KR20030025200A - Substances extracted from corn which can inhibit the activities of amylase, pharmaceutical compositions and food additives containing the same extracts for treatment or prevention of obesity and diabetes mellitus, and processes for their preparation - Google Patents

Abstract

PURPOSE: A process of preparing a corn extract by extracting a corn kernel or corn husk in war, a polar organic solvent or a mixture thereof and then removing the extraction solvent from the extract is provided. Therefore, a composition containing the corn extract is effectively used as a pharmaceutical composition for the prophylaxis and treatment of obesity or diabetics and a food additive for regulating blood sugar levels. CONSTITUTION: A corn kernel or corn husk is extracted at 0 to 100deg.C in a polar organic solvent such as C1-4 lower alcohol, the extract is dissolved or suspended in water, an aqueous solution or aqueous suspension of the extract is brought into contact with a polar organic solvent or nonpolar organic solvent to disperse the extract component. Thereafter, the polar organic solvent solution or aqueous solution thereof is recovered, the polar organic solvent or water is removed therefrom to produce a partially purified corn extract.

Description

Corn extract capable of inhibiting the activity of amylase, pharmaceutical compositions and food additives for the treatment or prevention of obesity or diabetes mellitus containing the same, and methods for their preparation.Substances extracted from corn which can inhibit the activities of amylase, pharmaceutical compositions and food additives containing the same extracts for treatment or prevention of obesity and diabetes mellitus, and processes for their preparation}

The present invention relates to a corn extract and a method for producing the same that can inhibit the activity of amylase. The present invention also relates to a pharmaceutical composition for treating or preventing obesity or diabetes mellitus containing the corn extract and a blood glucose control food additive containing the corn extract.

According to the evidence report: clinical guideline on the identification, evaluation, and treatment of overweight and obesity in adults (1999, NIH), approximately 97 million Americans are obese. There are about 15.7 million people with type 2 diabetes who are in a condition and one of the diseases associated with obesity.

In addition, according to the World Health Organization (WHO), the number of diabetics is estimated to be about 300 million by 2025. It is known to reach about 5.9% of the population (Current Pharmaceutical Design, 2001, 7: 417-450). Rapidly increasing obesity and diabetes, especially diabetes with obesity, are one of the chronic diseases that cause enormous economic and social damage.

The exact mechanism of the development of obesity is not known to date, but obesity is thought to be caused by the imbalance between energy intake and consumption. Thus, the development of therapeutics for the treatment of obesity to date is mainly divided into three (Nutrition, 2000, 16: 953-960). The first is drugs that reduce appetite by reducing appetite, primarily drugs of the sympathetic nervous system. Drugs approved by the FDA for the treatment of this class of obesity include phentermine, mazindol and sibutramine, and fenproprex has not yet been approved. Second, it affects the digestion and intestinal absorption of the ingested food, and related drugs include orlist (Xenical (TM), Roche pharmaceuticals), which inhibit the digestion of fat. There are also hormones, such as testosterone, that have been approved for use in the treatment of obesity after being approved by the FDA for use other than for the treatment of obesity and diabetes. The third and the like ephedrine (ephedrine), caffeine (caffeine) as a drug that increases the consumption of the energy accumulated in the body, β ₃ that are still under development - has adrenergic receptor (β ₃ -adrenoreceptor) antagonist. As a result, drug therapies are now actively seeking to treat or prevent diabetes associated with obesity or obesity by agents that reduce blood sugar, inhibit sugar absorption, enhance insulin action, or cause loss of appetite. Is being studied.

In addition, drugs for treating type 2 diabetes include sulfonylureas, rosiglitazone, and metformin, which increase the action of insulin, a glycemic control hormone, and alpha, which inhibits the absorption of sugar. -Glucosidase inhibitors, amylase inhibitors, and dietary fiber.

Accordingly, it is an object of the present invention to provide a corn extract having an effect of inhibiting the activity of amylase, an intestinal digestive enzyme, which greatly reduces the breakdown of carbohydrates into low saccharide substances that can be absorbed into the body, that is, inhibiting amylase activity. .

It is also an object of the present invention to provide a method for producing a corn extract capable of inhibiting amylase activity from corn.

It is also an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of diabetes or obesity by administering a pharmaceutically effective amount of the above extract to inhibit the production of low sugars absorbed into the body.

It is also an object of the present invention to provide a food additive comprising corn extract, which is added to foods such as bread, noodles, rice, etc., and exhibits a prophylactic effect of diabetes or obesity.

FIG. 1 is a graph showing changes in blood glucose levels after administration of starch and corn extracts to Spraue-Dawley rats.

Figure 2 is a graph showing the blood glucose level change measured after the administration of starch and corn extract to the child seed (ICR) mice.

FIG. 3 is a graph showing the change in blood glucose levels after administration of Active Compound 3 obtained by further purification of starch and corn extracts to IC eggs (ICR) mice.

Figure 4 is a graph showing the change in blood glucose levels after administration of corn starch and corn extract to Spraue-Dawley rats.

FIG. 5 is a graph showing changes in blood glucose levels after administration of flour and corn extracts to Spraue-Dawley rats.

Figure 6 is a graph showing the inhibitory effect on porcine pancreatic amylase of corn extract obtained by purifying the crude corn bran crude extract by an ion exchange elution method using an anion exchange resin (elution solution: acid aqueous solution of pH 2 ~ 6.5).

Figure 7 is a graph showing the inhibitory effect on porcine pancreatic amylase of corn extract obtained by purifying the crude corn bran crude extract by an ion exchange elution method (elution solution: aqueous NaCl solution of 0.01 ~ 0.5 M concentration) by an anion exchange resin.

The present invention provides a corn extract capable of inhibiting the activity of amylase.

Corn extract according to the present invention is a step of extracting the corn with an extraction solvent consisting of water or a polar organic solvent or a mixed solvent thereof, removing the solids from the extract obtained in the extraction step, and removing the extract solvent from the extract It can be obtained by the extraction process comprising the step of obtaining a corn extract.

Corn used in the present invention is preferably corn kernels (corn kernel) or corn husk (corn husk), the extraction solvent may be a lower alcohol having 1 to 4 carbon atoms. Extraction may be carried out at a temperature of 0 ~ 100 ℃, solids removal may be carried out by centrifugation or filtration.

