KR20190018175A - The Black Ginseng extract including alcoholic liver injury prevention functional ingredients and method for manufacturing it - Google Patents

Abstract

A purpose of the present invention is to provide a method for extracting an extract from a black ginseng which contains a functional component for preventing alcoholic liver damage. More specifically, a manufacturing method of a black ginseng extract according to the present invention comprises steps of: putting a primary steamed and dried ginseng into a drying and steaming device; rapidly raising the temperature up to 60-70°C at room temperature and maintaining for 10 minutes; raising the temperature up to the target temperature (90-98°C) and manufacturing the black ginseng extract by repeating a steaming process for 3-5 times that each process takes 2-5 hours. Therefore, it is possible to increase a yield of total ginsenoside which can be obtained from the black ginseng, and thus can enhance an effect of preventing liver cell damage by having an increased yield of the total ginsenoside and antioxidant potency. Furthermore, the manufacturing method of the black ginseng extract is more effective in terms of time, economy and efficiency than a manufacturing method of nine times steaming and drying.

Description

Technical Field [0001] The present invention relates to a black ginseng extract containing a functional ingredient for preventing alcoholic liver damage and a method for preparing the same,

It is an object of the present invention to provide a method for extracting an extract containing an alcoholic liver injury-preventing functional ingredient from black ginseng.

Korean ginseng has a historical record of about 1,000 years ago and is now a unique traditional traditional medicinal herb or health functional food, which is shaped by a global market size of about $ 3.5 billion and domestic venture companies can enter the global market. Ginseng is a perennial semisynthetic oily plant belonging to the acanthaceae family. Its efficacy has been widely known in various fields of China and Korea and has been used as herbal medicine in many fields since the past. The ingredients of ginseng include carbohydrate (60 ~ 70%), (12 to 16 percent), saponin (3 to 6 percent), fat soluble ingredients (1-2 percent), ash (4-6 percent) and vitamins (0.005 percent). The physiological activity of ginseng has recently been systematically pharmacologically approached, and it has been recognized that the ginseng root effect, sexual function and improvement of reproductive dysfunction, anti-hypertension and anti-arteriosclerosis effect, hematopoietic function and anemia treatment effect, blood glucose metabolism and diabetes improvement effect, Improvement of dysfunction, hangovers effect, prevention of parasitic infection, and analgesic action. It is widely known that ginseng contains a large amount of ginsenoside, and it has recently been used as a food for health food by applying it to foods in recent years.

The ginseng saponin is an alcoholic OH of protopanaxadiol (protopanaxadiol or protopanaxatriol) of R1, R2 and R3, which is known as ginseng saponin. The active ingredient in ginseng is generally known as ginseng saponin The present invention relates to a compound having a structure in which saccharides such as glucose, rhamnose, xylose and arabinose are ester-linked, and more than 30 kinds of compounds have been found to date. These compounds have anticancer activity, antidiabetic activity, inhibition of central nervous system, It is known that the compound exhibits a pharmacological activity effect such as prevention, promotion of liver function and elimination of hangover, anti-fatigue and anti-stress action, antioxidative action, anti-inflammatory activity, protein synthesis promoting action and immunity enhancing action.

These ginseng saponins are generally known to exist in ginseng, but it is known that certain ginseng saponin exists only in processed ginseng. In recent years, many studies have been conducted to further increase the content of ginseng saponin present only in these processed ginseng. As a result, processed ginseng such as taeguk ginseng (taeguk ginseng), black ginseng (black ginseng) . In particular, black ginseng means that the ginseng has turned black after a few times of boiling and drying, and it has been found that not only the ginseng saponin which is basically present in the course of several times of proliferation and drying is lost, It is known that the content of ginseng saponin is increased. Therefore, research on a method for more effectively producing such ginseng is being actively carried out. The problem of the black ginseng is that the content of the ginseng saponin component contained in the black ginseng does not show a remarkable difference from that of the red ginseng. Therefore, the content of the ginseng saponin component contained in the black ginseng, especially the content of the ginseng saponin component present only in the processed ginseng Can be significantly increased compared to that of red ginseng, it is expected that the above-mentioned problem of black ginseng can be solved, but no achievement has been reported yet.

