KR100481880B1 - A Recrptor Analog against Helicobacter pylori and its extraction Method from Auricularia auricula-judae - Google Patents

Abstract

The present invention comprises the steps of pulverizing the dried soybean mushroom and removing the ethanol soluble components using ethanol; Extracting the necessary components from the crushed woody mushroom crushed ethanol soluble components first by using hot water, adding a sufficient amount of ethanol to the hot water extract and removing the ethanol soluble components secondly and ethanol from which the ethanol soluble components are removed A method of extracting the receptor analog component of Helicobacter pylori from a wood mushroom, which comprises dissolving an insoluble portion in a sufficient amount of distilled water to remove the distilled water insoluble component and separating the remaining distilled water insoluble component. It was found that the extract of thirsty mushroom, which has no toxicity and has excellent receptor analog performance, was superior to the previously known receptor analog.

Description

A recrptor analog against helicobacter pylori and its extraction method from Auricularia auricula-judae}

The present invention relates to a method of extracting a component having a function of inhibiting gastric wall adhesion of Helicobacter pylori from mushrooms, and an adhesion inhibitory component of the Helicobacter pylori , and more specifically, to the stomach epithelial cells from the soybean fungi belonging to the soybean fungus Helicobacter pylori bacteria found to cause various gastric diseases The present invention relates to a method for extracting an adhesion inhibitory ingredient that inhibits adhesion to the stomach wall and an adhesion inhibitory extract extracted by the method.

Helicobacter pylori has been identified as a major causative agent of chronic gastritis and peptic ulcer since it was first isolated from human gastric mucosa by marshall and warren in Australia in 1982.In 1994, helicobacter pylori was identified as a carcinogen by an international cancer research organization under the World Health Organization. It was identified. The fungus is a Gram-negative bacillus with several flagella, which grows in the surface layer or mucus of the gastric mucosa. The most characteristic microbiological features are strong motility and urease enzyme activity. Strong motility is to live freely in the mucus of the stomach, the main role is the flagella. In addition, urease activity can effectively break down urea present in the gastric mucosa, and it is estimated that ammonia produced thereby counteracts the attack by gastric acid by neutralizing the surrounding environment of bacteria. Helicobacter pylori , which has these characteristics, is a causative agent of chronic gastritis, stomach, duodenal ulcer, and gastric cancer. About 60-70% of the adult population in Korea is infected, and chronic gastritis patients have an infection rate of more than 90%. . The method used to eliminate the Helicobacter pylori bacteria known as the cause of gastrointestinal diseases is to administer antibiotics directly. The most commonly used antibiotics are amoxycillin, clarithromysin, etc. There are some problems with the use of antibiotics. Due to the low pH of the stomach, the titer decreases and it is difficult to reach gastric epithelial cells, and there are many problems such as the emergence of antibiotic resistant strains and the simultaneous killing of harmful bacteria by antibiotics.

Therefore, the present inventors have extensively conducted a screening study of Helicobacter pylori adhesion inhibitors from edible and medicinal plants, and in particular, the present invention was completed by extracting excellent adhesion inhibitors from mushrooms.

Different mushrooms have different components and compositions, and their physiological activities vary widely. Among the mushrooms known to date, more than 350 mushrooms are currently used for food. These mushrooms contain large amounts of proteins, polysaccharides, various enzymes, and vitamins. It has high nutritional value and high digestibility, and is considered to be very valuable food. In addition, many studies have been published, such as anti-cancer activity of polysaccharides that destroy anti-tumor cells by stimulating the immune system, lowering blood pressure of various enzymes, inhibiting anemia and antibacterial activity, and preventing hypertension by potassium, which is a large amount of minerals. Recently, antithrombotic activity of mushroom-derived substances has also been reported.

In place of antibiotics with this problem, several types of treatments are recommended. The use of antimicrobial peptides, methods of using lactobacilli probiotics that adhere to the epithelial cells competitively with Helicobacter pylori bacteria, immunization with immunized substances, methods of inhibiting urease enzyme activity, and methods of using adhesion inhibitors. These methods are difficult to achieve satisfactory results in only one way, and it has been found that merging multiple methods in combination is effective. Recently, products using these complex materials have been developed, and their application is particularly active in the fermented milk market. Among them, Helicobacter pylori is competitive A method of inhibiting adhesion of Helicobacter pylori to the epithelial cells by using mainly live lactobacilli which adheres to the epithelial cells is used.

