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WO2023219376A2 - Composition naturelle pour inhiber la production de n-nitrosamine carcinogène dans l'estomac et les intestins et favoriser la production d'oxyde nitrique dans les intestins, et son utilisation - Google Patents

Composition naturelle pour inhiber la production de n-nitrosamine carcinogène dans l'estomac et les intestins et favoriser la production d'oxyde nitrique dans les intestins, et son utilisation Download PDF

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WO2023219376A2
WO2023219376A2 PCT/KR2023/006244 KR2023006244W WO2023219376A2 WO 2023219376 A2 WO2023219376 A2 WO 2023219376A2 KR 2023006244 W KR2023006244 W KR 2023006244W WO 2023219376 A2 WO2023219376 A2 WO 2023219376A2
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nitric oxide
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composition
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천현수
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to a natural composition for inhibiting the production of carcinogenic N-nitrosamine in the stomach and promoting the production of nitric oxide in the intestine and its use. More specifically, it relates to blocking the production of carcinogenic N-nitrosamine in strongly acidic conditions such as the stomach and the intestinal environment. It relates to a natural composition that produces a certain amount of nitric oxide and allows absorption of nitric oxide metabolites in the body.
  • Nitric oxide (hereinafter referred to as NO) is a gaseous substance produced when NOS (NO synthase), which exists in bacteria, plants, and animals, decomposes arginine (L-arginine), a known amino acid, and is a highly reactive radical ( (Refer to Scheme 1-1), it is hydrophobic and is an unstable substance with chemical properties that are rapidly oxidized and changed into hydrophilic NO metabolites (nitrite ions [NO 2 - ], nitrate ions [NO 3 - ], etc.) (Reaction Scheme 1-2 and 1-3). Because NO is very small and hydrophobic, it can move quickly in aqueous solutions such as blood or body fluids, and in particular, can move through blood vessel barriers, cell membranes, etc.
  • the NO produced is highly reactive, it reacts with oxygen dissolved in blood or body fluids or oxygen bound to red blood cells, and is transformed into NO metabolites that are water-soluble and have a longer existence time (see Schemes 1-2 and 1-3). By being (oxidized) and dissolved in water-soluble blood, it can move to all parts of the body and be excreted out of the body.
  • NO 2 - is a hydrophilic and relatively stable ionic substance, and can be dissolved in blood and body fluids and moved to all parts of the body, but cannot pass through organ barriers, blood vessel barriers, and cell membranes.
  • NO 2 - is reduced to hydrophobic NO under acidic and oxygen-poor conditions (e.g., venous blood conditions) and passes through organ barriers, blood vessel barriers, and cell membranes, thereby exhibiting NO's unique physiological functions and pharmacological effects (Scheme 2 -1).
  • NO 2 - can be reduced to NO by reacting with vitamin C (ascorbic acid/ascorbate, hereinafter referred to as Asc) dissolved in blood or body fluids (see Schemes 2-2 and 2-3).
  • NO is mainly produced in vascular endothelial cells through the process of Scheme 1-1) and is used to regulate the physiological function of relaxing nearby blood vessels, and part of the generated NO is converted to NO 2 - through the process of Scheme 1-2)
  • NO By being oxidized and dissolved in the blood, it moves to all parts of the body and is reduced to NO through the process of Scheme 2-1) or Scheme 2-2) in places where NO is needed, thereby manifesting the physiological functions and pharmacological effects of NO.
  • NO generated in excess by NOS or NO generated by reduction of excess NO 2 - is oxidized to NO 3 - through the process of Reaction Formula 1-3) and is discharged out of the human body through urine or sweat.
  • NO generated in Scheme 1-1) or 2-1) reaches the cytoplasm of the target cell and chemically combines with glutathione (hereinafter referred to as GSH) abundantly present in the cytoplasm to form S-nitrosoglutathione ( S- After generating nitrosoglutathione (hereinafter referred to as GSNO), NO is stored inside the cell in the form of GSNO for a certain period of time (see Scheme 3-1).
  • GSH glutathione
  • GSNO S-nitrosoglutathione
  • NO is stored inside the cell in the form of GSNO for a certain period of time (see Scheme 3-1).
  • GSSG glutathione disulfide
  • Scheme 3-2 when GSNO exists in excess inside the cell, the excess GSNO is reduced to GSH and NH 3 by GSNO reductase (hereinafter referred to as GSNOR), thereby reducing the potential toxicity of NO.
  • the amount of NO in the human body can be predicted as a total amount by the combined amount of NO 2 - and NO 3 - , but the actual usable NO concentration can be predicted by the concentration of NO in the blood by the concentration of NO 2 - that can be converted to NO. It is known that about 100-500 nM (average 300 nM) of NO 2 - exists in the blood of normal people without disease, but in people with diseases such as diabetes, the concentration of NO 2 - in the blood is sometimes observed to be lower than that of normal people. There are many. There may be several ways to raise the reduced concentration of NO 2 - to the normal range, but it is considered the most reasonable method to maintain the concentration of NO 2 - in the blood within the normal range by consuming NO metabolites through food or food. .
  • NO 3 - reductase nitrate reductase, hereinafter referred to as NaRase
  • NO 2 present in the periplasm between the outer membrane and the cell membrane of bacteria is reduced to NO by nitrite reductase (hereinafter referred to as NiRase)
  • NiRase nitrite reductase
  • some of the reduced NO is absorbed into the body, and some of the NO is Through several stages of reduction, it is converted to N 2 and eliminated from the intestines (see Scheme 5). Therefore, in order to produce a sufficient amount of NO using NO 3 - , it is necessary to consume a large amount of NO 3 - .
  • N-nitrosamine N -nitrosamine, hereinafter referred to as R 2 N-NO
  • R 2 N-NO secondary amines
  • NO 3 - ingested orally cannot be reduced in the stomach, but is reduced to NO in the small and large intestines and absorbed into the body.
  • the degree of NO reduction varies depending on the individual's intestinal bacterial environment and eating habits, and the NO 2 - amount is higher than the amount. You must consume a certain amount of NO 3 - . Therefore, a certain amount of effective NO body absorption cannot be expected through NO 3 - intake (see Scheme 5).
  • Scheme 7-2) and Scheme 7-3) may have an advantage in that they block the production of N-nitrosamine, but NO production from ingested NO 2 - is suppressed, which ultimately makes NO absorption in the body difficult. There may be some bad aspects.
  • PhO-NO shown in Scheme 7-2) or GSNO shown in Scheme 7-3) produces NO in the intestinal environment such as the small and large intestines, NO absorption in the body is possible.
  • GSH which is abundant in the small intestine, to generate NO (see Scheme 8).
  • Scheme 7-3) and Scheme 8-1 if GSH is taken together when taking NO 2 - , the production of N-nitrosamine is blocked in the stomach and NO is produced in the intestine, which ultimately facilitates NO absorption in the body. there is.
