Compositions for inhibiting the production of carcinogenic n-nitrosamines in the stomach and uses thereof

Abstract

The present invention relates to a composition for preventing, improving or treating hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury or dermatitis, which comprises a natural ferment 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 yellow lac extract as an effective ingredient, and the composition of the present invention generates S-nitrosoglutathione by gastric juice (strong acid conditions) of the stomach, and after being delivered to the small intestine or large intestine, can generate a prescribed concentration of nitric oxide within a prescribed time, thereby enabling a variety of applications.

Description

Technical Field

[0001] This invention relates to a composition for inhibiting the production of carcinogenic N-nitrosamines in the stomach and its use therein, and more specifically, to a composition that blocks the production of carcinogenic N-nitrosamines under strongly acidic conditions such as the stomach and generates a prescribed amount of nitric oxide in the intestinal environment, thereby enabling the absorption of nitric oxide metabolites in the body. Background Technology

[0002] Nitric oxide (NO) is a gaseous substance produced by the decomposition of NOS (NO synthase) found in bacteria, plants, and animals into the amino acid arginine. It is a highly reactive free radical, an unstable substance, hydrophobic, and rapidly oxidized into hydrophilic NO metabolites (referring to nitrite ions [NO2]). - ], nitrate ions [NO3] - The chemical properties of NO (see reactions 1-2 and 1-3) are as follows. Due to its extremely small size and hydrophobic nature, NO can move rapidly in aqueous solutions such as blood or body fluids. In particular, it can cross vascular barriers and cell membranes within a given duration (estimated to be less than one second). Furthermore, because the produced NO is highly reactive, it reacts with oxygen dissolved in blood or body fluids, or with oxygen bound to red blood cells, to convert (oxidize) water-soluble NO metabolites that have a longer duration of existence (see reactions 1-2 and 1-3). These metabolites then dissolve in water-soluble blood, allowing them to move to all parts of the body and be excreted.

[0003] NO2 is a NO metabolite. - NO2 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 it cannot cross organ barriers, vascular barriers, cell membranes, etc. However, NO2... - Under acidic and hypoxic conditions (e.g., venous blood conditions), NO2 is reduced to hydrophobic NO, thereby crossing organ barriers, vascular barriers, cell membranes, etc., and exhibiting the inherent physiological functions and pharmacological effects of NO (refer to reaction formula 2-1). Furthermore, NO2... - It reacts with vitamin C (ascorbic acid / ascorbate, hereinafter referred to as Asc) dissolved in blood or body fluids, and can thus be reduced to NO (see reaction formulas 2-2 and 2-3).

[0004] In the human body, NO is mainly produced in vascular endothelial cells through process 1-1), which is used to regulate the physiological function of dilating adjacent blood vessels. A portion of the NO produced is oxidized to NO2 through process 1-2). - It dissolves in the blood, thus moving to all parts of the body, and is reduced to NO at sites where NO is needed through processes in reaction 2-1) or reaction 2-2), thereby exhibiting the physiological and pharmacological effects of NO. Excess NO produced due to NOS or excessive NO2... - The NO produced during reduction is oxidized to NO3 through the process described in reactions 1-3). - These substances are then excreted through urine or sweat. Therefore, it can be considered that there was no excessive NO or NO2. - This results in significant toxicity to humans. However, during reactions 1-3), cyanosis may occur as red blood cells lose oxygen, but this symptom quickly disappears as the red blood cells re-bind to oxygen.

[0005] The NO produced in reaction 1-1) or reaction 2-1) reaches the cytoplasm of the target cell and chemically binds with the abundant glutathione (GSH) in the cytoplasm to produce S-nitrosoglutathione (GSH). S NO is stored intracellularly as glutathione (GSNO) for a specified period (see Reaction 3-1). When the cell needs NO, the stored GSNO combines with GSH to produce NO and glutathione disulfide (GSSG) (see Reaction 3-2). On the other hand, when there is an excess of GSNO intracellularly, the excess GSNO is reduced to GSH and NH3 by GSNO reductase (GSNOR), thereby reducing the potential toxicity of NO.

[0006] The amount of NO in the human body can be determined by NO2. - and NO3 - The total sum is used for prediction, but the actual usable NO concentration can be determined based on the amount of NO2 that can be converted into NO. - The concentration is used to predict the NO concentration in the blood. It is known that the blood of healthy individuals without disease contains approximately 100-500 nM (average 300 nM) of NO2. - However, in most cases, NO2 levels have been observed in the blood of people with diseases such as diabetes. - The concentration is lower than normal. There are several ways to reduce NO2 levels. -The concentration has increased to the normal range, but the most reasonable method is considered to be to reduce the NO2 in the blood through the intake of NO metabolites via diet or food. - The concentration remains within the normal range. In fact, humans and animals reduce their blood NO2 levels through diet. - The concentration was maintained within the normal range. Furthermore, an analysis of various reports on the effects of NO metabolites in laboratory animals showed that restricting NO metabolite intake led to diabetic symptoms in the animals, but conversely, oral administration of NO metabolites to diabetic animals improved their diabetes (Kina-Tanada). et al ., Diabetologia. 2017, 60(6): 1138-1151).

[0007] Humans and laboratory animals cannot directly absorb NO metabolites into their bodies. When humans ingest NO metabolites, the NO2 in the NO metabolites... - In the stomach, under the strong acidic conditions of gastric juice, NO is reduced to NO. The reduced NO then passes through the stomach wall and reaches the blood or body fluids, where it is oxidized to NO2. - This allows for absorption within the body (see reaction formula 4).

[0008] On the other hand, the ingested NO3 - (or other NO metabolites) are different from NO2 - NO3- can be produced by bacteria that coexist in the small and large intestines. - The reductase (NaRase) reduces NO to NO2. - Then, it is absorbed by the NO2 present in the periplasm between the bacterial outer membrane and the cell membrane. - NiRase (nitrite reductase) reduces NO to N2. Some of the reduced NO is absorbed into the body, while some NO undergoes a multi-step reduction process to become N2 and is excreted from the intestines (see reaction 5). Therefore, in order to utilize NO3... - Producing a sufficient amount of NO requires the intake of a large amount of NO3. - .

[0009] To increase the low NO (or NO2) levels in the blood - If NO metabolites are ingested orally at certain concentrations, the following three health problems may occur.

[0010] First, due to the highly acidic conditions of gastric juice, orally ingested NO2... -This can lead to the body absorbing excessive amounts of NO in a short period of time, which may cause a rapid drop in blood pressure, hypoglycemia, cyanosis, and other symptoms.

[0011] Second, due to the highly acidic conditions of gastric juice, orally ingested NO2... - It reacts with secondary amines (hereinafter referred to as R2N-H) to potentially produce N-nitrosamines, which are known carcinogens. N -nitrosamine, hereinafter referred to as R2N-NO (refer to reaction 6).

[0012] Third, orally ingested NO3 - It cannot be reduced in the stomach, but is reduced to NO in the small and large intestines and absorbed into the body. However, the degree of NO reduction varies depending on the individual's intestinal bacterial environment and dietary habits, and requires an intake of more than NO2. - More NO3 - Therefore, through the intake of NO3 - One cannot expect the body to absorb a prescribed amount of effective NO (refer to reaction formula 5).

[0013] Various studies have been conducted to suppress NO2 in the stomach, which is a highly acidic condition. - This leads to the formation of the potentially carcinogenic N-nitrosamine (R2N-NO). Many studies, particularly one (Shenoy et al., Cancer Lett. 1992; 65(3): 227-232), have shown that polyphenols (PhOH), cysteine ​​(Cys), and glutathione (GSH), which contains cysteine, in food materials react with NO2 better than secondary amines (R2N). - This combination can block the production of R2N-NO (refer to reaction 7-2 and reaction 7-3).

