KR101487541B1 - Analytical method of ginseng polysaccharide - Google Patents

Abstract

A method for analyzing active components of ginseng polysaccharide of the present invention is characterized by including the steps of (I) selectively extracting ginseng polysaccharide contained in ginseng; (II) manufacturing a sample by low temperature aging and pulverizing the extracted ginseng polysaccharide; (III) performing qualitative and quantitative analysis of the manufactured sample; and (IV) finding glucose, galactose, arabinose, glucoronate, and galacturonic acid as active components of ginseng polysaccharide by using the CG and HPLC analysis data, wherein the (I) and (II) steps comprise the steps of (1) a preparing step of moving white ginseng or fresh ginseng washed with water to an extraction tank; (2) extracting by using purified water; (3) filtering by using a filter and cooling the filtered extract; (4) secondarily filtering the cooled extract through the step by using a filter; (5) concentrating the secondarily filtered extract and obtaining the concentrate; (6) inputting the concentrate to anyone selected among ethanol, propanol, butanol, pentanol, and acetone, soaking ginseng polysaccharide substance, and then separating and removing saponin layer of a supernatant; (7) applying purified water to the saponin layer removed ginseng polysaccharide, dissolving with water, and concentrating; (8) aging the concentration at low temperature; (9) stirring the concentrate to stabilize and killing bacteria by high temperature sterilization; and (10) drying the concentrate by freezing or spraying in hot air, and manufacturing a powder-shaped sample.

Description

[Technical Field] The present invention relates to an effective ingredient of a ginseng polysaccharide and an analytical method of ginseng polysaccharide,

The present invention relates to a method for analyzing a ginseng polysaccharide (junsan). More particularly, the present invention relates to an active ingredient of ginseng polysaccharide and a human body reaction assay method for evaluating efficacy and safety by conducting qualitative and quantitative analysis of an active ingredient contained in a ginseng polysaccharide and a human body application test.

Herbal medicine raw materials such as ginseng, Hwanggi, and myeonmundong have been widely used for the purpose of relieving diseases and healing in general, and ginseng has been used for many years as the most noble herbal medicine ingredient in the Far East. Ginseng is the broadest concept that includes both ginseng, white ginseng, red ginseng, wild ginseng, camphor, ginseng, raw ginseng, and taekguk ginseng. Through the history of long use, ginseng has been listed as one of the most important health functional food in Korea.

Ginseng contains 50-70% of carbohydrates, 10-15% of crude protein, 3-8% of crude saponin and 3-7% of ash, and the major physiologically active ingredients are saponin, phenolic component, polyacetylene component, Alkaloid components, polysaccharides and the like are known. Among them, ginseng saponin, which is called "ginsenoside", is named among the saponins of ginseng among the various saponins of plants. Ginseng saponin is a glycoside composed of glycone and aglycone. Up to now, about 30 species have been isolated and their structure is known.

Recently, it has been found that various non-saponin fractions other than the saponin component have various pharmacological activities. Therefore, researches on pharmacological activity focusing on these active fractions have been variously developed.

Non-saponins include essential oils, phytosterols, polyacetylenes, polyphenols, flavonoids, polysaccharides, alkaloids, vitamins, minerals and enzymes. .

The polysaccharide is classified into Ginsan and Panaxan, and Jinsan is named Ginsan (ginsan / ginseng polysaccharide) by attaching -an, which means ginseng and polysaccharide of phosphoric acid. Jinsan is a pure polysaccharide composed of glucan (polysaccharide composed of glucose) and fructan (polysaccharide composed of fructan) and is a nano-polymer substance with a molecular weight of about 5,000 ~ 15,000. As a result of animal experiments, Jinsan promotes regeneration and differentiation of peripheral blood cells as well as bone marrow cells, thereby promoting hematopoiesis and antimicrobial action and reducing side effects caused by radiation therapy. In addition, activation of macrophages promotes the production of interleukin-12, thereby activating the production of interferon by Th1-lymphocytes, thereby increasing the ability of natural killer cells (NK-Cell) and enhancing the cellular immune response (CTL) . In addition, it has been reported that it is effective for diseases such as diabetes by lowering blood sugar. In addition, there are 21 kinds of Pak-Nak-san from Pak-Naksan A to U, and its anti-cancer effect is good.

