KR100492034B1 - Antibacterial agent and oral composition for preventing and treating caries and periodontal disease containing panduratin derivatives - Google Patents

Abstract

The present invention relates to an antimicrobial agent and oral composition for preventing and treating tooth decay and periodontitis. The antimicrobial agent and oral composition of the present invention is characterized by containing a panduratin derivative as an active ingredient. Panduratin derivatives such as isopanduratin A have very high antibacterial activity against caries-inducing Streptococcus mutans and periodontal disease-causing Porphyromonas gingivalis . In addition, it has no side effects and is stable at high temperatures, and can be processed at a wide range of temperatures. Therefore, panduratin derivatives are effective for the prevention and treatment of tooth decay and periodontitis, and can be used in oral compositions of various formulations such as toothpaste compositions, mouthwashes, gums, candy, jelly, chocolate, and the like. Can be used.

Description

Antibacterial agent and oral composition for preventing and treating caries and periodontal disease containing panduratin derivatives}

Tooth decay is a disease that accounts for a large part of dental diseases, from children to adults. According to the report of the Korean Dental Association, more than 90% of Korean children have experienced dental caries. In addition, more than 80% of adults have periodontitis.

Although there are various causes of tooth decay and periodontitis, in general, the decay caused by the breakdown of carbohydrates such as sugar or starch of food residues attached to the teeth by the fermentation of bacteria resident in the mouth causes decay of the dental hard tissues do. Accordingly, the anaerobic pathogens multiply and the gum tissue is destroyed by the toxin component generated by the periodontitis strains, resulting in a phenomenon that teeth shake and drop out.

Several species are known as pathogenic microorganisms that cause tooth decay and periodontitis, but Streptococcus mutans and Porphyromonas gingivalis are the main pathogens for caries and periodontitis. 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 based on sucrose in food. 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 bacterial membrane, 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. This high concentration of acid causes Ca ²⁺ to dissolve in calcium phosphate, which is a component of the tooth, and dissolves the enamel, which is the outer membrane of the tooth, causing tooth decay (Sigmund, SS, et al., J. et al. Periodontol., 63: 322-331, 1992).

As a method used to suppress tooth decay and periodontitis, a method using antibiotics such as sporamycin, vancomycin, chlorohexidine, and inhibition of pathogenic bacteria by organic or inorganic fluorine Methods are used (Mellberg, JR, et al., Quintessence, 41-80, 1983).

However, the method of using antibiotics has the disadvantage that microorganisms become resistant or cause side effects such as diarrhea and vomiting, and there is a problem that the growth of the flora in the oral cavity is inhibited due to the abuse of these antibiotics. . On the other hand, antimicrobial substances derived from natural products known to date are mostly inferior in antimicrobial activity, poor thermal stability, there is a problem that the antimicrobial activity is sharply reduced.

Therefore, the technical problem to be achieved by the present invention is to solve the above problems, the antibacterial activity against the caries causing bacteria Streptococcus mutans and periodontal disease causing bacteria Porphyromonas gingivalis very high Furthermore, to provide an antimicrobial agent and oral composition for the prevention and treatment of caries and periodontitis that can be processed at a wide range of temperatures without any side effects and stable at high temperatures.

In order to achieve the above technical problem, the present invention provides an antibacterial agent and oral composition for caries and periodontitis prevention and treatment, characterized in that it contains a panduratin derivative represented by the following general formula (1) as an active ingredient;

<Formula 1>

In Formula 1, R ₁ and R ₂ (R ₁ = , R ₂ = ), (R ₁ = , R ₂ = ), (R ₁ = , R ₂ = ) And (R ₁ = , R ₂ = ) Is any one selected from the group consisting of.

In the antimicrobial and oral compositions of the present invention, panduratin derivatives can be used extracted from Kaempferia pandurata , such panduratin derivatives can be used together with pharmaceutically acceptable carriers, e.g. tablets, It can be prepared as an antimicrobial agent in various formulations such as capsules, soft capsules, liquids, pills, injections and the like. It can also be added to a variety of oral compositions such as gums, candies, jelly, foods such as chocolate, mouthwashes, toothpastes and the like.

Hereinafter, the antimicrobial agent and oral composition for preventing and treating tooth decay and periodontitis according to the present invention will be described in detail.

The panduratin derivative as an active ingredient of the antimicrobial agent and oral composition for caries and periodontitis prevention and treatment according to the present invention is a compound represented by the following formula (1).

<Formula 1>

In Formula 1, R ₁ and R ₂ (R ₁ = , R ₂ = ), (R ₁ = , R ₂ = ), (R ₁ = , R ₂ = ) And (R ₁ = , R ₂ = ) Is any one selected from the group consisting of.

The panduratin derivative may be extracted from a plant such as Kaempferia pandurata Roxb., But is not limited thereto. Kaempferia pandurata Roxb. Is a plant belonging to the zingiberaceae family. The root is widely used for colds, enteritis, skin diseases and urethral pain. Caemperia pandurata includes pinocembrin chalcone, cardamonin, pinocembrin, pinostrobin, 4-hydropanduratin A, and panduratin A. (panduratin A) and the like, these components have been reported to have anti-cancer effects (Trakoontivakorn, G., et al ., J. Agric. Food Chem ., 49, 3046-3050, 2001), flavonoids dihydro kelkon (dihydrochalcone) compounds of the strains have been reported to exhibit an insecticidal effect (see: Pandji, C., such as, phytochemistry, 34, 415-419, 1993 ). However, no antimicrobial activity of panduratin derivatives such as isopanduratin A against oral pathogenic bacteria has been reported.

The present inventors have tested the antimicrobial activity against caries-inducing bacteria Streptococcus mutans and periodontal disease-inducing bacteria Porphyromonas gingivalis using panduratin derivatives. It was found to exhibit a very strong antimicrobial activity against. Therefore, when the panduratin derivative is added to an antibacterial agent such as tablets, capsules, soft capsules, liquids, pills, injections, or the like, oral compositions such as gum, candy, jelly, chocolate, mouthwashes, toothpastes, etc. It can prevent and treat periodontitis.

A method of extracting panduratin derivatives from Kaemperia pandurata will be described in detail by taking an isopanduratin A extraction method.

First, the dried Kaemperia pandurata is pulverized and mixed with 75% methanol to extract solvent, then the solvent is removed and the extract is concentrated. The crude crude extract and ethyl acetate were mixed to extract the ethyl acetate soluble component. This extraction process is preferably repeated two or more times. Although crude extract and ethyl acetate soluble extract component obtained through the extraction process is not a single component, it exhibits strong antibacterial activity against Streptococcus mutans and Popiromonas gingivalis which are representative oral pathogenic strains.

