KR102602347B1 - Natural composition for inhibiting formation of carcinogenic N-nitrosamine in the stomach and promoting production of nitric oxide in the intestine and uses thereof - Google Patents

Abstract

The present invention relates to a natural composition for inhibiting the production of carcinogenic N-nitrosamine in the stomach and promoting the production of nitric oxide in the intestine and its use. The natural composition of the present invention is a natural composition in which sodium nitrite (NaNO ₂ ) or a nitric oxide metabolite is immobilized and stabilized. It contains fermented product and glutathione-containing yeast extract as active ingredients, and S-nitrosoglutathione is produced by gastric juice in the stomach (strong acid conditions), and is delivered to the small or large intestine and then produces nitric oxide at a certain concentration for a certain period of time. It is expected to be widely applied in fields related to the treatment of high blood pressure, diabetes, enteritis, stress bowel syndrome, arthritis, or atopy.

Description

Natural composition for inhibiting formation of carcinogenic N-nitrosamine in the stomach and promoting production of nitric oxide in the intestine and uses thereof}

The present invention relates to a natural composition for inhibiting the production of carcinogenic N-nitrosamine in the stomach and promoting the production of nitric oxide in the intestine and its use. More specifically, it relates to blocking the production of carcinogenic N-nitrosamine in strongly acidic conditions such as the stomach and the intestinal environment. It relates to a natural composition that produces a certain amount of nitric oxide and allows absorption of nitric oxide metabolites in the body.

Nitric oxide (hereinafter referred to as NO) is a gaseous substance produced when NOS (NO synthase), which exists in bacteria, plants, and animals, decomposes arginine (L-arginine), a known amino acid, and is a highly reactive radical ( (Refer to Scheme 1-1), it is hydrophobic and is an unstable substance with chemical properties that are rapidly oxidized and changed into hydrophilic NO metabolites (nitrite ions [NO ₂ ^- ], nitrate ions [NO ₃ ^- ], etc.) (Reaction Scheme 1-2 and 1-3). Because NO is very small and hydrophobic, it can move quickly in aqueous solutions such as blood or body fluids, and in particular, can move through blood vessel barriers, cell membranes, etc. within a given existence time (estimated to be about 1 second or less). there is. In addition, because the NO produced is highly reactive, it reacts with oxygen dissolved in blood or body fluids or oxygen bound to red blood cells, and is transformed into NO metabolites that are water-soluble and have a longer existence time (see Schemes 1-2 and 1-3). By being (oxidized) and dissolved in water-soluble blood, it can move to all parts of the body and be excreted out of the body.

As an NO metabolite, NO ₂ ^- is a hydrophilic and relatively stable ionic substance, and can be dissolved in blood and body fluids and moved to all parts of the body, but cannot pass through organ barriers, blood vessel barriers, and cell membranes. However, NO ₂ ^- is reduced to hydrophobic NO under acidic and oxygen-poor conditions (e.g., venous blood conditions) and passes through organ barriers, blood vessel barriers, and cell membranes, thereby exhibiting NO's unique physiological functions and pharmacological effects (Scheme 2 -1). Additionally, NO ₂ ^- can be reduced to NO by reacting with vitamin C (ascorbic acid/ascorbate, hereinafter referred to as Asc) dissolved in blood or body fluids (see Schemes 2-2 and 2-3).

In the human body, NO is mainly produced in vascular endothelial cells through the process of Scheme 1-1) and is used to regulate the physiological function of relaxing nearby blood vessels, and part of the generated NO is converted to NO ₂ ^- through the process of Scheme 1-2) By being oxidized and dissolved in the blood, it moves to all parts of the body and is reduced to NO through the process of Scheme 2-1) or Scheme 2-2) in places where NO is needed, thereby manifesting the physiological functions and pharmacological effects of NO. NO generated in excess by NOS or NO generated by reduction of excess NO ₂ ^- is oxidized to NO ₃ ^- through the process of Reaction Formula 1-3) and is discharged out of the human body through urine or sweat. Therefore, it can be said that there is no significant human toxicity caused by excessive amounts of NO or NO ₂ ^- . However, cyanosis may appear as red blood cells lose oxygen in the process of Scheme 1-3), but this symptom is quickly resolved as red blood cells combine with oxygen again.

NO generated in Scheme 1-1) or 2-1) reaches the cytoplasm of the target cell and chemically combines with glutathione (hereinafter referred to as GSH) abundantly present in the cytoplasm to form S-nitrosoglutathione ( S- After generating nitrosoglutathione (hereinafter referred to as GSNO), NO is stored inside the cell in the form of GSNO for a certain period of time (see Scheme 3-1). When cells require NO, they combine stored GSNO and GSH to produce NO and glutathione disulfide (hereinafter referred to as GSSG) (see Scheme 3-2). Meanwhile, when GSNO exists in excess inside the cell, the excess GSNO is reduced to GSH and NH ₃ by GSNO reductase (hereinafter referred to as GSNOR), thereby reducing the potential toxicity of NO.

The amount of NO in the human body can be predicted as a total amount by the combined amount of NO ₂ ^- and NO ₃ ^- , but the actual usable NO concentration can be predicted by the concentration of NO in the blood by the concentration of NO ₂ ^- that can be converted to NO. It is known that about 100-500 nM (average 300 nM) of NO ₂ ^- exists in the blood of normal people without disease, but in people with diseases such as diabetes, the concentration of NO ₂ ^- in the blood is sometimes observed to be lower than that of normal people. There are many. There may be several ways to raise the reduced concentration of NO ₂ ^- to the normal range, but it is considered the most reasonable method to maintain the concentration of NO ₂ ^- in the blood within the normal range by consuming NO metabolites through food or food. . In fact, humans and animals maintain their blood NO ₂ ^- concentration in the normal range through food. In addition, when analyzing several reports that studied the effects of NO metabolites in experimental animals, diabetes symptoms appeared in experimental animals when NO metabolite intake was restricted, but on the contrary, when NO metabolites were orally administered to diabetic experimental animals. Diabetes was improved (Kina-Tanada et al ., Diabetologia. 2017, 60(6): 1138-1151).

Humans and laboratory animals cannot directly absorb NO metabolites into the body. When humans ingest NO metabolites, the NO ₂ ^- of the NO metabolites are reduced to NO by the strong acid conditions of gastric juice in the stomach, and then the reduced NO passes through the stomach wall and reaches the blood or body fluids, where it is converted to NO ₂ ^- . Absorption into the body is completed through oxidation (see Scheme 4).

Meanwhile, ingested NO ₃ ^- (another NO metabolite), unlike NO ₂ ^-, is reduced to NO ₂ ^- by NO ₃ ^- reductase (nitrate reductase, hereinafter referred to as NaRase) of bacteria living in the small and large intestines. Next, NO ₂ present in the periplasm between the outer membrane and the cell membrane ^of bacteria is reduced to NO by nitrite reductase (hereinafter referred to as NiRase), some of the reduced NO is absorbed into the body, and some of the NO is Through several stages of reduction, it is converted to N ₂ and eliminated from the intestines (see Scheme 5). Therefore, in order to produce a sufficient amount of NO using NO ₃ ^- , it is necessary to consume a large amount of NO ₃ ^- .

When NO metabolites are taken orally to increase low blood NO (or NO ₂ ^- ) levels, three harmful health problems may occur:

First, due to the strong acid condition of gastric juice, excessive NO ₂ ^- taken orally can be absorbed into the human body in a short period of time, resulting in a rapid decrease in blood pressure, hypoglycemia, and cyanosis.

Second, due to the strong acid condition of gastric juice, orally ingested NO ₂ ^- reacts with secondary amines (hereinafter referred to as R ₂ NH) to form N-nitrosamine ( N -nitrosamine, hereinafter referred to as R ₂ N-NO), a known carcinogen. notation) can be generated (see Scheme 6).

Third, NO ₃ ^- ingested orally cannot be reduced in the stomach, but is reduced to NO in the small and large intestines and absorbed into the body. However, the degree of NO reduction varies depending on the individual's intestinal bacterial environment and eating habits, and the NO ₂ ^- amount is higher than the amount. You must consume a certain amount of NO ₃ ^- . Therefore, a certain amount of effective NO body absorption cannot be expected through NO ₃ ^- intake (see Scheme 5).