  • NO metabolites are reduced to NO under oxygen-poor or acidic conditions and regulate blood circulation by dilating blood vessels or promoting capillary formation (Lundberg et al., Nat Rev Drug Discov 2008, 7:156-167).
  • the four important physiological functions of blood circulation can be seen as 1 supply of oxygen and nutrients, 2 excretion of carbon dioxide and waste products, 3 maintenance of body temperature, and 4 performance of immune function. Therefore, NO can be seen to affect the absorption function of nutrients, influence the excretion function of waste products, and regulate body temperature and immune function.
  • NO can return high blood pressure to normal blood pressure by dilating blood vessels and regulating blood flow (Amaral et al., Redox Biol. 2015, 5:340-346).
  • NO metabolite that can be converted to NO could be developed as a treatment for hypertension with fewer side effects.
  • several recent research reports have shown the blood sugar reducing effect of NO metabolites (Khalifi et al., Nitric Oxide. 2015, 44:24-30) and the insulin secretion promoting effect of beta cells (Nystrom et al., Free Radic Biol Med. 2012, 53:1017-1023), effect of resolving insulin resistance (Ohtake et al., Nitric Oxide. 2015, 44:31-38), effect of improving diabetes complications (Bahadoran et al., Nutr Metab (Lond). 2015, 12 :16. doi:10.1186/ s12986-015-0013-6), etc.
  • Korean Patent No. 2256793 discloses 'a method for producing a natural fermentation composition containing fixed nitrogen oxide precursors and a natural fermentation composition prepared by this method'
  • Korean Patent Publication No. 2019-0084592 discloses '
  • a method for producing a natural fermentation composition that immobilizes nitric oxide in a natural fermentation material and a natural fermentation composition prepared by this method are disclosed, the present invention is used for 'inhibiting the production of carcinogenic N-nitrosamine in the stomach and promoting the production of nitric oxide in the intestine.
  • ‘natural compositions and their uses’ There is no description regarding ‘natural compositions and their uses’.
  • the present invention has been developed in response to the above-mentioned needs, and the present inventors have developed a material containing sodium nitrite and sulfur; Or, as a result of oral administration of a mixture of natural fermented products and sulfur-containing substances in which nitric oxide metabolites were fixed and stabilized to experimental animal models of diabetes, blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, and atopic dermatitis, sodium nitrite alone or The present invention was completed by confirming that the mixture of sulfur-containing substances had excellent improvement effects on blood sugar, blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, and atopic dermatitis compared to the natural fermentation alone experimental group.
  • the present invention provides a natural fermentation product in which sodium nitrite (NaNO 2 ) or a nitric oxide metabolite is immobilized and stabilized; and sulfur-containing substances as active ingredients, and provides a composition that inhibits the production of carcinogenic en-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.
  • NaNO 2 sodium nitrite
  • sulfur-containing substances as active ingredients
  • the present invention provides a pharmaceutical composition containing the composition as an active ingredient.
  • the present invention provides a health functional food composition containing the composition as an active ingredient.
  • the composition of the present invention is a mixture of nitric oxide metabolites and sulfur-containing substances to block the production of carcinogenic N-nitrosamine in the gastric juice of the stomach (strong acid conditions), and to produce a certain amount of nitric oxide in the small or large intestine to oxidize it in the body. It was developed to enable absorption of nitrogen metabolites, and is expected to be widely applied in related fields such as treatment and symptom improvement of high blood pressure, diabetes, enteritis, arthritis, liver disease, and atopy, which are known to be diseases that can be improved by nitric oxide.
  • Figure 1 shows that in the gastrointestinal environment, nitric oxide metabolites (NO 2 - ) and glutathione (GSH) generate s-nitrosoglutathione (GSNO) by gastric acid to block the production of carcinogens, and in the intestinal environment, nitric oxide (SNO) (SNO)
  • GSNO s-nitrosoglutathione
  • Figure 2 is an absorption spectrum showing that s-nitrosoglutathione (GSNO) is produced from a natural fermentation product containing nitric oxide metabolites (NO 2 - ) and a yeast extract containing glutathione (GSH) under strong acid conditions.
  • GSNO s-nitrosoglutathione
  • Figure 3 shows that when a diabetic experimental animal model consumes a sample mixed with a natural fermentation product containing nitric oxide metabolites (NO 2 - ) and a yeast extract containing glutathione (GSH), the total amount of nitric oxide metabolites in the blood (NO x ) increases. This is a result that shows what is being done.
  • Figure 4 shows the results showing the anti-diabetic effect of reducing fasting blood sugar when a sample of a mixture of natural fermentation product containing nitric oxide metabolites (NO 2 - ) and yeast extract containing glutathione (GSH) was treated in a diabetic experimental animal model for 4 weeks. am.
  • Figure 5 is a graph analyzing the degree of disease activation according to sample administration in the ulcerative colitis model. *: p ⁇ 0.05 (compared to control group), #: p ⁇ 0.05 (lettuce fermentation liquid and lettuce fermentation liquid+GSH experimental group).
  • Figure 6 is a graph analyzing the change in intestinal length according to sample administration in an ulcerative colitis model. *: p ⁇ 0.05 (compared to control group), #: p ⁇ 0.05.
  • Figure 7 is a graph analyzing the level of TNF- ⁇ in serum according to sample administration in an ulcerative colitis model. *: p ⁇ 0.05 (compared to control group), #: p ⁇ 0.05.
  • Figure 8 is a graph analyzing the level of IL-1 ⁇ in serum according to sample administration in an ulcerative colitis model. *: p ⁇ 0.05 (compared to control group), #: p ⁇ 0.05.
  • FIG. 9 is a schematic diagram of an experiment to confirm the effect of improving stressful bowel syndrome.
  • WAS stands for water avoidance stress, and the treated samples are shown in Table 3.
  • Figure 10 shows the results of measuring the number of stools for each experimental group according to sample administration in the stress bowel syndrome model.
  • A shows the average number of stools per day
  • B shows the accumulated number of stools until the end date of the experiment.
  • Figure 11 shows the results of measuring blood pressure changes before and after sample administration in a hypertension model.
  • Figure 12 shows the results of measuring the change in joint edema thickness after sample administration in an arthritis-inducing model using MIA (monosodium iodoacetate). *: p ⁇ 0.05 (compared to control group).
  • Figure 13 shows the results of evaluating the arthritis index in an arthritis induction model using MIA. *: p ⁇ 0.05 (compared to control group).
  • Figure 14 shows the results of measuring ALT and AST levels in serum after administering samples in an acute liver injury model. *: p ⁇ 0.05 (compared to control group).
  • Figure 15 shows the results of analyzing morphological changes in lesions by observing the right ear area after administering a sample in an atopic dermatitis model. *: p ⁇ 0.05 (compared to control group).
  • Figure 16 shows the results of measuring the change in thickness of the right ear area after administering a sample in an atopic dermatitis model.