Next, ethyl acetate is removed to concentrate the ethyl acetate soluble component, and then separated by each component according to the polarity difference of each component. At this time, it is preferable to use silica gel column chromatography as a separation method. For example, a solvent in which hexane and ethyl acetate are mixed at a ratio of 5: 1 (v / v) is used. The impurities are removed by developing by using a solvent, and hexane, chloroform, and ethyl acetate are separated by using a solvent mixed at a ratio of 10: 5: 1.5 (v / v), respectively, and finally a single antimicrobial activity The material can be separated.

The antimicrobial active ingredient obtained through such extraction and separation process has the structure of Formula I (R ₁ = , R ₂ = Isopanduratin A).

In particular, isopanduratin A has very low antimicrobial activity due to extremely low concentrations of inhibitory concentrations against oral pathogenic strains Streptococcus mutans and Popyromonas jinjibaris.

The panduratin derivatives such as isopanduratin A thus obtained may be prepared together with pharmaceutically acceptable carriers, for example, as antibacterial agents in various formulations such as tablets, capsules, soft capsules, solutions, pills, injections and the like.

In particular, various oral compositions such as foods such as gum, candy, jelly, chocolate, mouthwashes, toothpaste, and the like may be added to all or a part of a composition commonly used together with panduratin derivatives or crude extracts containing the same. For example, oral cleaning agents include abrasives, fluorine compounds as medicinal agents, binders, foaming agents, flavoring agents, sweeteners, buffers, alcohol components and other ingredients, and toothpaste compositions include abrasives, fluorine compounds as agents, binders. In the gum composition, gum base, sugar powder, glucose, starch hydrolyzate, glycerin, fragrance, etc., sugar, starch syrup, sorbitol, emulsifier, shortening ) Oils, flavors, pigments, acidulants, etc., jelly, ordinary sugar, gelling agent, purified water, citric acid, liquid fructose, etc., chocolate Of it can be prepared with sugar and cocoa scalpel, milk powder, oral composition by the addition of cocoa butter, and the like as an emulsifying agent.

     Hereinafter, the present invention will be described in detail with reference to Examples. However, embodiments according to the present invention can be modified in many different forms, the scope of the present invention should not be construed as limited to the embodiments described below. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

In addition, in the following Examples and Experimental Examples, the% not specifically indicated as% by weight represents the weight%.

Example of Extracting Crude Extract Containing Isopanduratin A

Primary extraction example

The dried Kaemperia pandurata was pulverized with a mixer, and then 100 g of the pulverized Kaemperia pandurata sample was extracted using 400 ml of 75% by volume methanol as an extraction solvent. The extracted sample was filtered with Whatman No. 2 filter paper, the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, and then lyophilized to remove the crude component.

Thermal Stability of Crude Extracts

The crude extracts prepared according to the first extraction example were heat-treated at 60 ° C., 70 ° C., 80 ° C., 90 ° C. and 100 ° C. for 30 minutes, at 121 ° C. for 15 minutes, and then the crude extract was 0.1 ml in each well. The antimicrobial activity against Streptococcus mutants was measured using a well diffusion assay, which was incubated at room temperature for at least 1 hour and incubated at 37 ° C for 16 hours or more. The stability was evaluated. The antimicrobial activity of crude extracts at different treatment temperatures is shown in Table 1 below.

Processing time (minutes) 30 15 Treatment temperature (℃) 60 70 80 90 100 121 Low ring diameter of plate (mm) 15 15 14 14 14 15

Referring to Table 1, it can be seen that the crude extract heat-treated at a high temperature from 60 ℃ to 121 ℃, the antibacterial activity is hardly reduced even after the heat treatment, the antimicrobial active ingredient is stable even at high temperatures.

Bactericidal Activity of Crude Extracts by Viable Cell Count Method

The crude extract prepared according to the first extraction example was dissolved in dimethyl sulfoxide to prepare samples at concentrations of 0.001%, 0.003% and 0.005%, respectively. Each prepared sample was reacted at 37 ° C. for 1 minute, 2 minutes, 5 minutes, and 10 minutes, and then diluted 10 times in sequence to perform a pour plate method, and then in a 37 ° C. incubator. The cultures were measured for viable cell numbers of Streptococcus mutants. The result is shown in FIG.

In Figure 1, ◆ indicates a control dimethyl sulfoxide, ▲ indicates a sample of 0.001% concentration, ● indicates a sample of 0.003% concentration, ■ indicates a sample of 0.005% concentration.

Referring to FIG. 1, the sample containing the crude extract significantly reduced the viable cell count of Streptococcus mutants within 1 minute, especially when the concentration of the extract was 0.005% (50 µg / ml). It was shown to kill. This suggests that the inhibitory activity of the antimicrobial substances according to the present invention is very excellent, considering that other medicinal plant extracts generally show effective inhibition at a concentration of 200-1000 ㎍ / ㎖, and between 1 and 2 minutes This means that they can kill enough germs during the brushing process, considering that people brush their teeth in about three minutes on average.

Second extraction example

The dried Kaemperia pandurata was pulverized with a mixer, and then 100 g of the pulverized Kaemperia pandurata sample was mixed with 400 ml of 75% by volume methanol and repeatedly extracted at room temperature for two days. The extracted sample was filtered through Whatman filter paper No. 2 to obtain a crude extract. The obtained crude extract was mixed with ethyl acetate at a ratio of 1: 1, and extracted by dissolving the dissolved component twice, and then removing the solvent component to obtain an ethyl acetate fraction.

Determination of Minimum Inhibitory Concentration According to Ethyl Acetate Fraction

The ethyl acetate fraction obtained according to the second extraction example was dissolved in 0.5% methanol, and 1 ml of each of the platelets of blood was placed in the prepared test tubes, and the crude extract obtained according to the first extraction example and the second extraction example were prepared only in the first test tube. The ethyl acetate fractions thus obtained were each added in 1 ml at 2% concentration, and then diluted twice in sequence.

100 μl of Streptococcus mutance and Popiromonas ginjibaris were added to each test tube to 2 × 10 ⁵ CFU / mL in this dilution solution. The results are shown in Table 2 below.

Minimum inhibitory concentration (㎍ / ㎖) Methanol Crude Extract Ethyl Acetate Fraction Streptococcus mutans 62.5 31.3 Popiromonas jinjibaris 62.5 31.3

Referring to Table 2, although the crude crude extract and ethyl acetate soluble components were not processed, the minimum inhibitory concentrations of the representative oral pathogenic strains Streptococcus mutans and Popiromonas ginjivalis were 62.5 µg / ml and 31.3 µg / ml, respectively, showed very good antimicrobial activity. This is much lower than the minimum inhibitory concentration of catechins.

