KR102597682B1 - Red ginseng with enhanced ginsennoside content and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a red ginseng concentrate enriched with ginsenoside Rg3 and a method for producing the same. Specifically, red ginseng with enhanced ginsenoside content has improved anti-inflammatory activity, suppresses free radical generation, and significantly improves cytotoxicity against cancer cells. It relates to concentrates and methods for producing them.

Description

Red ginseng concentrate with enhanced ginsenoside content and manufacturing method thereof {RED GINSENG WITH ENHANCED GINSENNOSIDE CONTENT AND MANUFACTURING METHOD THEREOF}

The present invention relates to a red ginseng concentrate with enhanced ginsenoside content and a method for manufacturing the same. Specifically, the ginsenoside content is enhanced, with improved anti-inflammatory activity, suppressed generation of active oxygen, and significantly improved cytotoxicity against cancer cells. It relates to red ginseng concentrate and its manufacturing method.

Recently, with the aging of the population, increasing interest in well-being or health care to improve quality of life, and social interest, health foods with effects such as improving immunity and restoring vitality, such as ginseng and red ginseng products The use of various health functional foods has increased. Ginseng is a traditional medicinal herb, a perennial food belonging to the red ginseng species of the genus Pseudomonas, and is also the most well-known tonic in both the East and the West. It also inhibits aging, prevents arteriosclerosis, improves hyperlipidemia, improves high blood pressure, strengthens liver function, has anti-stress effects, etc. It has various effects, including increasing immunity. The main ingredients and contents of ginseng are 4-6% of minerals, 0.05% of vitamins, 60-70% of carbohydrates such as monosaccharides, polysaccharides, and pectins, 1-2% of fat-soluble components such as fatty acids, rectified substances, and phenolic compounds, proteins, amino acids, Nitrogen compounds such as nucleic acids and alkaloids are known to be 12-16%.

Red ginseng is red ginseng made by steaming and drying fresh ginseng. The main components of red ginseng include glycosides, flavonoids, B vitamins, trace elements, enzymes, antioxidants, organic acids and amino acids, but the main effective component known is saponin. . Saponins in ginseng and red ginseng are a type of glycoside and have a unique chemical structure that is different from saponins found in other plants. Their efficacy is also greatly different, and they have various types and various effects. In order to distinguish saponins from ginseng and red ginseng from other plant saponins, they are called ginsenosides, meaning ginseng glycosides.

Ginsenoside Rg3, which is contained in trace amounts only in red ginseng, has vascular relaxation, platelet aggregation inhibition, anti-inflammatory activity, inhibition of free radicals, anticancer activity and anticancer drug resistance inhibition, and brain nerve cell protection, while ginsenoside Rh2 has anticancer and immune enhancing effects. Ginsenoa sheet Rg1 has the effect of strengthening memory, preventing dementia, controlling diabetes, preventing obesity, and relieving fatigue. Ginsenoside Rg2 has the effect of enhancing memory, preventing dementia, anticancer activity, and inhibiting platelet aggregation, Re has the effect of improving liver function, promoting bone marrow cell production, strengthening memory, and preventing dementia, and Rb1 has the effect of expanding blood vessels, strengthening immunity, and anti-stress. and blood pressure control effect, C-K has anti-cancer effect, increased immunity, and hair growth effect, and Rg5 has antioxidant activity, anti-cancer activity, platelet aggregation inhibitory effect, anti-allergic activity, memory improvement and nerve cell protection effect, anti-inflammatory activity, and dementia. There is a preventive effect, etc. In addition, Rk1 has anticancer activity, memory improvement and neuron protection effects, dementia prevention effect, cell regeneration activity, and anti-aging effect, and Rh1 is known to be a substance with liver protection, antitumor activity, and platelet aggregation inhibitory activity. . In addition, it has been reported that Rb1 enhances the synthesis of testosterone in the human body, promotes the regeneration of skin tissue, improves blood flow through cerebrovascular regeneration and reconstruction, and also has the effect of suppressing secondary degeneration of nervous tissue, RC is known to have excellent sedative properties, and is especially known to inhibit the metastasis or proliferation of breast cancer. In addition, Rf, Ro, Rd, etc. are known to be effective in suppressing lipid peroxidation, analgesic effects on brain nerve cells, detoxifying alcohol, treating inflammation, promoting protein and lipid activity, and promoting secretion of adrenal corticosteroids.

Among them, ginsenoside Rg3 is a red ginseng ingredient that has recently attracted attention, but red ginseng concentrate extracted through a general method had the disadvantage of having an excessively low content of ginsenoside Rg3. Therefore, in order to further enhance the effect of ginsenoside Rg3 described above, the development of a method for concentrating the content of ginsenoside Rg3 is required.

Korean Patent Publication No. 10-2100453 (registered on April 7, 2020)

The present invention was created to solve the above-mentioned problems, and the problem to be solved by the present invention is to improve the content of ginsenoside to have high anti-inflammatory activity and suppressed oxygen generation amount, and significantly improved cytotoxicity against cancer cells. To provide a red ginseng concentrate with increased ginsenoside content and a method for manufacturing the same.

The method for producing red ginseng concentrate with increased ginsenoside content of the present invention to solve the above problems includes the steps of 1) diluting the red ginseng concentrate to form a diluted solution; 2) natural precipitation or centrifugation of the diluted solution to obtain a water-soluble material; 3) preparing an acidified solution by adding and mixing an organic acid to the water-soluble material; and 4) obtaining an insoluble solid by heating and cooling the acidified solution and subjecting it to natural precipitation or centrifugation; may include.