[0014] Reaction formulas 7-2) and 7-3) may have some advantages in blocking the formation of N-nitrosamines, but due to the intake of NO2... - Inhibiting NO production, which ultimately leads to poor NO absorption, may have a downside. However, if PhO-NO (represented in reaction 7-2) or GSNO (represented in reaction 7-3) produces NO in the intestinal environment, such as the small and large intestines, then NO absorption in the body can be achieved. GSNO produced in the stomach and reaching the intestines can react with GSH, which is abundant in the small intestine, to produce NO (see reaction 8). (Refer to reactions 7-3) and 8-1). If NO2 is ingested...- When GSH is ingested together, the production of N-nitrosamines in the stomach is blocked, and NO is produced in the intestines, which ultimately leads to better NO absorption in the body.

[0015] In our bodies, NO metabolites are reduced to NO under hypoxic or acidic conditions, regulating blood circulation by dilating blood vessels or promoting capillary formation (Lundberg et al., Nat RevDrug Discov. 2008, 7:156-167). The four major physiological functions of blood circulation are: ① oxygen and nutrient supply, ② carbon dioxide and waste excretion, ③ maintaining body temperature, and ④ immune function. Therefore, NO can be considered to affect nutrient absorption, waste excretion, and regulate body temperature and immune function. For example, NO can regulate blood flow by dilating blood vessels, thereby helping to restore high blood pressure to normal levels (Amaral et al., RedoxBiol. 2015, 5:340-346). Therefore, NO metabolites that can be converted into NO could be developed as a treatment for hypertension with fewer side effects. Furthermore, several recent studies have reported that NO metabolites have effects such as lowering blood sugar (Khalifi et al., Nitric Oxide. 2015, 44:24-30), promoting insulin secretion from β cells (Nystrom et al., FreeRadic Biol Med. 2012, 53:1017-1023), alleviating insulin resistance (Ohtake et al., Nitric Oxide. 2015, 44:31-38), and improving diabetic complications (Bahadoran et al., Nutr Metab (Lond). 2015, 12:16. doi:10.1186 / s12986-015-0013-6). Therefore, multiple studies are underway to develop NO metabolites into therapeutic agents for diabetes and its complications (Ghasemi et al., Nitric Oxide. 2017, 70:9-24). However, if NO metabolites are ingested orally for the purpose of treating various diseases, the three health problems mentioned above may occur.

[0016] On the other hand, Korean Patent No. 2256793 discloses "a method for preparing a natural fermentation composition containing a fixed nitric oxide precursor and a natural fermentation composition prepared by the method", and Korean Patent Publication No. 2019-0084592 discloses "a method for preparing a natural fermentation composition in which nitric oxide is fixed in a natural fermentation substance and a natural fermentation composition prepared by the method", but does not describe the "composition for inhibiting the production of carcinogenic N-nitrosamines in the stomach and its use" of the present invention. Summary of the Invention

[0017] (a) Technical problems to be solved This invention is made in accordance with the requirements described above. The inventors mix glutathione, caffeic acid, chlorogenic acid, coffee extract, and yellow lacquer into a natural fermentation product in which nitric oxide metabolites have been immobilized and stabilized. Dendropanax morbifera The results of oral administration of a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 (cyanocobalamin) to experimental animal models of hypertension, enteritis, irritable bowel syndrome, arthritis, liver disease, and atopic dermatitis confirmed that, compared with the experimental group administered natural ferments alone, the experimental group administered a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 had superior effects in improving blood pressure, enteritis, irritable bowel syndrome, arthritis, liver disease, and atopic dermatitis, thus completing the present invention.

[0018] (II) Technical Solution To address the aforementioned problems, the present invention provides a composition for the prevention, improvement, or treatment of hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury, or dermatitis, the composition comprising a natural fermentation 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 safflower extract as an active ingredient.

[0019] (III) Beneficial Effects The composition of the present invention mixes nitric oxide metabolites with glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 to block the production of carcinogenic N-nitrosamines in gastric juice (under strong acid conditions) and to produce a specified amount of nitric oxide in the small or large intestine, thereby enabling the absorption of nitric oxide metabolites in the body. Therefore, it is expected to have wide applications in related fields, such as the treatment and symptom improvement of diseases known to be improveable by nitric oxide, such as hypertension, enteritis, arthritis, liver disease, and atopic dermatitis. Attached Figure Description

[0020] Figure 1 A graph showing the disease activity index after administration of natural fermentation broth and glutathione samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0021] Figure 2 A graph to analyze the disease activity index after administration of natural fermentation broth and caffeic acid samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0022] Figure 3 A graph to analyze the disease activity index after administration of natural fermentation broth and chlorogenic acid samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0023] Figure 4 A graph to analyze the disease activity index after administration of natural fermentation broth and coffee extract samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0024] Figure 5 A graph showing the disease activity index after administration of natural fermentation broth and lacquer extract samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0025] Figure 6 A graph to analyze the disease activity index after administration of natural fermentation broth and vitamin B12 (cyanocobalamin) samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0026] Figure 7 A graph showing changes in intestinal length after administration of natural fermentation broth and glutathione samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0027] Figure 8 A graph showing changes in intestinal length after administration of natural fermentation broth and caffeic acid samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0028] Figure 9 A graph showing changes in intestinal length after administration of natural fermentation broth and chlorogenic acid samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0029] Figure 10 A graph showing changes in intestinal length after administration of natural fermentation broth and coffee extract samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0030] Figure 11 A graph showing changes in intestinal length after administration of natural fermentation broth and lacquer extract samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0031] Figure 12 A graph showing changes in intestinal length after administration of natural fermentation broth and vitamin B12 samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0032] Figure 13 A graph showing the serum levels of TNF-α(a) and IL-1β(b) after administration of natural fermentation broth and glutathione samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0033] Figure 14 A graph showing the serum levels of TNF-α(a) and IL-1β(b) after administration of natural fermentation broth and caffeic acid samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0034] Figure 15 A graph showing the serum levels of TNF-α(a) and IL-1β(b) after administration of natural fermentation broth and chlorogenic acid samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0035] Figure 16A graph showing the serum levels of TNF-α(a) and IL-1β(b) after administration of natural fermentation broth and coffee extract samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0036] Figure 17 A graph showing the serum levels of TNF-α(a) and IL-1β(b) after administration of natural fermentation broth and lacquer extract samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0037] Figure 18 A graph showing the serum levels of TNF-α(a) and IL-1β(b) after administration of natural fermentation broth and vitamin B12 samples in an ulcerative colitis model. p<0.05 (compared to the control group).

[0038] Figure 19 This is a schematic diagram of an experiment used to confirm the improvement effect of irritable bowel syndrome. WAS refers to water avoidance stress.

[0039] Figure 20 The results of fecal quantity measurements in each experimental group after administration of natural fermentation broth and glutathione samples in an irritable bowel syndrome model are shown. (a) shows the average daily fecal quantity, and (b) shows the cumulative fecal quantity up to the end of the experiment.

[0040] Figure 21 The results of fecal quantity measurements in each experimental group after administration of natural fermentation broth and caffeic acid samples in an irritable bowel syndrome model are shown. (a) shows the average daily fecal quantity, and (b) shows the cumulative fecal quantity up to the end of the experiment.

[0041] Figure 22 The results of fecal quantity measurements in each experimental group after administration of natural fermentation broth and chlorogenic acid samples in an irritable bowel syndrome model are shown. (a) shows the average daily fecal quantity, and (b) shows the cumulative fecal quantity up to the end of the experiment.

[0042] Figure 23 The results of fecal quantity measurements in each experimental group after administration of samples of natural fermentation broth and coffee extract in an irritable bowel syndrome model are shown. (a) shows the average daily fecal quantity, and (b) shows the cumulative fecal quantity up to the end of the experiment.

