KR101744481B1 - Composition for improving, treating or preventing gastric ulcer comprising Barley fermented by lactic bacteria as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for improving, treating or preventing gastric ulcer comprising a fermented product of barley as an active ingredient, and more particularly, to a composition for fermenting a fermented product of fermentation of barley, Or a fermented product of fermented lactic acid after fermentation of yeast; and a fermented product of barley obtained as an active ingredient, thereby exhibiting excellent gastric mucosal protection effect through antioxidative effect, through a pharmaceutical composition for treating or preventing gastric ulcer, and antioxidative effect To a method for producing a fermented barley exhibiting gastric mucosal protective effect.

Description

[0001] The present invention relates to a composition for improving, treating or preventing gastric ulcer comprising, as an active ingredient, a fermented barley fermented product,

The present invention relates to a health functional food for improving or preventing gastric ulcer comprising a fermented barley as an active ingredient, a pharmaceutical composition for treating or preventing gastric ulcer, and a method for producing fermented barley showing gastric mucosal protective activity.

The fermented natural product obtained by fermenting natural product is known to increase the physiological activity of natural product due to biotransformation or probiotic effect by fermentation. Therefore, fermented natural product can be used as a new functional food raw material, It is popular as a material.

In particular, it is known that harmless microorganisms which can be edible are more effective than the raw natural materials which do not ferment fermented natural products.

Barley ( Hourdeum vulgare L) is a typical grain, and it contains a wide variety of phenolic compounds. It is known that it exhibits various physiological activities based on antioxidative actions such as anticancer and gastrointestinal protective effects. In fermentation process, Absorption is increased, and the physiological activity based on the antioxidative effect is further increased, so that it is known to be very effective for improving health.

To date, it has been known that the fermentation significantly increases the antioxidant effect, uric acid reduction effect, atopic dermatitis improvement effect, liver protection effect and immune activity effect of the barley extract.

However, there have been no reports of application of barley extracts to improve stomach health or to treat gastric-related diseases.

Korean Patent No. 1391441 Korean Patent No. 1468698

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an object of the present invention is to provide a health functional food for improving or preventing gastric ulcer by using fermented barley.

Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of gastric ulcer by utilizing fermented barley.

It is still another object of the present invention to provide a method for producing a fermented barley product which is effective for improving, preventing or treating gastric ulcer.

One aspect of the present invention relates to a health functional food for improving or preventing gastric ulcer comprising a fermented barley as an active ingredient.

Another aspect of the present invention relates to a pharmaceutical composition for treating or preventing gastric ulcer comprising a fermented barley as an active ingredient.

Another aspect of the present invention relates to a method for producing a fermented barley comprising a yeast fermentation step in which yeast is inoculated into a saccharified saccharide solution.

The barley fermented product of the present invention has an effect of protecting the gastric mucosa by the antioxidant effect.

FIG. 1 is a schematic diagram showing a series of procedures for observing the gastric mucosal protective effect of each experimental group after administering the test substances prescribed in each experimental group to the experimental rats of Experimental Example 3. FIG.
Fig. 2 shows the results of visual observation of changes in gastric mucosal lesions in each experimental group of Experimental Example 3. Fig.
FIG. 3 is a graph showing the gross lesion area of each experimental group in Experimental Example 3. FIG.
FIG. 4 is a representative histological image of the fundic mucosa obtained from each experimental group of Experimental Example 3. FIG.
FIG. 5 is a schematic view showing a series of procedures for observing the gastric mucosal protective effect of each experimental group after administering the test substances prescribed in each experimental group to the experimental rats of Experimental Example 4. FIG.
6 is a graph showing the results of visual observation of changes in gastric mucosal lesions in each experimental group of Experimental Example 4. FIG.
7 is a graph showing the gross lesion area of each experimental group in Experimental Example 4. FIG.
8 is a graph showing the content of MPO (Myeloperoxidase) in each experimental group of Experimental Example 4. FIG.
FIG. 9 is a representative histological image of the fundic mucosa obtained from each experimental group of Experimental Example 4. FIG.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

The present invention relates to a health functional food for improving or preventing gastric ulcer comprising an active ingredient of fermented barley, and the present invention also relates to a pharmaceutical composition for treating or preventing gastric ulcer comprising fermented barley as an active ingredient, The present invention relates to a method for producing a fermented barley comprising a yeast fermentation step of fermenting yeast by inoculating yeast into a saccharified solution of barley. The barley fermented product may further comprise a lactic acid fermentation step of fermenting the fermented product obtained in the yeast fermentation step by inoculating the lactic acid bacteria.

The weight ratio of 1,3-1,4-beta-glucan and 1,3-1,6-beta-glucan in the barley fermentation product may be 5: 1 to 15: 1 in consideration of the gastric mucosal protective effect due to the antioxidative effect, May be from 5: 1 to 10: 1. The weight ratio is based on the solids weight.

The barley fermented product may contain 10 to 15% by weight, preferably 11 to 14% by weight, of the polysaccharide in the total fermented barley, taking into consideration the gastric mucosal protective effect by the antioxidant effect. The weight is based on the solids weight.

The barley fermented product may be a barley yeast fermented product obtained by fermenting the barley saccharified solution with yeast, or a barley fermented product obtained by fermenting the yeast fermented product with the lactic acid bacteria.

Wherein the barley saccharified liquid is prepared by immersing barley in water and then gelatinizing it; And saccharification by adding at least one selected from maltose, amylase and yeast to the gelled barley.

The smoothing step may be performed by adding 1 to 1.5 times water of the barley weight to the barley from which the husks have been removed, immersing the barley for 11 to 13 hours, removing water, and allowing the barley to be softened at 80 to 100 DEG C for 1 to 3 hours .

The saccharification may be performed by adding 2 to 4 times as much water as the barley weight to the barley after cooling, adding at least one selected from the group consisting of malt, amylase and koji, and saccharifying at 45 to 65 ° C for 5 to 7 hours .

The thus-prepared barley saccharide solution may be sterilized at 110 to 130 DEG C for 10 to 20 minutes before yeast fermentation described later.

The step of fermenting the saccharification solution with yeast may include fermenting the saccharomyces cerevisiae such as Saccharomyces cerevisiae by inoculating the yeast Saccharomyces sp. And fermentation may be carried out at a constant fermentation (0 rpm) at a temperature of 10 to 40 ° C for 20 to 100 hours, preferably at 20 to 40 ° C for 20 to 50 hours, and at 50 rpm or less. The temperature, time, and rpm at the yeast fermentation were optimized considering that the content of 1,3-1,6-glucan in the fermented product after yeast fermentation is significantly included in the improvement, prevention, and treatment of gastric ulcer.

The thus-produced fermented product after fermentation of yeast may be sterilized at 110 to 130 ° C for 10 to 20 minutes before fermentation of lactic acid bacteria described later.

The step of fermenting the fermented product obtained after the fermentation of the yeast may be carried out by inoculating the fermented product with lactic acid bacteria, wherein the lactic acid bacteria are selected from the group consisting of Weissella sp. , Lactobacillus sp. May be at least one selected from the group consisting of Bifidobacterium sp. , Pediococcus sp. , Leuconostoc sp. And Lactococcus sp . The temperature, time, and rpm at the time of fermentation of the lactic acid bacteria are fermented at a constant fermentation (0 rpm) or 50 rpm or less at 10 to 40 ° C for 20 to 80 hours, preferably 20 to 40 ° C for 20 to 50 hours , Which is optimized considering that the content of 1,3-1,6-glucan in the fermentation product produced after fermentation of lactic acid bacteria is significantly included in the improvement, prevention and treatment of gastric ulcer.

Specifically, the lactic acid bacteria are selected from the group consisting of Weissella cibaria , Lactobacillus paraplantarum , Lactobacillus acidoohilus , Bifidobacterium longum , Bifidobacterium, It may be one or more selected from the group consisting of Bifidobacterium brevis , Pediococcus pentosaceus , Leuconostoc mesenteroides and Lactococcus lactis .

The thus produced fermented product after fermentation of lactic acid bacteria may be sterilized at 110 to 130 ° C for 10 to 20 minutes before fermentation of lactic acid bacteria described later.

Meanwhile, the fermented barley according to the present invention may be dried by a conventional method in the art including freeze drying and hot air drying.

As used herein, the term " comprising as an active ingredient " is meant to include an amount sufficient to achieve an improvement, treatment or prophylactic efficacy or activity of a barley fermentation product of the present invention.

The health functional food may be a preparation of the fermented barley in the form of a capsule, tablet, powder, granule, liquid, ring, flake, paste, syrup, gel, jelly or bar, , Spices, gum, confectionery, etc., and it is produced in the form of general food. When it is taken, it means bringing about a specific effect on health. Unlike general medicine, however, There is no side effect that can occur in the case.

