JP4457035B2 - Oral composition for antibacterial against throat inflammation-causing bacteria (hemolytic streptococci) and for inhibiting hemolytic toxins produced by this species - Google Patents

Description

The present invention relates to antibacterial, and oral compositions for inhibition of hemolysin produced according species against throat inflammation pathogenic bacteria (hemolytic streptococcus).

  Streptococcus pyogenes (Streptococcus pyogenes) is a facultative anaerobic Gram-positive cocci that are distributed in the human oral cavity and pharynx. This bacterium is the most pathogenic among streptococci and is known to cause pharyngitis, tonsillitis, otitis media, etc. in the mucosa, and various suppurative inflammations in the skin. Proliferate in

  Known virulence factors of this bacterium include hemolysin (streptolysin O), dick toxin, streptokinase, DNA-degrading enzyme, hyaluronidase, etc., etiology of respiratory diseases such as pharyngitis, tonsillitis, bronchitis Plays an important role.

  Needless to say, in order to prevent and treat these respiratory diseases, it is important to inhibit the growth of this fungus in the lesion and to suppress the pathogenic factor of the fungus.

  From the viewpoint of controlling the fungus, it is effective to apply an antibacterial substance effective against the fungus. Conventionally, penicillin, cephem, and cephalosporin antibiotics were used, but they are powerful, but they are suitable for daily use due to the appearance of resistant bacteria and side effects. I can't say that.

  Therefore, as an antibacterial substance effective for the fungus suitable for daily use, fruits, vegetables, and other natural products derived from daily food are suitable. Conventionally, karin fruit has attracted attention as an example of such a thing.

  Japanese Patent Application Laid-Open Nos. 59-187769 and 2-312565 disclose use of fruit juice or extract obtained by extracting karin with honey for the purpose of promoting health, but the object is extracted with honey. It is limited to what has been done, and its effect is very abstract.

  In addition, Japanese Patent Application Laid-Open Nos. 62-61538 and 62-228230, both of which are filed by the present applicant, disclose that the solvent extract of Karin has antibacterial activity against Streptococcus mutans and Staphylococcus aureus. However, antibacterial activity against Streptococcus pyogenes has not been clarified.

  Further, Japanese Patent Publication No. 63-26083 and Japanese Patent Application Laid-Open No. 1-290619 have an anti-cariogenic effect on pentacyclic acidic triterpene compounds such as oleanolic acid, which are said to exist widely in nature, Discloses specific antibacterial activity against mutans, but karin fruit contains oleanolic acid and other specific pentacyclic acidic triterpene compounds and these have antibacterial activity against Streptococcus pyogenes That is not disclosed.

JP 59-187769 A Japanese Patent Laid-Open No. 2-312565 JP 62-61538 A JP-A-62-2228230 Japanese Patent Publication No. 63-26083 JP-A-1-290619

  As a result of screening using natural solvent extract as a sample, the present inventors conducted screening using antibacterial activity against Streptococcus pyogenes and hemolytic toxin (streptolysin O) inhibitory activity produced by this bacterium as an index. The inventors have found that the extract has these effects, and have completed the present invention.

The present invention is 50-100% lower alcohol solution Karin fruit, acetone, ethyl acetate or n- hexane or containing the extracted extract and triterpene derivatives obtained from the extract in mixtures of two or more solvents Ri Na a antibacterial, and oral compositions for inhibition of hemolysin produced according species against throat inflammation pathogenic bacteria (hemolytic streptococcus).

  Karin (Chaenomeles sinensis, Cydonis sinensis, Pseudocydinis sinensis), which is the raw material of the product of the present invention, is a deciduous high tree cultivated in various parts of the world, a plant native to the Rosaceae family, and its fruit is used as a food material In Chinese medicine, it is called mokka, and medicinal liquor made from shochu is used for coughing and expectoration. However, the active ingredient is unknown, and it is also unclear what action mechanism exerts antitussive and expectorant effects.

  The product of the present invention can be extracted and produced from karin fruit as follows.

  First, the carin fruit is left as it is or dried, and then mechanically pulverized by an appropriate means, and then 50 to 100% lower alcohol aqueous solution, preferably ethanol aqueous solution, acetone, ethyl acetate or n-hexane or two of them. The extraction operation is performed by applying a sufficient amount of the above mixed solvent. These solvents may be ordinary ones that are commercially available, and two or more of these solvent mixtures may be arbitrarily selected and mixed at an arbitrary mixing ratio. The extraction operation may be performed by immersing the pulverized quince fruit in the above-mentioned solvent at room temperature for an appropriate period of time, but is preferably heated to reflux at a temperature of 50 to 100 ° C. for 1 to 5 hours, more preferably about 3 hours. By the way.

