KR100747640B1 - Composition comprising the stamen extract of Nelumbo nucifera or the compound isolated therefrom for preventing or treating diabetic complication - Google Patents

Abstract

The present invention is trained in Nelumbo nucifera stamen) extract or a composition for the prevention and treatment of diabetic complications containing the compound isolated therefrom as an active ingredient, the soft water extract of the present invention or a compound isolated therefrom is a potent inhibitory activity against aldose reductase Since the composition, the composition can be usefully used as a pharmaceutical composition and health functional food for preventing or treating diabetic complications.

Training, aldose reductase, diabetic complications, pharmaceutical composition, dietary supplement

Description

Composition comprising the stamen extract of Nelumbo nucifera or the compound isolated there from for preventing or treating diabetic complication

The present invention relates to a composition for preventing or treating diabetic complications, comprising a soft water extract or a compound represented by the following formula (1) isolated therefrom.

Diabetes mellitus is one of the most common chronic adult diseases, and it is estimated that about 5% of the total population in Korea is at least 2.5 million. In advanced countries, the number of diabetics is increasing every year, and in Korea, the number of patients is expected to increase gradually with the improvement of living standard and westernized lifestyle. Diabetes is a metabolic disease in which the body's ability to use glucose normally impairs its blood sugar levels and increases its glucose in the blood, causing excess sugar to be excreted in the urine. Diabetes is caused by a lack of insulin. Insulin deficiency occurs when the absolute amount of insulin is low or when a relatively low amount of hormones other than insulin are released. Insulin, a polypeptide hormone, is produced from beta cells of the island of Langerhans in the pancreas. When pancreatic hormone insulin is balanced in the body with a hormone that acts against the insulin secreted by the pituitary gland, the adrenal gland and the thyroid gland, the glycemic control function is normal. maintain.

About 10% of diabetics are classified as Type I. Insulin dependent diabetes mellitus (IDDM) and are usually hereditary because they develop in younger age groups under 20 years of age, also known as juvenile onset diavetes. . It is a disease with many acute forms and high incidence in girls, but can be treated by insulin administration. It occurs due to specific human lymphocyte antigen (HLA) and viral infection, which causes the cells of Langerhans to be destroyed to prevent insulin from secreting. In general, people lose weight and are prone to ketonacidosis.

Type II.Non-insulin dependent diabetes mellitus NIDDM is also called adult onset diabetes, which develops after age 40. Peripheral tissues such as muscles and adipose tissue due to lack of exercise, obesity, overeating and stress, etc. In the United States, susceptibility to insulin is slowed, and the metabolic disorder develops for 4-5 years. Type 2 diabetes is called insulin-independent diabetes because it can be cured by weight loss by diet and exercise therapy, and 50-80% can be cured.

Malnutrition-related diabetes (type 3 diabetes) is a young diabetes with a high incidence in the so-called Third World, especially in the tropics, which is slightly different from the previous two forms of diabetes, which has a history of malnutrition in childhood. The instrument separated it into a separate form.

Diabetes is a greater risk for complications than it is for itself, and one of the greatest goals in its treatment today is to inhibit the development or progression of diabetic complications. Diabetic complications are long-standing symptoms of diabetes, and chronic complications usually occur 10 to 15 years after the onset of diabetes. Representative diseases include diabetic retinopathy, diabetic nephropathy, and diabetic neurosis.

Diabetic neuropathy is a disorder of the nervous system due to diabetes, which is caused by disorders of the peripheral nerves, loss of the keyboard, paralysis of motor nerves, autonomic nerve disorders, etc., resulting in tingling, burning, painful and sexual dysfunction. It can also cause your stool to go away.

Diabetic nephropathy is a microvascular complication caused by sclerotic lesions of the renal glomerular capillaries. Elevated blood pressure also acts to make diabetic nephropathy worse, with diabetic nephropathy usually affecting about 5% of people who have had diabetes for more than 10 to 15 years.

Diabetic retinopathy is a microvascular complication that is a serious complication that causes blindness in diabetics.

A recent study on the relationship between diabetes control and complications showed that normalizing blood glucose levels with enhanced insulin treatment significantly reduced the incidence of diabetes complications (Seaquist ER et al. , New Eng . J. Haematol , 320 , pp1161-1165, 1989). ).

