KR20230090840A - manufacturing method of edible gintonin-enriched fraction, ginseng polysaccharide fraction and crude ginseng total saponin fraction from ginseng - Google Patents

Abstract

The present invention relates to a method for preparing a gintonin-enriched fraction, a ginseng polysaccharide fraction, and a ginseng ginseng saccharide fraction from ginseng, and more particularly, without using an organic solvent harmful to the human body, alcohol ( It relates to a method for mass-producing three edible fractions from ginseng in high yield using only fermented ethanol) and water without discarding active ingredients, and the present invention, beyond the conventional method of separating each according to the type of active ingredient, As it can be prepared through a series of processes at once by the ginpoline method [separation method of ginseng tonin fraction (GEF) fraction, ginseng polysaccharide fraction, and ginseng saccharide fraction fraction], three different kinds of It is possible to mass-produce active ingredients of ginseng in a short time with high yield, and to utilize not only ginseng root, but also ginseng fruit, ginseng night, red ginseng night discarded after red ginseng extraction, and ginseng stem or leaves discarded after ginseng harvest. It is economical because it can utilize waste resources and has price competitiveness because it can reduce costs.

Description

Manufacturing method of edible gintonin-enriched fraction, ginseng polysaccharide fraction and crude ginseng total saponin fraction from ginseng}

The present invention relates to a method for preparing a gintonin-enriched fraction, a ginseng polysaccharide fraction, and a ginseng ginseng saccharide fraction from ginseng, and more particularly, without using an organic solvent harmful to the human body, alcohol ( It relates to a method for mass-producing three edible fractions from ginseng in high yield using only fermented ethanol) and water without discarding active ingredients.

Ginseng is generally used as an adaptogen or tonic for life extension. do. The ginseng has been used for hundreds of years not only in Korea but also in China and Japan, and is still one of the most famous medicinal herbs consumed in the world.

The ginseng is known to contain several components. Ginsenoside, which was first discovered in ginseng, was isolated in the early 1960s, and at this time, other components other than ginsenoside were not found. It has been cited a lot. Following the isolation of ginsenosides, it has been reported that ginseng contains ginseng polysaccharides with immunoactive activity. Recently, it has been reported that ginseng also contains gintonin, a lysophosphatidic acid receptor ligand linked to G protein, and it has been found that other unknown components are also contained.

The amount of individual ginsenoside components of ginseng is small, the separation process is complicated, and pure isolated individual ginsenosides are expensive. ginseng total saponin fraction, cGSF) has been used.

Organic solvents such as methanol and butanol are harmful to the human body, so the crude saponin fraction extracted using methanol and butanol has been mainly used for research purposes rather than food, and furthermore, other physiologically active components except for the extraction target, the irradiated saponin fraction Most of them were discarded in the separation process because organic solvents were used.

On the other hand, most conventional methods of separating ginseng polysaccharide fractions or ginseng tonin fractions (GEF) also use solvents such as ethanol and water. After separation, the remaining physiologically active ingredients are discarded without being utilized.

In order to use ginseng as a raw material for functional foods or medicines, it must be cultivated for at least 4 to 6 years. The increase in production cost due to long-term cultivation is one of the reasons why the price of raw ginseng is set very high compared to other natural medicines.

Therefore, there is a need for a simple, economical, and novel separation method that can solve the above-mentioned problems of using harmful organic solvents, prevent the disposal of active ingredients of ginseng, and minimize the loss of raw ginseng.

Republic of Korea Patent No. 10-1603294

An object of the present invention is to prepare a ginseng alcohol extract (step 1); Concentrating and drying the ginseng alcohol extract (step 2); Dissolving the concentrated and dried alcohol extract in water and precipitating at low temperature to separate a first supernatant and a first lower layer (step 3); obtaining a gintonin-enriched fraction (GEF) from the first lower layer (step 4); Adding alcohol to the first supernatant and precipitating at a low temperature to separate the second supernatant and the second lower layer (step 5); obtaining a ginseng polysaccharide fraction from the second lower layer (step 6); and obtaining a ginseng irradiation fraction from the second supernatant (step 7).

Another object of the present invention is to provide a ginseng tonin fraction, a ginseng polysaccharide fraction, or a ginseng irradiation fraction prepared by the above preparation method.

Another object of the present invention is to provide a health functional food composition for preventing or improving thrombotic diseases containing the gin and tonin fraction.

Another object of the present invention is to provide a health functional food composition for increasing antioxidant or immune activity containing the ginseng polysaccharide fraction.

The technical problem to be achieved by the present invention is not limited to the above-mentioned technical problem, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to solve the above problem,

The present invention comprises the steps of preparing a ginseng alcohol extract (step 1); Concentrating and drying the ginseng alcohol extract (step 2); Dissolving the concentrated and dried alcohol extract in water and precipitating at low temperature to separate a first supernatant and a first lower layer (step 3); obtaining a gintonin-enriched fraction (GEF) from the first lower layer (step 4); Adding alcohol to the first supernatant and precipitating at a low temperature to separate the second supernatant and the second lower layer (step 5); obtaining a ginseng polysaccharide fraction from the second lower layer (step 6); and obtaining a ginseng irradiation fraction from the second supernatant (step 7).

In addition, the present invention provides a ginseng tonin fraction, a ginseng polysaccharide fraction, or a ginseng irradiation fraction prepared by the above preparation method.

In addition, the present invention provides a health functional food composition for preventing or improving thrombotic diseases containing the gintonin fraction.

In addition, the present invention provides a health functional food composition for increasing antioxidant or immune activity containing the ginseng polysaccharide fraction.

The present invention separates the second ginseng active ingredient using the remaining fraction after separating the first ginseng active ingredient, and separates the third ginseng active ingredient using the remaining fraction after separating the second ginseng active ingredient. It is possible to solve the problem of wasting the active ingredient of ginseng by separating one active ingredient and discarding the remaining fraction.

In addition, it is possible to manufacture ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng ginseng ginseng saccharide fraction through a series of processes at once, rather than the conventional method of separating them according to the type of active ingredient. Three types of ginseng active ingredients can be mass-produced in a short time with high yield, and not only ginseng root, but also ginseng fruit, ginseng meal, red ginseng meal discarded after red ginseng extraction, and ginseng stem or leaf discarded after ginseng harvest It is economical because it can utilize waste resources and has price competitiveness because it can reduce costs.