Disclosure of the Invention The object of the present invention is to provide a method for efficiently producing black ginseng extract. More specifically, by extracting an extract from black ginseng, the total content of ginsenoside is increased, And to provide a black ginseng extract and an extraction method. It is another object of the present invention to provide a black ginseng extract having a hepatocyte protective effect against alcohol and a method for producing the same.

 In order to accomplish the above object, the present invention provides a method for extracting black ginseng extract comprising an alcoholic liver injury-preventing functional ingredient, comprising the steps of: a) drying ginseng; b) raising the temperature of the dried ginseng at 60 to 70 ° C at room temperature; c) raising the temperature of the ginseng steamed at 60 to 70 ° C to 70 to 120 ° C to reflux; d) repeating steps b) and c) three to five times to produce black ginseng; And e) extracting the black ginseng extract from the black ginseng using solvent extraction.

The present invention also provides a black ginseng extract prepared by the above-mentioned method.

The present invention also provides a pharmaceutical composition for prevention or treatment of liver injury comprising the black ginseng extract prepared by the above-mentioned method.

The present invention also provides a health functional food composition for prevention or improvement of liver damage comprising a black ginseng extract prepared by the above-described method.

According to the present invention, the ginseng extract of the present invention is prepared by firstly adding ginseng to a boil, heating the mixture rapidly at 60 to 70 ° C at room temperature, holding it for 10 minutes, raising the temperature to 90 to 98 ° C for 2 to 5 hours, Is repeated three to five times, it is possible to improve the yield of total ginsenosides obtained from black ginseng. In addition, the total ginsenosides are improved in yield and antioxidant ability, so that the effect of preventing damage of hepatocytes can be enhanced. Furthermore, it is more effective in terms of time, economy and efficiency than the method of manufacturing corduroy.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram for producing black ginseng according to the present invention. FIG.
2 is UPLC-QTOF / MS comparison data of Example 1, Comparative Example 1 and Comparative Example 2. Fig.
FIG. 3 is a graph showing blood concentrations of OT / AST, GPT / ALT, LDH and SOD after ethanol administration after administration of the black ginseng extract or pretreatment.
FIG. 4 is a bar graph showing the liver and spleen weights after administration of ethanol extract and administration of black ginseng extract.

Hereinafter, preferred embodiments of the present invention will be described in detail. It should be understood, however, that the invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. It should be noted that the embodiments of the present invention described below are intended to sufficiently convey the spirit of the present invention to those skilled in the art.

According to an embodiment of the present invention, there is provided a method of preparing ginseng comprising: a) drying ginseng; b) raising the temperature of the dried ginseng at 60 to 70 ° C at room temperature; c) raising the temperature of the ginseng steamed at 60 to 70 ° C to 70 to 120 ° C to reflux; d) repeating steps b) and c) three to five times to produce black ginseng; And e) extracting the black ginseng extract from the black ginseng using solvent extraction.

In the present invention, the step b) may be performed for 1 minute to 60 minutes, preferably 1 minute to 30 minutes, and most preferably 10 minutes.

In the present invention, the step c) may be carried out for 1 hour to 10 hours, preferably 1 hour to 8 hours, and most preferably 2 hours to 5 hours.

In the present invention, the extraction solvent is selected from the group consisting of water, ethyl acetate (EtOAc), alcohols having 1 to 4 carbon atoms, butanol having 1 to 4 carbon atoms, and mixtures thereof. , And most preferably ethyl acetate (EtOAc).

The black ginseng extract according to the present invention is composed of ginsenosides Rk1, Rh4, Rg5, F4, Rk3, Rg3-S, Rg3-R, Rg4, MC, compound Y, compound O, Rh3, Rk2, F2, Any one component selected from the group can be increased compared to the extract of corn guppies and preferably can be ginsenosides Rk1, Rh4, Rg5, F4 and Rg3-S.