Hyperglycemia, a symptom of diabetes, causes the diabetic complications (Sato Y. and Rifkin DB, J. Cell. Biol . , 109 , pp309-315, 1989), oxidative stress ( Williamson JR et al. , Diabets , 42 , pp801-813, 1993), reduction of myoinositol and decrease of Na ⁺ , K ⁺ -ATPase activity (Greene DA et al. , New Eng . J. Med . , 316 , pp 599-606, 1987). Among them, the polyol pathway is one of glucose metabolism pathways, which consists of two reaction systems in which glucose is converted into fructose via sorbitol and two enzymes (Gabbay KH). and O'Sullivan JB, Diabetes , 17 , pp239, 1968; Dvornik D. et al. , Science , 182 , pp1146, 1973). Glucose is an important energy source and is normally absorbed into cells and is then metabolized in the glycolysis pathway by metabolizing glycosyl-6-phosphate by hexokinase. It is metabolized through a path no more than just a few% (Collins JG and Corder CN, . Invest. Ophthalmol. Visual Sci, 16, pp242, 1977). However, cell membrane permeation of glucose in tissues where glucose uptake into cells does not depend on insulin depends on extracellular glucose concentration. Therefore, in diabetes, hyperglycemic state, lens, retina, cornea, peripheral nerve, blood vessel, renal glomeruli, erythrocyte, etc. Intracellular glucose concentration of insulin-independent tissues of the rats increases, and glucose metabolism is enhanced by aldose reductase, an essential enzyme of the polyol pathway, which is present in large quantities in the cells of these tissues. .. Collins JG and Corder CN, Invest Ophthalmol Visual Sci, 16, pp242, 1977;.. Thus the Dvornik D. et al, Science, 182 , pp1146, 1973), as a result of the onset of symptoms sites of sorbitol accumulation diabetic Diabetes complications such as cataracts, diabetic retinopathy, diabetic keratosis, diabetic neurosis, and diabetic nephropathy (Heath H. and Hamlett YC, Diabetologia , 12 , pp 43, 1976). The reduction of glucose to sorbitol catalyzed by aldose reductase does not cause a reverse reaction at its equilibrium. Furthermore, since the activity of sorbitol dehydrogenase is lower than that of aldose reductase, the conversion of sorbitol to fructose is slightly, and consequently, the enhancement of the polyol pathway accumulates sorbitol (Collins JG and Corder CN, Invest. Ophthalmol. Visual Sci., 16, pp242, 1977). In addition, sorbitol, which is a polysaccharide alcohol, has high polarity and is difficult to diffuse out of the cell membrane, which also causes intracellular accumulation of sorbitol. That is, when glucose concentration in the lens increases due to diabetes, sorbitol production is enhanced by aldose reductase, and accumulation of sorbitol raises intracellular osmotic pressure and promotes water inflow into the lens, causing cell swelling ( Gabbay KH and O'Sullivan JB, Diabetes , 17 , pp239, 1968; Dvornik D. et al. , Science , 182 , pp1146, 1973). This swelling enhances the permeability of lens fibroblasts, the influx of sodium (Na ⁺ ) and chlorine (Cl ⁻ ) ions, potassium ions (K ⁺ ), amino acids, peptides, adenosine triphosphate (ATP), myo-inositol ) Causes spillage. Electrolytic equilibrium destruction, such as sodium ions and potassium ions, further increases osmotic pressure, further promotes cell swelling, and prevents the cell membrane from maintaining normal. At the same time, intracellular protein degeneration proceeds and the lens becomes turbid. Cataract symptoms, which appear as the lens becomes turbid, are used as an indicator of diabetic progression. Therefore, a substance that inhibits the production of sorbitol is able to prevent the development of diabetic cataract, and theoretically, it is possible by inhibiting the enzyme aldose reductase producing sorbitol. Indeed, many reports have shown that, in vitro experiments, substances that inhibit aldose reductase inhibit the formation of cataracts in galactosemic rats, a conditional model of experimental diabetic cataracts. have. Okda J. et al. , Chem . Pharm . Bull ., 33 , pp2990, 1985) described 1-[(para-bromo-phenyl) -sulfonyl] hydan with inhibitory activity against aldose reductase. Toin (1-[(p-bromo-phenyl) -sulfonyl] hydantoin) has been shown to inhibit cataract formation in galactosemic rats and to suggest a link between flavonoids with aldose reductase inhibitory activity and diabetic cataract suppression. There is also a report (Varma SD and Kinoshit JH, Biochem . Pharmacol ., 25 , pp 2505, 1976; Parmar NS and Ghosh MN, Exp . Eye. Res ., 29 , pp 229, 1979). In addition, Shin et al. (Shin KH et al. , Kor . J. Pharmacogn ., 25 , pp41, 1994) reported that the flavonoids contained in gold were found in the It has been reported to inhibit activity and at the same time inhibit cataract formation in galactosemic rats. Aida et al. (Aida K. et al. , Planta Med ., 53 , pp121, 1987) reported that these extracts inhibited aldose reductase activity in in vitro experiments on herbal medicines that have long been used to treat diabetic neuropathy in Japan. It has been reported that iso-liquiritigenin has potent inhibitory activity against aldose reductase (Aida K. et al. , Planta Med ., 56 , pp254, 1989).

Diabetic complications vary, and due to direct hyperglycemia, peripheral neuropathy, retinopathy, nephropathy, cataracts, keratosis, and the like, one of the mechanisms of the onset of the polyol pathway is mentioned. Thus, aldose reductase inhibitors have attracted attention as agents for the prevention and treatment of diabetic complications such as cataracts, and some aldose reductase inhibitors have been reported to improve diabetic complications in animal and clinical trials. Active research has been conducted to identify inhibitors of aldose reductase from substances, and flavonoids, tannins, coumarins and essential oils have inhibitory effects on aldose reductase activity extracted from various tissues of cattle, rats or humans. This was reported.

Smoking ( Nelumbo nucifera Gaertner) is a perennial aquatic plant belonging to the water lily family, widely distributed all over the world, used for ornamental, edible and medicinal purposes, and uses almost all parts such as leaves, flowers, lotus and lotus roots for food (Luo X). et al ., Analytica Chemica Acta , 538, pp 129-133, 2005). Traditionally, the leaves have been used for fever and detoxification because they are lower lobes. Seeds and flesh have been used for tonic, hemostatic drugs, nocturnal enuresis, and gynecological diseases. (KHP), pp500, 2000; Kim Chang-min, Chinese Medicinal Dictionary, pp5956-5959, 1998).

Training ( Nelumbo nucifera stamen) the lotus ( Nelumbo nucifera ), which is harvested in full bloom in summer and dried.

Known as components of each part of the lotus, seed embryos contain alkaloids such as lienesine, isoliensinine, lotusine, neferine and pronuciferine. Isolated on the leaves: anonaine, pronuciferine, N- nornuciferine, liriodenine, di-methylcoclaurine, Merlin furnace (roemerine), nusi Perrin (nuciferine), five-nolnu when Perrin (O-nornuciferine), DL-American pabin (dl -armepavine), Al-Kokura We (R -coclaurine), S-nolko Curacao We ( Alkaloids such as S- norcoclaurine and flavonoid compounds such as quercetin glycosides have been identified. Ple This compound is known, such as flavonoids. Physiological activities include hepatoprotective and antioxidant activities of lotus roots (Sohn DH et al , Phytomedicine , 10, pp165-169, 2003), and pharmacological activities of lotus root were antibacterial and antidiarrheal activity, psychopharmacological activity, and voyage fever. Activity, antibacterial activity, antifungal activity, anti-inflammatory activity, antidiabetic activity, antioxidant activity (Mukherjee PK et al, Phytotherapy Research , 9, pp522-524, 1995; Mukherjee PK et al ., Journal of Ethnopharmacology , 54, pp63-67 , 1996; Mukherjee PK et al ., Indian Journal of Experimental Biology , 82, pp274-276, 1996; Mukherjee PK et al ., Indian Journal of Pharmacolology , 27, pp 262-364, 1995; Mukherjee PK et al ., Indian Drugs , 32, pp274-276, 1995; Mukherjee PK et al ., Planta Med . , 63, pp 367-370, 1995; Lee MW et al , Natural Product Sciences , 7, 107-109, 2001; Hu M and Skibsted LH, Food Chemistry , 76, pp327-333, 2002). Lotus leaf has antioxidant and anti-aze activity (Cho EJ et al , Phytomedicine , 10, pp 544-551, 2003; Wu MJ et al , The American Journal of Chinese Medicine , 31, pp 687-698, 2003; Kashiwada Y et al , Bioorg . Med . Chem . , 13, pp443-448, 2005), and soft water is known to have antioxidant activity (Jung HA et al , Arch. Pharm . Res ., 26, pp279-285, 2003; Korean Patent Registration No. 0514916, 2005). have.

However, there has been no report on the inhibitory effect of aldose reductase of soft water extracts and compounds isolated therefrom, and no improvement effect on diabetes and its complications has been reported. Therefore, the present inventors have completed the present invention by inhibiting aldose reductase in soft water, separating and identifying extracts and compounds for preventing and treating diabetic complications, and then confirming the inhibitory effect on aldose reductase.