As a conventional method for analyzing ginseng polysaccharide, Patent Document 10-2013-0010987 (2013.01.30) extracts ginsenoside polysaccharide from ginseng

Determination of crude saponin content using water saturated butanol method, measurement of total sugar content using DNS (dinitro salicylic acid) reagent, measurement of acid polysaccharide content using carbazole-sulfuric acid method and measurement of molecular weight distribution using GPC . In addition, although it is not a method for analyzing ginseng polysaccharide, Japanese Patent Laid-Open No. 2003-0085918 (2003.11.07) relates to a method for simultaneously quantitatively analyzing active ingredients contained in ginseng, red ginseng or processed products thereof, Rb, Rc, Rd, Re, and Rf, which are the main quality reference materials of ginseng, red ginseng or their work, were injected into an ion trap type LC / MS equipped with SSI (sonic spray ionization) , And Rg 1 at the same time.

Therefore, studies on the molecular weight and composition of the ginseng polysaccharide have been carried out to date. However,

The method of analyzing the content and thus the effect of the immune enhancement and regulating function is insignificant. The research for evaluating the efficacy and safety of the ginseng polysaccharide by applying the ginseng polysaccharide to the human body has not been made at all. Is required.

Published Patent No. 10-2013-0010987 (Jan. 30, 2013) Published Patent Application No. 2003-0085918 (Nov. 2003)

The present invention has been made in order to solve the above problems of the prior art. According to the present invention, a ginseng polysaccharide is extracted from ginseng and subjected to GC and HPLC analysis to quantitate and qualitatively analyze effective components of ginseng polysaccharide, .

Another object of the present invention is to provide a method for analyzing the human body reaction of extracted ginseng polysaccharide, thereby providing the immunostimulatory effect and safety of the ginseng polysaccharide.

To achieve the above object, the present invention provides a method for producing ginseng, comprising: (I) selectively extracting ginseng polysaccharide contained in ginseng by extraction, filtration, concentration, precipitation and removal; (II) preparing a sample by powdering the extracted ginseng polysaccharide at low temperature; (III) Qualitative and quantitative analysis of the prepared sample through GC and HPLC analysis; And (IV) identifying the active ingredient of the ginseng polysaccharide using the GC and HPLC analysis data.

In one embodiment, D - (+) - galacturonic acid monohydrate, D-Glucuronic acid, D - (+) - Galactose, D - ) -Arabinose can be used.

In one embodiment, the HPLC may be a mixture of potassium dihydrogen phosphate (ACN) = 55: 45 to 85:15, and the mixture may be a mobile phase, Can be diluted in purified water and used as an internal standard solution for quantitative analysis through a calibration curve.

In the present invention, the active ingredient of the ginseng polysaccharide analyzed by the above-described assay method may be glucose, galactose, arabinose, glucuronic acid, galacturonic acid.

The present invention also provides a method for producing ginseng, comprising the steps of: (i) extracting ginseng polysaccharide contained in ginseng, selectively extracting it by filtration, concentration, sparging and removal; (Ii) preparing a sample by powdering the extracted ginseng polysaccharide at low temperature; (Iii) taking a manufactured sample to a subject to be tested and performing a human body reaction test; And (iv) evaluating the efficacy and safety of the immune enhancement according to the active ingredient of the ginseng polysaccharide using the human body reaction analysis data.

In one embodiment, the active ingredient of the ginseng polysaccharide may be glucose, galactose, arabinose, glucuronic acid, galacturonic acid.

In one embodiment, the ginseng polysaccharide human body application assay may evaluate any one or more of NK cell activity, phagocytic activity, monocyte-derived mediators, gastrointestinal infections and safety factors, It is intended for healthy adults aged 3 to 75 years old and may be taken orally for 10 to 15 weeks.