As another example of the present invention, the black ginseng extract prepared by the above-mentioned contents can be provided.

Another example of the present invention is to provide a pharmaceutical composition for preventing or treating liver damage comprising the black ginseng extract prepared by the above-mentioned contents. The present invention relates to a health supplement containing a black ginseng extract rich in ginsenoside and having high hepatic cell protection effect, wherein the health supplement is provided as a liquid beverage formulation . As an example of the health supplements, the black ginseng extract may be pulverized using a method such as spray drying, and the powder may be provided as one or more solid formulations selected from tablets, pills, powders, tablets and capsules .

Example 1. Preparation of black ginseng extract

The washed ginseng is dried in a hot air dryer at 40 ° C for 3 to 10 hours. The first ginseng and dried ginseng were put into a steamer and rapidly heated to 60 ~ 70 ℃ at room temperature and maintained for 10 minutes. The temperature was raised to the target temperature (90 ~ 98 ℃) and the ginseng was repeated 3-5 times for 2 ~ 5 hours. Was repeatedly extracted three times at room temperature while immersed in an aqueous 80% MeOH solution for 24 hours, and the resulting filtrates were combined and concentrated under reduced pressure to obtain a MeOH extract. The obtained MeOH extracts were compared using thin layer chromatography (TLC), and the extracts were combined to obtain a total of 300 g of a concentrate. The concentrated extract was partitioned three times with EtOAc (3 L) / H ₂ O (3 L), and the H ₂ O layer was partitioned three times with n-BuOH (2.7 L). The layers were concentrated under reduced pressure to obtain an EtOAc fraction, an n-BuOH fraction and an H ₂ O fraction. Of these, the EtOAc fraction was used as Example 1.

Example 2 Isolation of Active Ingredient from Black Ginseng Extract

The ethyl acetate distribution extract obtained in Example 1 was purified by SiO2 column chromatography (SiO2 cc) (φ14 × 16 cm, CHCl3-MeOH-H2O = 15: 3: 1) → CHCl3 -MeOH-H 2 O = 7: 3: 1), and 100 ml portions thereof were aliquoted to obtain an aliquot. The aliquots were identified by TLC (CHCl3-MeOH-H2O = 9: 3: 1) and similar fractions were pooled together and concentrated using a vacuum concentrator to yield 10 fractions (BGE1 to BGE10).

Thereafter, the 10 fractions were used to separate and purify the compound.

(1) Octadecyl Silicas column chromatography (ODS cc, manufactured by Waters; Φ 4 × 9 cm, MeOH-1) was applied to the first fraction (BGE, 1 g) H2O = 3: 1) to give a total of five fractions (BGE1-1 to BGE1-5).

The first fraction (BGE1-1-1, 300 mg) of the five fractions was subjected to ODS column chromatography (Φ 2.5 × 7 cm, MeOH-H2O = 3: 1) to give a total of 8 fractions (BGE1-1 -1-1 to BGE1-1-1-8) were finally obtained. Compound O (8 mg), Rg4 (11 mg) and Rk3 (12 mg) were finally separated.

(2) Next, the third fraction (BGE3, 1.5 g) out of the 10 fractions was subjected to ODS column chromatography (Φ 3 × 8 cm, MeOH-H2O = 3: 1) (BGE 3-5, 500 mg) was subjected to ODS column chromatography (Φ 3 × 7 cm, MeOH-H 2 O = 2.5: 1) to obtain a total of 10 fractions (BGE3-5-1 to BGE3-5-10) were obtained and finally ginsenoside F4 (8 mg), Rg4 (20 mg), F3 (23 mg), Rg3-S (5 mg).

