KR20150078773A - Composition comprising broussonetia kazinoki extract for preventing and treating diabetic complication - Google Patents

Abstract

The present invention relates to a composition comprising a Broussonetia kazinoki extract as an active ingredient for preventing and treating diabetic complications and, more specifically, to a pharmaceutical composition comprising a Broussonetia kazinoki extract as an active ingredient for preventing and treating, or alleviating diabetic complications and to a functional health food including the same. The Broussonetia kazinoki extract of the present invention has anti-oxidant effect to control the induction of diabetic complications due to an oxidative stress caused by the self-oxidation of glucose, exhibits inhibitory efficacy against aldose reductase and has excellent inhibitory ability against advanced glycation end products to have outstanding therapeutic or alleviating effects on diabetic complications caused by the generation of advanced glycation end products, thereby being used as an active ingredient in a composition for preventing and treating various diabetic complications.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for preventing and treating diabetic complications,

The present invention relates to a method for producing Broussonetia The present invention also relates to a pharmaceutical composition for preventing and treating diabetic complications using the extract of the present invention.

Diabetes mellitus is one of the most important diabetes mellitus in the world. Recently, the prevalence of diabetes mellitus has reached 10% in Korea with rapid economic growth. Currently, diabetes mellitus has more than 240 million diabetic patients and 2025 In 2009, the American Physicians Association (JAMA) announced that 60% of the cases will occur in Asia, with a worldwide increase to 380 million. In particular, the onset of diabetes mellitus was drawn to the elderly, and it was inevitable that complications would be caused by prolonged life expectancy. That is, generally, 10 to 20 years after the onset of diabetes, almost all the organs in the body are damaged, causing diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, Diabetic neuropathy, heart disease, cancer or osteoporosis. Chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end-stage renal failure, and diabetic cataract and retinopathy cause blindness and eventually death. In the United States, diabetes is the cause of blindness in the 25- to 74-year-old age group, and after 15 to 20 years after the onset of diabetes, 60% are blind. Therefore, even if the duration of complications in diabetic patients is delayed by 5 to 10 years, the quality of life of patients and their families will be different, and will have a big impact on national finances.

The mechanism of this diabetic complication is largely explained by (1) the nonenzymatic glycation of protein, (2) the polyol pathway, and (3) the oxidative stress. .

Non-enzymatic glycation of proteins is a condensation reaction in which amino acid groups such as lysine residues of proteins and reducing sugars do not have enzymatic action, that is, by a milliad reaction. As a result of this reaction, advanced glycation endproducts (AGEs) do. The non-enzymatic glycosylation of proteins involves the following steps: (1) an amino acid group such as a lysine residue of a protein and an aldehyde or ketone of a reducing sugar undergo nucleophilic addition reaction without enzymatic action to form a schiff base as an initial step product, (2) a step in which a reversible Amadori-type early glycation end product is produced by condensation of ketoamine adducts adjacent to the base, and (2) a step in which a reversible ampholytic early glycation end product is decomposed The final glycation products, which are irreversible products, are produced, and the final glycation products thus formed are bound or cross-linked with proteins or lipids, thereby producing irreversible products such as glycated proteins or glycated lipids . Unlike the reversible amaryllis type early glycation products, the final glycation products are irreversible reaction products, and once formed, they are not degraded even when the blood glucose is restored to normal, accumulated during the survival period of the protein or lipid bound to the final glycation products, (Vinson, JA et al., 1996, J. Nutritinal Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur. J. Biochem. , ≪ / RTI > 210: 729-739). For example, glycated albumin, one of the final glycation products produced by the reaction of glucose with various proteins, is an important factor in causing chronic diabetic nephropathy. Glycated albumin enters the ganglion cell more easily than normal albumin without glycosylation, and high glucose stimulates mesangial cells to increase synthesis of extracellular matrix. Overgrowth of glycosylated albumin and increased extracellular matrix cause fibrosis of the glomerulus. Such a mechanism would continue to damage the shrine sphere, leading to a stage where extreme treatment methods, such as hemodialysis or organ transplantation, are inevitable. In addition, it has been reported that collagen in the arterial wall due to chronic diabetes and basement membrane protein in the ganglion cell are combined with the final glycation products and accumulate in tissues (Brownlee, M., et al., 1986, Sciences, 232, 1629- 1632).

