KR100824365B1 - Composition comprising the extracts, fractions and the isolated compounds of phyllostachys nigra munro for prevention or treatment of diabetic complications - Google Patents

Abstract

A composition comprising an extract of Phyllostachys nigra MUNRO, a fraction thereof or a compound isolated from the fraction is provided to inhibit accumulation of sorbitol, a fundamental cause of nephropathy and neuropathy shown as diabetic complications, by inhibiting aldose reductase, thereby being used as a pharmaceutical composition or a health food for preventing diseases related to the diabetic complications. A method for isolating a compound from Phyllostachys nigra MUNRO comprises the steps of: (a) extracting the Phyllostachys nigra MUNRO with ethanol and filtering the extract; (b) concentrating and freeze-drying the filtrate to obtain an extract; (c) fractionating an ethylacetate layer from the extract using a separation funnel; and (d) subjecting the fraction to silica gel column chromatography using CH2Cl2-MeOH as a solvent to obtain orientin, luteolin 6-C-(6''-O-transcaffeoylglucoside), tricetin 3',4',5'-trimethyl ether 7-O-b-D-glucopyranoside, tricine, p-coumaric acid or cis-coumaric acid. A pharmaceutical composition for preventing or treating diabetic retinopathy, diabetic neuropathy or diabetic nephropathy comprises the obtained compound as an effective ingredient.

Description

Composition comprising the extracts, fractions and the isolated compounds of Phyllostachys nigra MUNRO for prevention or treatment of diabetic complications}

1 is a structural formula of eight compounds isolated from the porridge fraction

The present invention relates to a composition for preventing or treating diabetic complications comprising the extract of porridge, and more particularly, an extract prepared by extracting porridge with an organic solvent of ethanol and the extract n-hexane, methylene chloride, ethyl acetate, n -Fractions obtained by fractionation with an organic solvent of butanol show an aldose reductase inhibitory effect and relates to a pharmaceutical composition or health food that can prevent or improve diabetic complications.

Diabetes is a chronic disease that causes glucose to be excreted in the urine due to a lack of insulin, resulting in higher levels of glucose (blood sugar) in the blood. There are many diseases associated with diabetes that may be present in almost all diseases other than diabetes. However, the incidence of complications is high and important include retinopathy, neuropathy and nephropathy.

As diabetes increases the body's blood sugar levels for a long time, chronic complications occur as glycation products invade the kidneys, nerves, retinas, and large and small blood vessels throughout the body. Over half of all diabetic complications are diabetic retinopathy and adult blindness. It is the biggest cause of onset. Symptoms include vision problems, glaucoma, and vision loss.

In addition, diabetic neuropathy causes peripheral neuropathy due to carbohydrate and lipid metabolism abnormalities. Symptoms include decreased keyboard loss, loss, perception disorders, and limb pain. In particular, the lower extremity may complain of symptoms such as nighttime muscle pain, and the burning of the soles of the feet, hyperalgesia and tactile blunts. Usually it is symmetrical and movement disorder is rare. In addition, autonomic neuropathy, left and right swelling of the pupil, sweating abnormalities, sores (陰 蔿), residual urination, delayed urination, urinary tract (폐), constipation and diarrhea can be seen.

Nephropathy is also very common and is considered the most important cause of death of complications of diabetes. Diabetes patients are found to be about 20 times more at risk of kidney failure than normal people. Symptoms include glomerulosclerosis, proteinuria, and kidney failure. Renal complications have been reported to be caused by excessive aldose reductase or oxidative stress.

Mechanisms leading to diabetic complications include osmotic stress caused by changes in the mechanism of non-enzymatic glycation of protein and polyol pathway and oxidative stress caused by free radicals. ) And the like.

In non-enzymatic glycosylation of proteins, amino acid groups, such as lysine residues, are condensed and cross-reacted with reducing sugars without enzymatic action to produce advanced glycation endproducts (AGEs). Final glycosylation products are irreversible reaction products, so once they are produced they do not degrade even when blood sugar returns to normal, but accumulate in tissues during protein survival, resulting in abnormal changes in tissue structure and function (Vinson JA et al., J Nutritinal). Biochemistry , 7, 559-663, 1996; Smith PR et al., Eur . J Biochem ., 210, 729-739, 1992). Non-enzymatic protein glycosylation results in glycation of proteins such as basement membrane, plasma albumin, lens protein, fibrin, and collagen, and the resulting glycosylated product abnormally alters the structure and function of tissues, resulting in diabetic retinopathy, It causes chronic diabetic complications such as cataract, diabetic nephropathy and diabetic neuropathy.

