KR102346863B1 - Composition for preventing or treating Kidney Disease Comprising Morin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating kidney disease comprising morin as an active ingredient. In addition, the present invention relates to a food composition and feed composition for preventing or improving kidney disease comprising morin as an active ingredient.
According to the present invention, morin or morin hydrate restores the survival rate, body weight, kidney weight and water consumption of CKD animal models, significantly improves serum biochemical parameters of CKD mice, and the pathology of kidney morphology and section. restore academic change. In addition, the morin or morin hydrate of the present invention restores the expression of pro-inflammatory and fibrosis-related markers to normal. Accordingly, it is expected that morin or morin hydrate according to the present invention can be utilized as a natural therapeutic agent effective for the treatment or prevention of chronic kidney disease.

Description

A composition for preventing or treating kidney disease comprising morin as an active ingredient {Composition for preventing or treating Kidney Disease Comprising Morin}

The present invention relates to a pharmaceutical composition for preventing or treating kidney disease comprising morin as an active ingredient.

In addition, the present invention relates to a food composition and feed composition for preventing or improving kidney disease comprising morin as an active ingredient.

Chronic kidney disease (CKD) is the leading cause of death in Asia and the United States. There are currently no effective treatments for this disease other than current treatment strategies that rely on blood pressure control by blocking the renin-angiotensin system. Such current treatment strategies serve only to delay the onset of end-stage renal disease, which can be associated with serious side effects. About 200 million people worldwide have CKD. In the United States, African Americans (AAs) have a fourfold risk compared to non-Hispanic whites, and worldwide, the highest rates are found in low-income countries in Asia and sub-Saharan Africa. In sub-Saharan Africa, more than half a million people develop end-stage renal disease (CKD stage 5) each year, the majority of whom die prematurely. The medical cost and economic burden of CKD is so great that it is unsustainable even in developed countries in the West.

The most common cause of CKD is diabetes, accounting for approximately 50%, followed by hypertension (25%), including glomerulonephritis and polycystic kidney disease. In addition, other risk factors are due to the accumulation of different toxic substances and metabolites, which may contribute to increased mortality in CKD. The most commonly used definition of CKD disease is based on a decreased glomerular filtration rate (GFR) (>15 ml/min, stage G5) over 3 months used for the diagnosis of CKD. A decrease in GFR indicates a persistently increased risk of death. Also, the majority of clinical studies use albuminuria and proteinuria, the overwhelming majority of urinary proteins, which are closely related to ESRD and increased mortality. Sclerosis of glomerulus and interstitial fibrosis are common histopathologic and structural features of CKD. Tubulointerstitial fibrosis is closely correlated with renal function and exhibits complex structural changes common in the kidney, including matrix metallo-proteinase (MMP) and collagen production by epithelial cells and activated myofibroblasts. Included. Fibrosis is a reactive process that develops primarily in response to epithelial damage and is almost always accompanied by inflammation, which is manifested by increased cytokine expression and accumulation of macrophages and inflammatory cells. Research on fibrosis has improved significantly over the past few years, providing new therapeutic goals.

Currently, there are no adequate therapies to treat most CKDs. It is associated with the potential for reversible renal failure, including renal sepsis, nephrotoxic drugs and urinary obstruction. Steroids and immunosuppressants have some potential to arrest or reverse kidney disease, but are ineffective in treating kidney disease in diabetic and hypertensive patients.

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

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

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

Accordingly, an object of the present invention is chronic comprising Morin, 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one, a salt thereof, or Morin hydrate as an active ingredient. To provide a pharmaceutical composition for preventing or treating kidney disease.

In addition, another object of the present invention is to include Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), a salt thereof or Morin hydrate as an active ingredient. To provide a food composition and feed composition for preventing or improving chronic kidney disease.

In order to achieve the above object, the present invention is morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), its salt or morin hydrate (Morin hydrate) as an active ingredient It provides a pharmaceutical composition for the prevention or treatment of chronic kidney disease, including.