The health functional foods are very useful because they can be ingested routinely. The added amount of the fermented barley in such a health functional food can not be uniformly determined depending on the kind of the health functional food to which it is added but may be added within a range that does not deteriorate the original taste of the food, To 50% by weight, preferably 0.1 to 20% by weight. In the case of health functional foods in the form of capsules, tablets, powders, granules, liquids, pellets, slices, pastes, syrups, gels, jellies or bars, the amount is usually 0.1 to 100% by weight, preferably 0.5 to 80% %.

The health functional food may contain not only barley fermented product as an active ingredient but also a component which is ordinarily added at the time of food production, and includes, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the health functional food of the present invention is prepared from a drink and a beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, various plant extracts and the like may be further added have.

In a pharmaceutical composition for the treatment or prevention of gastric ulcer comprising the fermented product of barley as an active ingredient, the fermented product of the fermented product is, for example, 0.001 mg / kg or more, preferably 0.1 mg / kg or more, more preferably 10 mg / kg or more, more preferably 100 mg / kg or more, even more preferably 250 mg / kg or more, and most preferably 0.1 g / kg or more. Since the barley fermented product is a natural product, there is no adverse effect on the human body even when it is administered in an excessive amount, so that the quantitative upper limit of the fermented barley contained in the composition of the present invention can be selected by a person skilled in the art within a suitable range.

The pharmaceutical composition may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A flavoring agent and the like can be used.

The pharmaceutical composition may be formulated to contain at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

The pharmaceutical form of the pharmaceutical composition may be a granule, a powder, a tablet, a coated tablet, a capsule, a suppository, a liquid, a syrup, a juice, a suspension, an emulsion, a drip agent or an injectable liquid agent. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like, preferably oral administration.

The appropriate dosage of the pharmaceutical composition will vary depending on factors such as the formulation method, the manner of administration, the age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, The physician can easily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment, the daily dosage of the pharmaceutical composition is 0.001-10 g / kg.

The pharmaceutical composition may be prepared in a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Into a capacity container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. In addition, it is apparent that, based on the teachings of the present invention including the following examples, those skilled in the art can easily carry out the present invention in which experimental results are not specifically shown.

Production Example 1: Production of barley glycoside

The barley was removed from the husks of domestic barley ( Hordeum vulgare L) purchased from Jeonnam Agricultural Cooperative Agricultural Cooperative (Hwasun Gun) and 1.3 times as much as the barley weight was added and immersed for 12 hours. The immersed water was removed and the barley was allowed to cool for 2 hours at 90 ° C.

Three times as much water as the above barley weight was added to the grated barley, and malt was added and saccharified at 55 占 폚 for 6 hours to prepare a saccharified saccharide solution.

Production Example 2: Yeast fermentation conditions of the saccharified saccharide solution

Experiments were conducted on fermentation temperature, time, and rpm in order to find the optimal yeast fermentation conditions of the saccharified saccharide solution prepared in Preparation Example 1.

In the barley saccharified solution prepared in Preparation Example 1, saccharomyces Saccharomyces cerevisiae, JKK091002 (KCCM 11056P) was inoculated and yeast fermentation was carried out at the temperature, time and rpm conditions shown in Table 1 below.

Since beta glucan from barley is generally 1,3-1,4beta glucan, the contents of 1,3-1,6beta glucan from yeast were measured to establish optimal yeast fermentation conditions. The content of the 1,3-1,6-beta-glucan is based on the solid content, and the content of 1,3-1,6-beta-glucan is measured per 1 g of the solid content of the yeast fermentation product.

The content of 1,3-1,6beta glucan in the fermented yeast was measured according to the experiment of the Health Functional Food Promotion (3.4.3 Beta Glucan) of the KFDA.

Yeast fermentation conditions In fermented yeast
1,3-1,6 beta-glucan content
(mg / solid content g) Temperature (℃) Time (hr) rpm 20 24 10 1.12 25 1.21 30 1.28 35 1.25 30 12 0 0.75 24 1.27 36 1.49 48 1.90 60 1.90 (Alcohol odor) 72 1.90 (Alcohol odor) 30 24 0 1.27 20 1.22 50 0.95 100 0.23

As a result, as shown in Table 1, when the yeast fermentation temperature was 25 ° C or higher, the content of 1,3-1,6beta glucan in the yeast fermentation product was high, and when the temperature was 30 ° C, 1,3-1,6beta glucan And the content was the highest.

When the yeast fermentation time was more than 24 hours, the content of 1,3-1,6beta glucan in the yeast fermentation product was high, and the content of 1,3-1,6beta glucan was the highest at 48 hours. When the yeast fermentation time was more than 60 hours, the content of 1,3-1,6beta glucan was found to be equivalent to that of 48 hours, but there was a problem that alcohol was consumed.

In addition, when the yeast fermentation rpm is 100 rpm, the content of 1,3-1,6-beta-glucan is significantly lowered, and thus the rpm is preferably 50 rpm or less. Respectively.

Therefore, based on the above results, yeast fermentation will be carried out at 30 ° C, 48 hours and 0 rpm in yeast fermentation in the following examples.

Production Example 3: Fermentation conditions of lactic acid bacteria in fermented barley yeast

The fermentation temperature, time and rpm of the barley yeast fermented product prepared in Preparation Example 2 were examined in order to find the optimal lactic acid fermentation conditions.

The yeast fermented product prepared in Preparation Example 2 was inoculated with Weissella cibaria (KCTC 18141P) and fermented with lactic acid bacteria at the temperature, time and rpm as shown in Table 2 below.

Since the lactic acid fermentation generally produces extracellular polysaccharides, the content of polysaccharides in the fermented lactic acid bacteria was measured to set the optimal lactic acid fermentation conditions. The content of the polysaccharide is based on the solid content, and the content of the polysaccharide per 1 g of the solid content of the fermented lactic acid bacteria is measured.

Fermentation conditions of lactic acid bacteria In fermented lactic acid bacteria
Polysaccharide content (mg / solid content g)) Temperature (℃) Time (hr) rpm 20 24 10 91 25 92 30 94 35 90 30 12 0 64 24 100 36 100 48 101 30 24 0 92 10 81 20 77 30 52

As a result, as shown in Table 2, when the yeast fermentation temperature was 25 ° C or higher, the content of 1,3-1,6beta glucan in the yeast fermentation product was high, and when the temperature was 30 ° C, 1,3-1,6beta glucan And the content was the highest.

When the yeast fermentation time was more than 24 hours, the content of 1,3-1,6beta glucan in yeast fermented product was high, and the content of 1,3-1,6beta glucan at 48 hours was the highest. When the yeast fermentation time was more than 60 hours, the content of 1,3-1,6beta glucan was found to be equivalent to that of 48 hours, but there was a problem that alcohol was consumed.

In addition, when the yeast fermentation rpm is 100 rpm, the content of 1,3-1,6-beta-glucan is significantly lowered, and thus the rpm is preferably 50 rpm or less. Respectively.

Therefore, based on the above results, yeast fermentation will be carried out at 30 ° C, 48 hours and 0 rpm in yeast fermentation in the following examples.

Comparative Example 2: Preparation of fermented barley yeast

In the barley saccharified solution prepared in Preparation Example 1, saccharomyces Saccharomyces cerevisiae (KCCM 11056P) was inoculated and fermented at 30 DEG C for 48 hours to obtain a yeast fermented product, which was then sterilized at 121 DEG C for 15 minutes.

Cells and impurities were removed from the fermented lactic acid bacteria using a filter press having a pore diameter of 1 mu m.

Thereafter, the mixture was sterilized at 90 DEG C for 2 hours, and spray-dried at a capacity of 180 L / h to prepare a barley yeast fermentation product.

Example 2: Preparation of fermented barley extracts (FBe)

Example 2-1: Preparation of wysseleibia ( Weissella cibaria : KCTC 18141P) Production of fermented barley using lactic acid bacteria

The barley yeast fermented product prepared in Preparation Example 1 was inoculated with Weissella cibaria (KCTC 18141P), and then fermented at 30 ° C for 48 hours to obtain a fermented lactic acid bacterium, And sterilized at 121 DEG C for 15 minutes.

Cells and impurities were removed from the fermented lactic acid bacteria using a filter press having a pore diameter of 1 mu m.

Thereafter, the mixture was sterilized at 90 DEG C for 2 hours and then spray-dried at a volume of 180 L / h to prepare a barley fermented product.

Example 2-2: Lactobacillus paraplaterum (KCTC 18142P) Production of fermented barley using lactic acid bacteria

A fermented product of barley was prepared in the same manner as in Comparative Example 2 except that Lactobacillus paraplatamil (KCTC 18142P) was used instead of Weissella cibaria (KCTC 18141P) as a lactic acid bacterium.

