KR20190080379A - Composition for preventing or treating Kidney Disease Comprising Morin - Google Patents

Abstract

The present invention relates to a composition for preventing or treating kidney diseases comprising morin as an active ingredient. The present invention also relates to a food and feed compositions for preventing or treating kidney diseases comprising morin as an active ingredient. According to the present invention, morin or a morin hydrate restores a survival rate, the weight, the kidney weight, and water consumption of a chronic kidney disease (CKD) animal model, significantly mitigates a serum biochemical parameter of a CKD mouse, and restores a histopathologic change in the shape and the segment of a kidney. Moreover, the morin or the morin hydrate restores expression of a marker related to proinflammatory properties and fiberization to a normal state. Accordingly, the morin or the morin hydrate can be used as an effective natural treatment in treatment or prevention of CKD.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating renal disease,

The present invention relates to a pharmaceutical composition for preventing or treating renal disease, which comprises maurine as an active ingredient.

The present invention also relates to a food composition and a feed composition for preventing or ameliorating a renal disease, which comprises mauren as an active ingredient.

Chronic kidney disease (CKD) is a leading cause of death in Asia and the United States. There are currently no effective treatments available to treat this disease other than current treatment strategies that rely on blood pressure control with renin-angiotensin system blockade. Such current therapeutic strategies only serve to delay the onset of end-stage renal disease, which may be associated with serious side effects. Approximately 200 million people worldwide suffer from CKD. In the United States, African Americans (AAs) have a fourfold increased risk compared to non-Hispanic whites, with the highest rates in low-income countries in Asia and sub-Saharan Africa. In sub-Saharan Africa each year, more than 500,000 people develop end-stage renal disease (CKD 5), with the majority of these patients leading to premature death. CKD's medical costs and economic burdens are enormous and can not be sustained in advanced Western countries.

The most common cause of CKD is diabetes, which accounts for about 50%, followed by hypertension (25%) including glomerulonephritis and polycystic kidney disease. In addition, other risk factors are due to the accumulation of different toxicants and metabolites, which can contribute to the increased mortality of CKD. The most commonly used definition for CKD disease is based on the glomerular filtration rate (GFR) (> 15 ml / min, stage G5), which is used for CKD diagnosis for more than 3 months. GFR reduction indicates an ongoing increase in the risk of death. Most clinical studies also use albuminuria and proteinuria, the overwhelming majority of urinary proteins, closely related to ESRD and mortality rates. Sclerosis of glomerulus and interstitial fibrosis are common histopathological and structural features of CKD. Tubulointerstitial fibrosis is closely related to kidney function and shows a common complex structural change in the kidney, including matrix metallo-proteinase (MMP) and collagen production by epithelial cells and activated myofibroblasts . Fibrosis is a reactive process that occurs primarily in response to epithelial damage and is almost always accompanied by inflammation, which is manifested by cytokine expression and increased accumulation of macrophages and inflammatory cells. Research on fibrosis has greatly improved over the past few years and has provided new therapeutic targets.

Currently, there is no proper treatment for most CKD. Kidney sepsis, renal toxic drugs, and urinary obstruction (urinary obstruction). Steroids and immunosuppressants are less likely to stop or reverse kidney disease, but have no effect on the treatment of kidney disease in patients with diabetes and hypertension.

 Accordingly, the present inventors have developed novel therapeutic candidates for chronic kidney disease using a well known flavonoid, i.e., a morin hydrate (MH).

In the present invention, a CKD mouse model was developed by administration of adenine (AD) via oral gavage, which increased the levels of serum creatinine, BUN, albumin and increased the expression level of pro-inflammatory markers . In addition, increased expression of cathepsin D and MMP was confirmed by histopathological and Western blot analysis.

In addition, it was confirmed that pre-treatment and post-treatment of MH in AD-induced CKD mouse model resulted in reduction of renal fibrosis by decreasing serum biochemical parameters and expression levels of cathepsin and MMP. Based on this, we proposed a new therapeutic agent, maureen hydrate, which can reduce renal fibrosis and slow the progression of CKD.

