Organ disorder amelioration agent

Abstract

The purpose of the present invention is to provide a preparation capable of effectively ameliorating organ disorder. The organ disorder amelioration agent according to the present invention is characterized by containing, as an active ingredient, an H-type ulvan or an alkali metal ion salt of ulvan.

Description

Organ damage improvement agent

[0001] The present invention relates to a preparation that can effectively improve organ damage.

[0002] Abnormal immune responses and damage to capillaries can cause organ damage. It is said that there are 100 trillion bacteria, representing over 1,000 different types, in the human intestine. Intestinal bacteria are primarily divided into good bacteria, bad bacteria, and opportunistic bacteria, and the balance of these bacteria varies greatly from person to person, affecting intestinal function.

[0003] The ideal intestinal environment is said to have a ratio of good bacteria to bad bacteria to opportunistic bacteria of 2:1:7. A gut with a high proportion of good bacteria maintains a slightly acidic environment and promotes good bowel movements. On the other hand, an increase in bad bacteria leads to the production of toxic substances in the intestine, which not only causes bowel movement abnormalities but can also lead to colon cancer. Opportunistic bacteria function in the same way as either the dominant good or bad bacteria, and as bad bacteria increase, they begin to produce toxic substances in the intestine. Because good bacteria maintain a slightly acidic environment and suppress the growth of bad bacteria, if bad bacteria increase and good bacteria decrease, they are no longer able to suppress their growth, leading to a vicious cycle in which the intestinal environment deteriorates further.

[0004] When the intestinal environment deteriorates due to an increase in bad bacteria, toxic substances are produced in the intestines, causing symptoms such as diarrhea, constipation, abdominal bloating, abdominal pain, and borborygmi. If the intestinal environment continues to deteriorate, it can lead to inflammatory bowel diseases such as colon cancer and Crohn's disease. Furthermore, toxic substances such as ammonia produced in the intestines are absorbed into the blood vessels of the intestinal mucosa and circulate throughout the body, causing diseases such as diabetes and arteriosclerosis, as well as rough skin. Furthermore, the activity of intestinal bacteria and inflammation can create tiny molecular-level gaps in the small intestinal wall, allowing foreign substances such as bacteria, viruses, and proteins that would normally not enter the bloodstream to leak into the bloodstream, causing various symptoms. This condition is known as leaky gut syndrome.

[0005] For example, the intestinal environment deteriorates due to an increase in bad bacteria such as Clostridium perfringens and Staphylococcus aureus. Causes of an increase in bad bacteria include a high-fat, high-protein diet, an irregular lifestyle including lack of sleep and stress, and chronic constipation. However, if these symptoms are severe, treatment with intestinal regulators is necessary in addition to improving lifestyle habits.

[0006] In addition, the intestinal mucosa has the function of producing immune cells that protect the body. It has been reported that when the intestinal environment deteriorates, this immune function also declines, weakening the immune system and making people more susceptible to infections.

[0007] In contrast, maintaining an ideal balance of intestinal bacteria is called intestinal regulation. Intestinal regulation requires improving lifestyle habits, but if that alone does not improve the condition, drug therapy using intestinal regulators is often used. Intestinal regulation also refers to improving bowel problems such as constipation and diarrhea, abdominal distension, and pain by improving the intestinal environment.

[0008] To regulate the intestines, it is important to increase the number of beneficial bacteria in the intestines and improve the intestinal environment. Beneficial bacteria that have a positive effect on the human body, as well as foods, beverages, and preparations that contain them, are called probiotics. Active ingredients in probiotics include the beneficial bacteria themselves, yogurt, natto, pickles, and other foods that increase beneficial bacteria, as well as oligosaccharides and dietary fiber, which serve as food for beneficial bacteria.

[0009] Some algae synthesize polysaccharides through photosynthesis and accumulate them in large quantities. For example, alginic acid produced by brown algae has the property of gelling in the presence of divalent metal ions such as calcium ions and magnesium ions, and is therefore used in the food, medical, and industrial fields as a thickener, stabilizer, gelling agent, dietary fiber material, and the like. Furthermore, for example, Enteromorpha spp., a large green algae of the genus Ulva, has one of the highest growth rates of all plants and is expected to be utilized as a biomass resource. Enteromorpha spp. contains approximately 5-15% starch and cellulose per dry weight of the algal cells, as well as approximately 20-30% of a polysaccharide called ulvan.

[0010] Various applications of naturally occurring polysaccharides have been investigated. For example, Patent Documents 1 and 2 disclose compositions containing anti-inflammatory agents and sulfated polysaccharides, which are described as exhibiting very strong and synergistic anti-inflammatory effects. Patent Document 3 describes that a combination of hyaluronic acid or its salts with sulfated ulvans is advantageous in anti-aging, tissue regeneration, and treatment of skin lesions.

[0011] Additionally, Non-Patent Document 1 describes that sulfated polysaccharides derived from algae have anti-inflammatory effects and change the bacterial flora of overweight patients, and Non-Patent Document 2 describes that ulvan, a polysaccharide derived from algae, changes the intestinal bacterial flora of mice.

[0012] Japanese Patent Application Laid-Open No. 2023-40008 Special Publication No. 2020-500945 Special Publication No. 2017-514825

[0013] Lauren A. Roach et al., Mar. Drugs, 2022, 20, 500; Kunal Pratap et al., Foods, 2022, 11, 565

[0014] As mentioned above, ulvan obtained from algae is known to have anti-inflammatory effects and to alter the intestinal flora. However, because organ failure can lead to death, there is a need for preparations that have a more effective effect of improving organ damage. Therefore, an object of the present invention is to provide a preparation that can effectively improve organ damage.

