KR101226881B1 - A composition comprising the extract of Proso millet as an active ingredient for preventing and treating inflammatory disease - Google Patents

Abstract

The present invention relates to a composition containing a millet extract as an active ingredient, and more particularly, to a method for preventing and treating diabetes mellitus by confirming the excellent inhibitory activity against α-amylase and α-glucosidase which are carbohydrate digesting enzymes of millet extract of the present invention A therapeutic pharmaceutical composition and a health functional food.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention and treatment of diabetes, which contains, as an active ingredient,

The present invention relates to a pharmaceutical composition for the prevention and treatment of diabetes mellitus comprising a millet extract as an active ingredient or a health functional food.

[Patent Document 1] Brands K, Colvin E, Williams LJ, Wang R, Lock RB, Tuch BE. (2008) Reduced immunogenicity of first-trimester human fetal pancreas. Diabetes 57: 627-634

Mediated changes in peroxisome proliferator-activated receptor γ-mediated changes in skeletal muscle fuel handling are independent of the treatment of direct thiazolidinedione action in isolated rats (Braskir B, Gras F, Neschen S, Roden M, Wagner L, gene expression. Diabetes 50: 2309-2315

[Literature 3] Chen X, Zheng Y, Shen Y. (2006) Voglibose (Basen, AO-128), one of the most important α-glucosidase inhibitors. Curr Med Chem. 13: 109-116

[4] Cryer DR, Nicholas SP, Henry DH, Mills DJ, Stadel BV. (2005) Comparative outcomes study of metformin intervention versus conventional approach The COSMIC Approach Study. Diabetes Care 28: 539-543

[Literature 5] Fineman MS, Bicsak TA, Shen LZ, Taylor K, Gaines E, Varns A, Kim D, Baron AD. (2003) Effect on glycemic control of exenatide (synthetic exendin-4) in patients with type 2 diabetes mellitus. Diabetes Care 26: 2370-2377

[Literature 6] Folin O, Denis W. (1912) On phosphotungstic-phosphomolybdic compounds as color reagents. J Biol Chem. 12: 239-243

[Reference 7] Jeong IK, Chung JH, Min YK, Lee MS, Kim KW, Chung YE, Park JY, Hong SK, Lee KU. (2002) Comparative study of the effects of acarbose and voglibose in type 2 diabetic patients. Korean Diabetes J. 26; 134-146

[Literature 8] Kim YM, Jeong YK, Wang MH, Lee WY, Rhee HI. (2005) Inhibitory effect of pine extract on alpha-glucosidase activity and postprandial hyperglycemia. Nutrition 21: 756-761

Large V, Beylot M. (1999) Modifications of citric acid cycle activity and gluconeogenesis in streptozotocin-induced diabetes and effects of metformin. Diabetes 48: 1251-1257

[Literature 10] Laube H. (2002) Acarbose: an update of its therapeutic use in diabetes treatment. Clin Drug Invest. 22: 141-156

[Literature 11] Lee, BB, Park, SR, Han, CS, Han, DY, Park, HY, Lee, SC. (2008) Antioxidant activity and inhibition activity against α-amylase and α-glucosidase of Viola mandshurica extracts. J Korean Soc Food Sci Nutr 37: 405-409

[Document 12] Moller DE. (2001) New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414, 821-827

[Literature 13] Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P, Rooyackers O, Zhou G, Williamson JM, Ljunqvist O, Efendic S, Moller DE, Thorella, Goodyear LJ. (2002) Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 51: 2074-2081

[14] Park H, Hwang KY, Kim YH, Oh KH, Lee JY, Kim K. (2008) Discovery and biological evaluation of novel alpha-glucosidase inhibitors with in vivo antidiabetic effect. Bioorg Med Chem Lett. 18: 3711-3715

[15] Soh H, Lee S, Ha Y. (2002) Total lipid content and fatty acid composition in Setaria italica , Panicum miliaceum and Sorghum bicolor . J. East Asian Soc. Dietary Life 12: 123-128

(2003) Combination treatment with metformin and glibenclamide versus single-drug therapies in type 2 diabetes mellitus (2003). Tosi F, Muggeo M, Brun E, : a randomized, double-blind, comparative study. Metabolism 52: 862-867

