JP2011046647A - CHOLESTEROL INHIBITOR CONTAINING POLY-gamma-GLUTAMIC ACID - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cholesterol inhibitor which is highly safe and is acceptable in use for food. <P>SOLUTION: Poly-γ-glutamic acid is made to act as the component of the cholesterol inhibitor. The cholesterol inhibitor is used for treating and preventing hypercholesterolemia. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cholesterol inhibitor containing poly-γ-glutamic acid (hereinafter also referred to as “γ-PGA”) as an active ingredient, and its use in medicine or food and drink.

  In recent years, in addition to lack of exercise, the Japanese lifestyle has become westernized, and hypercholesterolemia has been frequently observed. The presence of this disease can cause serious cardiovascular diseases such as ischemic heart diseases such as arteriosclerosis, myocardial infarction, and cerebral infarction, and blood cholesterol inhibitors are effective for the prevention and treatment of these diseases. Something has been revealed.

  Dietary cholesterol and endogenous cholesterol excreted from the gallbladder with food intake are taken up into mixed micelles of bile acids, monoglycerides and fatty acids, and the cholesterol transporter (Niemann-Pick C1) present in jejunal absorptive epithelial cell villi Like 1: Captured by NPC1L1) Then, it is esterified by cholesterol ester acyltransferase in the jejunal absorptive epithelial cells and transferred to blood in the form of cholesterol ester. It is pointed out that when dietary intake continually consumes excessive cholesterol, the level of LDL cholesterol in the blood increases, leading to the development of hypertension and atherosclerotic diseases such as ischemic heart disease Has been.

  Drugs that suppress cholesterol in the blood include drugs that act on the cholesterol synthesis system in the liver (statins, etc.), drugs that suppress the reabsorption of bile acids (anion exchange resin system), and recently, absorption of cholesterol. An NPC1L1 inhibitor (ezetimibe; SCH58235) has also been developed.

  On the other hand, poly-γ-glutamic acid, which is the main component of natto mucilage, is a substance that has the ability to solubilize minerals in the intestinal tract and promotes mineral absorption. Poly-γ-glutamic acid has a mineral solubilizing ability due to the action of many carboxyl groups in the molecule. Furthermore, since they are bound by γ bonds, they are not degraded by digestive enzymes present in the small intestine, and are believed to retain activity in the intestinal tract. When this poly-γ-glutamic acid is used as a feed, it has been reported to promote the absorption of minerals and reduce the amount of accumulated body fat (see, for example, Patent Document 1). The effect on humans is not clear.

  Further, oral hyperlipidemia therapeutic agents containing acidic polyamino acids having a molecular weight of 10,000 to 100,000 as pharmacologically effective amounts are known, and α-polyglutamic acid is also described as an acidic polyamino acid ( For example, see Patent Document 2).

JP 09-28309 A Japanese Patent Laid-Open No. 52-28947

  Since there are so many subjects who are at risk of hypercholesterolemia, blood cholesterol can be prevented by inhibiting the absorption of dietary cholesterol in preventing diseases such as arteriosclerosis and ischemic heart disease. It is very important to reduce the value. However, most of the pharmaceuticals currently used inevitably have side effects. An object of the present invention is to provide a highly safe cholesterol inhibitor that is acceptable for use as a food.

The present inventors analyzed various components in blood and organs when poly-γ-glutamic acid was administered using a model animal raised on a high cholesterol diet. It was found that cholesterol elevation in the middle and liver was suppressed by administration of poly-γ-glutamic acid, and that this action was based on cholesterol absorption inhibition, and the present invention was completed based on these findings.
It has been found that poly-γ-glutamic acid suppresses fat absorption (“Fat Absorption Inhibitor” Japanese Patent Application No. 2007-332363). However, it is described that this is based on suppressing pancreatic lipase, an essential enzyme for the digestion of neutral fat. Since pancreatic lipase does not affect cholesterol absorption, this finding cannot easily explain the cholesterol-suppressing action of poly-γ-glutamic acid.
Patent Document 2 ("oral hyperlipidemia therapeutic agent, Japanese Patent Application Laid-Open No. 52-28947)" describes that α-polyglutamic acid exhibits cholesterol suppression. This shows a polymerized product. On the other hand, the present invention relates to poly-γ-glutamic acid obtained by polymerizing D-glutamic acid mainly derived from natto by γ bond.
In α-polyglutamic acid described in Patent Document 2, a carboxyl group at the α-position is polymerized with an amino group to form a polymer main chain. α-polyglutamic acid does not exist in nature and can be obtained by chemical synthesis. Since glutamic acid has two carboxyl groups, there is a high possibility that branching will occur when attempting to polymerize chemically, and it is extremely difficult to synthesize a polymer that does not contain branches, and even if it can be synthesized, its cost is high It is extremely difficult to use industrially. Therefore, α-polyglutamic acid is commercially available as a reagent and used for various studies, but at present, it is hardly used industrially.
On the other hand, poly-γ-glutamic acid is a natural polymer, as described above, and is synthesized by an enzyme with high substrate specificity, so that the molecular structure is linear. Furthermore, as described above, it is considered that it is not degraded by digestive enzymes due to γ-binding. This poly-γ-glutamic acid is completely different from the α-type substance, and its reactivity, properties and functions are completely different.

