KR101607188B1 - Soybean stalks of the high-content isoflavone derivatives and preparing method thereof - Google Patents

Abstract

The present invention relates to a soybean stem having a high content of isoflavone derivatives and a method for producing the same, and more particularly, to a soybean stem having a high content of the isoflavone derivative, The production method and the high content of isoflavone derivatives produced by the use of the bean stalks are useful as functional foods and medicinal materials for diseases caused by estrogen imbalance and deficiency of antioxidant ability.

Description

TECHNICAL FIELD The present invention relates to soybean stems, and more particularly, to soybean stems having a high content of isoflavone derivatives,

The present invention relates to a method for producing a high-content soybean stem of an isoflavone derivative, a high-content soybean stem of an isoflavone derivative prepared by the method, a use of a high-content soybean stem of the isoflavone derivative, and a mass production method of an isoflavone derivative from a soybean stem .

Isoflavone derivatives are representative flavonoids with excellent pharmacological efficacy, and a great deal of capital is invested in the development of functional plants with high content in the world. Isoflavones are nonsteroidal estrogens, which are known to be phytoestrogenic because they have a chemical structure and physiological characteristics similar to those of estrogen. These are similar to estrogen, a female hormone, and show useful physiological activities expected in estrogen. It has been reported that it reduces the risk of osteoporosis especially after menopause and lowers plasma cholesterol. It is also reported that the antioxidant activity and antioxidant activity of coronary heart disease are lowered.

Isoflavones exist in the form of a glycoside linked to a beta-glycoside or in the form of a non-glycosylated sugar. Examples of non-glycosides include genistein, daidzein and glycitein, and glycosides include genistin, daidzin, and glycitin. . Soybean-derived non-fermented foods contain many glycosides, while fermented foods mainly contain non-glycosides as the sugar is decomposed from the glycosides by bacteria involved in fermentation.

Most of the isoflavones used as food and medicinal materials are extracted from beans and red clover. Many breeding studies have been carried out for the development of soybean containing high levels of isoflavones, but soybeans containing 4000 ㎍ / g total isoflavone have been reported at the highest level ( J. Agric . Food Cehm . 2012, 60, 6045-6055). Korean Patent Laid-Open Publication No. 2003-93025 discloses a method for producing germinated soybean containing high concentration of isoflavone in which washed soybean is soaked in purified water and then germinated to increase the isoflavone content by 40 to 70% The content is low.

Therefore, the present inventors have made efforts to develop a soybean stem containing high levels of isoflavone derivatives. As a result, when ethylene or ethylene donor ethephon (2-chloroethylphosphonic acid) was treated during soybean cultivation, high concentrations of isoflavone derivatives And confirming accumulation in bean stalks.

It is an object of the present invention to provide a process for producing a soybean stem having a high content of isoflavone derivatives, which comprises the step of treating bean stalks with ethylene, an ethylene donor or an ethylene generator.

It is still another object of the present invention to provide a high content soybean stem of an isoflavone derivative produced by the above-described method.

It is another object of the present invention to provide a food and pharmaceutical material containing a high content of an isoflavone derivative, a bean stem, an extract of bean stem, or a fraction of the extract.

It is still another object of the present invention to provide a method for mass production of isoflavone derivatives from soybean stem using ethylene, ethylene donor or ethylene generator.

In order to accomplish the object of the present invention, the present invention provides a method for producing a high-content soybean stem of an isoflavone derivative represented by the following formula (1), comprising the step of treating ethylene oxide, ethylene donor or ethylene generator on a bean stem Method;

[Chemical Formula 1]

.

.

Wherein R ₁ is independently H or OH, and R ₂ is independently H, glucose or glucose malonate.

The present invention also provides a high content soybean stem of an isoflavone derivative prepared by the above-described method.

The present invention also provides a composition comprising a high content of an isoflavone derivative bean stem, an extract of the bean stem, or a fraction of the extract of the present invention.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diseases caused by estrogen deficiency comprising as an active ingredient an isoflavone derivative high-content soybean stem of the present invention, an extract of bean stem, or a fraction of the above extract do.

In addition, the present invention provides a health food for prevention and improvement of diseases caused by estrogen deficiency containing an isoflavone derivative high-content soybean stem, an extract of bean stem, or a fraction of the extract as an active ingredient according to the present invention .

The present invention also provides a pharmaceutical composition for antioxidant containing an isoflavone derivative high-content bean stem according to the present invention, an extract of the bean stem, or a fraction of the extract as an active ingredient.

The present invention also provides an antioxidant health food containing as an active ingredient an isoflavone derivative high-content bean stem according to the present invention, an extract of the bean stem, or a fraction of the extract.

In addition, the present invention provides a pharmaceutical composition for skin care comprising an isoflavone derivative high-content soybean stem according to the present invention, an extract of bean stem, or a fraction of the extract as an active ingredient.

The present invention also provides a cosmetic composition for skin care comprising an isoflavone derivative high-content bean stem according to the present invention, an extract of bean stem, or a fraction of the extract as an active ingredient.

In addition, the present invention provides a health food for skin care containing an isoflavone derivative high-content bean stem according to the present invention, an extract of bean stem, or a fraction of the extract as an active ingredient.

In addition, the present invention provides a tea using a high-content soybean stem of an isoflavone derivative according to the present invention.

