KR101226824B1 - A composition comprising the extract of Sorghum bicolor L. Moench as an active ingredient for preventing and treating inflammatory disease - Google Patents

Abstract

The present invention relates to a composition containing a sorghum extract as an active ingredient, and specifically, an excellent inhibitory activity against a-amylase and α-glucosidase, which are carbohydrate digestive enzymes of the sorghum extract of the present invention, and a post-prandial blood sugar increase alleviating effect. By confirming, it can be usefully used for the provision of a pharmaceutical composition for preventing and treating diabetes and a dietary supplement.

Description

A composition comprising the extract of Sorghum bicolor L. Moench as an active ingredient for preventing and treating inflammatory disease}

The present invention relates to a pharmaceutical composition or health functional food for preventing and treating diabetes containing sorghum extract as an active ingredient.

References 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

Brunmair B, Gras F, Neschen S, Roden M, Wagner L, Waldhausl W, Furnsinn C. (2001) Direct thiazolidinedione action on isolated rat skeletal muscle fuel handling is independent of peroxisome proliferator-activated receptorγ-mediated changes in gene expression. Diabetes 50: 2309-2315

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

[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) additive to existing metformin and / or sulfonylurea treatment in patients with type 2 diabetes. Diabetes Care 26: 2370-2377

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, Lee MK, Kim KW, Chung YE, Park JY, Hong SK, Lee KU. (2002) Comparative study about the effects of acarbose and voglibose in type 2 diabetic patients. Korean Diabetes J. 26; 134-146

[Reference 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

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

[Document 11] Lee BB, Park SR, Han CS, Han DY, Park E, 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

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

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

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

[16] Tosi F, Muggeo M, Brun E, Spiazzi G, Perobelli L, Zanolin E, Gori M, Coppini A, Moghetti P. (2003) Combination treatment with metformin and glibenclamide versus single-drug therapies in type 2 diabetes mellitus : 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 treatment and risk of complications in subjects with type 2 diabetes (UKPDS 33). Lancet 352: 837-853

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

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

[Document 20] Crop Information Center, National Institute of Crop Science, Rural Development Administration

Diabetes is a metabolic disease characterized by high blood glucose levels. Diabetes affects 180 million people worldwide, and the number of patients is expected to more than double in 2030. Recently, the number of diabetic patients is increasing rapidly, and the age of onset is gradually decreasing. Diabetes is classified into two types, and type 1 diabetes is caused by the fact that β cells, which are responsible for the production and secretion of insulin in the pancreas, are damaged by autoimmune diseases and thus do not secrete insulin. Therefore, 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 all diabetes, normally secretes insulin by the β-cells of the pancreas, but the action of insulin or the ability of insulin to lower liver or muscle cells lowers blood glucose. It is a disease that occurs because it does not happen properly. Type 1 diabetes is called insulin dependent diabetes and type 2 diabetes is insulin independent diabetes (Moller, 2001; Vidal-Puig et al., 2001).

Current clinical treatments for diabetes include sulfonylurea, biguanide, thiazolidinedione, and α-amylase inhibitors and α-glucosidase. Inhibitors are being used. As the mechanism of action, sulfonylurea (Sulfonylurea) The agent of the system is increased the Ca ^{2 +} in the cell in combination with a receptor of the β cells of the pancreas and as a result leakage of insulin granules out affects the Saito skeleton (cytoskeleton) cells Stimulate insulin secretion to increase insulin secretion (UKPDS group, 1998; Fineman et al., 2003). Biguanide-based drugs play a role in promoting glucose consumption by reducing gluconeogenesis in the liver and enhancing AMP kinase (AMPK) activity in muscle cells ( Large et al., 1999; Cryer et al., 2005; Musi et al., 2002). Thiazolidinedione-based drugs bind to PPAR-γ, a transcription factor present in the nucleus of insulin target cells, and promote the action of insulin by promoting the synthesis of several proteins that respond to insulin ( Brunmair et al., 2001; Moller, 2001). This improves insulin resistance, the leading cause of type 2 diabetes. In addition, drugs of the α-amylase and α-glucosidase inhibitor systems act to inhibit carbohydrates from hydrolyzing into monosaccharides by digestive enzymes, thus slowing blood sugar elevation after meals (Chen et al., 2006; Moller, 2001). In most cases of diabetes treatment, two or more of these drugs are used in combination at the same time, and as a result, the blood sugar is effectively lowered by synergistic effects between the drugs (Tosi et al., 2003).