It is an object of the present invention to provide a composition useful for the prevention and treatment of diabetic complications comprising a soft water extract having excellent aldose reductase inhibitory activity or a compound isolated therefrom as an active ingredient.

In order to achieve the above object, the present invention ( Nelumbo) It provides a pharmaceutical composition for the prevention and treatment of diabetic complications containing nucifera stamen) extract as an active ingredient, and containing a pharmaceutically acceptable carrier or excipient.

The extract is a crude extract or a non-polar solvent soluble extract, the crude extract includes an extract soluble in water, ethanol, methanol, butanol or a mixed solvent thereof, preferably methanol, the non-polar solvent soluble extract is dichloromethane, chloroform or Nonpolar solvents selected from ethyl acetate, preferably extracts soluble in ethyl acetate.

The present invention is trained in Nelumbo nucifera stamen) A pharmaceutical composition for the prevention and treatment of diabetic complications comprising an extract or a compound having the structure of the following general formula (I) separated therefrom as an active ingredient and containing a pharmaceutically acceptable carrier or excipient do.

(I)

(I)

Where

R ₁ represents a hydrogen atom, a galactosyl group, a glucosyl group, a glucuronyl group, a rhamnose- (1 → 6) -glucosyl group, a rhamnose- (1 → 2) -glucuronyl group, a rhamnose- (1 → 2) a substituent selected from -glucosyl group and glucuron-methyl group;

R ₂ is a hydrogen atom or a glucosyl group;

R ₃ and R ₄ are each independently a hydrogen atom, -OH or O-R 'group, and R' is a hydrogen atom

Or a lower alkyl group.

Preferred compounds of the above general formula (I) compounds include the following compounds.

Camperol (1), Camperol-3- O- β-D-galactopyranoside (2), Camperol-7- O- β-D-glucopyranoside (3), Camperol-3- Ο -β-D- glucopyranoside (4), camp Ferrol -3- Ο -α-L- ramno pyrazol nosil - (1 → 6) -β- D- glucopyranoside (5), camp Ferrol - 3- O- α-L-Ram nopyranosyl- (1 → 2) -β-D-glucuronopyranoside (6), Camperol-3- O- α-L-Rhamnopyranosyl- (1 → 2) -β-D-glucopyranoside (7), camperol-3- O- β-D-glucuronopyranoside (8), camphorol-3- o- β-D-glucu Lonofyranosyl methyl ester (9), myricetin-3 ', 5'-dimethylether-3- O- β-D-glucopyranoside (10), quercetin-3- o- β-D-gluco Lonofyranoside (11), Isolamnetin-3- O- β-D-glucopyranoside (12), Isoramnetin-3- O- α-L-Ranmopyranosyl- (1 → 6)- β-D-glucopyranoside (13).

Diabetic complications as defined herein include diabetic cataracts, diabetic retinopathy, diabetic keratosis, diabetic neurosis or diabetic nephropathy.

The pharmaceutical composition for the treatment and prevention of diabetic complications containing the soft water extract of the present invention and the compound isolated therefrom comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition.

Hereinafter, the present invention will be described in detail.

The soft water crude extract of the present invention, the nonpolar solvent soluble extract and the compound separated therefrom can be obtained as follows.

The soft water crude extract of the present invention is pulverized after softening and pulverizing the soft water, and the volume of water, methanol, ethanol and butanol of about 3 to 20 times, preferably about 3 to 5 times the weight of the soft water dry. Lower alcohol or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably about 0.5 hours to 2 days, preferably at an extraction temperature of 20 to 100 ° C., preferably 20 to 70 ° C., with methanol It is preferably obtained by extracting from 1 to 5 times, preferably 3 times consecutively by extraction methods such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 1 hour to 1 day, and then filtered under reduced pressure and rotating the filtrate under vacuum Concentrated under reduced pressure at 20 to 100 ℃, preferably 40 to 70 ℃ by a concentrator to obtain a soft water crude extract soluble in water, lower alcohol or a mixed solvent thereof.

The soft water crude extract is suspended in water and then extracted with a solvent in the order of dichloromethane, ethyl acetate, n -butanol to obtain a soft nonpolar solvent soluble extract of the present invention, preferably an ethyl acetate soluble fraction. More specifically, dichloromethane: water: methanol may be mixed in a crude soft extract, ie, soft methanol extract, in a ratio, preferably 10: 9: 1, to obtain a dichloromethane fraction and a water-soluble fraction. Ethyl acetate can be added to the soluble fraction to obtain ethyl acetate soluble fraction and water soluble fraction, and finally the water soluble fraction can be extracted with butanol to obtain butanol soluble fraction and water soluble fraction.

For the non-polar solvent soluble fraction, preferably ethylacetate soluble fraction can be subjected to Sephadex LH-20 column chromatography, with a fixed amount per hour with methanol, preferably 1,000 ml per hour Column fractionation was carried out at a rate of 5 to obtain five fractions F1 to F5, of which F2 fractions were subjected to Sephadex LH-20 column chromatography using methanol. Fractions may be obtained to obtain the compounds of (5), (6), (7), (8), and (11), respectively, and the F3 fraction may be subjected to silica gel column chromatography. Ethyl acetate: methanol = 10: 1 to obtain a fraction by a mixed solvent to obtain the compounds of the above (2), (3), (4), respectively, the F4 and F5 fractions It can be recrystallized Isolation and Identification of a compound of the above (1), 9, 10, 12, 13.

(1) Kaempferol: R ₁ = H, R ₂ = H

2 Camp Ferrol -3- Ο -β-D- galacto-pyrano seed (Kaempferol 3- O- β-D- galactopyranoside): R 1 = Gal, R 2 = H

3 Camp Ferrol -7- Ο -β-D- glucopyranoside (Kaempferol 7- O- β-D- glucopyranoside): R 1 = H, R 2 = Glc

4 Camp Ferrol -3- Ο -β-D- glucopyranoside (Kaempferol 3- O- β-D- glucopyranoside): R 1 = Glc, R 2 = H

(5) Camperol-3- O- α-L-lamnopyranosyl- (1 → 6) -β-D-glucopyranoside (Kaempferol 3- O- α-L-rhamnopyranosyl-(1 → 6)- β-D-glucopyranoside)

R ₁ = Rha- (1 → 6) -Glc, R ₂ = H

(6) Camperol-3- O- α-L-Rhamnosyl- (1 → 2) -β-D-glucuronopyranoside (Kaempferol 3- O- α-L-rhamnopyranosyl-(1 → 2) β-D-glucuronopyranoside): R ₁ = Rha- (1 → 2) -Gln, R ₂ = H

(7) Camperol-3- O- α-L-lamnopyranosyl- (1 → 2) -β-D-glucopyranoside (Kaempferol 3- O- α-L-rhamnopyranosyl-(1 → 2)- β-D-glucopyranoside)

