Composition for inhibiting production of carcinogenic n-nitrosamine in stomach and use thereof

Abstract

The present invention relates to a composition for prevention, alleviation, or treatment of hypertension, hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver damage, or dermatitis, comprising, as active ingredients, a natural fermentate in which nitric oxide metabolites are immobilized and stabilized and at least one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and Dendropanax extract. The composition of the present invention, upon exposure to gastric acid (strong acidic conditions), generates S-nitrosoglutathione, which is subsequently delivered to the small intestine or colon, where it can continuously release nitric oxide at a controlled concentration for a certain period. Thus, the composition can be variously utilized.

Description

Composition for inhibiting gastric carcinogenic N-nitrosamine production and use thereof

[0001] The present invention relates to a composition for inhibiting the production of carcinogenic N-nitrosamines in the stomach and its use, and more particularly, to a composition that blocks the production of carcinogenic N-nitrosamines in a strongly acidic condition such as the stomach and produces a certain amount of nitric oxide in the intestinal environment, thereby enabling the absorption of nitric oxide metabolites in the body.

[0002] Nitric oxide (NO) is a gaseous substance that is produced by NOS (NO synthase) present in bacteria, plants, and animals when it breaks down arginine (L-arginine), an amino acid. It is a highly reactive radical (see reaction formula 1-1). It is hydrophobic and quickly oxidizes to form hydrophilic NO metabolites (nitrite ions [NO2 - ], nitrate ion [NO3 - ] etc.) is an unstable substance with chemical properties that change (see Reaction Schemes 1-2 and 1-3). Because NO is very small in size and hydrophobic, it can move quickly in aqueous solutions such as blood or body fluids, and in particular, it can pass through blood vessel barriers, cell membranes, etc. within a given existence time (estimated to be less than about 1 second). In addition, because the generated NO is highly reactive, it reacts with oxygen dissolved in blood or body fluids or oxygen bound to red blood cells, and is transformed (oxidized) into water-soluble NO metabolites (see Reaction Schemes 1-2 and 1-3) that have a longer existence time and are dissolved in water-soluble blood, so that they can move to all parts of the body and be excreted from the body.

[0003]

[0004] NO2 as a NO metabolite - It is a hydrophilic and relatively stable ionic substance that dissolves in blood and body fluids and can move to all parts of the body, but cannot pass through organ barriers, blood vessel barriers, cell membranes, etc. However, NO2- NO is reduced to hydrophobic NO under acidic conditions and oxygen-deficient conditions [e.g., venous blood conditions], thereby passing through organ barriers, blood vessel barriers, and cell membranes, and exhibiting the unique physiological functions and pharmacological effects of NO (see Scheme 2-1). In addition, NO2 - can be reduced to NO by reacting with vitamin C (ascorbic acid / ascorbate, hereinafter referred to as Asc) dissolved in blood or body fluid (see reaction formulas 2-2 and 2-3).

[0005]

[0006] In the human body, NO is mainly produced in vascular endothelial cells by the process of reaction formula 1-1) and is used to regulate the physiological function of relaxing nearby blood vessels, and some of the produced NO is converted to NO2 by the process of reaction formula 1-2). - By being oxidized and dissolved in the blood, NO moves to all parts of the body and is reduced to NO by the process of Reaction 2-1) or Reaction 2-2) at the place where NO is needed, thereby showing the physiological function and pharmacological effect of NO. NO produced in excess by NOS or excess NO2 - The NO produced in the reduction is NO3 through the process of reaction equation 1-3. - It is excreted from the body through urine or sweat by being oxidized. Therefore, excess NO or NO2 - It can be seen that there is no significant human toxicity due to it. However, cyanosis may appear as red blood cells lose oxygen during the process of reaction 1-3), but this symptom is quickly resolved as the red blood cells re-combine with oxygen.

[0007] The NO generated in Reaction 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 generate S-nitrosoglutathione (hereinafter referred to as GSNO), and then stores NO inside the cell in the form of GSNO for a certain period of time (see Reaction Scheme 3-1)). When the cell needs NO, it combines the stored GSNO with GSH to generate NO and glutathione disulfide (hereinafter referred to as GSSG) (see Reaction Scheme 3-2)). Meanwhile, when GSNO exists in excess inside the cell, the excess GSNO is reduced to GSH and NH3 by GSNO reductase (hereinafter referred to as GSNOR), thereby reducing the potential toxicity of NO.

[0008]

[0009] The amount of NO in the human body is NO2 - Wow NO3 - The total amount can be predicted by the sum of the NO2 that can be converted to NO, but the actual usable NO concentration is - The blood NO concentration can be predicted by the concentration. The blood of a normal person without disease contains about 100-500 nM (average 300 nM) of NO2. - Although it is known that NO2 exists in the blood, in people with diseases such as diabetes, - Concentrations are often observed to be lower than normal. Reduced NO2 - There may be several ways to raise the concentration of NO2 to the normal range, but one is to take NO metabolites through food or food products to increase blood NO2 - Maintaining the concentration within the normal range is considered the most reasonable method. In fact, humans and animals can increase their blood NO2 levels through food. -The concentration is maintained within the normal range. In addition, an analysis of several reports studying the effects of NO metabolites in laboratory animals showed that when the intake of NO metabolites was restricted, diabetic symptoms appeared in the laboratory animals, but conversely, when NO metabolites were orally administered to diabetic laboratory animals, diabetes was improved (Kina-Tanada et al., Diabetologia. 2017, 60(6): 1138-1151).

[0010] Humans and experimental animals cannot directly absorb NO metabolites into their bodies. When humans ingest NO metabolites, the NO2 of the NO metabolites - After being reduced to NO by the strong acid conditions of gastric juice in the stomach, the reduced NO passes through the stomach wall and reaches the blood or body fluid to produce NO2. - Absorption in the body is completed by oxidation (see reaction scheme 4).

[0011]

[0012] Meanwhile, the NO3 consumed - (another NO metabolite) is NO2 - Unlike NO3 from bacteria that live in the small and large intestines - NO2 by nitrate reductase (hereinafter referred to as NaRase) - After being reduced to NO2, which exists in the periplasm between the outer membrane and the cell membrane of the bacteria - It 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 converted to N2 through several reduction steps and eliminated in the intestine (see reaction scheme 5). Therefore, NO3 - To produce sufficient amount of NO using a large amount of NO3 - Consumption is required.

[0013]

[0014] Low blood NO (or NO2)- ) To increase the concentration, taking NO metabolites orally can cause three harmful health problems:

[0015] First, NO2 taken orally - Due to the strong acidity of gastric juice, excessive NO can be absorbed into the body in a short period of time, which can cause rapid decrease in blood pressure, hypoglycemia, and cyanosis.

[0016] Second, NO2 taken orally is absorbed due to the strong acidity of gastric juice. - It can react with a secondary amine (hereinafter referred to as R2N-H) to produce N-nitrosamine (hereinafter referred to as R2N-NO), which is known to be a carcinogen (see reaction scheme 6).

[0017] Third, NO3 taken orally - is not reduced in the stomach but is reduced to NO in the small and large intestines and absorbed into the body, but the degree of NO reduction varies depending on the individual's intestinal bacterial environment and eating habits, and also NO2 - NO3 in greater quantities than the amount - You need to consume NO3. - A certain amount of effective NO absorption in the body cannot be expected through ingestion (see Scheme 5).

[0018]

[0019] NO2 in the stomach, which is under highly acidic conditions - Several studies have been conducted to suppress the production of potential carcinogens, N-nitrosamines (R2N-NO). Among many studies, one study in particular (Shenoy et al., Cancer Lett. 1992; 65(3): 227-232) showed that polyphenols (hereinafter referred to as PhOH), cysteine ​​(hereinafter referred to as Cys), and glutathione (GSH) containing cysteine ​​contained in food ingredients were more effective in producing NO2 than secondary amines (R2N). -It was found that R2N-NO production can be blocked by combining with (see Scheme 7-2) and Scheme 7-3).

[0020]

[0021] Reaction formulas 7-2) and 7-3) may have a positive aspect in that they block the production of N-nitrosamines, but the ingested NO2 - There may be a negative aspect in that NO production is suppressed, and ultimately NO is not well absorbed in the body. However, if PhO-NO as seen in Reaction Scheme 7-2) or GSNO as seen in Reaction 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 produce NO (see Reaction Scheme 8)). Looking at Reaction Schemes 7-3) and 8-1), NO2 - When GSH is taken together with food, the production of N-nitrosamine is blocked in the stomach and NO is produced in the intestines, ultimately allowing for better absorption of NO in the body.

[0022]

[0023] In our body, NO metabolites are reduced to NO under oxygen-deficient or acidic conditions, which dilate blood vessels or promote capillary formation, thereby regulating blood circulation (Lundberg et al., Nat Rev Drug Discov. 2008, 7:156-167). The four major physiological functions of blood circulation can be viewed as ① supplying oxygen and nutrients, ② excreting carbon dioxide and waste products, ③ maintaining body temperature, and ④ performing immune functions. Therefore, NO can be seen to affect the absorption of nutrients, influence the excretion of waste products, and regulate body temperature and immune functions. For example, NO can return high blood pressure to normal by dilating blood vessels and regulating blood flow (Amaral et al., Redox Biol. 2015, 5:340-346). Therefore, NO metabolites that can be converted to NO could be developed as a treatment for hypertension with fewer side effects. In addition, recent research reports have reported the blood sugar-lowering effect of NO metabolites (Khalifi et al., Nitric Oxide. 2015, 44:24-30), the insulin secretion-promoting effect of beta cells (Nystrom et al., Free Radic Biol Med. 2012, 53:1017-1023), the insulin resistance-relieving effect (Ohtake et al., Nitric Oxide. 2015, 44:31-38), and the diabetic complication-improving effect (Bahadoran et al., Nutr Metab (Lond). 2015, 12:16. doi:10.1186 / s12986-015-0013-6), and various studies are being conducted to develop NO metabolites as a treatment for diabetes and diabetic complications (Ghasemi et al., Nitric Oxide. 2017, 70:9-24). However, when taking NO metabolites orally for the purpose of treating various diseases, there may be three health problems as mentioned above.