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

In addition, the present invention is 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; Preferably, it provides a food composition for preventing or improving chronic kidney disease.

Furthermore, the present invention relates to 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, preferably It provides a feed composition for preventing or improving chronic kidney disease.

According to the present invention, morin or morin hydrate restores the survival rate, body weight, kidney weight and water consumption of CKD animal models, significantly improves serum biochemical parameters of CKD mice, and the pathology of kidney morphology and section. restore academic change. In addition, the morin or morin hydrate of the present invention restores the expression of pro-inflammatory and fibrosis-related markers to normal.

Accordingly, it is expected that morin or morin hydrate according to the present invention can be utilized as a natural therapeutic agent effective for the treatment or prevention of chronic kidney disease.

1 shows the survival rate and change in body weight of an animal model.
(A) It is a figure showing the survival rate of animals treated with adenine, morin hydrate, and adenine + morin hydrate at the indicated concentrations after 15 days of adenine treatment.
(B) A diagram showing the survival rate of animals treated with adenine, morin hydrate, and adenine + morin hydrate 21 days after adenine treatment.
(C) It is a diagram showing the change in body weight of animals 15 days after adenine treatment, after treatment with adenine, morin hydrate, and adenine + morin hydrate as a percentage.
(D) A diagram showing changes in body weight of animals as a percentage after 21 days of adenine treatment, treatment with adenine, morin hydrate, and adenine + morin hydrate.
Figure 2 shows the rate of change of relative kidney weight and water consumption in CKD mice.
(A) Shows the changes in the relative kidney weight of animals after 15 days of adenine treatment and after treatment with the indicated concentrations of adenine, morine hydrate and adenine + morine hydrate.
(B) Shows the changes in relative kidney weight of animals after treatment with adenine, morine hydrate, and adenine + morine hydrate after 21 days of adenine treatment.
(C) Percentage of water consumption of animals after 15 days of adenine treatment, after treatment with adenine, morine hydrate, and adenine + morine hydrate.
(D) Percentage of water consumption of animals after treatment with adenine, morine hydrate and adenine + morine hydrate after 21 days of treatment with adenine.
Data are presented as mean ± SD (n = 8). Values with different letters (ad) were significantly different from each other (p <0.05).
3 shows changes in serum biochemical parameters of CKD mice after treatment with morin hydrate.
(A) A diagram showing changes in the amount of serum albumin in mice after treatment with adenine, morin hydrate, and adenine + morin hydrate at the indicated concentrations after 15 days of adenine treatment.
(B) A diagram showing changes in the amount of serum BUN in mice.
(C) A diagram showing changes in the amount of serum creatinine in mice.
(D) A diagram showing the change in the amount of serum albumin in mice after 21 days of adenine treatment, adenine, morin hydrate, and adenine + morin hydrate treatment.
(E) A diagram showing changes in serum BUN in mice.
(F) is a diagram showing changes in the amount of serum creatinine in mice.
Data are presented as mean ± SD (n = 8). Values with different letters (ad) were significantly different from each other (p <0.05).
4 is a view showing a change in the shape of the kidney.
(A) Shows the morphology of mouse kidneys after treatment with adenine, morine hydrate and adenine + morine hydrate at the indicated concentrations after 15 days of adenine treatment.
(B) Shows the morphology of mouse kidneys after treatment with adenine, morin hydrate, and adenine + morin hydrate 21 days after adenine treatment.
5 is a diagram showing the results of histopathological analysis of H & E-stained kidney sections (original magnification, 200 x) of each group after successive experimental treatments.
(A) After 15 days of adenine treatment, the morphology associated with pathological tissue damage, swelling, inflammation and fibrosis in the groups treated with adenine, morine hydrate and adenine + morine hydrate at the indicated concentrations.
(B) After 21 days of adenine treatment, the morphology associated with pathological tissue damage, swelling, inflammation and fibrosis in the groups treated with adenine, morin hydrate, and adenine + morin hydrate.
6 is a diagram showing the preventive effect of morin hydrate on the expression of mouse kidney protein after continuous treatment.
(A) Western blot analysis was performed using specific pro-inflammatory antibodies to cox-2 and (B) MCP-1. Relative expression was analyzed by densitometry analysis using ImageJ software in mouse kidneys after morin hydrate treatment.
Western blot analysis was performed using specific fibrosis-associated antibodies to (C) MMP-9 and (D) cathepsin D. Relative expression was analyzed by densitometry analysis using ImageJ software in mouse kidneys after morin hydrate treatment. β-actin was used as an internal control. Data are presented as mean ± SD (n = 8). Values with different letters (ad) were significantly different from each other (p <0.05).
7 is a diagram showing the effect of morin hydrate treatment on kidney protein expression.
(A) Western blot analysis was performed using specific pro-inflammatory antibodies to cox-2 and (B) MCP-1, and relative expression was analyzed by densitometry analysis using ImageJ software in mouse kidneys after morin hydrate treatment .
(C) Western blot analysis was performed using sperm fibrosis-associated antibodies to MMP-9 and (D) cathepsin D. Relative expression was analyzed by densitometry analysis using ImageJ software in mouse kidneys after morin hydrate treatment, and β-actin was used as an internal control. Data are presented as mean ± SD (n = 8). Values with different letters (ae) were significantly different from each other (p <0.05).
8 is a diagram showing the preventive and therapeutic effects of morin hydrate on kidney protein in mice
(A & B) Relative expression of MMP-2 was analyzed by densitometry analysis using ImageJ software in mouse kidneys after treatment with morin hydrate through gelatin gyrography.
Data are presented as mean ± SD (n = 8). Values with different letters (ad) were significantly different from each other (p <0.05).