Accordingly, an object of the present invention is to provide a method for preparing a compound of formula (I), which is effective for the prophylactic and / or therapeutic treatment of chronic (chronic) inflammatory diseases comprising morin, 2- (2,4- dihydroxyphenyl) -3,5,7- trihydroxychromen- And to provide a pharmaceutical composition for preventing or treating renal disease.

Another object of the present invention is to provide a pharmaceutical composition containing morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, a salt thereof or morin hydrate And to provide a food composition and a feed composition for preventing or ameliorating chronic renal disease.

In order to achieve the above object, the present invention provides a pharmaceutical composition containing morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, a salt thereof or morin hydrate as an active ingredient Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of chronic kidney disease.

In addition, Morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate activates AKT expression, and the resistance factor Myd88 And NF-kB mediated inflammatory response.

The present invention also relates to a pharmaceutical composition for the treatment of kidney diseases comprising morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, a salt thereof or morin hydrate as an active ingredient, Preferably a food composition for preventing or improving chronic kidney disease.

Further, the present invention relates to a pharmaceutical composition for treating a kidney disease comprising morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, a salt thereof or morin hydrate as an active ingredient, Thereby providing a feed composition for preventing or improving chronic kidney disease.

According to the present invention, the morin or maureen hydrate regains the survival rate, body weight, kidney weight and water consumption of CKD animal models, significantly improves serum biochemical parameters of CKD mice, Restore the change of science. In addition, the morin or maureen hydrate of the present invention restores the expression of proinflammatory and fibrosis-related markers to normal.

Accordingly, it is expected that the present invention can be used as a therapeutic agent for natural products effective for the treatment or prevention of chronic kidney disease.