[17] United Kingdom Prospective Diabetes Study (UKPDS) Group (1998) Intensive blood glucose control with sulphonylureas or insulin compared with conventional treatments and subjects with type 2 diabetes (UKPDS 33). Lancet 352: 837-853

[Literature 18] Vidal-Puig A, O'Rahilly S. (2001) Metabolism. Controlling the glucose factory. Nature 413: 125-126

[Literature 19] Wilson JJ, Ingledew WM. (1982) Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes. Appl Environ Microbiol. 44: 301-307

[Document 20] Rural Development Administration Culinary Information Center, National Institute of Food Science and Technology

Diabetes is a metabolic disorder characterized by high blood glucose levels. Globally, diabetes mellitus is estimated to reach 180 million people, and by 2030, the number of patients is expected to increase by more than double. In recent years, the number of diabetic patients has rapidly increased, and the age of onset is gradually decreasing. Diabetes is categorized into two types, type 1 diabetes is responsible for the production and secretion of insulin in the pancreas is caused by autoimmune diseases that are damaged by insulin secretion is caused by the cells. Thus, blood glucose is not absorbed into cells and leads to diabetes (Brands et al., 2008). On the other hand, type 2 diabetes, which accounts for about 90% of total diabetes mellitus, normally produces insulin secretion by the pancreatic β-cells, but insulin action or insulin action on the liver or muscle cells decreases, It is a disease caused by not being able to get up properly. Type 1 diabetes is called insulin-dependent diabetes and type 2 diabetes is called non-insulin dependent diabetes (Moller, 2001; Vidal-Puig et al., 2001).

Currently, clinical trials for diabetes include sulfonylurea, biguanide, thiazolidinedione, and amylase inhibitors and glucosidase inhibitors, Inhibitor-based drugs are being used. As a mechanism of action, a sulfonylurea-based drug binds to the pancreatic β-cell receptor to increase Ca ^{2 +} in the cell, and as a result, it affects the cytoskeleton so that the outflow of insulin granules out of the cell (UKPDS group, 1998; Fineman et al., 2003). The Biguanide system mediates the reduction of gluconeogenesis in the liver and promotes AMP kinase (AMPK) activity in muscle cells, thereby promoting the consumption of glucose Large et al., 1999; Cryer et al., 2005; Musi et al., 2002). Thiazolidinedione-based drugs bind to PPAR-γ, a transcription factor in the nucleus of insulin-target cells, and promote the action of insulin by promoting the synthesis of various kinds of proteins that respond to insulin ( Brumanir et al., 2001; Moller, 2001). This improves insulin resistance, a major cause of type 2 diabetes. In addition, drugs such as? -Amylase inhibitor and? -Glucosidase inhibitor drugs inhibit the hydrolysis of carbohydrates into monosaccharides by digestive enzymes, resulting in a gradual increase in postprandial blood glucose levels (Chen et al al., 2006; Moller, 2001). Most of these drugs are used simultaneously in combination with two or more of these drugs in the diabetes treatment process, resulting in effective reduction of blood sugar by the synergistic effect between the drugs (Tosi et al., 2003).

On the other hand, inhibitors of carbohydrate digestion such as? -Amylase inhibitors and? -Glucosidase inhibitors alleviate the rapid rise of postprandial blood glucose levels and are known to be useful drugs for the treatment of diabetic patients. Currently known carbohydrate digestion inhibitors for the treatment of diabetes are acarbose (see FIG. 1A) and voglibose (see FIG. 1B). Acarbose is a type of pseudotetrasaccharide that has a structure similar to that of oligosaccharides and therefore has a structure similar to that of carbohydrase enzymes such as α-amylase, glucoamylase, invertase, , Dextranase,? -Glucosidase, and maltase, and affinity with each of the enzymes is inhibited by? -Amylase of the left And the right is the weakest in the case of α-glucosidase (maltase). In addition, acarbose has an affinity for invertase of 10 ⁴ to 10 ⁵ times stronger than that of sucrose, but has affinity for isomaltase and β-glucosidase Very low or absent, the inhibitory effect on isomaltase and β-glucosidase is not significant (Laube, 2002). On the other hand, voglibose is a kind of valiolamine derivative which has a structure similar to monosaccharide and effectively inhibits? -Glucosidase but not? -Amylase (Chen et al., 2006).

