KR101017447B1 - Antibacterial composition and oral composition for preventing caries containing the extract of fennel - Google Patents

Abstract

The present invention relates to an oral composition for preventing caries and an antimicrobial composition, and more particularly, to an oral cavity composition for preventing caries and an antimicrobial composition containing a fennel extract as an active ingredient. The fennel extract of the present invention has an inhibitory activity of glucosyltransferase, a causative agent of tooth decay, and antimicrobial activity against Streptococcus mutans , a decay-causing bacterium, as well as no side effects and stable at high temperatures. Can be used in the oral cavity composition for preventing tooth decay and antimicrobial composition that can be processed even in the temperature range.

Streptococcus mutans, Glucosyltransferase, Fennel, Extract

Description

Antibacterial composition and oral composition for preventing caries containing the extract of fennel

1 is a graph showing the inhibition activity of glucosyltransferase by the fennel hot water extract of the present invention according to the change of temperature and time.

◆: 1 hour heating

■: 6 hours heating

▲: 12 hours heating

Figure 2 is a graph showing the degree of plaque formation by the fennel hot water extract of the present invention compared with the case of administering distilled water.

Before Use: Average Plaque Index of Subjects Before Clinical Trials

Fennel: Plaque index of the group using fennel hot water extract

Distilled water: plaque index of the group using distilled water

Figure 3 is a graph showing the antimicrobial activity against Streptococcus mutans ( Streptococcus mutans ) by the fennel ethanol extract of the present invention compared to the control.

■: control

●: Add 1mg / ml fennel ethanol extract

Figure 4 is a Streptococcus mutans ( Streptococcus) by the fennel ethanol extract of the present invention mutans ) is a graph showing the change in pH of the growth medium compared to the control.

■: control

●: Add 1mg / ml fennel ethanol extract

5 is a graph showing the antimicrobial activity of Streptococcus mutans by concentration of fennel ethanol extract.

The present invention relates to an oral composition for preventing caries and an antimicrobial composition, and more particularly, to an oral cavity composition for preventing caries and an antimicrobial composition containing a fennel extract as an active ingredient.

The cause of dental caries is diverse, but lactic acid, which is caused by the breakdown of carbohydrates such as sugars and starch of food debris attached to teeth by fermentation of bacteria, which resides in the oral cavity, is usually used to demineralize dental hard tissue. Tooth decay occurs. Several species are known as pathogenic microorganisms that cause this tooth decay, but Streptococcus mutans is a major pathogen causing caries (see Liljemark WF et al., Crit. Rev. Oral Bio.Med. 7). : 180-198, 1996). Streptococcus mutans secrete an enzyme called glucosyltransferase (GTase) to extracellular or cell surface to produce glucose and fructose using sucrose as a substrate. Insoluble glucan, a polymer of glucose, forms on the tooth surface. The glucan causes other microorganisms in the oral cavity to adhere to the tooth surface, forming a dental plaque, or dental plaque. Within this plaque, lactic acid bacteria, including Streptococcus mutans bacteria, multiply to produce lactic acid as a carbon source, and lactic acid is collected and concentrated in adherent glucan. So a high concentration of a concentrated acid of the calcium phosphate composition of the tooth melts the Ca ^{2 +} will be unloaded by causing dissolution of the tooth decay sikimeuroseo enamel (enamel) of the tooth outer film (see: Sigmund, such as SS, J. Periodontol. 63: 322-331, 1992).

As a method used to suppress such caries, a method using antibiotics such as sporamycin, vancomycin and chlorhexidine, and a method for inhibiting pathogenic bacteria by organic or inorganic fluorine, etc. (Mcbain, AJ et al., Appl. Environ. Microbiol. 69: 4770-4776, 2003). However, if the antibiotics are used continuously, the microorganisms may become resistant or cause side effects such as diarrhea and vomiting. In addition, the abuse of these antibiotics inhibits the growth of flora in the oral cavity. have.

Therefore, it is necessary to separate anti-cavities without side effects from natural products such as medicinal plants, and to kill germs that cause tooth decay. It is stable at high temperature and can be processed at a wide range of temperatures, so it can be used in various formulations Development is urgently needed.