This non-enzymatic protein glycosylation leads to saccharification in proteins such as basement membrane, plasma albumin, lens protein, fibrin, collagen, etc., and the resulting final glycation products abnormally change the structure and function of the tissue to cause diabetic retinopathy retinopathy, retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy. In addition, it has been reported that lipid metabolism abnormality occurs in the process of producing a final glycation end product in a hyperglycemic state, and a defensive system function against harmful oxygen free radicals generated at the same time is lowered, resulting in oxidative stress (Yokozawa, T., et al., 2001, J. of Trad. Med., 18: 107-112). These non-enzymatic glycation reactions and oxidative stress mechanisms are related to each other.

The polyol pathway includes the steps of (1) reducing aldose or ketose to form sorbitol by the action of aldose reductase (AR), and (2) step of producing sorbitol by oxidation with sorbitol dehydrogenase to produce fructose . Although the aldose reductase has a very low affinity for glucose in the normal state, the aldose reductase, which is the first enzyme of the polyol pathway, is excessively activated by the hyperglycemic state, resulting in excessive hyperglycemia being converted to sorbitol and fructose And the osmotic balance is disrupted to cause complications. That is, due to the increased osmotic pressure, water is drawn into diabetic retinopathy, diabetic cataract, and diabetic neuropathy (Kim, EJ, et al., Diabetes Mellitus, Korean Diabetes Society, Korea Medicine, 483; Soulis-Liparota, T., et al., 1995, Diabetologia, 38: 357-394). (Nakamura, N., et al., 2000, Free Radic Biol. Med., 29: 293-32), which has been reported to activate the major enzyme of the polyol pathway in human microvascular endothelial cells 17-25). At this time, fructose is about 10 times faster in non-enzymatic glycosylation of protein than glucose. Thus, a high concentration of fructose binds to the protein and ultimately accelerates the formation of the final glycation end product. Thus, nonenzymatic glycation, polyol pathway, and oxidative stress mechanisms are associated with each other, leading to diabetic complications. It has been reported that lipid metabolism abnormality occurs during the production of a final glycation end product in a hyperglycemic state, and oxidative stress is induced by deterioration of the defensive system function against harmful oxygen free radicals generated at the same time (Yokozawa, T., et al, 2001, J. of Trad. Med., 18: 107-112). Therefore, the non-enzymatic glycation reaction and the oxidative stress mechanism are related to each other. Thus, it has been found that inhibition of the final glycation endogenesis is very important for delaying, preventing or treating the onset of diabetic complications (Brownlee, M., et al., 1988, N. Engl. Med., 318, 1315-1321 ).

Currently, aminoguanidine, a synthetic glycoprotein inhibitor, is a nucleophilic hydrazine that binds with lipid products to prevent cross-linking with proteins, thereby inhibiting the production of final glycation products and leading to complications (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, which has progressed to Phase III clinical trials, but it has been discontinued due to toxic effects caused by long-term administration. Therefore, there is a demand for the development of safe and efficacious natural medicines.

Recently, many studies have been published to prevent / treat diabetic and diabetic complications of natural products. Korean Patent Laid-Open Publication No. 2013-0096588 describes a method for producing a fermented product of acid garlic using a solid phase fermentation method and a composition for preventing or treating diabetic or diabetic complication including fermented product of an acid garlic, and Korean Registration No. 10-1126677 A composition for preventing and / or treating diabetic and / or diabetic complications containing Codonopsis lanceolata extract or a fraction of the above extract as an active ingredient has been described. In Korean Registration No. 10-1142154, Opuntia ficus- The present invention relates to a composition for preventing and / or treating diabetes and / or diabetic complications comprising an extract of a plant, an indica root or a fraction of the above extract as an active ingredient. Korean Registration No. 10-0393908 discloses a composition for preventing or treating diabetic complications Rubus coranus Miquel) extract, which is effective in inhibiting or preventing diabetic complications, A composition comprising Vitamin E or Vitamin C is described.