  • Figure 17 shows the results showing the spleen index at the end of the experiment for each experimental group after administering the sample in the atopic dermatitis model. *: p ⁇ 0.05 (compared to control group).
  • Figure 18 shows the results of measuring the level of TNF- ⁇ in the serum at the end of the experiment for each experimental group after administering the sample in the atopic dermatitis model. *: p ⁇ 0.05 (compared to control group).
  • Figure 19 shows the results of measuring the level of IL-4 in the serum at the end of the experiment for each experimental group after administering the sample in the atopic dermatitis model. *: p ⁇ 0.05 (compared to control group).
  • Figure 20 shows the results of measuring blood pressure changes after administration of a sample of lettuce fermentation broth (LF) mixed with NAC, GSH, MSM, ALA, or GSH-containing yeast extract (BY) in a model of hypertension.
  • NAC n-acetyl cysteine
  • GSH glutathione
  • MSM methylsulfonylmethane
  • ALA alpha-lipoic acid.
  • Figure 21 shows the results of measuring changes in blood pressure after administration of a sample containing NAC, GSH, MSM, ALA, or GSH-containing yeast extract (BY) mixed with soybean fermentation broth (BF) in a model of hypertension.
  • Figure 22 shows the results of measuring blood pressure changes after administration of a sample containing NAC, GSH, MSM, ALA, or GSH-containing yeast extract (BY) mixed with garlic fermentation broth (GF) in a model of hypertension.
  • Figure 23 shows the results of measuring blood pressure changes after administration of a sample of yeast extract (BY) containing NAC, GSH, MSM, ALA, or GSH mixed with soybean sprout fermentation broth (SSF) in a model of hypertension.
  • Figure 24 shows the results of measuring blood pressure changes after administration of a sample of sodium nitrite (NaNO 2 ) mixed with NAC, GSH, MSM, ALA, or GSH-containing yeast extract (BY) in a model of hypertension.
  • the present invention provides a natural fermentation product in which sodium nitrite (NaNO 2 ) or a nitric oxide metabolite is immobilized and stabilized; and sulfur-containing substances as active ingredients, and provides a composition that inhibits the production of carcinogenic en-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.
  • NaNO 2 sodium nitrite
  • sulfur-containing substances as active ingredients
  • the sulfur-containing substances include glutathione, N-acetyl cysteine, and methylsulfonyl. It may be, but is not limited to, yeast extract containing methane (methylsulfonylmethane), alpha-lipoic acid, or glutathione.
  • the glutathione-containing yeast extract may have a glutathione content of 7 to 15% by weight based on the total weight of the extract, but is not limited thereto.
  • Yeast extract containing glutathione contains not only glutathione but also various other beneficial ingredients for health.
  • the sulfur-containing material functions to help maintain a long nitric oxide (NO) release time.
  • glutathione is a small-sized peptide composed of three amino acids, glutamic acid, cysteine, and glycine, and is an endogenous reducing substance produced in plants, animals, fungi, and some bacteria and archaea. In the body or within cells, glutathione prevents cell damage by combining with free radicals, peroxides, toxic substances, and heavy metals. Additionally, GSH can react with nitric oxide (NO) to produce s-nitrosoglutathione (GSNO). GSNO generated through this process delivers NO to where it is needed through blood vessels in the body, stores NO within cells for a certain period of time, and decomposes into NO when needed, thereby demonstrating the pharmacological effects of NO.
  • NO nitric oxide
  • GSNO s-nitrosoglutathione
  • N-acetyl cysteine is a synthetic N-acetyl derivative and prodrug of the endogenous amino acid L-cysteine, which is a precursor of GSH, and has mucopolysaccharide hydrolysis, antioxidant, and cell activity. It is a substance with protective, anti-cancer and anti-inflammatory activities.
  • methylsulfonylmethane is a naturally occurring organic sulfur-containing substance that exhibits strong antioxidant and anti-inflammatory activities, and ingestion of MSM is known to increase GSH levels in the body.
  • alpha-lipoic acid is a naturally occurring micronutrient synthesized in small amounts by plants and animals (including humans), and is a substance with antioxidant and potential chemical protection activities.
  • ALA is known to act as a free radical scavenger, help repair oxidative damage, and promote GSH synthesis.
  • the natural fermented product in which the nitric oxide metabolite is immobilized and stabilized is obtained by adding fermentation bacteria to a nitrogen-containing natural product and fermenting 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 minutes. It may be manufactured by a manufacturing method including fermentation for one day, but is not limited thereto, and detailed manufacturing methods can be referred to Korean Patent No. 2129038.
  • composition according to the present invention which inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine, may further include polyphenols in addition to natural fermented products and sulfur-containing substances in which nitric oxide metabolites are fixed and stabilized.
  • the polyphenol functions to promote the reduction of nitric oxide metabolites in the natural fermentation product to nitric oxide, but is not limited thereto, and may be resveratrol, quercetin, silymarin, anthocyanin, or catechin acid.
  • the polyphenol may be used in the form of a compound or a powder form of a natural material containing each polyphenol compound (e.g., aronia powder, barley sprout powder, etc.), but is not limited thereto.
  • the natural fermented product according to the present invention can be fermented by adding 0.01 to 3 parts by weight of fermentation bacteria to 100 parts by weight of the mixture of nitrogen-containing natural product and water, preferably 1:0.5 to 1 of the nitrogen-containing natural product and water. After mixing at a weight ratio of , fermentation may be performed by adding 0.01 to 3 parts by weight of fermentation bacteria based on 100 parts by weight of the mixture, but is not limited thereto.
  • the fermentation may be carried out at a temperature of 10 to 40°C, preferably at a temperature of 18 to 35°C, more preferably at a temperature of 20 to 33°C, and even more preferably at a temperature of 25 to 33°C. , but is not limited to this.
  • the fermentation may be performed under dissolved oxygen concentration conditions of 0.01 to 0.1 mg/L, preferably 0.03 to 0.1 mg/L, but is not limited thereto.
  • the fermentation may be performed for 2 to 30 days, but is not limited thereto.
  • the fermentation may be terminated when the chemical oxygen demand is 200 to 700 mg/L, but is not limited thereto.
  • the fermentation bacteria are Bacillus sp., Bifidobacterium sp., Enterococcus sp., Lactobacillus sp., and Lactococcus sp. ) or Weissella sp. strains, preferably Bacillus subtilis, B. amyloliquefaciens , B. natto , and Bacillus licheni. Formis ( B. licheniformis ), Bifidobacterium bifidum ( Bifidobacterium infantis ) , Bifidobacterium longum ( B. longum ), Enterococcus faecium ), Enterococcus faecalis ( E.
