KR20150135746A - Pharmaceutical composition for prevention or treatment of diabetic complications and angioedema comprising Euphorbia pekinensis - Google Patents

Abstract

The Euphorbia pekinensis extract of the present invention or its purified fraction inhibits excessive production of the final glycation endproduct, inhibits the production of final glycation products in human retinal pigment epithelium, and inhibits hyperglycemia in the animal model zebrafish diabetic ophthalmic model To prevent the widening of the vitreoretinal vascular diameter due to VEGF, to inhibit the damage of blood-retinal barrier (BRB) by VEGF, to inhibit blood retinal barrier damage in the type 2 diabetic model, It has been shown to inhibit (repair) diabetic ocular disease by inhibiting the damage of occludin, one of the closely related proteins in the retina. At the same time, it reduces proteinuria, reduces the accumulation of 8-OHdG in urine and kidney, Inhibition of renal damage by inhibiting accumulation of the final glycation end product in the kidney and inhibition of lower limb in the animal model of the lower limb, A preventive and therapeutic pharmaceutical composition for the authentication and angioedema may be useful.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention and treatment of diabetic complications and angioedema containing an antipyretic extract or a purified fraction thereof as an active ingredient and a pharmaceutical composition for prevention and treatment of diabetic complications and angioedema comprising Euphorbia pekinensis

The present invention relates to a composition for preventing and treating diabetic complications and angioedema containing an antipark extract or a purified fraction thereof as an active ingredient.

The number of people with diabetes now exceeds 240 million worldwide and will rise to 380 million by 2025 and 60% will be in Asia, announced by the American Medical Association (JAMA) in 2009, The incidence of diabetic complications is rapidly increasing with the entry into the aging society and the onset of diabetes mellitus. In Korea, the prevalence of diabetes reaches 10%. Especially, diabetic complication patients are exploding due to the fact that the onset of diabetes is drawn to the younger generation and the life span is prolonged.

In the past five years, diabetic complications have increased by 60% and medical expenses have increased by 54% to KRW 203.5 billion (National Health Insurance Corporation, Aug. 2011). In other words, after 10 to 15 years of the onset of diabetes, almost all the organs of the body are damaged, resulting in retinopathy (diabetic retinopathy), diabetic cataract, diabetic nephropathy, diabetic neuropathy, Diabetic foot ulcers, diabetic heart disease, diabetic osteoposis, or diabetic arteriosclerosis.

Although diabetic medicines (metformin, rosiglitazone, gemiglo, and januvia) that are currently being administered are used to regulate the blood sugar level in the early stage of diabetes, there is a possibility that diabetic complications such as diabetic eye disease, , Neuropathy, foot ulceration, etc.) can not be fundamentally prevented or treated. Although diabetic complications start with diabetes, the mechanism of transition to diabetic complication is different from diabetes. Therefore, it is necessary to delay, prevent, or treat diabetic complications by using drugs that can be delayed or prevented by diabetic complications while controlling with a blood sugar modulating agent.

Chronic diabetic nephropathy requires hemodialysis and organ transplantation. Diabetic cataracts and retinopathy cause blindness. In the United States, diabetes is the leading cause of blindness in the 25 to 74 year old age group. A diabetic foot ulcer causes a terrible situation that requires the limb to be severed, and diabetic neuropathy is accompanied by knife-like pain. Diabetic heart disease causes sudden death. Therefore, even if the complication of diabetes mellitus is delayed for several years, the quality of life of the patient and the family will be changed, and it will greatly contribute to the national finances.

Representative factors inducing such diabetic complications have been reported, including production of the final glycation end product, activation of aldose reductase, activation of PKC isomer, and increase of hexosamine pathway ( Nature , 414, 813-820, 2000; Diabetologia , 38: 357-3941, 1995). The irreversible reaction of these factors accelerates the oxidative stress, making diabetic complications worse.

Nonenzymatic glycation of proteins is a process in which amino acid groups such as lysine residues of proteins and reducing sugars produce advanced glycation endproducts (AGEs) by a condensation reaction (milliad reaction) without enzymatic action It says. That is, the Schiff base, which is an early stage product, is formed, and the Schiff base and the adjacent ketoamine adduct are condensed with each other to produce an amadori-type early glycation product, , The reversible amorphous early glycosylation product is rearranged without degradation, resulting in a final glycation end product, which is an irreversible product. The resulting final glycation products bind or cross-link with proteins or lipids to produce irreversible products of glycated proteins or glycosylated lipids.

This final glycation product binds (crosses) with proteins or lipids such as basement membrane, plasma albumin, lens protein, fibrin, collagen and accumulates in tissues during the lifetime of the protein or lipid to change the structure and function of the tissue abnormally It causes complications. In addition, blood glucose is restored to the normal state, and the reaction with the already generated final glycation end product and protein or lipid continues, resulting in complications ( N. Engl. Med. , 1988, 318, 1315-1321). Also, in this state, the defensive system function against oxygen free radicals is degraded and oxidative stress is induced ( J. of Trad. Med . 2001, 18: 107-112). Based on these mechanisms, it has been reported that it is important to inhibit the production of final glycation products in order to delay or prevent the onset of diabetic complications ( N. Engl. Med . 1998, 318, 1315-1321).

Diabetic retinopathy leads to non-proliferative retinopathy and eventually to proliferative retinopathy, resulting in blindness and blindness of the retinal blood vessels and nerve cells in the chronic hyperglycemic state. In the hyperglycemic environment, the pericytes surrounding the capillaries of the retina begin to be damaged, leading to microaneurysm, leading to endothelial damage, and to the acellular capillary, which can not function as a blood vessel ). These abnormal neovascular vessels are very fragile due to their very weak walls, causing blood components to leak out, eventually leading to blindness. Several pathological phenomena occur because the intercellular tight-junction proteins that connect the endothelial cells with surrounding cells such as occludin or claudin are damaged. In order to prevent such diabetic retinopathy, it is necessary to prevent peripheral cell damage, which is an early symptom. Fenofibrate (Lipidil, Abott), approved by the FDA in December 2013, has been approved as a PPARα agonist for the treatment of diabetic retinopathy. The ACCORD study and the FILD study reported that 33% of strictly controlled diabetic patients prevented metastatic diabetic retinopathy, but delayed the transition to diabetic retinopathy by approximately 40%.