Several studies have been conducted to suppress the production of the potential carcinogen N-nitrosamine (R ₂ N-NO) by NO ₂ ^- in the stomach under strongly acidic conditions. Among many studies, one study in particular (Shenoy et al., Cancer Lett. 1992; 65(3): 227-232) found that polyphenol (hereinafter referred to as PhOH) and cysteine (hereinafter referred to as Cys) contained in food ingredients. It was found that glutathione (GSH) containing cysteine (denoted as) and cysteine can bind to NO ₂ ^- more than secondary amine (R ₂ N) and block R ₂ N-NO production (Scheme 7-2) and Scheme 7 See -3)).

Scheme 7-2) and Scheme 7-3) may have an advantage in that they block the production of N-nitrosamine, but NO production from ingested NO ₂ ^- is suppressed, which ultimately makes NO absorption in the body difficult. There may be some bad aspects. However, if PhO-NO shown in Scheme 7-2) or GSNO shown in Scheme 7-3) produces NO in the intestinal environment such as the small and large intestines, NO absorption in the body is possible. In the case of GSNO produced in the stomach and reaching the intestines, it can react with GSH, which is abundant in the small intestine, to generate NO (see Scheme 8). Looking at Scheme 7-3) and Scheme 8-1), if GSH is taken together when taking NO ₂ ^- , the production of N-nitrosamine is blocked in the stomach and NO is produced in the intestine, which ultimately facilitates NO absorption in the body. there is.

In our body, NO metabolites are reduced to NO under oxygen-poor or acidic conditions and regulate blood circulation by dilating blood vessels or promoting capillary formation (Lundberg et al., Nat Rev Drug Discov 2008, 7:156-167). The four important physiological functions of blood circulation can be seen as ① supply of oxygen and nutrients, ② excretion of carbon dioxide and waste products, ③ maintenance of body temperature, and ④ performance of immune function. Therefore, NO can be seen to affect the absorption function of nutrients, influence the excretion function of waste products, and regulate body temperature and immune function. For example, NO can return high blood pressure to normal blood pressure by dilating blood vessels and regulating blood flow (Amaral et al., Redox Biol. 2015, 5:340-346). Therefore, a NO metabolite that can be converted to NO could be developed as a treatment for hypertension with fewer side effects. In addition, several recent research reports have shown the blood sugar reducing effect of NO metabolites (Khalifi et al., Nitric Oxide. 2015, 44:24-30) and the insulin secretion promoting effect of beta cells (Nystrom et al., Free Radic Biol Med. 2012, 53:1017-1023), effect of resolving insulin resistance (Ohtake et al., Nitric Oxide. 2015, 44:31-38), effect of improving diabetes complications (Bahadoran et al., Nutr Metab (Lond). 2015, 12 :16. doi:10.1186/ s12986-015-0013-6), etc. have been reported, and various studies are underway to develop NO metabolites as a treatment for diabetes and diabetic complications (Ghasemi et al., Nitric Oxide. 2017, 70 :9-24). However, when NO metabolites are taken orally for the purpose of treating various diseases, there may be three harmful health problems as mentioned above.

Meanwhile, Korean Patent No. 2256793 discloses 'a method for producing a natural fermentation composition containing fixed nitrogen oxide precursors and a natural fermentation composition prepared by this method', and Korean Patent Publication No. 2019-0084592 discloses ' Although a method for producing a natural fermentation composition that immobilizes nitric oxide in a natural fermentation material and a natural fermentation composition prepared by this method are disclosed, the present invention is used for 'inhibiting the production of carcinogenic N-nitrosamine in the stomach and promoting the production of nitric oxide in the intestine. There is no description regarding ‘natural compositions and their uses’.

The present invention has been derived from the above needs, and the present inventors have developed a mixture of yeast extract containing sodium nitrite and glutathione; Alternatively, a mixture of natural fermented product with immobilized and stabilized nitric oxide metabolites and yeast extract containing glutathione was orally administered to experimental animal models of diabetes, blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, and atopic dermatitis. As a result, sodium nitrite alone Alternatively, the present invention was completed by confirming that the mixture of glutathione-containing yeast extract had an excellent improvement effect on blood sugar, blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, and atopic dermatitis compared to the natural fermentation alone experimental group.

In order to solve the above problem, the present invention provides a natural fermentation product in which sodium nitrite (NaNO ₂ ) or a nitric oxide metabolite is immobilized and stabilized; and glutathione as an active ingredient, and provides a composition that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.

Additionally, the present invention provides a pharmaceutical composition containing the composition as an active ingredient.

Additionally, the present invention provides a health functional food composition containing the composition as an active ingredient.

The composition of the present invention mixes nitric oxide metabolites and glutathione to block the production of carcinogenic N-nitrosamine in the gastric juice of the stomach (strong acid conditions), and produces a certain amount of nitric oxide in the small or large intestine, thereby improving the absorption of nitric oxide metabolites in the body. It has been developed to enable this, and is expected to be widely applied in related fields such as treatment and symptom improvement of high blood pressure, diabetes, enteritis, arthritis, liver disease, and atopy, which are known to be diseases that can be improved by nitric oxide.

Figure 1 shows that in the gastrointestinal environment, nitric oxide metabolites (NO ₂ ^- ) and glutathione (GSH) generate s-nitrosoglutathione (GSNO) by gastric acid to block the production of carcinogens, and in the intestinal environment, nitric oxide ( This diagram describes the process by which nitric oxide metabolites are absorbed into the body by producing NO).
Figure 2 is an absorption spectrum showing that s-nitrosoglutathione (GSNO) is produced from a natural fermentation product containing nitric oxide metabolites (NO ₂ ^- ) and a yeast extract containing glutathione (GSH) under strong acid conditions.
Figure 3 shows that when a diabetic experimental animal model consumes a sample mixed with a natural fermentation product containing nitric oxide metabolites (NO ₂ ^- ) and a yeast extract containing glutathione (GSH), the total amount of nitric oxide metabolites in the blood (NO _x ) increases. This is a result that shows what is being done.
Figure 4 shows the results showing the anti-diabetic effect of reducing fasting blood sugar when a sample of a mixture of natural fermentation product containing nitric oxide metabolites (NO ₂ ^- ) and yeast extract containing glutathione (GSH) was treated in a diabetic experimental animal model for 4 weeks. am.
Figure 5 is a graph analyzing the degree of disease activation according to sample administration in the ulcerative colitis model. *: p<0.05 (compared to control group), #: p<0.05 (lettuce fermentation liquid and lettuce fermentation liquid+GSH experimental group).
Figure 6 is a graph analyzing the change in intestinal length according to sample administration in an ulcerative colitis model. *: p<0.05 (compared to control group), #: p<0.05.
Figure 7 is a graph analyzing the level of TNF-α in serum according to sample administration in an ulcerative colitis model. *: p<0.05 (compared to control group), #: p<0.05.
Figure 8 is a graph analyzing the level of IL-1β in serum according to sample administration in an ulcerative colitis model. *: p<0.05 (compared to control group), #: p<0.05.
Figure 9 is a schematic diagram of an experiment to confirm the effect of improving stressful bowel syndrome. WAS stands for water avoidance stress, and the treated samples are shown in Table 3.
Figure 10 shows the results of measuring the number of stools for each experimental group according to sample administration in the stress bowel syndrome model. (A) shows the average number of stools per day, and (B) shows the accumulated number of stools until the end date of the experiment.
Figure 11 shows the results of measuring blood pressure changes before and after sample administration in a hypertension model.
Figure 12 shows the results of measuring the change in joint edema thickness after sample administration in an arthritis-inducing model using MIA (monosodium iodoacetate). *: p<0.05 (compared to control group).
Figure 13 shows the results of evaluating the arthritis index in an arthritis induction model using MIA. *: p<0.05 (compared to control group).
Figure 14 shows the results of measuring ALT and AST levels in serum after administering samples in an acute liver injury model. *: p<0.05 (compared to control group).
Figure 15 shows the results of analyzing morphological changes in lesions by observing the right ear area after administering a sample in an atopic dermatitis model. *: p<0.05 (compared to control group).
Figure 16 shows the results of measuring the change in thickness of the right ear area after administering a sample in an atopic dermatitis model.
Figure 17 shows the results showing the spleen index at the end of the experiment for each experimental group after administering the sample in the atopic dermatitis model. *: p<0.05 (compared to control group).
Figure 18 shows the results of measuring the level of TNF-α in the serum at the end of the experiment for each experimental group after administering the sample in the atopic dermatitis model. *: p<0.05 (compared to control group).
Figure 19 shows the results of measuring the level of IL-4 in the serum at the end of the experiment for each experimental group after administering the sample in the atopic dermatitis model. *: p<0.05 (compared to control group).

