KR101954770B1 - A pharmaceutical composition for treating and preventing liver demage and a functional food for protecting liver comprising the soybean sprout extract by high hydrostatic pressure - Google Patents

Abstract

The present invention relates to a health functional food for liver protection comprising an extract of bean sprouts treated with ultra high pressure, a pharmaceutical composition for prevention and treatment of liver diseases, and a method for extracting bean sprouts treated with ultra high pressure. The extract of bean sprouts of the present invention, Compared with extracts and bean sprouts juices, asparagine is significantly extracted and at the same time isoflavone content, particularly the amount of extract of non-glycosylated isoflavone, which is easily absorbed, is high, and hepatocyte growth inhibition activity by hydrogen peroxide is excellent in HepG2 cells.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating and preventing liver diseases containing an ultra-high pressure processed soybean sprout extract, and a pharmaceutical composition for protecting liver from hepatic diseases and a functional food for protecting the liver from sprout extract by high hydrostatic pressure.

The present invention relates to a health functional food for liver protection comprising a natural plant extract as an active ingredient, or a pharmaceutical composition for the treatment and prevention of liver diseases.

Bean sprout is a germinated soybean which can be easily grown in a short time regardless of the season, making it an economical and nutritional supple food. The color of bean sprouts is white or light yellow, and it contains a lot of vitamin B 1, vitamin B 2 and vitamin C.

As a prior art that includes bean sprouts in some compositions, Korean Patent Publication No. 2011-0108686 discloses antioxidant activity of extracts obtained by treating a complex of natural plant extracts containing glucosidase as one of the alternative components of soybean sprout extract Korean Patent No. 098109 discloses a food composition for removing hangover which comprises an extract of more than a dozen kinds of natural plants including calcium compounds and bean sprouts. In Korean Patent No. 0504351, other plant extracts Together with a bean sprout juice in some compositions.

As a prior art that includes bean sprouts as a main constituent, Korean Patent Registration No. 0989869 discloses a hangover relieving effect of a fermented soybean sprout which is naturally fermented by adding rice extract and sugar to bean sprouts.

Thus far, the prior art has been used as a simple extract of raw bean sprouts, hot water of dried bean sprouts, or alcohol, and has not been shown to have sufficient hepatoprotective effect only by bean sprouts juice or bean sprouts extract. Therefore, In the present invention, it has been found that a component having an effect on liver protection contained in bean sprouts can be extracted through ultrahigh pressure to achieve a remarkably excellent liver protecting effect, thus completing the present invention.

It is an object of the present invention to provide a health functional food for liver protection containing an extract of bean sprouts treated with ultra-high pressure as an active ingredient.

It is also an object of the present invention to provide a pharmaceutical composition for preventing and treating liver diseases, which comprises an extract of bean sprouts of ultra-high pressure as an active ingredient.

The present invention also provides a method for extracting bean sprouts of ultrahigh pressure.

The present invention provides a health functional food for liver protection comprising an extract of bean sprouts treated with ultra high pressure as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for prevention and treatment of liver disease, which comprises an extract of bean sprouts of ultra-high pressure as an active ingredient.

In the present invention, it is preferable that the bean sprouts are treated at a pressure of 200 to 600 MPa, preferably 400 to 550 MPa, at a temperature of 15 to 35 DEG C for 1 to 30 minutes, preferably 2 to 10 minutes. When the ultrahigh pressure treatment pressure is lower than the above range, the extraction amount of asparagine and non-glycoside isoflavone is lowered and the hepatocyte protective activity is lowered. When the ultrahigh pressure treatment pressure exceeds the above range, the extraction amount of the asparagine and the non-glycoside isoflavone does not increase any more, and the hepatocyte protective activity is slightly lowered. When the ultra-high pressure treatment temperature is lower than the above range, the extraction amount of asparagine and non-glycoside isoflavone is lowered, and when the upper limit is exceeded, the hepatocyte protective activity is lowered. It is difficult to control the ultrahigh-pressure treatment time constantly below the above-mentioned range, and when the above-mentioned range is exceeded, the extraction amount of asparagine and non-glycoside isoflavone is no longer increased.

