KR101115505B1 - The composition for the prevention and treatment of diabetic complications containing the extracts or fractions of herbal medicine as active ingredient - Google Patents

Abstract

The present invention is a frond riparia Lour.) The present invention relates to a composition for preventing and treating diabetic complications containing the extract or fractions thereof as an active ingredient. More specifically, the amphibian extract or fractions thereof are used for the production and aldose of the final glycated product which is an indicator of diabetic complications. It inhibits the activity of the reductase and exhibits potent anti-diabetic complications through anti-cataract, anti-retinal and anti-neoplastic effects, and thus can be usefully used as an active ingredient for preventing and treating diabetic complications.

Weeping water (Homonoia riparia Lour.), Diabetes complications, Final glycation end products,

Description

The composition for the prevention and treatment of diabetic complications containing the extracts or fractions of herbal medicine as active ingredient}

The present invention relates to a composition for the prevention and treatment of diabetic complications containing the amphibian extract or a fraction thereof as an active ingredient.

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, there are more than 240 million diabetics worldwide. 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. That is, almost 10 to 20 years after diabetes, almost all organs in the body are damaged, diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic Diabetic neuropathy, heart disease, cancer or osteoporosis may appear. Chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end stage renal failure, with diabetic cataracts and retinopathy leading to blindness and eventually death. In the United States, diabetes is the leading cause of blindness for ages 25-74, and 60% of blindness occurs 15 to 20 years after the onset of diabetes. Therefore, delaying the onset of complications in diabetic patients by 5 to 10 years will affect the quality of life of patients and their families and will have a major impact on national finances.

Mechanisms that induce diabetic complications are largely explained by nonenzymatic glycation of protein, polyol pathway, and oxidative stress. Nonenzymatic glycation of protein refers to the condensation reaction of amino acids such as lysine residues of proteins and reducing sugars due to enzymatic condensation reactions, that is, milliard reactions. glycation endproducts (AGEs). The non-enzymatic glycosylation of protein (1) an amino acid group such as a lysine residue of the protein and an aldehyde or ketone of a reducing sugar undergo a nucleophilic addition reaction without enzymatic action to form a Schiff base, which is an early product, The base and the adjacent ketoamine adduct condensate with each other to produce a reversible Amadori type of early glycosylated product, and (2) the hyperglycemic state is continued to degrade the reversible Amadori type of early glycated product. The final glycosylated product, which is an irreversible product, is produced by rearrangement, and the resulting glycosylated products are combined or cross-linked with proteins or lipids to produce an irreversible glycosylated protein or glycosylated lipid. Can be divided into the stages generated. Unlike the reversible amadori type of early glycosylated products, the final glycosylated product is an irreversible reaction product. Once produced, blood sugar does not decompose when recovered to normal, but accumulates in the tissue during the survival of the protein or lipid to which the final glycated product is bound. Abnormally alters the structure and function of the organs and causes 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 final glycation products produced by the reaction of glucose and various proteins, is an important factor in causing chronic diabetic nephropathy. Glycosylated albumin is more easily introduced into renal glomerular cells than normal albumin without glycosylation, and high concentrations of glucose stimulate mesangium cells to increase extracellular matrix synthesis. Excessively 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, chronic diabetes has been reported to accumulate collagen in the arterial wall and basal membrane protein in the renal glomeruli and to accumulate in tissues (Brownlee, M., et al., 1986, Sciences , 232, 1629-). 1632). As described above, glycosylation occurs in proteins such as the basement membrane, plasma albumin, lens protein, fibrin, and collagen by non-enzymatic protein glycosylation reactions. chronic diabetes complications such as retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy. In addition, it has been reported that lipid metabolism abnormality occurs in the process of producing the final glycated product in hyperglycemic state and oxidative stress is induced by deterioration of 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 glycosylation and oxidative stress is related.

The polyol pathway is (1) reduced by aldose reductase (AR) action from aldose or ketose to form sorbitol, and (2) sorbitol is oxidized by sorbitol dehydrogenase to produce fructose. The process consists of. At steady state, aldose reductase has a very low affinity for glucose, but the hyperglycemic state causes excessive activation of aldose reductase, the first enzyme in the polyol pathway, resulting in the conversion of excess hyperglycemia into sorbitol and fructose. Accumulation in the osmotic 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 (Kim Eung-jin et al., Diabetes, Korean Diabetes Association, Korea Medicine, p. 483; Soulis-Liparota, T., et al., 1995, Diabetologia , 38: 357-394). Final glycation end 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). At this time, fructose is about 10 times faster than non-enzymatic glycosylation of protein. Thus, high concentrations of fructose bind to the protein and eventually accelerate the formation of the final glycated product. As such, non-enzymatic glycosylation, polyol pathways, and oxidative stress mechanisms of action 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, non-enzymatic glycosylation and oxidative stress mechanisms of action are interrelated. Therefore, in order to delay, prevent or treat the development of diabetic complications, the final glycated product Inhibition of formation has been found to be very important (Brownlee, M., et al., 1988, N. Engl . Med . , 318, 1315-1321).

Currently, aminoguanidine, a synthetic agent as a protein glycosylation inhibitor, is a nucleophilic hydrazine, which binds with the amadori product and prevents cross-linking with the protein, thereby inhibiting the production 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 and has been conducted until phase III clinical trials. Therefore, the development of safe and effective natural medicine is desired.

Homonoia riparia Lour. Is an evergreen shrub belonging to Euphorbiaceae, with a height of 0.5 to 3 m and flowers bloom around January to May. It is distributed in Korea, India, China, Taiwan, and New Guinea. In Chinese medicine, bitter taste is high and the sex of medicine is mild. Dissipating heat and detoxifying, diuretic effect is known. Examples of amphibians include gallic acid, taraxerone, quercetin-3-Ob-glucopyranosyl (1-6) -OaL-rhamnoside (quercetin-3-ObD-glucopyranosyl (1-6)) -OaL-rhamnoside} (Chinese Herb, Shanghai Science and Technology Press, Vol. 4, 3619; ISBN 7-5323-5106-8 / R.1287). However, modern medical pharmacological action and continuous ingredient studies have not been reported, and the efficacy of the amphibians on diabetes or diabetic complications has not been revealed.

Therefore, the present inventors are developing a safe and effective natural medicine for the diabetic complications, there is little toxicity and side effects, while the amphibian extract or its fraction inhibits the production of the final glycation product and the aldose reductase, thereby enhancing the potent antidiabetic complication. The present invention was completed by confirming the anti-aging or anti-cancer effect.