As a preferred embodiment of the present invention, the organic acid may be added in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the water-soluble material.

As a preferred embodiment of the present invention, the organic acid may include one or more selected from citric acid, ascorbic acid, oxalic acid, and lactic acid.

In a preferred embodiment of the present invention, centrifugation in steps 2) and 4) can be performed for 5 to 20 minutes at a rotation speed of 4,000 to 6,000 rpm, respectively.

In a preferred embodiment of the present invention, purified water may be added in an amount of 8 to 10 times the weight of the red ginseng concentrate in the dilution process.

As a preferred embodiment of the present invention, the heating process can be performed at 70 to 95°C for 6 to 10 hours.

As a preferred embodiment of the present invention, the cooling process can be performed until the final temperature reaches 10 to 30°C.

For another purpose of the present invention, a red ginseng concentrate with improved ginsenoside content, which is prepared by the above-described production method and has an increase rate of 5,000 to 15,000% in ginsenoside Rg3 content measured through the following relational equation 1, can be prepared.

[Relationship 1]

In equation 1, the ginsenoside Rg3 content means the sum of the ginsenoside Rg3(R) and the enantiomer Rg3(S) content of the ginsenoside Rg3(R).

As a preferred embodiment of the present invention, the red ginseng concentrate may have an increase rate of ginsenoside Rh4 content of 800 to 2,500% as measured through equation 2 below.

[Relational Expression 2]

The red ginseng concentrate with increased ginsenoside content prepared according to the present invention has excellent anti-inflammatory activity and significantly low cytotoxicity by suppressing the incidence of nitric oxide (NO) and the amount of reactive oxygen species generated, which cause inflammation, even at high concentrations.

Figure 1 is a diagram showing each step of the method for producing red ginseng concentrate with increased ginsenoside content according to a preferred embodiment of the present invention.
Figure 2 shows the red ginseng concentrate (blue), which is a concentrate preparation, and the red ginseng concentrate (red) with increased ginsenoside content prepared according to a preferred embodiment of the present invention, in the production method according to a preferred embodiment of the present invention. This is a comparison graph of major ginsenosides.
Figure 3 (a) is a graph showing the cytotoxicity (cytotoxicity) according to the concentration of red ginseng concentrate before enhancement of the ginsenoside component prepared according to a preferred embodiment of the present invention, and Figure 3 (b) is a graph showing a preferred embodiment of the present invention. This is a graph showing the cytotoxicity (cytotoxicity) according to the concentration of red ginseng concentrate with increased ginsenoside content according to the example.
Figure 4 a) is a graph showing cytotoxicity (cytotoxicity) according to concentration after treating red ginseng concentrate before ginsenoside component enhancement with inflammatory substances (LPS) according to a preferred embodiment of the present invention, Figure 4 b ) is a graph showing cytotoxicity (cytotoxicity) according to concentration after treating red ginseng concentrate with increased ginsenoside content with inflammatory substances (LPS) according to a preferred embodiment of the present invention.
Figure 5 a) is a graph showing the intracellular nitric oxide (NO) generation rate according to the concentration of red ginseng concentrate before enhancement of the ginsenoside component according to a preferred embodiment of the present invention, and Figure 5 b) is a preferred embodiment of the present invention. This is a graph showing the intracellular nitric oxide (NO) generation rate according to the concentration of red ginseng concentrate with increased ginsenoside content according to the example.
Figure 6 a) is a graph showing the production rate of reactive oxygen species (ROS) according to the concentration of red ginseng concentrate before enhancement of ginsenoside components according to a preferred embodiment of the present invention, and Figure 6 b) is a graph showing the preferred embodiment of the present invention in cells. This is a graph showing the production rate of reactive oxygen species (ROS) according to the concentration of ginsenoside Rg3 concentrated red ginseng concentrate according to the example.

As described above, the conventional red ginseng concentrate had an excessively low content of ginsenoside Rg3, which had the problem of poor efficacy such as anti-inflammatory activity and inhibition of active oxygen radicals. In particular, ginsenoside Rg3 is a component that does not exist in ginseng. If the content of ginsenoside Rg3 can be increased, the occurrence of nitric oxide (NO) and the amount of active oxygen generated can be suppressed, resulting in excellent anti-inflammatory activity and significantly lowering cytotoxicity. This has the advantage of enhancing the special effects of red ginseng concentrate.

Before explaining the present invention, the red ginseng concentrate used in the present invention will first be described.

Red ginseng concentrate according to an embodiment of the present invention includes the steps of preparing an extract obtained by adding red ginseng to an alcohol dilution solution; It can be prepared including the step of concentrating the extract under reduced pressure to prepare a red ginseng concentrate.

First, the red ginseng may be steamed 3- to 10-year-old ginseng, and preferably 4 to 9-year-old ginseng.

On the other hand, the alcohol dilution is made by diluting alcohol with purified water, and the alcohol dilution may contain the alcohol at a concentration of 60 to 80% by weight (w/w), preferably 65 to 75% by weight (w). It can be included at a concentration of /w).

Meanwhile, the extraction can be repeated 1 to 5 times at 60 to 90°C at a pressure of 1 to 2 atm, and preferably 2 to 4 times at 65 to 85°C at a pressure of 1.5 to 1.7 atm. there is.