In order to achieve the object of the present invention, the present invention provides a natural fermentation product in which sodium nitrite (NaNO ₂ ) or a nitric oxide metabolite is immobilized and stabilized; and glutathione as an active ingredient, and provides a composition that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.

In the composition according to the present invention that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine, the glutathione may be pure glutathione or an extract containing glutathione, preferably a yeast extract containing glutathione. However, it is not limited to this.

Additionally, in the present invention, the glutathione-containing yeast extract may have a glutathione content of 7 to 15% by weight based on the total weight of the extract, but is not limited thereto. Yeast extract containing glutathione contains not only glutathione but also various other beneficial ingredients for health.

As used herein, the term “glutathione (GSH)” is a small-sized peptide composed of three amino acids: glutamic acid, cysteine, and glycine, and is an endogenous reducing substance produced in plants, animals, fungi, and some bacteria and archaea. In the body or within cells, glutathione prevents cell damage by combining with free radicals, peroxides, toxic substances, and heavy metals. Additionally, GSH can react with nitric oxide (NO) to produce s-nitrosoglutathione (GSNO). GSNO generated through this process delivers NO to where it is needed through blood vessels in the body, stores NO within cells for a certain period of time, and decomposes into NO when needed, thereby demonstrating the pharmacological effects of NO.

In the present invention, the natural fermented product in which the nitric oxide metabolite is immobilized and stabilized is prepared by adding fermentation bacteria to a nitrogen-containing natural product and culturing it at a temperature of 18 to 35°C and a dissolved oxygen concentration of 0.03 to 0.1 mg/L for 2 to 30 days. It may be manufactured by a manufacturing method including a fermentation step, but is not limited thereto, and detailed manufacturing methods can be referred to Korean Patent No. 2129038.

The composition according to the present invention, which inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine, may further include polyphenols in addition to natural fermented products and glutathione in which nitric oxide metabolites are immobilized and stabilized. The polyphenol functions to promote the reduction of nitric oxide metabolites in the natural fermentation product to nitric oxide, but is not limited thereto, and may be resveratrol, quercetin, silymarin, anthocyanin, or catechin acid. The polyphenol may be used in the form of a compound or a powder form of a natural material containing each polyphenol compound (e.g., aronia powder, barley sprout powder, etc.), but is not limited thereto.

The natural fermented product according to the present invention can be fermented by adding 0.01 to 3 parts by weight of fermentation bacteria to 100 parts by weight of the mixture of nitrogen-containing natural product and water, preferably 1:0.5 to 1 of the nitrogen-containing natural product and water. After mixing at a weight ratio of , fermentation may be performed by adding 0.01 to 3 parts by weight of fermentation bacteria based on 100 parts by weight of the mixture, but is not limited thereto.

In addition, the fermentation may be carried out at a temperature of 10 to 40°C, preferably at a temperature of 18 to 35°C, more preferably at a temperature of 20 to 33°C, and even more preferably at a temperature of 25 to 33°C. , but is not limited to this.

Additionally, the fermentation may be performed under dissolved oxygen concentration conditions of 0.01 to 0.1 mg/L, preferably 0.03 to 0.1 mg/L, but is not limited thereto.

Additionally, the fermentation may be performed for 2 to 30 days, but is not limited thereto.

Additionally, the fermentation may be terminated when the chemical oxygen demand is 200 to 700 mg/L, but is not limited thereto.

In addition, the fermentation bacteria are Bacillus sp., Bifidobacterium sp., Enterococcus sp., Lactobacillus sp., and Lactococcus sp. ) or Weissella sp. strains, preferably Bacillus subtilis, B. amyloliquefaciens , B. natto , and Bacillus licheni. Formis ( B. licheniformis ), Bifidobacterium bifidum ( Bifidobacterium infantis ) , Bifidobacterium longum ( B. longum ), Enterococcus faecium ), Enterococcus faecalis ( E. faecalis ), Lactobacillus acidopilus ( Lactobacillus acidopilus ), Lactobacillus alimentarius ( L. alimentarius ), Lactobacillus bulgaricus ( L. bulgaricus ), Lactobacillus casei ( L. casei ), Lactobacillus curvatus ( L. curvatus ), Lactobacillus delbrukii ( L. delbrukii ), Lactobacillus johnsonii ( L. johnsonii ), Lactobacillus farciminus ( L. farciminus ), Lactobacillus Gasseri ( L. gasseri ), Lactobacillus helveticus ( L. helveticus ), Lactobacillus rhamnosus ( L. rhamnosus ), Lactobacillus reuteri ( L. reuteri ), Lactobacillus sakei ( L. sakei ), Lactococcus Any one selected from the group consisting of Lactococcus lactis and Weissella cibaria can be used, preferably Bacillus subtilis with the accession number KCTC12501BP - Chun Hyun Su. However, it is not limited to this.

In addition, the nitrogen-containing natural products are not limited to this, but include lettuce, pork sprouts, spinach, blueberries, dandelions, pomegranates, cabbage, garlic, noni, onions, beans, bean sprouts, mulberry leaves, bitter melon, bokbunja, Houttuynia cordata, aronia, Yulcho, One selected from the group consisting of yellow lotus, hijiki, tangerine, mushrooms, calamus, seaweed, Chinese cabbage, kelp, salmon milt, abalone shell (seokgyeolmyeong), crustacean shell, cricket, apple, mugwort, tangerine, silkworm, rehmannia glutinosa, and sipjeondaebotang. It could be more than that.

The human body's nitric oxide concentration can be predicted by the total amount of nitrite ions (NO ₂ ^- ) and nitrate ions (NO ₃ ^- ), but the actual usable nitric oxide concentration is NO ₂ which can be converted to nitric oxide (NO). ^- NO concentration in blood can be predicted by concentration. As a nitric oxide metabolite, NO ₂ ^- is a hydrophilic and relatively stable ionic substance, and can dissolve in blood and body fluids and move throughout the body, but cannot pass through organ barriers, blood vessel barriers, and cell membranes. However, when NO ₂ ^- is reduced to fat-soluble NO by nitrite reductase or acidic conditions, it can pass through organ barriers, blood vessel barriers, and cell membranes. Therefore, in order for NO ₂ ^- to reach target cells and exert a specific pharmacological effect or to be absorbed into the body from the stomach or intestines, it must be reduced to NO.

If nitric oxide metabolites (especially NO ₂ ^- ) are ingested orally to increase the low concentration of nitric oxide metabolites in the blood or to obtain the pharmacological effects of nitric oxide, the following two harmful health problems may occur. . First, due to the strong acid condition of gastric juice, excessive NO ₂ ^- ingested orally can be absorbed into the body in a short period of time, resulting in a rapid decrease in blood pressure, hypoglycemia, and cyanosis. Second, because of the strong acid conditions of gastric juice, orally ingested NO ₂ ^- can react with secondary amines to produce carcinogenic N-nitrosamines.

The composition according to the present invention produces s-nitrosoglutathione (GSNO) through a chemical reaction between the nitric oxide metabolite (NO ₂ ^- ) contained in the natural fermentation product and the glutathione (GSH) contained in the yeast extract under the strong acid conditions of the stomach. It was developed as a composition that blocks the production of N-nitrosamine (R ₂ N-NO) and generates a certain amount of nitric oxide (NO) in the intestinal environment, enabling absorption of nitric oxide metabolites in the body.

In addition, in the composition according to the present invention, the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized may preferably contain 0.1 to 10 mM based on NO ₂ ^- , but is not limited thereto.

As used herein, the term “nitric oxide metabolite” refers to nitrite (NO ₂ ^- ), R-NO ₂ ^- , R-NO, RO-NO, nitrate (NO ₃ ^- ), R-NO ₃ ^- , GS-NO (or A general term for GSNO), GS-NO ₂ , etc., where R- means one H atom has been removed from the carbon chain of polyphenol, fatty acid, etc., and RO- means that one H atom has been removed from ROH, and in the case of GS-, it means that one H atom has been removed from the cysteine residue of glutathione (GSH). In other words, it means a functional group to which nitric oxide or nitrite ions can bind. Nitric oxide metabolites are converted to nitric oxide under acidic and reducing conditions. In strongly acidic conditions such as the stomach, nitric oxide metabolites are reduced by hydrogen ions (H ⁺ ) to generate nitric oxide, and in weakly acidic conditions such as the intestines (small intestine, large intestine), polyphenols, glutathione, vitamin C, and bacterial nitric oxide reductase are produced. Nitric oxide metabolites can be reduced to nitric oxide by reducing substances such as .

