WO2012023803A2

WO2012023803A2 - Composition for preventing and treating complications of diabetes mellitus, containing brandisia hancei hook. f. extract or fraction thereof as active ingredient

Info

Publication number: WO2012023803A2
Application number: PCT/KR2011/006046
Authority: WO
Inventors: 김진숙; 김정현; 김찬식; 김영숙; 김온순; 김기모; 손은진; 정동호; 이윤미
Original assignee: 한국한의학연구원
Priority date: 2010-08-17
Filing date: 2011-08-17
Publication date: 2012-02-23
Also published as: WO2012023803A3

Abstract

The present invention relates to a composition for preventing and treating complications of diabetes mellitus, containing a Brandisia hancei Hook. f. extract or a fraction thereof as an active ingredient. More specifically, a Brandisia hancei Hook. f. extract or a fraction thereof inhibits the generation of an advanced glycation end product, which is an indicator of diabetes mellitus complications, and the activity of aldose reductase, and is strongly effective for complications of diabetes mellitus including diabetic retinopathy,and thus can be useful as an active ingredient of a composition for preventing and treating complications of diabetes mellitus.

Description

_χχ

【Specification】

[Name of invention]

Composition for preventing and treating diabetic complications containing wheatgrass extract or fractions thereof as an active ingredient

Technical Field

The present invention relates to a composition for the prevention and treatment of diabetic complications, specifically, ^^^ Brandisia hancei Hook, f.) Pharmaceutical composition for the prevention and treatment of diabetic complications using extract, pharmaceutical for preventing and treating diabetic cancer A composition and a composition for health food.

Background Art

Diabetes mellitus is one of the most important adult diseases in the world. In recent years, with the rapid economic growth in Korea, the prevalence of diabetes has reached 10%. Currently, the world has more than 240 million people with diabetes. It will grow to 380 million worldwide, of which 60 percent will occur in Asia, according to the 2009 American Medical Association (JAMA). In particular, the onset of diabetes mellitus has been extended to middle age, and the prolonged lifespan has led to complications. In general, almost 10 to 20 years after diabetes, almost all organs in the body are damaged, resulting in diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetes. Diabetic neuropathy, heart disease, cancer or osteoporosis may occur. Chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end stage renal failure. Diabetic cataracts and retinopathy lead to blindness and eventually death. In the United States, diabetes is the leading cause of blindness for ages 25 to 74 years, 60% after 15 to 20 years of diabetes. Leads to blindness. Therefore, delaying the onset of complications in diabetic patients by only 5 to 10 years will change the quality of life of patients and their families and will have a major impact on national finances.

Mechanisms for inducing diabetic complications are largely explained by nonenzymatic glycat ion of protein, polyol pathway and oxidative stress. A nonenzymatic glycat ion of protein is a group of amino acids such as lysine residues of a protein and reducing sugars due to enzymatic condensation reactions, that is, milliard reactions. Glycation products (advanced glycat ion endproducts, AGEs) are produced. The non-enzymatic glycosylation reaction of the protein comprises: (1) amino acid groups such as lysine residues of the protein and aldehydes or ketones of reducing sugars react with nucleophilic addition without enzymatic action to form a Schiff base, an initial product. The base and the adjacent ketoamine adduct condense to each other to produce a reversible Amador i preglycosylated product, and (2) the hyperglycemic state persists, leading to reversible premature glycosylation. The product is rearranged without being degraded to produce a final glycosylated product, which is an irreversible product, and the resulting glycosylated products are combined or cross-linked with proteins or lipids to irreversible glycosylated proteins or glycosylated lipids. It can be divided into stages in which the product of is produced. Unlike the reversible Amadori type of early glycosylated products, the final glycosylated products are irreversible reaction products, so once they are produced, they are not degraded even when blood sugar returns to normal. Abnormally alters the structure and function of the organ, causing complications throughout the tissue (Vinson, JA et al., 1996, J. Nutritinal Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur. J. Biochem., 210: 729-739). For example, glycated albumin, one of the end glycosylated products produced by reaction of glucose and various proteins, Acts as a critical factor in causing chronic diabetic nephropathy. Glycosylated albumin enters renal glomeruli more easily than normal albumin without glycosylation, and high concentrations of glucose stimulate mesangium cells to increase extracellular matrix synthesis. Excessly introduced glycated albumin and increased extracellular matrix result in fibrosis of the glomeruli. These mechanisms continue to damage the glomerulus, leading to the stage of extreme treatment methods such as hemodialysis or organ transplantation. In addition, it has been reported that collagen accumulates in the arterial wall and basal membrane proteins in the renal glomeruli are accumulated in tissues due to chronic diabetes mellitus (Brownlee, M., et al., 1986, Sciences, 232, 1629). -1632). The glycosylation occurs in the basement membrane, plasma albumin, lens protein, fibrin, and collagen by non-enzymatic protein glycosylation. The resulting glycosylated product abnormally changes the structure and function of the diabetic diabetic retinopathy. It causes chronic diabetic complications such as diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabet ic neuropathy. In addition, it has been reported that lipid metabolism abnormalities occur in the process of producing the final sugar volcano in hyperglycemic state and oxidative stress is caused by deterioration of the defense system function against harmful oxygen free radicals (Yokozawa, T.). Et al, 2001, J. of Trad. Med., 18: 107-112). As such, the mechanism of action of non-enzymatic glycation reaction and oxidative stress is related.

