KR102403429B1 - Enteric coated tablet comprising natural fermented product with fixed and stabilized nitric oxide metabolite as effective component and producing nitric oxide in intestinal environment - Google Patents

Abstract

The present invention relates to an enteric-coated tablet containing a natural fermented product with fixed and stabilized nitric oxide metabolite as an active component, and producing nitric oxide in intestinal environment. The enteric-coated tablet of the present invention is developed to deliver nitric oxide metabolites to the small intestine or large intestine without exposing the same to gastric juice (strongly acidic conditions) and then generate nitric oxide at a certain concentration for a certain period of time. Therefore, the enteric-coated tablet is expected to be widely applied in fields related to the treatment of hypertension, diabetes, enteritis, stress-induced bowel syndrome, arthritis, or atopy.

Description

Enteric coated tablet comprising natural fermented product with fixed and stabilized nitric oxide metabolite as effective component and producing nitric oxide in intestinal environment environment}

The present invention relates to an enteric tablet containing a natural fermented product in which a nitric oxide metabolite is immobilized and stabilized as an active ingredient and produces nitric oxide in the intestinal environment, and more particularly, a nitric oxide metabolite obtained through microbial fermentation is immobilized and stabilized It relates to an enteric tablet that contains natural fermentation products and polyphenols and can produce nitric oxide that can be absorbed by the human body for a certain period of time even in the intestinal environment, which is a weak acidic or weakly alkaline condition other than a strong acidic condition.

Nitric oxide (hereinafter referred to as NO) is a gaseous substance produced by decomposing arginine, known as an amino acid, by NOS (NO synthase), which is present in bacteria, plants, and animals, and is a highly reactive radical (Scheme 1) -1), it is hydrophobic and is an unstable substance with chemical properties that is rapidly oxidized and changed into hydrophilic NO metabolites (referring to nitrite ions [NO ₂ ^- ], nitrate ions [NO ₃ ^- ], etc.) (Scheme 1- 2 and 1-3). Because NO is very small in size and hydrophobic, it can move rapidly in aqueous solutions such as blood or body fluids. In particular, it can pass through blood vessel barriers and cell membranes for a given existence time (estimated to be less than about 1 second). have. In addition, since the generated NO is highly reactive, it reacts with oxygen dissolved in blood or body fluid or oxygen bound to red blood cells, and is transformed into a water-soluble and longer-lived NO metabolite (refer to Schemes 1-2 and 1-3). By being (oxidized) and dissolved in water-soluble blood, it can be transported anywhere in the body and excreted out of the body.

As a NO metabolite, NO ₂ ^- is a hydrophilic and relatively stable ionic substance, dissolved in blood and body fluids, and can move to all parts of the body, but cannot pass through organ barriers, blood vessel barriers, cell membranes, etc. However, NO ₂ ⁻ is reduced to hydrophobic NO under acidic conditions and oxygen deficiency and passes through organ barriers, blood vessel barriers, cell membranes, etc., thereby exhibiting the intrinsic physiological function and pharmacological effects of NO (refer to Scheme 2-1). Also, NO ₂ ⁻ may be reduced to NO by reacting with vitamin C (ascorbic acid/ascorbate, hereinafter referred to as Asc) dissolved in blood or body fluid (refer to Schemes 2-2 and 2-3).

In the human body, NO is mainly produced by the process of Reaction Scheme 1-1) in vascular endothelial cells and is used for regulating the physiological function of relaxing adjacent blood vessels, and a part of the generated NO is converted to NO ₂ ^- by the process of Scheme 1-2). By being oxidized and dissolved in blood, it moves to all places in the human body and is reduced to NO by the process of Reaction Scheme 2-1) or Scheme 2-2) in places where NO is needed, thereby showing the physiological functions and pharmacological effects of NO. NO generated in excess by NOS or NO generated in the reduction of excess NO ₂ ⁻ is oxidized to NO ₃ ⁻ by the process of Reaction Equation 1-3) and is excreted out of the human body through urine. Therefore, it can be seen that there is no significant human toxicity due to excess NO or NO ₂ ^- . However, in the process of Scheme 1-3), cyanosis may appear as erythrocytes lose oxygen, but this symptom is quickly resolved as erythrocytes are combined with oxygen again.

The amount of NO in the human body can be predicted as the total amount by the sum of NO ₂ ^- and NO ₃ ^- , but the actual usable NO concentration can predict the blood NO concentration by the NO ₂ ^- concentration 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 blood NO ₂ ⁻ concentration is sometimes observed to be lower than that of normal people. There are many. There may be several ways to raise the reduced NO ₂ ⁻ concentration to the normal range, but maintaining the blood NO ₂ ⁻ concentration in the normal range by ingesting NO metabolites through food or food is the most reasonable method. . In fact, humans and animals maintain their blood NO ₂ ⁻ levels in the normal range through food. In addition, when analyzing several reports on the effects of NO metabolites in experimental animals, when NO metabolite intake was restricted, diabetes symptoms appeared in experimental animals, but conversely, when NO metabolites were orally administered to diabetic experimental animals Diabetes improved.

Humans and laboratory animals cannot directly absorb NO metabolites into the body. When humans ingest NO metabolite, NO ₂ ⁻ of NO metabolite is reduced to NO by the strong acid condition of gastric juice in the stomach, and then the reduced NO passes through the stomach wall to reach blood or body fluid and is converted to NO ₂ ⁻ Oxidation completes absorption into the body (refer to Scheme 3).

On the other hand, ingested NO ₃ ^- (another NO metabolite) is different from NO ₂ ^- reduced to NO ₂ ^- by NO ₃ ^- reductase (nitrate reductase, hereinafter referred to as NRase) of bacteria symbiotic in the small intestine and large intestine. Next, it reacts with polyphenol (hereinafter referred to as Ph-OH) abundantly present in the intestine and is reduced to NO, so that it can be absorbed into the body (refer to Scheme 4).

Taking NO metabolites orally to raise low blood NO (or NO ₂ ⁻ ) levels or to lower high blood sugar levels can cause three unhealthy problems:

First, orally ingested NO ₂ ⁻ may cause excessive NO to be absorbed into the human body in a short time due to the strong acid condition of gastric juice, resulting in a rapid decrease in blood pressure, hypoglycemia, and cyanosis.

Second, because of the strong acid condition of gastric juice, NO ₂ ^- taken orally reacts with secondary amine (hereinafter referred to as R2N-H) and N-nitrosamine (hereinafter referred to as R2N-N=O), which is known as a carcinogen. notation) can be generated (see Scheme 5).

Third, orally ingested NO ₃ ⁻ cannot be reduced in the stomach but is reduced to NO in the small and large intestine and absorbed into the body, but the degree of NO reduction depends on the individual’s intestinal bacterial environment as well as vegetable (polyphenol) eating habits. Therefore, effective NO absorption of a certain amount cannot be expected.

In our body, NO metabolites are reduced to NO under oxygen-deficient or acidic conditions, thereby regulating blood flow by dilating blood vessels or promoting capillary formation (Lundberg et al., Nat Rev Drug Discov. 2008, 7). :156-167). The blood flow regulation action of these NO metabolites suppresses vascular damage caused by hypertension by returning high blood pressure to normal blood pressure (Amaral et al., Redox Biol. 2015, 5:340-346). Therefore, NO metabolite can be developed as one of the antihypertensive drugs with few side effects. In addition, several recent research reports have reported the hypoglycemic 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), insulin resistance relief effect (Ohtake et al., Nitric Oxide. 2015, 44:31-38), diabetic complications improvement effect (Bahadoran et al., Nutr Metab (Lond). 2015, 12 :16. doi:10.1186/ s12986-015-0013-6), etc. have been reported, and various studies are underway to develop NO metabolites as therapeutic agents for diabetes and diabetic complications (Ghasemi et al., Nitric Oxide. 2017, 70 :9-24). However, for the purpose of treating high blood pressure or diabetes, oral intake of NO metabolites can have three adverse health problems, as mentioned above.

