KR20150034024A - A method for preparing extracts of wheat sprouts, oat sprouts or rye sprouts having increased content of policosanol - Google Patents

Abstract

The present invention relates to a mass production method of a policosanol compound comprising a step of extracting the top of barley sprouts which have been grown for 5-30 days after sowing and have been collected; and to a pharmaceutical composition and a health food for prevention or treatment of metabolic disorder-induced diseases or cardiovascular disorders, which comprise an extract of barley sprouts which have been grown for 5-30 days after sowing and have been collected as an active ingredient. According to the present invention, a wheat sprout extract, an oat sprout extract and a rye sprout extract have been manufactured from edible plants so the extracts are mostly free from side effects such as toxicity or the like and contain policosanol as an active ingredient. A period when wheat sprouts, oat sprouts and rye sprouts contain the policosanol to the maximum is investigated, and extracts obtained from the wheat sprouts, oat sprouts and rye sprouts harvested in the period have excellent effects in increasing the activity of AMPK involved in metabolism and/or cholesterol biosynthesis and inhibiting the activity of HMG-CoA reductase. Therefore, the extracts can be practically used for prevention or treatment of metabolic disorder-induced diseases or cardiovascular disorders.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing policosanol-containing sprout wheat, sprout oat or roots rye extract,

The present invention relates to a method for mass production of a policosanol compound, which comprises extracting a top part of a bud sprout collected for 5 to 30 days after sowing, a method for mass production of a policosanol compound by extracting the extract of a barley sprout grown for 5 to 30 days after sowing, A pharmaceutical composition for the prevention or treatment of diseases or cardiovascular diseases caused by a metabolic disorder, and a health functional food.

The modern society is becoming aged due to the rapid development of medical technology, the awareness of health is spreading due to the economic development, and the tendency to pursue high quality of life is a trend not only in Korea but also in the global trend. It is expected to accelerate. Therefore, development of new biomedical materials, development of functional food materials and development of well - being materials are required to meet the rapidly changing social phenomena and increasingly diverse consumer expectations.

The sprout wheat, sprout wheat, sprout oats, and bud sprouts, which are the top parts of the barley crops including the young leaves after sowing, have long been known as nutritionally superior food sources with high content of vitamins and minerals. Containing secondary metabolites, industrial availability of health-functional foods and pharmaceuticals is increasing

On the other hand, AMP-activated protein kinase (AMPK) is an enzyme that acts on cellular energy homeostasis. The three subunits of a, b, and g are heterotrimers and are known to be variously distributed in tissues such as musculoskeletal, liver, and brain in the body, which are functional enzymes stored from yeast to human . Activation of AMPK has been shown to promote hepatic fatty acid oxidation and ketogenesis, to inhibit cholesterol synthesis, lipogenesis and triglyceride synthesis, to inhibit fat cell lipolysis and lipid production, to skeletal muscle fatty acid oxidation and to muscle glucose uptake And regulate insulin secretion by splenic < RTI ID = 0.0 > ss-cells. ≪ / RTI > When energy homeostasis involving AMPK is broken and energy imbalance occurs, dysfunctions such as muscle, liver and fat cells are caused and various metabolic diseases are caused thereby.

HMG-CoA reductase is an enzyme known to be involved in the pathway of cholesterol biosynthesis. By inhibiting its activity, HMG-CoA reductase can lower the total cholesterol concentration and the LDL level, which is known as bad cholesterol, It is known.

Accordingly, the present inventors have made intensive studies to find a substance effective for the prevention or treatment of diseases or cardiovascular diseases caused by metabolic disorders from natural products secured in the human body. As a result, it has been found that the extracts of the ground beet pulp harvested at 5-30 days after sowing Policosanol, which is a component that regulates the activity of enzymes involved in metabolic regulation and / or cholesterol biosynthesis, contains a large amount and exhibits excellent AMPK phosphorylation activity and HMG-CoA reductase inhibitory activity, thus completing the present invention.

An object of the present invention is to provide a method for mass production of a policosanol compound, which comprises extracting a ground part of a bud bud which is raised for 5 to 30 days after sowing.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by a metabolic disorder or a cardiovascular disease, comprising an extract of a barley sprout collected for 5 to 30 days after sowing as an active ingredient .

Another object of the present invention is to provide a health functional food for preventing or ameliorating a disease caused by a metabolic disorder or a cardiovascular disease, which comprises an extract of a barley sprout collected for 5 to 30 days after sowing as an active ingredient .