An object of the present invention is to provide a composition for the prevention and treatment of diabetic complications containing an amphibian extract or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention is a flock ( Homomonia) riparia Lour.) It provides a composition for preventing and treating diabetic complications containing the extract as an active ingredient.

The present invention also provides a composition for the prevention and treatment of diabetic complications containing the fraction prepared by further fractionating the amphibian extract with an organic solvent as an active ingredient.

The present invention also provides a dietary supplement for preventing and improving diabetic complications, which contains the amphibian extract or a fraction thereof as an active ingredient.

In another aspect, the present invention provides a functional feed additive for preventing and improving diabetic complications containing a water extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a composition for inhibiting the final glycation product containing the amphibian extract or a fraction thereof as an active ingredient.

The present invention also provides an anti-aging and delaying pharmaceutical composition containing an amphibian extract or a fraction thereof as an active ingredient.

In addition, the present invention provides an anti-aging and delayed health functional food containing the amphibian extract or a fraction thereof as an active ingredient.

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

In addition, the present invention provides a health functional food for cancer prevention and improvement containing the amphibian extract or a fraction thereof as an active ingredient.

The amphibian extract of the present invention or fractions thereof inhibits the production of the final glycation end products, which are indicative of diabetic complications, and the activity of aldose reductase, and the potent antidiabetic inhibitory activity through anti-cataract, anti-retinal and anti-neoplastic effects, and antioxidant. Since it shows activity, it can be usefully used as an active ingredient of a composition for preventing and treating diabetic complications.

Hereinafter, the present invention will be described in detail.

The present invention is a frond riparia Lour.) It provides a composition for preventing and treating diabetic complications containing the extract as an active ingredient.

The present invention also provides a composition for the prevention and treatment of diabetic complications containing the fraction prepared by further fractionating the amphibian extract with an organic solvent 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, heart disease, cancer, osteoporosis, and atherosclerosis. 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 amphibian 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 the amphibian Homonoia riparia Lour .;

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

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

In the above method, the amphibian of step 1) can be used without limitation, such as cultivated or commercially available. The amphibian may use the whole plant, and preferably uses a leaf or a branch, but is not limited thereto.

The extraction solvent is preferably water, alcohol or a mixture thereof. It is preferable to use C ₁ to C ₄ lower alcohol as the alcohol, it is preferable to use ethanol or methanol as the lower alcohol, more preferably 80% ethanol, but is not limited thereto. Alcohols having a water content of 0.1% to 50% can also be used. As extraction methods, conventional methods in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction, can be used, and the extraction is preferably performed once to five times by hot water extraction, and three times to extract repeatedly. More preferably, but is not limited thereto. The extracting solvent may be added 1 to 10 times the weight of the dried weeping leaf and eggplant, preferably 1 to 5 times. The extraction temperature is preferably 20 to 30 ° C., but is not limited thereto. In addition, the extraction time is preferably 24 to 48 hours, 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. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In a specific embodiment of the invention, the amphibian leaves and branches are washed with water and then dried in the shade. The dried weeping leaf and eggplant were crushed and placed in an extraction container, and extracted repeatedly at room temperature with an extraction solvent. After the amphibian extract was obtained, the solids were removed using a filter paper or the like and filtered. The extract was concentrated under reduced pressure to prepare a amphibian extract.

The fraction of the amphibian extract is preferably prepared by a manufacturing method including adding an organic solvent to the amphibian extract to prepare an organic solvent fraction, but not always limited thereto.

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

The fraction is obtained by suspending the amphibian extract in water, and then sequentially fractionating the mixture with normal-hexane, ethyl acetate, normal-butanol, and water. It is preferably one of them, but is not limited thereto.

In a specific embodiment of the present invention, the solvent was evaporated from the amphibian extract and water and normal-hexane were added to the obtained residue to separate a normal-hexane layer to obtain a normal-hexane fraction. Ethyl acetate was mixed with the water layer except for the hexane layer, and the ethyl acetate layer was separated to obtain an ethyl acetate fraction. Also, after removing the ethyl acetate layer, normal-butanol was mixed and the normal-butanol layer was separated to obtain a normal-butanol fraction. Finally, after removing the normal-butanol layer, a water fraction of the water layer was obtained (see FIG. 1).

The present inventors used bovine serum albumin (BSA) as a protein to analyze the degree of binding of fructose and glucose in order to analyze the inhibitory effect of the production of the final glycated product, which is an indicator of diabetic complications and an evaluation of therapeutic efficacy. It was used as an indicator. In addition, as a positive control group was used aminoguanidine (aminoguanidine) is known to have a very good inhibitory effect on the final glycated product. As a result, it was confirmed that the test group treated with the amphibian extract or fractions thereof exhibited the effect of inhibiting the end glycated product far more effectively than the previously known aminoguanidine (see Table 1).

In order to more accurately confirm the function of the amphibian extract or fractions thereof, the present inventors confirmed the cleavage efficacy for crosslinking of the final glycated product and BSA. ALT-711 (Alteon Inc., Ramsey, NJ), a drug known as an AGEs cross-linker breaker, was compared with an extract or fractions of the amphibian. (cross-linker breaking) effect was confirmed to be excellent (see Table 2).

The inventors have measured the inhibition of the activity of aldose reductase, another indicator of diabetic complications of the amphibian extract or fractions thereof. As a comparative control, it was measured using 3,3-tetramethylene glutaric acid. As a result, the amphibian 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 3).

The present inventors confirmed the prophylactic and therapeutic effects of the amphibian extract on retinopathy and nephropathy in a type 2 diabetic animal model, SDT (spontaneous diabetic torii) mice. As a result, treatment with the amphibian extract significantly reduced blood retinal barrier damage, perivascular cell loss, and acellular capillary formation in diabetic groups (see FIGS. 2 and 3), and in retinal vessels and retinal nerve cells. Accumulation of final glycation end products was reduced (see FIG. 4), and apoptosis was inhibited (see FIG. 5), and it was confirmed that the amphibian extract had an effect of inhibiting diabetic retinopathy. In addition, the treatment of the amphibian extract showed a significant decrease in the renal function index and the amount of AGE and CML in the urine or kidney (see FIGS. 6, 7 and 8), and the thickening of the glomerular basement membrane and the mesangial matrix. Since expansion, glomerular hypertrophy, glomerulosclerosis, degree of collagen infiltration and podocyte loss were significantly reduced (FIGS. 9, 10 and 11), it was confirmed to effectively suppress diabetic nephropathy.