In addition, since it is prepared using only alcohol (ethanol) and water, it is edible, can be put into practical use without a separate stability (toxicity) test, and can be used as a health functional food composition.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a flow chart of a method for preparing a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng ginseng saccharide fraction of the present invention.
Figure 2 schematically shows a method for preparing a gintonin fraction, a ginseng polysaccharide fraction, and a ginseng ginsenoside saccharide fraction (= ginseng method) according to an embodiment of the present invention.
3 shows the contents of galatouronic acid (a) and gallic acid (b) contained in a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng ginsenoside saccharide fraction according to an embodiment of the present invention.
FIG. 4 shows the result of comparing the cytoplasmic transient calcium mobilization ([Ca ²⁺ ] _i transient) abilities of a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng irradiation fraction of an embodiment of the present invention.
FIG. 5 shows the results of comparing the ABTS ⁺ radical scavenging abilities of a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng saccharide fraction of an embodiment of the present invention.
Figure 6 shows the results of comparing the anti-platelet activity of the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng xoxoponin fraction according to an embodiment of the present invention.
FIG. 7 shows the results of comparing the immune activation abilities of a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng xoxoponin fraction according to an embodiment of the present invention.
FIG. 8 shows the results of comparing the effects of ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng irradiation fraction on cell survival and protection against cell death by ROS according to an embodiment of the present invention.
9 shows the content of arginine contained in a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng ginseng saccharide fraction according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail. However, the present invention can be implemented in many different forms, and the present invention is not limited by the embodiments described herein, and the present invention is only defined by the claims to be described later.

In addition, terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. In the entire specification of the present invention, 'include' a certain element means that other elements may be further included without excluding other elements unless otherwise stated. In addition, the 'include' also means that the component is used alone.

One aspect of the present invention provides a method for preparing a ginseng fraction, a polysaccharide fraction of ginseng, and a fraction of a ginseng irradiated ginseng, and a ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng ginseng saccharide fraction prepared using the same.

1 is a flowchart of a method for preparing a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng ginseng saccharide fraction of the present invention.

Referring to FIG. 1 , the method for producing a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng ginsenoside fraction of the present invention comprises the steps of preparing a ginseng alcohol extract (step 1); Concentrating and drying the ginseng alcohol extract (step 2); Dissolving the concentrated and dried alcohol extract in water and precipitating at low temperature to separate a first supernatant and a first lower layer (step 3); obtaining a gintonin-enriched fraction (GEF) from the first lower layer (step 4); Adding alcohol to the first supernatant and precipitating at a low temperature to separate the second supernatant and the second lower layer (step 5); obtaining a ginseng polysaccharide fraction from the second lower layer (step 6); and obtaining a ginseng irradiation fraction from the second supernatant (step 7).

The present invention is a method for obtaining a gintonin fraction, a ginseng polysaccharide fraction, and a ginseng saccharide fraction by minimizing the loss of active ingredients through a series of processes without discarding active ingredients from a ginseng alcohol extract. ”, “Pol” in polysaccharide and “in” in saponin.

In one embodiment of the present invention, the "gintonin fraction, ginseng polysaccharide fraction, and ginseng saccharide fraction" may refer to three types of fractions extracted from ginseng and containing different components and characteristics.

First, the Ginpoline method of the present invention includes a step of preparing a ginseng alcohol extract (step 1) and a step of concentrating and drying the ginseng alcohol extract (step 2).

In the present invention, "extract" includes all extracts, purified products, diluted solutions, concentrated solutions, and dried products.

In one embodiment of the present invention, the ginseng is selected from the group consisting of wild ginseng, camphor ginseng, cultivated ginseng, American ginseng, western ginseng, Chinese ginseng, red ginseng, fresh ginseng, white ginseng, cultured ginseng, and other ginseng by-products including ginseng meal and red ginseng meal. It may be any one or more, for example, 4- to 6-year-old white ginseng (Panax ginseng C. A. Meyer), and the ginseng may be one or more selected from the roots, leaves, stems, fruits, and flowers of ginseng.

The ginseng method of the present invention can be performed using not only ginseng root, but also ginseng fruit, ginseng meal, red ginseng meal discarded after red ginseng extraction, and ginseng stem or leaves discarded after ginseng harvest, thereby reducing waste resources. It has the advantage of being economical.

In one embodiment of the present invention, in steps 1 and 2, the dried ginseng powder is mixed with alcohol in a volume of about 1 to 20 times, for example, 1 to 10 times, for example, 1 to 5 times the weight of the sample. 60 ℃ to 120 ℃, for example, 70 ℃ to 100 ℃, for example, 70 ℃ to 90 ℃, for example, from about 0.1 to 48 hours at 75 ℃ to 85 ℃, for example, 1 to 24 hours, for example, for 5 to 12 hours, using any one of the following extraction methods, for example, reflux cooling extraction method, among stirring extraction, hot water extraction, cold brew extraction, warming extraction, reflux cooling extraction and ultrasonic extraction After extraction, it is recovered by filtration, and the above extraction method is repeated several times, for example, 1 to 10 times, for example, 2 to 5 times, and then the supernatant is collected and concentrated under reduced pressure. .

In one embodiment of the present invention, the alcohol is a mixed solvent of water and ethanol having a mixing ratio of about 1: 0.1 to 10, for example, 50% to 90%, for example, 60% to 80%, for example For example, it may mean an alcohol having 60% to 70% ethanol or an ethanol content equivalent thereto.

Next, dissolving the concentrated and dried alcohol extract of the present invention in water and precipitating at a low temperature to separate the first supernatant and the first lower layer (step 3); and obtaining a gintonin-enriched fraction (GEF) from the first lower layer (step 4).

In one embodiment of the present invention, steps 3 and 4 relate to a method for producing a gin and tonin fraction (GEF) using only alcohol and water, using an organic solvent that is not available for conventional food or expensive This corresponds to a method capable of obtaining a gintonin fraction (GEF) without performing anion exchange resin chromatography.

By carrying out the processes of steps 3 and 4, it is possible to mass-produce edible gintonin fraction (GEF) in a fast, inexpensive, and high yield.