Therefore, the object of the present invention is to solve the above problems, there is an inhibitory activity of glucosyltransferase which is the causative agent of tooth decay and antimicrobial activity against Streptococcus mutans , which is a caries-causing bacterium, is very high as well as side effects. It is to provide an oral composition for preventing tooth decay and antimicrobial composition that can be processed at a wide range of temperatures without being stable at high temperatures.

In order to achieve the object of the present invention as described above, the present invention provides a tooth decay oral composition or antimicrobial composition comprising a fennel extract as an active ingredient.

Fennel or Foeniculum vulgare ) is a perennial herb of hawthorn hawthorn and has a distinctive scent, is used as a dry, dry, insecticide and expectorant in Chinese medicine. However, there is no known antimicrobial effect against the prevention of caries and against Streptococcus mutans.

The present inventors prepared hot water and organic solvent extracts from about 420 herbal medicines, and tested the antibacterial activity against Streptococcus mutans , the inhibitory activity of glucosyltransferase and the caries-inducing bacteria, and fennel extract. It was confirmed that there is an inhibitory activity of glucosyltransferase which is much stronger than other extracts (see Example 1), and it was found that the temperature was excellent by maintaining the activity even at a high temperature (see Example 3). Fennel extract also showed antimicrobial activity against Streptococcus mutans (see Example 6) and significantly reduced plaque accumulation through actual clinical trials (see Example 4) to prevent caries. It can be seen that it can be used as an antimicrobial composition for oral compositions for Streptococcus mutans.

Therefore, the fennel extract of the present invention can be used as an oral composition or an antimicrobial composition.

The composition for oral cavity of the present invention is not limited to the fennel extract as an active ingredient, but refers to foods such as gum, candy, jelly, chocolate, mouthwashes, toothpaste, and the like, and components of the composition commonly used with the fennel extract. It may be prepared according to a known method by adding all or a part of it, for example, in the case of mouthwash, abrasives, fluorine compounds as a medicinal agent, binders, foaming agents, flavors, sweeteners, buffers, alcohol components and other ingredients, toothpaste In the case of an abrasive, a fluorine compound as a pharmacological agent, a binder, a foaming agent, a flavoring agent, a sweetening agent, a buffer, etc., a gum base, a sugar powder, glucose, starch hydrolyzate, a glycerin, a flavoring agent, etc. in the case of a gum, a conventional Sugar, starch syrup, sorbitol, emulsifier, shortening fat, flavoring, coloring, acidulant, etc. In the case of chocolate, purified water, citric acid, liquid fructose, and the like, in the case of chocolate, can be prepared by adding cocoa butter or the like as a normal sugar, cocoa mass, milk powder, or an emulsifier. In an amount of ˜5% by weight.

The antimicrobial composition of the present invention is a fennel extract as an active ingredient, but is not limited thereto, and is prepared in various formulations such as tablets, capsules, soft capsules, liquids, pills, injections, etc. together with pharmaceutically acceptable carriers. It may be applied differently depending on the intended use.

Fennel extract used in the present invention can be extracted using hot water or an organic solvent. The organic solvent is C1-C6 alcohol, acetone (acetone), ether (ether), benzene, chloroform (chloroform), ethyl acetate (ethyl acetate), methylene chloride, hexane (hexane) , Various solvents such as cyclohexane and petroleum ether may be used alone or in combination. Preferably it can be extracted with hot water or ethanol.

In the hot water extraction method of fennel used in the embodiment of the present invention, cold water is added to the fennel, stirred and filtered to remove unnecessary components, and then concentrated with a condenser by adding hot water, followed by stirring with ethanol at room temperature and centrifugation. It is a method of producing and extracting the supernatant by stirring and centrifuging by adding water to the precipitate again, the extracted supernatant may be prepared in powder form or concentrated liquid form by lyophilization or spray drying (see Example 2). ).