In 2014, the US FDA approved IIuvien (fluocinolone acetonide intravitreal implant, Alimera Science) was approved for treatment of diabetic macular edema.

Diabetic nephropathy is an important factor causing chronic diabetic nephropathy due to glycated albumin combined with a final glycoprotein and protein. Glycated albumin enters the ganglion cell more easily than normal albumin, and chronic high concentration of glucose stimulates mesangial cells to increase extracellular matrix synthesis. Glycosylated albumin and increased extracellular matrix result in fibrosis of the spermatid sphere. If the spermatozoa continue to be damaged by such a mechanism, extreme treatment methods such as hemodialysis or organ transplantation can only be used It will be.

Angioedema refers to a disease in which blood vessels that are deep in the skin, under the skin, or under the mucous membrane are increased in permeability and fluid is drained from the blood vessels and accumulated in the surrounding tissues (i.e., edema occurs) (Retina) edema or macular degeneration due to angioedema caused by damage to the retinal blood barrier, or the macular edema due to retinal hemorrhage. Varicose veins, etc. occur.

Age-related macular degeneration (AMD) is an irreversible disease that affects many elderly people and is a leading cause of blindness. It is increasingly prevalent due to the global aging of the population. In the United States, macular degeneration is a major cause of blindness, environmental and genetic factors also affect the onset, and smoking has been reported to be the most fatal disease. In addition, obesity, excessive antioxidant, and dietary fat intake also cause macular degeneration and have a more progressive effect. Therefore, eating a healthy diet, weight control, proper exercise, smoking cessation will reduce the incidence of macular degeneration. The prevalence of premature (dry) macular degeneration in the United States was 3.9% in the 40-50 age group, but 22.8% in the age group 75 and older (Beaver Dam Eye Study). Elderly patients over 75 years old had a 5.4% incidence and 7.1% had end stage AMD. 1.9% of Caucasians living in Australia are terminal Macular Degenerative, and during the 5-year period, the incidence was 0% in the young age group below 55 and 18.5% in the elderly group above 85 years. It is also similar for Asian Malay peoples (Blue Mountain Eye Study, Progress in Retinal and Eye Research , 1-15, 2014).

In Korea, the number of patients with mild macular degeneration has increased 7.4-fold in the past year, and the incidence in the 40-50 age group has increased nine-fold (2011). However, the most serious of these is the lack of macular degeneration. Lucentis of Switzerland Novartis is an antibody treatment and is very expensive and has a disadvantage that its vision can not be restored because it stops the progress of the disease.

Macular changes are classified as dry macular degeneration and wet macular degeneration. Dry macular degeneration is caused by the accumulation of a waste substance drusen in the maculae, which damages the macular connection between the choroid and the macular upper part, resulting in damage to the macula. As this progresses, it progresses to wet AMD. In other words, the blood vessels do not function normally due to accumulation of waste materials in the macular area, and nutrients and oxygen are not supplied, so new abnormal blood vessels start to be formed (choroidal neovascularization: CNV). The resulting blood vessels are very weak in the walls, causing proteins or red blood cells in the blood vessels to seep into the macula and the retina, eventually resulting in various factors such as photoreceptor (rod-cone photoreceptor) and retinal pigment epithelial layer death ( Nutrition Research , 34, 95-105, 2014; Plos one , 8, e71064, 2013).

Retinal pigment epithelial cells (RPEs) play an important role in maintaining healthy eyes by supporting the Bruchs membrane (BrM) while maintaining a non-proliferative state. Cystatin C secreted from the retinal pigment epithelium is a powerful cysteine proteinase inhibitor that plays an important role in normal regulation of protein circulation in BrM. However, excessive accumulation of the final glycation end product results in a decrease in the expression and secretion of cystatin C protein, leading to an imbalance of proteolytic activity in the RPE basal part, resulting in macular degeneration. Thus, it has been reported that macular degeneration can also be prevented (inhibited) when inhibiting the production of the final glycation end product (Kay P, et al., IOVS , 2014, 55 (2), 926-34).

VEGF (Vascular Endothelial Growth Factor) is secreted from the retinal pigment epithelium layer to regulate the periphery of the Bruchs membrane (BrM) and regulate the growth and density of the capillary endothelial cells. In steady state, VEGF secretion is very tightly controlled to prevent neovascularization. However, if VEGF secretion is not tightly regulated, it is a crucial factor in reaching late stages of macular degeneration. Abnormal elevation of VEGF secretion results in abnormal and weak blood vessels and destruction of blood vessels ( J. Cell. Mol. Med. , 17, 7, 833-843, 2013).

The varicose vein is a disease in which the vein is visible outside the skin. Veins distributed in the limbs include a large deep vein between the muscles, a superficial vein that is directly under the skin, The vein wall is weakened when the pressure in the lower limb is increased, and the flow of blood in the vein is reduced. It is always caused by the backflow of the blood to the heart and the veins stretching, which can damage the valve that keeps it constant toward the heart.

Recently, it has been disclosed that Euphorbia ebracteolata belonging to the major pole can be used as a composition for prevention and treatment of arthritis. Ingenol-3-palmitate or Ingenol-3-myristinate compounds, which are extracted from the Korean counterpart, exhibit a potent anti-inflammatory activity against arthritis and are toxic And no side effects (Korea, Application No. 10-2011-0125873).

Accordingly, the present inventors have conducted intensive studies for the prevention and treatment of diabetic complications and angioedema, and found that the antipark extract or its purified fraction inhibits excessive production of ultrafine glycoconjugates that occur in the chronic diabetic state, Inhibits the production of glycoconjugates, prevents the widening of the diameter of the vitreous retinal vessels due to hyperglycemia in the animal model zebrafish diabetic ophthalmic model, causes end-stage symptoms of diabetic retinopathy or retinitis (macular) edema Inhibition of blood-retinal barrier breakage (BRB) by VEGF, inhibition of blood retinal barrier in type 2 diabetic model, inhibition of occludin damage, It has been shown to prevent (cure) the disease, reduce proteinuria, reduce the accumulation of 8-OHdG in urine and kidneys, Inhibiting renal injury by inhibiting accumulation of glycation products, and exhibiting an effect of suppressing lower limb edema in the animal model of the lower limb. Thus, the present invention has been completed upon confirming that diabetic complications and angioedema are effectively prevented and treated.