In the present invention, the extract of bean sprouts of ultra-high pressure is an extract of water, a lower alcohol having 2 to 4 carbon atoms or an alcohol aqueous solution thereof, preferably water or ethanol, because it can be used as a raw material for food without an additional solvent removal step . Extract preparation is carried out at 15 to 35 DEG C, preferably at 15 to 25 DEG C for 0.5 to 24 hours, preferably 1 to 6 hours. Since the bean sprouts tissues are changed to be easily extracted through the ultra-high pressure treatment, the extraction amount of asparagine is increased as compared with the conventional hot water extraction or alcohol reflux extraction under the above conditions.

The liver protection effect or the liver disease treatment and prevention effect of the present invention can be interpreted as a result of an increase in the content of the asparagine, the non-glycoside isoflavone, or the ingredient not specified in the present invention contained in the extract.

The pharmaceutical dosage form of the pharmaceutical composition for the treatment and prevention of liver diseases may be used in the form of a pharmaceutically acceptable salt thereof and may be used alone or in combination with other pharmacologically active compounds as well as in a suitable aggregate form.

The pharmaceutical composition for the treatment and prevention of liver diseases according to the present invention can be administered orally or parenterally in the form of oral preparations such as powders, granules, suspensions, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, And may contain suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or the excipient or diluent includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a commonly used filler, a weight agent, a binder, a wetting agent, a disintegrant or a surfactant may be used.

Solid preparations for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the above extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, 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. Witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used as a base for suppositories.

The preferred dosage of the pharmaceutical composition for treating and preventing liver disease according to the present invention may be appropriately selected by those skilled in the art depending on the condition of the patient, the weight, the degree of disease, the drug form, the administration route and the period. However, for a desired effect, the dose may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for the treatment and prevention of liver diseases according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In addition, the health functional food showing the liver protecting effect of the present invention can be used as it is or can be used in combination with other food or food ingredients, and can be appropriately used according to a conventional method. The amount of the active ingredient to be mixed may be suitably determined according to each use purpose such as prevention, health, or treatment.

Generally, the extract of bean sprouts of ultra-high pressure according to the present invention can be added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material. 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. Further, since the present invention uses an extract from a natural product, Or more.

There is no particular limitation on the form or type of the health functional food. Examples of the food to which the above-mentioned substance can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, , Various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes, and may include foods in the conventional sense.

The beverage food of the health functional food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the functional food according to the present invention.

In addition to the above, the health functional food for liver protection according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, , Glycerin, alcohol, carbonated beverages. In addition, the liver-protecting health functional food of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the composition of the present invention.

Since the extract of bean sprouts of the present invention has a high abundance of asparagine and a high content of isoflavone, particularly an extractable amount of unglycosylated isoflavone, which is easily absorbed, and an excellent hepatocyte-inhibitory activity by hydrogen peroxide in HepG2 cells, A liver-protecting food composition or a pharmaceutical composition for the treatment and prevention of liver disease.

FIG. 1 is a chromatogram of an amino acid analysis chromatogram of Experimental Example 1, showing the peak of asparagine used as a standard substance in a chromatogram.
FIG. 2 is a graph showing the results of the isoflavone analysis of Example 2, in which daidzin (DI) and genistin (GI), which are used as standards, and daidzein (DE) and genistein GE).
FIG. 3 is a graph showing the survival rate of HepG2 cells in each group in Experimental Example 3 as a relative cell viability relative to the group not treated with hydrogen peroxide.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent, however, to those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereby.

Example  1: Manufacture of ultra high pressure bean sprouts extract

The bean sprouts were germinated with 'Junjiruri' varieties and grown in the usual manner for 7 to 10 days and grown to 5 to 6 cm.

100 g of the bean sprouts were ground with a blender and treated at a high temperature of 20 ° C and 500 MPa for 3 minutes. 400 ml of water was added to the ultra-high pressure treated bean sprouts, followed by extraction at room temperature for 3 hours at 20 ° C. And the extract was taken and the extract powder of bean sprouts treated with ultrahigh pressure was prepared using a freezer vacuum dryer.

Comparative Example  1: bean sprouts Heat number  Preparation of extract

400 mL of hot water at 95 ° C was added to 100 g of the same soybean sprout as in Example 1, and the mixture was extracted for 6 hours. The extract was centrifuged and filtered to obtain an extract, and a hot-water extract powder of soybean sprouts was prepared using a freeze-dried vacuum dryer.

Comparative Example  2: Preparation of ethanol extract of bean sprouts

400 mL of 70% ethanol was added to 100 g of the same soybean sprout as in Example 1, and the mixture was extracted at 50 DEG C for 3 hours. The extract was centrifuged and filtered, and an extract was obtained and ethanol extract powder of soybean sprouts was prepared using a freeze- .