In addition, the present invention can be purified to the above extract in order to include an active ingredient that can inhibit the activity of amylase in a higher ratio.

Purifying process of the extract is a step of dissolving or suspending the extract in water, by contacting the extract aqueous solution or aqueous suspension with a non-polar organic solvent that is not mixed with water to have no amylase inhibitory activity in the aqueous solution or aqueous suspension of the extract Dispersing into the non-polar organic solvent, removing the solution of the non-polar organic solvent, contacting the extract aqueous solution or aqueous suspension with a polar organic solvent which is not mixed with water to inhibit amylase in the aqueous extract or aqueous suspension Distributing an active ingredient having the polar organic solvent, and recovering the solution of the polar organic solvent, and removing the polar organic solvent therefrom to obtain a partially purified corn extract.

In addition, the purification process of the extract may be performed by chromatography. That is, the component which does not have amylase inhibitory activity mixed in the said extract can be removed by chromatography.

The present invention also provides a pharmaceutical composition capable of treating or preventing a disease associated with amylase activity, comprising a corn extract extracted by the above method and a pharmaceutically acceptable carrier. Here, the disease associated with amylase activity is in particular obesity or diabetes, and it is preferable that the pharmaceutical composition of the present invention has a form suitable for medicinal oral administration.

The present invention also provides a food additive comprising a corn extract extracted by the above method. The food additive of the present invention is for controlling the blood glucose of the food intake when the food intake. Therefore, the food additive of the present invention should be added to a food containing polysaccharide or more of disaccharides.

Hereinafter, the present invention will be described in detail.

The present invention provides corn extract and extraction method which can inhibit the activity of amylase extracted from corn.

Corn to be extracted in the present invention is known as poaceae (corn), corn (corn), maize (meize), Indian (mealie), Indian corn, etc. (Zea mays) [Cyperralus Order Cyperales, family Zea, genus mays. In other words, the corn that can be used in the present invention may be any type as long as it can be classified as geese. In addition, the amylase inhibitory corn extract of the present invention was extracted from corn kernels or corn seeds or corn husks, but also from corn stalks and corn cobs. It can be extracted.

The extract of the present invention includes an active ingredient having an activity capable of inhibiting the activity of amylase, and is extracted from corn (Zea mays). At this time, it is preferable to use water or a polar organic solvent or a mixture thereof as the extraction solvent. As a preferred extraction solvent, water or alcohol, preferably, lower alcohols having 1 to 4 carbon atoms, more preferably methanol or ethanol may be used, in addition, a polar organic solvent having an appropriate polarity may be used. Such polar organic solvents may include diethyl ether, dichloromethane (also called methylene chloride), acetone, ethyl acetate, acetonitrile and the like. These extraction solvents may be used alone or in combination.

Extraction may be performed according to conventional methods. Extraction is typically carried out by stirring corn and extractant in a closed container. The extraction can be carried out at a temperature of 0 to 100 ° C., preferably 30 to 55 ° C., for a time such that the extract can be sufficiently extracted, typically for 1 hour to 1 month. At this time, if the extraction temperature is increased, the extraction time can be shortened, but if the temperature is too high, the active ingredient may be decomposed or changed by a chemical reaction, it is good to maintain a suitable temperature. The corn used for extraction, for example corn kernels or bran, may be used as a whole, but in order to proceed with the extraction efficiently, it is preferable to be used in a pulverized state.

After extraction, the solids are removed from the extract containing the extraction solvent. As a means for removing solids, generally known means can be used, for example, centrifugation, filtration, decantation, and the like. If the extraction solvent is removed from the extract from which the solids are removed, the corn extract of the present invention can be obtained. At this time, removal of the extraction solvent may also be performed by a conventional method. Extraction solvent removal method may include exposure to vacuum evaporation, freeze drying, dry nitrogen or dry air by atmospheric evaporation, vacuum centrifugal evaporator and the like. When removing the extraction solvent, heat may or may not be added depending on the conditions. However, applying excessive heat is undesirable.

In order to remove components that do not have an inhibitory activity on the activity of amylase from the corn extract obtained by the above extraction, further purification can be carried out.

One such purification method is chromatography. Such chromatography can be carried out by selecting the appropriate type of chromatography, elution solvent, and elution conditions. For example, silica gel column chromatography (Merck, 9385, 70-230 mesh) can be used to remove inert components from the corn extract obtained above using a suitable solvent such as a mixed solvent of methylene chloride, methanol and distilled water. Can be. At this time, the eluting solvent may be set such that the polarity gradually increases with the dissolution time. In addition, in order to further purify this partially purified corn extract or to obtain a single active ingredient, for example, the active fraction of the corn extract obtained above may be used as, e.g., methanol as the elution solvent, using Sephadex LH20 (pharmacia, 17-0090-01), followed by reverse phase silica gel chromatography using 30% to 10% acetonitrile as the eluting solvent with the active fraction showing activity. To obtain a single active ingredient, the pure active compound can be obtained by finally recrystallization using a solvent such as a mixed solvent of acetonitrile and methanol, for example.

In addition, in order to increase the safety in the further purification process of the corn extract of the present invention can be purified using only ethanol (alcohol) and water as a solvent without using an organic solvent harmful to the human body. One such example is the use of anion exchange resins. Some of the substances that inhibit amylase activity in the corn extract of the present invention have a carboxyl group or similar functional groups, so an anion exchange resin can be used to separate them. More specifically, the corn extract of the present invention may be adjusted to have a weak alkaline hydrogen ion concentration (pH), for example, pH 8-9. At this time, ethanol in the same volume and volume of the extract may be used to dissolve the corn extract. The corn extract solution may be passed through an anion exchange resin equilibrated with an aqueous solution having a neutral or weakly alkaline hydrogen ion concentration, for example, pH 7-8, to bind the active ingredients having the corresponding functional groups to the anion exchange resin. have. The anion exchange resin may then be washed with distilled or tap water to remove components that are not bound to the resin. Thereafter, the active ingredients bound to the anion exchange resin can be eluted with an aqueous solution of acid or salt at a suitable concentration. At this time, the pH of the acid aqueous solution is not particularly limited as long as it is in the acidic range, 4 ~ 6 is the most preferred considering only the activity. The aqueous salt solution depends on the type of salt used, but may have a range of 0.001 to 0.5 M. Further purification using anion exchange resin can generally be carried out by column chromatography, but can also be carried out in other ways.