In addition, it has been reported that abnormal glycation metabolism occurs in the process of producing the final glycated product in hyperglycemic state and oxidative stress is caused by deterioration of defense system function against harmful oxygen free radicals (Yokozawa T et al., J of Trad. Med ., 18, 107-112, 2001). As such, the mechanism of action of non-enzymatic glycosylation and oxidative stress is related.

The polyol refers to an alcohol reduced by aldose reductase (AR) from aldose or ketose. In general, when glucose enters the cell, hexokinase converts it into 6-glucose and then breaks down the pathway. However, when the concentration of glucose increases, hexokinase that binds to it is saturated, and excess glucose binds to aldose reductase, which is less affinity than hexokinase, and is metabolized to sorbitol, thereby accumulating sorbitol in cells. Aldose reductase is present in the retina, sclera, lens, kidney, erythrocytes, brain, muscle, liver and peripheral nerves, especially in the peripheral nerves can be a problem because glucose permeation is not affected by insulin. In other words, when hyperglycemia causes excessive accumulation of sorbitol in Schumann cells, osmotic pressure may be increased, resulting in edema, necrosis and collapse of myelin sheath, and water is introduced due to the continuously increased osmotic pressure, leading to diabetic complications ( Diabetes, Eung-Jin Kim et al., The Korean Diabetes Association, Korea Medicine, p. 483; Soulis-Liparota T et al., Diabetologia , 38, 357-394, 1995).

Final glycation end products have been reported to activate aldose reductase (AR), the main enzyme of the polyol pathway, in human microvascular endothelial cells (Nakamura N, et al., Free Radic Biol . Med ., 29, 17 -25, 2000). In a steady state, aldose reductase has a very low affinity for glucose, but at high concentrations of glucose, aldose reductase is activated to excessively reduce glucose to sorbitol, and this sorbitol is converted to fructose by sorbitol dehydrogenase. This fructose is about 10 times faster than the non-enzymatic glycosylation of proteins. Thus, high concentrations of fructose bind to the protein and eventually accelerate the formation of the final glycated product.

Renal complications are the most common cause of death of complications of diabetes. In addition, diabetics have been found to have a 20-fold higher risk of kidney failure than normal patients. Aldose reductase inhibitors AS-3201, Tollestat, Fornalrestrat and Epalrestat have been developed as treatments for nephropathy, but studies on their therapeutic effects are still ongoing. In addition, as part of the development of a therapeutic agent for diabetic complications, aldose reductase inhibitory activity has been shown, and at the same time, studies on natural substances having anti-diabetic, antioxidant and hypoglycemic effects have been actively conducted.

Once diabetes develops and blood sugar does not drop quickly, diabetic complications should be taken to prevent diabetic complications. Currently, aminoguanidine HCl, the only synthetic agent for protein glycosylation inhibitors, is a nucleophilic hydrazine, which binds to the product of condensation reactions and prevents cross-linking with the protein, thereby inhibiting the formation of the final glycation product as a complication. Delay or prevent progression (Brownlee M et al., Sciences , 232, 1629-1632, 1986; Edelstein D et al., Diabetes , 41, 26-29, 1992). Aminoguanidine HCl is the most promising synthetic drug for the prevention or treatment of diabetic complications, but it has been conducted up to Phase III clinical trials, but there is a problem that toxicity is induced upon prolonged administration.

Meanwhile, Phyllostachys nigra MUNRO) is a genus name of succulent, bamboo, and yak-juk. It is an evergreen herbaceous plant with a flower, about 10 cm in height, and 5-8 cm in diameter. At first it is covered with white powder, but the stem is black. Flowers blossom in June-July, flowers are pale yellow, bamboo shoots come in April-May, light reddish brown. In October, the fruit matures and is spirit and fruit. Used for edible, ornamental, industrial, medicinal, edible young shoots, ornamental stems and ornamental materials, for construction, and for dying. It is used as medicine for medicine, tetanus, sweating, labor, paralysis.

According to the herbaceous tree, the bamboo leaves are cool, tasteless and nontoxic. It helps to sleep better, stops to get rid of alcohol, dissolves heat and removes sweat. It is said that it is better to speak with a stroke.

