KR20140062680A - Compositions and functional food for prevention or treatment of diabetic complications comprising using extract of carpinus cordata - Google Patents

Abstract

The present invention relates to a therapeutic agent for diabetic complications and a method for treating diabetic complications by using the same in which a Capinus cordata extract or a fraction thereof inhibits generation of advanced glycation end-products which are indicators of an outbreak of diabetic complications, and which can be used as a therapeutic agent for preventing or treating diabetic complications by having superior effects for preventing or treating diabetic ophthalmic diseases in animal diabetic model tests.

Description

Technical Field [0001] The present invention relates to a pharmaceutical composition for preventing or treating diabetic complications including an extract of the black magpie, and a health functional food comprising the extract of Carpinus cordata,

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating diabetic complications comprising an extract of a magpie bark, or a fraction thereof as an active ingredient.

Diabetes is one of the most important geriatric diseases in the world. In recent years, with the rapid economic growth, the prevalence of diabetes in Korea has reached 10%, now more than 240 million people worldwide, and 308 million in 2025 And 60% of them will develop in Asia, according to the American Medical Association (JAMA) in 2009. In particular, the onset of diabetes was elicited by the elderly, and the prolongation of life span led to complications. In general, almost 10 to 20 years after the onset of diabetes, almost all organs in the body are damaged, causing diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy diabetic neuropathy), heart disease, cancer, and osteoporosis. Chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end-stage renal failure, and diabetic cataract and retinopathy cause blindness and eventually death.

In all age groups, the rate of diabetic complications increased, rather than diabetes. In particular, in the 40s and 50s, the rate of peripheral circulatory disorder complications increased 6.5 times in diabetic patients and 2.2 times in diabetic retinopathy patients. The cost of treatment for peripheral circulatory disorder and diabetes complications increased 54.4% from KRW 80.7 billion in 2006 to KRW 153 billion in 2010 and diabetic retinopathy increased from KRW 32.7 billion to KRW 50.5 billion (Aug. 29, 2011). This is because the average life expectancy is increasing and the number of older patients with diabetes is increasing. Therefore, it is important to take measures in advance to prevent diabetic patients from developing diabetic complications.

In the United States, diabetes is the cause of blindness in the 25- to 74-year-old age group, and 60% of blindness after 15 or 20 years of diabetes develops blindness. Therefore, even if the duration of diabetic complication is delayed by 5 or 10 years, the quality of life of the patient and his / her family will be different, and it will have a big impact on national finances.

One of the typical factors that cause such diabetic complications is explained by the nonenzymatic glycation of proin. The nonenzymatic glycation of protein is a condensation reaction in which amino acid groups such as lysine residues of proteins and reducing sugars do not have enzyme activity, that is, by a milliad reaction. As a result of this reaction, glycation endproducts, AGEs) are generated. The non-enzymatic glycosylation of proteins involves (1) nucleophilic addition reaction of an amino acid group such as a lysine residue of a protein and an aldehyde or ketone of a reducing sugar without enzymatic action to form a schiff base as an initial step product, (2) an amadori-type early glycation end product which is reversible due to the persistence of hyperglycemia, and (3) a step of producing an amyloid-type early glycation end product which is reversible by condensation of a ketoamine adduct and an adjacent ketoamine adduct, Is rearranged without decomposition to produce a final glycation product which is an irreversible product. And the resulting final glycation products are bound or cross-linked with proteins or lipids to produce irreversible products such as glycated proteins or glycosylated lipids.

Unlike the reversible amaryllis type early glycation products, the final glycation products are irreversible reaction products, and once formed, they are not degraded even if the blood glucose is restored to normal, and accumulated in tissues during the survival period of the protein or lipid bound to the final glycation end product (Vinson, JA et al., 1996, J. Nutritinal Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur. Biochem., 210: 729-739).

For example, glycated albumin, one of the final glycation products produced by the reaction of glucose with various proteins, is an important factor in causing chronic diabetic nephropathy. Glycated albumin enters the ganglion cell more easily than normal albumin without glycosylation, and high glucose stimulates mesangial cells to increase synthesis of extracellular matrix. Overgrowth of glycosylated albumin and increased extracellular matrix cause fibrosis of the glomerulus. Such a mechanism would continue to damage the shrine sphere, leading to a stage where extreme treatment methods, such as hemodialysis or organ transplantation, are inevitable. In addition, it has been reported that collagen in the arterial wall due to chronic diabetes and basement membrane protein in the ganglion cell are combined with the final glycation products and accumulate in tissues (Brownlee, M., et al., 1986, Sciences, 232, 1629- 1632).

This non-enzymatic protein glycosylation leads to saccharification in proteins such as basement membrane, plasma albumin, lens protein, fibrin, collagen, etc., and the resulting final glycation products abnormally change the structure and function of the tissue to cause diabetic retinopathy retinopathy, retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy.