On the other hand, carbohydrate digestion inhibitors, such as α-amylase inhibitors and α-glucosidase inhibitors, are known to be useful agents for the treatment of diabetics because they alleviate the rapid rise in blood sugar levels after eating. Carbohydrate digestion inhibitors currently available for the treatment of diabetes are known as acarbose (see FIG. 1A) and boglibose (see FIG. 1B). Acarbose is a kind of pseudotetrasaccharide and has a structure similar to oligosaccharide, so it is a carbolytic enzyme α-amylase, glucoamylase, and invertase. Competitively inhibit enzymatic action on the substrates of dextranase, α-glucosidase and maltase, and their affinity for the enzymes on the left side of α-amylase The case is the strongest and the right α-glucosidase (maltase) is the weakest. In addition, affinity than acarbose (acarbose) is the only kinase Berta affinity for (invertase) is strong to 10 ⁴ to 10 ⁵ times that of sucrose (sucrose), iso maltase (isomaltase) and β- glucosidase (glucosidase) is Very low or no inhibitory effects on isomaltase and β-glucosidase (Laube, 2002). On the other hand, voglibose is a kind of valoliolamine derivative, which is similar in structure to monosaccharides and effectively inhibits α-glucosidase but does not inhibit α-amylase. (Chen et al., 2006).

Ingestion of α-amylase inhibitors and α-glucosidase inhibitors to alleviate the sudden increase in blood sugar after meals results in poor digestion and absorption of carbohydrates, resulting in intestinal bacteria that use undigested carbohydrates. The number of is increased. Indigestible carbohydrates and sugars are also broken down by colon-derived enzymes and metabolized to produce organic acids such as acetic acid, butyric acid, and lactic acid. . The organic acids produced may lower the pH of the intestine and increase the osmotic pressure to cause diarrhea and abdominal pain, and other metabolic products such as carbon dioxide and methane may cause side effects such as bloating (Jeong et al., 2002). ).

Sorghum ( Sorghum bicolor L. Moench) is an annual or perennial herbaceous plant, the seed of which is young and enclosed in a hard, glossy base. One root is one and the root is generated from the bottom of the fourth lobe elongated, myocardial, absorbent, dry resistance is strong. Stems and leaves are short stems of about 1m of soy sauce, and long stems of 2-3m. Saving water is 8 ~ 13 nodes, usually about 10 nodes, and inside of stem is cold. The surface of the stem has a significant acupressure, and reddish-brown pigment is formed at the place where it is damaged or damaged. The leaf is large, 1m long, the butterfly is about 5cm, the middle is white and distinct. There is no leaf, and lobe is dark brown rim membrane piece with lip hair. Isaac has a large number of hydroponic, about 10 nodes, 5 to 6 diameters in each node, and 2 to 3 diameters grow again. The daily number is 1,462 ~ 3,985 grains and the average number is 2,747 grains.

The varieties are not arranged and the native species are grown. It is relatively tall, and it is from Isaac's estimate to smuggling. Golden Sorghum (黄 金) was bred in 2004 as a breed separated from the native species. It is a brackish water, belonging to the early spring species, and its height is about 160cm, the bicolor is smuggled, and the color of the seed is red. Dafeng Shui was cultivated in 2007 as a cultivar isolated from the native species. Its shape is similar to that of golden wax sorghum but it is about 20cm tall (National Institute of Crop Science, RDA, http://www.nics.go.kr/).

Since Korea is currently farming mainly on paddy fields, the cultivation and harvesting of traditional grains is weak, and grains are used for mixing meals and stocks (Soh et al., 2002). Recently, as interest in health functional well-being foods has increased, there has been an increasing interest in the functionality of grains, cultivation and consumption of grains. However, systematic studies on the biological activity of cereal grains are very lacking, and in particular, studies on the efficacy of cereal grains related to obesity and diabetes have been rarely performed.

Accordingly, the present inventors have tried to find a substance excellent in inhibiting the rapid blood sugar rise after eating without side effects in the living body, and tested the pharmacological effect of sorghum extract, the excellent α-amylase (amylase) and α-glucosidase of sorghum extract (glucosidase) inhibitory activity and post-prandial blood sugar elevation inhibitory effect was confirmed by completing the present invention.

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

In addition, the present invention provides a dietary supplement for the prevention and improvement of diabetes mellitus containing sorghum extract as an active ingredient.

Sorghum as defined herein bicolor L. Moench) is chalsusu (Sorghum bicolor L. Moench var. Chal), Golden Swordsman ( Sorghum) bicolor L. Moench var. Hwanggeumchal and White-tailed Sorghum ( Sorghum) bicolor L. Moench var. Huinchal) is characterized in that the crude extract of sorghum, polar solvent soluble extract or non-polar solvent soluble extract.

A "crude extract" as defined herein is a solvent selected from water containing purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol or the like, or a mixed solvent thereof, preferably a water and ethanol mixed solvent, more preferably Contains extracts available in 60-90% ethanol.

