TW200902046A - Agents for preventing renal failure - Google Patents

Abstract

The present invention provides agents having inhibiting function for kidney glomerular diseases and kidney arteriosclerosis, agents for preventing renal failure, and functional foods promising effects for preventing renal failure. The inhibitor agent for kidney glomerular diseases and kidney arteriosclerosis of the present invention contains Xaa Pro Pro as an effective component.

Description

200902046 • Nine, invention description: . [Technical field to which the invention belongs] is a preventive agent for inhibiting kidney spheroid disease and renal arteriosclerosis, a renal failure preventive agent containing the active ingredient, and an active ingredient of the product. A functional food for the effect of renal failure. Further, the present invention relates to a method for inhibiting hardening. Furthermore, Taisu. 糸 Sphere disease and renal artery ^ [Prior Art] w is a method for preventing renal failure. Second =: points::;:: recognizes, disease. = change more ~ ~ failure:: ==: insurance: or pet animals and other humans have a close relationship with animals: _ = = treatment effective In the case of an agent, it is an inhibitor of angiotensin-converting enzyme inhibitor which can inhibit the action of angiotensin π from the blood vessel = tube = fermenting stimulating hormone π (', Lv tight & ' /, soil with Lapuli (-. In addition / two ^ _ 〇.., the purchase pointed out that the lowering of the blood in the lower two:: change:, the progress of the renal barrier. But in another - aspect, relative to the self-efficiency On the contrary, it is pointed out that == two: all the diseases have a hundred ^ ^ 昼 昼 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 320 070 070 070 070 070 070 070 070 070 070 070 070 070 070 070 070 070 320 320 070 Wide and new (take new medicine, 48:1404 to 1409, 1993). - That is, the antihypertensive agent is not only in the prevention of heart failure, but also the possibility of inducing renal failure. Fully 'transfers well to the heart system (four) dirty becomes unable to remove the metabolic waste in the blood well when passing the blood. The kidney is caused by various kidney diseases The function of the ruined disease, the eagle eagle 31 cattle such as diabetic nephropathy, chronic kidney spheroid: month 'f sclerosis, multiple cystic kidney (polycystic kidney ΤζΙ ^ (chr〇nic —-^is) ^ Month... 脰 kidney fire, systemic lupus erythematosus (systemic lupus 7 kidney movement jit ΓΤ X, renal squamous sclerosis, renal arteriosclerosis caused by == incomplete, etc. In addition, ACE inhibitors are effective for kidney failure' but It is also indicated that there is a possibility that the acute kidney: i::F early deterioration may occur, and the usefulness of these agents is not sufficient (latest medicine, 48:14〇4 ^ * ^ ^ ^ ^ ^ ,). As mentioned above, caused by various factors such as mil sugar or drug disorders, it is not sufficient to prevent all the deaths from the primary diseases such as the above. On the other hand, Kaizi points to the side of the kidney spheroid disease, which is a common cause of renal failure. In addition, 'month arteriosclerosis is a disease associated with renal failure. From this perspective, the compound that has the inhibitory effect of renal spheroid disease, Or a compound having an inhibitory effect on renal arteriosclerosis, which can be considered as a kidney It is useful to prevent and treat it. Although there are reports that the peptide derived from food materials such as casein has (10) inhibitory activity, these blood stasis reduction effects are known as 320070 6 200902046, but not directly. Whether it has a renal spheroid disease inhibitory effect or an arteriosclerosis inhibitory effect (Japanese Patent No. 142, a kidney)

Am> J* ACE The effect of prevention or treatment of renal failure is qualitative as described above, so it is expected that there will be prevention or treatment of diseases that inhibit ACE. SUMMARY OF THE INVENTION According to the present invention, there is provided an inhibitory activity independent of A C E and having a renal glomerular disease to be additionally heated or a composition. In the present invention, the present invention provides a preventive agent comprising the above-mentioned compound or composition, and a functional food object which is expected to have an effect of preventing renal failure. The compound or composition is administered to a method for producing a kidney spheroid disease, and the renal artery is inhibited. Therefore, according to the present invention, the compound or composition comprising the above-mentioned compound or composition is provided. The above-mentioned compound or composition is also used in the manufacture of a medicine for inhibiting the sclerosing of kidney spheroid diseases. The invention department is also a compound or composition of the above-mentioned cages used to prevent kidney shoots. The inventors of the present invention found that the tripeptide peptide having a specific structure called Xaa Pr0 Pr0 (xaa is an arbitrary 320070 7 200902046 \ natural amino acid) has an inhibitory effect on kidney spheroids. Inhibition of the role of nephrosclerosis, and the invention of medicines and functional foods effective in preventing renal failure. The specific content is as follows. The present invention provides an inhibitor of kidney spheroid diseases and renal arteriosclerosis comprising Xaa Pro Pro as an active ingredient. Further, the present invention provides a renal failure preventive agent comprising the above-described action of inhibiting kidney spheroid diseases and renal arteriosclerosis. Further, the present invention provides a functional food comprising the above-described kidney spheroid disease and an inhibitor of renal arteriosclerosis. Furthermore, the present invention provides a method for inhibiting kidney spheroid diseases and renal arteriosclerosis comprising administering Xaa Pro Pro or a composition containing Xaa 'Pro Pro to a subject. Accordingly, the present invention also provides a method of preventing renal failure comprising administering a composition of Xaa Pro Pro or Xaa Pro Pro to a subject. Further, the present invention is also a composition using Xaa Pro Pro or Xaa Pro Pro in the manufacture of a medicine for inhibiting kidney spheroid diseases and renal arteriosclerosis. In particular, the present invention is also a composition using Xaa Pro Pro or Xaa Pro Pro in the manufacture of a medicine for preventing renal failure.