It is an object of the present invention to provide a pharmaceutical composition for preventing and treating diabetic complications and angioedema containing an extract of Euphorbia pekinensis or a purified fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for prevention and treatment of diabetic complications and angioedema containing an extract of Euphorbia pekinensis or a purified fraction thereof as an active ingredient.

The present invention also provides a diabetic complication and a dietary supplement for prevention and improvement of angioedema, which comprise a dipole extract or a purified fraction thereof as an active ingredient.

The Euphorbia pekinensis extract of the present invention or its purified fraction inhibits excessive production of the final glycation endproduct, prevents the widening of the vitreous retinal blood vessel diameter due to hyperglycemia in the zebrafish diabetic ophthalmic model, It inhibits the damage of the blood-retinal barrier (BRB), inhibits the damage of the blood retinal barrier in the type 2 diabetes model, and prevents the occlusion of the diabetic ocular disease ), Which is effective in reducing proteinuria, reducing the accumulation of 8-OHdG in urine and kidney, inhibiting the accumulation of the final glycation end product in the kidney, and thus preventing the renal damage. Therefore, the pharmaceutical composition for preventing and treating diabetic complications , And the dipivalent extract or its purified fraction exhibits the effect of breaking the cross-linking of the final glycosylated product and protein In addition, it has been confirmed that the human retinal pigment epithelium cell line exhibits an inhibitory effect on the final glycation endogenesis and exhibits an effect of inhibiting the blood retinal barrier that induces angioedema in an animal model. Therefore, the antipark extract of the present invention, or its purified fraction, And a pharmaceutical composition for prevention and treatment of angioedema.

Brief Description of the Drawings Fig. 1 is a diagram showing a method for producing a purified fraction (EP70C) from an Euphorbia pekinensis extract (EP70).
FIG. 2 is a graph showing the effect of inhibiting the production of final glycation products in the human retinal pigment epithelium cell line under hyperglycemic conditions:
HG: hyperglycemic treated group;
BSA: bovine serum albumin treated group;
EP70C: Experimental group treated with 10, 20, 50 μg / ml of the antipyretic fraction (EP70C); And
AG: Positive control group, aminoguanidine.
3 is a graph showing the vitreous blood vessel image, blood vessel diameter and vasodilation inhibitory effect in a zebrafish model:
Normal group: positive control group;
Diabetic group: hyperglycemic group;
EP70C-5: Experimental group treated with 5 占 퐂 / ml of fractionated fraction (EP70C); And
EP70C-10: Experimental group treated with 10 占 퐂 / ml of fractionated fraction (EP70C).
4 is a graph showing the amount of 8-OHdG (8-Oxo-2'-deoxyguanosine) in urine and proteinuria of a mouse model for the prevention and treatment of diabetic nephropathy.
NOR: positive normal group;
DM: diabetic group;
Feno: Fenofibrate 100 mg / kg administered group;
EP70C-100: 100 mg / kg administration group of antipeptor fraction (EP70C); And
EP70C-250: 250 mg / kg of the antipyretic fraction (EP70C).
Figure 5 is a diagram showing the accumulation of 8-OHdG (8-Oxo-2'-deoxyguanosine) accumulation in the shrine sphere:
Feno: Fenofibrate 100 mg / kg administered group;
EP70C-100: 100 mg / kg administration group of antipeptor fraction (EP70C); And
EP70C-250: 250 mg / kg of the antipyretic fraction (EP70C).
FIG. 6 is a diagram showing the accumulation of final saccharides in the shrine sphere by dyeing:
Feno: Fenofibrate 100 mg / kg administered group;
EP70C-100: 100 mg / kg administration group of antipeptor fraction (EP70C); And
EP70C-250: 250 mg / kg of the antipyretic fraction (EP70C).
FIG. 7 is a graph showing the efficacy of the antipyretic retinopathy prevention efficacy in the prevention of blood retinal barrier damage after administration of the antipyretic fraction (EP70C) in db / db mouse of type 2 diabetes model for 6 weeks:
NOR: normal animal group (nondiabetic heterozygote db / + mouse);
DM: diabetic animal group (C57BL / KsJ-Leprdb / db diabetic mouse);
FENO: fenofibrate 100 mg / kg / day group;
EP70C-100: 100 mg / kg administration group of antipeptor fraction (EP70C); And
EP70C-250: 250 mg / kg of the antipyretic fraction (EP70C).
FIG. 8 is a graph showing the efficacy of the prevention of acellular damage, a vaginal protein in the diabetic retinopathy prevention efficacy after administering the fractionated tablet fraction (EP70C) for 6 weeks in type 2 diabetic model db / db mice:
NOR: normal animal group (nondiabetic heterozygote db / + mouse);
DM: diabetic animal group (C57BL / KsJ-Leprdb / db diabetic mouse);
FENO: 100 mg / kg / day of phenobibrate; And
EP70C-100: 100 mg / kg administration group of antipeptor fraction (EP70C);
EP70C-250: 250 mg / kg of the antipyretic fraction (EP70C).
FIG. 9A is a graph showing fluorescence intensity measured by inhibiting the damage of the blood retinal barrier in a mouse model in which collapse of a blood-retinal barrier (BRB)
NOR: retinas of positive control mice;
VEGF: Vascular endothelial growth factor (VEGF) -treated mouse retina;
EP70: retinas of mice of the antiprotozoal (EP70) -treated group; And
EP70C: retinas of mouse treated with fractionated refinement fraction (EP70C).
FIG. 9B is a graph showing an effect of inhibiting damage to the blood retinal barrier in a mouse model in which collapse of blood retinal barrier is induced.
NOR: retinas of positive control mice;
VEGF: retinas of VEGF-treated mice;
EP70: retinas of mice of the antiprotozoal (EP70) -treated group; And
EP70C: retinas of mouse treated with fractionated refinement fraction (EP70C).
FIG. 10 shows the effect of preventing and treating lower limb vein in an animal model that induced formalin as a lower limb vein.
Normal: normal animals (SD rat);
Edema: animal group causing lower limb swelling;
EP70C-100: 100 mg / kg administration group of antipeptor fraction (EP70C); And
EP70C-250: 250 mg / kg of the antipyretic fraction (EP70C).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for prevention and treatment of diabetic complications and angioedema containing an extract of a bipolar plate or a purified fraction thereof as an active ingredient.