Next, the reduced pressure concentration can be performed by reducing the pressure to a pressure of 0.5 to 2 atm at 30 to 60°C, and preferably can be performed by reducing the pressure to a pressure of 0.75 to 1 atm at 40 to 45°C.

On the other hand, the red ginseng concentrate prepared by the above-described production method may contain solid content of 40% by weight or more, preferably 50 to 90% by weight, of the total weight.

In addition, compared to other conventional red ginseng concentrates, the red ginseng concentrate is extracted and extracted at a high concentration without decomposing the ginsenoside glycoside, which has two or more sugars bonded to carbons 3, 6, or 20 of the ginsenoside chemical structure. It has a concentrating property, and by having this property, it is possible to obtain the effect of increasing the content of ginsenoside Rg3 or ginsenoside Rh4.

Hereinafter, the present invention will be described in more detail through a method for producing red ginseng concentrate with increased ginsenoside content, which will be described later.

The method for producing red ginseng concentrate with increased ginsenoside content of the present invention includes the steps of: 1) diluting the red ginseng concentrate to form a diluted solution; 2) natural precipitation or centrifugation of the diluted solution to obtain a water-soluble material; 3) preparing an acidified solution by adding and mixing an organic acid to the water-soluble material; and 4) obtaining an insoluble solid by heating and cooling the acidified solution and subjecting it to natural precipitation or centrifugation; may include.

Specifically, the dilution process in step 1) can be performed by adding purified water at 8 to 10 times the weight of the red ginseng concentrate, and preferably at 8.5 to 9.5 times.

If the purified water is added in less than 8 times, dilution may not be done properly, which may increase the destruction and deformation of the active ingredient during the acidification or heating step. If it is added in more than 10 times, it will take a lot of time for concentration. There is a risk that productivity may decrease, and as the diluent becomes excessively diluted, the activity of hydrogen ions increases in the acidification step, causing the sugar bonded to carbon 3 of ginsenoside Rg3 to decompose or the hydroxyl group at carbon 20 to undergo a condensation reaction. Problems may arise where the content of ginsenoside Rg3 is lowered and the sugar bonded to carbon 6 of ginsenoside Rh4 is decomposed or the hydroxyl group at carbon 20 undergoes a condensation reaction, lowering the content of ginsenoside Rh4.

Next, the natural precipitation or centrifugation in step 2) is preferably performed for a time when the content of residue (precipitate) excluding the water-soluble substances in the diluted solution is less than 1% by weight, and more preferably, the content of the residue is less than 1% by weight. This can be carried out until it becomes less than 0.1% by weight.

Specifically, the natural precipitation can be performed under conditions known in the art, and is preferably performed for a time when the content of the residue (precipitate) in the diluted solution is less than 1% by weight. More preferably, it is desirable to carry out the process until the content of the residue is less than 0.1% by weight.

Meanwhile, the centrifugation can be used without limitation as long as it is a centrifugation method known in the art. Preferably, it can be performed for 5 to 20 minutes at a rotation speed of 4,000 to 6,000 rpm, and preferably at a rotation speed of 4,500 to 5,500 rpm. It can be performed at any speed for 10 to 15 minutes.

If the centrifugation is performed at a rotation speed of less than 4,000 rpm or for less than 5 minutes, there may be a problem that residues (sediment) other than water-soluble substances may not be sufficiently removed as centrifugation is not sufficiently performed. If centrifugation is performed at a rotation speed exceeding 6,000 rpm or for more than 20 minutes, the heat generated during the centrifugation process causes ginsenoside Rb1 or Rg1 to change to ginsenosides other than ginsenoside Rg3 or Rh4. There may be problems that can be decomposed into .

In addition, when the red ginseng concentrate is acidified without performing the natural precipitation or centrifugation, there may be a problem in that the precipitate is decomposed and the conversion of ginsenoside Rb1 or Rg1 to ginsenoside Rg3 or Rh4 is prevented and the content is lowered. .

At this time, the precipitate may be a substance dissolved in the diluted solution, and preferably may be a substance (residue) other than the ginsenoside component.

Next, the organic acid of step 3) can be added and mixed in an amount of 0.1 to 5 parts by weight, preferably 0.3 to 3 parts by weight, based on 100 parts by weight of the supernatant.

If the organic acid is added in less than 0.1 parts by weight, the ginsenoside content may not be sufficiently increased, and if it is added in more than 5 parts by weight, the pH concentration of the diluted solution is lowered too much, destroying the active ingredients of the final manufactured red ginseng concentrate. There may be problems with it becoming or deforming.

Additionally, the organic acid may include one or more selected from citric acid (citric acid), ascorbic acid, oxalic acid, and lactic acid, and more preferably citric acid.

The acidified solution prepared in step 3) described above may have a pH concentration of 1.5 to 4.5, and preferably may have a pH concentration of 2 to 4. If the pH concentration is less than 1.5, the solution may become excessively acidic and there may be a problem of decomposition of ginsenoside Rg3 or Rh4, and if it exceeds 4.5, the ginsenoside Rg3 or Rh4 content will decrease and other undesired ginseng may be added. There may be a problem with senocides being created.

Next, the heating process in step 4) can be performed at 70 to 95°C for 6 to 10 hours, preferably at 80 to 95°C for 7.5 to 9.5 hours, and more preferably at 80 to 95°C. It can be performed for 7.0 to 9.0 hours at ℃.