  • Lactobacillus acidopilus Lactobacillus acidopilus
  • Lactobacillus alimentarius Lactobacillus alimentarius
  • Lactobacillus bulgaricus Lactobacillus casei ( L. casei )
  • Lactobacillus curvatus Lactobacillus delbrukii
  • Lactobacillus johnsonii L. johnsonii
  • Lactobacillus farciminus L. farciminus
  • Lactobacillus Gasseri L. gasseri
  • Lactobacillus helveticus L.
  • Lactococcus Any one selected from the group consisting of Lactococcus lactis and Weissella cibaria can be used, preferably Bacillus subtilis with the accession number KCTC12501BP - Chun Hyun Su. However, it is not limited to this.
  • the nitrogen-containing natural products are not limited to this, but include lettuce, pork sprouts, spinach, blueberries, dandelions, pomegranates, cabbage, garlic, noni, onions, beans, bean sprouts, mulberry leaves, bitter melon, bokbunja, Houttuynia cordata, aronia, Yulcho, One selected from the group consisting of yellow lotus, hijiki, tangerine, mushrooms, calamus, seaweed, Chinese cabbage, kelp, salmon milt, abalone shell (seokgyeolmyeong), crustacean shell, cricket, apple, mugwort, tangerine, silkworm, rehmannia glutinosa, and sipjeondaebotang. It could be more than that.
  • the human body's nitric oxide concentration can be predicted by the total amount of nitrite ions (NO 2 - ) and nitrate ions (NO 3 - ), but the actual usable nitric oxide concentration is NO 2 which can be converted to nitric oxide (NO).
  • - NO concentration in blood can be predicted by concentration.
  • NO 2 - is a hydrophilic and relatively stable ionic substance, and can dissolve in blood and body fluids and move throughout the body, but cannot pass through organ barriers, blood vessel barriers, and cell membranes.
  • NO 2 - is reduced to fat-soluble NO by nitrite reductase or acidic conditions, it can pass through organ barriers, blood vessel barriers, and cell membranes. Therefore, in order for NO 2 - to reach target cells and exert a specific pharmacological effect or to be absorbed into the body from the stomach or intestines, it must be reduced to NO.
  • nitric oxide metabolites (especially NO 2 - ) are ingested orally to increase the low concentration of nitric oxide metabolites in the blood or to obtain the pharmacological effects of nitric oxide, the following two harmful health problems may occur.
  • NO 2 - ingested orally due to the strong acid condition of gastric juice, excessive NO 2 - ingested orally can be absorbed into the body in a short period of time, resulting in a rapid decrease in blood pressure, hypoglycemia, and cyanosis.
  • the composition according to the present invention contains the nitric oxide metabolite (NO 2 - ) contained in natural fermentation and the sulfur-containing substances glutathione (GSH), N-acetyl cysteine (NAC), or glutathione contained in glutathione-containing yeast extract.
  • GSH glutathione
  • NAC N-acetyl cysteine
  • glutathione glutathione-containing yeast extract.
  • the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized may preferably contain 0.1 to 10 mM based on NO 2 - , but is not limited thereto.
  • nitric oxide metabolite refers to nitrite (NO 2 - ), R-NO 2 - , R-NO, RO-NO, nitrate (NO 3 - ), R-NO 3 - , GS-NO (or A general term for GSNO), GS-NO 2 , etc., where R- means one H atom has been removed from the carbon chain of polyphenol, fatty acid, etc., and RO- means that one H atom has been removed from ROH, and in the case of GS-, it means that one H atom has been removed from the cysteine residue of glutathione (GSH).
  • GSH cysteine residue of glutathione
  • Nitric oxide metabolites are converted to nitric oxide under acidic and reducing conditions. In strongly acidic conditions such as the stomach, nitric oxide metabolites are reduced by hydrogen ions (H + ) to generate nitric oxide, and in weakly acidic conditions such as the intestines (small intestine, large intestine), polyphenols, glutathione, vitamin C, and bacterial nitric oxide reductase are produced. Nitric oxide metabolites can be reduced to nitric oxide by reducing substances such as .
  • GSNO nitric oxide metabolite or nitric oxide conjugate
  • GSH glutathione
  • Scheme 8-1 nitric oxide (NO) generated in the intestinal environment penetrates into the body from the intestinal environment and is oxidized into nitric oxide metabolites (NO 2 - ), reaching body fluids or blood vessels (see Scheme 8-3).
  • the present invention also provides a pharmaceutical composition containing as an active ingredient the composition of the present invention that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.
  • the composition that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestines includes sodium nitrite (NaNO 2 ) or a natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized; and a sulfur-containing material as active ingredients, the details of which are the same as those described above.
  • the pharmaceutical composition of the present invention may be used for preventing or treating diabetes, high blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, or atopic dermatitis, but is not limited thereto.
  • the pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.
  • composition of the present invention can be formulated and used in the form of oral preparations such as granules, tablets, capsules, etc. according to conventional methods, but is not limited thereto.
  • a natural fermentation product in which the sodium nitrite or nitric oxide metabolite of the present invention is immobilized and stabilized; and sulfur-containing substances; carriers, excipients, and diluents that may be included in a pharmaceutical composition comprising a composition containing as an active ingredient 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 and mineral oil, etc.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include the fermented slug extract with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups.
  • ком ⁇ онентs such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used as a base for suppositories.
  • the pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, and all methods of administration can be expected, but are preferably administered orally or rectally. Not limited.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the pharmaceutically effective amount of the pharmaceutical composition containing the natural fermentation product in which the sodium nitrite or nitric oxide metabolite according to the present invention is immobilized and stabilized and a sulfur-containing substance may vary depending on the patient's age, gender, and weight, and generally 2 mg to 4 mg per kg of body weight, preferably 3 mg, can be administered every day or every other day, divided into 1 to 3 times per day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.
  • the present invention also provides a health functional food composition containing as an active ingredient a composition according to the present invention that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine.
  • the composition that inhibits the production of carcinogenic N-nitrosamine in the stomach and promotes the production of nitric oxide in the intestine is sodium nitrite (NaNO 2 ) or a natural fermented product in which the nitric oxide metabolite is immobilized and stabilized. ; and a sulfur-containing material as active ingredients, the details of which are the same as those described above.
  • the health functional food composition of the present invention may be used to prevent or improve diabetes, high blood pressure, enteritis, stress bowel syndrome, arthritis, liver disease, or atopic dermatitis, but is not limited thereto.
  • Health functional foods Naturally fermented products in which the sodium nitrite or nitric oxide metabolites are immobilized and stabilized; and sulfur-containing substances;
  • Examples of health functional foods containing a composition containing as an active ingredient include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, rice cakes, and ice cream. It includes dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in the conventional sense.
  • the health functional food may be manufactured in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, effervescent tablet, and beverage, but is not limited thereto.
  • the active ingredient can be used appropriately depending on its purpose of use (prevention or improvement).
  • natural fermentation products in which the sodium nitrite or nitric oxide metabolites of the present invention are immobilized and stabilized during the production of food or beverages; and sulfur-containing substances as active ingredients.