Determination of Antimicrobial Activity of Soluble Components of Ethyl Acetate

The ethyl acetate soluble component prepared according to the second extraction example was dissolved in dimethyl sulfoxide to prepare an ethyl acetate soluble component solution at 1% concentration, and diluted 10-fold to prepare an ethyl acetate soluble component solution at 0.1% concentration.

The ethyl acetate soluble component solutions of the prepared 1% and 0.1% concentrations were measured for the antimicrobial activity against Streptococcus mutants by the well diffusion analysis.

As a control for control, the antimicrobial activity was measured by the same method using dimethyl sulfoxide, which is a solvent that does not contain a sample, and as a control for comparing the antimicrobial activity, 1% green tea extract solution dissolved in dimethyl sulfoxide. The antimicrobial activity was measured by the same method.

The antimicrobial activity of ethyl acetate soluble component solutions, control and control at concentrations of 1% and 0.1% were compared with the diameter (mm) of the clear zone formed around the well after incubation, and the results are shown in FIG. 2.

In the photograph of Fig. 2, a is the antimicrobial activity of the ethyl acetate soluble component solution at 0.1% concentration, b is the antimicrobial activity of the ethyl acetate soluble component solution at 1% concentration, c is the antimicrobial activity of dimethyl sulfoxide as a control, d is The antimicrobial activity of the green tea extract at the concentration of 1%, respectively, was shown.

In the case of the well diffusion assay, the activity may vary depending on the extent of diffusion if the sample is viscous. However, since the ethyl acetate soluble component sample and the green tea extract show little viscosity in solution, the activity can be compared as the size of the low ring. There is a number. Referring to FIG. 2, the control group dimethyl sulfoxide did not affect the bacteria, and when compared with the low ring size of the green tea extract at the same concentration of 1%, the size of the low ring size of the ethyl acetate soluble component solution sample was larger. That is, it can be seen that the ethyl acetate soluble component solution has better antibacterial activity than the green tea extract, which is known to be effective in preventing caries.

Third extraction example

The ethyl acetate soluble component prepared according to the second extraction example was inert using a solvent system containing hexane and ethyl acetate in a ratio of 5: 1 (v / v) in a column packed with silica gel at 6 × 15 cm. Subsequently, hexane, chloroform, and ethyl acetate were separated by thin layer chromatography (TLC, silica gel 60F ₂₅₄ , Merck) with a developing solvent of 10: 5: 1.5 (v / v), respectively. Accordingly, the fractions were concentrated and dried into four fractions from fraction 1 to fraction 4, and the antimicrobial activity of each fraction was investigated by the following method. 1 ml each of the platelets were prepared in the prepared test tube, 1 ml of the sample of each purified fraction was added only to the test tube for the first time, and then diluted twice by dilution. 100 μl of the bacterium was added to each test tube to be 2 × 10 ⁵ CFU / mL, followed by incubation for at least 16 hours at 37 ° C., and the minimum inhibitory concentration without turbidity was measured. The minimum inhibitory concentration of each fraction measured is shown in Table 3 below.

Fraction Minimum inhibitory concentration (㎍ / ㎖) One 250 2 7 3 62.5 4 500

Referring to Table 3, the minimum inhibitory concentration value of fraction 2 was found to be low as 7 ㎍ / ㎖. Fraction 2 was again subjected to thin layer chromatography (TLC, silica gel 60F ₂₅₄ , Merck) using hexane, chloroform and ethyl acetate as a developing solvent of 10: 5: 1.5 (v / v), respectively, to give a value of Rf = 0.23. In the ultraviolet (254, 365 nm, VL-6-LC, Vilber lourmat) single material having a strong absorption band was isolated.

The molecular weight of the single substance having antimicrobial activity purified by the above method was measured by EI-MS, and ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured at 500 MHz (solvent: MeOD), respectively. ¹³ C-NMR spectrum, ¹ H-NMR spectrum and EI-MS spectrum are shown in FIGS. 3, 4 and 5, respectively.

EI / MS ( m / z , rel. Int.): 406 ([M] ⁺ , 5), 337 (6), 272 (19), 271 (100), 167 (97), ¹³ C-NMR (125 MHz, CD ₃ OD) 205.89 (s, C-7), 167.06 (s, C-6), 164.41 (s, C-4), 162.81 (s, C-2), 146.98 (s, C-1 " ), 136.89 (s, C-3 '), 130.97 (s, C-9'), 127.68 (d, C-3 ", 5"), 126.74 (d, C-2 ", 6"), 125.00 ( d, C-4 "), 124.12 (d, C-8 '), 120.58 (d, C-4'), 105.71 (s, C-1), 95.71 (d, C-5), 90.69 (d, C-3), 56.40 (q, OMe), 55.69 (d, C-1 '), (1H, dd, J = 4.5, 11.5 Hz, H-1'), 3.80 (3H, s, -OMe), 3.28 (1H, ddd, 44.23 (d, C-2 '), 38.68 (d, C-6'), 37.30 (t, C-5 '), 30.04 (t, C-7'), 26.06 (q, C-11 '), 23.31 (q, C-12'), 18.17 (q, C-10 '), ¹ H-NMR (500 MHz, CD ₃ OD) 7.07 (2H, dd, J = 7.2, 8.2 Hz , H-3 ", 5"), 7.05 (2H, d, J = 8.2 Hz, H-2 ", 6"), 6.95 (1H, dd, J = 7.2, 7.2 Hz, H-4 "), 5.88 (1H, d, J = 2.0 Hz, H-3), 5.70 (1H, d, J = 2.0 Hz, H-5), 5.33 (1H, br.s, H-4 '), 4.77 (1H, m , H-8 '), 4.42 J = 11.7, 10.5, 6.4 Hz, H-6'), 2.43 (1H, m, H-2 '), 2.25 (1H, br.d, J = 5.2 Hz, H- 5 '), 2.15 (1H, br.d, J = 17.7 Hz, H-7'), 1.98 (1H, ddd, J = 15.0, 7.0, 7.0 Hz, H-7 '), 1.92 (1H, m, H-5'), 1.69 (3H, s, H-12 '), 1.40 (6H, s, H-10', 11 ')

When the above separated single material was recrystallized from EtOAc-MeOH, a sticky pale orange oil component was obtained.

Compound isopanduratin A has a molecular weight of 406 as [M] ⁺ is observed at m / z 406 in EI / MS, and the molecular formula is C ₂₆ H ₃₀ O ₄ .