If the heating process is performed at a temperature below 70°C or for less than 6 hours, the manufacturing time is extended, so the productivity of the red ginseng concentrate with increased ginsenoside content is reduced or the desired ginsenoside component is lost. There is a risk that sufficient production may not be produced, and if the process is performed at a temperature exceeding 95°C or for more than 10 hours, not only will the process cost increase, but other active ingredients in the red ginseng concentrate will be destroyed. In particular, 20% of ginsenoside Rh4 will be destroyed. The hydroxyl group bonded to this carbon may undergo a condensation reaction and decompose into other ginsenosides, causing a decrease in the content of the ingredient.

Next, the cooling process in step 4) can be performed until the final temperature reaches 10 to 30°C, and preferably, it can be performed until the final temperature reaches room temperature of 15 to 25°C.

If the cooling process is performed so that the final temperature is less than 10℃, there may be a problem in which ginsenoside Rg3 or Rh4 forms a precipitate that is insoluble in water, so the insoluble solid material does not contain sufficient ginsenoside Rg3 and Rh4 components. There may be a problem that it is not properly detected, and if the process is performed until the final temperature exceeds 30°C, there may be a problem of generating an intermediate that is converted to ginsenoside Rg3 or Rh4, that is, an impurity.

Next, the precipitation process of step 4) can be performed through natural precipitation or centrifugation, similar to the precipitation process of step 2), and the content of the residue (precipitate) in the acidified solution after performing the heating and cooling process It is preferable to carry out the process for a time that is less than 1% by weight, and more preferably, it can be carried out until the content of the residue is less than 0.1% by weight.

Specifically, the natural precipitation can be performed under conditions known in the art, and is preferably performed for a time such that the content of the residue (precipitate) in the acidified solution subjected to the heating and cooling process is less than 1% by weight. do. More preferably, it is desirable to carry out the process until the content of the residue is less than 0.1% by weight.

Meanwhile, the centrifugation can be performed by a centrifugation method known in the art, and can be performed for 5 to 20 minutes at a rotation speed of 4,000 to 6,000 rpm, preferably for 10 minutes at a rotation speed of 4,500 to 5,500 rpm. Can be performed for ~15 minutes.

If the centrifugation is performed at a rotation speed of less than 4,000 rpm or for less than 5 minutes, there may be a problem that the residue may not be sufficiently removed as the centrifugation is not sufficiently performed. If performed at a rotation speed exceeding , or performed for more than 20 minutes, there may be a problem in which ginsenoside Rg3 or Rh4 is decomposed due to the heat generated during the centrifugation process.

At this time, the insoluble solid material prepared in step 4) may be an acidic insoluble material that precipitates without dissolving in the acidified solution.

Next, after performing step 4), step 5) of drying the insoluble solid and then powdering it may be further included.

Red ginseng concentrate with increased ginsenoside content prepared by the above-described method may be solid type.

The second method for producing a red ginseng concentrate with increased ginsenoside content of the present invention includes the steps of 1) diluting the red ginseng concentrate to form a diluted solution; 2) centrifuging the diluted solution to obtain a supernatant; and 3) heating and cooling the supernatant to obtain a liquid red ginseng concentrate with increased ginsenoside content.

In the second manufacturing method, all processes except the acidification process of adding organic acid and the process of centrifuging the acidified solution are performed under the same conditions as in the first manufacturing method described above, so detailed description will be omitted.

Red ginseng concentrate with increased ginsenoside content prepared by the second method described above may be of liquid type.

The red ginseng concentrate with increased ginsenoside content prepared by the above-described method may have an increase rate of ginsenoside Rg3 content of 5,000 to 15,000%, preferably 7,000 to 13,000%, as measured through equation 1 below.

[Relationship 1]

In equation 1, the ginsenoside Rg3 content means the sum of the ginsenoside Rg3(R) and the enantiomer Rg3(S) content of the ginsenoside Rg3(R).

In addition, the red ginseng concentrate with increased ginsenoside content may have a sum of ginsenoside Rg3(R) and ginsenoside Rg3(S) content of 50 to 150 mg/g, preferably 60 to 140 mg/g. It may be g.

If the sum of the contents of the ginsenoside Rg3(R) and ginsenoside Rg3(S) is less than 50 mg/g, the anti-inflammatory activity and oxygen suppressing ability of the red ginseng concentrate at high concentrations may not be sufficient, and conversely, the content In order to reach 150 mg/g, the conditions of the above-described concentration process must become more severe, so manufacturing costs increase, and problems such as changes in the content composition of other ingredients or structure destruction during the manufacturing process may occur. .

In addition, the red ginseng concentrate with increased ginsenoside content may have an increase rate of ginsenoside Rh4 content of 1,000 to 3,000%, preferably 1,300 to 2,700%, as measured through Equation 2 below.

[Relational Expression 2]

Meanwhile, the red ginseng concentrate with increased ginsenoside content may further include 6 to 13 mg/g ginsenoside Rg2 and 70 to 120 mg/g ginsenoside Rg4 as active ingredients, preferably may further include ginsenoside Rg2 at a content of 7 to 12 mg/g and ginsenoside Rg4 at a content of 75 to 110 mg/g as active ingredients.

Meanwhile, the red ginseng concentrate with increased ginsenoside content can be applied to various fields such as food, cosmetics, and pharmaceuticals. Preferred examples include health functional foods, functional cosmetics, or natural medicines.