  • the composition is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials.
  • it can be used in amounts within a range that poses no safety problems.
  • the health functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings.
  • natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient.
  • the natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) are preferred.
  • the flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
  • a natural flavoring agent e.g., thaumatin, stevia extract, etc.
  • a synthetic flavoring agent e.g., saccharin, aspartame, etc.
  • fertilization broth is used to mean “a natural fermentation product in which nitric oxide metabolites have been immobilized and stabilized.”
  • Sodium nitrite (NaNO 2 ), nitrate reductase, hydrochloric acid (HCl), and streptozotocin (hereinafter referred to as STZ) used in the present invention were purchased from Sigma (USA), and were obtained in powder form from 7.
  • Yeast extract containing % to 15% glutathione was purchased from Vision Biocam Co., Ltd. (manufactured in China).
  • a composition sample can be prepared by mixing the powder of glutathione-containing yeast extract with lettuce or garlic fermentation broth manufactured by Human Enos.
  • the sample used in the example of this embodiment was 1 mM sodium nitrite (NaNO 2 ), 1 mM garlic or lettuce fermentation broth based on the contained nitric oxide metabolite NO 2 - concentration, and based on the contained glutathione (GSH) concentration. It is a mixture of 1 mM yeast extract mixed with the sodium nitrite in a ratio of 1:1, or mixed with the garlic or lettuce fermentation broth in a ratio of 1:1.
  • the lettuce or garlic fermentation broth means concentrating each fermented product prepared through the production method disclosed in Korean Patent No. 2129038 at 20 to 30 ° C. for 24 to 48 hours at a stirring speed of 100 to 300 rpm.
  • the total amount of nitric oxide metabolites contained in fermentation broth or blood meaning the combined concentration of NO 2 - and NO 3 - , hereinafter referred to as NO All were reduced to NO 2 - and the total concentration of NO 2 - was measured to determine the total content or concentration of nitric oxide metabolites.
  • the total NO 2 - content is determined by first adding nitrate reductase to reduce the fermentation broth for 1 hour, diluting it 1,000 times with distilled water, and centrifuging at 3,000 rpm for 3 minutes to remove insoluble polyphenols and dietary fiber. After removal, 50 ⁇ l of the clear supernatant was taken and used as a measurement sample.
  • the collected blood was centrifuged at 3,000 rpm for 3 minutes to precipitate blood cells, then 40 ⁇ l of supernatant plasma was taken and mixed with 10 ⁇ l of buffer solution containing nitrate reductase, reacted for 1 hour, and then used as a measurement sample. .
  • sample NO 2 - + GSH
  • GSNO s-nitrosoglutathione
  • the sample's unique absorption spectrum (absorption) spectrum was analyzed.
  • 0.1 mM HCl solution was added to the fermentation broth to remove all NO 2 - and then yeast extract containing glutathione (GSH) was mixed to prepare reference cell sample R1.
  • a composition sample containing a mixture of fermentation broth and yeast extract was dissolved in distilled water to prepare sample cell sample S1, and 0.1 mM HCl was added thereto and reacted at room temperature for 5 minutes to prepare sample cell sample S2.
  • the generation of GSNO was determined by comparing the maximum absorption wavelength ( ⁇ max ).
  • the 6-week-old male Balb/c mice used in this animal experiment were purchased from Samtaco (Korea) and were used in the experiment after an acclimatization period of one week.
  • the experimental animals were divided into 4 groups of 5 each, with each group being a control group that did not process any samples, an experimental group 1 treated with a fermentation broth sample (NO 2 - 1 mM based on concentration), and a yeast extract sample (1 mM based on GSH concentration).
  • the samples were divided into Experimental Group 2 treated with (mM) and Experimental Group 3 treated with a mixed sample of fermentation broth and yeast extract (1mM NO 2 - + 1mM GSH). Each sample was administered orally at regular intervals three times a day for three days.
  • Example 1 Production of GSNO by a mixed sample of fermentation broth containing nitric oxide metabolites and yeast extract containing glutathione under strong acid conditions.
  • composition sample mixed sample of garlic fermentation broth [0.1mM as NO 2 - ] and yeast extract [0.1mM as GSH]
  • yeast extract prepared in the experimental method 1-1) described above produces GSNO in strong acid conditions such as the stomach.
  • an experiment was performed according to the experimental method 1-3) described above, and the results shown in FIG. 2 were obtained.
  • Example 2 Body absorption of nitric oxide metabolites by mixed sample of fermentation broth containing nitric oxide metabolites and yeast extract containing glutathione
  • composition sample mixed sample of garlic fermentation broth [0.1mM as NO 2 - ] and yeast extract [0.1mM as GSH]
  • yeast extract prepared in the experimental method 1-1) described above
  • nitric oxide metabolites In order to investigate whether it is absorbed into the body, an experiment was performed according to test method 1-4), and the results shown in Figure 3 were obtained.
  • Example 3 Antidiabetic effect by mixed sample of fermentation broth containing nitric oxide metabolite and yeast extract containing glutathione
  • composition sample mixed sample of garlic fermentation broth [0.1mM as NO 2 - ] and yeast extract [0.1mM as GSH]
  • nitric oxide metabolites It was found that absorption into the body was possible (see Example 2).
  • the present invention demonstrates that nitric oxide metabolites absorbed into the body have a preventive and improvement effect on high blood pressure, diabetes, enteritis, stress bowel syndrome, arthritis, liver disease, and atopic dermatitis. It seemed.
  • nitric oxide metabolites absorbed into the body by ingestion of a sample mixed with nitric oxide-containing fermentation broth and glutathione-containing yeast extract have the pharmacological effects claimed in the prior invention
  • various pharmacological effects were tested. Among them, diabetes, known as a representative disease, was selected and the anti-diabetic effect of the sample prepared according to experimental method 1-1) was investigated.
  • diabetic experimental animals were prepared according to the following experimental method, and then treated three times a day for 4 weeks with samples consisting of only fermentation broth, samples consisting of yeast extract only, and samples consisting of a mixture of fermentation broth and yeast extract. And the anti-diabetic effect of each sample was investigated.
  • mice used as a diabetes experimental animal model were purchased from Samtaco (Korea), underwent an acclimatization period for one week, and were then used for the purpose of the experiment.
  • Diabetic experimental animals were created by injecting the diabetes-inducing substance STZ into the abdominal cavity of the experimental animals. Specifically, diabetes was induced by injecting once into the abdominal cavity of experimental animals 120 mg/kg STZ dissolved in 0.1 M citrate buffer (pH 4.5), and two weeks later, a small amount was injected from the tail vein of the fasting experimental animals. Blood was collected and measured using a GlucotrendTM blood glucose meter from Roche, Germany. Experimental animals with measured blood sugar levels of 200 mg/dL or less were excluded, and fasting blood sugar levels were measured again one week later, and experimental animals whose blood sugar levels remained above 200 mg/dL were selected and used as diabetes test animals.