^{In the 13} C-NMR (125 MHz) spectrum, 26 signals were observed. Analysis of the signal along with the DEPT spectrum revealed one ketone (δ _C 205.89) and three olefin quaternary carbons (δ _C 167.06, 164.41, 162.81) bound to oxygen. In addition, four olefin quaternary carbons (δ _C 146.98, 136.89, 130.97, 105.71) bonded with carbon were observed, and nine olefine methine carbon {δ _C 127.68 (x2), 126.74 (x2), 125.00, 124.12, 120.58, 95.71, and 90.69), of which two pairs of signals were duplicated, suggesting the presence of mono- or para-disubstituted benzene rings. In the high magnetic region, one methoxy (δ _C 56.40), three methines (δ _C 55.69, 44.23, 38.68), two methylenes (δ _C 37.30, 30.04), three methyls (δ _C 26.06, 23.31, 18.17) Since a carbon signal was observed, it was assumed to be a compound having two benzene rings and two pairs of double bonds.

^{In the 1} H-NMR (500 MHz) spectrum, signals derived from monocyclic benzene rings {δ7.07 (2H, dd, J = 7.2, 8.2 Hz), δ7.05 (2H, d, J = 8.2 Hz), δ6 .95 (1H, dd, J = 7.2, 7.2 Hz)} and signals derived from 1,2,4,6 tetrasubstituted benzene rings {δ5.88 (1H, d, J = 2.0 Hz), δ5.70 ( 1H, d, J = 2.0 Hz). In addition, the presence of one methoxy (δ3.80, 3H, s) and three aryl methyl {δ1.69 (3H, s), δ1.40 (6H, s)} was also confirmed. In addition, in the high magnetic field, the integration of each signal and the data of ¹³ C-NMR were examined together. As a result, three methines {δ4.42 (1H, dd, J = 4.5, 11.5 Hz), δ3.28 (1H, ddd) , J = 11.7, 10.5, 6.4 Hz), δ2.43 (1H, m)} and two methylene {δ2.25 (1H, br.d, J = 5.2 Hz), δ2.15 (1H, br.d , J = 17.7 Hz), δ 1.98 (1H, ddd, J = 15.0, 7.0, 7.0 Hz), and δ 1.92 (1H, m)} were confirmed. Each proton signal was observed for each correlation in the ¹ H- ¹ H COSY spectrum. That is, the connection of the proton signal from H-2 "to H-6", the connection of H-1 '->H-6'-> H-5 '->H-4', H-1 '->H-2'-> The connection of H-7 'to H-8' and the long range coupling of H-3 and H-5 were also confirmed.

The above results are summarized and compared with the spectral data, the compound is (2-methoxy-4,6-dihydroxyphenyl)-[3'-methyl-2 '-(3 "-methylbut-2 &quot; -enyl) -6'- It was structured with phenylcyclo-hex-3'-enyl] methanone and isopanduratin A because it was already an isomer with panduratin A isolated from fingerroot ( Boesenbergia pandurata ).

Based on the above results, it was found that the isolated single substance is isopanduratin A represented by the formula (1).

Determination of Antimicrobial Activity of Isopanduratin A

Dissolve the isopanduratin A separated according to the third extraction example in 0.5% methanol, put 1 ml of blood plate medium into the prepared test tubes, and 1% of isopanduratin A at 2% concentration in the first test tube only. ㎖ was added, and then diluted two times sequentially.

100 μl of Streptococcus mutance and Popiromonas ginjibaris were added to each test tube to 2 × 10 ⁵ CFU / mL in this dilution solution. The results are shown in Table 4 below.

Minimum inhibitory concentration (㎍ / ㎖) Streptococcus mutans 4 Popiromonas jinjibaris 4

      As shown in Table 4, isopanduratin A, a kind of panduratin derivative, showed a very low inhibitory concentration value of 4 µg / ml for representative oral pathogenic bacteria Streptococcus mutans and Popiromonas jinjibaris.

      In addition, in addition to Streptococcus mutans, the minimum of isopanduratin A against other caries-inducing bacteria Streptococcus sobrinus, Streptococcus sanguis, Streptococcus salivarius Inhibition concentration was measured by the same method as described above, the minimum inhibitory concentration value was also very low as about 4 ㎍ / ㎖. The minimum inhibitory concentration values of isopanduratin A are green tea extract with a minimum inhibitory concentration value of 125 µg / ml, carvacrol, isoeugenol with a minimum inhibitory concentration value of 250 µg / ml, and a minimum inhibitory concentration. Compared with the industrial oral-microbial antimicrobial agents used conventionally, such as thymol and eucalyptol whose concentration value is 500 micrograms / mL, it is the low value. In addition, the values close to the minimum inhibitory concentration of 2 ㎍ / ㎖ of the powerful antibiotics (biocomtics vancomycin, hexamedine), considering the side effects of these antibiotics are separated from natural products with little side effects It can be seen that the usefulness of isopanduratin A is much greater.

Bactericidal Activity of Isopanduratin A by Viable Cell Count Method

Isopanduratin A isolated according to the third extraction example was used to measure the antimicrobial activity against Streptococcus mutans, a representative oral pathogenic bacterium. That is, isopanduratin A was dissolved in dimethyl sulfoxide to prepare a sample having a concentration of 0.0005%, 0.001%, and 0.002%, and the prepared sample was mixed with Streptococcus mutants bacteria solution at 37 ° C. for 1 minute and 2 minutes, respectively. After reacting for 5 minutes and 10 minutes, 10-fold dilutions were performed sequentially to carry out a pour plate method, which was cultured in an incubator at 37 ° C. to measure the number of viable cells. The results are shown in FIG.

In Figure 6, ◆ represents a control 0.02% methanol, ● is a sample of 0.0005% concentration, ▲ is a sample of 0.001% concentration, ■ represents a sample of 0.002% concentration.

Referring to FIG. 6, the sample containing isopanduratin A significantly reduced the viable cell count of Streptococcus mutants within 1 minute, especially when the concentration was 0.002% (20 µg / ml). It was almost killed. This suggests that effective antimicrobial activity can be achieved even at very low concentrations compared to other medicinal plant extracts, and the killing of bacteria in about one minute means that people brush their teeth in about three minutes on average. It means you can kill them enough.

Changes in Cell Type with Addition of Isopanduratin A

Isopanduratin A prepared according to the third extraction example was dissolved in 0.08% methanol, adjusted to a final concentration of 0.001%, and then reacted with Streptococcus mutans for 30 minutes in a 37 ° C. incubator to transmit a transmission electron microscope (TEM). The morphology of the cells was observed.