"Polar solvent soluble extract" as defined herein includes extracts soluble in water, methanol, butanol or a mixed solvent thereof, preferably water or butanol, more preferably butanol.

"Non-polar solvent soluble extract" as defined herein includes extracts soluble in hexane, methylene chloride, chloroform, or ethyl acetate, preferably hexane, methylene chloride or ethyl acetate, more preferably in hexane or 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.

Sorghum extract of the present invention may be prepared as follows. After washing and sintering the millet, it is ground for 1 minute to 30 minutes, preferably 5 minutes to 20 minutes, and mixed with 60% to 90% ethanol several times, and then at a temperature of 30 ° C to 150 ° C, preferably 50 ° C to 100 ° C. The extract obtained by reflux extraction was centrifuged for 5 minutes to 30 minutes, preferably 10 minutes to 25 minutes at a speed of 1,000rpm to 20,000rpm, preferably 5,000rpm to 15,000rpm to collect only the supernatant, and concentrated under reduced pressure and dried. The sorghum crude extract of the present invention can be obtained.

In addition, the polar solvent or non-polar solvent soluble extract of the present invention is about 0.0005 to 0.005 times the weight of the crude extract, preferably 60 to 90% ethanol crude extract, preferably 0.05 to 0.5 times the volume (v / w% Non-polar solvent soluble extract fractions which are available in non-polar solvents such as n-hexane, methylene chloride, ethyl acetate by carrying out a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate and butanol after addition of water; And polar solvent soluble extract fractions soluble in polar solvents such as butanol, water and the like.

The inventors of the present invention confirmed the excellent inhibitory activity of α-amylase and α-glucosidase of sorghum extract obtained by the preparation method and the effect of inhibiting post-prandial blood sugar elevation prevention and treatment of diabetes. It has been found to be useful for the provision of pharmaceutical compositions and dietary supplements useful for the study.

Therefore, the present invention provides a pharmaceutical composition for the prevention and treatment of diabetes mellitus containing the sorghum extract obtained by the above method as an active ingredient.

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

The pharmaceutical composition for the prevention and treatment of diabetes mellitus containing sorghum extract of the present invention comprises 0.1 to 50% by weight of the extract relative to the total weight of the composition.

Pharmaceutical compositions containing sorghum extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions containing sorghum extract according to the present invention may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents which may be used in the formulation comprising fractions, may 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. 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 form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations contain at least one excipient such as starch, calcium carbonate and sucrose in the fraction. Or 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. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

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.

The present invention provides a dietary supplement for the prevention and improvement of diabetes mellitus containing sorghum extract as an active ingredient.

The composition comprising the extract of the present invention can be used in various ways, such as drugs, foods and drinks for the 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 may generally be added to 1 to 5% by weight of the total food weight of the health food composition of the present invention, the health beverage composition is 0.02 to 10 g, preferably based on 100 ml Can be added in a ratio of 0.3 to 1 g.

In addition to containing the sorghum extract as an essential ingredient in the indicated ratio, the health beverage composition of the present invention has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . 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. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates 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 its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable beverages. 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 sorghum extract of the present invention has an excellent effect of inhibiting the enzymatic action of α-amylase and α-glucosidase, which are digestive enzymes of carbohydrates, and relieving postprandial blood sugar increase. It can be used as a useful pharmaceutical composition or health functional food for the treatment and prevention of diabetes.

1 is a view showing the structure of acarbose (acarbose) and boglibose (voglibose),
2 is a diagram showing a classification scheme of 80% ethanol extract using various solvents,
3 is a diagram showing the inhibitory activity of 80% ethanol extract of various grains against α-amylase,
Figure 4 is a diagram showing the inhibitory activity of the 80% ethanol extract of (A) Chaksu and millet (B) Whitestalk and meso against α-amylase at a concentration of 0.125 ~ 1 mg / ml,
5 is a diagram showing the inhibitory activity of the organic solvent fractions of (A) waxy water and millet (B) white waxy water and meso against α-amylase,
6 is a diagram showing the stability of the heat of the methylene chloride (methylene chloride) fraction of the brine water,
7 is a diagram showing the stability of the methylene chloride (methylene chloride) fraction of the water in the acid,
8 is a diagram showing the inhibitory activity of ethanol extracts of sorghum and various grains against α-glucosidase,
9 is a diagram showing the inhibitory activity of the ethanol extract of golden wax sorghum against glucosidase at various concentrations,
10 is a diagram showing the inhibitory activity of the organic solvent fraction of golden wax sorghum against α-glucosidase,
FIG. 11 is a diagram showing the inhibitory activity of the organic solvent fractions of water and edible blood on α-glucosidase,
12 is a diagram showing the quantification of total phenolic compounds in (A) 80% ethanol extracts of various grains (B) organic solvent fractions of waxy water, golden waxy water, edible blood, meso,
Figure 13 is a diagram showing the post-prandial blood sugar level of 80% ethanol extract of golden wax water.