R ₁ = Rha- (1 → 2) -Glc, R ₂ = H

8 camp Ferrol -3- Ο -β-D- glucuronic nopi pyrano seeded with (Kaempferol 3- O- β-D- glucuronopyranoside): R 1 = Gln, R 2 = H

9 camp Ferrol -3- Ο -β-D- glucuronic furnace pyrazol nosil methyl ester (Kaempferol 3- O- β-D- glucuronopyranosyl methyl ester): R 1 = Gln-Me, R 2 = H

10 in advance paroxetine 3 ', 5'-dimethyl ether -3- Ο -β-D- glucopyranoside (Myricetin 3', 5'-dimethylether 3- O- β-D-glucopyranoside): R 1 = Glc

11. quercetin -3- Ο -β-D- glucuronic in nopi pyrano seed (Quercetin 3- O- β-D- glucuronopyranoside): R 1 = Gln

12 iso person netin -3- Ο -β-D- glucopyranoside (Isorhamnetin 3- O -β-D- glucopyranoside): R 1 = Glc

13 iso person netin -3- Ο -α-L- ramno pyrazol nosil - (1 → 6) -β- D- glucopyranoside (Isorhamnetin 3- O- α-L- rhamnopyranosyl - (1 → 6) -β-D-glucopyranoside): R ₁ = Rha- (1 → 6) -Glc

The present invention provides a method for separating and purifying crude extracts, nonpolar solvent soluble extracts and compounds separated therefrom from soft water.

As a result of investigating the prophylactic and therapeutic effects of diabetic complications of the soft water extracts obtained from the above processes or the compounds isolated therefrom, the aldose reductase was inhibited prominently, and as a pharmaceutical composition for the prevention and treatment of diabetic complications. It was confirmed that it can be usefully used.

Therefore, the present invention comprises a soft water extract obtained by the above method and a compound isolated therefrom as an active ingredient, and a pharmaceutical composition for preventing and treating diabetic complications containing a pharmaceutically acceptable carrier or excipient. to provide.

Soft water extract of the present invention or a compound isolated therefrom has little toxicity and side effects, so can be used with confidence even for long-term use for the purpose of prevention.

The application amount and application method of the pharmaceutical composition comprising the soft water extract of the present invention and the compound separated therefrom may vary depending on the formulation and the purpose of use.

In addition, the composition comprising the soft water extract of the present invention and a compound isolated therefrom may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the composition comprising the soft water extract of the present invention and the compounds isolated therefrom include, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia Rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be.

Compositions comprising soft water extracts according to the present invention and compounds isolated therefrom are prepared according to conventional methods, respectively, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and It can be formulated and used in the form of sterile injectable solutions. When formulated, it is prepared using conventional diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient, for example, starch, calcium carbonate, sucrose ( Prepare by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, 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 amount of the soft water extract of the present invention and the compound separated therefrom may vary depending on the age, sex, and weight of the patient, but the amount of 0.1 to 100 mg / kg may be administered once to several times daily. The dosage of the compound can also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention also provides a dietary supplement for the prevention and improvement of diabetic complications containing the soft water extract and a compound isolated therefrom as an active ingredient.

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

The dietary supplement for the prevention and improvement of diabetic complications of the present invention comprises the extract or compound in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition.

In addition, it is possible to manufacture and process as a health functional food in the form of a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups or health drinks for the purpose of preventing and improving diabetic complications. Do.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for having the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

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

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

Example  1. Training Crude extract  Produce

The soft water used in the present invention was purchased from a medicinal herb in Busan. After soft freeze-drying, 10 ℓ of methanol was added to the powder obtained by grinding and pulverized. The mixture was refluxed and extracted three times at a predetermined time interval (12h, 6h, 3h) at 70 ° C. The extract obtained after filtration was applied to a vacuum rotary concentrator to remove methanol at 40 ° C., and then 240 g of crude water extract was obtained as an extracted residue, of which 2 g was used as a sample for screening aldose reductase inhibitory activity. It was.

Example  2. Preparation of Soft Dichloromethane Soluble Extract

       The crude extract obtained in Example 1 was dissolved in 2 L of water, and 2 L of dichloromethane was added to 2 L of the water-soluble part, followed by mixing 3-4 times to obtain 2 L of a water-soluble fraction layer and 2 L of a dichloromethane-soluble fraction. Thereafter, the dichloromethane soluble fraction was dried to obtain 53 g of a dichloromethane soluble extract, of which 2 g was used as a sample for searching for aldose reductase inhibitory activity.

Example  3. Preparation of Soft Ethyl Acetate Soluble Extract

2 L of ethyl acetate was added to 2 L of the water-soluble fraction layer obtained in Example 2, followed by mixing three to four times to obtain 2 L of the water-soluble fraction and 2 L of the ethyl acetate-soluble fraction, and then dried the ethyl acetate fraction. 21 g of ethyl acetate soluble extract was obtained and used as a sample, of which 2 g was used as a sample for searching for aldose reductase inhibitory activity.

Example  4. Training Butanol  Preparation of Soluble Extracts

2 L of butanol was added to 2 L of the water-soluble fraction obtained in Example 3, followed by mixing 3-4 times to obtain 2 L of the water-soluble fraction and 2 L of the butanol-soluble fraction, and then the water-soluble fraction and the butanol-soluble fraction. After drying, 29 g of soluble extract and soluble extract of butanol were obtained and used as a sample, of which 2 g was used as a sample for searching for aldose reductase inhibitory activity.

Example  5. Flavonoids  Isolation of the compound

21 g of the soft water ethyl acetate extract of Example 4 was subjected to Sephadex LH-20 column chromatography. At this time, a methanol solvent was used as a developing solvent, and the fixed phase was 1,000 ml per hour using a lipophilic Sephadex LH-20 (25-100 μ, Sigma Chem. Co., St. Louis, USA). 16,500 ml of the fractions (F1-F5) obtained by the respective fractions were concentrated, and 3,000 ml of the F1-F4 fractions and 3,300 ml of the F5 fractions, respectively, were 3.5 g, 5.7 g, 8.3 g, 1.5 g, 2.0 of the five lower fractions. g was obtained.