[0043] Figure 24The results of fecal quantity measurements in each experimental group after administration of natural fermentation broth and yellow lacquer extract samples in an irritable bowel syndrome model are shown. (a) shows the average daily fecal quantity, and (b) shows the cumulative fecal quantity up to the end of the experiment.

[0044] Figure 25 The results of fecal quantity measurements in each experimental group after administration of natural fermentation broth and vitamin B12 samples in an irritable bowel syndrome model are shown. (a) shows the average daily fecal quantity, and (b) shows the cumulative fecal quantity up to the end of the experiment.

[0045] Figure 26 The results show the changes in blood pressure before and after administration of natural fermentation broth and glutathione samples in a hypertension model.

[0046] Figure 27 The results show the changes in blood pressure before and after administration of natural fermentation broth and caffeic acid samples in a hypertension model.

[0047] Figure 28 The results show the changes in blood pressure before and after administration of natural fermentation broth and chlorogenic acid samples in a hypertension model.

[0048] Figure 29 The results show the changes in blood pressure before and after administration of natural fermentation broth and coffee extract samples in a hypertension model.

[0049] Figure 30 The results show the changes in blood pressure before and after administration of natural fermentation broth and lacquer extract samples in a hypertension model.

[0050] Figure 31 The results show the changes in blood pressure before and after administration of natural fermentation broth and vitamin B12 samples in a hypertension model.

[0051] Figure 32 The results show the changes in joint swelling thickness after administration of natural fermentation broth and glutathione samples in an arthritis-inducing model using monosodium iodoacetate (MIA). p<0.05 (compared to the control group).

[0052] Figure 33 The results show the changes in joint swelling thickness after administration of natural fermentation broth and caffeic acid samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0053] Figure 34The results show the changes in joint swelling thickness after administration of natural fermentation broth and chlorogenic acid samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0054] Figure 35 The results show the changes in joint swelling thickness after administration of natural fermentation broth and coffee extract samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0055] Figure 36 The results show the changes in joint swelling thickness after administration of natural fermentation broth and lacquer extract samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0056] Figure 37 The results show the changes in joint swelling thickness after administration of natural fermentation broth and vitamin B12 samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0057] Figure 38 The results show the arthritis index after administration of natural fermentation broth and glutathione samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0058] Figure 39 The results show the arthritis index after administration of natural fermentation broth and caffeic acid samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0059] Figure 40 The results show the arthritis index after administration of natural fermentation broth and chlorogenic acid samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0060] Figure 41 The results show the arthritis index after administration of natural fermentation broth and coffee extract samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0061] Figure 42 The results show the arthritis index after administration of natural fermentation broth and lacquer extract samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0062] Figure 43 The results show the arthritis index after administration of natural fermentation broth and vitamin B12 samples in an arthritis-inducing model using MIA. p<0.05 (compared to the control group).

[0063] Figure 44 A graph showing the serum levels of TNF-α (a), metallopeptidase-9 (b), and prostaglandin E2 (PGE2) (c) after administration of natural fermentation broth and glutathione samples in an arthritis-inducing model using MIA.

[0064] Figure 45 A graph showing the serum levels of TNF-α (a), metallopeptidase-9 (b), and PGE2 (c) after administration of natural fermentation broth and caffeic acid samples in an arthritis-inducing model using MIA.

[0065] Figure 46 A graph showing the serum levels of TNF-α (a), metallopeptidase-9 (b), and PGE2 (c) after administration of natural fermentation broth and chlorogenic acid samples in an arthritis-inducing model using MIA.

[0066] Figure 47 A graph showing the serum levels of TNF-α (a), metallopeptidase-9 (b), and PGE2 (c) after administration of natural fermentation broth and coffee extract samples in an arthritis-inducing model using MIA.

[0067] Figure 48 A graph showing the serum levels of TNF-α (a), metallopeptidase-9 (b), and PGE2 (c) after administration of natural fermentation broth and lacquer extract samples in an arthritis-inducing model using MIA.

[0068] Figure 49 A graph showing the serum levels of TNF-α (a), metallopeptidase-9 (b), and PGE2 (c) after administration of natural fermentation broth and vitamin B12 samples in an arthritis-inducing model using MIA.

[0069] Figure 50 The results show the serum ALT and AST levels measured in an acute liver injury model after administration of natural fermentation broth and glutathione samples. p<0.05 (compared to the control group).

[0070] Figure 51 The results show the serum ALT and AST levels measured in an acute liver injury model after administration of natural fermentation broth and caffeic acid samples. p<0.05 (compared to the control group).

[0071] Figure 52 The results show the serum ALT and AST levels measured in an acute liver injury model after administration of natural fermentation broth and chlorogenic acid samples. p<0.05 (compared to the control group).

[0072] Figure 53 The results show the serum ALT and AST levels measured after administration of natural fermentation broth and coffee extract samples in an acute liver injury model. p<0.05 (compared to the control group).

[0073] Figure 54 The results show the serum ALT and AST levels measured in an acute liver injury model after administration of natural fermentation broth and lacquer extract samples. p<0.05 (compared to the control group).

[0074] Figure 55 The results show the serum ALT and AST levels after administration of natural fermentation broth and vitamin B12 samples in an acute liver injury model. p<0.05 (compared to the control group).

[0075] Figure 56 The results show the morphological changes observed in the right ear after administration of natural fermentation broth and glutathione samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0076] Figure 57 The results show the morphological changes observed in the right ear after administration of natural fermentation broth and caffeic acid samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0077] Figure 58 The results show the morphological changes observed in the right ear after administration of natural fermentation broth and chlorogenic acid samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0078] Figure 59The results show the morphological changes observed in the right ear after administration of samples of natural fermentation broth and coffee extract in an atopic dermatitis model. p<0.05 (compared to the control group).

[0079] Figure 60 The results show the morphological changes observed in the right ear after administration of samples of natural fermentation broth and yellow lacquer extract in an atopic dermatitis model. p<0.05 (compared to the control group).

[0080] Figure 61 The results show the morphological changes observed in the right ear after administration of natural fermentation broth and vitamin B12 samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0081] Figure 62 The results show the changes in thickness of the right ear after administration of natural fermentation broth and glutathione samples in an atopic dermatitis model.

[0082] Figure 63 The results show the changes in thickness of the right ear after administration of natural fermentation broth and caffeic acid samples in an atopic dermatitis model.

[0083] Figure 64 The results show the changes in thickness of the right ear after administration of natural fermentation broth and chlorogenic acid samples in an atopic dermatitis model.

[0084] Figure 65 The results show the changes in thickness of the right ear after administration of natural fermentation broth and coffee extract samples in an atopic dermatitis model.

[0085] Figure 66 The results show the changes in thickness of the right ear after administration of natural fermentation broth and lacquer extract samples in an atopic dermatitis model.

[0086] Figure 67 The results show the changes in thickness of the right ear after administration of natural fermentation broth and vitamin B12 samples in an atopic dermatitis model.

[0087] Figure 68 The results of spleen index at the end of the experiment for each experimental group after administration of natural fermentation broth and glutathione samples in an atopic dermatitis model are shown. p<0.05 (compared to the control group).

[0088] Figure 69 The results of spleen index at the end of the experiment for each experimental group after administration of natural fermentation broth and caffeic acid sample in an atopic dermatitis model are shown. p<0.05 (compared to the control group).

[0089] Figure 70 The results of spleen index at the end of the experiment for each experimental group after administration of natural fermentation broth and chlorogenic acid samples in an atopic dermatitis model are shown. p<0.05 (compared to the control group).

[0090] Figure 71 The results of spleen index at the end of the experiment for each experimental group after administration of natural fermentation broth and coffee extract samples in an atopic dermatitis model are shown. p<0.05 (compared to the control group).