[0015] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that a specific derivative of natural ulvan exhibits excellent organ damage-improving activity, and have completed the present invention. The present invention is described below.

[0016] [1] An organ damage ameliorating agent comprising, as an active ingredient, H-type ulvan or an alkali metal ion salt of ulvan. [2] The organ damage ameliorating agent according to [1], wherein the alkali metal is Na and / or K. [3] The organ damage ameliorating agent according to [1] or [2], wherein 30 mol % or more of the carboxyl groups and sulfate groups of the H-type ulvan are protonated. [4] The organ damage ameliorating agent according to any one of [1] to [3], wherein 40 mol % or more of the carboxyl groups and sulfate groups of the alkali metal ion salt of ulvan form salts with alkali metal ions. [5] The organ damage ameliorating agent according to any one of [1] to [4], which improves intestinal bacterial flora. [6] The organ damage ameliorating agent according to any one of [1] to [5], which improves leaky gut.

[0017] [7] Use of H-type ulvan or an alkali metal ion salt of ulvan for improving organ damage. [8] The use according to [7] above, wherein the alkali metal is Na and / or K. [9] The use according to [7] or [8] above, wherein 30 mol % or more of the carboxyl groups and sulfate groups of the H-type ulvan are protonated.

[10] The use according to any one of [7] to [9] above, wherein 40 mol % or more of the carboxyl groups and sulfate groups of the alkali metal ion salt of ulvan form salts with alkali metal ions.

[11] The use according to any one of [7] to

[10] above, for improving intestinal bacterial flora.

[12] The use according to any one of [7] to

[11] above, for improving leaky gut.

[0018]

[13] A method for ameliorating organ damage, comprising a step of administering H-type ulvan or an alkali metal ion salt of ulvan.

[14] The method according to

[13] above, wherein the alkali metal is Na and / or K.

[15] The method according to

[13] or

[14] above, wherein 30 mol % or more of the carboxyl groups and sulfate groups of the H-type ulvan are protonated.

[16] The method according to any one of

[13] to

[15] above, wherein 40 mol % or more of the carboxyl groups and sulfate groups of the alkali metal ion salt of ulvan form salts with alkali metal ions.

[17] The method according to any one of

[13] to

[16] above, wherein the intestinal bacterial flora is improved.

[18] The method according to any one of

[13] to

[17] above, wherein the leaky gut is improved.

[0019] The organ damage ameliorating agent according to the present invention can effectively ameliorate organ damage, and therefore the present invention is industrially extremely advantageous as a technology relating to an excellent organ damage ameliorating agent.

[0020] FIG. 1(1) is a graph comparing the bowel movement frequency between young and old mice, FIG. 1(2) is a graph showing the change in bowel movement frequency when H-type ulvan was administered, and FIG. 1(3) is a graph showing the change in bowel movement frequency when ulvan Na salt was administered. FIG. 2(1) is a graph showing the change in the amount of intestinal bacteria in young and old mice, and FIG. 2(2) is a graph showing the change in the amount of intestinal bacteria when H-type ulvan was administered. FIG. 3 is a graph showing the change in the intestinal bacterial flora in mice administered ulvan Na salt. FIG. 4 is a graph showing the change in the barrier function of the small intestine when endotoxin (LPS) and / or ulvan or ulvan Na salt was administered. FIG. 5(1) is a graph showing the change in the expression level of the inflammatory cytokine IL-6 by intestinal epithelial cells when endotoxin (LPS) and / or ulvan or ulvan Na salt was administered, and FIG. 5(2) is a graph showing the change in the expression level of IL-1β. Figure 6 is a graph showing changes in the expression levels of inflammatory cytokines IL-1β, IL-6, and TNFα by macrophages when endotoxin (LPS) and / or ulvan, H-type ulvan, or ulvan Na salt was administered. Figure 7 (1) is a graph comparing the amount of dextran leaked from the intestinal tract into the blood when purified water, natural ulvan, or ulvan Na salt was administered, and Figure 7 (2) is a graph comparing the amount of LPS leaked from the intestinal tract into the blood. Figure 8 is a graph showing changes in serum ALT activity in mice administered ulvan or ulvan Na salt. Figure 9 (1) is a graph showing the expression level of Claudin-1 in the colonic tissue of mice administered ulvan or ulvan Na salt, and Figure 9 (2) is a graph showing the expression level of Claudin-2 in the colonic tissue of mice administered ulvan or ulvan Na salt.

[0021] The organ damage ameliorating agent according to the present invention contains, as an active ingredient, H-type ulvan or an alkali metal ion salt of ulvan. The ulvan derivative as the active ingredient can be used alone or in combination of two or more. That is, the active ingredient of the organ damage ameliorating agent according to the present invention may be one or more selected from the group consisting of H-type ulvan and alkali metal ion salts of ulvan. For example, the active ingredient of the organ damage ameliorating agent according to the present invention may be a combination of H-type ulvan and an alkali metal ion salt of ulvan.