Figure 1 shows the survival rate and weight change of animal models.
(A) Diagram showing the survival rate of animals treated with adenine, maureen hydrate and adenine + maureen hydrate at the indicated concentrations after 15 days of adenine treatment.
(B) After 21 days of treatment with adenine, survival rates of animals treated with adenine, maureen hydrate and adenine + maureen hydrate are shown.
(C) After 15 days of treatment with adenine, weight change of animals after treatment with adenine, maureen hydrate and adenine + maureen hydrate is shown in percentage.
(D) Treatment of Adenine After 21 days, adenine, maureen hydrate and adenine + maureen hydrate are shown as a percentage change in animal weight after treatment.
Figure 2 shows the relative kidney weight and rate of change of water consumption in CKD mice.
(A) Adenine, maureen hydrate and adenine + maureen hydrate at the indicated concentrations after 15 days of treatment with adenine.
(B) Adenine, maureen hydrate, and adenine + maureen hydrate after 21 days of adenine treatment.
(C) After 15 days of adenine treatment, it represents the percentage of water consumed by animals after treatment with adenine, maize hydrate and adenine + maize hydrate.
(D) Adenine, maureen hydrate and adenine + maureen hydrate after 21 days of adenine treatment.
Data are presented as mean ± SD (n = 8). Values with different letters (ad) are significantly different from each other (p <0.05).
Figure 3 shows the change in serum biochemical parameters of CKD mice after treatment with maureen hydrate.
(A) Changes in serum albumin levels in mice after treatment with adenine, maureen hydrate and adenine + maureen hydrate at the indicated concentrations after 15 days of adenine treatment.
(B) the amount of serum BUN in the mouse.
(C) changes in the serum creatinine level in the mouse.
(D) Changes in serum albumin levels in mice after adenine, maureen hydrate and adenine + maureen hydrate treatment 21 days after adenine treatment.
(E) shows the change in serum BUN of the mouse.
(F) changes in serum creatinine levels in mice.
Data are presented as mean ± SD (n = 8). Values with different letters (ad) are significantly different from each other (p <0.05).
Fig. 4 is a diagram showing a change in the shape of the kidney. Fig.
(A) Shows mouse kidney morphology after treatment with adenine, maureen hydrate and adenine + maureen hydrate at the indicated concentrations after 15 days of adenine treatment.
(B) After 21 days of adenine treatment, it shows the shape of mouse kidney after treatment with adenine, maureen hydrate and adenine + maureen hydrate.
Figure 5 shows the histopathological analysis results of H & E-stained kidney sections (original magnification, 200 x) of each group after continuous experimental treatment.
(A) After 15 days of adenine treatment, adenine, maureen hydrate and adenine + maureen hydrate were treated at the indicated concentrations, indicating patterns associated with pathological tissue damage, swelling, inflammation and fibrosis.
(B) After 21 days of adenine treatment, adenine, morin hydrate and adenine + morin hydrate treated groups exhibit pathological tissue damage, swelling, inflammation and fibrosis.
FIG. 6 is a graph showing a preventive effect on the expression of mouse kidney protein by continuous treatment of the maize hydrate. FIG.
(A) Western blot analysis was performed using specific proinflammatory antibodies against cox-2 and (B) MCP-1. Relative expression was analyzed by densitometry analysis using ImageJ software at mouse kidney after treatment with maureen hydrate.
(C) MMP-9, and (D) cathepsin D were used to perform Western blot analysis. Relative expression was analyzed by density gauge analysis using ImageJ software at mouse kidney after treatment with maureen hydrate. beta -actin was used as an internal control. Data are presented as mean ± SD (n = 8). Values with different letters (ad) are significantly different from each other (p <0.05).
FIG. 7 shows the effect of treatment with the maize hydrate for expression of the kidney protein. FIG.
(A) Western blot analysis was performed using specific proinflammatory antibodies against cox-2 and (B) MCP-1, and relative expression was analyzed by densitometric analysis using Mouse Image Strength .
(C) Western blot analysis was performed using antibodies directed against fibrosis to MMP-9 and (D) cathepsin D. Relative expression was analyzed by densitometric analysis using ImageJ software at mouse kidney after treatment with maureen hydrate, and β-actin was used as an internal control. Data are presented as mean ± SD (n = 8). Values with different letters (ae) are significantly different from each other (p <0.05).
Fig. 8 is a graph showing a preventive and curative effect on the kidney protein of the rats by the case of the maize
(A & B) MMP-2, relative expression was analyzed by densitometry using ImageJ software on mouse kidney after treatment with morin hydrate.
Data are presented as mean ± SD (n = 8). Values with different letters (ad) are significantly different from each other (p <0.05).

Hereinafter, the present invention will be described in detail.

The present invention relates to the prophylactic or therapeutic treatment of nephropathy comprising morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate as an active ingredient, A pharmaceutical composition for therapeutic use is provided.

Morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate (3,5,7,2 ', 4'- pentahydroxyflavone) ) May be extracted from fruit, leaves, etc. of Maclura pomifera (Osage orange), Maclura tinctoria (old fustic), Morus alba (Mulberry) or Psidium guajava (common guava) And does not limit the type of plant.

The morin of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like. Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, But are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, halides, halides, halides, halides, halides, halides, But are not limited to, lactose, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene Sulfonates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, and the like, as well as sulfonates such as benzyl sulfonate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, -Sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method. For example, a morpholine derivative is dissolved in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., , Or may be prepared by drying, or the solvent and excess acid may be distilled off under reduced pressure, followed by drying and crystallization in an organic solvent.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

Further, the present invention includes all of the above-mentioned morin and pharmaceutically acceptable salts thereof, as well as solvates, optical isomers, hydrates and the like, which can be prepared therefrom.

In addition, the above-mentioned maize or maize hydrate can be synthesized chemically or biologically, and the synthesis method thereof is not limited.

In the present invention, the morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate can be used for the treatment or prevention of a renal disease have.