In particular, GSNO, known as a nitric oxide metabolite or nitric oxide conjugate, can safely produce nitric oxide by reacting with glutathione (GSH) secreted by the liver through the bile duct or GSH secreted by intestinal bacteria (Scheme 8-1)). In this way, nitric oxide (NO) generated in the intestinal environment penetrates into the body from the intestinal environment and is oxidized into nitric oxide metabolites (NO ₂ ^- ), reaching body fluids or blood vessels (see Scheme 8-3). The above process is summarized graphically as shown in Figure 1.

The present invention also provides a pharmaceutical composition containing as an active ingredient the composition of the present invention that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.

In the pharmaceutical composition according to the present invention, the composition that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestines includes sodium nitrite (NaNO ₂ ) or a natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized; and glutathione as active ingredients, the details of which are the same as those described above.

The pharmaceutical composition of the present invention may be used for preventing or treating diabetes, high blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, or atopic dermatitis, but is not limited thereto.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.

In addition, the pharmaceutical composition of the present invention can be formulated and used in the form of oral preparations such as granules, tablets, capsules, etc. according to conventional methods, but is not limited thereto.

A natural fermentation product in which the sodium nitrite or nitric oxide metabolite of the present invention is immobilized and stabilized; and glutathione as an active ingredient. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, and acacia gum. , alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Various compounds or mixtures may be mentioned. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include the fermented slug extract with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, and all methods of administration can be expected, but are preferably administered orally or rectally. Not limited.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Specifically, the pharmaceutically effective amount of the pharmaceutical composition containing the natural fermentation product with immobilized and stabilized sodium nitrite or nitric oxide metabolite according to the present invention and the yeast extract containing glutathione may vary depending on the patient's age, gender, and weight. Generally, 2 mg to 4 mg per kg of body weight, preferably 3 mg, is administered every day or every other day, and can be administered in divided doses from once to three times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The present invention also provides a health functional food composition containing as an active ingredient a composition according to the present invention that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.

In the health functional food composition according to the present invention, the composition that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine is sodium nitrite (NaNO ₂ ) or a natural fermented product in which the nitric oxide metabolite is immobilized and stabilized. ; and glutathione as active ingredients, the details of which are the same as those described above.

The health functional food composition of the present invention may be used to prevent or improve diabetes, high blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, or atopic dermatitis, but is not limited thereto.

There are no particular restrictions on the types of health functional foods. Naturally fermented products in which the sodium nitrite or nitric oxide metabolites are immobilized and stabilized; Examples of health functional foods containing a composition containing glutathione as an active ingredient include dairy products including meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, rice cakes, and ice cream. , various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

The health functional food may be manufactured in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, effervescent tablet, and beverage, but is not limited thereto.

A natural fermentation product in which the sodium nitrite or nitric oxide metabolite of the present invention is immobilized and stabilized; When a composition containing glutathione as an active ingredient is used as a food additive, a natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized; and glutathione as active ingredients may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The active ingredient can be used appropriately depending on its purpose of use (prevention or improvement). In general, natural fermentation products in which the sodium nitrite or nitric oxide metabolites of the present invention are immobilized and stabilized during the production of food or beverages; and glutathione as active ingredients are added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health control, it can be used in amounts within a range that poses no safety problems.

The health functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufacturing a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) are preferred. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.). In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages.

In this specification, the term “fermentation broth” is used to mean “a natural fermentation product in which nitric oxide metabolites have been immobilized and stabilized.”

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

1. Experimental method

1-1) Preparation of reagents and samples

Sodium nitrite (NaNO ₂ ), nitrate reductase, hydrochloric acid (HCl), and streptozotocin (hereinafter referred to as STZ) used in the present invention were purchased from Sigma (USA), and were obtained in powder form from 7. Yeast extract containing % to 15% glutathione was purchased from Vision Biocam Co., Ltd. (manufactured in China). A composition sample can be prepared by mixing the powder of glutathione-containing yeast extract with lettuce or garlic fermentation broth manufactured by Human Enos. The sample used in the example of this embodiment was 1 mM sodium nitrite (NaNO ₂ ), 1 mM garlic or lettuce fermentation broth based on the contained nitric oxide metabolite NO ₂ ^- concentration, and based on the contained glutathione (GSH) concentration. It is a mixture of 1 mM yeast extract mixed with the sodium nitrite in a ratio of 1:1, or mixed with the garlic or lettuce fermentation broth in a ratio of 1:1. The lettuce or garlic fermentation broth means concentrating each fermented product prepared through the production method disclosed in Korean Patent No. 2129038 at 20 to 30 ° C. for 24 to 48 hours at a stirring speed of 100 to 300 rpm.

1-2) Measurement of nitric oxide metabolite concentration

The total amount of nitric oxide metabolites contained in fermentation broth or blood (meaning the combined concentration _of ^NO ₂ ^- and NO ₃ ^- , hereinafter referred to as _NO All were reduced to NO ₂ ^- and the total concentration of NO ₂ ^- was measured to determine the total content or concentration of nitric oxide metabolites.

More specifically, in the case of fermentation broth, the total NO ₂ ^- content is determined by first adding nitrate reductase to reduce the fermentation broth for 1 hour, diluting it 1,000 times with distilled water, and centrifuging at 3,000 rpm for 3 minutes to remove insoluble polyphenols and dietary fiber. After removal, 50 ㎕ of the clear supernatant was taken and used as a measurement sample. In the case of blood, the collected blood was centrifuged at 3,000 rpm for 3 minutes to precipitate blood cells, then 40 ㎕ of supernatant plasma was taken and mixed with 10 ㎕ of buffer solution containing nitrate reductase, reacted for 1 hour, and then used as a measurement sample. . Dispense 50 ㎕ of NO ₂ ^- standard solution and 50 ㎕ of measurement sample into each 96-well plate, and add 100 ㎕ of test solution [30% acetic acid solution containing 1% sulfanilamide and 0.1% N -(1-naphthyl)ethylenediamine A solution containing a 60% acetic acid solution mixed in a ratio of 1:2] was added and left at room temperature for 10 minutes to induce a color development reaction. Next, the absorbance value of each well was measured at a wavelength of 570 nm using a multi-plate reader, and then the NO _x concentration was calculated by comparing the absorbance value of the standard solution and the absorbance value of the sample.

1-3) Absorbance analysis

To investigate whether the sample (NO ₂ ^- + GSH) prepared in the above experimental method 1-1) can produce s-nitrosoglutathione (GSNO) in strongly acidic conditions such as the stomach, the sample's unique absorption spectrum (absorption) spectrum) was analyzed. To briefly describe the measurement process, first, 0.1 mM HCl solution was added to the fermentation broth to remove all NO ₂ ^- and then yeast extract containing glutathione (GSH) was mixed to prepare reference cell sample R1. Second, a composition sample containing a mixture of fermentation broth and yeast extract was dissolved in distilled water to prepare sample cell sample S1, and 0.1 mM HCl was added thereto and reacted at room temperature for 5 minutes to prepare sample cell sample S2. Insert R1 into the reference cell of the UV-Vis spectrophotometer, insert S1 or S2 into the sample cell, and then obtain the unique spectrum shown by the S1 and S2 samples at a wavelength of 300 nm to 700 nm for each sample. The generation of GSNO was determined by comparing the maximum absorption wavelength (λ _max ).

1-4) Analysis of the total amount of nitric oxide metabolites in the blood of experimental animals

The 6-week-old male Balb/c mice used in this animal experiment were purchased from Samtaco (Korea) and were used in the experiment after an acclimatization period of one week. The experimental animals were divided into 4 groups of 5 each, with each group being a control group that did not process any samples, an experimental group 1 treated with a fermentation broth sample (NO ₂ ^- 1mM based on concentration), and a yeast extract sample (1mM based on GSH concentration). The samples were divided into Experimental Group 2 treated with (mM) and Experimental Group 3 treated with a mixed sample of fermentation broth and yeast extract (1mM NO ₂ ^- + 1mM GSH). Each sample was administered orally at regular intervals three times a day for three days. On the last day of the experiment, all experimental animals participating in the experiment were anesthetized by inhalation using ether, and then blood was collected from the heart using a syringe. The total amount of nitric oxide metabolites (NO _x ) in the collected blood was calculated using the experimental method described in Experimental Method 1-2) above.