The polyol pathway is (1) reduced by aldose reductase (AR) action from aldose or ketose to form sorbbi, and (2) sorbbi is oxidized by dehydrogenase to fructose The process consists of creating a step. At steady state, aldose reductase has a very low affinity for glucose, but due to hyperglycemia, aldose reductase, the first enzyme in the polyol pathway, is excessively activated, which causes excessive hyperglycemia to be converted into fructose. 3 accumulated in tissue The pressure balance is broken, causing complications. In other words, due to the increased osmotic pressure, water is introduced and progresses to diabetic retinopathy, diabetic cataract, diabetic neuropathy, etc. (Woong-Jin Kim et al. Diabetes Society, Korean Medicine, p. 483; Soulis-Liparota, T., et al., 1995, Diabetologia, 38: 357-394). Final glycosylation products have been reported to activate aldose reductase, a major enzyme of the polyol pathway, in human microvascular endothelial cells (Nakamura, N., et al., 2000, Free Radic Biol. Med., 29 ： 17-25). Fructose is about 10 times faster than non-enzymatic glycosylation of glucose. Therefore, high concentrations of fructose bind to the protein and eventually accelerate the formation of the final glycated product. As such, non-enzymatic glycosylation reactions, polyl pathways and oxidative stress mechanisms are linked to each other to cause diabetic complications. It has been reported that lipid metabolism abnormalities occur in the process of producing the final glycated product in hyperglycemic state and oxidative stress is caused by deterioration of defense system function against harmful oxygen free radicals (Yokozawa, T., et al, 2001, J. of Trad. Med., 18: 107-112. Therefore, the mechanism of action of non-enzymatic glycation reaction and oxidative stress is related to each other. Therefore, in order to delay, prevent or treat the development of diabetic complications, it has been found that the inhibition of final glycation end product formation is very important (Brownlee, M., et al., 1988, N. Engl. Med., 318, 1315-1321). ). At present, aminoguanidine, a synthetic glycosylation inhibitor, is a nucleophilic hydrazine, which prevents cross-linking with proteins by binding to the amadori product, thereby inhibiting the formation of the final glycation product and progressing to complications. Delay or prevent (Brownlee, M., et al., 1986, Sciences, 232, 1629-1632; Edelstein, D. et al., 1992, Diabetes, 41, 26-29). Aminoguanidine is the most promising synthetic drug for the prevention and treatment of diabetic complications. There was a problem that caused toxicity when administered. Therefore, the development of safe and effective natural medicine is desired.

^^^ ¥ d) Brandisia hancei Hook. /. ) Is a Chinese herb belonging to Scrophulariaceae. Wheat germ is a bitter taste of Chinese medicine, cold in nature, hepat it is, hyperlipidemia, hypercholesterol. It has been used as a folk remedy for hypercholesteromia, rheumatoid arthritis (Zheng—Dan He et al., J. of Ethnopharmacology 71 (2000) 483-486). Component studies have reported brandioside, acetoside, 2'-acetylacetoside, pol iumside, etc. (Zheng-Dan He et al., Phytochemistry, 30, 2, 701-702, 1991). (Zheng-Dan He et al., J. of Ethnopharmacology 7i (2000) 483-486), no further pharmacological and component studies have been reported. Accordingly, the present inventors are developing a safe and effective natural medicine for diabetic complications with little toxicity and side effects, and the whole glycoside extract or its fractions are produced through in vitro (//? Vitro) experiments and aldose production. The present invention has been completed by inhibiting reductase activity, showing potent antidiabetic complications including diabetic retinopathy, and revealing anti-cancer effects.

[Detailed Description of the Invention]

[Technical problem]

The object of the present invention is the currency 0? s / a hancei Hook, f.) To provide a pharmaceutical composition for the prevention and treatment of diabetic complications containing an extract or a fraction thereof as an active ingredient. Technical Solution

In order to achieve the above object, the present invention provides a wheat call ^{_{(Λ3 / 7 '5/3}} hancei Hook. F.) Diabetic complications, prevention and treatment of pharmaceutical composition containing extracts or fractions thereof as an active ingredient.

In addition, the present invention provides a composition for preventing and improving diabetic complications containing wheat flour extract or fractions thereof as an active ingredient.

In addition, the present invention provides a method for treating diabetic complications comprising administering a pharmaceutically effective amount of wheatgrass extract or a fraction thereof to an individual suffering from diabetic complications.

The present invention also provides a method for preventing diabetic complications comprising administering a pharmaceutically effective amount of wheatgrass extract or a fraction thereof to a subject.

In addition, the present invention provides a wheat flour extract or a fraction thereof for use as a pharmaceutical composition for preventing and treating diabetic complications.

In addition, the present invention provides a wheat flour extract or fractions thereof for use as a composition for preventing and improving diabetic complications. Hereinafter, the present invention will be described in detail. The present invention is a call 09 / 7i / s / a hancei Hook. /.) Provides a pharmaceutical composition for preventing and treating diabetic complications containing the extract as an active ingredient.

The diabetic complication is any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic cancer, diabetic osteoporosis, and diabetic atherosclerosis Preferred but not limited to this. Diabetes complications are symptoms caused by long-term diabetes mellitus. _? It is different from the onset criteria and judgment criteria of, and the treatment for diabetes complications can be used separately from the treatment for diabetes.

The wheat flour extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) extracting by adding an extraction solvent to wheat flour;

2) cooling the extract of step 1) and then filtering; And

3) drying the filtered extract of step 2) under reduced pressure.

