KR20140062681A - Compositions and functional food for prevention or treatment of diabetic complications comprising using extract of paulownia coreana - 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 Paulownia 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 on 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 paulowniae extract,

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of diabetic complications comprising an extract of Tungus yedoensis 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 < RTI ID = 0.0 > Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur . J. Biochem . , ≪ / RTI > 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, when studying natural medicines for preventing or treating diabetic complications as described above, the extracts of paulownia forest or their fractions inhibit the production of final glycation products and hypertrophy of eye veins in zebrafish due to hyperglycemia Prevention or treatment of diabetes mellitus, and has completed the present invention.

The present invention is to provide a pharmaceutical composition and a health functional food comprising an extract of Tungsten (Pavlenberg) or a fraction thereof as an active ingredient, which can prevent or treat diabetic complications.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating diabetic complications comprising an extract of Tungsten (Pb) or its fractions as an active ingredient.

The term "Tung" used in the present invention, scientific name is Paulownia Coreana is a Korean specialty plant. Circular or pentagonal leaves are about 25 ㎝ long and have star - shaped brown hairs on the back side. Leaf edges are flat. The purple flower runs on a cone flower at the end of May to June, with 5 petals and calyx each. Inside and outside of the petals are star-shaped fur and glandular hair.

The term "extract" as used in the present invention means a specific component separated from a pine tree using a liquid solvent. As the solvent, water, methanol, ethanol, butanol, ethyl acetate 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 tungstate extract of the present invention can be obtained by extracting flowers, branches, leaves or roots of a paulownia tree. As the extraction method, any one selected from the group consisting of room temperature extraction, hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction and steam extraction can be used.

In addition, in the present invention, the tungstate extract can be further fractionated, and the ethyl acetate fraction obtained by suspending the tungstate ethanol extract in water and then fractionating it with ethyl acetate is preferable. It is also preferred that the water fraction is a butanol fraction or a water fraction obtained by fractionating a water fraction obtained by suspending a paulownia tree ethanol extract in water and then fractionating with ethyl acetate by butanol. It is also preferred that the extract is a butanol fraction or a water fraction obtained by suspending a paulownia tree ethanol extract in water and then fractionating with 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 diabetes complication is caused by abnormal activity of aldose reductase and accelerated oxidative stress. Therefore, the activity of aldose reductase and the antioxidative activity of aldose reductase are measured in the development of diabetic complication, It can be judged. 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 has been confirmed that the extracts of Tungus pentobacterium, or fractions thereof, are effective for the prevention or treatment of diabetic complications. According to one embodiment of the present invention, it has been confirmed that the inhibitory effect of aldose reductase, which is an index of diabetic complication treatment, has an effect of preventing or treating diabetic complications.

The term "prophylactic " as used in the present invention means any action that inhibits or delays disease by administration of the composition comprising the above-mentioned Tungsten 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 containing the Tungsten Extract or its fraction.

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 admixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like to the Tung minu extract or the 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 Tungsten Extract or its fraction is preferably 1 mg / kg to 500 mg / kg, and may be administered once or several times a day, if necessary.

Further, the present invention provides a health functional food for preventing or ameliorating diabetic complications, comprising the extract of Tungus yedoensis 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 Tunga extract according to an embodiment of the present invention.
FIG. 2 is a graph showing changes in blood vessel thickness in zebrafish of the ethanol extract of paulowniae and fractions thereof 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. PCE-1 and PCE-5 were obtained by using ethyl acetate fraction of 1 μg / ml and 5 μg / ml, respectively, in the high glucose group, PCW-1 and PCW-5 contained 1 μg / ml and 5 μg / ml, respectively, of paulowniae fraction, 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 : Tung tree extract, and its object Fraction  Produce

North Chungcheong Province collected from the Jade Spring Geum and identified a paulownia (Paulownia coreana ), the following extracts and fractions were prepared.

1) Extraction step

Shade, three-folded paulownia ( Paulownia coreana ) was pulverized and 1.0 L of 80% ethanol was added. The mixture was repeatedly extracted from the extraction container at room temperature for 48 hours for 3 hours, and then concentrated under reduced pressure at 40 ° C. to obtain an ethanol extract.

2) Concentration, drying step

The 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 paulowniae were prepared. First, 500 ml of water and ethyl acetate (500 ml) were added to the paulowniae ethanol extract, and the ethyl acetate fraction obtained by separating into the water layer and the ethyl acetate layer using a separatory funnel was concentrated under reduced pressure using a vacuum distillation apparatus to obtain an ethyl acetate fraction Respectively.

The water layer was further fractionated into a water layer and a butanol layer by adding 500 ml of n-butanol, using a separatory funnel, and the obtained butanol fraction and water fraction were concentrated under reduced pressure using a vacuum distillation apparatus to obtain n-butanol fraction and water Fractions were obtained.