On the other hand, the mulberry (Broussonetia kazinoki) is called a deciduous tree in Chinese characters. It is a deciduous broad-leaved shrub of Broussonetia kazinoki SIEB., Moraceae. Its name is Brousonetia kazinoki SIEB. It is a deciduous broad-leaved shrub that can be seen from the margin of the mountain. It has short hairs on small branches but disappears when growing. Its bark is grayish brown and its height is about 3m. Leaves are egg-shaped or oval-shaped, with sharp sawtooth on the edge, and they are torn in different shapes as the worms eat. The flower is reddish purple in June to June, and the fruit is spherical in shape and mature in September. The mulberry leaves are cut in the center of the skins and the fruit is called 楮 实 子. The leaves are collected in summer and autumn with young branches and leaves. Leaves are horny, egg-shaped or egg-shaped, long acuminate, original or hypodermic, 5-20 cm long, sharp at the end, sometimes deeply cracked, with sharp sawtooth on the edge. Some young trees have two or three acute angle. The surface is rough and the back has hairs at first. The petiole is 1 to 2 cm long, with curly hairs but gradually disappears (National Biology Specification Information System, http://www.nature.go.kr/plant/plantGuide/). Mackerel was once used to weave a type of fabric called foil, which is used as paper mill raw material. In order to process paper mulberry into papermaking raw materials, the harvested stem is first cut into a length of 1 to 2 m, put in a sealed pot, and steamed. If you steam it for about two hours, you smell the first soil and you smell a sweet flavor. At this time, take out and peel. The dried ones are called black skin or rough skin and can be processed again to obtain white skin. The black peel that is scratched is used as a raw material for the production of white wool (Nakamura et al., 1962), the cultivation and use of special forest products (1983), Korea Forestry Research Institute (1966), Mulberry Cultivation and Utilization (Kim Young-Ryun, Forestry, Forestry Cooperatives) Association, 1973). It is known that the pharmacological effect is effective for humidification, diuretic, hibernation, rheumatism, petechiae, species, bruises, and dermatitis. Chemical components include Kazinol A, Kazinol B, Kazinol D, Kazinol E, Kazinol F, Kazinol K, Kazinol Broussonetine J, broussonetine J, broussonetine R, broussonetine S, broussonetine T, broussonetine V, broussonetine O, broussonetine P, broussonetine Q, broussonetine Q, rosinol Q, kazinol R, broussonetine W, broussonetine X, broussonol B, broussonol C, broussonol D, and broussonol E, broussonetinine A, broussonetinine B, 7,4'-dihydroxyflavan, and oleanolic acid. However, such mackerel has not been reported to be useful for diabetic complications.

Accordingly, the inventors of the present invention confirmed the efficacy of the compound for the treatment of martenberry diabetic complications and completed the present invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating diabetic complications containing mulberry extract as an active ingredient, and a health food.

In order to accomplish the above object, the present invention provides a method for producing Broussonetia kazinoki) provides a preventive and therapeutic pharmaceutical composition for diabetic complications, containing the extract as an active ingredient.

The present invention also provides a health food for prevention and improvement of diabetic complications containing mulberry extract as an active ingredient.

The mackerel extract of the present invention exhibits an antioxidative effect and can control the induction of diabetic complications due to oxidative stress caused by autoxidation of glucose, exhibits an aldose reductase inhibitory effect, Can exert excellent effects on the treatment or amelioration of diabetic complications caused by the production of glycation products and can be usefully used as an effective ingredient of a composition for preventing or treating various diabetic complications.