On the other hand, Korean Patent Application Laid-Open No. 2015-0118160 discloses an 'enteric tablet' containing a large amount of active ingredient, does not thicken the film, and has sufficient impact resistance, and Korea Patent Publication No. 2010-0063059 discloses 'Mypoletic enteric tablet' A sustained-release composition containing and a method for preparing the same are disclosed, but an enteric tablet containing a natural fermented product in which the nitric oxide metabolite of the present invention is immobilized and stabilized as an active ingredient and produces nitric oxide in the intestinal environment has been described. none.

The present invention has been derived from the above needs, and the present inventors provide a natural substance in which nitric oxide metabolites are immobilized and stabilized so that nitric oxide at an effective concentration for lowering blood pressure or blood sugar can be generated for a certain period of time in the environment of the small and large intestine. An enteric tablet composition comprising a fermented product or a mixture of the natural fermented product and polyphenol was developed, and the composition gradually produces nitric oxide in the intestinal environment of the experimental animal, resulting in hypertension, diabetes, enteritis, stress bowel syndrome, arthritis, By confirming that liver disease, atopic dermatitis, etc. can be improved, the present invention has been completed.

In order to solve the above problems, the present invention provides an enteric tablet containing sodium nitrite (NaNO ₂ ) or a natural fermentation product in which a nitric oxide metabolite is immobilized and stabilized as an active ingredient, and generates nitric oxide in the intestinal environment.

In addition, the present invention provides a pharmaceutical composition comprising as an active ingredient a natural fermentation product in which the enteric tablet or nitric oxide metabolite is immobilized and stabilized.

In addition, the present invention provides a health functional food composition comprising, as an active ingredient, a natural fermented product in which the enteric tablet or nitric oxide metabolite is immobilized and stabilized.

The enteric tablet of the present invention was developed so that nitric oxide metabolites can be delivered to the small or large intestine without exposing them to gastric juice (strong acid conditions) in the stomach and then generate nitric oxide at a certain concentration for a certain period of time. Hypertension, diabetes, enteritis , is expected to be widely applied in fields related to the treatment of stress bowel syndrome, arthritis, liver disease, and atopic dermatitis.

1 is a diagram comparing the intestinal nitric oxide production of an enteric tablet containing a fermented product mixed with nitric oxide metabolites and polyphenols with the nitric oxide production in the stomach.
2 is a schematic diagram of an experiment for confirming the effect of improving stress bowel syndrome. WAS means water avoidance stress, and the treated samples are shown in Table 3.
3 is a graph analyzing the degree of disease activation according to sample administration in an ulcerative colitis model.
4 is a graph analyzing changes in intestinal length according to sample administration in an ulcerative colitis model.
5 is a graph analyzing TNF-α and IL-1β levels in serum according to sample administration in an ulcerative colitis model.
6 is a result of measuring the number of variables for each experimental group according to the sample administration in the stress bowel syndrome model, (A) is the daily average number of variables, (B) shows the accumulated number of variables until the end of the experiment.
7 is a result of measuring blood pressure changes before and after administration of a sample in a hypertensive model.
8 is a result of measuring blood glucose change according to sample administration in a diabetes model.
9 is a result of measuring the levels of TNF-α, IL-1β and PGE2 in serum after administering a sample in an arthritis model.
10 is a result of measuring ALT and AST levels in serum after administering a sample in an acute liver injury model.
11 is a result of analyzing the morphological change of the lesion by observing the right ear region after administering the sample in the atopic dermatitis model.
12 is a result of measuring the thickness change of the right ear region after administering the sample in the atopic dermatitis model.
13 is a result of measuring the weight of the spleen at the end of the experiment for each experimental group after the administration of the sample in the atopic dermatitis model.
14 is a result of measuring the levels of TNF-α and IL-4 in serum at the end of the experiment for each experimental group after administering the sample in the atopic dermatitis model.
15 is a result of measuring the change in body temperature for each experimental group after administration of a natural fermented product sample.
16 is a result of confirming the change in the wound area for each experimental group after induction of the wound. WHD: a control group in which distilled water was applied to the wound site after induction of a wound, WHN: a group treated with 1 mM nitrite coating solution on the wound site, WHNCA: a group treated with 1 mM nitrite and 1 mM citric acid coating solution on the wound site, WHCA: wound area Group treated with 1 mM citric acid coating solution on the site, WHNP: group treated with 1 mM citric acid and sample (fermented cabbage) coating solution on the wound site, WHP: group treated with sample (fermented cabbage) coating solution on the wound site.
17 is a view of an experimental animal in which an electrode for EEG (electroencephalogram) recording is inserted and fixed.
18 is a cortical EEG test result, showing the recording of LFPs (local field potentials). Bofore: before sample administration, After: after sample administration.
19 is an analysis result of changes in cerebral blood flow in experimental animals measured 30 minutes after intestinal administration of garlic fermented broth.

In order to achieve the object of the present invention, the present invention provides an enteric tablet containing sodium nitrite (NaNO ₂ ) or a natural fermented product in which a nitric oxide metabolite is immobilized and stabilized as an active ingredient, and generates nitric oxide in the intestinal environment.

The enteric tablet according to the present invention may further include polyphenols in addition to the natural fermentation products in which sodium nitrite (NaNO ₂ ) or nitric oxide metabolites are immobilized and stabilized. The polyphenol functions to promote the reduction of nitric oxide metabolites in natural fermentation to nitric oxide, but is not limited thereto, but may be resveratrol, quercetin, silymarin, anthocyanin or catechinic acid. The polyphenol may be used in the form of a compound or in the form of a powder of a natural material containing each polyphenol compound (eg, aronia powder, barley sprout powder, etc.), but is not limited thereto.

In the enteric tablet according to the present invention, the natural fermented product in which the nitric oxide metabolite is immobilized and stabilized is obtained by adding a fermented strain to a nitrogen-containing natural product at a temperature of 18 to 35° C. and a dissolved oxygen concentration of 0.03 to 0.1 mg/L from 2 to It may be prepared by a manufacturing method comprising a step of fermenting for 30 days, but is not limited thereto.

For a method for preparing a natural fermented product in which the nitric oxide metabolite is immobilized and stabilized according to the present invention, reference may be made to Korean Patent Registration No. 2129038.

As used herein, the term "enteric-coated tablet" means to prevent disintegration in the acidic stomach and to disintegrate in the alkaline intestine, and the gastric mucosa is stimulated and vomiting It means a tablet to prevent decomposition into gastric acid, which interferes with digestive enzymes, and to expect action in the intestine.

Materials used for enteric coating of tablets and capsules include fat, fatty acids, wax, shellac, Cellulose acetate phthalate or Hydroxypropylmethylcellulose phthalate (HPMCP), but are not limited thereto, Conventional coating materials used in the manufacture of enteric-coated tablets may be selected and used.

The enteric tablet according to an embodiment of the present invention is preferably prepared by sequentially coating a natural fermented product in which nitric oxide metabolites are immobilized and stabilized with stearic acid, glycerin, glycerin fatty acid ester (enteric coating), and HPMCP (enteric coating). may be, but is not limited thereto. The natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized may be used in the form of a fermented product or a concentrate, or a powdered form through freeze-drying or hot air drying after concentrating the fermentation broth, but is not limited thereto.