Hereinafter, the present invention will be described in detail. However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Reference Example 1. Reagents and Instruments

1-1. Reagents and Instruments Used to Extract Sorghum Crude Extracts

The solvent used for extraction and fractionation of sorghum was (95% ethanol (Duksan), 99.5% methylene chloride, 99.5% ethyl acetate, 96.0% n-hexane, 99.0% n-butanol (Dongyang Steel Chemical)). Rotary vacuum evaporator (Heidolph LR 4000, Germany), aspirator (Eyela A-3S, Japan) was used.

1-2. Obtaining Crops Used in Experiments

The sorghum used for the experiment was provided by the Department of Functional Crop (Kyongnam Miryang). Sorghum was classified into the form of grains with seeds and blood, and the form of grains with only the seeds peeled off the blood. 1 kg of rice flour (junggok), golden rice flour (gongsu), and white rice flour (gongsu) were each provided (see Table 1).

No Crop name Sample type Sample amount (kg) One millet Jeonggok One 2 Yellow Seal Field Gorge One 3 Chimney Jeonggok One 4 Golden Swordsman Gorge One 5 White horse Gorge One 6 Yellow tea Jeonggok 0.4 7 Mezzo Jeonggok One 8 Green tea Jeonggok One 9 Green tea Gorge One 10 Golden tone Gorge One 11 Edible Blood Gorge 0.1 12 Yulmu Jeonggok One 13 red bean Jeonggok One

Example  1. Extraction and fractionation of sorghum

1-1. Sorghum Crude extract  detach

Wash 250 ~ 500g of sorghum seed provided by National Institute of Crop Science (Myongyang Milyang) and wash it with Blender 7012 (Dynamics Corporation, USA) for 10 minutes, and then use 1000ml of 80% ethanol 3 times (3 times each time). After mixing, the extract obtained by reflux extraction using a reflux condenser at a temperature of 80 ° C. (Corning 2560-400, USA) was centrifuged at a speed of 10,000 rpm for 20 minutes to collect only the supernatant and a rotary vacuum concentrator. evaporator, Heidolph LR 4000, Germany) was concentrated under reduced pressure and dried to obtain 23g (yield 5.75%) of 80% ethanol sorghum extract (hereinafter referred to as "SS-2") (Fig. 2; Table 2).

1-2. Polar solvents and Nonpolar Solvent  Fraction of soluble extract

Leave a small amount of the 80% ethanol extract obtained in Example 1-1 and dissolve the remainder in 500 ml of water (the undissolved portion was dissolved in n-hexane, placed in an aqueous layer and fractionated). N-hexane (500 ml × 3 times) After pouring, the n-hexane layer was concentrated after fractionation to obtain an n-hexane fraction (hereinafter referred to as “SS-2HE”). The aqueous layer was then extracted with methylene chloride (500 ml × 3 times) and the methylene chloride layer was concentrated to give a methylene chloride fraction (hereinafter referred to as “SS-2MC”). The aqueous layer was then extracted with ethyl acetate (500 ml × 3 times) and the extract was concentrated to give ethyl acetate fractions (hereinafter referred to as “SS-2EA”), respectively. The aqueous layer was then extracted with n-butanol (500 ml × 3 times) and concentrated to give an n-butanol fraction (hereinafter referred to as “SS-2BU”). Finally, the remaining aqueous layer was also concentrated to obtain an aqueous layer fraction (hereinafter referred to as “SS-2WA”) (FIG. 2; see Table 2).

** Dry weight of ethanol extracts and various organic solvent fractions (gram) No Grain Extraction amount 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 Valley 500 26.48 3.08 0.75 0.97 0.80 1.10 3 Waxu Jeongok 400 23.31 1.82 1.25 0.18 0.83 0.66 4 Golden Blind Shooter 400 23.24 2.28 0.34 0.77 1.52 4.49 5 Whitewater gorge 500 22.17 2.45 1.75 0.27 1.40 2.71 6 Yellow Chajo Jeongok 250 15.01 1.23 0.79 0.67 2.61 1.28 7 Mezzo Jeonggok 500 34.3 2.86 7.18 1.69 1.99 1.09 8 Cheongchajo Jeongok 500 40.34 3.04 3.68 0.60 9.32 5.93 9 Green tea 500 17.63 2.29 0.85 0.33 3.19 3.16 10 Golden tones 500 29.01 1.47 0.51 0.31 1.37 1.79 11 Edible blood grain 63 2.24 0.58 0.27 0.13 0.54 0.77 12 Yulmu Jeongok 500 37.96 0.89 1.65 0.72 0.68 2.32 13 Red beans 500 24.75 0.15 6.34 0.19 1.57 10.2