[0022] Ulvan is a polysaccharide produced by green algae of the genus Enteromorpha (Enteromorpha sp.), such as Enteromorpha mianensis, and by green algae of the genus Ulva (Ulva sp.), such as Ulva mianensis, and is a sulfated anionic polysaccharide containing uronic acids, such as glucuronic acid and iduronic acid, and sulfated rhamnose, galactose, xylose, glucose, and the like. Approximately two-thirds to three-quarters of the ulvan is composed of alternating polymerized uronic acids and sulfated rhamnose, and the remaining portion is mainly composed of polymerized sulfated rhamnose, etc. Hereinafter, naturally occurring ulvan may be referred to as natural ulvan.

[0023]

[0024] There are two main structural units of natural ulvans: urbanobiuronic acid 3-sulfate type A, in which β-D-glucuronic acid and 3-sulfate α-L-rhamnose are linked by a 1→4 bond; urbanobiuronic acid 3-sulfate type B, in which α-L-iduronic acid and 3-sulfate α-L-rhamnose are linked by a 1→4 bond.

[0025] Natural ulvan mainly contains magnesium ions, calcium ions, sodium ions, and potassium ions, with magnesium ions being the most abundant of the cations. However, ulvan is less likely to gel than alginic acid, and even if it contains a large amount of magnesium ions, it often remains in a sol state.

[0026] Natural ulvan can be extracted from ulvan-containing algae, such as Enteromorpha spp., by conventional methods. For example, since ulvan is water-soluble, after the raw algae is micronized, water or the like is added as a solvent and the extraction is carried out at a temperature of about 80°C or higher and 120°C or lower. Since ulvan is insoluble in common organic solvents, it precipitates when a water-miscible organic solvent, such as methanol, ethanol, or 2-propanol, is added to the extract. The precipitated ulvan can be separated from the solvent by filtration, centrifugation, or the like, and then dried.

[0027] The H-type ulvan, which is the active ingredient of the organ dysfunction improving agent according to the present invention, refers to an ulvan in which at least a portion of the carboxyl groups and sulfate groups of a natural ulvan are protonated, i.e., in the form of —COH and —OSOH. Hereinafter, the carboxyl groups and sulfate groups in an ulvan may be collectively referred to as “acidic groups.” Specifically, this refers to an ulvan in which the ratio of the total number of moles of protonated carboxyl groups (—COH) and protonated sulfate groups (—OSOH) to the total number of moles of acidic groups contained in the ulvan is 10 mol% or more. This ratio is preferably 20 mol% or more, more preferably 30 mol% or more. There is no particular upper limit to this ratio, and it may be 100 mol%. However, since it may be difficult to protonate all of the carboxyl groups and sulfate groups of a natural ulvan, this ratio is preferably 95 mol% or less or 90 mol% or less, and more preferably 80 mol% or less or 70 mol% or less. For example, the acid dissociation constant pKa of formic acid is 3.75, while the pKa of sulfuric acid is -3.0. 6.75 Since sulfate groups are stronger acids than carboxy groups, for example, the protonation rate is 200 s, it is thought that in H-type ulvan, carboxy groups are preferentially protonated over sulfate groups. The proportion of protonated acid groups can be determined by quantifying the cations in an ulvan sample using inductively coupled plasma (ICP) atomic emission spectrometry, estimating the proton amount from the obtained data, and assuming that all cations and protons are present in the acid groups.

[0028] The ulvan alkali metal ion salt, which is the active ingredient of the organ damage improving agent according to the present invention, refers to an ulvan in which at least a portion of the divalent metal ions, such as calcium ions or magnesium ions, which form salts with the carboxyl or sulfate groups of a natural ulvan, have been substituted with alkali metal ions. Specifically, this refers to an ulvan in which the ratio of the number of moles of divalent ions to the total number of moles of acidic groups contained in the ulvan is 30 mol% or less and the ratio of the number of moles of alkali metal ions is 20 mol% or more. The ratio of divalent ions is preferably 20 mol% or less, more preferably 10 mol% or less. The ratio of alkali metal ions is preferably 30 mol% or more, more preferably 40 mol% or more. There is no particular upper limit to this ratio, and it may be 100 mol%, but because it may be difficult to convert all of the acidic groups of a natural ulvan into alkali metal ion salts, the ratio is preferably 95 mol% or less or 90 mol% or less, more preferably 80 mol% or less. The proportion of acidic groups that form salts with alkali metal ions can be determined, for example, by quantifying the cations contained in the ulvan sample using inductively coupled plasma (ICP) atomic emission spectroscopy, estimating the amount of protons from the obtained data, and assuming that all alkali metal ions are present in the acidic groups.

[0029] Examples of alkali metal ions include lithium ions, sodium ions, potassium ions, rubidium ions, and cesium ions. One or more alkali metal ions selected from the group consisting of lithium ions, sodium ions, and potassium ions are preferred, sodium ions and / or potassium ions are more preferred, and sodium ions are even more preferred.

[0030] The molecular weight of H-type ulvan and ulvan alkali metal ion salt, which are active ingredients of the organ damage improving agent according to the present invention, is not particularly limited, but can be, for example, 10,000 or more. The molecular weight is preferably 100,000 or more, more preferably 200,000 or more, and even more preferably 500,000 or more. Although there is no particular upper limit to the molecular weight, an excessively large molecular weight may make handling difficult, so the molecular weight is preferably 2,000,000 or less, more preferably 1,500,000 or less, and even more preferably 1,000,000 or less.

