Patent Application
20100022748
The present invention relates to an active ingredient having an action for preventing kidney glomerular diseases and renal arteriosclerosis, a prophylactic agent for renal failure containing the active ingredient, as well as functional foods containing the active ingredient, which functional food are expected to have a prophylactic effect on renal failure.
In addition, the present invention relates to a method for preventing kidney glomerular diseases and renal arteriosclerosis. Further, the present invention relates to a method for preventing renal failure.
Recently, the number of those who have a risk factor considered to cause heart diseases or renal diseases, such smoking, hypertension, hyperglycemia, and hyperlipidemia, have increased in some countries. There is an increasing trend in the number of patients suffering from heart failure or renal failure. In addition, these pathologic states are not limited to humans and the increasing trend in morbidity of such diseases are seen among animals that closely interact with humans, such as dogs, cats, other companion animals and pet animals.
An example of an effective drug for treating heart failure is an angiotensin converting enzyme inhibitor (ACEI), such as Enalapril, which inhibits an enzyme converting angiotensin I to angiotensin II having a hypertensive action (i.e. angiotensin converting enzyme; ACE), and has a hypotensive action. In addition, the antihypertensive drug reportedly improves the progression of renal damage at the same time as lowering blood pressure (J. Clin. Invest., 77, 1993-2000, 1986). Yet on the other hand, for heart failure induced by various primary diseases, the ACE inhibitor is not effective for all cases. Rather there is a risk of developing acute renal failure accompanying lowering blood pressure. It has been pointed out that careful administration is required (Saishin Igaku, 48:1404 to 1409, 1993), That is, the antihypertensive drug can be insufficient for the prophylaxis of heart failure but also could rather induce renal failure.
Renal failure generally refers to conditions where the kidney cannot filter blood to remove metabolic wastes in the blood successfully.
Renal failure is the pathologic state where renal functions are deteriorated by various renal diseases. Examples of the primary disease include diabetic nephropathy, chronic glomerulonephritis, nephrosclerosis, polycystic kidney, chronic pyelonephritis, rapidly progressive glomerulonephritis, SLE nephritis, glomerulosclerosis, and renal artery blood flow failure by renal arteriosclerosis. It has been reported that ACE inhibitors are effective against renal failure, which has drawn attention. Yet it has pointed out that the ACE inhibitors could cause acute renal failure and aggravation of renal damage. And thus it cannot be said that usefulness of these drugs is satisfactory (Saishin Igaku, 48:1404-1409, 1993). As described above, because renal failure is caused by various factors including hyperglycemia and drug damage, improving hypertension alone is not sufficient to prevent all renal failures originated from the above described primary disease. On the other hand, glomerular disease is a common lesion of renal failure and renal arteriosclerosis is a lesion associated with renal failure. Under such circumstances, a compound having a suppressive action for glomerular disease or a compound additionally having a suppressive action for renal arteriosclerosis can be said to be useful for prophylaxis and/or treatment of renal failure.
Meanwhile, it has been reported that a peptide originated from food materials, such as casein has the ACE inhibitory activity and such peptides are known to have a hypotensive action. However, whether or not the peptides have an action for preventing kidney glomerular diseases and an action for suppressing renal arteriosclerosis has not been directly demonstrated (Japanese Patent Publication No. 2782142, J Dairy Sci. 1995, 78:777-783, J. Dairy Sci. 1995, 78:1253-1257, Am. J. Clin. Nutr. 1996, 64:767-771). Additionally, as described above, since the prophylactic and therapeutic effect for renal failure mediated by ACE inhibition is limited, prophylaxis and treatment of the disease which are not dependent on the ACE inhibition are desired.
The present invention provides a compound or composition having a suppressive action against kidney glomerular disease and a suppressive action against renal arteriosclerosis independently of the ACE inhibitory activity.
In addition, the present invention provides a prophylactic agent for renal failure and functional foods expected to have a prophylactic effect on renal failure containing the above described compound or composition.
The present invention provides a method for suppressing kidney glomerular disease and a method for suppressing renal arteriosclerosis, which method includes administration of the above described compound or composition to a subject.
Accordingly, the present invention provides a method for preventing renal failure, which method includes administration of the above described compound or composition to a subject.
The present invention is also a use of the above described compound or composition in producing a pharmaceutical for suppressing kidney glomerular disease and for suppressing renal arteriosclerosis. In particular, the present invention is a use of the above described compound or composition in producing a pharmaceutical for preventing renal failure.
The present inventors have discovered that a tripeptide having a specific structure Xaa Pro Pro (wherein Xaa is any naturally occurring amino acid) has a suppressive action against kidney glomerular disease and suppressive action against renal arteriosclerosis, and have reached to invent a pharmaceutical and functional foods useful for prophylaxis of renal failure. Concrete contents of the present invention are as follows:
The present invention provides a suppressive agent against kidney glomerular disease and renal arteriosclerosis containing Xaa Pro Pro as an active ingredient.
the present invention also provides a prophylactic agent for renal failure having the above described suppressive action against kidney glomerular disease and renal arteriosclerosis.
In addition, the present invention provides functional foods containing the above described suppressive action against kidney glomerular disease and renal arteriosclerosis.