The kidney disease may be an acute kidney disease or a chronic kidney disease. The renal disease may include diabetic nephropathy, hypertensive nephropathy, glomerulonephritis, pyelonephritis, interstitial nephritis, lupus nephritis, polycystic kidney disease, renal failure, and the like

Particularly, the morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate of the present invention is useful as a prophylactic or therapeutic agent for chronic renal diseases Can be used.

Chronic kidney disease (CKD) is a condition in which proteinuria develops continuously, evidence of kidney damage such as hematuria, or kidney function is continuously decreased for more than 3 months. It is accompanied by symptoms of anemia and hypertension, and the child has a slow development. The child is suffering from anemia, diarrhea, skin irritation and bad breath (bad breath of ammonia). It can be accompanied by a symptom that is worse and worse.

Chronic renal failure refers to a condition in which a kidney is damaged for more than 3 months or a decrease in renal function is continuously observed, and generally refers to a case where the kidney functions to 50% or less of the normal kidney. In such a case, kidney does not perform the function of filtering the waste materials, so that the level of creatinine and blood urine nitrogen (BUN) in plasma is increased, and protein including albumin is detected in urine. In the case of chronic renal failure, a portion of the kidney is not functioning properly, so that the normally functioning kidney is overloaded and the normally functioning part is continuously damaged, resulting in chronic renal failure. Therefore, dialysis or kidney transplantation may finally be necessary. Chronic renal failure is a concept included in chronic kidney disease.

In addition, the above-mentioned chronic renal disease includes not only the disease but also various complications derived from chronic kidney disease.

As used herein, the term "prevention" refers to any act that inhibits a kidney disease or delays the onset of a kidney disease by administration of the pharmaceutical composition. The term "treatment" refers to any action that alters or alleviates symptoms of a renal disease by administration of the pharmaceutical composition.

In the present invention, the morin or maureen hydrates are useful for restoring survival rate, body weight, kidney weight and water consumption of CKD animal models, significantly improving serum biochemical parameters of CKD mice, and histopathological changes in kidney morphology and sections . In addition, the morin or maureen hydrate of the present invention restores the expression of proinflammatory and fibrosis-related markers to normal.

In the present invention, the pharmaceutical composition may be used in a method of preventing or treating a kidney disease, and specifically, the method of preventing or treating a kidney disease may include administration to a subject in which a kidney disease is expected to occur or to be developed .

The term "administration" of the present invention means introducing the composition into a subject in an appropriate manner.

The term "individual" of the present invention means all animals such as rats, mice, livestock and the like, including humans that have developed or may develop renal disease. Specific examples include, but are not limited to, mammals including humans .

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is determined by the kind and severity of the subject, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. For example, the composition may be administered as an active ingredient at a dose of 0.01 to 500 mg / kg per day, specifically 10 to 100 mg / kg, and the administration may be administered once a day or divided into several times . In addition, the pharmaceutical composition of the present invention may contain from 0.001 to 50% by weight of the maize or maize hydrate of the present invention, based on the total weight of the composition.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by a person skilled in the art.

The pharmaceutical composition for preventing or treating renal disease of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredient. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have. Specifically, when formulating, it can be prepared by using diluents or excipients such as fillers, weights, binders, humectants, disintegrants, surfactants and the like commonly used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be determined depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition may be used alone, for improving, alleviating, treating or preventing chronic renal failure, or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers.

The present invention relates to a method for the prevention of kidney diseases comprising morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, a salt thereof or morin hydrate as an active ingredient Or improving food composition.

The term "improvement" of the present invention means all the actions for improving or alleviating renal disease upon administration of the composition.

The term "food" of the present invention is intended to encompass all kinds of foods, such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams, Vitamin complex, health functional food, and health food, all of which include foods in a conventional sense.

The term "functional food" as used herein means the same term as "food for special health use" (FoSHU). In addition to nutrition, It means food. Here, the term "function (surname)" means that the structure and function of the human body have a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the formulations of the foods can also be produced without restrictions as long as they are formulations recognized as food. The composition for food of the present invention can be manufactured in various forms, and unlike general drugs, it has advantages of being free from side effects that may occur when a food is used as a raw material for a long period of time, and is excellent in portability, Can be ingested as an adjuvant to promote the effect of preventing or ameliorating renal disease.