Hereinafter, the present invention will be described in detail.

The present invention is Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), its salt or Morin hydrate (Morin hydrate) as an active ingredient for preventing or preventing kidney disease A therapeutic pharmaceutical composition 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 the fruits and leaves of Maclura pomifera (Osage orange), Maclura tinctoria (old fustic), Morus alba (Mulberry) or Psidium guajava (common guava), and the morin or morin hydrate can be extracted from various plants. and does not limit the type of the plant.

Morin of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, and the like, and 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, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Late, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-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 morine derivative is dissolved in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., and an organic acid or inorganic acid is added to filter the resulting precipitate. , it can be prepared by drying, or it can be prepared by distilling the solvent and excess acid under reduced pressure and then drying it and crystallizing it in an organic solvent.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

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

In addition, the morin or morin hydrate may be chemically or biologically synthesized, and the synthesis method is not limited thereto.

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 treating or preventing kidney disease. have.

The kidney disease may be acute kidney disease or chronic kidney disease. The kidney disease may include diabetic nephropathy, hypertensive nephropathy, glomerulonephritis, pyelonephritis, interstitial nephritis, lupus nephritis, polycystic kidney disease, renal failure, etc.

In particular, the Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), salt or Morin hydrate of the present invention is used as a preventive or therapeutic agent for chronic kidney disease. can be used

Chronic kidney disease (CKD)" refers to a condition in which proteinuria is continuously present, there is evidence of kidney damage such as hematuria, or there is a continuous decrease in kidney function for more than 3 months. Swelling around and lower extremities, drowsiness, easy fatigue, lack of appetite, nausea, itchy skin, bad breath (symptom of ammonia odor), etc. It may be accompanied by symptoms of deterioration of the complexion.