The amount of nitric oxide in the human body can be predicted by the sum of NO ₂ ^- and NO ₃ ^- , but the actual usable nitric oxide concentration can predict the blood NO concentration by the NO ₂ ^- concentration that can be converted to nitric oxide. As a nitric oxide metabolite, NO ₂ ^- is a hydrophilic and relatively stable ionic substance, dissolved in blood and body fluids, and can move to all parts of the body, but cannot pass through organ barriers, blood vessel barriers, cell membranes, etc. Taking nitric oxide metabolites orally to increase the concentration of low blood nitric oxide metabolites (NO ₂ ⁻ ) or to lower high blood sugar levels can cause three adverse health problems:

First, orally ingested NO ₂ ⁻ may cause excessive nitric oxide to be absorbed into the human body in a short time due to the strong acid condition of gastric juice, resulting in a rapid decrease in blood pressure, hypoglycemia, and cyanosis. Second, because of the strong acid condition of gastric juice, NO ₂ ^- taken orally reacts with secondary amines (hereinafter referred to as R ₂ NH) and N-nitrosamine (hereinafter R ₂ NN=O), which is known to be a carcinogen. ) can be created. Third, oral NO ₃ ⁻ cannot be reduced in the stomach, but is reduced to nitric oxide in the small and large intestine and absorbed into the body. Therefore, an effective absorption of a certain amount of nitrogen oxide cannot be expected.

The enteric tablet according to the present invention delivers nitric oxide metabolites contained in natural fermented products to the small intestine or large intestine without exposing the nitric oxide metabolites contained in the natural fermented product to the strong acid conditions of the stomach. It may be developed by encapsulating a composition in which a metabolite and polyphenol are mixed.

In addition, in the enteric tablet according to the present invention, the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized may preferably contain 100 ppm or more based on NO ₂ ⁻ , but is not limited thereto.

As used herein, the term "nitric oxide metabolite" refers to nitrite (NO ₂ ^- ), R-NO ₂ ^- , R-NO, nitrate (NO ₃ ^- ), and R-NO ₃ ^- , where R is polyphenols. (polyphenols), including fatty acids (fatty acid), etc., refers to a functional group to which nitric oxide or nitrite ions can be bound. Nitric oxide metabolites are converted to nitric oxide under acidic and reducing conditions. In acidic conditions such as stomach and skin, hydrogen ions (H ⁺ ) oxidize nitric oxide metabolites to generate nitric oxide, and under reducing conditions such as intestines (small intestine, large intestine), polyphenols or vitamin C are reduced Substances reduce the nitric oxide metabolite to form nitric oxide.

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

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

In addition, the fermentation may be performed at a dissolved oxygen concentration of 0.01 to 0.1 mg/L, preferably 0.03 to 0.1 mg/L, but is not limited thereto.

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

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

In addition, the fermentation strain is Bacillus genus ( Bacillus sp.), Bifidobacterium sp., Enterococcus genus ( Enterococcus sp.), Lactobacillus genus ( Lactobacillus sp.), Lactococcus sp. ) or Weissella genus ( Weissella sp.) may be a strain, preferably Bacillus subtilis ( Bacillus subtilis ), Bacillus amyloliquefaciens ( B. amyloliquefaciens ), Bacillus natto ( B. natto ), Bacillus licheni Formis ( B. licheniformis ), Bifidobacterium bifidum ( Bifidobacterium bifidum ), Bifidobacterium Infantis ( B. infantis ), Bifidobacterium longum ( B. longum ), Enterococcus faecium ( Enterococcus ) faecium ), Enterococcus faecalis ( E. faecalis ), Lactobacillus acidopilus ( Lactobacillus acidopilus ), Lactobacillus alimentarius ( L. alimentarius ), Lactobacillus bulgaricus ( L. bulgaricus ), bulgaricus ( L. bulgaricus ) L. casei ), Lactobacillus curvatus ( L. curvatus ), Lactobacillus delbrukii ( L. delbrukii ), Lactobacillus johnsonii ( L. johnsonii ), Lactobacillus parsiminus ( L. farciminus ), Lactobacillus Gasseri ( L. gasseri ), Lactobacillus helveticus ( L. helveticus ), Lactobacillus rhamnosus ( L. rhamnosus ), Lactobacillus reuteri ( L. reuteri ), Lactobacillus sakei ( L. sakei ), Lactococcus Any one selected from the group consisting of lactis ( Lactococcus lactis ) and Weissella cibaria may be used, and preferably, the accession number is KCTC12501BP Basil Ruth subtilis ( Bacillus subtilis ) - may be Chun Hyun Su, but is not limited thereto.

In addition, the nitrogen-containing natural products are not limited thereto, but lettuce, donuts, spinach, blueberries, dandelions, pomegranates, cabbage, garlic, noni, onions, beans, bean sprouts, peanuts, peanut sprouts, velvet beans, mulberry leaves, bitter gourd, bokbunja , Eosongcho, Aronia, Yulcho, Yellow lily, Golden, Hwangbaek, Hibiscus, Samchae, Mushrooms, Calamus, Seomcho, Chinese cabbage, Kelp, Salmon milt, Abalone shell (Seokgyeolmyeong), Crustacean shell, Cricket, Lavender, Chamomile, Graviola, Basil , apple, mugwort, tangerine, silkworm, Myongmok Rehmannia and Sipjeondaebotang may be at least one selected from the group consisting of.

The present invention also provides a pharmaceutical composition comprising, as an active ingredient, a natural fermentation product in which the enteric tablet or nitric oxide metabolite is immobilized and stabilized.

In the pharmaceutical composition according to the present invention, the natural fermented product in which the nitric oxide metabolite is immobilized and stabilized is at a temperature of 18 to 35° C. and dissolved oxygen concentration of 0.03 to 0.1 mg/L by adding a fermented strain to the nitrogen-containing natural product. It may be prepared by a manufacturing method comprising a step of fermentation for 2 to 30 days, but is not limited thereto, and nitrogen-containing natural products and fermentation strains are the same as described above.

In addition, in the pharmaceutical composition, the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized may further include a plant containing a polyphenol compound, and the plant containing the polyphenol compound is not limited thereto , aronia or sprouted barley. The plant may be included in the form of powder or extract, but is not limited thereto.

The pharmaceutical composition according to the present invention may be used for the prevention or treatment of hypertension, diabetes, enteritis, stress bowel syndrome, arthritis, liver disease, atopic dermatitis, sleep disturbance, or cerebrovascular disease, but is not limited thereto. The sleep disorder may be non-organic sleep disorder including non-organic insomnia, nightmares, sleepwalking, etc. and organic sleep disorder including organic insomnia, narcolepsy, and sleep apnea, and the cerebrovascular disease is preferably a cerebral blood flow disorder disease. may be a stroke, but is not limited thereto.

In addition, in the pharmaceutical composition according to the present invention, the natural fermentation product may be used in the form of a concentrate or in the form of a solid, but is not limited thereto. In addition, the concentrate or solidified product of the natural fermentation is not limited thereto, but it may be more preferable to use it after coating.

In the pharmaceutical composition of the present invention, when the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized contains 5 ppm or more and less than 10 ppm of nitric oxide metabolite based on NO ₂ ^- , there is a disease prevention effect, When the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized contains 10 ppm or more and less than 40 ppm of nitric oxide metabolite based on NO ₂ ^- Fermented product is based on NO ₂ ^- If it contains more than 40 ppm of nitric oxide metabolite, it can have a therapeutic effect on diseases.

In the pharmaceutical composition according to the present invention, the natural fermented product may be preferably included in an amount of 40 mg/g to 140 mg/g based on the total amount of the pharmaceutical composition, and more preferably included in an amount of 60 mg/g to 120 mg/g Preferably, it is more preferably included in an amount of 90 mg/g to 110 mg/g.