The health food refers to a food having an active health promotion or promotion effect compared with a general food, and a health supplement food refers to a food for health assistance. In some cases, the terms health functional foods, health foods, and health supplements may be used interchangeably.

Since the food composition of the present invention can be routinely ingested, it can be very usefully used because it can be expected to have a high effect on preventing or improving kidney disease.

The food composition may further comprise a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier conventionally used in the art can be used.

In addition, the food composition may contain additional components that are commonly used in food compositions and can improve odor, taste, visual appearance, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like.

In addition, the food composition may contain at least one kind selected from the group consisting of preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate), disinfectants (such as bleaching powder and highly bleached white powder, sodium hypochlorite), antioxidants (butylhydroxyanisole (BHA) (Sodium nitrite), bleach (sodium sulfite), seasoning (sodium MSG glutamate, etc.), sweeteners (dicin, cyclamate, saccharin, etc.), coloring agents , Sodium, etc.), perfume (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate), emulsifiers, thickeners (foams), encapsulating agents, gum bases, foam inhibitors, solvents, And may include food additives. The additives may be selected and used in appropriate amounts depending on the type of food.

The morin or salt thereof of the present invention, or the maize hydrate can be used as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the food composition of the present invention may be added in an amount of not more than 50 parts by weight, specifically not more than 20 parts by weight, based on the food or beverage, when the food or drink is prepared. However, in case of long-term ingestion for health and hygiene purposes, the active ingredient may be contained in an amount not exceeding the above range and there is no problem in terms of safety.

As an example of the food composition of the present invention, it can be used as a health beverage composition. In this case, various flavors or natural carbohydrates can be added as an additional ingredient like ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjuster, stabilizer, Alcohols or carbonating agents, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

The food composition of the present invention may be contained at various weight percentages as long as it can exhibit a preventive or ameliorative effect on kidney diseases. Specifically, the food composition of the present invention may contain the morin or its salt or the monohydrate of the present invention in an amount of 0.00001 to 100 wt. %, Or 0.01 to 80% by weight.

The present invention also relates to a pharmaceutical composition for the treatment or prophylaxis of kidney diseases comprising morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, a salt thereof or morin hydrate Thereby providing a feed composition for prevention or remediation.

The term "livestock" refers to domestic animals, such as mammals, poultry, and the like. In the present invention, the mammal may be, for example, a dog, a cat, a cow, a pig, a goat, a sheep or a horse. Examples of the poultry include a chicken, a duck and a turkey.

The feed composition may include general basic feeds fed according to the type of livestock. These basic feeds are known in the art and are commercially available depending on the type of livestock, so that any person skilled in the art will be able to manufacture or purchase them very easily.

Specifically, the basic feed may include some or all of a starch-containing substance, a protein-containing substance, a fat-containing substance, a vitamin-containing substance, an inorganic substance-containing substance, an antioxidant substance, an antibiotic,

The starch-containing material may be included in the feedstuff of the rabbit as long as it can sustain the growth of the animal. Such starch-containing material is generally obtained from corn, soybean, wheat, sorghum, barley, oats and the like. Examples of suitable starch-containing substances include corn flour or powder, oat flour or powder, bean flour or powder, wheat flour or powder. Suitable suitable starch containing materials are oat flour, corn flour, wheat flour, soy flour, and the like. The concentration of such a starch-containing substance is not particularly limited as long as it can effectively maintain the growth of the animal. Generally, the starch-containing material may be included in the basic feed in a range of about 30 to 80% by weight.

Protein-containing materials can also be included in animal feed as long as they can sustain the growth of the animal. For economic reasons, protein-containing substances such as fish meal and soybean powder have been used. Others include soy protein concentrate, blood meal, plasma protein, skim milk dry product, milk protein concentrate, corn gluten powder, wheat gluten Powder, yeast, sunflower seed powder and the like. Preferred protein-containing substances are fish meal, blood meal, plasma protein, soybean powder and the like. As long as the protein-containing material can effectively maintain the growth of the animal, its concentration in the animal feed is not important. Generally, the protein-containing material can be included in the basic feed in a range of about 10 to 50% by weight.