In order to achieve the above object, the present invention provides a method for mass production of a policosanol compound, which comprises extracting a top part of a bud of a barley sprout collected for 5 to 30 days after sowing.

In the present invention, the term "wheat sprout" means the young leaf of a wheat crop, and the sprout is also called wheat sprout. Preferably the barley may be wheat, oats and rye. As in the specific examples of the present invention, the wheat, oats, and rye seeds are evenly sprayed on the soil and soil using a box for cultivation and cultivation at room temperature, and the soil is covered with the soil. Then, the soil is sprayed without drying I raised it. After several days to germinate (germination), the barley can be harvested and used. Preferably 2 to 40 days after sowing, more preferably 5 to 30 days may be used. Extracts of sprout wheat, germinated oats, and bud rye collected at this time have the highest content of policosanol, which is the active ingredient, and can exhibit more excellent activity. In the present invention, sprout wheat, sprout oats, bud sprouts can be purchased commercially, sold or harvested in nature.

When the wheat germ is grown from the wheat seed to obtain the wheat germ, the wheat germ can be grown at a temperature of 15 to 28 ° C, more preferably at a temperature of 20 to 25 ° C, after sprouting. When growing under the above-mentioned conditions, the length of the upper part reaches 15 to 30 cm though it may be different depending on the kind of grains, kinds of cultivars, etc., so it can be collected and used.

In the present invention, the term "above ground part " means a part of the foliage above the surface of the earth, centering on an indicator in the plant. That is, it refers to the upper surface of the surface except for the roots of the ripening buds, and may include leaves, stems, flowers, etc., but preferably refers to the buds of the root of the ripening root. When about 5 to 30 days have elapsed after sowing, since much more various physiologically active ingredients, especially policosanol compounds, are contained in the ground part at this time, it is preferable to collect them at this time.

The term "extraction" in the present invention means a process of separating an active ingredient from a sample using an appropriate leaching solution. The leach solution is also referred to as an extraction solvent. As the extraction solvent, a solvent capable of dissolving the components to be extracted is used. The extraction solvent may be, but is not limited to, water, an organic solvent, or a mixed solvent thereof. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dichloromethane Non-polar solvents or mixed solvents thereof may be used. Further, n-hexane, fermented alcohol, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof may be preferably used. The specific extraction can be carried out at a temperature of 20 to 100 DEG C, preferably room temperature, for several hours to several days by adding a solvent of about 2 to 50 times, preferably about 5 to 20 times the volume of the sample, have. Non-limiting examples of extraction methods include hot water extraction, cold extraction, reflux cooling extraction and ultrasonic extraction. In a specific embodiment of the present invention, an extract was prepared using water as the solvent, particularly hot water at 60 to 90 ° C. The extraction was carried out for about 12 hours with stirring at room temperature.

The term " policosanol or polycosanol "in the present invention is a generic term for a natural extract of plant waxes. The policosanol is a mixture of several fatty alcohols derived from beeswax of plants such as sugar cane and yam, the most abundant being octacosanol and the next being triacontanol. It may also be used to lower the LDL level, which is a bad cholesterol, to increase HDL, a good or healthy cholesterol, and to help prevent atherosclerosis. However, the effect is not clearly known until now. Non-limiting examples of the policosanol compounds include docosanol (C ₂₂ H ₄₆ O), tetracosanol (C ₂₄ H ₅₀ O), hexacosanol (C ₂₆ H ₅₄ O), heptacosanol (C ₂₇ H ₅₆ O) Octacosanol (C ₂₈ H ₅₈ O) and triacontanol (C ₃₀ H ₆₂ O).

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating a disease or a cardiovascular disease caused by a metabolic disorder comprising, as an active ingredient, an extract of a barley sprout collected for 5 to 30 days after sowing .

In another aspect, the present invention provides a method for preventing or ameliorating a disease caused by a metabolic disorder comprising an extract of a barley sprout grown for 5 to 30 days after sowing as an active ingredient, or a health functional food for preventing or ameliorating a cardiovascular disease to provide.

