KR20230046711A - COMPOSITION FOR PROMOTING BIOFILM FORMATION COMPRISING EXTRACT OF Polygonati Rhizoma - Google Patents

Abstract

The present invention relates to a composition for promoting biofilm formation containing a Polygonatum sibirium extract as an active ingredient, and when the Polygonatum sibirium extract according to the present invention is used, it is very economical in in vitro cell testing of oral anaerobic bacteria, and can reduce the effort and cost used in animal testing, making it useful in the development of various oral products, oral disease medicines and the like.

Description

A composition for promoting biofilm formation containing hwangjeong extract {COMPOSITION FOR PROMOTING BIOFILM FORMATION COMPRISING EXTRACT OF Polygonati Rhizoma}

The present invention relates to a composition for accelerating biofilm formation comprising a hwangjeong extract.

A biofilm, also called a biofilm, refers to a state in which microorganisms produce polysaccharides around cells and aggregate with adjacent microorganisms through the medium to form a film on a solid or biological surface.

Biofilm is known to play an important role in the pathogenesis of various diseases such as dental caries, bacterial endocarditis, chronic sinusitis, food poisoning, pneumonia, etc., and by oral bacteria represented by Porphyromonas gingivalis mainly formed

However, since these oral bacteria are generally anaerobic bacteria and are cultured only under specific conditions, the experimental design using them is very difficult and requires a lot of expert know-how, which is costly and time-consuming for bacteria to sufficiently propagate. there is a problem.

For this reason, the development of a natural product-derived material that promotes the formation of anaerobic bacteria (biofilm) at the laboratory level is essential and requires continuous research.

KR 10-1908411 B1

The present inventors have intensively researched to develop a natural product-derived biofilm formation promoter with fewer side effects. As a result, the present invention was completed by confirming that Polygonati Rhizoma has an excellent ability to promote biofilm formation.

Accordingly, an object of the present invention is to provide a composition for promoting biofilm formation comprising Polygonati Rhizoma extract as an active ingredient.

Another object of the present invention is to provide a composition for culturing oral bacteria comprising a hwangjeong extract as an active ingredient.

The present inventors have intensively researched to develop a natural product-derived biofilm formation promoter with fewer side effects. As a result, it was confirmed that Hwangjeong ( Polygonati Rhizoma ) has an excellent ability to promote biofilm formation.

The present invention relates to a composition for promoting biofilm formation and a composition for culturing oral bacteria, comprising an extract of Hwangjeong as an active ingredient.

The present invention confirmed a significant result in the biofilm formation promoting effect on the titanium surface using the Hwangjeong ( Polygonati Rhizoma ) extract.

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 promoting biofilm formation comprising Polygonati Rhizoma extract as an active ingredient.

In the present invention, "Hwangjeong ( Polygonati Rhizoma )" is a dried rhizome of Polygonatum sibiricum Rehd., a plant of the lily family, and has blood pressure lowering action, blood sugar lowering action, anti-arteriosclerosis action, liver fat deposition prevention action, etc. is known to have

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, Hwangjeong 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. The fraction obtained through the purification method is also included in the Hwangjeong extract of the present invention.

The Hwangjeong extract of the present invention may 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.

Hwangjeong 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 promoting biofilm formation may include the Hwangjeong 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, "promoting biofilm formation" means increasing the rate of biofilm formation, which means improving the rate of increase in the number of viable cells per unit time from the start of culturing 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).

In addition, the promotion of biofilm formation may be to promote the growth of the biofilm-forming microorganisms, that is, oral bacteria, when they are to be cultured in a composition (medium) for culturing oral bacteria. In order to promote the growth of microorganisms in a culture medium, the composition of the present application may be used by being added to a composition (medium) for culturing oral bacteria.

According to another aspect of the present invention, the present invention relates to a composition for culturing oral bacteria comprising a hwangjeong extract as an active ingredient.

In the present invention, the description of the Hwangjeong extract is the same as described above, and the description regarding this uses the above description.

In the present invention, the composition for culturing oral bacteria refers to a composition (medium) for the purpose of culturing biofilm-forming microorganisms, that is, oral bacteria, and can help the growth of oral bacteria, which is common in the art of the present application. It may contain ingredients known as

The oral bacterial culture composition may be in liquid or solid form, and may be solidified by mixing a liquid medium with agar.

In the present invention, the composition for promoting biofilm formation may include the Hwangjeong extract at a concentration of 1 mg/mL.

The composition for culturing oral bacteria may further include one or more selected from the group consisting of a carbon source, a nitrogen source, and an inorganic salt, and may help the growth of oral bacteria, or a minimum component required for culturing oral bacteria. Any ingredient that is present may be included without limitation.