The present invention also provides a method for producing tea using a high-content soybean stem of an isoflavone derivative according to the present invention.

The present invention also provides a method for mass production of isoflavone derivatives from soybean stem using ethylene, ethylene donor or ethylene generator.

In addition,

1) treating soybean stem with ethylene, ethylene donor or ethylene generator to prepare bean stem; And

2) extracting the soybean stem of step 1) and isolating the isoflavone derivative. The present invention also provides a method for mass production of an isoflavone derivative.

The present invention confirms that the isoflavone derivative is accumulated at a high concentration in the soybean stem by treating the soybean plant with 10 to 90 days after sowing with ethylene or an ethylene donor, Beans stem, bean stem extract and extract fractions containing a high content of isoflavone derivatives as well as a method of producing a stem, can be usefully used as food for diseases and medicinal materials caused by estrogen imbalance and deficiency of antioxidant ability.

FIG. 1 is an HPLC chromatogram showing an increase in the content of isoflavone derivatives before and after treatment with ethylene. FIG.
FIG. 2 shows HPLC chromatogram results of extracts obtained by extracting bean stem before ethanol treatment with ethanol. FIG.
Fig. 3 is a chart showing the HPLC chromatogram results of an extract obtained by extracting soybean stem after ethanol treatment with ethanol. Fig.
FIG. 4 is a diagram showing a mass spectrometry (Q-TOF MS) result in which the peak 1 of FIG. 3 is identified as daidzin.
FIG. 5 is a diagram showing a mass spectrometry (Q-TOF MS) result in which peak 2 in FIG. 3 is genistin.
FIG. 6 is a graph showing a mass spectrometry (Q-TOF MS) result showing that peak 3 in FIG. 3 is malonyldaidzin.
FIG. 7 is a graph showing a mass spectrometry (Q-TOF MS) result showing that peak 4 in FIG. 3 is malonylgenistin.
FIG. 8 is a graph showing UPLC chromatogram results of an extract obtained by treating ethylene glycol with an ethanol-extracted bean stem followed by treatment with beta-glucosidase. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing a soybean stem having a high content of isoflavone derivatives, comprising the step of treating bean stem with ethylene, ethylene donor or ethylene generator.

The soybean stem is preferably a soybean stem that has passed 10 days or more after sowing, more preferably a soybean stem that has passed 40 days after sowing, but is not limited thereto.

The ethylene donor is preferably ethephon but is not limited thereto. The ethylene is the simplest olefinic hydrocarbon (chemical formula: C ₂ H ₄ ) which is colorless and sweet. As a kind of plant hormone, it has a function of promoting the maturation of plant tissues such as the change of physiology after harvest of fruits and vegetables, especially the maturation of horticultural crops and the yellowing of leafy vegetables. Ethephon (2-chloroethylphosphonic acid) is a kind of ethylene generator, which generates ethylene under neutral and alkaline conditions of pH 4.1 or higher. When an aqueous solution of 1000-2000 times in a liquid phase is sprayed on fruits and vegetables Decomposes and generates ethylene to promote maturation, chilling and pigmentation.

The ethylene, ethylene donor or ethylene generator is preferably treated at a concentration of 5 to 500 ppm, more preferably at a concentration of 5 to 100 ppm, but not always limited thereto. The ethylene, ethylene donor or ethylene generator can be treated before or after harvesting the bean stem.

The isoflavone derivative is preferably a compound represented by the following formula (1), more preferably any one of the compounds represented by the following formulas (2) to (7), but is not limited thereto.

Wherein R ₁ is independently H or OH, and R ₂ is independently H, glucose or glucose malonate.

The present invention also provides an isoflavone derivative high-content soybean stem prepared by the above-described method.

The isoflavone derivative is preferably a compound represented by the formula (1), more preferably any one of the compounds represented by the formulas (2) to (7), but is not limited thereto.

In a specific example of the present invention, the inventors of the present invention found that when soybean plants treated with ethylene (100-1000 ppm) for 40 days (growth period R4) after soybean sowing had significantly increased isoflavone derivatives in soybean stem (See Fig. 1). In addition, isoflavone derivatives were not detected in soybean stem not treated with ethylene. However, in the soybean stem treated with ethylene, it was confirmed that four kinds of isoflavone derivatives daidzin, genistin, malonyldaidzin, and malonyl genistein were rapidly accumulated in bean stalks (see Table 1) . The soybean stem treated with ethylene was extracted with ethanol to obtain an extract. The soybean stem extract was treated with beta-glucosidase to isolate and purify the six kinds of isoflavone derivatives. (See FIG. 3 to FIG. 8). In addition, the present invention provides a method for producing the above-mentioned microorganisms.

The present inventors also quantitatively analyzed four isoflavone derivatives, daidzin, genistin, malonylidenechin and malonyl genistein isolated from soybean stem harvested after ethylene treatment and soybean stem after ethylene treatment, respectively, after ethylene treatment As a result, it was confirmed that the content of isoflavone derivatives in the case of bean stem harvested after ethylene treatment was remarkably increased, so that the method of harvesting ethylene after treatment of bean stalks was the method of producing isoflavone derivative high content soybean stem (See Table 1).

The present invention also provides a method for mass production of isoflavone derivatives from soybean stem using ethylene, ethylene donor or ethylene generator.

