KR101817186B1 - Composition for inhibiting quorum sensing comprising Citrus junos extact as effective component - Google Patents

Abstract

The present invention relates to a composition for inhibiting the recognition of quorum water comprising an extract of citrus peel as an active ingredient. The citron extract according to the present invention has no toxicity, and has a quorum recognition-inhibiting activity which hinders communication between microorganisms, thereby effectively preventing the growth of microorganisms and the formation of a biofilm. Therefore, the composition of the present invention can be widely used in a variety of life tools and medical instruments that need to prevent infection of microorganisms or prevent biofilm formation, and can be used as a therapeutic agent or feed additive for diseases caused by antimicrobial agents and microorganisms Lt; / RTI >

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting the recognition of quorum in citrus junos as an active ingredient,

The invention citron (Citrus junos extract as an active ingredient.

When a certain level of cell concentration is reached, microorganisms use their chemical language to transmit signals to each other and thereby regulate the expression of specific genes. The mechanism by which gene expression depends on the density of individuals is called quorum sensing (QS). The quorum sensing mechanism is a mechanism in which each microorganism accumulates a low molecular signaling substance such as an autoinducer (AI) in the cell environment depending on the cell concentration to induce active proliferation, fill a certain quorum, It is a kind of regulatory mechanism that induces expression. Microbes regulate diverse physiological phenomena such as biofilm formation, virulence, bioluminescence, antibiotic production or gene transfer by conjugation, motility through the quorum sensing mechanism.

A typical phenomenon represented by the communication of microorganisms is the formation of a biofilm. Biofilm refers to a smooth mucous layer on the surface formed by the combination of microbes and high molecular substances of cells that are produced during the metabolism of microorganisms. When microorganisms attach to various inanimate organisms and surfaces of organisms to form a biofilm and form clusters, resistance to environmental stresses such as lack of nutrients, exposure to toxic substances, changes in acidity, oxidative shock, and exposure to strong antibiotics are significantly increased And biofilms produced by pathogens or potential pathogens are considered to be a major cause of chronic infections and are believed to be important sources of infection due to their ability to form and tolerate biofilms on the surface of medical equipment and in the holding devices inside the human body. Therefore, the development of an antibacterial agent for inhibiting the formation of biofilm is required.

Meanwhile, Citrus junos ) is a plant belonging to the genus of manganese and citrus. It is cultivated mainly in the southern part of the country in the southern part of the country. The fruit is soft and succulent. It is medicinal in the effect of strengthening the camouflage in the herbal medicine and the private.

It is disclosed in Korean Patent No. 0637366 that citron extract can be used as an external preparation for skin because of its excellent effect of decomposing subcutaneous fat of adipocytes and cellulite removal effect, and Korean Patent No. 0543122 is effective in the treatment of inflammatory diseases .

As described above, various physiological activity effects of citron extract are known. It is not known at all that the citron extract has a quorum-sensing inhibitory activity.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a quan- tity-recognition-inhibiting composition comprising the citron extract of the present invention as an active ingredient, which inhibits microbial communication, Thereby completing the present invention.

In order to accomplish the above object, the present invention provides a composition for inhibiting the recognition of quorum water comprising an extract of citrus peel as an active ingredient.

In addition, the present invention provides an antimicrobial composition comprising an extract of citrus peel as an active ingredient.

The present invention also provides a feed additive composition comprising an citron extract as an active ingredient.

The present invention relates to a composition for inhibiting the recognition of a quorum of the instant invention comprising an extract of citrus peel as an active ingredient. The citrus active ingredient of the present invention has no toxicity and is capable of being edible and has a quorum sensing inhibitory activity which hinders communication between microorganisms. It is possible to effectively block the growth of biofilm and the formation of biofilm, and thus it can be widely used for a variety of life tools and medical instruments which need to prevent infection of microorganisms or prevent formation of biofilm. A therapeutic agent for diseases, a feed additive, and the like.

Figure 1 shows the quercetin recognition inhibitory activity of the citron extract against the chromobacterium bioreceptor strain (1, 2. Citron methanol extract; 3. Residue after methanol extraction with MTBE (methyl tertiary butyl ether) 4. Extracts extracted; 4. MTBE extract; N. negative control (DMSO); PC. Positive control (Resveratrol)).
Fig. 2 is a graph showing the effect of inhibiting the formation of biofilm of Erysiphe grisea, Pseudomonas aeruginosa and Pseudomonas aeruginosa by concentration of citron extract.
Fig. 3 is a graph showing the results of evaluation of motility inhibitory ability of Pseudomonas aeruginosa extract of citron extract. Fig.
FIG. 4 is a graph showing the results of confirming antimicrobial activity of citrus fruit extract on Yusinia enterocolitica and Pseudomonas aeruginosa.