[0024] Meanwhile, Korean Patent Registration No. 2256793 discloses 'a method for producing a natural fermentation composition containing a fixed nitric oxide precursor and a natural fermentation composition produced by the method', and Korean Publication Patent No. 2019-0084592 discloses 'a method for producing a natural fermentation composition that fixes nitric oxide in a natural fermentation material and a natural fermentation composition produced by the method', but 'a composition for inhibiting the production of carcinogenic N-nitrosamines in the stomach and its use' of the present invention are not described.

[0025] The present invention was derived from the above-mentioned needs, and the inventors of the present invention completed the present invention by orally administering a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, Dendropanax morbifera extract or vitamin B12 (cyanocobalamin) to a natural fermented product in which nitric oxide metabolites are fixed and stabilized, to an experimental animal model for blood pressure, enteritis, stress-related bowel syndrome, arthritis, liver disease and atopic dermatitis, and confirming that the experimental group administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, Dendropanax morbifera extract or vitamin B12 had a superior effect on improving blood pressure, enteritis, stress-related bowel syndrome, arthritis, liver disease and atopic dermatitis compared to the experimental group administered the natural fermented product alone.

[0026] In order to solve the above problem, the present invention provides a composition for preventing, improving or treating hypertension, enteritis, stress-related bowel syndrome, arthritis, acute liver damage or dermatitis, comprising as active ingredients a natural fermented product in which a nitric oxide metabolite is immobilized and stabilized; and any one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract and phellodendron amurense extract.

[0027] The composition of the present invention is a mixture of nitric oxide metabolites and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron amurense extract or vitamin B12, which blocks the production of carcinogenic N-nitrosamines in the gastric juice (strong acid conditions) of the stomach and produces a certain amount of nitric oxide in the small or large intestine to enable absorption of nitric oxide metabolites in the body. It is expected to be widely applied in related fields such as the treatment and symptom improvement of hypertension, enteritis, arthritis, liver disease, atopy, etc., which are known diseases that can be improved by nitric oxide.

[0028] Figure 1 is a graph analyzing the degree of disease activation according to the administration of natural fermented liquid and glutathione samples in an ulcerative colitis model. *: p<0.05 (vs. control group).

[0029] Figure 2 is a graph analyzing the degree of disease activation according to the administration of natural fermented liquid and caffeic acid samples in an ulcerative colitis model. *: p<0.05 (vs. control group).

[0030] Figure 3 is a graph analyzing the degree of disease activation according to the administration of natural fermented liquid and chlorogenic acid samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0031] Figure 4 is a graph analyzing the degree of disease activation according to the administration of natural fermented liquid and coffee extract samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0032] Figure 5 is a graph analyzing the degree of disease activation according to the administration of natural fermented liquid and Hwangchil extract samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0033] Figure 6 is a graph analyzing the degree of disease activation according to the administration of natural fermented liquid and vitamin B12 (cyanocobalamin) samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0034] Figure 7 is a graph analyzing changes in intestinal length according to administration of natural fermented liquid and glutathione samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0035] Figure 8 is a graph analyzing changes in intestinal length according to administration of natural fermented liquid and caffeic acid samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0036] Figure 9 is a graph analyzing changes in intestinal length according to administration of natural fermented liquid and chlorogenic acid samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0037] Figure 10 is a graph analyzing changes in intestinal length according to administration of natural fermented liquid and coffee extract samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0038] Figure 11 is a graph analyzing changes in intestinal length according to administration of natural fermented liquid and Hwangchil extract samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0039] Figure 12 is a graph analyzing changes in intestinal length according to administration of natural fermented liquid and vitamin B12 samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0040] Figure 13 is a graph analyzing the levels of TNF-α(a) and IL-1β(b) in serum following administration of natural fermented liquid and glutathione samples in an ulcerative colitis model. *: p<0.05 (vs. control group).

[0041] Figure 14 is a graph analyzing the serum TNF-α(a) and IL-1β(b) levels according to administration of natural fermented liquid and caffeic acid samples in an ulcerative colitis model. *: p<0.05 (vs. control group).

[0042] Figure 15 is a graph analyzing the levels of TNF-α(a) and IL-1β(b) in serum according to administration of natural fermented liquid and chlorogenic acid samples in an ulcerative colitis model. *: p<0.05 (vs. control group).

[0043] Figure 16 is a graph analyzing the levels of TNF-α(a) and IL-1β(b) in serum according to administration of natural fermented liquid and coffee extract samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0044] Figure 17 is a graph analyzing the levels of TNF-α(a) and IL-1β(b) in serum according to administration of natural fermented liquid and Hwangchil extract samples in an ulcerative colitis model. *: p<0.05 (compared to the control group).

[0045] Figure 18 is a graph analyzing the levels of TNF-α(a) and IL-1β(b) in serum following administration of a natural fermented liquid and vitamin B12 sample in an ulcerative colitis model. *: p<0.05 (vs. control group).

[0046] Figure 19 is a schematic diagram of an experiment to confirm the effect of improving stress-induced bowel syndrome. WAS stands for water avoidance stress.

[0047] Figure 20 shows the results of measuring the number of stools in each experimental group according to the administration of natural fermented liquid and glutathione samples in a stress-induced bowel syndrome model. (a) shows the average number of stools per day, and (b) shows the accumulated number of stools until the end of the experiment.

[0048] Figure 21 shows the results of measuring the number of stools in each experimental group according to the administration of natural fermented liquid and caffeic acid samples in a stress-induced bowel syndrome model. (a) shows the average number of stools per day, and (b) shows the accumulated number of stools until the end of the experiment.

[0049] Figure 22 shows the results of measuring the number of stools in each experimental group according to the administration of natural fermented liquid and chlorogenic acid samples in a stress-induced bowel syndrome model. (a) shows the average number of stools per day, and (b) shows the accumulated number of stools until the end of the experiment.

[0050] Figure 23 shows the results of measuring the number of stools in each experimental group according to the administration of natural fermentation liquid and coffee extract samples in a stress-induced bowel syndrome model. (a) shows the average number of stools per day, and (b) shows the accumulated number of stools until the end of the experiment.

[0051] Figure 24 shows the results of measuring the number of stools by experimental group according to the administration of natural fermented liquid and Hwangchil extract samples in a stress-induced bowel syndrome model. (a) shows the average number of stools per day, and (b) shows the accumulated number of stools until the end of the experiment.

[0052] Figure 25 shows the results of measuring the number of stools in each experimental group according to the administration of natural fermented liquid and vitamin B12 samples in a stress-induced bowel syndrome model. (a) shows the average number of stools per day, and (b) shows the accumulated number of stools until the end of the experiment.

[0053] Figure 26 shows the results of measuring blood pressure changes before and after administration of natural fermented liquid and glutathione samples in a hypertensive model.

[0054] Figure 27 shows the results of measuring blood pressure changes before and after administration of natural fermented liquid and caffeic acid samples in a hypertensive model.

[0055] Figure 28 shows the results of measuring blood pressure changes before and after administration of natural fermentation liquid and chlorogenic acid samples in a hypertensive model.

[0056] Figure 29 shows the results of measuring blood pressure changes before and after administration of natural fermentation liquid and coffee extract samples in a hypertensive model.

[0057] Figure 30 shows the results of measuring blood pressure changes before and after administration of natural fermented liquid and Hwangchil extract samples in a hypertensive model.

[0058] Figure 31 shows the results of measuring blood pressure changes before and after administration of natural fermented liquid and vitamin B12 samples in a hypertensive model.

[0059] Figure 32 shows the results of measuring the change in joint swelling thickness after administration of natural fermentation liquid and glutathione sample in an arthritis-induced model using MIA (monosodium iodoacetate). *: p<0.05 (vs. control group).

[0060] Figure 33 shows the results of measuring the change in joint swelling thickness after administration of natural fermented liquid and caffeic acid samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0061] Figure 34 shows the results of measuring the change in joint swelling thickness after administration of natural fermented liquid and chlorogenic acid samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0062] Figure 35 shows the results of measuring the change in joint swelling thickness after administration of natural fermentation liquid and coffee extract samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0063] Figure 36 shows the results of measuring the change in joint swelling thickness after administration of natural fermented liquid and Hwangchil extract samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0064] Figure 37 shows the results of measuring the change in joint swelling thickness after administration of natural fermented liquid and vitamin B12 samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0065] Figure 38 shows the results of evaluating the arthritis index after administration of natural fermented liquid and glutathione samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0066] Figure 39 shows the results of evaluating the arthritis index after administration of natural fermented liquid and caffeic acid samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0067] Figure 40 shows the results of evaluating the arthritis index after administration of natural fermented liquid and chlorogenic acid samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0068] Figure 41 shows the results of evaluating the arthritis index after administration of natural fermentation liquid and coffee extract samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0069] Figure 42 shows the results of evaluating the arthritis index after administration of natural fermented liquid and Hwangchil extract samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0070] Figure 43 shows the results of evaluating the arthritis index after administration of natural fermented liquid and vitamin B12 samples in an arthritis-induced model using MIA. *: p<0.05 (compared to the control group).

[0071] Figure 44 is a graph analyzing the levels of TNF-α (a), Metallopeptidase-9 (b), and Prostaglandin E2 (PGE2) (c) in serum after administration of natural fermented liquid and glutathione sample in an arthritis-induced model using MIA.

[0072] Figure 45 is a graph analyzing the levels of TNF-α (a), Metallopeptidase-9 (b), and PGE2 (c) in serum after administration of natural fermentation liquid and caffeic acid samples in an arthritis-induced model using MIA.

[0073] Figure 46 is a graph analyzing the levels of TNF-α (a), Metallopeptidase-9 (b), and PGE2 (c) in serum after administration of natural fermentation liquid and chlorogenic acid samples in an arthritis-induced model using MIA.

[0074] Figure 47 is a graph analyzing the levels of TNF-α (a), Metallopeptidase-9 (b), and PGE2 (c) in serum after administration of natural fermentation liquid and coffee extract samples in an arthritis-induced model using MIA.