2. Statistical analysis

The results obtained in each experiment were expressed as mean ± standard deviation (SD). The significance between two experimental groups was analyzed through a t-test, and in the case of more than two experimental groups, the significance was analyzed through one-way ANOVA followed by Duncan's post hoc test. If the P-value was not greater than 0.05 (p < 0.05), it was considered statistically significant.

Example 1. Production of GSNO by a mixed sample of fermentation broth containing nitric oxide metabolites and yeast extract containing glutathione under strong acid conditions.

Investigate whether the composition sample (mixed sample of garlic fermentation broth [0.1mM as NO ₂ ^- ] and yeast extract [0.1mM as GSH]) prepared in the experimental method 1-1) described above produces GSNO in strong acid conditions such as the stomach. To this end, an experiment was performed according to the experimental method 1-3) described above, and the results shown in FIG. 2 were obtained.

Looking at the results in Figure 2, under neutral or weak acidic conditions rather than strong acid, the composition sample shows a unique absorption band spectrum (λmax = 354 nm) of NO ₂ ^- , but under strong acid conditions such as stomach acid, a unique absorption band spectrum indicating the production of GSNO ( λmax = 334 nm). What these experimental results mean is that a sample composed of a mixture of fermentation broth containing nitric oxide metabolites and yeast extract containing glutathione produces GSNO in strong acid conditions such as the stomach.

Example 2. Body absorption of nitric oxide metabolites by mixed sample of fermentation broth containing nitric oxide metabolites and yeast extract containing glutathione

When an experimental animal ingests the composition sample (mixed sample of garlic fermentation broth [0.1mM as NO ₂ ^- ] and yeast extract [0.1mM as GSH]) prepared in the experimental method 1-1) described above, nitric oxide metabolites In order to investigate whether it is absorbed into the body, an experiment was performed according to test method 1-4), and the results shown in Figure 3 were obtained.

Looking at the results in Figure 3, the total amount of nitric oxide metabolites ( _NO increased significantly. However, no significant changes in _NOx were observed in the experimental group that consumed samples consisting of only yeast extract. From these results, it can be seen that even if the nitric oxide metabolite is converted to GSNO in the stomach, it is decomposed in the small intestine and the nitric oxide metabolite is eventually absorbed (see Schemes 8-1) and 8-3). In other words, it was found that absorption of nitric oxide metabolites in the body was possible for samples composed of a mixture of fermented broth containing nitric oxide metabolites and yeast extract containing glutathione, just as in samples composed only of fermented broth containing nitric oxide metabolites.

Example 3. Antidiabetic effect by mixed sample of fermentation broth containing nitric oxide metabolite and yeast extract containing glutathione

When an experimental animal ingests the composition sample (a mixed sample of garlic fermentation broth [NO ₂ ^- rosso 0.1mM] and yeast extract [GSH 0.1mM]) prepared in the experimental method 1-1) described above, nitric oxide metabolites It was found that absorption into the body was possible (see Example 2).

In the prior invention (Application No. 10-2021-0035653), the present invention demonstrates that nitric oxide metabolites absorbed into the body have a preventive and improvement effect on high blood pressure, diabetes, enteritis, stress bowel syndrome, arthritis, liver disease, and atopic dermatitis. It seemed.

In 'Example 3' of the present invention, in order to investigate whether nitric oxide metabolites absorbed into the body by ingestion of a sample mixed with nitric oxide-containing fermentation broth and glutathione-containing yeast extract have the pharmacological effects claimed in the prior invention, various pharmacological effects were tested. Among them, diabetes, known as a representative disease, was selected and the anti-diabetic effect of the sample prepared according to experimental method 1-1) was investigated.

In order to achieve the purpose of this experiment, diabetic experimental animals were prepared according to the following experimental method, and then treated three times a day for 4 weeks with samples consisting of only fermentation broth, samples consisting of yeast extract only, and samples consisting of a mixture of fermentation broth and yeast extract. And the anti-diabetic effect of each sample was investigated.

3-1. Production of diabetic experimental animals and administration of samples

Six-week-old male Balb/c mice used as a diabetes experimental animal model were purchased from Samtaco (Korea), underwent an acclimatization period for one week, and were then used for the purpose of the experiment. Diabetic experimental animals were created by injecting the diabetes-inducing substance STZ into the abdominal cavity of the experimental animals. Specifically, diabetes was induced by injecting once into the abdominal cavity of experimental animals 120 mg/kg STZ dissolved in 0.1 M citrate buffer (pH 4.5), and two weeks later, a small amount was injected from the tail vein of the fasting experimental animals. Blood was collected and measured using a Glucotrend™ blood glucose meter from Roche, Germany. Experimental animals with measured blood sugar levels of 200 mg/dL or less were excluded, and fasting blood sugar levels were measured again one week later, and experimental animals whose blood sugar levels remained above 200 mg/dL were selected and used as diabetes test animals.

Diabetic experimental animals were divided into 4 groups of 5 animals each. Each group consisted of a diabetic experimental group that did not treat the sample, experimental group 1 that treated the fermentation broth sample (NO ₂ ^- 1 mM based on concentration), and yeast extract sample (based on GSH concentration). They were divided into experimental group 2, which was treated with 1 mM) and experimental group 3, which was treated with a mixed sample of fermentation broth and yeast extract (1mM NO ₂ ^- + 1mM GSH). Each sample was administered orally at regular intervals three times a day for 4 weeks. After fasting for 16 hours every week, a small amount of blood was collected from the tail vein of the diabetic experimental animals, and fasting blood glucose was measured using a Glucotrend blood glucose meter.

Additionally, on the last day of the experiment, all experimental animals participating in the experiment were anesthetized by inhalation using ether, and then blood was collected from the heart using a syringe. The total amount of nitric oxide metabolites (NO _x ) in the collected blood was calculated using the experimental method described in Experimental Method 1-2) above.

3-2. Anti-diabetic effect analysis

Looking at the results in Figure 4, no significant change in fasting blood sugar was observed in the experimental group that consumed a sample composed of only yeast extract, but the experimental group that consumed a sample composed of only fermented broth and the experimental group that consumed a sample composed of a mixture of fermented broth and yeast extract It was confirmed that fasting blood sugar level was significantly decreased at 4 weeks compared to the control group that did not process the sample. In addition, it was observed that the fasting blood sugar of the experimental group that consumed a sample composed of a mixture of fermented broth and yeast extract was significantly lower than the fasting blood sugar of the experimental group that consumed a sample composed of only the fermented broth. From these results, it was found that the sample consisting of a mixture of fermentation broth and yeast extract had excellent anti-diabetic effect and was more effective than the fermentation broth alone.

Example 4. Effect of improving enteritis by a mixed sample of fermented broth containing nitric oxide metabolites and yeast extract containing glutathione

The experimental animals used in this experiment were 6-week-old male Balb/c mice purchased from Samtaco (Korea) and used after an acclimatization period of one week. The average weight of Balb/c mice was 21.40 ± 1.20 g, and samples were administered orally once daily for 3 weeks. In the experimental animal breeding room, light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23±2°C and humidity at 50-60%.

4-1. Group composition and induction of ulcerative colitis

The experimental animals were divided into 6 groups: a normal group treated with no treatment and administered distilled water (DW), a control group induced with enteritis and administered DW, a group induced with enteritis and administered 1mM sodium nitrite (NaNO ₂ ), and a group treated with enteritis. They were classified into a group that induced enteritis and administered 1mM of fermented lettuce liquid, a group that induced enteritis and administered 1mM NaNO ₂ + glutathione-containing yeast extract (hereinafter referred to as GSH), and a group that induced enteritis and administered 1mM of fermented lettuce liquid + GSH (Table 1). Ulcerative colitis was induced by freely ingesting 3.0% DSS (Dextran sulfate sodium) for 5 days in all experimental groups except the normal group. To calculate the number of animals in a group, the minimum number that determines statistical significance was used in accordance with the 3R principle.