In the present invention, when only water and ethanol (alcohol) are used as a solvent in the extraction and additional purification of corn, the safety of the obtained corn extract becomes very high, so this method uses the corn extract of the present invention as an additive of food. It can be particularly suitable when used, and it is also economical since no increase in costs such as strict process control to ensure safety occurs.

Another purification method is to use the difference in partition coefficient according to polarity and solubility characteristics. A component (active ingredient) having an inhibitory activity on the activity of amylase contained in the corn extract of the present invention is more soluble in a polar solvent having a moderate polarity therebetween than water and a nonpolar solvent. In addition, the corn extract of the present invention contains components (inactive ingredients) that do not have an inhibitory activity on the activity of amylase, among these components are well dissolved in water or nonpolar solvents. Therefore, based on the property that the solubility in polar solvents and nonpolar solvents varies depending on the polarity of the chemical, it is well soluble in water and inactive ingredients that are well soluble in nonpolar solvents from the corn extract of the present invention using a fractionator. Inactive ingredients can be removed.

In order to remove inactive ingredients that are well soluble in nonpolar solvents, the corn extract obtained by the extraction process is first completely dissolved or suspended in water. At this time, in order to completely dissolve the extract in water, a large amount of water is required or heated, but it is accompanied by inconvenience of handling, so the next step may be performed even if the extract is not completely dissolved. Next, the non-polar organic solvent which is not mixed with water is brought into contact with the extract aqueous solution or the aqueous suspension. This process can be carried out in a fractionator. Then, an inactive ingredient having nonpolar characteristics contained in the extract aqueous solution or the water suspension is dissolved in the nonpolar organic solvent. That is, more is distributed into the nonpolar solvent. Thereafter, the solution of the non-polar organic solvent is removed and water is removed from the aqueous solution to obtain a partially purified corn extract of the present invention. By this process, it is possible to remove the inactive ingredients well dissolved in the non-polar organic solvent from the corn extract obtained by the extraction process.

As the non-polar organic solvent that can be used in the present invention, it is generally preferable to use an organic solvent having a polarity of less than petroleum ether in the polarity table provided for the fraction and not mixed with water. Particularly preferred nonpolar solvent is hexane. In general, the polarity order of the organic solvents shown in the polarity table provided for fractionation is as follows.

Cyclohexane <n-pentane <Hexane <Isooctane <Petrolether <Xylol <Isopropyl ether <Toluene <Diethyl ether <Chloroform <Dichloromethane <Tetrahydrofuran <Acetone <Dioxane <Ethyl acetate < n-propanol <n-butanol <ethanol <methanol <water (listed in nonpolar to polar order). For solvents not described herein, polarity can be found in a more detailed table.

More detailed and organized solvent polarity is suggested by Dimroth and Reichardt (see Christian Reichant, "Solvents and solvent effects in organic chemistry", 2nd Ed., 1988 published by VCH Verlagsgesellschaft mbH). Solvent polarity parameters, in particular standardized solvent polarity parameters, can also be obtained. The solvent polarity parameter (E _T ) is a long wavelength-wavelength absorption band (longest-wavelength) of pyridinium-N-phenoxide betain dyes of formulas (1) and (2) solvatochromic absorption band) is calculated by calculating the energy of the equation (1).

Formula 1

Formula 2

Equation 1

In the above equation, h is Planck's constant, c is the speed of light, Is the wavenumber of the photon producing electronic excitation, and N _A is the avogadro number.

The mean E _T value of 55.4 for methanol means that the transition energy required to make one mole of the dye dissolved in methanol from the electronic ground state to the first excited state is 55.4. It is called kcal. The measurement is typically carried out at 25 ° C. and 1 bar conditions.

On the other hand, the pyridinium-N-phenoxide betaine dye of formula (1) is slightly soluble in water, insoluble in polar solvents, and insoluble in nonpolar solvents. Therefore, in order to solve this solubility problem, a hydrophobic betaine dye of formula (2) in which five tert-butyl groups are substituted is further used as a secondary reference probe.

Further, it is a polar solvent in the Figure, it is convenient to use a parameter _(T E) a polar solvent Standardization Standardization also parameters (normalized _T E ^N) according to the equation (2). This standardization used water and tetramethylsilane (TMS) as an extreme reference solvent.

Equation 2

The solvent polarity parameter for various solvents can be obtained by the above calculation, and the solvent polarity parameter for the main solvents is shown in the following table.

Table 1

menstruum E _T (kcalmol- ¹ ) E _T ^N (standardized value) Tetramethylsilane 30.7 0.000 n-pentane 31.0 0.009 n-hexane 31.0 0.009 n-octane 31.1 0.012 Cyclohexane 30.9 0.006 Dichloromethane 40.7 0.309 1,2-dichloroethane 41.3 0.327 benzene 34.3 0.111 toluene 33.9 0.099 Methanol 55.4 0.762 ethanol 51.9 0.654 1-propanol 50.7 0.617 1-butanol 50.2 0.602 1-pentanol 49.1 0.568 1-hexanol 48.8 0.559 1,2-ethanediol 56.3 0.790 phenol 61.4 (40 ℃) 0.948 Diethyl ether 34.5 0.117 Tetrahydrofuran 37.4 0.207 1,4-dioxane 36.0 0.164 Acetone 42.2 0.355 2-hexanone 40.1 0.290 Acetic acid 51.7 0.648 Ethyl acetate 38.1 0.228 Pyrrolidin-2-one 48.3 0.543 Pyridine 40.5 0.302 water 63.1 1.000

Non-polar solvents that can be used in the present invention include experimental experiments on the transition energy of long-wavelength solvatochromic absorption bands of pyridinium-N-phenoxide betain dyes. The value of said solvent polarity parameter obtained by measuring by 0.15 or less, Preferably it can use 0.1 or less. Examples of preferred nonpolar solvents include n-hexane, cyclohexane, n-pentane, n-octane, petroleum ether, toluene and the like.