When taking α-amylase inhibitors and α-glucosidase inhibitors to relieve the sudden increase in postprandial blood glucose, digestion and absorption of carbohydrates is not smooth due to the effects of the inhibitors. Therefore, intestinal bacteria using unabsorbed carbohydrates . In addition, digestible carbohydrates and saccharides are degraded by enzymes derived from bacteria in the colon and then metabolized to participate in the production of organic acids such as acetic acid, butyric acid, and lactic acid . The resulting organic acids may lower the pH of the intestine and increase osmotic pressure to induce diarrhea and abdominal pain, and other metabolic by-products such as carbon dioxide and methane may cause side effects such as abdominal bloating (Jeong et al., 2002 ).

Panicum miliaceum ) is a short parasitic inflorescences and has a weight of 1000-500 grams of seedlings and a weight of 500-530 grams of seeds. Grass seeds are 2.25 to 2.5 mm long, 2 mm wide, ovate, and round on the back side. The root is one root and the root is relatively myocardial (deep root), it is resistant to dryness and strong in absorptive capacity. The number of tillering plants is two to three, and most of them are effective tillering. Depending on the breed, the plant length ranges from 30 to 120 cm, and the stem is sturdy and upright or the base is lying skewed. Stems and leaves are covered with hair and are resistant to pests. The surface of the stem and seedling The green rice husk is green and turns into yellow green or red green as the seedling is ripe. The leaf sheath is open and covered with hairs on the very small ridge. The leaf tip is short and thick. There is no Leaflet (Rural Development Administration, National Institute of Food and Agriculture, Crop Information Center http://www.nics.go.kr/).

In Korea, the cultivation and harvesting of traditional rice grains is weak, and rice grains are used for mixed rations (Soh et al., 2002). In recent years, interest in health functional and well-being foods has increased, interest in the functionality of the grains, and the growing and consumption of grains have also been on the rise. However, systematic research on the physiological activities of the grains is not well studied, and studies on the efficacy of the grains related to obesity and diabetes have been rarely conducted.

Therefore, the inventors of the present invention found that a substance having excellent effect of suppressing rapid blood glucose elevation after a meal without adverse effect on the living body was tested for its pharmacological effect. As a result, excellent α- (glucosidase) inhibitory activity, thereby completing the present invention.

 In order to achieve the above object, the present invention provides a pharmaceutical composition for prevention and treatment of diabetes mellitus comprising a millet extract as an active ingredient.

The present invention also provides a health functional food for prevention and improvement of diabetes mellitus containing millet extract as an active ingredient.

The term " Panicum " miliaceum ) is the captain ( Panicum miliaceum ), yellow sticks ( Panicum miliaceum there is. Norangchal), a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

As used herein, the term " crude extract " means water containing purified water, a solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol and butanol, or a mixed solvent thereof, preferably water and an ethanol mixed solvent, And extracts soluble in 60 to 90% ethanol.

The " polar solvent-soluble extract " as defined herein includes extracts which are soluble in water, methanol, butanol or a solvent mixture thereof, preferably water or butanol, more preferably butanol.

As used herein, "nonpolar solvent-soluble extract" includes extracts that are soluble in hexane, methylene chloride, chloroform, or ethyl acetate, preferably hexane, methylene chloride or ethyl acetate, more preferably methylene chloride solvent.

Diabetes, as defined herein, includes Type 1 or Type 2 diabetes, preferably Type 2 diabetes.

Hereinafter, the present invention will be described in detail.

The millet extract of the present invention can be prepared as follows. After the flask is washed and sieved, it is ground for 1 to 30 minutes, preferably 5 to 20 minutes, and then 60 to 90% ethanol is mixed several times. Thereafter, the mixture is heated at a temperature of 30 to 150 캜, preferably 50 to 100 캜 The extract obtained by the reflux extraction is centrifuged at a speed of 1,000 rpm to 20,000 rpm, preferably 5,000 rpm to 15,000 rpm for 5 minutes to 30 minutes, preferably 10 minutes to 25 minutes. Only the supernatant is collected separately, The millet extract of the present invention can be obtained.