Reference Example  2. Reagents and Instruments

2-1. Materials and Devices Used in Carbohydrate Digestive Enzyme Testing

α-Amylase (from human salivary, A1031), α-glucosidase (from Brewer's yeast, G4634), a soluble starch used as a substrate, p-nitrophenyl-aD-glucopyranoside, and acarbose, a standard known as an inhibitor of both enzymes. A8980) was purchased from Sigma (St, Louis, MO, USA). Folin-Ciocalteu's phenol reagent was used for the quantification of polyphenol compounds in sorghum extracts from Fluka (Sigma-Aldrich, Schweiz, Switzerland), and tannic acid used as the polyphenol compound standard was Avondale Laboratories (Oxon, England). Purchased from

2-2. Preparation of Experimental Animals for Experiments on Inhibitory Effect on Elevated Blood Glucose Levels

Male 6-week-old ICR mice were purchased from Hyochang Bioscience. Animals used in the experiment were fasted for 16 hours before fasting blood glucose measurement. Drinking water was freely available during fasting. The animals used in the experiment were divided into three experimental groups as follows. Each experimental group consisted of 5 mice per group.

Group 1: control group (soluble starch: 2 g / kg)

Group 2: Ethanol Extracts of Soluble Starch (2 g / kg) and Golden Sorghum (Grain)

            (100 mg / kg) administration group

Group 3: soluble starch (2 g / kg) and acarbose (100 mg / kg).

Experimental Example  1. α- Amylase  Measurement of inhibitory activity

In order to measure the α-amylase inhibitory activity of the samples obtained in Example 1, the experiment was performed by applying the method disclosed in the literature as follows (Wilson et al., 1982).

1-1. Α- of sorghum ethanol extract Amylase  Inhibitory activity

Human saliva-derived α-amylase was dissolved in PBS (Gibco 21600-010, USA) at 40 unit / ml, and soluble starch was dissolved in PBS at a concentration of 1% as a substrate of α-amylase. As an inhibitor, ethanol extract and each organic solvent fraction of sorghum were dissolved in dimethylsulfoxide (Dimethylsulfoxide; DMSO, Kanto chemical 10378-73, Japan) at a concentration of 1 mg / ml. To measure the inhibitory activity against α-amylase, 290 μl PBS, 10 μl α-amylase solution (40 unit / ml) and 50 μl sorghum extract (1 mg / ml) were mixed and pre-incubated at 37 ° C. for 10 minutes. (preincubation) was performed. Thereafter, 350 μl of a 1% soluble starch solution, which is a substrate, was added, and reacted at a temperature of 37 ° C. for 30 minutes. In order to measure the amount of soluble starch remaining after the reaction, iodine (I ₂ ) was dissolved in a 5% potassium iodide solution in a reaction solution (700 μl) to 0.5%, and then 50 in 0.05 N HCl solution. 300 μl of iodine solution (0.1% KI + 0.01% I2 / 0.05N HCl) diluted in pears was added and developed, and the optical density was measured at 620 nm using a spectrophotometer (Shimadzu UV-1650PC, Japan). Amylase inhibitory activity was investigated (see Table 3). In the control group, soluble starch as a substrate, α-amylase as an enzyme, and ethanol extract derived from sorghum and DMSO, a solvent used to dissolve the extract instead of each organic solvent fraction, were added, and soluble starch as a substrate was added to the sample group. And an enzyme, α-amylase, sorghum-derived ethanol extract and each organic solvent fraction were added. In addition, blank 1 was added with 80% EtOH extract derived from sorghum and sorghum as a substrate, and DMSO, a solvent used to dissolve the extract, in place of each organic solvent fraction, and blank 2 was added with soluble starch as a substrate. A sorghum-derived ethanol extract and each organic solvent fraction were added. On the other hand, acarbose was dissolved in DMSO at the concentrations of 50, 100 and 200 μg / ml as a standard of the α-amylase inhibitor, and added instead of the sorghum-derived sample, and the α-amylase inhibitory activity was compared with the samples. As a result, α-amylase inhibitory activity was calculated by the absorbance of the sample (blank) and the absorbance of the blank (blank 2) for the difference between the absorbance of the control (control) and the absorbance of blank 1 (blank 1) The ratio of the difference is calculated first, then the value is subtracted from 1 and multiplied by 100 to express it 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 ℃ for 10 min 1% Soluble starch solution 350 μl 350 μl 350 μl 350 μl Incubate at 37 ℃ 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 experiment, the ethanol extract (1 mg / ml) of the brine water showed the highest inhibitory activity against α-amylase compared to the control group added only DMSO (7 mg%) (see FIG. 3).