[0031] H-type ulvan and ulvan alkali metal ion salts can be prepared by converting the cations contained in natural ulvan into protons (H + ) or alkali metal ions. For example, an OH-type anion exchange resin is added to an aqueous solution of natural ulvan, and the solution is treated at 0°C or higher and 100°C or lower for 10 minutes to 1 hour, followed by filtration or centrifugation to remove anions contained in the natural ulvan, such as sulfate ions and chloride ions. Next, an alkali ion-type cation exchange resin is added to the ulvan solution, and the solution is treated at 0°C or higher and 100°C or lower for 10 minutes to 1 hour, followed by filtration or centrifugation to remove the cation exchange resin, thereby producing an alkali metal ion salt of ulvan. Furthermore, an H-type cation exchange resin is added to an aqueous solution of ulvan alkali metal ion salt, and the solution is treated at 0°C or higher and 100°C or lower for 10 minutes to 1 hour, followed by filtration or centrifugation to remove the cation exchange resin. H-type ulvan and ulvan alkali metal ion salt can be isolated from the aqueous solution of H-type ulvan and ulvan alkali metal ion salt by lyophilization or the like. Hereinafter, H-type ulvan and ulvan alkali metal ion salts may be collectively referred to as ulvan derivatives.

[0032] The organ disorder to be improved by the organ disorder improving agent according to the present invention is not particularly limited, but examples thereof include disorders of the digestive tract such as the large intestine and stomach, liver failure, renal failure, respiratory failure, and cardiac failure, with large intestine disorders and liver failure being particularly preferred. That is, the organ disorder improving agent according to the present invention may be an agent for regulating intestinal function.

[0033] The ulvan derivatives of the present invention can improve the intestinal microbiota. More specifically, the intestinal microbiota differ between young and old mice, and the abundance of certain bacterial species can differ significantly. However, administration of the ulvan derivatives of the present invention can bring the intestinal microbiota of old mice closer to that of young mice. Disturbances in the intestinal microbiota can increase the number of harmful bacteria, and opportunistic bacteria can begin to act in a manner similar to that of harmful bacteria, leading to various physical disorders and diseases. In particular, symptoms such as bowel irregularities such as diarrhea and constipation, abdominal distension, abdominal pain, and borborygmus often appear. Probiotics are live microorganisms that provide beneficial effects when ingested in appropriate amounts. Ingestion of probiotics can improve the intestinal microbiota. The ulvan derivatives of the present invention can also be used as probiotics. In fact, the present inventors have experimentally found that ingestion of the ulvan derivatives of the present invention improves bowel irregularities in old mice.

[0034] Furthermore, a condition in which the balance of the intestinal flora is disrupted or holes form in the intestinal mucosa due to defects in the cells that form the intestinal wall or the intestinal mucosa layer, causing foreign substances such as bacteria, viruses, and proteins to leak into the bloodstream, and the accompanying symptoms are sometimes referred to as leaky gut syndrome. The present inventors have experimentally found that the ulvan derivatives according to the present invention improve the intestinal barrier function and suppress inflammation of enterocytes.

[0035] In addition, the organ damage ameliorating agent according to the present invention can restore or bring decreased liver function close to normal. Also, it can restore damaged colon tissue. Therefore, the organ damage ameliorating agent according to the present invention may be a liver function improving agent or a colon tissue improving agent.

[0036] The organ damage-ameliorating agent of the present invention contains an ulvan derivative as an active ingredient. The active ingredient refers to a component contained in the organ damage-ameliorating agent of the present invention that exhibits organ damage-ameliorating activity. In other words, the organ damage-ameliorating agent of the present invention contains an ulvan derivative in an amount sufficient to exert the organ damage-ameliorating effect. Specifically, the proportion of the ulvan derivative in the organ damage-ameliorating agent of the present invention is not particularly limited, but for example, the proportion of the ulvan derivative in the organ damage-ameliorating agent of the present invention can be 10% by mass or more and 100% by mass or less.

[0037] The method for ameliorating organ damage according to the present invention comprises the step of administering to a subject the organ damage-ameliorating agent according to the present invention, namely, H-type ulvan or an alkali metal ion salt of ulvan.

[0038] The administration frequency and dose of the organ damage-ameliorating agent according to the present invention may be appropriately adjusted depending on the age, sex, condition, etc. of the subject, and an amount sufficient to exert an organ damage-ameliorating effect is administered to the subject. For example, the daily dose of H-type ulvan and / or alkali metal ion salt of ulvan can be 0.05 mg / kg body weight or more and 1 g / kg body weight or less.

[0039] The organ damage improving agent according to the present invention can be administered not only to humans but also to animals other than humans. Examples of animals to which the agent can be administered include livestock such as horses, cows, pigs, sheep, goats, camels, and llamas; sports animals such as racehorses; pets such as dogs and cats; laboratory animals such as mice, rats, guinea pigs, and rabbits; and poultry such as chickens, ducks, turkeys, and ostriches.

[0040] The dosage form of the organ damage ameliorating agent of the present invention is not particularly limited, and may be, for example, the ulvan derivative itself, a composition in combination with other components, or a solution or suspension thereof. The dosage form of the organ damage ameliorating agent of the present invention is not particularly limited, and examples thereof include tablets, powders, capsules, sugar-coated tablets, granules, and liquids. The organ damage ameliorating agent of the present invention may contain pharmaceutically acceptable additives depending on the dosage form. Examples of such additives include excipients, disintegrants, lubricants, binders, antioxidants, colorants, sweeteners, anti-aggregating agents, preservatives, solubilizers for the active ingredient, and stabilizers.