The polar solvent or the non-polar solvent soluble extract of the present invention may further contain about 0.0005 to 0.005 times, preferably 0.05 to 0.5 times the volume (v / w%) of the crude extract, preferably 60 to 90% ), Followed by fractionation using n-hexane, methylene chloride, ethyl acetate and butanol to obtain non-polar solvent-soluble extract fractions, which are dissolved in a nonpolar solvent such as n-hexane, methylene chloride or ethyl acetate; And polar solvent-soluble extract fractions soluble in polar solvents such as butanol and water can be obtained.

The inventors of the present invention confirmed the excellent inhibitory activity of? -Amylase and? -Glucosidase in the millet extract obtained by the above-described method, thereby providing a pharmaceutical composition useful for prevention and treatment of diabetes and a health composition It is confirmed that it is useful for providing food.

Accordingly, the present invention provides a pharmaceutical composition for preventing and treating diabetes mellitus containing the millet extract obtained as described above as an active ingredient.

The present invention also provides a health functional food for prevention and improvement of diabetes mellitus containing the millet extract obtained by the above production method as an active ingredient.

The pharmaceutical composition for prevention and treatment of diabetes containing the millet extract of the present invention comprises the above extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

The pharmaceutical compositions containing the millet extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical composition containing the millet extract according to the present invention can be administered orally or parenterally in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, Examples of the carrier, excipient and diluent which can be contained in the composition including the fraction include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate , Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. 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 are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.5 g / kg to 5 g / kg, preferably 1 g / kg to 3 g / kg per day. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

There is provided a health functional food for preventing and improving diabetes mellitus containing the millet extract of the present invention as an active ingredient.

The composition containing the extract of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of diabetes.

Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powders, granules, tablets, capsules or beverages have.

The amount of the extract in the food or beverage of the present invention is generally from 1 to 5% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is preferably 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional component such as ordinary beverages, in addition to containing the above extract as an essential ingredient at the indicated ratio, . Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As described above, the millet extract of the present invention exerts an excellent effect of inhibiting the enzymatic action of amylase and? -Glucosidase, which are digestive enzymes of carbohydrates, and is useful for the treatment and prevention of diabetes A pharmaceutical composition or a health functional food.

1 is a view showing the structure of acarbose and voglibose,
FIG. 2 is a view showing a classification scheme of an 80% ethanol extract using various solvents,
FIG. 3 is a graph showing the inhibitory activity of 80% ethanol extract of various grains against? -Amylase,
FIG. 4 is a graph showing the inhibition activity of 80% ethanol extracts of (A) pollen and millet (B) white bolls and meso for α-amylase at a concentration of 0.125 to 1 mg /
FIG. 5 is a graph showing the inhibition activity of organic solvent fractions of (A) brix and millet (B) white scales and meso to amylase,
FIG. 6 is a chart showing the heat stability of the methylene chloride fraction of the millet,
7 is a diagram showing an investigation of the stability of the methylene chloride fraction of the millet against the acid,
8 is a graph showing the inhibitory activity of 80% ethanol extract of various grains including edible blood against? -Glucosidase,
Fig. 9 (A) is a diagram showing the determination of total phenolic compounds in the organic solvent fractions of 80% ethanol extract (B), gold wax, edible bark and millet of various grains.

Hereinafter, the present invention will be described in detail. However, the following examples, reference examples and experimental examples are illustrative of the present invention, and the present invention is not limited thereto.

Reference example  1. Reagents and devices

1-1. Reagents and devices used for extracting crude extract

(95% ethanol (Duksan), 99.5% methylene chloride, 99.5% ethyl acetate, 96.0% n-hexane and 99.0% n-butanol) were used for the extraction and fractionation of the millet. (Heidolph LR 4000, Germany) and an aspirator (Eyela A-3S, Japan) were used as a rotary vacuum evaporator.

1-2. Acquisition of crops used in the experiment

The pots used for the experiment were provided by the Department of Functional Crops of the National Institute of Food Science and Technology (Milyang, Kyungnam). The capitals were classified into the form of the seeds and blood with the blood, and the form of the sutures that only the seeds were left. 1 kilogram each of a badge (yellow), a yellow stick (yellow) (see Table 1).