In the present invention, the term "extract" means a preparation in which a sample is squeezed with a suitable leaching solution and the leaching solution is concentrated by evaporation. Without being limited thereto, the extract solution, the diluted solution, A dried product to be obtained, a controlled preparation thereof or a purified product thereof. The extract of the present invention can be prepared by extracting with an extraction solvent or extracting with an extraction solvent and fractionating the extract with a fraction solvent. The extraction solvent may be, but is not limited to, water, an organic solvent, or a mixed solvent thereof. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dichloromethane Non-polar solvents or mixed solvents thereof may be used. Further, n-hexane, fermented alcohol, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof may be preferably used. Specifically, a solvent having a volume of about 2 to 50 times, preferably about 5 to 20 times the dry weight may be added and extracted at a temperature of 20 to 100 ° C, preferably room temperature, for several hours to several days. Non-limiting examples of extraction methods include hot water extraction, cold extraction, reflux cooling extraction and ultrasonic extraction. In a specific embodiment of the present invention, an extract was prepared using an organic solvent such as hexane or fermented alcohol as the solvent. The extraction was carried out with stirring at room temperature for about 48 hours.

Preferably, the policosanol compound is selected from the group consisting of docosanol (C ₂₂ H ₄₆ O), tetracosanol (C ₂₄ H ₅₀ O), hexacosanol (C ₂₆ H ₅₄ O), heptacosanol (C ₂₇ H ₅₆ O) Octacosanol (C ₂₈ H ₅₈ O) and triacontanol (C ₃₀ H ₆₂ O).

Preferably, the composition of the present invention has an effect of enhancing the phosphorylation activity of AMPK or inhibiting the activity of HMG-CoA reductase, so that the metabolic activity involved in the enzymes or the prevention or treatment of diseases resulting from abnormal secretion of cholesterol Lt; / RTI >

The term "AMP-activated protein kinase " (AMPK) in the present invention is a transcription factor that plays a pivotal role in the regulation of intracellular energy balance and nutrient metabolism, thereby regulating phosphorylation of various enzymes, thereby providing a variety of enzymes such as glucose transport, fatty acid synthesis and cholesterol biosynthesis It is known to affect physiological function. The AMPK plays a crucial role in metabolism of carbohydrates and fats as a major regulator of metabolic conversion in response to intracellular energy changes. For example, acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutril-CoA reductase (3-hydroxy -3-methylglutryl-CoA reductase (HMGCR), inhibiting the enzyme that fatty acid enters into mitochondria, inhibiting the production of malonyl CoA (malonyl CoA), and thus inhibiting lipid synthesis And improves cholesterol.

In the present invention, the term "3-hydroxy-3-methylglutryl-CoA reductase" is a kind of enzyme involved in cholesterol biosynthesis. Twenty to twenty-five percent of the cholesterol biosynthesized in the body is produced in the liver. Cholesterol biosynthesis begins when acetyl-CoA and acetoacetyl-CoA are converted to HMG-CoA. HMG-CoA is reduced to mevalonate by HMG-CoA reductase (HMGCR). The reaction is irreversible as a rate limiting reaction. Generally, in mammalian cells, the enzyme is inhibited by cholesterol-induced cholesterol as well as by the internalization and degradation of LDL through low density lipoprotein (LDL) receptors. The competitive inhibitor of the reductase induces LDL receptor expression in the liver, thereby increasing the catabolism of plasma LDL and lowering the plasma concentration of cholesterol. These cholesterol causes various cardiovascular diseases. Thus, statin, a cholesterol lowering drug known in the art, is targeted to HMGCR and is known to act as an inhibitor of HMGCR in this reaction, thereby reducing cholesterol synthesis. It is also known that AMPK regulates the activity of HMGCR by regulating phosphorylation as described above.

In the present invention, the term "a disease caused by metabolic disorders" refers to diseases caused by abnormal metabolic processes, which are congenital due to abnormality of genetic enzymes, diseases caused by endocrine organs, It may be acquired. Diseases caused by congenital metabolic disorders are induced by the loss of a single gene. Diseases caused by metabolic disorders can cause relatively less important physical characteristics or skeletal abnormalities, but can even lead to serious diseases or death. Non-limiting examples of diseases caused by metabolic disorders include obesity, type 2 diabetes, aging, metabolic syndrome, insulin resistance, diabetes mellitus, Diabetes mellitus, diabetic myopathy, hypertension, lipotoxicity, hyperinsulinemia, Huntington's disease, Alzheimer's disease and Parkinson's disease.