Example 2 -3: Bifidobacterium Long Sword ( Bifidobacterium longum , ATCC  15707) Barley using lactic acid bacteria Fermentation product  Produce

A barley fermented product was prepared in the same manner as in Comparative Example 2 except that Bifidobacterium longum (ATCC 15707) was used instead of Weissella cibaria ( KCTC 18141P) as a lactic acid bacterium.

Comparative Example 1: Production of fermented barley by lactic acid fermentation

Weissella cibaria ( KCTC 18141P) was inoculated into the saccharified saccharide solution prepared in Preparation Example 1, and then fermented at 30 ° C for 48 hours to obtain a fermentation product of lactic acid bacteria, And sterilized at 121 DEG C for 15 minutes.

Cells and impurities were removed from the fermented lactic acid bacteria using a filter press having a pore diameter of 1 mu m.

Thereafter, the mixture was sterilized at 90 DEG C for 2 hours and then spray-dried at a volume of 180 L / h to prepare a barley fermented product.

Experimental Example 1  : Depending on the fermentation method Fermented  Component analysis

The following Tables 3 to 5 show the results of measuring the contents of major components in the products of each step in the preparation of the fermented barley. That is, the content of major components in the saccharified solution after barley saccharification in Production Example 1, the barley yeast fermentation product of Comparative Example 2, and the barley fermentation obtained by fermentation of yeast and lactic acid bacteria of Example 2 were shown. The content is based on solids.

As shown in Examples 2-1 to 2-3, the content of the major components of the lactic acid bacteria fermented according to the kinds of the lactic acid bacteria when fermenting the lactic acid bacteria was measured.

In the following Table 3, the pH was measured using a pH meter, and the sugar content was measured using a sugar content meter.

Production Example 1 Comparative Example 2 Example 2-1 Example 2-2 Example 2-3 Comparative Example 1 Luxury Glycation leaven
Fermentation Lactobacillus
Fermentation
(Waissel Shivaria) Lactobacillus
Fermentation
(Lactobacillus paraplanaceum) Lactobacillus
Fermentation
(Bifidobacterium lemongi) Lactobacillus
Fermentation pH 4.60 4.36 4.26 4.20 4.10 4.11 4.30 Sugar content 7.5 18.4 19.2 19.1 19.1 19.0 18.6

Table 4 below shows the results of measurement of the content of beta-glucan in the fermentation product. The content of beta-glucan was measured by the method of the Health Functional Food Revolution (3.4.3 beta-glucan) and measured on the basis of the solid content.

Production Example 1 Comparative Example 2 Example 2-1 Example 2-2 Example 2-3 Comparative Example 1 Luxury Glycation leaven
Fermentation Lactobacillus
Fermentation
(Waissel Shivaria) Lactobacillus
Fermentation
(Lactobacillus paraplanaceum) Lactobacillus
Fermentation
(Bifidobacterium lemongi) Lactobacillus
Fermentation 1,3-1,4beta glucan
(A) 7.8 mg 15.5 mg 16.0 mg 15.9 mg 15.9 mg 15.9 mg 15.7 mg 1,3-1,6
Beta Glucan
(B) 0.4 mg 0.6 mg 1.6 mg 3.1 mg 2.9 mg 2.8 mg 0.6 mg A: B 19.5: 1 25.8: 1 10: 1 5.1: 1 5.5: 1 5.7: 1 26.2: 1

Table 5 below shows the content of polysaccharides in the total saccharified liquid or fermentation product, and the polysaccharide was measured by using the dry weight method and is based on the solid content.

Production Example 1 Comparative Example 2 Example 2-1 Example 2-2 Example 2-3 Comparative Example 1 Luxury Glycation leaven
Fermentation Lactobacillus
Fermentation
(Waissel Shivaria) Lactobacillus
Fermentation
(Lactobacillus paraplanaceum) Lactobacillus
Fermentation
(Bifidobacterium lemongi) Lactobacillus
Fermentation Polysaccharide
(weight%) 3.3 8.8 10.4 13.0 12.9 12.8 9.1

As shown in Tables 3 to 5, the barley yeast fermentation product of Comparative Example 2 prepared by yeast fermentation and the barley fermentation products of Examples 2-1 to 2-3 prepared by yeast fermentation and lactic acid fermentation The content of 1,3-1,6-beta-glucan and the polysaccharide content were higher than those of the fermented barley lactic acid bacteria of Comparative Example 1.

Experimental Example 2: In Vitro ( in vitro ) For gastric mucosal protection

Six-week-old ICR male mice were purchased from Orient Bio (Seongnam), purified for 10 days, fasted for 24 hours, and then subjected to ether staining, and then stomach tissues were collected.

In order to examine the gastric mucosal protective effect of the test substances described in each test group as shown in Table 6 below, the gastric mucosal injury was induced using the gastric mucosal damage inducer for the collected stomach tissues, and nitrate / nitrite contents were measured.

As a gastric mucosal injury inducer, a mixture of HCl / EtOH mixture (HE, 98% EtOH (Merck, Germany) containing 150 mM HCl (Merck, Germany)) was used. (RA) (Ranitidine hydrochloride, Sigma-Aldrich, USA), Comparative Example 2, the FBe prepared in Examples 2-1 to 2-3 and the fermentation products used in Comparative Example 1 were used.

Nitric oxide (NO) is one of the typical mediators responsible for the defense mechanism of the gastrointestinal mucosa. It is produced by NO-synthase and is known to be an important factor controlling the blood flow and microcirculation in stomach. Currently, drugs that stimulate the secretion of NO are effective in ethanol-induced gastric ulcers, while drugs that inhibit the secretion of NO are known to increase gastric mucosal sensitivity to ethanol. NO is an antioxidant that inhibits lipid peroxidation . Thus, the nitrate / nitrite content of the gastric mucosa indicates the secretion of NO, which is responsible for the defense mechanism of the gastrointestinal mucosa (Morais et al ., "Protective effect of anacardic acids from cashew (Anacardium occidentale) Chem Biol Interact, vol.183 (2010), p264-9).

Experimental group Experimental material Method of administration Nitrate / nitrite content (M / g protein) compare
Experimental Example
2-1 normal
Control group Sterile distilled water Single application of sterilized distilled water 10 mg / kg 2.43 ± 0.73 compare
Experimental Example
2-2 HE
Control group - sterile distilled water
- HCl / EtOH mixture Single dose of sterile distilled water 10 mg / kg was applied and after 1 hour, 5 ml / kg of HCl / EtOH mixture was applied 1.03 + - 0.25 ^a compare
Experimental Example
2-3 RA - RA
- HCl / EtOH mixture RA 100 mg / kg was dissolved in sterilized distilled water and applied at a dose of 10 ml / kg. After 1 hour, 5 ml / kg of HCl / EtOH mixture was applied 1.76 ± 0.61 ^bc compare
Experimental Example
2-4 Lactobacillus
Fermentation product
(Comparative Example 1) - fermented lactic acid bacteria
(Comparative Example 1)
- HCl / EtOH mixture 100 mg / kg of fermented lactic acid fermentation product (Comparative Example 1) was dissolved in sterilized distilled water and applied at a rate of 10 ml / kg. After 1 hour, 5 ml / kg of HCl / EtOH mixture was applied 1.12 ± 0.28 ^bd Experimental Example
2-1 leaven
Fermentation product
(Comparative Example 2) - yeast fermentation product
(Comparative Example 2)
- HCl / EtOH mixture 100 mg / kg of fermented yeast fermentation product (Comparative Example 2) was dissolved in sterilized distilled water and applied at a rate of 10 ml / kg. After 1 hour, 5 ml / kg of HCl / EtOH mixture was applied 1.44 ± 0.28 ^c Experimental Example
2-2 FBe (Example 2-1) - FBe
(Example 2-1) 100 mg / kg
- HCl / EtOH mixture 100 ml / kg of FBe (Example 2-1) was dissolved in sterilized distilled water and applied at a rate of 10 ml / kg. After 1 hour, 5 ml / kg of HCl / EtOH mixture was applied 1.53 + - 0.25 ^c Experimental Example
2-3 FBe (Example 2-2) - FBe
(Example 2-2) 100 mg / kg
- HCl / EtOH mixture 100 ml / kg of FBe (Example 2-2) was dissolved in sterilized distilled water, and 10 ml / kg was applied once. After 1 hour, 5 ml / kg of HCl / EtOH mixture 1.47 ± 0.28 ^ac Experimental Example
2-4 FBe (Example 2-3) - FBe
(Example 2-3) 100 mg / kg
- HCl / EtOH mixture 100 ml / kg of FBe (Example 2-3) was dissolved in sterilized distilled water and applied at a rate of 10 ml / kg. After 1 hour, 5 ml / kg of HCl / EtOH mixture was applied 1.46 ± 0.25 ^ac

^a p < 0.01 and ^b p < 0.05 are values compared with normal control by MW (Mann-Whitney U).