Chronic renal failure refers to a case in which the kidneys have been damaged for more than 3 months or the renal function continues to decrease, and generally refers to a case in which the kidney functions at less than 50% of the normal kidney. In this case, the kidneys do not perform the function of filtering wastes properly, so the levels of creatinine and blood urine nitrogen (BUN) in the plasma increase, and proteins including albumin are detected in the urine. In the case of chronic kidney failure, a part of the kidney does not function properly, so the normally functioning kidney is overloaded and the normally functioning portion is continuously damaged, and as a result, it progresses to chronic kidney failure. Therefore, it may eventually lead to a condition that requires dialysis or a kidney transplant. Chronic kidney failure is a concept included in chronic kidney disease.

In addition, the chronic kidney disease is a concept that includes not only the disease, but also various complications derived from the chronic kidney disease.

As used herein, the term "prevention" refers to any action of inhibiting kidney disease or delaying the onset of kidney disease by administering the pharmaceutical composition. The term "treatment" refers to any action that improves or beneficially changes the symptoms of kidney disease by administration of the pharmaceutical composition.

In the present invention, morin or morin hydrate restores survival rate, body weight, kidney weight and water consumption in CKD animal models, significantly improves serum biochemical parameters in CKD mice, and histopathological changes in kidney shape and section to restore In addition, the morin or morin hydrate of the present invention restores the expression of pro-inflammatory and fibrosis-related markers to normal.

In the present invention, the pharmaceutical composition may be used in a method for preventing or treating kidney disease, and specifically, the method for preventing or treating kidney disease may include administering to an individual suffering from or expected to develop kidney disease. .

As used herein, the term “administration” means introducing the composition to a subject by an appropriate method.

As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, including humans, that have or can develop kidney disease, and specifically, may be mammals including humans, but is not limited thereto. .

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, sex, drug activity, sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. For example, the composition may be administered at a dose of 0.01 to 500 mg/kg per day, specifically 10 to 100 mg/kg as an active ingredient, and the administration may be administered once or divided several times a day. . In addition, the pharmaceutical composition of the present invention may contain morin or morin hydrate of the present invention in a weight percentage of 0.001 to 50% 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 may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition for preventing or treating kidney disease of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredients described above. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. have. Specifically, in the case of formulation, it can be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, and a surfactant commonly used. Solid preparations 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 or more excipients, 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. It can be prepared by adding various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to liquids for oral use and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition may be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the improvement, alleviation, treatment or prevention of chronic renal failure.

The present invention includes Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), its salt or Morin hydrate as an active ingredient, prevention of kidney disease Or it provides a food composition for improvement.

As used herein, the term "improvement" refers to any action in which renal disease is improved or is advantageously changed by administration of the composition.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, health functional foods, and health foods, and includes all foods in a conventional sense.

The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of drugs using food as a raw material, and is excellent in portability, so the present invention Foods of can be ingested as an adjuvant to enhance the effect of prevention or improvement of kidney disease.

The health food means food having an active health maintenance or promotion effect compared to general food, and health supplement food means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement may be used interchangeably.

Since the food composition of the present invention can be consumed on a daily basis, a high effect can be expected for the prevention or improvement of kidney disease, and thus it can be very usefully used.

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

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

In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (foaming agents), film agents, gum base agents, foam inhibitors, solvents, improving agents, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

Morin or a salt thereof of the present invention, or Morin hydrate may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the food composition of the present invention may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less with respect to the food or beverage. However, when ingested for a long period of time for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

As an example of the food composition of the present invention, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may 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 drink composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, or the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it 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 contain various weight % as long as it can exhibit an effect of preventing or improving kidney disease, but specifically 0.00001 to 100 wt. % or 0.01 to 80% by weight, but is not limited thereto.

In addition, the present invention is 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. It provides a feed composition for prevention or improvement.

The feed is a general feed fed to livestock, and the term "livestock" refers to domestic animals, ie, mammals, poultry, and the like. In the present invention, the mammal may include dogs, cats, cattle, pigs, goats, sheep, horses, and the like, and poultry may include chickens, ducks, turkeys, and the like, but is not limited thereto.