The present invention provides a method for suppressing kidney glomerular disease and renal arteriosclerosis, which method includes administration of Xaa Pro Pro or a composition containing Xaa Pro Pro to a subject.
In addition, the present invention provides a method for preventing renal failure, which method includes administration of Xaa Pro Pro or a composition containing Xaa Pro Pro to a subject.
The present invention is also a use of Xaa Pro Pro or a composition containing Xaa Pro Pro in producing a pharmaceutical for suppressing kidney glomerular disease and renal arteriosclerosis. In particular, the present invention is also a use of Xaa Pro Pro or a composition containing Xaa Pro Pro in producing a pharmaceutical for preventing renal failure
Preferably Xaa Pro Pro is Val Pro Pro and/or Ile Pro Pro.
In other embodiment of the present invention, the active ingredient Xaa Pro Pro is derived from an animal milk casein hydrolysate or a concentrate thereof.
In other further embodiment of the present invention, the active ingredient Xaa Pro Pro is originated from a fermented product obtained by fermenting a raw material containing a milk protein with a bacterium belonging to the species Lactobacillus helveticus.
Preferably the bacterium belonging to the species Lactobacillus helveticus is Lactobacillus helveticus CM4 strain (FERM BP-6060).
The agent for preventing kidney glomerular disease and renal arteriosclerosis according to the present invention comprises a tripeptide with a specific structure of Xaa Pro Pro as an active ingredient. The term “agent” herein is not restricted to a pharmaceutical but refers to a composition such as a pharmaceutical composition or food composition, or a compound such as a food additive. The term “agent for preventing kidney glomerular disease and renal arteriosclerosis” used in the present invention refers to a compound or composition having an action to suppress progression of kidney glomerular disease and renal arteriosclerosis lesions, an action to improve kidney glomerular disease and renal arteriosclerosis lesions, or a prophylactic effect on the onset of kidney glomerular disease and renal arteriosclerosis. In addition, the “glomerular disease” used in the present invention refers to a disease with lesions occurred in the glomerulus of the kidney, and histologically, refers to conditions where atrophy of the glomerulus, glomerulus deterioration, glomerulosclerosis, alterations in Bowman's capsule, or the like is observed. Concrete examples of the name of disease include minimal change nephrotic syndrome, focal glomerular sclerosis, endocapillary proliferative glomerulonephritis (so-called acute nephritis), IgA glomerulonephritis (mesangial proliferative glomerulonephritis), membranous nephropathy, membranoproliferative glomerulonephritis, and crescentic glomerulonephritis.
The Xaa in the active ingredient Xaa Pro Pro according to the present invention may be any naturally occurring amino acid. Concrete examples include Val Pro Pro (valine proline proline), lie Pro Pro (isoleucine proline proline), Ser Pro Pro (serine proline proline), and Leu Pro Pro (leucine proline proline). Preferred are Val Pro Pro and Ile Pro Pro with Val Pro Pro being more preferred. As the active ingredient, the agent may contain a combination of two or more of the tripeptides Xaa Pro Pro, as exemplified by a combination of Val Pro Pro and Ile Pro Pro.
The active ingredient Xaa Pro Pro may be an chemically synthesized tripeptide or naturally occurring tripeptide.
As an chemical synthetic method of tripeptide Xaa Pro Pro, a common method such as solid phase method (t-Boc method and Fmoc method) and liquid phase method can be employed. For instance, the peptide may be synthesized using an automatic peptide synthesizer such as a peptide synthesizer (PSSM-8type) manufactured by Shimadzu Corporation. With regard to reaction conditions for the peptide synthesis and the like, those skilled in the art can arbitrarily set appropriate reaction conditions and the like, based on their technological common knowledge depending on the synthesis method to be selected and desired tripeptide Xaa Pro Pro.
Alternatively, as the naturally occurring peptide, the tripeptide may be originated from an animal milk casein hydrolysate or a concentrate thereof, as well as may be originated from a fermented product obtained by fermenting a food material containing a protein with fungi or bacteria such as Aspergillus and Lactobacillus.
In cases where the animal milk casein hydrolysate or the concentrate thereof, or the fermented product obtained by fermenting the raw material containing the milk protein with the bacterium belonging to the species Lactobacillus helveticus, besides the tripeptide Xaa Pro Pro which is the active ingredient according to the present invention, free amino acids may be contained. Furthermore, in addition to the above described peptide and free amino acids, for example, lipids, ash, carbohydrates, dietary fibers, water and the like, all of which are normally contained in commercially available animal milk caseins or milk proteins may be contained. Additionally, as required, a part or all of the appropriate components among these may be taken out.
The active ingredient Xaa Pro Pro according to the present invention may be originated from an animal milk casein hydrolysate or a concentrate thereof obtained by a method of hydrolyzing an animal milk casein with a group of enzymes yielding Xaa Pro Pro, in particular Val Pro Pro and Ile Pro Pro and/or a method of fermenting animal milk with Aspergillus.
Examples of the animal milk casein include cow milk, horse milk, goat milk, and ewe milk. In particular, cow milk casein being preferably used.
A concentration of casein when the animal milk casein is hydrolyzed or fermented is not restricted but is preferably 1 to 19% by weight in order to efficiently produce the animal milk casein degraded product.