The present inventors can prevent and treat gastritis, gastric and duodenal ulcer disease treatment by taking a long-term safe and long-term treatment through daily life rather than antibiotic treatment, and concomitantly, a new concept for preventing and treating gastrointestinal diseases. A food development study was conducted. In particular, the present inventors focused on the selection of a substance capable of inhibiting the adhesion of Helicobacter pylori from edible natural substances. Bacterial infection is caused by the production of pathogens such as toxins after the pathogenic bacteria reach and settle at the site of infection. Thus, the first stage of infection is adherence. Many pathogenic bacteria bind to and attach to receptor molecules in the host cell. Although helicobacter pylori has not been clearly identified as a mechanism or receptor for attaching to gastric parietal cells, it has been reported to bind glycocomplexes. In particular, sialyated oligosaccharides have been reported to effectively inhibit the adhesion of Helicobacter pylori to the epithelial cells obtained directly from patients, suggesting the possibility that the inhibitor can be developed as an effective treatment for gastrointestinal diseases.

Accordingly, the present inventors have studied and studied the adhesion inhibitory components of Helicobacter pylori bacteria from mushrooms in particular. It was confirmed that the extracts of the woody mushrooms showed excellent adhesion inhibitory effect on the phase epithelial cells of Helicobacter pylori bacteria. Therefore, an object of the present invention is to provide a method for extracting a component having an excellent anti-adhesion effect against Helicobacter pylori bacteria from a fungus and a food for improving gastrointestinal diseases using the extract.

The present invention for achieving the above object comprises the steps of pulverizing the dried wood mushrooms; Removing ethanol soluble components using ethanol, extracting necessary components from neutralized alkaline hot water from crushed ethanol soluble components first, and adding sufficient ethanol to hot water extract. Helicobacter pylori bacteria from the soybean mushroom, which comprises the steps of removing the ethanol soluble component secondly after standing for 24 hours and removing residual ethanol and water from the ethanol insoluble portion from which the ethanol soluble component is removed and obtaining the remaining water-soluble polysaccharide component. The present invention relates to a method for extracting the anti-adhesion component of, a safe and effective extract extracted by such a method and the use of the extract.

Hereinafter, the present invention will be described in detail.

The dried wood mushroom sample is ground with a grinder. In order to increase the extraction yield in the next step of extraction, it is desirable to make the particle size fine during grinding.In this case, the lower the moisture content of the soybean mushroom, the easier the grinding process is. It is necessary to carry out hot air drying at a temperature below 60 ℃ to lower the moisture content as much as possible.

In order to increase the extraction yield, the method may further include homogenizing the size of the pulverized particles, and in order to homogenize the pulverized particles, screening is preferable. When screening, it is preferable to use a screen of 40 mesh or more. If the screen is larger than the mesh size, the surface area of the particles is relatively small. Therefore, the yield to extract the components to be removed is removed in the next step of removing the ethanol-soluble components. Because it is lowered.

Removing ethanol-soluble components from the crushed mushrooms is added to 60-100% ethanol to the crushed mushrooms, and stirred for 6 to 24 hours at a temperature below the boiling point. It is appropriate that the ratio of the finely ground material is 1:30 to 1: 1 (w / w), and preferably 1:20 to 1:10. The higher the amount of ethanol is added, the easier the extraction of ethanol soluble components is. If the cost of separation and recovery process is high and the ethanol content is too low, there is a problem that the extraction yield of the soluble components is lowered. In addition, the ethanol concentration of 60 to 100% is appropriate, but using a lower concentration of ethanol will lower the extraction yield of ethanol soluble substances. The extracted thirsty ethanol and ethanol mixtures are separated from the pulverized mushrooms and ethanol by centrifugation or filtration.In this case, the separated ethanol contains pigment components, monosaccharides, and contaminants on the surface of the thirsty mushrooms. It contains.

Thirst mushroom pulverized from ethanol-soluble components is extracted with hot water for 2 to 5 hours. At this time, the amount of distilled water added is suitable for 10 to 80 times the weight of the dry mushroom, and the temperature of the hot water is 70 to 100 ℃. If the amount of distilled water is less than 10 times, it is difficult to extract the desired components sufficiently, on the contrary, if more than 80 times, it is difficult to recover the extract. Even if the extraction temperature is less than 70 ℃ extraction rate is significantly reduced, and if the extraction temperature is more than 100 ℃ may occur a change in the extraction component. In order to prevent evaporation of water during hot water extraction, it is preferable to use a sealed extractor and to increase the yield of extracted components and to extract the components evenly, it is preferable to repeat the extraction process several times under the above conditions. If the extraction yield is not good according to the cultivation or harvesting time of the wood mushroom, it is preferable to extract with sodium hydroxide solution of 0.01N to 0.5N instead of using water as an extraction solvent to improve the extraction yield. Neutralization process is needed.