Accordingly, the present inventors have found that, in studying natural medicines for preventing or treating diabetic complications, the present inventors found that the extracts or fractions thereof inhibit the production of final glycation products and prevent thickening of eye veins of zebrafish caused by hyperglycemia Or treatment of diabetes mellitus, the present inventors have completed the present invention.

The present invention is to provide a pharmaceutical composition and a health functional food comprising an extract of a magpie bark extract or a fraction thereof as an active ingredient capable of preventing or treating diabetic complications.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating diabetic complications, comprising the extract of Shark bark extract or a fraction thereof as an active ingredient.

As used herein, the term "extract" means a particular ingredient extracted from a magpie with a liquid solvent. As the solvent, water, methanol, ethanol, butanol, ethyl acetoate or a mixture thereof may be used. It is preferable to extract methanol, ethanol or a mixture thereof with water in order to efficiently extract the active ingredient. The concentration of methanol and ethanol is preferably 10 to 90%, but is not limited thereto.

The magpie root extract of the present invention can be obtained by extracting branches, leaves or roots of a magpie bark. As the extraction method, any one selected from the group consisting of hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction and steam extraction can be used.

Also, in the present invention, it is preferable that the fraction is an ethyl acetate fraction obtained by suspending the magpie extract in water and fractionating it with ethyl acetate. It is also preferred that the water extract is a butanol fraction or a water fraction obtained by suspending the ethanol extract of the magpie bark and then fractionating the water fraction obtained by fractionation with ethyl acetate into butanol.

The term "diabetic complication" used in the present invention means a symptom caused when the diabetes persists for a long period of time. Diabetic complications are different from the criteria for the onset and judgment of diabetes, and diabetic complications are used separately from diabetic agents.

The cause of the diabetic complication is abnormally excessive production of the final glycation product, which causes complications in various organs. Therefore, the efficacy of the therapeutic agent can be determined by measuring the inhibitory effect on the final glycation endproduct during development as a therapeutic agent for diabetic complications. In addition, since diabetic complications are caused by diabetic retinopathy, diabetic cataract, nephropathy, neuropathy, diabetic heart disease and the like, it directly inhibits retinopathy, cataract, nephropathy and neuropathy and thus exhibits a direct therapeutic effect.

In the present invention, it was confirmed that the black currant extract, or a fraction thereof, is effective for preventing or treating diabetic complications. According to one embodiment of the present invention, it is possible to confirm the efficacy of inhibiting the production of final glycation products, which is an index of the treatment of diabetic complications, and to prevent or treat hypertrophy of the blood vessels in the zebrafish caused by hyperglycemia, .

As used herein, the term "prevention" means any act that inhibits or delays disease by the administration of a composition comprising said magpie extract, or a fraction thereof. The term "treatment" as used in the present invention means all the actions of improving or ameliorating symptoms of a disease by administration of the composition comprising the extract or the fractions thereof.

The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredients. 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 composition of the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method. In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin and the like, in the magpie extract, or a fraction thereof. 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 composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art. The daily dose of the magpie extract, or a fraction thereof, is preferably 1 mg / kg to 500 mg / kg, and may be administered once to several times per day as needed.

The present invention also provides a health functional food for preventing or ameliorating diabetic complications comprising an extract of a magpie bark, or a fraction thereof, as an active ingredient.

The health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. In addition, the health functional food may be in the form of any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, Lt; / RTI >

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a "food additive" Standards and standards.

Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, coloring matter, licorice extract, crystalline cellulosic, high- , A sodium L-glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent, and the like.

At this time, the extract according to the present invention, which is added to foods containing beverages in the process of manufacturing a health functional food, can be appropriately increased or decreased as required, and preferably 1 to 15% by weight based on 100% Is preferably added.

INDUSTRIAL APPLICABILITY The present invention is a natural extract effective for prevention or treatment of diabetic complications, which can be used not only as a pharmaceutical composition for preventing or treating diabetic complications, but also as a health functional food.

FIG. 1 shows a phylogenetic fractionation step of a magpie root extract according to an embodiment of the present invention.
FIG. 2 is a graph showing changes in blood vessel thickness in zebrafish of an extract of a magpie supernatant according to an embodiment of the present invention. Figure 2a shows a fluorescence image, and Figure 2b is a graphical representation of changes in blood vessel thickness.
FIG. 3 shows changes in blood vessel thickness in a zebrafish of a magpie buckwheat fraction according to an embodiment of the present invention. FIG. 3A shows a fluorescence image, and FIG. 3B is a graph showing changes in blood vessel thickness. 24E-1 and 24E-5, respectively, were treated with 1 ㎍ / ㎖ and 5 ㎍ / ㎖ of Shattuckov-Dalmatian ethanol extract, respectively, and 24B-1 and 24B- 5 means 1 ㎍ / ㎖ and 5 ㎍ / ㎖ treated group of magpie buckwheat fraction, and 24W-1 and 24W-5 means 1 ㎍ / ㎖ and 5 ㎍ / ㎖ treated group respectively.