[0041] As shown in the following examples, the ulvan derivatives according to the present invention exhibit intestinal regulating effects, such as improving the intestinal flora, improving bowel movements, improving the intestinal barrier function, and anti-inflammatory effects on enterocytes. Furthermore, the ulvan derivatives according to the present invention also exhibit organ damage ameliorating effects, such as improving liver function and large intestine tissue. The ulvan derivatives according to the present invention are obtained by converting algae-derived polysaccharides into free forms or alkali metal ion salts, and are therefore considered to be highly safe. Therefore, the organ damage ameliorating agents according to the present invention may be regularly ingested as health foods that are excellent in organ damage ameliorating effects, such as intestinal regulating effects.

[0042] For example, the ulvan derivatives of the present invention can be incorporated into common foods and beverages as an active ingredient of an organ damage improving agent, such as an intestinal regulator. The foods and beverages to which the ulvan derivatives of the present invention can be added are not particularly limited, and examples thereof include beverages such as milk drinks, soft drinks, sports drinks, nutritional drinks, beauty drinks, and liquid nutrients; confectioneries such as chewing gum, chocolate, candy, jelly, cakes, biscuits, and crackers; frozen desserts such as ice cream and frozen desserts; noodles such as udon, Chinese noodles, spaghetti, and instant noodles; paste products such as kamaboko, chikuwa, and half pieces; condiments such as dressings, mayonnaise, and sauces; bread, ham, rice porridge, cooked rice, soup, various retort foods, and various frozen foods. The foods and beverages containing the ulvan derivatives of the present invention can be used in applications such as so-called health foods, supplements, functional foods, foods with functional claims, nutritional supplements, foods for specified health uses, foods with nutrient functions, nursing care foods, smile care foods, chewing and swallowing supplements, thick liquid foods, and foods for the sick.

[0043] This application claims the benefit of priority based on Japanese Patent Application No. 2023-108242, filed on June 30, 2023. The entire contents of the specification of Japanese Patent Application No. 2023-108242, filed on June 30, 2023, are incorporated herein by reference.

[0044] The present invention will be explained in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is of course possible to carry out the invention by making appropriate modifications within the scope of the above and below-described aims, and all such modifications are included in the technical scope of the present invention.

[0045] Example 1: Production of ulvan AMI salt (1) Production of natural extract ulvan After thoroughly washing and freeze-drying, Minami Aonori (Enteromorpha) was pulverized in a blender. Distilled water (20 mL) was added to the obtained dry powder (1 g), and hot water extraction was performed at 90°C. The supernatant was collected, and distilled water (20 mL) was added to the residue, followed by hot water extraction again. This procedure was repeated twice. The collected aqueous solution (approximately 60 mL) was concentrated, and ethanol was added to adjust the ethanol concentration to 75%, causing ulvan to precipitate. The resulting precipitate was collected by centrifugation and freeze-dried to obtain natural extract ulvan.

[0046] (2) Preparation of Ulvan AMI Salt Using an aqueous NaOH solution, an anion exchange resin (Cl type "Amberlite") was used. TM IRA410Cl (manufactured by Organo Corporation) was used as an OH-type anion exchange resin. The obtained OH-type anion exchange resin (0.5 g) was added to a 0.5% aqueous solution (100 g) of naturally extracted ulvan, and the mixture was stirred at room temperature for 30 minutes. The supernatant was collected, and fresh OH-type anion exchange resin was added again and treated in the same manner. The treatment with the OH-type anion exchange resin and the collection of the supernatant were repeated five times in total to remove sulfate (SO4 2- ) and chloride ions (Cl - The resulting aqueous ulvan solution (approximately 100 g) was treated with a sodium cation exchange resin (Amberlite TMUsing 0.5 g of "IR120BNa" (manufactured by Organo Corporation), the cations at the cation sites of ulvan were partially exchanged with Na ions, followed by freeze-drying. The molecular weight of the resulting ulvan AMI salt was measured by gel permeation chromatography (GPC). The cation concentration contained in the resulting ulvan AMI salt was measured by inductively coupled plasma (ICP) atomic emission spectroscopy. The results are shown in Table 1. In Table 1, the proton concentration is an estimated value based on the results of ICP atomic emission spectroscopy, and the "ratio of alkali metal ions (AMI) + protons" is calculated by calculating the acidic group concentration from the results of ICP atomic emission spectroscopy using the following formula, and then calculating the ratio of alkali metal ions and H to the calculated acidic group concentration. + Since the obtained ulvan Na salt contains almost no protons, the ratio of alkali metal ions to acidic groups can be said to be 41 mol %. Acidic group concentration = (Na + Concentration + K + Concentration + H + concentration)+2×(Ca 2+ Concentration + Mg 2+ concentration)

[0047] Example 2: Preparation of H-type ulvan The OH-type anion exchange resin (0.5 g) was added to a 0.5% aqueous solution (100 g) of naturally extracted ulvan, and the mixture was stirred at room temperature for 30 minutes. The supernatant was collected, and fresh OH-type anion exchange resin was added again and treated in the same manner. The treatment with the OH-type anion exchange resin and the collection of the supernatant were repeated five times in total to remove sulfate (SO4 2- ) and chloride ions (Cl - The resulting aqueous ulvan solution (approximately 100 g) was treated with a sodium cation exchange resin (Amberlite TM IR120BNa (Organo Corporation) (0.5 g) was added and stirred at room temperature for 30 minutes. Further, H-type cation exchange resin (Amberlite TM IR120BH (Organo Corporation) (0.45 g) was added and stirred at room temperature for 30 minutes to partially exchange the cations at the cationic sites of ulvan with H ions, followed by lyophilization. The resulting H-type ulvan was analyzed in the same manner as in Example 1(2). The results are shown in Table 1.