^c p < 0.01 and ^d p < 0.05 are values compared to HE control by MW.

Referring to Table 6 above, the nitrate / nitrite content in the stomach tissues was significantly decreased (p <0.01) in the HE control group as compared with the control group, and that in the fermented lactic acid fermentation product (Comparative Example 1) (P <0.05, p <0.05), respectively. On the other hand, in the yeast fermentation test of Comparative Example 2 and in the FBe test groups of Examples 2-1 to 2-3, the nitrate / nitrite content was significantly (p <0.05) lower than that of the HE control, And nitrate / nitrite content was significantly decreased (p <0.01) compared to the control group.

Thus, as shown in Table 6, the barley fermented product obtained in Example 2-1 having a high content of 1,3-1,6 beta-glucan should be subjected to animal experiments for the gastric mucosal protection effect in the following Experimental Example .

How to measure

In the following Experimental Examples 3 and 4, the measurement method for each item for confirming the gastric mucosal protective effect is as follows.

(1) Changes in gastric mucosal lesions

Mice from each experimental group were sacrificed by cervical dislocation and the abdomen was removed by cervical dislocation and the stomach was cut off.

The abdomen was opened with a median incision and then the stomach was cut off. The cut stomach was opened along a large curvature and fixed in 10% neutral buffered formalin for 24 hours before digital images were obtained.

A visual check of the ulcer sites on the surface, and then, automatic computer image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Quebec, Canada) S ㆌ leyman et al using the., Morais et al. . and Oyagi et al method was measured in accordance with the (S ㆌ leyman et al;. "Comparative study on the gastroprotective potential of some antidepressants in indomethacin-induced ulcer in rats"; Chem Biol Interact, vol.180 (2009), p318 -24); Morais et al .; "Protective effect of anacardic acids from cashew (Anacardium occidentale) on ethanol-induced gastric damage in mice"; Chem Biol Interact, vol. 183 (2010), p 264-9; Oyagi et al .; "Protective effects of a gastrointestinal agent containing Korean red ginseng on gastric ulcer models in mice &quot;; BMC Complement Altern Med., Vol. 10 (2010), p45)

Visually observable lesions were measured and the damage score was calculated. For this purpose, the total area of the gastric ulcer was calculated as the area of the gastric mucosa (mm 2).

(2) Changes in lipid peroxidation in gastric tissues

The level of gastric mucosal lipid peroxidation was determined by measuring MDA using the thiobarbituric acid test.

The mouse was excised from the stomach and washed with cold saline. To minimize hemoglobin ' s interference by free radicals, all blood stuck to the mucosa was removed.

The corpus mucosa was scraped and weighed and homogenized using 10 mL of 100 g / l KCl (Sigma-Aldrich, USA).

0.5 mL of homogenate was diluted with 0.2 mL of 80 g / L sodium lauryl sulfate (Sigma-Aldrich, USA), 1.5 mL of 200 g / L acetic acid (Merck, Germany) 2-thiobarbiturate (Sigma-Aldrich, USA) and 0.3 mL of distilled water.

The mixed solution was heated at 98 ° C for 1 hour, cooled, added with 5 mL of n-butanol: pyridine (15: 1, Sigma-Aldrich, USA), mixed for 1 min, centrifuged at 4,000 rpm for 30 min And the absorbance of the supernatant was measured. The absorbance of the supernatant was measured at a wavelength of 532 nm using a UV / Vis spectrometer (OPTIZEN POP, Mecasys, Daejeon, Korea).

Standard curves were obtained using 1,1,3,3-tetramethoxypropane (Sigma-Aldrich, USA) and the results were expressed as the concentration of MDA per unit tissue weight (nM / g tissue).

(3) Changes in CAT (Catalase) activity in gastric tissues

CAT activity was measured by the method described by Evans and Diplock (Evans RC and Diplock AT; " Laboratory Techniques in Biochemistry and Molecular Biology. In Techniques in Free Radical Research (Burtin RH and Knippenberg PH, Eds) 1991, 199-201).

To obtain an enzyme dilution, a homogenous suspension of mouse gastric mucosa was diluted using a buffer as described above. Then, 2 ml of the enzyme dilution was added to the cuvette, mixed with 1 ml of 30 mM H ₂ O _2, and absorbance was measured at 240 nm wavelength for 10 seconds using a UV / Vis spectrometer.

The initial absorbance of the reaction mixture was about 0.5. The enzyme activity was expressed as a first order constant (mM / min / mg tissue) indicating decomposition of H ₂ O ₂ at room temperature.

(4) Changes in SOD (superoxide dismutase) activity in gastric tissues

SOD activity in the stomach tissues was measured using the method of Minami and Yoshikawa (Minami M, Yoshikawa H.; "A simplified assay method of superoxide dismutase activity for clinical use"; Clinica Chimica Acta, vol.92 (1979), p337 -42).

15 μl of the stomach suspension was mixed with 450 μl of cold deionized water, 125 μl of chloroform and 250 μl of ethanol and centrifuged at 4 ° C for 2 min at 8,000 ㅧ g.

Then, 500 μl of the extract was added to the reaction mixture containing 500 μl of 72.4 mM triscacodylate buffer with 3.5 mM diethylene pentaacetic acid (pH 8.2; Sigma-Aldrich, USA).

Then 10 μl of 9 mM pyrogallol (Sigma-Aldrich, USA) dissolved in 10 mM HCl was added and 100 μl of 16% reaction mixture was incubated at 37 ° C for 5 min.

The culture was terminated by the addition of 300 μl of 2 M formic buffer (pH 3.5) containing 16% Triton X-100 (Sigma-Aldrich, USA) and the absorbance at 540 nm was measured in the spectrophotometer.

It is equal to the amount of enzyme which reduces the initial absorbance of nitroblue tetrazolium of SOD enzyme activity by 50%.

(5) Changes in Nitrate / nitrite content in stomach tissue

Analysis of nitrate / nitrite content in gastric mucosa was performed using Griess reagent (Green et al ., "Analysis of nitrate, and [15N] nitrate in biological fluids"; Anal Biochem.vol.10 (1982) 8).

Stomach tissues were homogenized in 50 mM potassium phosphate buffer (pH 7.8; Sigma-Aldrich, St. Louis, Mo., USA) and centrifuged at 11,000 ㅧ g for 15 min at 4 ° C. 100 μl of the supernatant was mixed with 100 μl of Griess reagent (0.1% N- (1-naphthyl) ethylenediamide dihydrochloride, 1% sulfanilamide in 5% phosphoric acid; all obtained from Sigma-Aldrich, St. Louis, Mo., USA) After 10 minutes, the absorbance was measured at a wavelength of 540 nm using a microplate reader (Tecan, M nnedorf, Switzerland).

A standard curve was obtained using sodium nitrite and the nitrate / nitrite content was expressed as μM nitrate / nitrite / g of protein. The protein concentration of each sample was determined by the Bradford assay (Bradford MM; "A rapid and sensitive method for quantitation of microgram quantities of proteins using the principle of protein-dye binding &quot;, Anal Biochem. Vol. 72 (1976) Lt; / RTI &gt;

(6) Histopathological changes

Samples were taken from the above sites (between cardiac and pylorus, fundus). And was obliquely cut based on the lumen. The cut fundus was fixed with 10% neutral buffered formalin (NBF) for 24 h.

After paraffin embedding, 3-4 μm sections were prepared. Sections were stained with H & E (hematoxylin and eosin) for optical microscopy.

Subsequently, individual fundus cut with oblique lines was observed in histological profiles.

The basal lamina thickness from the luminal mucosal surface of the periulcerative area of oblique cut tissue samples to the muscularis mucosa was measured. ., As described in Ku et al as measured, it was measured using automatic computer image analyzer (Model Eclipse 80i, Nikon, Japan ) (Ku et al: "Effect of Lonicerae Flos extracts on reflux esophagitis with antioxidant activity";. World J Gastroenterol, vol. 15 (2009), p. 4799-805).

In addition, the invasiveness rate (%) of the fundus was calculated using the following Equation 1, and the semi-quantitative score was classified into four levels.

0: Normal

1: mucosal surface damage

2: mucous membrane severity damage

3: Complete mucosal damage

<Formula 1>

Invaded percentages of lesions (%)

= (Depth of ulcerative lesion in gastric mucosa / thickness of total gastric mucosa) x 100

(7) Changes in MPO (Myeloperoxidase) content in gastric tissues

0.2 g of stomach tissue samples were homogenized in potassium phosphate buffer (50 mM K ₂ HPO ₄ , pH 6.0; Sigma-Aldrich, USA) containing hexadecyltrimethylammonium bromide (HETAB; 0.5% w / v; Sigma-Aldrich, USA) .