In one embodiment of the present invention, step 3 is performed by dissolving the concentrated and dried alcohol extract obtained in step 2 in about 10 to 20 times the amount of water, for example, tertiary distilled water, 12 hours to 120 hours, eg 24 hours or 120 hours, eg 48 hours to 96 hours, at 2 °C to 6 °C, eg 3 °C to 5 °C. It may be stored for a period of time, inducing precipitation, and separated into a first supernatant and a first lower layer, and at this time, the first lower layer may include the first lower layer precipitate.

In one embodiment of the present invention, step 4 may be performed by centrifuging the first lower layer obtained in step 3 to obtain a first lower layer precipitate and freeze-drying it.

Next, the ginpoline method of the present invention includes the steps of adding alcohol to the first supernatant separated in step 3 and precipitating at a low temperature to separate the second supernatant and the second lower layer (step 5); obtaining a ginseng polysaccharide fraction from the second lower layer (step 6); and obtaining a ginseng irradiation fraction from the second supernatant (step 7).

The first supernatant remains after obtaining the gin and tonin fraction in step 4, and conventionally corresponds to a material that is discarded after obtaining the gin and tonin fraction.

The inventors of the present invention found that the effective physiologically active components of ginseng could be separated from the remaining material after obtaining the ginseng and tonin fraction, and completed the present invention.

In one embodiment of the present invention, in step 5, the alcohol of the first supernatant obtained in step 3 is added, and the temperature is 0 ° C to 8 ° C, for example, 2 ° C to 6 ° C, for example, 3 ° C. to 5° C. for 12 to 120 hours, such as 24 hours or 120 hours, such as 48 to 96 hours, to induce precipitation and to separate into a second supernatant and a second lower layer. In this case, the second lower layer solution may include the second lower layer precipitate.

In one embodiment of the present invention, the step 5 may be performed by adding the alcohol to the first supernatant so that the alcohol content is 80% or more, for example, 80% to 99%.

In step 5, when the alcohol content in the first supernatant is less than 80%, precipitation may not occur easily, and thus separation of the second supernatant and the second lower layer may not occur.

In one embodiment of the present invention, step 6 may be performed by dissolving the second lower layer precipitate obtained in step 5 in water, for example, tertiary distilled water, and then freeze-drying.

Also, in one embodiment of the present invention, step 7 may be performed by concentrating the second supernatant obtained in step 5 under reduced pressure.

In one embodiment of the present invention, steps 6 and 7 may be performed first. For example, after the second supernatant and the second lower layer are separated in step 5, step 7 may be performed first, then step 6 may be performed, or steps 6 and 7 may be performed simultaneously. free

As described above, the ginfoline method of the present invention is obtained from ginseng through a series of processes without discarded active ingredients, and three types of fractions containing different components and characteristics, namely, gintonin fraction, ginseng polysaccharide fraction, and ginseng Since all of the sasaponin fractions can be obtained, the ginseng tonin fraction obtained in step 4, the ginseng polysaccharide fraction obtained in step 6, and the ginseng sasaponin fraction obtained in step 7 may have different components and characteristics, respectively.

In one embodiment of the present invention, the ginseng and tonin fraction obtained in step 4 is a diol containing Rb1, Rb2, Rc and Rd compared to the ginseng polysaccharide fraction and the ginseng saccharide fraction obtained in steps 6 and 7, respectively. (diol) may contain more ginsenosides.

In addition, the gintonin fraction may contain a component having an anti-platelet action or a component capable of protecting cells from oxidative stress, and thus may be used as a health functional food composition for preventing or improving thrombotic diseases in the following aspects. there is. A description in this regard will be given later.

In addition, in one embodiment of the present invention, the ginseng irradiation fraction obtained in step 7 is a triol (triol containing Rg1 and Re) compared to the ginseng tonin fraction and ginseng polysaccharide fraction obtained in steps 4 and 6, respectively. ) can contain more ginsenosides.

In addition, in one embodiment of the present invention, the ginseng polysaccharide fraction obtained in step 6 may contain more arginine than the ginseng tonin fraction and ginseng saccharide fraction obtained in steps 4 and 7, respectively. .

In addition, the ginseng polysaccharide fraction may contain an acidic polysaccharide component, an antioxidant component, and an immune active component, and thus may be used as a health functional food composition for antioxidant or immune activity enhancement in the following aspects. A description in this regard will be given later.

One aspect of the present invention provides a health food composition for preventing or improving thrombotic diseases containing the gintonin fraction obtained in the above aspect.

In one embodiment of the present invention, the health food composition may include food additives acceptable food additives.

In one embodiment of the present invention, health foods to which the gin and tonin fraction can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, and health functional foods.

In addition, it may be added to foods or beverages for the purpose of preventing thrombotic diseases. At this time, the amount of the gin and tonin fraction in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, and the health drink composition is 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 mL. can be applied

The health beverage composition of the present invention is not particularly limited in other ingredients except for containing the gin and tonin fraction as an essential ingredient in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates like conventional beverages. there is. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of flavoring agents other than those described above include natural flavoring agents such as thaumatin and stevia extracts such as rebaudioside A and glycyrrhizin; and synthetic flavoring agents such as saccharin, aspartame, and the like can advantageously be used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above, the gintonin fraction of the present invention is various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, It may contain a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, the gintonin fraction of the present invention may contain natural fruit juice, fruit juice beverages, and fruit flesh for producing vegetable beverages. These components may be used independently or in combination. At this time, the ratio of additives is not very important, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the gin and tonin fraction of the present invention.

One aspect of the present invention provides a health food composition for increasing antioxidant or immune activity containing the ginseng polysaccharide fraction obtained in the above aspect.

In one embodiment of the present invention, the health food composition may include food additives acceptable food additives.

In one embodiment of the present invention, health foods to which the ginseng polysaccharide fraction can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, and health functional foods.

In addition, it can be added to food or beverages for the purpose of antioxidant or immune activity increasing effect. At this time, the amount of the ginseng polysaccharide fraction in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, and the health drink composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 mL. can be applied

The health beverage composition of the present invention is not particularly limited in other ingredients except for containing the ginseng polysaccharide fraction as an essential ingredient in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates like conventional beverages. there is. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of flavoring agents other than those described above include natural flavoring agents such as thaumatin and stevia extracts such as rebaudioside A and glycyrrhizin; and synthetic flavoring agents such as saccharin, aspartame, and the like can advantageously be used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above, the ginseng polysaccharide fraction of the present invention is various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, It may contain a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, the ginseng polysaccharide fraction of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. At this time, the ratio of the additive is not very important, but it is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the ginseng polysaccharide fraction of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited and interpreted by these examples.