Ulcerative colitis experimental group composition Group DSS ¹⁾ Material Dose n One Jeongsang-gun No distilled water - 5 2 control group Yes distilled water - 5 3 NaNO ₂ Yes Sodium nitrite 1mM 5 4 lettuce fermentation liquid Yes Lettuce fermented extract 1mM 5 5 NaNO ₂ + GSH ²⁾ Yes Sodium nitrite + GSH 1mM 5 6 Lettuce fermentation liquid + GSH Yes Lettuce fermented extract + GSH 1mM 5 ¹⁾ DSS: Dextran sulfate sodium
²⁾ GSH: Yeast extract containing glutathione

4-2. Measurement of disease activation level

Intestinal disease is measured by signs such as weight loss, diarrhea accompanied by bleeding and mucus, and shortening of the colon. Previous studies have shown that there are three main clinical signs of disease activity index (DAI): weight loss, diarrhea, and rectal bleeding. Weight loss is calculated as the difference between initial and current weight. Diarrhea is defined as the presence of persistent soft stool in the rectum without fecal pellet formation. Rectal bleeding is distinct from bloody diarrhea, total rectal bleeding, or other diarrhea. In the present invention, DAI was calculated as follows.

DAI = (weight loss score) + (diarrhea score) + (rectal bleeding score)

The medical variables used here are comprehensive functional measures that are similar to the medical symptoms that occur when ulcerative colitis occurs in humans (Table 2).

Disease activity index Score weight loss (%) Diarrhea Rectal bleeding 0 none None Normal One 1-5 Mild Occult 2 6-10 3 11-15 4 16-20 5 >20 Severe watery Gross bleeding

4-3. Measurement of intestinal length and cytokines in serum

After the experiment was completed for all experimental groups, blood was collected from the orbital vein under respiratory anesthesia, serum was separated, the abdomen was incised, the intestine was removed from the end to the cecum, and the intestinal length was measured. For cytokines in serum, the production of TNF-α and IL-1β was measured using an ELISA kit (R&D Systems, USA). After adding 50 ㎕ of each ADS (assay diluent solution) to each well with antibodies attached, 50 ㎕ of each sample was added, left at room temperature for 2 hours, and then washed 4 times with 300 ㎕ of washing buffer. After washing, 100 ㎕ of mouse TNF-α and IL-1β antibodies were added to each well and incubated at room temperature for 2 hours, then removed and washed four times. 100 ㎕ of substrate solution was added to each well and left at room temperature for 30 minutes, then 100 ㎕ of reaction stop solution was added, and the absorbance was measured at 450 nm with an ELISA reader (SPECTRA max M2, USA) from Molecular Devices.

4-4. Effect of natural fermentation and glutathione mixture in ulcerative colitis model

The DSS colitis model exhibits clinical signs characterized by weight loss, diarrhea, and bloody stool. The results of measuring the impact on these clinical symptoms using DAI are as follows. No symptoms were measured in the normal group, and the disease activity index increased in all groups except the normal group. It was confirmed that the sample administration group showed an overall decrease compared to the DSS group. In particular, there was a significant difference between the lettuce fermentation liquid administration group and the lettuce fermentation liquid + GSH mixed administration group (Figure 5).

In addition, the colon of mice sacrificed on the 5th day after DSS administration was removed and its length was checked. As a result, the intestine length was longer in all groups administered the sample compared to the control group, and the NaNO ₂ group, the lettuce fermentation solution group, and the NaNO ₂ + GSH mixture were found to be longer than the control group. This was confirmed in the following order: administration group, lettuce fermentation solution + GSH mixed administration group (Figure 6).

In addition, as a result of measuring inflammatory cytokines in the blood in a colitis-induced animal model, the levels of TNF-α and IL-1β, which are indicators of inflammation, decreased in all sample administration groups compared to the control group, especially in the lettuce fermentation solution + GSH mixed administration group. The lowest values were observed (Figures 7 and 8).

Example 5. Effect of improving stressful bowel syndrome by a mixed sample of fermented broth containing nitric oxide metabolites and yeast extract containing glutathione

The experimental animals used in this experiment were 8-week-old female Wistar rats purchased from Samtaco and used after an acclimatization period of one week. The average weight of Wistar rats was 180.5±0.5 g, and the samples were orally administered once a day. In the experimental animal breeding room, light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23±2°C and humidity at 50-60%.

5-1. Force Composition and Water Avoidance Stress (WAS)

To observe changes in colonic motility caused by natural fermentation products containing nitric oxide metabolites and yeast extracts containing glutathione, the remaining groups, excluding the normal group, were filled with water in each WAS tank, placed on an escape platform, and subjected to intermittent stress. Then, the number of stools was measured. was measured. The experiment measures drinking water and feed intake every day while administering each sample. In addition, WAS was conducted for a total of 8 days, divided into 2 sessions of 15 animals per hour, taking into account the intermediate replacement time of 60 minutes each day (Figure 9), and the number of stools was measured.

The group consists of 6 groups with 6 animals in each group: a normal group (No-stress) that was not exposed to WAS, a control group that performed WAS and was stressed, a group that performed WAS and was administered sodium nitrite (NaNO ₂ ), and a group that performed WAS and lettuce fermentation liquid. The administration group consisted of a group administered WAS and a mixture of NaNO ₂ + GSH, and a group administered WAS and a mixture of lettuce fermentation liquid + GSH (Table 3). Exposure to WAS caused diarrhea-like irritable growth syndrome-like symptoms (increased defecation) and increased mucosal immunity, and it was observed whether administration of each sample could reduce these symptoms.

Stress Bowel Syndrome Experimental Group Composition Group WAS Material Dose n One Jeongsang-gun NO distilled water - 6 2 control group Yes distilled water - 6 3 NaNO ₂ Yes Sodium nitrite 1mM 6 4 lettuce fermentation liquid Yes Lettuce fermented extract 1mM 6 5 NaNO ₂ + GSH Yes Sodium nitrite + GSH 1mM 6 6 Lettuce fermentation liquid + GSH Yes Lettuce fermented extract + GSH 1mM 6

5-2. Effect of natural fermentation and glutathione mixture in a stressed bowel syndrome model

The results of measuring the number of stools for each sample when stress is induced through WAS are shown in Figure 10. In all sample administration groups, the number of stools decreased compared to the control group, and the decrease was observed in the following order: NaNO ₂ administration group, NaNO ₂ + GSH mixture administration group, lettuce fermentation liquid administration group, and lettuce fermentation liquid + GSH mixture administration group.

Example 6. Blood pressure improvement effect by mixed sample of fermentation broth containing nitric oxide metabolite and yeast extract containing glutathione

The experimental animals used in this experiment were 7-week-old male Sprague Dawley rats (hereinafter referred to as SD rats) purchased from Samtaco and used after an acclimatization period of one week. The average weight of SD rats was 198.95 ± 2.10 g, and the samples were administered orally before the experiment. In the experimental animal breeding room, light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23±2°C and humidity at 50-60%.

6-1. DOCA-Salt hypertension rat model production and group composition

To create a model of hypertension, DOCA (Deoxycorticosterone acetate; TCI, Japan) was intraperitoneally administered to 8-week-old male SD rats at a concentration of 25 mg/kg twice a week for 4 weeks, and 1% sodium chloride aqueous solution was used as a drinking water to induce hypertension. At the same time, distilled water (DW) was administered to the control group and each sample was administered to the experimental group. To confirm the induction of hypertension, blood pressure was measured using a mouse tail blood pressure system (MK-2000STS, MUROMACHI KIKAI, Japan), and animals with systolic blood pressure of 130 mmHg or higher were selected and used. Blood pressure of experimental animals was measured in real time for 55 minutes per animal by administering samples to each group. After the rat was placed in a correction frame and fixed, a piezo electric pulse sensor and an occlusion cuff were placed in its tail, and the pulse signal connected to the computer was observed at an appropriate level. At this time, the device was operated to observe systolic blood pressure.

The experimental animals were separated into 5 groups. A control group was administered distilled water to a blood pressure model in which DOCA was intraperitoneally administered and fed regular feed and 1% sodium chloride aqueous solution. In the same manner as the control group, DOCA was administered intraperitoneally and fed regular feed and 1% sodium chloride aqueous solution. One blood pressure model was divided into NaNO ₂ administration group, lettuce fermentation liquid administration group, NaNO ₂ + GSH mixture administration group, and lettuce fermentation liquid + GSH mixture administration group (Table 4).