  Next, the extract obtained in this way is concentrated under reduced pressure to obtain a preparation contained in the oral cavity composition and food and drink of the present invention.

  The composition thus obtained is further subjected to column chromatography for separation and purification. The column chromatography to be used is arbitrary, and a technique such as adsorption chromatography, partition chromatography, high performance liquid chromatography, thin layer chromatography or the like may be used. The developing solution used may be a lower alcohol such as methanol, an aqueous solution thereof, acetone, ethyl acetate or n-hexane. The obtained separated and purified product is a triterpene derivative that constitutes another oral composition of the present invention and a preparation contained in food and drink.

  The triterpene derivative according to the present invention has the following structure.

  In the oral cavity composition of the present invention and the preparation contained in the food or drink, any one or more of these derivatives is used as an active ingredient.

  The thus obtained solvent extraction composition and the oral composition of the present invention, which is a triterpene derivative, and the preparation contained in the food and drink can be used for a wide range of applications. Considering that it is excellent and high in safety, it can be blended into chewing gum, candy, tablet confectionery, gargle and the like for daily use. When used for such applications, it is preferable to add the preparation so as to be contained in the product at a ratio of 0.01 to 10% by weight.

  The product of the present invention not only has a particularly strong growth inhibitory effect against Streptococcus pyogenes, which is a throat inflammation-causing bacterium, but also against the hemolytic toxin (Streptridine O) produced by this species. It also has an inhibitory effect and effectively acts on various infections including throat inflammation and purulent diseases.

  Hereinafter, examples, test examples, and application examples of the present invention will be described.

Example 1
5 kg of 50% ethanol was added to 1 kg of the pulverized dried fruits of karin, and the mixture was subjected to reflux extraction at 100 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 324 g of a preparation contained in the product of the present invention which is a reddish brown solid having a sour taste and aroma.

Example 2
5 kg of 100% ethanol was added to 1 kg of pulverized dried fruits of karin, and the mixture was subjected to reflux extraction at 80 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 77 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 3
5 kg of acetone was added to 1 kg of the pulverized dried karin fruit, and the mixture was refluxed and extracted at 80 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 45 g of a preparation contained in the product of the present invention, which is a reddish brown solid having a sour taste and aroma.

Example 4
5 kg of ethyl acetate was added to 1 kg of the pulverized dried karin fruit, and the mixture was subjected to reflux extraction at 80 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 35 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 5
5 liters of n-hexane was added to 1 kg of the pulverized dried fruit of karin, and the mixture was heated and refluxed at 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 5 g of a preparation contained in the product of the present invention, which was a light green solid having a sour taste and aroma.

Example 6
A mixed solvent of 3 liters of 50% ethanol and 2 liters of acetone was added to 1 kg of ground quince fruits, and extraction under heating was performed at a temperature of 100 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 280 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 7
A mixed solvent of 3 liters of 100% ethanol and 2 liters of ethyl acetate was added to 1 kg of ground quince fruits, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 82 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 8
A mixed solvent of 3 liters of 100% ethanol and 2 liters of n-hexane was added to 1 kg of ground quince fruits, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 72 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 9
A mixed solvent of 3 liters of acetone and 2 liters of ethyl acetate was added to 1 kg of pulverized dried fruits of karin, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 58 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 10
A mixed solvent of 3 liters of acetone and 2 liters of n-hexane was added to 1 kg of pulverized dried fruits of karin, and the mixture was heated and refluxed at 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 42 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 11
A mixed solvent of 3 liters of ethyl acetate and 2 liters of n-hexane was added to 1 kg of pulverized dried fruits of karin, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 35 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 12
A mixed solvent of 1 liter of 100% ethanol, 1 liter of acetone, 1 liter of ethyl acetate and 2 liters of n-hexane was added to 1 kg of pulverized dried fruits of karin, and the mixture was refluxed and heated at 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 63 g of a preparation contained in the product of the present invention as a reddish brown solid having a sour taste and aroma.

Example 13
Water was added to and dispersed in 60 g of the preparation contained in the product of the present invention obtained in Example 2, and then partitioned with ethyl acetate to obtain an ethyl acetate-soluble fraction (17.1 g).

  This ethyl acetate soluble fraction was adsorbed on a column packed with 1 kg of silica gel (Wakogel C-200, Wako Pure Chemical Industries). This column was eluted with n-hexane ethyl acetate (10: 1 to 1: 1) and fractionated. Fraction that shows blue or purple spots with 10% sulfuric acid around Rf value 0.2-0.5 when developed with n-hexane-ethyl acetate (2: 1) using TCL plate (60F254, Merck) Collected.

