KR20230046792A - COMPOSITION FOR PREVENTING BIOFILM FORMATION COMPRISING EXTRACT OF Piperis Nigri Fructus - Google Patents

Abstract

The present invention relates to a composition for inhibiting biofilm formation comprising an extract of Piper nigrum as an active ingredient, and when using the extract of Piper nigrum extract according to the present invention, proliferation of oral bacteria and formation of a biofilm can be suppressed, and thus, the composition can be usefully used in the development of various oral products and treatment agents for oral diseases.

Description

Composition for inhibiting biofilm formation containing a Hocho extract {COMPOSITION FOR PREVENTING BIOFILM FORMATION COMPRISING EXTRACT OF Piperis Nigri Fructus}

The present invention relates to a composition for inhibiting biofilm formation comprising Hocho extract as an active ingredient.

Dental cavities (cavities) and periodontal disease (periodontal disease) cause various clinical symptoms such as pain, masticatory dysfunction, destruction of periodontal tissues, bad breath and cold teeth, and are the main factors that cause tooth loss. It is known, and the causative factors of these oral diseases are increasing due to changes in dietary life.

It is known that about 600 to 800 or more microorganisms exist in the human oral cavity, and these microorganisms are controlled by enzymes such as lysozyme secreted by saliva. However, the oral environment rich in nutrients and moisture is a favorable condition for the growth of microorganisms, and the tongue or dental plaque provides an excellent habitat for microorganisms. Some of these microorganisms are opportunistic pathogens and cause diseases such as dental caries, periodontal disease, and tooth decay (dentin hypersensitivity) and bad breath.

On the other hand, a biofilm is also called a biofilm, and means a state in which microorganisms produce polysaccharides around cells and aggregate with adjacent microorganisms through this to form a film on a solid or biological surface.

Biofilm formation is known to play an important role in the pathogenesis of various diseases such as dental caries, bacterial endocarditis, chronic sinusitis, food poisoning, and pneumonia.

In particular, in oral diseases such as dental caries, pathogens settle in the oral cavity by forming plaque (biofilm) on the tooth surface to exhibit pathogenicity. Therefore, the control of these biofilms is considered very important for the prevention and treatment of oral diseases.

For this reason, the development of natural product-derived preventive and/or therapeutic agents with fewer side effects is essential and requires continuous research.

KR 10-2020-0093514 A

The present inventors have made intensive research efforts to develop natural product-derived biofilm formation inhibitors with fewer side effects. As a result, the present invention was completed by confirming that Piperis Nigri Fructus has an excellent ability to inhibit biofilm formation.

Accordingly, an object of the present invention is to provide a composition for inhibiting biofilm formation comprising an extract of Hocho ( Piperis Nigri Fructus ) as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for oral use containing Hocho extract as an active ingredient.

The present inventors have made intensive research efforts to develop natural product-derived biofilm formation inhibitors with fewer side effects. As a result, it was confirmed that Hocho ( Piperis Nigri Fructus ) has excellent ability to inhibit biofilm formation.

The present invention relates to a composition for inhibiting biofilm formation and a quasi-drug composition for oral use containing an extract of Hochoe as an active ingredient.

In the present invention, significant results were confirmed in the inhibitory effect of biofilm formation on the titanium surface using the extract of Hocho (Piperis Nigri Fructus) .

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention relates to a composition for inhibiting biofilm formation comprising an extract of Piperis Nigri Fructus as an active ingredient.

In the present invention, " Piper nigri Fructus " is a fruit close to maturity of Piper nigrium, which is collected and dried when the fruit is dark green from autumn to next spring. Hocho oil extracted from Hocho is linolenic acid It has been reported to have an excellent effect on diseases and treatment of circulatory system such as arteriosclerosis, but there has been no report on biofilm formation inhibitory activity.

The term 'extract' used herein has a meaning commonly used in the art as a crude extract, but also includes a fraction obtained by further fractionating the extract in a broad sense. That is, the Hocho extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through the purification method are also included in the hocho extract of the present invention.

The hocho extract of the present invention can be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In the present invention, the extraction solvent is at least one selected from the group consisting of water, straight chain or branched alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol, hexane and diethyl ether It may be a solvent, but is not limited thereto.