'Ojuk tea' is said to help liver detoxification and relieve fatigue and have an excellent effect on high blood pressure, and thus, Oju tea and its manufacturing method have been developed and reported (Korean Patent No. 2001-0011491). In addition, it was reported that the extract of Ojuk showed LDL oxidation inhibitory effect, inhibited NF-κB activity, and inhibited the expression of ICAM, VCAM, and MCP-1, thereby reducing the risk of atherosclerosis and could be used as an anti-arterial agent Salmonella typhimurium TA 100 ( Salmonella) at a concentration of 0.25 to 5.00 mg / plate typhimurium TA 100) showed no toxicity when treated, it was reported that animal experiments using bamboo extract (Korean patent 2003-0014155).

However, there is no patent on a composition containing the bamboo porridge extract that can treat or prevent diabetic complications by inhibiting aldose reductase.

Thus, the present inventors while searching for aldose reductase inhibitors in natural products to overcome the problems such as side effects of the conventional therapeutics, Ojuk extract and fractions exhibit the inhibitory effect of aldose reductase on diabetes complications to prevent or prevent diabetes complications The present invention has been completed by revealing that it can be used as a therapeutic agent.

It is an object of the present invention to provide a bamboo extract, fractions and isolated compounds that can be effectively used for the inhibition of aldose reductase to treat or prevent diabetes complications caused by changes in the mechanism of the polyol pathway.

In order to achieve the above object, the present invention provides a porridge extract for preventing or treating complications of diabetes.

The present invention also provides a fraction of the porridge extract for preventing or treating diabetic complications.

The present invention also provides a method for separating a compound for preventing or treating diabetic complications from porridge.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating diabetic complications comprising the Ojuk extract, a fraction of the extract or a compound isolated from the fraction as an active ingredient.

In addition, the present invention provides a health food for preventing and improving complications of diabetic extract, the fraction of the extract or a compound separated from the fraction as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides bamboo extracts and fractions for the prevention or treatment of diabetic complications.

The porridge can be used without limitation, such as cultivated or commercially available, the solvent for extraction is ethanol. The present inventors extracted and filtered the porridge, and then the filtrate was concentrated and lyophilized to obtain the porridge extract.

The fractional solvent for obtaining the fraction of the 5-axis extract is an organic solvent, preferably selected from the group of n-hexane, methylene chloride, ethyl acetate and n-butanol, preferably ethyl acetate, usually using a separatory funnel Fractionation. Fractions were obtained by separation purification using silica gel chromatography and HPLC.

In addition, the present invention comprises the steps of 1) obtaining an extract from the bamboo porridge; 2) fractionating the ethyl acetate layer from the extract of step 1; And 3) adding CH ₂ Cl ₂ -MeOH to the fraction of step 2 to separate the compound for preventing or treating diabetes complications from the porridge.

Ethyl acetate was added to the fraction of step 2, and the fractions obtained by silica gel column chromatography with CH ₂ Cl ₂ -MeOH solvent were separated from the eight compounds, and the structure was determined through instrumental analysis (see FIG. 1).

The eight compounds isolated were isorientin, orientin, p-coumaric acid, cis-coumaric acid, and luteolin 6-C- (6 ' -O -trans-cafeoylglucoside) [Luteolin 6-C- (6'' -O- trans-caffeoylglucoside)], tricetin 3', 4 ', 5'-trimethyl ester 7-O-β-D Glucopyranoids (tricetin 3 ', 4', 5'-trimethyl ether 7-O-β-D-glucopyranoside), tricin and bitexin.

The present invention also provides a pharmaceutical composition for the prevention or treatment of diabetic complications containing the fraction of Ojuk extract as an active ingredient.

The present inventors conducted experiments on the enzyme precipitate of the lens of the rat in order to determine the effect of the fraction of Ojuk extract on aldose reductase inhibition. Specifically, the lens of the rat was extracted to prepare an aldose reductase source, and each fraction was added to the prepared aldose reductase source, and then DL-glyceraldehyde was reacted with substrate to measure NADPH absorbance reduction rate at 340 nm. It was.

As a result, it was confirmed that the fraction treated with ethyl acetate had the highest inhibitory effect among the fractions fractionated by adding various organic solvents (see Table 2).