As used herein, the term "diabetic complication" is a complication that may be caused by diabetes, including diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic retinopathy, Diabetic neuropathy, heart disease, cancer or osteoporosis, and the like.

As used herein, the term "prophylactic " means any action that inhibits or slows the progression of diabetic complications by administration of the composition of the present invention.

As used herein, the terms " treatment "and" improvement "refer to all actions by which administration of the composition of the invention improves or alleviates symptoms of diabetic complications.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

The term "individual" as used herein means all animals such as a human, a monkey, a dog, a goat, a pig or a mouse having a disease in which symptoms of diabetic complication can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of diabetic complications containing mulberry extract as an active ingredient.

The diabetic complication is preferably one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic neuropathy, heart disease, cancer or osteoporosis, but is not limited thereto.

The mulberry extract is preferably, but not limited to, prepared by a process comprising the steps of:

1) extracting the extract by adding an extraction solvent to the mulberry tree, preferably the mulberry tree branch;

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 mulberry of step 1) can be used without limitation such as cultivated or commercially available.

The extraction solvent is preferably an alcohol or a mixture of water and alcohol. As the alcohol, a C ₁ to C ₆ lower alcohol is preferably used, more preferably methanol and ethanol, and most preferably ethanol. As the extraction method, any extraction known in the art can be used, but preferably by cold extraction. It is preferable that the extraction solvent is added by 1 to 10 times the amount of dried mulberry tree extract. The extraction time is preferably 10 to 96 hours, more preferably 15 to 72 hours, but is not limited thereto. The extraction frequency is preferably 2 to 5 times, more preferably 1 to 3 times, or 3 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 is not limited thereto.

The mulberry extract of the present invention is also prepared by a manufacturing method comprising the following steps:

1 ') extracting an extractant, preferably an alcohol or a mixture of water and alcohol, on a branch of a mulberry tree, preferably a mulberry tree;

2 ') filtering the extract of step 1');

3 ') Step 2') filtration of the filtered extract under reduced pressure and drying;

4 ') suspending the extract of step 3') in water and then sequentially extracting with hexane, methylene chloride, ethyl acetate, and butanol to obtain respective fractions; And

5 ') Step 4 ') of each of the fractions obtained is concentrated under reduced pressure and then dried.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration of the steps 3 ') and 5'), but is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but is not limited thereto.

The present inventors have found that the mackerel extract has an antioxidative effect and thus can control the induction of diabetic complications due to oxidative stress caused by autoxidation of glucose, exhibits an aldose reductase inhibitory effect, It is possible to excel in the treatment or improvement of diabetic complications caused by the production of glycoconjugates (see Tables 1 to 3).

The composition containing the extract of Miscanthus officinalis or the fraction of the present invention may further contain one or more active ingredients which exhibit 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, . ≪ / RTI > Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and 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 simple diluents commonly used . 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 can be administered orally or parenterally according to the desired method, and the parenteral administration can be selected by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, . 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, The dose is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and can 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 for the prevention and treatment of diabetic complications.

The present invention also provides a method of treating diabetic complications comprising administering to a subject suffering from diabetic complications a composition containing a pharmaceutically effective amount of a mulberry extract as an active ingredient.

The present invention also provides a method for preventing diabetic complications, comprising administering to a subject a composition comprising a pharmaceutically effective amount of a mulberry extract as an active ingredient.

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

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

The above-mentioned administration method may be oral or parenteral administration. In the case of parenteral administration, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, intramural injection, intracerebroventricular injection, Can be administered by injection.

The diabetic complication is preferably any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic neuropathy, heart disease, cancer or osteoporosis, but is not limited thereto.

The mackerel extract of the present invention exhibits an antioxidative effect and can control the induction of diabetic complications due to oxidative stress caused by autoxidation of glucose, exhibits an aldose reductase inhibitory effect, Can exert excellent effects on the treatment or amelioration of diabetic complications caused by the production of glycation products and can be usefully used as an effective ingredient of a composition for preventing or treating various diabetic complications.