An example of the above mentioned enzyme group is preferably an enzyme group (X) including a peptidase capable of cleaving between Pro and Xaa residues at the carboxyl terminus of the Xaa Pro Pro Xaa sequence.
The enzyme group (X) is preferably a serine type proteinase having serine in the active center or a metal proteinase having a metal in the active center. Examples of the metal proteinase include neutral protease I, neutral protease II and leucyl aminopeptidase. It is preferred that at least one type of these metal proteinases be additionally included in that the desired hydrolysates can be efficiently obtained in a short time, and even in a one-step reaction. Additionally, the peptidase capable of cleaving the above described Pro Xaa sequence is preferably an enzyme showing the isoelectric point in the acidic region.
An example of the above described enzyme group or enzyme group (X) is an enzyme group originated from Aspergillus such as Aspergillus oryzae. Such an enzyme group includes an enzyme group which fungus cells were cultured in an appropriate culture medium and an enzyme produced was extracted with water. Among the enzyme groups originated from Aspergillus oryzae, an enzyme group showing the isoelectric point in the acidic region is in particular preferred.
As the enzyme group originated from Aspergillus oryzae, commercially available products can be used. Examples thereof are Sumizyme FP, LP or MP (all registered trademark, manufactured by SHINNIHON CHEMICALS Corporation), Umamizyme (registered trademark, manufactured by Amano Enzyme Inc.), Sternzyme B11024, PROHIDROXY AMPL (all trade names, manufactured by Higuchi Shoukai Co., Ltd.), Orientase ONS (registered trademark, manufactured by Hankyu Bioindustry CO., Ltd.), Denatyme AP (registered trademark, manufactured by Nagase ChemteX Corporation) with Sumizyme FP(registered trademark, SHINNIHON CHEMICALS Corporation) being preferably used.
When these commercially available enzyme groups are used, optimum conditions are usually pre-determined. Yet, conditions such as an amount of enzyme to be used and reaction time can be appropriately altered depending on an enzyme group to be used such that the above described casein hydrolysate can be obtained.
An amount of the enzyme group to be added when the above described animal milk casein is hydrolyzed is, for instance, such that the weight ratio of enzyme group/animal milk casein in a aqueous solution in which the animal milk casein is dissolved is not less than 1/1000, preferably 1/1000 to 1/10, especially preferably 1/100 to 1/10, further preferably 1/40 to 1/10.
The reaction conditions can be appropriately selected depending on an enzyme group to be used such that an intended casein hydrolysate is obtained. A temperature is usually 25 to 60° C., preferably 45 to 55° C. And pH is 3 to 10, preferably 5 to 9, particularly preferably 5 to 8. The enzyme reaction time is usually 2 to 48 hours, preferably 7 to 15 hours.
Termination of the enzyme reaction can be achieved by inactivating the enzyme. Usually the enzyme is inactivated at 60 to 110° C. to stop the reaction.
After the enzyme reaction is terminated, as required, it is preferred to remove precipitates by centrifugation removal and various filter treatments.
Additionally, as required, a peptide having bitterness and/or smell can be removed from the obtained hydrolysate. The removal of such a bitterness component and/or smell component can be done using activated charcoals, hydrophobic resins or the like. For instance, the removal can be carried out by adding the activated charcoals to the obtained hydrolysate in 1 to 20% by weight based on an amount of casein used and by allowing to react the resulting mixture for 1 to 10 hours. Removal of the activated charcoals can be carried out by a known method such as centrifugation and membrane treatment process.
The thus obtained reaction mixture containing the animal milk casein hydrolysate or the concentrate thereof can be added as is to a liquid product such as beverage to be use in the functional foods. In order to improve versatility of the animal milk casein hydrolysate, the above described reaction mixture can be, after concentrated, dried to yield a powdered form.
The content ratio of Xaa Pro Pro contained in the animal milk casein hydrolysate or the concentrate thereof is usually not less than 1% by weight, preferably 1 to 5% by weight based on a total amount of peptides and free amino acids in the animal milk casein hydrolysate or the concentrate thereof. By having the content ratio of not less than 1% by weight, higher actions are expected. Additionally, either when the content ratio of each of Ile Pro Pro or Val Pro Pro contained in the animal milk casein hydrolysate or the concentrate thereof is not less than 0.3% by weight based on a total amount of peptides and free amino acids in the animal milk casein hydrolysate or the concentrate thereof, or when the content ration of a total of Ile Pro Pro and Val Pro Pro is not less than 0.3% by weight, high effects are expected. Further, when 0.3% by weight or more of each of lie Pro Pro and Val Pro Pro is contained, higher effects are expected.
The active ingredient Xaa Pro Pro according to the present invention may be also originated from a fermented product obtained by fermenting a raw material containing a milk protein with a bacterium belonging to the species Lactobacillus helveticus. Although the bacterium belonging to the species Lactobacillus helveticus is preferably used individually in the fermentation, other Lactobacillus or the like may be included/contained to the extent that the desired effects according to the present invention is not adversely affected.