(3) The seventh fraction (BGE7, 3.30 g) of the first 10 fractions was subjected to ODS column chromatography (Φ 3 × 10 cm, MeOH-H2O = 4: 1) BGE7-8) was obtained by dissolving about 100 mg of each BGE7-5 fraction (400 mg) in prepolymer in 1 ml of a solvent. The mixture was diluted with CH2Cl2-MeOH (5: 1) And flowed at a flow rate of 1.5 ml. The detector used was a Refractive Index Detector. The detector was checked while checking the signal from the detector. The above procedure was repeated and the same fractions were collected and confirmed by TLC. Finally, (5 mg), Compound Y (6 mg), Compound K (3 mg), ginsenoside Rk1 (18 mg), Rg 5 (12 mg) and Rh2 (4 mg).

(4) The ninth fractions (BGE9, 250 mg) of the first 10 fractions were subjected to ODS column chromatography (Φ 2 × 8 cm, MeOH-H2O = 4: 1) BGE9-3) was obtained by dissolving about 50 mg in 1 ml of the solvent using prep LC (150 mg) for the BGE9-1 fraction, and eluted with a ratio of 8: 1 in CH2Cl2-MeOH (8: And flowed at a flow rate of 1.5 ml. The detector used was a Refractive Index Detector. The detector was checked while checking the signal from the detector. The above procedure was repeated and the same fractions were collected and confirmed by TLC. Finally, ginsenoside Rk2 (8 mg) and Rh3 (12 mg) were isolated.

Comparative Example 1 and Comparative Example 2. Extract of corn gupo black ginseng

The black ginseng of Gujingugupo was purchased from two commonly-manufactured black ginseng (Ginseng Korea Ginseng; Comparative Example 1 and Daedong Ginseng; Comparative Example 2) and used as a control.

Experimental Example 1. Analysis of components of Example 1, Comparative Example 1 and Comparative Example 2.

Experimental Example 1-1. Establish UPLC analysis conditions

Waters ACQUITY UPLC ™ system (Waters Corp., MA, USA). Thermo Hypersil Gold (2.1 X 100 mm 1.9 μm) column was used and the mobile phase was acetonitrile with 0.1% formic acid added and 0.1% formic acid. B) was used. The column oven was maintained at 40 DEG C and the mobile phase consisted of solvent A (0.1% formic acid in water, v / v) and solvent B (0.1% formic acid in acetonitrile, v / v). Optimal LC elution conditions were applied as follows: 0-0.5 min, 15-15% B; 0.5-1 min, 15-20% B; 1-6 minutes, 20-20% B; 6-13 min, 20-30% B; 13-23 minutes, 30-35% B; 23-24 min, 35-38% B; 24-27 minutes, 38-60% B, and 27-31 minutes, 60-90% B. Flow rate was 0.5 mL / min. The 2 mu F fraction was injected into the column using an auto-sampler.

Experimental Example 1-2. Establishment of Q-TOF / MS condition

Optimal conditions for metabolism analysis of black ginseng in negative and positive modes were established using a Q-TOF micro mass detector (Waters, Manchester, UK). As a result of comparison, various metabolites were effectively analyzed in an anion electron spraying mode (Negative mode), and the analysis conditions were as follows; capillary voltage 3000, cone voltage 40, collision energy 6, desolvation temperature 550 캜, source temperature 120 캜, cone gas flow 30 L / h, desolvation gas flow 800 L / h. Precursor ion information was collected between 100 and 2,000 m / z.

The components of the samples of Example 1, Comparative Example 1 and Comparative Example 2 were analyzed by the above method (Fig. 2). 2A is a result of UPLC-QTOF / MS, and FIG. 2B is a result of quantifying the UPLC-QTOF / MS. Example 1 of the present invention was compared with Comparative Example 1 and Comparative Example 2 to find that Rk1, Rh4, Rg5, F4, Rk3, Rg3-R, Rg3-R, Rg4, MC, compound Y, compound O, Rh3, F2 , Rh2, and Ck components were increased

EXPERIMENTAL EXAMPLE 2 Experiment for Inducing Hepatotoxicity by Ethanol Administration