As a result, the amphibian extract of the present invention or a fraction thereof is the cause of diabetic complications, and the inhibitory effect on the production of the final glycation product, which is a measure of the efficacy of the treatment for diabetic complications, the inhibitory effect of aldose reductase activity and the diabetic animal model. Since the efficacy for diabetic complications was shown to be excellent, it was confirmed that it can be usefully used as an active ingredient for preventing and treating diabetic complications.

It contains 0.1 to 99.9% by weight of the amphibian extract of the present invention or a fraction thereof based on the total weight of the composition of the present invention as an active ingredient, 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 fillers, 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 (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, 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 externally or intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrauterine epidural or cerebrovascular injection methods for parenteral administration. For skin use externally.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease of the patient, the daily dosage is the amount of the amphibian 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 response modifiers.

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

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably an ape-like animal such as a chimpanzee or gorilla.

The method of administration may be administered orally or parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, intracerebroventricular injection or intrathoracic injection It can be administered by injection.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. Dosage may vary depending on the weight, age, sex, health status, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of a particular patient.

The diabetic complication is preferably any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, heart disease, cancer, osteoporosis, and atherosclerosis.

The amphibian extract or fractions thereof of the present invention inhibit the production of the final glycation end product and the activity of aldose reductase, and have the effect of decomposing crosslinking of the final glycation end product and the protein, as well as in the type 2 diabetic animal model. by showing the inhibitory effect on diabetic cataract, diabetic retinopathy and diabetic nephropathy in vitro (in vitro) and in vivo (in In vivo ) it was confirmed that effectively suppresses diabetic complications, it can be usefully used for the prevention and treatment of diabetic complications.

The present invention also provides a dietary supplement for preventing and improving diabetic complications, which contains the amphibian extract or a fraction 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, heart disease, cancer, osteoporosis, and atherosclerosis.

The amphibian extract or fractions thereof of the present invention inhibit the production of the final glycation end product and the activity of aldose reductase, and have the effect of decomposing crosslinking of the final glycation end product and the protein, as well as in the type 2 diabetic animal model. by showing the inhibitory effect on diabetic cataract, diabetic retinopathy and diabetic nephropathy in vitro (in vitro) and in vivo (in In vivo ) it was confirmed that effectively inhibits diabetic complications, can be usefully used in the health functional food for the prevention and improvement of diabetic complications.

The functional food of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients. Such natural carbohydrates are 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 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, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the functional food of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it 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 another aspect, the present invention provides a functional feed additive for preventing and improving diabetic complications containing a water extract or a fraction 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, heart disease, cancer, osteoporosis, and atherosclerosis.

The amphibian extract or fractions thereof of the present invention inhibit the production of the final glycation end product and the activity of aldose reductase, and have the effect of decomposing crosslinking of the final glycation end product and the protein, as well as in the type 2 diabetic animal model. by showing the inhibitory effect on diabetic cataract, diabetic retinopathy and diabetic nephropathy in vitro (in vitro) and in vivo (in In vivo ) it was confirmed that effectively inhibit the diabetic complications, can be usefully used in the functional feed additive for the prevention and improvement of diabetic complications.

The feed additives can be prevented from diabetic complications by steadily ingesting poultry, livestock, and the like, and can treat already occurring diabetic complications.

In the feed additive of the present invention, the amount of the amphibian extract or its fraction is 0.1-20% by weight, lipase is 0.001-0.01% by weight, tertiary calcium phosphate is 1-20% by weight, and vitamin E is 0.01 to 0.1% by weight of enzyme powder, 1 to 10% by weight of enzyme powder, 0.1 to 10% by weight of lactic acid bacteria, 0.01 to 10% by weight of Bacillus culture medium and 20 to 90% by weight of glucose. It is preferable, but not limited thereto, and if an amphibian extract or a fraction thereof is added in an effective amount, it can be used as a feed additive of the present invention.

The effective amount refers to an amount capable of preventing diabetic complications or treating already occurring diabetic complications by continuously ingesting poultry and livestock. Moreover, it is preferable that the quantity which does not produce the bad influence beyond the benefit by addition is preferable.

In addition, the feed additive may additionally contain a carrier that is acceptable to poultry and livestock. In the present invention, the feed additive may be added as it is or a known carrier, stabilizer and the like, and various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives may be added as necessary. The shape may be in a suitable state such as powder, granules, pellets, suspension, or the like. When supplying the feed additive of the present invention can be supplied alone or mixed in the feed for poultry and livestock.

In addition, the present invention provides a composition for inhibiting the final glycation product containing the amphibian extract or a fraction thereof as an active ingredient.

The amphibian extract or fractions thereof of the present invention inhibit the production of the final glycation end product and the activity of aldose reductase, and have the effect of decomposing crosslinking of the final glycation end product and the protein, as well as in the type 2 diabetic animal model. by showing the inhibitory effect on diabetic cataract, diabetic retinopathy and diabetic nephropathy in vitro (in vitro) and in vivo (in In vivo ) it was confirmed that effectively inhibit the diabetic complications, it can be usefully used as an active ingredient of the composition for inhibiting the final glycation product which is an indicator of diabetic complications.

The present invention also provides an anti-aging and delaying pharmaceutical composition containing an amphibian extract or a fraction thereof as an active ingredient.

In addition, the present invention provides an anti-aging and delayed health functional food containing the amphibian extract or a fraction thereof as an active ingredient.

It has been reported that lipid metabolism abnormalities occur during the production of final glycation end products, and that oxidative stress is induced by deterioration of defense system function against harmful oxygen free radicals (Yokozawa, T., et al, 2001, J.). of Trad . Med ., 18: 107-112), the amphibian extract of the present invention or a fraction thereof effectively inhibits the production of the final glycated product, and thus may be usefully used in anti-aging and delayed pharmaceutical compositions or health functional foods.

The present invention also provides a method for preventing and delaying aging, comprising administering to a subject suffering from diabetes the amphibian extract or a fraction thereof.

The amphibian extract of the present invention or a fraction thereof effectively inhibits the production of the final glycated product, and thus can be usefully used for anti-aging and delay.