Hereinafter, the present invention will be described in more detail with reference to the following Production Examples and Examples. However, the following Preparation Examples and Examples are for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example : Magpie bark extract, and its objection Fraction  Produce

The following extracts and fractions were obtained from the shrub mites collected and identified in the Okcheon Geum River, Chungbuk Province.

1) Extraction step

After shredding the shrubs of three shaded shrubs, shrubs of 80% ethanol were repeatedly extracted from the extraction vessel at room temperature and concentrated under reduced pressure to prepare an ethanol extract.

2) Concentration, drying step

The ethanol extract was filtered and concentrated under reduced pressure. At this time, the temperature during the concentration was kept at 40 캜 to 45 캜 so as to prevent decomposition and hydrolysis of the constituents.

3) System fractionation stage

As shown in Fig. 1, fractions of magpie bark were prepared. First, 500 mL of water and ethyl acetate (500 mL) were added to the ethanol extract of M. magenta, and the ethyl acetate fraction obtained by separating into the water layer and the ethyl acetate layer was separated by using a separatory funnel under reduced pressure to obtain an ethyl acetate fraction.

Further, 500 ml of n-butanol was added to the water layer, and the water layer and the butanol layer were separated by using a separatory funnel. The obtained butanol fraction and water fraction were concentrated under reduced pressure to obtain n-butanol fraction and water fraction, respectively.

Experimental Example  One: The final glycation product  Evaluation of generation inhibition efficacy

The inhibitory effect on the production of the final glycation end product in the test tube of the magpie bark extract and its systematic fractions prepared in Example 1 was analyzed as follows.

1) Experimental method

Bovine serum albumin (BSA) (Sigma, USA) was used as a protein source. BSA was added to 50 mM phosphate buffer (pH 7.4) to a concentration of 10 mg / ml. A solution of 0.2 M fructose and 0.2 M glucose was used as a source.

To the prepared BSA solution, a mixed solution of fructose and glucose was added. The extracts and their fractions were prepared at a concentration of 1 ㎍ / ㎖ to 100 ㎍ / ㎖, respectively. The extracts and their fractions were dissolved in DMSO and then 15% tween 80. The total DMSO content 0.2%), and the mixture was added to the mixture of BSA and sugar, followed by incubation at 37 ° C for 7 days.

At this time, 0.02% sodium azide and antimycotics were added as an antibacterial agent and an antifungal agent, respectively. The control group was a culture of BSA and sugar mixture, and the blank group of the test group and the control group was not cultured after each preparation. On the other hand, aminoguanidine was used as a positive control, which is an index that can compare excellent efficacy. All four cultures were prepared to minimize errors. After 7 days, the content of the final glycation products in the culture broth was analyzed and the results were measured. The final glycation products are fluorescent, brownish, and have physicochemical properties that allow them to cross-link, as well as possessing ligands that can be recognized by cell membrane receptors. (Vinson, JA et al., J. Nutr. Biochem., 7: 1), the amount of the final glycation end product having such characteristics was measured by a microplate reader (Excitation: 350 nm, 659-663, 1996), and the degree of inhibition was calculated by the following equation.

AGE-BSA (%) = (100 - (fluorescence intensity of sample group - fluorescence intensity of sample blank group)) / (fluorescence intensity of control group - fluorescence intensity of control group) × 100

On the other hand, the test for inhibiting the production of aminoguanidine, which is a positive control group, was performed by dissolving aminoguanidine in distilled water at a concentration of 55.5 μg / ml, 74 μg / ml and 92.5 μg / ml at 37 ° C. For 7 days, and the amount of the final glycation products produced in the respective culture media was measured by a microplate reader (Excitation: 350 nm, emission: 450 nm).

2) Experimental results

The extracts and fractions thereof of the magpie bark extract were tested for their inhibitory effect on the final glycation end product in the test tube according to the above test methods. The results are shown in Table 1 below.