The ethanol extraction method of the fennel used in the embodiment of the present invention is 10 times the amount of 70% ethanol added to the fennel and stirred at room temperature, potassium hydroxide is added to the extracted extract extracted by adding n-hexane to extract the lower layer solution The supernatant may be prepared by evaporating and drying the supernatant by distillation under reduced pressure distillation by adding hydrochloric acid to n-hexane followed by partition extraction. However, the manufacturing method of the fennel extract is not necessarily limited to the above method.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

Fennel Hydrothermal  Or organic solvent extracts Glucosyltransferase  Inhibitory activity measurement

10 times (w / v) of distilled water was added to the sample of finely crushed fennel with a domestic mixer, and extracted at 100 ° C. for 50 minutes to prepare a hydrothermal extract. In addition, 10 times of 70% or 100% ethanol, 70% or 100% methanol, 50% or 100% acetone, ethyl acetate, dichloromethane, n-hexane and the like were added to the sample, and extracted by stirring at room temperature for 24 hours. After removing the extraction solvent with nitrogen gas dissolved in 70% ethanol to prepare an organic solvent extract of fennel.

Inoculate Streptococcus mutans into 2 L of BHI (brain heart infusion) medium, anaerobically incubate for 48 hours at 37 ℃, centrifugation (4 ℃, 5000 g, 10 minutes) to remove the cells supernatant Biomax Ultrafiltration with 8 (Millipore, Billerica. Mass., USA), concentrated to 200 ml and then dialyzed overnight at 4 ℃ in a buffer solution (potassium phosphate, 50 mM, pH 6.9) to prepare a glucosyltransferase coenzyme Dilute to 1.4 unit / ml.

150 μl of the fennel extract prepared above was mixed with 600 μl of the enzyme solution prepared above, pre-incubated at 37 ° C., and 150 μl of 8.4% sucrose was reacted for 1 hour, and then 900 μl of methanol was added to the reaction solution. Was added to stop the reaction and centrifuged (10000 rpm, 10 minutes) to obtain a precipitate (insoluble glucan). The precipitate was washed sequentially with 1 ml of distilled water and 1 ml of 70% ethanol, followed by 200 µl of distilled water and 800 µl of anthrone color reagent (anthrone 200 mg / H ₂ SO ₄ 100 ml), followed by boiling for 10 minutes. After heating and color development, the mixture was cooled for 3 minutes and absorbance was measured at 550 nm with a spectrophotometer. If the absorbance decreases at the wavelength, the glucosyltransferase inhibitory activity means that the production of glucan is inhibited. Glucosyltransferase (GTase) inhibitory activity was calculated as follows and listed in Table 1 below.

However, the absorbance of the control solution is the absorbance of the reaction solution except for the plant extract.

As shown in Table 1, hot water and organic solvent extracts of fennel have high glucosyltransferase inhibitory activity, and thus they can be used as an oral composition for preventing tooth decay by glucosyltransferase.

Measurement Results of Glucosyltransferase Inhibitory Activity of Fennel Extract Extraction solvent % Inhibition cold water 10.1 Hydrothermal 61.0 70% ethanol 57.5 100% ethanol 70.1 70% methanol 51.2 100% methanol 69.2 50% acetone 26.0 100% acetone 63.2 Ethyl acetate 70.9 Dichloromethane 68.0 Hexane 73.7

< Example  2>

Preparation of Fennel Hydrothermal Extract

The hot water extract of fennel of <Example 1> has a strong aroma and color, so it can not be used as it is, 1500 L of cold water is added to 100 kg of fennel and stirred for 1 hour, filtered and treated the same method once more Unnecessary components including water soluble sugars were removed. 1200 L of water was added to the treated fennel and extracted at 100 ° C. for 60 minutes, concentrated to 100 L with a concentrator, and then 10% of 30% ethanol was added to the concentrate, stirred at room temperature for 30 minutes, and centrifuged to the resulting precipitate. 100 liters of water was added, stirred for 30 minutes, centrifuged, and the supernatant was lyophilized or spray-dried to prepare fennel hydrothermal extract with almost no color or odor of powdery or concentrated liquid form.

< Example  3>

fennel Hydrothermal  Temperature stability of extract

In order to analyze the glucosyltransferase inhibitory activity change according to the temperature of the fennel hot water extract, the fennel hot water extract of <Example 2> was heated at 60 ° C., 80 ° C. or 100 ° C. for 1 hour, 6 hours and 12 hours, respectively. After glucosyltransferase inhibitory activity was measured in the same manner as described in <Example 1>, the results are shown in FIG. 1.