[0048] Example 3: Production of ulvan AMI salt The OH-type anion exchange resin (0.5 g) was added to a 0.5% aqueous solution (100 g) of naturally extracted ulvan, and the mixture was stirred at room temperature for 30 minutes. The supernatant was collected, and fresh OH-type anion exchange resin was added again and treated in the same manner. The treatment with the OH-type anion exchange resin and the collection of the supernatant were repeated five times in total to remove free sulfate ions (SO4 2- ) and chloride ions (Cl - Next, the resulting aqueous ulvan solution (approximately 100 g) was treated with an H-type cation exchange resin ("Amberlite"). TM IR120BH (Organo Corporation) (0.5 g) was added, and the mixture was stirred at room temperature for 30 minutes. The resin was removed, and the mixture was stirred again for 30 minutes. Furthermore, a Na-type cation exchange resin (Amberlite TM Most of the cations at the cationic sites of ulvan were exchanged with Na ions using IR120BNa (Organo Corporation) (47.3 g), followed by freeze-drying. The resulting ulvan AMI salt was analyzed in the same manner as in Example 1(2). The results are shown in Table 1. Note that the resulting ulvan Na salt contains almost no protons, so the ratio of alkali metal ions (AMI) to acidic groups can be said to be 93 mol%.

[0049]

[0050] Example 4: Change in fecal frequency Five standard male inbred mice, C57BL / 6j, were raised with free access to standard feed and water. The time it took for 10-week-old and 100-week-old mice to produce 200 mg of feces was measured. The results are shown in Figure 1 (1). As shown in Figure 1 (1), compared to the 10-week-old mice, the 100-week-old mice tended to have a decline in digestive function due to aging, resulting in a decrease in fecal output. Furthermore, when the measurement results were tested using a two-way analysis of variance followed by Tukey's test, there was a significant difference between the 10-week-old mice and the 100-week-old mice at p = 0.008.

[0051] Based on the above results, nine 54-week-old male C57BL / 6j mice were randomly divided into two groups. A 5 mg / mL aqueous solution of either the H-type ulvan of Example 2 or the ulvan AMI salt of Example 1 was dissolved in purified water and orally administered once daily at a dose of 50 mg / kg body weight using an oral administration probe. Standard chow and water were provided ad libitum. The time required to produce 200 mg of stool was measured immediately before the start of administration and on the 28th day after the start of administration. The results of administration of the H-type ulvan are shown in Figure 1 (2), and the results of administration of the ulvan AMI salt are shown in Figure 1 (3). As shown in Figure 1, although bowel movements tend to become impaired with aging, it was demonstrated that the H-type ulvan and ulvan AMI salt of the present invention can improve the function of the digestive tract and promote bowel movements.

[0052] Example 5: Changes in the intestinal microbiota Total genomic DNA was extracted from the feces of the 10-week-old and 100-week-old mice collected in Example 4 using a QIAamp Fast DNA Stool Mini Kit (Qiagen) according to the manufacturer's protocol. DNA concentration was monitored using a Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific). Next-generation sequencing library construction and Illumina sequencing were performed by the same company. 16S MetaVx TM A sequencing library was constructed using a Mammalian Library Preparation kit (GENEWIZ). Briefly, 20-50 ng of DNA was used to generate PCR amplification products (amplicons) covering the V3 and V4 hypervariable regions of the bacterial 16s rRNA gene. DNA concentration was detected using a microplate reader ("Infinite 200 Pro" Tecan), and fragment size was detected by 1.5% agarose gel electrophoresis. As a result, a significant difference (p = 0.049) was observed in the amount of Clostridia_vadinBB60_group-derived DNA contained in the feces of 10-week-old and 100-week-old mice, revealing that the amount was low in the feces of 100-week-old old mice and high in the feces of 10-week-old young mice (Figure 2(1)).

[0053] Furthermore, in Example 4, feces collected from mice administered with H-type ulvan were analyzed in the same manner. As a result, the feces 28 days after the start of administration contained significantly more Clostridia_vadinBB60_group-derived DNA than those before administration (p=0.001) (Figure 2(2)). These results suggest that administration of H-type ulvan changed the intestinal environment of aged mice to one similar to that of young mice.

[0054] Furthermore, in Example 4, feces collected from mice administered ulvan Na salt were similarly analyzed to examine a wider range of bacteria. Significant changes in the intestinal flora were observed in the feces 28 days after the start of administration compared to before administration. Although there was a significant difference in DNA content, the results are shown in Figure 3. As shown in Figure 3, significant changes were observed in the DNA content of Clostridia_vadinBB60_group, Lachnospiraceae NK4A136, and Turicibacter in the feces 28 days after the start of administration of ulvan Na salt compared to before administration. As shown in the above results, Clostridia_vadinBB60_group was found in significantly higher amounts in the intestines of young mice compared to old mice, suggesting that administration of ulvan Na salt changed the intestinal environment of old mice to one similar to that of young mice. It is said that the amount of Lachnospiraceae NK4A136 present decreases in inflammatory bowel disease. The administration of ulvan Na salt significantly increased the amount, which is considered to have improved the intestinal environment. Turicibacter increases with the ingestion of lard or a high-fat diet, but the administration of ulvan Na salt significantly reduced the amount, which is considered to have improved the intestinal environment.