The hot water extract is separated only from the supernatant by centrifugation or filter press, and the filtrate obtained by filtering the separated supernatant is concentrated under reduced pressure to increase the solid content. At this time, since the concentrate is very high in viscosity, the solid content of 3% to 20% is suitable for the next process.

In order to fractionate the ethanol insoluble components from the hot water extract concentrate, ethanol is added to the hot water extract concentrate, and the final concentration of ethanol is adjusted to 70 to 90%, followed by standing at 4 to 25 ° C for 12 to 24 hours. In order to shorten the settling time, it is preferable to heat to 60 degrees C or less in the hermetic extractor with a stirrer, and to stir gradually. After standing, the ethanol insoluble component is centrifuged or filtered by a filter press to recover the insoluble components.

The ethanol insoluble extract thus obtained is dried under reduced pressure to completely remove residual ethanol, and then lyophilized to obtain a final extract of which the inhibitory substance of Helicobacter pylori is the main ingredient.

The extract of the soybean mushroom showed an excellent inhibitory effect on the adhesion of Helicobacter pylori bacteria in vitro in vitro using epithelial cells. In particular, the extract is a natural food ingredient, as well as the advantage that there is no side effect at all in vivo, as a completely water-soluble ingredient can be applied to a variety of food fields without being limited to the application of the product. In addition, when used in combination with the antimicrobial peptide and IgY used for the bacterium bacterium Helicobacter pylori shows excellent synergistic effect. Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the invention and do not limit the scope of the invention.

(Example 1)

400 g of the dry wood mushroom was pulverized to 40 mesh or less, added to 2L of 80% ethanol, and stirred at room temperature for 24 hours. The stirred mixture was centrifuged and the precipitate was collected, added to 10 L of distilled water, extracted in a closed extractor while heated to a different temperature in the range of 40 to 100 ° C., and then filtered through a filter to obtain a filtrate. The solids content of is shown in Table 1. Each filtrate was concentrated under reduced pressure up to 10% solids content at 50 ° C., and ethanol was added to each concentrated extract to adjust the final ethanol concentration differently in the range of 40 to 90%, and then left at 10 ° C. for 24 hours. After standing, ethanol insoluble solids were filtered through a filter, and the dry mass of the obtained ethanol insoluble component was measured. As shown in Table 1, when the temperature of the hot water is lower than 70 ° C., the content of the extracted solids is significantly reduced. This means that the adhesion inhibitory component of Helicobacter pylori bacteria to be extracted from the woody mushroom is a high molecular weight polysaccharide and does not leach well at low temperatures. In addition, looking at the ethanol insoluble solids content obtained from the hot water extract concentrate in Table 1 it can be seen that the yield is significantly lower when the final ethanol concentration after ethanol mixing is less than 70%.

Meanwhile, instead of using hot water as an extraction solvent during the extraction step, 0.1N sodium hydroxide solution was used as the extraction solvent, extracted at 80 ° C. for 2 hours, and then neutralized with 1N hydrochloric acid. Yield was improved by about 10%.

Hydrothermal extraction Ethanol precipitation Hot water extraction temperature (℃) Solid content of hot water extract (%) Final Ethanol Concentration (%) Final solids yield (g) 40 0.32 40 5.0 60 9.0 70 25 80 28 90 27 50 0.38 40 7.0 60 12 70 29 80 32 90 33 60 0.49 40 9.0 60 13 70 38 80 44 90 43 70 0.73 40 32 60 51 70 67 80 68 90 70 80 0.76 40 36 60 53 70 71 80 73 90 73 90 0.8 40 40 60 52 70 74 80 76 90 75 100 0.8 40 40 60 52 70 72 80 74 90 73

(Example 2)

500 g of the dried charcoal mushrooms were pulverized to 40 mesh or less, added to 10 L of 85% ethanol, and stirred at room temperature for 24 hours. The stirred mixture was centrifuged and the precipitate was collected, added to 20 L of distilled water, extracted with heating at a temperature of 90 ° C. for 2 hours, and compressed and filtered to obtain 15 L of the filtrate. The filtrate was concentrated under reduced pressure so that the solid content was 10%, and ethanol was added thereto at a final concentration of 80%, and the mixture was left at 10 ° C for 24 hours. After filtration of ethanol precipitates to recover ethanol insoluble solids, residual ethanol was completely removed in a vacuum oven at 50 ° C., and residual moisture was lyophilized to obtain 95 g of a final extract having a water content of 4%.