Xaa Pro Pro should be Val Pro Pro and/or lie Pro Pro. In other aspects of the invention, the active ingredient Xaa Pro Pro is derived from a casein hydrolysate or a concentrate thereof. In another aspect of the present invention, the active ingredient Xaa Pro Pro is derived from a fermentation product obtained by fermenting a raw material containing milk protein by a bacterium belonging to the species Lactobacillus helveticus. 320070 200902046 ; belongs to the Swiss Lactobacillus species * in the ' strain (CCRC 910IH). Niobium ore, preferably Lactobacillus bulgaricus CM4 [Embodiment] The kidney spheroid disease of the present invention includes an inhibitor such as Xaa Pro Pr and: arteriosclerosis, and the "agent" is not an active ingredient. In the food or food composition, etc. _ thing two: two = for example, a pharmaceutical composition. In this issue, the so-called "two inhibitors" refers to the role of inhibition and resistance to sclerosing lesions. "The eye layer and the kidney movement ^ « or /, improve the kidney spheroid disease and the lunar artery A compound or composition that has the effect of sclerosing a disease, or an effect of preventing kidney and kidney movement: sclerosis. In addition, the term "spheroid disease" as used in the present invention refers to a disease in which a spheroid of the kidney is diseased. The upper system has been changed. It can be confirmed that the spheroid atrophy, the spheroidal ruin, the spheroid hardening, and the change of the sac. In terms of the disease name of the body, for example, a small-changing renal disease group, local spheroid sclerosis, intraductal proliferative spheroid nephritis (also known as acute nephritis), and IgA nephropathy (mesis proliferative spheroid nephritis) may be mentioned. , membranous nephropathy, membranous proliferative spheroid nephritis, and crescentic spheroid nephritis. The Xaa of the active ingredient Xaa Pro Pro of the present invention may be any natural amino acid. Specific examples thereof include Val Pr〇 Pr〇 (proline-proline _ valine), lie Pro Pro (isorecine-proline 脯-proline), and Ser Pr〇.

Pro (serine-proline acid/proline), Leu Pro Pro (leucine acid/proline _ 脯月女酸) special 'Val Pro Pro, lie Pro Pro, preferably Val Pro 320070 9 200902046 :.Pro. Further, as the active ingredient, a combination of one or more kinds of the three peptides XaaPr〇pr〇2 such as 1 p(7)卩(7) and ne pr〇·.Pro may be contained. An active ingredient (10) Pro Pro can be a biochemically synthesized dipeptide - or a natural tripeptide. For the organic chemical synthesis method of the three-peptide Xaa Pro Pro, a general method such as a solid phase method (B〇c method, Fmoc method) or a liquid phase method can be used, and for example, the use of Shimadzu Corporation can be used. A peptide synthesizing device (psSM_8 type), which is synthesized by an automatic peptide synthesizing device. Regarding the reaction conditions for peptide synthesis, etc., appropriate reaction conditions can be arbitrarily set according to the technical knowledge of the manufacturer, in accordance with the selected synthesis method or the desired dipeptide Xaa Pro Pro. Or, in the case of a natural person, it may be derived from the animal milk road protein water (4) or its concentrate, or may be derived from a fermentation of a raw material containing milk protein by a so-called sputum or lactic acid bacteria. The resulting fermenter. In the use of animal milk to ferment protein hydrolysate or its discriminant, or to make the milk containing L!! by fermentation of the bacteria belonging to the Lactobacillus species of Switzerland, the % 'except for the active ingredient of the present invention Other than h: package: free amino acid, in addition to the above-mentioned peptide and free amino acid, may also contain commercially available mammalian milk road protein, lipids contained in milk protein, ash, carbon water Compounds, dietary fiber, moisture, etc. ' = in addition to the appropriate components of these components - part or all of the active ingredient Xaa Pr of the present invention. Pr. It can also be the source of the beast or its concentrate, the hydrolyzate or the enzyme 320070 which makes the two to h...(especially Val Pr〇Pr〇: used abalone and 1ie pr〇Pro) 10 200902046 • Group, and obtained by the method of fermenting animal cheese protein AA+丄. The method of knowing or making the mammalian milk protein by casein such as 羊 goat milk, 干 马 、, goat milk, especially suitable for the use of bovine cheese protein. The protein of the demulsification pathway is limited in the case of proteolysis or fermentation, but it is preferably not particularly limited in weight for efficient protein/length. Sheep also produce protoplasts of animal milk, from 1 to 19 f for the above enzyme groups.

The Pn>I>r〇Xaa sequence, and the bovine, for example, includes a Xaa-containing enzyme group (X). ^^ Ρ Ρ 〇 〇 Xaa residue between the peptide enzyme group (X) is a protein containing active protease, 戋 爷 爷 具有 具有 具有 具有 具有 具有 , , , , , , , , , , , , 爷 爷Protease is a protease II such as gold and leucine amine-peptidase, and in a short time, and further: to make the hydrolysate efficiently, to see, to include these enzymes to ^ From the point of view of the U-segment reaction to the peptide sequence of the heart sequence = Ray: outside: it is better to cut off the enzyme. The isoelectric sputum is shown in the acidic region as the above-mentioned enzyme group or enzyme group (, Aspereil 丨w, & °, for example, rice noodle 麴 1 pg us 〇ryzae), etc. For the L group, the enzyme group obtained by cultivating the enzyme in an appropriate medium, etc., especially the yeast produced by the strain, and the sputum derived from the rice sputum The enzyme group indicated by the acid region is derived from the enzyme group of the rice sputum bacterium, '' can be sold in the market for 320070 11 200902046 - a' or MP (above, registration: P butyl, chemistry ( Stock system); Umamizyme (registered trademark, Amano

Enzyme(^)S); sternzyme Bu〇24 ^ pR〇HiDR〇xy trade name 'Jukou Chamber of Commerce Co., Ltd.);0rientase urgent moINDUSTRY (share) system; D_(four) (and recorded by 濑, Changshengsheng Chemical Co., Ltd. .