Example

Example 1. Preparation of ginseng tonin fraction (GEF), ginseng polysaccharide fraction, and ginseng irradiation fraction from ginseng

Figure 2 is a schematic representation of the ginpholine method of the present invention.

Hereinafter, referring to FIG. 2, the ginpoline method of the present invention will be described.

- Preparation of ginseng alcohol extract

5 L of 60 to 70% (w/v) alcohol was added to 1,000 g of white ginseng (Panax ginseng C.A. Meyer) powder, a type of 4-year-old ginseng purchased from Korea Ginseng Corporation (Daejeon, Korea), and refluxed at about 80 ° C for 8 hours. Extraction was cooled, filtered (repeated 3 times), concentrated in a vacuum concentrator, dissolved in tertiary distilled water and freeze-dried to obtain 330 g of alcohol extract (70% alcohol extract, average value of 4 runs).

- Preparation of ginseng tonin fraction (GEF), ginseng polysaccharide fraction, and ginseng ginseng irradiation fraction

About 10 to 20 times of tertiary distilled water was added to the alcohol extract, and the lyophilized extract was mixed so that it was completely dissolved in water. The alcohol extract dissolved in the tertiary distilled water was refrigerated at 4 ° C. for 2 to 4 days, naturally inducing precipitation by gravity to separate the first supernatant and the first lower layer, and centrifugation to obtain a first lower layer. A precipitate was obtained.

After re-centrifuging the first lower layer precipitate, 14.8 g of gintonin fraction (average value of 4 runs) was obtained by freeze-drying.

Add 4 times the alcohol to the first supernatant, mix to make 80% alcohol compared to the first supernatant, and refrigerate at 4 ℃ for 2 to 4 days to naturally induce precipitation by gravity to obtain a second supernatant and It was separated into a second lower layer liquid, and a second lower layer precipitate was obtained through centrifugation.

The second lower layer precipitate was dissolved in tertiary distilled water and then freeze-dried to obtain 54.2 g of ginseng polysaccharide fraction (average value of 4 runs).

In addition, the second supernatant was concentrated under reduced pressure to obtain a final irradiation fraction of 185 g (an average value of 4 runs).

The total yield of the ginseng tonin fraction (GEF), ginseng polysaccharide fraction, and ginseng ginsenoside saponin fraction relative to 1 kg of ginseng was 254.3 g, and the yield was 25.4%. Ginseng polysaccharide fraction (5.42%) > gintonin fraction (1.48%) followed in order.

In the case of the yield of 330 g of the alcohol extract, it was confirmed that a high yield of about 77% or more was exhibited.

Experimental example

Measurement example 1. Quantitative measurement of galacturonic acid

Galacturonic acid contained in the ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng saxophonin fraction was quantified using galacturonic acid (ginseng acid polysaccharide indicator) as a standard material.

The standard galacturonic acid was diluted to 0, 10, 20, 30, 40, and 50 μg/mL in tertiary distilled water at a concentration of 1 mg/mL. After adding 20 μL of each concentration standard prepared in advance to a 2 mL tube, add 80 μL of tertiary distilled water, add 50 μL of 0.1% carbazol (carbazole) solution prepared using 100% EtOH, and finally A standard material was prepared by mixing 600 μL of a sulfuric acid (H ₂ SO ₄ ) solution.

In order to quantify the galacturonic acid contained in the ginseng tonin fraction, the ginseng polysaccharide fraction, or the ginseng saccharide fraction, stock solutions were first prepared at a concentration of 100 mg/mL for each of the three samples. Add 20 μL of each of the three samples to a 2 mL tube, add 80 μL of tertiary distilled water, add 50 μL of 0.1% carbazol (carbazole) solution made using 100% EtOH, and finally add 600 μL of sulfuric acid solution. mixed.

After reacting the standards for each concentration and three samples together at 85 ° C for 15 minutes, each standard substance and each sample were added at 100 μL / well to a 96 well plate, and ELISA reader (Molecular Devices Co., CA, USA) ) was calculated using the optical density (OD) value obtained after measuring the absorbance at a wavelength of 525 nm. The r2 value of the standard curve for the standard material was 0.99 or more.

Measurement example 2. Quantitative measurement of gallic acid

Gallic acid (ginseng total polyphenol indicator) was used as a standard material. The standard substance gallic acid (gallic acid) was diluted to 0, 10, 20, 30, 40, and 50 μg/mL by making a concentration of 1 mg/mL in tertiary distilled water. Standard materials for each concentration diluted in a 2 mL tube were prepared.

In order to quantify the gallic acid contained in the ginseng tonin fraction, the ginseng polysaccharide fraction, or the ginseng saccharide fraction, stock solutions were first prepared at a concentration of 100 mg/mL for each of the three samples. After adding 80 μL of the standard material for each concentration diluted in a 2 mL tube and the ginseng tonin fraction, ginseng polysaccharide fraction, or ginseng saccharide fraction, 20 μL of Folin-Ciocalteu reagent was added, mixed, and reacted at room temperature for 5 minutes. After that, 100 μL of 2% sodium carbonate solution (NaCO ₃ ) was added and reacted at room temperature for 1 hour. Standard substances and samples were added at 100 μL/well to a 96 well plate and then read through an ELISA reader (Molecular Devices Co., CA, USA). The absorbance at a wavelength of 750 nm was measured and calculated using the optical density (OD) value. The r2 value of the standard curve for the standard material was 0.99 or more.

Measurement Example 3. Intracellular free calcium ([Ca] in cultured hippocampal neurons (HT22 cell) ²⁺ ] _i ) measures the transient increase in

Hippocampus-derived neurons (HT 22 cells) were cultured according to the method of Cho et al (2021).