The feed composition may include general basic feeds fed according to the type of livestock. All of these basic feeds are known in the art for their necessary components and compositions and suitable composition ratios depending on the type of livestock, and since they are also commercially available, anyone skilled in the art will be able to very easily manufacture or purchase them.

Specifically, the basic feed may be composed of all or part of a suitable starch-containing material, protein-containing material, fat-containing material, vitamin-containing material, mineral-containing material, antioxidant material, antibiotics, etc. depending on the type of livestock.

All of the starch-containing materials may be included in the basic feed of breeding animals as long as they can maintain the growth of breeding animals. These starch-containing materials are generally obtained from corn, soybeans, wheat, sorghum, barley, oats, and the like. Suitable starch-containing substances include ground or powder of corn, ground or powder of oats, ground or powder of soybean, and ground or powder of wheat. Preferred suitable starch-containing materials are oat meal, corn meal, wheat meal, soy meal and the like. The concentration of the starch-containing material is not particularly limited as long as it can effectively maintain the growth of domesticated animals. In general, the starch-containing material may be included in the basic feed in the range of about 30 to 80% by weight.

Protein-containing substances may also be included in the basic feed of livestock as long as they can maintain the growth of domestic animals. For economic reasons, protein-containing substances such as fish meal, soy flour, etc. are used, others being soy protein concentrate, blood meal, plasma protein, dried skim milk, 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 meal and the like. As long as the protein-containing material can effectively maintain the growth of the breeding animals, its concentration in the basic feed of the breeding animals is not important. In general, the protein-containing material may be included in the basic feed in the range of about 10 to 50% by weight.

Examples of the fat-containing material include, but are not limited to, lard (Lard), tallow, soybean oil, lecithin, coconut oil, and the like. The concentration of the fat-containing material is not limited as long as it can maintain the growth of breeding animals, and may be included in the basic feed in the range of generally about 2 to 20 weight.

In addition, the basic feed may contain water-soluble and fat-soluble vitamins and trace minerals. The vitamins may include vitamin A, vitamin D, vitamin E, vitamin K, riboflavin, pantothenic acid, niacin, vitamin B12, folic acid, biotin, vitamin C, and the like. Copper, zinc, iodine, selenium, manganese, iron, cobalt, etc. may be used as trace minerals. As used herein, "trace" means that the amount of these components used in the basic feed for livestock is substantially less than the amount of other components. In general, when vitamins or minerals are included in the basic feed in trace amounts, it may be included in an amount of about 0.0001 to 5% by weight in the total weight of the composition.

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

Hereinafter, the present invention will be described in more detail based on examples. The examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the invention is not limited by these examples according to the gist of the present invention.

Experimental example

Experimental Example 1: Animal model

All animal experiments performed during the study were approved by the Laboratory Animal Care Committee and Daegu University. All techniques were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male BALB/c mice (initially 5 to 6 weeks old, about 25 g) were obtained from Oriental Biotechnology (Korea) and fed ad libitum with standard pellet feed and tap water at 22° C., relative humidity of about 30%, and a light-dark cycle of 12 hours.

Experimental Example 2: Experimental Design

After a 1 week acclimatization 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). The second group was treated with AD (150 mg/kg body weight) by oral gavage for 21 days. Groups 3 and 4 were treated with AD for 15 days and then MH was administered for 1 week at 20 mg/kg and 40 mg/kg doses, respectively, by oral gavage. The fifth group was administered only MH (40 mg/kg) via oral gavage. In addition, groups 6 and 7 were administered MH at 20 mg/kg and 40 mg/kg through oral gavage, respectively, 21 days after AD treatment. Doses of AD and MH were normalized based on their effects. Mice were weighed weekly and total water consumption was measured daily for the duration of the experiment. All animals were sacrificed by sacvical dislocation after the indicated treatment period. Whole blood was collected from the tail vein or abdominal aorta with an EDTA-tube (Soyagreentec co., Korea), centrifuged at 2500 g for 15 min at 4 °C to separate the serum, and stored at -80 °C for further analysis. Kidneys were removed, blotted with filter paper and weighed. A small piece of the kidney was immersed in 4% formalin for subsequent histopathological examination. The remaining kidneys were frozen at -80 °C for biochemical and molecular analysis and western blot. The kidneys were homogenized with cold RIPA buffer to obtain a 10 mg/ml homogenate. Kidney homogenate was centrifuged at 15,000 g for 10 min at 4 °C and the resulting supernatant was used to measure several indicators.