7 is a photograph of Streptococcus mutans treated with 0.08% methanol as a control, and FIG. 8 is a photograph of Streptococcus mutans after treatment with 0.001% isopanduratin A. FIG. In the control of FIG. 8, there was no change in cell morphology, whereas in the photograph of FIG. 9, the cell membrane of Streptococcus mutans isolates and the cell wall was also outlined despite the treatment of very low isopanduratin A of 0.001%. It can be seen that it is blurred and destroyed. This means that isopanduratin A damages the cell walls and cell membranes of Streptococcus mutans bacteria, thereby destroying the cell membranes and losing osmotic function, thereby stopping the physiology of microorganisms and inhibiting growth.

Preparation of Oral Compositions Containing Crude Extracts

Examples 1-4

The crude extract obtained according to the first extraction example was added, and an oral cleanser was prepared using the ingredients and contents shown in Table 5 below.

Major Components Example 1 Example 2 Example 3 Example 4 Crude extract 5.0% Crude extract 1.0% Crude extract 0.1% Crude extract 0.01% Sodium fluoride 0.02% Sodium fluoride 0.02% Sodium fluoride 0.02% Sodium fluoride 0.02% Ethanol 6.6% Ethanol 6.6% Ethanol 6.6% Ethanol 6.6% Glycerin 6.0% Glycerin 6.0% Glycerin 6.0% Glycerin 6.0% l-menthol 0.005% l-menthol 0.005% l-menthol 0.005% l-menthol 0.005% Saccharin sodium Saccharin sodium Saccharin sodium Saccharin sodium Citric acid Citric acid Citric acid Citric acid Pigment amount Pigment amount Pigment amount Pigment amount Peppermint Essence Peppermint Essence Peppermint Essence Peppermint Essence Spearmint Essence Spearmint Essence Spearmint Essence Spearmint Essence

Determination of Anti-Cavity Activity of Oral Cleaners Containing Crude Extracts

    To the test tube containing 4 ml of the oral cleanser containing the crude extracts prepared in Examples 1 to 4, 1 ml of the solution containing the oral pathogenic bacteria was added, incubated while shaking the tube, and then over time. The antimicrobial activity was measured by measuring the number of viable cells, and the results are shown in Table 6 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 1 4.0 × 10 ⁵ <One Example 2 4.0 × 10 ⁵ <One Example 3 4.0 × 10 ⁵ <One Example 4 4.0 × 10 ⁵ 1.1 × 10 ² Mouthwashes without Crude Extracts 4.0 × 10 ⁵ 2.5 × 10 ⁴ Porphyromonas gingivalis Example 1 3.5 × 10 ⁵ <One Example 2 3.5 × 10 ⁵ <One Example 3 3.5 × 10 ⁵ <One Example 4 3.5 × 10 ⁵ 1.1 × 10 ² Mouthwashes without Crude Extracts 3.5 × 10 ⁵ 1.2 × 10 ⁴

Examples 5-8

The crude extract obtained according to the first extraction example was added, and toothpaste was prepared using the ingredients and contents shown in Table 7 below.

Major Components Example 5 Example 6 Example 7 Example 8 Crude extract 5.0% Crude extract 1.0% Crude extract 0.1% Crude extract 0.01% Sorbitol liquid 19.2% Sorbitol liquid 19.2% Sorbitol liquid 19.2% Sorbitol liquid 19.2% Glycerin 22.0% Glycerin 22.0% Glycerin 22.0% Glycerin 22.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Sucrose Fatty Acid Ester 1.7% Sucrose Fatty Acid Ester 1.7% Sucrose Fatty Acid Ester 1.7% Sucrose Fatty Acid Ester 1.7% Alkyl sodium sulfate 1.0% Alkyl sodium sulfate 1.0% Alkyl sodium sulfate 1.0% Alkyl sodium sulfate 1.0% Sodium chloride 1.0% Sodium chloride 1.0% Sodium chloride 1.0% Sodium chloride 1.0% Spices 1.1% Spices 1.1% Spices 1.1% Spices 1.1% Carboxymethyl Cellulose Sodium 0.4% Carboxymethyl Cellulose Sodium 0.4% Carboxymethyl Cellulose Sodium 0.4% Carboxymethyl Cellulose Sodium 0.4% Purified water balance Purified water balance Purified water balance Purified water balance

Determination of Anti-Cavity Activity of Toothpaste Containing Crude Extracts

    Except for using the toothpaste containing the crude extract prepared according to Examples 5 to 8 according to the same method as in Example 1 was measured and the results are shown in Table 8.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 5 4.0 × 10 ⁵ <One Example 6 4.0 × 10 ⁵ <One Example 7 4.0 × 10 ⁵ <One Example 8 4.0 × 10 ⁵ <One Toothpaste without crude extract 4.0 × 10 ⁵ 3.1 × 10 ⁴ Porphyromonas gingivalis Example 5 3.5 × 10 ⁵ <One Example 6 3.5 × 10 ⁵ <One Example 7 3.5 × 10 ⁵ <One Example 8 3.5 × 10 ⁵ <One Toothpaste without crude extract 3.5 × 10 ⁵ 6.7 × 10 ⁴

Examples 9-12

The crude extract obtained according to the first extraction example was added, and a gum was prepared using the ingredients and contents shown in Table 9 below.

Major Components ㅣ Example 9 Example 10 Example 11 Example 12 Crude extract 5.0% Crude extract 1.0% Crude extract 0.1% Crude extract 0.01% Gum base 20% Gum base 20% Gum base 20% Gum base 20% Sugar powder 54% Sugar powder 54% Sugar powder 54% Sugar powder 54% 10% glucose 10% glucose 10% glucose 10% glucose Starch hydrolyzate 10% Starch hydrolyzate 10% Starch hydrolyzate 10% Starch hydrolyzate 10% Glycerin 5% Glycerin 5% Glycerin 5% Glycerin 5% Spices Spices Spices Spices Purified water Purified water Purified water Purified water

Determination of Anti-Cavity Activity of Gum Containing Crude Extracts

    Except for using the gum containing the crude extract prepared according to Examples 9 to 12 was measured according to the same method as in Example 1 and the results are shown in Table 10 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 9 4.0 × 10 ⁵ <One Example 10 4.0 × 10 ⁵ <One Example 11 4.0 × 10 ⁵ <One Example 12 4.0 × 10 ⁵ 1.8 × 10 ² Gum without crude extract 4.0 × 10 ⁵ 2.7 × 10 ⁴ Porphyromonas gingivalis Example 9 3.5 × 10 ⁵ <One Example 10 3.5 × 10 ⁵ <One Example 11 3.5 × 10 ⁵ 2.1 × 10 ³ Example 12 3.5 × 10 ⁵ 1.1 × 10 ⁴ Gum without crude extract 3.5 × 10 ⁵ 2.7 × 10 ⁴

Examples 13-16

The crude extract obtained according to the first extraction example was added, and a candy was prepared in the ingredients and contents shown in Table 11 below.