The supernatant was discarded and the pellet was homogenized again with 50 mM K ₂ HPO containing 0.5% (w / v) HETAB and 10 mM EDTA (Sigma-Aldrich, USA) Respectively.

o-dianizidine 2HCl by measuring H ₂ O ₂ -dependent oxidation.

One unit of enzyme activity was determined by using a UV / Vis spectrophotometer (OPTIZEN POP, Mecasys, Korea) at a wavelength of 460 nm and an MPO content per unit tissue weight (nM / mg tissue )

(8) Changes in GSH (Glutathione) content in stomach tissue

GHS content of the gastric mucosa was determined according to the method of Sedlak and Lindsay (Sedlak J, Lindsay RH; "Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent"; Anal Biochem, vol. ), p192-205).

The gastric mucosal surface was scraped off, collected, weighed and homogenized in 2 mL of 50 mM Tris-HCl buffer containing 20 mM EDTA (Sigma-Aldrich, USA) and 0.2 mM sucrose (Merck, Germany) at pH 7.5 .

A homogeneous suspension was precipitated with 0.1 mL of 25% trichloroacetic acid (Merck, Germany), centrifuged at 4 ° C for 42 min at 4200 rpm, and the precipitate was removed. The supernatant was used to determine GSH concentration using 5,5'-dithiobis (2-nitrobenzoic acid; Sigma-Aldrich, USA).

Absorbance was measured at 412 nm using a UV / Vis spectrophotometer and the GHS content of the gastric mucosa was expressed as "nM / mg tissue".

Experimental Example 3: Animal test - HCl / EtOH induced gastric mucosal injury

3-1. Experimental animals and experimental group

The animals were sacrificed for 10 days and then fasted for 24 hours. The body weight of mice (mean: 38.27 ± 1.81 g, , 25.60 ~ 41.80g) were used for the experiment.

As a gastric mucosal injury inducer, a mixture of HCl / EtOH mixture (HE, 98% EtOH (Merck, Germany) containing 150 mM HCl (Merck, Germany)) was used. (RA) (Ranitidine hydrochloride, Sigma-Aldrich, USA) and FBe prepared in Example 2-1 were used. Here, each test substance (RA, FBe300, FBe200, and FBe100) was dissolved in sterilized distilled water at a dose of 10 ml / kg in 100 ml / kg.

Experimental group Experimental material Method of administration compare
Experimental Example
3-1 normal
Control group Sterile distilled water Single oral administration of sterile distilled water 10 mg / kg compare
Experimental Example
3-2 HE
Control group - sterile distilled water
- HCl / EtOH mixture Sterilized distilled water 10 mg / kg was administered once orally, and after 1 hour, 5 ml / kg of HCl / EtOH mixture compare
Experimental Example
3-3 RA
Administered group - RA
- HCl / EtOH mixture RA 100 mg / kg was dissolved in sterilized distilled water to give a single oral dose of 10 ml / kg, followed by oral administration of HCl / EtOH mixture 5 ml / kg after 1 hour Experimental Example
3-1 FBe300
Administered group - FBE 300 mg / kg
- HCl / EtOH mixture FBe 300 ml / kg was dissolved in sterilized distilled water to give a single oral dose of 10 ml / kg and after 1 hour, 5 ml / kg of HCl / EtOH mixture Experimental Example
3-2 FBe200
Administered group - FBE 200 mg / kg
- HCl / EtOH mixture 200 mg / kg of FBe was dissolved in sterilized distilled water to give a single oral dose of 10 ml / kg. After 1 hour, 5 ml / kg of HCl / EtOH mixture Experimental Example
3-3 FBe100
Administered group - FBE 100 mg / kg
- HCl / EtOH mixture A single oral dose of 10 ml / kg of FBe 100 mg / kg was dissolved in sterile distilled water and after 1 hour, 5 ml / kg of HCl / EtOH mixture

FIG. 1 is a schematic diagram showing a series of procedures for observing the gastric mucosal protection effect of each experimental group after administering the test substances prescribed in each experimental group to the mice to be tested.

As shown in Fig. 1, 6-week-old ICR male mice, which were experimental animals, were purified for 10 days, then fasted for 24 hours, and then grouped into 6 groups of 8 mice per group.

Thereafter, according to each test group and administration method as shown in Table 7, a mixture of HCl / EtOH, which is a substance inducing gastric mucosal injury, was administered to each test substance.

Comparative Experimental Example 3-1 is a normal control group to which 10 mg / kg of sterilized distilled water was orally administered.

Comparative Experimental Example 3-2 is a HE control in which 10 mg / kg of sterilized distilled water was administered orally once, and then the HCl / EtOH mixture was orally administered after 1 hour.

Comparative Experimental Example 3-3 is a RA-administered group in which 100 mg / kg of RA was dissolved in sterilized distilled water, and a single oral administration was performed at a dose of 10 ml / kg, followed by oral administration of the HCl / EtOH mixture after 1 hour.

Experimental Examples 3-1 to 3-3 were prepared by dissolving FBE 300 mg / kg, 200 mg / kg and 100 mg / kg in sterilized distilled water, respectively, in a single oral dose at a dose of 10 ml / kg, FBe300, FBe300 and FBe300 administered orally.

1-2. Observation of gastric mucosal protective effect

In Experiment 1-1, HCl / EtOH mixture was administered to each experimental group, and gastric mucosal protection effect was observed in each experimental group after 1 hour. The gastric mucosal protective effect was evaluated by changes in gastric mucosal lesions, changes in lipid peroxidation in gastric tissues, changes in CAT (catalase) activity in stomach tissues, changes in SOD (superoxide dismutase) activity in stomach tissues, And histopathologic changes.

end. Changes in gastric mucosal lesions

Figure 2 shows the changes of gastric mucosal lesions in each experimental group (F: Fundus regions of stomach; PY: Pylorus regions of stomach, Scale bars = 5 mm).

FIG. 3 is a graph showing the Gross Lesion Area of each experimental group. The area indicates the area where the hemorrhagic gastric mucosa lesion was observed, and the average area observed in 8 individuals included in each experimental group . In FIG. 3, ^a p < 0.01 is a value compared to a normal control by MW (Mann-Whitney U), and ^b p < 0.01 is a value compared to HE control by MW.

2, hemorrhagic ulcerative findings were scattered throughout the gastric mucosa in experimental groups that caused gastric mucosal damage with all HCl / EtOH mixtures, such as HE control, RA treatment, FBe 300 treatment, FBe 200 treatment and FBe 100 treatment groups (PU: Pylorus regions of stomach, Scale bars = 5 mm).

3, there was a significant (p <0.01) increase in the visual field area in the HE control group compared to the normal control group. However, in the FBe300, FBe200, and FBe100 groups, <0.01 or p <0.05), respectively.

Compared with the HE control group, the RA treated group showed a similar decrease (p <0.01) in the area of the visceral ulcer lesion similar to that of the FBe200 treated group.

The gastric mucocutaneous lesion area showed 4098.56% point change in HE control group compared to HE control group, but it was -39.47, -52.52, -39.23 compared with HE control group in RA, FBe 300, FBe 200 and FBe 100 treatment groups And -28.78% point, respectively.

I. Changes in lipid peroxidation in stomach tissues

Table 8 below shows the changes in lipid peroxidation (gastric tissue MDA (malondialdehyde) content) in gastric tissues of normal control or HE-treated experimental groups, such as HE control, RA treatment group, FBe300 treatment group, FBe200 treatment group and FBe100 treatment group, Changes in CAT (catalase) activity, changes in superoxide dismutase (SOD) activity in stomach tissues, and changes in nitrite / nitrite content in stomach tissues. Here, the change of the antioxidant system through the increase of the lipid peroxidation, the change of the CAT and the SOD activity can be seen. For example, when the activity of endogenous antioxidants SOD and CAT decreases, free radicals accumulate. These free radicals cause damage to the gastric mucosa (Saravanan et al ., 2003) It is known to destroy tissue (Freeman and Carpo, 1982; Comporti, 1985)

Lipid peroxidation
(Tissue MDA concentration, nM of MDA / g tissue) Nitrate / nitrite content (M / g protein) Enzyme activity Catalase
(mM / min / g tissue) SOD
(mM / min / g tissue) Normal control group 2.16 ± 0.70 2.74 ± 0.75 624.25 + - 123.89 731.50 ± 146.04 HE control 6.70 + 0.90 ^a 1.06 + - 0.25 ^e 265.63 + - 53.87 ^a 326.75 ± 67.09 ^a RA-treated group 3.68 ± 0.68 ^ac 1.79 ± 0.26 ^fg 391.63 ± 50.81 ^ac 465.75 ± 50.70 ^ac FBe300 treated group 2.89 ± 0.68 ^c 3.32 ± 0.61 ^g 638.38 ± 117.29 ^c 624.75 ± 111.80 ^bc FBe200 treated group 3.81 ± 0.87 ^ac 1.77 ± 0.24 e ^g 386.25 + - 71.39 ^ac 465.38 ± 55.73 ^ac FBe100 treated group 4.92 + 0.63 ^ac 1.47 ± 0.28 ^eh 350.00 ± 45.28 ^ad 428.25 ± 43.11 ^ad

^a p < 0.01 and ^b p < 0.05 are values compared with the normal control group by LSD (Least-significant differences multi-comparison).