Specifically,

1) treating soybean stem with ethylene, ethylene donor or ethylene generator to prepare bean stem; And

2) extracting the soybean stem of step 1) and isolating the isoflavone derivative.

In the above method, the soybean stem of step 1) is preferably a soybean stem that has passed 10 days or more after sowing, more preferably a soybean stem that has passed 40 days after sowing, more preferably a soybean stem that has passed 10 to 90 days after sowing Is most preferable.

In this method, the ethylene, ethylene donor or ethylene generator of step 1) can be treated before or after harvesting the bean stalks.

In the above method, the ethylene donor of step 1) is preferably ethephon, but not limited thereto, and all the substances which induce or generate ethylene can be used.

In this method, the ethylene, ethylene donor or ethylene generator in step 1) is preferably treated at a concentration of 100 to 1000 ppm, more preferably at a concentration of 100 to 500 ppm.

In the above method, the isoflavone derivative is preferably a compound represented by the above formula (1), and more preferably at least one of the compounds represented by the above formulas (2) to (7).

The present invention can be used as a method for mass production of isoflavone derivatives by producing bean stalks highly containing isoflavone derivatives by treating beef stalks with ethylene and ethylene donor esters.

The present invention also provides a composition comprising a soybean stem having a high content of isoflavone derivative prepared by treating ethylene glycol, an ethylene donor or an ethylene generator in a bean stem, an extract of the bean stem, or a fraction of the extract.

Preferably, the bean stem extract or the fraction thereof is produced by a manufacturing method comprising the following steps, but not limited thereto:

1) extracting the bean stem with an extraction solvent;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying to prepare a bean stem extract; And

4) Extracting the bean stem extract of step 3) with an organic solvent to prepare bean stem fractions.

In the above method, the bean stem of step 1) is collected from a soybean plant having a high concentration of isoflavone after ethylene treatment and used. The ethylene treatment may be performed after the plant is harvested, but it is more preferable to treat the plant before harvesting. The soybean plants treated with ethylene may be leaves, stalks or roots, but it is not limited thereto. According to a preferred embodiment of the present invention, bean stalks are preferably used.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. The extraction solvent is preferably added by 1 to 10 times the amount of the dried soybean stem, more preferably by adding 4 to 6 times. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but is not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the above method, the bean stem extract of step 4) is preferably a dark brown crude extract which is a concentrated bean stem extract, but not limited thereto, and the organic solvent is n-hexane, chloroform, ethyl acetate or butanol According to a preferred embodiment of the present invention, chloroform is more preferable, but not limited thereto. The fractions are n-hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction or water fraction obtained by suspending the bean stem extract in water and then sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, But is not limited thereto. The fraction may be obtained by repeating the fractionation process from the bean stem extract for 1 to 5 times, preferably 3 times, followed by fractionation and concentration under reduced pressure, but it is not limited thereto.

In addition, the present invention relates to a method for producing an isoflavone derivative, which is produced by treating a soybean stem with ethylene, an ethylene donor or an ethylene generator, A pharmaceutical composition for the prevention and treatment of a disease is provided.

The disease caused by the estrogen deficiency is preferably one selected from the group consisting of osteoporosis, heart disease, breast cancer, external sound disease, hyperlipidemia, skin aging and facial flushing, but is not limited thereto.

Since the isoflavone derivatives isolated from the high-content isoflavone bean stalks of the present invention have established physiological activity, the bean stem extract and the fractions thereof containing the compounds are useful as pharmaceutical compositions for the prevention and treatment of diseases caused by estrogen deficiency Can be used.

The composition of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The composition of the present invention can be administered orally or parenterally, and it is preferable to select an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, But is not limited thereto.

The composition of the present invention can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to conventional methods have. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a dose of 0.0001 to 1 g / kg, preferably 0.001 to 200 mg / kg, per day, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In addition, the present invention relates to a method for producing an isoflavone derivative, which is produced by treating a soybean stem with ethylene, an ethylene donor or an ethylene generator, And provides a health food for prevention and improvement of a disease.

The disease caused by the estrogen deficiency is preferably one selected from the group consisting of osteoporosis, heart disease, breast cancer, external sound disease, hyperlipidemia, skin aging and facial flushing, but is not limited thereto.

The isoflavone derivatives isolated from the soybean stem of Isoflavone of the present invention have physiological activity, so that the soybean stem extract and the fractions thereof containing the compounds are useful as a health food for prevention and improvement of diseases caused by estrogen deficiency Can be used.

The health food of the present invention can be added to the soybean stem of the soybean stem, the extract of the soybean stem, or the fraction of the above extract directly, or it can be used together with other foods or food ingredients, and can be suitably used according to a conventional method have.