The carbon source is glucose, maltose, lactose, cellobiose, mannose, trehalose, fructose, melibiose, Galactose, raffinose, sucrose, stachyose, mannitol, arabinose, sorbitol, xylose, esculin It may be sugars such as esculin, rhamnose, salicin, and starch, and/or organic acids such as citric acid, tartaric acid, and malic acid, and/or low molecular weight fatty acids.

The nitrogen source may be casein, beef extract, tryptone, peptone, soypeptone, and/or yeast extract.

The inorganic salt may be P, Na, K, Mg, S, and/or Mn, and the culture medium may further contain trace amounts of other inorganic salts. The inorganic salt is, for example, polysorbate 80, dibasic potassium phosphate (K ₂ HPO ₄ ), Na-acetate, ammonium citrate ((NH ₄ ) ₂ citrate), magnesium sulfate (MgSO ₄ Х 7H ₂ O), manganese sulfate ( MnSO ₄ Х H ₂ O), etc., and the type and content of the inorganic salt may be appropriately selected depending on the pH and osmotic pressure of the culture medium. In addition, when natural ingredients such as beef extract, peptone, and yeast extract are used in the medium, additional inorganic salts may not be added or the content thereof may be set to be small in consideration of the inorganic salts contained in the natural materials.

The content of the carbon source, nitrogen source, and inorganic salt component may be set according to the purpose of culturing oral bacteria, the target number of bacteria, etc. within a range that does not inhibit the biofilm formation promoting effect (promoting the growth of oral bacteria).

The composition for culturing oral bacteria may further include a component that can help promote the growth of oral bacteria, and the type and content of the component does not inhibit the biofilm formation promoting effect (promoting the growth of oral bacteria). It can be set according to the purpose of culturing oral bacteria, the target number of bacteria, etc. within the range.

The oral bacteria are Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , Tannerella forsythia , 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 selected from the group consisting of, but is not limited thereto.

The present invention relates to a composition for promoting biofilm formation containing Hwangjeong extract as an active ingredient. When using the Hwangjeong extract according to the present invention, it is very economical for in vitro cell experiments of oral anaerobic bacteria and used for animal experiments Since it can reduce the effort and cost, it can be usefully used in the development of various oral products and oral disease treatments.

Figure 1 shows the biofilm formation promoting ability evaluation results of the Hwangjeong extract of the present invention according to an embodiment of the present invention.
Figure 2 shows the biofilm formation promoting ability evaluation results of the Hwangjeong 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. Evaluation of biofilm formation promoting effect of Hwangjeong extract

Hwangjeong ( Polygonati Rhizoma ) hot water extract (21.26 mg/mL) used in the experiment was provided by Korea Plant Extract Bank (KPEB), and the distribution number of Hwangjeong extract is CW04-096.

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, and 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 (0.229; OD dilution value - 0.065; OD medium) - 20.258

= (1153.3916Х10 ⁶ CFU/mL) Х 20

= 23067.832Х10 ⁶ CFU/mL

= 230.67832Х10 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)/(230.67832Х10 ⁸ CFU/mL) Х 4 mL

= (1Х10 ⁸ CFU/mL)/(230.67832Х10 ⁸ CFU/mL) Х 4,000 μL

= 17.34013436

= 17.34 µL

In addition, the input volume of the Hwangjeong extract was calculated using Equation 1 below.

[Calculation 1]

In 4 mL of BHI medium, 17.34 μL, the input volume of P. gingivlais calculated above, and 11.76 μL (1 mg/mL), the input volume of Hwangjeong extract, were respectively removed. The titanium disc prepared above, 17.34 μL of P. gingivlais culture medium, and 11.76 μL of Hwangjeong extract (1 mg/mL) were added to the medium from which each volume was removed, respectively, followed by vortexing. Then, 1 mL was added to each well of the 24-well plate and gently shaken in an ∞ pattern. 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.

As can be seen in Figures 1 and 2, 1mg / mL of Hwangjeong extract promotes the formation of a biofilm of P. gingivalis , thereby saving time and money spent on culturing gum causative bacteria, and creating a stable experimental environment It became. Through this, it is expected that the development of oral health products can be promoted.

Claims (5)

Hwangjeong ( Polygonati Rhizoma ) A composition for promoting biofilm formation comprising an extract as an active ingredient. The method of claim 1, wherein the Hwangjeong 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 promoting biofilm formation that is extracted with at least 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 promoting biofilm formation. Hwangjeong ( Polygonati Rhizoma ) Oral bacterial culture composition containing an extract. The method of claim 4, wherein the oral bacteria are Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , Tannerella forsythia , Treponema denti 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 ) Which is selected from the group consisting of, Oral bacterial culture composition.

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