The present invention also provides a health food for prevention and improvement of diabetic complications containing mulberry extract as an active ingredient.

The diabetic complication is preferably any one selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic neuropathy, heart disease, cancer or osteoporosis, but is not limited thereto.

The mackerel extract of the present invention exhibits an antioxidative effect and can control the induction of diabetic complications due to oxidative stress caused by autoxidation of glucose, exhibits an aldose reductase inhibitory effect, Can excel in the treatment or amelioration of diabetic complications caused by the production of glycation products and can be usefully used as an effective ingredient of health foods for prevention and improvement of various diabetic complications.

The health food of the present invention may be used as it is, or may be used in combination with other food or food ingredients, and suitably used according to conventional methods.

There is no particular limitation on the kind of the health food. Examples of the foods to which the mulberry extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, drinks, tea, , An alcoholic beverage and a vitamin complex, and includes all the health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and 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 generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. 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 composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Production Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by Examples, Experimental Examples and Production Examples.

< Example  1> Qing tea mulberry  Preparation of bark extract

300 g of mulberry leaves were immersed in 2.5 L of 80% EtOH aqueous solution for 3 days at room temperature. The same procedure was repeated two times in total, and the resulting extract was concentrated by using a vacuum concentrator to obtain an ethanol extract (16 g, 5.3%).

The resulting extract was suspended in water and then sequentially fractionated with n-hexane, methylene chloride, ethyl acetate, and butanol. The fractions were concentrated by using a vacuum concentrator and then lyophilized to obtain a fraction (n-hexane) ), Methylene chloride fractions (2.37 g, 14.8%), ethyl acetate fractions (1.51 g, 9.4%), butanol fractions (2.10 g, 13.1%) and H ₂ O fractions (8.0 g, 50%).

< Experimental Example  1> Measure diabetes complications or preventive effects 1 - Measure antioxidant effect

In order to measure the inhibitory effect of oxidative stress on the incidence of diabetic complications, the antioxidative effect of the extracts of mulberry tree branches and fractions was investigated in order to measure the inhibitory effect on the diabetic complications of the mulberry tree branch extracts and fractions prepared in Example 1 Respectively.

The antioxidant effect was confirmed by ABTS free radical scavenging activity and DPPH scavenging activity. In the experiment for confirming the antioxidant effect described below, methanol extract was added to the obtained extract to prepare an extract at a concentration of 1 mg / ml in order to use the mulberry branch extract of the present invention and each fraction for the experiment.

The ABTS free radical scavenging activity was measured by mixing 2 mM ABTS and 3.5 mM potassium persulfate in 10 ml of tertiary distilled water, adding 290 ml of tertiary distilled water, diluting 30 times, overnight) to induce ABTS radical formation. The extracts of mulberry tree branch and fraction 10 prepared in Example 1 and 290 AB of ABTS radical solution were added thereto, reacted at room temperature for 10 minutes, and absorbance was measured at 750 nm. Trolox, a known antioxidant, was used as a control in order to compare the inhibition rate of ABTS radicals of the mulberry extract and fractions. The radical scavenging activity of the extracts and fractions of the mulberry leaves of the present invention was calculated as the concentration required for 50% elimination of ABTS radicals and converted into IC ₅₀ values.

ABTS radical inhibition IC ₅₀ ([mu] g / ml) 80% ethanol extract of mulberry leaves 20.1 Hexane fraction 67.2 Methylene chloride fraction 11.0 Ethyl acetate fraction 3.1 Butanol fraction 18.6 Trolox (control) 3.5

As shown in Table 1, it was confirmed that the extracts of mulberry tree branches and the respective fractions had antioxidative activity. From the above results, it was confirmed that the extract of the present invention has antioxidative effect and can control the induction of diabetic complications due to oxidative stress caused by autoxidation of glucose.