Fp (registered trademark, New Japan Chemical Co., Ltd.) is preferred. When the enzyme group is sold in the market, it is usually set as the best strip of the material, and the condition is changed by the method of the above casein hydrolyzate. In order to use the enzyme 罝 and reaction time, etc.), the heart: the amount of the enzyme group added to the above animal protein, such as Λ# . , ^ 尺, in the trough, to make the enzyme group / animal cheese protein ratio In 1/1000 or more, it should be in the curry to (four), especially in the 1 to 10, and more preferably in 1Μ. The reaction conditions to the ratio of (10) can be adapted to the manner in which the protein hydrolysate is used; In addition, the enzyme is reversed v=5f; 'especially for 15 hours k to 2 to 48 hours, preferably 7 is usually the end of the reaction, can be carried out by inactivating the enzyme, hang: in (four) Fu The enzyme is inactivated and the reaction is stopped. After the reaction of the prime reaction, the precipitate is centrifuged according to the needs of the environment. 320070 12 200902046 *. Removed or treated with various filters. In addition, the peptides obtained from the two cylinders or odor can be obtained as needed. . The bitterness component or the barium dioxide removal has a bitter taste by using activated carbon or a hydrophobic resin. , the removal of the knife, can be made in the solution, relative to the used road egg <11 if it can be obtained by the water %, and .... In an hour, it can be carried out by means of centrifugation or removal of activated carbon from the membrane, in such a manner as to obtain a package 4 = known method. The reaction solution can be directly added to the digestive substance and the physical food. In addition, in order to improve the mouth of the West, it is used as a function, and it can be used to reduce the V/W. The general use of the protein hydrolysate is also a form in which the powder is dried after being made into a powder. The content of the U-containing protein hydrolysate or its concentrate is based on the total amount of the veterinary protein hydrolysate or the i-concentrate VT peptide and the free amino acid, usually! More than one percent by weight! Up to 5% by weight. By making the content ratio i weight, a higher efficiency can be expected. In addition, the content ratio of the IleP P breakwater protein hydrolyzate contained in the above or its concentrate, 3 Pro Pro or the Val Pr〇Pr〇, is in the protein hydrolysate or its concentrate. And the total amount of the free amino acid is set to be 0.3% by weight or more, or the total amount of nePr〇Pr〇Ρπ> is g. 3% by weight or more, and both of them can be expected. In the case of IJe Pro Pro and Va]Pro Pr〇, when it is 3% by weight or more, higher efficacy can be expected. In addition, the active ingredient of the present invention, XaaPrQPf, can be derived from a strain belonging to the Lactobacillus species of Switzerland. Fermenting a raw material containing milk protein; 320070 13 200902046 *. The fermented product is obtained. : · The strain of acid rod _ bacteria ' is preferably used alone, but does not damage the present invention. Other lactic acid bacteria, etc. may be included in the range of desired effects. For the bacteria belonging to the Lactobacillus species of Switzerland, it is preferable to produce a large amount of proteases: for example: and / or VaiPr〇Pr〇. For example,