There is no particular limitation on the kind of the food. Examples of the food include dairy products including functional mixed tea, drink, meat, sausage, bread, biscuit, rice cake, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The isoflavone derivative high-content soybean stem, the extract of the bean stem or the fraction of the extract according to the present invention can be added directly to the food or can be used together with other foods or food ingredients and can be suitably used according to a conventional method . The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). In general, the amount of the soybean stem, the extract of the bean stem, or the fraction of the extract of the present invention may be 0.01 to 15 wt% of the total weight of the whole food, and the health beverage composition may be added in an amount of 100 ml May be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention has no particular limitation on the components other than the bean stalks, the bean stalks, or the fractions of the extract, which contain the high content of the isoflavone derivatives as essential components in the indicated ratios, As well as various flavors or natural carbohydrates as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above-mentioned foods, the food of the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the high content isoflavone derivative bean stalk of the present invention, the bean stem extract, or the fraction of the above extract may contain natural fruit juice and pulp for producing fruit juice beverage and vegetable beverage. These components may be used independently or in combination. Although the proportion of such an additive is not so important, it is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the high content of the isoflavone derivative of the present invention bean stalk, the bean stem extract, or the fraction of the above extract.

The present invention also relates to an antioxidant pharmaceutical composition comprising, as an active ingredient, an isoflavone derivative high-content soybean stem prepared by treating ethylene glycol, an ethylene donor or an ethylene generator in a soybean stem, or an extract of the soybean stem, Lt; / RTI >

The present invention also relates to an antioxidant health food containing an isoflavone derivative high content soybean stem prepared by treating ethylene oxide, ethylene donor or ethylene generator in bean stalks, an extract of bean stalks or a fraction of the above extract as an active ingredient .

The isoflavone derivatives isolated from the soybean stem of Isoflavone of the present invention have physiological activity, so that the soybean stem extract containing the compounds and fractions thereof can be usefully used as an antioxidant composition.

The present invention also relates to a pharmaceutical composition for skin care comprising an isoflavone derivative high-content soybean stem prepared by treating ethylene glycol, ethylene donor or ethylene generator in a bean stem, an extract of the soybean stem, or a fraction of the extract, Gt;

The present invention also relates to a cosmetic composition for skin care comprising an isoflavone derivative high-content soybean stem produced by treating ethylene glycol, ethylene donor or ethylene generator in bean stalks, an extract of bean stalks or a fraction thereof, Lt; / RTI >

In addition, the present invention relates to a pharmaceutical composition for treating skin health, comprising an isoflavone derivative high-content soybean stem prepared by treating ethylene glycol, an ethylene donor or an ethylene generator in a bean stem, an extract of the bean stem, Provide food.

Since the physiological activity of the isoflavone derivatives isolated from the soybean stem of the present invention containing isoflavone is established, the bean stem extract and the fractions thereof containing the compounds can be usefully used as a skin cosmetic composition.

The cosmetic composition may include, but is not limited to, lotions, ointments, gels, creams, patches or sprays. In producing a cosmetic composition containing a high content of an isoflavone derivative of the present invention, an extract of the bean stem, or a fraction of the extract, a composition containing a high content of the isoflavone derivative of the present invention, , The bean stem extract, or the fraction of the extract may be added in an amount of 3 to 30 parts by weight, preferably 5 or 20 parts by weight.

The cosmetic composition of the present invention may further contain a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, an antioxidant, an antioxidant, A preservative, a stabilizer, a foaming agent, a perfume, a surfactant, water, an ionic or nonionic emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, , Pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient conventionally used in cosmetic compositions.

The components can also be introduced in amounts commonly used in the field of dermatology.

In addition, the present invention provides tea using a high-content soybean stem of an isoflavone derivative according to the present invention.

The present invention also provides a mixed tea obtained by mixing a high-content soybean stem of the isoflavone derivative according to the present invention and a herb plant having a flavor.

The car using the bean stem

1) washing and crushing the high-content soybean stem of the isoflavone derivative according to the present invention;

2) roasting the shredded stem; And

3) drying the roasted stem.

In this method, the crushing of step 1) is preferably finely crushed to 1 to 5 mm using a crusher, but the size is not limited thereto.

In the above method, the roasting of step 2) is preferably roasted at a temperature of 250 to 300 DEG C for 30 to 60 minutes in a roaster, but the temperature and time are not limited thereto.

In the above method, the drying of step 3) may be carried out by using a natural dryer or a dryer, and drying is preferably performed at a temperature of 30 to 50 ° C for 24 to 36 hours, but the temperature and time are not limited thereto.

The herb plant is preferably at least one selected from the group consisting of chemomail, lemongrass, rosehip, lavender, peppermint, fennel, rosemary, jasmine, hibiscus, rose flower, apple fruit, strawberry fruit, lemon fruit, Hubs are all available.

The tea or mixed tea may be processed into a form selected from the group consisting of a flavored tea, a tea bag, an instant tea, and a can.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

EXAMPLES The following examples and preparative examples are merely illustrative of the present invention, and the present invention is not limited by the following examples and preparative examples.

< Example  1> Isoflavone derivatives High content  Manufacture of bean stalks

The RDA is distributing soybean seeds (Glycine max) and cultivating 10 to 90 days after sowing in a soybean plant with 5-100 ppm concentration of ethephon (2-chloroethylphosphonic acid) or 100 -1.00 ppm of ethylene was treated to produce high content isoflavone derivative bean stalks. The accumulation of isoflavones in bean stalks may vary depending on the concentration of ethylene or ethylene to be treated and the growing period of the plant being treated.

Specifically, ethylene was periodically treated with soybean plants at a concentration of 200 ppm for 40 days after sowing.