Experimental Example 3: Biochemical method

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

Experimental Example 4: Pathological analysis

Renal tissue was fixed in 4% buffered formalin, treated with step-by-volume ethyl alcohol, embedded in paraffin wax, cut to a thickness of 4-5 μm, and evaluated for inflammation, fibrosis, basement membrane atrophy and tubular dilatation. Stained with 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

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

For western blot analysis, equal amounts of protein lysates (20 μg per lane) were mixed with 5x sample buffer (50 mM Tris, 2% SDS, 10% glycerol, 1% DTT and 0.1% bromophenol blue at pH 6.8). , and after heating at 95° C. for 5 minutes, the protein was separated by 12% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the separated protein was transferred to a polyvinyldenefluoride (PVDF) membrane (Roche Diagnostics, Indianapolis, IN, USA) through electroplating. Then, the membrane was probed with a primary antibody, followed by immersion with a horseradish peroxidase (HRP)-conjugated secondary antibody, followed by enhanced chemiluminescence (ECL) according to the recommended procedure (Amersham Pharmacia, Piscataway, NJ, USA). visualized as

Experimental Example 6: Gelatin Zymography

The enzymatic activity of MMP-2 was determined through gelatin zymography. Briefly, kidney tissue homogenates from all groups 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 in incubation buffer (50 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl2, 0.02 % triton X100) at 37°C for 24 h. incubated. Gels were stained with 0.5% (w/v) Coomassie Brilliant Blue in 30% (v/v) methanol and 10% (v/v) acetic acid.

Experimental Example 7: Drugs 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-β-actin and HRP-conjugated anti-rabbit immunoglobulins are manufactured by 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: Confirmation of the effect of increasing the survival rate of MH in AD-induced CKD model

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

To evaluate the effects of MH on animal survival and physiological disturbances in the AD-induced CKD model, survival and mortality rates were calculated in each group of animals after treatment at the indicated doses. As a result, it showed 100% survival rate in control group and MH (40 mg/kg body weight) treated mice. The 21-day mortality rate in the AD-treated animals group was 100%. On the other hand, in mice 15 and 21 days old after AD pretreatment, the high-dose MH-treated group confirmed a survival rate of nearly 60% ( FIGS. 1A and B). Moreover, AD-treated mice observed significant reductions in body weight and kidney weight. Water intake also increased significantly. As a result of 7 days of MH treatment after 15 and 21 days of AD pretreatment, body weight and kidney weight were significantly increased compared to mice in the AD alone group. Water intake was significantly decreased after 7 days of MH treatment (Fig. 1 C &D; Fig. 2 A, B, C & D).

Example 2: Confirmation of Effect of MH on Serum Parameters of AD Induced CKD Model

Next, we observed serum biochemical parameters related to renal function. It was observed that AD-treated mice significantly increased creatinine, blood urea nitrogen (BUN) and albumin in serum. However, administration of MH after 15 and 21 days of AD pretreatment significantly improved all indicators compared to mice treated with AD alone (Fig. 3A, B, C, D, E & F).

It was objectively determined that the general appearance of mice was also improved by MH, especially at high doses (40 mg/kg body weight). The height of the control group and the MH-administered group appeared normal. However, the kidneys of the AD treated group were pale in color, contracted, and showed a rough surface. The appearance of the kidneys of mice treated with AD+MH for 7 days after 15 and 21 days of AD exposure was significantly improved compared to those of mice treated with AD alone ( FIGS. 4A & B).