In the present invention, the extraction temperature may be, for example, 0 °C to 150 °C, specifically 50 °C to 100 °C, but is not limited thereto.

In the present invention, the extraction time may be, for example, 1 hour to 10 hours, specifically 3 hours to 8 hours, and more specifically 5 hours to 7 hours, but is not limited thereto.

Hocho extract of the present invention can be extracted by a known natural product extraction method. For example, it can be extracted by cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, and heating extraction. Specifically, it can be extracted by hot water extraction or reflux cooling extraction, 1 to 10 times, more specifically 2 Extraction can be repeated 7 times to 7 times.

In the present invention, the composition for inhibiting biofilm formation may include the Hocho extract at a concentration of 1 mg/ml.

In the present invention, "biofilm" refers to a thin film composed of a large amount of macromolecular compounds in which microorganisms accumulate on the surface and are associated with cells, and organic substances and inorganic substances attached to the surface, and can be used interchangeably with biofilms. there is. Bacteria in biofilms can increase their resistance to antibiotics 10 to 1000 times more than in the suspended state, and the morphology of bacteria is changed so that antibiotics are inactivated by extracellular polysaccharide or oxygen, or by biofilm formation. It slows growth and acquires tolerance.

The biofilm is Porphyromonas gingivalis ( Porphyromonas gingivalis ), Aggregatibacter actinomycetem comitans ( Aggregatibacter actinomycetemcomitans ), Tannerella forsythia ( Tannerella forsythia ), Treponema denticola ( Treponema denticola ), Prevotella nigrescens , Eubacterium nodatum, Parvimonas micra , Eikenella corrodens, Campylobacter rectus , Fusobacterium nucleatum ( Fusobacterium nucleatum ), Streptococcus sobrinus ( Streptococcus sobrinus ), Streptococcus salivarius ( Streptococcus salivarius ), Streptococcus anginasus ( Streptococcus anginasus ), Streptococcus mutans ( Streptococcus mutans ) and Prevotella intermedia ( Prevotella intermedia ) It may be formed by a microorganism selected from the group consisting of, but is not limited thereto.

In the present invention, "inhibition of biofilm formation" means to reduce the rate of biofilm formation, which means a decrease in the rate of increase in the number of viable cells per unit time from the start of the culture of the biofilm-forming microorganisms, that is, oral bacteria. . The number of viable cells of the microorganisms may be measured by counting the number of colonies of the microorganisms appearing after culturing, and may be expressed as the number of viable cells per mL (CFU/mL).

According to another aspect of the present invention, the present invention relates to a quasi-drug composition for oral cavity comprising a Hocho extract as an active ingredient.

Ingredients included in the quasi-drug composition of the present invention may include ingredients commonly used in oral quasi-drug compositions in addition to the above active ingredients as active ingredients, such as abrasives, wetting agents, binders, foaming agents, sweeteners, preservatives, medicinal ingredients, flavors agents, pigments, solvents, brighteners, solubilizers or pH adjusters.

The quasi-drug composition for oral use of the present invention can be prepared in any formulation commonly prepared in the art, for example, toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral ointment, and oral varnish. , oral rinse and gum massage cream, but may have formulations such as, but are not limited thereto.

As an example, when the oral quasi-drug composition of the present invention is a toothpaste formulation, it may contain a wetting agent, an abrasive, a binder, a foaming agent, a flavoring agent, a sweetening agent, a coloring agent, a preservative, a medicinal component, a solvent, a pH adjusting agent, and the like.

The humectant is to make the powder of toothpaste ingredients paste and prevent the toothpaste from hardening in the air, and glycerin, sorbitol, propylene glycol, polyethylene glycol, etc. alone or in combination of two or more, 1 to 60 weight of the total weight of the composition %, specifically, 10 to 50% by weight may be used.

The foaming agent diffuses the toothpaste into the oral cavity to increase the cleaning effect and acts as a surfactant to clean oral contamination. Alternatively, 0.5 to 10% by weight, specifically 0.5 to 5% by weight of the total weight of the composition may be used by mixing two or more kinds.