  • Diabetic experimental animals were divided into 4 groups of 5 animals each. Each group consisted of a diabetic experimental group that did not treat the sample, experimental group 1 that treated the fermentation broth sample (NO 2 - 1 mM based on concentration), and yeast extract sample (based on GSH concentration). They were divided into experimental group 2, which was treated with 1 mM) and experimental group 3, which was treated with a mixed sample of fermentation broth and yeast extract (1mM NO 2 - + 1mM GSH). Each sample was administered orally at regular intervals three times a day for 4 weeks. After fasting for 16 hours every week, a small amount of blood was collected from the tail vein of the diabetic experimental animals, and fasting blood glucose was measured using a Glucotrend blood glucose meter.
  • Example 4 Effect of improving enteritis by a mixed sample of fermented broth containing nitric oxide metabolites and yeast extract containing glutathione
  • the experimental animals used in this experiment were 6-week-old male Balb/c mice purchased from Samtaco (Korea) and used after an acclimatization period of one week.
  • the average weight of Balb/c mice was 21.40 ⁇ 1.20 g, and samples were administered orally once daily for 3 weeks.
  • light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23 ⁇ 2°C and humidity at 50-60%.
  • the experimental animals were divided into 6 groups: a normal group treated with no treatment and administered distilled water (DW), a control group induced with enteritis and administered DW, a group induced with enteritis and administered 1mM sodium nitrite (NaNO 2 ), and a group treated with enteritis. They were classified into a group that induced enteritis and administered 1mM of fermented lettuce liquid, a group that induced enteritis and administered 1mM NaNO 2 + glutathione-containing yeast extract (hereinafter referred to as GSH), and a group that induced enteritis and administered 1mM of fermented lettuce liquid + GSH (Table 1).
  • GSH glutathione-containing yeast extract
  • Intestinal disease is measured by signs such as weight loss, diarrhea accompanied by bleeding and mucus, and a shortened colon.
  • Previous studies have shown that there are three main clinical signs of disease activity index (DAI): weight loss, diarrhea, and rectal bleeding.
  • Weight loss is calculated as the difference between initial and current weight.
  • Diarrhea is defined as the presence of persistent soft stool in the rectum without fecal pellet formation.
  • the appearance of rectal bleeding is separate from bloody diarrhea, total rectal bleeding, or other diarrhea.
  • DAI was calculated as follows.
  • DAI (weight loss score) + (diarrhea score) + (rectal bleeding score)
  • the medical variables used here are comprehensive functional measures that are similar to the medical symptoms that occur when ulcerative colitis occurs in humans (Table 2).
  • mice TNF- ⁇ and IL-1 ⁇ antibodies were added to each well and incubated at room temperature for 2 hours, then removed and washed four times.
  • 100 ⁇ l of substrate solution was added to each well and left at room temperature for 30 minutes, then 100 ⁇ l of reaction stop solution was added, and the absorbance was measured at 450 nm with an ELISA reader (SPECTRA max M2, USA) from Molecular Devices.
  • the DSS colitis model exhibits clinical signs characterized by weight loss, diarrhea, and bloody stool.
  • the results of measuring the impact on these clinical symptoms using DAI are as follows. No symptoms were measured in the normal group, and the disease activity index increased in all groups except the normal group. It was confirmed that the sample administration group showed an overall decrease compared to the DSS group. In particular, there was a significant difference between the lettuce fermentation liquid administration group and the lettuce fermentation liquid + GSH mixed administration group ( Figure 5).
  • mice sacrificed on the 5th day after DSS administration was removed and its length was checked.
  • the intestine length was longer in all groups administered the sample compared to the control group, and the NaNO 2 group, the lettuce fermentation solution group, and the NaNO 2 + GSH mixture were found to be longer than the control group. This was confirmed in the following order: administration group, lettuce fermentation solution + GSH mixed administration group ( Figure 6).
  • Example 5 Effect of improving stressful bowel syndrome by a mixed sample of fermented broth containing nitric oxide metabolites and yeast extract containing glutathione
  • the experimental animals used in this experiment were 8-week-old female Wistar rats purchased from Samtaco and used after an acclimatization period of one week.
  • the average weight of Wistar rats was 180.5 ⁇ 0.5 g, and the samples were orally administered once a day.
  • light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23 ⁇ 2°C and humidity at 50-60%.
  • the group consists of 6 groups with 6 animals in each group: a normal group (No-stress) that was not exposed to WAS, a control group that performed WAS and was stressed, a group that performed WAS and was administered sodium nitrite (NaNO 2 ), and a group that performed WAS and lettuce fermentation liquid.
  • the administration group consisted of a group administered WAS and a mixture of NaNO 2 + GSH, and a group administered WAS and a mixture of lettuce fermentation liquid + GSH (Table 3). Exposure to WAS caused diarrhea-like irritable growth syndrome-like symptoms (increased defecation) and increased mucosal immunity, and it was observed whether administration of each sample could reduce these symptoms.
  • Example 6 Blood pressure improvement effect by mixed sample of fermentation broth containing nitric oxide metabolite and yeast extract containing glutathione
  • the experimental animals used in this experiment were 7-week-old male Sprague Dawley rats (hereinafter referred to as SD rats) purchased from Samtaco and used after an acclimatization period of one week.
  • the average weight of SD rats was 198.95 ⁇ 2.10 g, and the samples were administered orally before the experiment.
  • light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23 ⁇ 2°C and humidity at 50-60%.
  • DOCA Deoxycorticosterone acetate; TCI, Japan
  • 1% sodium chloride aqueous solution was used as a drinking water to induce hypertension.
  • distilled water (DW) was administered to the control group and each sample was administered to the experimental group.
  • blood pressure was measured using a mouse tail blood pressure system (MK-2000STS, MUROMACHI KIKAI, Japan), and animals with systolic blood pressure of 130 mmHg or higher were selected and used.
  • Blood pressure of experimental animals was measured in real time for 55 minutes per animal by administering samples to each group. After the rat was placed in a correction frame and fixed, a piezo electric pulse sensor and an occlusion cuff were placed in its tail, and the pulse signal connected to the computer was observed at an appropriate level. At this time, the device was operated to observe systolic blood pressure.
  • the experimental animals were separated into 5 groups.
  • a control group was administered distilled water to a blood pressure model in which DOCA was intraperitoneally administered and fed regular feed and 1% sodium chloride aqueous solution.
  • DOCA was administered intraperitoneally and fed regular feed and 1% sodium chloride aqueous solution.
  • One blood pressure model was divided into NaNO 2 administration group, lettuce fermentation liquid administration group, NaNO 2 + GSH mixture administration group, and lettuce fermentation liquid + GSH mixture administration group (Table 4).