[0091] Figure 72 The results of spleen index at the end of the experiment for each experimental group after administration of natural fermentation broth and lacquer extract samples in an atopic dermatitis model are shown. p<0.05 (compared to the control group).

[0092] Figure 73 The results of spleen index at the end of the experiment for each experimental group after administration of natural fermentation broth and vitamin B12 samples in an atopic dermatitis model are shown. p<0.05 (compared to the control group).

[0093] Figure 74 The results show the serum levels of TNF-α(a) and IL-4(b) at the end of the experiment in each experimental group after administration of natural fermentation broth and glutathione samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0094] Figure 75 The results show the serum levels of TNF-α(a) and IL-4(b) at the end of the experiment in each experimental group after administration of natural fermentation broth and caffeic acid samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0095] Figure 76 The results show the serum levels of TNF-α(a) and IL-4(b) at the end of the experiment in each experimental group after administration of natural fermentation broth and chlorogenic acid samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0096] Figure 77 The results show the serum levels of TNF-α(a) and IL-4(b) at the end of the experiment in each experimental group after administration of natural fermentation broth and coffee extract samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0097] Figure 78 The results show the serum levels of TNF-α(a) and IL-4(b) at the end of the experiment in each experimental group after administration of natural fermentation broth and lacquer extract samples in an atopic dermatitis model. p<0.05 (compared to the control group).

[0098] Figure 79 The results show the serum levels of TNF-α(a) and IL-4(b) at the end of the experiment in each experimental group after administration of natural fermentation broth and vitamin B12 samples in an atopic dermatitis model. p<0.05 (compared to the control group). Detailed Implementation

[0099] This invention provides a pharmaceutical composition for the prevention or treatment of hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury, or dermatitis, the composition comprising a natural fermentation 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 safflower extract as an active ingredient.

[0100] In the pharmaceutical composition of the present invention, the active ingredient can inhibit the production of carcinogenic N-nitrosamines in the stomach and promote the production of nitric oxide in the intestine.

[0101] The concentration of nitric oxide in the human body can be determined based on nitrite ions (NO2). - ) and nitrate ions (NO3) - The total amount of nitric oxide can be predicted by adding the two components, but the actual usable concentration of nitric oxide can be determined based on NO2, which can be converted into nitric oxide (NO). - Concentration is used to predict NO concentration in the blood. NO2 is a metabolite of nitric oxide. - NO2 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 it cannot cross organ barriers, vascular barriers, cell membranes, etc. However, if NO2... - When NO2 is reduced to lipid-soluble NO by nitrite reductase or under acidic conditions, it can cross organ barriers, vascular barriers, cell membranes, etc. Therefore, in order to reduce NO2... - To reach target cells and exhibit specific pharmacological effects, or to be absorbed into the body in the stomach or intestines, it must be reduced to NO.

[0102] To increase the concentration of low-level nitric oxide metabolites in the blood or to achieve the pharmacological effects of nitric oxide, oral ingestion of nitric oxide metabolites (especially NO2) is recommended. - If NO2 is ingested orally, the following two health problems may occur. First, due to the highly acidic conditions of gastric juice, the ingested NO2 may cause adverse reactions. - This can lead to excessive NO absorption in a short period, potentially causing a rapid drop in blood pressure, hypoglycemia, and cyanosis. Secondly, due to the highly acidic conditions of gastric juices, orally ingested NO2... - It can react with secondary amines to produce carcinogenic N-nitrosamines.

[0103] The pharmaceutical composition of the present invention utilizes nitric oxide metabolites (NO2) contained in natural fermentation products. - A composition is developed that allows for the absorption of nitric oxide by the body through a chemical reaction between glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and lacquer extract, under the strongly acidic conditions of the stomach, to form S-nitrosoglutathione (GSNO), thereby blocking the production of N-nitrosamines (R2N-NO) and generating a specified amount of nitric oxide (NO) in the intestinal environment. In this invention, "coffee extract" is used to refer to the same substance as coffee extract, and "lacquer extract" refers to lacquer tree extract.

[0104] In the pharmaceutical composition of the present invention, the immobilized and stabilized natural ferment of the nitric oxide metabolite can be prepared by a method comprising the steps of adding a fermentation strain to a nitrogen-containing natural material and fermenting it for 2 to 30 days at a temperature of 18°C ​​to 35°C and a dissolved oxygen concentration of 0.03 mg / L to 0.1 mg / L, but not limited thereto. The nitrogen-containing natural material can be soybean, garlic, bean sprouts, cabbage, or whey, but is not limited thereto. A detailed method for preparing the natural ferment of the present invention can be found in Korean Patent No. 2129038.

[0105] The natural ferment of the present invention can be fermented with 0.01 to 3 parts by weight of a fermentation strain relative to 100 parts by weight of a mixture of nitrogen-containing natural material and water. Preferably, the nitrogen-containing natural material and water can be mixed at a weight ratio of 1:0.5 to 1, and then 0.01 to 3 parts by weight of a fermentation strain can be added relative to 100 parts by weight of the mixture for fermentation, but it is not limited thereto.

[0106] Furthermore, the fermentation can be carried out at a temperature of 10°C to 40°C, preferably at a temperature of 18°C ​​to 35°C, more preferably at a temperature of 20°C to 33°C, and even more preferably at a temperature of 25°C to 33°C, but is not limited thereto.

[0107] Furthermore, the fermentation can be carried out under dissolved oxygen concentration conditions of 0.01 mg / L to 0.1 mg / L, preferably, under dissolved oxygen concentration conditions of 0.03 mg / L to 0.1 mg / L, but is not limited thereto.

[0108] Furthermore, the fermentation can take anywhere from 2 to 30 days, but is not limited to this.

[0109] Furthermore, the fermentation can be terminated when the chemical oxygen demand is 200–700 mg / L, but is not limited to this.

[0110] Furthermore, preferably, the fermentation strain can be Bacillus subtilis with the preservation number KCTC12501BP (…). Bacillus subtilis )-Chun Hyun Su, but not limited to this.

[0111] Furthermore, in the pharmaceutical composition of the present invention, NO2 - It is estimated that the immobilized and stabilized natural fermentation product of the nitric oxide metabolite preferably contains 0.1 mM to 10 mM, but is not limited thereto.

[0112] In this specification, the term "nitric oxide metabolite" refers to nitrite (NO2). - ), R-NO2 - R-NO, RO-NO, nitrates (NO3) - R-NO3 - GS-NO (or GSNO), GS-NO2, etc., are collective terms. R- refers to the removal of a hydrogen atom from the carbon chain of polyphenols, fatty acids, etc.; RO- refers to the removal of a hydrogen atom from ROH; and GS- refers to the removal of a hydrogen atom from the cysteine ​​residue of glutathione (GSH). In other words, it refers to the functional group that nitric oxide or nitrite ions can bind to. Nitric oxide metabolites are converted to nitric oxide under acidic and reducing conditions. Under strongly acidic conditions such as those in the stomach, nitric oxide metabolites can be converted by hydrogen ions (H+). + Nitric oxide is produced by reduction. Under weakly acidic conditions such as the intestines (small intestine and 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.

[0113] In particular, GSNO, known as a nitric oxide metabolite or nitric oxide conjugate, reacts with glutathione (GSH) secreted from the liver via the bile duct or from intestinal bacteria, thereby safely producing nitric oxide (refer to reaction formula 8-1). As described above, nitric oxide (NO) produced in the intestinal environment permeates into the body from the intestinal environment and is oxidized to nitric oxide metabolites (NO2). - ), thereby reaching body fluids or blood vessels (refer to reaction formula 8-3)).