No Crop name Sample form Sample amount (kg) One millet Round One 2 Yellow sticker Bedside One 3 Chimney Round One 4 A gold rally Bedside One 5 White chimney Bedside One 6 Yellow carpet Round 0.4 7 Meso Round One 8 A la carte Round One 9 A la carte Bedside One 10 Golden Joe Bedside One 11 Edible blood Bedside 0.1 12 Yulmu Round One 13 red bean Round One

Example  1. Extraction and fractionation of millet

1-1. millet Crude extract  detach

500 g of seeds from the National Institute of Advanced Industrial Science and Technology (Milyang, Kyungnam) were washed and cut with Blender 7012 (Dynamics Corporation, USA) for 10 minutes, and then 1000 ml of 80% ethanol (3 hours at a time) (Corning 2560-400, USA) at a temperature of 80 ° C. The resulting extract was centrifuged at a speed of 10,000 rpm for 20 minutes to collect only the supernatant, and the concentrate was centrifuged using a rotary vacuum concentrator (yield: 5.6%) of 80% ethanolic extract (hereinafter referred to as " PM-2 ") was obtained by concentrating the supernatant by evaporator (Heidolph LR 4000, Germany) and drying.

1-2. Polar solvent and Nonpolar solvent  Fraction of soluble extract

The remaining portion was dissolved in 500 ml of water (the unmelted portion was dissolved in n-hexane and the aqueous layer was further fractionated), leaving a small amount of the 80% ethanol extract obtained in Example 1-1. N-Hexane (500 ml × 3 times) After pouring and separating, the n-hexane layer was concentrated to obtain an n-hexane fraction (hereinafter referred to as "PM-2HE"). The aqueous layer was then extracted with methylene chloride (500 ml × 3 times), and the methylene chloride layer was concentrated to obtain a methylene chloride fraction (hereinafter referred to as "PM-2MC"). Then, the aqueous layer was extracted with ethyl acetate (500 ml × 3 times) and the extract was concentrated to obtain ethyl acetate fraction (hereinafter referred to as "PM-2EA"). Subsequently, the aqueous layer was extracted with n-butanol (500 ml x 3 times) and concentrated to obtain an n-butanol fraction (hereinafter referred to as "PM-2BU"). The water layer finally remaining was also concentrated to obtain an aqueous layer fraction (hereinafter referred to as " PM-2WA ") (see FIG.

** Dry weight of ethanol extracts and various organic solvent fractions (grams) No Grain Extraction quantity Ethanol Hexane Methylene chloride Ethyl acetate n-Butanol d'H ₂ O One Millet 500 28.3 - 3.81 0.05 0.48 0.62 2 Yellow sticky balloons 500 26.48 3.08 0.75 0.97 0.80 1.10 3 Chopping block 400 23.31 1.82 1.25 0.18 0.83 0.66 4 Golden Chopsticks Suite 400 23.24 2.28 0.34 0.77 1.52 4.49 5 White chimney 500 22.17 2.45 1.75 0.27 1.40 2.71 6 A yellow carpet 250 15.01 1.23 0.79 0.67 2.61 1.28 7 Mezzo 500 34.3 2.86 7.18 1.69 1.99 1.09 8 A la carte 500 40.34 3.04 3.68 0.60 9.32 5.93 9 A la carte 500 17.63 2.29 0.85 0.33 3.19 3.16 10 Golden Joe Suite 500 29.01 1.47 0.51 0.31 1.37 1.79 11 Edible bean curd 63 2.24 0.58 0.27 0.13 0.54 0.77 12 Yulmu Song 500 37.96 0.89 1.65 0.72 0.68 2.32 13 Red bean curd 500 24.75 0.15 6.34 0.19 1.57 10.2

Reference Example 2 . Materials and devices used in carbohydrate digestion experiments

α-amylase (from human salivary, A1031), α-glucosidase (from Brewer's yeast, G4634), soluble starch, p-nitrophenyl-aD-glucopyranoside and acarbose, A8980) were purchased from Sigma (St. Louis, MO, USA). The folin-Ciocalteu's phenol reagent used for the determination of the polyphenol compounds contained in the extract was purchased from Fluka (Sigma-Aldrich, Schweiz, Switzerland) and the tannic acid used as a standard polyphenol compound was purchased from Avondale Laboratories (Oxon, England) Lt; / RTI >

Experimental Example  1. α- Amylase  Measurement of inhibitory activity

In order to measure the? -Amylase inhibitory activity of the samples obtained in Example 1, experiments were conducted as described below (Wilson et al., 1982).