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

In addition, the present invention provides a health functional food for preventing and improving cancer containing the amphibian extract or a fraction thereof as an active ingredient.

It has already been reported that the final glycation end products are cancer-causing (Tokuda H. et al., 2005, Book of Abstract of 53rd GA Congress joint with SIF, P076), the amphibian extract of the present invention or a fraction thereof is the final glycation end product Because it effectively inhibits the production of, and has the effect of decomposing the cross-linking of the final glycation product and protein, it can be usefully used in pharmaceutical compositions for the treatment and prevention of cancer or health functional food for the treatment and improvement of cancer.

In addition, the present invention provides a method for preventing and treating cancer, comprising administering to a subject having cancer the amphibian extract or a fraction thereof.

Since the extract of the present invention or a fraction thereof effectively inhibits the final glycation end product, and has the effect of decomposing the crosslinking of the final glycation end product and the protein, it can be usefully used for cancer prevention and treatment.

Hereinafter, the present invention will be described in detail with reference to 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> Weeping  Preparation of Extract

<1-1> Weeping  Ethanol extract

The specimen (no. TBRC-VN-118) used leaves and branches of the Homonoia riparia Lour. Stored in the specimen room of the Diabetes Complication Research Center of the Korean Institute of Oriental Medicine. After crushing the leaf and eggplant of finely cultivated weeping and cultivated in 80% ethanol, and repeatedly extracted at room temperature in an extraction container, the extract is filtered and then prevent the decomposition and hydrolysis of the components. Concentration under reduced pressure while maintaining the temperature at 40 ~ 45 ℃ or less so as to give 190 g of the ethanol extract.

<1-2> Weeping  Methanol extract

170 g of the amphibian methanol extract was prepared according to the method of Example <1-1>, wherein methanol was used instead of ethanol.

<1-3> Weeping  Water extract

According to the method of Example <1-1>, 195 g of amphibian water extract was prepared, and water was used instead of ethanol.

< Example  2> Weeping Fraction  Produce

<2-1> Weeping  Normal Hexane Fraction

0.5 g of water and 0.5 l of normal-hexane were added to 12 g of the amphibian ethanol extract obtained in Example <1-1>, and the normal-hexane layer was separated. The method was repeated three times, and then concentrated under reduced pressure at 40 ° C. to obtain 3.98 g of a normal-hexane fraction.

<2-2> Weeping  Ethyl acetate Fraction

0.5 L of ethyl acetate was added to the water layer obtained in Example <2-1>, and the ethyl acetate layer was separated. The method was repeated three times, and then concentrated under reduced pressure at 40 ° C. to obtain 56.0 g of ethyl acetate fraction.

<2-3> Weeping  Normal Butanol Fraction

0.5 L of normal-butanol was added to the water layer obtained in Example <2-2>, and the normal-butanol layer was separated. The process was repeated three times and then concentrated under reduced pressure at 40 ° C. to give 41.4 g of normal-butanol fraction.

<2-4> Weeping  water Fraction

The water layer obtained in Example <2-3> was concentrated under reduced pressure at 40 ° C. to obtain 88.6 g of a water fraction.

Experimental Example 1 Final Glycation Product Inhibition Effect Analysis

Inhibition of the production of final glycation product in vitro of the amphibian extract obtained in Example 1 and the normal-hexane, ethyl acetate, normal-butanol or water fraction obtained in Example 2 was analyzed. Bovine serum albumin (BSA, Sigma, USA) was selected as a protein source. BSA was prepared by adding 50 mM phosphate buffer (pH 7.4) to a concentration of 10 mg / mL. As a sugar source, a solution containing 0.2 M fructose and 0.2 M glucose was used. A fructose and glucose mixture was added to the prepared BSA solution. The amphibian ethanol extract was prepared at a concentration of 2.5 μg / ml, 5 μg / ml or 10 μg / ml, the normal-hexane fraction was 10 μg / ml, 25 μg / ml or 50 μg / ml and the ethyl acetate fraction was 1.25 μg. / Ml, 2.5 μg / ml or 5 μg / ml, normal-butanol fractions were prepared at concentrations of 1.25 μg / ml, 2.5 μg / ml or 5 μg / ml. Water fractions were prepared at concentrations of 2.5 μg / ml, 5 μg / ml or 10 μg / ml, respectively. After dissolving all the compounds in dimethylsulfoxide (DMSO), 15% tween 80 was added, and the total DMSO content was 0.2%. The prepared extract or a fraction thereof was added to the mixture of BSA and sugar and incubated at 37 ° C. for 14 days. 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 of the test and control groups. On the other hand, aminoguanidine was used as a positive control group which can be compared with the excellent efficacy. Specifically, aminoguanidine was dissolved in distilled water and incubated for 14 days at 37 ° C. at a concentration of 18.5 μg / ml, 37 μg / ml or 55.5 μg / ml by the method described above. All cultures were prepared by four to avoid errors as much as possible. After 14 days of culture, the contents of the final glycated product produced in the culture solution were analyzed and the results are shown. The final glycosylated product was fluorescence, brown, and not only possessed physicochemical properties that could cross-link, but also had ligands recognized by cell membrane receptors. The amount of the final glycated product having this property was measured by a microplate reader (Excitation: 350 nm, Emission: 450 nm) to analyze the degree of inhibition of production of the final glycated product (Vinson, JA et al., J. Nutr . Biochem . , 7: 659-663, 1996). The production inhibition rate of the final glycosylated product was calculated according to the following [Equation 1].