In the present invention, the term "cardiovascular disease" is a generic term for a disease that occurs in blood vessels such as arteries, capillaries and veins, heart or both blood vessels and heart, and is also referred to as heart disease. The cardiovascular diseases refer to all diseases that affect cardiovascular, for example, heart diseases, vascular diseases of the brain and kidney, and peripheral artery diseases. The causes of cardiovascular disease vary, but atherosclerosis and / or hypertension are the most common. Physiological and morphological changes due to aging can also alter vascular function and increase the risk of cardiovascular disease. Other causes include elevated serum total cholesterol and / or low density lipoprotein cholesterol levels. Non-limiting examples of such cardiovascular diseases include hypertension, atherosclerosis, cardiac hypertrophy, ischemic heart disease, stroke, peripheral vascular disease, cardiac function Heart failure, rheumatic heart disease, congenital heart disease, and myocardial ischemia-reperfusion injury.

According to a specific embodiment of the present invention, the extracts of barley sprout, such as sprout wheat, sprout oats and bud rye extract, have the activity of activating AMPK phosphorylation and inhibiting HMG-CoA reductase, especially harvested at around 10 days after sowing Extracts of sprout wheat, buds oats, and shoot ryegrass showed remarkably excellent activity. Thus, by facilitating the AMPK phosphorylation and inhibiting the HMG-CoA reductase activity according to the present invention, the metabolism can be facilitated and the cholesterol can be improved, resulting in a reduction in AMPK expression, a decrease in AMPK phosphorylation or inhibition of AMPK activity and / Or a metabolic disorder caused by overexpression or hyperactivity of the < RTI ID = 0.0 > hyperglycaemia < / RTI >

The term "prevention" in the present invention means a disease caused by metabolic disturbance comprising the extract of sprouts of the pulverulent according to the present invention or a pharmaceutical composition for the prevention or treatment of cardiovascular diseases, Or any action that inhibits or delays the onset of cardiovascular disease.

The term " treatment "or" improvement "in the present invention refers to the administration of a pharmaceutical composition for preventing or treating a disease or a cardiovascular disease caused by metabolic disorders comprising, as an active ingredient, Of the disease or of the cardiovascular disease symptom is improved or benefited.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants.

In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels. Examples of carriers, excipients and diluents that can be included in the composition of the present invention 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 or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension agents, solutions, emulsions and syrups have. 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.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administering" of the present invention means introducing a predetermined substance into an individual by an appropriate method, and the administration route of the composition of the present invention can be administered through any ordinary route as long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal.

The term "individual" refers to all animals such as mice, mice, livestock, and the like, including humans who have developed or are capable of developing a disease or cardiovascular disease caused by metabolic disorders. Preferably, it may be a mammal, including a human. Since the composition of the present invention activates AMPK phosphorylation, an enzyme involved in metabolism, and inhibits the activity of HMG-CoA reductase, the composition can be administered to an individual to effectively prevent or treat diseases or cardiovascular diseases caused by metabolic disorders . The composition of the present invention may be administered in combination with a therapeutic agent for diseases or cardiovascular diseases caused by existing metabolic disorders.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. Generally, the active substance may be administered at a dose of from about 0.01 mg / kg / day to 1000 mg / kg / day. For oral administration, a range of 50 to 500 mg / kg may be suitable. The administration may be carried out once per day, or divided into several doses.

The term "health functional food" of the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, mixing or the like a specific ingredient contained in a food raw material, Refers to foods that are designed and processed so that the body control function such as bio-defense, regulation of bodily rhythm, prevention and recovery of disease can be sufficiently exhibited to the living body, and the composition for health food is used for prevention of diseases, Can perform related functions.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition comprising the sprout wheat, the sprout oats, and the sprout rye extract of the present invention as an active ingredient can be prepared by mixing other suitable auxiliary ingredients that can be contained in the food according to the selection of a person skilled in the art, and known additives. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component. It also includes foods used as feed for animals.

Examples of foods that can be used in the present invention include special nutritional foods such as crude oil, milk, baby food, meat products, fish meat products, tofu, mushrooms, noodles such as ramen noodles, (Such as soy sauce, doenjang, kochujang, mixed potatoes), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickles ), Beverages (such as fruit, vegetable beverages, beverages, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. The food may also be prepared in the form of tablets, granules, powders, capsules, solutions in liquid form, and the like according to known production methods. There are no particular restrictions on other ingredients other than those containing the sprout wheat, sprout oats, and roasted rye extract of the present invention as an effective ingredient, and various other flavors or natural carbohydrates may be included as an additional ingredient.