1-1. The α- Amylase  Inhibitory activity

Amylase derived from human saliva was dissolved in PBS (Gibco 21600-010, USA) at 40 unit / ml, soluble starch was dissolved in PBS at a concentration of 1% as a substrate of? -Amylase, and? -Amylase Were dissolved in dimethylsulfoxide (DMSO, Kanto chemical 10378-73, Japan) at a concentration of 1 mg / ml. To measure the inhibitory activity against α-amylase, 290 μl of PBS, 10 μl of α-amylase solution (40 units / ml) and 50 μl of millet extract (1 mg / ml) were mixed and incubated at 37 ° C. for 10 minutes Preincubation was performed. Then, 350 μl of a 1% soluble starch solution, which is a substrate, was added and reacted at 37 ° C for 30 minutes. To measure the amount of soluble starch remaining after the reaction, 0.5% iodine (I ₂ ) was dissolved in a 5% potassium iodide solution in a reaction solution (700 μl) 300 μl of diluted iodine solution (0.1% KI + 0.01% I2 / 0.05N HCl) was added to the solution and the optical density was measured at 620 nm using a spectrophotometer (Shimadzu UV-1650PC, Japan) Amylase inhibitory activity was investigated (see Table 3). The control (control) was prepared by adding soluble starch as substrate,? -Amylase, which is an enzyme, and DMSO, which is a solvent used for dissolving the extract, in place of the ethanol extract and each organic solvent fraction. Amylase, and enzyme-derived ethanol extracts, and the respective organic solvent fractions were added. Blank 1 was prepared by adding soluble starch as a substrate and 80% EtOH extract from a capillary and DMSO as a solvent used for dissolving the extract in place of each organic solvent fraction. Blank 2 contained soluble starch, And ethanol extracts from each plant and each organic solvent fraction were added. On the other hand, as a standard substance of α-amylase inhibitor, acabos was also dissolved in DMSO at a concentration of 50, 100 and 200 μg / ml, and the result was compared with the samples in terms of α-amylase inhibitory activity in addition to the sample obtained from the millet. As a result, the? -Amylase inhibitory activity was calculated from the absorbance of the sample group and the absorbance of blank 2 (blank 2) relative to the difference between the absorbance of the control (control) and the absorbance of blank 1 (blank 1) , The value is subtracted from 1, multiplied by 100, and expressed as a percentage.

Control Sample Blank 1 Blank 2 α-Amylase solution 10 μl 10 μl - - PBS 290 μl 290 μl 300 μl 300 μl Inhibitor (Cereal extract) - 50 μl - 50 μl DMSO 50 μl - 50 μl - Preincubate at 37 ° C for 10 min 1% Soluble starch solution 350 μl 350 μl 350 μl 350 μl Incubate at 37 ° C for 30 min I ₂ solution
(0.1% KI + 0.01% I 2 /0.05N HCl) 300 μl 300 μl 300 μl 300 μl Read OD at 620 nm

As a result of the above experiment, the ethanol extract of the millet (squirrel) showed an inhibitory activity of 55.6% in? -Amylase compared to the control group containing only DMSO (see FIG. 3).

The inhibitory activity of α-amylase was investigated for each concentration by treating the extracts of Cryptomeria japonica with concentrations ranging from 0.125 mg / ml to 1 mg / ml. In the case of millet, the inhibitory activity was 0.3% at 0.125 mg / ml, 9.9% at 0.25 mg / ml, 23.8% at 0.5 mg / ml and 48.2% at 1 mg / ml (see FIG.

1-2. Millet Fraction  α- Amylase  Inhibitory activity

In the experiment using the ethanol extract, the highest intact α-amylase inhibition ratio was fractionated by organic solvent, and the fraction was treated at a concentration of 1 mg / ml.