Five-week-old male Balb / c mice were orally administered 25% ethanol at a dose of 5 g / kg once a day for a total of 7 days to induce alcoholic liver disease. FIG. 4 is a graph showing blood concentrations of GOT / AST, GPT / ALT and LDH, which are indexes of liver toxicity, by inducing liver toxicity by ethanol administration. FIG. 4A is a graph showing the blood concentration of GOT / AST (U / I), FIG. 4B is a graph indicating the blood concentration of GPT / ALT (U / I) U / I). ≪ / RTI > 4, the blood concentrations of GOT / AST, GPT / ALT, and LDH in the experimental group (EtOH only) administered with 25% ethanol compared to the control group (NONE) in which 25% ethanol was not administered at 5 g / . Accordingly, it can be confirmed that the hepatocytes are damaged and the toxicity is expressed by administering 25% ethanol. 3 days before ethanol administration, GOT / AST and GPT / ALT were quantified in the serum of the mice which were orally administered in Example 1 at a dose of 0.5 mg / mouse and 2 mg / mouse once a day for 10 times, Respectively. At this time, blood serum GPT / ALT, GOT / AST and LDH were quantified using the serum samples 1 day after completion of the ethanol administration. As a result, blood AST / ALT elevated by ethanol administration showed an inhibitory effect at the dose of 2 mg / mouse of Example 1 (FIGS. 4A and 4B). In addition, a significant inhibitory effect was observed by the administration of Example 1 in the increase of LDH blood concentration by ethanol administration (Fig. 4C).

Experimental Example 3. Analysis of antioxidant activity in liver tissue

To investigate the effect of Example 1 on the expression of SOD (Superoxide dismutase), an antioxidative activity enzyme present in liver, expression levels of these genes in liver tissues were quantified. The conditions of the experiment are the same as those of Experimental Example 3 above. SOD was determined by quantifying SOD in liver tissues by lysing liver tissue on the first day after completion of administration of ethanol. From the dose of 0.5 mg / mouse of Example 1, the amount of SOD expression was increased, and a significant increase was observed up to a dose of 2 mg / mouse (Fig. 4D). It was found that Example 1 was effective for antioxidation.

Experimental Example 4. Results of Spleen and Liver Weighing Results

In order to confirm the safety of Example 1, spleen and liver weight changes of the experimental animals according to the administration of Example 1 were measured. As a result, it was found that the weight of organs did not significantly affect the weight reduction of the organs as well as the reduction of the organ weight by the administration of Example 1. There was also no change in weight gain during the study period.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention.

Claims (8)

a) drying ginseng;
b) raising the temperature of the dried ginseng at 60 to 70 ° C at room temperature;
c) raising the temperature of the ginseng steamed at 60 to 70 ° C to 70 to 120 ° C to reflux;
d) repeating steps b) and c) three to five times to produce black ginseng; And
e) extracting the black ginseng extract from the black ginseng using an extraction solvent;
≪ / RTI > The method according to claim 1,
Wherein the step b) is carried out for 1 minute to 60 minutes. The method according to claim 1,
Wherein the step c) is performed for 1 to 10 hours. The method of claim 1, wherein the extraction solvent is selected from the group consisting of water, ethyl acetate (EtOAc), alcohols having 1 to 4 carbon atoms, butanol having 1 to 4 carbon atoms and C4 Lt; / RTI > of the black ginseng extract. The method of claim 1, wherein the black ginseng extract is selected from the group consisting of ginsenosides Rk1, Rh4, Rg5, F4, Rk3, Rg3-S, Rg3-R, Rg4, MC, compound Y, compound O, Rh3, Rk2, F2, k is higher than that of the black ginseng extract. A black ginseng extract prepared by the method of claim 1. A pharmaceutical composition for prevention or treatment of liver injury comprising a black ginseng extract prepared by the method of claim 1. A health functional food composition comprising the black ginseng extract prepared by the method of claim 1 for preventing or improving liver damage.

Images