Hereinafter, the configuration and effects will be described in more detail with reference to preferred embodiments of the present invention. However, this does not mean that the scope of the present invention is limited to the scope of the following examples. The present invention can be implemented by changing or deleting the configuration of the embodiments or adding other configurations within the range known to those skilled in the art, and the following examples should be understood as merely illustrating preferred embodiments.

[Example]

Preparation example: Preparation of red ginseng concentrate

100 kg of red ginseng (5-year-old ginseng, Geumsan, Chungcheongnam-do) was extracted three times at 70°C with 70% alcohol dilution (900 kg, w/w) and concentrated under reduced pressure at 60°C to produce red ginseng with a solid content of about 60% by weight. A concentrate (90 kg) was prepared.

Example 1: Preparation of red ginseng concentrate with increased ginsenoside content (Method 1)

The red ginseng concentrate (90kg) prepared in Preparation Example 1 was diluted 9.0 times with purified water (810kg) to prepare a 10% by weight (w/w) dilution.

Next, the diluted solution was centrifuged at a rotation speed of 5,000 rpm to obtain residue (precipitate) and water-soluble material.

Next, 1 part by weight (9 kg) of citric acid was added to 100 parts by weight of the water-soluble material for acidification to obtain an acidified solution with a pH concentration of 3.0.

Next, the acidified solution was heated at 85°C for 8 hours and cooled to room temperature (25°C), centrifuged at a rotation speed of 5,000 rpm, the supernatant was discarded, and red ginseng with increased ginsenoside content, which is an insoluble solid, was obtained. A concentrate was prepared.

Experimental Example 1: Ingredient Analysis

The components of the red ginseng concentrate of Example 1 and Preparation Example were analyzed using high-performance liquid chromatography (HPLC), and the results are shown in Figure 2.

Looking at Figure 2, it was confirmed that most components were improved in Example 1 compared to the preparation example, especially ginsenoside Rh4, ginsenoside Rg4, ginsenoside Rg3(R), and ginsenoside Rg3. The increase in content of (S) was most noticeable.

As a control, medium (Cont) without inflammatory factors (LPS) and nothing added, and red ginseng concentrate according to Example 1 and Preparation Example were used at 10 μg/ml, 25 μg/ml, 50 μg/ml, and 100 μg/ml, respectively. When treating mouse-derived macrophages (RAW264.7 cells) at concentrations of 250 μg/ml, 500 μg/ml, and 1,000 μg/ml, the cell survival rate in each sample after 24 hours is shown in a in Figure 3. ) are shown in ~b).

Looking at a) to b) of Figure 3, the preparation example (a) of Figure 3) has low cytotoxicity when 250 μg/ml or less and has excellent cell safety, while Example 1 (b) of Figure 3) has 25 μg/ml. It was confirmed that cell safety was excellent due to low cytotoxicity when the concentration was below / ㎖.

Experimental Example 3: Cytotoxicity evaluation on LPS treated cells

As a control, medium (Cont) without any inflammatory factors (LPS) and nothing added, and mice derived from mice treated with inflammation-inducing substances (LPS 100 ng/ml) using different concentrations of the red ginseng concentrate according to Example 1 and Preparation Example. When treated with macrophages (RAW264.7 cells), the cell survival rate in each sample after 24 hours is shown in Figure 4 a) to b).

Looking at a) to b) of Figure 4, the preparation example (a) of Figure 4) has low cytotoxicity when 250 μg/ml or less and has excellent cell safety, while Example 1 (b) of Figure 4) has low cytotoxicity at 250 μg/ml or less. It was confirmed that cell safety was excellent due to low cytotoxicity when the concentration was below / ㎖.

Experimental Example 4: Measurement of nitrogen monoxide (NO) generation

As a control, the medium (Cont) with no inflammatory factors (LPS) and nothing added, and the red ginseng concentrate according to Example 1 and Preparation Example were mixed at different concentrations as shown in Table 1 below to produce an inflammation-inducing substance (LPS 100 ng/ml). When treated with mouse-derived macrophages (RAW264.7 cells), the nitric oxide (NO) inhibition rate (amount generated) in each sample after 24 hours was measured and shown in Figures 5 a) to b) and Table 1. shown in

At this time, each experiment was repeated three times, and the average and deviation values were calculated and shown in Table 1 below.

Looking at a) to b) of Figure 5, the preparation example (a) of Figure 5) must be treated at a high concentration of 250 μg/ml or more to reduce cytotoxicity and reduce the amount of nitric oxide (NO) generated, thereby providing excellent cell safety. 1 (b) in Figure 5) was confirmed to have low cytotoxicity and excellent cell safety, as the amount of nitric oxide (NO) generated was significantly low even at a low concentration of about 25 μg/ml.

(μg/ml) Cont LPS 10 25 50 100 Preparation example
(% of LPS) average 4.99 100 93.12 80.41 64.24 35.56 Deviation 0.56 3.66 2.12 1.48 1.28 0.85 Example 1
(% of LPS) average 3.97 100 42.46 19.17 9.80 4.80 Deviation 0.19 2.83 1.54 2.03 0.22 0.11

Looking at Table 1, it was confirmed that Example 1 showed excellent nitrogen monoxide (NO) generation even at a low concentration of 10 to 100 μg/ml, while the preparation example showed a high nitrogen monoxide generation amount of more than 60% of LPS. In particular, , at the same concentration of 25 μg/ml, the amount of nitrogen monoxide (NO) generated is 80.41% of LPS in the preparation example, and 19.17% of LPS in Example 1, and the difference in the amount of nitrogen monoxide (NO) generated is more than 60% of LPS. It was found that there was a very big difference.