[0114] Furthermore, in the pharmaceutical composition of 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. It is an endogenous reducing agent produced in plants, animals, fungi, and some bacteria and archaea. In vivo or intracellularly, glutathione prevents cell damage by binding to free radicals, peroxides, toxic substances, heavy metals, etc. Furthermore, GSH can react with nitric oxide (NO) to produce S-nitrosoglutathione (GSNO). GSNO produced through this process transports NO to the required sites in the body via blood vessels and stores NO in cells for a specified time, breaking it down into NO when needed to exert the pharmacological effects of NO.

[0115] Furthermore, in the pharmaceutical composition of the present invention, the caffeic acid or chlorogenic acid is a phenolic compound that promotes the reduction of nitric oxide metabolites in natural ferments to nitric oxide. Coffee extract is known to be rich in caffeic acid and chlorogenic acid, and yellow lacquer ( Dendropanax morbifera The extract also contains a variety of polyphenolic compounds. Furthermore, when cyanocobalamin, the vitamin B12 compound used in this invention, enters the body, cobalt is reduced from a trivalent state to a divalent or monovalent state, and then converted into adenosylcobalamin or methylcobalamin, which acts as a coenzyme.

[0116] The pharmaceutical compositions of the present invention may also contain suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions.

[0117] Furthermore, the pharmaceutical compositions of the present invention can be formulated into oral dosage forms such as granules, tablets, and capsules using conventional methods, but are not limited thereto.

[0118] The carrier, excipients, and diluents that may be included in the pharmaceutical compositions of the present invention can be various compounds or mixtures including lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, sodium alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. During formulation, commonly used fillers, expanders, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients are used. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., which are formulated by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the fermented larval extract. Furthermore, 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, and syrups. Besides commonly used simple diluents such as water and liquid paraffin, they can contain various excipients, such as wetting agents, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used as non-aqueous solvents or suspensions. Semi-synthetic fatty acid esters, polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, and glycerin gelatin can be used as the base for suppositories.

[0119] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All routes of administration are conceivable, and preferably, administration can be carried out orally or rectally, but is not limited thereto.

[0120] The pharmaceutical compositions of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered once or multiple times. Considering all the above factors, it is important to administer the drug in the smallest amount possible to achieve the greatest effect without side effects, which can be readily determined by those skilled in the art.

[0121] Specifically, the pharmaceutically effective amount of the pharmaceutical composition of the present invention can vary depending on the patient's age, sex, and weight, and is usually administered at a dose of 2 to 4 mg per kg of body weight daily or every other day, preferably 3 mg, which can be divided into 1 to 3 doses per day. However, since the dosage can be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage does not limit the scope of the present invention in any way.

[0122] This invention also provides a health-promoting functional food composition for preventing or improving hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury, or dermatitis, the composition comprising a natural fermentation 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 safflower extract as an active ingredient.

[0123] In the health-functional food composition of the present invention, the nitric oxide metabolite is immobilized and stabilized by natural fermentation products and glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract and lacquer extract as described above.

[0124] There are no particular limitations on the types of health functional foods mentioned. Examples of health functional foods containing the aforementioned health functional food composition include meat, sausage, bread, chocolate, candy, snacks, biscuits, pizza, ramen, other noodles, chewing gum, rice cakes, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and multivitamins, covering all health functional foods in the traditional sense.

[0125] The aforementioned health functional foods can be prepared in any dosage form selected from powders, granules, pills, tablets, capsules, candies, syrups, effervescent tablets, and beverages, but are not limited thereto.

[0126] When using the health-functional food composition of the present invention as a food additive, the composition can be added directly or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The active ingredient can be used appropriately according to its intended use (prevention or improvement). Generally, in the preparation of food or beverages, the amount of the composition of the present invention added is 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw materials. However, in cases of long-term intake for health control purposes, amounts within a range where there are no safety concerns can be used.

[0127] The health-functional food of the present invention comprises ingredients commonly added during food preparation, such as proteins, carbohydrates, fats, nutrients, and flavoring agents. For example, when prepared as a beverage, in addition to the active ingredients, it may contain natural carbohydrates or flavoring agents as additional 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., sematriol, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.). In addition to the health-functional food composition, it may also contain various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages, etc.

[0128] The present invention also provides a veterinary composition for the prevention or treatment of hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury, or dermatitis, said composition comprising a natural fermentation 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 safflower extract as an active ingredient.

[0129] In the veterinary composition of the present invention, the nitric oxide metabolite is an immobilized and stabilized natural ferment, and glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and lacquer extract are as described above.

[0130] The veterinary compositions 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 compositions of the present invention may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, sodium alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, cetyl alcohol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

[0131] The veterinary composition of the present invention may further include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. The veterinary composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to animals. The dosage form may be powder, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

[0132] The effective amount of the veterinary composition of the present invention can be appropriately selected according to the individual animal. The effective ingredient of the present invention, depending on the severity of the disease or symptom, the individual's age, weight, health status, or sex, can be determined based on factors including sensitivity, route of administration, duration of administration, other compositions used in combination with or concurrently with the stated composition, and other factors known in the physiological or veterinary fields.

[0133] In this specification, the term "fermentation broth" is used interchangeably with "natural fermentation product in which nitric oxide metabolites are immobilized and stabilized".

[0134] The present invention will now be described in detail through embodiments. However, the following embodiments are merely examples of the present invention, and the scope of the present invention is not limited to the following embodiments.

[0135] 1. Experimental Methods 1-1) Preparation of reagents and samples The experiment used fermented broths of lettuce, soybeans, garlic, bean sprouts, cabbage, or whey prepared by HumanEnos Co., Ltd., which contained 1 mM of NO2. -The fermentation broth was prepared using 1 mM sodium nitrite (NaNO2) as a control group for the natural fermentation product. 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 all purchased from Sigma-Aldrich and prepared at a concentration of 1 mM, then mixed with the natural fermentation broth or sodium nitrite at a 1:1 weight ratio. Dried yeast containing glutathione was purchased from Vision Biochem Co., Ltd., and used at a concentration of 1 mM based on glutathione levels. For the coffee extract, 20 L of purified water was added to 500 g of ground green coffee beans, and the mixture was extracted at 94 °C for 10 minutes, followed by centrifugation at 4500 rpm for 5 minutes. The supernatant was then used for the experiment. For the yellow lacquer extract, 4 L of fermented alcohol was added to 200 g of crushed yellow lacquer tree stems and leaves, and extraction was carried out at 88°C for 2 hours. The extract was then centrifuged at 4500 rpm for 5 minutes, and the supernatant was used for experiments. The coffee extract and yellow lacquer extract were mixed with the natural ferment at a 1:1 weight ratio and used in experiments.

[0136] The lettuce, soybean, garlic, bean sprouts, cabbage, or whey fermentation liquid mentioned refers to a concentrated fermentation liquid prepared by the preparation method disclosed in Korean Patent No. 2129038. In short, each raw material and water are mixed at a weight ratio of 1:0.5 to 1, and then 0.01 to 3 parts by weight of fermentation strain are added relative to 100 parts by weight of the mixture. Fermentation is carried out 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. Solid-liquid separation is performed using a centrifuge, and the resulting supernatant is concentrated at a temperature of 20 to 30°C with a stirring speed of 100 to 300 rpm for 24 to 48 hours.

[0137] 1-2) Determination of nitric oxide metabolite concentration The total amount of nitric oxide metabolites (referring to NO2) contained in the fermentation broth - and NO3 - The concentration of the sum of the concentrations of NO and NH3 is denoted as NO. x By adding nitrate reductase, all NO3 contained in the concentrate is removed. - Reduced to NO2 - and measure NO2 - The total concentration is used to determine the total content or concentration of nitric oxide metabolites.

