KR100454226B1 - Processing method of licorice extract and its' utilization on developing anti-cariogenic products - Google Patents

Abstract

The present invention relates to a method for preparing licorice extract and a functional anti-cardium substance containing licorice extract.

Licorice extract manufacturing method of the present invention comprises the steps of mixing the licorice and licorice weight 10 to 30 times the water and extracting hot water 3 to 5 times for 10 minutes to 3 hours at a temperature of 40 ~ 150 ℃; Filtering the hot water extracted licorice to remove sweet components of licorice; Adding a 5-30-fold organic solvent to the residue of licorice remaining after filtration, and extracting under reduced pressure for 10 minutes to 4 hours at a temperature of 30 to 100 ℃ and filtration; After the step, it is characterized in that it comprises a step of lyophilization or hot air drying after the concentration by adding an excipient of 1 to 100% of the fine office crab.

An object of the present invention is to provide a substance which can suppress tooth caries (cavities) by preparing a licorice extract and providing a toothpaste, chewing gum or candy containing the licorice extract as an active ingredient.

Description

Processing method of licorice extract and its' utilization on developing anti-cariogenic products}

The present invention relates to a method for preparing licorice extract and a functional anti-caustic substance containing licorice extract, and more particularly, a method for preparing licorice extract comprising extracting hot liquor from a licorice extract and adding an excipient and extracting it with an organic solvent. The licorice extract extracted by the method relates to an anti-cardium substance contained in toothpaste, gum or candy.

As dietary patterns are gradually diversified, consumption of sugars is on the rise, while immune function in the human body is weakened by stress and disease, and in particular, oral microorganisms are on the rise.

According to a survey by the Korean Dental Association (1992), more than 90% of Korean children have experienced dental caries (aka tooth decay) and 80% of adults have gum disease.

Tooth decay is a bacterial dental hard tissue disease in which microorganisms in the oral cavity, in particular Streptococcus mutans , are the main cause of demineralization of minerals in teeth and destruction of dentin, leading to defects in dental tissues.

First of all, the cause of human caries is briefly described. Tooth decay occurs when three conditions exist: tooth, mutans streptococci and carbohydrates. Mutans such as Streptococcus mutans (about 80% of isolates) and Streptococcus sorbitus (about 20%) decompose sucrose, an efficient energy source, to use post-lactic acid used for growth. Produces an organic acid that is mainly used. By the action of these organic acids, hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), which is a tooth enamel chemical, is decomposed. This is called decalcification.

In addition, mutans produces glucose and fructose using sucrose as a substrate, and glucose produced from these is insoluble glucan (hereinafter referred to as glucosyltransferase). Glucan is formed on the tooth surface. Tooth decay bacteria and the like are attached to the insoluble glucan attached to the tooth surface, and the attachment group of the microorganisms thus formed is called plaque. Therefore, the effective anti-cavities can inhibit the use of sucrose substitute sweeteners that are not available to cavities, materials that break down or dissolve insoluble glucans, and inhibit the growth of cavities or materials that can lower the activity of GTase to inhibit the synthesis of insoluble glucans. If you can find a material with a function to make it possible.

The statistical comparison of dental caries is expressed by the DMFT index of 12-year-old children, which is an index of the average number of caries experienced by 12-year-old children in permanent teeth. In the late 20's, 2.5's in the early 1990's and 3.0 in the early 1990's, the rate of dental caries in Korean children has increased dramatically. Japan is currently reported to be 4.7 to 4.9. In the United States, tooth decay is gradually decreasing in the United States to 7.6 in 1960, 5.8 in 1976, 4.0 in 1979, 3.1 in 1986 and 2.68 in 1987.