In addition, the pharmaceutical composition may be formulated and used in the form of oral dosage forms such as granules, tablets, and capsules according to a conventional method according to the purpose of use, and may be administered orally or rectal, but not limited thereto.

The pharmaceutical composition of the present invention may be formulated using a pharmaceutically acceptable carrier or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Examples of suitable carriers, excipients and diluents that may be included in such pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition may further include a filler, an anti-aggregant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

In addition, the pharmaceutical composition according to the present invention is high blood pressure, diabetes, enteritis by administering a pharmaceutically effective amount to a patient with hypertension, diabetes, enteritis, stress bowel syndrome, arthritis, liver disease, atopic dermatitis, sleep disorder or cerebrovascular disease. , stress bowel syndrome, arthritis, liver disease, atopic dermatitis, sleep disturbance or cerebrovascular disease can be prevented and treated. In addition, the pharmaceutical composition comprising an enteric tablet or a natural fermented product according to the present invention has excellent skin cell regeneration effect, and thus can be effectively used in the treatment of wounds and burns.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the pharmaceutically effective amount of an enteric tablet or a pharmaceutical composition comprising a natural fermentation product in which a nitric oxide metabolite is immobilized and stabilized according to the present invention may vary depending on the age, sex, and weight of the patient, and generally per kg of body weight. 2 mg to 4 mg, preferably 3 mg, is administered daily or every other day, and may be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

The present invention also provides a health functional food composition comprising, as an active ingredient, a natural fermented product in which the enteric tablet or nitric oxide metabolite is immobilized and stabilized.

In the health functional food composition according to the present invention, the natural fermented product in which the nitric oxide metabolite is immobilized and stabilized is obtained by adding a fermented strain to a nitrogen-containing natural product at a temperature of 18 to 35° C. and dissolved oxygen of 0.03 to 0.1 mg/L. It may be prepared by a manufacturing method comprising a step of fermenting for 2 to 30 days at a concentration, but is not limited thereto, and the nitrogen-containing natural products and fermented strains are the same as described above.

In addition, in the health functional food composition, the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized may further include a plant containing a polyphenol compound, and the plant containing the polyphenol compound is limited thereto However, it may be aronia or sprouted barley. The plant may be included in the form of powder or extract, but is not limited thereto.

The health functional food composition according to the present invention may be for prevention or improvement of hypertension, diabetes, enteritis, stress bowel syndrome, arthritis, liver disease, atopic dermatitis, sleep disturbance, or cerebrovascular disease, but is not limited thereto.

The health functional food composition of the present invention includes all health functional food, health supplement, and the like, and the health functional food composition may be prepared in the form of powder, granule, pill, tablet, capsule, candy, syrup or beverage. , but not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient may be appropriately used depending on the purpose of its use (prevention or improvement). In general, in the production of food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the total raw materials. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols (eg, , xylitol, sorbitol, erythritol, etc.) is preferable. As the flavoring agent, natural flavoring agents (eg, taumartin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

For example, when the health functional food composition of the present invention is a food composition such as a functional beverage, the food composition may include a sweetener, a flavoring agent, a physiologically active ingredient, a mineral, etc. in addition to the active ingredient thereof.

The sweetener may be used in an amount to give the food a suitable sweetness, and may be natural or synthetic. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents may be used to improve taste or aroma, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural product, the purpose of nutritional enhancement in addition to flavor may be concurrently used. The natural flavoring agent may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or obtained from green tea leaves, horseradish leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. Optionally, a synthetic flavoring agent may be used, and the synthetic flavoring agent may be an ester, an alcohol, an aldehyde, a terpene, or the like.

As the physiologically active substance, catechins such as catechin, epicatechin, gallogatechin, and epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, and riboflavin can be used. As minerals, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc, and the like can be used.

In addition, the health functional food composition of the present invention may include a preservative, an emulsifier, an acidulant, a thickener, etc. as necessary in addition to the sweetener. These preservatives, emulsifiers, etc. are preferably added in a trace amount as long as the use to which they are added can be achieved. Trace amount means a range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition when expressed numerically. Examples of the preservative include sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), and the like. Examples of the emulsifier include gum acacia, carboxymethylcellulose, and xanthan gum. , pectin, and the like, and examples of the acidulant include acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like, but is not limited thereto. Such acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste. In addition, examples of the thickener include a suspending agent, a settling agent, a gel-forming agent, a bulking agent, and the like.

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

1. Experimental Materials

The materials used in this experiment were sodium nitrite (NaNO ₂ ) and (U) Human Enos fermented soybean broth, lettuce fermented cabbage, fermented cabbage, and garlic fermented aronia and sprouted barley powder, each of which contains polyphenols. By mixing, the nitric oxide metabolite content was adjusted to be the same based on the NO ₂ ^{-concentration} , and then used in the experiment.

The soybean, lettuce, cabbage or garlic fermented broth was concentrated at a stirring speed of 100 to 300 rpm for 24 to 48 hours at 20 to 30 ° C. it means.

NO ₂ ^- and NO ₃ ^- contained in the fermentation broth are converted to NO ₂ ^- by adding zinc powder (the solid state does not cause a change in the volume of the liquid and thus does not decrease the concentration ⁾ _. The total content of nitric oxide metabolites was determined by reducing and measuring the concentration of total NO ₂ ⁻ .

To be more specific, the total NO ₂ ⁻ content is first diluted 1,000-fold with distilled water and then centrifuged at 3,000 rpm to remove insoluble polyphenols and dietary fiber from the concentrated solution reduced for 1 hour by adding zinc powder, then clear supernatant 50 μl was taken and used as a measurement sample. 50 μl of NO ₂ ^- standard solution and 50 μl of the measurement sample are dispensed into 96-well plates, respectively, and 100 μl of test solution [30% acetic acid solution containing 1% sulfanilamide and 0.1% N-( A solution of a 60% acetic acid solution containing 1-naphthyl)ethylenediamine in a ratio of 1:2] was added, and the mixture was left at room temperature for 10 minutes to induce a color reaction. Then, the absorbance value of each well was measured at a wavelength of 570 nm using a multi-plate reader, and then the absorbance value of the standard solution and the absorbance value of the sample were compared to calculate NO ₂ ^{-concentration} .

In the present invention, the types of nitric oxide metabolite-containing natural fermentation products prepared according to the method of the Korean patent registration and the productivity of nitric oxide metabolites in each fermented product are summarized in Table 1 below.

2. Statistical processing

The results obtained in each experiment were expressed as mean ± standard deviation (mean ± SD). Statistics on the significance of each test group were analyzed using One-way Anova SPSS v.12 for statistical comparison between the control group and the experimental group, and Duncan's post-hoc test was performed to determine statistically significant when p < 0.05. .

Example 1. Enteritis improvement effect of natural fermented product containing nitric oxide metabolite

As the experimental animals used in this experiment, 6-week-old male Balb/c mice were purchased from Samtaco (Korea) and were used after undergoing a one-week acclimatization period. The average weight of Balb/c mice was 20.25±1.51 g, and the sample was administered directly (rectally) into the intestine once a day. Contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2℃ and the humidity was maintained at 50-60%.

1-1. Group composition and induction of ulcerative colitis

Experimental animals were classified into 6 groups, and the normal group administered with distilled water (DW) without any treatment, the control group that induced enteritis and was administered DW, the group that induced enteritis and administered 235 ppm of sodium nitrite (NaNO ₂ ), and 235 ppm of enteritis It was classified into a group administered with 235 ppm of soybean fermented solution, a group administered with fermented soybean solution + aronia mixed solution 235 ppm, and a group administered with fermented soybean solution + 235 ppm of mixed solution of sprouted barley that induced enteritis (Table 2). Ulcerative colitis was induced by ad libitum ingestion of 3.0% DSS (Dxtran sulfate sodium) for 5 days in all experimental groups except the normal group. For the calculation of the number of animals in a group, the minimum number covering statistical significance was used in accordance with the 3R principle.