Major Components Example 13 Example 14 Example 15 Example 16 Crude extract 5.0% Crude extract 1.0% Crude extract 0.1% Crude extract 0.01% Starch syrup 12.0% Starch syrup 12.0% Starch syrup 12.0% Starch syrup 12.0% Sorbitol 65.0% Sorbitol 65.0% Sorbitol 65.0% Sorbitol 65.0%  Emulsifier 0.5%  Emulsifier 0.5%  Emulsifier 0.5%  Emulsifier 0.5% Shortening 2.5% Shortening 2.5% Shortening 2.5% Shortening 2.5% 11.0% sugar 11.0% sugar 11.0% sugar 11.0% sugar Spice balance Spice balance Spice balance Spice balance Pigment residue Pigment residue Pigment residue Pigment residue Purified water balance Purified water balance Purified water balance Purified water balance

Determination of Anti-Cavity Activity of Candy Containing Crude Extracts

    Except for using the candy containing the crude extract prepared according to Examples 13 to 16 according to the same method as in Example 1 measured the number of viable cells and the results are shown in Table 12 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 13 4.0 × 10 ⁵ <One Example 14 4.0 × 10 ⁵ <One Example 15 4.0 × 10 ⁵ <One Example 16 4.0 × 10 ⁵ 4.1 × 10 ² Candy without crude extract 4.0 × 10 ⁵ 3.7 × 10 ⁴ Porphyromonas gingivalis Example 13 3.5 × 10 ⁵ <One Example 14 3.5 × 10 ⁵ <One Example 15 3.5 × 10 ⁵ <One Example 16 3.5 × 10 ⁵ 3.3 × 10 ² Candy without crude extract 3.5 × 10 ⁵ 3.1 × 10 ⁴

Examples 17-20

To the crude extract obtained according to the first extraction example was added to prepare a jelly with the ingredients and contents shown in Table 13.

Major Components Example 17 Example 18 Example 19 Example 20 Crude extract 5.0% Crude extract 1.0% Crude extract 0.1% Crude extract 0.01% Gelling agent 2.5% Gelling agent 2.5% Gelling agent 2.5% Gelling agent 2.5% Citric Acid 1.0% Citric Acid 1.0% Citric Acid 1.0% Citric Acid 1.0% Liquid Fructose 19.5% Liquid Fructose 19.5% Liquid Fructose 19.5% Liquid Fructose 19.5% Purified water 56.0% Purified water 56.0% Purified water 56.0% Purified water 56.0% 12% sugar 12% sugar 12% sugar 12% sugar Spice balance Spice balance Spice balance Spice balance

Determination of Anti-Cavity Activity of Jelly Containing Crude Extracts

    Except for using the jelly containing the crude extract prepared according to Examples 17 to 20 according to the same method as in Example 1 was measured and the results are shown in Table 14.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 17 4.0 × 10 ⁵ <One Example 18 4.0 × 10 ⁵ <One Example 19 4.0 × 10 ⁵ <One Example 20 4.0 × 10 ⁵ 3.3 × 10 ² Jelly without extract 4.0 × 10 ⁵ 3.9 × 10 ⁴ Porphyromonas gingivalis Example 17 3.5 × 10 ⁵ <One Example 18 3.5 × 10 ⁵ <One Example 19 3.5 × 10 ⁵ <One Example 20 3.5 × 10 ⁵ 4.2 × 10 ² Jelly without extract 3.5 × 10 ⁵ 3.3 × 10 ⁴

Examples 21-24

The crude extract obtained according to the first extraction example was added, and chocolate was prepared using the ingredients and contents shown in Table 15 below.

Major Components Example 21 Example 22 Example 23 Example 24 Crude extract 5.0% Crude extract 1.0% Crude extract 0.1% Crude extract 0.01% Coco Ames 23.0% Coco Ames 23.0% Coco Ames 23.0% Coco Ames 23.0% Milk powder 15.0% Milk powder 15.0% Milk powder 15.0% Milk powder 15.0% 37.0% sugar 37.0% sugar 37.0% sugar 37.0% sugar Cocoa Butter 23.0% Cocoa Butter 23.0% Cocoa Butter 23.0% Cocoa Butter 23.0% Spice balance Spice balance Spice balance Spice balance Purified water balance Purified water balance Purified water balance Purified water balance

Determination of Anti-Cavity Activity of Jelly Containing Crude Extracts

    Except that the chocolate containing crude extract prepared according to Examples 21 to 24 was used to measure the number of viable cells in the same manner as in Example 1 and the results are shown in Table 16 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 21 4.0 × 10 ⁵ <One Example 22 4.0 × 10 ⁵ <One Example 23 4.0 × 10 ⁵ <One Example 24 4.0 × 10 ⁵ 1.8 × 10 ² Chocolate without crude extract 4.0 × 10 ⁵ 3.4 × 10 ⁴ Porphyromonas gingivalis Example 21 3.5 × 10 ⁵ <One Example 22 3.5 × 10 ⁵ <One Example 23 3.5 × 10 ⁵ <One Example 24 3.5 × 10 ⁵ 2.6 × 10 ² Chocolate without crude extract 3.5 × 10 ⁵ 3.1 × 10 ⁴

Preparation of Oral Compositions Containing Isopanduratin A

Examples 25-28

Isopanduratin A obtained according to the third extraction example was added, and an oral cleanser was prepared using the ingredients and contents shown in Table 17 below.