As a result, as shown in Fig. 1, the concentration of the target isoflavone derivative of the bean stem was increased after the treatment. In addition, it was confirmed that soybean stem gradually changed to yellow due to the influence of ethylene, which is a plant aging hormone (Fig. 1). Therefore, the above results show that soybean stem which has been rapidly diluted with 5-100 ppm diluted esters or treated with 100-1000 ppm of ethylene and has been rapidly accumulated in soybean plants obtained from 10 to 90 days after sowing Respectively.

< Example  2> Isoflavone derivatives High content  Production of bean stem extract

The bean stalks obtained by the method of Example 1 were dried and then extracted with 50-100% alcohol at 20-100 ° C for 3-10 days. The extracted solvent was concentrated under reduced pressure to obtain an extract at a yield of 8-15%.

Specifically, bean stalks obtained by the method described in Example 1 were dried, and 1 kg of the dried bean stalks were extracted with 80% ethanol at room temperature for 7 days. The solvent extracted was concentrated under reduced pressure to obtain 86 g of an extract . The dried soybean stem was extracted with pure water at 40-100 ℃. The extracted solvent was concentrated under reduced pressure to obtain an extract at a yield of 10-17%.

As a result, as shown in FIG. 2 and FIG. 3, it was found that the content of isoflavone derivatives in the extract obtained by extracting ethylene-treated bean stalks with ethanol was remarkably high compared with the extract obtained by extracting bean stem before ethanol treatment with ethanol 2 and Fig. 3).

< Example  3> Purification of isoflavone derivatives from soybean stem

Isoflavone derivatives were separated and purified from the high-content isoflavone ethanol extract of soybean stem obtained by the method described in Example 2 above using MPLC (Teledyne Isco CombiFlash Companion). C18 silica gel (YMC, 50 μm) was used as the column material. The elution conditions were gradient elution of acetonitrile solvent to 0-90% in water / acetonitrile mixed solution, Respectively.

Further, in order to separate and purify the isoflavone derivative from the extract by treating beta-glucosidase with the high-content isoflavone-derived ethanol extract obtained by the method described in Example 2 above, C18 silica gel Chromatography was performed to isolate two isoflavone derivatives of the four isoflavone glycosides converted by beta-glucosidase. Thus, a total of six isoflavone derivatives were isolated.

< Example  4> Structural analysis of isolated isoflavone derivatives

UPLC / Q-TOF MS and NMR spectroscopy analysis were performed to analyze the structure of the six isoflavone derivative compounds isolated by the method described in Example 3 above.

As a result, as shown in FIG. 4, it was confirmed that peak 1 of the six isoflavone derivatives was daidzin represented by the following formula (2) (FIG. 4).

(2)

Daidzin (peak 1): ESI-MS (Q -TOF) [M + H] + 417.1186 (. Calc 417.1186, C 21 H 21 O 9, 0.0 ppm); ^{1 H NMR (500MHz, DMSO- d} 6) δ 3.30 (2H, m, H-4 ", 2"), 3.47 (3H, m, H-3 ", H-5", 6˝b), 3.73 ( (2H, d, J = 8.6 Hz, H-3 ', 5'), 6.12 (1H, d, J = 14.2, 7.4 Hz, 7.15 (1H, dd, J = 8.6, 2.1 Hz, H-6), 7.24 (1H, d, J = 2.1 Hz, H-8), 7.41 (2H, d, J = 8.6 Hz, H-2', 6 '), 8.04 (1H, d, J = 8.9 Hz, H-5), 8.39 (1H, s, H-2), and 9.56 (1H, s, 4'-OH); ^{13 C NMR (125 MHz, DMSO-} d 6) δ 61.1 (C-6˝), 70.1 (C-4˝), 73.6 (C-2˝), 76.9 (C-3˝), 77.7 (C-5 1), 115.6 (C-3 ', 5'), 116.0 (C-6), 118.9 (C-4a), 122.8 (C-1), 127.4 (C-5), 130.6 (C-2 &apos;, 6 &apos;), 153.8 C-7), 175.2 (C-4).

Further, as shown in Fig. 5, it was confirmed that Peak 2 was genistin represented by the following Chemical Formula 3 (Fig. 5).

(3)

Genistin (peak 2): ESI-MS (Q -TOF) [M + H] + 433.1139 (. Calc 433.1135, C 21 H 21 O 10, 0.9 ppm); ^{1 H NMR (500 MHz, DMSO-} d 6) δ 3.19 (1H, dd J = 9.9, 4.8 Hz, H-4 "), 3.29 (1H, dd, m, J = 13.4, 7.4 Hz, H-2" ), 3.32 (1H, dd, J = 13.4, 4.8 Hz, H-3 "), 3.45 (1H, m, H-5"), 3.48 (1H, dd, J = 11.4, 5.5 Hz, H-6 " b), 3.73 (1H, dd , J = 11.4, 4.8 Hz, H-6 "a), 5.06 (1H, d, J = 7.4 Hz, H-1"), 6.49 (1H, d, J = 2.1 Hz , H-6), 6.72 ( 1H, d, J = 2.1 Hz, H-8), 6.84 (2H, d, J = 6.6 Hz, H-3', 5'), 7.41 (2H, d, J = 6.6 Hz, H-2 ', 6'), 8.41 (1H, s, H-2), 9.53 (1H, s, 4'-OH), and 12.9 (1H, s, 5-OH). ^{13 C NMR (125 MHz, DMSO-} d 6) δ 61.1 (C-6 "), 70.1 (C-4"), 73.6 (C-2 "), 76.9 (C-3"), 77.6 (C-5 ), 95.0 (C-8), 100.0 (C-6), 100.4 (C-1) C-1 '), 130.5 (C-2', 6 '), 154.9 (C-2), 157.6 C-7), 180.9 (C-4).