The fat-containing material includes, but is not limited to, lard, tallow, soybean oil, lecithin, coconut oil and the like. The concentration of the fat-containing substance is not limited as long as it can maintain the growth of the animal, and it can be included in the basic feed in the range of about 2 to 20 wt%.

The base feed can also contain water-soluble, fat-soluble vitamins and trace minerals. Examples of the vitamins include vitamin A, vitamin D, vitamin E, vitamin K, riboflavin, pantothenic acid, niacin, vitamin B12, folic acid, biotin and vitamin C. Copper, zinc, iodine, selenium, manganese, iron, cobalt and the like can be used as a trace amount of an inorganic substance. Here, "trace" means that the amount of these ingredients used in the livestock base feed is substantially less than the amount of the other ingredients. Generally, when a small amount of vitamin or mineral is included in the basic feed, it may be contained in an amount of about 0.0001 to 5% by weight based on the total weight of the composition.

In addition, the base feed may include antioxidants such as BHT (Butylated Hydroxytoluene), BHA (Butylated Hydroxyanisole), vitamin E and ascorbic acid, and these antioxidants may be contained in a very small amount of about 0.0001 to 1% by weight.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

Experimental Example

Experimental Example  1: Animal model

All animal experiments conducted during the study are subject to approval by the Laboratory Animal Care Committee and Daegu University. All of the techniques were performed in accordance with the guidelines for the management and use of laboratory animals published by the National Institutes of Health. Male BALB / c mice (initial 5 to 6 weeks old, initially about 25 g) were obtained from Oriental Biotechnology (Korea) and freeze-fed standard pelleted feeds and tap water under light and dark conditions at 22 ° C and 30% relative humidity for 12 hours.

Experimental Example  2: Experimental design

After a one week acclimation period, mice (n = 56) were randomly divided into 7 equal groups and treated for 6 consecutive weeks. The first group continued to receive the same diet without treatment until the end of the study (control group). Group 2 received AD (150 mg / kg body weight) for 21 days by oral gavage. Group 3 and Group 4 received AD for 15 days and MH for 20 mg / kg and 40 mg / kg, respectively, for 1 week by oral gavage. Group 5 received only MH (40 mg / kg) via oral gavage. In group 6 and 7, MH was administered at 20 mg / kg and 40 mg / kg, respectively, via oral gavage administration 21 days after AD treatment. Capacity of AD and MH was standardized based on the effect. The mice were weighed weekly for the duration of the experiment and the total water consumption was measured daily. After the indicated treatment period, all animals were sacrificed by sacvical dislocation. Whole blood was collected from the tail vein or abdominal aorta with EDTA-tube (Soyagreentec co., Korea), and serum was separated by centrifugation at 2500 g for 15 minutes at 4 ° C and stored at -80 ° C for further analysis. The kidneys were removed, blotted with filter paper and weighed. A small piece of kidney was soaked in 4% formalin for subsequent histopathological examination. The remaining kidneys were frozen at -80 ° C for biochemical and molecular analysis and western blotting. The kidneys were homogenized with cold RIPA buffer to obtain a 10 mg / ml homogenate. The kidney homogenate was centrifuged at 15,000 g for 10 min at 4 ° C and the supernatant obtained was used to measure various indicators.

Experimental Example  3: Biochemical method

Serum was separated by centrifugation (2500 g, 15 min) and stored at -80 ° C for further analysis. Normal serum biochemical characterization was performed using commercially available ELISA kits for various renal pathology markers such as creatinine, BUN and albumin in all mice. Biochemical amounts were measured and expressed as g / dL or mg / dL, and measurements were performed according to the method provided by the diagnostic kit (BioVision, Inc., Milipitas, CA, USA).