Since the sprout wheat, the sprout oats, and the sprout rye extract of the present invention are used as a food composition or a pharmaceutical composition, they can be used safely because they are less harmful than general synthetic compounds.

The sprout wheat, sprout oats, and sprout rye extract of the present invention are produced from edible plants, so that there is little concern about side effects such as toxicity and contains policosanol as an active ingredient. Extracts extracted from sprout wheat, sprout oats, and bud rye harvested at this time to search for a period containing the maximum amount of policosanol were used to enhance AMPK activity involved in metabolism and / or cholesterol biosynthesis, and to enhance the activity of HMG-CoA reductase And thus can be effectively used for the prevention or treatment of diseases caused by metabolic disorders or cardiovascular diseases.

Fig. 1 is a diagram showing a real image of sprout wheat, sprout oats, and bud rye harvested several to ten days after sowing.
Fig. 2 is a diagram showing the TLC development of six policosanol compounds derived from sprout wheat, bud oats, and bud rye extract.
Figure 3 is a GC-MS chromatogram showing a standard policosanol mixture and policosanol from bud mill, sprout oats, and bud rye extract.
FIG. 4 is a graph showing the AMPK phosphorylation activity of sprout wheat, sprout oats, and bud rye extracts at different harvesting times.
FIG. 5 shows HMG-CoA reductase inhibitory activity of sprout wheat, bud extract, and bud rye extract at different harvesting times.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  One: Sprout wheat , Sprout oats, sprouts from rye Policosanol  Extraction, Separation and Purification of Compounds

In order to uniformly germinate wheat, oats and rye seeds of various varieties, it is immersed in water at room temperature for 6 to 12 hours, taken out and allowed to stand at room temperature for about 12 hours to release water, The wheat, oats, and rye seeds were evenly sprinkled on the soil and soil and covered with soil. He then raised the ground of wheat, oats, and rye, giving water periodically so that the soil did not dry out. When seeds were sowed and grown for 3, 5, 10, 20, 30 days, grown sprout wheat, sprout oats, sprouts harvested above the ground of sprout wheat, sprout oats and bud rye when the length of the rye reached 5 to 30 cm Respectively. The harvested sprout wheat, sprout oats, and bud rye were washed with water, drained and shaded. Shrimp oats, and bud rye were crushed using a crusher. 200 g of powdery sprout wheat, sprout oats, and bud rye were divided into 1,000 ml of n-hexane Or a fermented alcohol was used to extract the active ingredient with stirring at room temperature for 48 hours. The extracted samples were filtered through a filter paper and concentrated under reduced pressure to obtain about 15 to 20 g of each extract. The content of active ingredients was determined by GC-MS (gas chromatography-mass-mass chromatography) analysis in the form of a crude extract without further purification such as sprout wheat, sprout oats, n-hexane extract of fermented rye and fermented spirulina extract .

Example  2: Sprout wheat , Sprout oats, sprout rye origin Policosanol  Identification of compounds

The molecular structure of the extract obtained in Example 1 was determined by comparing the retention time, the molecular weight and the daughter ion of the molecule using a GC-MS analyzer, and the molecular structure was determined. same. Compound 2 was tetracosanol, compound 3 was hexacosanol, compound 4 was heptacosanol, and compound 4 was in the order of low molecular weight. , Compound 5 was identified as octacosanol, and compound 6 was identified as triacontanol. The structures of the separated policosanol compounds are shown in Table 1.

[Compound 1]: docosanol

 1) Properties: White powder

 2) Molecular weight: 326

3) Molecular formula: C ₂₂ H ₄₆ O

[Compound 2]: tetracosanol

 1) Properties: White powder

 2) Molecular weight: 354

3) Molecular formula: C ₂₄ H ₅₀ O

[Compound 3]: Hexacosanol

 1) Properties: White powder

 2) Molecular weight: 382

3) Molecular formula: C ₂₆ H ₅₄ O

[Compound 4]: Heptacosanol

 1) Properties: White powder

 2) Molecular weight: 396

3) Molecular formula: C ₂₇ H ₅₆ O

[Compound 5]: Octacosanol

 1) Properties: White powder

 2) Molecular weight: 410

3) Molecular formula: C ₂₈ H ₅₈ O

[Compound 6]: Triacontanol

 1) Properties: White powder

 2) Molecular weight: 438

3) Molecular formula: C ₃₀ H ₆₂ O

GC-MS spectra of six kinds of policosanol compounds, such as hexacosanol and octacosanol, which have high content in the content of sprout wheat, sprout oats, and bud roe extract and excellent physiological activity, are shown in FIG.