143 ?4?〇Τ&quot;1ηίη' S· AnaL 〇 984)) The value of 9〇 determined by the method of Yamam〇to et al., N. et al. J. Biochem. (1993) 114, 74〇) It is not a strain of 4〇〇 or more. For these suitable strains (10) strains (Tongshang filial piety workers...: for example, the Swiss Lactobacillus vegetable industry technology 卩元生命工工工所', Japan's Katsura City, Tsukuba City, East J Ding', 々九奶 (now In the case of the Industrial Technology and Technology Co., Ltd., the company is located in the city of Tsukuba, the city of Tsukuba, the center of the Tsukuba Center, and the second is the 305-8566. 1997. δ. Institute, registered number (10) C91 xian) (hereinafter referred to as the vegetable development research nine strains according to the patent procedures on the microbial deposit on the plant: this (10) 莽 is a genus, and this strain has been patented. The fermented material is contending with the raw material of the hole protein and containing the ingredients of Switzerland, which are added to the raw material containing milk protein, and are appropriately selected, each fermenting yeast, fermenting, and specializing in the fermentation. Fermentation conditions can be fermented to obtain 320070 14 200902046 ·. In this case, the hair obtained in this way can also be used as a powder by freeze drying, spray drying, etc. Bacillus species弁 is preferably used as a sufficiently high-activity bacterium. It is cultured before k仃 and is about 1个9/ml. The initial solid number is preferably 1〇5 to be contained by a strain belonging to the Lactobacillus species of Switzerland. Fermented product obtained by fermenting, for example, in order to make the taste good, preference, health food, etc. in the use of the functional food of the protein shell. = The strain belonging to the Lactobacillus species of Switzerland and the yeast 2:: The parent strain is not particularly limited. For example, the yeast is suitable for fermentation, and the yeast yeast such as sacch_yces cerevisiae is suitable for the purpose (10). Milk, just: For the raw material of milk protein, for example, animals such as rituals and goat's milk can be used; • de-waxed milk of processed milk of such milk, reduced milk = substance:; Milk, soy glutinous rice, condensed milk, etc., i biting cow m and other milk processing milk is better, and bovine yak ritual processing milk is especially good. ton of cow's milk solids concentration and helmet situation The concentration of the non-fat milk solids under the system:: general; = use, milk right, on the production and ^ 6 to 15 Amount of 2. 2 to 15% by weight of the above fermentation system is usually prepared by a static fermentation temperature of 25, for example, by 72 hours, preferably 2 2 W, preferably 30 to 45 t' fermentation time is 3 ', ', to 36 hours, in the lactic acid acidity of 5% to 320070 15 200902046 :: on: the method of stopping the fermentation to stop the fermentation. a from the fermented by the Lactobacillus species of Switzerland N (3) Pro Pro (I should be IIe Pr〇: g: converted to 10mg« is preferred, preferably in terms of dosage or intake in one case, the active ingredient of p preparation xaaProPr〇mp is ' Contains (and is Ile Pro Pro and/or Val Pro Pr〇) Right ^ Use lmg i 5g 'more preferably 3 calls to lg or so), can also be administered or ingested on the 1st. (4) During the period of material ailment and renal arteriosclerosis, it can be abused; bucket + E 1 , w and 卞 or photo pair ^ "the age of the person or animal taken, or the kidney failure of the person or animal Dangerousness, for example, 1 wi = ordering various adjustments from 7 days to 365 days. Active ingredient. 'The renal failure prophylactic agent of the present invention is the administration amount of the above-mentioned three peptides as a prophylactic agent for kidney failure Or the amount of intake, per ~μ and /ΓΓ, containing the active ingredient xaa pr〇pr〇 (preferably ^ 5 0 and or ValProPro) is generally 1〇_i〇g (preferably _ to foot and 3mg Use or ingestion to the lg or so. In the case of the person or animal at the time of the administration of the preventive agent for the renal failure on the 1st, or the age of the animal may be considered. The environment and the like are variously adjusted. For example, the renal failure of the animal may be generally more than one day, 320070 16 200902046 ·. It is preferably from 7 days to 365 days. The kidney spheroid of the present invention is sown - and renal failure Precautions: The disease and the inhibition of the arteriosclerosis of the lupus are good, and the method of ingesting or ingesting is based on the oral prescription. In the case of the renal magnetic agent of the present invention, and the preventive agent for renal failure: the disease and the inhibition of renal arteriosclerosis, the shape of the drug is administered (4) g, which is a capsule, a soft capsule, and a capsule. , hard two microcapsules, powder, granules, liquid agents, and the like. When the clothing is used as a medicine, for example, a pharmaceutically acceptable carrier, an adjuvant, a shape, a toxin, a stabilizer, a binder, a sputum agent, an anti-chemical agent, a preservative , anti-oxidation transfer: Bu buffer, increase the peak required by the unit dosage form for manufacturing. The slave of the present invention contains the inhibitor of arteriosclerosis of the H material of the present invention as an active ingredient, = inhibition of spheroid diseases and renal arteriosclerosis, and pre-second;:; functional foods such as health foods for specific efficacy. In order to obtain such an effective intake, if there is a daily, continuous, or intermittent long-term intake of food (for example, functional food) in the human condition, the amount of active ingredient Xaa W Pro per day is Or the amount of pr〇pr〇 and/or Va1ProPro is generally (four) to %, preferably _ to 5g, more preferably 3 to lg, depending on the number of intakes per i day, may cause food (eg The amount of intake per j times of functional foods is lower than the above amount. 