As a result of the above-mentioned experiment, the n-hexane fraction inhibition activity test was not carried out because the n-hexane fraction was not applied in the case of the millet. The ethanol extracts of Kjanggol showed 55.5% inhibition activity, 95.3% of methylene chloride fraction and 32.9% of ethyl acetate fraction. On the other hand, the butanol fraction showed an inhibition rate of 20.9%, indicating that the inhibitory activity was somewhat lower than that of the two preceding fractions, and the inhibitory activity was hardly observed in the aqueous layer. As a result of the measurement of inhibitory activity by fraction, it was confirmed that the methylene chloride fraction showed the highest activity in the millet, and the component that inhibits the? -Amylase was the most affinity substance to methylene chloride (see FIG. 5).

1-3. Methylene chloride Fraction  Stability study for heat and acid

The inhibition rate of α-amylase was found to be higher than that of the control group, which was heat-treated for 15 to 60 minutes by methylene chloride fraction and acarbose (200 μg / ml), which had the highest inhibitory activity in ethanol fraction and fractionation by organic solvent, Change.

As a result of the above experiment, it was found that the methylene chloride fraction of the millet showed high inhibitory activity irrespective of the heat treatment time, and the heat treatment did not affect the? -Amylase inhibiting activity of the methylene chloride fraction.

Amylase inhibition activity of the methylene chloride fraction of the millet (pH 2) treated with methylene chloride (200 μg / ml) was measured at 37 ° C for 1 hour or 2 hours, After the treatment, the reaction was carried out. As a result of measuring the inhibitory activity of? -Amylase by an acid treatment time, it was confirmed that the activity of? -Amylase was not reduced by acid treatment. As a result, it was found that the methylene chloride fractions of the capillary and acarbose did not lose the inhibitory activity against? -Amylase even under strong acids and at high temperatures (see FIG. 7).

Experimental Example  2. α- Glucosidase  ( glucosidase ) Inhibition activity measurement

In order to measure the α-glucosidase inhibitory activity of the samples obtained in Example 1, experiments were conducted by applying the method disclosed in the following literature (Kim et al., 2005; Park et al., 2008; Lee et al., 2008).

2-1. The α- Glucosidase  Inhibitory activity

α-glucosidase was prepared in a concentration of 10 unit / ml in 50 mM sodium phosphate buffer (pH 6.8) (manufactured by Dawson et al., Data for biochimecal research) and diluted to a concentration of 0.25 unit / ml Followed by 20 μl each. The substrate was prepared by dissolving p-nitrophenyl-α-D-glucopyranoside in sodium phosphate buffer (50 mM, pH 6.8) at a concentration of 3 mM. As an inhibitor against α-glucosidase, ethanol extract of capricorn was dissolved in DMSO at a concentration of 10 mg / ml. First, 20 μl α-glucosidase dilution in a 96-well plate (Corning CLS3595, USA), 65 μl sodium phosphate buffer (50 mM, pH 6.8) and 15 μl long-cell ethanol extract were mixed and incubated for 10 min at 37 ° C After the addition, 100 μl of a 3 mM p-nitrophenyl-α-D-glucopyranoside solution was added and reacted at 37 ° C. for 30 minutes (see Table 4). After the reaction, the inhibition rates were compared by measuring the absorbance at 405 nm using a microplate reader (Molecular devices Thermo max, USA). At this time, the control (control) was a mixture of nitrophenyl-α-D-glucopyranoside, α-glucosidase as an enzyme, and caproic ethanol extract and each organic solvent fraction as a solvent DMSO was added to the sample. To the sample, p-nitrophenyl-α-D-glucopyranoside, α-glucosidase, and caponeic ethanol extract were added. In the blank 1, nitrophenyl-α-D-glucopyranoside and Ganoderma ethanol extract were added as a substrate and DMSO as a solvent instead of each organic solvent fraction. Blank 2 2), p-nitrophenyl-α-D-glucopyranoside, which is a substrate, and millet ethanol extract were added. On the other hand, acarbose was also dissolved in DMSO at a concentration of 5 mg / ml and 10 mg / ml as a standard substance of the α-glucosidase inhibitor and added to the sample instead of the grain-derived sample to compare α-glucosidase inhibitory activity with the samples . At this time, the ratio of the absorbance difference of the absorbance blank 2 (blank 2) of the sample group to the difference between the absorbance of the control (control) and the absorbance of the blank 1 (blank 1) was calculated first Then, the value was subtracted from 1, multiplied by 100, and the inhibitory activity was calculated by calculating the percentage as shown in the following formula (2).