- Mix of Fura 2-AM and brain hippocampus-derived nerve cells (HT22 cells)

In the present invention, in order to confirm whether the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng irradiation fraction show an effect on the transient increase in [Ca ²⁺ ] _i in hippocampal HT22 cells, HT22 cells (2 to 4 ×10 ⁶ /mL) was suspended in a 1.5 mM Ca ²⁺ -containing buffer or Ca ²⁺ -free buffer, incubated at 37 °C for 30 minutes, and then each fraction (gintonin fraction, ginseng polysaccharide fraction, and ginseng saccharide fraction) Fraction) An increase in intracellular calcium [Ca ²⁺ ] _i in brain hippocampus-derived neurons was tested.

Specifically, according to the method of Jø et al., a Ca ²⁺ containing buffer (pH 7.4) composed of NaCl 120 mM, KCl 5 mM, MgCl ₂ 1 mM, CaCl ₂ 1.5 mM, glucose 10 mM, and HEPES 25 mM and NaCl 120 mM , KCl 5 mM, MgCl ₂ 1 mM, EGTA 0.2 mM, glucose 10 mM, and HEPES 25 mM Ca ²⁺ -free buffer (pH 7.4) was used, and 2.5 μM Fura 2- AM was mixed with astrocytes or brain hippocampus-derived neuronal HT22 cells (1~2×10 ⁶ /mL), and excess fura-2AM was removed by washing the cells three times with Ca ²⁺ containing buffer or Ca ²⁺ non-containing buffer. did

- [Ca in the cell suspension ²⁺ ] _i Fluorescence measurement of

[Ca ²⁺ ] _i was measured in a Fura 2-loaded cell using an RF-5300PC Intracellular Ion Measurement System (Shimadzu Corporation, Japan). Specifically, the Fura 2-loaded cells were finally diluted in 2-4×10 ⁶ /mL test buffer and then transferred to a polystyrene cuvette (Elkay Ultra-UV). The cells were agitated using a Teflon-coated magnet, and the cuvette housing was adjusted to 37 °C. Excitation wavelengths were alternated between 340 nm and 380 nm under computer control, and emission was detected at 510 nm. At this time, the width of the excitation and emission gap was set to 3 nm, and the background correction was performed according to the method of Jø et al., but digitonin and EGTA ensured that fura-2AM was completely combined with Ca ²⁺ and dissociated from Ca ²⁺ It was used as a Ca ²⁺ concentration adjustment reagent to create a state of being.

- [Ca from Fura 2 measurements ²⁺ ] _i estimation of

The measured ratio value of 340 nm to 380 nm was converted to the value _of [Ca ²⁺ ] i using the equation of Hounsell et al., represented by Equation 1 below:

[Equation 1]

[Ca ²⁺ ] = K _d [(R - R _min )/(R _max - R)](S _f380 /S _b380 )

In the above formula, K _d is the dissociation constant (224 nM),

R is the measured fluorescence ratio of 340 nm to 380 nm;

R _max and R _min are the R value at a Ca ²⁺ saturated concentration with 50 μg/ml digitonin added and the R value in a Ca ²⁺ -free medium with 20 mM EGTA added, respectively;

S _f380 and S _b380 are Fura-2AM fluorescence intensities at 380 nm when digitonin and EGTA are added, respectively.

- Data analysis

In order to obtain a concentration response curve including the ginseng tonin fraction, the ginseng polysaccharide fraction, or the ginseng saccharide fraction, the amplitude of the observed peak was normalized and plotted, and then using Origin software (Northampton, MA) to obtain the following formula 2 Corresponded to the Hill equation, denoted by

[Equation 2]

y/y _max = [A] ⁿ /([A] ⁿ + [EC ₅₀ ] ⁿ )

In the above formula, y is the % activity at a given concentration of the ginseng fraction, the ginseng polysaccharide fraction, or the ginseng saccharide fraction,

y _max is the maximum peak intracellular calcium mobilization,

[EC ₅₀ ] is the concentration of each fraction of the alcohol extraction that can exhibit 50% of the maximum response,

[A] is the concentration of each fraction of alcohol extraction,

n is the interaction coefficient.

All values were expressed as mean ± standard error, and the average difference between the data of the control group and each fraction treatment group of alcohol extraction was analyzed using unpaired Student's t test. Statistical significance was tested at p<0.05.

Measurement example 4. Verification of cell viability

Hippocampus-derived neurons (HT 22 cells) were cultured according to the method of Cho et al (2021).

The cytotoxicity of the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng saccharide fraction was analyzed using the WST assay. In addition, the cell viability and protective effects of ginseng tonin fraction, ginseng polysaccharide fraction, or ginseng polysaccharide fraction or ginseng ginsenoside fraction by oxidative stress induced by iodoacetic acid (IAA) were also verified using the WST assay.

The cells were dispensed at 1X10 ⁴ cells/mL in a 96 well plate and the cells were cultured for 24 hours. Thereafter, each sample (0.1, 0.3, 1, 3, 10, 30, 100 and 300 μg/mL) or IAA (2 μg/mL) was treated and incubated for 24 hours, and then 1/10 WST solution was added. medium was replaced. After 2 hours, the absorbance was measured at a wavelength of 570 nm using an ELISA reader (Molecular Devices Co., CA, USA) and calculated using the optical density (OD) value obtained.

Measurement example 5. Nitric oxide (NO) production measurement

NO production by the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng saccharide fraction was measured.

RAW 264.7 cells were dispensed at 1X10 ⁵ cells/mL into a 96 well plate and allowed to stand for 24 hours to stabilize. After that, each sample (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 μg/mL) or LPS (2 μg/mL) was further incubated for 24 hours, and 200 μL of After collecting the culture medium and centrifuging the culture medium (12,000 rpm, 3 minutes), the supernatant was taken, the same amount of Griess reagent (Sigma) was added, reacted at room temperature for 15 minutes, and the absorbance was measured at 540 nm.

The r2 value of the standard curve for standard NO (sodium nitrite) was more than 0.99.

Measurement example 6. ABTS+ radical scavenging activity

ABTS ⁺ antioxidant activities by ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng ginseng saccharide fraction were all measured using the supernatant obtained after adding ethanol at a concentration of 100 mg/mL and then centrifuging at 3,000 rpm for 10 minutes using the supernatant as a sample. . This was measured by a modified method by referring to the method of Shiddhuraju P (2002) et al.

