showing the results of measuring the quorum detection inhibition of chromobacterium violaseum CV026 ( Chromobacterium violaceum CV026) of the vanilla planifolia extract of the present invention by a liquid culture incubation assay (flask incubation assay) ego,

Figure 2 is a graph showing the result of measuring the quorum detection inhibition of chloromobacterium violaseum CV026 ( Chromobacterium violaceum CV026) of vanillin of the present invention by a liquid incubation assay (flask incubation assay),

Figure 3 is a photograph showing the results of measuring the inhibition of quorum detection for chromobacterium violaseum CV026 ( Chromobacterium violaceum CV026) of vanillin of the present invention by a liquid incubation assay,

Figure 4 is a graph showing the results of measuring the concentration dependence of the quorum detection inhibitory ability of the vanillin ( Chromobacterium violaceum CV026) of vanillin (vanillin) by the liquid culture incubation assay (flask incubation assay).

The present invention relates to a novel antimicrobial agent using the vanilla planifolia extract and vanillin's quorum detection inhibitory action.

It has been shown that signaling between cells and cells that have been thought to occur only in cellular organisms also exists in microorganisms. Microorganisms send and receive signals through a substance called autoinducer (AI), which is used to find a better environment, move or spore, biofilm, bioluminescence, Activities include the synthesis of viral factors. The concentration of self-inducing substances released from microorganisms is a function of sensing the concentration of microorganisms, and after a certain concentration has been formed, a group activity (see Miller and Bassler, Annu. Rev. Microbiol. 55: 165-199, 2001). This series of microbial signaling is called quorum sensing.

For Gram-negative bacteria, N- acyl homoserine lactone (AHL) is used as a self-inducing substance (see Schaffer et al., Proc. Natl. Acad. Sci. USA, 93: 9505-9509). , 1996). AHL is synthesized by the LuxI family of proteins, and is recognized by the LuxR family of proteins and then acts as a transcription factor for specific gene expression. AHL has various structures depending on the length of the hydrocarbon chain forming the acyl group, and the number of carbon atoms used for each species is known to be different. In addition to AHL, the gram-negative bacteria have been identified as 3-hydroxy-palmitic acid methyl ester, heptyl-hydroxy-quinolone (PQS), etc. (see Podbielski and Kreikemeyer, Int. J. Infect. Dis. 8: 81-95, 2004).

In the case of Gram-positive bacteria, oligopeptide is used instead of AHL as a self-inducing substance (Dunny and Leonard, Annu. Rev. Microbiol. 51: 527-564, 1997). Oligopeptides are released into the body after the polypeptide precursors are degraded by a specific protease, and then recognized by the receptor, which is then sequenced through a series of phosphorylation processes, whereby transcription factors bind to the gene to produce specific gene expression. It is known. In addition to oligopeptides, Gram-positive bacteria have been found to use many of the above mentioned substances as signaling agents.

The first attempted method of inhibiting quorum detection was to apply a signal from one microbial species to another. In the absence of autoinduced substances of the same species, the microorganisms were found to have poor quorum detection. Homogeneous lactonases have also been isolated from various microorganisms (Dong et al., Proc. Natl. Acad. Sci. USA 97: 3526-3531, 2001). Furanone and its derivatives isolated from Delisea pulchra have been reported to block quorum detection in most Gram-negative bacteria (Kjelleberg et al., Auqat.Microb.Ecol. 13: 85-93). , 1997).

To date, most antibiotics have been used to control microorganisms, but they have been a serious problem by causing genetic modifications to allow bacteria to have resistance (see Therrien and Levesque, FEMS Microbiol. Rev. 24: 251). -262, 2000).

Therefore, there is a great need for a new concept of antimicrobial agent that provides antimicrobial activity by not killing microorganisms but causing disease, that is, inhibiting quorum detection.

 Therefore, the technical problem to be achieved by the present invention is to provide a new antimicrobial agent through the effective suppression of quorum detection in order to solve the problems of the general antibiotics.

In order to achieve the above technical problem, the present invention provides a novel antimicrobial agent using the vanilla planifolia extract and the quorum detection inhibitory activity of vanillin (vanillin) represented by the following formula (1).