Experimental Example 5: Measurement of reactive oxygen species (ROS) production

As a control, medium (Cont) without any inflammatory factors (LPS) and nothing added, and mice derived from mice treated with an inflammation-inducing substance (LPS 100 ng/ml) using different concentrations of the red ginseng concentrate according to Example 1 and Preparation Example. When treated with macrophages (RAW264.7 cells), the amount of reactive oxygen species (ROS) generated in each sample was measured after 24 hours, and is shown in Figure 6 a) to b) and Table 2 below.

At this time, each experiment was repeated three times, and the average and deviation values were calculated and shown in Table 2 below.

Looking at a) to b) of FIG. 6, the preparation example (a) of FIG. 6) must be treated at a high concentration of 250 μg/ml or more to have low cytotoxicity and low production of reactive oxygen species (ROS), thereby providing excellent cell safety. 1 (b) in Figure 5) was confirmed to have low cytotoxicity and excellent cell safety due to the significantly low production of reactive oxygen species (ROS) even at a low concentration of about 25 μg/ml.

(μg/ml) Cont LPS 10 25 50 100 Preparation example
(% of LPS) average 31.24 100.0 87.35 82.70 73.38 58.01 Deviation 3.80 4.17 3.98 2.17 4.78 1.55 Example 1
(% of LPS) average 24.31 100.0 64.33 40.09 32.32 13.75 Deviation 1.09 3.30 0.82 1.04 2.27 0.77

Looking at Table 2, it was confirmed that Example 1 showed excellent reactive oxygen species (ROS) production even at a low concentration of 10 to 100 μg/ml, while Preparation Example showed a high nitrogen monoxide production of 60% of LPS.

In addition, in particular, at the same concentration of 25 μg/ml, the amount of reactive oxygen species (ROS) generated was 82.70% of LPS in the preparation example, and 40.09% of LPS in Example 1, showing a difference in the amount of reactive oxygen species (ROS) generated by more than 40% of LPS. I was able to.

Example 2: Preparation of red ginseng concentrate with increased ginsenoside content (Method 2)

The red ginseng concentrate (90 kg) prepared in Preparation Example 1 was diluted with purified water (810 kg) to prepare a 10% (w/w) dilution.

Next, the diluted solution was centrifuged at a rotation speed of 5,000 rpm to obtain residue (precipitate) and supernatant.

Next, the supernatant was heated at 85°C for 8 hours and cooled to room temperature (25°C) to prepare a liquid type red ginseng concentrate with increased ginsenoside content.

Example 3 to Example 12 and Comparative Example 1 to Comparative Example 11: Preparation of red ginseng concentrate with increased ginsenoside content

Red ginseng concentrate was prepared in the same manner as in Example 1, but Examples 3 to 12 and Comparative Examples 1 to 11 were performed under the conditions shown in Tables 3 to 8 below.

Experimental Example 6: Evaluation of physical properties of red ginseng concentrate with increased ginsenoside content

Red ginseng concentrates with increased ginsenoside content of Preparation Example, Examples 1 to 12, and Comparative Examples 1 to 11 were prepared in the following manner, and the results are shown in Tables 3 to 8 below. .

(1) Ingredient analysis

Components were analyzed in the same manner as in Experimental Example 1 above. In addition, the rate of increase in the ginsenoside Rg3 content was measured using the following equation 1, and the increase rate of the ginsenoside Rh4 content was measured using the following equation 2.

[Relationship 1]

In equation 1, the ginsenoside Rg3 content means the sum of the ginsenoside Rg3(R) and the enantiomer Rg3(S) content of the ginsenoside Rg3(R).

[Relational Expression 2]

(2) Cytotoxicity evaluation according to concentration

The experiment was performed in the same manner as in Experimental Example 2, except that the concentration for treating mouse-derived macrophages (RAW264.7 cells) was 100 μg/ml.

(3) Cytotoxicity evaluation on LPS-treated cells

The experiment was performed in the same manner as in Experimental Example 3, except that the concentration for treating mouse-derived macrophages (RAW264.7 cells) was 100 μg/ml. The lower the cytotoxicity, the less harmful it was judged to be.

(4) Measurement of nitrogen monoxide (NO) generation

The experiment was performed in the same manner as in Experimental Example 4, except that the concentration for treating mouse-derived macrophages (RAW264.7 cells) was 25 μg/ml.

(5) Measurement of reactive oxygen species (ROS) production

The experiment was performed in the same manner as in Experimental Example 5, except that the concentration for treating mouse-derived macrophages (RAW264.7 cells) was 25 μg/ml.