Among them, the extracts of Chaksu (Jeonggok) and White Chaksu (Geok) were treated at concentrations of 0.125 mg / ml to 1 mg / ml, respectively, and the inhibitory activity of α-amylase was examined. In the case of ethanol extract of Chaksu (Junggok), the inhibitory activity of 3.9% at 0.125 mg / ml, 11.7% at 0.25 mg / ml, 29.6% at 0.5 mg / ml and 53.0% at 1 mg / ml When the ethanol extract of the grains) was also tested by concentration, the white waxy water (grain) had 4.3%, 8.6% and 19.7% of inhibitory activity at 0.125 mg / ml, 0.25 mg / ml, 0.5 mg / ml and 1 mg / ml, respectively. , 42.3% (see FIG. 4B).

1-2. Sorghum Organic Solvents Fraction  α- Amylase  Inhibitory activity

In the experiment using the ethanol extract, the water of the highest number of chaksu (jeonggok), which was the highest inhibitory rate of α-amylase, was fractionated by organic solvent, and the fraction was treated at a concentration of 1 mg / ml.

As a result of the experiment, the ethanol extract showed 61.7% inhibitory activity in the experiments of each fraction of the organic solvents of the water of Chamulsu (jeonggok) at a concentration of 1 mg / ml, and the fractions of n-hexane and methylene chloride were 97.7% and 99.5. % Of the two fractions strongly inhibited the activity of α-amylase, but the ethyl acetate (EtOAc) and n-butanol (BuOH) fractions showed 25.9% and 5.2%, respectively, than the n-hexane and methylene chloride fractions. It showed much lower inhibitory activity, and the aqueous layer had no inhibitory activity (see FIG. 5).

In addition, α-amylase inhibition experiments were also conducted at 1 mg / ml concentration of white waxy water (grain). The inhibitory activity of ethanol, n-hexane, methylene chloride, ethyl acetate, and butanol fractions was 46.6%, 93.5%, 95.9%, 63.2%, and 13.8%, respectively. There was no inhibitory activity against α-amylase (see FIG. 5B).

1-3. Methylene chloride Fraction  Investigation of stability against heat and acid

The methylene chloride fraction and the acarbose 200 μg / ml of the waxy water (jeonggok), which showed the highest inhibitory activity in the ethanol extract and the organic solvent fractionation experiments, were heat treated for 15 to 60 minutes and compared with the control group without heat treatment. The change was tested.

As a result of the experiment, it was found that the heat treatment did not affect the α-amylase inhibitory activity of the sorghum methylene chloride fraction regardless of the time of heat treatment of the methylene chloride fraction of waxy water (see Fig. 6).

To determine the changes in α-amylase inhibitory activity of methylene chloride fractions in brine water (pH 2) with acid treatment (pH 2), the methylene chloride fractions and 200 μg / ml of acarbose, respectively, were acidified at 37 ° C. for 1 or 2 hours. The reaction was carried out after the treatment. As a result of measuring the inhibitory activity of α-amylase at each acid treatment time, it was confirmed that the activity of α-amylase was not reduced by acid treatment. As a result, it was found that methylene chloride fraction and acarbose of the brine water did not lose their inhibitory activity against α-amylase even in strong acid and high temperature (see FIG. 7).

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

In order to determine the α-glucosidase inhibitory activity of the samples obtained in Example 1, the experiment was conducted by applying the method disclosed in the literature as follows (Kim et al., 2005; Park et al., 2008; Lee et al., 2008).

2-1. Α- of sorghum ethanol extract Glucosidase  Inhibitory activity

α-glucosidase was prepared at 10 unit / ml in 50 mM sodium phosphate buffer (pH 6.8) (manufactured by Dawson et al., Data for biochimecal research) and diluted to 0.25 unit / ml at the time of treatment. Then 20 μl each. The substrate was reacted by dissolving p-nitrophenyl-α-D-glucopyranoside in sodium phosphate buffer (50 mM, pH 6.8) at a concentration of 3 mM. In addition, ethanol extract of sorghum was dissolved in DMSO at a concentration of 10 mg / ml as an inhibitor of α-glucosidase. First, mix 20 μl α-glucosidase dilution with 65 μl sodium phosphate buffer (50 mM, pH 6.8) and 15 μl ethanol extract in a 96-well plate (Corning CLS3595, USA). After incubation, 100 μl of 3 mM p-nitrophenyl-α-D-glucopyranoside solution, which was a substrate, was added thereto, and reacted for 30 minutes at a temperature of 37 ° C. (see Table 4). After the reaction, the absorbance was measured by using a microplate leader (Molecular devices Thermo max, USA) at 405 nm wavelength to compare the inhibition rate. At this time, the control (control) is a substrate instead of p-nitrophenyl-α-D-glucopyranoside (enzyme α-glucosidase, and sorghum ethanol extract and each organic solvent fraction) DMSO was added, and sample was added with p-nitrophenyl-α-D-glucopyranoside, an enzyme, α-glucosidase, and sorghum ethanol extract. In addition, blank 1 was added with p-nitrophenyl-α-D-glucopyranoside, sorghum ethanol extract, and solvent DMSO instead of each organic solvent fraction, and blank 2 (blank). 2) p-nitrophenyl-α-D-glucopyranoside and sorghum ethanol extract were added. Meanwhile, as a standard of α-glucosidase inhibitor, acarbose was dissolved in DMSO at concentrations of 5 mg / ml and 10 mg / ml, and added to the sample instead of the grain-derived sample, and the α-glucosidase inhibitory activity was compared with the samples. . In this case, the α-glucosidase inhibitory ability of the sample is calculated first of the ratio of the difference in absorbance of the absorbance blank 2 (blank) of the sample to the difference between the absorbance of the control (blank) and the absorbance of the blank (blank 1). Then, the value was subtracted from 1 and multiplied by 100 to calculate the percentage as shown in Equation 2 below to show the inhibitory activity.