Example 3: Confirmation of the effect of MH on histopathological symptoms in AD-induced CKD model

Next, we performed histopathological examination of kidney sections from different groups of mice to evaluate the pathological status after AD treatment. Kidney sections from control mice showed no signs of tissue damage, and kidneys from AD treated mice showed various pathological signs such as extensive damage, inflammation, fibrosis and swelling. Treatment of MH did not affect the morphologic morphology of the kidney, whereas treatment of 7 days of MH after 15 days and 21 days after AD exposure alleviated the pathological symptoms to steady state (Fig. 5A & B).

Example 4: Confirmation of the effect of MH on the expression of markers related to renal inflammation and fibrosis

To evaluate the effect of MH on the expression levels of various renal inflammation and fibrosis related markers, immunoblotting analysis was performed. Significantly increased expression levels of pro-inflammatory markers such as MCP-1 and cox-2 were identified in AD treated mice, and both groups (after 15 and 21 days) treated with MH compared with mice treated with AD alone was significantly normalized in a concentration-dependent manner ( FIGS. 6A & B and 7A & B). In addition, it was confirmed that the expression levels of major fibrosis-related markers such as cathepsin-D and MMP-9 were increased in AD-treated mice. However, the expression of these markers was significantly decreased after 15 and 21 days of AD pre-treated mice, and 7 days after MH treatment, compared with mice treated with AD alone (Fig. 6C &D; Fig. 7C & D).

Example 5: Gelatin zymography MMP using -2 expression and activity MH for check the effect

In addition, the expression level of MMP 2 was evaluated using gelatin zymography. The expression levels of pro and active forms of MMP-2 were significantly increased in AD-treated mice, and significantly less observed in control and MH-treated animals. The expression level of MMP-2 was significantly decreased on the 7th day of the MH administration group after 15 and 21 days AD administration compared with the AD administration group alone (FIGS. 8A and B).

Research on CKD has been slow despite advances in knowledge and technology, as studies on the definitive molecular mechanisms contributing to the onset and pathogenesis of chronic kidney disease and the lack of novel drugs are lacking. In the past few years, the concept of dietary chemotherapy has received attention and numerous new natural compounds have been proposed as potential therapeutics. In addition, MH is a potent molecule with therapeutic efficacy. One of the hallmarks of MH is its strong antioxidant and free radical scavenging properties. Unlike other flavonoids that can generate ROS through auto-oxidation, MH retains its antioxidant properties even at high concentrations (300 mg/kg body weight per day). ROS can regulate the functions of several transcription factors by influencing genes involved in the synthesis of proinflammatory cytokines, and can regulate cell survival and cell proliferation. As chronic oxidative stress and inflammation contribute to the pathogenesis of renal fibrosis and ultimately induce CKD, MH may be a powerful drug to treat this fatal disease.

Claims (6)

Prevention of renal failure containing Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), a pharmaceutically acceptable salt thereof, or Morin hydrate as an active ingredient or a therapeutic pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein Morin, a pharmaceutically acceptable salt thereof, or Morin hydrate reduces the amounts of creatinine, blood urea nitrogen (BUN) and albumin in plasma. Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), a salt thereof, or a food composition for preventing or improving renal failure comprising Morin hydrate as an active ingredient . The food composition according to claim 3, wherein morin, a salt thereof or morin hydrate reduces the amount of creatinine, blood urea nitrogen (BUN, blood urea nitrogen) and albumin in plasma. Feed composition for preventing or improving renal failure comprising Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one), a salt thereof, or Morin hydrate as an active ingredient . The feed composition according to claim 5, wherein morin, a salt thereof, or morin hydrate reduces the amount of creatinine, blood urea nitrogen (BUN, blood urea nitrogen) and albumin in plasma.

Images