320070 17 200902046 . When directly using the animal cheese protein hydrolysate or its concentrate containing the active ingredient Xaa Pro Pro, in human cases, the hydrolyzate or its concentrate is generally 1111 § to 1 〇〇g per day. Especially 1〇〇1^ to 1〇邑 left ^ is better. When directly using the fermented product containing the active ingredient Xaa Pr〇 pr〇 (4) = dry matter, in the case of humans, the fermented product; the dried knot of the east knot is dried, intraoperatively every! It is better to take i to i (10) g, especially 2 to ^. The food of the present invention (for example, the function + raw σ, AA * W - οι- » ι U j force is not particularly special during the ingestion period of the raw. The second is the long-term intake of J is good, but in order to obtain the above effects, for example, For more than 1 day, it should be between 7 and 365 days. Effective foods (for example, functional foods contain the Xaa Pro Pro containing the invention) (should be Val Pr〇Pr〇 and (5) work with ml heart and stomach The inhibition of arteriosclerosis, for example, the obtained animal cheese protein hydrolysate or its concentrate, or the fermented product containing the milk protein raw material, can be added to the various foods in a straight shape: In addition, the granules, 兮 艮 艮 艮 肀〇 肀〇 , 因 因 因 因 因 因 因 因 因 因 因 因 因 因 因 因 因 因 因 库 库 库 库 库 库 库 库 库 库 库 库 库 库 库 库 库 库 库For example, sugars, proteins, lipids, /, flavors, such as each of the mountains, "',, minerals, flavoring agents, spices, pigments, -," to improve the balance of nutrition or flavor. Open... For example, fermentation such as lactic acid bacteria beverage 320070 18 200902046 Σσ, various processed foods, dry powders, lozenges, capsules, granules, etc., further can be various beverages, yogurt, flowing food, jelly, candy, ret〇rt food, Ingot type candy, cookie, honey cake (casteua), bread, biscuit (^(6) cent), chocolate, etc. The present invention will be described in more detail below using examples, but the scope of the invention is not limited to the examples. (Example) f [Strong peptide synthesis example] The active ingredients of the present invention, lie Pro Pro and Val Pro Pro, were synthesized by the organic chemical synthesis method (Fmoc method) shown below. The synthesis system was synthesized using Shimadzu Corporation. The synthesis apparatus (PSSM-8 type) is carried out by a solid phase method. In the case of a solid phase carrier, a 2-Chlorotrityl type polybenzonitrile resin is used in combination with a ruthenium group. 50 mg (hereinafter referred to as Fmoc) resin which protects the amino group of valine acid (registered trademark SynProPep Resin, manufactured by Shimadzu Corporation). According to the above amino acid sequence, Fmoc-Ile which protects the amine group with an Fmoc group, Fmoc-Pro, and Fmoc-Val each 1 ΟΟμ ιοΙ, according to the usual method, according to the peptide sequence in order to obtain the peptide binding resin. Then suspend the peptide binding resin in 1 ml of the reaction solution 10 (10 vol% acetic acid, 10 vol% trifluoroethanol, 80 vol % dichloromethane), reacted at room temperature for 30 to 60 minutes, and the peptide was cleaved from the resin, and the reaction liquid was filtered through a glass filter. After removing the solvent of the filtrate under reduced pressure, immediately add 1 ml of the reaction liquid B ( 82.5% by volume of trifluoroacetic acid, 3 vol% of ethyl methyl sulfide 19 320070 200902046 (ethyl methyl sulfide), 5 vol% pure water, 5 vol% phenylmethyl sulphur _ (thioanisole), 2.5 vol. Dithiol, 2% by volume sulfur test, 'reacted at room temperature for 6 hours and the side chain protecting group was removed. 10 ml of anhydrous ether was added thereto to precipitate a peptide, which was centrifuged at 3000 rpm for 5 minutes for separation. The precipitate was washed with anhydrous diethyl ether several times, and then dried by blowing nitrogen gas. All of the unpurified synthetic peptides thus obtained were dissolved in 2 ml of a 0.1 N aqueous hydrochloric acid solution, and then purified by HPLC using a reverse column of C18 under the following conditions. Pump: Model L6200 intelligent pump (Intelligent pump), testing machine: UV absorption at 215 nm by model L4000 UV detector (Hitachi, Ltd.); column: Bondasphere 5pC18 (made by Waters - company); eluent: A The solution is 0.1 wt% / 〇 TFA aqueous solution, and the B solution contains 0.1 wt% butyl? Octaacetonitrile; (Β/Α+Β)χ100°/〇 : 0-40% (60 minutes); flow rate: 1 ml/min. The eluted fraction showing the maximum absorption was taken out in portions, and 5.7 mg and 6.5 mg of the desired synthetic peptides lie Pro Pro and Val Pro Pro were respectively obtained by drying and kneading. The purified peptide was analyzed from the N-terminus of the peptide by a fully automated protein primary structure analyzer (model PPSQ-10, manufactured by Shimadzu Corporation), and further, an amino acid analyzer (model 800 series, manufactured by JASCO Corporation) Analysis can confirm that the results are as designed. [Preparation example of animal milk protein hydrolyzate] lg of casein (manufactured by Nippon NZMP Co., Ltd.) derived from cow's milk was added to 99 g of distilled water adjusted to about 80 ° C and thoroughly stirred, and then 1 N sodium hydroxide was added (and The solution manufactured by Wako Pure Chemical Industries Co., Ltd. was pH 7.0, and the temperature was adjusted to 20 320070 200902046 to 20 ° C to prepare a matrix solution. In the dibasic solution, the ratio of the weight of the enzyme/luline protein is reduced: at least the metalloprotease, the 2 chymotrypsin, the neutral protease, the neutral protease η, and the leucine = peptidase The commercially available enzyme (registered trademark %: Jing" '5.. called ^ = eight" knife clock in the south press to treat the enzyme inactivated, get the road protein two broad solution solution. Then, make the resulting enzyme The decomposition product solution is dried by a spray dryer to prepare a powder. The analysis of the components contained in the powder of the lamp is carried out. The protein is determined by the XjeMahl method, and the amino acid is an amino acid analyzer. In the evening, the amount of amino acid is subtracted from the amount of the protein as the amount of the peptide. Further, the lipid is determined by an acid decomposition method, the ash is determined by direct ashing, and the water is heated by atmospheric pressure. In addition, the residual amount of each compound was subtracted from _% as the amount of carbohydrate. As a result, the amino acid was 35.8%, the S' peptide was 45.7 wt%, and the water content was 6.6% by weight. 0.2 weight. 7°, ash content 4.1% by weight, carbon hydrated The amount of the substance A is 7.6 wt%. <Measurement of the peptide-constituting amino acid> The powder prepared by the above-mentioned preparation is dissolved in an appropriate amount of distilled water, and analyzed by an automatic peptide analyzer (trade name: PPSQ) (manufactured by Shimadzu Corporation). In the powder, the amino acid was sequentially ordered from the N-terminal side. In addition, the automatic peptide analyzer did not detect the free amino acid. In addition, the total of the amino acid of the fifth residue was 12 〇pm. 〇1, the total of the amino acid of the sixth residue is 1〇〇pm〇l. Based on these results, it is found that the amino acid of the above-mentioned 320070 21 200902046 powder containing almost 2 residues of the peptide is Pr〇. 49.5%. Furthermore, the amine of the third residue is 29.8%. It is a win or a triple win. In addition, the ratio of the peptide is significantly increased to a higher ratio of the base acid to the peptide of Pro. The above powder contains a large amount of Xaa Pro