<Formula 1>

As a novel quorum detection inhibitor of the present invention, vanillin is isolated from a plant such as Vanilla planifolia as a single substance, or chemically synthesized. Vanillin may be prepared with a pharmaceutically acceptable carrier, for example, as an antimicrobial agent in various formulations, such as tablets, capsules, soft capsules, solutions, pills, injections and the like.

Hereinafter, the antimicrobial activity by the quorum detection inhibition of vanilla panifolia extract and vanillin according to the present invention will be described in detail.

Vanillin, the active ingredient of the quorum-inhibiting activity according to the present invention, is a compound represented by the following formula (1).

<Formula 1>

Vanillin, a novel quorum detection inhibitor according to the present invention, is a colorless fragrance that is naturally contained in vanilla planifolia and provides various biological activities such as anticancer, antioxidant, antiviral, ice cream, It is well known as a natural physiologically active substance and flavor component applied to confectionery, coffee, fragrances and the like.

The inventors of the present invention found that the vanilla panifolia extract and vanillin, the main component of the plant, are quorum of Gram-negative bacteria such as Chromobacterium violaceum CV026, Pseudomonas aeruginosa , and the like. It was found to exhibit antimicrobial activity by inhibiting detection.

Vanillin, a quorum detection inhibitor according to the present invention, is not limited to that contained in vanilla flanifolia, but is isolated from other plants and synthesized from 4-hydroxybenzaldehyde, or eugenol It includes all chemical methods, such as those obtained from decomposition.

Vanillin, a quorum detection inhibitor according to the present invention, can be separated and purified from an organic solvent extract of vanilla flanifolia or an oil obtained by compressing it. Solvents from which the active ingredient can be extracted include purified water, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, ether, Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether, alone or in combination Can be used. Separation and purification of vanillin from extracts of vanilla panifolia are performed by column chromatography and high-performance liquid chromatography (HPLC) filled with various synthetic resins such as silica gel and activated alumina. Or can be used in parallel. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

The preparation method of the extract of vanilla flanifolia is demonstrated in detail, for example.

First, the dried vanilla flanifolia was ground by a mixer, and then 100 g of the ground vanilla flanifolia sample was extracted using 400 ml of 75% (v / v) methanol as an extraction solvent. The extracted sample was filtered using Whatman No. 2 filter paper, the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, and then freeze-dried to remove the water component, thereby extracting vanilla flanifolia extract (18.6 g). Got it.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided to more completely describe the present invention to those skilled in the art.

Example 1 Inhibitory Effect of Vanilla Planifolia Extract and Vanillin on Chromobacterium Violase CV026 on Chromobacterium violaceum CV026

The Gram-negative bacterium, Chromobacterium violaceum , produces a purple pigment called violacein (violacein) when quorum detection occurs due to autoinduced substances. Chromobacterium violaceum CV026, a mutant of the bacterium, is modified to prevent normal expression of CviI, which synthesizes autoinducers. Therefore, quorum detection can not be observed without artificially inducing self-inducing substances, and this principle can be used to test the inhibition of quorum detection of specific components (see McClean et al. Microbiology 143: 3703-3711, 1997). That is, the quorum detection inhibition of the vanilla panifolia extract and vanillin chromobacterium violaseum CV026 can be determined by measuring the degree of production of the violasane pigment. In the present invention, the quorum detection inhibitory activity of vanilla flanifolia extract and vanillin was determined by the liquid culture method of Experimental Example 1 and Experimental Example 2 below.

Experimental Example 1: Inhibitory Activity of the Quorum Detection of Vanilla Planifolia Extract by Liquid Incubation Assay

The chromobacterium violaseum CV026 bacteria were incubated in 20 ml of Luria-Bertani (LB) liquid medium at 30 ° C. for 12-14 hours and then inoculated into the liquid medium prepared according to the composition of Table 1 below.

flask Furtherance A LB Badge + CV026 B LB Badge + CV026 + HHL C LB Medium + CV026 + HHL + Vanilla Planifolia Extract