Further, as shown in Fig. 6, among the six isoflavone derivatives, Peak 3 was found to be malonyldaidzin represented by the following formula (4) (Fig. 6).

[Chemical Formula 4]

Malonyl daidzin (peak 3): ESI-MS (Q -TOF) [M + H] + 503.1190 (calc 503.1190, C 24 H 23 O 12, 0.0 ppm.); ^{1 H NMR (500 MHz, DMSO-} d 6) δ 3.17 (2H, m, malonyl-CH 2), 3.19 (1H, m, H-4 "), 3.34 (1H, m, H-2"), 3.38 (1H, m, H-6 "), 3.75 (H-5"), 4.14 , J = 7.6 Hz, H- 1 "), 6.52 (2H, d, J = 8.2 Hz, H-3', 5'), 6.74 (1H, d, J = 8.9 Hz, H-6), 7.04 ( 1H, s, H-8), 7.38 (2H, d, J = 8.2 Hz, H-2 ', 6'), 7.95 (1H, d, J = 8.9 Hz, , H-2). ⁴ C NMR (125 MHz, DMSO- d ₆ ) ? 41.9 (malonyl-CH ₂ ), 64.2 (C-6?), 67.3 (C-4a), &lt; / RTI &gt; 75.6 (C-3) , 120.4 (C-3), 121.9 (C-1 '), 130.2 (C-2', 6 '), 157.6 (C-7), 166.8 (malonyl-COOR), 169.5 (malonyl-COOH), 179.9 (C-4).

Further, as shown in Fig. 7, among the six isoflavone derivatives, Peak 4 was found to be malonylgenistin represented by the following formula (5) (Fig. 7).

[Chemical Formula 5]

Malonyl genistein (Peak 4): ESI-MS (Q-TOF) [M + H] ⁺ 519.1138 (calc. 519.1139, C ₂₄ H ₂₃ O ₁₃ , -0.2 ppm); ^{1 H NMR (500 MHz, DMSO-} d 6) δ 3.19 (2H, m, malonyl-CH 2), 3.20 (1H, m, H-4 "), 3.28 (1H, m, H-2"), 3.33 (1H, m, H-3 "), 4.12 (1H, m, H-6" b), 4.35 (1H, m, H-6 "a), 5.12 (1H, d, J = 7.7 Hz, H- (2H, d, J = 8.1 Hz, H-3 ', 5'), 7.39 (2H, d, J = 8.1 Hz, H-2 ', 6'), 8.40 (1H, s, H-2). ^{13 C NMR (125 MHz, DMSO-} d 6) δ 41.6 (malonyl-CH 2), 63.9 (C-6 "), 69.4 (C-4"), 72.7 (C-2 "), 73.6 (C-5 (C-3), 94.3 (C-8), 99.3 (C-1 "), 99.4 (C-6), 106.0 , 120.9 (C-1 '), 122.5 (C-3), 130.2 (C-2', 6 '), 154.6 (C-5), 162.6 (C-7), 167.0 (malonyl-COOR), 167.5 (malonyl-COOH), 180.5 (C-4).

Further, as shown in FIG. 8, it was confirmed that one of the isoflavone derivatives converted from the four isoflavone derivatives was a daidzein represented by the following formula (6) (FIG. 8).

[Chemical Formula 6]

Daidzein: ESI-MS (Q-TOF ) [M + H] + 255.0658 (calc 255.0657, C 15 H 11 O 4, 0.4 ppm.); ^{1 H NMR (500 MHz, DMSO-} d 6) δ 6.84 (2H, d J = 8.6 Hz, H-3', 5') , 6.85 (1H, d, J = 2.0 Hz, H-8), 6.96 (1H, dd, J = 8.8, 2.2 Hz, H-6), 7.38 (2H, d J = 8.6 Hz, H-2 ', 6'), 8.07 (1H, d, J = 8.8 Hz, H-5), 8.15 (1H, s, H-2). ^{13 C NMR (125 MHz, DMSO-} d 6) δ 102.5 (C-8), 115.4 (C-6), 115.6 (C-3', 5'), 117.0 (C-4a), 123.0 (C-1 ), 123.9 (C-3), 127.7 (C-5), 130.5 (C-2 ', 6'), 153.2 163.1 (C-7), 175.1 (C-4).

Further, as shown in Fig. 8, it was confirmed that one of the isoflavone derivatives converted from the four isoflavone derivatives was genistein represented by the following formula (7) (Fig. 8).