[0138] To be more specific, the NO2 in the fermentation broth -The total content determination method is as follows: First, nitrate reductase is added to the fermentation broth for 1 hour for reduction. The broth is then diluted 1000 times with distilled water and centrifuged at 3000 rpm for 3 minutes to remove insoluble polyphenols and dietary fiber. Then, 50 μl of the clear supernatant is taken as the test sample. 50 μl of NO2... - Standard solution and 50 μl of test sample were inoculated into 96-well plates, and 100 μl of test solution was added [a 30% acetic acid solution containing 1% sulfanilamide and a solution containing 0.1% N-(1-naphthyl)ethylenediamine (... N A solution of 60% acetic acid (1-naphthyl)ethylenediamine was mixed in a 1:2 ratio and allowed to stand at room temperature for 10 minutes to induce a colorimetric reaction. Next, the absorbance of each well was measured at 570 nm using a multi-plate reader, and NO was calculated by comparing the absorbance of the standard solution with that of the sample. x concentration.

[0139] 2. Statistical Analysis All results obtained in each experiment are expressed as mean ± standard deviation (SD). The significance between two experimental groups was analyzed using a t-test. In cases with more than two experimental groups, significance was analyzed using one-way ANOVA, followed by a Duncan post-hoc test. A p-value not greater than 0.05 (p < 0.05) was considered statistically significant.

[0140] Example 1. The effect of fermentation broth containing nitric oxide metabolites and mixed samples containing glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 on improving enteritis. The experimental animals used in this experiment were 6-week-old male Balb / c mice purchased from Samtako (Korea) and acclimatized for one week. The average weight of the Balb / c mice was 20–24 g. The samples were administered orally once daily for 3 weeks. The animal housing was maintained with regulated light and darkness at 12-hour intervals, a temperature of 23±2℃, and humidity of 50–60%.

[0141] 1-1. Composition and Induction of Ulcerative Colitis The laboratory animals were classified as shown in Tables 1 and 2 below. The experimental group containing the mixture of chlorogenic acid, vitamin B12, coffee extract, and safflower extract had the same composition as the caffeic acid mixture in Table 2. As mentioned above, the coffee extract and safflower extract were mixed at the same weight ratio as the natural fermentation broth, and 1 mM of dose refers to NO2 in the natural fermentation broth. -concentration.

[0142] Ulcerative colitis was induced by allowing all experimental groups, except the control group, to freely ingest 3.0% dextran sulfate sodium (DSS) for 5 days. Following the 3R principle, the minimum number of animals required to form a group was used to calculate the number of animals in each group.

[0143] Table 1 The experimental group for ulcerative colitis consisted of a mixture of natural fermentation broth and glutathione (GSH). Table 2 Composition of the experimental group for ulcerative colitis using a mixture of natural fermentation broth and caffeic acid. 1-2. Measuring the disease activity index Intestinal diseases are assessed by symptoms such as weight loss, diarrhea accompanied by bleeding and mucus, and shortening of the colon. Based on previous research, the Disease Activity Index (DAI) has three main clinical symptoms: 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 granular stool in the rectum and the persistent presence of soft stool. Rectal bleeding differs from diarrhea with bleeding or complete rectal bleeding, and other types of diarrhea. In this invention, the DAI is calculated as follows.

[0144] DAI = Weight loss score + Diarrhea score + Rectal bleeding score The medical variables used were comprehensive functional tests that were similar to the medical symptoms of a person suffering from ulcerative colitis (Table 3).

[0145] Table 3 Disease activity index 1-3. Measuring intestinal length and determining serum cytokines After the experiment, all experimental groups underwent orbital vein blood collection and serum separation under respiratory anesthesia. The abdomen was then incised, and the portion of the large intestine extending to the cecum was cut and removed to determine intestinal length. Serum cytokine levels were measured using an ELISA kit (R&D Systems, USA) to determine TNF-α and IL-1β production. 50 μl of assay diluent (ADS) was added to each well containing antibody, followed by 50 μl of sample. The wells were incubated at room temperature for 2 hours, then washed four times with 300 μl of wash buffer. After washing, 100 μl of mouse TNF-α and IL-1β antibodies were added to each well, and the wells were incubated at room temperature for 2 hours before being removed and washed four times. Add 100 μl of substrate solution to each well, let stand at room temperature for 30 minutes, then add 100 μl of stop solution, and measure the absorbance at 450 nm using an ELISA reader (SPECTRA max M2, USA) from Molecular Devices.

[0146] 1-4. Effects of the mixture on the ulcerative colitis model The DSS colitis model exhibits clinical symptoms characterized by weight loss, diarrhea, and bloody stools. The results of DAI assays on the impact on these clinical symptoms are as follows: Figures 1 to 6 As shown. No symptoms were detected in the normal group, and the overall DAI was lower in the sample-treated groups compared to the control group (DSS group). In particular, it was confirmed that the DAI was significantly lower in the groups treated with a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 compared to the groups treated with natural fermentation broth alone.

[0147] Furthermore, the results of removing the large intestine of mice sacrificed on day 5 after DSS administration and confirming its length showed that the intestinal length of all administered samples was longer than that of the control group. Compared with the NaNO2 administration group, the intestinal length of the natural fermentation broth administration group was longer. Compared with the natural fermentation broth administration group alone, the intestinal length of the mixed administration groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 was longer, closer to the normal group. Figures 7 to 12 ).

[0148] Furthermore, in an animal model of colitis induction, the results of measuring inflammatory cytokines in the blood showed that, compared with the control group, the levels of TNF-α and IL-1β, indicators of inflammation, were reduced in all sample treatment groups. In particular, the lowest levels were observed in the mixed treatment groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 compared with the natural fermentation broth treatment groups alone. Figures 13 to 18 ).

[0149] Example 2. Improvement of irritable bowel syndrome based on fermentation broth containing nitric oxide metabolites and mixed samples containing glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12. The experimental animals used in this experiment were 8-week-old female Wistar rats purchased from the Samtako family and acclimatized for one week before use. The average weight of the Wistar rats was 180.5 ± 0.5 g. The samples were administered orally once daily. The light and darkness in the animal housing were adjusted at 12-hour intervals, and the temperature was maintained at 23 ± 2℃, with humidity maintained at 50–60%.

[0150] 2-1. Composition and Water Avoidance Stress (WAS) To observe changes in colonic motility induced by the mixture of the present invention, for the remaining groups other than the normal group, the animals were placed on a platform after the WAS tank was filled with water to apply intermittent stress, and then the amount of feces was measured. Daily water intake and feed intake were measured for each sample during the administration period. Furthermore, the WAS was conducted for a total of 8 days, divided into two sessions, with 15 animals per session for 1 hour, taking into account a 60-minute interval between daily changes. Figure 19 Under these circumstances, the amount of feces is measured.

[0151] The composition of the groups is shown in Tables 4 and 5 below. The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and safflower extract had the same composition as the caffeic acid mixture in Table 5. Exposure to WAS was used to induce symptoms similar to diarrhea-like irritable bowel syndrome (increased bowel movements) and enhanced mucosal immunity, and it was observed whether administration of each sample could alleviate these symptoms.

[0152] Table 4 Experimental composition of irritable bowel syndrome using a mixture of natural fermentation broth and glutathione (GSH) Table 5 Experimental composition of irritable bowel syndrome using a mixture of natural fermentation broth and caffeic acid. 2-2. Effects of the mixture on the irritable bowel syndrome model The results of fecal quantity measurements for each sample under WAS-induced stress are as follows: Figures 20 to 25As shown in the figure. Compared with the control group, the amount of stool was reduced in all sample treatment groups. The amount of stool was even reduced in the mixed treatment groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract or vitamin B12 compared with the natural fermentation broth treatment group alone.