[0075] Figure 48 is a graph analyzing the levels of TNF-α (a), Metallopeptidase-9 (b), and PGE2 (c) in serum after administration of natural fermented liquid and Hwangchil extract samples in an arthritis-induced model using MIA.

[0076] Figure 49 is a graph analyzing the levels of TNF-α (a), Metallopeptidase-9 (b), and PGE2 (c) in serum after administration of natural fermented liquid and vitamin B12 samples in an arthritis-induced model using MIA.

[0077] Figure 50 shows the results of measuring serum ALT and AST levels after administration of natural fermented liquid and glutathione samples in an acute liver injury model. *: p<0.05 (vs. control group).

[0078] Figure 51 shows the results of measuring serum ALT and AST levels after administration of natural fermentation liquid and caffeic acid samples in an acute liver injury model. *: p<0.05 (vs. control group).

[0079] Figure 52 shows the results of measuring serum ALT and AST levels after administration of natural fermentation liquid and chlorogenic acid samples in an acute liver injury model. *: p<0.05 (vs. control group).

[0080] Figure 53 shows the results of measuring serum ALT and AST levels after administration of natural fermentation liquid and coffee extract samples in an acute liver injury model. *: p<0.05 (vs. control group).

[0081] Figure 54 shows the results of measuring ALT and AST levels in serum after administration of natural fermented liquid and Hwangchil extract samples in an acute liver injury model. *: p<0.05 (vs. control group).

[0082] Figure 55 shows the results of measuring serum ALT and AST levels after administration of natural fermentation liquid and vitamin B12 samples in an acute liver injury model. *: p<0.05 (vs. control group).

[0083] Figure 56 shows the results of analyzing the morphological changes in the lesion by observing the right ear area after administering natural fermented liquid and glutathione samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0084] Figure 57 shows the results of analyzing the morphological changes in the lesion by observing the right ear area after administering natural fermented liquid and caffeic acid samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0085] Figure 58 shows the results of analyzing the morphological changes in the lesion by observing the right ear area after administering natural fermentation liquid and chlorogenic acid samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0086] Figure 59 shows the results of analyzing the morphological changes in the lesion by observing the right ear area after administering natural fermentation liquid and coffee extract samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0087] Figure 60 shows the results of analyzing the morphological changes in the lesion by observing the right ear area after administering natural fermented liquid and Hwangchil extract samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0088] Figure 61 shows the results of analyzing the morphological changes in the lesion by observing the right ear area after administering a natural fermented liquid and vitamin B12 sample to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0089] Figure 62 shows the results of measuring the change in thickness of the right ear area after administering a natural fermented liquid and glutathione sample to an atopic dermatitis model.

[0090] Figure 63 shows the results of measuring the change in thickness of the right ear area after administering a natural fermented liquid and caffeic acid sample to an atopic dermatitis model.

[0091] Figure 64 shows the results of measuring the change in thickness of the right ear area after administering natural fermentation liquid and chlorogenic acid samples to an atopic dermatitis model.

[0092] Figure 65 shows the results of measuring the change in thickness of the right ear area after administering natural fermentation liquid and coffee extract samples to an atopic dermatitis model.

[0093] Figure 66 shows the results of measuring the change in thickness of the right ear area after administering natural fermented liquid and Hwangchil extract samples to an atopic dermatitis model.

[0094] Figure 67 shows the results of measuring the change in thickness of the right ear area after administering a natural fermented liquid and vitamin B12 sample to an atopic dermatitis model.

[0095] Figure 68 shows the results of the spleen index at the end of the experiment for each experimental group after administering natural fermented liquid and glutathione samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0096] Figure 69 shows the results of the spleen index at the end of the experiment for each experimental group after administering natural fermented liquid and caffeic acid samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0097] Figure 70 shows the results of the spleen index at the end of the experiment for each experimental group after administering natural fermented liquid and chlorogenic acid samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0098] Figure 71 shows the results of the spleen index at the end of the experiment for each experimental group after administering natural fermentation liquid and coffee extract samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0099] Figure 72 shows the results of the spleen index at the end of the experiment for each experimental group after administering natural fermented liquid and Hwangchil extract samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0100] Figure 73 shows the results of the spleen index at the end of the experiment for each experimental group after administering natural fermented liquid and vitamin B12 samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0101] Figure 74 shows the results of measuring the levels of TNF-α(a) and IL-4(b) in the serum at the end of the experiment for each experimental group after administering natural fermented liquid and glutathione samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0102] Figure 75 shows the results of measuring the levels of TNF-α(a) and IL-4(b) in the serum at the end of the experiment for each experimental group after administering natural fermentation liquid and caffeic acid samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0103] Figure 76 shows the results of measuring the levels of TNF-α(a) and IL-4(b) in the serum at the end of the experiment for each experimental group after administering natural fermentation liquid and chlorogenic acid samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0104] Figure 77 shows the results of measuring the levels of TNF-α(a) and IL-4(b) in the serum at the end of the experiment for each experimental group after administering natural fermentation liquid and coffee extract samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0105] Figure 78 shows the results of measuring the levels of TNF-α(a) and IL-4(b) in the serum at the end of the experiment for each experimental group after administering natural fermented liquid and Hwangchil extract samples to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0106] Figure 79 shows the results of measuring the levels of TNF-α(a) and IL-4(b) in the serum at the end of the experiment for each experimental group after administering a natural fermented liquid and vitamin B12 sample to an atopic dermatitis model. *: p<0.05 (compared to the control group).

[0107] The present invention provides a pharmaceutical composition for preventing or treating hypertension, enteritis, stress-related bowel syndrome, arthritis, acute liver damage or dermatitis, comprising as an active ingredient a natural fermented product in which a nitric oxide metabolite is immobilized and stabilized; and any one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract and phellodendron extract.

[0108] In the pharmaceutical composition of the present invention, the effective ingredient may suppress the production of carcinogenic N-nitrosamines in the stomach and promote the production of nitric oxide in the intestines.

[0109] The concentration of nitric oxide in the human body is nitrite ion (NO2 - ) and nitrate ion (NO3 - ) can be predicted as the total amount by adding up, but the actual usable nitric oxide concentration is NO2 that can be converted to nitric oxide (NO). - Blood NO concentration can be predicted by the concentration of NO2 as a nitric oxide metabolite. - It is a hydrophilic and relatively stable ionic substance, and can be dissolved in blood and body fluids and move to all parts of the body, but cannot pass through organ barriers, blood vessel barriers, cell membranes, etc. However, NO2 - When NO is reduced to lipid-soluble NO by nitrite reductase or acidic conditions, it can pass through organ barriers, blood vessel barriers, cell membranes, etc. Therefore, NO2 - In order for it to reach the target cells and exert a specific pharmacological effect or to be absorbed into the body through the stomach or intestines, it must be reduced to NO.

[0110] To increase the concentration of low blood nitric oxide metabolites or to obtain the pharmacological effects of nitric oxide, orally administered nitric oxide metabolites (especially NO2 - ) can cause two harmful health problems: First, NO2 taken orally - Due to the strong acidity of gastric juice, excessive NO can be absorbed into the body in a short period of time, which can cause rapid decrease in blood pressure, hypoglycemia, cyanosis, etc. Second, due to the strong acidity of gastric juice, NO2 taken orally - can react with secondary amines to form carcinogenic N-nitrosamines.

[0111] The pharmaceutical composition according to the present invention comprises a nitric oxide metabolite (NO2) contained in a natural fermentation product. - ) and glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and Phellodendron amurense extract react chemically under the strong acid conditions of the stomach to form S-nitrosoglutathione (GSNO), thereby blocking the production of N-nitrosamine (R2N-NO) and generating a certain amount of nitric oxide (NO) in the intestinal environment, thereby enabling the body to absorb nitric oxide metabolites. In the present invention, coffee extract is used in the same meaning as coffee extract, and Phellodendron amurense extract means an extract of the Phellodendron amurense tree.

[0112] In the pharmaceutical composition of the present invention, the natural fermented product in which the nitric oxide metabolite is immobilized and stabilized may be produced by a manufacturing method including a step of adding a fermentation strain to a nitrogen-containing natural product and fermenting the product 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, but is not limited thereto, and the nitrogen-containing natural product may be, but is not limited to, soybeans, garlic, soybean sprouts, cabbage, or whey. For a detailed manufacturing method of the natural fermented product according to the present invention, reference may be made to Korean Patent No. 2129038.

[0113] The natural fermentation product according to the present invention can be fermented by adding 0.01 to 3 parts by weight of a fermentation strain to 100 parts by weight of a mixture of a nitrogen-containing natural product and water, and preferably, the nitrogen-containing natural product and water are mixed in a weight ratio of 1:0.5 to 1, and then fermented by adding 0.01 to 3 parts by weight of a fermentation strain to 100 parts by weight of the mixture, but is not limited thereto.

[0114] In addition, the fermentation may be performed 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 thereto.

[0115] Additionally, the fermentation may be performed under conditions of a dissolved oxygen concentration of 0.01 to 0.1 mg / L, preferably 0.03 to 0.1 mg / L, but is not limited thereto.

[0116] Additionally, the fermentation may be performed for 2 to 30 days, but is not limited thereto.

[0117] Additionally, the fermentation may be terminated when the chemical oxygen demand is 200 to 700 mg / L, but is not limited thereto.

[0118] In addition, the fermentation strain may preferably be Bacillus subtilis - Chun Hyun Su, whose deposit number is KCTC12501BP, but is not limited thereto.

[0119] In addition, in the pharmaceutical composition according to the present invention, the natural fermentation product in which the nitric oxide metabolite is fixed and stabilized is preferably NO2. - It may contain 0.1 to 10 mM based on, but is not limited to,

[0120] The term "nitric oxide metabolite" as used herein refers to nitrite (NO2 -), R-NO2 - , R-NO, RO-NO, nitrate (NO3 - ), R-NO3 - , GS-NO (or GSNO), GS-NO2, etc., where R- means that one H atom has been removed from the carbon chain of polyphenols, fatty acids, etc., RO- means that one H atom has been removed from ROH, and in the case of GS-, 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, hydrogen ions (H + ) is reduced to nitric oxide, and in slightly acidic conditions such as the intestines (small intestine, large intestine), nitric oxide metabolites can be reduced to nitric oxide by reducing substances such as polyphenols, glutathione, vitamin C, and bacterial nitric oxide reductase.