^c p < 0.01 and ^d p < 0.05 are values compared with HE control by LSD.

^e p < 0.01 and ^f p < 0.05 are values compared with normal control by MW (Mann-Whitney U).

^g p < 0.01 and ^h p < 0.05 are values compared with HE control by MW.

Referring to Table 8, an increase in MDA (malondialdehyde) content in the stomach tissue, that is, an increase in lipid peroxidation, was observed in the HE control group as compared with the normal control group (p <0.01). In addition, the MDA content in stomach tissues was significantly decreased (p <0.01) in RA, FBe300, FBe200, and FBe100 groups compared to HE control group. Compared with HE control group, RA 100 mg / kg group A significant (p <0.01) decrease in lipid peroxidation in the stomach tissues was observed similar to that in the FBe 200 mg / kg group.

Lipid peroxidation in the stomach tissues showed 210.43% point change compared to the normal control in the HE control group, but was -45.02 in RA 100 mg / kg, FBe 300, 200 and 100 mg / 56.92, -43.09 and -26.61%, respectively.

All. Changes in gastric CAT activity

As shown in Table 8, in the HE control group, CAT activity in the stomach tissues was significantly decreased (p <0.01) as compared with the normal control group. In the FBe300, FBe200 and FBe100 treated groups, (p <0.05) increased CAT activity in the stomach tissues. In comparison with the HE control group, RA-treated group also showed an increase in CAT activity in stomach tissues similar to that of FBe200 I could confirm.

The CAT activity in gastric tissues was -61.24% in the HE group compared with the control group. In the RA, FBe300, FBe200 and FBe100 groups, the CAT activities were 68.40, 212.97, 67.22 and 38.44% Change has appeared.

la. Changes in SOD (superoxide dismutase) activity in stomach tissues

As shown in Table 8, in the HE control group, SOD activity was decreased in the stomach tissues (p <0.01) compared to the normal control group, but in the FBe300, FBe200 and FBe100 treated groups, 0.01 or p <0.05), the increase in SOD activity in the stomach tissues was dose-dependent. Compared with the HE control group, the RA-treated group had similar SOD activity (p <0.01) Increase in the number of workers.

SOD activity in the stomach tissues was -57.45% in the HE group compared to the normal control group. However, in the RA, FBe300, FBe200 and FBe100 groups, the SOD activity was 47.44, 140.33, 45.41 and 31.76% .

In conclusion, the FBe300, FBe200 and FBe100 treated groups significantly inhibited the inhibition of the antioxidant defense system by HCl / EtOH, that is, the increase of lipid peroxidation and the decrease of SOD and CAT activity. Therefore, it can be seen that the gastric mucosal protective effect by FBe is based on the antioxidative effect. In particular, the FBe200-treated group showed similar antioxidative effects to the RA-treated group in the HCl / EtOH-induced gastric mucosal injury mice.

hemp. Changes in Nitrate / nitrite Content in Stomach Tissue

Nitric oxide (NO) is one of the typical mediators responsible for the defense mechanism of the gastrointestinal mucosa. It is produced by NO-synthase and is known to be an important factor controlling the blood flow and microcirculation in stomach. Currently, drugs that stimulate the secretion of NO are effective in ethanol-induced gastric ulcers, while drugs that inhibit the secretion of NO are known to increase gastric mucosal sensitivity to ethanol. NO is an antioxidant that inhibits lipid peroxidation .

Thus, the nitrate / nitrite content of the gastric mucosa indicates the secretion of NO, which is responsible for the defense mechanism of the gastrointestinal mucosa (Morais et al ., "Protective effect of anacardic acids from cashew (Anacardium occidentale) Chem Biol Interact, vol.183 (2010), p264-9).

As shown in Table 8, nitrate / nitrite content in the stomach tissues was significantly decreased (p <0.01) in the HE control group than in the HE control group. However, in the FBe300, FBe200 and FBe100 treated groups, <0.01 or p <0.05). The increase in nitrate / nitrite content in the stomach tissues was dose-dependent. Compared with the HE control group, the RA-treated group also had significantly similar (p <0.01) And nitrate / nitrite contents were increased.

The concentration of nitrate / nitrite in the stomach tissues was 55.33% in the HE group compared to the control group, but 42.54, 91.20, 42.43 and 31.06% in the RA, FBe300, FBe200 and FBe100 groups Change has appeared.

bar. Histopathological change

Table 9 below shows Histomorphometric Analysis of Fundus from normal control or HE treated experimental groups, e.g., RA, FBe300, FBe200 and FBe100 treated groups.

Figure 4 is a representative histological image of the fundic mucosa obtained from normal control or HE treated experimental groups, such as RA, FBe300, FBe200 and FBe100 treated groups (MM: Muscularis mucosa; LU: Lumen; SM: Submucosa, ML: Muscular layer, MU: Mucosa layer, Scale bars = 180 μm).

division Histomorphometry Invasion rate of gastric mucosal injury (%) ^{Note 1)} Average mucosal thickness around the ulcer (m) Semi-quantitative score ^{Note 2)} Normal control group 1.29 ± 1.17 748.72 ± 126.79 0.50 ± 0.53 HE control 71.43 + - 11.96 ^e 304.81 ± 58.72 ^a 2.63 ± 0.52 ^a RA-treated group 28.39 ± 7.18 ^ef 524.57 ± 82.22 ^ac 1.50 + 0.76 ^ac FBe300 treated group 18.97 ± 5.90 ^ef 604.91 + - 89.74 ^ac 1.25 ± 0.46 ^bc FBe200 treated group 29.86 ± 10.03 ^ef 522.83 + 51.24 ^ac 1.63 ± 0.52 ^ac FBe100 treated group 45.71 ± 10.7 ^ef 411.43 ± 51.14 ^ad 1.88 ± 0.64 ^ad

Note 1) Invaded percentages of lesions,%

= (Depth of ulcerative lesion in gastric mucosa / thickness of total gastric mucosa) x 100

Note 2) Semiquantative scores are divided into four grades:

0: Normal

1: mucosal surface damage

2: mucous membrane severity damage

3: Complete mucosal damage

^a p < 0.01 and ^b p < 0.05 are values compared with the normal control group by LSD.

^c p < 0.01 and ^d p < 0.05 are values compared with HE control by LSD.

^e p <0.01 was compared with MW (Mann-Whitney U) compared with the normal control group.

^f p < 0.01 is a value compared with HE control by MW.

Referring to Table 9 and FIG. 4, typical hemorrhagic gastric ulcer symptoms such as mucosal dislocation, necrosis and hyperemia were observed in the HE control group, and the invasion rate of gastric mucosal damage, (P <0.01 or p <0.05) in the histopathologic hemorrhagic gastric ulcer was significantly inhibited in RA, FBe300, FBe200, and FBe100 treated groups, while FBe300, FBe200 and FBe100 There was a clear dose-dependent rate of invasion of gastric mucosal damage, a decrease in mean mucosal thickness around the ulcer and a semi-quantitative score in the treated group.

The histopathologic and semi-quantitative scores on gastric mucosal damage incidence, mean peri-ulcerative mucosa thickness (μm) and gastric mucosal damage were 5453.35, -59.29 and 425.00 % point. However, in RA, FBe300, FBe200 and FBe100 treated groups, the invasion rate of gastric mucosal damage was -60.26, -73.44, -58.19 and -36.01% 98.45, 71.53, and 34.98% points, respectively, and histopathologic semiquantitative scores of -42.86, -52.38, -38.10, and -28.57% points were obtained, respectively.

Experimental Example 4: Animal Experiments - Indomethacin-Induced Gastric Mucosa Injury in Rats

4-1. Experimental animals and experimental group

The experimental animals were purchased from Orient Bio (Seongnam), 6 weeks old SD male rats, fasted for 10 days, fasted for 24 hours, and then weighed (average 258.58 ± 15.62 g , 229.00 ~ 283.00g) were used for the experiment.

IND (Sigma-Aldrich, USA) was used as an inducer of gastric mucosal injury. OM (Omeprazole, Sigma-Aldrich, USA), a therapeutic agent for ulcers, Were used. Here, each test substance (OM, FBe300, FBe200 and FBe100) was dissolved in sterilized distilled water and used at a dose of 5 ml / kg.