As described above, the analytical technique of the ginseng polysaccharide can be established by analyzing the qualitative and quantitative analysis of the active ingredients of the ginseng polysaccharide extracted from ginseng through the GC, HPLC and human application tests of the present invention and by analyzing the efficacy and safety.

In addition, it can be used for the development and commercialization of health functional food using the molecular weight of ginseng polysaccharide, the enhancing effect of immune function, and the new active ingredient for enhancing immune function of ginseng.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flowchart showing a method of producing and analyzing ginseng polysaccharide for immune enhancement according to an embodiment of the present invention; FIG.
FIG. 2 illustrates gas chromatography (GC) of an effective ingredient of a ginseng polysaccharide according to an embodiment of the present invention.
Figure 3 shows high performance liquid chromatography (HPLC) of the active ingredients of ginseng polysaccharide according to one embodiment of the present invention.
Figure 4 shows high performance liquid chromatography (HPLC) and calibration curves of galactose and arabinose in ginseng polysaccharide according to one embodiment of the present invention.
FIG. 5 shows the amount of change in the natural killer cell (NK cell) activity according to an embodiment of the present invention.
Figure 6 shows the amount of phagocytic activity change according to one embodiment of the present invention.
FIG. 7 shows the average amount of change for a monocyte-derived mediator (LPS stimulation: IL-12, TNF-?) According to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the drawings of the present invention, the sizes and dimensions of the structures are enlarged or reduced from the actual size in order to clarify the present invention, and the known structures are omitted so as to reveal the characteristic features, and the present invention is not limited to the drawings . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail to avoid obscuring the subject matter of the present invention.

In the present invention, the analysis of ginseng polysaccharide (I) is performed by extracting ginseng polysaccharide contained in ginseng, extracting the ginseng polysaccharide selectively by filtration, concentration, precipitation and removal, (II) extracting ginseng polysaccharide at low temperature and pulverizing Preparing a sample; (III) qualitatively and quantitatively analyzing the prepared sample through GC and HPLC analysis; and (IV) confirming the active ingredient of the ginseng polysaccharide using the GC and HPLC analysis data.

(1) preparation step of transferring white ginseng or water-washed ginseng to an extraction tank, (2) preparation of a ginseng extraction tank, and (3) (3) filtration using a 1 탆 filter, and then cooling the filtered extract to 10 to 20 캜; (4) 4) Secondary filtration using a 0.6 ㎛ filter (5) Concentration to 30 Brix to obtain a concentrated liquid; (6) Concentrate is added to any one of ethanol, propanol, butanol, pentanol, Separating and removing the saponin layer of the supernatant after the precipitation of the polysaccharide substance, (7) adding ginseng polysaccharide from which the saponin layer has been removed to the supernatant, dissolving the saponin in water, and concentrating it to 50 Brix, and (8) The step of aging (9) Stirring the concentrate to stabilize it, and then sterilizing the suspension through sterilization. In addition, the ginseng polysaccharide concentrate obtained through the ginseng polysaccharide extraction method is prepared by lyophilization or hot air spray drying to be used in the analysis of ginseng polysaccharide.

The sample prepared in the present invention is subjected to qualitative and quantitative analysis by GC and HPLC in the step (III). The column used for the GC was Supelco SP-2380 Silica, the detector used for the flame ionization detector (FID), and the carrier gas used for the nitrogen gas. The temperature was raised at 4 ° C / min from 200 ° C to 250 ° C can do. The column used for HPLC was Eclipse XDB C18, the detector was PDA detector, and the mobile phase was a mixture of potassium dihydrogen phosphate and acetonitrile.

According to the present invention, in the GC and HPLC analysis methods, the reference material can be analyzed by comparing the retention time and the effective component of the ginseng polysaccharide. D - (+) - Glucose monohydrate and L - (+) - Arabinose were used as D - (+) - Galacturonic acid monohydrate, D-Glucuronic acid, D- Preferably, each of the above five standard substances is dissolved in distilled water and used as a standard solution.