The above-mentioned dipole extract is preferably, but not necessarily, prepared by a manufacturing method comprising the following steps:

1) extracting the counter electrode with an extraction solvent;

2) filtering the extract of step 1); And

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

In the above method, the counter electrode of step 1) can be used without limitation such as grown or commercially available.

The extraction solvent is preferably water, alcohol or a mixture thereof. As the alcohol, C1 to C2 lower alcohol is preferably used, and as the lower alcohol, 30% ethanol, 50% ethanol, 70% ethanol or methanol is preferably used. The extraction method is preferably, but not limited to, decompression high-temperature extraction, hot water extraction, reflux extraction, hot water extraction, cold extraction, room temperature extraction, ultrasonic extraction or steam extraction. It is preferable that the extraction solvent is added in an amount of 1 to 10 times the amount of the counter electrode to be extracted. The extraction temperature is preferably 30 to 100 DEG C, but is not limited thereto. The extraction time is preferably from 2 to 48 hours, but is not limited thereto. In addition, the number of times of extraction is preferably 2 to 5 times, but is not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

The counter-electrode fraction was obtained by dissolving the counter electrode extract obtained from the above-described preparation method in 100% primary distilled water, filtering the resulting filtrate through an ion exchange resin such as Amberlite XAD-2, Diaion MClgel HP20 or Kogel BG4600, Subsequently, the remaining organic matter components adsorbed on the column were desorbed again with ethanol, hexane, chloroform, ethyl acetate or butanol, preferably 50% ethanol solution to prepare soluble fractions, and then, using a vacuum decompression concentrator or a vacuum rotary evaporator But it is not limited thereto.

The diabetic complication may be any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic neuropathy, diabetic foot ulcer, diabetic heart disease, diabetic osteoporosis or diabetic atherosclerosis , But is not limited thereto.

The angioedema may be any one selected from the group consisting of retinal edema, macular degeneration, macular edema, retinal degeneration, and lower limb vein, but is not limited thereto.

In a specific example of the present invention, the present inventors confirmed the effect of inhibiting the production of final glycation endproducts of the extract of the major pole, the purified fraction and the reflux extract, and the fraction of the antipark (EP70C) (EP70) was 25.8 times higher than aminoguanidine and 150 times higher than that of the antifouling fraction (EP70C). In addition, the reflux of 30%, 50% and 70% ethanol was 3.3 times, 3.5 times and 1.8 times higher than that of the antiprotozoal extract (EP70) and 84.5 times, 89.3 times and 47.3 times higher than that of aminoguanidine, respectively (See Table 1), and it was confirmed that human RPE cell line also significantly inhibited the production of the final glycation end product (see FIG. 2).

In order to examine the therapeutic effect of diabetic retinopathy in the zebrafish model of the fractional fraction (EP70C), the diabetic zebrafish embryos were raised in a 130 mM glucose environment and the diameter of vitreous blood vessels in the hyperglycemic group was 13.86 ± 0.42 AU was significantly increased (P <0.001), but in the 5 ㎍ / ㎖ antiprotozoal fraction (EP70C) treated group was 12.33 ± 0.73 AU and in the 10 ㎍ / ㎖ antiprotozoal fraction (EP70C) treated group was 11.52 ± 0.53 AU (P < 0.01) and inhibited vasodilation (see FIG. 3).

Six-week-old male (db / db) mice were divided into two groups: normal (NOR), diabetic (DM), antipyretic (DM) (EP70C-100), 100 mg / kg of fraction (EP70C), 250 mg / kg of EP70C, and 100 mg / kg of Fenofibrate , And the amount of 8-OHdG in urine and proteinuria was significantly decreased in the 250 mg / kg administration group (EP70C-250) of the antifouling fraction (EP70C) (see FIG. 4). In addition, the accumulation of 8-OHdG in the shrines was found to be decreased in the Feno-treated group and the 100 mg / kg-administered group (EP70C-100) of the antifouling fraction (EP70C-100) In the group treated with ㎎ / kg (EP70C-250), it was confirmed that the accumulation of 8-OHdG was restored to the normal group level (see FIG. 5).

In addition, the present inventors confirmed that the accumulation of the final glycation products in the shrines was reduced in the Feno-administered group and the 100 mg / kg administered group (EP70C-100) of the antiprotozoal fraction (EP70C-100) EP70C) 250 mg / kg administration group (EP70C-250) confirmed that the accumulation of the final glycation products recovered to the normal group level (see FIG. 6).

In addition, the present inventors analyzed the degree of leakage by injecting a fluorescent substance at the time of autopsy in order to confirm whether or not the fractional fraction (EP70C) was prevented and treated for blood retinal barrier damage. As a result, And the effect of preventing and treating retinal vascular damage was confirmed (see FIG. 7). In addition, a dose-dependent significant inhibition of the damage and the therapeutic effect were confirmed in the administration group of occludin (EP70C) of occludin, which is one of the tightly linked proteins of the retina (see FIG. 8).

In order to confirm the effect of retinal (macular) edema (degeneration) effect in the animal model of the major pole extract (EP70) and the major pole fraction (EP70C), the present inventors measured the VEGF protein (P <0.05) due to blood retinal barrier damage in all subjects of the VEGF-treated group, which was 2.8 times more efflux than that of the control group (P <0.05) (EP70C) treated group did not prevent the fluorescent substance from leaking out of the blood vessel due to damage of the blood retinal barrier. However, the fluorescein leakage due to VEGF was significantly (P < 0.05) to inhibit the damage of the blood retinal barrier (see FIG. 9).