Experimental group Experimental material Method of administration Comparative Experimental Example
4-1 normal
Control group Sterile distilled water Sterilized distilled water 5 ml / kg twice at intervals of 30 minutes Comparative Experimental Example
4-2 IND
Control group - sterile distilled water
-IND 5 ml / kg of sterilized distilled water was administered in a single oral administration, and 30 minutes later, IND 25 mg / kg orally Comparative Experimental Example
4-3 OM
Administered group -OM
-IND OM 10 mg / kg was dissolved in sterilized distilled water and administered once at a dose of 5 ml / kg. After 30 minutes, 25 mg of IND was orally administered Experimental Example
4-1 FBe300
Administered group -FBe 300 mg / kg
-IND FBe 300 mg / kg was dissolved in sterilized distilled water and was administered once at a dose of 5 ml / kg. After 30 minutes, IND 25 mg / kg orally Experimental Example
2-2 FBe200
Administered group -FBe 200 mg / kg
-IND FBe 200 mg / kg was dissolved in sterilized distilled water and administered once at a dose of 5 ml / kg. After 30 minutes, IND 25 mg / kg orally Experimental Example
2-3 FBe100
Administered group -FBe 100 mg / kg
-IND FBe 100 mg / kg was dissolved in sterilized distilled water and administered once at a dose of 5 ml / kg. After 30 minutes, IND 25 mg / kg orally

FIG. 5 is a schematic diagram showing a series of procedures for observing the gastric mucosal protective effect of each experimental group after administering the prescribed test substances to the experimental rats.

As shown in FIG. 5, 6-week-old SD male rats were fasted for 10 days and then fasted for 24 hours. Eight mice per group were selected as a total of six groups.

Then, each experimental material and IND was administered according to each experimental group and administration method as shown in Table 10 above.

Comparative Experimental Example 4-1 is a normal control group in which 5 ml / kg of sterilized distilled water was administered twice at intervals of 30 minutes.

Comparative Experimental Example 4-2 is an IND control group, which is a gastric mucosal injury-induced control group in which 5 ml / kg of sterilized distilled water is administered in a single oral dose and the IND 25 mg / kg is administered orally 30 minutes later.

Comparative Experimental Example 4-3 is OM administration group in which 10 mg / kg of OM was dissolved in sterilized distilled water and single oral administration was performed at a dose of 5 ml / kg and IND was administered at 25 mg / kg after 30 minutes.

In Experimental Examples 4-1 to 4-3, 300 mg / kg, 200 mg / kg and 100 mg / kg of FBe were respectively dissolved in sterilized distilled water to give a single oral dose of 5 ml / kg. After 30 minutes, 25 mg of IND Administered group of FBe300, FBe200 and FBe100.

2-2. Observation of gastric mucosal protective effect

In the above 2-1, IND was administered to each experimental group, and 6 hours later, gastric mucosal protection effect was observed in each experimental group. The gastric mucosal protective effect was assessed by the changes of gastric mucosal lesions, changes of MPO (Myeloperoxidase) content in the stomach, changes in lipid peroxidation in gastric tissues, changes in GSH (Glutathione) content in the stomach tissues, Changes in SOD activity and histopathological changes in stomach tissues were observed.

end. Changes in gastric mucosal lesions

FIG. 6 is a photograph showing the change of gastric mucosal lesion 6 hours after administration of IND in each experimental group (CA: PU: Pylorus regions of stomach, Scale bars = 11 mm).

Referring to FIG. 6, hemorrhagic ulcer findings were scattered throughout the gastric mucosa in all IND-induced gastric mucosal injury experimental groups.

FIG. 7 is a graph showing the gross lesion area of each experimental group. The area represents the area where hemorrhagic gastric mucosal lesion was observed, and the average area observed in 8 individuals included in each experimental group do. In FIG. 7, ^a p < 0.01 is a value compared to a normal control group by MW, and ^b p < 0.01 is a value compared with an IND control by MW.

7, there was a significant (p <0.01) increase in the visual field area in the IND control group as compared with the normal control group. However, in the FBe300, FBe200, and FBe100 treated groups, The reduction of visual lesion area was dose-dependent. Compared with the IND control group, OM-treated group also showed a similar decrease (p <0.01) of visual ulcer lesion area as the FBe200 treated group.

In the OM, FBe300, FBe200 and FBe100 treated groups, the gastric mucosal lesion area showed a difference of -45.29, -67.29, -43.94 and -28.35% points compared with the IND control group, Change.

I. Changes in MPO (Myeloperoxidase) content in stomach tissue

MPO is an enzyme secreted by neutrophil. Increasing the activity of MPO in the gastric mucosal tissues is used as an indicator of the increase of neutrophilic leukocytes in the gastric mucosa during various gastric mucosal damage (Konaka et al . , 2005), and increased MPO activity due to neutrophilic leukocyte infiltration in the ulcer lesions caused by gastric mucosal damage by NSAIDs (Whittle, 2004).

FIG. 8 is ^a graph showing the MPO content after 6 hours of administration of IND in each experimental group, where ^a p < 0.01 is a value compared to the normal control by MW, ^b p < to be.

8, there was a significant increase in the MPO content in the IND control group (p <0.01) compared to the inactivation control group. However, in the FBe300, FBe200, and FBe100 treated groups, <0.01). The decrease of MDA content in stomach tissue was dose-dependent. In addition, compared with the IND control group, the OM-treated group showed a similar (p < 0.01) decrease in the MPO content in the stomach tissues as the FBe200 treated group.

MPO content in the stomach tissues showed a 401.32% point change in the IND control group compared to the control group, but -43.94, -67.51, -43.57 and -28.56% points in the OM, FBe300, FBe200 and FBe100- Change has appeared.

All. Changes in lipid peroxidation in stomach tissues

Table 11 shows changes in lipid peroxidation in gastric tissues, changes in GSH content in stomach tissues, changes in CAT activity in stomach tissues of normal control group or IND treated experimental groups such as IND control group, OM administration group, FBe300 administration group, FBe200 administration group and FBe100 administration group And SOD activity in stomach tissues.

The change in lipid peroxidation was observed with an increase in MDA, the product of lipid peroxidation. The increase in MDA was known to be an important cause of gastric mucosal damage by NSAIDs including IND (Frei et al ., 1991) The toxic products formed are known to destroy surrounding tissues (Freeman and Crapo, 1982; Comporti, 1985).

Lipid peroxidation
(Tissue MDA concentration, nM of MDA / g tissue) GSH content (nM / mg tissue) Enzyme activity Catalase
(mM / min / g tissue) SOD
(mM / min / g tissue) Normal control group 2.73 ± 1.15 5.21 ± 1.34 87.75 ± 17.29 151.50 ± 16.58 IND control group 20.02 ± 3.60 ^a 1.49 ± 0.22 ^d 174.25 + 26.86 ^a 75.75 ± 12.63 ^a OM treated group 12.91 + 2.96 ^ac 2.37 ± 0.42 ^de 126.63 ± 14.92 ^ac 113.88 +/- 10.74 ^ac FBe300 treated group 6.11 + 1.79 ^ac 3.37 ± 0.54 ^de 109.63 ± 16.40 ^bc 131.00 ± 15.15 ^ac FBe200 treated group 13.01 ± 2.16 ^ac 2.56 ± 0.68 ^de 124.75 ± 19.31 ^ac 115.00 ± 11.99 ^ac FBe100 treated group 15.56 ± 1.95 ^ac 2.05 ± 0.34 ^de 142.13 + - 13.42 ^ac 100.38 ± 18.91 ^ac

^a p < 0.01 and ^b p < 0.05 are values compared with the normal control group by LSD.

^c p <0.01 compared with the IND control by LSD.

^d p <0.01 is the value of MW compared with the normal control group.

^e p <0.01 is the value compared to the IND control by MW.

In the IND control group, the increase of the MDA content in the stomach, that is, the increase in the lipid peroxidation, was significant (p <0.01) as compared with the normal medium control group. In the FBe300, FBe200 and FBe100 treatment groups, (P <0.01) decreased lipid peroxidation in the stomach tissues similar to that of the FBe200-treated group, as compared with the IND control group.

Lipid peroxidation in the stomach tissues showed 634.05% point change compared to the normal medium control in the IND control group, but -35.51, -69.50, -35.00 and -22.28% points in the OM, FBe300, FBe200 and FBe100- Respectively.

la. Changes in GSH content in gastric tissues

GSH is a typical endogenous antioxidant, and there is a marked decrease in SOD activity and GSH content when gastric mucosal damage is caused by NSAIDs (Odabasoglu et al ., 2006; S ㆌ leyman et al., 2009). When ROS is formed by IND administration, it is known that SOD, which is a typical endogenous antioxidant enzyme, is activated and GSH is counteracting gastric mucosal damage by ROS (El-Missiry et al ., 2001). Secondary SOD activity Reduction and consumption of GSH are known to cause marked gastric mucosal damage (Robert, 1975; Suleyman et al., 2009).