The binder prevents separation between powder and liquid components in toothpaste, and is a mixture of cellulose derivatives such as sodium carboxymethyl cellulose, methyl cellulose, and hydroxypropyl cellulose, sodium alginate, carrageenan, xanthan gum, etc. alone or in combination of two or more. 0.1 to 5% by weight, specifically 0.3 to 2% by weight of the total weight of the composition may be used.

The abrasive is calcium carbonate (CaCO ₃ ), calcium phosphate dibasic (CaHPO ₄ , CaHPO ₄ 2H ₂ O), silicic anhydride (SiO ₂ 2H ₂ O), aluminum hydroxide (Al(OH) ₃ ), potassium pyrophosphate, magnesium carbonate, etc. alone or in combination of two or more, and 1 to 60% by weight, specifically 10 to 50% by weight of the total weight of the composition can be used

The flavoring agent is to enhance the feeling of use by imparting a refreshing feeling and smell to the toothpaste, and peppermint oil, spearmint oil, menthol, etc. alone or in combination of two or more are used in an amount of 1 to 60% by weight, specifically 0.01 to 60% by weight of the total weight of the composition. 5% by weight can be used.

The sweetener is to remove the unpleasant taste caused by raw materials for toothpaste and improve the refreshing feeling, and 1 to 60% by weight of the total weight of the composition by mixing saccharic acid, aspartame, xylitol, licorice acid, etc. alone or in combination of two or more, specifically 0.01 to 5% by weight can be used.

The active ingredients are for the prevention of dental caries, periodontal disease, toothache prevention, cold lice prevention, bad breath removal, etc. Triclosan, tocopherol acetate, sodium monofluorophosphate, etc. may be used alone or in combination of two or more. In the present invention, forsitoside ratio can be used as an additional active ingredient.

The quasi-drug composition for oral use of the present invention may be used alone or in combination, or may be used in combination with other quasi-drug compositions for oral use other than the present invention.

According to another aspect of the present invention, the present invention relates to a food composition for oral cavity containing a Hocho extract as an active ingredient. The food composition may be used in the form of health functional food, but is not limited thereto.

The extract included in the food composition of the present invention may be included in the form of a fraction thereof or a processed product thereof. In addition, the composition may include a food additive that is acceptable in food science in addition to the active ingredient.

In the present invention, "food supplement additive" refers to a component that can be added to food supplementally, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.

In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionalities for the human body. Here, "functionality" means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time using food as a raw material, and has excellent portability, so that the composition of the present invention Health functional food can be consumed as a supplement to prevent or improve oral diseases.

There is no limit to the form that the health functional food of the present invention can take, and it can include all foods in a conventional sense, and can be used interchangeably with terms known in the art, such as functional foods. In addition, the health functional food of the present invention can be prepared by mixing suitable other auxiliary ingredients and known additives that can be included in food according to the selection of those skilled in the art. Examples of food that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., and it can be prepared by adding the forsitoside ratio according to the present invention to juice, tea, jelly, juice, etc. prepared as a main component. It also includes food used as feed for animals.

The present invention relates to a composition for inhibiting biofilm formation comprising a Hocho extract as an active ingredient. It can be usefully used for the development of oral disease treatment, etc.

1 shows the evaluation results of the biofilm formation inhibitory ability of the Hocho extract of the present invention according to an embodiment of the present invention.
Figure 2 shows the evaluation results of the biofilm formation inhibitory ability of the Hocho extract of the present invention according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

The results of all experiments were expressed as Mean±Standard error, and the statistical analysis of the experimental data was performed with Student's t-test, and it was judged to be statistically significant when the p value was less than 0.001.

Experimental Example 1. Evaluation of biofilm formation inhibitory effect of Hocho extract

Hocho ( Piperis Nigri Fructus ) hot water extract (23.2 mg/mL) used in the experiment was provided by the Korea Plant Extract Bank (KPEB), and the distribution number of Hocho extract is CW04-080.

After spraying 200 μl of filtered saliva on a titanium disk (10 mm in diameter, Dentium), it was gently shaken in an ∞ shape. After incubation at 37° C. for 4 hours, saliva was removed.