Example  3: By kinds of cultivars, by growing season Sprout wheat , Sprout oats, sprout rye extract Policosanol  Content analysis

The following experiments were carried out to determine the maximum content of policosanol in each cultivar and growth period of sprout wheat, sprout oats, roots - derived extracts or policosanol compounds isolated therefrom. The seeds were prepared, seeded and germinated in the same manner as in Example 1, and then the top portion of the sprout wheat, the sprout oats, and the bud rye was harvested when the sprout wheat, the sprout oats, and the bud rye were grown for 3, 5, 10, . After shading, the mixture was stirred at room temperature with n-hexane and fermented juice, extracted three times, and the solvent was concentrated under reduced pressure. 0.5 ml of chloroform was added to each sample and transferred to a 1 ml volumetric flask and 250 μl of a derivative reagent MSTFA (N-methyl-N- (trimethylsiyl) trifluoroacetamide, Sigma) of GC-MS / MS (Agilent 7890) Was added and reacted at 60 ° C for 20 minutes. Then, chloroform was added until the volume became 1 ml. 2 μl of the sample was injected into a GC-MS / MS system (Waters). The column was DB-5 (length: 30 m, inner diameter: 250 μm, thickness: 0.25 μm, Agilent). As the mobile phase gas, helium gas was injected at a flow rate of 1.0 ml / min. The temperature was increased from the initial temperature of 150 ° C (1 minute) to 320 ° C at 20 ° C / min and 15 ° C / min. Waters TQD GC-Mass was used for detection. In order to quantify policosanol in sprout wheat, sprout oats, and bud rye extracts, different concentrations were prepared and the standard calibration curves were obtained from the standard samples by the same method as described above and injected into the GC-MS system. As shown in FIG. 2, it was confirmed that the sprout wheat, the sprout oats, and the sprout rye extract contained six kinds of policosanol compounds. The content of policosanol by the kinds of cultivars, And analyzed by GC-MS spectrometer.

The sprout wheat, sprout oats, sprout rye extract, or policosanol compounds isolated therefrom of the present invention are wheat varieties such as Kumkang wheat and landscape wheat, oats as oat varieties, ginseng oats, ginseng oats and chestnut oats, The seeds of rye were sowed and germinated at 20 to 25 캜 growth conditions. When the sprouts grew until 3, 5, 10, 20 and 30 days, the top part was harvested and the policosanol content of the extract prepared by the method described in Example 1 was measured As a result, the policosanol contents of 412.6 mg / 100g and 460.4 mg / 100g were shown in the case of sprout wheat and the case of wheat and landscape wheat were respectively harvested within 5 to 10 days, respectively. In the case of sprout oat, When all oats were harvested between 5 and 10 days, they showed policosanol contents of 430.2 mg / 100 g, 386.2 mg / 100 g, 425.2 mg / 100 g and 406.5 mg / 100 g, respectively. Also, in the case of bud rye, the content of policosanol was 389.2 mg / 100g and 405.2 mg / 100g, respectively, when harvested in the period between 5 and 10 days

The contents of policosanol compounds in the cultivars of sprout wheat, sprouted oats, bud rye, and growth period are summarized in Table 3 below.

As shown in Table 3, it was confirmed that policosanol was contained in wheat, oats, and rye in the highest content of sprout wheat, sprouted oats, and bud rye grown for 5 to 10 days regardless of the cultivars.

Example  4: Sprout wheat , Sprout oats, sprouts from rye Policosanol  Optimal extraction of compounds

The seeds of the sprout wheat, the sprout oats, and the sprout rye used in Examples 1 and 3 were cultivated after 5 to 10 days of sowing, which was the highest policosanol content, 1 g of the powder prepared by the method described in the above was added to 20 ml of hexane, fermented juice and water, respectively. The mixture was stirred and extracted at 50 DEG C for 24 hours, and the supernatant was separated with a centrifuge (10,000 rpm, 4 DEG C) lt; / RTI > filter (Advantec MFS, Inc, CA, USA). After the filtrate was concentrated under reduced pressure, the content of policosanol such as hexacosanol and octacosanol was determined by GC-MS / MS according to the method described in Example 3. The content of the active ingredient according to the extraction solvent was compared and analyzed, Respectively. The results are shown in Table 4 below.