In addition, the present inventors confirmed the anti-lower limb edema effect in an animal model of a fractionated fraction (EP70C) of a counter electrode extract of the present invention (see FIG. 10).

Therefore, the Euphorbia pekinensis extract of the present invention or its purified fraction inhibits excessive production of the final glycation products, inhibits the production of ultimate glycation products in human retinal pigment epithelium cell lines, and inhibits the production of zebrafish diabetic ophthalmic model To prevent the widening of the vitreoretinal vascular diameter due to hyperglycemia, to inhibit the damage of the blood-retinal barrier (BRB) by VEGF, and to inhibit the damage of the blood retinal barrier in the type 2 diabetic model (Treatment) of diabetic ocular disease by inhibiting the damage of occludin, which is one of the close connective proteins of the retina. At the same time, it decreased proteinuria and decreased the accumulation of 8-OHdG in urine and kidney Inhibition of kidney damage by inhibiting the accumulation of the final glycation end product in the kidney, Diabetic complications and angioedema. The present invention also provides a pharmaceutical composition for prevention and treatment of diabetic complications and angioedema.

The composition containing the counter electrode extract of the present invention or the purified fraction thereof may further contain one or more active ingredients showing the same or similar functions in addition to the above components.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . &Lt; / RTI &gt; Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, Sucrose, Lactose, Gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are commonly used simple diluents . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally in accordance with the intended method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, . The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and the dose may be administered 1 to 6 times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

Further, the present invention provides a diabetic complication and a health functional food for preventing or ameliorating angioedema, which comprises a major pole extract or a purified fraction thereof as an active ingredient.

The Euphorbia pekinensis extract of the present invention or its purified fraction inhibits excessive production of the final glycation endproduct, inhibits the production of final glycation products in human retinal pigment epithelium, and inhibits hyperglycemia in the animal model zebrafish diabetic ophthalmic model To prevent the widening of the vitreoretinal vascular diameter due to VEGF, to inhibit the damage of blood-retinal barrier (BRB) by VEGF, to inhibit blood retinal barrier damage in the type 2 diabetic model, It has been shown to inhibit (repair) diabetic ocular disease by inhibiting the damage of occludin, one of the closely related proteins in the retina. At the same time, it reduces proteinuria, reduces the accumulation of 8-OHdG in urine and kidney, Inhibition of renal damage by inhibiting accumulation of the final glycation end product in the kidney and inhibition of lower limb in the animal model of the lower limb, It can be effectively used to increase and angioedema prevention and improved health functional food for.

The health functional food of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be selected from the range of 0.01 to 0.04 part by weight, specifically about 0.02 to 0.03 part by weight per 100 parts by weight of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, protective colloid thickener, pH adjuster, stabilizer, preservative, glycerin, A carbonating agent used in a carbonated beverage, and the like. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

&Lt; Example 1 > Preparation of a dipole extract and its fractions

Euphorbia pekinensis was purchased from Kyungdong Market, Seoul, Korea for the preparation of the extract of the present invention, and is being kept at the Korean Institute of Oriental Medicine (Daejeon, Korea) for complications of diabetes mellitus.

&Lt; 1-1 > Preparation of antiproto extract

3 kg of the counter electrode was repeatedly extracted with 70% ethanol (30 L) at room temperature. The extract was filtered and concentrated under reduced pressure at about 50 ° C and dried to obtain a counter electrode extract (EP70).

&Lt; 1-2 > Production of fractionated tablet fraction

The mobile phase and stationary phase conditions were used to prepare the counter-electrode fraction from the counter electrode extract (40 g) prepared in Example <1-1>.

Specifically, 50% ethanol (5 L) was added to 100% primary distilled water (3 L) and then the eluted solution was concentrated under reduced pressure and lyophilized to obtain 12 g of a counter-current fraction (EP70C) (Fig. 1).

&Lt; 1-3 > Preparation of antigorous reflux extract

30 g of 30% ethanol, 30 ml of 50% ethanol and 30 ml of 70% ethanol were added to 5 g of the dried counter electrode, respectively, and the mixture was subjected to reflux extraction at about 50 ° C. The extract was filtered and concentrated under reduced pressure and lyophilized to obtain 0.46 g of a 30% ethanol reflux extract (EP30R), 0.55 g of a 50% ethanol reflux extract (EP50R) and 0.46 g of a 70% ethanol reflux extract (EP70R) Respectively.

Experimental Example 1 Confirmation of Inhibitory Effect on Formation of Advanced Glycation Endproducts (AGEs)

The nonenzymatic glycation of protein is the result of the condensation reaction (milliad reaction) of amino acid groups such as lysine residues of proteins and reducing sugars without enzymatic action, resulting in final glycation products. Unlike the reversible amido type early saccharide, the final glycation end product produced in the chronic hyperglycemic environment is an irreversible reaction product. Once formed, it does not decompose when the blood glucose is restored to normal, but accumulates in the tissue during the protein survival period Gene, and so on, thereby causing diabetic complications by abnormally changing the structure and function of the tissue. In the present invention, the following experiment was conducted in order to confirm the inhibitory effect of the extract of the counter electrode, the purified fraction and the reflux extract on the final glycation endogenesis.

Specifically, the protein source was 10 mg / ml bovine serum albumin (Sigma, St. Louis, Mo., USA) dissolved in 50 mM phosphate buffer (pH 7.4) M glucose were each used. The compositions of the present invention prepared in Example 1 and aminoguanidine as a positive control were dissolved in 30 μl of 0.2% DMSO and dissolved again in 15% tween 80. The protein source, the sugar source, and the compositions of the present invention thus prepared were mixed to prepare a total of 1 ml, and the mixture was incubated at 37 ° C for 7 days. At that time, 0.02% sodium azide was added to prevent bacterial growth during the incubation period. After the incubation, the fluorescence was measured at 450 nm (Spectrofluorometric detector: Bio-TEK, Synergy HT, USA; Ex: 350 nm; Em).