The present invention relates to a composition for inhibiting the recognition of quorum water comprising an extract of citrus peel as an active ingredient.

The citron extract is preferably extracted with at least one solvent selected from water, alcohols having 1 to 4 carbon atoms, ether and hexane, but is not limited thereto.

The strains showing the quorum sensing inhibitory activity of the citron extract include gram-positive bacteria and gram-negative bacteria, preferably C. violaceum , Y. enterocolitica and Pseudomonas sp . But are not limited to, one or more strains selected from P. aeruginosa .

The citron extract according to the present invention is free from toxicity and has a quorum sensing inhibiting activity which hinders communication between microorganisms and is capable of effectively blocking the growth of microorganisms and the formation of biofilm. Accordingly, the citrus peel extract of the present invention can be used in a variety of life tools and medical instruments that need to prevent infection of bacteria or prevent formation of a biofilm.

In addition to the citron extract, a carrier, excipient or diluent may further be included. Examples of the carrier, excipient or diluent that can be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, and magnesium stearate.

The present invention also relates to an antimicrobial composition comprising an extract of citrus peel as an active ingredient. Strains are the citrus extract showing the antibacterial activity is Gram-positive bacteria and Gram-negative bacteria, and include, preferably Bacillus cereus (Bacillus cereus , Staphylococcus aureus , and Listeria monocytogenes . However, the present invention is not limited thereto.

The present invention also relates to a feed additive composition comprising an citron extract as an active ingredient.

When the composition of the present invention is used as an antimicrobial agent, a therapeutic agent for diseases caused by a strain, or an additive for feed, it may be administered orally or parenterally in a conventional manner. In the case of formulation, a filler, A binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples. It is to be understood by those skilled in the art that these preparations and examples are merely intended to explain the present invention more specifically and that the scope of the present invention is not limited thereto.

Manufacturing example  1. Preparation of citron extract

Citrus junos ) were purchased and cultivated in Goheung-gun, Jeonnam. The citron was cut into a suitable size for the skin and then lyophilized to powder, 700 ml of 70% methanol per g of dry weight was extracted with shaking at a rate of 150 rpm at room temperature for 24 hours, filtered through a vacuum evaporator, After the solvent was removed by drying under reduced pressure, the extract was recovered and concentrated to prepare a crude extract.

In order to investigate the activity of the volatile components, 30 ml of MTBE (Methyl Tertiary Butyl Ether) was added per 0.5 g of dry powder of each plant. The extracts were also concentrated and lyophilized and dissolved in DMSO Activity measurement.

Example  1. Verification of quorum recognition inhibition activity of citron extract

In order to confirm quorum sensing inhibition activity of citron extracts, quorum sensing inhibition activity was confirmed by using C. violaceum , which is known to have a quorum sensing function by a magnetic induction substance. First, LB (Luria-Bertani) agar medium was inoculated with 5 × 10 ⁶ cfu / ml of chromobacterium biorecemium and then poured into Petri dishes to prepare LB agar plates. For the activity measurement, wells were prepared on the surface using a borer on an agar plate, citron extracts of various concentrations (5 - 100 mg / ml) were dropped in the wells, Lt; / RTI >

The size of the purple pigment inhibiting ring around the agar wells was then measured. Resveratrol was used as a positive control and DMSO was used as a negative control.

As a result of measuring the quercetin recognition inhibitory activity of the citron extract of the present invention against quorum bacterium biorecem, the quercetin recognition inhibitory activity of chromoprotein bioreceptor in the methanol extract of citron was similar to that of the positive control group resveratrol 1). In addition, the pigment inhibition rings of the citron extracts were measured to determine the correct quercetin inhibition activity, and the inhibitory activity of the citron extract was similar to that of the positive control (Table 1).

Concentration-dependent quorum sensing inhibitory activity of citron extract on chromobacterium violosium
Concentration (mg / ml) Diameter of inhibitory zone (mm) Methanol Negative control (NC, DMSO) Positive control (PC, Resveratrol) 100 25 10 25 20 17 - 20 10 15 - 17 5 10 - 14

Example  2. Citron extract Biofilm  Evaluation of inhibition against formation

In order to verify the quercetin inhibitory activity of citron extracts, the inhibitory effect of citron extract on the biofilm formation was evaluated by modifying O'Tool and Kolter 's method. Sikjungdokgyun the yeosi California Enterococcus coli urticae (Y. enterocolitica) and industrial Rouge Pseudomonas surface adhesion properties of strong indicator organism (P. aeruginosa) 24 hours ago from 30 ℃ After incubation, polyvinyl chloride so that 0.01 at OD ₆₀₀ ( Polyvinyl Chloride), and then cultured at 30 ° C at 50 rpm. After 24 hours, the culture medium was discarded, washed with PBS (phosphate-buffered saline) buffer, stained with 0.3% crystal violet for 30 minutes, and washed twice with PBS. The remaining crystal violet was dissolved in 95% ethanol and absorbance was measured at 595 nm.