As shown in FIG. 1, 100% of inhibitory activity was observed when heated for 1 hour at all temperatures, and 95% or more of glucosyltransferase inhibitory activity was observed when heated for 6 hours or 12 hours. Nevertheless, it can be used as an oral composition for the prevention of tooth decay by a stable glucosyltransferase (see Fig. 1).

< Example  4>

fennel Hydrothermal  Clinical Trial on the Inhibition of Plasma Bacterial Membrane Extracts

All five teeth of each male and female volunteer in their 20's were cleaned by removing the bacterial membrane from the tooth surface, and the same toothpaste and toothbrush were provided. To measure the plaque index at a fixed time the next afternoon, brushing teeth after breakfast the day before plaque measurement, and then brushing the teeth afterwards. Five groups (No. 1, 3, 5, 7, 9) were assigned to group A, and five persons (No. 2, 4, 6, 8, 10) were placed on group B. After measuring plaque accumulation, wipe the tooth surface of the subject by using prophlaxis, and after the dinner on the same day, the hot water extract solution of fennel of <Example 2> was prepared in group A and control group B. A solution (distilled water) was used. Specifically, the solution was used twice as a method of rinsing the mouth for 10 minutes in 1 minute within 3 minutes after a meal. The concentration of the experimental solution was 50 mg / ml and repeated until each meal was finished. It was carried out by.

The criteria and calculation method for the adhesion of the bacterial surface of the dental plaque is to hit the buccal surface of the tooth by a specialist at the appointed time in the afternoon after the end of the period, and then stain the tartar with the disclosing solution and divide the surface into 5 equal parts. If the bacterial membrane is not attached, it is scored as 0 point. When the bacterial membrane is attached, the degree of attachment of bacteria is evaluated. After the first experiment, the above process was repeated about 2 weeks, but the control solution was given to group A and the hot water extract solution of fennel was used to group B, and the result was analyzed by T-test using the SPSS program. Treated.

As a result, the cumulative degree of dental plaque in the case of oral care using fennel hot water extract was decreased by 1.8 points in comparison with the case of no oral care, and increased by 3 points in case of distilled water. When the fennel hydrothermal extract was used in all groups, plaque decreased and showed reproducible results. The results were significant at the 95% confidence interval. As a result, when the fennel hot water extract was used, the cumulative accumulation of about 4.8 points was reduced compared to the case where the fennel hot water extract was used, and the fennel hot water extract could be used as an oral composition for preventing tooth decay by removing plaque. (See FIG. 2).

< Example  5>

Preparation of Fennel Ethanol Extract

10 g of 70% ethanol was added to 100 g of fennel and stirred at room temperature for 24 hours to adjust the pH to 10 by adding 1 N potassium hydroxide to the extracted extract, followed by two-part extraction by adding 1 L of n-hexane. 1 N hydrochloric acid was added to the lower layer to adjust the pH to 2, and then, 1 L of n-hexane was added and extracted twice.

< Example  6>

Of fennel ethanol extract Streptococcus Mutans  Growth Inhibition Activity Evaluation

<6-1> Fennel Ethanol Extract Streptococcus Mutans  Growth Inhibition Activity Assay

In order to analyze the growth inhibitory activity against Streptococcus mutans of the fennel ethanol extract prepared in <Example 5>, 100 mg of 70% ethanol extract of <Example 5> was dissolved in distilled water and ethanol, respectively, and then Streptococcus Inoculated with 100 ml of BHI medium with 1 ml of mutans culture (OD ₆₀₀ = 1) and incubated for 30 hours while proliferation of Streptococcus mutans was measured by turbidity at 600 nm for each time period. It was.

As shown in FIG. 3, the growth of Streptococcus mutans was not significantly different from the control group in the addition group of the fennel ethanol extract until 15 hours after the culture, but the growth of Streptococcus mutans was significantly increased in the control group after 15 hours after the culture. In contrast, the addition of fennel ethanol extract significantly inhibited the growth of Streptococcus mutans up to 30 hours after incubation (see FIG. 3).

On the other hand, as a result of measuring the pH change of the culture medium according to the growth of Streptococcus mutans, the pH change was not changed in all groups until 15 hours after the initial culture, but the control and fennel hot water extracts were added after 15 hours after the culture. In the group, the pH was significantly lowered to 5.3, while in the addition group of fennel ethanol extract, the pH was maintained at 7 (see FIG. 4).