As the bacterium belonging to the species Lactobacillus helveticus, a proteinase-producing bacterium capable of highly producing Ile Pro Pro and/or Val Pro Pro is preferred. For instance, a bacterium strain showing a U/OD590 value measured in accordance with a method by Yamamoto et al. (Yamamoto, N. et al. J. Biochem.) (1993) 114, 740), which is based on a method by Twining et al. (Twining, S. Anal. Biochem.) 143 3410 (1984)) of not less than 400 is preferred.
An example of such a preferred bacterium strain is Lactobacillus helveticus CM4 strain (Ministry of International Trade and Industry, National Institute of Bioscience and Human-Technology, 1-1-3 Higashi, Tsukuba, Ibaraki, Japan, Zip code 305, (currently, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, AIST, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, Zip code 305-8566), Accession No.: FERM BP-6060, Date of deposit: Aug. 15, 1997) (hereinafter referred to as CM4 strain). CM4 strain has been registered under the above described accession number under the Budapest Treaty concerning an international approval of microorganism depository on patent procedures, and already patented.
The fermented product obtained by fermenting a raw material containing milk protein with the bacterium belonging to the species Lactobacillus helveticus can be obtained by adding a fermented milk starter containing the bacterium strain belonging to the species Lactobacillus helveticus to a raw material containing the milk protein and by fermenting the mixture on appropriately selected conditions such as fermentation temperature.
A concentrate of the thus obtained fermented product or the like may be powdered with freeze drying, spray drying, or the like and may be used as a powder.
The bacterium belonging to the species Lactobacillus helveticus is preferably used as a starter with sufficiently high activities, which is preliminarily pre-cultured. The number of the bacteria at the beginning is preferably about 105 to 109 cells/ml.
The fermented product obtained by fermenting a raw material containing a milk protein with the bacterium belonging to the species Lactobacillus helveticus, for example, when used for functional foods such as Foods for Specified Health Uses, can be also fermented with yeast in conjunction with the above described bacterium strain belonging to the species Lactobacillus helveticus in order to have better flavor and better palatability. An example of the strain of yeast is preferably the genus Saccharomyces such as Saccharomyces cerevisiae but not restricted thereto. The content ratio of the yeast can be appropriately selected depending on a purpose.
Examples of the raw material containing the milk protein include animal milk such as cow milk, horse milk, ewe milk, goat milk, vegetable milk such as soybean milk, and processed milk of these milk including skimmed milk, reconstituted milk, powdered milk, and condensed milk. Cow milk, soybean milk and processed milk of these milk is preferred and cow milk or processed milk thereof is in particular preferred.
Although the solid content concentration of the milk is not particularly restricted, when the skimmed milk is for instance used, the solid content concentration of non-fat milk is usually about 3 to 15% by weight and preferably 6 to 15% by weight for better productivity.
The above described fermentation is usually carried out by standing or stirring fermentation, for example, by a method of fermenting at a temperature of 25 to 45° C., preferably 30 to 45° C., with a fermentation time of 3 to 72 hours, preferably 12 to 36 hours, wherein the fermentation is terminated when lactic acid acidity reaches 1.5% or more.
The content ratio of Xaa Pro Pro, preferably Ile Pro Pro and/or Val Pro Pro in the fermented product obtained by fermenting the raw material containing the milk protein with the bacterium belonging to the species Lactobacillus helveticus is preferably 10 mg or more, preferably 15 mg or more, in terms of 100 g of a freeze dried product of the fermented product.
As for a dose or intake per day of the agent for preventing kidney glomerular disease and renal arteriosclerosis according to the present invention, in the case of human, usually a dose of about 10 μg to 10 g. preferably 1 mg to 5 g, further preferably 3 mg to 1 g of the active ingredient Xaa Pro Pro, preferably Val Pro Pro and/or Ile Pro Pro may be administrated or taken dividedly in several times in a day.
A period of administration or intake of the agent for preventing kidney glomerular disease and renal arteriosclerosis can be adjusted in many ways in view of age of humans or animals to be administrated or to ingest and/or environments for a risk factor against kidney glomerular disease and renal arteriosclerosis of the humans or animals. The period can be for example usually not less than one day, preferably 7 days to 365 days.
The prophylactic agent for renal failure according to the present invention comprises the above described tripeptide as an active ingredient.
As for a dose or intake per day of the prophylactic agent for renal failure according to the present invention, in the case of human, usually a dose of about 10 μg to 10 g. preferably 1 mg to 5 g, further preferably 3 mg to 1 g of the active ingredient Xaa Pro Pro, preferably Val Pro Pro and/or Ile Pro Pro may be administrated or taken dividedly in several times in a day.
A period of administration or intake of the prophylactic agent for renal failure can be adjusted in many ways in view of age of human or animals to be administrated or ingest and/or environments for a risk factor against renal failure of the human or animals. The period can be for example usually 1 day or more, preferably 7 days to 365 days.
A method for administrating or taking in the agent for preventing kidney glomerular disease and renal arteriosclerosis, as well as a prophylactic agent for renal failure according to the present invention is preferably oral administration.
A form of the agent for preventing kidney glomerular disease and renal arteriosclerosis, as well as a prophylactic agent for renal failure according to the present invention, when used as a pharmaceutical, can be a form of formulation for oral administration. Examples thereof include tablet, pill, hard capsules, soft capsules, microcapsule, powder, pellet, and solution.