[0055] Example 6: Effect on leaky gut (1) Intestinal epithelial barrier function F-hisec cells (Wako), which are intestinal epithelial cells prepared from human iPS cells, were seeded on a Matrigel-coated plastic culture plate ("Transwell #3413" manufactured by Corning). Then, the cells were cultured in a dedicated medium for 9 days. Next, as shown in Figure 4, endotoxin (LPS) and / or each ulvan was added to the medium and cultured. After 24 hours, the epithelial barrier function was measured using a resistance measurement system ("Millicell-ERS" manufactured by Merck). As the ulvan Na salt, the ulvan AMI salt (AMI + H) of Example 1 was used. + 41 mol%) was used. The results are shown in Figure 4. In Figure 4, "*" indicates that there is a significant difference at p<0.05 according to two-way analysis of variance followed by Tukey's test, and "**" indicates that there is a significant difference at p<0.01 according to two-way analysis of variance followed by Tukey's test.

[0056] As shown in Figure 4, LPS treatment significantly decreased the intestinal epithelial barrier function, but treatment with natural ulvan significantly improved it. Treatment with ulvan Na salt in addition to LPS significantly improved the intestinal epithelial barrier function compared with treatment with LPS and natural ulvan.

[0057] (2) Inflammatory Cytokines The cultured cells were harvested, and gene expression of the inflammatory cytokines IL-6 and IL-1β was quantified using a high-efficiency real-time PCR master mix (THUNDERBIRD® Next SYBR® qPCR Mix, manufactured by Toyobo Co., Ltd.). Total RNA was normalized for each reaction using β-actin complementary DNA as an internal standard. The quantification results for IL-6 mRNA are shown in Figure 5(1), and the quantification results for IL-1β mRNA are shown in Figure 5(2). In Figure 5, "*" indicates a significant difference at p<0.05 by two-way ANOVA followed by Tukey's test, and "**" indicates a significant difference at p<0.01 by two-way ANOVA followed by Tukey's test.

[0058] As shown in Figure 5, LPS treatment increased the expression level of inflammatory cytokines, whereas natural ulvan treatment tended to decrease the expression level of inflammatory cytokines, although no significant difference was observed. Furthermore, treatment with ulvan Na salt in addition to LPS significantly suppressed the expression level of inflammatory cytokines by intestinal epithelial cells, more significantly than treatment with LPS plus natural ulvan, suggesting that the intestinal epithelial barrier function was improved.

[0059] Example 7: Effect on macrophages Cultured macrophage cells RAW236.7 were cultured and treated with endotoxin (LPS) or with each ulvan in addition to LPS for 24 hours. As the ulvan Na salt, the ulvan AMI salt (AMI+H) of Example 1 was used. + : 41 mol%) or the ulvan Na salt of Example 3 (AMI + H + : 93 mol%). The cultured cells were then harvested, and mRNA levels of the inflammatory cytokines IL-1β, IL-6, and TNFα were quantified using a high-efficiency real-time PCR master mix (THUNDERBIRD® Next SYBR® qPCR Mix, Toyobo Co., Ltd.). Total RNA was normalized for each reaction using β-actin complementary DNA as an internal standard. The results are shown in Figure 6. In Figure 6, "*" indicates a significant difference at p<0.05 by two-way ANOVA followed by Tukey's test, and "**" indicates a significant difference at p<0.01 by two-way ANOVA followed by Tukey's test.

[0060] As shown in Figure 6, when macrophages were treated with LPS, the expression levels of each inflammatory cytokine increased. Furthermore, when treated with natural ulvan, the expression levels of each inflammatory cytokine tended to increase or decrease slightly, without any significant difference. Furthermore, when treated with H-type ulvan or ulvan Na salt in addition to LPS, the expression levels of each inflammatory cytokine significantly decreased. Thus, these experiments suggest that ion exchange can produce effects not found in natural ulvan.

[0061] Example 8: Effect on leaky gut - in vivo experiment (1) Measurement of administered dextran Twelve male C57BL / 6j mice aged 76 weeks were randomly divided into three groups of four mice each. + FITC-dextran 4kDa (41 mol%) was dissolved in purified water to prepare a 5 mg / mL aqueous solution, and the solution was orally administered once daily at a dose of 50 mg / kg body weight using an oral administration probe. The control group received the same amount of purified water. Standard feed and water were provided ad libitum. On the 26th day after the start of administration, after a 4-hour fast, FITC-dextran 4kDa was dissolved in purified water to prepare a 20 mg / mL aqueous solution, and the solution was orally administered at a dose of 200 mg / kg body weight using an oral administration probe. After 2 hours, the animals were euthanized, and blood was collected to obtain serum. The FITC fluorescence intensity of the obtained serum samples was measured using a fluorescence plate reader ("Infinite 200," manufactured by TECAN) at emission: 485 nm, excitation: 535 nm. The results are shown in Figure 7(1). In FIG. 7, "**" indicates that there is a significant difference at p<0.01 according to two-way analysis of variance followed by Tukey's test.