(7)

Genistein: ESI-MS (Q-TOF ) [M + H] + 271.0600 (. Calc 271.0606, C 15 H 11 O 5, -2.2 ppm); ^{1 H NMR (500 MHz, DMSO-} d 6) δ 6.21 (1H, d J = 1.6 Hz, H-6), 6.33 (1H, d, J = 1.6 Hz, H-8), 6.86 (1H, dd, J = 6.7, 1.8 Hz, H-3', 5'), 7.38 (1H, dd, J = 6.7, 1.8 Hz, H-2', 6') , 8.06 (1H, s, H-2). ¹³ C (125 MHz, DMSO- d ₆ ) ? 94.0 (C-8), 99.3 (C-6), 104.8 ), 122.7 (C-1 '), 130.5 (C-2', 6 '), 154.3 (C-2), 157.8 164.7 (C-7), 180.6 (C-4).

< Example  5> HPLC Quantitative analysis of isoflavone derivatives using

Quantitative analysis of the four isoflavone compounds isolated by the method described in Example 3 above was performed using HPLC.

Specifically, HPLC was performed using a HPLC Series 200 system manufactured by Perkinelmer (USA), and a column of ODS series Zolbox Bonus-RP (150 × 4.6 mm) was used. The mobile phase was acetonitrile containing 0.1% acetic acid and 0.1% acetic acid, and gradient elution was employed in which the acetonitrile solvent was increased from 95% for 85 minutes at the initial 5%. The flow rate was adjusted to 1 ml / min, the amount of the extracted solution was 10 μl, and the detection wavelength was 254 nm. Four isoflavone compounds isolated by the method described in Example 3 were dissolved in methanol to prepare a standard solution in the range of 1-100 μg / ml, subjected to HPLC analysis, and a calibration curve was drawn from the peak area Respectively. The isoflavone standard used in the analysis was four kinds of didegene, genistin, malonyl didezin and malonyl genistein, all of which contained 98% or more of high purity standards (Table 1).

As a result, as shown in Table 1, the isoflavone derivatives were not detected in the ethanol fraction of the soybean stem without ethylene treatment, whereas the isoflavone derivatives were not detected in the ethanol fraction of the soybean stem treated with 100-1000 ppm of ethylene, It was confirmed that the content of the derivative increased sharply. In particular, the total isoflavone content after ethylene treatment was 11951 ㎍ / g, which was the highest (Table 1).

Isoflavone Content (/ / g) No treatment After processing Daid Jean ( 1 ) Not detected 1931.7 ± 71.2 Genevieve ( 2 ) Not detected 2004.6 ± 86.1 Malonylide jean ( 3 ) Not detected 3982.0 + - 129.2 Maloney Genestin ( 4 ) Not detected 4033.1 + - 156.5 Total isoflavone - 11951.3 ± 443

The preparation examples for the composition of the present invention are illustrated below.

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

The isoflavone derivatives of the present invention,

0.1 g of the extract or the fraction of the extract

Lactose 1.5 g

Talc 0.5 g

The above ingredients were mixed and filled in an airtight container to prepare powders.

<1-2> Manufacture of tablets

The isoflavone derivatives of the present invention,

0.1 g of the extract or the fraction of the extract

Lactose 7.9 g

Crystalline cellulose 1.5 g

0.5 g of magnesium stearate

After mixing the above ingredients, tablets were prepared by direct tableting method.

<1-3> Preparation of capsules

The isoflavone derivatives of the present invention,

0.1 g of the extract or the fraction of the extract

Corn starch 5 g

4.9 g of carboxycellulose

After mixing the above components to prepare a powder, the powder was filled in a hard capsule according to a conventional preparation method of a capsule to prepare a capsule.

&Lt; 1-4 > Preparation of injection

The isoflavone derivatives of the present invention,

0.1 g of the extract or the fraction of the extract

Sterile sterilized water for injection

pH adjuster

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

<1-5> Liquid  Produce

The isoflavone derivatives of the present invention,

0.1 g of the extract or the fraction of the extract

10 g per isomer

5 g mannitol

Purified water quantity

Each component was dissolved in purified water in accordance with a conventional method for producing a liquid agent, and the lemon flavor was added in an appropriate amount, followed by mixing the above components. Then, purified water was added to adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

< Manufacturing example  2> Manufacture of health food

<2-1> Mixed car  Produce

The isoflavone derivatives of the present invention were mixed with high-content bean stalks, herb extracts or fractions of herb extracts and flavor herb plants to prepare mixed tea.

<2-2> Production of flour food

Bread, cakes, cookies, crackers and noodles were prepared by adding 0.5 to 5.0 parts by weight of the high content isoflavone derivatives of the present invention to the wheat flour and adding the mixture to the flour.

<2-3> soup  And juicy ( gravies )

0.1 to 5.0 parts by weight of a high content of an isoflavone derivative of the present invention, a bean stem, an extract thereof, or an extract fraction was added to soups and juices to prepare health promotion meat products, noodle soups and juices.

<2-4> Ground Beef  Produce

A ground beef for health promotion was prepared by adding 10 parts by weight of the high content isoflavone derivative of the present invention, bean stem, its extract or the fraction of the extract to ground beef.

<2-5> Dairy products ( dairy products )

5 to 10 parts by weight of a high content isoflavone derivative of the present invention soybean stem, its extract or a fraction of the extract was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-6> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were alfalted by a known method, dried and dispersed, and granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a known method, and then they were prepared into powders having a particle size of 60 mesh by a grinder.