In the above method, the bulking of step 1) can be used without limitation, such as being grown or commercially available. The wheat currency may use the whole wheat currency, preferably, but not limited to using branches, stems, or roots.

The extraction solvent is preferably water, alcohols, their mixtures or water-soluble alcohols. It is preferable to use d to C ₂ lower alcohol as the alcohol, it is preferable to use ethane or methane as the lower alcohol, it is more preferable to use an 80% ethanol aqueous solution, but is not limited thereto. As the extraction method, conventional methods in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction, can be used. The hot water extraction method is preferably performed one to five times, and three times to extract repeatedly. More preferred but not limited thereto. The extraction solvent may be added 0.1 to 10 times to the dried wheat bran, it is preferable to add 0.3 to 5 times. Extraction temperature is preferably 20 to 30 ^° C, but is not limited thereto. In addition, the extraction time is preferably 12 to 48 hours, but is not limited thereto.

In the above method, the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto. ₀

o In a specific embodiment of the present invention, the whole branch, stem or root is washed with water and then dried in the shade. The dried whole wheat flour, stems or roots were crushed and placed in an extraction container, and extracted repeatedly at room temperature with an extraction solvent. After obtaining a wheat flour extract, the solids were removed using a filter paper or the like and filtered. The extract was concentrated under reduced pressure to prepare a wheatgrass extract. In addition, the present invention provides a pharmaceutical composition for preventing and treating diabetic complications containing the fraction prepared by further fractionating the wheat flour extract with an organic solvent as an active ingredient.

Fractions of the wheat flour extract is preferably prepared as follows, but not always limited thereto.

1) suspending the whole wheat extract in water, and then separating the n-nucleic acid layer by adding n-nucleic acid;

2) separating the ethyl acetate layer by adding ethyl acetate to the remaining water layer of step 1);

3) separating butane layer by adding butanol to the remaining water layer of step 2); And

4) separating the water fraction in the remaining water layer of step 3).

In the above method, the organic solvent is preferably, but not limited to, nucleic acid, ethyl acetate or butanol. The fraction may be obtained by repeating the fractionation process from 1 to 5 times, preferably 3 times from the wheat flour extract, and preferably concentrated under reduced pressure after the fraction, but is not limited thereto.

The organic solvent is more preferably ethyl acetate or butanol. The diabetic complication is any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic cancer, diabetic osteoporosis, and diabetic atherosclerosis Preferably but not limited to _g does not. Diabetes complications are symptoms caused by long-term diabetes mellitus, but differ from the onset criteria and judgment criteria of diabetes mellitus, and the treatment for diabetic complications can be used separately from the diabetes treatment.

The fraction was obtained by evaporating the solvent from the wheat extract and adding the nucleic acid with water to the obtained residue to separate the nucleic acid layer to obtain a nucleic acid fraction. Ethyl acetate was mixed with the water layer except for the nucleic acid layer, and the ethyl acetate layer was separated to obtain an ethyl acetate fraction. Further, after removing the ethyl acetate layer, butanol was mixed and the butane layer was separated to obtain a fraction of butane. Finally, after removing the butanol layer, a water fraction of the water layer was obtained (see FIG. 1).

In a specific embodiment of the present invention, the present inventors used bovine serum albumin (BSA) as a protein to analyze the inhibitory effect of the production of the final glycated product, which is an indicator of diabetic complications and an evaluation of treatment efficacy. The degree of binding to fructose and glue course was used as an index. In addition, as a positive control group, aminoguanidine, which is known to have an excellent inhibitory effect on the final glycosylated product, was used. As a result, it was confirmed that the wheat flour extract or the test group treated with fractions thereof showed about 4.2 times more effective inhibition of the end glycated product than aminoguanidine, a positive control (see Table 1).

In a specific embodiment of the present invention, the inventors have measured the inhibition of the activity of aldose reductase, which is another indicator of diabetic complications of wheatgrass extract or fractions thereof. 3,3-tetramethylene glutaric acid as a comparative control

It was measured using (Tetramethyleneglutaric acid) ol. As a result, the wheat currency extract or fractions thereof of the present invention was found to inhibit aldose reductase activity more effectively than the known aldose reductase inhibitors (see Table 2). In a specific embodiment of the present invention, the inventors of the present invention, in order to confirm the efficacy of diabetic retinopathy for diabetic retinopathy, using a straptozotocin (streptozotocin) As a result of confirming the efficacy on diabetic retinopathy using type 1 diabetic rats, the wheat flour extract of the present invention prevents the accumulation of the final glycation product retinal tissue (see FIG. 2), and apoptosis of retinal tissue (apoptosis) (See FIG. 3), prevent the loss of retinal neurons (see FIG. 4), inhibit the activation of Muller cells (see FIG. 5), inhibit the nuclear migration of NF-kB, Decreasing bax and increasing be 12 were found to have a significant effect on diabetic retinopathy.