In addition, in the case of HPLC, the mobile phase can be used by mixing a used potassium dihydrogen phosphate, which is a buffer solution, with an organic solvent, and more specifically, a ratio of potassium dihydrogen phosphate: acetonitrile (ACN) = 55:45 to 85:15 Can be used. If the organic solvent ratio exceeds 45, precipitation of the buffer solution may occur. If the organic solvent ratio is less than 15, the peak may be widened, so that it is preferable to maintain the above ratio range.

At this time, since the small particles of the buffer solution may affect the column, it is preferable to filter the buffer solution and adjust the pH (pH 7.0) of the buffer solution before mixing with the organic solvent.

The internal standard substance of the HPLC analysis of the present invention can be used as an internal standard solution by diluting allose to 10 to 30 times with purified water. Using the internal standard solution, the ginseng polysaccharide active ingredient is quantitatively analyzed can do.

In the present invention, the effective components of the ginseng polysaccharide analyzed through the aforementioned active ingredient analysis method may be glucose, galactose, arabinose, glucuronic acid and galacturonic acid, preferably galactose, arabinose. Since the retention time of the ginseng polysaccharide may vary depending on the conditions under which the ginseng polysaccharide is analyzed, it is preferable to establish the analysis conditions through various experiments.

In the present invention, the analysis of the human body polysaccharide body reaction comprises the steps of (i) extracting ginseng polysaccharide contained in ginseng by extraction, filtration, concentration, precipitation and removal, (ii) extracting the extracted ginseng polysaccharide at low temperature, (Iii) subjecting the prepared sample to a subject to be tested for a human body reaction, and (iv) using the human body reaction analysis data to determine the effect of the active ingredient of the ginseng polysaccharide And evaluating the safety.

The method of preparing the ginseng polysaccharide sample in steps (i) and (ii) above is the same as described in the step of confirming the effective ingredient of the ginseng polysaccharide mentioned above, and the active ingredient of the ginseng polysaccharide obtained through the same Glucose, galactose, arabinose, glucuronic acid, and galacturonic acid.

The ginseng polysaccharide human body application analysis of the present invention can evaluate any one or more of NK cell activity, phagocytic activity, monocyte-derived mediators, gastrointestinal infections and safety factors. Examples of the safety items include adverse reactions, Laboratory tests, electrocardiograms, and physical examinations.

The analysis of the human ginseng polysaccharide of the present invention was conducted for 10 to 15 weeks in healthy adult male and female patients aged 50 to 75 years. The analysis was performed by double-blind, randomized, placebo-controlled , And the subject should be able to evaluate the suitability and participate in the experiment.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Production Example: Preparation of Samples by Extraction of Ginseng Polysaccharide

3 kg of ginseng is added 8 times water, and water-soluble extract is obtained 3 times at 80 ° C. The obtained extract is filtered and concentrated to 30 Brix, and the saponin layer of the supernatant is separated by precipitating ginseng polysaccharide by using ethanol in the concentrate. The separated pure ginseng polysaccharide precipitate is dissolved by adding 3 times of water, and then concentrated at 50 ° C at 50 Brix. Thereafter, a concentrated ginseng polysaccharide is obtained through a low-temperature aging process, stabilization through agitation, and high-temperature sterilization process. The ginseng polysaccharide concentrate is powdered by freeze drying.

Example 1: GC analysis

Gas chromatography (GC) analysis of ginseng polysaccharide was carried out using Supelco SP-2380 Silica column, FID detector for carrier gas, nitrogen gas for carrier gas and temperature of 4 ℃ / min at 200 ~ 250 ℃. .

The standard solution is prepared by dissolving 20 mg of 5 standard substances in 10 ml of tertiary distilled water. D - (+) - Galactose, D - (+) - Glucose monohydrate and L - (+) - Arabinose were used for neutral sugars and D - (+) - Galacturonic acid monohydrate and D-Glucuronic acid were used for acidic sugars do. The test solution is prepared by dissolving 2 ml of 2M TFA in 10 mg of ginseng polysaccharide sample and reacting at 121 ° C for 1.5 hours.