Production Inhibition Rate (%) = 100- (Fluorescence Intensity of Sample Group-Fluorescence Intensity of Sample Blank Test Group) / (Fluorescence Intensity of Control Group-Fluorescent Intensity of Control Blank Test Group) × 100

As a result, as shown in the following [Table 1], weaning ethanol extract, methanol extract, water extract, normal-hexane fraction of the ethanol extract, ethyl acetate fraction, normal-butanol fraction and the final glycation product inhibitory effect of water fraction IC ₅₀ values of were 5.67 μg / ml, 6.59 μg / ml, 6.75 μg / ml, at least 50 μg / ml, 4.68 μg / ml, 4.53 μg / ml, and 8.49 μg / ml, respectively. It was proved to be remarkably superior to the positive control aminoguanidine (IC ₅₀ value: 52.96 ㎍ / ml) at 9.3, 8.0, 7.8, 11.3, 11.7, and 6.2 times, respectively. The effect was shown to be the most effective (Table 1). Thus, the amphibian extract or fractions thereof was found to inhibit the binding of protein and sugar to inhibit the production of the final glycation end products, and the ethanol extract having the highest inhibitory effect on the end glycation end products was used for the following diabetic complication efficacy analysis.

density
(Μg / ml) Inhibition
(%) IC ₅₀
(Μg / ml) Day 14 Day 14 Day 14 Weeping Ethanol Extract 2.5
5
10 26.26 ± 2.64
47.55 ± 1.03
79.52 ± 1.46
5.67 ± 0.24 Weeping methanol extract 2.5
5
10 23.20 ± 1.04
44.35 ± 1.08
76.71 ± 1.33
6.59 ± 0.21 Weeping Water Extract 2.5
5
10 22.16 ± 1.61
43.11 ± 1.56
75.13 ± 1.07
6.75 ± 1.12 Normal-hexane Fraction of Weeping Extract 10
25
50 13.06 ± 2.46
17.62 ± 2.05
23.77 ± 3.09
> 50 Ethyl Acetate Fraction of Aqueous Extract 1.25
2.5
5 19.58 ± 3.60
33.59 ± 1.62
52.06 ± 2.85
4.68 ± 0.28 Normal-Butanol Fraction of Aqueous Extract 1.25
2.5
5 21.86 ± 1.29
34.61 ± 3.12
53.45 ± 1.73
4.53 ± 0.25 Water fraction of the amphibian extract 2.5
5
10 24.02 ± 3.03
35.40 ± 2.51
56.32 ± 1.40
8.49 ± 0.38 Aminoguanidine 18.5
37
55.5 15.50 ± 1.79
33.56 ± 0.51
52.81 ± 2.81
52.96 ± 1.95

< Experimental Example  2> Final Glycation Product ( AGE ) To evaluate the cleavage efficacy of crosslinking In vitro ( in in vitro )  analysis

1.0 μg AGE-BSA was dispensed into collagen coated 96 well microtitre plates and incubated at 37 ° C. for 4 hours. After washing three times with Tween added to 0.05% PBS to remove unbound AGE-BSA, ALT-711 (4, a drug known as an amphibian extract or AGEs cross-linking breaker) 5-dimethyl-3- (2-oxo-2-phenylethyl) -thiazolium chloride; Alteon Inc., Ramsey, NJ) was prepared by serial dilution from 1000 μg / ml to 1 μg / ml, and then in each well. Three times (triplicate) aliquots and incubated for 4 hours at 37 ℃. After incubation, each well was washed three times with Tween added to 0.05% PBS, and rabbit polyclonal anti-AGE-BSA antibody to detect remaining AGE-BSA crosslinked with collagen. Antibody; MBL international, Woburn, MA) was diluted 1: 250 and dispensed into each well, and then incubated for 1 hour at 37 ℃. After 1 hour of incubation, washed three times with TBS added to 0.05% PBS, horseradish peroxidase-linked goat anti-rabbit antibody (Sigma, USA) After application, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine, TMB) was used as a substrate and the color was measured at 450 nm. The percentage of breaking of AGE-BSA was calculated according to Equation 2 below.

AGE-BSA (%) = absorbance of wells without sample / absorbance of wells without sample × 100

As a result, as shown in the following [Table 2], it was confirmed that the AGE-BSA cleavage efficacy of the amphibian extract is more than 38 times better than the control ALT-711 (Table 2).

Concentration (μg / ml) IC ₅₀
(Μg / ml) 0 10 50 100 1000 Weeping Ethanol Extract 100.00 ± 7.43 73.00 ± 4.79 24.70 ± 1.09 18.45 ± 0.94 17.10 ± 1.92 26.13 ± 2.74 ALT-711 100.00 ± 26.69 100.18 ± 18.4 100.13 ± 4.42 100.21 ± 5.46 51.77 ± 9.54 > 1000

< Experimental Example  3> Aldoz  Reductase ( aldose reductase , AR Activity Inhibition Effect Analysis

The efficacy of inhibiting aldose reductase activity in vitro of the amphibian extract, or its normal-hexane, ethyl acetate, normal-butanol and water fractions, was analyzed. In order to obtain natural aldose reductase from the eyes of SD rats (Sprague-Dawley rat, 250-280 g) according to the Dufrane (1984) method, 135 mM Na, K-phosphate buffer solution (K-phosphate) buffer; pH 7.0) and 10 mM 2-mercaptoethanol were pulverized 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 used after filtering through a 0.2 μm filter. The process was all performed at 4 ℃. Enzyme protein source was quantified by a lowry method using BSA. 135 mM Na, K-phosphate buffer (pH 7.0), 100 mM lithium sulfate, 0.03 mM NADPH, 0.04 mM DL-glyceraldehyde and 100 μg / ml of enzyme The mixture was dissolved in 0.1% DMSO and added to 50 µl of the sample diluted to each concentration to a total volume of 1 ml, followed by reaction at 37 ° C. for 10 minutes. At this time, the test group was a mixture without addition of 0.04 mM DL- glyceraldehyde (DL-glycealdehyde), STD is 135 mM Na, K-phosphate buffer (pH 7.0), 100 mM lithium sulfate (Lithium 50 μl NADP (0.2-5 μM) was added to the sulfate. After completion of the reaction by adding 0.3 ml of 0.5 N HCl sample, 1 ml of 6 M NaOH added with 10 mM imidazole was added and reacted at 60 ° C. for 10 minutes to convert NADP to a fluorescent product. Was measured. Samples were triplicated. Efficacy was measured by using a spectrofluorophotometric detector (Bio-TEK, Synergy HT, USA). 360 nm, Em. Measured at 460 nm, indicated as IC ₅₀ value. The amphibian extract was prepared at a concentration of 1 μg / ml, 2.5 μg / ml or 5 μg / ml, the normal-hexane fraction at a concentration of 2.5 μg / ml, 5 μg / ml or 10 μg / ml, and the ethyl acetate fraction At concentrations of 0.5 μg / ml, 1 μg / ml, 2.5 μg / ml, the normal-butanol fraction is at a concentration of 1 μg / ml, 2.5 μg / ml or 5.0 μg / ml, and the water fraction is 2.5 μg / ml, 5 Prepared at concentrations of μg / ml or 10 μg / ml, respectively. The control group included 3,3-tetramethyleneglutaric acid, one of the excellent aldose reductase inhibitors, at a concentration of 3.72 μg / ml, 4.66 μg / ml or 5.59 μg / ml. After preparation, the inhibitory effect of aldose reductase activity was measured.