  That is, the cholesterol inhibitor, the cholesterol-suppressing composition, and the cholesterol-suppressing method of the present invention are characterized by containing poly-γ-glutamic acid as an active ingredient. In one embodiment, the cholesterol inhibitor is preferably administered to a subject who has hypercholesterolemia or is likely to have it. That is, it can be used as a therapeutic or prophylactic agent for hypercholesterolemia. In addition, it is more preferable to administer poly-γ-glutamic acid orally between 2 hours before meal and 4 hours after meal when eating a high cholesterol content.

  In another aspect of the present invention, there is provided a pharmaceutical composition or a food / beverage composition comprising the cholesterol inhibitor.

  The cholesterol-suppressing agent of the present invention is used as a pharmaceutical product or food / beverage product composition to prevent or improve these subjects for subjects who need to suppress cholesterol absorption, particularly subjects with hypercholesterolemia. Useful. Moreover, even in normal people, it is useful as a health functional food for subjects who are concerned about an increase in blood cholesterol.

It is a graph which shows transition of the plasma total cholesterol density | concentration of mouse | mouth γ-PGA long-term drinking administration. Data are shown as mean ± standard deviation (n = 8, ** p <0.01 (comparison with control group)). It is a graph which shows the amount of liver lipid of a mouse | mouth γ-PGA long-term drinking administration, the triglyceride in liver, a free fatty acid, and a cholesterol level. Data are shown as mean ± standard deviation (n = 8, * p <0.05, ** p <0.01 (comparison with control group)). It is a graph which shows transition of the plasma total cholesterol density | concentration of mouse | mouth γ-PGA long-term drinking administration. Data are shown as mean ± standard deviation (n = 8, ** p <0.01 (comparison with control group)). It is a graph which shows the amount of liver lipid of a mouse | mouth γ-PGA long-term drinking administration, the triglyceride in liver, a free fatty acid, and a cholesterol level. Data are shown as mean ± standard deviation (n = 8, ** p <0.01 (comparison with control group)). 1 is a graph showing the daily dry feces weight, total fecal lipid content, triglycerides, free fatty acids, cholesterol, neutral sterols, and total bile acid content after long-term administration of mouse γ-PGA. Data are shown as mean ± standard deviation (n = 8, * p <0.05, ** p <0.01, *** P <0.001 (comparison with control group)).

  The cholesterol absorption inhibitory action in the present invention refers to an action that improves the collapse of blood cholesterol balance mainly by suppressing the absorption of lipids from the intestinal tract. In particular, it refers to suppressing or delaying the treatment, improvement, and onset of hypercholesterolemia by suppressing the absorption of cholesterol. In addition, “hypercholesterolemia” generally refers to a subject whose blood total cholesterol level is 220 mg / dl and LDL cholesterol level is 140 mg / dL or more. According to a national nutrition survey conducted in 1997, 27% of men were diagnosed with hypercholesterolemia and 33% were women.

  The γ-PGA used in the present invention may be used after extracting and purifying γ-PGA in the natto mucilage, and extracting and purifying γ-PGA secreted outside the cells of the genus Bacillus such as Bacillus natto. May be used. Although high purity may be used, it is not always necessary to have high purity, and there is no problem even if other substances such as levan, which is a natto sticky product or a secreted product of Bacillus natto, are included.

  The average molecular weight of γ-PGA used in the present invention is not particularly limited from the viewpoint of its effectiveness, and γ-PGA having a weight average molecular weight of 3000 to 3 million when usually separated from natto or natto bacteria is used. Can be used. In addition, when the molecular weight is low, it is easily decomposed during storage of the solution, and when the molecular weight is too high, the viscosity of the aqueous solution is too high and handling becomes difficult. Therefore, it is preferable to use 10,000 to 1,000,000 γ-PGA from the viewpoint of handling as a pharmaceutical composition or a food / beverage product composition. In addition, the weight average molecular weight of (gamma) -PGA is measured by the light-scattering method, for example. At this time, for example, dn / dc (refractive index concentration increment, solvent 100 mM Tris-HCl (pH 8.6) and 0.3 M NaCl) at the wavelength of 633 nm is 0.191.