Therefore, the wheat flour extract or fractions thereof of the present invention inhibit the accumulation of glycation products occurring in the long-term diabetic state, inhibit the activity of aldose reductase, and have a powerful effect on diabetic complications including diabetic retinopathy. Therefore, it was confirmed that it can be usefully used as an active ingredient of the pharmaceutical composition for preventing and treating diabetic complications. It contains 0.1 to 99.9% by weight of the wheat flour extract or fractions thereof as an active ingredient with respect to the total weight of the composition of the present invention, and may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various oral or parenteral formulations. When formulated, diluents or excipients such as layering agents, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. Lubricants such as stearic acid magnesium, talc and the like are also used in addition to the simple brothers. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. This may be included. Formulations for parenteral administration include _{1 χ} sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized preparation, suppository. As the non-aqueous solvent and the suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as evolved oil, injectable esters such as ethyl acrylate, etc. may be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select external or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine epidural or cerebrovascular injection mode for parenteral administration. For skin use externally. The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate and severity of the disease, the daily dosage is the amount of wheat flour extract 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and may be administered 1 to 6 times per day.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological reaction modifiers. In addition, the present invention provides a composition for preventing and improving diabetic complications containing wheatgrass extract or fractions thereof as an active ingredient.

The diabetic complication is preferably any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease diabetic cancer, diabetic osteoporosis, and diabetic atherosclerosis One is not limited to this.

The wheat flour extract or fractions thereof of the present invention inhibits the production of the final glycation end products and the activity of aldose reductase, and exhibits a strong effect on diabetic complications including diabetic retinopathy, thereby preventing and improving diabetic complications. Oil in food composition Can be used.

The functional food of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients. The above-mentioned natural carbohydrates include sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, xyl, sorbitol and erythritol. to be. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is preferably selected in the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention.

In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols. And carbonizing agents used in carbonated beverages. In addition, the functional food of the present invention may contain a pulp for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The ratio of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

In addition, the present invention provides a pharmaceutical composition for preventing and treating diabetic cancer, which contains the wheatgrass extract or a fraction thereof as an active ingredient.

In another aspect, the present invention provides a composition for preventing and improving diabetic cancer containing wheat flour extract or fractions thereof as an active ingredient.

It has already been reported that the final glycated product causes cancer (Tokuda H. et al., 2005, Book of Abstract of 53rd GA Congress joint with SIF, P076). The wheatgrass extract or fractions thereof effectively inhibit the production of the final glycation end products, and thus may be useful as an active ingredient for treating and improving diabetic cancer or as a composition for health food for treating and improving diabetic cancer. In addition, the present invention provides a method for treating diabetic complications comprising administering a pharmaceutically effective amount of wheatgrass extract or a fraction thereof to an individual suffering from diabetic complications.

The pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit or risk ratio applicable to the medical treatment, including the type, severity, activity of the drug, sensitivity to the drug and time of administration of the individual. The route of administration and the rate of ^excretion can be determined by the duration of treatment, factors including the drug being used simultaneously, and other factors well known in the medical field.

The wheat flour extract or fractions thereof of the present invention is useful in the prevention and treatment of diabetic complications, since it exhibits a potent effect on the production of the final glycation end products and the inhibition of aldose reductase, and on diabetic complications including diabetic retinopathy. Can be used. In addition, the present invention provides a method for treating diabetic cancer comprising administering a pharmaceutically effective amount of wheatgrass extract or a fraction thereof to a subject with diabetic cancer.

In addition, the present invention provides a method for preventing diabetic cancer comprising administering to the individual a pharmaceutically effective amount of wheatgrass extract or a fraction thereof.

It has been reported that the final glycation end products are allergenic (Tokuda H. et al., 2005, Book of Abstract of 53rd GA Congress joint with SIF, P076), the wheat flour extract or fractions thereof of the present invention effectively inhibits the production of the final glycated product, it can be useful in the prevention and treatment of diabetic cancer. In addition, the present invention provides a wheat flour extract or a fraction thereof for use as a pharmaceutical composition for preventing and treating diabetic complications.

In addition, the present invention provides a wheat flour extract or fractions thereof for use as a composition for preventing and improving diabetic complications.

The wheat flour extract or fractions thereof of the present invention inhibit the production of the final glycation end products and the activity of aldose reductase, and have a strong effect on diabetic complications including diabetic retinopathy, thus preventing and treating diabetic complications. It can be usefully used as an active ingredient of the composition, and a composition for health food. In addition, the present invention provides a wheat flour extract or a fraction thereof for use as a pharmaceutical composition for preventing and treating diabetic cancer.

In addition, the present invention provides a wheat flour extract or fractions thereof for use as a composition for preventing and improving diabetic cancer.

It has already been reported that final glycation end products are cancer-causing (Tokuda H. et al., 2005, Book of Abstract of 53rd GA Congress joint with SIF, P076), the wheat flour extract or fractions thereof of the present invention Since effectively inhibits the production of, can be usefully used as an active ingredient in the pharmaceutical composition for preventing and treating diabetic cancer, and the composition for health food.

Advantageous Effects

Hancei Hook, f.) Extract of the present invention or fractions thereof ,

15 It is useful as an active ingredient in the pharmaceutical composition for preventing and treating diabetic complications because it inhibits the production of the final glycated product and the aldose reductase activity, which are indicators of diabetic complications, and exhibits potent antidiabetic complications including diabetic retinopathy. Can be used. [Brief Description of Drawings]

1 is a schematic diagram of a process for preparing wheat flour fractions.

FIG. 2 is a diagram showing the effect of wheatgrass extract on the final glycation product accumulation in the retinal tissue:

NOR: normal group;

DM: diabetic group;

MET: metformin treated group;

BH-50: whole wheat extract 50 mg / kg treated group; And

BH-250: Whole wheat extract 250 mg / kg treated group.