  • DOCA- injection DOCA dose material Dose One control group ⁇ 25mg/kg distilled water - 5 2 NaNO 2 ⁇ 25mg/kg Sodium nitrite 1mM 5 3 lettuce fermentation liquid ⁇ 25mg/kg Lettuce fermented extract 1mM 5 4 NaNO 2 + GSH ⁇ 25mg/kg Sodium nitrite + GSH 1mM 5 5 Lettuce fermentation liquid + GSH ⁇ 25mg/kg Lettuce fermented extract + GSH 1mM 5
  • Example 7 Effect of improving arthritis by a mixed sample of fermented broth containing nitric oxide metabolites and yeast extract containing glutathione
  • the experimental animals used in this experiment were 7-week-old male SD rats purchased from Samtaco and used after an acclimatization period of one week. Samples were administered orally once daily for 4 weeks. In the experimental animal breeding room, light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23 ⁇ 2°C and humidity at 50-60%.
  • MIA Monosodium iodoacetate
  • the experimental animals were divided into six groups: a normal group administered distilled water without any treatment, a control group administered distilled water after inducing osteoarthritis through MIA, a group administered 1 mM NaNO 2 , a group administered 1 mM fermented lettuce solution, and NaNO 2 + GSH. They were divided into a 1mM administration group and a lettuce fermentation solution + GSH 1mM administration group, and were orally administered for 4 weeks (Table 5). To calculate the number of animals in a group, the minimum number that determines statistical significance was used in accordance with the 3R principle.
  • the left knee joint area of the rat was measured to measure changes in leg edema thickness after inducing arthritis through MIA.
  • Leg edema thickness was measured after anesthesia using vernier calipers (Mitutoyo Co., Japan).
  • the arthritis index was assessed visually by three experimenters after photographing each animal before sacrificing the animal model. The evaluation was performed independently by three experimenters three times by modifying the method of Kim et al. (Korean J Orient Physiol Pathol, Effects of Acanthopanax senticosus and onion mixture extract on the collagen-induced arthritis in rat model. 2011, 25;1000-1007). Each score was assigned from 1 to 5 points and expressed as the average value (Table 6).
  • TNF- ⁇ and Metallopeptidase-9 were measured using an ELISA kit (R&D Systems, USA). After adding 50 ⁇ l of ADS (assay diluent solution) to each well with antibodies attached, 50 ⁇ l of each sample was added, left at room temperature for 2 hours, and then washed 4 times with 300 ⁇ l of washing buffer. After washing, 100 ⁇ l of mouse TNF- ⁇ and MMP-9 antibodies were added to each well and incubated at room temperature for 2 hours, then removed and washed four times.
  • ADS assay diluent solution
  • the amount of PGE 2 produced in serum was measured using an ELISA kit. After adding 50 ⁇ l of ADS to each well with antibodies attached, 50 ⁇ l of sample was added to each well, left at room temperature for 2 hours, and then washed four times with 300 ⁇ l of washing buffer. After washing, 50 ⁇ l of Proteoglycan E2 conjugate as an antibody was added to each well and incubated at room temperature for 2 hours, then removed and washed four times. 200 ⁇ l of substrate solution was added to each well and left at room temperature for 30 minutes, then 100 ⁇ l of reaction stop solution was added, and the absorbance was measured at 450 nm using an ELISA reader from Molecular Devices.
  • the serum was separated and the levels of TNF- ⁇ , MMP-9, and PGE 2 , which are major indicators of inflammation, were measured.
  • the levels of all sample administration groups decreased compared to the control group, and the NaNO 2 administration group, lettuce fermentation solution, NaNO 2 + GSH mixed administration group, and lettuce The fermentation broth + GSH mixed administration group showed a significant decrease in that order (Table 7).
  • TNF- ⁇ , MMP-9, and PGE 2 Changes in TNF- ⁇ , MMP-9, and PGE 2 according to sample treatment in an arthritis induction model using MIA Measurement Group Jeongsang-gun control group NaNO 2 lettuce fermentation liquid NaNO 2 +GSH lettuce fermentation liquid +GSH TNF- ⁇ (pg/mL) 15.42 ⁇ 0.65 65.15 ⁇ 5.45 54.48 ⁇ 2.85 49.65 ⁇ 4.22 50.22 ⁇ 2.15 46.38 ⁇ 2.12 MMP-9 (ng/mL) 0.25 ⁇ 0.03 0.82 ⁇ 0.04 0.58 ⁇ 0.04 0.54 ⁇ 0.07 0.46 ⁇ 0.03 0.45 ⁇ 0.03 PEG 2 (ng/mL) 0.71 ⁇ 0.03 1.08 ⁇ 0.03 0.97 ⁇ 0.02 0.91 ⁇ 0.03 0.89 ⁇ 0.03 0.85 ⁇ 0.03
  • Example 8 Liver protection effect by mixed sample of fermentation broth containing nitric oxide metabolites and yeast extract containing glutathione
  • the experimental animals used in this experiment were 7-week-old male Sprague Dawley rats (SD rats) purchased from Samtaco and used after an acclimatization period of one week.
  • the average weight of SD rats was 210.45 ⁇ 1.65 g.
  • the light and dark were adjusted every 12 hours in the laboratory animal breeding room, and the temperature was maintained at 23 ⁇ 2°C and humidity at 50-60%.
  • Rats used in the experiment were fasted for 12 hours, anesthetized with isoflurane, and blood was collected from the abdominal vena cava. Serum was separated by centrifugation at 3,000 rpm for 20 minutes. Blood AST (Aspartic acid transaminase) and ALT (Alanine transaminase) were measured using AST and ALT measurement reagents (Asan pharmaceutical) applying the Reitman-Frankel enzymatic method.
  • Serum ALT and AST levels are commonly measured clinically as biomarkers for liver health.
  • serum ALT and AST levels decreased in all sample administration groups compared to the control group, especially in the NaNO 2 administration group, lettuce fermentation solution administration group, and NaNO 2 + GSH mixed administration group, lettuce fermentation liquid + GSH mixed administration group showed a decrease in that order (FIG. 14).
  • Example 9 Effect of improving contact dermatitis by mixed sample of fermentation broth containing nitric oxide metabolite and yeast extract containing glutathione
  • the experimental animals used in this experiment were 5-week-old male Balb/c mice purchased from Samtaco and used after an acclimatization period of one week.
  • the average weight of Balb/c mice was 20.80 ⁇ 0.82 g, and the samples were orally administered once daily for 12 days.
  • light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23 ⁇ 2°C and humidity at 50-60%.
  • DNFB 1-fluoro-2,4-dinitrobenzene
  • reagent was prepared by diluting 0.5% DNFB in a solution of acetone:olive oil mixed at a ratio of 4:1.
  • 40 ⁇ l of 0.5% DNFB was applied to the back area for 4 days to induce skin sensitization.