Until now, research on food-related anti tooth decay has mainly focused on sweeteners that replace sucrose, which is not easily used by mutans bacteria, and the separation of substances having anti-cartococcus activity from natural substances. have. Among alternative sweeteners, especially palatinose, has been reported to inhibit the formation of plaque by strongly inhibiting the activity of GTase. The catechins in green tea and oolong tea have been shown to have potent inhibitory activity against GTase, and the glycoprotein called mutastein is isolated from the culture of Aspergillus terreus, a fungus isolated from soil. Has been reported to have GTase inhibitory activity. Attempts have also been made to obtain antiseptic bacteria directly from food by obtaining antibodies from S. mutans from the IgY of eggs. However, most of them have not been commercialized and the development of anti-cavity materials with industrial value is insufficient.

Therefore, the present inventors tried to find a material that can inhibit the growth of cavities and decrease GTase activity for effective anti-cavities, and extract licorice residues from the licorice by extracting the licorice components with organic solvent after excipients. Concentrated licorice extract was found to inhibit the growth of caries and reduce the activity of GTase. Accordingly, an object of the present invention is to provide a substance which can suppress tooth caries (cavities) by preparing a licorice extract as described above and providing a toothpaste, gum or candy containing the licorice extract as an active ingredient.

1 is a manufacturing process of the licorice extract of the present invention.

Figure 2 is a silica gel column chromatography of the licorice extract of the present invention.

FIG. 3 is HPLC chromatography comparing each fraction of FIG. 2 on HPLC.

Figure 4 is a photograph showing the growth inhibition against caries bacteria when the licorice extract of the present invention is used in toothpaste (control, no addition, 0.025% licorice extract group, 0.05% licorice extract group, 0.1% licorice from the top clockwise) Extract addition group).

Figure 5 is a photograph showing the growth inhibition against caries bacteria when the licorice extract of the present invention is used in gum (control, no addition, 1.0% licorice extract group, 3.0% licorice extract group, 2.0% licorice from the top clockwise) Extract addition group).

Licorice extract manufacturing method of the present invention comprises the steps of mixing the licorice and licorice weight 10 to 30 times water as shown in Figure 1 3 to 5 times hot water extraction for 10 minutes to 3 hours at a temperature of 40 ~ 150 ℃;

Filtering the hot water extracted licorice to remove sweet components of licorice;

Adding an organic solvent of licorice residues 5-30 times to the residue of licorice remaining after filtration and extracting and filtering under reduced pressure for 10 minutes to 4 hours at a temperature of 30 to 100 캜;

Concentrating the extract by adding an excipient of 1-20% licorice crab after the above step and then lyophilizing or hot-air drying in a general manner for 2-4 days at a temperature of -50 to -40 ℃, 15-30 Torr It is characterized by. On the other hand, it is possible to provide an anti tooth decay material of toothpaste, gum or candy containing licorice extract prepared by the above method as an active ingredient.

In the above water, licorice is not sufficiently submerged in water when used less than 10 times with respect to licorice weight, and when it is used in excess of 30 times, licorice is excessively diluted in water, thereby decreasing efficiency. It is good to maintain 10-30 times.

In the hot water extraction step, experiments were carried out with various methods. The best licorice extract was obtained when hot water extraction was carried out 3 to 5 times for 10 minutes to 3 hours at a temperature of 40 to 150 ° C.

The organic solvent may be ethanol, methanol, butanol, propanol, ethyl acetate, acetone, chloroform, nucleic acid, and when using the organic solvent less than 5 times licorice residue, there is a problem that it is not enough extraction, organic solvent When using more than 30 times of licorice residues, there is a problem that the amount of solvent is excessively increased without increasing the extraction efficiency, so that only the cost increases, and organic solvents should maintain the licorice residues 5 to 30 times.

Excipients may use Carageenan, lactose, dextrin, cyclodextrin, casein, pectin, xanthan gum, or pectin, and when the excipient is used in less than 1% licorice residue, it will help to improve the solubility of the extract in water. If the excipient is used more than 100% licorice bean curd, the anti-cariogenic effect of the extract is lowered, so the excipient is preferably maintained 1 to 100% licorice bean curd.

Hereinafter, the present invention will be described by the following examples and test examples. However, these do not limit the technical scope of the present invention.