In order to analyze the components of the final extract, 10% of the sample was placed in a vial with a Teflon-lined screw cap, and 72% (w / w) sulfuric acid was blown with nitrogen gas. 125 μl was added. After adding sulfuric acid, the screw cap was closed and left at room temperature for 45 minutes. Nitrogen gas was blown again, and the third distilled water was added to 1.35 ml, and the screw cap was closed, followed by hydrolysis at 100 ° C. for 3 hours.

After cooling the hydrolyzed sample to room temperature, 0.32ml of 15M ammonia solution was added and neutralized with 50µl of myo-inositol at a concentration of 20mg / ml to prepare a final solution.

Separately, 2 g of sodium borohydride was added to 100 ml of anhydrous dimethylsulfoxide anhydride, and the mixture was heated to 100 ° C. to prepare a sodium hydrogen boride solution.

1 ml of sodium hydrogen boride solution was added to 0.1 ml of the final solution and left at 40 ° C. for 90 minutes to reduce the excess. The excess sodium hydrogen boride remaining after reduction was decomposed by adding 0.1 ml of 18 M acetic acid. After decomposition, add 0.2 ml of 1-methylimidazole and 2 ml of acetic anhydride, mix, and leave for 10 minutes at room temperature for acetylation. After cooling to room temperature.

After cooling, 1 ml of dichloromethane was added and mixed vigorously, homogenized, and when the layers were separated, dichloromethane, which was taken down, was stored at -20 ° C and used for analysis.

In order to perform GC-MS and GC analysis on the sample pretreated as above, J & W Science's DB-225 capillary column was used. At this time, the oven was maintained at 195 ° C. for 3 minutes and then raised to 200 ° C. at a rate of 2 ° C./min for 10 minutes, and held at 210 ° C. at a rate of 5 ° C./min for 30 minutes. The temperature of the sample inlet and the detector was 220 degreeC.

5 μl of the pretreated sample was injected into a dedicated syringe, and then a peak corresponding to each sugar was identified using a gas chromatography-mass spectrometry (hereinafter referred to as “GC-MS”). And gas chromatography (hereinafter referred to as "GC").

At this time, the standard reagents of arabic sugar (arabinose), fucose (fucose), galactose (galactose), glucose (glucose), mannose (rhamnose), ribose (ribose), xylose ( Standard monosaccharide, a mixed sample of xylose) was used, and myoinositol was used as an internal standard.

The results of analyzing the components as described above are shown in FIG. The results of the repeated analysis of the final extract extracted from the sour mushrooms were repeated five times: on average, fucose at 13.756 minutes, xylose at 19.962 minutes, mannose at 32.154 minutes, galactose at 34.526 minutes, glucose at 37.535 minutes, internal at 40.329 minutes Peaks in the standard were detected. Based on the area of the peak, it was found that the monosaccharide composition of ethanol insoluble component was 0.2-0.4% of fucose, 22.2-25.0% of xylose, 38.1-41.9% of mannose, 3.9-4.5% of glucose, and 25.7-33.8% of glucose. . Therefore, it was confirmed that the anti-adhesion component of Helicobacter pylori bacteria extracted from soybean mushroom was polysaccharide composed of several kinds of sugars.

(Example 3)

In order to examine the inhibitory properties of Helicobacter pylori from the final extract extracted by the method of Example 2, 100 μl of gastric epithelial cells (human gastric adenocarcinoma, AGS) in a 96 well plate at a concentration of 2 × 10 5 / ml Aliquots were precultured for 24 hours to form a monolayer. Suspended Helicobacter pylori was incubated in physiological saline (PBS) to which phosphate buffer was added at a concentration of 108 CFU / ml, and then the extract of thirsty mushrooms was prepared at various concentrations (0 mg / ml, 1 mg / ml, 10 mg / ml, 20 mg / ml). ml), each was incubated for 15 minutes, and then added to AGS and reacted in 37 ° C. and 10% CO 2 incubator for 2 hours. After the reaction, the cells were washed three times with PBS to remove Helicobacter pylori bacteria that did not adhere to AGS. 100-200 μl of urease activity measurement medium was added to the 96 well plates thus prepared, followed by reaction for 30 minutes at 37 ° C., and the color development was measured at 540 nm. At this time, the more Helicobacter pylori bacteria attached to AGS cells, the higher the absorbance because of the higher urease activity. In order to compare the antiadhesive properties of Helicobacter pylori bacterium from the extracts of the fungus, two concentrations of 50mg / ml and 10mg / ml of sialic lactose reported as excellent anti-adhesive agents of Helicobacter pylori were reported in the literature. The urease activity of the added control was also measured (FIG. 2). In FIG. 2, "M50", "M10" and "M1" represent 50 mg / ml, 10 mg / ml and 1 mg / ml, respectively, and the fungus extracts obtained in Example 2, and "SL50" and "SL10" represent cyanic lactose ( sialic lactose) 50 mg / ml and 10 mg / ml, respectively.