In addition, in order to remove the ineffective components that are well soluble in water, a polar organic solvent which is not mixed with water is contacted with the aqueous solution or the water suspension of the corn extract obtained in the extraction process in the above manner. Then, an inactive ingredient that is well soluble in water is more distributed in water, while an active ingredient that is well soluble in polar organic solvent is more distributed in polar organic solvent. Thus, by removing the aqueous solution and removing the polar organic solvent from the solution of the polar organic solvent, a partially purified corn extract can be obtained. By this process, it is possible to remove the inactive ingredients well soluble in water from the corn extract obtained by the extraction process.

As the polar organic solvent that can be used in the present invention, it is preferable to use an organic solvent or a mixed solvent thereof, which generally has a polarity of at least diethyl ether and at most butanol in the polarity table provided for fractionation, and is not mixed with water. It is preferable to use. Particularly preferred polar organic solvents are ethyl acetate, butanol and mixed solvents thereof. In addition, a polar solvent that can be used in the present invention is a transition energy for the longest-wavelength solvatochromic absorption band of pyridinium-N-phenoxide betain dyes. The value of the above-mentioned solvent polarity parameter obtained by experimentally measuring may be in the range of 0.1 to 0.9, preferably in the range of 0.2 to 0.8. Examples of preferred polar solvents include dichloromethane (MC), chloroform, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol, 1-dodecanol, diethyl ether, tetrahydrofuran, ethyl acetate, ethyl aceto Acetate, ethyl benzoate, etc. are mentioned.

The removal of the inactive ingredients well dissolved in the non-polar organic solvent described above and the removal of the inactive ingredients well dissolved in water are preferably carried out sequentially, and any one may be performed as necessary. In addition, in order to remove the inactive ingredients that are well soluble in water, the corn extract may be dissolved in a polar organic solvent and then contacted with water.

Corn extract or partially purified corn extract obtained by the method as described above may inhibit the absorption of low sugars by inhibiting the activity of amylase in the intestine to prevent the rise of blood sugar and induce satiety due to undigested carbohydrates. Can be. In addition, it is extracted from edible plants and is harmless to humans or animals.

Thus, the corn extract of the present invention can be used for the treatment or prevention of diseases associated with amylase activity. That is, the present invention provides a pharmaceutical composition capable of treating or preventing a disease associated with amylase activity, comprising a corn extract or partially purified corn extract obtained by the above method and a pharmaceutically acceptable carrier. Here, the disease associated with amylase activity is obesity or diabetes, in particular diabetes with obesity, or hyperglycemia and the like.

The pharmaceutical composition of the present invention is an active ingredient that inhibits amylase activity, and includes a corn extract extracted from corn. Such corn extracts are typically formulated in combination with a carrier. The pharmaceutical composition of the present invention can also be used in the form of solids, such as particles or tablets (Tablet) form, can also be used in liquid form.

In preparing a formulation of a pharmaceutical composition containing a corn extract of the present invention, the composition prepared by mixing or diluting the corn extract with a carrier, excipient and diluent in the form of a capsule, sachet or other container It is preferable to enclose it in a carrier or the like. Such formulations may be, for example, in the form of tablets, pills, granules, powders, assays, elecils, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatine capsules, sterile powders and the like. In particular, a form suitable for oral administration is preferred.

Examples of suitable carriers, excipients and diluents for use in the pharmaceutical compositions of the present invention include aluminum salts, phenoxyethylethanol, water, saline, lactose, dextrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose , Amorphous cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the formulation of the pharmaceutical composition of the present invention may further comprise a filler, anti-coagulant, lubricant, wetting agent, fragrance, emulsifier, preservative and the like.

The effective amount referred to herein means the minimum amount that can reduce the amount of low sugars absorbed into the body in the intestine of the mammal, and the amount of the extract of the present invention administered into the body can be adjusted in consideration of the route of administration and the subject of administration. have.

The pharmaceutical composition of the present invention may be administered to the subject to be administered one or more times daily. Unit dosage refers to physically discrete units suitable for unit administration for human subjects and other mammals, each containing a suitable pharmaceutical carrier and an extract of the present invention exhibiting a therapeutic effect.

For adults, the dosage unit for oral administration preferably contains at least 0.1 g of the corn extract of the present invention, and the pharmaceutically effective amount of the corn extract of the present invention is oral dosage of 0.1 to 10 g at a time, preferably 0.5 to 5 g. However, the dosage varies depending on the patient's weight, obesity and diabetes severity, and the extract and supplemental active ingredient used. In addition, the total daily dose can be divided into several divided doses and administered continuously as needed. Thus, the range of dosage does not limit the scope of the invention in any way.

In addition, the corn extract of the present invention can suppress the rise of blood sugar by reducing the amount of low saccharides absorbed into the body by inhibiting the digestion of carbohydrates when edible with a disaccharide or more polysaccharides, especially carbohydrate-containing foods. Therefore, the corn extract of the present invention can be used as a food additive for controlling the blood sugar of the food intake when the food intake. That is, the present invention is to provide a food additive for controlling the blood sugar of the food intake at the time of food intake, including corn extract or partially purified corn extract extracted from corn.

The food additive of the present invention may be added in the process of preparing a food containing two or more polysaccharides, especially carbohydrates such as bread, cookies, soft drinks, chewing gum, toothpaste, mouthwash, butter, jam, noodles, rice and the like. The food additive of the present invention does not necessarily have to be a food or a form added to the food, and may be a form that can be separately ingested before or after ingestion of the food. That is, even if the food additive of the present invention is not in the form added to the food, the intake of the food at the time of ingestion of the food is added to the food and edible together, or ingested separately from the food at the time of or before the intake of the food, as a result If used in a form that can inhibit the digestion and absorption of food, especially carbohydrates in the intestinal tract of the ingestor is within the scope of the present invention. In other words, the food additive of the present invention includes those formulated to exert substantially the same effect as the form added to the food.

On the other hand, the composition comprising a corn extract or partially purified corn extract of the present invention does not necessarily mean that the drug is approved, it can be understood as a concept that includes a conventional functional food or even health supplements.

Hereinafter, the present invention is specifically illustrated through specific examples (and experimental examples). However, the following examples are merely illustrative of specific embodiments of the present invention, and are not intended to limit the scope of the present invention.

Example 1

Extraction of Amylase Inhibitory Active Substances from Corn

Corn kernels (C) and bran (corn husk or corn bran (CH)) were purchased from corn to obtain an active extract with inhibitory effect against alpha-amylase, a carbohydrate digestive enzyme. The flakes were made into powder and used as the next step.