For neutral sugars, take 300 μl of 5 test solutions and standard solutions, mix with 2 ml of 1M NH ₄ OH, add 100 mg of NaBH ₄ and stir for 4 hours. After the addition of acetic anhydride, concentrate, add 5 ml of methanol, concentrate, and repeat this process three times. The concentrated sample is dissolved in 10 ml of water and then filtered. Concentration of the obtained filtrate is followed by addition of 1 ml of acetic anhydride and 1 ml of pyridine, followed by GC analysis.

In the case of acidic saccharide, the same procedure as in the case of the neutral saccharide is carried out, and the filtrate is concentrated using a filtrate using 1 M HCl.

GC analysis is shown in FIG. 2, and peaks of arabinose, galactose, and glucose can be confirmed in the test solution of neutral sugar in FIG. 2 (a), while peaks of arabinose, galactose and glucose in the test solution of 2 (b) . In the case of GC analysis, a supplementary experiment should be carried out.

Example 2. High performance liquid chromatography (HPLC)

HPLC analysis was performed on column Eclipse XDB C18, detector PDA detector, and mobile phase using a mixed solvent of potassium dihydrogen phosphate and acetonitrile.

The standard solution is prepared by dissolving 20 mg of the ginseng polysaccharide of the example in 10 ml of purified water. The internal standard solution is prepared by dissolving in 5 ml of purified water and 10 mg of Allose, and then diluting 20 times with purified water.

2 mg of the ginseng polysaccharide of the example is added to 2 ml of 2M TFA, and the test solution is hydrolyzed at 110 ° C for 1.5 hours under nitrogen gas filling. Hydrolysis is followed by neutralization to pH 7.0 using NaOH and HCl and pretreatment.

Add 0.1 ml of the prepared internal standard solution, 2 ml of the standard solution, 0.9 ml of polymethyl pentene (PMP) and 0.9 ml of 0.3 M NaOH to the test tube, and mix. After mixing, the reaction was carried out in a water bath at 70 ° C for 30 minutes, and then neutralized by the addition of 0.9 ml of 0.3 M HCl. After that, 2 ml of chloroform is added and separated from the water layer. The extraction is carried out three times in total. The extracted water layer was used for analysis. The total amount of ginseng polysaccharide was calculated by HPLC analysis three times in total. The content of constitutional components of the five active ingredients is calculated by the following formula.

The HPLC analysis of the ginseng polysaccharide was shown in FIG. 3, and as a result, ginseng sugar analysis revealed that glucuronic acid (17.418 min), galacturonic acid (18.768 min), glucose (21.178 min), galactose 25.831 min) was detected. Also, it was confirmed that the average content (mg / g) of each constituent was found to be 172.745, 17.226, 12.874, 0.915, and 25.290 by the composition formula.

In the present invention, the analytical conditions for HPLC were established by setting the index components of the above five active ingredients as galactose and arabinose.

2 ml of each of D - (+) - Galactose and L - (+) - Arabinose is used as a reference material.

The test solution is prepared by adding 2 mL of 2M TFA to 2 mg of the sample, and hydrolyzing the mixture at 110 ° C. for 1.5 hours under nitrogen gas filling. After hydrolysis, neutralize to pH 7.0 using NaOH and HCl, and add 5 ml of distilled water.

Add 2 ml of each of the standard solution and the test solution into a test tube, add 0.9 ml of polymethylpentene and 0.9 ml of 0.3 M NaOH, and mix. After mixing, the reaction was carried out in a water bath at 70 ° C for 30 minutes, and then neutralized by the addition of 0.9 ml of 0.3 M HCl. Then, 2 ml of chloroform was added thereto, and the mixture was separated from the water layer. The extraction was carried out three times in total, and the extracted water layer was used for the analysis. The result is shown in FIG. Also, the calibration curve was confirmed by using the internal standard material, and the average value was calculated by repeating the experiment six times.

As can be seen from FIG. 4 (a), it was confirmed that the galactose peak and 25.46 minutes of arabinose peak were detected at 22.94 min, and the results are shown in FIG. 4 (b) The average contents of these components were 16. 874 mg / g and 12.425 mg / g, respectively.