As shown in FIG. 2, the addition of 1 mg of soybean mushroom extract resulted in a 50% reduction in the number of Helicobacter pylori bacteria attached to the phase epithelial cells compared to the case where no addition was made. It showed more than% adhesion inhibitory effect. In addition, as a result of comparing the anti-adhesion ability with the same amount of cyanactic lactose, it was found that the neck mushroom extract had a very good anti-adhesion property against Helicobacter pylori bacteria.

(Example 4)

According to the method of (Example 2), the wood mushroom extract lyophilized powder was prepared. The powder was added 10g to the wire mixed with 300g brown rice powder and 700g yulmul powder, and in addition to 10g of lactic acid bacteria of 1012 CFU / g, 50g of egg yolk powder containing 0.5% IgY was mixed to prepare a wire to protect the stomach.

Recently, the next generation of treatment for gastrointestinal or respiratory infectious diseases has been in the spotlight is a method of blocking receptors on the bacterial surface. The selective attachment of Helicobacter pylori to the epithelial cells can be explained by the fact that adhesin on the surface of bacteria recognizes and attaches the adhesin receptor on the surface of the phase epithelial cells. Therefore, the use of a receptor analog (receptor analog) as an inhibitor of adhesion can inhibit the adhesion of Helicobacter pylori to the epithelial cells, and effectively prevent and treat gastrointestinal diseases. As revealed in the present invention, the extract of the soybean mushroom had an excellent effect of significantly inhibiting the attachment of Helicobacter pylori to the epithelial cells. The thirsty mushroom extract in the present invention is extracted from only thirsty mushrooms, which have been widely used as edible mushrooms for many years, using only distilled water and ethanol. Most of the receptors of host cells to which microorganisms have been identified so far are glycoconjugates. The fungus extract of the present invention is a polysaccharide composed of fucose, xylose, mannose, galactose and glucose, and thus exhibits a receptor analog characteristic of Helicobacter pylori bacteria, thereby effectively inhibiting Helicobacter pylori from attaching to epithelial cells. It seems to be.

Therefore, the extract of the soybean mushroom of the present invention in the form of powder or liquid concentrate is added to foods and various functional food materials or to various beverages by using excellent solubility and thermal stability of Helicobacter pylori bacteria causing the various stomach diseases It is possible to produce a new concept of dietary supplements that can prevent adherence to epithelial cells. In addition, it is difficult to effectively prevent and treat infectious gastrointestinal diseases by using only known formulations and bacteria lactic acid bacteria disinfecting Helicobacter pylori can increase the effect when used in combination with these.

1 is a graph showing the results of the GC-MS for the extract of the present invention,

Figure 2 is a graph showing the receptor analog properties of Helicobacter pylori bacteria of the extract of the present invention

Claims (4)

Grinding dried mushrooms; Using ethanol to remove unwanted components from the mill; Dissolving the ethanol insoluble component in a sufficient amount of hot water or alkaline solution to extract the water-soluble component;  Recovering the ethanol insoluble component by adding a sufficient amount of ethanol to the water soluble component; Method of extracting the adhesion inhibitory component of the Helicobacter pylori bacteria from the soybean mushroom comprising the step of removing residual ethanol and water in the ethanol insoluble portion. Grind the dried wood mushroom and remove the unnecessary ethanol-soluble components from the ground using 60-100% ethanol, add sufficient water or 0.01-0.5N sodium hydroxide solution to the ethanol-insoluble component and heat it to below boiling point Extract the water-soluble component, and then add a sufficient amount of ethanol to the water-soluble component to adjust the final concentration of 60 to 100%, leave for 12-24 hours and then remove the residual ethanol and water in the ethanol insoluble portion, It has a monosaccharide composition of 0.1-1.0% of cose, 20-30% of xylose, 35-45% of mannose, 2-7% of galactose and 25-35% of glucose, and inhibits the adhesion of Helicobacter pylori to phase epithelial cells. Oyster mushroom extract. delete Health supplement food comprising the extract of claim 1 or 2 as an active ingredient.