Use concentration (/ / ml) Inhibition (%) IC ₅₀ ([mu] g / ml) Culture period 7 days Culture period 7 days Culture period 7 days Magpie root ethanol extract 5
10
25 26.62 ± 3.21
46.97 ± 1.75
52.28 ± 1.54
10.61 ± 0.50
Ethyl acetate fraction One
5
10 9.34 ± 2.35
45.48 ± 0.26
59.60 ± 1.23
6.61 + 0.14
Butanol fraction 5
10
25 39.22 ± 0.89
47.48 ± 2.98
60.78 ± 3.13
30.19 + - 4.05
Water fraction 25
50
100 22.59 ± 1.54
38.70 ± 0.59
52.82 + - 0.51
90.06 ± 1.15
Aminoguanidine 55.5
74
92.5 39.93 + 1.74
49.97 ± 3.22
56.62 ± 2.25
77.04 + - 3.23

As shown in Table 1, IC ₅₀ value of the inhibitory effects produced advanced glycation end products of blackcurrant birch extract according to the present invention was confirmed to 10.61 ㎍ / ㎖. It was proved that the efficacy is about seven times higher than that of aminoguanidine (IC ₅₀ value: 77.04 / / ml), which is a positive control synthetic single compound. In addition, the efficacy of ethyl acetate fraction and butanol fraction was also excellent. That is, the ethyl acetate fraction was about 12 times more effective than the positive control and about 3 times more effective than the butanol fraction.

Therefore, it was confirmed that the extracts and fractions thereof inhibit the binding of protein and sugar to strongly inhibit the production of final glycation products.

Experimental Example  2: of the magpie bark extract in vivo  On the system Anti-diabetic  Complication efficacy experiment

Zebrafish is an in vivo system model that is one of the vertebrate animals, and has a relatively short experimental period and a relatively low price compared with rodents such as mice and mice. Mechanisms, 3, 236-245 (2010); Journal of Molecular Endocrinology (2007) 38, 433-440). Hereinafter, zebrafish was used to measure the antidiabetic complication efficacy in the in vivo system of the black currant extract.

1) Experimental method

① Preparation of zebrafish breeding ship

Embryos were obtained by crossing male and female transgenic zebrafish (Tg (kdr: EGFP)) expressing a fluorescent protein (green fluorescence protein) specifically in vascular endothelial cells.

② Hyperglycemia induction and drug treatment

Twenty - four hours after fertilization, the embryos expressing the fluorescence were selected, and 5 individuals were divided into 24 well plates and hyperglycemia was induced using 30 mM glucose. At this time, the drug to be identified (magpie bark extract or its fraction) was diluted with glucose solution. Replace with fresh solution for 3 days after drug treatment.

③ Analysis of vitreous blood vessel change

After 5 days of treatment under hyperglycemic conditions, they were fixed with 4% paraformaldehyde for one day. The changes of the hyaloid vasculature were analyzed under fluorescent microscope by separating the lens from the fixed body.

2) Experimental results

The efficacy of ethanol extract of magpie bark

Fig. 2 shows the result of treating the extract of the black magpie with ethanol. As shown in FIG. 2, the vitreous blood vessels of hyperglycemia group (HG) cultured in 30 mM glucose solution for 5 days were significantly expanded compared with the control group. The hyperglycemic - induced vasodilatation was significantly inhibited in the experimental group (+ ㎍ / ㎖, + 5 ㎍ / ㎖) treated with both hyperglycemia and.

② The efficacy of the fraction of the extract of the magpie bark extract

The results of the treatment of ethyl acetate, butanol and water fractions fractionated from the ethanol extract of the black magpie bark are shown in FIG. As shown in FIG. 3, it was demonstrated that the ethylacetate and butanol fractions, except for the water fraction, significantly inhibited hyperglycemia-induced retinal vascular dilatation. Especially, 5 ㎍ / ㎖ of ethyl acetate fraction was recovered to almost normal level.

Claims (8)

A pharmaceutical composition for preventing or treating diabetic complications, comprising the extract of Aspergillus oryzae, or a fraction thereof as an active ingredient.
[Claim 3] The pharmaceutical composition according to claim 1, wherein the magpie extract is extracted from the branches, leaves or roots of the magpie.
The pharmaceutical composition according to claim 1, wherein the magpie extract is extracted with water, methanol, ethanol, butanol, ethyl acetate or a mixture thereof.
The pharmaceutical composition according to claim 1, wherein the extraction is at room temperature extraction, hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction or steam extraction.
2. The pharmaceutical composition according to claim 1, wherein the fraction is an ethyl acetate fraction obtained by suspending an epidermis ethanol extract in water and then fractionating it with ethyl acetate.
The pharmaceutical composition according to claim 1, wherein the fraction is a butanol fraction or a water fraction obtained by fractionating a water fraction obtained by suspending an epidermidum ethanol extract in water, followed by fractionation with ethyl acetate, with butanol.
The pharmaceutical composition according to claim 1, wherein the diabetic complication is diabetic retinopathy, diabetic cataract, nephropathy, neuropathy or diabetic heart disease.
A health functional food for preventing or ameliorating diabetic complications, comprising as an active ingredient, an extract of V. vulnificus, or a fraction thereof.

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