Pro, it is speculated that these peptides are resistant peptides to the enzyme decomposition by proteases in Iu^Jb 4 in the living body. _ &lt;Measurement of the peptide contained in the enzyme decomposition product> For the powder of the above-mentioned enzyme decomposition product, three kinds of chemically synthesized standard peptides are used, and the usual method of obtaining the powder is shown in Table 3 The amount of peptide. The results are shown in Table 1. [Table 1] Sequence of peptides Ser 3 Pro in Pro Pro Pro Pro Pro Pro --

Further, the amount of the peptide and the free amino acid in the solution in which the above powder was diluted with distilled water was 815 mg/ml, the amount of the peptide was 4 57 mg/ml, and the amount of Xaa Pro in the peptide was 514.5 pg, The ratio of Xaa Pr〇 to the total amount of the peptide in the powder and the free amino acid is 63% by weight, and further, since the amount of Xaa Pr〇Pr〇 in the peptide is 116.5 jug, The total amount of the peptide and the free amino acid in the powder, the ratio of Xaa ΡΓ〇Pro was confirmed to be 1.4% by weight. 320070 22 200902046 [Preparation example of CM4 fermented milk feed] The fermented milk obtained by fermenting a raw material containing milk protein by the CM4 strain was used to prepare an animal material containing the active ingredient XaaPr〇Pr〇 of the present invention. Dissolve the commercially available j skim milk powder with distilled water at a solid rate of 9% (w/w), and heat it at high temperature for 1Q5t and 1Q minutes at high (4), then cool to room temperature and inoculate 3% (v). /w) The CM4 strain fermentation broth (strain 5x1 〇, /ml) was fermented in a static state for 24 hours to obtain CM4 fermented milk. The obtained C Μ 4 fermented milk is subjected to 8 〇 t instant temperature sterilization (that is, the sterilization method of cooling immediately after heating to 8 〇t), and then obtained by drying (four) knots, powder, and. The obtained dry powder was mixed with a commercially available powdered product (trade name "CE_2", manufactured by Japan CLEA Co., Ltd.) at a mass ratio of 10:90 to prepare a solid feed, which was used as a CM4 fermented milk feed. Herein, MM Pro 34. lmg/kg derived from (10) fermented milk and 17.1 mg/kg in Pro Pro were contained. [Experiment 1 : Val Pro Pro (VPP, 欧欧时'm 丄, ) kidney spheroid disease and inhibition of renal dysfunction] Lemai In order to evaluate the three peptides Val Prn T&gt; Mmn,