These results generally indicate that the pH of the Streptococcus mutans is lowered by the organic acid produced as it grows, but the growth of Streptococcus mutans is inhibited by the fennel ethanol extract and the pH remains constant.

Therefore, the fennel ethanol extract inhibits the growth of Streptococcus mutans, which is the causative agent of caries, and thus has the effect of eliminating the cause of caries. Therefore, the fennel ethanol extract has antibacterial activity against Streptococcus mutans. It can be used in the oral composition for the prevention of tooth decay caused by Streptococcus mutans.

<6-2> Fennel Ethanol Extract Streptococcus Mutans  Concentration Determination for Growth Inhibitory Activity

As can be seen from the results of <Example 6-1>, the ethanol extract of fennel has growth inhibitory activity against Streptococcus mutans, and the appropriate concentration for growth inhibition activity was measured using a paper disk method. . Since the fennel ethanol extract prepared in Example 5 is non-aqueous, it is difficult to diffuse in the medium, so that the ethanol extract was dissolved in 0.1 N sodium hydroxide at 25 mg / ml, 50 mg / ml, and 100 mg / ml, respectively. After loading 30 μl each, the growth inhibitory activity of Streptococcus mutans was examined by a paper disc method on a solid medium, compared with 0.1 N sodium hydroxide as a control.

As a result, in the control group, no growth inhibitory ring was found, while the fennel ethanol extract was found to form a growth inhibitory ring at a concentration of 50 mg / ml or more (see FIG. 5). A fennel ethanol extract of 50 mg / ml or more effectively prevented growth of Streptococcus mutans. It was found to inhibit.

< Production Example >

Preparation of Oral Compositions Containing Fennel Extract

<Manufacture example 1-4>

The extract of the fennel prepared in the <Example 2> or <Example 5> was added to prepare a mouthwash with the ingredients and contents shown in Table 2 below.

<Production example 5-8>

The extract of fennel prepared in <Example 2> or <Example 5> was added and a toothpaste was prepared using the ingredients and contents shown in Table 3 below.

<Manufacture example 9-12>

An extract of the fennel prepared in <Example 2> or <Example 5> was added thereto, and a gum was prepared using the ingredients and contents shown in Table 4 below.

<Production example 13-16>

The extract of the fennel prepared in <Example 2> or <Example 5> was added to prepare a candy with the ingredients and contents shown in Table 5 below.

<Production Example 17-20>

The extract of the fennel prepared in the <Example 2> or <Example 5> was added to prepare a jelly with the ingredients and contents shown in Table 6.

<Production Example 21-24>

The extract of the fennel prepared in <Example 2> or <Example 5> was added to prepare a chocolate with the ingredients and contents shown in Table 7.

As described above, the fennel extract has excellent antimicrobial activity against the inhibitory activity of glucosyltransferase and tooth decay-causing Streptococcus mutans , and is isolated from natural products and has no side effects and is stable at high temperatures to prevent tooth decay. It can be used for oral compositions and antimicrobial compositions.

Claims (5)

(a) adding water to the fennel, stirring and filtering to pretreat the fennel; (b) extracting the pretreated fennel with hot water; (c) adding 30% ethanol to the extract, stirring and centrifuging to obtain a precipitate; And (D) oral cavity composition for preventing tooth decay containing hot water extract of fennel prepared by the method comprising the step of dissolving the precipitate in water as an active ingredient. According to claim 1, wherein the oral composition is a mouthwash for preventing tooth decay, characterized in that consisting of any one of the formulations selected from the group consisting of a mouthwash, toothpaste, gum, candy, jelly, and chocolate. delete (a) adding water to the fennel, stirring and filtering to pretreat the fennel; (b) extracting the pretreated fennel with hot water; (c) adding 30% ethanol to the extract, stirring and centrifuging to obtain a precipitate; And (d) Antimicrobial composition, characterized in that it has an antimicrobial activity against Streptococcus mutans by containing the hot water extract of fennel prepared by the method comprising the step of dissolving the precipitate in water as an active ingredient. delete

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