The present invention also provides a pharmaceutical composition for preventing or treating diabetic complications containing the compound of the Oju fraction as an active ingredient.

The present inventors conducted an experiment targeting the enzyme precipitate of the lens of the rat in order to determine the effect of the compound of Ojuk extract on aldose reductase inhibition. Specifically, after adding each compound to the aldose reductase source prepared by the above method, DL-glyceraldehyde was reacted with a substrate to measure the rate of decrease of NADPH absorbance at 340 nm.

As a result, it was confirmed that luteolin 6- C- (6 " -O -trans-cafeoylglucoside) had the highest inhibitory effect among the eight compounds separated from the ethyl acetate layer (see Table 3).

Compounds of the Oju fraction separated by the above method have an inhibitory effect on alpha glucosidase and aldose reductase, and examples of diabetic complications that can be prevented or treated with Oju extract of the present invention are due to changes in the mechanism of the polyol pathway. Diabetic retinopathy, diabetic neuropathy, nephropathy, and the like, but are not limited thereto.

Neuropathy is peripheral neuropathy or autonomic neuropathy, and nephropathy is characterized in that it is selected from the group comprising glomerulosclerosis, proteinuria or renal failure. Diabetes complications, neuropathy and nephropathy are characterized by osmotic stress due to changes in the mechanism of the polyol pathway.

The pharmaceutical composition for preventing or treating diabetic complications of the present invention contains the Oju extract, a fraction thereof or a compound separated from the fraction as an active ingredient. The composition may be administered in some cases at the time of clinical administration, and may be used in the form of a general pharmaceutical formulation. Preferred pharmaceutical preparations include oral preparations such as tablets, hard or soft capsules, solutions, suspensions and the like, which pharmaceutical preparations are conventionally pharmaceutically acceptable carriers such as excipients, binders, disintegrants, glidants It can be prepared using an oral preparation such as a solubilizer, a suspending agent, a preservative or an extender.

In the pharmaceutical composition for the prevention or treatment of diabetic complications of the present invention, the dosage of the porridge extract may be determined by a specialist depending on various factors such as the severity of the disease, the patient's condition, age, sex, and complications, but generally adult It can be administered at a dose of 10 mg to 2 g per day (preferably about 50 kg body weight), preferably 100 mg to 0.5 g. In addition, it is intended to contain a daily dose or 1/2, 1/3 or 1/4 of the above-mentioned porridge extract per unit dosage form, and may be administered 1 to 6 times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for the prevention or treatment of diabetes and complication related diseases.

In addition, the present invention provides a health food for preventing and improving diseases related to diabetes and complications.

When the composition of the present invention is used as a food additive, each component described above or a mixture thereof can be added as it is or used with other food or food ingredients, and can be suitably used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, the porridge extract of the present invention may be added in an amount of 1 to 50% by weight, preferably 10 to 30% by weight based on the raw materials in preparing food or beverage. The effective dose of the porridge extract of the present invention can be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control, It is evident that the component can be used in an amount above this range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of foods to which the composition may be added include dairy, meat, sausages, bread, chocolate, candy, snacks, confectionery, dairy products including pizza, ramen, other noodles, gums, ice cream, various soups, beverages, alcoholic beverages. And vitamin complexes and the like, and include all of the health foods in a conventional sense.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention, but not to limit the content of the present invention.

< Example  1> Cucumber Extract of  Produce

The dried Ojuk (Gangneung Ojuk Farm) was ground to an appropriate size, 2.0 kg of Oju and 2.0 L of ethanol (50-95%) were added to the extraction container, and the mixture was left at room temperature for 7 days and filtered through a filter paper to obtain an extract. The extraction process was repeated five times, after which the solvent was concentrated under reduced pressure to obtain 255.0 g of ethanol extract.

< Example  2> from bamboo porridge extract Fraction  Produce

Using the separatory funnel in Example 1, n-hexane (106.0 g), methylene chloride (11.0 g), ethyl acetate (5.0 g), n-butanol (60.0 g) and water (72.6 g). Fractionated into fractions.