As a result, as shown in the following [Table 3], the efficacy of inhibiting the aldose reductase activity of the amphibian extract, the normal-hexane fraction of the extract, the ethyl acetate fraction, the normal-butanol fraction or the water fraction was IC ₅₀ value of 2.60, respectively. Μg / ml, 5.58 µg / ml, 1.20 µg / ml, 3.35 µg / ml, 7.19 µg / ml. Compared to the positive control 3,3-tetramethyleneglutaric acid (IC ₅₀ value: 5.41 μg / ml), the extract, ethyl acetate fraction and normal-butanol fraction were 2.3, 4.5 and 1.6 times better, respectively ( Table 3). Thus, it was confirmed that the efficacy of inhibiting aldose reductase activity of the amphibian extract, the ethyl acetate fraction, and the normal-butanol fraction was significantly superior to that of the synthetic product which is a single synthetic compound.

Extract ＼ Efficacy density
(Μg / ml) Inhibitory effect
(%) Inhibitory effect (IC _{50 value} )
(Μg / ml) Weeping Ethanol Extract One
2.5
5 33.60 ± 2.50
48.40 ± 6.35
75.60 ± 3.67
2.60 ± 0.37 Normal-hexane Fraction of Weeping Extract 2.5
5
10 37.50 ± 2.54
49.04 ± 6.93
65.71 ± 1.47
5.58 ± 0.60 Ethyl Acetate Fraction of Aqueous Extract 0.5
One
2.5 33.62 ± 6.76
48.94 ± 2.21
75.32 ± 6.06
1.20 ± 0.09 Normal-Butanol Fraction of Aqueous Extract One
2.5
5 19.58 ± 5.96
40.91 ± 3.97
69.93 ± 0.61
3.35 ± 0.06 Water fraction of the amphibian extract 2.5
5
10 22.03 ± 6.82
42.31 ± 5.84
63.64 ± 3.97
7.19 ± 0.83 3,3-tetramethylene glutaric acid
(3.3-Tetramethyleneglutaric acid)
3.72
4.66
5.59 33.03 ± 4.36
40.27 ± 1.36
51.58 ± 0.78
5.41 ± 0.13

< Experimental Example  4> Confirmation of efficacy in type 2 diabetes animal model

<4-1> Breeding of Type 2 Diabetic Animal Model Weeping  Administration of Extract

To verify the prevention and treatment of diabetic complications, the efficacy of the amphibian extract was analyzed in spontaneous diabetic Torii (SDT) rats. Animals were spontaneous diabetic Torii (SDT) mice, type 2 diabetes model animals. Ten-week-old male SDT rats (CLEA Japan Inc., Tokyo, Japan) were acclimated for one week and then bred for 15 weeks until the induction of hyperglycemia at 300 mg / dl. Feed and drinking water were fed freely. After 15 weeks, 8 rats per group were divided into 5 groups, and the amphibian extract (HR) and the control drug metformin (MET) were orally administered once daily. The test group was fed to normal group (NOR), diabetes group (DM), metformin 350 mg / kg administration group (MET) to the diabetic group, and to the diabetic group 100 mg / kg administration group (HR-100), diabetic group Liu Extract 250 mg / kg administration group (HR-250). Each group was orally administered once a day for 16 weeks. Urine was collected for 24 hours one day before autopsy, and organs harvested at autopsy were stored at -80 ℃.

<4-2> Inhibition of Diabetic Retinopathy in Type 2 Diabetic Animal Models

<4-2-1> Damage to the blood retinal barrier ( Blood - retinal barrier breakage )

If hyperglycemia persists, blood retinal barriers are damaged due to abnormal function of the retinal blood vessels of the eye, and the effect of the amphibian extract on the prevention of blood retinal barrier damage was confirmed. At necropsy of the rats of Example <4-1>, pentobarbital sodium (25 mg / kg body weight) was anesthetized by intraperitoneal injection. The abdominal cavity and thoracic cavity of anesthetized rats were opened to secure the heart, and 50 mg / ml fluorescein-dextran (2 × 10 ⁶ molecular weight) prepared by dissolving in 1 ml / ml of sterile PBS was injected into the left ventricle. . After 10 minutes, the eye was removed, and for the left eye, the retina was detached from the eyecup. The detached retina was placed on a slide and mounted with an aqueous mounting medium. After drying sufficiently, it was observed under a fluorescence microscope.

As a result, as shown in FIG. 2, the outflow of fluorescence was not observed in the normal group (NOR), but in the diabetic group (DM), the fluorescent substance leaked out of the blood vessel due to damage of the blood retinal barrier (arrow). ) And non-perfusion areas (arrows) caused by narrowing of some blood vessels. In the metformin-administered group (MET), a slight but rare fluorescence leakage was observed, whereas in the amphibian extract-administered group (HR-100 or HR-250), these symptoms were not observed.

<4-2-2> Peripheral cell loss ( pericyte loss ) And acellular capillary formation (acellular capillary formation Preventive effect

Representative early symptoms of diabetic retinopathy include pericyte loss and acellular capillary formation, one of the constituent cells of retinal blood vessels. Acellular capillary formation was confirmed. The retina was removed from the right eye of the rat, washed with running water, and incubated for 1 hour at 37 ° C in 3% trypsin. The degraded retina was transferred to PBS and the internal membrane was removed. The vascular frame was separated from the retinal background using a glass rod, placed on a slide and dried. PAS staining and hematoxylin staining observed changes in cell walls and nuclei.

As a result, as shown in FIG. 3, acellular capillary formation with a loss of perivascular cells and loss of vascular endothelial cells, a closed vessel with narrowing of blood vessels, and the like were observed. Peripheral cell loss, acellular capillary formation, and closed vessels were significantly increased in the diabetic group, and metformin (MET) or amphibian extract administration group (HR-100 or HR-250). The change was shown to be markedly reduced (FIG. 3).