Composition of the Ulcerative Colitis Experimental Group Group DSS ¹⁾ Material Dose n One normal group No DW ²⁾ - 5 2 control Yes DW - 5 3 NaNO ₂ Yes sodium nitrogen dioxide 235 ppm 5 4 fermented soybean Yes soybean fermented extract 235 ppm 5 5 Soybean Fermented Liquid + Aronia Yes soybean fermented extract + Aronia 235 ppm 5 6 Soybean fermented liquid + sprouted barley Yes soybean fermented extract + Barley Sprout 235 ppm 5 ¹⁾ DSS: Dxtran sulfate sodium
²⁾ DW: distilled water

1-2. Measuring the degree of disease activation

Bowel disease is measured by signs such as weight loss, diarrhea with bleeding and mucus, and shortening of the colon. According to the results of previous studies, 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 the initial and present weights. Diarrhea is defined as the absence of granules of feces in the rectum and persistent soft feces in the rectum. Visible rectal bleeding is independent of diarrhea with bleeding or gross rectal bleeding or other diarrhea. In the present invention, DAI was calculated as follows.

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

The medical parameters used here are comprehensive functional measures similar to the medical symptoms of ulcerative colitis in humans (Table 3).

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

1-3. Measurement of intestinal length and measurement of cytokines in serum

After completion of the experiment, all of the experimental groups were bled from the orbital vein under respiratory anesthesia, and after serum separation, an abdominal incision was made, excised from the distal end of the large intestine to the cecum, and the length of the intestine was measured. For cytokines in serum, the production of TNF-α and IL-1β was measured using an ELISA kit (R&D Systems, USA). After 50 μl of each ADS (assay diluent solution) was added to each well to which the antibody was attached, 50 μl of each sample was added, left at room temperature for 2 hours, and washed 4 times with 300 μl of wash buffer. After washing, 100 μl of mouse TNF-α and IL-1β as an antibody was added to each well, incubated for 2 hours at room temperature, removed and washed 4 times. 100 μl of the substrate solution was put into each well, left at room temperature for 30 minutes, 100 μl of a stop solution was added, and absorbance was measured at 450 nm with an ELISA reader (SPECTRA max M2, USA) from Molecular Devices.

1-4. Effect of Natural Fermentation in Ulcerative Colitis Model

The DSS colitis model presents clinical signs characterized by weight loss, diarrhea, and bloody stools. The results of measuring the effect on these clinical symptoms using DAI are as follows. No symptoms were measured in the normal group, and the disease activity index increased in the remaining groups except for the normal group. It was confirmed that the sample administration group decreased overall compared to the DSS group. In particular, the group administered with fermented soybean solution + aronia mixed solution and the group administered with fermented soybean solution + barley sprouted solution showed a significant difference compared to the control group (FIG. 3).

In addition, on the 5th day after DSS administration, the large intestine of the sacrificed mice was excised and the length was checked. As a result, the length of the intestine was longer in all groups to which the sample was administered than in the control group. There was a significant difference in the group administered with the sprouted barley mixture compared to the control group (FIG. 4).

In addition, as a result of measuring inflammatory cytokines in the blood in the colitis-induced animal model, the levels of TNF-α and IL-1β, which are indicators of inflammation, were decreased in both the sample administration groups compared to the control group. A significant difference was observed in the fermented soybean solution + barley sprout mixture administration group compared to the control group (FIG. 5). Through the above results, it was found that the natural fermented product directly administered into the intestine produced nitric oxide from the nitric oxide metabolite to induce the improvement of colitis, and the use of the nitric oxide metabolite-containing natural fermented tablet according to the present invention as an enteric tablet I could see the possibilities directly.

Example 2. Stress bowel syndrome improvement effect of natural fermented products containing nitric oxide metabolites

For the experimental animals used in this experiment, 8-week-old female Wistar rats were purchased from Samtaco and were used after acclimatization for one week. Contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2℃ and humidity at 50-60%.

2-1. Military composition and Water Avoidance Stress (WAS)

To observe changes in colonic motility caused by natural fermentation containing nitric oxide metabolites, 40 mg of famotidine, a gastric acid inhibitor, was administered to 1 ㎖, and 30 minutes later, 1 ㎖ of sample was orally administered to each group. After filling each WAS tank with water in the rest of the groups except for the normal group, place the animals on an escape platform to give them intermittent stress, and then measure the number of stools. WAS was performed for a total of 8 days for 60 minutes every day (FIG. 2).

The group consists of 6 groups of 6 animals in each group. Normal group not exposed to WAS (No-stress), control group subjected to stress while performing WAS, sodium nitrite (NaNO ₂ ) administered group performing WAS, and lettuce fermentation broth performing WAS. The administration group consisted of a group administered with fermented lettuce + aronia mixed solution administered with WAS, and a group administered with fermented lettuce + barley sprouted solution administered with WAS. Exposure to WAS induced diarrhea-type irritable bowel syndrome-like symptoms (increased defecation) and mucosal immunity, and it was observed whether administration of each sample could reduce it.

Composition of the stress bowel syndrome experimental group Group WAS Material Dose n One normal group NO D.W. - 6 2 control Yes D.W. - 6 3 NaNO ₂ Yes sodium nitrogen dioxide 135 ppm 6 4 Lettuce Fermented Liquid Yes Lactuca sativa fermented extract 135 ppm 6 5 Fermented Lettuce + Aronia Yes Lactuca sativa fermented extract + Aronia 135 ppm 6 6 Fermented lettuce + barley sprouts Yes Lactuca sativa fermented extract + Barley Sprout 135 ppm 6

2-2. Effects of Natural Fermentation in Stress Bowel Syndrome Model

The results of measuring the number of variables for each sample when stress is induced through WAS are shown in FIG. 6 . The number of stools was decreased in all of the sample administration groups compared to the control group, and in particular, the group administered with fermented lettuce + aronia mixed solution and group administered with fermented lettuce + barley sprouted solution showed a significant difference compared to the control group.

Example 3. Blood pressure improvement effect of natural fermented products containing nitric oxide metabolites

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 acclimatized for one week before use. The average weight of SD rats was 250-300 g, and the contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2° C., and the humidity was maintained at 50-60%.

3-1. Preparation and group composition of DOCA-Salt hypertensive rat model

For the preparation of the hypertension model, DOCA (Deoxycorticosterone acetate; TCI, Japan) was administered intraperitoneally to 8-week-old male SD rats twice a week at a concentration of 25 mg/kg for 4 weeks, and 1% sodium chloride aqueous solution was used as a negative number to induce hypertension. At the same time, distilled water (DW) was administered to the control group, and each fermented product was administered to the experimental group. To check the induction of hypertension, blood pressure was measured using a mouse tail blood pressure system (MK-2000STS, MUROMACHI KIKAI, Japan), and animals with a systolic blood pressure of 130 mmHg or higher were selected and used.

Blood pressure of the experimental animals was measured in real time for 50 minutes per animal by administering a sample to each group. After the rat was placed in a calibrating frame and fixed, a piezo electricpulse sensor and an occlusion cuff were put on the tail to fix it, and the pulse signal connected to the computer was observed to an appropriate degree. At this time, the systolic blood pressure was observed by operating the device.