< Example  3> From fractions Chemical  Produce

The ethyl acetate fraction of <Example 2> was subjected to silica gel column chromatography with CH ₂ Cl ₂ -MeOH solvent to separate eight compounds. The molecular weight of the eight isolated compounds was measured, and the structure was determined by instrumental analysis of NMR and IR (FIG. 1). NMR results were obtained by dissolving 8 compounds in DMSO- d 6, and recording ¹ H- and ¹³ C-NMR at 500 and 25 MHz with a spectrometer (Varian Unity Inova 500) using a cryoprobe. Six of them were flavonoid compounds, and two compounds were phenylpropanoids compounds. The structure of each compound identified is: (1) Isoorientin, (2) Orientin, (3) Vitexin, (4) Luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) [Luteolin 6-C- (6 '' -O- trans-caffeoylglucoside)], (5) Tricetin 3 ', 4', 5'-trimethyl ester 7-O-β- D-glucopyranoid (tricetin 3 ', 4', 5'-trimethyl ether 7-O-β-D-glucopyranoside), (6) Tricin, (7) p-coumaric acid), (8) cis-coumaric acid. Among these, luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) and tricetin 3 ', 4', 5'-trimethyl ester 7-O-β-D-glucopyranoid It was the first compound reported to separate from.

Of isolated compounds NMR  result location One 2 3 ¹ H ( δ, mult , J, Hz ) ¹³ C ( δ ) ¹ H ( δ, mult , J, Hz ) ¹³ C ( δ ) ¹ H ( δ, mult , J, Hz ) ¹³ C ( δ ) 2 164.3 164.7 164.0 3 6.67 (1 H, s ) 103.5 6.57 (1 H, s ) 103.1 6.77 (1 H, s) 102.6 4 182.7 182.7 181.9 5 161.4 161.1 160.9 6 109.6 6.20 (1 H, s ) 98.8 6.27 (1 H, s) 98.9 7 164.1 163.4 162.5 8 6.48 (1 H, s ) 94.2 105.2 104.2 9 156.9 156.7 155.8 10 104.0 104.7 104.2 One' 122.1 122.7 121.8 2' 7.40 (1 H, d, 2.1) 113.9 7.41 (1 H, s ) 114.7 8.03 (1H, d, 8.7) 128.5 3 ' 146.5 146.5 6.90 (1H, d, 8.7) 116.0 4' 150.5 150.3 160.9 5 ' 6.89 (1H, d, 7.8) 116.8 6.80 (1 H, d , 8.3) 116.3 6.90 (1H, d, 8.7) 116.0 6 ' 7.42 (1H, doublet of 7.8, 2.1) 119.7 7.47 (1 H, d , 8.3) 120.1 8.03 (1H, d, 8.7) 128.5 One" 4.60 (1 H, d , 9.8) 73.7 4.62 (1H, d , 9.9) 74.1 4.71 (1H, d, 10.0) 2" 71.3 71.4 3 " 79.6 79.4 4" 70.9 71.4 5 " 82.3 82.7 6 " 62.2 62.3 5-OH 13.57 (1 H, s ) 13.1 (1 H, s )

location One 2 3 ¹ H ( δ , mult, J, Hz) ¹³ C ( δ ) ¹ H ( δ, mult , J, Hz ) ¹³ C ( δ ) ¹ H ( δ, mult , J, Hz ) ¹³ C ( δ ) 2 164.2 165.9 164.9 3 6.68 (1H, s ) 103.3 7.13 (1 H, s ) 105.7 6.98 (1 H, s ) 104.4 4 182.4 182.8 182.5 5 161.3 161.8 162.1 6 109.1 6.86 (1 H, d , 2.0) 98.8 6.55 (1H, d , 2.0) 99.5 7 163.8 163.9 164.3 8 6.49 (1H, s ) 94.0 6.39 (1 H, d , 2.0) 93.6 6.20 (1 H, d , 2.0) 94.9 9 156.8 158.0 158.0 10 103.9 104.0 104.3 One' 121.9 126.2 121.1 2' 7.40 (1 H, s ) 113.8 7.37 (2H, s ) 105.5 7.32 (2H, s ) 105.0 3 ' 146.0 153.6 148.9 4' 150.2 138.4 140.5 5 ' 7.04 (1 H, s ) 116.6 5.16 (1H, doublet , 7.5) 102.6 12.96 (1H, s ) 6 ' 7.42 (1 H, d , 8.4) 119.5 74.8 3.88 (6H, s ) 57.1 One" 4.57 (1H, doublet , 9.8) 73.6 77.3 2" 4.05 (1H, m ) 70.4 70.6 3 " 3.20 (1 H, m ) 79.2 78.1 4" 3.17 (1H, m ) 71.0 61.5 5 " 3.43 (1 H, m ) 78.7 12.86 (1H, s ) 6 " 4.41 (1H, d , 11.3), 4.00 (1H, m ) 65.0 3.89 (6H, s ) 56.8 One'" 126.0 3.88 (3H, s ) 57.4 2'" 7.01 (1H, d , 8.3) 115.5 3 '" 145.8 4'" 148.9 5 '" 6.75 (1 H, d , 8.3) 116.2 6 '" 6.89 (1 H, d , 8.3) 121.8 7 '" 7.47 (1 H, d , 15.8) 146.3 8'" 6.29 (1 H, d , 15.8) 114.4 9 '" 167.2 5-OH 13.62 (1 H, s )