Example 1 Example 2 Example 3 Example 4




manufacturing
method Dilution process
(Purified water input amount) 9 multiples 9 multiples 9 multiples 9 multiples primary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 5,000 5,000 Organic acid input amount (parts by weight) One - 0.3 3 Acidification solution pH concentration 3.0 - 4.0 2.0 heating temperature 85 85 85 85 hour 8 8 8 8 secondary precipitation division centrifugation - centrifugation centrifugation rotation speed
(rpm) 5,000 - 5,000 5,000

Jinseno
side
enhanced
red ginseng
concentrate Ginsenoside
content
(mg/g) Rg3(R) 55.33 30.52 44.29 48.96 Rg3(S) 72.99 58.41 61.47 66.54 Rh4 84.32 69.77 72.63 75.97 Rg5 54 58 42 44 Rg3 content increase rate (%) 9,770 5027 8035 8784 Rh4 content increase rate (%) 1,364 1113 1163 1221 Cytotoxicity (%) 30 42 29 31 Cytotoxicity (%) after treatment with inflammation-inducing substances 25 28 24 26 NO generation (% of LPS) 19.17 21.64 20.94 18.63 ROS production (% of LPS) 40.09 29.55 41.69 39.58

Example 5 Example 6 Example 7 Example 8




manufacturing
method Dilution process
(Purified water input amount) 8.5 multiple 9.5 multiple 9 multiples 9 multiples primary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 5,000 5,000 Organic acid input amount (parts by weight) One One One One Acidification solution pH concentration 3.0 3.0 3.0 3.0 heating temperature 85 85 75 90 hour 8 8 8 8 secondary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 5,000 5,000 Jinseno
side
Rg3
content has been improved
red ginseng
concentrate Ginsenoside
content
(mg/g) Rg3(R) 52.96 49.24 42.18 56.98 Rg3(S) 65.16 60.35 59.76 73.54 Rh4 79.85 71.53 68.56 71.61 Rg5 51 40 52 54 Rg3 content increase rate (%) 8986 8330 7741 9940 Rh4 content increase rate (%) 1288 1144 1092 1145 Cytotoxicity (%) 38 29 30 30 Cytotoxicity (%) after treatment with inflammation-inducing substances 27 24 25 25 NO generation (% of LPS) 21.91 18.86 19.52 18.99 ROS production (% of LPS) 41.52 39.91 40.38 39.56

Example 9 Example 10 Example 11 Example 12



manufacturing
method Dilution process (purified water input amount) 9 multiples 9 multiples 9 multiples 9 multiples primary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 4,500 5,500 Organic acid input amount (parts by weight) One One One One Acidification solution pH concentration 3.0 3.0 3.0 3.0 heating temperature 85 85 85 85 hour 7.5 9.5 8 8 secondary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 4,500 5,500 Jinseno
side
Rg3
content has been improved
red ginseng
concentrate Ginsenoside
content
(mg/g) Rg3(R) 42.09 58.74 54.39 42.89 Rg3(S) 61.48 76.11 71.85 60.15 Rh4 70.35 72.53 83.96 79.14 Rg5 52 55 54 52 Rg3 content increase rate (%) 7866 10273 9610 7826 Rh4 content increase rate (%) 1123 1161 1360 1276 Cytotoxicity (%) 30 30 35 30 Cytotoxicity (%) after treatment with inflammation-inducing substances 25 25 28 25 NO generation (% of LPS) 20.33 18.06 21.36 18.68 ROS production (% of LPS) 40.56 39.05 42.50 39.58

Preparation example 1 Comparative Example 1 Comparative example 2 Comparative example 3




manufacturing
method Dilution process (purified water input amount)






- 9 multiples 9 multiples 9 multiples primary precipitation division centrifugation centrifugation - rotation speed
(rpm) 5,000 5,000 - Time (minutes) --- --- - Organic acid input amount (parts by weight) 0.001 8 One Acidification solution pH concentration 5.0 1.2 3.0 heating temperature 85 85 85 hour 8 8 8 secondary precipitation division centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 5,000 Jinseno
side
Rg3
content has been improved
red ginseng
concentrate Ginsenoside
content
(mg/g) Rg3(R) 0.48 10.8 29.64 20.59 Rg3(S) 0.82 28.92 35.78 25.87 Rh4 5.75 36.45 41.92 34.55 Rg5 54.0 26 29 24 Rg3 content increase rate (%) - 2955 4932 3473 Rh4 content increase rate (%) - 534 629 500 Cytotoxicity (%) 95 26 33 50 Cytotoxicity (%) after treatment with inflammation-inducing substances 95 22 28 39 NO generation (% of LPS) 80.41 21.63 17.74 32.88 ROS production (% of LPS) 82.70 41.69 38.44 52.34

Comparative example 4 Comparative Example 5 Comparative Example 6 Comparative example 7




manufacturing
method Dilution process
(Purified water input amount) multiple of 5 13 multiples 9 multiples 9 multiples primary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 5,000 5,000 Organic acid input amount (parts by weight) One One One One Acidification solution pH concentration 3.0 3.0 3.0 3.0 heating temperature 85 85 55 100 hour 8 8 8 8 secondary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 5,000 5,000 Jinseno
side
Rg3
content has been improved
red ginseng
concentrate Ginsenoside
content
(mg/g) Rg3(R) 47.49 31.58 18.49 57.81 Rg3(S) 59.58 40.25 26.33 74.93 Rh4 71.33 39.85 27.61 18.49 Rg5 46 32 49 56 Rg3 content increase rate (%) 8136 5425 3347 10110 Rh4 content increase rate (%) 1140 593 380 221 Cytotoxicity (%) 57 27 30 30 Cytotoxicity (%) after treatment with inflammation-inducing substances 35 22 25 25 NO generation (% of LPS) 29.53 18.05 19.65 18.67 ROS production (% of LPS) 49.65 38.49 40.97 39.14