< Experimental Example  2> Measures to prevent diabetes complications 2 - Aldos  Reducing enzyme inhibitory activity

In order to measure the inhibitory effect on the onset of diabetic complication caused by the inhibition of aldose reductase, the extracts of mulberry tree branch extract and the fractions thereof, Reduced enzyme inhibitory activity was measured. The inhibitory activity of the aldose reductase was determined by extracting the lens of the rat. In order to confirm the inhibitory effect of the aldose reductase described below, the extract of the mulberry tree branch of the present invention and each of the fractions were added to DMSO to prepare an extract at a concentration of 1 mg / ml.

 Seven male WKY (Wistar Kyoto), 10 weeks old, were used in the experimental animals (Korea). After the animal was anesthetized with ether, the lens was removed from the experimental animal, and a certain amount of sodium phosphate buffer (pH 6.2) was added and crushed according to the wet weight of the extracted lens to homogenize the lens. The hybridized lens was centrifuged at 4 ° C, and the supernatant was saturated with ammonium sulfate to 40%. The supernatant obtained by centrifugation was again taken, and ammonium sulfate was further added to 70% After stirring, the pellet obtained by centrifugation was suspended in a minimum amount of buffer solution, dialyzed for 1 day, and then used as an enzyme source.

100 μl of the enzyme source, 700 μl of a phosphate buffer (pH 7.0) as a reaction buffer, 100 μl of DL-glyceraldehyde (0.1 M) as a substrate and 100 μl of a coenzyme NADPH (1.6 mM) After the enzyme solution was added and reacted, the decrease rate of NADPH absorbance at 340 nm was measured with a spectrophotometer. Quercetin, a known aldose reductase inhibitor, was used as a control in order to compare the aldose reductase inhibitory activity of the mulberry tree branch extract and each of the fractions. The aldose reductase inhibitory activity of the mulberry tree branch extract and fractions of the present invention is shown in Table 2 below in terms of the IC ₅₀ value at the time when the activity of aldose reductase was inhibited by 50%.

Concentration (/ / ml) % Inhibition IC ₅₀ ([mu] g / ml) 80% ethanol extract of mulberry leaves 10 65.26 7.24 5 32.9 One 27.00 Hexane fraction 10 41.93 - 5 - One - Methylene chloride fraction 10 88.84 5.06 5 49.62 One 18.08 Ethyl acetate fraction 10 70.56 6.52 5 38.16 One 3.77 Butanol fraction 10 63.27 2.09 5 40.65 One 25.17 Quercetin 10 66.07 1.33 5 65.61 One 30.26

As shown in Table 2, the 80% ethanol extracts and fractions of mulberry leaves showed excellent efficacy of extracts and fractions compared to quercetin, a single compound used as a positive control, in reducing the aldose reductase activity (Table 2).

< Experimental Example  3> Measure diabetes complications or preventive effects 3 - The final glycation product  Production inhibitory effect

In order to measure the inhibitory effect on the onset of diabetic complications due to the inhibition of final glycation endogenesis in relation to the treatment or preventive effect of diabetic complications of the mulberry branch extract and each of the fractions prepared in Example 1, The effect of inhibiting product formation was measured. In order to confirm the effect of inhibiting the formation of the final glycation end product, the extracts of the mulberry tree branch of the present invention and the respective fractions were added to the extract obtained in Example 1, .

A solution of 50 mM bovine serum albumin (BSA) and 5 mM methyl glyoxal in solution was dissolved in 0.1 M sodium phosphate buffer (pH 7.4) , And 0.02% of sodium azide as an antimicrobial agent to prevent bacterial growth during the reaction period. The extracts of mulberry tree branches and fractions were added to the reaction solution and reacted at 37 ° C for 5 days. Then, fluorescence (excitation (Ex): 350 nm, emission (EM): 450 nm) was measured using a fluorescence spectrophotometer And the results are shown in Table 3 below. In order to compare the inhibition of the final glycation endogenous production of the mulberry tree branch extract and each of the fractions, aminoguanidine, a known inhibitor of final glycation endogenesis, was used as a control.