Referring to Table 1, Flask A is a control for culturing bacteria in a liquid medium, and Flask B is a purple pigment given 10 μM of N -hexanoyl homoserine lactone (HHL), an autoinducer of chromobacterium violaseum CV026. It is a group that induces the synthesis of the substance violacein (violacein). Flask C was an experimental group containing 10 μM of autoinducer and 1% (w / v) of vanilla flanifolia extract. The contents of flask B and violacein were compared to determine the quorum-inhibiting activity of vanilla flanifolia extract. . Each culture was collected 500 μl every 12 hours and mixed with 500 μl of dimethyl sulfoxide (DMSO) and centrifuged for 3 minutes at 10000 rpm. When the cells precipitate after centrifugation, 200 μl of the supernatant of the solution is dispensed into four wells of a 96-well plate, and the degree of purple color development can be measured with an ELISA microplate reader. Absorbance was measured at a wavelength of 585 nm as shown in FIG. 1.

Referring to Figure 1, the absorbance of the violacein synthesized in each flask according to the incubation time can be observed. Flask A did not show absorbance at a wavelength of 585 nm because violacein, which is a purple pigment, was not synthesized because the bacteria were cultured in a liquid medium containing no self-inducing substance. Flask B was synthesized by artificially-induced self-inducing substance, violet pigment, violacein, and showed high absorbance at a wavelength of 585 nm. Flask C is an experimental group in which both self-inducing substance and vanilla flanifolia extract were put together, and compared with Flask B, the absorbance measured by violacein was significantly decreased. These results indicate that vanilla flanifolia extract reduced the production of violasane by efficiently inhibiting chromobacterium violaseum CV026 quorum detection. The vanilla flanifolia extract did not affect the viability of the chromobacterium violaseum CV026, as measured by the bioburden method. The results showed that the vanilla flanifolia extract did not detect the quorum purely regardless of the survival of the fungus. It can be interpreted as inhibited.

Experimental Example 2: Inhibitory Activity of the Quorum Detection of Vanillin by Flask Incubation Assay

Chromobacterium violaseum CV026 bacteria were incubated in 20 ml of Luria-Bertani (LB) liquid medium at 30 ° C. for 12-14 hours and then inoculated into the liquid medium prepared according to the composition of Table 2 below.

flask Furtherance A LB Badge + CV026 B LB Badge + CV026 + HHL C LB Medium + CV026 + HHL + Vanillin

Referring to Table 2 above, Flask A is a control for culturing bacteria in a liquid medium, and Flask B is a purple pigment given 10 μM of N- hexanoyl homoserine lactone (HHL), an autoinducer of chromobacterium violaseum CV026. It is a group that induces the synthesis of the substance violacein (violacein). Flask C was an experimental group in which 10 μM of autoinducer and 1000 μM of vanillin were put together to compare the contents of flask B and violacein to determine the quorum detection inhibitory activity of vanillin. Each culture was collected 500 μl every 12 hours and mixed with 500 μl DMSO and centrifuged for 3 minutes at 10000 rpm. When the cells precipitate after centrifugation, 200 μl of the supernatant of the solution is dispensed into four wells of a 96-well plate, and the degree of purple color development can be measured with an ELISA microplate reader. Absorbance was measured at a wavelength of 585 nm as shown in FIG. 2.

2, the absorbance of the violacein synthesized in each flask according to the incubation time can be observed. Flask A did not show absorbance at a wavelength of 585 nm because violacein, which is a purple pigment, was not synthesized because the bacteria were cultured in a liquid medium containing no self-inducing substance. Flask B was synthesized by artificially-induced self-inducing substance, violet pigment, violacein, and showed high absorbance at a wavelength of 585 nm. Flask C is an experimental group in which both self-inducing substances and vanillin are added, and compared with Flask B, the absorbance measured by violacein decreases. These results indicate that vanillin reduced violacein production by efficiently inhibiting chromobacterium violaseum CV026 quorum detection.

Figure 3 is a photograph showing that 1000 μM of vanillin of the experiment reduced the synthesis of violacein in the flask to significantly reduce the production of purple pigment. Flask A is not cultured as a culture medium of chromobacterium violaseum CV026, and flask B is active in detecting quorum by adding autoinducer, and violasane is synthesized to have a dark purple color. On the other hand, Flask C added with 1000 μM of vanillin inhibits quorum detection, which decreases the expression of violacein, which can be observed to be pale in purple.