Experimental animals were divided into 5 groups, and DOCA was administered intraperitoneally, and distilled water was administered to the blood pressure model fed with general feed and 1% sodium chloride aqueous solution. In a blood pressure model in which sodium nitrite (NaNO ₂ ) was administered, DOCA was intraperitoneally administered as in the control group, and lettuce fermented solution was administered to a blood pressure model in which normal feed and 1% sodium chloride aqueous solution were fed, and DOCA was intraperitoneally administered as in the control group, In the blood pressure model fed with 1% sodium chloride aqueous solution, fermented lettuce + aronia mixed solution was administered, and DOCA was administered intraperitoneally as in the control group. . 40 mg of famotidine, a gastric acid inhibitor, was administered to 1 ㎖, and 30 minutes later, 1 ㎖ of sample was orally administered to each group. In the experiment of the present invention, the famotidine is a drug known to inhibit gastric acid secretion through a number of previous reports, and the sample that is post-processed by blocking gastric acid secretion in the gastric mucosa in advance through the pretreatment of famotidine is Helps avoid exposure to acidic conditions in the stomach. Therefore, the experimental conditions for pretreatment with famotidine as described above can be used as a method to indirectly verify the effect of the sample as an enteric tablet.

Blood pressure experimental group composition Group Treatment n DOCA injection Dose material Dose One control ○ 25 mg/kg DW - 5 2 Lettuce Fermented Liquid ○ 25 mg/kg Lactuca sativa fermented extract 135 ppm 5 3 NaNO ₂ ○ 25 mg/kg sodium nitrogen dioxide 135 ppm 5 4 Fermented Lettuce + Aronia ○ 25 mg/kg Lactuca sativa fermented extract + Aronia 135 ppm 5 5 Fermented lettuce + barley sprouts ○ 25 mg/kg Lactuca sativa fermented extract + Barley Sprout 135 ppm 5

3-2. Effects of Natural Fermentation in DOCA-induced Hypertension Model

The results of blood pressure measurement before and after administration of each fermentation broth in rats induced with hypertension by DOCA-salt are shown in FIG. 7 . In particular, a decrease in blood pressure was observed between 10 and 20 minutes after oral administration of the natural fermentation sample. could confirm that The above result can be inferred as an effect induced by the natural fermented product according to the present invention generating nitric oxide from nitric oxide metabolites in the intestinal environment. could be judged to be

Example 4. Diabetes improvement effect of natural fermentation products containing nitric oxide metabolites

As the experimental animals used in this experiment, 8-week-old male C57BL/6 mice were purchased from Samtaco and acclimatized for one week before use. Contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2℃ and the humidity was maintained at 50-60%.

4-1. Type 2 diabetes induction and grouping

Diabetes was induced by intraperitoneal injection of 120 mg/kg of STZ (streptozotocin; Sigma, USA) dissolved in 0.1 M citrate buffer (pH 4.5) to the experimental animals once. Animals with a blood sugar of 200 mg/dL or less as measured by hepatic hemoglobin (Glucotrend, Roche, Germany) by collecting blood from the tail vein of fasting state experimental animals 2 weeks after STZ administration were excluded. After one week, blood glucose was measured again, and animals with a blood glucose level of 200 mg/dL or higher were selected and used.

The experimental group was constituted as shown in Table 5 below, and the blood glucose lowering effect was analyzed by performing a glucose tolerance test. Specifically, in the glucose tolerance test, after fasting for more than 12 hours before the experiment, blood was collected from the tail vein of the experimental animal to measure the fasting blood glucose level. After administering a sample to each group orally and enterally, 2 g/kg of glucose was orally administered 30 minutes later, and a blood glucose meter (Accu -check, Roche, Berlin, Germany) was used to measure blood glucose. For oral administration, 40 mg of famotidine, a gastric acid inhibitor, was administered in 1 ㎖, and each sample was administered 30 minutes later.

Diabetes experimental group composition Group Meterial Dose Meterial Dose n One normal group D.W. - Glucose 2 g/kg 5 2 drug control group metformin 200 mg/kg Glucose 2 g/kg 5 3 NaNO ₂ sodium nitrogen dioxide 235 ppm Glucose 2 g/kg 5 4 fermented soybean Soybean fermented extract 235 ppm Glucose 2 g/kg 5 5 Soybean Fermented Liquid + Aronia Soybean fermented extract + Aronia 235 ppm Glucose 2 g/kg 5 6 Soybean fermented liquid + sprouted barley Soybean fermented extract + Barley Sprout 235 ppm Glucose 2 g/kg 5

4-2. Effect of Natural Fermentation in Diabetes Model

As a result of the glucose tolerance test, when the natural fermentation was administered to the mice induced with diabetes by STZ, the blood sugar reduction effect was shown in all the fermented solution groups. In the group administered with the barley mixture, a better blood sugar reduction effect was observed (FIG. 8).

Example 5. Arthritis improvement effect of natural fermented product containing nitric oxide metabolite

As the experimental animals used in this experiment, 5-week-old male Balb/c mice were purchased from Samtaco and acclimatized for a week before use. The average weight of Balb/c mice was 22.78±1.25 g, and the sample was orally administered once daily. Contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2℃ and humidity at 50-60%.

5-1. Induction and grouping of experimental arthritis

Induction of experimental arthritis was performed by anesthetizing the experimental animals in each group except for the normal group, removing hair around the knee of Balb/c mice, diluting LPS (Lipopolysaccharide), an osteoarthritis-inducing substance, in distilled water, and then diluting 300 μg each /kg each was injected into the knee joint. After 3 days of drug injection, the degree of swelling in the joints and feet and the walking condition of the experimental animals were observed to confirm the induction of arthritis, and then the experiment was carried out.

Experimental animals were classified into 6 groups, the normal group administered with distilled water without any treatment, the control group administered with distilled water and induced arthritis through LPS, the sodium nitrite (NaNO ₂ ) 235 ppm administration group, 235 ppm fermented lettuce solution group, Each sample was classified into a fermented lettuce solution + aronia mixture administered at 235 ppm, and a fermented lettuce solution + barley sprout mixture administered at 235 ppm, and administered orally every day for 4 weeks. For oral administration, 40 mg of famotidine, a gastric acid inhibitor, was administered in 1 ㎖, and each sample was administered 30 minutes later. For the calculation of the number of animals in a group, the minimum number covering statistical significance was used in accordance with the 3R principle.

Arthritis experimental group composition Group LPS Meterial Dose n One normal group No D.W. - 5 2 control Yes D.W. - 5 3 NaNO ₂ Yes sodium nitrogen dioxide 235 ppm 5 4 Lettuce Fermented Liquid Yes Lactuca sativa fermented extract 235 ppm 5 5 Fermented Lettuce + Aronia Yes Lactuca sativa fermented extract + Aronia 235 ppm 5 6 Fermented lettuce + sprouted barley Yes Lactuca sativa fermented extract + Barley Sprout 235 ppm 5

5-2. Serum cytokines and PGE2 (Prostaglandin E ₂ ) measurement

The levels of TNF-α and IL-1β in serum and the amount of PGE2 production were measured using an ELISA kit (R&D Systems, USA), and the specific method is the same as in Examples 1-3.

5-3. Effects of Natural Fermentation in Arthritis Model

As a result of measuring TNF-α, IL-1β and PGE2 in serum after administering each sample in the arthritis induction model through LPS, TNF-α, IL-1β and PGE2 were all treated with LPS in the fermented solution-treated group (LPS) ), and in particular, the group administered with fermented lettuce + aronia mixed solution and group administered with fermented lettuce + barley sprouted solution showed a significant difference compared to the control group (FIG. 9).

Example 6. Hepatoprotective effect of natural fermented products containing nitric oxide metabolites

For the experimental animals used in this experiment, 7-week-old male Sprague Dawley rats (SD rats) were purchased from Samtaco and acclimatized for one week before use. The average weight of SD rats was 250-300 g, and the contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2° C., and the humidity was maintained at 50-60%.