As shown in Fig. 2 and Table 2, both microbes of Yusinia enterocolitica and Pseudomonas aeruginosa inhibited the biofilm formation-inhibiting ability of the citron extract in a concentration-dependent manner. Thus, the citron extract inhibited the quorum recognition of two microorganisms Respectively.

Determination of concentration-dependent activity of citron extract on biofilm
density(%) Absorbance for biofilm formation (mean ± standard deviation), 595 nm Jessinia enterrocolitica
( Y. enterocolitica ) Pseudomonas aeruginosa
( P. aeruginosa ) One 0.182 + 0.023 0.198 + 0.026 0.8 0.312 + 0.045 0.388 + 0.048 0.5 0.545 + 0.043 0.564 + 0.078 0.2 0.867 ± 0.063 0.858 + 0.049 DMSO (Negative control) 0.954 + 0.042 0.983 + 0.032

Example  3. Assessment of inhibition of motility of citron extract

Another method of confirming the quorum-recognition inhibitory activity was carried out on Pseudomonas aeruginosa reported to be associated with biofilm formation. Various concentrations (0.5% (w / v), 1% (w / v)) of citron extract were added to LB agar (0.3% (w / v)) containing 10 mM glucose and 1% made. A single colony of Pseudomonas aeruginosa was stuck in the center of the plate and inoculated and cultured at 30 캜 for 48 hours. LB agar plates containing no citron extract were used as a control.

As shown in Fig. 3, the citron extract showed a quercetin recognition inhibiting activity inhibiting the migration of Pseudomonas aeruginosa in a concentration-dependent manner.

Example  4. Yushinia Entero Colittica and  Pseudomonas To Erusinosa  Evaluation of antimicrobial activity of citron extract

In order to confirm whether the inhibition effect of citron extract on the biofilm formation is due to the quercetin inhibition activity of microorganisms or simply the antimicrobial activity of the citron extract, Y. enterocolitica and Pseudomonas aeruginosa The antimicrobial activity of P. aeruginosa was measured. First, microorganisms corresponding to Mcfarland 0.5 were inoculated into Mueller-Hinton agar medium, and then poured into Petri dishes. Citron extracts at various concentrations (0.5-2%) were dropped on a sterilized paper disc (= 6 mm), dried, placed on agar plates, and cultured at 30 ° C for 24 hours.

As shown in Fig. 4, the antimicrobial activity was not exhibited even in the citron extract of 2% (w / v) concentration. Therefore, it was confirmed that the inhibitory activity on the biofilm formation of Pseudomonas aeruginosa and Pseudomonas aeruginosa was due to the quorum- .

Example  5. Verification of antimicrobial activity of citron extract

Have the same citron extract used in the quorum sensing inhibition activity verify the Bacillus cereus (B. cereus), Staphylococcus aureus, minimum growth inhibitory concentration (MIC) for (S. aureus), L. monocytogenes Jeans (L. monocytogenes) Were measured according to the guidelines of the Laboratory Standards Association (CLSI, 2007). The minimum growth inhibitory concentration (MIC) of citron extracts was measured according to Disc diffusion method. The target microorganisms of Mcfarland 0.5 were plated on Mueller-Hinton agar and the extract was diluted to 100 mg-1 mg. After incubation for 30 to 24 hours, the presence of antimicrobial activity was observed under the same conditions, and the minimum growth inhibitory concentration (MIC), which showed antibacterial activity, Were measured. At this time, the inhibition rings were determined based on 7 mm.

    As a result, the minimum inhibitory concentration (1-10 mg / ml) of MTBE extract of citron was the lowest, indicating that the antimicrobial activity was the highest, indicating that the antimicrobial activity of the volatile component is good (Table 3).

Bacillus cereus by the citrus extract (B. cereus), antibacterial activity (MIC) of L. monocytogenes Jeans (L. monocytogenes), Staphylococcus aureus (S. aureus)
Concentration (mg / ml) Minimum inhibitory concentration (mg / ml) methanol MTBE extraction after methanol treatment MTBE B. cereus 2 10 One L. monocytogenes 20 100 10 S. aureus 10 50 5

Claims (3)

Which is characterized by exhibiting a quorum sensing inhibitory activity against at least one strain selected from the group consisting of Y. enterocolitica and Pseudomonas aeruginosa , . delete The antimicrobial composition for inhibiting biofilm formation according to claim 1, wherein the citrus methanol extract further comprises a carrier, an excipient or a diluent.

Images