[0121] In particular, GSNO, known as nitric oxide metabolite or nitric oxide conjugate, can safely produce nitric oxide by reacting with glutathione (GSH) secreted from the liver through the bile duct or GSH secreted by intestinal bacteria (see Scheme 8-1). Nitric oxide (NO) produced in the intestinal environment in this way penetrates the body from the intestinal environment and forms nitric oxide metabolite (NO2 - ) and reaches the body fluid or blood vessels by being oxidized (see Reaction Formula 8-3).

[0122] In addition, in the pharmaceutical composition according to the present invention, the glutathione may be in the form of pure glutathione or an extract containing glutathione, but is not limited thereto. "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 cells, glutathione combines with free radicals, peroxides, toxic substances, heavy metals, etc. to prevent cell damage. In addition, GSH can react with nitric oxide (NO) to produce S-nitrosoglutathione (GSNO). GSNO produced through this process delivers NO to where it is needed through blood vessels in the body, and stores NO for a certain period of time within cells, and when needed, breaks down into NO to exhibit the pharmacological action of NO.

[0123] In addition, in the pharmaceutical composition of the present invention, the caffeic acid or chlorogenic acid is a phenol compound that promotes the process of reducing nitric oxide metabolites in natural fermentation to nitric oxide. Coffee extract is known to be an extract rich in caffeic acid and chlorogenic acid, and Dendropanax morbifera extract is also an extract containing various polyphenol compounds. In addition, cyanocobalamin, a vitamin B12 compound used in the present invention, when it enters the body, cobalt is reduced from a trivalent state to a divalent or monovalent state, and then converted to adenosylcobalamin or methylcobalamin to function as a coenzyme.

[0124] The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

[0125] In addition, the pharmaceutical composition of the present invention can be formulated and used in the form of oral preparations such as granules, tablets, and capsules according to conventional methods, but is not limited thereto.

[0126] Carriers, excipients, and diluents that may be included in the pharmaceutical composition of the present invention include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, the composition 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 are prepared by mixing the fermented earthworm extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include Wetepsol, Macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0127] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes, and all modes of administration can be anticipated, preferably oral or rectal administration, but is not limited thereto.

[0128] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0129] Specifically, the pharmaceutically effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally administered at 2 mg to 4 mg per kg of body weight, preferably 3 mg, daily or every other day, and may be administered once to three times a day. However, the dosage may increase or decrease depending on the route of administration, severity of the disease, sex, body weight, age, etc., and thus the above dosage does not limit the scope of the present invention in any way.

[0130] The present invention also provides a health functional food composition for preventing or improving hypertension, enteritis, stress-related bowel syndrome, arthritis, acute liver damage, or dermatitis, comprising as active ingredients a natural fermented product in which a nitric oxide metabolite is immobilized and stabilized; and any one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and phellodendron amurense extract.

[0131] In the health functional food composition according to the present invention, the natural fermented product in which the nitric oxide metabolite is fixed and stabilized, and glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and phellodendron extract are as described above.

[0132] There are no special restrictions on the types of the above health functional foods. Examples of health functional foods containing the above health functional food composition include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, rice cakes, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0133] The above health functional food may be manufactured in any one of the following forms: powder, granules, pills, tablets, capsules, candies, syrups, effervescent tablets, and beverages, but is not limited thereto.

[0134] When the health functional food composition of the present invention is used as a food additive, the composition 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 may be used appropriately depending on its intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw material. However, in the case of long-term intake for the purpose of health management, it may be used in an amount that does not pose any safety issues.

[0135] The health functional food of the present invention includes ingredients typically added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredients. The natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agents may be natural flavoring agents (e.g., thaumatin, stevia extract, etc.) or synthetic flavoring agents (e.g., saccharin, aspartame, etc.). In addition to the above health functional food composition, it may further contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0136] The present invention also provides a veterinary composition for preventing or treating hypertension, enteritis, stress-related bowel syndrome, arthritis, acute liver damage or dermatitis, comprising as active ingredients a natural fermented product in which a nitric oxide metabolite is immobilized and stabilized; and any one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract and phellodendron extract.

[0137] In the veterinary composition according to the present invention, the natural fermented product in which the nitric oxide metabolite is fixed and stabilized, and glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract and phellodendron extract are as described above.

[0138] The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

[0139] The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

[0140] The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. This may be determined based on factors including the severity of the disease or condition, the individual's age, weight, health status, or sex, sensitivity to the active ingredient of the present invention, the route of administration, the duration of administration, other compositions combined with or used concurrently with the composition, and other factors well known in the fields of physiology and veterinary medicine.

[0141] In this specification, the term "fermented liquid" is used interchangeably to mean "natural fermented product in which nitric oxide metabolites are fixed and stabilized."

[0142]

[0143] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0144]

[0145] 1. Experimental method

[0146] 1-1) Preparation of reagents and samples

[0147] Lettuce, soybeans, garlic, bean sprouts, cabbage or whey fermentation solution manufactured by Human Enos Co., Ltd. contains 1 mM of NO2 - Fermented broth containing was used in the experiment. 1 mM of sodium nitrite (NaNO2) was used as a comparison group for natural fermented products, and caffeic acid (CAS No. 331-39-5), chlorogenic acid (CAS No. 327-97-9), vitamin B12 (cyanocobalamin, CAS No. 68-19-9), and glutathione (CAS No. 70-18-8) were purchased from Sigma-Aldrich and prepared at a concentration of 1 mM, and then mixed with natural fermented broth or sodium nitrite at a weight ratio of 1:1 for use. In addition, dried yeast containing glutathione was purchased from Vision Biochem Co., Ltd. and 1 mM was used as the standard for glutathione concentration. The coffee extract was prepared by adding 20 L of purified water to 500 g of ground coffee beans and extracting them at 94°C for 10 minutes, centrifuging at 4,500 rpm for 5 minutes, and using the supernatant in the experiment. The Hwangchil extract was prepared by adding 4 L of fermented alcohol to 200 g of the ground stem and leaves of the Hwangchil tree and extracting them at 88°C for 2 hours. Afterwards, the extract was centrifuged at 4,500 rpm for 5 minutes, and the supernatant was used in the experiment. The coffee extract and Hwangchil extract were mixed with a natural fermentation product at a weight ratio of 1:1 for the experiment.

[0148] The above lettuce, soybean, garlic, soybean sprout, cabbage or whey fermentation liquid refers to a concentrate of a fermented product manufactured through the manufacturing method disclosed in Korean Patent No. 2129038. Simply put, each raw material and water are mixed in a weight ratio of 1:0.5 to 1, and then 0.01 to 3 parts by weight of a fermentation strain is added to 100 parts by weight of the mixture, and fermented 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, and the supernatant obtained by solid-liquid separation using a centrifuge is concentrated at 20 to 30°C for 24 to 48 hours at a stirring speed of 100 to 300 rpm.

[0149]

[0150] 1-2) Measurement of nitric oxide metabolite concentration

[0151] Total amount of nitric oxide metabolites (NO2) contained in the fermented solution - Wow NO3 - It means the concentration of the sum of the concentrations of NO x ) is added with nitrate reductase to remove NO3 contained in the concentrate. - All of them are NO2 - and reduced to total NO2 - The total content or concentration of nitric oxide metabolites was determined by measuring the concentration of .

[0152] More specifically, in the case of fermentation liquid, total NO2 - The content was first reduced for 1 hour by adding nitrate reductase, diluted 1,000 times with distilled water, centrifuged at 3,000 rpm for 3 minutes to remove insoluble polyphenols and dietary fiber, and then 50 ㎕ of the clear supernatant was taken and used as a measurement sample. 50 ㎕ of NO2 -The standard solution and 50 μl of the measurement sample were dispensed into each 96-well plate, and 100 μl of the test solution [a solution of 30% acetic acid solution containing 1% sulfanilamide and 60% acetic acid solution containing 0.1% N-(1-naphthyl)ethylenediamine mixed in a 1:2 ratio] was added, and the color reaction was induced by leaving it at room temperature for 10 minutes. Then, the absorbance value of each well was measured at a wavelength of 570 nm using a multi-plate reader, and then the absorbance value of the standard solution and the absorbance value of the sample were compared to determine NO x The concentration was calculated.

[0153]

[0154] 2. Statistical analysis

[0155] The results obtained from each experiment were expressed as the mean ± standard deviation (SD). The significance between two experimental groups was analyzed using a t-test. For more than two experimental groups, significance was analyzed using a one-way ANOVA followed by a Duncan post hoc test. A p-value less than 0.05 (p < 0.05) was considered statistically significant.

[0156]

[0157] Example 1. Effect of improving enteritis by a mixed sample of fermented liquid containing nitric oxide metabolites and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12.

[0158] The experimental animals used in this experiment were 6-week-old male Balb / c mice purchased from Samtaco (Korea) and used after a one-week acclimation period. The average weight of the Balb / c mice was 20–24 g, and the sample was administered orally once daily for 3 weeks. The experimental animal breeding room was maintained at a temperature of 23±2°C and a humidity of 50–60%, with a light / dark cycle controlled at 12-hour intervals.

[0159]

[0160] 1-1. Military composition and the induction of ulcerative colitis

[0161] The experimental animals were classified as shown in Tables 1 and 2 below. The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and Phellodendron amurense extract was composed identically to the caffeic acid mixture in Table 2. As mentioned above, the coffee extract and Phellodendron amurense extract were mixed in the same weight ratio as the natural fermentation liquid, and 1 mM of the dose corresponds to the NO2 in the natural fermentation liquid. - It means concentration.

[0162] Ulcerative colitis was induced by administering 3.0% DSS (Dextran sulfate sodium) ad libitum to all experimental groups except the control group for 5 days. The number of animals in each group was calculated using the minimum number that obscured statistical significance according to the 3R principle.