The F2 (5.7 g) fraction was purified by Sephadex LH-20 column chromatography using 3,100 ml of methanol solvent, and the compound (5 mg, 25 mg) as described below in 10 lower fractions (F2I-F2 '). , F2III), 6 (15 mg, F2IV), 7 (20 mg, F2VI), 8 (350 mg, F2VIII), 11 (35 mg, F2VIII). In addition, the F3 (8.3 g) fraction was applied to silica gel column chromatography, where a mixed solvent of ethyl acetate: methanol = 10: 1 was used as a developing solvent, and as a stationary phase, Kiesel gel 60 (Kiesel gel 60, 230). 2 (200 mg, F3II), 3 (50 mg) as described below in the eight lower fractions (F3I-F3 #) obtained by purification with 4,100 ml mixed solvent using -400 mesh, Merck, Germany). , F3III), 4 (280 mg, F3V) were isolated. In addition, the F4 (1.5 g, F4I-F4 ') and F5 (2.0 g, F5I-F5') fractions were recrystallized to give the compounds 1 (260 mg, F4I), 9 (20 mg, F4IV), 10 (45 mg, F5IV), 12 (10 mg, F5V), 13 (5 mg, F5VIII) were isolated. Analysis of each compound by NMR analyzer is as follows.

(1) Compound 1: Kaempferol

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₁₅ H ₁₀ O ₆

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 6.19 (1H, d, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.92 (2H , d, J = 8.0 Hz, H-3 ', 5'), 8.06 (2H, d, J = 8.0 Hz, H-2 ', 6'), 12.47 (1H, brs, 5-OH)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 175.9 (C-4), 163.9 (C-7), 160.7 (C-5), 159.2 (C-4 ′), 156.2 (C-9) , 146.8 (C-2), 135.7 (C-3), 129.5 (C-2 ', 6'), 121.7 (C-1 '), 115.4 (C-3', 5 '), 103.0 (C-10 ), 98.2 (C-6), 93.5 (C-8).

(2) Compound 2: camp Ferrol -3- Ο -β-D- galacto-pyrano seed (Kaempferol 3- O- β-D- galactopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₁ H ₂₀ O ₁₁

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 5.41 (1H, d, J = 7.7 Hz, H-1 '), 6.21 (1H, J = 2.0 Hz, H-6), 6.43 (1H , d, J = 2.0 Hz, H-8), 6.86 (2H, d, J = 8.0 Hz, H-3 ′, 5 ′), 8.08 (2H, d, J = 8.0 Hz, H-2 ′, 6 ′), 12.63 (1H, broad singlet, 5-OH)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.2 (C-5), 160.0 (C-4 ′), 156.4 (C-9) , 156.4 (C-2), 133.2 (C-3), 131.0 (C-2 ', 6'), 120.9 (C-1 '), 115.1 (C-3', 5 '), 104.0 (C-10 ), 101.7 (C-1´´), 98.7 (C-6), 93.7 (C-8), 75.8 (C-5´´), 73.1 (C-3´´), 71.2 (C-2´´ ), 67.9 (C-4´´), 60.2 (C-6´´).

(3) Compound 3: Camp Ferrol -7- Ο -β-D- glucopyranoside (Kaempferol 7- O- β-D- glucopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₁ H ₂₀ O ₁₁

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 6.44 (1H, d, J = 7.7 Hz, H-1 '), 6.81 (1H, d, J = 1.7 Hz, H-8), 8.10 (2H, d, J = 8.8 Hz, H-2 ', 6'), 6.94 (2H, d, J = 8.8 Hz, H-3 ', 5'), 5.42 (1H, d, J = 7.2 Hz, H-1´´)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 176.1 (C-4), 162.7 (C-7), 160.4 (C-5), 159.4 (C-4 ′), 155.8 (C-9) , 147.5 (C-2), 136.1 (C-3), 129.6 (C-2 ′, 6 ′), 121.5 (C-1 ′), 115.5 (C-3 ′, 5 ′), 104.7 (C-10 ), 99.9 (C-1´´), 98.8 (C-6), 94.4 (C-8), 77.2 (C-5´´), 76.4 (C-3´´), 73.1 (C-2´´ ), 69.69 (c-4´´), 60.6 (C-6´´).

(4) Compound 4: Camp Ferrol -3- Ο -β-D- glucopyranoside (Kaempferol 3- O- β-D- glucopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₁ H ₂₀ O ₁₁

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 5.47 (1H, d, J = 7.3 Hz, H-1 '), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H , d, J = 2.0 Hz, H-8), 6.88 (2H, d, J = 8.0 Hz, H-3 ', 5'), 8.05 (2H, d, J = 8.0 Hz, H-2 ', 6 ′), 12.62 (1H, broad singlet, 5-OH)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 160.0 (C-4 ′), 156.5 (C-9) , 156.4 (C-2), 133.3 (C-3), 131.0 (C-2 ′, 6 ′), 121.0 (C-1 ′), 115.2 (C-3 ′, 5 ′), 104.1 (C-10 ), 100.9 (C-1´´), 98.8 (C-6), 93.7 (C-8), 77.6 (C-5´´), 76.5 (C-3´´), 74.3 (C-2´´ ), 70.0 (C-4´´), 60.9 (C-6´´).

(5) Compound 5: Camperol-3- O- α-L-lamnopyranosyl- (1 → 6) -β-D-glucopyranoside (Kaempferol 3- O- α-L-rhamnopyranosyl-(1 → 6) -β-D-glucopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₇ H ₃₀ O ₁₅

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 0.97 (3H, s, J = 6.2 Hz, CH 3), 4.38 (1H, s, H-1´´), 5.30 (1H, d, J = 7.4 Hz, H-1´), 6.23 (1H, J = 2.1 Hz, H-6), 6.45 (1H, d, J = 2.1 Hz, H-8), 6.89 (2H, d, J = 8.8 Hz, H-3 ′, 5 ′), 7.97 (2H, d, = 8.8 Hz, H-2 ′, 6 ′), 10.22 (1H, brs, OH), 11.01 (1H, brs, OH), 12.55 (1H, brs, 5-OH)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.4 (C-4), 164.2 (C-7), 161.1 (C-5), 160.0 (C-4 ′), 156.8 (C-9) , 156.4 (C-2), 133.2 (C-3), 130.8 (C-2 ', 6'), 120.8 (C-1 '), 115.1 (C-3', 5 '), 103.9 (C-10 ), 101.3 (C-1´´), 100.7 (C-1´´´), 98.7 (C-6), 93.7 (C-8), 75.1 (C-5´´), 76.4 (C-3´ ´), 74.1 (C-2´´), 71.8 (C-4´´´), 70.6 (C-2´´´), 70.3 (C-3´´´), 69.9 (C-4´´) , 68.2 (C-5´´´), 66.9 (C-6´´), 17.7 (C-3´´´).