Pro Pro (VPP) kidney spheroid and renal arteriosclerosis inhibition effect, 斟 铬 铬 M ^ ^ 于 于 于 于 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾 肾Frequency, 9 to 12, a group of 7-week-old wisi male rats (Japan SLC Co., Ltd.) enters the Ning Ning large free intake dissolved in lg / L concentration 320070 23 200902046 \ L-NAME (Sigma Drinking water, dissolved in L-NAME and VPP at concentrations of lg/L and 0.3g/L*, respectively, and dissolved in L-NAME at concentrations of lg/L and 0.5mg/L, respectively Drinking water for enalapril of angiotensin-converting enzyme (ACE) inhibitor. Here, the dosage of enalapril was set in such a manner that its ACE inhibitory activity was equal to VPP. The rats were sacrificed by exsanguination under diethyl ether anesthesia, and after removing the kidney, 10% neutral buffered fumarin solution was used. fixed. The tissue was sliced from the fixed kidney parallel to the short axis (including the tip of the kidney nipple). The tissue was subjected to paraffin embedding and then thinly cut by microtomy to 2.0 to 2.5 μm. Thickness. Two to five sections were made for each rat. The thinly sectioned sections were subjected to hematoxylin-eosin staining or periodic acid methenamine silver. Staining, also known as PAM staining. Histopathology is performed according to the method of Foglieni et al. (Chiara Foglieni, et al., &quot;Protective effect of EDTA preadministration on renal ischemia&quot; BMC Nephrology 2006, 7:5, [on line] , March 15, 2006, BioMed Central Ltd, http://www.biomedcentral.com/bmcnephrol/) 5 by pathologists unrelated to the trial without the name of the sample.禝本真, Lin Yuzao, and Tanaka Shouko, "The pathological organization of experimental animals" SOFT SIENCE, p. 380-396), the definition of spheroid lesions, under the microscope observation, It can be confirmed that the spheroids of atrophic lesions, spheroidal scum, spheroid hardening, and changes in the Bow's sac are identified as mitochondrial lesions. Atrophic lesions refer to the shrinkage of the spheroids due to atrophy of 24 320070 200902046 c The ratio of the size of the sac is changed, the microvessel cavity of the spheroid disappears and the state of the uterus is lost, and the spheroid hardening refers to a state in which the mesangial matrix can be confirmed (the angial is increased without accompanying the proliferation of collagen fibers, abalone) The sac == can confirm the presence of hypertrophy of epithelial cells or the intake of fat droplets or egg particles. In one slice as a whole, the frequency of occurrence of these spheroid lesions is scored as 〇 to 3, 廿The scores of the individual bodies of the five-seven-seven-seven-seven-story rulings. The scores of the individual bodies determined by the formula are statistically processed. The scores of the Fuxi 砰 砰 〇: physiology within 1 to 2 of the spheroid lesions The situation caused by sexual change. The section of Piai, the 'knife in the observation area to the lesion area of 1 to 2 spheroids, is a local and small range. ° Scoring 2: 2 to 4 virtual silk card was observed Range to 4 skeins The lesion area 'is a score of 3: when the lesion area of the spheroid is observed to be more than $, and it can be confirmed that it is a wide range. The area of the lesion refers to the whole slice of the work, and it can be confirmed within a certain range. There are multiple lesions. The thickness of the small arterial wall in the renal cortex was also evaluated by histopathological examination with spheroid disease. In the 』 =::::: two, all the arteries can be confirmed, and the large breast sputum is compared with the arteries at the same position. The fat thickness of the middle and small arterial wall is scored as 0 to 3 below. , decide each 320070 25 200902046, individual score. Rating 〇: 1 ^ 'In the case of the same fat, the whole blade is only confirmed to be equal to the normal rat or lightly identifiable. It can be confirmed that the medium or medium frequency can be confirmed by two degrees or a high frequency. Right 0 Γ 1 : Hypertrophy compared to normal atmosphere Score 2: Hypertrophy compared with normal rats Score 3: Hypertrophy compared with normal rats and only: shown in Figure 1 and Figure 2. As shown in Fig. 1, the small movements are compared to the clusters that are smeared because of the effect in giving VPP. The decrease in the thickness of the two fertilizers indicates that νρρ has a frequency of inhibition of renal arteriosclerosis, which is caused by two uptakes and the spheroidal disease is suppressed. Therefore, the kidney spheroids of νρρ are shown, π 士 』 because VPP is not only It is effective to inhibit the appearance of spheroid lesions while inhibiting the small and medium arterial wall. In addition, it was found that it was not possible to confirm the effect of renal artery P and spheroid lesion inhibition in enalapril, and it was not caused by inhibition of ACE. 8 The correct effect and [: test 2: lie Pn) PrG (IPP) kidney spheroid disease and kidney sclerosis inhibition effect] In order to evaluate the kidney spheroid, disease for the three peptides Ile Pro Pro (Ipp) As well as the inhibitory effect of renal arteriosclerosis, the kidney state test was performed. A 7-week-old male genus 320070 26 200902046 rats (Japan SLC Co., Ltd.) with a total of 3 groups of 9 to 12 groups was tested. After one week of domestication, the rats were allowed to ingest L-NAME (Sigma) drinking water at a concentration of lg/L for 8 weeks, at concentrations of lg/L and 0.3 g/L, respectively. Drinking water in which L-NAME and IPP are dissolved, and drinking water of L-NAME and enalapril which is an angiotensin-converting enzyme (ACE) inhibitor are dissolved at a concentration of lg/L and 0.5 mg/L, respectively. Here, the dosage of enalapril is set such that the ACE inhibitory activity is equal to the IPP. The rats were sacrificed by exsanguination under diethyl ether anesthesia, and the kidneys were removed and fixed with 10% neutral buffered formalin. The tissue piece was prepared by cutting from the fixed kidney parallel to the short axis to include the tip of the kidney nipple. The tissue was embedded in paraffin and then thinly cut by a sectioning method to a thickness of 2.0 to 2.5 μm. Two to five sections were made for each rat. The thinly sectioned sections were stained with hematoxylin and eosin or silver perhexylide tetraamine silver (sputum staining). Histopathological examination was performed according to the method of Foglieni et al. (Chiara Foglieni, et al. 5 &quot;Protective effect of EDTA preadministration on renal ischemia&quot; BMC Nephrology 2006, 7:5 ? [on line]5 March 15, 2006 BioMed Central Ltd, http://www.biomedcentral.com/bmcnephrol/), was performed by a pathologist who was not involved in the trial and examination without notifying the sample name. Under the observation of the microscope, it was confirmed that the atrophic lesion, the spheroidal scum, the spheroid hardening, the change of the Bowman's sac, etc. were identified as those having a globular lesion. The definition of various spheroid lesions is based on the writings of the essays (Sui Benzhen, Lin Yuzao, and Tanaka Shouko, "The Pathology of Experimental Animals" 320070 27 200902046 [as texel society, ρ3δ0 to 396), atrophic Lesion refers to: 4, the state of the sputum body contraction and the ratio of the size of the spheroid and the bursa of the sac, the scum of the spheroid refers to the disappearance of the microvascular cavity of the spheroid and the state of function = the state of the spheroid hardening Refers to the state in which the enhancement of the mesangial matrix is recognized and the hyperplasia of the meridian is recognized. The change in the Bowman's capsule refers to the fact that it can be confirmed, Tian Yue, the hypertrophy or the intake of fat droplets or protein particles: . To the slasher: the body scores the frequency of these spheroid lesions to 3, and determines the score of each body. Rating 〇: A situation caused by biochemicals in which the spheroid lesion is ί to 2 or less. The door "is physiologically changed and the lesion area of the spheroid at 1 to 2 is observed, which is a small and small range. Equine score 2: In the case of the lesions ', ν, ' in the range of 2 to 4 imaginary, the middle score is 3: when the disease of the spheroid is observed, it can be confirmed as a wide range (4) π ° is 5 Above, and here, the lesion area refers to the thickness of the small arterial wall in the renal pelvic motility of the renal medulla of the medulla, also: the extent of the sputum, and the existence of the pathological examination for the ping Z, /, 4 spheroidal lesions can be confirmed by the whole slice by the whole slice. The observation of the microscope will be almost the same position in the 1st specimen::::: compared with the normal thickness of the normal rat of the same age and The sputum of the small and medium arterial wall and the degree of renal wall arterial wall were scored as the following Q to 320070 28 200902046 3 The score of each body was determined. , the same fertilizer:: two in the whole of one slice, only confirmed that the light or light frequency can be confirmed with the normal rat, the medium or medium frequency can be confirmed to the degree or high frequency can be confirmed to § bisector 1 : Hypertrophy compared with normal rats. Score 2: Hypertrophy compared with normal rats. Score 3: Hypertrophy compared with normal rats. These spheroid lesions and renal hilar arteries The thickness of the wall fertilizer and the thickness of the medium 1 = wall thickness of the fertilizer, and will be evaluated in all three items;: the second upper sputum is determined to be morbid, based on the number of morbid and non-pathological rats. Comparison than the test. 2] As for the evaluation of rat kidney pathology, 7 L-NAME and ipp (" *p&lt;0.05 based on the results, turtle + ΤΧ) ϋ + A 上 not IPP also inhibits the spheroid: == It is found that it is possible to confirm the prevention of renal failure. 4. It can be confirmed that the silk material (4) (four) sees the inhibition of the kidney spheroid disease and renal arteriosclerosis of the present invention, 320070 29 200902046, even if the angiotensin-converting enzyme (ACE) cannot be confirmed. In the case of the inhibitory action of the inhibitor, it also shows the effectiveness, and therefore, it can be used as a preventive and/or therapeutic medicine for renal failure caused by ACE inhibitory activity, especially as a preventive agent. In addition, since the active ingredient is derived from a naturally occurring tripeptide of food or the like, a drug having a small side effect and high efficacy can be expected. Further, a functional food is provided by The kidney spheroid disease and the inhibitor of renal arteriosclerosis of the present invention are used or added to a beverage or a food, and can be taken safely and daily, and can be expected to prevent renal failure. References) • 1. Japanese Patent No. 2782142 2. J. Clin. Invest, 77, 1993-2000, 1986 3. Latest Medicine, 48: 1404 to 1409, 1993 4. J. Dairy Sci. 1995, 78 :777-783 5. J. Dairy Sci. 1995, 78:1253-1257 6. Am. J. Clin. Nutr. 1996, 64:767-771 [Simple description of the diagram] Figure 1 shows the test 1 Val Pro Pro (VPP) was used to test the effect of hypertrophy on the small arterial wall of rat kidney. A group of 11 L, NAME, L-NAME and enalapril were administered as a group of 9 L-NAME. The NAME and VPP dosing groups were tested for a group of 12, and the graphs were expressed as mean ± standard error. The comparison was performed by the Mann-Whitney test. The mark (*) in the figure indicates significant 30 320070 200902046 difference (p&lt; 0.05 Fig. 2 is a graph showing the results of the test for the inhibition of the appearance of rat kidney spheroid lesions of Val Pro Pro (VPP) of Test 1. The comparison was made by the Mann-Whitney test. The symbol (*) in the figure is shown. Indicates significant difference (ρ &lt; 0·05). [Main component symbol description] None 31 320070