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 ℃ for 10 min 3 mM p-Nitrophenyl-a-
D-glucopyranoside 100 μl 100 μl 100 μl 100 μl Incubate at 37 ℃ for 30 min Read OD at 405 nm

As a result of the experiment, the 80% ethanol extract was treated with 10 mg / ml to compare the α-glucosidase inhibitory activity. As a result, the α-glucosidase inhibitory activity of the ethanol extract of golden wax sorghum (grain) was 90.8%. It showed about 1.75 times higher inhibitory activity than the 51.6% inhibition rate of the same amount of acarbose (10 mg / ml). Inhibition rate was determined by treating the ethanol extract of golden wax sorghum (grain) at a concentration of 5 mg / ml and showed an inhibition rate of 47.0%, similar to that of 44.1% of acarbose 5 mg / ml (see FIG. 8).

The ethanol extract of golden wax sorghum (grain), which was the most active in the ethanol extract experiment, was treated at concentrations of 1.25, 2.5, 5, and 10 mg / ml, respectively, to examine α-glucosidase inhibitory activity. Acarbose, a standard, was also treated at a concentration of 5 mg / ml and 10 mg / ml, respectively. As a result, the inhibition rate of 9.4%, 2.5 mg / ml was 30.4%, 5 mg / ml was 39.0%, and 10 mg / ml was 76.0% at 1.25 mg / ml concentration. % And 10 mg / ml showed an inhibition rate of 51.1%. Ethanol extract of golden wax sorghum (grain) had about 1.5 times higher inhibitory activity on α-glucosidase than 10 mg / ml of acarbose, and 5 mg / ml of acarbose at 5 mg / ml It showed almost similar inhibitory activity (see FIG. 9).

2-2. Sorghum Organic Solvent Fraction  α- Glucosidase  Inhibitory activity

Ethanol extract of golden wax sorghum (grain) and fractions of each organic solvent were treated with 120, 60, 30, and 15 ㎍ / ml to confirm the inhibitory activity of each fraction. At 120 ㎍ / mL, all fractions showed more than 60% of all α-glucosidase inhibitory activity, so that the fractions were sequentially diluted to 60, 30 15 ㎍ / mL to confirm which fraction was the most active. Compared.

As a result of the experiment, the activity was compared with 30 ㎍ / mL, and the inhibitory activity of the n-hexane fraction was 86.8% and the methylene chloride fraction was 82.1%, which was about 170 times higher than 5 mg of acarbose. It showed about 3 times stronger activity than 24.3% of inhibitory activity of / ml. In the experiments in which each organic solvent fraction was diluted to 15 mg / ml, the inhibitory activity of n-hexane and methylene chloride fractions was 31.9% and 30.8%, respectively. It showed similar inhibitory activity. As a result, the organic solvent fraction of the golden wax sorghum showed that it is about 340 times more potent α-glucosidase inhibitory activity than a single substance, acarbose, even though the mixture of various substances (see FIG. 10).

2-3. Α- of the organic solvent fraction of brine water Glucosidase  Inhibitory activity

Experiments were carried out by treating the organic solvent fraction of the waxy water (jeonggok) with 10, 5 and 2.5 mg / ml, respectively, which showed the highest inhibitory activity in the α-amylase inhibitory activity experiment.

As a result of the experiment, 80% ethanol extract of Chaksu (jeonggok) showed 18.3% of inhibitory activity at 10 mg / ml, whereas the same concentrations of n-hexane, methylene chloride, and ethyl acetate fractions were 89.3%, 81.4%, and 91.2, respectively. As a%, it showed about 1.5 times stronger activity than 58.9% of the inhibitory activity of 10 mg / ml of acarbose, the same substance. Butanol and aqueous fractions showed 14.7% and 24.5% of inhibitory activity, respectively, showing low inhibitory activity (see FIG. 11).