As a result, as shown in Table 1, the antifouling fraction (EP70C) had a 5.8-fold higher inhibitory effect on the final glycation endproduct than the major fraction extract (EP70) It was confirmed that the purified fraction (EP70C) was 150 times higher. The reflux of 30%, 50%, and 70% ethanol was 3.3, 3.5 and 1.8 times higher than that of the major extract (EP70) and 84.5 times, 89.3 times and 47.3 times higher than that of aminoguanidine, respectively. (EP70C) and counter-current reflux extracts (EP30R, EP50R and EP70R) were superior to each other in inhibiting the production of final glycation products, considering that aminoguanidine is a synthetic single compound Table 1).

IC ₅₀ ([mu] g / ml) Efficacy comparison Contrast Extract (EP70) In contrast to positive control (AG) Room temperature extraction method Dipole extract (EP70) 3.08 ± 0.02 × 25.8 The major fraction refined fraction (EP70C) 0.53 + 0.02 × 5.8 × 150.0 Reflux extraction Antipolar reflux extract (EP30R) 0.94 + - 0.01 × 3.3 × 84.5 Antipolar reflux extract (EP50R) 0.89 + 0.02 × 3.5 × 89.3 Antipolar reflux extract (EP70R) 1.68 ± 0.05 × 1.8 × 47.3 Aminoguanidine (positive control) 79.47 ± 1.43

<Experimental Example 2> Inhibitory effect of endogenous glycoprotein production on human retinal pigment epithelial cell line in hyperglycemic environment

The inhibitory effect of EP70C produced in Example 1 on human glycoprotein epithelial cell line in the hyperglycemic environment was confirmed.

Specifically, human retinal pigment epithelial cell line (ARPE-19: ATCC No. CRL-2302) was cultured in Dulbeccos modified Eagles medium (DMEM, Gibco, USA) in a 5% CO2 incubator in a hyperglycemic environment. BSA at a final concentration of 500 占 퐂 / ml and hyperglycemia at 25 mM, and the EP70C of the present invention was treated with concentration (10, 20, 50 占 퐂 / ml). The positive control group also treated aminoguanidine (AG, 10 mM). After washing with 1 × PBS, the samples were treated with a buffer solution (Laemmli Sample Buffer, Cat. No. 161-0737, Bio-Rad Laboratories, CA, USA) and boiled at 100 ° C for 5 minutes. Then, BCA (Pierce Biotechnology, IL, USA). Proteins were electrophoresed (120 V, 2 hrs) in 10% polyacrylamide gels (PAGE) containing SDS and transferred to a PVDF membrane (Bio-Rad Laboratories) with transfer buffer (0.25 M Tris, 1.92 M Glycine, pH 8.3-8.4) , CA, USA) (250 mA, 1.5). The cells were blocked with 5% non fat milk in TBS-T (200 mM Tris, 1.37 M NaCl, 0.05% Tween 20) and reacted at 4 ° C with AGEs antibody (Anti-AGEs monoclonal Ab, Clone No. 6D12). After washing, the HRP-conjugated secondary antibody was reacted, washed again, and analyzed with enhanced chemiliuminescence (ECL), analyzed with LAS-3000 (Fuji film, JPN), and statistically treated with GraphPad Prism 5 (San Diego).

As a result, as shown in Fig. 2, the EP70C of the present invention inhibited the production of final glycation products even in human retinal pigment epithelial cells in a hyperglycemic environment in a concentration-dependent manner (10 μg / ml, 20 μg / ml, 50 μg / ml) (Fig. 2).

<Experimental Example 3> The effect of the crossing-linked AGE and protein complex on the final glycated product was confirmed.

In order to confirm the breakage effect on the substrate protein and the cross-linking of the final glycation products of the major pole extract and the purified fraction thereof, the following experiment was conducted.

In the positive control group, ALT-711 (Alteon Inc., Ramsey, NJ), which is in a clinical stage and has excellent cross-linking effect of the final glycosylated product and the protein substrate, was used. The cross- Was carried out by modifying the experimental method of Vasan.

Specifically, 1.0 μg of AGE-BSA (Transgenic Inc. Kobe, Japan) was dispensed into a collagen-coated 96-well microtiter plate (Greiner Bio-One, Germany) And cross-linked with AGE-BSA and collagen. Thereafter, the unbound AGE-BSA was washed three times with 0.05% PBST, and the mixed extract of the present invention and ALT-711, which was a positive control, were added, followed by incubation at 37 ° C for 4 hours. BSA (6D12, Transgenic Inc., Kobe, Japan) was used to wash the cells with 0.05% PBST and cross-link with collagen to detect residual AGE-BSA. Mouse monoclonal anti- Linked goat anti-mouse IgG antibody (1: 250) was diluted 1: 250 and then incubated at 37 ° C for 1 hour. After 1 hour, the cells were washed with 0.05% PBST and washed with HRP- (Santa Cruz, CA, USA), and the absorbance was measured (450 nm) after color development using TMB (3.3 ', 5,5'-tetramethylbenzidine) as a substrate. Cross-linking breaking of AGE-BSA was calculated using the following equation (2).

As shown in Table 2, the ethanol extracts of 30%, 50% and 70% of the antipodes were superior to the positive control by 102.5, 164.2 and 100.4 times, respectively (Table 2).

sample IC ₅₀ ([mu] g / ml) Efficacy comparison Compared to ALT-711 Antipolar reflux extract (EP30R) 166.50 ± 36.39 × 102.5 Antipolar reflux extract (EP50R) 103.90 ± 14.87 × 164.2 Antipolar reflux extract (EP70R) 169.95 ± 9.84 × 100.4 ALT-711 (positive control) 17,060 ± 2.35 -

<Experimental Example 4> Anti-diabetic retinopathy effect in a zebrafish model

Zebrafish belongs to the vertebrate and is in the spotlight as an in vivo system that is similar to humans and is fertile and can be quickly tested for efficacy at relatively low prices compared to rodents (Disease models & Mechanism, 236-245, 2010; J. of Molecular Endocrinology 38, 433-440, 2007). In the present invention, a transformed zebrafish (Tg (kdr: EGFP)) expressing a fluorescent protein specifically in vascular endothelial cells was used.