Each corn kernel powder and 5 kg and 15 kg of bran were soaked overnight in 10 L and 30 L of methanol, respectively. It was warmed at 37 ° C. and stirred using a stirrer for effective extraction. After 24 hours the solids were allowed to settle and the cotton wool was filtered out of the funnel to remove the final float. Methanol of the filtrate was evaporated with a rotary evaporator, and the amount of the obtained extract was measured and recovered with 500 mL of tertiary distilled water per 100 g of the extract.

The extract recovered with water was first mixed with the same amount of hexane (DAEJUNG chemicals & metals Co. Ltd., Korea, hereinafter H), placed in a separation funnel, and left for 24 hours after stirring to separate only the hexane layer of the upper layer. It was. Re-fractionated 2-3 times with hexane, concentrated the hexane fractions using a distillation, and dried by completely removing the solvent in a vacuum oven.

The remaining water layer is secondly mixed with the same amount of methylene chloride (MC) (DAEJUNG) and placed in a separator, left to stir for 24 hours after stirring to separate only the lower MC layer. The fractions were re-distilled 2-3 times with MC, the MC fractions were concentrated using a distillation, and then dried by completely removing the solvent in a vacuum oven.

The remaining water layer is thirdly mixed with the same amount of ethyl acetate (EA) (DAEJUNG) and placed in a separator, and left to stir for 24 hours after stirring to separate only the EA layer of the upper layer. The fractions were re-distilled 2-3 more times with EA, the EA fractions were concentrated using a distillation and then dried by completely removing the solvent in a vacuum oven.

The remaining water layer was mixed with the same amount of butanol (Bu) (DAEJUNG) in a fourth order, and placed in a separator, and left alone for 24 hours after stirring to separate only the upper Bu layer. The fractions were re-distilled 2-3 times more, and the Bu fractions were concentrated using a distiller and then dried by completely removing the solvent in a vacuum oven.

Finally, the remaining water layer was concentrated using a distillation, and then dried by removing moisture in a vacuum oven.

Each fraction was dissolved in dimethyl sulfoxide (Sigma, D-5879) at a concentration of 800 mg / ml for inhibition of in vitro amylase and animal experiments, and stored at -20 ° C.

Example 2

Further Purification of Corn Extracts

The EA fraction obtained in Example 1 was chromatographed using silica gel (Merckel, Merck, 9385, 70-230mesh) by gradually increasing the polarity of methylene chloride, methanol and distilled water at a ratio of 50: 3: 0.2. It was.

EA fractions were divided into seven subfractions by polarity, and each subfraction was found to increase amylase inhibitory activity in fractions 3 and 4 through in vitro amylase inhibitory activity tests and animal experiments.

The active subfractions were re-fractionated again by chromatography using Sephadex LH20 (sephadex LH20; pharmacia, 17-0090-01) using methanol as a solvent, respectively, and showed activity through in vitro amylase activity test and animal experiment. The active fraction is prepared by reverse phase silica gel chromatography using 30% to 10% acetonitrile (Acetonitril, MeCN) (DAEJUNG) as a solvent, and recrystallization using 5-20% MeCN and methanol. Separated them.

The small fractions produced during the purification process were conducted mainly on the active fractions through the inhibitory activity against amylase and animal experiments, and each of the small fractions and the active compounds were 100 mg / ml for the inhibitory activity against in vitro amylase and the animal experiments. Was dissolved in dimethyl sulfoxide (Sigma, D-5879) and stored separately at -20 ℃ was used.

Example 3

In vitro amylase activity test

Inhibition of amylase activity of the fractions according to the solubility of each organic solvent of the extract from corn kernels and corn husk was tested. Amylase was used as porcine pancreatic amylase (sigma, A-6255, USA), and para-nitrophenyl-Ad-maltoside as a substrate (sigma, N-5885, PNPM) was used. Substrates pNPM and extracts of 40 mM final concentration in amylase action buffer (20 mM phosphate buffer, pH 6.9, 20 mM NaCl, 0.1 mM CaCl ₂ ) were placed on a microtiter plate and left at 37 ° C. for one hour. After that, the pig pancreatic amylase was added to a final concentration of 56 ug / ml and reacted at 37 ° C for 1 hour. After 1 hour, the absorbance was measured at 405 nm (reference 650 nm) using an absorbance analyzer (spectrophotometry, ELISAreader; Molecular Device Inc., USA). The above experiments were performed in triplicates.

Inhibitory activity in the present specification was calculated as follows.

Amylase inhibitory activity (%) = [(absorbance by substrate-absorbance by substrate and extract) / (absorbance by substrate)] X 100.

Amylase inhibition activity of corn kernel and corn husk extract fractions and isolated active compounds was shown in Table 2, Table 3, and ethyl acetate fraction (C-) compared to other organic solvents. EA, CH-EA) showed high inhibitory activity, and showed about 50% and 73% inhibitory activity against corn kernel and bran, respectively. In addition, the yield of ethyl acetate fraction (CH-EA, hereinafter corn extract) of the bran was about 0.1% (w / w) relative to the dry weight of the starting bran.

As a result, the corn extract of the present invention was found to be effective in inhibiting the activity of amylase, a carbohydrate digestive enzyme.

Table 2. Amylase Inhibitory Activity of Organic Solvent Fraction Extract from Corn Kernels

C-H C-MC C-EA C-Bu C-W Concentration (mg / ml) 2.5 2.5 2.5 2.5 2.5 Inhibitory activity (%) 8.8 11.8 50.8 27.3 0.4

CH: Hexane fraction for the extract of corn kernels, C-MC: Methylene chloride fraction for the extract of corn kernels, C-EA: Ethyl acetate fraction for the extract of corn kernels, C-Bu: Ethyl for extract of the corn kernels Acetate fraction, CW: water fraction for extract of corn kernels, amylase: use of pig pancreatic amylase.

Table 3. Amylase Inhibitory Activity of Organic Solvent Fraction Extract from Corn husk

CH-H CH-MC CH-EA CH-Bu CH-W Concentration (mg / ml) 2.5 2.5 2.5 2.5 2.5 Inhibitory activity (%) 18.7 33.1 73.0 42.2 8.1

CH-H: Hexane fraction for extract of corn husk, CH-MC: Hexane fraction for extract of corn husk, CH-EA: Ethyl acetate fraction for extract of corn husk, CH-Bu: For extract of corn husk Butanol fraction, CH-W: Water fraction for extract of corn husk, Amylase: Porcine pancreatic amylase.