After mixing ABTS 14 mM dissolved in distilled water with Potassium persulfate 4.9 mM dissolved in distilled water, it was left in the dark for 12-16 hours. The ABTS ⁺ radical-generated solution was measured at 734 nm and adjusted to have an absorbance of 0.700 (±0.02). 180 μL of this solution and 20 μL of the sample solution were mixed and the absorbance change was measured at 734 nm at 1 minute intervals for 6 minutes did

Measurement Example 7. Measurement of Cytokines Production

The ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng ginsenoside fraction diluted with the culture medium were treated at different concentrations (0.1, 0.3, 1, 3, 10,30, 100, and 300 μg/mL) in a 37 °C, 5% CO ₂ incubator. After culturing for 24 hours, the amount of IL-6 and Tumor Necrosis Factor (TNF)-α secreted into the supernatant was measured using an ELISA kit (BD Biosciences, San Diego, CA, USA and Invitrogen, San Diego, CA, USA). was measured.

After dispensing at 1X10 ⁵ cells/mL in a 96-well plate, let it stand for 24 hours to stabilize, and then treat the ginseng tonin fraction, ginseng polysaccharide fraction, ginseng polysaccharide fraction, or LPS (2 μg/mL) for another 24 hours. After culturing, collecting the culture medium and centrifuging the culture medium (12,000 rpm, 3 minutes), the supernatant was taken.

For IL-6 quantification, 50 μL of sample and standard were put into each well, left at room temperature for 2 hours, and washed 5 times with wash buffer (300 μL/well). Then, 100 μL of the working detector was added, left at room temperature for 1 hour, washed 7 times, and substrate solution (TMB One-Step Substrate Reagent) was added at 100 μL/well and left in the dark for 30 minutes. . After stopping the reaction by adding 50 μL of Stop solution, the value measured at 570 nm was subtracted from the value measured at 450 nm using an ELISA reader and quantified.

For TNF-α quantification, 50 μL of sample and standard were put into each well, 50 μL of biotin-conjugate was added, left at room temperature for 2 hours, washed 6 times with 400 μL/well, and 100 μL of Streptavidin-HRP was added and left in the dark at room temperature for 1 hour. After additional washing six times, 100 μL of TMB substrate solution was added, left in the dark for 30 minutes, 100 μL of stop solution was added, and absorbance was measured at 450 nm using an ELISA reader.

Measurement Example 8. Phagocytosis (Neutral Red Uptake)

The ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng ginsenoside fraction diluted with medium were treated at concentrations (0.1, 0.3, 1, 3, 10, 30, 100, and 300 μg/mL) in a 37 °C, 5% CO ₂ incubator. After culturing for 24 hours, add 100 μL of neutral red solution (N4638-1G, Sigma) dissolved in 10 mM PBS to a final concentration of 0.075%, leave it at room temperature for 1 hour, remove the supernatant, and wash twice with PBS. After that, 100 μL/well of EtOH: 0.01% acetic acid (1:1) solution was added, left at room temperature for 16 to 18 hours, and absorbance was measured at 540 nm with an ELISA reader.

Measurement Example 9. Anti-platelet aggregation action measurement

Seven-week-old rats were used. After anesthetizing the white paper, blood was collected from the white paper's heart to obtain platelets. Acid citrate dextrose (ACD) was used as an anticoagulant. The collected blood was centrifuged at 1,000 rpm for 7 minutes to obtain platelet rich plasma (PRP), and the PRP was further centrifuged at 2,000 rpm for 7 minutes to obtain pre-settling platelets (pelleted platelets). The pelleted platelets were suspended in Tyrode's buffer (137 mM NaCl, 12 mM NaHCO ₃ , 5.5 mM glucose, 2 mM KCl, 1 mM MgCl ₂ and 0.3 mM NaHPO ₄ , pH 7.4) to obtain washed platelets. was obtained.

The platelet aggregation test was performed using a platelet aggregation measuring device (Aggregometer, Chrono-log, Havertown, PA, USA). First, after adding 1 mM CaCl ₂ , each of the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng ginsenoside saccharide fraction was added to cells containing platelets. Collagen (2.5 μg/mL) was added to induce platelet aggregation while stirring the platelets at 37° C. for 1 minute, and aggregation was allowed to occur for 5 minutes. After 5 minutes, the degree of aggregation was measured.

Measurement Example 10. Quantification of free arginine

Free arginine contained in each fraction was analyzed and quantified using Amino Acid Annalyzer (SYKAM, No. S433, Sykam GmbH, Gewerbering 15, 86922 Eresing, Germany). A stock solution (100 μM, Sykam Catalog No. S000031) was used as the standard amino acid (Kim et al., 2019).

Each fraction sample was dissolved at 100 mg/10 mL in tertiary distilled water and then dissolved at 25 °C for 3 hours while shaking. Thereafter, centrifugation was performed, and the supernatant was filtered using a 0.22 μm filter and freeze-dried. Each freeze-dried powder was further diluted by adding 5 mL of tertiary distilled water. 200 μL of each fraction was used for analysis. For chromatographic analysis, three different concentrations of standard arginine and each sample were dissolved in lithium citrate buffer and used as a mobile phase. The ninhydrine reagent operated a separation pump at room temperature at a rate of 0.35 mL/min. Amino acid detection was performed at 570 nm and 440 nm. The column used was TLCA K07/Li, 4.6ⅹ mm. The arginine content of each of the three fractions was expressed as % for each fraction.

Experimental Example 1. LPA C18: 2 and phosphatidic acid (PA C16: 0-C18: 2) content confirmation

Analysis of the LPA C18:2 and PA content contained in the ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng ginseng saccharide fraction obtained in Example 1 and the PA content was performed using a liquid chromatography-mass spectrometry/mass spectrometry system (LC-MS/MS system) was quantified according to the method of Cho et al (2019), and the results are shown in Table 1 below (average value of 4 runs):

LPA (μ/mg) PA (μg/mg) Gintonin Fraction (GEF) 1.951±0.106 10.6±0.649 Ginseng polysaccharide fraction (GPF) N.D. (Not detected) N.D. (Not detected) Razoponin fraction (cGTSF) 0.659±0.0020 0.837±0.067

Referring to Table 1 above, it was confirmed that the gintonin fraction contained LPA C18:2 and PA the most, and in the ginseng sagoponin fraction, the two components contained an amount corresponding to about 30% of the gintonin fraction, It was found that the ginseng polysaccharide fraction contained almost none of the above two components.