As shown in Table 9, GSH content in the stomach tissues of the IND control group was significantly (p <0.01) lower than that of the normal control group. However, in the FBe300, FBe200 and FBe100 treated groups, p <0.01). The increase of GSH content in gastric tissues was dose-dependent. In addition, compared with the IND control group, the OM administration group showed an increase in the GSH content in the stomach tissue similar to that of the FBe administration group (p <0.01).

hemp. Changes in gastric CAT activity

As shown in Table 11, CAT activity in stomach tissues showed a -71.39% point change in the IND control group compared to the normal control group. In the OM, FBe300, FBe200 and FBe100 treated groups, 59.31, 125.84 and 71.73 And 37.58% point, respectively.

In the IND control group, CAT activity in the stomach tissues was significantly increased (p <0.01) as compared to the normal medium control group, but in the FBe300, FBe200 and FBe100 treated groups, significant (p <0.01) The decrease in activity was dose-dependent. In addition, compared with the IND control group, the OM-treated group also showed a significant (p < 0.01) decrease in CAT activity in the stomach tissue similar to that of the FBe200-treated group.

CAT activity in stomach tissues showed 98.58% point change compared to the normal control in the IND control group, but was -27.33, -37.09, -28.41 and -18.44% in the OM, FBe300, FBe200 and FBe100 treated groups, respectively Change.

bar. Changes in gastric CAT activity

As shown in Table 11, there was a significant (p <0.01) decrease in SOD activity in the stomach tissues in the IND control group compared to the normal control group, but significant (p <0.01) Increased SOD activity in the tissues.

In particular, in the FBe-treated group, an increase in dose-dependent SOD activity was observed. Compared with the IND control group, the OM treated group showed an increase in SOD activity in the stomach tissue similar to that of the FBe200 treated group.

SOD activity in the stomach tissues showed -50.00% point change compared to the normal medium control group in the IND control group but 50.33, 72.94, 51.81 and 32.51% point change in the OM, FBe300, FBe200 and FBe100 treated groups compared with the IND control group Respectively.

four. Changes in SOD Activity and Histopathological Changes in Stomach Tissue

Table 12 below shows the results of a histomorphometric analysis of the fundus obtained from the normal control group or the IND treated groups.

Figure 9 is a representative histological image of a fundic mucosa from a normal or HE-treated experimental group, such as the IND control, OM treated group, FBe 300, FBe 200 and FBe 100 (MM: Muscularis mucosa; LU: Lumen; SM: Submucosa; ML: Muscular layer; MU: Mucosa layer, Scale bars = 180 μm).

division Histomorphometry Invasion rate of gastric mucosal injury ^{Note 1)} Average mucosal thickness around ulcer Semi-quantitative score ^{Note 2)} Normal control group 4.27 ± 2.87 902.66 ± 131.22 0.38 + - 0.52 IND control group 68.53 ± 12.68 ^e 281.50 ± 87.64 ^a 2.75 + 0.46 ^a OM treated group 26.65 ± 15.66 ^eF 538.88 ± 87.26 ^ab 1.38 ± 0.52 ^ab FBe300 treated group 13.00 ± 5.46 ^f 635.59 +/- 129.75 ^ab 1.13 ± 0.64 ^ab FBe200 treated group 25.63 ± 10.67 ^ff 521.04 ± 90.25 ^ab 1.50 ± 0.53 ^ab FBe100 treated group 32.59 ± 14.44 ^eF 410.09 ± 58.15 ^ac 2.00 ± 0.53 ^ab

Note 1) Invaded percentages of lesions,%

= (Depth of ulcerative lesion in gastric mucosa / thickness of total gastric mucosa) x 100

Note 2) Semiquantative scores are divided into four grades:

0: Normal

1: mucosal surface damage

2: mucous membrane severity damage

3: Complete mucosal damage

^a p <0.01 is the value compared with the normal control by LSD.

^b p < 0.01 and ^c p < 0.05 are the values compared to the IND control by LSD.

^d p <0.01 is the value of MW compared with the normal control group.

^e p <0.01 is the value compared to the IND control by MW.

Referring to Table 12 and FIG. 9, in the IND control, typical gastric ulcer symptoms such as surface mucosal dislocation, necrosis and hyperemia were recognized, and the invasion rate of gastric mucosal damage (p <0.01) Mucosal thickness and semi-quantitative scores were increased, but histopathological gastric ulcer was significantly inhibited (p <0.01 or p <0.05) in OM, FBe300, FBe200 and FBe100 treated groups. In the FBe300, FBe200, and FBe100 groups, the dose-dependent gastric mucosal damage incidence, ulcer surrounding mean mucosal thickness, and semi-quantitative scores were decreased. In the FBe200 group, gastric mucosal protection similar to OM was observed And histopathologically.

In the OM, FBe300, FBe200 and FBe100 treated groups, the invasion rate of gastric mucosal damage was -61.12, -81.04, -62.61 and -52.45%, respectively, compared with the IND control, point.

The average mucosal thickness around the ulcer showed a change of 68.81% point in the IND control group compared to the control group. In the OM, FBe300, FBe200 and FBe100 treated groups, 91.43, 125.78, 85.09 and 45.68% .

The histopathologic and semi-quantitative scores for gastric mucosal damage were 633.33% points in the IND control group compared to the control group. In the OM, FBe300, FBe200 and FBe100 treated groups, 45.45 and -27.27%, respectively.

It will be apparent to those skilled in the art that the present invention is not limited to the embodiments described above and that various changes and modifications may be made without departing from the spirit and scope of the present invention as defined by the appended claims. As shown in FIG.

It will be understood by those skilled in the art that the present invention may be embodied in many other specific forms without departing from the spirit or scope of the invention as defined by the appended claims and their equivalents. .

Claims (20)

Wherein the weight ratio of 1,3-1,4beta glucan and 1,3-1,6beta glucan in the barley fermented product is 5: 1 to 5.7: 1, and the polysaccharide content of the barley fermented product Is from 11 to 14% by weight. delete delete The health functional food for improving or preventing gastric ulcer according to claim 1, wherein the barley fermented product is a fermented barley yeast obtained by fermenting yeast of a barley glycoside. The functional food for improving or preventing gastric ulcer according to claim 1, wherein the barley fermented product is obtained by fermenting a saccharified barley glyceride with yeast and fermenting lactic acid bacteria. Wherein the weight ratio of 1,3-1,4beta glucan and 1,3-1,6beta glucan in the barley fermented product is 5: 1 to 5.7: 1, and the polysaccharide content of the barley fermented product Is from 11 to 14% by weight. delete delete [Claim 7] The pharmaceutical composition for treating or preventing gastric ulcer according to claim 6, wherein the barley fermented product is a fermented barley yeast obtained by fermenting yeast of a barley glycoside. [Claim 7] The pharmaceutical composition for treating or preventing gastric ulcer according to claim 6, wherein the barley fermented product is obtained by fermenting yeast with a saccharified saccharide solution, followed by fermentation with lactic acid bacteria. A yeast fermentation step in which yeast is inoculated into a saccharified barley liquid and fermented; And
And fermenting the fermented product obtained in the yeast fermentation step by inoculating the fermented product with lactic acid bacteria, wherein the fermented product is fermented,
The weight ratio of 1,3-1,4beta glucan and 1,3-1,6beta glucan in the barley fermented product is 5: 1 to 5.7: 1, and the content of the polysaccharide in the barley fermented product is 11-14% Wherein the fermentation product has a gastric mucosal protective activity. delete 12. The method of claim 11,
The above-
Immersing the barley in water and gelatinizing it; And
Adding at least one selected from the group consisting of maltose, amylase and Nuruk to the enriched barley, and saccharifying the barley fermentation product. 12. The method according to claim 11, wherein the yeast is Saccharomyces sp . 12. The method of claim 11, wherein the lactic acid bacterium is selected from the group consisting of Weissella sp. , Lactobacillus sp. , Bifidobacterium sp. , Pediococcus sp. Wherein at least one selected from the group consisting of Leuconostoc sp. And Lactococcus sp. Is produced. 12. The method according to claim 11, wherein the yeast fermentation is performed at 10 to 40 DEG C for 20 to 100 hours. The method of producing a fermented barley according to claim 16, wherein the yeast fermentation is fermentation under static fermentation or 50 rpm or less. [12] The method of claim 11, wherein the lactic acid fermentation is performed at 10 ~ 40 &lt; [deg.] &Gt; C for 20 ~ 80 hours. 19. The method of producing a fermented barley according to claim 18, wherein the lactic acid fermentation is fermentation under static fermentation or 50 rpm or less. delete

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