In the case of preparing as a pharmaceutical, for example, pharmaceutically acceptable carrier, adjuvant, vehicle, excipient, antiseptics, stabilizing agent, binder, pH adjusting agent, buffer agent, thickener, gelatinizer, preservatives, anti-oxidant or the like can be, as required, used. The pharmaceutical can be produced in a unit dose form required pharmaceutical formulation administration generally accepted.
The foods according to the present invention comprises the agent for preventing kidney glomerular disease and renal arteriosclerosis according to the present invention as an active ingredient, and can be, for example, functional foods, such as Foods for Specified Health Uses, which has efficacies such as the suppressive action against kidney glomerular disease and renal arteriosclerosis and prophylaxis for renal failure.
An intake to attain such efficacies, for example, in the light of the fact that the functional foods are daily, continuously or intermittently taken for a long period of time, in the case of human, is usually about 10 μg to 10 g per day in terms of an amount of the active ingredient Xaa Pro Pro or an amount of Val Pro Pro and/or Ile Pro Pro, preferably 1 mg to 5 g, more preferably 3 mg to 1 g. Depending on the number of intake per day, an intake of the foods such as the functional foods per once can be reduced further to a lower amount than the above described amount.
In cases where the animal milk casein hydrolysate or the concentrate containing the active ingredient Xaa Pro Pro is used as is, in the case of human, the hydrolysate or the concentrate thereof is preferably taken usually in 1 mg to 100 g per day, particularly in about 100 mg to 10 g.
In cases where the freeze dried product of the fermented product containing the active ingredient Xaa Pro Pro is used as is, in the case of human, usually 1 to 100 g per day in terms of a dried amount of the freeze dried product of the fermented product, in particular, about 2 to 50 g is preferably taken.
A period of the intake of the foods according to the present invention, for example the functional foods, is not particularly restricted. The intake for a long period of time is preferred. In order to attain the above described efficacies, for example, the period can be usually not less than one day, preferably 7 days to 365 days.
The foods according to the present invention, for example the functional foods, comprise the agent for preventing kidney glomerular disease and renal arteriosclerosis containing the active ingredient Xaa Pro Pro, preferably Val Pro Pro and/or Ile Pro Pro. For instance, the animal milk casein hydrolysate or the concentrate thereof and the fermented product, all of which were obtained in the above described manner, can be added to a variety of foods as they are or in the form of powder or granule. If necessary, a fermented product by Lactobacillus other than Lactobacillus helveticus, other ingredients used in foods including sugars, proteins, lipids, vitamins, minerals, or flavors, or additives such as various carbohydrates, lipids, vitamins, minerals, sweeteners, flavors, coloring agents, texture improving agents or a mixture thereof may be added to improve nutritional balance, flavor, and/or the like.
The foods according to the present invention, for example the functional foods, can be any form of solids, gels, or liquids. Examples thereof include fermented dairy products such as lactobacillus beverages, a variety of processed foods and beverages, dried powders, tablets, capsules, and granule. Further, additional examples can be various beverage, yogurts, fluid diets, jerrys, candies, retort-packed foods, tablet candies, cookies, Castella (or Japanese sponge cake), breads, biscuits, and chocolates.
The present invention will now be described in detail by way of examples thereof, but the scope of the present invention is by no means limited by the examples.
The active ingredient according to the present invention Ile Pro Pro and Val Pro Pro were synthesized by the following chemical synthesizing method (Fmoc method). The synthesis was carried out by solid phase method using an automatic peptide synthesizer (PSSM-8type) manufactured by Shimadzu Corporation.
As a solid phase carrier, 50 mg of a resin (SynProPep Resin®, manufactured by Shimadzu Corporation) which is a 2-chlorotrityl(2-Chlorotrityl)type polystyrene resin in which proline protected its amino group with a fluorenymethyloxy carbonyl group (hereinafter referred to as Fmoc for short) is bound, was used. According to the above described amino acid sequence, Fmoc-Ile, Fmoc-Pro, and Fmoc-Val (100 μmol each) which are protected their amino group with the Fmoc group were reacted in the order of the peptide sequence to obtain a peptide-bound resin in accordance with a conventional method.
This peptide-bound resin was then suspended in 1 ml of a reaction mixture A (10 volume % acetic acid, 10 volume % trifluoroethanol, 80 volume % dichloromethane), allowed to react at room temperature for 30 to 60 minutes. Thereafter the peptides were separated from the resin and the reaction mixture A was filtered with a glass filter. Immediately after the solvent in the filtrate was removed under reduced pressure, 1 ml of reaction mixture B (82.5 volume % trifluoroacetic acid, 3 volume % ethyl methyl sulfide, 5 volume % purified water, 5 volume % thioanisole, 2.5 volume % ethanedithiol, 2 volume % thiophenol) was added and the mixture was allowed to react at room temperature for six hours to uncouple the side chain protecting group. To the resultant 10 ml of anhydrous ether was added to precipitate the peptide, and centrifuged at 3000 revolutions for five minutes to separate. The precipitate was washed with anhydrous ether was several times and then dried by spraying nitrogen gas. The entire volume of the thus obtained unpurified synthetic peptide was dissolved in 2 ml of 0.1N hydrochloric acid aqueous solution and then HPLC using a C18 reverse phase column was carried out in accordance with the conditions below.