Experimental Example  4: Pathological analysis

The renal tissue was fixed in 4% buffered formalin, treated with a stepwise volume of ethyl alcohol, embedded in paraffin wax, cut to a thickness of 4-5 μm and evaluated for inflammation, fibrosis, basement membrane atrophy and coronary swelling Hematoxylin and eosin (HE). Histological evaluation was performed under an optical microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan).

Experimental Example  5: Western Blot  analysis

Mouse kidney tissue lysates were prepared with radioimmunoprecipitation assay RIPA buffer (Sigma-Aldrich), homogenized and centrifuged at 15,000 g for 10 min at 4 ° C. Protein samples were prepared from each kidney tissue sample in each group and quantified by the Bradford method.

For Western blot analysis, an equal volume of protein lysate (20 μg per lane) was resuspended in 5 × sample buffer (50 mM Tris, 2% SDS, 10% glycerol, 1% DTT and 0.1% Bromophenol blue) , Heated at 95 ° C for 5 minutes, and then proteins were separated by 12% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the separated proteins were electroplated to a polyvinyldenefluoride (PVDF) membrane (Roche Diagnostics, Indianapolis, IN, USA). The primary antibody was then probed on the membrane and then the secondary antibody conjugated to horseradish peroxidase (HRP) was touched and subsequently ECL (enhanced chemiluminescence, ECL) was performed according to the recommended procedure (Amersham Pharmacia, Piscataway, NJ, USA) .

Experimental Example  6: Gelatin Zymography  (Gelatin Zymography )

Enzyme activity of MMP-2 was determined by gelatinization. Briefly, all groups of kidney tissue homogenates were mixed with non-reducing sample buffer and electrophoresed on a 10% polyacrylamide gel containing 0.1% (w / v) gelatin. The gel was washed with wash buffer containing 2.5% Triton X-100 and incubated for 24 hours at 37 C in incubation buffer (50 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl2, 0.02% triton X100) Lt; / RTI &gt; The gel was stained with 0.5% (w / v) Coomassie Brilliant Blue in 30% (v / v) methanol and 10% (v / v)

Experimental Example  7: Drug and chemical reagents

Adenine (AD) and morin hydrate (MH) were obtained from Sigma (St. Louis, Mo., USA). Anti-cathepsin D, anti-MMP-9, anti-MCP-1, anti-cox-2 and anti-beta-actin and HRP-conjugated anti-rabbit immunoglobulins were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.

Example

Example  1: in the AD-induced CKD model Of MH Survival rate Synergistic effect  Confirm

Previous studies have shown that inflammation induced by oxidative stress is closely related to the onset of kidney as fibrosis progresses. Therefore, renal inflammation and fibrosis induced by oxidative stress should be considered as potential targets for the prevention and treatment of CKD. The use of natural products such as flavonoid MH can be a safe, effective and inexpensive option to protect kidneys from aggressive factors and prevent dialysis or transplantation from progressing to kidney failure, the only option. Dialysis and transplantation are costly and difficult to use in developing countries.

To assess the effect of MH on physiological disorders in animal survival and AD-induced CKD models, survival rates and mortality were calculated in each group of animals after treatment of the indicated doses. As a result, 100% survival rate was observed in the control and MH (40 mg / kg body weight) treated mice. The mortality rate on day 21 of AD treated animals was 100%. On the other hand, high doses of MH treatment group on days 15 and 21 after AD pretreatment showed a survival rate of almost 60% (FIGS. 1A and B). Furthermore, a significant reduction in body weight and kidney weight was observed in AD treated mice. Water intake also increased significantly. As a result of treatment with MH for 15 days after AD treatment and 21 days after AD treatment, weight and height of kidney were significantly increased compared with mice treated with AD alone. Water intake was significantly decreased after 7 days of MH treatment (Fig. 1 C &D; Figs. 2A, B, C & D).