Experimental Example 2. Confirmation of ginsenoside content

Analysis of the individual ginsenoside contents contained in the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng ginseng saccharide fraction obtained in Example 1 was carried out according to the method of Park et al (2017), and the results are shown in Table 2 below. is plotted (average of 4 runs):

Gintonin Fraction (GEF) Ginseng polysaccharide fraction (GPF) Ginseng Josaponin Fraction (cGTSF) Rg1 (mg/g) 6.54±0.78 0.14±0.01 11.73±0.50 Rb1 (mg/g) 60.17±10.02 0.25±0.04 15.04±2.4 Rg3 (mg/g) 1.94±1.04 N.D. (Not detected) 0.17±0.12 Re (mg/g) 4.18±0.04 N.D. (Not detected) 8.04±0.20 RF (mg/g) 4.53±0.01 N.D. (Not detected) 2.74±0.25 Rg2 (mg/g) 1.01±0.06 N.D. (Not detected) 0.29±0.04 Rc (mg/g) 24.45±3.90 N.D. (Not detected) 5.53±1.58 Rb2 (mg/g) 19.95±2.14 N.D. (Not detected) 3.76±1.34 Rb3 (mg/g) 2.56±0.26 N.D. (Not detected) 0.63±0.01 Rd (mg/g) 6.01±0.99 N.D. (Not detected) 1.48±0.17

Referring to Table 2, it was confirmed that most representative ginsenosides of ginseng were present in the ginseng tonin fraction and the ginseng ginsenoside fraction, and only trace amounts were detected in the ginseng polysaccharide fraction.

In particular, it was found that the ginsenoside diols of ginseng, Rb1, Rb2, Rc and Rd, were contained more in the ginsenoside fraction than in the ginseng ginsenoside fraction, and the order of their content was in the order of Rb1 > Rc > Rb2. It was confirmed that, when the production method of the present invention was used, diol-type ginseng ginsenosides were highly concentrated in the gintonin fraction.

On the other hand, it was confirmed that the ginseng saxophonin fraction contained more Rg1 and Re, which are ginsenoside triols, than the gintonin fraction.

Experimental Example 3. Confirmation of galacturonic acid and gallic acid content

The content of galacturonic acid contained in the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng saccharide fraction obtained in Example 1 was comparatively quantified according to the method of Do et al (1993), and the results are shown in FIG. 3 a) shown in

Referring to a) of FIG. 3, it was confirmed that galacturonic acid, a ginseng acidic polysaccharide indicator substance, was contained in the ginseng polysaccharide fraction more than twice as much as the gintonin fraction and the ginseng ginseng saccharide fraction. It was confirmed that the acidic polysaccharide component was most concentrated in the ginseng polysaccharide fraction.

The total phenol content contained in the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng saccharide fraction obtained in Example 1 was comparatively quantified using gallic acid as an indicator substance according to the method of Shin et al (2001), , the results are shown in b) of FIG. 3 .

Referring to b) of FIG. 3 , the gallic acid content was found to be the highest in the ginseng and tonin fraction, and it could be confirmed that the gallic acid content was similar in the ginseng polysaccharide fraction and the ginseng ginsenoside ponin fraction.

Experimental Example 4. [Ca ²⁺ ] _i transient induction comparison

Hippocampal neurons (HT22 cells) known to have abundantly expressed LPA receptors by the ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng irradiation fraction obtained in Example 1 [Ca ²⁺ ] _i transient induction ability was confirmed, Results are shown in FIG. 4 .

Referring to a) of FIG. 4, when 0.3 μg/ml of each of the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng irradiation fraction was treated, there was no effect on the ginseng polysaccharide fraction and the ginsenoside fraction, but in the gintonin fraction, It was confirmed that [Ca ²⁺ ] _i transient was induced.

Referring to b) of FIG. 4, it was confirmed that [Ca ²⁺ ] _i transient was induced in each fraction in a concentration-dependent manner, but in the case of the ginseng polysaccharide fraction, [Ca ²⁺ ] _i It was confirmed that transient was not induced.

In the case of the irradiated ponin fraction, [Ca ²⁺ ] _i transient was not induced as much as the gintonin fraction, but [Ca ²⁺ ] _i transient was induced at 1 μg/ml, and saturation was confirmed at that concentration.

It was confirmed that the degree of [Ca ²⁺ ] _i transient induction ability of the ginseng tonin fraction, ginseng polysaccharide fraction, and irradiation fraction was proportional to the LPA C18:2 content of each fraction shown in Table 1 above.

Experimental Example 5. Comparison of antioxidant action

Ginseng polysaccharide components are known to have antioxidant activity (Ha et al., 2017).

ABTS ⁺ radical removal levels of the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng saccharide fraction obtained in Example 1 were confirmed, and the results are shown in FIG. 5 .

Referring to FIG. 5 , it was confirmed that the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng ginsenoside fraction all removed radicals in a concentration-dependent manner.

However, the ginseng polysaccharide fraction showed the highest ABTS ⁺ radical scavenging ability, and it was confirmed that it was more than twice as high as the ginseng tonin fraction and the ginseng irradiation fraction at the same concentration.

Experimental Example 6. Comparison of anti-platelet action

Ginseng is known to have anti-platelet activity (Irfan et al., 2019).

In order to compare the anti-platelet action of the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng irradiation fraction obtained in Example 1, collagen was used as a platelet aggregation-inducing substance (Irfan et al. 2019), and the anti-platelet activity - The platelet activity was compared and the results are shown in FIG. 6 .

Referring to FIG. 6, when platelets were treated with collagen alone or collagen and each fraction at the same time, in the case of the gintonin fraction, platelet aggregation by collagen was statistically inhibited, but the ginseng polysaccharide fraction and the ginseng irradiation fraction In the case of , it was confirmed that platelet aggregation by collagen was not inhibited, and only the gintonin fraction among the three types of fractions had an anti-platelet action.

Experimental Example 7. Comparison of indicators related to immune activity

Ginseng polysaccharide components are known to induce immune activity (Kim et al., 2002).