Table 4. IC ₅₀ Values for Compounds Isolated from the EA Fraction from Oxygen

Inhibitor Extract 1 Extract 3 Extract 4 Extract 5 Extract 7 Extract 10 IC ₅₀ (mM) 7.1 7.2 7.3 8.7 3.5 6.1

Extracts 1-10: Compounds having a molecular weight of 100-300 or less (hereinafter referred to as active compounds) isolated from CH-EA, Amylase: Use of pig pancreatic amylase.

On the other hand, 302 g of the corn extract extracted from the corn husk by the extraction process to obtain a fraction of each organic solvent in the same manner as in Example 1, 64.4 g of hexane (H) fraction, 12.84 of methylene chloride (MC) fraction g, ethyl acetate (EA) fraction 3.94g, butanol (Bu) fraction 114g, water (W) fraction 106.65g each. From this result and the inhibitory activity results of Table 3, it can be seen that the inactive ingredients that do not inhibit amylase activity in the water layer and the hexane layer are considerably contained, and the active ingredients that inhibit amylase activity are contained in the polar organic solvent. It can be seen that it is quite included. Therefore, the above results, the present invention shows that the corn extract of the present invention can be further purified by using the difference in the solubility in each solvent and the polarity of the active ingredients and inactive ingredients contained in the corn extract. In addition, when evaluated roughly, it can be seen that ethyl acetate has good selectivity for the active ingredient, but does not have high solubility, butanol has lower selectivity for the active ingredient than ethyl acetate, but has high solubility. Methylene chloride can be seen that both selectivity and solubility are between ethyl acetate and butanol. Therefore, the efficiency of removing inactive ingredients for inhibiting amylase activity from corn extracts can be achieved by using their mixed solvents.

Example 4

Effect of Corn Extracts on Lowering Postprandial Blood Sugar in Spraue-Dawley Rats

Spraue-Dawley (SD) rats (DAEHAN Biolink Co. Ltd., Korea) used in this example are 6-week old, males, between 150-200 g. The breeding environment was provided with 12 hours of periodic lighting (lighting from 09:00 to 21:00, shading from 21:00 to 09:00), and temperature and humidity were maintained at 22 to 24 ℃ and 40 to 60%, respectively.

In order to evaluate postprandial hypoglycemic effect by administering corn extract (CH-EA) of the present invention with starch, the ethyl acetate fraction extract having excellent in vitro amylase inhibitory activity was used. Starch was dissolved by dipping potato starch (sigma, S-2630) in physiological saline (0.9% NaCl), followed by excipient (2% Tween-80), and corn extract with starch and excipient. Was used.

18 hours before the administration of the composition of the present invention prepared by the above method by fasting the SD rats by only supplying water, the capillary tube with anticoagulant was cut by cutting the tail-tip to measure the blood glucose level of the fasted state Blood was collected to obtain plasma. In order to see the effect of the hypoglycemic effect on the post-prandial composition of the present invention, the starch and corn extracts were separated into four groups, each containing only starch, and a group administered with a mixture of corn extract and starch. It was administered orally so that. Blood was collected in the same manner as above 30 and 60 minutes after administration.

Blood glucose levels were measured at 490 nm using an absorbance analyzer using a Tinder kit (Trinder kit, sigma, 315-500). Blood glucose was calculated based on the standard solution. Differences between the two groups of blood glucose levels were tested by the unpaired Student's t- test.

The change in blood glucose level obtained in this example is as shown in FIG. 1, and the blood glucose level drop was significant (p <0.001) at 30 and 60 minutes after the corn extract (CH-EA) was administered to SD rats in an amount of 400 mg / kg. It showed an effect.

Example 5

Effect of Corn Extract on Hypoglycemic Effect in Child Seed (ICR) Mice

The ICR mouse (DAEHAN Biolink Co. Ltd., Korea) used in this example is 6 weeks old, male, between 25 and 30 g, and the breeding environment is the same as in Example 4.

To prepare a composition for oral administration as in Example 4 to evaluate the hypoglycemic effect of postprandial administration by administering the corn extract (CH-EA) of the present invention and the active compound 3 isolated by further purification with starch, respectively.

20 hours before the administration of the composition of the present invention prepared by the above method to fast the ICR mice by water only, and then to measure the blood glucose level of the fasted state using a capillary tube with an anticoagulant in the retroorbital venous plexus Blood was collected to obtain plasma. In order to see the effect of the hypoglycemic effect on the post-prandial composition of the present invention, starch and corn were separated into groups administered with only starch, mixed with corn extract and starch, and mixed with and administered with active compound 3 and starch. The extract was administered orally to 1 g, 100 ~ 400 mg per kg body weight of ICR mice, respectively. Blood was collected in the same manner as in Example 4 at 30, 60, and 120 minutes after administration. Blood glucose levels were carried out in the same manner as in Example 4 using enzyme colorimetry, and the results were analyzed in the same manner as in Example 4.

Changes in blood glucose levels obtained in this Example are the same as those in FIGS. 2 and 3, and the corn extract (CH-EA) and the active compound 3 were treated in ICR mice in an amount of 100-400 mg / kg, which was significant at 30 minutes ( p <0.001) showed a hypoglycemic effect.

Example 6

Hypoglycemic Effects of Corn Extracts Using Various Grains

SD rats and a breeding environment used in this example are the same as in Example 4. In order to evaluate the hypoglycemic effect of post-prandial blood sugar by administering the extract of the present invention with grains, corn starch and wheat flour sold as foods were used, and a pharmaceutical composition for oral administration as in Example 4 was prepared.

In order to see the effect of hypoglycemic hypoglycemic effect of the composition of the present invention, after separating five grains into the group to be administered by mixing grains, corn extract and grains, the grains and corn extracts to 0.5g, 400mg per kg of SD rat body weight, respectively It was administered orally. Blood was collected in the same manner as above at 30, 60 and 120 minutes after administration. Blood glucose levels were carried out in the same manner as in Example 4 using enzyme colorimetry, and the results were analyzed in the same manner as in Example 4.