Example 3. Chemical properties of active ingredients

To confirm the chemical properties of the active ingredients, 6 fractions (G-1 to G-6) were subjected to Sephacryl S-500 HR chromatography to obtain ginseng polysaccharide extracts and DEAE Sephadex A-25 gromat 7 fractions (G1-1 to G1-7) were obtained. Table 1 shows the ginseng polysaccharide (hereinafter, referred to as 'Y-75' in the analysis result) extracted and prepared and the constituent sugars of G-1 and G1-1 having high sugar content in the above fractions.

Glucose Galactose Arabinose Etc Y-75 78.7 16.0 2.0 3.3 G-1 84.2 11.7 2.2 1.9 G1-1 91.7 5.8 0.6 1.9

3.1 Lymphocyte proliferative ability

Lymphocytes isolated from the blood were cultured with ConA (concanavalin A, Sigma) or a sample. The degree of proliferation of lymphocytes was measured by the ³ H-thymidine (TdR) uptake method on the second day after the incubation, as shown in Table 2.

As shown in Table 2, spleen cells (hereinafter, referred to as 'SC') were assayed by varying the content of humic acid. As a result, the splenic cells of the mice proliferated greatly and the G1-1 The fraction is relatively high in lymphocyte proliferation.

3.2 BAK cell production ability

BAK cell production and cancer cell killing activity were measured by the ⁵¹ Cr glass method for 4 hours, which is shown in Table 3. As a result, it was confirmed that when the spleen cells were cultured in the medium containing Jin-San, the cancer cell killing ability was higher and the G-1 fraction was relatively higher than that of the spleen cell cultured in the medium without the ginseng .

Culture Dose (/ / ml) 3H-Thymidine incorporation (cpm) SC - 2.443 ± 198 SC + ConA 2.5 211,591 ± 12,311 SC + LPS 10 74,942 ± 8,908

SC + 75% EtOH inner part 1000 110,123 ± 7,021 500 84,582 ± 6,665 250 81,175 ± 5,687 125 77,820 + 4,560 62.5 66,139 ± 2,235 31.3 59,074 ± 4,562

SC + G-1 1000 141,374 ± 10,346 500 113,786 ± 11,120 250 106,102 ± 8,800 125 93,231 ± 9,925 62.5 79,603 + 7,580 31.3 64,218 ± 9,256

SC + G1-1 1000 151,078 ± 10,213 500 125,534 + 9,001 250 106,225 ± 8,948 125 94,750 ± 6,541 62.5 83,960 + 5,658 31.3 74,541 ± 3,431

Culture
Dose (/ / ml) % cytotoxicity
(effector: Target) 100: 1 30: 1 SC - 1.65 0.37 SC + rIL-2 30u / ml 71.11 51.46
SC + OK432 100 6.45 2.44 50 14.42 3.93 10 3.24 1.01
SC + 75% EtOH
inner part 200 16.21 5.14 100 18.46 9.00 50 7.71 2.64 10 3.34 1.85

SC + G-1 200 3.57 2.07 100 6.89 2.13 50 12.14 6.10 10 19.34 7.91 2 7.57 2.34

SC + G1-1 200 4.46 1.82 100 5.05 2.61 50 19.82 7.61 10 22.25 9.75 2 9.56 3.55

Example 4. Human body application test

The ginseng polysaccharide was administered to a general hospital in Korea. The ginseng polysaccharide extract (hereinafter referred to as "Y-75") was administered to healthy adults aged 50 to 75 years old for 14 weeks The efficacy and safety of immunization were evaluated after taking 3 times a day, morning and evening after meal. In addition, the human body application test can be carried out on random assignment days, 8 weeks, and 14 weeks.

The statistical analysis of human body test was analyzed in the form of ITT (intent-to-treat) analysis group and PP (per protocol) analysis group.