Major Components Example 25 Example 26 Example 27 Example 28 Isopandu Latin A 1.0% Isopanduratin 0.1% Isopanduratin 0.01% Isopandulatin A 0.001% Sodium fluoride 0.02% Sodium fluoride 0.02% Sodium fluoride 0.02% Sodium fluoride 0.02% Ethanol 6.6% Ethanol 6.6% Ethanol 6.6% Ethanol 6.6% Glycerin 6.0% Glycerin 6.0% Glycerin 6.0% Glycerin 6.0% l-menthol 0.005% l-menthol 0.005% l-menthol 0.005% l-menthol 0.005% Saccharin sodium Saccharin sodium Saccharin sodium Saccharin sodium Citric acid Citric acid Citric acid Citric acid Pigment amount Pigment amount Pigment amount Pigment amount Peppermint Essence Peppermint Essence Peppermint Essence Peppermint Essence Spearmint Essence Spearmint Essence Spearmint Essence Spearmint Essence

Determination of Anti-Cavity Activity of Mouthwashes Containing Isopanduratin A

    Except for using the oral cleaning agent containing isopanduratin A prepared according to Examples 25 to 28 according to the same method as in Example 1 measured the number of viable cells and the results are shown in Table 18 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 25 4.0 × 10 ⁵ <One Example 26 4.0 × 10 ⁵ <One Example 27 4.0 × 10 ⁵ <One Example 28 4.0 × 10 ⁵ 3.2 × 10 ² Mouthwashes without Isopanduratin 4.0 × 10 ⁵ 2.5 × 10 ⁴ Porphyromonas gingivalis Example 25 3.5 × 10 ⁵ <One Example 26 3.5 × 10 ⁵ <One Example 27 3.5 × 10 ⁵ <One Example 28 3.5 × 10 ⁵ 5.5 × 10 ² Mouthwashes without Isopanduratin 3.5 × 10 ⁵ 2.5 × 10 ⁴

Examples 29-32

Isopanduratin A obtained according to the third extraction example was added, and toothpaste was prepared using the ingredients and contents shown in Table 19 below.

Major Components Example 29 Example 30 Example 31 Example 32 Isopandu Latin A 1.0% Isopanduratin 0.1% Isopanduratin 0.01% Isopandulatin A 0.001% Sorbitol liquid 19.2% Sorbitol liquid 19.2% Sorbitol liquid 19.2% Sorbitol liquid 19.2% Glycerin 22.0% Glycerin 22.0% Glycerin 22.0% Glycerin 22.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Sucrose Fatty Acid Ester 1.7% Sucrose Fatty Acid Ester 1.7% Sucrose Fatty Acid Ester 1.7% Sucrose Fatty Acid Ester 1.7% Alkyl sodium sulfate 1.0% Alkyl sodium sulfate 1.0% Alkyl sodium sulfate 1.0% Alkyl sodium sulfate 1.0% Sodium chloride 1.0% Sodium chloride 1.0% Sodium chloride 1.0% Sodium chloride 1.0% Spices 1.1% Spices 1.1% Spices 1.1% Spices 1.1% Carboxymethyl Cellulose Sodium 0.4% Carboxymethyl Cellulose Sodium 0.4% Carboxymethyl Cellulose Sodium 0.4% Carboxymethyl Cellulose Sodium 0.4% Purified water balance Purified water balance Purified water balance Purified water balance

Determination of anti-cavity activity of toothpaste containing isopanduratin

    Except for using a toothpaste containing isopanduratin A prepared according to Examples 29 to 32 was measured according to the same method as in Example 1 and the results are shown in Table 20 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 29 4.0 × 10 ⁵ <One Example 30 4.0 × 10 ⁵ <One Example 31 4.0 × 10 ⁵ <One Example 32 4.0 × 10 ⁵ <One Toothpaste without Isopanduratin 4.0 × 10 ⁵ 3.1 × 10 ⁴ Porphyromonas gingivalis Example 29 3.5 × 10 ⁵ <One Example 30 3.5 × 10 ⁵ <One Example 31 3.5 × 10 ⁵ <One Example 32 3.5 × 10 ⁵ <One Toothpaste without Isopanduratin 3.5 × 10 ⁵ 3.1 × 10 ⁴

Examples 33-36

Isopanduratin A obtained according to the third extraction example was added thereto, and a gum was prepared using the ingredients and contents shown in Table 21 below.

Major Components Example 33 Example 34 Example 35 Example 36 Isopandu Latin A 1.0% Isopanduratin 0.1% Isopanduratin 0.01% Isopandulatin A 0.001% Gum base 20% Gum base 20% Gum base 20% Gum base 20% Sugar powder 54% Sugar powder 54% Sugar powder 54% Sugar powder 54% 10% glucose 10% glucose 10% glucose 10% glucose Starch hydrolyzate 10% Starch hydrolyzate 10% Starch hydrolyzate 10% Starch hydrolyzate 10% Glycerin 5% Glycerin 5% Glycerin 5% Glycerin 5% Spices Spices Spices Spices Purified water Purified water Purified water Purified water

Determination of Anti-Cavity Activity of Gum Containing Isopanduratin A

    Except for using a gum containing isopanduratin A prepared according to Examples 33 to 36 was measured according to the same method as in Example 1 and the results are shown in Table 22 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 33 4.0 × 10 ⁵ <One Example 34 4.0 × 10 ⁵ <One Example 35 4.0 × 10 ⁵ <One Example 36 4.0 × 10 ⁵ <One Isopanduratin A minus gum 4.0 × 10 ⁵ 2.5 × 10 ⁴ Porphyromonas gingivalis Example 33 3.5 × 10 ⁵ <One Example 34 3.5 × 10 ⁵ <One Example 35 3.5 × 10 ⁵ <One Example 36 3.5 × 10 ⁵ <One Isopanduratin A minus gum 3.5 × 10 ⁵ 2.8 × 10 ⁴

Examples 37-40

Isopanduratin A obtained according to the third extraction example was added, and a candy was prepared using the ingredients and contents shown in Table 23 below.

Major Components Example 37 Example 38 Example 39 Example 40 Isopandu Latin A 1.0% Isopanduratin 0.1% Isopanduratin 0.01% Isopandulatin A 0.001% Starch syrup 12.0% Starch syrup 12.0% Starch syrup 12.0% Starch syrup 12.0% Sorbitol 65.0% Sorbitol 65.0% Sorbitol 65.0% Sorbitol 65.0%  Emulsifier 0.5%  Emulsifier 0.5%  Emulsifier 0.5%  Emulsifier 0.5% Shortening 2.5% Shortening 2.5% Shortening 2.5% Shortening 2.5% 11.0% sugar 11.0% sugar 11.0% sugar 11.0% sugar Spice balance Spice balance Spice balance Spice balance Pigment residue Pigment residue Pigment residue Pigment residue Purified water balance Purified water balance Purified water balance Purified water balance

Determination of Anti-Cavity Activity of Candies Containing Isopanduratin A

    Except for using the candy containing isopanduratin A prepared according to Examples 37 to 40 according to the same method as in Example 1 was measured the number of viable cells and the results are shown in Table 24 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 37 4.0 × 10 ⁵ <One Example 38 4.0 × 10 ⁵ <One Example 39 4.0 × 10 ⁵ <One Example 40 4.0 × 10 ⁵ <One Isopanduratin candy minus 4.0 × 10 ⁵ 3.2 × 10 ⁴ Porphyromonas gingivalis Example 37 3.5 × 10 ⁵ <One Example 38 3.5 × 10 ⁵ <One Example 39 3.5 × 10 ⁵ <One Example 40 3.5 × 10 ⁵ <One Isopanduratin candy minus 3.5 × 10 ⁵ 2.9 × 10 ⁴

Examples 41-44

Isopanduratin A obtained according to the third extraction example was added, and a jelly was prepared using the ingredients and contents shown in Table 25 below.