[0153] Example 3. Blood pressure improvement effects of fermentation broth containing nitric oxide metabolites and mixed samples containing glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12. The experimental animals used in this experiment were 7-week-old male Sprague Dawley rats (hereinafter referred to as SD rats) purchased from the Samtako family and acclimatized for one week before use. The average weight of the SD rats was 198.95±2.10g. Samples were administered orally before the experiment. The light and darkness in the animal housing were adjusted at 12-hour intervals, the temperature was maintained at 23±2℃, and the humidity was maintained at 50-60%.

[0154] 3-1. Construction of a DOCA-salt hypertensive rat model and its composition For the establishment of a hypertension model, 8-week-old male SD rats were administered deoxycorticosterone acetate (DOCA; TCI, Japan) twice weekly via intraperitoneal administration for 4 weeks. Hypertension was induced using 1% sodium chloride aqueous solution as drinking water. Distilled water (DW) was administered to the control group, while each sample was administered to the experimental groups. To confirm the induction of hypertension, blood pressure was measured using a mouse tail blood pressure system (MK-2000STS, Muromachi Machinery Co., Ltd., Japan), selecting animals with systolic blood pressure above 130 mmHg. Blood pressure was measured in real-time for 55 minutes per animal after administering the sample to each group. Rats were placed in a restraint frame and fixed. A piezoelectric pulse sensor and occlusion cuff were inserted and fixed to the tail. The pulse signal connected to the computer was observed to be at an appropriate level. The device was then activated, and systolic blood pressure was observed. The composition of the experimental groups is shown in Tables 6 and 7 below. The experimental group containing the mixture of chlorogenic acid, vitamin B12, coffee extract, and safflower extract has the same composition as the caffeic acid mixture in Table 7.

[0155] Table 6 The blood pressure experimental group consisted of a mixture of natural fermentation broth and glutathione (GSH). Table 7 Composition of the blood pressure experimental group of natural fermentation broth and caffeic acid mixture 3-2. Effects of the mixture on the DOCA-induced hypertension model Blood pressure measurements were taken before and after administration of each fermentation broth to rats with hypertension induced by DOCA-salt (e.g.) Figures 26 to 31 As shown, a decrease in blood pressure was confirmed between 10 and 20 minutes after oral administration, particularly in the groups receiving a mixture of natural fermentation broth and glutathione, caffeic acid, chlorogenic acid, coffee extract, lavender extract, or vitamin B12.

[0156] Example 4. Arthritis improvement effects of fermentation broth containing nitric oxide metabolites and mixed samples containing glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12. The experimental animals used in this experiment were 7-week-old male SD rats from the Samtako family, which were acclimatized for one week before use. The rats were administered the drug orally once daily for four weeks. The animal housing was maintained with regulated light and darkness at 12-hour intervals, a temperature of 23±2℃, and humidity of 50–60%.

[0157] 4-1. Induction of Arthritis and Composition For the induction of arthritis, after anesthetizing each experimental group (excluding the normal group), the hair around the left knee of the SD rats was completely removed. 50 μl (60–80 mg / ml) of monosodium iodoacetate (MIA), an osteoarthritis inducer, was injected into the left knee joint using a 31G insulin syringe (0.25 × 8, BD Medical-Diabetes Care, USA). MIA was diluted with 0.9% saline, and each experimental sample was administered to each experimental group 7 days after MIA injection. The composition of the experimental groups is shown in Tables 8 and 9 below. The composition of the mixture of chlorogenic acid, vitamin B12, coffee extract, and safflower extract in the experimental group was the same as that of the caffeic acid mixture in Table 9.

[0158] Table 8 The experimental group of arthritis was composed of a mixture of natural fermentation broth and glutathione (GSH). Table 9 Arthritis experimental group composition of natural fermentation broth and caffeic acid mixture 4-2. Measure the thickness of leg swelling. To determine changes in leg swelling thickness following MIA-induced arthritis, the left knee joint of rats was measured. After anesthesia, leg swelling thickness was measured using vernier calipers (Mitutoyo Co., Japan).

[0159] 4-3. Severity Score Assessment For the assessment of the arthritis index, images of each animal were taken before the animal model was euthanized, and then visual assessment was performed by three researchers. The assessment was a modification of the method of Kim et al. (Korean J Orient Physiol Pathol, Effects of Acanthopanax senticosus and onion mixture extract on the collagen-induced arthritis in rat model. 2011, 25; 1000-1007), with three researchers making three independent observations and assigning scores from 1 to 5, which were expressed as the average (Table 10).

[0160] Table 10 Clinical assessment of arthritis 4-4. Measurement of serum inflammatory cytokines and the production of inflammatory mediators. For serum cytokines, the production of TNF-α and metallopeptidase-9 (MMP-9) was measured using an ELISA kit (R&D Systems, USA). 50 μl of assay diluent (ADS) was added to each well containing antibody, followed by 50 μl of sample. The wells were incubated at room temperature for 2 hours, then washed four times with 300 μl of wash buffer. After washing, 100 μl of mouse TNF-α and MMP-9 antibodies were added to each well, and the wells were incubated at room temperature for 2 hours to remove the antibodies, followed by four washes. 100 μl of substrate solution was added to each well, and the wells were incubated at room temperature for 30 minutes. Then, 100 μl of stop solution was added, and the absorbance was measured at 450 nm using a Molecular Devices ELISA reader (SPECTRA max M2, USA).

[0161] 4-5. Determine serum prostaglandin E2 (PGE2) levels. The production of PGE2 in serum was determined using an ELISA kit. 50 μl of ADS was added to each well containing antibody, followed by 50 μl of sample. The wells were incubated at room temperature for 2 hours, then washed four times with 300 μl of wash buffer. After washing, 50 μl of proteoglycan E2 conjugate was added to each well as antibody, and the wells were incubated at room temperature for 2 hours to remove the antibody, followed by four washes. 200 μl of substrate solution was added to each well, and the wells were incubated 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 from Molecular Devices.

[0162] 4-6. Effects of the mixture on an arthritis model After the experiment, the morphological changes in the left knee joint of each experimental group were observed to confirm the degree of arthritis relief. Compared with the normal group without any treatment, changes in leg thickness caused by swelling were confirmed in the control group treated only with MIA, and the thickness decreased with sample treatment. Figures 32 to 37 ).

[0163] Furthermore, the assessment results of the degree of arthritis in each group of animal models of osteoarthritis induced by MIA are as follows: Figures 38 to 43As shown, no special symptoms such as swelling were observed in the normal group without MIA injection. However, in the control group (MIA) where arthritis was induced by MIA, swelling was observed in the left leg with induced osteoarthritis, indicating that the swelling was relieved by sample treatment.

[0164] After the experiment, serum was separated and the levels of TNF-α, MMP-9, and PGE2, which are the main indicators of inflammation, were measured. Compared with the control group, the levels of all sample treatment groups decreased. Compared with the natural fermentation broth treatment group alone, the treatment groups with a mixture of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 showed a more significant decrease. Figures 44 to 49 ).

[0165] Example 5. Liver protective effects of fermentation broth containing nitric oxide metabolites and mixed samples containing glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12. The experimental animals used in this experiment were 7-week-old male Sprague-Dawley rats (SD rats) purchased from the Samtako family and acclimatized for one week before use. The average weight of the SD rats was 210.45±1.65g. The light and darkness in the animal housing were adjusted at 12-hour intervals, the temperature was maintained at 23±2℃, and the humidity was maintained at 50-60%.

[0166] 5-1. Induction of liver injury and composition For the carbon tetrachloride (CCl4)-induced hepatotoxicity model, acute liver injury was induced by intraperitoneal administration of a mixture of CCl4 and olive oil at a dose of 0.1 ml per 100 g body weight. The control group received only intraperitoneal injection of olive oil instead of CCl4. Prior to CCl4 injection, all groups were pretreated with a 1 mM sample orally three times every other day, except for the control group which received only distilled water (DW) orally. Following the 3R principle, the minimum statistically significant number of animals was used to calculate the group composition.