<4-2-3> Final glycation product ( Advanced glycation end products , Retinal vessels of AGEs) Retinal nerve cells  Accumulation Prevention Effect

The deparaffinized and hydrolyzed slides were reacted with 3% hydrogen peroxide solution for 10 minutes to remove endogenous peroxidase activity, and then washed three times with PBS containing 0.05% Tween 20. After blocking with 5% casein to eliminate non-specific reactions, the primary antibodies, anti-AGEs antibody (Cosmo Bio) and anti-GFAP antibody (Santacurz) were diluted 1: 200 to 1 Treated for hours. After washing with PBS for 1 hour and treated with labeled streptoavidin biotin (LSAB) kit (Dako, USA) or FITC-bound secondary antibody, color development with DAB was performed to observe changes in AGEs under fluorescence and optical microscopy.

As a result, as shown in FIG. 4, the accumulation of the final glycation end products of diabetic complications in the retinal vessels and retinal nerve cells was increased in the diabetic group (DM) compared to the normal group (NOR), and the amphibian extract In the group administered, especially high concentration group (HR-250), the final glycation product accumulation was significantly reduced, which was significantly better than the MET group (MET) (Fig. 4).

<4-2-4> retinal blood cells and Retinal nerve cell  Apoptosis ( apoptosis Preventive effect

The tissue sections were deparaffinized and hydrated, and then treated with proteinase K solution at 20 ㎍ / ml, washed with PBS for 15 minutes at 37 ° C, followed by washing with PBS. After reaction at 37 ° C. for 1 hour with a TUNEL reation mixture solution (In situ cell death detection kit, AP, Roche, Germany), the presence of perivascular and retinal neurons in the retinal vessels was observed under a fluorescence microscope. In addition, TUNEL staining was performed to confirm whether the loss was caused by apoptosis.

As a result, as shown in FIG. 5, the number of TUNEL positive cells was significantly increased in the diabetic group compared to the normal group, and in the amphibian extract administration group (HR-100 or HR-250), Apoptosis was significantly reduced (FIG. 5).

<4-3> Diabetic in Type 2 Diabetic Animal Model Nephropathy  Related analysis

<4-3-1> New Functional Indicator, AGEs  And CML  Positive analysis

To investigate the effects of the amphibian extract on kidney function due to chronic diabetes, renal function indicators (proteinuria, albuminuria, glomerular filtration rate (Ccr)) and urine collected for 24 hours in the last week of autopsy The amount of glycation products {AGEs and carboxymethyllysine (CML)} was quantified. ELISA analysis was performed to measure proteinuria, albuminuria, glomerular filtration rate, AGEs, CML, podocytes (synaptopodin and WT-1). Urine was mixed in a 96 well plate with a coating buffer (50 mM carbonate buffer, pH 9.6) in a ratio of 1: 2 so that the final dose was 100 μl, and then left at 37 ° C. for 3 hours. After washing three times with 0.05% PBST and blocked with 3% skim milk (skim milk). After washing for 1 hour, AGEs (Transgenic, Japan), CML (Transgenic, Japan), albumin (albumin), synaptopodin, and WT-1 (Santa cruz, USA) antibodies were each 1: 1000. After dilution, 100 μl was loaded per well, and then left at 37 ° C. for 2 hours. After washing with 0.05% PBST, diluted with HRP-bound secondary antibody, 100 μl was loaded and left at 37 ° C. for 1 hour. 100 μl of substrate 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine; TMB) was added in 0.05% PBST. After standing for 5 minutes, 100 μl of a reaction stop solution (1M H ₂ SO ₄ ) was added thereto, and the absorbance was measured by an ELISA reader.

In addition, immunological staining analysis was performed according to the following method. The deparaffinized and hydrolyzed slides were reacted with 3% H ₂ O ₂ solution for 10 minutes to remove endogenous peroxidase activity, and then washed three times with PBS containing 0.05% Tween 20. . Blocking with 5% AFB (animal free bock) to remove nonspecific reactions, then diluting primary antibody AGEs (Transgenic, Japan) and CML (Transgenic, Japan) 1: 100 each for 1 hour or overnight It was. After washing with PBS for 1 hour and treated with labeled streptavidin biotin (LSAB) kit (Dako, USA), DAB (Dako, USA) color development was observed under an optical microscope, respectively.

As a result, as shown in FIG. 6, proteinuria and CCr changes were all significantly increased in the diabetic group (DM) compared to the normal group (NOR). In the case of proteinuria, the MET group showed higher levels than the diabetic group, and in the amphibian extract group (HR-100 or HR-250), there was a significant decrease compared to the diabetic group (DM). In the case of albuminuria (albuminuria), a significant increase was observed in the diabetic group (DM) compared to the normal group (NOR), and both the MET group (MET) or the amphibian extract group (HR-100 or HR-250) were diabetic group ( A significant decrease can be observed compared to DM). Efficacy similar to the experimental results of the indicator of glomerular filtration rate (Ccr) could be observed (FIG. 6). In addition, as shown in FIG. 7, the final glycated products AGEs and CML in urine were measured in urine, and the levels of the diabetic group were significantly increased in comparison with the normal group, respectively, and significantly in the MET group (MET). No decrease was seen. On the other hand, the amphibian extract administration group (HR-100 or HR-250) showed a significant decrease (Fig. 7). In addition, the histological staining and blotting of renal glomeruli significantly increased the accumulation of AGEs and CML in the diabetic group (DM) compared to the normal group (NOR). Or HR-250), a significant decrease in concentration-dependent manner was observed (FIG. 8).

<4-3-2> Pathological changes analysis of glomeruli

Kidneys were extracted at rat necropsy and fixed in 10% neutralized formalin overnight, then dehydrated three times with xylene and embedded in paraffin. The embedded tissue block was used to prepare a continuous section having a thickness of 4 μm on a slide. To observe the morphological changes, periodic acid shiff (PAS) and Masson's trichrome staining were performed. Stained slides were observed under an optical microscope.

As a result, as shown in FIG. 9, the thickening of the glomerular basement membrane by the extracellular matrix deposition in the diabetic group (DM) glomeruli and the expansion of the mesialial matrix and the glomeruli of the glomeruli, as compared to the normal group (NOR). Hypertrophy and glomerulosclerosis were marked. However, in the amphibian extract-administered group (HR-100 or HR-250), the concentration of glomeruli and the expansion of vascular matrix were significantly reduced. In the MET group (MET), the morphological changes were reduced compared to the diabetic group (DM), but no significant decrease was observed (FIG. 9). In addition, Masson's trichrome was performed to evaluate the fibrosis caused by collagen infiltrating the cortex of the kidney. The MET administration group (MET) also did not show a significant difference from the diabetic group (DM). However, in the low or high concentration group (HR-100 or HR-250) of the amphibian extract, the degree of collagen deposition was significantly lower than that of the diabetic group (DM) (FIG. 10).