Specifically, as a diabetic group, transgenic zebrafish embryos were raised in a 130 mM glucose environment. In the treatment group of the present invention, the 130 mM glucose and the counterpart fraction (EP70C) were simultaneously treated at two concentrations (5 ㎍ / ㎖, 10 ㎍ / ㎖) and contacted for a certain period. The thickness of the vitreous retina was measured at about 30 μm around the optic disc and analyzed with GraphPad Prizm 5 software.

As a result, as shown in Fig. 3, the vitreous diameter of the normal group was 8.59 ± 0.26 AU, and the vitreous diameter of the hyperglycemic group was significantly expanded to 13.86 ± 0.42 AU (P <0.001), but the 5 ㎍ / It was found that 12.33 ± 0.73 AU of the purified fraction (EP70C) treated group and 11.52 ± 0.53 AU of the 10 ㎍ / ㎖ treated fraction (EP70C) were significantly inhibited (P <0.01). (EP70C) treated group significantly (P <0.01) inhibited the vitreoretinal vasodilation by 29.06 ± 13.86% and 44.48 ± 9.97%, respectively, on the basis of hyperglycemic treatment group Respectively. It was confirmed that the antifouling fraction (EP70C) was excellent in the prevention and treatment of ocular diseases caused by diabetes (Fig. 3).

Experimental Example 5 Anti-diabetic complication effect of the fractionated tablet fraction (EP70C) in the type 2 diabetic animal model

<5-1> Experimental animal breeding and experimental design

In order to confirm the therapeutic effect of anti-diabetic complication in the type 2 diabetic animal model of the composition of the present invention (EP70C), the following experiment was conducted.

Specifically, we used a 6 - week - old male (db / db) mouse after 1 week of adaptation. After one week, only individuals with blood glucose level of 350 ㎎ / ㎗ were selected and randomly divided into groups. Feed and drinking water were fed free. (NOR), diabetic group (DM), antipyretic fraction (EP70C) 100 ㎎ / ㎏ (control group) were administered orally for 6 weeks. (EP70C-100), a 250 mg / kg administration group (EP70C), and a 100 mg / kg administration group (Feno) of fenofibrate. Body weight, water, and feed intake were measured once a week, and autopsy was performed at 6 and 12 weeks. Urine samples were collected 24 hours before the autopsy and stored at -80 ° C. From the blood, urine and tissues of each group, blood biochemical factors, ELISA measurements, nerve conduction measurements, neurofibrillary tangles, histopathological examination, Western blotting, immunohistochemical staining, The effect of anti - diabetic complication was confirmed by quantitative and qualitative analysis such as barrier damage measurement, measurement of disappearance of tight junction protein, and VEGF measurement.

<5-2> Identification of anti-diabetic nephropathy effect by reduction of proteinuria in urine and antioxidant effect

To assess the efficacy of prophylaxis and treatment of chronic renal failure, we analyzed the amount of 8-OHdG (8-Oxo-2'-deoxyguanosine) in urine, proteinuria and urine 24 hours a day. As a result, as shown in FIG. 4, these indicators were significantly increased in the diabetic group as compared with the normal group. The positive control group was not effective and the amount of 8-OHdG in urine and proteinuria was significantly decreased in the 250 ㎎ / ㎏ administration group (EP70C-250) (Fig. 4).

<5-3> Confirmation of anti-diabetic nephropathy effect by 8-OHdG accumulation inhibitory effect on the shrine spheres

The accumulation inhibitory effect of 8-OHdG on renal glomeruli of the fractional fraction (EP70C) was analyzed by immunohistochemical staining.

As a result, as shown in FIG. 5, the accumulation of 8-OHdG was decreased in the Feno-treated group and the 100 mg / kg-administered group (EP70C-100) , And the accumulation of 8-OHdG was recovered to the normal level, especially in the 250 mg / kg administration group (EP70C-250) of the antifouling fraction fraction (FIG. 5).

<5-4> Confirmation of anti-diabetic effect by inhibiting the accumulation of the final glycation products in the shrine sphere

The inhibitory effect on the accumulation of the final glycation products in the renal glomeruli of the antifouling fraction (EP70C) of the present invention was analyzed by immunohistochemical staining.

As a result, as shown in FIG. 6, the accumulation of the final glycation products was decreased in the Feno-treated group and the 100 mg / kg-treated group (EP70C-100) In particular, it was confirmed that the accumulation of the final glycation products was restored to the normal group level in the 250 mg / kg administration group (EP70C-250) of the purified fraction (FIG. 6). As a result, it was confirmed that the antifouling fraction fraction (EP70C) was excellent in the prevention and treatment of diabetic nephropathy.

<5-5> Anti-diabetic retinopathy effect by prevention of blood retinal barrier damage

 In order to confirm whether the antifouling fraction (EP70C) of the present invention prevents and treats blood-retinal barrier breakage (BRB) damage, the degree of leakage was analyzed by injecting fluorescent material at the time of autopsy.

As a result, as shown in FIG. 7, no fluorescence leakage was observed in the normal group, but fluorescence leakage of the diabetic group was significantly increased 2-fold compared with that in the normal group, and retinal tissue was observed brightly, , But the fluorescence efflux was significantly decreased in the group treated with the antifouling fraction (EP70C), thus confirming the preventive and therapeutic effect of retinal vascular injury. However, it has been confirmed that fenofibrate, which is approved as a diabetic retinopathy drug, does not prevent and treat damage to the blood retinal barrier (Fig. 7).

<5-6> Anti-diabetic retinopathy effect by inhibiting the damage of occludin (occludin) in the retina

The inhibitory effect of occludin, which is one of the retinal adhesion-linked proteins of EP70C of the present invention, and its therapeutic effect were confirmed.

As a result, as shown in FIG. 8, ochrodine was remarkably degraded in the diabetic group, but it was confirmed that the group treated with the counter-electrode fraction (EP70C) was significantly inhibited and treated in a concentration-dependent manner. As a result, it was confirmed that the effluent fraction fraction (EP70C) was almost normal and excellent in efficacy. However, it has been confirmed that fenofibrate, which is approved as a diabetic retinopathy drug, can not prevent and treat damage to the blood retinal barrier (FIG. 8).