(6) Compound 6: Camperol-3- O- α-L-lamnopyranosyl- (1 → 2) -β-D-glucuronopyranoside (Kaempferol 3- O- α-L-hamnopyranosyl-( 1 → 2) -β-D-glucuronopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₇ H ₂₈ O ₁₆

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 12.51 (H, s, 5-OH), 7.98 (2H, d, J = 8.9 Hz, H-2 ′, 6 ′), 6.84 (2H, d, J = 8.9 Hz, H-3 ', 5'), 6.21 (1H, d, J = 1.9 Hz, H-8), 5.99 (1H, d, J = 1.9 Hz, H-6), 5.73 ( 1H, d, J = 7.0 Hz, H-1´´), 3.70 (1H, m, H-3´´´), 3.65 (1H, m, H-5´´´), 3.42 (1H, m, H-2´´´, 3.41 (1H, m, H-2´´), 3.26 (1H, m, H-5´´), 3.09 (1H, m, H-4´´´), 0.71 ( 3H, d, J = 6.2 Hz, CH ₃ )

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.3 (C-4), 172.2 (C-6´´), 164.6 (C-7), 161.0 (C-5), 159.8 (C-4 ′), 156.2 (C-9), 155.9 (C-2), 132.5 (C-3), 130.8 (C-2 ′, 6 ′), 121.1 (C-1 ′), 114.9 (C-3 ′, 5 ′), 103.5 (C-10), 100.5 (C-1´´´), 98.5 (C-6), 98.2 (C-1´´), 93.7 (C-8), 77.4 (C-3´ ´), 77.3 (C-2´´), 73.8 (C-5´´), 72.3 (C-4´´), 71.8 (C-4´´´), 70.6 (C-3´´´), 70.5 (C-2´´´), 68.3 (C-5´´´), 17.2 (C-6´´´)

(7) Compound 7: Camperol-3- O- α-L-lamnopyranosyl- (1 → 2) -β-D-glucopyranoside (Kaempferol 3- O- α-L-hamnopyranosyl-(1 → 2) -β-D-glucopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₇ H ₃₀ O ₁₅

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 12.64 (1H, s, 5-OH), 8.03 (2H, d, J = 8.9 Hz, H-2 ′, 6 ′), 6.88 (2H, d, J = 8.9 Hz, H-3 ', 5'), 6.42 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 2.1 Hz, H-6), 5.65 ( 1H, d, J = 7.3 Hz, H-1´´), 5.07 (1H, brs, H-1´´´). 0.76 (3H, d, J = 6.2 Hz, H-6´´´)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.3 (C-4), 164.2 (C-7), 161.2 (C-5), 159.9 (C-4 ′), 156.3 (C-9) , 156.1 (C-2), 132.7 (C-3), 130.7 (C-2 ′, 6 ′), 120.9 (C-1 ′), 115.1 (C-3 ′, 5 ′), 103.9 (C-10 ), 98.3 (C-1´´), 100.6 (C-1´´´), 98.7 (C-6), 93.6 (C-8), 77.5 (C-2´´, C-5´´), 77.2 (C-3´´), 71.8 (C-4´´´), 70.6 (C-3´´´), 70.5 (C-2´´´), 70.2 (C-4´´), 68.3 ( C-5´´´), 60.8 (C-6´´), 17.2 (C-6´´´)

(8) Compound 8: Camp Ferrol -3- Ο -β-D- glucuronic nopi pyrano seeded with (Kaempferol 3- O- β-D- glucuronopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₁ H ₁₈ O ₁₂

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 5.43 (1H, d, J = 7.2 Hz, H-1 '), 6.15 (1H, d, J = 2.0 Hz, H-6), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.86 (2H, d, J = 8.0 Hz, H-3 ′, 5 ′), 8.02 (2H, d, J = 8.0 Hz, H-2 ′ , 6 ′), 12.48 (1H, brs, 5-OH)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.4 (C-4), 171.4 (C-6´), 164.5 (C-7), 161.1 (C-5), 160.1 (C-4 ′), 156.4 (C-9), 156.4 (C-2), 133.2 (C-3), 131.0 (C-2 ′, 6 ′), 120.7 (C-1 ′), 115.1 (C-3 ′, 5 ′), 103.8 (C-10), 101.3 (C-1´´), 98.8 (C-6), 93.8 (C-8), 76.1 (C-5´´), 75.2 (C-3´´ ), 73.9 (C-2´´), 71.8 (C-4´´).

(9) Compound 9: Camp Ferrol -3- Ο -β-D- glucuronic furnace pyrazol nosil methyl ester (Kaempferol 3- O- β-D-glucuronopyranosyl methyl ester)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₂ H ₂₀ O ₁₂

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 3.57 (1H, s, OMe), 5.47 (1H, d, J = 7.4 Hz, H-1´), 6.21 (1H, d, J = 2.0 Hz, H-6), 6.43 (1H, d, J = 2.0 Hz, H-8), 6.89 (2H, d, J = 8.0 Hz, H-3 ′, 5 ′), 8.02 (2H, d, J = 8.0 Hz, H-2 ', 6'), 12.51 (1H, brs, 5-OH)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.2 (C-4), 169.0 (C-6´), 164.3 (C-7), 161.2 (C-5), 160.2 (C-4 ′), 156.6 (C-9), 156.4 (C-2), 133.0 (C-3), 130.9 (C-2 ′, 6 ′), 120.6 (C-1 ′), 115.1 (C-3 ′, 5 ′), 104.0 (C-10), 101.4 (C-1´´), 98.9 (C-6), 93.8 (C-8), 75.6 (C-5´´), 75.5 (C-3´´ ), 73.9 (C-2´´), 71.5 (C-4´´), 51.9 (OMe).

(10) Compound 10: pre-paroxetine 3 ', 5'-dimethyl ether -3- Ο -β-D- glucopyranoside (Myricetin 3', 5'-dimethylether 3- O- β-D-glucopyranoside)

Form of material: amorphous yellow powder.

Molecular formula of substance: C ₂₃ H ₂₄ O ₁₃

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 3.84 (6H, s, OCH 3), 5.59 (1H, d, J = 7.3 Hz, H-1´´), 6.22 (1H, J = 2.1 Hz , H-6), 6.50 (1H, d, J = 2.1 Hz, H-8), 7.49 (2H, s, H-2 ′, 6 ′), 9.17 (1H, brs, OH), 10.87 (1H, brs, OH), 12.60 (1H, brs, 5-OH)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.4 (C-4), 164.1 (C-7), 161.2 (C-5), 156.3 (C-9), 156.3 (C-2), 147.4 (C-3 ', 5'), 138.6 (C-4 '), 133.1 (C-3), 119.8 (C-1'), 106.9 (C-2 ', 6'), 104.0 (C-10 ), 100.7 (C-1´´), 98.7 (C-6), 93.8 (C-8), 77.4 (C-5´´), 76.4 (C-3´´), 74.3 (C-2´´ ), 69.8 (C-4´´), 60.5 (C-6´´), 56.2 (OCH ₃ ).