Figure 3 is a diagram showing the effect on the loss of retinal neurons of wheat flour extract:

NOR: normal group;

DM: diabetic group;

MET: metformin treated group;

BH-50: whole wheat extract 50 mg / kg treated group; And

BH-250: Whole wheat extract 250 mg / kg treated group.

Figure 4 is a diagram showing the effect on the activation of Muller cells of wheatgrass extract:

NO: normal group;

DM: diabetic group;

MET: metformin treated group; BH-50: whole wheat extract 50 mg / kg treated group; And

BH-250: Whole wheat extract 250 mg / kg treated group.

5 is a diagram showing the effect on the activation and nuclear transfer of NF-kB of wheat flour extract:

NOR: normal group;

DM: diabetic group;

MET: metformin treated group;

BH-50: whole wheat extract 50 mg / kg treated group; And

BH-250: Whole wheat extract 250 mg / kg treated group.

Figure 6 is a diagram showing the effect on the expression of Bax and Be 1-2 of wheatgrass extract:

NOR: normal group;

DM: diabetic group;

MET: metformin treated group;

BH-50: whole wheat extract 50 mg / kg treated group; And

BH-250: Whole wheat extract 250 mg / kg treated group.

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples and Preparation Examples.

Example 1 Preparation of an Aqueous Extract of Wheat Flour Ethane

After crushing 196 g of dry and fine wheat flour and leaves at 70:30, respectively, add 1.0 L of 80% ethane in an aqueous solution, and repeatedly extract at room temperature from an extraction container. _χ? After filtering the extract, the concentration at the time of concentration was maintained at 40 ~ 451 or less to prevent decomposition and hydrolysis of the constituents and concentrated under reduced pressure to give 14.5 g of ethanol extract. In addition, specimens of evidence (no. Diab-2008-85) were stored in the Korean Diabetes Association Center for Diabetic Complications Research Center.

Example 2 Preparation of Wheat Flour Fraction

To prepare the wheat flour fraction, the solvent was evaporated from the wheat flour extract of <Example 1> and water and nucleic acid were added to the obtained residue to separate the hexane layer to obtain a nucleic acid fraction (6.7%). Ethyl acetate was added to the water layer except for the nucleic acid layer, and the ethyl acetate layer was separated to obtain an ethyl acetate fraction (K) (7%). In addition, butane was separated after removing the ethyl acetate layer, and the butanol layer was separated to give a butanol fraction (49.8%). Finally, after removing the butanol layer, a water fraction (26.9%) of the water layer was obtained. Experimental Example 1 Analysis of Inhibitory Effect on Final Glycolic Acid Production

In vitro final glycation product production inhibitory effect of the wheatgrass extract obtained in <Example 1> was analyzed.

Specifically, bovine serum albumin (BSA, Sigma, USA) was selected as a protein source. BSA was prepared by adding 50 ιτιΜ phosphate buffer (pH 7.4) to a concentration of 10 rag /. As a sugar source, a solution containing 0.2 M fructose and 0.2 M glucose was used. To the prepared BSA solution, fructose and glucose mixtures were added. Whole Currency Ethanol Extract 5

Or 25 concentrations. After dissolving all the compounds in dimethyl sulfoxide (DMS0), 15% tween 80 was added, and the total DMS0 content was 0.2%. The extract obtained above was added to the mixture of BSA and sugar. Incubated for 14 days at 37 ^° C by _{1 Q.} At this time, 0.02% sodium azide and antimycotics were added as antibacterial and antifungal agents. Cultures of BSA and sugar mixtures were used as controls, and extracts or fractions thereof were prepared, and those not cultured were used as blanks for the test and control groups. On the other hand, aminoguanidine was used as a positive control group as an index to compare the superiority of efficacy. Specifically, aminoguanidine is dissolved in distilled water and 55.5 at 37 ^° C. by the method described above.

Or 92.5; each was incubated for 14 days at a concentration of «g / ^. All cultures were prepared by four to avoid errors as much as possible. After 14 days of culture, the final glycosylated product content was analyzed. The final glycosylated product was fluorescent and brown in color, and possessed physicochemical properties that could cross-link as well as cell membrane receptors. Had a perceivable ligand. Microplate detection of final glycation end products with these properties

7} (Microplate reader; Excitation: 350 nm, Emission: 450 nm) was analyzed to determine the degree of inhibition of the production of the final sugar volcanic acid (Vinson, JA et al., J. Nutr. Biochem., 7: 659-663, 1996). The production inhibition rate of the final glycosylated product was calculated according to Equation 1 below.

[Equation 1]

As a result, as shown in the following [Table 1], the IC ₅₀ value of the final sugar water production inhibitory effect of the wheat ethanol extract was 15.23 g /. This is a positive control _ig aminoguanidine (IC ₅₀ value: 63.40 // g / m «, 4.2 times more potent than the efficacy was proved (Table 1). It was confirmed to inhibit the formation of [Table 1].

Experimental Example 2 Analysis of Effect of Inhibiting Aldose Reductase (AR) Activity

The inhibitory effect of aldose reductase activity on wheatgrass extract, or its nucleic acid, ethyl acetate, butane and water fractions in vitro was analyzed.