[0163] Composition of the ulcerative colitis experimental group with a mixture of natural fermented liquid and glutathione (GSH) Group D S Material Dose 1 Normal group No Distilled water-5 2 Control group Yes Distilled water-5 3 NaNO2 Yes Sodium nitrite 1 mM 5 4 NaNO2 + 50% GSH Yes Sodium nitrite + Yeast extract 50% glutathione 1 mM 5 5 NaNO2 + 98% GSH Yes Sodium nitrite + 98% glutathione 1 mM 5 6 Lettuce fermented liquid Yes Lactuca sativa fermented extract 1 mM 5 7 Lettuce fermented liquid + 50% GSH Yes Lactuca sativa fermented extract + Yeast extract 50% glutathione 1 mM 5 8 Lettuce fermented liquid + 98% GSH Yes Lactuca sativa fermented extract + 98% glutathione 1 mM 5 9 Soybean fermented liquid Yes Bean fermented extract 1 mM510 soybean fermented solution+50% GSHYesbean fermented extract +Yeast extract 50% glutathione1 mM514Garlic fermented extract+98% GSHYesGarlic fermented extract +98% glutathione1mM515Soybean sprout fermented extract1mM516Soybean sprout fermented extract+50% GSHYessoybean sprout fermented extract + Yeast extract 50% glutathione1mM517Bean sprout fermented extract+98% GSHYessoybean sprout fermented extract +98% glutathione 1 mM 5 18 Cabbage fermented extract 1 mM 5 19 Cabbage fermented extract + 50% GSH 50% glutathione 1 mM 5 20 Cabbage fermented extract + 98% GSH 50% glutathione 1 mM 5 21 Whey fermented extract 1 mM 5 22 Whey fermented extract + 50% GSH 50% glutathione 1 mM 5 23 Whey fermented extract + 98% GSH 50% glutathione 1 mM 5

[0164] Composition of the experimental group for ulcerative colitis with a mixture of natural fermented liquid and caffeic acid Group D S Material Dose 1 Normal group No Distilled water-3 2 Control group Yes Distilled water-3 3 NaNO2 Yes Sodium nitrite 1 mM 3 4 NaNO2 + caffeic acid Yes Sodium nitrite + caffeic acid 1 mM 3 5 Lettuce fermented liquid Yes Lactuca sativa fermented extract 1 mM 3 6 Lettuce fermented liquid + caffeic acid Yes Lactuca sativa fermented extract + caffeic acid 1 mM 3 7 Soybean fermented liquid Yes Bean fermented extract 1 mM 3 8 Soybean fermented liquid + caffeic acid Yes Bean fermented extract + caffeic acid 1 mM 3 9 Garlic fermented liquid Yes Garlic fermented extract 1 mM 3 10 Garlic fermented liquid + caffeic acid Yes Garlic fermented extract + caffeic acid 1 mM 3 11 Soybean sprout fermented liquid Yes Soybean sprout fermented extract 1 mM312 bean sprout fermentation solution+caffeic acidYessoybean sprout fermented extract + caffeic acid1 mM313 cabbage fermentation solutionYesCabbage fermented extract1 mM314 cabbage fermentation solution+caffeic acidYesCabbage fermented extract +caffeic acid1 caffeic acid1mM3

[0165]

[0166] 1-2. Measurement of disease activation level

[0167] Bowel disease is measured by signs such as weight loss, diarrhea with blood and mucus, and a shortened colon. Previous studies have shown that the disease activity index (DAI) is composed of three major clinical signs: weight loss, diarrhea, and rectal bleeding. Weight loss is calculated as the difference between initial and current weight. Diarrhea is defined as the absence of fecal pellets in the rectum and the presence of persistent, soft stool. Rectal bleeding is distinct from diarrhea with blood, gross rectal bleeding, and other types of diarrhea. In the present invention, the DAI was calculated as follows.

[0168] DAI = (weight loss score) + (diarrhea score) + (rectal bleeding score)

[0169] The medical variables used here are comprehensive functional measures similar to the medical symptoms observed in ulcerative colitis in humans (Table 3).

[0170] Disease activity indexScoreweight loss (%)DiarrheaRectal bleeding0noneNoneNormal11-5MildOccult26-10311-15416-205>20Severe wateryGross bleeding

[0171]

[0172] 1-3. Measurement of intestinal length and serum cytokines

[0173] After the experiment, all experimental groups were subjected to orbital vein blood collection under inhaled anesthesia, serum separation, abdominal incision, and resection from the terminal colon to the cecum to measure the intestinal length. Serum cytokines were measured for the production of TNF-α and IL-1β using an ELISA kit (R&D Systems, USA). 50 μl of each ADS (assay diluent solution) was added to each well with antibodies, and 50 μl of each sample was added, left at room temperature for 2 hours, and then washed four times with 300 μl of washing buffer. After washing, 100 μl of mouse TNF-α and IL-1β antibodies were added to each well, incubated at room temperature for 2 hours, removed, and washed four times. 100 μl of substrate solution was added to each well and left at room temperature for 30 minutes. Then, 100 μl of stop solution was added and the absorbance was measured at 450 nm using an ELISA reader (SPECTRA max M2, USA) from Molecular Devices.

[0174]

[0175] 1-4. Effect of the mixture in an ulcerative colitis model

[0176] The DSS colitis model presents with clinical symptoms characterized by weight loss, diarrhea, and bloody stool. The results of measuring the effects on these clinical symptoms using the DAI are shown in Figures 1 to 6. No symptoms were observed in the normal group, while the sample-administered groups showed overall decreases compared to the control group (DSS group). In particular, the DAI was more significantly reduced in the groups administered a combination of glutathione, caffeic acid, chlorogenic acid, coffee extract, dendropanax extract, or vitamin B12 compared to the group administered the natural fermented liquid alone.

[0177] In addition, the length of the large intestine of mice sacrificed on the 5th day after DSS administration was checked by extracting the large intestine, and it was confirmed that the intestinal length was longer in all groups administered the sample than in the control group, and the intestinal length was longer in the group administered natural fermented liquid than in the group administered NaNO2, and the intestinal length was confirmed to be closer to the normal group in the group administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12 than in the group administered natural fermented liquid alone (Figs. 7 to 12).

[0178] In addition, when measuring inflammatory cytokines in the blood in an animal model induced with colitis, the levels of TNF-α and IL-1β, which are indicators of inflammation, were reduced in all groups administered with the sample compared to the control group, and in particular, the lowest levels were observed in the group administered with a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, dendritic stem extract, or vitamin B12 compared to the group administered with only natural fermented liquid (Figs. 13 to 18).

[0179]

[0180] Example 2. Effect of a mixture of a fermented solution containing nitric oxide metabolites and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron amurense extract, or vitamin B12 on improving stress-induced bowel syndrome.

[0181] The experimental animals used in this experiment were 8-week-old female Wistar rats purchased from Samtaco and used after a one-week acclimatization period. The average weight of the Wistar rats was 180.5±0.5 g, and the sample was administered orally once daily. The experimental animal breeding room was maintained at a 12-hour light / dark cycle, with a temperature of 23±2°C and a humidity of 50–60%.

[0182]

[0183] 2-1. Military Composition and Water Avoidance Stress (WAS)

[0184] In order to observe the changes in colonic movement due to the mixture according to the present invention, the WAS tanks were filled with water for each group except the normal group, and the animals were placed on an escape platform to provide intermittent stress, and then the number of feces was measured. The experiment measured the water and feed intake every day while administering each sample. In addition, the WAS was divided into two sessions of 15 animals per hour, taking into account the intermediate change time, for a total of 8 days (Fig. 19), and the number of feces was measured during this time.

[0185] The group composition was as shown in Tables 4 and 5 below. The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and phellodendron amurense extract was composed identically to the caffeic acid mixture in Table 5. Exposure to WAS induced diarrheal irritable bowel syndrome-like symptoms (increased stool) and enhanced mucosal immunity, and it was observed whether administration of each sample could reduce these symptoms.

[0186] Stress-induced bowel syndrome experimental composition of a mixture of natural fermented liquid and glutathione (GSH) GroupWASMaterialDosen1Normal groupNodistilled water-52Control groupYesdistilled water-53NaNO2Yessodium nitrite1 mM54NaNO2+50% GSHYessodium nitrite + Yeast extract 50% glutathione1 mM55NaNO2+98% GSHYessodium nitrite + 98% glutathione1 mM56Lettuce fermented liquidYesLactuca sativafermented extract1 mM57Lettuce fermented liquid+50% GSHYesLactuca sativafermented extract + Yeast extract 50% glutathione1 mM58Lettuce fermented liquid+98% GSHYesLactuca sativafermented extract +98% glutathione1 mM59Soybean fermented liquidYesbean fermented extract1 mM510 soybean fermented solution+50% GSHYesbean fermented extract +Yeast extract 50% glutathione1 mM514Garlic fermented extract+98% GSHYesGarlic fermented extract +98% glutathione1 mM515Soybean sprout fermented extract1mM516Soybean sprout fermented extract+50% GSHYessoybean sprout fermented extract + Yeast extract 50% glutathione1mM517Bean sprout fermented extract+98% GSHYessoybean sprout fermented extract+ 98% glutathione 1 mM 5 18 Cabbage fermented extract Yes Cabbage fermented extract 1 mM 5 19 Cabbage fermented extract + 50% GSH Yes Cabbage fermented extract + Yeast extract 50% glutathione 1 mM 5 20 Cabbage fermented extract + 98% GSH Yes Cabbage fermented extract + 98% glutathione 1 mM 5 21 Whey fermented extract Yes Whey fermented extract 1 mM 5 22 Whey fermented extract + 50% GSH Yes Whey fermented extract + Yeast extract 50% glutathione 1 mM 5 23 Whey fermented extract + 98% GSH Yes Whey fermented extract + 98% glutathione 1 mM 5

[0187] Stress-induced bowel syndrome experimental configuration with a mixture of natural fermented liquid and caffeic acid GroupWASMaterialDosen1Normal groupNodistilled water-32Control groupYesdistilled water-33NaNO2Yessodium nitrite1 mM34NaNO2+caffeic acidYessodium nitrite + caffeic acid1 mM35Lettuce fermented liquidYesLactuca sativafermented extract1 mM36Lettuce fermented liquid+caffeic acidYesLactuca sativafermented extract+caffeic acid1 mM37Soybean fermented liquidYesbean fermented extract1 mM38Soybean fermented liquid+caffeic acidYesbean fermented extract +caffeic acid1 mM39Garlic fermented liquidYesGarlic fermented extract1 mM310Garlic fermented liquid+caffeic acidYesGarlic fermented extract +caffeic acid1 mM311Soybean sprout fermented liquidYessoybean sprout fermented extract1 mM312 bean sprout fermentation solution+caffeic acidYessoybean sprout fermented extract + caffeic acid1 mM313 cabbage fermentation solutionYesCabbage fermented extract1 mM314 cabbage fermentation solution+caffeic acidYesCabbage fermented extract +caffeic acid1 caffeic acid1mM3

[0188]

[0189] 2-2. Effect of the mixture in a stress-induced bowel syndrome model

[0190] The results of measuring the number of stools for each sample when stress was induced through WAS are as shown in Figures 20 to 25. In all sample administration groups, the number of stools decreased compared to the control group, and the number of stools tended to decrease more in the group administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12 than in the group administered only natural fermented liquid.