(11) Compound 11: quercetin -3- Ο -β-D- glucuronic nopi pyrano seeded with (Quercetin 3- O- β-D- glucuronopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₁ H ₁₈ O ₁₃

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 12.30 (1H, s, 5-OH), 9.72 (1H, brs, H-3 ′), 8.10 (1H, s, H-2), 7.40 (1H, dd, J = 2.0, 8.4 Hz, H-6 '), 6.82 (1H, d, J = 8.6 Hz, H-5'), 6.34 (1H, d, J = 1.8 Hz, H-8) , 6.15 (1H, d, J = 2.1 Hz, H-6), 5.26 (1H, d, J = 6.5 Hz, H-1´´)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.4 (C-4), 172.6 (C-6´), 165.2 (C-7), 160.9 (C-5), 157.2 (C-9 ), 156.5 (C-2), 148.5 (C-4 ′), 144.8 (C-3 ′), 133.9 (C-3), 120.9 (C-1 ′), 120.5 (C-6 ′), 117.6 ( C-5 ′), 115.4 (C-2 ′), 103.5 (C-10), 102.7 (C-1´´), 99.0 (C-6), 93.8 (C-8), 76.5 (C-3´ ´), 74.3 (C-5´´), 74.0 (C-2´´), 71.8 (C-4´´).

(12) Compound 12: isopropyl person netin -3- Ο -β-D- glucopyranoside (Isorhamnetin 3- O -β-D- glucopyranoside)

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₂ H ₂₂ O ₁₂

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 12.62 (1H, s, OH), 7.93 (1H, d, J = 1.9 Hz, H-2 ′), 7.58 (1H, dd, J = 1.8 , 8.6 Hz, H-6 ′), 6.93 (1H, d, J = 8.6 Hz, H-5 ′), 6.45 (1H, d, J = 1.9 Hz, H-8), 6.20 (1H, d, J = 1.9 Hz, H-6), 5.46 (1H, d, J = 7.2 Hz, H-1´), 3.85 (3H, s)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.1 (C-4), 164.6 (C-7), 161.2 (C-5), 149.5 (C-4 ′), 156.4 (C-9) , 155.9 (C-2), 133.2 (C-3), 113.3 (C-2 '), 122.3 (C-6'), 120.9 (C-1 '), 146.9 (C-3'), 115.3 (C -5 ′), 104.0 (C-10), 100.5 (C-1´´), 98.8 (C-6), 93.7 (C-8), 77.5 (C-5´´), 76.4 (C-3´ ´), 74.21 (C-2´´), 69.9 (C-4´´), 60.8 (C-6´´), 55.6 (OCH ₃ )

(13) Compound 13: isopropyl person netin -3- O- α-L- ramno pyrazol nosil - (1 → 6) -β- D- glucopyranoside (Isorhamnetin 3- O- α-L- rhamnopyranosyl - (1 6) -β-D-glucopyranoside

Form of material: amorphous yellow powder.

Molecular Formula of Material: C ₂₈ H ₃₂ O ₁₆

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 12.51 (1H, s, OH), 7.79 (1H, d, J = 2.2 Hz, H-2 ′), 7.52 (1H, dd, J = 2.2 , 8.6 Hz, H-6 ′), 6.89 (1H, d, J = 8.6 Hz, H-5 ′), 6.22 (1H, d, J = 2.2 Hz, H-8), 6.01 (1H, d, J = 2.2 Hz, H-6), 5.46 (1H, d, J = 7.3 Hz, H-1´), 3.83 (3H, s), 0.97 (3H, d, J = 6.2 Hz)

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 177.1 (C-4), 164.6 (C-7), 161.2 (C-5), 149.5 (C-4 ′), 156.4 (C-9) , 155.9 (C-2), 133.2 (C-3), 113.3 (C-2 '), 122.3 (C-6'), 120.9 (C-1 '), 146.9 (C-3'), 115.3 (C -5 ′), 104.0 (C-10), 101.3 (C-1´´), 100.8 (C-1´´´), 98.7 (C-6), 93.8 (C-8), 76.4 (C-3 ´´), 75.8 (C-5´´), 74.2 (C-2´´), 71.8 (C-4´´´), 70.6 (C-4´´), 70.3 (C-3´´´) , 69.9 (C-2´´´), 68.2 (C-5´´´), 66.9 (C-6´´), 55.6 (C-OCH3), 17.7 (C-6´´´)

Experimental Example  1. Soft Water Extract Aldoz  Reductase Inhibitory Activity Experiment

Preparation of enzyme source to measure aldose reductase inhibitory activity of soft water crude extract and nonpolar solvent soluble extract (Hayman S. and Kinoshite IH, J. Biol . Chem . , 240 , pp877-882 , 1965).

First, for the preparation of the enzyme source, the lens of the rat (Sprague Dawley, Samtako, Korea) was extracted and homogenized by adding about 0.5 ml of phosphate buffer (pH 6.2) per lens. After centrifugation at 10,000 rpm for 20 minutes at 4 ° C, the supernatant was taken up to 40% with ammonium sulfate, and the supernatant was centrifuged and ammonium sulfate was added to 70% again for 1 hour. The pellet obtained by stirring was centrifuged and suspended in a minimum amount of phosphate buffer, dialyzed for about 1 day, and then used as an enzyme source. 690 μl of potassium phosphate buffer (pH 7.0), 100 μl of enzyme source (0.04 unit), 100 μl of NADPH (1.6 mM; Sigma, St. Louis, MO, USA), dimethyl sulfoxide (DMSO 10 μl of each soft water extract sample dissolved in 100 μl and 100 μl of DL-glyceraldehyde (DL-glyceraldehyde, 10 mM: Sigma, St. Louis, MO, USA) as substrates. The reduction rate of NADPH absorbance at 340 nm was measured using a photometer (U / V spectrophotometer; ultrospec 2100pro, Amersham Biosciences, Sweden). The blinds were measured by the reaction buffer instead of the sample and the substrate, the control was measured by adding dimethyl sulfoxide instead of the sample, and the positive control was measured by concentration using quercetin (Quercetin: Sigma, St. Louis, MO, USA). . The% aldose reductase inhibitory activity was calculated by Equation 1 below and statistically processed using a regression equation.

        % Inhibition = 1- (absorbance of sample-absorbance of control) / standard absorbance × 100

sample Application concentration (µg / ml) Inhibition Rate (%) IC ₅₀ (μg / ml) Soft water extract 50 60.90 Soft water dichloromethane soluble fraction 50 55.51 Soft Water Ethyl Acetate Soluble Fraction 50 97.24 10.14 10 56.60 5 23.24 Soft Butanol Soluble Fraction 50 54.73 Soft Water Soluble Fraction 50 41.80 Control group (quercetin) 10 82.53 1.37

As a result of the experiment, as shown in Table 1, the IC ₅₀ value for aldose reductase of the soft water ethyl acetate soluble fraction was 10.14 µg / ml, which was the most excellent inhibitory activity compared to other soft water extracts.