Claims (1)

200902046 ' X. Patent application scope: _ 1. An inhibitor of kidney spheroid disease and renal arteriosclerosis, containing: Xaa Pro Pro as an active ingredient. 2. The inhibitor of kidney spheroid disease and renal artery hardening according to claim 1 of the patent scope, wherein Xaa Pr〇 Pr〇 is and/or I] e Pro Pro. .: A 24 patent range! Or (4) a spheroidal disease; an inhibitor of lunar arterialization, wherein the Μ h is derived from a mammalian protein hydrolysate or a concentrate thereof. 4"; the spheroidal disease of the kidney in the third paragraph of the patent scope and the renal arterial calculus casein hydrolysate is a fermented product obtained by fermenting the veterinary protein protein by sputum. 5. The suppression of the remaining, in which the body disease and the renal arteries are hard, apply for a specialized decomposition product. Inhibitors, kidney spheroid diseases, and enzymes of renal arteriosclerosis. 〃,, the enzyme derived from sputum is derived from rice noodles 麴 7 · If you apply for full-time inhibition of renal arteriosclerosis, χ, kidney spheroid disease and: protein raw materials by: Pr. It is derived from those who have the fermented product. , the bacterium of the bacterium of the bacterium of the bacterium of the genus Phytophthora. 8. In the case of the patent-scoped inhibitor, the corpse of the visceral spheroid disease and the renal arterial hard * is based on the Swiss Lactobacillus species. Switzerland 320070 32 200902046 : Lactobacillus CM4 strain (CCRC 910111). A preventive agent for renal failure, comprising an inhibitor of kidney spheroid disease and renal arteriosclerosis as an active ingredient of any one of claims VIII to 8. 10. A functional food&apos; includes a kidney spheroid disease and a suppression of renal arteriosclerosis according to the scope of patent application y*r-, a functional food as claimed in claim 10, 2. The function of preventing kidney failure. '', showing damage 32〇〇7〇33 200902046 Sequence Listing &lt;110> Calpis &lt;120> Kidney Failure Preventive Agent &lt;130&gt; 0P08032-1 &lt;150&gt; JP2007-81638 &lt;;151&gt; 2007-03-27 <160> 1 &lt;170&gt; Patent In version 3. 3 &lt;210&gt; 1 &lt;211> 4 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt; Recognition sequence of peptide &lt;220> &lt;221> mi sc feature &lt;222> (1).7(1) &lt;223> Xaa can be any natural amino acid &lt;220> &lt;221> mi Sc feature &lt;222> (4).7(4) &lt;223> Xaa can be any natural amino acid &lt;400> 1 Xaa Pro Pro Xaa 320070