Pump: Type L6200 intelligent pump (Hitachi, Ltd); detector: Type L4000UV detector (Hitachi, Ltd) for detection of ultra-violet absorption at 215 nm; column: Micro Bondashere5 μC18 (Waters Corp.); eluting solutions: solution A; 0.1% by weight TFA aqueous solution, solution B; acetonitrile with 0.1% by weight TFA aqueous solution; (B/A+B)×100(%): 0→40% (60 minutes); and flow rate: 1 ml/minute. The eluted fraction showing the maximum absorbance was collected and then freeze-dried to obtain the intended synthetic peptide Ile Pro Pro and Val Pro Pro (5.7 mg and 6.5 mg, respectively). The purified peptide was analyzed using an automatic protein primary structure analyzer (Type PPSQ-10, manufactured by Shimadzu Corporation) from the N-terminal of the peptide. The purified peptide was further analyzed an amino acid analyzer (Type 800 series, manufactured by JASCO corporation) to confirm that the peptide was the one as designed.
To Casein originated from cow milk (Nippon NZMP Ltd.) (1 g) 99 g of distilled water adjusted to about 80° C. was added and the mixture was stirred well. Subsequently, 1N sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) solution was added. The mixture was then adjusted to pH 7.0 and a temperature of 20° C. to prepare a substrate solution.
To the obtained substrate solution a commercially available enzyme (registered trademark “Sumizyme FP”, manufactured by SHINNIHON CHEMICALS Corporation) was added, which was derived from Aspergillus oryzae and contains at least metal protease, serine protease, neutral protease I, neutral protease II and leucyl aminopeptidase such that the enzyme/casein weight ratio of 1/25 was attained. The mixture was allowed to react at 50° C. for 14 hours. Subsequently, the enzymes were inactivated by autoclave at 110° C. for ten minutes, thereby obtaining a casein enzymolytic product solution. Then, the obtained enzymolytic product solution was dried by spray drying to prepare a powder.
Components contained in the obtained powder were analyzed. Proteins were determined by Kjeldahi method and amino acids were measured by an amino acid analyzer. In addition, a difference after subtracting an amount of the amino acids from an amount of the proteins was defined as an amount of peptides. Further, lipids, ash content and water were determined by acid decomposition method, direct ashing method, and drying method by heating under atmospheric pressure, respectively. The remainder after subtracting the amount of each component from 100% was defined as an amount of carbohydrates. The results showed that the powder contained 35.8% by weight amino acids, 45.7% by weight peptides, 6.6% by weight water contents, 0.2% by weight lipids, 4.1% by weight ash contents and 7.6% by weight carbohydrates.
The powder prepared above was dissolved in an appropriate amount of distilled water and analyzed with an automatic peptide analyzer (trade name PPSQ-10 manufactured by Shimadzu Corporation) to check in what order amino acid is located from the N-terminal in the powder. The automatic peptide analyzer does not detect any free amino acids.
The total amount of the amino acid located at the 5th residue position was 120 pmol and the total amount of the amino acid located at the 6th residue position was 100 pmol. Based on these results, most peptides in the above described powder were found to be dipetptides or tripeptides. Additionally, a proportion of the peptides having Pro as the amino acid at the 2nd residue position markedly increased to 49.5%. A proportion of the peptides having Pro as the amino acid at the 3rd residue position was as high as 29.8%.
Hence the above described powder contains many tripeptides of Xaa Pro Pro. Thus these peptides were presumably a peptide highly resistant to enzymatic degradation actions of proteases in living bodies.
For the above described powder of the enzymolytic product, amounts of the tripeptides shown in Table 1 contained in the powder was determined, in accordance with a conventional method, using various chemically synthesized standard peptides. Results are shown in Table 1.
The amount of peptides and free amino acids in a solution in which the above described powder was dissolved and diluted in distilled water was 8.15 mg/ml, and an amount of the peptides was 4.57 mg/ml, an amount of Xaa Pro in the peptides was 514.5 μg. The proportion of Xaa Pro based on a total amount of the peptides and free amino acids in the powder was therefore 6.3% by weight.
Further, an amount of Xaa Pro Pro in the peptide was 116.5 μg and it was thus confirmed a proportion of Xaa Pro Pro based on a total amount of the peptides and free amino acids in the powder was 1.4% by weight.
Using fermented milk obtained by fermenting a raw material containing a milk protein with the CM4 strain, animal feeds containing the active ingredient Xaa Pro Pro according to the present invention was prepared.
Commercially available powdered non-fat milk was dissolved in distilled water to the solid content of 9% (w/w) and then autoclaved at 105° C. for ten minutes to sterilize under heating at high temperature. After cooled to room temperature, the resulting solution was inoculated with a CM4 strain starter fermentation solution (the number of the bacteria: 5×108/ml) at 3% (v/w) and left to stand at 37° C. for 24 hours to yield CM4 fermented milk.