<Example>

800L of licorice was mixed with 40 kg of licorice and extracted with hot water three times for two hours at a temperature of 90 ° C. under normal pressure, and the hot water extracted licorice was filtered to completely remove glycyrrhizin, which is a sweet component representing sweetness of licorice. After the filtration, the remaining licorice residue (Liquorice thin) was added with 75% ethanol residue of 15 times of licorice with organic solvent and extracted for 2 hours under reduced pressure and 85 ℃. After filtering the extract, 10% of licorice residue with excipient Carrageenan was added and concentrated to obtain an extract in the form of a paste, which was lyophilized for 3 days at -50 ℃, 15 Torr to obtain a licorice extract in the powder state.

<Test Example 1>

In order to measure the antimicrobial effect of the licorice bacteria of the licorice extract prepared by the above Example ( Streptococcus mutans , KFRI 1171, 1172, 1175) was used as a strain and growth medium brain heart infusion broth (BHI broth ) Was inoculated with Streptococcus mutans (KFRI 1171, 1172, 1175) to recover the activity of the strain by three passages in a 37 ° C rotating incubator (shaking incubator). The recovered strains were aliquoted into agar plate medium and incubated at 37 ° C. for 24 hours to identify colonies.

Add 0.1 ml of the culture strain and 0.1 ml of 4-20 ppm licorice extract to a test tube, and add 2.5 ml of top agar to the BHI agar. After incubating in the incubator for 24 hours, the number of colonies was measured and the results are shown in Table 1 below.

Table 1 shows the antimicrobial activity against Streptococcus mutans by concentration of licorice extract powder. The growth inhibition of Streptococcus mutans strains of 99.6 (KFRI 1171) to 64.3% (KFRI 1175) was observed even with the addition of lyophilized licorice extract powder at a concentration of 4 ppm, indicating that the licorice extract powder had no effect on Streptococcus mutans. Minimal Inhibitory Concentration is estimated to be 4 ppm or less.

Table 1. Antibacterial Effect of Licorice Extract Powder against Streptococcus Mutans

Item Powder concentration (ppm) Colonies per plate KFRI 1171 KFRI 1172 KFRI 1175 Control group 0 7.72 × 10 ⁷ 3.0 × 10 ⁷ 2.1 × 10 ⁷ Licorice extract group 42040 2.78 × 10 ⁵ 1.90 × 10 ³ 2.00 × 10 ² 7.8 × 10 ⁶ 4.5 × 10 ⁴ 1.8 × 10 ⁴ 7.5 × 10 ⁶ 1.8 × 10 ⁵ 2.5 × 10 ⁴

On the other hand, when the licorice extract of 20 ppm concentration was added, Streptococcus mutans Growth inhibitory effects of 99.9, 99.8 and 99.1% were observed on KFRI 1171, KFRI 1172, and KFRI 1175, respectively. Streptococcus mutans The inhibitory effect of growth was 99.99, 99.94 and 99.88% against KFRI 1171, KFRI 1172 and KFRI 1175, respectively.

<Test Example 2>

In order to isolate and identify the causative agent exhibiting the anti-cartococcal effect, the licorice extract prepared by the method of the above example was subjected to solvent fractions in the order of hexane, chloroform, ethylacetate and butanol. It was done. When the chloroform fraction having the strongest antibacterial activity among the solvent fractions was loaded into a silica gel column, the methanol fraction was fractionated by increasing the methanol concentration from 0% (CHCl ₃ 100%) to 100% step by step. Four fractions were fractionated as shown in Table 2 and their inhibitory effects on the Streptococcus mutans were shown in Table 2.

Table 2. Growth Inhibitory Effects on Streptococcus Mutans of Fractions Obtained from Silica Gel Columns

Fraction I II III IV Inhibitory zone diameter (mm) * 16.3 9.0 8.0 8.0

* paper disc diameter = 8.0mm

Antimicrobial activity against Streptococcus mutans Almost all of the fractions (I) and the composition of each fraction by using the YMC-Pack column on the HPLC (see Fig. 3), fraction (I) has a low polarity (hydrophobic) compared to the other fractions ), The ratio of constituents is very high, and these components may play an important role in the expression of carcass growth inhibitory ability.