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

The extracts of Tungusces brachyus and its fractions prepared in Example 1 were analyzed in the following manner for inhibiting the final glycation endogenesis in vitro.

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 Pu tree extract and its fractions were prepared at a concentration of 1 / / ㎖, 5 / / ㎖ and 10 / / ㎖, respectively (the extracts of Puerariae Radix and its fractions were dissolved in DMSO, and then 15% tween 80 was added, The total DMSO content was adjusted to 0.2%), which was added to the mixture of BSA and glucose and incubated 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 of Tungus pentaphyllum and its fractions were measured for their inhibitory effect on the final glycation end product in a 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 Tung tree ethanol extract One
5
10 0.96 3.59
27.72 ± 3.85
57.49 + - 2.96
8.72 ± 0.51 Ethyl acetate fraction One
5
10 18.93 ± 1.09
38.53 + - 4.36
70.34 ± 1.00
6.63 + 0.24 Butanol fraction One
5
10 1.31 ± 5.96
24.34 + - 2.57
68.37 + - 2.60
7.81 + - 0.37 Water fraction One
5
10 13.87 + - 4.52
28.71 ± 2.09
46.02 + - 2.15
> 100 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, according to the present invention, the IC ₅₀ value of the inhibitory effect on the final glycation end product of the pine bark extract was confirmed to be 8.72 / / ml. It was proved that the efficacy is about 9 times better than aminoguanidine (IC ₅₀ value: 77.04 / / ml), which is a positive control synthetic single compound. The efficacy of the ethyl acetate fraction and the butanol fraction was also confirmed to be about 12 times higher and about 10 times higher than that of aminoguanidine, respectively.

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

Experimental Example  2: Tungus extract and its Fraction in vivo  On the system Anti-diabetic  Complication efficacy experiment

Zebrafish (Zebrafish) has the advantage that one is able to quickly determine the efficacy can also be purchased at a relatively low price compared to rodents in vertebrates in vivo efficacy search system (Disease models & Mechanism, 3, 236-245 (2010); Journal of Molecular Endocrinology , (2007) 38, 433-440).

When zebrafish was cultured in hyperglycemic state for 30 days, various pathophysiological features were observed similar to those seen in the early stages of non-proliferative diabetic retinopathy (NPDR) (Disease Models & Mechanisms (2010) 3, 236-245). In the hyperglycemic state, the retina of the zebrafish was changed into an abnormal pathological condition in which the surface blood vessels become thicker, the cell junction between the vascular endothelial cells becomes weaker, and the vessel basement membrane becomes thicker than that of the normal retina . Hereinafter, zebrafish was used to measure the antidiabetic complication efficacy in the in vivo system of Tungus extract and its fractions.

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 - five hours after fertilization, the blastocysts expressing the fluorescence were selected, and 5 individuals were divided into 24 well plates and hyperglycemia was induced by using 30 mM glucose. Ethanol extract (PC), ethyl acetate fraction (PCE), butanol fraction (PCB) and water fraction (PCW) were separately diluted in glucose solution at 1 ㎍ / After 3 days of drug treatment, the solution was replaced with fresh solution.

③ Analysis of vitreous blood vessel change

After 5 days of treatment in 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 results of the treatment of the extracts of paulownia tree and its fractions are shown in Fig. 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. (P <0.01), hyperglycemic vasodilatation was significantly inhibited in the experimental groups (PC-1, PC-5) treated with 1 ㎍ / ㎖ and 5 ㎍ / . Ethyl acetate fractions (PCE) and butanol fractions (PCB) fractionated from ethanol extracts were also proved to have excellent anti - vascular effects.

Thus, the pungent extract and its fractions are highly effective in inhibiting (treating or preventing) vascular enlargement, a pathological symptom that occurs in the early stages of non-proliferative diabetic retinopathy (NPDR) in a person suffering from hyperglycemia .

Claims (9)

A composition for preventing or treating diabetic complications comprising an extract of Tungus yedoensis or a fraction thereof as an active ingredient.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the Tunga extract is extracted from flowers, branches, leaves or roots of Tunga.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the extract of Tungusces is extracted with water, methanol, ethanol, butanol, ethyl acetate or a mixture thereof.
The pharmaceutical composition according to claim 1, wherein the extraction is any one selected from the group consisting of room temperature extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, and steam extraction.
2. The pharmaceutical composition according to claim 1, wherein the fraction is an ethyl acetate fraction obtained by suspending a paulowniae ethanol extract in water followed by fractionation with ethyl acetate.
The pharmaceutical composition according to claim 1, wherein the fraction is a butanol fraction or a water fraction obtained by suspending a paulownia tree ethanol extract in water, and then fractionating the water fraction obtained by fractionation with ethyl acetate with butanol.
2. The pharmaceutical composition according to claim 1, wherein the fraction is a butanol fraction or a water fraction obtained by suspending a pupa extract in water and then fractionating the extract 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 an extract of Tungus tree or a fraction thereof as an active ingredient.

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