Changes in blood glucose levels obtained in this example are the same as those of FIGS. 4 and 5, and the corn glucose extract was administered to SD rats in an amount of 400 mg / kg, which was significantly (p <0.001, p <0.01) lower than that of the control group. Showed.

Example 7

Further purification by anion exchange resin (elution with acidic solution)

In order to obtain an active extract having the ability to inhibit the activity of the carbohydrate digestive enzyme alpha-amylase, 10 kg of corn husk was extracted with a mixed solvent of water and 95% ethanol (alcohol) for 12 hours at 40 ℃. The inhibitory effect on the porcine pancreatic amylase of corn bran crude extract according to the mixing ratio of water and 95% ethanol is shown in Table 5 below. The corn extract thus obtained was obtained from Whatman No. Filtration with 1 filter paper and evaporation of water and ethanol using a rotary evaporator to obtain crude crude extract (crude extracts). To this corn crude extract, 1 volume of primary distilled water or tap water was added and stirred at 40 to 80 ° C. for 1 hour. It filtered with 1 filter paper. The filtrate was concentrated and 1 volume of alcohol (95% ethanol) was added to the concentrated extract, which was stirred for 1 hour. After filtering with 1 filter paper, the hydrogen ion concentration (pH) of the filtrate was adjusted to 8-9 with dilute 1N NaOH aqueous solution. The pH-adjusted extract was brought into contact with anion exchange resin (Diaion SA 10 AP resin) equilibrated with an aqueous solution of pH 7-8 in the reactor, and then washed with primary distilled water or tap water to remove substances not bound to the resin. The resin was washed and the solution removed from the reactor. Then, an aqueous solution whose pH was adjusted to 2 to 6.5 with hydrochloric acid (0.5 N HCl) diluted in an reactor of anion exchange resin was added, stirred for 2 hours, and then allowed to stand for 1 hour before recovering the aqueous solution. Subsequently, this aqueous solution was neutralized with dilute sodium hydroxide (1N NaOH), filtered through a filter paper (Watman No. 1), and concentrated to obtain an bran extract. At this time, when the aqueous solution of pH 2 was used, the yield of the bran extract was 1%. The degree of inhibition of porcine pancreatic amylase of the extract thus obtained is shown in FIG. 6.

Table 5

Inhibitory Effects of Corn Oat Bran Extracts on Porcine Pancreatic Amylase with Water and Alcohol Mixture Ratio

Water and Ethanol Mixing Ratio Amylase inhibition% 100% of water 10 90% water + 10% ethanol 12 75% water + 25% ethanol 15 50% water + 50% ethanol 19 25% water + 75% ethanol 25 10% water + 90% ethanol 30 Ethanol 100% 35

Ethanol in the table is alcohol (ethanol 95%).

Example 8

Further purification by anion exchange resin (elution with brine solution)

In order to obtain an active extract having the ability to inhibit the activity of the carbohydrate digestive enzyme alpha-amylase, 10 kg of corn husk was extracted with a mixed solvent of water and 95% ethanol (alcohol) for 12 hours at 40 ℃. Table 5 shows the inhibitory effect of crude corn bran crude extract on porcine pancreatic amylase according to the mixing ratio of water and 95% ethanol. The corn extract thus obtained was obtained from Whatman No. Filtration with 1 filter paper and evaporation of water and ethanol using a rotary evaporator to obtain crude crude extract (crude extracts). To this corn crude extract, 1 volume of primary distilled water or tap water was added and stirred at 40 to 80 ° C. for 1 hour. It filtered with 1 filter paper. The filtrate was concentrated and 1 volume of alcohol (95% ethanol) was added to the concentrated extract, which was stirred for 1 hour. After filtering with 1 filter paper, the hydrogen ion concentration (pH) of the filtrate was adjusted to 8-9 with dilute 1N NaOH aqueous solution. The pH-adjusted extract was brought into contact with anion exchange resin (Diaion SA 10 AP resin) equilibrated with an aqueous solution of pH 7-8 in the reactor, and then washed with primary distilled water or tap water to remove substances not bound to the resin. The resin was washed and the solution removed from the reactor. Then, 0.01-0.5 M NaCl aqueous solution was added to the reactor of anion exchange resin, stirred for 2 hours, and then allowed to stand for 1 hour, and then the aqueous solution was recovered. Subsequently, this aqueous solution was filtered through filter paper (Watman No. 1), and concentrated to obtain an bran extract. At this time, in the case of using an aqueous solution of 0.5 M NaCl, the yield of the bran extract was 1%. The degree of inhibition of porcine pancreatic amylase of the extract thus obtained is shown in FIG. 7.

Corn extract of the present invention inhibits the activity of amylase, a carbohydrate digestive enzyme, inhibits the absorption of low sugars in the body, prevents the rise of blood sugar, reduces the excessive intake of nutrients, and shows effects in the prevention and treatment of diabetes or obesity. Not carbohydrates can lead to a feeling of satiety can lead to a dietary effect, as well as extracts from edible plants are harmless to the human body.

Therefore, the corn extract of the present invention can be used as a medicinal use for the prevention and treatment of obesity or diabetes mellitus, and also, by adding to food or food to suppress the digestion of carbohydrates to control the blood sugar of the intake and induces satiety Can be used as food additives.

Claims (29)