In order to compare the immune cell activity by the ginseng tonin fraction, ginseng polysaccharide fraction and ginseng irradiation fraction obtained in Example 1, nitric oxide (NO) production, IL6, TNF-α production and phagocytosis in macrophages (phagocytosis) ability was compared, and the results are shown in FIG. 7 . At this time, lipopolysaccharide (LPS) was used as a positive control.

Referring to a) of FIG. 7, in the case of NO production ability, the ginseng polysaccharide fraction (300 μg/ml) showed the highest level, and the gintonin fraction (GEF) had little NO production ability, and the ginseng polysaccharide fraction showed ginseng polysaccharide It was lower than the fraction, but it was confirmed that there was a little.

In addition, referring to b) of FIG. 7, in the case of IL-6 production ability, the ginseng polysaccharide fraction (300 μg/ml) increased IL-6 production, but the same concentration of gintonin fraction was suppressed, and ginseng crude It was confirmed that the saponin fraction did not show an ability to increase IL-6 production.

In addition, referring to c) of FIG. 7, in the case of the ability to produce TNF-α, ginseng polysaccharide was found to have the greatest effect in a concentration-dependent manner, but it was confirmed that there was no effect on the ginseng tonin fraction and the ginseng ginsenoside fraction. . It was confirmed that LPS used as a positive control increased TNF-α production.

However, referring to d) of FIG. 7 , macrophage phagocytosis was found to be greatest in the ginseng irradiation fraction, followed by the ginseng polysaccharide fraction and the gintonin fraction.

Overall, it was confirmed that the index study results for immune cell activity were the highest in the ginseng polysaccharide fraction.

Experimental Example 8. Comparison of cell protection ability by cell death defense and reactive oxygen species (ROS) generating substances

Experiments were performed using hippocampal-derived HT-22 neurons in order to compare the effects of ginseng tonin fraction, ginseng polysaccharide fraction, and ginseng irradiation fraction obtained in Example 1 on cell survival and defense against cell death by ROS. And the results are shown in Figure 8.

Referring to a) of FIG. 8 , it can be confirmed that the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng ginseng saccharide fraction do not affect cell death.

Referring to b) of FIG. 8, when iodoacetic acid (IAA) (3 μM), known as a substance that kills cells by generating ROS in mitochondria in nerve cells and blocking ATP production, was treated alone for 24 hours (control group , control), it was confirmed that the cells died, but when the gintonin fraction was simultaneously treated with IAA, it was confirmed that the effect of preventing cell death increased for each treatment concentration. In the case of the ginseng polysaccharide fraction, when treated simultaneously with IAA, there is an effect of preventing cell death by IAA treatment alone, but in the case of the ginseng polysaccharide fraction, it is confirmed that the effect is weaker than that of the ginseng tonin fraction and the ginseng irradiation fraction. could

Experimental Example 9. Comparison of arginine content

Arginine is one of the amino acids, and ginseng is known to contain more free arginine than other amino acids (Yoon et al., 2015), and white ginseng is known to contain about 1% of the arginine (Cho et al., 2008).

In the cardiovascular system, ginseng arginine acts as a NO donor to dilate blood vessels and lowers blood pressure (Lee et al., 2016). et al., 2018) has been reported.

The arginine content of the ginseng tonin fraction, the ginseng polysaccharide fraction, and the ginseng saccharide fraction obtained in Example 1 was measured, and the results are shown in FIG. 9 .

Referring to FIG. 9 , it was confirmed that the ginseng polysaccharide fraction contained 4 to 5 times more arginine compared to the ginseng tonin fraction and the ginseng ginsenoside fraction.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (15)

Preparing a ginseng alcohol extract (step 1);
Concentrating and drying the ginseng alcohol extract (step 2);
Dissolving the concentrated and dried alcohol extract in water and precipitating at low temperature to separate a first supernatant and a first lower layer (step 3);
obtaining a gintonin-enriched fraction (GEF) from the first lower layer (step 4);
Adding alcohol to the first supernatant and precipitating at a low temperature to separate the second supernatant and the second lower layer (step 5);
obtaining a ginseng polysaccharide fraction from the second lower layer (step 6); and
Obtaining a ginseng irradiation fraction from the second supernatant (step 7);
A method for producing a ginseng tonin fraction, a ginseng polysaccharide fraction, and a ginseng irradiation fraction comprising:
According to claim 1,
Wherein step 3 is performed at 0 ° C. to 8 ° C. for 12 hours to 120 hours.
According to claim 1,
Step 5 is characterized in that the alcohol is added to the first supernatant so that the alcohol content is 80% to 99%.
According to claim 1,
Wherein step 5 is performed at 0 ° C to 8 ° C for 12 hours to 120 hours.
According to claim 1,
The ginseng is at least one selected from wild ginseng, camphor ginseng, cultivated ginseng, American ginseng, Western ginseng, Chinese ginseng, red ginseng, fresh ginseng, white ginseng and cultured ginseng.
According to claim 5,
The method characterized in that the ginseng is at least one selected from the roots, leaves, stems, fruits and flowers of ginseng.
According to claim 1,
The method characterized in that the ginseng is at least one ginseng by-product selected from ginseng meal and red ginseng meal.
According to claim 1,
The method characterized in that the ginseng tonin fraction obtained in step 4 contains more diol ginsenosides than the ginseng polysaccharide fraction and ginseng saxophonin fraction obtained in steps 6 and 7, respectively.
According to claim 8,
The method of claim 1, wherein the diol ginsenoside comprises Rb1, Rb2, Rc and Rd.
According to claim 1,
The method characterized in that the ginseng irradiation fraction obtained in step 7 contains more triol ginsenosides than the gintonin fraction and ginseng polysaccharide fraction obtained in steps 4 and 6, respectively.
According to claim 10,
The method of claim 1, wherein the triol ginsenoside comprises Rg1 and Re.
According to claim 1,
The method characterized in that the ginseng polysaccharide fraction obtained in step 6 contains more arginine than the ginseng tonine fraction and ginseng saccharide fraction obtained in steps 4 and 7, respectively.
A ginseng tonin fraction, a ginseng polysaccharide fraction, or a ginseng irradiation fraction prepared by the method of claim 1.
A health food composition for preventing or improving thrombotic diseases containing the gin and tonin fraction of claim 13.
A health food composition for increasing antioxidant or immune activity containing the ginseng polysaccharide fraction of claim 13.

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