  Glutamic acid that constitutes γ-PGA generally includes both D-form and L-form, and it is said that the D / L ratio varies depending on the fungus body or medium composition. For example, γ-PGA disclosed in JP-A-3-47087 is said to have 90% or more of L-form glutamic acid. The D / L ratio of γ-PGA used in the present invention is not limited, and γ-PGA having any D / L ratio can be used. In addition, it is said that (gamma) -PGA contained in natto contains about 80% of D body.

  In order to produce γ-PGA having a specific molecular weight, a method of reducing the molecular weight of γ-PGA having a molecular weight higher than that by acid or a bacterium that degrades γ bond or an enzyme derived from an organ, and culturing of Bacillus natto, etc. There is a method of secreting γ-PGA, and either γ-PGA may be used.

  γ-PGA is generally obtained as a sodium salt, but using other pharmaceutically acceptable or edible salts or free γ-PGA has no effect.

  The cholesterol inhibitor containing the γ-PGA of the present invention as an active ingredient is used for a pharmaceutical, veterinary or food / drink for the prevention, treatment and / or improvement of hypercholesterolemia by using a composition containing the same. It can be used as a composition. Moreover, those forms are not limited, for example, foods and drinks such as health functional foods (food for specified health use, nutritional functional foods), health foods, nutritional supplements, etc., or easily available pharmaceuticals or quasi-drugs such as OTC It can be used as goods and veterinary drugs As used herein, “prevention” is to completely or partially prevent hypercholesterolemia, and “treatment” refers to the prevention of hypercholesterolemia as well as the part of adverse effects caused by these diseases. Including complete or complete healing.

  The cholesterol inhibitor containing γ-PGA of the present invention as an active ingredient and a composition containing the same can also be directly ingested. In addition, it can be ingested after being molded into a form that is easy to take, such as capsules, tablets, granules, using known additives such as carriers and auxiliaries. For the purpose of enhancing nutrition, various vitamins such as vitamins A, C, D, and E, and minerals such as calcium and iron can be added and used in combination. In particular, since γ-PGA has a mineral absorption promoting action, it is preferably used in combination with these. Subjects who need to prevent or ameliorate high cholesterol are included in the subjects to which the cholesterol suppressant of the present invention is administered. However, dietary restrictions to prevent obesity include osteoporosis and anemia due to decreased absorption of calcium and iron. Such side effects are often accompanied. Therefore, the cholesterol inhibitor of the present invention is useful as a cholesterol-lowering agent by containing minerals such as calcium and iron.

  The content of the cholesterol inhibitor of the present invention in these molding agents is preferably 0.1 to 100% by weight, more preferably 10 to 90% by weight. Furthermore, mixed with food and drink ingredients, confectionery such as chewing gum, chocolate, candy, jelly, biscuits and crackers; frozen confectionery such as ice cream and ice confectionery; beverages such as tea, soft drinks, energy drinks and beauty drinks; udon Noodles such as Chinese noodles, spaghetti and instant noodles; kneaded products such as rice cakes, bamboo rings and hampen; seasonings such as dressings, mayonnaise and sauces; fats and oils such as margarine, butter and salad oil; bread, ham, soup, retort food It can be used for all foods and drinks such as frozen foods.

  When used as a pharmaceutical, the dosage form is not particularly limited, and examples thereof include capsules, tablets, granules, injections, suppositories, and patches. In formulation, other pharmaceutically acceptable carriers, such as excipients, disintegrants, lubricants, binders, antioxidants, colorants, anti-aggregation agents, absorption enhancers, solubilizers, It can be prepared by appropriately adding a stabilizer or the like. When used as a quasi-drug, other additives are added as necessary, for example, ointments, liniments, aerosols, creams, soaps, facial cleansers, whole body cleansers, lotions, lotions, baths It can be used for an agent etc., and can also be used locally.

  The administration method of the cholesterol inhibitor of the present invention is 10 mg to 5 g in terms of the amount of γ-PGA per one meal intake in humans for the purpose of effectively exerting the effect of the cholesterol inhibitor of the present invention. It is characterized by administering so that it may become the dosage of this. It is preferably 100 mg to 1 g with a diet high in cholesterol. Administration of 10 mg or less of γ-PGA is not preferable because a sufficient cholesterol inhibitory effect cannot be exhibited. Ingestion of 5 g or more of γ-PGA is not preferable because the intake is a large amount and it is a burden to ingest, leading to a decrease in quality of life. In addition, the method for ingesting the cholesterol inhibitor of the present invention may be performed before meal, during meal, or after meal. However, in order to moderately inhibit the absorption of lipids in the meal, it is carried out for about 2 hours before the meal, preferably about 1 hour before and at most 4 hours after the meal, preferably within 2 hours, most preferably within 1 hour. It is preferable to do.