Specifically, in order to obtain a natural aldose reductase from the eyes of SD rats (Spr ague-Daw ley rat, 250-280 g) according to the Dufrane (1984) method, 135 mM Na, K-phosphate The solution (K-phosphate buffer; pH 7.0) and 10 mM 2-mercaptoethan (2-mercaptoethanol) were ground together with the extracted lens using a homogenizer and an ultrasonic grinder (Sonicater). After centrifugation at 14,000 rpm for 30 minutes, the supernatant was filtered through a 2 / m filter and used. The procedure was all performed at 4 ^° C. It was quantified by the Iowry method using BSA as the protein source of the enzyme (Enzyme). 135 mM Na, K-phosphate buffer solution (pH 7.0), 100 mM lithium sulfate (Lithium) sulfate), 0.03 mM NADPH, 0.04 mM DL- glyceraldehyde (DL-glycealdehyde) and 100 / m £ of enzyme mixture dissolved in 0.1% DMS0 and added 50 Η samples of dilution at each concentration. 1 was reacted at 37 ^° C for 10 minutes. At this time, the test group was a mixture without addition of 0.04 niM DL-glyceride (DL-glycealdehyde), STD was 50 to 135 mM Na, K-phosphate buffer solution (pH 7.0), 100 mM Lithium sulfate (Lithium sulfate) Samples with fit NADP (0.2-5 μΜ) were used. After the reaction was terminated by adding 0.5 N HC1 of sample 0.3, 6 M NaOH 1 m «with 10 mM imidazole was added thereto, and the reaction was repeated at 60 ^° C. for 10 minutes to give NADP as a fluorescent product. The degree of conversion was measured. Samples were run three times. The degree of efficacy was determined using a Spectrof luorophotometric detector (Bio-TEK, Synergy HT, USA). 360 rail, Em. Measured at 460 ππι, expressed as an IC ₅₀ value. Wheat flour extract was prepared at a concentration of 0.25 ug / n, 0.5 us / mi or 1.0 us / mt, nucleic acid fraction at a concentration of 2.5 μ / mi, 5 β / mt or 10 / g /, and ethyl acetate fraction was 2.5 ug / i, 5 g / l, 10 / / ^, butane fraction is 2.5 ug / ml, 5 μ / ηύ or 10

Water fractions were prepared at concentrations of 2.5 ug / m, 5 i / mi or 10 / «/ ΐΗ respectively. The control group included 3,3-tetramethylene glutaric acid (3,3—tetramethyleneglutaric acid), one of the good aldose reductase inhibitors.

The inhibitory effect of aldose reductase activity was measured after preparation at the concentration of 4.66 iig / mi or 5.59 / 111 £.

As a result, as shown in the following [Table 2], the wheat ₅₀ extract, the nucleic acid fraction, the ethyl acetate fraction, butanol fraction or water fraction of the Aldose reductase activity inhibitory effect of IC ₅₀ value of 0.86 ^ g / mi, 10 // g / m «or more, and 6.26 zg / mi, and 3.57 µ / ηύ. Positive control 3,3-tetramethyleneglutaric acid (IC ₅₀ value: 5

When comparing the efficacy in consideration of the monosynthetic compound, the activity inhibitory effect of the fractions of the present invention was excellent (Table 2). Wheat flour extract or powder thereof _{The Q1} fragment was found to inhibit aldose reductase activity.

Table 2

Concentration Suppression ICso (μg / m ^) (%) (%)

0.25 13.23 土 3.75

Whole Currency Extract 0.5 37.74 士 1.35 0.86 士 0.03

5 55.647 士 2.33

2.5 -12.42 of buckwheat extract 4.62

Nucleic Acid 5 6.38 士 3.54> 10 Fraction 10 2.68 士 2.53

2.5 of whole currency extract 44.85 士 0.91

Ethyl acetate 5 56.73 土 3.74 6.26 士 0.29 Fraction 10 73.61 士 3.90

2.5 21.88 土 0.88 of wheatgrass extract

Wong Tao 5 26.72 土 3.97 3.57 士 0.49 Fraction 10 52.67 士 3.90

2.5 of whole currency extract 22.62 1.90

5 24.10 土 2.04 9.66 土 0.41 ΐΙΓ "a 百 10 29.39 士 4.08

3,3-tetramethylene glutaric acid 3.72 33.03 士 4.36

(3.3-4.66 40.27 土 1.36 5.41 土 0.13

Tetramethyleneglutar ic 5.99 51.58 士 0.78

acid) Experimental Example 3 Efficacy Analysis of Antidiabetic Complications of Whole Wheat Extract

To determine the efficacy of wheatgrass extract on the complications of diabetes, the effect of diabetic retinopathy was evaluated by oral administration of wheatgrass extract for 12 weeks to type 1 diabetic rats induced with streptozotocin (streptozotocin).

<3-1> Experimental animal preparation

Experimental animals were used after 6 weeks of age male SD rats (Orien Bio, Korea) for one week. After injecting streptozotocin for diabetes induction at 60 mg / kg into the intraperitoneal injection, one week after the blood glucose test, only fasting blood glucose over 350 mg / dl was selected as a diabetic and diabetic group. Feed and drinking water were freely fed. After inducing diabetes, the test drug and the control drug metformin (MET) were orally administered daily to the following concentrations (whole flour extract 50 mg / kg, 250 mg / kg, metformin 350 mg / kg). The test group was divided into (1) normal group, (2) diabetic induction group, (3) metformin administration group, (4) wheatgrass extract 50 mg / kg administration group, and (5) wheatgrass extract 250 mg / kg administration group. It was. Drugs were orally administered for 12 weeks in each group. Fasting was performed 16 hours a day before necropsy, and the harvested organs were stored at -80 ^° C. <3-2> Confirmation of retinal tissue accumulation prevention effect of final glycated products (AGEs)

Immunohistochemical staining was performed to observe the degree of accumulation of final glycation end products (AGEs) known as the causative agent of diabetic complications.