location 7 8 ¹ H ( δ , mult , J, Hz ) ¹³ C ( δ ) ¹ H ( δ , mult , J, Hz ) ¹³ C ( δ ) One 126.0 126.5 2, 6 7.51 (2H, d , 8.6) 130.8 7.61 (2 H, d , 8.4) 133.2 3, 5 6.80 (2H, doublet , 8.6) 116.5 6.72 (2H, doublet , 8.4) 115.3 4 160.3 159.2 7 7.50 (1 H, d , 16.0) 144.7 6.68 (1H, d , 12.8) 141.6 8 6.30 (1H, d , 16.0) 116.2 5.72 (1H, d , 12.8) 118.2 9 168.7 168.6

< Experimental Example  1> of bamboo Fraction Aldos  Reductase inhibitory effect measurement

The present inventors performed an experiment on the enzyme precipitate of the lens of the rat in order to measure the aldose reductase inhibitory effect of the composition. First, in order to obtain an aldose reductase, the lens of the rat was extracted, and a certain amount of phosphate buffer was added according to the amount of wet growth, and the lens was homogenized. The homogenate was centrifuged at 4 ° C. to obtain a supernatant, and ammonium sulphate was added to the supernatant so that the final concentration was 40%. Thereafter, the supernatant was collected by centrifugation again, and then ammonium sulfate was added thereto at a final concentration of 70%, followed by mixing for about one hour. The precipitate obtained by centrifugation was suspended in PBS buffer and dialyzed for 1 day to obtain an aldose reductase source. (Please indicate whether the dialysate was used as an enzyme source.) Then, each prepared aldose reductase source was prepared. Fraction of was added and DL-glyceraldehyde was reacted with the substrate to measure the rate of decrease of NADPH absorbance at 340 nm. As a control group, tetramethylene glutaric acid (Sigma, USA) was used.

As shown in Table 2, it was confirmed that the ethyl acetate fraction of the fractions of <Example 2> has the highest inhibitory effect.

Extracts and Organic Solvents Fraction  Each Aldos  Reductase inhibition rate (%)  Composition  Concentration (μg / mL)  Inhibition Rate (%) IC ₅₀ ^* (μg / mL)  Control  TMG * 10 1 0.1 90.4 56.5 11.7  0.85  Example 1  EtOH Extract 10 1 0.5 53.2 25.3 6.4  58.9        Example 2  n-Hexane fraction 10 1 0.5 68.9 26.9 22.1  12.2  CH2Cl2 Fraction 10 1 0.1 99.4 44.6 22.1  1.3  EtOAc fraction water 10 1 0.1 100.0 87.8 39.4  0.083  n-BuOH fraction 10 1 0.1 100.0 63.8 10.3  1.12  Water fraction  10  49.6  -

* IC ₅₀ : 50% inhibitory concentration of aldose reductase

* TMG: tetramethylene glutaric acid control

< Experimental Example  2> extraction of porridge and Fraction  Measurement of aldose reductase inhibitory effect

Eight compounds isolated by the method of Example 3 were added to the aldose reductase source prepared in the same manner as in Experimental Example 1, and the rate of decrease of NADPH absorbance at 340 nm was measured by reacting DL-glyceraldehyde with a substrate. .

As a result, as shown in Table 3, it was confirmed that luteolin 6- C- (6 " -O -trans-cafeoylglucoside) among the eight compounds had the highest aldose reductase inhibitory effect.