4-1. Validation

4-1-1. Natural killer cell (NK cell) activity

The activity of NK cells is measured using K562 cells, in which peripheral blood mononuclear cells (PBMC) isolated from blood are labeled with efficacious cells and Na ₂ ⁵¹ CrO ₄ (200 Ci / ml) fh as target cells. Based on the changes at baseline and 8 weeks and 14 weeks after the start of the study, normal distribution is tested and compared using the independent t-test or Wilcoxon's rank sum test.

FIG. 5 shows changes in NK cell activity. The test group of FIG. 5 (a) showed an increase of 13.57 ± 117.78% at the 8th week from the baseline and 15.45 ± 14.55% at the 14th week, Figure 5 (b) shows that the control group in the ITT group increased 0.15 ± 11.41% at 8 weeks and 0.98 ± 14.21% at 14 weeks.

5 (c), the average change of NK cell activity was increased by 12.32 ± 11.94% at the 8-week time point compared to the base point of the PP test group, and the control group of the PP-treated group was 0.46 ± Respectively. In addition, at 14 weeks from baseline, the test group increased 14.92 ± 14.67% and the control group increased 1.11 ± 14.63%.

The difference between the experimental group and the control group was similar at 8 and 14 weeks compared to the baseline.

4-2-2. Phagocytic activity

Macrophages and polymorphonuclear leukocytes (PMNs) in peripheral blood are stimulated with FITC-labeled E. coli and phagocytic activity is measured by flow cytometry (FACS). Based on the changes at baseline and 8 weeks and 14 weeks after the start of the study, normal distribution is tested and compared using the independent t-test or Wilcoxon's rank sum test.

FIG. 6 shows changes in the phagocytic activity. In FIG. 6 (a), the test group of the ITT analysis group showed 4953.19 ± 8789.30% increase at 8 weeks and 7734.08 ± 7959.73% at 14 weeks, (b) The control group in the ITT group increased 1465.03 ± 8027.02% at 8 weeks and increased 1763.08 ± 7325.98% at 14 weeks.

6 (c) PP test group increased by 5061.25 ± 9784.41% at the 8th week from the baseline and increased by 7081.38 ± 8910.39% at the 14th week, and FIG. 6 (d) Increased 1825.59 ± 8809.46% at 8 weeks from the baseline and increased 2488.00 ± 8198.96% at 14 weeks.

The difference between the experimental group and the control group was similar at 8 and 14 weeks compared to the baseline.

4-2-3. Monocyte-derived mediator (LPS stimulation: IL-12, TNF-α)

The peripheral blood mononuclear cells (PBMC) isolated from the molds were stimulated with LPS (lipopolysaccharide) and cultured for 24 hours or 48 hours. The amount of cytokine contained in the culture solution was measured by an ELISA kit.

Figure 7 shows the mean change in IL-12 (Interleukin-12) and TNF-alpha (Tumor mecrosis factor-alpha). 7 (a), there were no significant differences in the control group of the ITT-analyzed test group for the IL-12 test and the ITT-treated control group for the IL-12 test shown in FIG. 7 (b) 7 (d) The ITT-treated group for TNF-α showed a decrease in the control group at 8 weeks compared to baseline, but increased again at 14 weeks .

7 (e), the control group of the PP analysis subject group for the IL-12 test and the control group of the PP analysis subject group for the IL-12 test of FIG. 7 (f) ) Of the test group of the PP-analyzed group against TNF-α was increased compared to the base point, and that of the control group of the PP-analyzed group of FIG. 7 (h)

The difference between the experimental group and the control group was similar at 8 and 14 weeks compared to the baseline.

4-2-4. The above-

After the random allocation date, the infectious disease and the number of infections are counted and recorded at regular intervals.

4-2. Stability observation

The stability of the test group was compared between the experimental group and the control group.

4-2-1. Physical examination

Physical examination is divided into two categories, normal and abnormal at baseline, 8 and 14 weeks. The items include allergies, cardiovascular, pulmonary and respiratory, gastrointestinal / liver and biliary systems, metabolic / endocrine, renal / urinary tract, reproductive system, musculoskeletal, skin and connective tissue, nervous system, .

The physical examination was performed with a Chi-square test or a Fisher's exact test. As a result, it was confirmed that both test and control groups were normal.