Experimental Example  1: Analysis of asparagine content

Since asparagine is known as an amino acid which helps hangover relief, the asparagine content of the extracts of Example 1 and Comparative Examples 1 and 2 was analyzed and compared by applying the methods described in the following documents. (Reference: Food and Drug Administration. 20. Food & Drug Specialization Notice No. 2012-1).

Amino acid content changes were analyzed using an automatic amino acid analyzer (HITACH L-8900). Standard solutions were prepared by diluting 2 mL of each sample with 0.02 N HCl from Wako Standard Amino Acid Mixture Standard Solution Type AN II and Type B, respectively. For the preparation and analysis of the test solution, samples and 16% trichloroacetic acid solution were added in the same amount, followed by shaking for 15 minutes, centrifugation at 3000 rpm for 15 minutes, and using the supernatant for analysis. The analytical column was used with a column for physiological Fluids Analysis # 2622 4.6 × 60 mm. Figure 1 is a chromatogram of the column, showing the peak of asparagine used as a reference material in chromatogram.

division Example 1 Comparative Example 1 Comparative Example 2 Asparagine (mg / 100g) 3652.8 ± 22.5 147.2 ± 31.5 666.4 ± 18.6

It can be seen that Example 1 contained significantly more asparagine extracted from the same amount of soybean sprouts than the Comparative Example, and the hot-water extracts and ethanol extracts of Comparative Examples 1 and 2 showed almost no difference.

Experimental Example  2: Analysis of isoflavone content

The asparagine contents of the extracts of Example 1 and Comparative Examples 1 and 2 were analyzed and compared by applying the methods described in the following documents (Reference: Food and Drug Administration, November 17, 2011. Standards and Specifications of Health Functional Foods 2011-68).

Isoflavones were extracted with methanol and analyzed using Agilent 1100 series. Quantitation was performed using Shiseido capcell pack C18 column 4.6 × 250 mm at 260 nm, the maximum absorption wavelength. For the preparation and analysis of the test solution, 0.5 g of the sample was dissolved in methanol in a 50 mL volumetric flask, and then ultrasonicated for 30 minutes and filtered through a 0.45 μm membrane filter. The mobile phase was used as solvent A (water: methanol: acetic acid = 88: 10: 2) and solvent B (methanol: acetic acid = 92: 2). Figure 2 is a chromatogram of the column showing that daidzin (DI) and genistin (GI), a glycoside used as a reference material, and daidzein (DE) and genistein (GE) ≪ / RTI >

division Example 1 Comparative Example 1 Comparative Example 2 DI (μg / g) 119.2 ± 28.5 12.8 ± 3.6 14.2 ± 2.6 GI (μg / g) 288.9 ± 33.2 16.0 + - 5.4 17.3 ± 4.5 DE (μg / g) 144.2 ± 15.6 55.6 ± 12.1 57.4 ± 14.91 GE (μg / g) 233.1 + - 42.1 182.8 ± 35.0 188.5 ± 32.2 Total isoflavone (μg / g) 785.4 267.2 277.4 Total non-glycosides (μg / g) 377.3 238.4 245.9

It can be seen that the content of isoflavone is 2.8 to 2.9 times more extracted and contained in Example 1 than those of Comparative Examples 1 and 2 and that the content of isoflavone is remarkably high even in the content of non-glycosides which is easily absorbed in vivo .

Experimental Example  3: Liver damage  Identify inhibitory activity

The liver damage inhibitory effect of the extracts of Example 1 and Comparative Examples 1 and 2 was measured by applying the method described in the literature as follows (Mba Gachou, C. and Dumenil, G. 1999. The protective activity of α- HepG2 cells by alkaline comet assay. Mutation Res./Genetic Toxicology and Environmental Mutagenesis 445 (1): 9-20).