The obtained CM4 fermented milk was sterilized at a reaching temperature of 80° C. and then freeze-dried to yield the powder. The obtained freeze dried powder was mixed with commercially available powdered feeds (trade name “CE-2”, manufactured by CLEA Japan, Inc.) at the mass ratio of 10:90. The mixture was formed into solid feeds to obtain CM4 fermented milk feeds. The feeds contained 34.1 mg/kg of Val Pro Pro and 17.1 mg/kg of lie Pro Pro, both of which peptides were originated from the CM4 fermented milk.
In order to evaluate the effect of the tripeptide Val Pro Pro (VPP) to suppress kidney glomerular disease and renal arteriosclerosis, a test on the frequency of occurrence of renal glomerular lesions and the degree of thickening of the small and middle renal artrey wall was carried out.
The test was carried out using three groups of male Wistar rats of seven weeks old (Japan SLC, Inc.), each of which group consist of 9 to 12 rats. After acclimation for a week, the animals received: water with L-NAME (manufactured by Sigma) being dissolved at a concentration of 1 g/L; water with L-NAME and VPP being dissolved at a concentration of 1 g/L and 0.3 g/L respectively; or water with L-NAME and angiotensin converting enzyme (ACE) inhibitor, Enalapril at a concentration of 1 g/L and 0.5 mg/L respectively, ad libitum for eight weeks. The dose of Enalapril was determined such that its activity to inhibit ACE was equivalent to VPP's.
The rats were sacrificed by exsanguination under diethylether anesthesia. The kidney was enucleated and then fixed with 10% neutral buffered formalin solution. Tissue samples were prepared from the fixed kidney by excising in parallel with the minor axis such that the tip of renal papilla was included. The specimen were embedded in paraffin and cut into thin sections with a thickness of 2.0 to 2.5 μm using a microtome. Two to five sections per rat were prepared. The thin sliced sections were subjected to hematoxylin eosin staining or periodic acid methenamine silver staining (PAM staining). In accordance with a method by Foglieni et al. (Chiara Foglieni, et al., “Protective effect of EDTA preadministration on renal ischemia” BMC Nephrology 2006, 7:5, [online], Mar. 15, 2006, BioMed Central Ltd, http://www.biomedcentral.com/bmcnephrol/), a histopathological examination was, with keeping the name of each sample concealed, carried out by a pathologist who was not involved in the test.
Under a microscopic observation, the glomerulus that atrophic lesions, deterioration of the glomerulus, glomerulosclerosis, alteration of Bowman's capsule or the like was observed was defined as the glomerulus with lesions, in accordance with the definition of the glomerular lesions described in a book by Enomoto et al. (edited by Makoto Enomoto, Yuzo Hayashi, and Hisako Tanaka, “Pathological tissues of experimental animals”, Softscience, pages 380-396). The atrophic lesion refers to conditions where the size ratio between the glomerulus and Bowman's capsule is altered due to glomerular atrophy. The deterioration of the glomerulus refers to conditions where the blood capillary cavity of the glomerulus is disappeared and glomerular functions are lost. The glomerulosclerosis refers to conditions where increased mesangial matrix was observed without accompanying hyperplasia of collagen fiber. The alteration of Bowman's capsule refers to conditions where hypertrophy of epithelial cells and uptake of lipid droplets and/or protein particles are observed. In one whole section, the frequency of occurrence of these glomerular lesions was scored from 0 to 3, and a score was determined for each individual rat. The thus determined score for each individual rat was subjected to statistical analysis and a score for each group was determined.
Score 0: A case where one or two glomerular lesions owing to physiologic alterations were observed.
Score 1: A case where one or two focal regions in the glomerulus were observed, which regions were a limited and small range.
Score 2: A case where two to four focal regions in the glomerulus were observed, which regions were a middle sized range.
Score 3: A case where five or more focal regions in the glomerulus were observed in a wide range.
The focal regions herein indicate that a plural of lesions are, on the basis of pathological findings, observed in a certain area in one whole section.
The thickening of the small-, and mid-sized artery wall existing in the renal cortex was also evaluated by a tissue pathological test in the same manner as the observation of the glomerular lesions. Under a microscopic observation, all arteries observed in one while section were compared with arteries observed at the virtually same location in the specimen from a normal rat of the same age in weeks. The thickness of the small- and mid-sized artery wall was scored from 0 to 3 as described below and a score for each individual rat was determined. The determined score for each individual rat was subjected to statistical analysis and a score for each group was determined.
Score 0: A case where only the thickening equal to the normal rat in one whole section was observed.
Score 1: A case where the thickening was observed in a mild degree or at low frequency, compared with the normal rat.
Score 2: A case where the thickening was observed in a moderate degree or at moderate frequency, compared with the normal rat.
Score 3: A case where the thickening was observed a high degree or at high frequency, compared with the normal rat.
The obtained results are shown in
The tripeptide Ile Pro Pro (IPP) was also tested for renal pathologic state, in order to evaluate its effect to suppress kidney glomerular disease and renal arteriosclerosis.
The test was carried out using three groups of male Wistar rats of seven weeks old (Japan SLC, Inc.), each of which group consist of 9 to 12 rats. After habituation breeding for a week, drinking water with L-NAME (manufactured by Sigma) being dissolved at a concentration of 1 g/L; drinking water with L-NAME and IPP being dissolved at a concentration of 1 g/L and 0.3 g/L respectively; drinking water with L-NAME and angiotensin converting enzyme (ACE) inhibitor, Enalapril at a concentration of 1 g/L and 0.5 mg/L respectively, were supplied ad libitum for eight weeks. The dose of Enalapril was determined such that its activity to inhibit ACE was equivalent to the activity of IPP to inhibit ACE.