% Inhibition of aldose reductase of each of the 8 compounds isolated from the fractions  Composition  Concentration (μM)  % Inhibition IC ₅₀ ^* (μM) Control  TMG * 10 1 0.1 83.1 52.8 21.8  0.92          Example 3  Isoorientin 10 1 0.1 61.4 40.9 20.5  1.91  Orientin  10  9.1  -  p-Coumaric acid 10 1 0.1 85.1 70.2 43.9  0.14  cis-Coumari c acid  10  34.1  - Luteolin 6-C- (6 "-O-trans-caffeoylglucoside) 10 1 0.1 0.01 100.0 75.8 72.7 39.8  0.0134  Vittariflav one  10  36.4  -  Tricin 10 1 0.1 93.2 47.7 3.3  1.48 Vitexin 10 1 0.1 68.2 47.7 22.7  2.03

* IC ₅₀ : 50% inhibitory concentration of aldose reductase

* TMG: tetramethylene glutaric acid control

Preparation Example 1 Preparation of Soft Capsule

100.0 mg of Ojuk extract prepared according to Example 1, soybean oil 175.0 mg, lead 45.0 mg, field hardened oil 127.5 mg, soybean phospholipid 21.0 mg, gelatin 212.0 mg, glycerin (specific gravity 1.24) 50.0 mg, di-sorbitol 76.0 mg, paraoxy 0.54 mg of methyl benzoate, 0.90 mg of paraoxybenzoic acid propyl, 0.56 mg of methylvanillin, and sulfuric acid No. 203 were prepared according to the manufacturing method of the soft capsule in the Pharmacopeia General Formulation so that an appropriate amount of ingredients was contained in one capsule.

Preparation Example 2 Preparation of Tablet

100.0 mg of the porridge extract prepared according to Example 1, corn starch 90.0 mg, lactose 175.0 mg, 15.0 mg L-hydroxypropylcellulose, 5.0 mg polyvinylpyrrolidone 90 and a suitable amount of ethanol were mixed homogeneously Granulated by the wet granulation method, 1.8 mg of magnesium stearate was added and mixed, and the tablets were compressed to 400 mg.

Preparation Example 3 Preparation of Capsule

The raw materials of 100.0 mg of the porridge extract, 83.2 mg of corn starch, 175.0 mg of lactose, and 1.8 mg of magnesium stearate were homogeneously mixed in Example 1, and the capsules were packed to contain 360 mg.

Preparation Example 4 Preparation of Drinks

The present inventors prepared a beverage composition containing the porridge extract prepared according to Example 1 as an active ingredient as follows.

Ojuk extract 0.48-1.28 mg, honey 522 mg, chitosanamide 5 mg, nicotinamide 10 mg, riboflavin sodium 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, orcinsin 50 mg, prepared according to Example 1, and A beverage was prepared using conventional methods with a composition and content of 200 ml of water.

The composition comprising the porridge extract, fractions or compounds isolated from the fractions of the present invention as an active ingredient accumulates sorbitol, which is a fundamental pathogenesis of renal failure and neuropathy (neuropathy), which is a common complication of diabetes by inhibiting aldose reductase. It can be used as a prophylactic and pharmaceutical composition or health food of diabetes-related complications.

Claims (12)

delete delete delete delete delete delete delete 1) extracting and filtering the porridge with ethanol; 2) concentrating and lyophilizing the filtrate to obtain an extract; 3) fractionating the ethyl acetate layer using the extract of step 2 with a separatory funnel; And 4) Silica gel column chromatography was performed on the fractions of step 3 with solvent CH ₂ Cl ₂ -MeOH to obtain Orientin, Luteolin 6-C- (6 "-O-trans-Capeoylglucoside) [Luteolin 6-C- (6 '' -O- trans-caffeoylglucoside)], tricetin 3 ', 4', 5'-trimethyl ester 7-O-β-D-glucopyranoside (tricetin 3 ', 4', Obtaining 5'-trimethyl ether 7- O- bD-glucopyranoside, Tricin, p-coumaric acid or cis-coumaric acid Including, the method of separating the compound from the porridge. delete The method of claim 8, wherein the compound is lutein 6-C- (6 '' -O -trans-cafeoylglucoside). A pharmaceutical composition for preventing or treating diabetic retinopathy, diabetic neuropathy or diabetic nephropathy, containing the compound obtained by the method of claim 8 as an active ingredient. A health food for preventing or improving diabetic retinopathy, diabetic neuropathy or diabetic nephropathy, containing the compound obtained by the method of claim 8 as an active ingredient.

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