Example  2: Serum parameters of AD-induced CKD model Of MH  Check the effect

Next, we observed serum biochemical parameters associated with kidney function. In AD-treated mice, creatinine, BUN (blood urea nitrogen) and albumin were significantly increased in the serum. However, administration of MH after 15 and 21 days of AD pretreatment significantly improves all indicators (Figures 3A, B, C, D, E & F) compared to mice treated with AD alone.

The general appearance of mice was also objectively judged to be improved by MH, especially at high doses (40 mg / kg body weight). The kidneys of control group and MH - treated group were normal. However, the elongation of the AD treatment group was light and shrunk with a rough surface. The kidneys treated with AD + MH for 7 days after 15 and 21 days of AD exposure significantly improved the kidneys of mice treated with AD alone (Figs. 4A & B).

Example  3: Pathologic Symptoms in AD-Induced CKD Model Of MH  Check the effect

Next, we performed histopathological examinations of kidney sections from different groups of mice to assess pathological status after AD treatment. The kidney sections of control mice did not show signs of tissue damage, and kidneys in AD-treated mice exhibited various pathological signs such as extensive damage, inflammation, fibrosis, and swelling. Treatment of MH did not affect the morphological morphology of the kidney, whereas treatment of MH for 7 days after 15 and 21 days after AD exposure relieved the pathological symptoms to steady state (FIGS. 5A & B).

Example  4: Kidney inflammation and fibrosis Marker  For expression Of MH  Check the effect

To assess the effect of MH on the expression levels of various renal inflammation and fibrosis-related markers, immunoblotting assays were performed. Significantly increased levels of proinflammatory markers such as MCP-1 and cox-2 were found in AD treated mice and compared to the two groups treated with MH (15 and 21 days) (Fig. 6A & B and Fig. 7A & B). In addition, expression levels of major fibrosis-related markers such as cathepsin-D and MMP-9 were found to increase in AD-treated mice. However, the expression of these markers was significantly reduced after 15 days and 21 days of AD pretreated mice and 7 days after MH treatment, compared to mice treated with AD alone (Fig. 6C &D; Fig. 7C & D).

Example  5: Gelatin Jamography Used MMP -2 &lt; / RTI &gt; MH of Korea  Check the effect

In addition, the level of expression of MMP2 was assessed using gelatin chomolysing. Expression levels of pro- and active forms of MMP-2 were significantly increased in AD-treated mice and significantly less in control and MH-treated animals. The expression level of MMP-2 was significantly decreased at day 15 of AD administration and at day 7 of MH-treated group after 21 days compared to AD alone (Figs. 8A and B).

Despite the lack of research on the lack of clear molecular mechanisms and new drugs that contribute to the onset and onset of chronic kidney disease, research on CKD has been slow to develop, despite improved knowledge and skills. Over the past few years, the concept of diet chemotherapy has attracted much attention and a number of new natural compounds have been proposed as potential treatments. In addition, MH is a potent molecule with therapeutic efficacy. One of the characteristics of MH is strong antioxidant and free radical scavenging properties. Unlike other flavonoids that can produce ROS through autoxidation, MH maintains its antioxidant properties even at high concentrations (300 mg / kg body weight per day). ROS affects genes involved in the synthesis of proinflammatory cytokines, thereby regulating the function of various transcription factors and regulating cell survival and cell proliferation. Since chronic oxidative stress and inflammation contribute to the onset of renal fibrosis and ultimately CKD, MH may be a potent drug that can treat these fatal diseases.

Claims (5)

A pharmaceutical composition for preventing or treating renal disease comprising morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate as an active ingredient Composition. The pharmaceutical composition according to claim 1, wherein the kidney disease is a chronic kidney disease. The method according to claim 1, wherein the morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate inhibits inflammation and fibrosis of the kidney . &Lt; / RTI &gt; A food composition for preventing or ameliorating a renal disease which comprises morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, its salt or morin hydrate as an active ingredient . A feed composition for preventing or ameliorating a renal disease comprising morin, 2- (2,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one, a salt thereof or morin hydrate as an active ingredient .

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