The rats were sacrificed by exsanguination under diethylether anesthesia.
The kidney was enucleated and then fixed with 10% neutral buffered formalin solution. Tissue samples were prepared from the fixed kidney by excising in parallel with the minor axis such that the tip of renal papilla was included. The specimen were embedded in paraffin and cut into thin sections with a thickness of 2.0 to 2.5 μm using a microtome. Two to five sections per rat were prepared.
The thin sliced sections were subjected to hematoxylin eosin staining or periodic acid methenamine silver staining (PAM staining). In accordance with a method by Foglieni et al. (Chiara Foglieni, et al., “Protective effect of EDTA preadministration on renal ischemia” BMC Nephrology 2006, 7:5, [online], Mar. 15, 2006, BioMed Central Ltd, http://www.biomedcentral.com/bmcnephrol/), a histopathological examination was, with keeping the name of each sample concealed, carried out by a pathologist who was not involved in the test.
Under a microscopic observation, the glomerulus that atrophic lesions, deterioration of the glomerulus, glomerulosclerosis, alteration of Bowman's capsule or the like was observed was defined as the glomerulus with lesions, in accordance with the definition of the glomerular lesions described in a book by Enomoto et al. (edited by Makoto Enomoto, Yuzo Hayashi, and Hisako Tanaka, “Pathological tissues of experimental animals”, Softscience, pages 380-396). The atrophic lesion refers to conditions where the size ratio between the glomerulus and Bowman's capsule is altered due to glomerular atrophy. The deterioration of the glomerulus refers to conditions where the blood capillary cavity of the glomerulus is disappeared and glomerular functions are lost. The glomerulosclerosis refers to conditions where increased mesangial matrix was observed without accompanying hyperplasia of collagen fiber. The alteration of Bowman's capsule refers to conditions where hypertrophy of epithelial cells and uptake of lipid droplets and/or protein particles are observed. In one whole section, the frequency of occurrence of these glomerular lesions was scored from 0 to 3, and a score was determined for each individual rat.
Score 0: A case where one or two glomerular lesions owing to physiologic alterations were observed.
Score 1: A case where one or two focal regions in the glomerulus were observed, which regions were a limited and small range.
Score 2: A case where two to four focal regions in the glomerulus were observed, which regions were a middle sized range.
Score 3: A case where not less than five focal regions in the glomerulus were observed in a wide range.
The focal regions herein indicate that a plural of lesions are, on the basis of pathological findings, observed in a certain area in one whole section.
The thickening of the renal artery wall at the hilum existing in the renal medulla and the thickening of the small and middle artery wall existing in the renal cortex were also evaluated by a tissue pathological test in the same manner as the observation of the glomerular lesions. Under a microscopic observation, all arteries observed in one whole section were compared with arteries observed at the virtually same location in the speciman from a normal rat of the same age in weeks. The thickening of the renal artery wall at the hilum and the thickening of the small- and mid-sized artery wall were scored from 0 to 3 as described below and a score for each individual rat was determined. The determined score for each individual rat was subjected to statistical analysis and a score for each group was determined.
Score 0: A case where only the thickening equal to the normal rat in one whole section was observed.
Score 1: A case where the thickening was observed in a mild degree or at low frequency, compared with the normal rat.
Score 2: A case where the thickening was observed in a moderate degree or at moderate frequency, compared with the normal rat.
Score 3: A case where the thickening was observed a high degree or at high frequency, compared with the normal rat.
These lesions of the glomerulus, the thickening of the renal artery wall at the hilum and the thickening of the small- and mid-sized artrey wall were individually scored. A rat with the score of not less than 1 in all three items was defined as one having the pathologic state. Using the number of the rats with and without the pathologic state, comparisons by likelihood-ratio test were made.
These results revealed that IPP also suppressed the onset of the pathologic state of the glomerulus and the renal artery, and thus IPP is effective on the prophylaxis of renal failure. In addition, like in Test 1, Enalapril herein did not exhibit such an effect. It was hence confirmed that the effect was not dependent on the ACE inhibition.
Since the agent for preventing kidney glomerular diseases and renal arteriosclerosis according to the present invention shows effectiveness, even when angiotensin converting enzyme (ACE) inhibitors do not exhibit the suppressive action, the agent is of great use as a pharmaceutical for the prophylaxis and/or treatment of renal failure, in particular as a prophylactic agent, which agent is not dependent on the ACE inhibitory activity. In addition, the active ingredient is a naturally occurring tripeptide originated from foods and the like, it is expected that the pharmaceutical with high efficacy and with little concern for side effects is provided.
Furthermore, by using or adding the agent for preventing kidney glomerular diseases and renal arteriosclerosis according to the present invention in foods or beverages, the functional foods expected to have the prophylactic effect for renal failure are provided.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-81638 | Mar 2007 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2008/055073 | Mar 2008 | US |
| Child | 12567628 | US |