Extracting corn with an extraction solvent of water or a polar organic solvent or a mixed solvent thereof, Removing solids from the extract obtained in the extraction step, and Method of producing a corn extract that can inhibit the activity of amylase comprising the step of removing the extract solvent from the extract to obtain a corn extract. The method of claim 1, The corn is a corn kernel (corn kernel) or corn husk (corn husk) method of producing a corn extract that can inhibit the activity of amylase, characterized in that. The method of claim 1, The polar organic solvent is a method of producing a corn extract that can inhibit the activity of amylase, characterized in that the lower alcohol having 1 to 4 carbon atoms. The method of claim 1, The extraction step is a method for producing a corn extract that can inhibit the activity of amylase, characterized in that carried out at a temperature of 0 ~ 100 ℃. The method of claim 1, Said solids removing step is to remove the solids by centrifugation or filtration to obtain a supernatant or filtrate, the method of producing a corn extract that can inhibit the activity of amylase. The method of claim 1, Method of producing a corn extract that can inhibit the activity of amylase characterized in that it further comprises the step of purifying the extract. The method of claim 6, Purifying step of the extract Dissolving or suspending the extract in water, Dispensing the components of the extract by contacting the aqueous solution or aqueous suspension with a polar or non-polar organic solvent that is not mixed with water, and Recovering the solution or the aqueous solution of the polar organic solvent, and removing the polar organic solvent or water therefrom to obtain a partially purified corn extract corn extract which can inhibit the activity of amylase Manufacturing method. The method of claim 6, Purifying step of the extract Dissolving or suspending the extract in water, Contacting said extract aqueous solution or aqueous suspension with a nonpolar organic solvent which is not mixed with water to distribute components having no amylase inhibitory activity in said aqueous solution or aqueous suspension to said nonpolar organic solvent, Removing the solution of the nonpolar organic solvent, Contacting the aqueous solution or aqueous suspension with a polar organic solvent that is not mixed with water to distribute an active ingredient having amylase inhibitory activity in the aqueous solution or aqueous suspension to the polar organic solvent, and Recovering the solution of the polar organic solvent, and removing the polar organic solvent therefrom to obtain a partially purified corn extract, the method of producing a corn extract capable of inhibiting the activity of amylase. The method according to claim 7 or 8, The polar organic solvent is used for the longest-wavelength solvatochromic absorption bands of pyridinium-N-phenoxide betain dyes of formulas (1) and (2). Preparation of a corn extract capable of inhibiting the activity of amylase, characterized in that the solvent polarity parameter obtained by experimentally measuring the transition energy has an polarity in the range of 0.1 to 0.9 and is an organic solvent which is not mixed with water. Way. The method of claim 9, The polar organic solvent is dichloromethane (MC), chloroform, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol, 1-dodecanol, diethyl ether, tetrahydrofuran, ethyl acetate, ethyl acetoacetate , Ethyl benzoate or a mixed solvent thereof, a method for producing a corn extract capable of inhibiting the activity of amylase. The method according to claim 7 or 8, The non-polar organic solvent is used for the longest-wavelength solvatochromic absorption band of pyridinium-N-phenoxide betain dyes of formulas (1) and (2). A method for producing a corn extract capable of inhibiting the activity of amylase, characterized in that the solvent polarity parameter obtained by experimentally measuring the transition energy has an polarity of 0.15 or less and is an organic solvent which is not mixed with water. The method of claim 11, The non-polar organic solvent is n-hexane, cyclohexane, n-pentane, n-octane, petroleum ether, toluene or a mixed solvent thereof, the method of producing a corn extract that can inhibit the activity of amylase. The method of claim 6, Purifying step of the extract is a method of producing a corn extract that can inhibit the activity of amylase, characterized in that to remove the components that do not have amylase inhibitory activity mixed in the extract by chromatography. The method of claim 13, The chromatography is a method of producing a corn extract that can inhibit the activity of amylase, characterized in that by using an anion exchange resin. The method of claim 14, Contacting the corn extract with the anion exchange resin, Washing the anion exchange resin with water to remove a substance not bound to the resin; Eluting the active ingredients bound to the anion exchange resin by an eluting solution, and Concentrating the eluted active ingredient to obtain a partially purified corn extract, the method of producing a corn extract that can inhibit the activity of amylase. The method of claim 15, The elution solution is a method of producing a corn extract that can inhibit the activity of amylase, characterized in that the acid or aqueous salt solution. Extracting corn with an extraction solvent of water or a polar organic solvent or a mixed solvent thereof, Removing solids from the extract obtained in the extraction step, and Removing the extraction solvent from the extract is prepared by a manufacturing method comprising the step of obtaining a corn extract, corn extract that can inhibit the activity of amylase. The method of claim 17, Corn is a corn extract (corn kernel) or corn husk (corn husk) Corn extract that can inhibit the activity of amylase, characterized in that. The method of claim 17, Dissolving or suspending the extract in water, Dispensing the components of the extract by contacting the aqueous solution or aqueous suspension with a polar or non-polar organic solvent that is not mixed with water, and Recovering the solution or the aqueous solution of the polar organic solvent, and removing the polar organic solvent or water therefrom to obtain a partially purified corn extract, which is obtained by partially purifying the extract by a further purification process, Corn extract that can inhibit the activity of amylase. The method of claim 17, Dissolving or suspending the extract in water, Contacting said extract aqueous solution or aqueous suspension with a nonpolar organic solvent which is not mixed with water to distribute components having no amylase inhibitory activity in said aqueous solution or aqueous suspension to said nonpolar organic solvent, Removing the solution of the nonpolar organic solvent, Contacting the aqueous solution or aqueous suspension with a polar organic solvent that is not mixed with water to distribute an active ingredient having amylase inhibitory activity in the aqueous solution or aqueous suspension to the polar organic solvent, and Inhibiting the activity of amylase obtained by partially purifying the extract by an additional purification process comprising recovering the solution of the polar organic solvent and removing the polar organic solvent therefrom to obtain a partially purified corn extract. Corn extract can. The method of claim 17, Corn extract which can inhibit the activity of amylase obtained by partially purifying the corn extract by an additional purification process using an anion exchange resin. The method of claim 21, Further purification process using the anion exchange resin Contacting the corn extract with the anion exchange resin, Washing the anion exchange resin with water to remove a substance not bound to the resin; Eluting the active ingredients bound to the anion exchange resin by an eluting solution, and Concentrating the eluted active ingredient to obtain a partially purified corn extract corn extract that can inhibit the activity of amylase. The method of claim 22, The elution solution is a corn extract that can inhibit the activity of amylase, characterized in that the acid or aqueous salt solution. A pharmaceutical composition capable of treating or preventing a disease associated with amylase activity, comprising the corn extract of any one of claims 17 to 23 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 24, wherein the disease associated with amylase activity is obesity or diabetes. The pharmaceutical composition according to claim 24, wherein the pharmaceutical composition has a form suitable for medicinal oral administration. A food additive for controlling blood sugar of a food intake upon ingestion of food, comprising the corn extract of any one of claims 17 to 23. The food additive according to claim 27, wherein the food additive is added to a food containing polysaccharide or more of disaccharides. 28. The food additive of claim 27 wherein the food additive is for preventing or inhibiting weight gain upon ingestion.