Add 500 μL of P. gingivalis (ATCC 33277, KCOM) to 9.5 mL of BHI medium (BHI, Hemin 5 μg/mL, Vitamin K1 1 μg/mL) and incubate at 37 °C for 24 hours. Each 1 mL was put into a tube and centrifuged at 12,000 rpm for 3 minutes. After discarding the supernatant, 1 mL of BHI medium was added to release the pellet. Then, 20 μL was taken and diluted 1/20 in 380 μL of BHI medium. 200 μL of the diluted culture medium was put into a 96-well plate, and OD ₆₀₀ was measured. Based on the measured OD ₆₀₀ value, the input volume of P. gingivalis was calculated at 1Х10 ⁸ CFU/mL using the following method.

[Total CFU of P. gingivalis in 1 mL, stock solution]

= {7156.4 (0.204; OD dilution value - 0.086; OD medium) - 20.258

= (824.1972 X 10 ⁶ CFU/mL) X 20

= 16483.944 X 10 ⁶ CFU/mL

= 164.83944 X 10 ⁸ CFU/mL

At this time, when the total volume is 4 mL (1 mL inoculated on 4 disks), the input volume (X below) is,

X μL = (1Х10 ⁸ CFU/mL)/(164.83944 Х 10 ⁸ CFU/mL) Х 4 mL

= (1Х10 ⁸ CFU/mL)/(164.83944 Х 10 ⁸ CFU/mL) Х 4,000 μL

= 24.2660373027...

= 24.27 µL

In addition, the input volume of the Hocho extract was calculated using the following formula 1.

[Calculation 1]

In 4 mL of BHI medium, 24.27 μL, the input volume of P. gingivlais calculated above, and 10.78 μL (1 mg/mL), the input volume of Hocho extract, were respectively removed. The titanium disc prepared above, 24.27 μL of P. gingivlais culture medium, and 10.78 μL of Hocho extract (1 mg/mL) were respectively added to the medium from which each volume was removed, followed by vortexing. Then, 1 mL was added to each well of the 24-well plate and gently shaken in an ∞ shape. It was incubated for 72 hours in an anaerobic chamber (anaerobic chamber, Whitley DG 250 Workstation 230v/50Hz; 80% N ₂ , 10% CO ₂ and 10% H ₂ and N ₂ 100%) at 37 °C.

Media in each well was aspirated and washed twice with DPBS. It was stained with crystal violet for 10 minutes at room temperature under light blocking. Washed 3 times with DPBS. After moving the titanium disk to a new plate, crystal violet was dissolved in a destaining buffer, put into each well of a 24-well plate, and OD ₅₉₅ was measured. Based on the measured OD ₅₉₅ value, the relative amount of biofilm (%) compared to the control group was calculated using Equation 2 below.

[Calculation 2]

As can be seen in Figures 1 and 2, Hocho extract at a concentration of 1 mg / mL or more inhibits the biofilm formation of P. gingivalis .

Claims (5)

A composition for inhibiting biofilm formation comprising an extract of Hocho ( Piperis Nigri Fructus ) as an active ingredient. The method of claim 1, wherein the Hocho extract is 1 selected from the group consisting of water, straight chain or branched alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol, hexane and diethyl ether A composition for inhibiting biofilm formation that is extracted with more than one solvent. The biofilm according to claim 1, wherein the biofilm is Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , Tannerella forsythia , Treponema denty Cola ( Treponema denticola ), Prevotella nigrescens , Eubacterium nodatum , Parvimonas micra , Eikenella corrodens , Campylobacter Rectus ( Campylobacter rectus ), Puzobacterium nucleatum ( Fusobacterium nucleatum ), Streptococcus sobrinus ( Streptococcus sobrinus ), Streptococcus salivarius ( Streptococcus salivarius ), Streptococcus anginasus ( Streptococcus anginasus ), Streptococcus mutans ( Streptococcus mutans ) And Prevotella intermedia ( Prevotella intermedia ) Formed by a microorganism selected from the group consisting of, a composition for inhibiting biofilm formation. A quasi-drug composition for oral use comprising a Hocho extract as an active ingredient. The method of claim 4, wherein the The dosage form of the quasi-drug composition is at least one dosage form selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, mouthwash, and gum massage cream. composition.

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