  • contact dermatitis was induced by applying 20 ⁇ l of 0.3% DNFB to the ears (challenge).
  • the experimental animals were divided into 6 groups.
  • the group that did not receive any treatment was set as the normal group, the control group in which DNFB was applied to the right ear and DW was administered, the group in which DNFB was applied to the right ear and NaNO 2 1mM administered, and the group in which DNFB was applied to the right ear.
  • They were divided into a group administered with 1mM of fermented lettuce liquid, a group administered with DNFB applied to the right ear and administered 1mM NaNO 2 + GSH, and a group administered with fermented lettuce liquid + 1mM GSH with DNFB applied to the right ear (Table 9).
  • To calculate the number of animals in a group the minimum number that determines statistical significance was used in accordance with the 3R principle.
  • the normal group (CON) and control group were each administered DW, and the remaining groups were orally administered each sample once a day.
  • Atopic dermatitis experimental group composition Group Left Right Meterial Dose n One Jeongsang-gun Normal Normal distilled water - 5 2 control group Normal 0.3% DNFB distilled water - 5 3 NaNO 2 Normal 0.3% DNFB Sodium nitrite 1mM 5 4 lettuce fermentation liquid Normal 0.3% DNFB Lettuce fermented extract 1mM 5 5 NaNO 2 +GSH Normal 0.3% DNFB Sodium nitrite + GSH 1mM 5 6 Lettuce fermentation liquid + GSH Normal 0.3% DNFB Lettuce fermented extract + GSH 1mM 5
  • Visual evaluation is a clinical evaluation method commonly used in atopic dermatitis, and is expressed as the total score of each of the five items to measure the severity of atopic dermatitis symptoms.
  • the evaluation items are erythema, pruritus & dry skin, edema & excoriation, erosion, and lichenification. For each item, no symptoms (0 points), It was scored as mild (1 point), moderate (2 points), and severe (3 points), and then totaled to give a score ranging from a minimum of 0 to a maximum of 15.
  • Ear thickness was measured using vernier calipers (Mitutoyo Co.) at intervals of 12 hours, 24 hours, and 48 hours for 3 days.
  • mice After measuring the ear thickness, Balb/c mice were sacrificed, their spleens were removed, and their weight was measured. The spleen index was calculated by dividing the spleen weight (mg) of Balb/c mice by body weight (g).
  • a mixture of yeast extract containing sodium nitrite and glutathione a mixture of yeast extract containing sodium nitrite and glutathione;
  • the composition of the present invention which consists of a mixture of fermented broth containing nitric oxide and yeast extract containing glutathione, not only has an anti-diabetic effect, but also has a preventive and improving effect on hypertension, enteritis, stress bowel syndrome, arthritis, liver disease, atopic dermatitis, etc., It can be usefully used in related industrial fields.
  • Example 10 Blood pressure improvement effect by mixed sample of fermentation broth containing nitric oxide metabolites and yeast extract containing pure glutathione, N-acetyl cysteine, methylsulfonylmethane, alpha-lipoic acid, or glutathione
  • GSH glutathione
  • NAC N-acetyl cysteine
  • MSM methylsulfonylmethane
  • composition of blood pressure experimental group for compositions containing sulfur-containing substances Group Treatment n DOCA- injection DOCA dose material Dose One ⁇ 25mg/kg fermented extract or Sodium nitrite 1mM 5 2 ⁇ 25mg/kg Group 1 + NAC 1mM 5 3 ⁇ 25mg/kg Group 1 + GSH 1mM 5 4 ⁇ 25mg/kg Group 1 + MSM 1mM 5 5 ⁇ 25mg/kg Group 1 + ALA 1mM 5 6 ⁇ 25mg/kg Group 1 + Yeast extract (GSH containing) 1mM 5

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Abstract

La présente invention concerne : une composition naturelle destinée à inhiber la production de N-nitrosamine carcinogène dans l'estomac et dans les intestins, et à favoriser la production d'oxyde nitrique dans les intestins ; et une utilisation de celle-ci. La composition naturelle selon la présente invention comprend un produit de fermentation naturelle, dans lequel un nitrite de sodium (NaNO2) ou un métabolite d'oxyde nitrique sont immobilisés et stabilisés, et une substance contenant du soufre en tant que principes actifs, produit du S-nitrosoglutathione au moyen d'acide gastrique (conditions acides fortes) dans l'estomac, et peut produire une concentration prédéterminée d'oxyde nitrique pendant une période de temps prédéterminée après avoir été administrée à l'intestin grêle et au côlon, et peut ainsi être largement appliqué dans des domaines liés au traitement de l'hypertension, du diabète, du syndrome du côlon irritable induit par le stress, de l'arthrite ou du traitement de la dermatite atopique.
PCT/KR2023/006244 2022-05-09 2023-05-09 Composition naturelle pour inhiber la production de n-nitrosamine carcinogène dans l'estomac et les intestins et favoriser la production d'oxyde nitrique dans les intestins, et son utilisation Ceased WO2023219376A2 (fr)

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KR1020220056809A KR102602347B1 (ko) 2022-05-09 2022-05-09 위장내 발암성 엔-니트로사민 생성 억제 및 장내 산화질소 생성 촉진용 천연 조성물 및 이의 용도
KR10-2022-0056809 2022-05-09

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WO2025100593A1 (fr) * 2023-11-09 2025-05-15 천현수 Composition pour inhiber la production de n-nitrosamine carcinogène dans l'estomac et son utilisation

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CA2650204A1 (fr) * 1996-12-31 1998-07-09 Harry B. Demopoulos Preparations pharmaceutiques de glutathion et modes d'administration de ces preparations
JP2004123707A (ja) * 2002-07-29 2004-04-22 Toyo Shinyaku:Kk 血流改善組成物
KR101150148B1 (ko) * 2010-04-23 2012-06-08 샘표식품 주식회사 글루타치온 함량이 증대된 신규 효모 변이주, 그 제조 방법 및 용도
KR101316806B1 (ko) * 2011-08-02 2013-10-10 (주)이뮤노텍 글루타치온과 실리마린을 유효성분으로 함유하는 항산화 및 간보호용 조성물
CN107613996A (zh) * 2015-05-29 2018-01-19 兴人生命科学株式会社 具有血管舒张作用的酵母抽提物
CN109069701A (zh) * 2016-02-25 2018-12-21 密歇根大学董事会 氧化氮产生制剂和试剂盒
KR102056338B1 (ko) * 2019-08-09 2019-12-16 주식회사 에브릿 홍삼 진세노사이드와 미생물 마늘발효에서 유래된 글루타치온 성분이 함유된 혈행개선효과를 가지는 건강기능성 조성물
KR102129038B1 (ko) * 2019-10-29 2020-07-01 천현수 질소 함유 천연물의 발효를 통한 산화질소 대사체의 고정화 및 안정화 방법

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WO2023219376A9 (fr) 2024-04-04

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