First, HepG2 cells were inoculated into a 24-well plate at 5 × 10 ⁴ cells / well in 1 mL MEM (Minimum Essential Medium, MEM, Hyclone, 01486) (ATCC, Manassas VA USA) . The medium (Minimum Essential Medium (MEM), Hyclone, 01486) containing 3% serum containing the sample was added to each well, and after 24 hours of culture, was reacted in an incubator at 37 ℃, remove the medium and the given contain 0.3 mM H ₂ O ₂ for The medium was incubated for 24 hours at 37 ° C in a medium containing 3% serum (Minimum Essential Medium (MEM), Hyclone, 01486), and the medium was removed and XTT-PMS solution (1 mg XTT (Sigma, P4625) / 10 g PMS (phenazine methosulfate, Sigma, P9625) / mL were added for 2 hours, and the protection effect from the liver damage by hydrogen peroxide was evaluated by the degree of formation of formazan Was measured using a microplate reader (BIO-TEK, 1010-1) at 450 nm using an instrument (BIO-TEK, 1010-1). The absorbance was measured using 0.3 mM H ₂ O ₂ And the negative control group was represented by the relative absorbance of the group treated with only the culture medium instead of the sample. Each of the samples of the examples and the comparative examples was added at 300 μg / ml, and 0.3 mM H ₂ O ₂ , And the positive control group was the relative absorbance A page acid as the relative absorbance of what was added to 40 μg / ml, and treated the 0.3 mM H ₂ O _2, it is shown in Table 3 and Fig.

division Negative control group Positive control group Example 1 Comparative Example 1 Comparative Example 2 Relative absorbance (%) 25.0 + - 2.4 ^a 76.1 ± 5.9 ^d 63.0 + - 3.5 ^c 41.3 ± 6.5 ^b 39.5 ± 4.4 ^b

As a result, it was confirmed that the extract of bean sprouts of Example 1 had a strong protective effect against hepatocyte injury by hydrogen peroxide compared with the negative control treated with hydrogen peroxide alone as well as Comparative Examples 1 and 2.

Experimental Example  4: Toxicity of hepatocytes

To determine the cytotoxicity of the extracts of Example 1 and Comparative Examples 1 and 2, the method of Rochem et al. Was modified as described below (Dai, Y. and AI Cederbaum 1995. Cytotoxicity of acetaminophen in human cytochrome P4502E1- transfected HepG2 cells, J. Pharmacol . Exp . Ther., 273: 1497-1505).

The medium (Minimum Essential Medium (MEM), Hyclone, 01486) was used for 24 hours after dispensing 2 × 10 ⁵ cells / well of HepG2 cell line (ATCC, Manassas VA USA) into a 24 wells plate for 24 hours. After the primary culture was completed, the culture medium (Minimum Essential Medium (MEM), Hyclone, 01486), fetal bovine serum, purchased from Hyclone and KTJ32092 (FBS) containing 3% 1 mL was dispensed and the re-culture was repeated for 5 days. On the 7th day after the incubation, 0.25 mL of XTT-PMS solution (1 mg XTT (XTT Sodium Cell Culture Test, Sigma, X4626) and 10 g PMS (phenazine methosulfate, , P9625) / mL of MEM (Minimum Essential Medium (MEM), Hyclone, 01486) mixed solution) was added and cultured again for 2 hours. The degree of cytotoxicity of the sample was measured by measuring the absorbance at 450 nm using a microplate reader (BIO-TEK) using a BIO-TEK instrument for the formation of formazan.

As a result, it was confirmed that the extracts of Comparative Examples 1 and 2 as well as the bean sprout extract of Example 1 of the present invention did not exhibit cytotoxicity up to a concentration of 1000 μg / mL.

Claims (11)

delete delete delete delete delete delete delete delete Treating the bean sprouts at an ultra-high pressure of 200 to 600 MPa, 15 to 35 DEG C for 1 to 30 minutes; And
And extracting the bean sprouts treated with ultrahigh pressure with water, a lower alcohol having 2 to 4 carbon atoms, or an aqueous alcohol solution containing these at 15 to 35 DEG C for 0.5 to 24 hours. Treating the bean sprouts at an ultra-high pressure of 200 to 600 MPa, 15 to 35 DEG C for 1 to 30 minutes; And
And extracting the bean sprouts treated with ultrahigh pressure at 15 to 35 ° C for 0.5 to 24 hours with water, a lower alcohol having 2 to 4 carbon atoms, or an aqueous alcohol solution containing them, for 0.5 to 24 hours.
Treating the bean sprouts at an ultra-high pressure of 200 to 600 MPa, 15 to 35 DEG C for 1 to 30 minutes; And
And extracting the bean sprouts treated with ultrahigh pressure at a temperature of 15 to 35 ° C for 0.5 to 24 hours with water, a lower alcohol having 2 to 4 carbon atoms or an alcohol aqueous solution containing them, for 0.5 to 24 hours. A method for producing a health functional food.

Images