Control Sample Blank 1 Blank 2 α-Glucosidase solution 20 μl 20 μl - - Phosphate buffer (pH 6.8) 65 μl 65 μl 85 μl 85 μl Inhibitor (Cereal extract) - 15 μl - 15 μl DMSO 15 μl - 15 μl - Preincubate at 37 ° C for 10 min 3 mM p-Nitrophenyl-a-
D-glucopyranoside 100 μl 100 μl 100 μl 100 μl Incubate at 37 ° C for 30 min Read OD at 405 nm

 As a result of the above experiment, the α-glucosidase inhibitory activity of the plant extract (80%) was compared with that of the plant extract (10 mg / ml), and the inhibitory activity of α-glucosidase was 18.6% And showed about 36% inhibitory activity compared to 51.6% of acarbose (10 mg / ml), a standard substance. Inhibition activity was not observed when ethanol extract of millet (5 g / ml) was treated at a concentration of 5 mg / ml (see Fig. 8).

Experimental Example  3. Determination of phenol compounds in millet extract

In order to confirm the amount of the phenol compound contained in the 80% ethanol extract of the samples obtained in Example 1, experiments were conducted as described below (Folin and Denis, 1912).

100 μl of 80% ethanol extract and each organic solvent fraction were added to each well and 500 μl of Folin-Ciocalteu's phenol reagent (Sigma-Aldrich, Schweiz, Switzerland) was added. After the reaction mixture was reacted at room temperature for 5 minutes, 7.5% Na ₂ CO ₃ (Hanawa 190-01825, Japan) was added in a volume of 400 μl. The resulting mixture was reacted at a temperature of 50 ° C for 5 minutes and centrifuged at 13,200 rpm for 2 minutes using a centrifuge (Eppendorf 5415R, Germany) to remove insoluble precipitates. The absorbance of the solution from which the precipitate was removed was measured with a spectrophotometer (Shimadzu UV-1650PC, Japan) at a wavelength of 760 nm. At this time, a standard curve was prepared using tannic acid as a standard material, and then the absorbance of the extract was substituted into the formula to quantify the phenolic compounds.

As a result of the experiment, it was found that the amount of the phenol compound in the millet (s) was 2.5 μg / mg, and the amount of the phenolic compound in the yellow sludge (sand bed) was 7.6 μg / mg, (26.6 μg / mg) compared to the control group (121.1 μg / mg), white pickles (16.5 μg / mg), green tea preparations (17.6 μg / Respectively. On the other hand, the organic solvent fractions of the millet (s) contained 36.3, 13.8, 25.8 and 11.6 μg / mg of phenol compounds in the order of methylene chloride, ethyl acetate fraction, butanol fraction and water layer fraction (see FIG. 9). Comparing phenol compound quantitation results with α-amylase and α-glucosidase inhibitory activity, the phenolic compound contents showed α-amylase and α-glucosidase inhibitory activity, even though they were relatively low in all fractions of the millet. These results suggest that a substance having an inhibitory activity of? -Amylase and? -Glucosidase as components contained in millet extract is not a kind of phenol compound (see FIG. 9).

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but will be specifically described.

Formulation example  One. Sanje  Produce

PM-2MC 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

PM-2 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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

Formulation example  3. Preparation of capsules

PM-2BU 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

PM-2HE 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

PM-2 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

PM-2WA 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

PM-2 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (8)

A pharmaceutical composition for the prevention and treatment of diabetes mellitus containing extracts of methylene chloride of Panicum miliaceum as an active ingredient. delete delete delete delete delete A health functional food for prevention and improvement of diabetes containing an extract of methylene chloride of Panicum miliaceum as an active ingredient. delete

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