[0191]

[0192] Example 3. Blood pressure improvement effect by a mixed sample of fermented liquid containing nitric oxide metabolites and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12.

[0193] The 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 a one-week acclimation period. The average weight of the SD rats was 198.95±2.10 g, and the samples were administered orally before the experiment. The animal breeding room was maintained at a temperature of 23±2℃ and a humidity of 50-60% with a light / dark cycle controlled at 12-hour intervals.

[0194]

[0195] 3-1. Production of DOCA-Salt Hypertensive Rat Model and Group Composition

[0196] A hypertensive model was created by administering DOCA (Deoxycorticosterone acetate; TCI, Japan) intraperitoneally twice a week at a concentration of 25 mg / kg for 4 weeks and inducing hypertension using 1% sodium chloride solution as drinking water in each group. At the same time, the control group was administered distilled water (DW) and the experimental group was administered the respective sample. To confirm the induction of hypertension, blood pressure was measured using a mouse tail blood pressure system (MK-2000STS, MUROMACHI KIKAI, Japan). Animals with a systolic blood pressure of 130 mmHg or higher were selected for use. Blood pressure of the experimental animals was measured in real time for 55 minutes per animal after administering the sample to each group. The rats were fixed in a correction frame, and a piezoelectric pulse sensor and an occlusion cuff were placed on the tail to secure them. The pulse signal connected to a computer was monitored at an appropriate level, and the device was operated to observe systolic blood pressure. The experimental groups were organized as shown in Tables 6 and 7 below. The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and Phellodendron amurense extract was composed identically to the caffeic acid mixture in Table 7.

[0197] Blood pressure experimental group composition of natural fermented liquid and glutathione (GSH) mixture Group Treatment n DOC ADose material Dose 1 Normal group No 25 mg / kg distilled water - 5 2 Control group Yes 25 mg / kg distilled water - 5 3 NaNO 2 Yes 25 mg / kg sodium nitrite 1 mM 5 4 NaNO 2 + 50% GSH Yes 25 mg / kg sodium nitrite + yeast extract 50% glutathione 1 mM 5 5 NaNO 2 + 98% GSH Yes 25 mg / kg sodium nitrite + 98% glutathione 1 mM 5 6 Lettuce fermented liquid Yes 25 mg / kg Lactuca sativa fermented extract 1 mM 5 7 Lettuce fermented liquid + 50% GSH Yes 25 mg / kg Lactuca sativa fermented extract + yeast extract 50% glutathione 1 mM 5 8 Lettuce fermented liquid + 98% GSH Yes 25 mg / kgLactuca sativafermented extract + 98% glutathione1 mM59 soybean fermented extractYes25 mg / kgbean fermented extract1 mM510 soybean fermented extract+50% GSHYes25 mg / kgbean fermented extract +Yeast extract 50% glutathione1 mM511 soybean fermented extract+98% GSHYes25 mg / kgbean fermented extract +98% glutathione1 mM512 Garlic fermented extractYes25 mg / kgGarlic fermented extract1mM513Garlic fermented extract+50% GSHYes25 mg / kgGarlic fermented extract +Yeast extract 50% glutathione1mM514Garlic fermented extract+98% GSHYes25 mg / kgGarlic fermented extract +98% glutathione1mM515Soy bean sproutYes25 mg / kgsoybean sprout fermented extract1 mM516 bean sprout fermentation solution +50%GSHYes25 mg / kgsoybean sprout fermented extract + Yeast extract 50% glutathione1 mM517콩나물발효액+98% GSHYes25 mg / kgsoybean sprout fermented extract + 98% glutathione1 mM518양배추발효액Yes25 mg / kgCabbage fermented extract1 mM519양배추발효액+50% GSHYes25 mg / kgCabbage fermented extract +Yeast extract 50% glutathione1 mM520양배추발효액+98% GSHYes25 mg / kgCabbage fermented extract +98% glutathione1 mM521유청발효액Yes25 mg / kgWhey fermented extract1 mM522유청발효액+50% GSHYes25 mg / kgWhey fermented extract +Yeast extract 50% glutathione1 mM523유청발효액+98% GSHYes25 mg / kgWhey fermented extract +98% glutathione1 mM5

[0198] Blood pressure experimental group composition of natural fermented liquid and caffeic acid mixture Group Treatment n DOC ADose material Dose 1 Normal group No 25 mg / kg distilled water - 3 2 Control group Yes 25 mg / kg distilled water - 3 3 NaNO 2 Yes 25 mg / kg sodium nitrite 1 mM 3 4 NaNO 2 + caffeic acid Yes 25 mg / kg sodium nitrite + caffeic acid 1 mM 3 5 Lettuce fermented liquid Yes 25 mg / kg Lactuca sativa fermented extract 1 mM 3 6 Lettuce fermented liquid + caffeic acid Yes 25 mg / kg Lactuca sativa fermented extract + caffeic acid 1 mM 3 7 Soybean fermented liquid Yes 25 mg / kg bean fermented extract 1 mM 3 8 Soybean fermented liquid + caffeic acid Yes 25 mg / kg bean fermented extract + caffeic acid 1 mM 3 9 Garlic fermented liquid Yes 25 mg / kg Garlic fermented extract 1 mM310garlic fermented extract+caffeic acidYes25 mg / kgGarlic fermented extract + caffeic acid1mM311soybean sprout fermented extract1mM312soybean sprout fermented extract1mM312soybean sprout fermented extract+caffeic acid1mM313cabbage fermented extractYes25 mg / kgCabbage fermented extract1 mM314Cabbage fermented extract+caffeic acidYes25 mg / kgCabbage fermented extract +caffeic acid1

[0199]

[0200] 3-2. Effect of the mixture in the DOCA-induced hypertension model

[0201] The results of measuring blood pressure before and after administration of each fermented liquid in rats in which hypertension was induced by DOCA-salt are as shown in Figures 26 to 31. A decrease in blood pressure was observed between 10 and 20 minutes after oral administration of the sample, and in particular, a high decrease in blood pressure was observed in the group administered a mixture of natural fermented liquid and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12.

[0202]

[0203] Example 4. Effect of improving arthritis by a mixture of fermented liquid containing nitric oxide metabolites and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12.

[0204] The animals used in this experiment were 7-week-old male SD rats purchased from Samtaco and acclimated for one week before use. Samples were administered orally once daily for four weeks. The animal housing room was maintained at a 12-hour light / dark cycle, with a temperature of 23±2°C and a humidity of 50-60%.

[0205]

[0206] 4-1. Causes and composition of arthritis

[0207] For the induction of arthritis, the animals in each experimental group except the normal group were anesthetized, and the area around the left knee of the SD rats was cleanly shaven. Monosodium iodoacetate (MIA), an osteoarthritis-inducing substance, was injected into the left knee joint at 50 ㎕ (60-80 mg / ㎖) using a 31G insulin syringe (0.25 × 8, BD Medical-Diabetes Care, USA). 0.9% saline was used for MIA dilution, and each experimental sample was administered to each experimental group 7 days after the MIA injection. The experimental groups were composed as shown in Tables 8 and 9 below. The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and macchamel extract was composed in the same way as the caffeic acid mixture in Table 9.

[0208] Composition of arthritis experimental group of natural fermented liquid and glutathione (GSH) mixture GroupMIAMaterialDosen1Normal groupNodistilled water-52Control groupYesdistilled water-53NaNO2Yessodium nitrite1 mM54NaNO2+50% GSHYessodium nitrite + Yeast extract 50% glutathione1 mM55NaNO2+98% GSHYessodium nitrite + 98% glutathione1 mM56Lettuce fermented liquidYesLactuca sativafermented extract1 mM57Lettuce fermented liquid+50% GSHYesLactuca sativafermented extract + Yeast extract 50% glutathione1 mM58Lettuce fermented liquid+98% GSHYesLactuca sativafermented extract + 98% glutathione1 mM59Soybean fermented liquidYesbean fermented extract1 mM510Soybean fermented liquid+50% GSHYesbean fermented extract +Yeast extract 50% glutathione1 mM511Soybean fermented extract+98% GSHYesbean fermented extract +98% glutathione1mM512Garlic fermented extractYesGarlic fermented extract1mM513Garlic fermented extract+50% GSHYesGarlic fermented extract +Yeast extract 50% glutathione1mM514Garlic fermented extract+98% GSHYesGarlic fermented extract +98% glutathione1 mM515Soybean sprout fermented extract1mM516Soybean sprout fermented extract+50% GSHYessoybean sprout fermented extract + Yeast extract 50% glutathione1mM517Soybean sprout fermented extract+98%Glutathione 1 mM5 18 Cabbage fermented extract Yes Cabbage fermented extract 1 mM5 19 Cabbage fermented extract + 50% GSH Yes Cabbage fermented extract + Yeast extract 50% glutathione 1 mM5 20 Cabbage fermented extract + 98% GSH Yes Cabbage fermented extract + 98% glutathione 1 mM5 21 Whey fermented extract Yes Whey fermented extract 1 mM5 22 Whey fermented extract + 50% GSH Yes Whey fermented extract + Yeast extract 50% glutathione 1 mM5 23 Whey fermented extract + 98% GSH Yes Whey fermented extract + 98% glutathione 1 mM5