6-1. Causes liver damage and group composition

In the experimental model of hepatotoxicity induction by carbon tetrachloride (CCl ₄ ), CCl ₄ was administered intraperitoneally with an equal amount of olive oil and 0.1 ㎖ per 100 g of body weight to induce acute liver damage. In the normal group, only olive oil was injected intraperitoneally instead of CCl ₄ . A sample of 235 ppm in each group was pretreated 3 times every other day by oral administration before injection of CCl ₄ , and only distilled water was orally administered to the normal group. For oral administration, 40 mg of famotidine, a gastric acid inhibitor, was administered in 1 ㎖, and each sample was administered 30 minutes later. For the calculation of the number of animals in a group, the minimum number covering statistical significance was used in accordance with the 3R principle.

CCl ₄ induced liver damage experimental group composition Group Meterial n One normal group D.W. 5 2 control D.W. 5 3 NaNO ₂ sodium nitrogen dioxide 5 4 Lettuce Fermented Liquid Lactuca sativa fermented extract 5 5 Fermented Lettuce + Aronia Lactuca sativa fermented extract + Aronia 5 6 Fermented lettuce + barley sprout Lactuca sativa fermented extract + Barley Sprout 5

6-2. Serum collection and serological testing

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

6-3. Effects of Natural Fermentation in Acute Liver Injury Model

ALT and AST levels in serum are commonly measured clinically as biomarkers for liver health. As a result of measuring ALT and AST after administration of each sample in the experimental model for inducing liver toxicity by CCl ₄ , ALT and AST levels in serum were decreased in both the sample administration groups compared to the control group. In the group administered with the sprouted barley mixture, there was a significant difference compared to the control group (FIG. 10).

Example 7. Contact dermatitis improvement effect of natural fermentation products containing nitric oxide metabolites

As the experimental animals used in this experiment, 5-week-old male Balb/c mice were purchased from Samtaco and acclimatized for a week before use. Samples are administered orally once daily. In the case of oral administration, 40 mg of famotidine, a gastric acid inhibitor, was administered in 1 ml, and each sample was administered 30 minutes later. Contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2℃ and humidity at 50-60%.

7-1. DNFB production and atopic dermatitis induction and group composition

After acclimatization for one week, Balb/c mice were acclimatized for 3 days by depilating the back area. DNFB (1-fluoro-2,4-dinitrobenzene; sigma, USA) reagent was prepared by diluting 0.5% DNFB in a solution in which acetone:olive oil was mixed in a ratio of 4:1. On the fourth day after hair removal, 40 μl of 0.5% DNFB was applied to the back for 4 days to induce skin sensitization. After skin sensitization, on the 12th day after sample administration, 20 μl of 0.3% DNFB was applied to the ear (challenge) to induce contact dermatitis.

Experimental animals were classified into 6 groups (Table 9), and distilled water was administered to the normal group (CON) and the control group, respectively, and each sample was orally administered to the remaining groups once a day. In the case of oral administration, 40 mg of famotidine, a gastric acid inhibitor, was administered in 1 ml, and each sample was administered 30 minutes later. For the calculation of the number of animals in a group, the minimum number covering statistical significance was used in accordance with the 3R principle.

Composition of atopic dermatitis experimental group Group left ear right ear Material n One normal group Normal Normal D.W. 5 2 control Normal 0.3% DNFB D.W. 5 3 NaNO ₂ Normal 0.3% DNFB sodium nitrogen dioxide 5 4 Cabbage Fermented Liquid Normal 0.3% DNFB cabbage fermented extract 5 5 Fermented Cabbage + Aronia Normal 0.3% DNFB cabbage fermented extract + Aronia 5 6 Cabbage fermented liquid + sprouted barley Normal 0.3% DNFB cabbage fermented extract + Barley Sprout 5

7-2. Visual evaluation

Visual evaluation is a clinical evaluation method commonly used in atopic dermatitis, and it is expressed as the sum of the scores for each of the five items to measure the severity of the symptoms of atopic dermatitis. Evaluation items were erythema, pruritus & dry skin, edema & excoriation, erosion and lichenification, and each item had no symptoms (0 points); Weak (1 point), normal (2 points), and severe (3 points) were scored, and the scores were summed up to give a score ranging from 0 to 15 points.

7-3. Measurement of ear swelling and spleen level

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

7-4. Effect of Natural Fermentation in Contact Dermatitis Model

After the end of the experiment, the morphological change of the atopic dermatitis lesion was confirmed by observing the region of the right ear treated with the sample for each experimental group. As a result, in the control group treated with only DNFB, atopic skin changes such as erythema, dry skin, erosion, edema and hematoma were confirmed compared to the left ear untreated (FIG. 11).

In addition, as a result of observing the right ear region treated with the samples for each experimental group after the end of the experiment, the ear thickness was decreased in all the sample-treated groups compared to the control group. It showed a significant difference compared to the control group (FIG. 12).

In addition, as a result of measuring the spleen weight by body weight for each experimental group after the end of the experiment, the spleen index decreased in all the sample administration groups compared to the control group. There was a significant difference (FIG. 13).

In addition, as a result of measuring the levels of TNF-α and IL-4 in the serum for each experimental group after the end of the experiment, the levels of TNF-α and IL-4 were decreased in both the sample administration groups compared to the control group. The group administered with the mixed solution and the group administered with the cabbage fermented solution + sprouted barley mixed solution showed a significant difference compared to the control group (FIG. 14).

Example 8. Effects of natural fermented products containing nitric oxide metabolites on changes in body temperature

For the experimental animals used in this experiment, 7-week-old male Sprague Dawley rats (SD rats) were purchased from Samtaco and acclimatized for one week before use. The average weight of SD rats was 250-300 g, and the contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2° C., and the humidity was maintained at 50-60%.

8-1. Group composition and body temperature change measurement

To measure body temperature change, block light after anesthesia before administering samples to each group, measure body temperature and thermal changes using a thermal imaging camera, and 0, 10, 20, 30 minutes after orally administering samples to each group. Measure the change in body temperature in each. For oral administration, 40 mg of famotidine, a gastric acid inhibitor, was administered in 1 ㎖, and each sample was administered 30 minutes later.

Composition of the body temperature change measurement experimental group Group material Dose n One control DW - 5 2 NaNO ₂ sodium nitrogen dioxide 235 ppm 5 3 Garlic Fermented Liquid Garlic fermented extract 235 ppm 5 4 Garlic Fermented Liquid + Aronia Garlic fermented extract + Aronia 235 ppm 5 5 Garlic fermented liquid + sprouted barley Garlic fermented extract + Barley Sprout 235 ppm 5

8-2. Analysis of changes in body temperature following administration of natural fermentation products

As a result of measuring changes in body temperature at 0, 10, 20, and 30 minutes after oral administration of the sample for each group, body temperature increased in all of the sample-administered groups compared to the control group. + There was a significant difference in the group administered with the sprouted barley mixture (FIG. 15).

Example 9. Skin cell regeneration effect of natural fermented product containing nitric oxide metabolite

Five-week-old male Balb/c mice were purchased from Samtaco and used after a one-week acclimatization period. Contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2℃ and the humidity was maintained at 50-60%. After one week of acclimatization, the experimental animals shaved the back area under isoflurane inhalation anesthesia, and then made two symmetrical circular defect wounds using a leather punch (4 mm, KaiMedical, China). induced. Experimental animals were classified into 6 groups. After induction of the wound, distilled water was applied to the wound area in the control group (WHD), the wound area was treated with 1 mM sodium nitrite solution (WHN), and the wound area was treated with 1 mM sodium nitrite and 1 mM sodium nitrite. Group treated with mM citric acid solution (WHNCA), group treated with 1 mM citric acid solution on the wound site (WHCA), group treated with 1 mM citric acid solution and cabbage fermented solution treated group (WHNP), applied cabbage fermented solution to the wound site It was classified into liquid treatment group (WHP).

