KR101906984B1 - A pharmaceutical composition for treating and preventing liver demage and a functional food for protecting liver comprising the soybean sprout extract by enzyme treatment - Google Patents

Abstract

The present invention relates to a health functional food for liver protection comprising an enzyme-treated bean sprouts extract as an active ingredient, a pharmaceutical composition for prevention and treatment of liver disease, and an enzyme-treated bean sprouts extract method, wherein the enzyme-treated bean sprouts extract of the present invention By extracting at room temperature, asparagine is extracted much more than ordinary bean sprouts extract or soybean soup juice, and at the same time, HepG2 cells are excellent in inhibiting liver damage by hydrogen peroxide.

Description

[0001] The present invention relates to a pharmaceutical composition for the treatment and prevention of liver diseases containing an enzyme-treated bean sprouts extract, and a pharmaceutical composition for preventing liver diseases,

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 exhibiting an effect on liver protection contained in bean sprouts can be obtained at a room temperature simultaneously with enzyme treatment, thereby achieving a remarkably excellent liver protecting effect.

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

It is also an object of the present invention to provide a pharmaceutical composition for prevention and treatment of liver disease comprising an enzyme-treated bean sprouts extract as an active ingredient.

The present invention also provides an enzyme-treated bean sprouts extraction method.

The present invention provides a health functional food for liver protection containing an enzyme-treated bean sprouts extract as an active ingredient.

The present invention also provides a pharmaceutical composition for prevention and treatment of liver diseases, which comprises an enzyme-treated bean sprouts extract as an active ingredient.

In the present invention, the enzyme treatment is performed with 1 to 10 parts by weight of pectinase per 100 parts by weight of bean sprouts. Extraction is carried out at room temperature in the state of mixing bean sprouts and enzymes, instead of performing extraction after enzymatic treatment. The pectinase is preferably a multifect pectinase FE extracted from an Aspergillus niger culture.

In the present invention, the enzyme-treated bean sprouts extract 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 into an easily extractable state through enzyme treatment, the extraction amount of asparagine is increased in comparison with ordinary 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.

The enzyme-treated bean sprouts extract of the present invention can be utilized as a food composition for liver protection or a pharmaceutical composition for the prevention and treatment of liver diseases, since a significant amount of asparagine is extracted and at the same time, the hepatic damage-inhibiting activity by hydrogen peroxide is excellent in HepG2 cells.

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 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 pulverized with a blender and then 20 g of multifect pectinase FE enzyme (Danisco, Denmark, 24000 U / g) was added at a temperature of 20 ° C. After adding 400 mL of water, The extract was centrifuged and filtered to obtain an extract, and an enzyme-treated bean sprout extract powder was prepared using a freeze-dried 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) 3434.9 ± 31.4 147.2 ± 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: 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 61.0 + - 6.5 ^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  3: 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 (9)

Characterized in that an enzyme-treated bean sprouts extract is used as an active ingredient and the enzyme treatment is performed with 1 to 10 parts by weight of pectinase per 100 parts by weight of bean sprouts.
delete The health functional food for liver protection according to claim 1, wherein the extract is an extract of water, a lower alcohol having 2 to 4 carbon atoms, or an alcohol aqueous solution thereof.
4. The health functional food for liver protection according to claim 3, wherein the extract is an extract extracted at 15 to 35 DEG C for 0.5 to 24 hours.
A pharmaceutical composition for the prevention and treatment of liver disease, comprising an extract of an enzyme-treated bean sprout as an active ingredient and treating the enzyme with 1 to 10 parts by weight of pectinase per 100 parts by weight of the bean sprouts.
delete [Claim 5] The pharmaceutical composition according to claim 5, wherein the extract is an extract of water, a lower alcohol having 2 to 4 carbon atoms, or an alcohol aqueous solution thereof.
[Claim 7] The pharmaceutical composition for preventing and treating liver diseases according to claim 7, wherein the extract is an extract extracted at 15 to 35 DEG C for 0.5 to 24 hours.
Mixing 1 to 10 parts by weight of pectinase with 100 parts by weight of bean sprouts; And
And extracting the bean sprouts mixed with the pectinase with water, a lower alcohol having 2 to 4 carbon atoms or an alcohol aqueous solution containing them at 15 to 35 DEG C for 0.5 to 24 hours.

Images