[0209] Composition of arthritis experimental group with mixture of natural fermented liquid and caffeic acid GroupMIAMaterialDosen1Normal groupNodistilled water-32Control groupYesdistilled water-33NaNO2Yessodium nitrite1 mM34NaNO2+caffeic acidYessodium nitrite + caffeic acid1 mM35Lettuce fermented liquidYesLactuca sativafermented extract1 mM36Lettuce fermented liquid+caffeic acidYesLactuca sativafermented extract+caffeic acid1 mM37Soybean fermented liquidYesbean fermented extract1 mM38Soybean fermented liquid+caffeic acidYesbean fermented extract +caffeic acid1 mM39Garlic fermented liquidYesGarlic fermented extract1 mM310Garlic fermented liquid+caffeic acidYesGarlic fermented extract +caffeic acid1 mM311Soybean sprout fermented liquidYessoybean sprout fermented extract1 mM312 bean sprout fermentation solution+caffeic acidYessoybean sprout fermented extract + caffeic acid1 mM313 cabbage fermentation solutionYesCabbage fermented extract1 mM314 cabbage fermentation solution+caffeic acidYesCabbage fermented extract +caffeic acid1 caffeic acid1mM3

[0210]

[0211] 4-2. Measurement of leg swelling thickness

[0212] To measure changes in leg edema thickness after arthritis was induced using MIA, the left knee joint area of ​​rats was measured. Leg edema thickness was measured after anesthesia using vernier calipers (Mitutoyo Co., Japan).

[0213]

[0214] 4-3. Measurement of arthritis severity score

[0215] The arthritis index was assessed visually by three researchers after photographing each animal before sacrificing the animal model. The assessment was conducted using a modified 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). Three researchers independently observed the animals three times, each scoring 1 to 5 points, and the average value was expressed (Table 10).

[0216] Clinical evaluation grade of arthritisClinical symptoms1No change2Mild erythema of the ankle joint3Mild swelling and erythema of the ankle joint4Moderate swelling and erythema of the ankle joint through the metatarsal bone5Severe swelling and erythema of the ankle joint through the digit

[0217]

[0218] 4-4. Measurement of inflammatory cytokines and inflammatory mediators in serum

[0219] Serum cytokine production was measured using an ELISA kit (R&D Systems, USA). 50 μl of each ADS (assay diluent solution) was added to each well with antibodies, and 50 μl of each sample was added. The wells were left at room temperature for 2 hours and then washed four times with 300 μl of washing buffer. After washing, 100 μl of mouse TNF-α and MMP-9 antibodies were added to each well and incubated at room temperature for 2 hours. The wells were then removed and washed four times. 100 μl of substrate solution was added to each well and left at room temperature for 30 minutes. 100 μl of stop solution was added and the absorbance was measured at 450 nm using an ELISA reader (SPECTRA max M2, USA) from Molecular Devices.

[0220]

[0221] 4-5. Measurement of PGE2 (Prostaglandin E2) in serum

[0222] The amount of PGE2 production in serum was measured using an ELISA kit. After adding 50 μl of ADS to each well containing antibodies, 50 μl of the sample was added to each well, incubated at room temperature for 2 hours, and then washed four times with 300 μl of washing buffer. After washing, 50 μl of Proteoglycan E2 conjugate as an antibody was added to each well, incubated at room temperature for 2 hours, removed, and washed four times. 200 μl of substrate solution was added to each well, incubated at room temperature for 30 minutes, and 100 μl of a reaction stop solution was added. The absorbance was measured at 450 nm using an ELISA reader from Molecular Devices.

[0223]

[0224] 4-6. Effect of the mixture in an arthritis model

[0225] After the experiment, the left knee joint area of ​​each experimental group was observed to determine morphological changes in the degree of arthritis relief. In the control group treated only with MIA, changes in leg thickness due to leg edema were observed compared to the normal group treated with no other treatment, and a decrease in leg thickness was confirmed according to sample treatment (Figs. 32 to 37).

[0226] In addition, as shown in Figures 38 to 43, the results of evaluating the degree of arthritis in each group for degenerative arthritis in an animal model induced by MIA were as follows: in the normal group that was not injected with MIA, no unusual symptoms such as edema were observed; in the control group (MIA) that induced arthritis by MIA, 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.

[0227] After the experiment, serum was separated and the levels of TNF-α, MMP-9, and PGE2, which are major indicators of inflammation, were measured. As a result, all groups administered the samples showed a decrease compared to the control group, and the groups administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12 showed a greater decrease than the group administered only natural fermented liquid (Figs. 44 to 49).

[0228]

[0229] Example 5. Hepatoprotective effect of a mixture of fermented liquid containing nitric oxide metabolites and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12.

[0230] The experimental animals used in this experiment were 7-week-old male Sprague Dawley rats (SD rats) purchased from Samtaco and used after a one-week acclimation period. The average weight of the SD rats was 210.45±1.65 g. The experimental animal breeding room was maintained at a 12-hour light / dark cycle, a temperature of 23±2°C, and a humidity of 50–60%.

[0231]

[0232] 5-1. Induction of liver damage and group composition

[0233] The experimental model of hepatotoxicity induced by carbon tetrachloride (CCl4) was induced by intraperitoneal injection of 0.1 ml per 100 g of body weight of CCl4 mixed with equal parts of olive oil, and the normal group was intraperitoneally injected with only olive oil instead of CCl4. Each group was pretreated orally three times every other day with a 1 mM sample before CCl4 injection, and the normal group was administered orally only DW (distilled water). The number of animals in the groups was calculated according to the principle of 3R, using the minimum number that could distinguish statistical significance.

[0234] Liver damage experimental group composition of natural fermented liquid and glutathione (GSH) mixture Group CCl 4 Material Dose 1 Normal group No Distilled water-5 2 Control group Yes Distilled water-5 3 NaNO 2 Yes Sodium nitrite 1 mM 5 4 NaNO 2 + 50% GSH Yes Sodium nitrite + Yeast extract 50% glutathione 1 mM 5 5 NaNO 2 + 98% GSH Yes Sodium nitrite + 98% glutathione 1 mM 5 6 Lettuce fermented liquid Yes Lactuca sativa fermented extract 1 mM 5 7 Lettuce fermented liquid + 50% GSH Yes Lactuca sativa fermented extract + Yeast extract 50% glutathione 1 mM 5 8 Lettuce fermented liquid + 98% GSH Yes Lactuca sativa fermented extract + 98% glutathione 1 mM 5 9 Soybean fermented liquid Yes Bean fermented extract 1 mM510 soybean fermented solution+50% GSHYesbean fermented extract +Yeast extract 50% glutathione1 mM514Garlic fermented extract+98% GSHYesGarlic fermented extract +98% glutathione1mM515Soybean sprout fermented extract1mM516Soybean sprout fermented extract+50% GSHYessoybean sprout fermented extract + Yeast extract 50% glutathione1mM517Bean sprout fermented extract+98% GSHYessoybean sprout fermented extract + 98%Glutathione 1 mM5 18 Cabbage fermented extract Yes Cabbage fermented extract 1 mM5 19 Cabbage fermented extract + 50% GSH Yes Cabbage fermented extract + Yeast extract 50% glutathione 1 mM5 20 Cabbage fermented extract + 98% GSH Yes Cabbage fermented extract + 98% glutathione 1 mM5 21 Whey fermented extract Yes Whey fermented extract 1 mM5 22 Whey fermented extract + 50% GSH Yes Whey fermented extract + Yeast extract 50% glutathione 1 mM5 23 Whey fermented extract + 98% GSH Yes Whey fermented extract + 98% glutathione 1 mM5

[0235] Liver damage experimental group composition of natural fermented liquid and caffeic acid mixture Group CCl 4 Material Dose 1 Normal group No Distilled water-3 2 Control group Yes Distilled water-3 3 NaNO 2 Yes Sodium nitrite 1 mM 3 4 NaNO 2 + caffeic acid Yes Sodium nitrite + caffeic acid 1 mM 3 5 Lettuce fermented liquid Yes Lactuca sativa fermented extract 1 mM 3 6 Lettuce fermented liquid + caffeic acid Yes Lactuca sativa fermented extract + caffeic acid 1 mM 3 7 Soybean fermented liquid Yes Bean fermented extract 1 mM 3 8 Soybean fermented liquid + caffeic acid Yes Bean fermented extract + caffeic acid 1 mM 3 9 Garlic fermented liquid Yes Garlic fermented extract 1 mM 3 10 Garlic fermented liquid + caffeic acid Yes Garlic fermented extract + caffeic acid 1 mM 3 11 Soybean sprout fermented liquid Yes Soybean sprout fermented extract 1 mM312 bean sprout fermentation solution+caffeic acidYessoybean sprout fermented extract + caffeic acid1 mM313 cabbage fermentation solutionYesCabbage fermented extract1 mM314 cabbage fermentation solution+caffeic acidYesCabbage fermented extract +caffeic acid1 caffeic acid1mM3

[0236] The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and Phellodendron amurense extract was composed identically to the caffeic acid mixture in Table 12.

[0237]

[0238] 5-2. Serum collection and serum testing

[0239] The 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. Measurement of AST (Aspartic acid transaminase) and ALT (Alanine transaminase) in the blood was performed using a reagent for measuring AST and ALT (Asan pharmaceutical) using the Reitman-Frankel enzymatic method.

[0240]

[0241] 5-3. Effect of the mixture in an acute liver injury model

[0242] Serum ALT and AST levels are commonly measured clinically as biomarkers for liver health. In an experimental model of liver toxicity induced by CCl4, ALT and AST were measured after administration of each sample. As a result, serum ALT and AST levels decreased in all sample administration groups compared to the control group. In particular, it was confirmed that serum ALT and AST levels decreased in the combined administration groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, dendritic cell extract, or vitamin B12 compared to the group administered natural fermented liquid alone (Figs. 50 to 55).