As shown in Table 4 above, policosanol compounds such as hexacosanol and octacosanol were detected in high content in organic solvents such as n-hexane and fermented alcohol extracts, but not in water extracts. Particularly, when n-hexane, which is a non-polar solvent, was used as an extraction solvent in the organic solvent, the policosanol content of the extract was the highest.

Example  5: By kind of cultivars, by growing season Sprout wheat , Sprout oats, sprout rye extract AMPK  Activity analysis

The effect of policosanol-containing seed mill, sprout oat, and bud rye extract on the AMPK (AMP-activated protein kinase) activity, which is a transcription factor that plays a pivotal role in regulation of intracellular energy balance and nutrient metabolism, was confirmed. Specifically, wheat, oats, and rye were cultivated with the highest concentration of policosanol, and the extracts of sprout wheat, sprout oats, and bud rye powder prepared by the growth period were extracted with an organic solvent, and treated with cells to confirm the activity of promoting phosphorylated AMPK expression Respectively.

5.1. Cell culture

HepG2 cells, a human hepatoma cell line, were purchased from Korea Cell Line Bank (KCLB). Cells were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 ° C and 5% Respectively.

5.2. In immunoblotting  Protein expression analysis

To the HepG2 cells treated with sprout wheat (ground wheat), sprout oats (light oats), sprout rye (duru rye), hexane and fermented alcohol extract prepared with the policosanol compound for each sowing period after seeding, dissolved buffer (10 mM Tris -HCl (pH 7.4), 0.1 M EDTA, 10 mM NaCl and 0.5% Triton X-100, protease inhibitor) and centrifuged at 13,000 rpm for 20 minutes at 4,000 rpm to obtain supernatant . The protein concentration was quantitated from the supernatant using the Bradford method and 40 μg of protein was taken. To the 40 μg of extracted protein was added 1 × SDS sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) Respectively. The prepared samples were electrophoresed on 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), transferred to PVDF membrane, and incubated in TBST buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) The skim milk powder was melted and blocked. The membranes were reacted with primary and secondary antibodies, respectively, for 1 hour at room temperature. After washing with TBST buffer, protein bands were visualized by chemiluminescence using ECL reagent. Protein bands were quantitated and quantified using software (Gel-Pro Analyzer 4.0) and standardized using α-tubulin as a reference gene. The percentage of phosphorylated AMPK and phosphorylated AMPK: AMPK was calculated by treating sprout wheat, bud oats, sprout rye-derived hexane and fermented alcohol extract, and plotted as a percentage of the control group, as shown in FIG.

As shown in FIG. 4, when the hepatocytes were cultured with the sprout wheat, sprout oats, and bud rye extract of the present invention, the changes in the expression levels of AMPK and phosphorylated AMPK protein were measured. The ratio of active p-AMPK to AMPK expression when treated with 200 μg / ml of harvested sprout wheat, sprout oats, and hexane extract of sprout rye at 3, 5, 10, 20 and 30 days after sowing, And 1.8 times and 2.3 times, respectively, in the group treated with the extracts of the sprouts harvested on days 3, 5, and 10, respectively. In the group treated with sprouts harvested after 20 days after seeding, Respectively. In other words, the shoot barley extract promoted AMPK phosphorylation, and this effect was found to be maximized in the sprout wheat, sprout oats, and bud rye extracts taken around 10 days after sowing. Since AMPK is a key transcriptional protein involved in anti-obesity, anti-diabetic, cholesterol improvement, anti-arteriosclerosis, endurance enhancement, functional cosmetic and antibacterial activity, these results indicate that the AMPK activates the metabolic disorder ≪ RTI ID = 0.0 > and / or < / RTI > cardiovascular disease.

Example  6: Varieties and Growth Period Sprout wheat , Sprout oats, sprout rye extract HMG -CoA reductase inhibitory activity assay

In the present invention, the polynosanol-containing buds wheat against HMG-CoA reductase (HMGCR), which is known to be involved in cholesterol biosynthesis, is measured using an HMG-CoA (3-hydroxymethylglutaryl Coenzyme A) expression measurement kit (Sigma, CS1090) Oat, and bud rye extracts. Wheat, oats and rye cultivars were cultivated with the highest policosanol content, and the extracts of sprout wheat, sprout oats, and bud rye powder were cultured and treated with the organic solvent to confirm HMGCR inhibitory activity.