Experimental Example  2. Compounds Isolated from Soft Ethyl Acetate Soluble Fraction Aldoz  Reductase Inhibitory Activity Experiment

Inhibitory activity of the aldose reductase of each compound obtained in Example 5 was carried out in the same manner as in Experimental Example 1, the results are shown in Table 2 below.

As shown in Table 2 below, the inhibitory activity against aldose reductase of all compounds isolated from the soft water ethyl acetate soluble fraction was generally good, and the above (5), (13), (4), (9), ( 3) The compounds showed IC ₅₀ values of 5.6, 9.4, 11, 11.6, and 14 μM for aldose reductase, showing superior inhibitory activity than quercetin (IC ₅₀ 16 μM), a well-known positive control for aldose reductase inhibitors. . 3-lamnopyranosyl- (1 → 6) -glucosyl group (3- O- α-L-rhamnopyranosyl- (1 → 6) -β-D-glucopyranosyl to flavonols such as camphorol or isoramnetine ) ( 5 , 13 : IC ₅₀ 5.6, 9.4, 11, 11.6, 14 μM) with the strongest inhibitory activity and compounds with glycosylation ( 4 , 3 , 2 ) with one sugar linked to camphorol : IC ₅₀ 11, 14, 18 μM), the compound ( 12 : IC ₅₀ 19 μM), which is a glycoside linked with one sugar to isoramnetine, and camphorol ( 1 : 24 μM of IC ₅₀ ) showed the inhibitory activity. In addition, camphorol-3-glucrunopyranosyl methyl ester was also very excellent in inhibitory activity ( 9 : IC ₅₀ 11.6 μM).

Serial number sample Concentration (μg / ml) Inhibition Rate (%) IC ₅₀ (Μg / ml) (μM) One Camperol 10 66.43  6.94  24 One 27.29 0.1 0.94 2 Camperol-3- O- β-D-galactopyranoside 10 56.13  8.26  18 5 40.12 One 2.04 3 Camperol-7- O- β-D-glucopyranoside 10 64.98  6.34  14 5 51.88 One 17.52 4 Camperol-3- O- β-D-glucopyranoside 10 82.84  5.05  11 One 27.74 0.5 15.08 5 Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside 5 67.99  3.32  5.6 One 26.33 0.5 18.18 6 Camperol-3- O- α-L-Rampnopyranosyl- (1 → 2) -β-D-glucuronopyranoside 10 30.10 7 Camperol-3- O- α-L-Rhamnopyranosyl- (1 → 2) -β-D-glucopyranoside 10 23.73 8 Camperol-3- O- β-D-glucuronopyranoside 10 51.89

9 Camperol-3- O- β-D-glucuronopyranosyl methyl ester 10 78.69  5.52  11.6 One 24.53 0.5 14.27 10 Myricetin-3 ', 5'-dimethylether-3- O- β-D-glucopyranoside 10 40.27 11 Quercetin-3- Ο- β-D-glucuronopyranoside 10 47.53  12 Isolamnetin-3- O- β-D-glucopyranoside 10 53.14  9.13  19 5 30.97 One 3.31 13 Isolamnetin-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside 10 72.02  5.86  9.4 5 50.83 One 17.83 Control Quercetin 10 75.62  5.49  16 One 35.67 0.1 6.92

Examples of the formulation of the composition will be described below, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation example  One. Powder  Produce

Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside of Example 5

                                                          20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside of Example 5

                                                        10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  3. Manufacture of capsule

Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside of Example 5

                                                       10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside of Example 5

                                                           10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside of Example 5

                                                         20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added thereto, the above components are mixed, and then, purified water is added to adjust the total volume to 100 ml, and then sterilized by filling into a brown bottle. do.

Formulation example  6. Manufacture of healthy food

Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside of Example 5

                                  ............. 1000 mg

Vitamin Blend ...............

Vitamin A Acetate ......... 70 μg

Vitamin E ............ 1.0 mg

Vitamin B1 ........................ 0.13 mg

Vitamin B2 ........................ 0.15 mg

Vitamin B6 ............... 0.5 mg

Vitamin B12 ............... 0.2 μg

Vitamin C ............ 10 mg

Biotin .............................. 10 ㎍

Nicotinamide ......... 1.7 mg

Folic acid ......................... 50 μg

Calcium Pantothenate ......... 0.5 mg

Inorganic mixtures ...............

Ferrous Sulfate ............... 1.75 mg

Zinc Oxide ............... 0.82 mg

Magnesium Carbonate ............... 25.3 mg

Potassium monophosphate ......................................... 15 mg

Dibasic calcium phosphate ............... 55 mg

Potassium Citrate ............... 90 mg

Calcium Carbonate ... 100 mg

Magnesium Chloride ........................... 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

Camperol-3- O- α-L-Ranmopyranosyl- (1 → 6) -β-D-glucopyranoside of Example 5

                                     ............. 1000 mg

Citric Acid .................................... 1000 mg

Oligosaccharide ......................................... 100 g

Plum concentrate ........................... 2 g

Taurine ......................................... 1 g

Purified water is added to the whole ..... 900 ㎖

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

Since the soft water extract of the present invention or the compound isolated therefrom shows excellent inhibitory activity against aldose reductase which is closely related to diabetic complications, it can be used as a pharmaceutical composition and health functional food for the prevention and treatment of diabetic complications. Can be.

Claims (10)

Training ( Nelumbo Nucifera stamen) A pharmaceutical composition for the prevention and treatment of diabetic complications comprising an extract as an active ingredient, and containing a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 1, wherein the extract is a crude extract available in water, ethanol, methanol, butanol or a mixed solvent thereof. delete The pharmaceutical composition of claim 1, wherein the extract is an extract available in a nonpolar solvent selected from dichloromethane, chloroform or ethyl acetate. delete Camperol-3- O- β-D-galactopyranoside, Camperol-3- O- β-D-glucopyranoside, Camperol-3- O- α-L-Rhamnopyranosyl- (1 → 2) -β-D-glucuronopyranoside, camperol-3- O- β-D-glucuronopyranoside, camperol-3- o- β-D-glucuronopyranosyl methyl ester, comprising: a pre-paroxetine 3 ', 5'-dimethyl ether -3- Ο -β-D- glucopyranoside or quercetin -3- Ο nopi pyrano seed in -β-D- glucuronidase, as an active ingredient A pharmaceutical composition for preventing and treating diabetic cataracts, diabetic retinopathy, diabetic keratosis or diabetic nephropathy, containing a pharmaceutically acceptable carrier or excipient. delete delete      Health functional food for the prevention and improvement of diabetic complications containing the soft water extract of claim 1 as an active ingredient. Functional food for the prevention and improvement of diabetic complications containing the compound of claim 6 as an active ingredient.