[0243]

[0244] Example 6. Effect of improving dermatitis by a mixture of fermented liquid containing nitric oxide metabolites and glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12.

[0245] The experimental animals used in this experiment were 5-week-old male Balb / c mice purchased from Samtaco and used after a one-week acclimation period. The average weight of the Balb / c mice was 20.80 ± 0.82 g, and the sample was administered orally once daily for 12 days. The experimental animal breeding room was maintained at 23 ± 2°C with a 12-hour light / dark cycle, and the temperature and humidity were maintained at 50–60%.

[0246]

[0247] 6-1. DNFB production and atopic dermatitis induction and group composition

[0248] After acclimatization for one week, Balb / c mice were shaved on the back and allowed to acclimatize for three days. DNFB (1-fluoro-2,4-dinitrobenzene; Sigma, USA) was prepared by diluting 0.5% DNFB in a 4:1 ratio of acetone and olive oil. On the fourth day after hair removal, 40 μl of 0.5% DNFB was applied to the back for four days to induce skin sensitization. On the 12th day after sample administration after skin sensitization, 20 μl of 0.3% DNFB was applied to the ears (challenge) to induce contact dermatitis.

[0249] The experimental groups are as shown in Tables 13 and 14 below, and the number of animals in the groups was calculated using the minimum number that obscured statistical significance in accordance with the 3R principle. The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and phellodendron extract was composed identically to the caffeic acid mixture in Table 14. The normal group (CON) and the control group were administered DW, respectively, and the remaining groups were administered each sample orally once a day.

[0250] Atopic dermatitis experimental group composition of natural fermented liquid and glutathione (GSH) mixture Group Treatment Left Right Material Dose Normal group Normal Normal distilled water - 5 Control group Normal 0.3% DNFB distilled water - 5 NaNO2 Normal 0.3% DNFB sodium nitrite 1 mM 5 NaNO2 + 50% GSH Normal 0.3% DNFB sodium nitrite + Yeast extract 50% glutathione 1 mM 5 NaNO2 + 98% GSH Normal 0.3% DNFB sodium nitrite + 98% glutathione 1 mM 5 Lettuce fermented liquid Normal 0.3% DNFB Actuca sativa fermented extract 1 mM 5 Lettuce fermented liquid + 50% GSH Normal 0.3% DNFB Actuca sativa fermented extract + Yeast extract 50% glutathione 1 mM5 lettuce fermented extract+98% GSHNormal0.3% DNFBLactuca sativafermented extract + 98% glutathione1 mM5 soybean fermented extractNormal0.3% DNFBbean fermented extract1 mM5 soybean fermented extract+50% GSHNormal0.3% DNFBbean fermented extract +Yeast extract 50% glutathione1 mM5 soybean fermented extract+98% GSHNormal0.3% DNFBbean fermented extract +98% glutathione1mM5garlicfermentedextractNormal0.3% DNFBGarlicfermented extract1mM5garlicfermentedextract+50% GSHNormal0.3% DNFBGarlicfermented extract +Yeast extract 50% glutathione1mM5garlicfermentedextract+98% GSHNormal0.3% DNFBGarlic fermented extract +98% Glutathione 1 mM5 bean sprout fermentation liquid Normal 0.3% DNFBsoybean sprout fermented extract1 mM5콩나물발효액+50% GSHNormal0.3% DNFBsoybean sprout fermented extract + Yeast extract 50% glutathione1 mM5콩나물발효액+98% GSHNormal0.3% DNFBsoybean sprout fermented extract + 98% glutathione1 mM5양배추발효액Normal0.3% DNFBCabbage fermented extract1 mM5양배추발효액+50% GSHNormal0.3% DNFBCabbage fermented extract +Yeast extract 50% glutathione1 mM5양배추발효액+98% GSHNormal0.3% DNFBCabbage fermented extract +98% glutathione1 mM5유청발효액Normal0.3% DNFBWhey fermented extract1 mM5유청발효액+50% GSHNormal0.3% DNFBWhey fermented extract +Yeast extract 50% glutathione1 mM5유청발효액+98% GSHNormal0.3% DNFBWhey fermented extract +98% glutathione1 mM5.

[0251] Atopic dermatitis experimental group composition of natural fermented liquid and caffeic acid mixture Group Treatment Left Right Material Dose Normal group Normal Normal distilled water-3 Control group Normal 0.3% DNFB distilled water-3 NaNO2 Normal 0.3% DNFB sodium nitrite 1 mM 3 NaNO2 + caffeic acid Normal 0.3% DNFB sodium nitrite + caffeic acid 1 mM 3 Lettuce fermented liquid Normal 0.3% DNFB Actuca sativa fermented extract 1 mM 3 Lettuce fermented liquid + caffeic acid Normal 0.3% DNFB Actuca sativa fermented extract + caffeic acid 1 mM 3 Soybean fermented liquid Normal 0.3% DNFB bean fermented extract 1 mM 3 soybean fermented liquid + caffeic acid Normal 0.3% DNFB bean fermented extract + caffeic acid 1 mM3 Garlic fermented extract Normal 0.3% DNFBGarlic fermented extract 1 mM3 Garlic fermented extract + caffeic acid Normal 0.3% DNFBGarlic fermented extract + caffeic acid 1 mM3 Soybean sprout fermented extract Normal 0.3% DNFBsoybean sprout fermented extract 1 mM3 Soybean sprout fermented extract + caffeic acid Normal 0.3% DNFBsoybean sprout fermented extract + caffeic acid 1 mM3 Cabbage fermented extract Normal 0.3% DNFBCabbage fermented extract 1 mM3 Cabbage fermented extract + caffeic acid Normal 0.3% DNFBCabbage fermented extract + caffeic acid 1 mM3 Whey fermented extract Normal 0.3% DNFBWhey fermented extract 1 mM3 Whey fermented extract + caffeic acid Normal 0.3% DNFBWhey fermented extract + caffeic acid1mM3.

[0252] 6-2. Visual evaluation

[0253] Visual assessment is a clinical evaluation method commonly used in atopic dermatitis. It measures the severity of atopic dermatitis symptoms by summing the scores of each of the five items. The evaluation items are erythema, pruritus and dry skin, edema and excoriation, erosion, and lichenification. Each item is scored as no symptom (0 points), mild (1 point), moderate (2 points), and severe (3 points), and the total score is then given from a minimum of 0 to a maximum of 15 points.

[0254]

[0255] 6-3. Measurement of ear swelling and spleen index

[0256] To measure changes in skin tissue after DNFB application, both ears of mice were measured. Ear thickness was measured using vernier calipers (Mitutoyo Co.) at 12, 24, and 48-hour intervals for 3 days.

[0257] After measuring ear thickness, Balb / c mice were sacrificed, and the spleens were removed and weighed. The spleen index was calculated by dividing the spleen weight (mg) of Balb / c mice by the body weight (g).

[0258]

[0259] 6-4. Effect of the mixture in a contact dermatitis model

[0260] After the experiment, the area of ​​the right ear treated with the sample of each experimental group was observed to confirm the morphological changes of the atopic dermatitis lesions. In the control group treated only with DNFB, atopic skin changes such as erythema, dry skin, erosion, edema, and hematoma were confirmed compared to the left ear that was not treated (Figs. 56 to 61). However, in the sample administration group, atopic skin changes such as erythema, dry skin, erosion, edema, and hematoma were confirmed to be milder than in the control group compared to the left ear that was not treated with anything, and it was found that atopic skin changes were less in the group administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, dendritic stem extract, or vitamin B12 than in the group administered only the natural fermented liquid.

[0261] In addition, after the experiment, the morphological changes in the atopic dermatitis lesions were confirmed by observing the right ear area treated with the sample of each experimental group. As a result, the ear thickness was reduced in all sample-administered groups compared to the control group, and it was confirmed that the reduction was greater in the group administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12 than in the group administered only natural fermented liquid (Figs. 62 to 67).

[0262] In addition, after the experiment was over, the spleen weight was measured by body weight in each experimental group, and the spleen index was reduced in all sample administration groups compared to the control group, and was reduced more in the group administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron extract, or vitamin B12 than in the group administered only natural fermented liquid (Figs. 68 to 73).

[0263] In addition, when measuring the levels of TNF-α and IL-4 in serum, the levels of TNF-α and IL-4 were reduced in all sample administration groups compared to the control group, and lower levels of TNF-α and IL-4 were shown in the mixed administration groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, phellodendron amurense extract, or vitamin B12 than in the group administered only natural fermented liquid (Figs. 74 to 79).

Claims

1. A pharmaceutical composition for preventing or treating hypertension, enteritis, stress-related bowel syndrome, arthritis, acute liver damage or dermatitis, comprising as an active ingredient a natural fermented product in which nitric oxide metabolites are immobilized and stabilized; and any one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract and phellodendron extract.

2. A pharmaceutical composition according to claim 1, wherein the effective ingredient inhibits the production of carcinogenic N-nitrosamines in the stomach and promotes the production of nitric oxide in the intestines.

3. A pharmaceutical composition characterized in that the natural fermented product in which the nitric oxide metabolite is fixed and stabilized in the first paragraph is manufactured by a manufacturing method including a step of adding a fermentation strain to a nitrogen-containing natural product and fermenting the product 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.

4. A pharmaceutical composition according to claim 3, characterized in that the nitrogen-containing natural product is soybean, garlic, soybean sprouts, cabbage or whey.

5. A pharmaceutical composition according to claim 1, characterized in that the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.

6. A health functional food composition for preventing or improving hypertension, enteritis, stress-related bowel syndrome, arthritis, acute liver damage, or dermatitis, comprising as active ingredients a natural fermented product in which nitric oxide metabolites are immobilized and stabilized; and any one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and phellodendron amurense extract.

7. A health functional food composition according to claim 6, characterized in that the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

8. A veterinary composition for preventing or treating hypertension, enteritis, stress-related bowel syndrome, arthritis, acute liver damage, or dermatitis, comprising as an active ingredient a natural fermented product in which nitric oxide metabolites are immobilized and stabilized; and any one selected from the group consisting of glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and phellodendron extract.

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