<4-3-3> Foot cell  Loss( Podocyte loss) Preventive effect against

In the early diabetic state, the loss of podocytes in the glomeruli occurs, and immunohistostaining method is used to analyze the efficacy of the amphibian extract on the loss of podocytes through synaptopodin and WT-1, known as podocyte markers. The amount of shedding cells in the urine was measured.

As a result, it was observed that the diabetic group (DM) was significantly increased compared to the normal group (NOR), and the podocyte discharge amount was significantly decreased in the group administered at the low or high concentration of the amphibian extract (HR-100 or HR-250). It was confirmed to prevent the loss of podocytes according to (Fig. 11).

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Powder  Produce

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

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of ethanol extract of Example <1-1> of the present invention

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of the normal-hexane fraction of Example <2-1> of the present invention

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

&Lt; 1-4 >

1 g of normal-hexane fraction of Example <2-1> of the present invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

<1-5> Preparation of granules

150 mg of the normal-hexane fraction of Example <2-1> of the present invention

Soybean Extract 50mg

Glucose 200 mg

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Manufacturing example  2> Manufacture of food

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

<2-1> Production of flour food

0.5-5.0 parts by weight of the ethanol extract of Example <1-1> of the present invention was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> soup  And juicy ( gravies Manufacturing

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

<2-3> ground Beef  Produce

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

<2-4> dairy products ( dairy products Manufacturing

5-10 parts by weight of the ethanol extract of Example <1-1> 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> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

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

The grains, seeds and the ethanol extracts of Example <1-1> prepared above were combined and prepared 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 of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

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

(0.5 part by weight),

Foxglove (0.5 part by weight)

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

Substances such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%) and 5 g of ethyl acetate fraction of Example 2 of the present invention. After homogeneous mixing and sterilization, it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of Vegetable Juice

5 g of the ethyl acetate fraction of Example <2-2> of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Preparation of Fruit Juice

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

< Manufacturing example  4> Preparation of feed additive

Using the normal-butanol fraction of Example <2-3> as an active ingredient, the present inventors prepared a feed additive with the following composition.

<The composition of the feed additive>

Normal-butanol fraction of Example <2-3> of the present invention: 0.1-20% by weight,

Lipase: 0.001 to 0.01% by weight,

Tertiary calcium phosphate: 1-20% by weight,

Vitamin E: 0.01-0.1% by weight,

Enzyme powder: 1 to 10% by weight,

Lactic acid bacteria: 0.1 to 10% by weight,

Bacillus culture medium: 0.01 to 10% by weight, and

Glucose: 20 to 90% by weight.

The amphibian extract of the present invention or a fraction thereof is effective in preventing and delaying diabetic complications, and is a natural extract effective in preventing aging, and is only pharmaceutically usable as a composition for diabetic complications, anti-aging and delaying, cancer prevention and treatment. In addition, it can be usefully used as a dietary supplement or a functional feed additive.

1 is a diagram showing the extraction and phylogenetic separation of the amphibian.

Figure 2 is a diagram showing the effect on the neovascularization of the amphibian extract in type 2 diabetes animal model (SDT).

3 is a diagram showing the effect on the paravascular cell loss and acellular capillary formation of the amphibian extract in type 2 diabetes animal model (SDT).

4 is a diagram showing the effect of the retinal vessels (left) and retinal nerve cells (right) final glycation product accumulation of the amphibian extract in type 2 diabetes animal model (SDT).

Figure 5 is a diagram showing the efficacy of the prevention of apoptosis of retinal vessels (left) and nerve cells (right) of the amphibian extract in type 2 diabetes animal model (SDT).

Figure 6 is a graph showing the proteinuria, albuminuria, glomerular filtration rate of renal function indicator of the amphibian extract in type 2 diabetes animal model (SDT).

Figure 7 is a diagram showing the effect on the final glycated product and CML content changes in the urine of the amphibian extract in type 2 diabetes animal model (SDT).

80 is a diagram showing the effect on the change in the final glycation product (AGEs) and CML content in the kidney of the amphibian extract in type 2 diabetes animal model (SDT).

9 is a diagram showing the effect on the morphological changes according to the accumulation of extracellular matrix material of the amphibian extract in type 2 diabetes animal model (SDT) through PAS staining.

FIG. 10 is a graph showing Masson's Trichrome staining of the effects of extracellular matrix accumulation of amphibian extract on type 2 diabetes animal model (SDT). FIG.

11 is a diagram showing the effect on the podocyte loss of the amphibian extract in type 2 diabetes animal model (SDT).

Claims (15)

Homonoia riparia Lour.) A composition for preventing and treating diabetic complications containing the extract as an active ingredient. The method of claim 1, wherein the amphibian extract is water, C ₁ A composition for preventing and treating diabetic complications, characterized in that extracted with a lower alcohol or a mixed solvent of C ₄ ~. 3. The composition for preventing and treating diabetic complications of claim 2, wherein the lower alcohol is ethanol or methanol. According to claim 2, wherein the lower alcohol is a composition for the prevention and treatment of diabetic complications, characterized in that the water content of 0.1% to 50%. The method of claim 1, wherein the amphibian extract is a composition for preventing and treating diabetic complications, characterized in that using the leaves or branches of the amphibian. A composition for preventing and treating diabetic complications, which contains a fraction prepared by further dividing the amphibian extract with an organic solvent as an active ingredient. The method of claim 6, wherein the fraction is a normal-hexane fraction, ethyl acetate fraction, normal-butanol fraction obtained by suspending the amphibian extract in water and then systematically fractionating the mixture into normal-hexane, ethyl acetate, normal-butanol and water. Or water fraction is any one composition for preventing and treating diabetic complications, characterized in that. 7. The diabetic complication of claim 1, wherein the diabetic complication is any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, heart disease and osteoporosis. A composition for preventing and treating diabetic complications. Health functional food for the prevention and improvement of diabetic complications containing the water extract or its fraction as an active ingredient. Functional feed additive for the prevention and improvement of diabetic complications containing the water extract or fractions thereof as an active ingredient. The composition for preventing and treating diabetic complications according to claim 1 or 6, wherein the amphibian extract or a fraction thereof inhibits the production of the final glycation end product. delete delete delete delete

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