Composition of blood pressure experimental group Group Treatment n DOCA-
injection DOCA dose material Dose One control group ○ 25mg/kg distilled water - 5 2 NaNO ₂ ○ 25mg/kg Sodium nitrite 1mM 5 3 lettuce fermentation liquid ○ 25mg/kg Lettuce fermented extract 1mM 5 4 NaNO ₂ + GSH ○ 25mg/kg Sodium nitrite + GSH 1mM 5 5 Lettuce fermentation liquid + GSH ○ 25mg/kg Lettuce fermented extract + GSH 1mM 5

6-2. Effect of natural ferment and glutathione mixture in DOCA-induced hypertension model

The results of measuring blood pressure before and after administration of each fermentation broth in rats with hypertension induced by DOCA-salt are shown in Figure 11. A decrease in blood pressure was confirmed between 10 and 20 minutes after oral administration of the sample. In particular, a high decrease in blood pressure was observed in the lettuce fermentation liquid and lettuce fermentation liquid + GSH mixed administration groups, and low blood pressure was maintained in the lettuce fermentation liquid administration group. In the GSH mixed administration group, blood pressure increased and then decreased again.

Example 7. Effect of improving arthritis by a mixed sample of fermented broth containing nitric oxide metabolites and yeast extract containing glutathione

The experimental animals used in this experiment were 7-week-old male SD rats purchased from Samtaco and used after an acclimatization period of one week. Samples were administered orally once daily for 4 weeks. In the experimental animal breeding room, light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23±2°C and humidity at 50-60%.

7-1. Causes and composition of arthritis

To induce arthritis, after anesthetizing the experimental animals in each group except the normal group, the area around the left knee of the SD rat was cleanly removed and the osteoarthritis-causing substance Monosodium iodoacetate (MIA) was injected into a 31G insulin syringe (0.25 × 8, BD). Medical-Diabetes Care, USA) was used to administer 50 ㎕ (60-80 mg/㎖) into the left knee joint. When diluting MIA, 0.9% saline was used, and each experimental sample for each experimental group was administered 7 days after MIA injection.

The experimental animals were divided into six groups: a normal group administered distilled water without any treatment, a control group administered distilled water after inducing osteoarthritis through MIA, a group administered 1 mM NaNO ₂ , a group administered 1 mM fermented lettuce solution, and NaNO ₂ + GSH. They were divided into a 1mM administration group and a lettuce fermentation solution + GSH 1mM administration group, and were orally administered for 4 weeks (Table 5). To calculate the number of animals in a group, the minimum number that determines statistical significance was used in accordance with the 3R principle.

Arthritis experimental group composition Group MIA Meterial Dose n One Jeongsang-gun No distilled water - 5 2 control group Yes distilled water - 5 3 NaNO ₂ Yes Sodium nitrite 1mM 5 4 lettuce fermentation liquid Yes Lettuce fermented extract 1mM 5 5 NaNO ₂ + GSH Yes Sodium nitrite + GSH 1mM 5 6 Lettuce fermentation liquid + GSH Yes Lettuce fermented extract + GSH 1mM 5

7-2. Leg swelling thickness measurement

The left knee joint area of the rat was measured to measure changes in leg edema thickness after inducing arthritis through MIA. Leg edema thickness was measured after anesthesia using vernier calipers (Mitutoyo Co., Japan).

7-3. Arthritis index assessment (severity score) measurement

The arthritis index was assessed visually by three experimenters after photographing each animal before sacrificing the animal model. The evaluation was performed independently by three experimenters three times by modifying the method of Kim et al. (Korean J Orient Physiol Pathol, Effects of Acanthopanax senticosus and onion mixture extract on the collagen-induced arthritis in rat model. 2011, 25;1000-1007). Each score was assigned from 1 to 5 points and expressed as the average value (Table 6).

Clinical evaluation of arthritis Rating clinical symptoms One No change 2 Mild erythema of the ankle joint 3 Mild swelling and erythema of the ankle joint 4 Moderate swelling and erythema of the ankle joint through the metatarsal bone 5 Severe swelling and erythema of the ankle joint through the digit

7-4. Measurement of production of inflammatory cytokines and inflammatory mediators in serum

For cytokines in serum, the production of TNF-α and Metallopeptidase-9 (MMP-9) was measured using an ELISA kit (R&D Systems, USA). After adding 50 ㎕ of ADS (assay diluent solution) to each well with antibodies attached, 50 ㎕ of each sample was added, left at room temperature for 2 hours, and then washed 4 times with 300 ㎕ of washing buffer. After washing, 100 ㎕ of mouse TNF-α and MMP-9 antibodies were added to each well and incubated at room temperature for 2 hours, then removed and washed four times. 100 ㎕ of substrate solution was added to each well and left at room temperature for 30 minutes, then 100 ㎕ of reaction stop solution was added, and the absorbance was measured at 450 nm with an ELISA reader (SPECTRA max M2, USA) from Molecular Devices.

7-5. PGE in serum ₂ (Prostaglandin E ₂ ) measurement

The amount of PGE ₂ produced in serum was measured using an ELISA kit. After adding 50 μl of ADS to each well with antibodies attached, 50 μl of sample was added to each well, left at room temperature for 2 hours, and then washed four times with 300 μl of washing buffer. After washing, 50 ㎕ of Proteoglycan E2 conjugate as an antibody was added to each well and incubated at room temperature for 2 hours, then removed and washed four times. 200 ㎕ of substrate solution was added to each well and left at room temperature for 30 minutes, then 100 ㎕ of reaction stop solution was added, and the absorbance was measured at 450 nm using an ELISA reader from Molecular Devices.

7-6. Effect of natural ferment and glutathione mixture in arthritis model

After completion of the experiment, the left knee joint area of each experimental group was observed to confirm morphological changes in the degree of arthritis relief. In the control group treated only with MIA, changes in thickness due to leg edema were confirmed compared to the legs of the normal group that was not treated with anything, and the thickness was confirmed to decrease with sample treatment (FIG. 12).

In addition, as shown in Figure 13, the results of assessing the degree of arthritis in each group for degenerative arthritis in an animal model inducing osteoarthritis through MIA showed that no unusual symptoms such as edema were observed in the normal group that was not injected with MIA, and MIA In the control group (MIA), in which arthritis was induced, symptoms accompanied by edema were observed in the left leg where degenerative arthritis was induced, and it was found that the edema was alleviated by sample treatment.

After the experiment was completed, the serum was separated and the levels of TNF-α, MMP-9, and PGE ₂ , which are major indicators of inflammation, were measured. As a result, the levels of all sample administration groups decreased compared to the control group, and the NaNO ₂ administration group, lettuce fermentation solution, NaNO ₂ + GSH mixed administration group, and lettuce The fermentation broth + GSH mixed administration group showed a significant decrease in that order (Table 7).

Changes in TNF-α, MMP-9, and PGE ₂ according to sample treatment in an arthritis induction model using MIA Measurement Group Jeongsang-gun control group NaNO ₂ lettuce fermentation liquid NaNO ₂
+GSH lettuce fermentation liquid
+GSH TNF-α (pg/mL) 15.42±0.65 65.15±5.45 54.48±2.85 49.65±4.22 50.22±2.15 46.38±2.12 MMP-9 (ng/mL) 0.25±0.03 0.82±0.04 0.58±0.04 0.54±0.07 0.46±0.03 0.45±0.03 PEG ₂ (ng/mL) 0.71±0.03 1.08±0.03 0.97±0.02 0.91±0.03 0.89±0.03 0.85±0.03

Example 8. Liver protection effect by mixed sample of fermentation broth containing nitric oxide metabolites and yeast extract containing glutathione

The experimental animals used in this experiment were 7-week-old male Sprague Dawley rats (SD rats) purchased from Samtaco and used after an acclimatization period of one week. The average weight of SD rats was 210.45 ± 1.65 g. The light and dark were adjusted every 12 hours in the laboratory animal breeding room, and the temperature was maintained at 23 ± 2°C and humidity at 50-60%.

8-1. Causing liver damage and forming a group

In the experimental model of hepatotoxicity induced by carbon tetrachloride (CCl ₄ ), acute liver damage was induced by intraperitoneally administering 0.1 ml per 100 g of body weight as a mixture of CCl ₄ and olive oil in equal amounts, and in the normal group, only olive oil was injected intraperitoneally instead of CCl ₄ . For each group, 1 mM samples were pretreated by oral administration three times every other day before CCl ₄ injection, and the normal group was orally administered only DW (distilled water). To calculate the number of animals in a group, the minimum number that determines statistical significance was used in accordance with the 3R principle.