Concentration (/ / ml) % Inhibition 80% ethanol extract of mulberry leaves 100 48.3 Hexane fraction 200 - Methylene chloride fraction 100 21.0 Ethyl acetate fraction 100 67.1 Butanol fraction 100 20.2 Aminoguanidine 100 39.1

As shown in the following Table 3, the extracts of mulberry tree branches and the fractions thereof were superior to the aminoguanidine used as a control at a concentration of 100 ㎍ / mL. It was confirmed that the extract of Mulberry tree branch of the present invention and each of the fractions were excellent in the treatment or improvement of diabetic complications caused by the production of the final glycation end product.

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Preparation of powder

The extract of Example 1 2 g

Lactose 1 g

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

<1-2> Preparation of tablets

The extract of Example 1       100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate           2 mg

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

&Lt; 1-3 > Preparation of capsules

The extract of Example 1       100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate   2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

<1-4> Production of pills

The extract of Example 1         1 g

Lactose 1.5 g

glycerin   1 g

Xylitol 0.5 g

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

The extract of Example 1 150 mg

Soybean extract        50 mg

glucose 200 mg

Starch       600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacturing of food

<2-1> Production of flour food

0.5 to 5.0 parts by weight of the extract of Example 1 of the present invention was added to wheat flour, and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<2-2> Production of soups and gravies

0.1 to 5.0 parts by weight of the extract of Example 1 of the present invention was added to the soup and the juice to prepare a health improvement meat product, noodle soup and juice.

<2-3> Preparation of ground beef

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

<2-4> Manufacture of dairy products

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

&Lt; 2-5 >

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 extract of Example 1 of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying, and dried with a hot air drier, and the dried product was pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds and the extract of Example 1 prepared above were blended in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 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)

The extract (3 parts by weight) of Example 1,

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  3> Manufacturing of beverage

<3-1> Production of health drinks

5 g of the extract of Example 1 of the present invention was homogeneously mixed with a material such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% Sterilized, and packaged in glass bottles, plastic bottles, and other small containers.

<3-2> Preparation of vegetable juice

5 g of the extract of Example 1 of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Production of fruit juice

1 g of the extract of Example 1 of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice.

Claims (8)

Mulberry ( Broussonetia The present invention relates to a pharmaceutical composition for the prevention and treatment of diabetic complications comprising, as an active ingredient,
The pharmaceutical composition for preventing and treating diabetic complications according to claim 1, wherein the mulberry extract is an extract obtained by extracting mulberry leaves with ethanol or a mixture of water and ethanol.
The method according to claim 1, wherein the extract of the mulberry extract is selected from the group consisting of hexane fraction, methylene chloride fraction, ethyl acetate fraction and butanol fraction obtained by extracting with ethanol or a mixture of water and ethanol and then sequentially extracting with hexane, methylene chloride, ethyl acetate and butanol Or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein the diabetic complication is selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, heart disease, cancer or osteoporosis &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof, for the prophylaxis and treatment of diabetic complications.
A health food for prevention and improvement of diabetic complications containing mulberry extract as an active ingredient.
[Claim 6] The health food for preventing and improving diabetic complications according to claim 5, wherein the mulberry extract is an extract obtained by extracting mulberry with ethanol or a mixture of water and ethanol.
The method according to claim 5, wherein the extract of the mulberry extract is selected from the group consisting of a hexane fraction, a methylene chloride fraction, an ethyl acetate fraction and a butanol fraction obtained by successively extracting with ethanol or a mixture of water and ethanol and then sequentially extracting with hexane, methylene chloride, ethyl acetate and butanol. Wherein the diabetic complication is a diabetic complication.
[Claim 6] The health food according to claim 5, wherein the diabetic complication is diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, heart disease, cancer or osteoporosis.