Experimental Example  3. Determination of Phenolic Compounds in Sorghum Extracts

In order to confirm the amount of the phenolic compound contained in the 80% ethanol extract of the samples obtained in Example 1, the experiment was conducted by applying the method disclosed in the literature as follows (Folin and Denis, 1912).

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

As a result of the experiment, it was found that the amount of phenolic compound of golden wax corn (grain) was 121.1 μg / mg, and the highest amount of phenolic compound was contained. 12A). The contents of phenolic compounds by fractions of organic solvents of golden waxy water (grain) and waxy water (corn) were high in ethyl acetate and butanol fractions. The content of phenolic compounds in the n-hexane and methylene chloride fractions of Golden Waxy Corn (Grain), which showed high activity in α-glucosidase inhibitory activity, was 56.2 μg / mg and 60.1 μg / mg, respectively. Ethyl acetate, butanol fraction of low inhibitory activity was 25-5 μg / mg, 244.1 μg / mg compared to 4 to 5 times lower content (see Figure 12B). The contents of phenolic compounds in the n-hexane and methyl chloride fractions of the waxy water with high α-amylase inhibitory activity were 7.6 μg / mg and 14.6 μg / mg, respectively, and 248.4 mg / mg of the ethyl acetate and butanol fractions, which had relatively low inhibitory activity. The phenolic compound content was about 10 to 30 times lower than 159.5 mg / mg. When quantitative results of phenol compounds were compared with α-amylase and α-glucosidase inhibitory activity, the fractions with high phenolic compound content and the fractions with high α-amylase and α-glucosidase inhibitory activity were inconsistent. These results suggest that the substance contained in the sorghum extract as an ingredient having inhibitory activity of α-amylase and α-glucosidase is not a kind of phenolic compound.

Experimental Example  4. Golden bristle  Effect of 80% Ethanol Extract on Postprandial Blood Glucose Inhibition

In order to confirm the effect of suppressing postprandial blood sugar elevation of the samples obtained in Example 1, experiments were conducted by applying the method disclosed in the literature (Heo et al., 2009; Bagri et al., 2009; Kim et al. , 2005).

80% ethanol extract (100 mg / kg body weight) and acarbose (100 mg / kg body weight) of golden wax sorghum (grain) were orally administered with soluble starch (2 g / kg body weight). Blood was collected from the tail vein of mice at 0, 30, 60, and 120 minutes after oral administration, and the change in postprandial blood glucose was measured. Blood glucose measurements were measured using a blood glucose meter (Accu Chek active Roche, Germany).

As a result of the experiment, the blood glucose level of the control group administered with only soluble starch rose to 190.6 mg / dl after 30 minutes, to 236.0 mg / dl after 60 minutes, and decreased to 143.8 mg / dl after 120 minutes. On the other hand, the blood glucose of the experimental group administered with ethanol extract of golden wax water (grain) was 139.3 mg / dl after 30 minutes, and rose to 183.0 mg / dl after 60 minutes, and 125.8 mg / day after 120 minutes Reduction to dl was significantly different from the control group orally administered only soluble starch. In the experimental group administered acarbose with soluble starch at a dose of 100 mg / kg body weight, the blood sugar level was 121.0 mg / dl after 30 minutes, and 130.3 mg / dl after 60 minutes, and 118.8 mg / dl after 120 minutes. It was confirmed that the hypoglycemic effect is strong (see FIG. 13).

Hereinafter, the preparation examples of the composition including the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  One. Sanje  Produce

SS-2 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

SS-2HE 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components and tableting according to the conventional tablet manufacturing method to prepare a tablet.

Formulation example  3. Preparation of capsules

SS-2EA 10 mg

3 mg of crystalline cellulose

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

SS-2MC 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

SS-2WA 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

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

SS-2 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

Magnesium chloride 24.8 mg

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

SS-2HE 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

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

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

Claims (8)

Extracts available in hexane, methylene chloride or ethyl acetate of Sorghum bicolr L. Moench var. Chal, Sorghum bicolr L. Moench var. Hwanggeumchal or Sorghum bicolor L. Moench var. Huinchal Pharmaceutical composition for the prevention and treatment of diabetes mellitus containing as an active ingredient. delete delete delete delete delete Extracts available in hexane, methylene chloride or ethyl acetate of Sorghum bicolr L. Moench var. Chal, Sorghum bicolr L. Moench var. Hwanggeumchal or Sorghum bicolor L. Moench var. Huinchal Health functional food for the prevention and improvement of diabetes containing as an active ingredient. delete

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