<Test Example 3>

The GTase activity inhibiting ability of the licorice extract prepared by the method of Example was examined and the results are summarized in Table 3 below.

GTase activity inhibiting ability of the licorice extract is Streptococcus mutans incubated in the same way as Test Example 1 KFRI 1171, KFRI 1172, and KFRI 1175 cultures were centrifuged at 8,000 rpm for 5 minutes at 4 ° C. Filtered to 101, the filtrate was adjusted to pH 7.0 using 1N-NaOH, and 1% sucrose and licorice extract were added to the crude enzyme solution prepared by adding 0.02% of sodium azide. GTase activity was determined by measuring the degree of inhibition of glucan production relative to the no additive group without licorice extract by measuring turbidity at 660nm after adding at ppm, 50 ppm and 100 ppm concentration and reacting at 37 ℃ for 24 hours. Inhibitory effect was compared.

As shown in Table 3, the addition of licorice extract at a concentration of 25 ppm significantly reduced the GTase activity of the cavities. The results showed that licorice extract not only inhibited the growth of cavities but also strongly reduced the activity of GTase. Shows.

Table 3. Inhibitory Effect of Licorice Extract on GTase Activity

sample Powder addition amount (㎍ / ㎖) O.D (660 nm) 1171 1172 1175 No furniture 0 78.02 47.7 74.02 Licorice extract group 2550100 101.92103.20103.85 91.9796.3493.42 104.02101.70101.65

<Test Example 4>

The licorice extract powder prepared by the method of the above example was added to an amount corresponding to 0.025%, 0.05%, and 0.1% of the existing toothpaste, and then the growth inhibitory effect on the caries was confirmed by Streptococcus mutans. 0.1 ml of KFRI 1171 culture was smeared on sterilized BHI agar, and a paper disc (diameter 8 mm) was placed thereon to absorb 50 µl of the extract and incubate at 37 ° C. for 24 hours (see FIG. 4). When 0.05% or more was added, a marked growth inhibition effect was observed.

<Test Example 5>

When the licorice extract powder prepared by the method of Example 1 is added in an amount corresponding to 1.0%, 2.0%, and 3.0% with respect to the existing gum, Streptococcus mutans which is a decayed fungus 0.1 ml of KFRI 1171 culture solution was smeared on sterilized BHI agar, placed on a paper disc (8 mm in diameter), and 50 µl of the extract was absorbed and incubated at 37 ° C. for 24 hours. As a result (see FIG. 5), when the licorice extract was added more than 1.0%, a distinct growth inhibition effect was observed.

The licorice extract prepared by the present invention as shown in the results of the test example had excellent growth inhibitory effect on caries and inhibitory effect on GTase activity, and the licorice extract according to the present invention was added to processed foods such as toothpaste, gum and candy. It is possible to supply food that can be curtailed so that consumers can eat without burdening tooth decay. In addition, by adding the licorice extract according to the present invention to a medical preparation that can be used for oral treatment can provide a medical preparation that can suppress tooth decay.

Claims (6)

In the method for producing licorice extract, Mixing licorice and licorice by weight 10 to 30 times and extracting hot water three to five times at a temperature of 40 to 150 ° C. for 10 minutes to 3 hours; Filtering hot water extracted licorice to remove sweet components of licorice; Adding licorice residues 5 to 30 times ethanol to the residue of licorice remaining after filtration and extracting and filtering under reduced pressure for 10 minutes to 4 hours at a temperature of 30 to 100 ° C .; After the step, the step of adding one or more excipients selected from carrageenan, lactose, dextrin, cyclodextrin, casein, pectin, xanthan gum or pectin to 1 to 100% compared to licorice residues, concentrate the extract and freeze-drying or hot-air drying Method for producing a licorice extract for GTase activity inhibition, comprising a delete delete Toothpaste containing licorice extract prepared by the method of claim 1 as an active ingredient Chewing gum containing licorice extract prepared by the method of claim 1 as an active ingredient Candy containing licorice extract prepared by the method of claim 1 as an active ingredient