As a result of observing the change in the area of the wound site of individuals randomly selected from the wound induction group up to 21 days after the induction of the wound, a large amount of exudate occurred at the site of the wound infection, and foreign substances such as litter were attached to the wound. A scab began to form at the inflamed site, and the difference in the area regenerated according to the sample occurred after 3 weeks, and the wound area was significantly reduced in both the sample (cabbage fermented broth)-applied group compared to the control group (FIG. 16).

Example 10. Cerebral blood flow improvement and sleep effect of natural fermented product containing nitric oxide metabolite

The natural fermentation products containing nitric oxide metabolites used in this experiment are lettuce fermented broth and garlic fermented broth manufactured by (U) Human Enos. The fermentation broth was used in the experiment after adjusting the content of nitric oxide metabolites to be the same based on the NO ₂ ^{-concentration} .

In addition, as the experimental animals, 7-week-old male SD rats were purchased from Samtaco (Korea) and used after acclimatization for one week. The average weight of SD rats was 195.5±1.51 g, and the sample was administered directly (rectally) into the intestine once a day. Contrast was controlled at 12-hour intervals in the experimental animal breeding room, and the temperature was maintained at 23±2℃ and the humidity was maintained at 50-60%.

10-1. Experimental group composition

Experimental animals were classified into 4 groups, and were classified into 1% isoflurane administered group (control group-1), isoflurane 3% administered group (control group-2), 300 ppm fermented lettuce solution group, and 200 ppm fermented garlic solution group. (Table 11). For the calculation of the number of animals in a group, the minimum number covering statistical significance was used in accordance with the 3R principle.

Composition of the cerebral blood flow improvement experimental group Group Material Dose n Control-1 isoflurane One% 5 control group-2 isoflurane 3% 5 Lettuce Fermented Liquid Lactuca sativa fermented extract 300 ppm 5 Garlic Fermented Liquid Garlic fermented extract 200 ppm 5

10-2. cortical electroencephalography

After anesthetizing SD rats with injection anesthetic (20% Urethane), fix the head in a stereotaxic instrument, cut the scalp subcutaneous connective tissue to expose the skull, and then connect one screw to the reference of the intan recording circuit in the front of the skull. After fixing and connecting to the ground, the electrode for EEG (electroencephalogram) recording is removed from the skull and placed on the dura mater and fixed with dental cement (FIG. 17). After connecting the data connection line to the Intan recording circuit, the signal data measured at a sampling rate of 1KHz from the EEG recording device (RHD2000, Intan Technologies) in freely moving SD rats in the awake state was removed from the power supply noise with a 60Hz notch filter, and the internal software Signal data between 0.2 and 1,000 Hz was recorded using a frequency filter.

10-3. Results of cortical EEG analysis

When the concentration of isoflurane anesthetic was administered to 1%, a wave occurred, and when the concentration was increased to 3%, the wave was observed remarkably. Wavelengths were issued from 3 minutes after administration of the fermentation broth, and it was observed that these wavelengths continued for more than 15 minutes (FIG. 18). It was confirmed that the spontaneous activity of a single neuron in the cerebral cortex was significantly reduced in the group administered with 3% isoflurane, and it was confirmed that the frequency of spontaneous activity of neurons was reduced in the group administered with lettuce and garlic fermented broth while the wavelength continued. The above results suggested that the natural fermented product containing the nitric oxide metabolite of the present invention can be utilized to improve or prevent sleep disorders.

10-4. Cerebral blood flow analysis results

In a brain hemodynamics test measured with a thermal imaging camera 30 minutes after the intestinal administration of 2 ml of 200 ppm garlic fermented solution to experimental animals, it was observed that the area of cerebral blood flow increased according to the administration of the fermented solution (Fig. 19). Through this, it was expected that the natural fermentation product containing the nitric oxide metabolite of the present invention could be used for the improvement or treatment of cerebral blood flow disorder diseases such as stroke.

Claims (12)

As an enteric tablet comprising a natural fermentation product in which sodium nitrite (NaNO ₂ ) or a nitric oxide metabolite is immobilized and stabilized in the natural fermentation product as an active ingredient, and produces nitric oxide in the intestinal environment,
The natural fermentation product is prepared by a manufacturing method comprising the step of adding a fermented strain to a nitrogen-containing natural product and fermenting it for 2 to 30 days at a temperature of 18 to 35° C. and a dissolved oxygen concentration of 0.03 to 0.1 mg/L. Characterized by Jang Yong-jeong. The enteric tablet according to claim 1, further comprising polyphenols in addition to the sodium nitrite or natural fermented product. The enteric tablet according to claim 2, wherein the polyphenol is resveratrol, quercetin, silymarin, anthocyanin or catechinic acid. delete The method according to claim 1, wherein the nitrogen-containing natural product is lettuce, pea sprouts, spinach, blueberry, dandelion, pomegranate, cabbage, garlic, noni, onion, soybean, bean sprout, peanut, peanut sprout, velvet bean, mulberry leaf, bitter melon, bokbunja, Eoseongcho, Aronia, Yulcho, Yellow lily, Golden, Hwangbaek, Hibiscus, Samchae, Mushrooms, Iris, Seomcho, Chinese cabbage, Kelp, Salmon milt, Abalone shell (Seokgyeolmyeong), Crustacean shell, Cricket, Lavender, Chamomile, Graviola, Basil, Jang Yongjeong, characterized in that at least one selected from the group consisting of apples, mugwort, tangerines, silkworms, Myongmok Rehmannia and Sipjeondaebotang. According to claim 1, wherein the fermentation strain is Bacillus genus ( Bacillus sp.), Bifidobacterium genus ( Bifidobacterium sp.), Enterococcus genus ( Enterococcus sp.), Lactobacillus genus ( Lactobacillus sp.), Lactococcus genus ( Lactococcus sp.) or Weissella sp. Enteric acid, characterized in that the strain. High blood pressure, diabetes, enteritis, stress bowel syndrome, arthritis, liver disease, atopic dermatitis, sleep disorder or cerebrovascular disease, wherein the enteric tablet of claim 1 or a nitric oxide metabolite is immobilized and stabilized in a natural fermentation product as an active ingredient As a pharmaceutical composition for the prevention or treatment of diseases,
The nitric oxide metabolite is immobilized and stabilized in the natural fermentation product is fermented for 2 to 30 days at a temperature of 18 to 35° C. and a dissolved oxygen concentration of 0.03 to 0.1 mg/L by adding a fermented strain to the nitrogen-containing natural product. A pharmaceutical composition, characterized in that it is prepared by a manufacturing method comprising the step of. The pharmaceutical composition according to claim 7, wherein the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized in the natural fermentation product further comprises a plant containing a polyphenol compound. delete High blood pressure, diabetes, enteritis, stress bowel syndrome, arthritis, liver disease, atopic dermatitis, sleep disorder or cerebrovascular disease, wherein the enteric tablet of claim 1 or a nitric oxide metabolite is immobilized and stabilized in a natural fermentation product as an active ingredient As a health functional food composition for preventing or improving diseases,
The nitric oxide metabolite is immobilized and stabilized in the natural fermentation product is fermented for 2 to 30 days at a temperature of 18 to 35° C. and a dissolved oxygen concentration of 0.03 to 0.1 mg/L by adding a fermented strain to the nitrogen-containing natural product. A health functional food composition prepared by a manufacturing method comprising the step of: The health functional food composition according to claim 10, wherein the natural fermentation product in which the nitric oxide metabolite is immobilized and stabilized in the natural fermentation product further comprises a plant containing a polyphenol compound. delete

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