Specifically, after diluting the test buffer concentrated five times with the third distilled water to 1-fold, a buffer solution containing NADPH was prepared, and then a HMG-CoA substrate and an HMG-CoA reductase were added to a 96-well plate in an amount of 60 μl And 5 μl each. After adding 20 μl of the prepared NADPH, 910 μl of each buffer solution was injected, and 10 μl of each of the extracted sprout wheat, sprout oat, and bud rye extract concentrate was added to each growth period, and the absorbance at 340 nm was measured. Their inhibition rates were calculated. Each sample was used for landscape wheat, light oats, and perilla rye, which were identified as the best varieties of policosanol expression by immunoblotting in Example 5.

12.44 is the product of the extinction coefficient for NADPH at 340 nm and the molar concentration (mM) of NADPH consumed in the reaction, i.e. epsilon [NADPH], the extinction coefficient is 6.22 / Is 2 mM. TV shows the total volume of reaction (1 ml per cuvette, 0.2 ml per plate), V is the volume of the enzyme used in the assay (ml), 0.6 is the mass of the enzyme per milliliter (mg protein) The concentration of the enzyme, LP, represents the light path (cm; 1 for the cuvette, 0.55 for the plate).

The results are shown in Fig. As shown in FIG. 5, the bud extract of all the crops showed HMG-CoA reductase inhibitory activity, and the extracts of sprout wheat, sprout oats and bud rye harvested about 10 days after sowing showed the most excellent inhibitory activity Respectively. These results were consistent with the highest content of policosanol, the active ingredient, in extracts of sprout wheat, germinated oats, and bud rye harvested around 10 days after sowing.

Claims (12)

A method for mass production of a policosanol compound, comprising the step of: extracting a ground part of a barley sprout grown for 5 to 30 days after sowing.
The method according to claim 1,
Wherein the pulpy stream is selected from the group consisting of wheat, oats and rye.
The method according to claim 1,
Wherein the extraction is carried out using n-hexane or a fermented spirit organic solvent.
The method according to claim 1,
The policosanol compound may be selected from the group consisting of docosanol (C ₂₂ H ₄₆ O), tetracosanol (C ₂₄ H ₅₀ O), hexacosanol (C ₂₆ H ₅₄ O), heptacosanol (C ₂₇ H ₅₆ O), octacosanol ₂₈ H ₅₈ O) and triacontanol (C ₃₀ H ₆₂ O).
The method according to claim 1,
Wherein the barley sprouts are grown at 15 to 25 DEG C and have a length of 15 to 30 cm above the ground.
A pharmaceutical composition for preventing or treating diseases or cardiovascular diseases caused by a metabolic disorder comprising, as an active ingredient, an extract of a barley sprout collected for 5 to 30 days after sowing.
8. The method of claim 7,
The extract is Toko sanol (C ₂₂ H ₄₆ O), tetra Kosa play (C ₂₄ H ₅₀ O), hexamethylene Kosa play (C ₂₆ H ₅₄ O), heptyl taco sanol (C ₂₇ H ₅₆ O), oktakosanol (C ₂₈ H ₅₈ O) and triacontanol (C ₃₀ H ₆₂ O).
The method according to claim 6,
Wherein said composition enhances the phosphorylation activity of AMPK or inhibits HMG-CoA reductase activity.
The method according to claim 6,
The diseases caused by the metabolic disorders include obesity, type 2 diabetes, aging, metabolic syndrome, insulin resistance, diabetic myopathy, ), Hypertension, lipotoxicity, hyperinsulinemia, and Huntington's disease, Alzheimer's disease, and Parkinson's disease. .
The method according to claim 6,
The cardiovascular diseases include hypertension, atherosclerosis, cardiac hypertrophy, ischemic heart disease, stroke, peripheral vascular disease, heart failure, Rheumatoid heart disease, congenital heart disease, and myocardial ischemia-reperfusion injury. ≪ RTI ID = 0.0 >
A health functional food for preventing or ameliorating a disease or cardiovascular disease caused by metabolic disorder comprising an extract of a barley sprout collected for 5 to 30 days after sowing as an active ingredient.
13. The method of claim 12,
The extract is Toko sanol (C ₂₂ H ₄₆ O), tetra Kosa play (C ₂₄ H ₅₀ O), hexamethylene Kosa play (C ₂₆ H ₅₄ O), heptyl taco sanol (C ₂₇ H ₅₆ O), oktakosanol (C ₂₈ H ₅₈ O) and triacontanol (C ₃₀ H ₆₂ O).

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