Comparative example 8 Comparative Example 9 Comparative Example 10 Comparative Example 11




manufacturing
method Dilution process (purified water input amount) 9 multiples 9 multiples 9 multiples 9 multiples primary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 3,500 6,500 Organic acid input amount (parts by weight) One One One One Acidification solution pH concentration 3.0 3.0 3.0 3.0 heating temperature 85 85 85 85 hour 4 14 8 8 secondary precipitation division centrifugation centrifugation centrifugation centrifugation rotation speed
(rpm) 5,000 5,000 3,500 6,500 Jinseno
side
Rg3
content has been improved
red ginseng
concentrate Ginsenoside
content
(mg/g) Rg3(R) 21.52 58.48 53.86 30.47 Rg3(S) 29.69 75.14 71.17 38.10 Rh4 31.56 20.55 83.04 57.56 Rg5 48 58 53 50 Rg3 content increase rate (%) 3839 10178 9517 5174 Rh4 content increase rate (%) 448 257 1344 901 Cytotoxicity (%) 30 30 47 29 Cytotoxicity (%) after treatment with inflammation-inducing substances 25 25 38 24 NO generation (% of LPS) 20.71 17.54 30.29 18.05 ROS production (% of LPS) 41.09 38.99 50.31 38.25

Looking at Tables 3 to 8 above, it was confirmed that Examples 1 to 12 according to the present invention not only had sufficiently improved ginsenoside content, but also had low cytotoxicity and low NO and ROS production. In addition, the liquid It was confirmed that Example 2 not only had a low content of effective ginsenosides but also had high cytotoxicity compared to Example 1 of the solid phase.

On the other hand, it was confirmed that in Comparative Examples 1 and 2, in which the organic acid was added in less than 0.1 parts by weight or in excess of 5 parts by weight, the rate of increase in the ginsenoside Rg3 and Rh4 content was very poor.

In addition, it was confirmed that Comparative Example 3, in which centrifugation was not performed, not only had a very poor rate of increase in Rg3 and Rh4 content, but also had problems with high cytotoxicity and high NO and ROS production. It is believed that this is because (impurities) were not removed.

In addition, it was confirmed that Comparative Example 4, where the purified water input was less than 8.0 times, not only had high cytotoxicity, but also high NO and ROS production, and in Comparative Example 5, where the purified water input was more than 10.0 times, it was confirmed that the rate of increase in Rg3 and Rh4 content was very poor. .

In addition, it was confirmed that the rate of increase in Rg3 and Rh4 content was very poor in Comparative Examples 6 and 8, where the heating temperature was less than 70°C or the heating time was less than 6 hours, and when the heating temperature exceeded 95°C or the heating time was 10 hours. It was confirmed that the Rh4 content increase rate was very poor in Comparative Examples 7 and 9, which were exceeded.

In addition, it can be seen that Comparative Example 10, where the centrifugation speed was less than 4,000 rpm, not only had high cytotoxicity but also high NO and ROS production, which was expected to be because the centrifugation speed was not sufficient and the active ingredient did not precipitate and remained in the supernatant. do.

In addition, it was confirmed that in Comparative Example 11, where the centrifugation speed exceeded 6,000 rpm, the rate of increase in Rg3 and Rh4 content was very poor.

Claims (9)

1) Diluting the red ginseng concentrate with purified water 8 to 10 times the weight of the red ginseng concentrate to form a diluted solution;
2) natural precipitation or centrifugation of the diluted solution to obtain a water-soluble material;
3) preparing an acidified solution with a pH of 1.5 to 4.5 by adding and mixing an organic acid in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the water-soluble material; and
4) performing a process including the step of heating and cooling the acidified solution at 70 to 95°C and subjecting it to natural precipitation or centrifugation to obtain an insoluble solid,
The organic acid includes one or more selected from citric acid, ascorbic acid, oxalic acid, and lactic acid,
The insoluble solid material has an increase rate of ginsenoside Rg3 content of 5,000 to 15,000% as measured using the following relational equation 1, and an increase rate of ginsenoside Rh4 content of 800 to 2,500% as measured using the following relational equation 2. Method for producing red ginseng concentrate with increased senoside content:
[Relationship 1]

In equation 1, the ginsenoside Rg3 content means the sum of the ginsenoside Rg3(R) and the enantiomer Rg3(S) content of the ginsenoside Rg3(R).
[Relational Expression 2]

delete delete According to paragraph 1,
A method for producing red ginseng concentrate with increased ginsenoside content, characterized in that the centrifugation in steps 2) and 4) is performed for 5 to 20 minutes at a rotation speed of 4,000 to 6,000 rpm, respectively.
delete According to paragraph 1,
A method for producing red ginseng concentrate with increased ginsenoside content, characterized in that the heating is performed for 6 to 10 hours.
According to paragraph 1,
A method for producing red ginseng concentrate with increased ginsenoside content, characterized in that the cooling is performed until the final temperature reaches 10 to 30°C.
Contains an insoluble solid material with a sum of ginsenoside Rg3(R) and ginsenoside Rg3(S) content of 50 to 150 mg/g,
The ginsenoside content increased, characterized in that the ginsenoside Rg3 content increase rate measured through the following relational equation 1 is 5,000 to 15,000%, and the ginsenoside Rh4 content increase rate measured through the following relational equation 2 is 800 to 2,500%. Red ginseng concentrate:
[Relationship 1]

In equation 1, the ginsenoside Rg3 content means the sum of the ginsenoside Rg3(R) and the enantiomer Rg3(S) content of the ginsenoside Rg3(R).
[Relational Expression 2]
delete