Experimental Example 6: Effect of anti-retinopathy on animal model inducing blood-retinal barrier (BRB) collapse

Blood edema causes damage to the retinal blood barrier, resulting in diabetic complications such as macular degeneration, retinopathy, retinal edema or diabetic retinopathy. In order to confirm the therapeutic effect of macular degeneration, retinopathy, retinal edema or antidiabetic retinopathy in an animal model of the composition of the present invention, the following experiment was conducted.

Specifically, the experimental animals were used after 7-week-old male (SD) rats were adapted, and feed and drinking water were fasted free. VEGF protein (Vascular endothelial growth factor, R & D research, USA) was administered to the left eye of the rat to induce the collapse of the blood-retinal barrier (BRB) The purified fraction (EP70C) was injected. After 24 hours of drug administration, anesthesia was performed, and 50 mg / ml of fluorescein-dextran was injected into the left ventricle. After 10 minutes, the retina was removed by removing the eyeball. The separated retina was mounted and observed with an aqueous mounting medium. For quantitative analysis, 50 mg / ml fluorescein-dextran was injected into the left ventricle, and blood was collected. Then, the fluorescein-dextran remaining in the blood vessel at a rate of 60 ml / After removing the eyeballs, the retina was removed. The separated retinas were weighed and centrifuged, and the supernatant was measured for fluorescence using an ELISA reader.

As a result, as shown in FIG. 9, fluorescence leakage was not observed in the normal group, but fluorescence was significantly (P <0.05) outflowed by about 2.8 times due to blood retinal barrier damage in all the subjects of the VEGF administration group (The more visible the blood retina is damaged). (EP70) treated group did not prevent the fluorescent substance from leaking out of the blood vessel due to damage of the blood retinal barrier. On the other hand, it was confirmed that the efflux of fluorescent substance by VEGF was significantly inhibited (P < 0.05) and the damage of the blood retinal barrier was inhibited (FIG. 9).

&Lt; Experimental Example 7 >

<7-1> Experimental animal breeding and experimental design

A 6-week-old male SD lette was purchased from Orient (Seongnam, South Korea) and used after purification for 1 week. After one week of refinement, each animal was randomly grouped as follows:

EP70C (50 mg / kg / day) and EP70C-100: lower limb induced group + EP70C (100 mg / kg / day) The administration group.

Each group was orally administered once daily in the morning from 3 days before induction of lower limb swelling, and induced edema in the afternoon after 4 days of pretreatment. EP70C was suspended in 0.5% CMC, and 0.5 CMC alone orally was administered to the normal group and the lower limb-induced group. The induction of lower limb edema was induced by administering 0.1 ml of saline containing 2.5% formalin in the hind paw of the right hind leg, and the same amount of physiological saline alone was injected into the normal group.

<9-2> Confirmation of changes in lower limb edema

The swelling status before and after induction of lower swelling was confirmed.

Specifically, the paw volume increase due to edema was measured using a pheythysmometer (Ugo Basile, Milan, Italy) immediately before and 1 minute after the administration of formalin. The edema index was calculated by the following equation (3).

As a result, as shown in Fig. 10, edema was observed before and 1 hour after induction of swelling in each group. As a result, slight increase in the base size was observed in the normal group by injection of sterile physiological saline, (100 mg / kg / day) was significantly (p <0.05) dependent on the dose of EP70C-50-100 (100 mg / kg / day) (Fig. 10).

Based on the above results, it was confirmed that EP70C was excellent in the prevention and treatment efficacy of the lower limb vein.

Claims (14)

A pharmaceutical composition for preventing and treating diabetic complications and angioedema containing an extract of Euphorbia pekinensis or a purified fraction thereof as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the extract is extracted using water, ethanol or methanol, or a mixture thereof as a solvent, for the prevention and treatment of diabetic complications and angioedema.
[Claim 3] The pharmaceutical composition according to claim 2, wherein the ethanol is 30 to 70% of a water-soluble alcohol.
The pharmaceutical composition according to claim 1, wherein the extract is extracted by a method selected from the group consisting of a low pressure high temperature extraction, a hot water extraction, a reflux extraction, a hot water extraction, a cold extraction, a room temperature extraction, an ultrasonic extraction, Gt;
[Claim 7] The pharmaceutical composition according to claim 1, wherein the purified fraction is purified and fractionated by column chromatography with 100% primary distilled water followed by 50% ethanol, and the pharmaceutical composition for the prevention and treatment of diabetic complications.
The pharmaceutical composition according to claim 1, wherein the diabetic complication is diabetic retinopathy, diabetic neuropathy, diabetic neuropathy, diabetic complications characterized by diabetic foot ulcers, and a pharmaceutical composition for the prevention and treatment of angioedema.
The pharmaceutical composition according to claim 1, wherein the angioedema is any one selected from the group consisting of macular degeneration, macular edema, retinal degeneration, and varicose veins.
A diabetic complication comprising a dipole extract or a purified fraction thereof as an active ingredient, and a health functional food for preventing and improving angioedema.
9. The dietary supplement according to claim 8, wherein the extract is extracted using water, ethanol or methanol or a mixture thereof as a solvent.
9. The dietary supplement according to claim 8, wherein the ethanol is a water-soluble alcohol in an amount of 30 to 70%.
9. The method according to claim 8, wherein the extract is extracted by a method selected from the group consisting of low-pressure high-temperature extraction, hot water extraction, reflux extraction, hot water extraction, cold extraction, room temperature extraction, ultrasonic extraction, Functional foods.
[Claim 9] The dietary supplement according to claim 8, wherein the purified fraction is purified by column chromatography with 100% primary distilled water followed by 50% ethanol, and the diabetic complication and health functional food for the prevention and improvement of angioedema.
9. The method of claim 8, wherein the diabetic complication is diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic neuropathy, diabetic foot ulcer, diabetic heart disease, diabetic osteoporosis or diabetic atherosclerosis Health functional food for prevention and improvement of complications and angioedema.
9. The dietary supplement according to claim 8, wherein the angioedema is any one selected from the group consisting of macular degeneration, macular edema, retinal degeneration and varicose veins.

Images