Specifically, during autopsy, organs were extracted, fixed in 10% neutralized formalin overnight, dehydrated three times with xylene, and embedded in paraffin. The embedded tissue block was used to make a continuous section of 4 urn thickness on a slide. All. Deparaffinization process and hydration All coarse slides were washed with PBS containing 0.05% tween 20 for 3 minutes in 3) ¾0 ₂ solution to remove endogenous peroxidase activity. Blocking with 5% casein was used to remove nonspecific reactions, followed by dilution with 1: 200 of the primary antibody, respectively, and applied for 1 hour or overnight. After washing with PBS for 1 hour, labeled streptoavidin biotin (LSAB) kit (Dako, USA) was applied, followed by color development with DAB and observed under an optical microscope. In the case of fluorescent staining, the FITC-conjugated secondary antibody was diluted 1: 200 and reacted for 1 hour, and then stained with DAPI and observed under a fluorescence microscope.

As a result, as shown in Figure 2, the accumulation of AGEs was increased in the diabetic group (DM) compared to the normal group (N0R), and showed a significant reduction effect in the MET group, even in the high concentration group wheat extract extract group It was confirmed that the results showed a significant reduction compared to (Fig. 2). <3-3> Confirmation of inhibitory effect on retinal neuronal cell loss

In order to confirm the inhibitory effect on the retinal neuronal cell loss of wheat bran extract, TUNEL staining was performed.

Specifically, the tissue sections were deparaffinized and hydrated, and then treated with proteinase K (proteinase K) solution at 20 ug / ml, washed with PBS at 37 ^° C for 15 minutes, and then washed with PBS again. TUNEL reation mixture solution (In situ cell death detection kit, AP, Roche, Germany) was reacted under the microscope for 1 hour at 37 ^° C.

As a result, as shown in Figure 3, apoptosis cells (apoptotic cells) were not observed mainly in the ganglial cell layer (ganglial cell layer) in the normal group, apoptotic cells were observed mainly in the ganglial cells (diabetic group) in the diabetic group. . But In the MET group and the wheat extract extract high concentration group, it was confirmed that apoptosis was significantly reduced compared to the diabetic group (FIG. 3).

<3-4> Confirmation of Activation Inhibition Effect of Muller Cells ¾Rell cells, which function in the retinal tissue and maintain their shape and function similar to those of inflammatory cells, are activated in diabetes Exacerbates retinopathy by releasing substances. In order to confirm the inhibitory effect of the wheat flour extract of the present invention according to cell activation, the activation of Müller cells was confirmed through staining of the labeling substance GFAP expressed according to Müller cell activation.

As a result, as shown in FIG. 4, the GFAP positive response was significantly increased in the diabetic group compared to the normal group, and significantly decreased in the MET group compared to the diabetic group. In addition, it was confirmed that at high concentration of wheat flour extract GFAP-positive reaction was significantly reduced compared to the diabetic group (Fig. 4). <3-5> Confirmation of NF-kB translocation inhibitory effect

In order to confirm the inhibitory effect of the wheat flour extract of the present invention against NF-kB nuclear migration, nuclear extract was prepared from retinal tissues and subjected to EMSA.

Specifically, the tissue was nuclearly isolated using a nuclear extract io kit (Pnom ic, Redwood city, CA), and the NF-kB probe (probe) was prepared based on the NF-kB consensus sequence (Dig oligonucleotide 3-end labeling kit, Roche, German). After the labeled probe was labeled (fluorescence), the sample was electrophoresed at 10 ug in 4% native gel and moved to 90V for 1 hour, and the fluorescence was observed through Odyseey.

As a result, as shown in FIG. 5, the intranuclear migration was significantly increased in the diabetic group compared to the normal group, whereas in the high concentration group of MET and wheat flour extract, It was confirmed that the decrease in volume (Fig. 5).

<3-6> Confirmation of effects on Bax and Be 12

In order to confirm the effect of the wheatgrass extract of the present invention on Bax, an apoptosis inducing factor, and Bcl2, an apoptosis inhibitor, immunohistochemical staining was performed in the same manner as in Experimental Example <3-2>.

As a result, as shown in FIG. 6, it was observed that the diabetic group had a significantly stronger Bax staining in the ganglion cell layer than the normal group, and the staining was weaker in the MET group than in the diabetic group. On the other hand, it was observed that staining was significantly reduced compared to the diabetic group at high concentration rather than low concentration of wheat flour extract. However, Bcl2 was significantly decreased in the diabetic group compared to the diabetic group and the normal group, and significantly increased in the MET group compared to the diabetic group. On the other hand, the high concentration and high drug treatment group showed a significant difference compared to the diabetic group rather than the low concentration of wheat flour extract (Fig. 6).

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of powder

2 g of ethanol extract of <Example 1> of the present invention

1 g lactose

The above ingredients were mixed and layered in an airtight cloth to prepare a powder.

<1-2> Preparation of Tablet

100 nig of ethane extract of <Example 1> of the present invention

Corn starch 100 nig

Lactose 100 nig Magnesium stearate 2 nig After the above components were mixed, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of the encapsulant

Nucleic acid fraction 100 mg corn starch 100 mg of <Example 2> of the present invention

100 mg magnesium stearate per mg 2 mg After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for preparing a capsule.