In addition, in order to observe the concentration dependence of vanillin in quorum-inhibiting activity, the concentration of vanillin in flask C of Table 2 was changed to 100, 316.2, and 1000 μM, respectively, and the absorbance was measured after 36 hours. Indicated.

Referring to FIG. 4, it can be seen that the synthesis of violacein decreases depending on the concentration of vanillin when incubated for 36 hours. The absorbance measured by violacein decreased by 16.2, 34.7, and 73.5% as the concentration of vanillin changed to 100, 316.2, and 1000 μM, respectively, indicating that vanillin concentration-dependently inhibits quorum detection of chromobacterium violaseum CV026. It can be seen that.

Vanillin was also found to have no effect on the viability of chromobacterium violaseum CV026, as measured by viable cell count in the same way as vanilla flanifolia extract. It can be interpreted that there is inhibition and concentration dependency.

Example 2: Inhibitory Effect of Vanillin on Pseudomonas aeruginosa PAO1 [ dsred , lasR - PlasB - gfp (ASV)]

The experimental rats were divided into four groups, as shown in Table 3, to measure quorum-sensing inhibitory activity against Pseudomonas aeruginosa PAO1 [ dsred , lasR - PlasB - gfp (ASV)], a Gram-negative bacterium of vanillin. . After mixing 1 ml of Pseudomonas aruzinosa PAO1 culture and 9 ml of 1.1% (w / v) alginate, 0.04 ml of the mixed solution was injected into the trachea of the rat. N- butanoyl homoserine lactone (BHL) and vanillin were induced intravenously by Pseudomonas aruginosa PAO1.

The quorum-sensing inhibitory activity of vanillin against Pseudomonas aruginosa PAO1 was determined by the content of Green Fluorescent Protein (GFP) and viable cell count (Wu et al. J. Antimicrob. Chemother. 53: 1054-1061, 2004) . The amount of GFP generated as a result of quorum detection of Pseudomonas aruzinosa PAO1 was measured by epifluorescence microscopy and expressed as relative to the control group. The viable cell count was measured by placing the lungs isolated from mice into PBS buffer to isolate the bacteria and incubating the cells at 37 ° C. for 24 hours using a pour plate method.

In Table 3, it can be seen that the addition of vanillin decreases GFP content, a measure of quorum detection of Pseudomonas aruzinosa PAO1, in a concentration-dependent manner. In addition, it can be seen from Table 3 that the number of bacteria of Pseudomonas aruzinosa PAO1 found in the lungs is greatly reduced by the administration of vanillin. These results indicate that vanillin provides antimicrobial activity by efficiently inhibiting quorum detection of Pseudomonas aruzinosa PAO1.

group Self-inducing substance (mg / kg) Vanillin (mg / kg) GFP concentration (%) Viable Count (CFU / Lung) Control 20 0 100 2.0 × 105 Experimental group 1 20 5 73.4 1.1 × 105 Experiment group 2 20 10 38.7 4.6 × 104 Experiment group 3 20 20 9.1 7.3 × 103

Vanilla planifolia extracts and vanillin are sensitive to Gram-negative bacteria, such as Chromobacterium violaceum CV026 and Pseudomonas aeruginosa . Antibacterial activity is shown through the inhibition of Vanilla Planifolia and Vanillin are ingredients extracted from natural products and consumed as foods, and have no side effects on the human body, and can provide antibacterial activity without side effects such as the production of resistant strains, the biggest problem of general antibiotics. Industrial utility is very high.

Claims (4)

Vanilla planifolia extract, a quorum sensing inhibitor Vanillin, a quorum sensing inhibitor represented by Formula 1; <Formula 1>

According to claim 1 and 2, Vanilla planifolia extract and vanillin represented by the formula (1) is Chromobacterium violaceum CV026 ( Chromobacterium violaceum CV026), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) Inhibition of quorum detection of Gram-negative bacteria, including The antimicrobial agent and antimicrobial composition according to claim 1 and 2, wherein the vanilla planifolia extract and the quorum sensing inhibitory action of vanillin represented by Chemical Formula 1 are used.

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