[Example 1] Drinking water administration test of γ-PGA to mice loaded with high cholesterol diet (1)
Seven-week-old male C57BL6 carried from Charles River, Japan was preliminarily raised for one week, and then divided into two groups (eight groups each) of control group and γ-PGA group. The diet is a Research Diet 4% (w / w) fat, 1.25% (w / w) high cholesterol diet (D12104), water for the control group, 2.5% for the γ-PGA group γ-PGA was ingested freely and bred for 4 weeks. Blood was collected from the orbital venous plexus once a week, plasma was separated from the blood, and the total cholesterol level was measured with Fuji Dry Chem (Fuji Film). 4 weeks after the start of administration, the liver was removed under fasting overnight, lipids in the liver were extracted by the Floch method, and the values of triglyceride, total cholesterol, and free fatty acids in the liver were measured with Test Wako kit (manufactured by Wako Pure Chemical Industries). It was measured. The test results are shown in FIGS. The significance test between each group was performed by the student's t test method, and the significance level was set to 5% or less.

  As shown in FIG. 1, it was found that the plasma cholesterol level was significantly suppressed in the group given γ-PGA by drinking water. Furthermore, as shown in FIG. 2, it was found that the cholesterol level in the liver was significantly reduced in the group given γ-PGA by drinking water.

[Example 2] Drinking water administration test of γ-PGA to mice loaded with high cholesterol diet (2)
A 7-week-old male C57BL6 carried from Charles River, Japan was preliminarily raised for 1 week, and then divided into two groups (8 mice each), a control group and a γ-PGA group. The diet was a Research Diet 20% (w / w) fat, 1.25% (w / w) cholesterol diet (D12108), the control group was water, and the γ-PGA group was 2.5% γ. -PGA was allowed to drink freely and reared for 4 weeks. Blood was collected from the orbital venous plexus once a week, plasma was separated from the blood, and the total cholesterol level was measured with Fuji Dry Chem (Fuji Film). Feces were collected 3 to 4 weeks after the start of administration, and the liver was excised overnight in 4 weeks, and the liver lipids were extracted by the Floch method, and the values of triglyceride, total cholesterol and free fatty acids in the liver were tested. It was measured with a kit (manufactured by Wako Pure Chemical Industries). The lipid content of the collected feces was extracted using Soxhlet (ACTAC), and the values of triglyceride and free fatty acid were measured with Test Wako kit (manufactured by Wako Pure Chemical Industries). Cholesterol content was measured using gas chromatography (Agilent technology). The amount of total bile acid in feces was extracted with alcohol, and the value of total bile acid was measured with a test Wako kit (manufactured by Wako Pure Chemical Industries). The test results are shown in FIGS. 3, 4, and 5 as mean values ± standard deviation. The significant difference test between groups was performed by the student's t test method, and the significance level was set to 5% or less.

  As shown in FIG. 3, it was found that the plasma cholesterol level significantly decreased in the group given γ-PGA by drinking water. As shown in FIG. 4, it was found that the cholesterol level in the liver was significantly reduced in the group given γ-PGA by drinking water. As shown in FIG. 5, the amount of cholesterol excreted in feces was significantly increased by the administration of γ-PGA. Furthermore, γ-PGA suppressed the amount of bile acids in feces. It has been reported that cholestyramine, which has a cholesterol lowering action by adsorbing bile acids, increases bile acids with increased fecal cholesterol compared to controls (Non-Patent Document, Br. J. Nutri. 2005, 94, 331-337), it was considered that γ-PGA exhibited cholesterol lowering action by suppressing absorption of cholesterol rather than bile acid adsorption.

  The cholesterol inhibitor of the present invention is highly safe and has an excellent cholesterol suppressing effect. Therefore, it is useful for use in foods and drinks such as health foods and health functional foods, or pharmaceuticals.

Claims (5)

  A cholesterol inhibitor comprising poly-γ-glutamic acid as an active ingredient.   The cholesterol-suppressing agent according to claim 1, which is used for treatment or prevention of hypercholesterolemia.   A pharmaceutical composition comprising the cholesterol inhibitor according to claim 1 or 2 and a pharmaceutically acceptable carrier.   A composition for suppressing cholesterol, comprising poly-γ-glutamic acid as an active ingredient.   A cholesterol-suppressing composition comprising 10 mg to 5 g of poly-γ-glutamic acid.

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