[0167] Table 11 The experimental group consisting of a mixture of natural fermentation broth and glutathione (GSH) for liver injury Table 12 Composition of the experimental group for liver injury from a mixture of natural fermentation broth and caffeic acid The experimental group containing a mixture of chlorogenic acid, vitamin B12, coffee extract, and lacquer extract had the same composition as the caffeic acid mixture in Table 12.

[0168] 5-2. Serum Collection and Serum Testing Rats used in the experiment were fasted for 12 hours, then anesthetized with isoflurane. Blood was collected from the abdominal vein, and serum was separated by centrifugation at 3000 rpm for 20 minutes. Aspartic acid transaminase (AST) and alanine transaminase (ALT) levels in the blood were measured using the Reitman-Frankel enzymatic method of AST and ALT assay (Asanpharmaceutical).

[0169] 5-3. Effects of the mixture on an acute liver injury model Serum ALT and AST levels are commonly measured clinically as biomarkers of liver health. In a CCl4-induced hepatotoxicity model, measurements of ALT and AST after administration of each sample confirmed that serum ALT and AST levels were decreased in all sample administration groups compared to the control group. In particular, serum ALT and AST levels were decreased in the combined administration groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 compared to the natural fermentation broth administration groups alone. Figures 50 to 55 ).

[0170] Example 6. The effect of fermentation broth containing nitric oxide metabolites and mixed samples containing glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 on improving dermatitis. The experimental animals used in this experiment were 5-week-old male Balb / c mice purchased from the Samtako family and acclimatized for one week. The average weight of the Balb / c mice was 20.80 ± 0.82 g. The samples were administered orally once daily for 12 days. The light and darkness in the animal housing were adjusted at 12-hour intervals, and the temperature was maintained at 23 ± 2℃, with humidity maintained at 50–60%.

[0171] 6-1. Preparation of DNFB and induction of atopic dermatitis and composition After acclimatizing Balb / c mice for one week, their back hair was removed, and the mice were allowed to acclimatize for three days. DNFB (1-fluoro-2,4-dinitrobenzene; Sigma, USA) reagent was prepared by diluting 0.5% DNFB in a solution of acetone and olive oil in a 4:1 ratio. On day 4 post-hair removal, 40 μl of 0.5% DNFB was applied to the back for four consecutive days to induce skin sensitization. On day 12 post-skin sensitization, 20 μl of 0.3% DNFB was applied to the ear to induce contact dermatitis.

[0172] The experimental groups are shown in Tables 13 and 14 below. Following the 3R principle, the minimum statistically significant number was used to calculate the number of animals in each group. The experimental groups containing the mixture of chlorogenic acid, vitamin B12, coffee extract, and safflower extract had the same composition as the caffeic acid mixture in Table 14. The normal control group (CON) and control group received daily doses (DW), while the remaining groups received each sample orally once daily.

[0173] Table 13 The experimental group for atopic dermatitis consisted of a mixture of natural fermentation broth and glutathione (GSH). Table 14 The experimental group of atopic dermatitis was composed of a mixture of natural fermentation broth and caffeic acid. 6-2. Visual Assessment Visual assessment is a commonly used clinical evaluation method for atopic dermatitis. To determine the severity of atopic dermatitis symptoms, it is expressed as the sum of scores for five assessment items. The assessment items include erythema, pruritus and dry skin, edema and excoriation, erosion, and lichenification. Each item is scored asymptomatic (0 points), mild (1 point), moderate (2 points), and severe (3 points), and the scores are added together to assign a score ranging from a minimum of 0 to a maximum of 15.

[0174] 6-3. Measure ear swelling and spleen index. To determine changes in skin tissue after DNFB application, the ears of mice were measured bilaterally. Ear thickness was measured using vernier calipers (Mitutoyo Co.) at 12, 24, and 48-hour intervals over 3 days.

[0175] After measuring ear thickness, Balb / c mice were euthanized and their spleens were removed and weighed. The spleen index was calculated by dividing the spleen weight (mg) of the Balb / c mouse by its body weight (g).

[0176] 6-4. Effects of the mixture on a contact dermatitis model After the experiment, the right ear areas treated with samples in each experimental group were observed to confirm the morphological changes of atopic dermatitis lesions. Compared with the untreated left ear, atopic skin changes such as erythema, dry skin, erosion, swelling, and hematoma were observed in the control group treated with only DNFB. Figures 56 to 61 However, it was confirmed that compared with the untreated left ear, the atopic skin changes such as erythema, dry skin, erosion, swelling and hematoma in the sample treatment group were milder than those in the control group. It can be seen that compared with the natural fermentation broth alone, the combination of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract or vitamin B12 had fewer atopic skin changes.

[0177] Furthermore, after the experiment, the morphological changes of the atopic dermatitis lesions in the right ear treated with samples in each experimental group were observed and confirmed. It was confirmed that, compared with the control group, the ear thickness was reduced in all sample-treated groups. This reduction was even more pronounced in the mixed-treatment groups containing glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 compared to the natural fermentation broth-only treatment group. Figures 62 to 67 ).

[0178] Furthermore, after the experiment, the spleen weight of different body weights in each experimental group was measured. Compared with the control group, the spleen index of all sample treatment groups decreased, and the decrease was even greater in the mixed treatment groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 compared with the natural fermentation broth treatment group alone. Figures 68 to 73 ).

[0179] Furthermore, the results of serum TNF-α and IL-4 level measurements showed that, compared with the control group, the levels of TNF-α and IL-4 in all sample treatment groups were decreased. Compared with the natural fermentation broth treatment groups, the mixed treatment groups of glutathione, caffeic acid, chlorogenic acid, coffee extract, safflower extract, or vitamin B12 showed low levels of TNF-α and IL-4. Figures 74 to 79 ).

Claims

1. A pharmaceutical composition for the prevention or treatment of hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury, or dermatitis, characterized in that, It contains a natural ferment containing immobilized and stabilized nitric oxide metabolites; and any one of the following groups as active ingredients: glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and safflower extract.

2. The pharmaceutical composition according to claim 1, characterized in that, The active ingredient inhibits the production of carcinogenic N-nitrosamines in the stomach and promotes the production of nitric oxide in the intestines.

3. The pharmaceutical composition according to claim 1, characterized in that, The immobilized and stabilized nitric oxide metabolite of the natural ferment is prepared by a method comprising the following steps: adding a fermentation strain to a nitrogen-containing natural product and fermenting for 2 to 30 days at a temperature of 18°C ​​to 35°C and a dissolved oxygen concentration of 0.03 mg / L to 0.1 mg / L.

4. The pharmaceutical composition according to claim 3, characterized in that, The nitrogen-containing natural substances are soybeans, garlic, bean sprouts, cabbage, or whey.

5. The pharmaceutical composition according to claim 1, characterized in that, In addition to the active ingredient, the composition may also contain a pharmaceutically acceptable carrier, excipient or diluent.

6. A health-functional food composition for preventing or improving hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury, or dermatitis, characterized in that, It contains a natural ferment containing immobilized and stabilized nitric oxide metabolites; and any one of the following groups as active ingredients: glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and safflower extract.

7. The health-functional food composition according to claim 6, characterized in that, The composition is prepared in any dosage form selected from powders, granules, pills, tablets, capsules, candies, syrups, and beverages.

8. A veterinary composition for the prevention or treatment of hypertension, enteritis, irritable bowel syndrome, arthritis, acute liver injury, or dermatitis, characterized in that, It contains a natural ferment containing immobilized and stabilized nitric oxide metabolites; and any one of the following groups as active ingredients: glutathione, caffeic acid, chlorogenic acid, vitamin B12, coffee extract, and safflower extract.

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