Major Components Example 41 Example 42 Example 43 Example 44 Isopandu Latin A 1.0% Isopanduratin 0.1% Isopanduratin 0.01% Isopandulatin A 0.001% Gelling agent 2.5% Gelling agent 2.5% Gelling agent 2.5% Gelling agent 2.5% Citric Acid 1.0% Citric Acid 1.0% Citric Acid 1.0% Citric Acid 1.0% Liquid Fructose 19.5% Liquid Fructose 19.5% Liquid Fructose 19.5% Liquid Fructose 19.5% Purified water 56.0% Purified water 56.0% Purified water 56.0% Purified water 56.0% 12% sugar 12% sugar 12% sugar 12% sugar Spice balance Spice balance Spice balance Spice balance

Determination of Anti-Cavity Activity of Jelly Containing Isopanduratin A

    Except for using a jelly containing isopanduratin A prepared according to Examples 41 to 44 was measured according to the same method as in Example 1 and the results are shown in Table 26 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 41 4.0 × 10 ⁵ <One Example 42 4.0 × 10 ⁵ <One Example 43 4.0 × 10 ⁵ <One Example 44 4.0 × 10 ⁵ <One Jelly minus isopanduratin 4.0 × 10 ⁵ 2.2 × 10 ⁴ Porphyromonas gingivalis Example 41 3.5 × 10 ⁵ <One Example 42 3.5 × 10 ⁵ <One Example 43 3.5 × 10 ⁵ <One Example 44 3.5 × 10 ⁵ <One Jelly minus isopanduratin 3.5 × 10 ⁵ 2.7 × 10 ⁴

Examples 45-48

Isopanduratin A obtained according to the third extraction example was added, and chocolate was prepared using the ingredients and contents shown in Table 27 below.

Major Components Example 45 Example 46 Example 47 Example 48 Isopandu Latin A 1.0% Isopanduratin 0.1% Isopanduratin 0.01% Isopandulatin A 0.001% Coco Ames 23.0% Coco Ames 23.0% Coco Ames 23.0% Coco Ames 23.0% Milk powder 15.0% Milk powder 15.0% Milk powder 15.0% Milk powder 15.0% 37.0% sugar 37.0% sugar 37.0% sugar 37.0% sugar Cocoa Butter 23.0% Cocoa Butter 23.0% Cocoa Butter 23.0% Cocoa Butter 23.0% Spice balance Spice balance Spice balance Spice balance Purified water balance Purified water balance Purified water balance Purified water balance

Determination of Anti-Cavity Activity of Chocolate Containing Isopanduratin A

    Except for using the chocolate containing isopanduratin A prepared according to Examples 45 to 48 was measured according to the same method as in Example 1 and the results are shown in Table 28 below.

Oral pathogenic bacteria Test solution Viable cell count (microbial count / ml) 0 hours 24 hours Streptococcus mutans Example 45 4.0 × 10 ⁵ <One Example 46 4.0 × 10 ⁵ <One Example 47 4.0 × 10 ⁵ <One Example 48 4.0 × 10 ⁵ <One Chocolate without Isopandu Latin A 4.0 × 10 ⁵ 3.2 × 10 ⁴ Porphyromonas gingivalis Example 45 3.5 × 10 ⁵ <One Example 46 3.5 × 10 ⁵ <One Example 47 3.5 × 10 ⁵ <One Example 48 3.5 × 10 ⁵ <One Chocolate without Isopandu Latin A 3.5 × 10 ⁵ 2.7 × 10 ⁴

Referring to Tables 5 to 28, the oral composition containing the crude extract that isolated and purified isopanduratin A was found to have excellent antibacterial activity, in particular, isopandu separated and purified from the crude extract It can be seen that the oral composition containing Latin A shows excellent antimicrobial activity even when the amount is small.

As such, panduratin derivatives, such as isopanduratin A, exhibit potent antimicrobial activity that completely kills Streptococcus mutants in 1-2 minutes even at very low concentrations of 20 μg / ml. This is because the components isolated from the conventional natural products have a second inhibitory activity that inhibits the activity of the enzymes produced by oral pathogenic bacteria, and thus have a stronger inhibitory activity because they have a primary inhibitory activity that kills the bacteria themselves. do. In addition, panduratin derivatives are components extracted from natural products, which can minimize side effects on the human body, and are excellent in high temperature stability and can be processed at any temperature, thus increasing extraction yield and shortening extraction time when extracting components. Productivity is high.

Therefore, panduratin derivatives are added to oral compositions of various formulations such as toothpaste compositions, mouthwashes, gums, candy, jelly, chocolates, etc. as well as antibacterial agents, for the purpose of preventing and treating caries and periodontitis. Can be effectively used.

1 is a graph showing the results of measuring the antimicrobial activity of Streptococcus mutants of the Kaemperia pandurata crude extract of the present invention by viable cell count measurement method,

Figure 2 is a photograph showing the results of the antimicrobial activity of Streptococcus mutants of the ethyl acetate fraction of the Kaemperia pandurata crude extract of the present invention confirmed by well-diffusion analysis,

Figure 3 is a ¹³ C-NMR spectrum of the isolated, purified isopanduratin A of the present invention,

Figure 4 is a ¹ H-NMR spectrum of the isolated purified isopanduratin A of the present invention,

5 is an EI-Mass spectrum of isolated, purified isopanduratin A of the present invention,

Figure 6 is a graph showing the results of measuring the antimicrobial activity of the isolated, purified isopanduratin A against the Streptococcus mutants by viable cell count method,

7 is an electron micrograph of Streptococcus mutans treated with 0.08% methanol as a control,

Figure 8 is an electron micrograph of Streptococcus mutants damaged by the isolated, purified isopanduratin A of the present invention.

Claims (4)

To prevent caries and periodontitis, characterized in that it contains isopanduratin A represented by the formula (1) as an active ingredient; <Formula 1> [Claim 2] The antimicrobial agent of claim 1, wherein the isopanduratin A is extracted from Kaempferia pandurata . An oral composition for preventing and treating tooth decay and periodontitis, comprising isopanduratin A of claim 1. The oral composition of claim 3, wherein the composition for oral cavity comprises any one formulation selected from the group consisting of a mouthwash, toothpaste, gum, candy, jelly, and chocolate.