Preparation of <1-4> Ring 1 g Lactose 1.5 g Glycerin 0.5 g of the nucleic acid fraction of Example 2 of the present invention

After mixing the components of the Λ group, it was prepared to be 4 g per ring according to a conventional method.

<1-5> Preparation of Granules Nucleic Acid Fraction 150 mg Soybean Extract 50 mg Glucose 200 mg Starch 600 rag After mixing the above components, 30% ethane was dried at 60 ^° C. by adding 100 ni to form granules, and then layered on fabric.

Preparation Example 2 Preparation of Food

Foods containing wheatgrass extract of the present invention were prepared as follows.

<2-1> Preparation of Flour Food

0.5 5.0 parts by weight of ethanol extract of <Example 2> of the present invention was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> Preparation of Soup and Gravy

0.1 to 5.0 parts by weight of ethanol extract of <Example 1> of the present invention was added to soups and broths to prepare meat products for health promotion, soups of noodles and broths.

<2-3> Preparation of ground beef

10 parts by weight of the ethanol extract of <Example 1> of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<2-4> Manufacture of dairy products

5 to 10 parts by weight of ethanol extract of <Example 1> of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Preparation of wire

Brown rice, barley, rice, and jujube were alphanized by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and sesame seeds were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The ethanol extract of <Example 1> of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by spraying and drying with a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

Cereals, seeds and the ethane of <Example 1> prepared above were prepared by combining the extract in the following ratio.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley) ，

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Ethanol extract (3 parts by weight) of <Example 1> of the present invention,

Manor (0.5 parts by weight) ，

Foxglove (0.5 part by weight)

Preparation Example 3 Preparation of Drinks

<3-1> Preparation of health drink

Homogeneously mixes minor ingredients such as liquid fructose (0.5%), oligosaccharide (>), sugar (2%), salt (0.5%), water (75¾) and 5 g of ethyl acetate fraction of Example 2 of the present invention. After sterilization, the product was packaged in a small packaging container such as a glass bottle, a plastic bottle, etc. <3-2> Preparation of Vegetable Juice

Vegetable juice was prepared by adding 5 g of ethyl acetate fraction of <Example 2> of the present invention to 1,000 m «of tomato or carrot juice.

<3-3> Preparation of Fruit Juice

Apple or grape juice of 1 g of ethyl acetate fraction of <Example 2> of the present invention , 000 ml, was added to prepare a fruit juice.

Claims

[Range of request]

[Claim 1]

Pharmaceutical composition for the prevention and treatment of diabetic complications containing the extract of ^ ^ Z / s / a hancei Hook, f. As an active ingredient.

[Claim 2]

The pharmaceutical composition for preventing and treating diabetic complications of claim 1, wherein the wheat flour extract uses branches, stems, or roots of wheat flour.

[Claim 3]

The pharmaceutical composition for preventing and treating diabetic complications of claim 1, wherein the wheat extract is extracted with water, water-soluble alcohol, or a mixed solvent thereof.

[Claim 4]

The pharmaceutical composition for preventing and treating diabetic complications of claim 3, wherein the water-soluble alcohol is ethane or methanol.

[Claim 5]

A pharmaceutical composition for preventing and treating diabetic complications, which contains a fraction prepared by adding an organic solvent to wheat flour extract as an active ingredient.

[Claim 6]

The method of claim 5, wherein the fraction is any one of nucleic acid fractions, ethyl acetate fractions, butanol fractions or water fractions obtained by suspending the wheat flour extract in water and then systematically fractionated with nucleic acid, ethyl acetate, butanol and water. Characteristic Pharmaceutical composition for preventing and treating diabetic complications.

[Claim 7]

The method according to any one of claims 1 to 5, wherein the diabetic complications are diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic cancer, diabetic osteoporosis, and A pharmaceutical composition for preventing and treating diabetic complications, characterized in that any one selected from the group consisting of diabetic atherosclerosis.

[Claim 8]

Health food composition for preventing and improving diabetic complications containing wheatgrass extract or fractions thereof as an active ingredient.

[Claim 9]

The method of claim 8, wherein the diabetic complication is selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease diabetic cancer, diabetic osteoporosis, and diabetic atherosclerosis Health food composition for preventing and improving diabetic complications, characterized in that any one.

[Claim 10]

A method of treating diabetic complications comprising administering a pharmaceutically effective amount of wheatgrass extract or a fraction thereof to an individual suffering from diabetic complications.

[Claim 111]

The method of claim 10, wherein the diabetic complications are diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic cancer, diabetic osteoporosis, and glucose A method of treatment, characterized in that any one selected from the group consisting of urinary atherosclerosis.

[Claim 12]

A method of preventing diabetic complications comprising administering to a subject a pharmaceutically effective amount of wheatgrass extract or a fraction thereof.

[Claim 13]

The method of claim 12, wherein the diabetic complication is a group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic cancer, diabetic osteoporosis, and diabetic atherosclerosis Prevention method, characterized in that any one selected from

[Claim 14]

Wheat flour extract or fractions thereof for use as a pharmaceutical composition for the prevention and treatment of diabetic complications.

[Claim 15]

Wheat flour extract or fractions thereof for use as a composition for the prevention and improvement of diabetic complications.

[Claim 16]

The method according to claim 14 or 15, wherein the diabetic complications are diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic cancer, diabetic osteoporosis, and diabetic atherosclerosis. Any one selected from the group consisting of atherosclerosis Wheat flour extract or fractions thereof