The isoflavone derivative high-content soybean stem, its extract or the fraction of the extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and dried with a hot-air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds, and the high content isoflavone derivatives of the present invention were prepared by blending the bean stalks, the extracts or the fractions of the extracts in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

(3 parts by weight) of the high content isoflavone derivative of the present invention, the extract or extract fraction thereof,

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  3> Manufacture of health drinks

The isoflavone derivatives of the present invention,

100 mg of the extract or the fraction of the extract

Citric acid 100 mg

100 mg of oligosaccharide

2 mg of plum concentrate

100 mg taurine

Purified water was added to 500 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered to obtain a sterilized 1 liter container which was sealed and sterilized, &Lt; / RTI &gt;

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

< Formulation example  4> Cosmetics  Preparation of composition

<4-1> Preparation of cream

The isoflavone derivatives of the present invention,

4.6 parts by weight of the extract or the fraction of the extract

Cetostearyl alcohol 2.8 parts by weight

Beeswax 2.6 parts by weight

Stearic acid 1.4 parts by weight

Glycerin monostearate 2 parts by weight

&Lt; tb &gt; &lt; tb &gt;

Sorbitol sesquioleate 1.4 parts by weight

Jojoba oil 4 parts by weight

Squalane 3.8 parts by weight

Polysorbate 60 1.1 parts by weight

Macadia oil 2 parts by weight

Tocopheryl acetate 0.2 part by weight

Methylpolysiloxane 0.4 part by weight

Ethylparaben 0.1 part by weight

Propylparaben 0.1 part by weight

Euxyl K-400 0.1 part by weight

1,3-butylene glycol 7 parts by weight

Methylparaben 0.05 part by weight

Glycerin 6 parts by weight

d-Pandenol 0.2 part by weight

Triethanolamine 0.2 part by weight

pt 41891 0.2 part by weight

p-H20 46.05 parts by weight

<4-2> Production of Lotion

The isoflavone derivatives of the present invention,

3.5 parts by weight of the extract or the fraction of the extract

Cetostearyl alcohol 1.6 parts by weight

Stearic acid 1.4 parts by weight

Glycerin monostearate of pro-type 1.8 parts by weight

&Lt; tb &gt; &lt; tb &gt;

Sorbitol sesquioleate 0.6 parts by weight

Squalene 4.8 parts by weight

Macadia oil 2 parts by weight

Jojoba oil 2 parts by weight

Tocopheryl acetate 0.4 part by weight

0.2 part by weight of methylpolysiloxane

Ethylparaben 0.1 part by weight

Propylparaben 0.1 part by weight

4 parts by weight of 1,3-butylene glycol

0.1 part by weight of methylparaben

Xanthan gum 0.1 part by weight

Glycerin 4 parts by weight

d-Pandenol 0.15 part by weight

Allantoin 0.1 part by weight

Cargar (2% aq. Sol) 4 parts by weight

Triethanolamine 0.15 part by weight

Ethanol 3 parts by weight

pt 41891 0.1 part by weight

p-H20 48.3 parts by weight

Claims (19)

Production of a high content of isoflavone derivative represented by the following formulas (2) to (7), comprising treating the soybean stem with ethylene, ethylene donor or ethylene generator at a concentration of 5 to 1000 ppm Way:
(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

.
The method according to claim 1, wherein the bean stem is a bean stalks 10 to 90 days after sowing, wherein the bean stalks are represented by the following formulas (2) to (5).
3. The method of claim 1, wherein the ethylene donor is ethephone. 2. The method according to claim 1, wherein the ethylene donor is ethephone.
2. The method according to claim 1, wherein the ethylene, ethylene donor or ethylene generator is treated at a concentration of 5 to 1000 ppm, wherein the isoflavone derivative is represented by the following formulas (2) to Way.
2. The method according to claim 1, wherein the isoflavone isoflavone derivative represented by the following formulas (2) to (5), which further comprises a step of treating beta-glucosidase, Gt;
The isoflavone derivative according to claim 5, wherein the soybean stem further comprises an isoflavone isoflavone derivative represented by the following formula (6) or (7) Production method of high content soybean stem:
[Chemical Formula 6]

(7)

A high-content soybean stem of an isoflavone derivative represented by the following formulas (2) to (5) prepared by the method of claim 1 or 5.
1) treating soybean stem with ethylene, ethylene donor or ethylene generator at a concentration of 5 to 1000 ppm to prepare bean stem; And
2) extracting an isoflavone derivative represented by the following formulas (2) to (5), which comprises extracting an isoflavone derivative represented by the following formulas (2) to (5) Mass production method.
A composition comprising a high content of an isoflavone derivative represented by the following general formulas (2) to (5), a soybean stem, an extract of the soybean stem, or a fraction of the extract.
An isoflavone derivative represented by the following formulas (2) to (5): (a) a high-content soybean stem, an extract of the bean stem or a fraction thereof;
11. The health food according to claim 10, which is used for prevention and improvement of any one selected from the group consisting of osteoporosis, heart disease, breast cancer, external quality disease, hyperlipidemia, skin aging and facial flushing.
delete delete delete delete delete A cosmetic composition comprising a high content of an isoflavone derivative represented by Formulas (2) to (5) of Claim 7, an extract of bean stem, or a fraction of the extract as an active ingredient.
delete An isoflavone derivative represented by the following formulas (2) to (5): (Tea) with a high content of soybean stem.

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