[0062] When leaky gut occurs, FITC-dextran 4kDa is promoted inflow into the bloodstream from the intestine, but when the intestinal barrier is fully functional, it is hardly absorbed into the bloodstream. The experimental results shown in Figure 7(1) confirmed that FITC-dextran 4kDa was detected in the serum of aged mice, suggesting that leaky gut occurs in an age-dependent manner. In the natural ulvan-administered group, there was a tendency for the amount of FITC-dextran 4 in the serum to decrease compared to the control group, but the amount of decrease was not significant. In contrast, a significant decrease in the amount of FITC-dextran 4 was confirmed in the ulvan Na salt-administered group, suggesting that the intestinal barrier function of aged mice had improved.

[0063] (2) Measurement of LPS. The LPS concentration in the serum samples was measured using a Pierce Chromogenic Endotoxin Quant Kit (Thermo Scientific). The results are shown in Figure 7(2). When leaky gut is present, LPS derived from intestinal bacteria is promoted in the bloodstream, but when the intestinal barrier is fully functional, it is hardly absorbed into the bloodstream. The experimental results shown in Figure 7(2) confirmed LPS in the serum of aged mice, suggesting that leaky gut is induced in an age-dependent manner. In the natural ulvan-treated group, serum LPS concentrations tended to be reduced compared to the control group, but the reduction was not significant. In contrast, a significant reduction in blood LPS levels was confirmed in the ulvan Na salt-treated group. This suggests that the ulvan Na salt-treated group improved intestinal barrier function in aged mice. Furthermore, since LPS is easily transferred into the blood when bad bacteria increase, it was suggested that the balance of intestinal bacteria was also improved in the ulvan Na salt-administered group.

[0064] Example 9: Effect on liver dysfunction - in vivo experiment Twelve 76-week-old male C57BL / 6j mice were randomly divided into three groups of four mice each. C57BL / 6j mice are the most commonly used strain for diet-induced obesity and develop metabolic diseases similar to those in humans. Naturally extracted ulvan (Example 1(1)) or ulvan Na salt (AMI+H) (Example 1(2)) were used in the experiments. +41 mol%) was dissolved in purified water to prepare a 5 mg / mL aqueous solution, and a dose of 50 mg / kg body weight was orally administered once daily using an oral administration probe. The control group received the same amount of purified water. Standard feed and water were available ad libitum. On the 26th day after the start of administration, the animals were euthanized, and blood was collected to obtain serum. ALT activity in the obtained serum samples was measured using an Alanine Transaminase Colorimetric Activity Assay Kit (Cayman Chemical Company). Serum ALT concentrations are shown in Figure 8. In Figure 8, "*" indicates a significant difference of p<0.05 using Tukey's test following two-way analysis of variance.

[0065] As shown in Figure 8 (1), the serum concentration of alanine aminotransferase (ALT), which leaks into the blood due to hepatocyte destruction, was not significantly reduced by ulvan, but was significantly reduced by ulvan Na salt. Therefore, it is believed that ulvan Na salt reduces liver tissue damage.

[0066] Example 10: Effect on colon tissue damage - in vivo experiment Twelve 76-week-old male C57BL / 6j mice were randomly divided into three groups of four mice each. The natural extract ulvan of Example 1(1) or ulvan Na salt (AMI+H) of Example 1(2) was administered. +41 mol%) was dissolved in purified water to prepare a 5 mg / mL aqueous solution, and the resulting solution was orally administered once daily at a dose of 50 mg / kg body weight using an oral administration probe. The control group received the same amount of purified water. Standard feed and water were available ad libitum. After euthanasia on day 26 after the start of administration, colonic tissue was collected, and gene expression of Claudin-1 and Claudin-2, which are involved in the formation of tight junctions between cells, was quantified using a high-efficiency real-time PCR master mix ("THUNDERBIRD® Next SYBR® qPCR Mix," manufactured by Toyobo Co., Ltd.). Total RNA was normalized for each reaction using β-actin complementary DNA as an internal standard. The results of Claudin-1 mRNA quantification are shown in Figure 9(1), and the results of Claudin-2 mRNA quantification are shown in Figure 9(2). In FIG. 9, "*" indicates that there is a significant difference at p<0.05 according to two-way analysis of variance followed by Tukey's test, and "ns" indicates that there is no significant difference.

[0067] Claudin-1 is important for the formation of strong intercellular tight junctions (tight junctions), while Claudin-2 forms weak tight junctions. Previous findings suggest that increasing Claudin-1 is effective in improving leaky gut. As shown in the results in Figure 9, the expression level of Claudin-2 decreased while the expression level of Claudin-1 increased in mice administered ulvan Na salt, suggesting that administration of ulvan Na salt restored barrier function via tight junctions in colonic tissue.

Claims

1. An organ damage improving agent characterized by containing H-type Urban or an alkali metal ion salt of Urban as an active ingredient.

2. The organ damage improving agent according to claim 1, wherein the alkali metal is Na and / or K.

3. The organ damage improving agent according to claim 1, wherein 30 mol% or more of the carboxyl groups and sulfate groups of the H-type urban are protonated.

4. The organ damage improving agent according to claim 1, wherein 40 mol% or more of the carboxyl groups and sulfate groups of the alkali metal ion salt of Urban form a salt with alkali metal ions.

5. An organ damage improving agent according to claim 1, which improves the intestinal microbiota.

6. The organ damage improving agent according to claim 1, which improves leaky gut.