Composition of CCl _4- induced liver injury experimental group Group CCl ₄ Meterial Dose n One Jeongsang-gun - distilled water - 2 control group o distilled water - 5 3 NaNO ₂ o Sodium nitrite 1mM 5 4 lettuce fermentation liquid o Lettuce fermented extract 1mM 5 5 NaNO ₂ + GSH o Sodium nitrite + GSH 1mM 5 6 Lettuce fermentation liquid + GSH o Lettuce fermented extract + GSH 1mM 5

8-2. Serum collection and serological testing

Rats used in the experiment were fasted for 12 hours, anesthetized with isoflurane, and blood was collected from the abdominal vena cava. Serum was separated by centrifugation at 3,000 rpm for 20 minutes. Blood AST (Aspartic acid transaminase) and ALT (Alanine transaminase) were measured using AST and ALT measurement reagents (Asan pharmaceutical) applying the Reitman-Frankel enzymatic method.

8-3. Effect of natural ferment and glutathione mixture in acute liver injury model

Serum ALT and AST levels are commonly measured clinically as biomarkers for liver health. As a result of measuring ALT and AST after administering each sample in an experimental model for liver toxicity induced by CCl ₄ , serum ALT and AST levels decreased in all sample administration groups compared to the control group, especially in the NaNO ₂ administration group, lettuce fermentation solution administration group, and NaNO ₂ + GSH mixed administration group, lettuce fermentation liquid + GSH mixed administration group showed a decrease in that order (FIG. 14).

Example 9. Effect of improving contact dermatitis by mixed sample of fermentation broth containing nitric oxide metabolite and yeast extract containing glutathione

The experimental animals used in this experiment were 5-week-old male Balb/c mice purchased from Samtaco and used after an acclimatization period of one week. The average weight of Balb/c mice was 20.80 ± 0.82 g, and the samples were orally administered once daily for 12 days. In the experimental animal breeding room, light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23±2°C and humidity at 50-60%.

9-1. DNFB manufacturing and atopic dermatitis induction and group composition

Balb/c mice were acclimatized for a week and then acclimatized for 3 days by removing hair from their backs. DNFB (1-fluoro-2,4-dinitrobenzene; Sigma, USA) reagent was prepared by diluting 0.5% DNFB in a solution of acetone:olive oil mixed at a ratio of 4:1. On the fourth day after hair removal, 40 μl of 0.5% DNFB was applied to the back area for 4 days to induce skin sensitization. On the 12th day after skin sensitization, contact dermatitis was induced by applying 20 ㎕ of 0.3% DNFB to the ears (challenge).

The experimental animals were divided into 6 groups. The group that did not receive any treatment was set as the normal group, the control group in which DNFB was applied to the right ear and DW was administered, the group in which DNFB was applied to the right ear and NaNO ₂ 1mM administered, and the group in which DNFB was applied to the right ear. They were divided into a group administered with 1mM of fermented lettuce liquid, a group administered with DNFB applied to the right ear and administered 1mM NaNO ₂ + GSH, and a group administered with fermented lettuce liquid + 1mM GSH with DNFB applied to the right ear (Table 9). To calculate the number of animals in a group, the minimum number that determines statistical significance was used in accordance with the 3R principle. The normal group (CON) and control group were each administered DW, and the remaining groups were orally administered each sample once a day.

Atopic dermatitis experimental group composition Group Left Right Meterial Dose n One Jeongsang-gun Normal Normal distilled water - 5 2 control group Normal 0.3% DNFB distilled water - 5 3 NaNO ₂ Normal 0.3% DNFB Sodium nitrite 1mM 5 4 lettuce fermentation liquid Normal 0.3% DNFB Lettuce fermented extract 1mM 5 5 NaNO ₂ +GSH Normal 0.3% DNFB Sodium nitrite + GSH 1mM 5 6 Lettuce fermentation liquid + GSH Normal 0.3% DNFB Lettuce fermented extract + GSH 1mM 5

9-2. Visual evaluation

Visual evaluation is a clinical evaluation method commonly used in atopic dermatitis, and is expressed as the total score of each of the five items to measure the severity of atopic dermatitis symptoms. The evaluation items are erythema, pruritus & dry skin, edema & excoriation, erosion, and lichenification. For each item, no symptoms (0 points), It was scored as mild (1 point), moderate (2 points), and severe (3 points), and then totaled to give a score ranging from a minimum of 0 to a maximum of 15.

9-3. Ear swelling and splenic index measurements

Both ears of the mouse were measured to measure changes in skin tissue after application of DNFB. Ear thickness was measured using vernier calipers (Mitutoyo Co.) at intervals of 12 hours, 24 hours, and 48 hours for 3 days.

After measuring the ear thickness, Balb/c mice were sacrificed, their spleens were removed, and their weight was measured. The spleen index was calculated by dividing the spleen weight (mg) of Balb/c mice by body weight (g).

9-4. Effect of natural fermentation in contact dermatitis model

After completion of the experiment, the right ear area where the samples from each experimental group were treated was observed to confirm morphological changes in atopic dermatitis lesions. In the control group treated with only DNFB, atopic skin changes such as erythema, dry skin, maceration, edema, and hematoma were confirmed compared to the left ear that was not treated with anything (Figure 15).

In addition, after the end of the experiment, the morphological changes in atopic dermatitis lesions were confirmed by observing the right ear area where the samples of each experimental group were treated. As a result, the ear thickness decreased in all sample administration groups compared to the control group, and the NaNO ₂ administration group, lettuce fermentation solution administration group, and NaNO A decrease was observed in the order of the ₂ + GSH mixed administration group and the lettuce fermentation liquid + GSH mixed administration group (Figure 16).

In addition, as a result of measuring the spleen weight by body weight for each experimental group after the end of the experiment, the spleen index decreased in all sample administration groups compared to the control group, and the NaNO ₂ administration group, the lettuce fermentation liquid administration group, the NaNO ₂ + GSH mixture administration group, and the lettuce fermentation liquid + GSH mixture administration group A decrease was observed in that order (Figure 17).

In addition, as a result of measuring the levels of TNF-α and IL-4 in serum, the levels of TNF-α and IL-4 were decreased in all sample administration groups compared to the control group. NaNO ₂ administration group, lettuce fermentation fluid administration group, NaNO ₂ + GSH mixture administration group, A decrease was observed in the order of the lettuce fermentation solution + GSH mixed administration group (Figures 18 and 19).

Overall, according to experimental method 1-1), a mixture of yeast extract containing sodium nitrite and glutathione; The composition of the present invention, which consists of a mixture of fermented broth containing nitric oxide and yeast extract containing glutathione, not only has an anti-diabetic effect, but also has a preventive and improving effect on hypertension, enteritis, stress bowel syndrome, arthritis, liver disease, atopic dermatitis, etc., It can be usefully used in related industrial fields.

Claims (8)

Fermented lettuce in which sodium nitrite (NaNO ₂ ) or nitric oxide metabolites are immobilized and stabilized; A pharmaceutical composition for preventing or treating diabetes, high blood pressure, enteritis, stress bowel syndrome, arthritis, acute liver damage, or atopic dermatitis, comprising as an active ingredient yeast extract containing glutathione,
The fermented lettuce product in which the nitric oxide metabolite is immobilized and stabilized is produced by adding fermentation bacteria to lettuce and fermenting for 2 to 30 days at a temperature of 18 to 35 ° C. and a dissolved oxygen concentration of 0.03 to 0.1 mg / L. A pharmaceutical composition, characterized in that it is manufactured by a method. The pharmaceutical composition according to claim 1, wherein the active ingredient inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine. The pharmaceutical composition according to claim 1, wherein the glutathione-containing yeast extract has a glutathione content of 7 to 15% by weight based on the total weight of the extract. delete delete delete Fermented lettuce in which sodium nitrite (NaNO ₂ ) or nitric oxide metabolites are immobilized and stabilized; A health functional food composition for preventing or improving diabetes, high blood pressure, enteritis, stress bowel syndrome, arthritis, acute liver damage, or atopic dermatitis, comprising yeast extract containing glutathione as an active ingredient,
The fermented lettuce product in which the nitric oxide metabolite is immobilized and stabilized is produced by adding fermentation bacteria to lettuce and fermenting for 2 to 30 days at a temperature of 18 to 35 ° C. and a dissolved oxygen concentration of 0.03 to 0.1 mg / L. A health functional food composition manufactured by a method. delete