  This fraction was eluted with methanol-water (90:10 to 100: 0) using an octadecylsilanol-type (ODS) preparative column (Senshu Scientific) for high-performance liquid chromatography (HPLC), and 205 nm UV Separate each peak while monitoring absorption.

  Each peak is further contained in the product of the present invention by repeatedly purifying with acetonitrile-water (95: 5 to 100: 0) and methanol-water (90:10 to 100: 0) using an ODS column for HPLC. Four types of colorless crystalline Compound 1 (2850 mg), Compound 2 (250 mg), Compound 3 (25 mg), and Compound 4 (25 mg) were obtained.

  These compounds were analyzed by melting point, mass spectrum, infrared absorption spectrum, and nuclear magnetic resonance spectrum. As a result, the compounds 1 to 4 were triterpene derivatives such as oleanolic acid, 3-acetyloleanolic acid, erythrodiol and ubaol. confirmed.

Test example 1
The antibacterial activity against the hemolytic streptococci, Staphylococcus aureus and caries-causing bacteria of the preparations contained in the products of the present invention prepared by the methods shown in Examples 1 to 5 and 13 was tested by the following method.

Test method 1) Test strain, medium used and culture conditions

2) Antibacterial activity test method Each strain is cultured for 24 hours in the respective medium and culture conditions to sufficiently grow the bacteria. Using a 96-well flat bottom microplate, prepare a 2-fold dilution series of sample solution (100 μl) in each well. Each culture is inoculated into a medium prepared at a double concentration, and 100 μl thereof is added to each well. After culturing for 24 hours, the turbidity (550 nm) of each well is measured using a microplate reader, the growth of the bacteria is determined, and the minimum growth inhibitory concentration (MIC) is obtained.

Test results The test results are shown in Table 2. The products of the present invention (Examples 1 to 5 and compounds 1 to 4) show antibacterial activity against Staphylococcus aureus and caries-causing bacteria, but the antibacterial activity against hemolytic streptococci is even stronger. Since antibacterial activity as in the product of the present invention was not observed in water of Karin or 30% ethanol extract, it is clear that the ethanol concentration when extracting Karin must be 50% or more.

Test example 2
The preparations contained in the products of the present invention prepared by the methods shown in Examples 1 to 5 and 13 were tested for hemolytic toxin (streptridine O) inhibitory activity by the following method.

Test method Rabbit defibrillated blood (25 ml) is washed 3 times with buffered saline (pH 6.5) (3000 rpm × 10 minutes), and erythrocytes are collected. Dilute to 200 ml with buffered saline to make a rabbit erythrocyte suspension. Dissolve the specified amount of sample in ethanol, and then dissolve in buffered saline (ethanol 2%) to obtain a sample solution. 600 μl of sample solution and 300 μl of streptolysin O (Eiken) aqueous solution (1 binding titer) are mixed in a 13 mm diameter test tube and reacted at 37 ° C. for 30 minutes. Add 300 μl of rabbit erythrocyte suspension and react at 37 ° C. for 45 minutes. After completion of the reaction, the mixture is centrifuged at 1500 rpm for 1 minute, and the degree of hemolysis of the supernatant is determined with the naked eye. Judgment criteria were as follows.

Judgment criteria-: No hemolysis, +: Incomplete hemolysis, ++: Complete hemolysis

Test results The test results are shown in Table 3. The preparations (Examples 1 to 5 and Compounds 1 to 4) contained in the products of the present invention showed hemolytic toxin inhibitory activity, and at a concentration of 1000 μg / ml, all samples completely inhibited hemolysis. No inhibitory activity was observed even at a concentration of 100 μg / ml, and no complete hemolysis was observed. Similarly to the antibacterial activity, the water concentration of Karin and the 30% ethanol extract did not show the hemolytic toxin inhibitory activity as in the preparation contained in the product of the present invention. It is clear that it must be greater than or equal to%.

Application Examples Chewing gum, candy, tablet confectionery, and gargle were prepared by the following formulation using the preparation contained in the product of the present invention prepared by the method shown in Examples 1, 2 and 13.

  Karin, which is the raw material of the product of the present invention, is used as an edible material and medicinal liquor, and there is no problem with its safety, so there is no problem with the safety of the product of the present invention, so only when antibiotics are applied. It is recognized that there are no worries about side effects.

  The product of the present invention is highly versatile and can be used for a wide range of uses, such as addition to foods, because there is no worry about side effects, and it has excellent aroma and taste.

Claims (1)

Triterpene derivatives obtained from karin fruit

Antibacterial, and oral compositions for inhibition of hemolysin produced according species against throat inflammation pathogenic bacteria shall be the active ingredient any one or more (hemolytic streptococcus) of.