4-2-2. Vital signs

The mean changes in the vital signs were compared at the 14th week based on the ITT group of 36 subjects in the test group and 36 subjects in the control group. Vital signs can measure blood pressure, pulse, and body temperature five minutes after sitting.

To determine whether there was a difference in the change in the test value between the test group and the control group, we compared the results with the independent t-test or wilcoxon's rank sum test. The paired t-test or wilcoxon's rank sum test Respectively.

As a result, there was no statistically significant difference in systolic blood pressure, diastolic blood pressure, mean change in body temperature, and mean change in pulse rate.

4-2-3. Experimental inspection

Screening visits (V1), 8 weeks (V3), and 14 weeks (V4). Laboratory tests are performed, and hematological, blood chemistry, and urine tests are performed.

As a result, there was no statistically significant difference between the 36 test groups and the 36 control group in all items at each time point compared to the baseline, and there were no clinically significant abnormal subjects.

4-2-4. cardiography

Screening visit (V1), 14 weeks (V4), and examines heart rate and ventricular hypertrophy, myocardial infarction, arrhythmia, pericarditis, reversal of drug and electrolyte metabolism. The chi-square test or Fisher's exact test was performed. As a result, it was confirmed that both test and control groups were normal.

4-2-5. Adverse reaction

The type and frequency of adverse events were determined by the Chi-square test or Fisher's exact test at the 14th week based on the 36 ITT group and 36 ITT group. As a result, 9 (25.00%) and 5 (13.89%) of the adverse events occurred in the test group and the control group, respectively. In addition, no subjects showed significant adverse reactions in the adverse reaction test.

In addition, safety observation, pregnancy test, urinalysis, and concomitant medication were observed. As a result, there were no clinically significant abnormal subjects. In the combined drug test, a total of 82 subjects, including 20 subjects in the test group and 20 subjects in the control group, were found to take the concurrent drug during the human body test period, and the Influenza and Purified Antigen among the total drugs were taken.

According to the present invention, the active ingredients of ginseng polysaccharide obtained from ginseng extract are analyzed by GC and HPLC, and the analysis conditions for evaluating the immunostimulating efficacy and safety after the administration of the ginseng polysaccharide extract for healthy subjects are established for 14 weeks And is effective in improving immunity enhancement and control function.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (9)

(I) selectively extracting ginseng polysaccharide contained in ginseng by extraction, filtration, concentration, precipitation and removal;
(II) preparing a sample by powdering the extracted ginseng polysaccharide at low temperature;
(III) Qualitative and quantitative analysis of the prepared sample through GC and HPLC analysis; And
(IV) Identifying glucose, galactose, arabinose, glucuronic acid, and galacturonic acid as effective components of the ginseng polysaccharide using the GC and HPLC analysis data; Lt; / RTI >
The steps (I) and (II)
(1) a step of transferring white ginseng or water-washed ginseng to an extraction tank, (2) a step of extracting at 70 to 80 ° C using 4 to 8 times of purified water relative to the mass of ginseng or white ginseng, (3) (4) a step of secondly filtering the cooled extract through a 0.6-μm filter (5) a step (5) of filtering the extracted second extract (6) adding a concentrate to any one of ethanol, propanol, butanol, pentanol, and acetone to precipitate the ginseng polysaccharide material, and separating and removing the saponin layer of the supernatant, (7) adding a purified water to the ginseng polysaccharide from which the saponin layer has been removed, dissolving in water and concentrating to 50 Brix, and (8) low temperature aging the concentrate at 10 ° C or less. (9) Stabilizing the concentrate by stirring, Through Step, 10 an active ingredient assay of ginseng polysaccharides characterized in that the concentrate by freeze drying or hot air drying a spray of the sample prepared in powder form to kill. The method according to claim 1, wherein the (Ⅲ) GC and HPLC analytical methods are D - (+) - Galacturonic acid monohydrate, D-Glucuronic acid, D - And L - (+) - Arabinose at a temperature ranging from 200 to 250 ° C at a rate of 4 ° C / min. The method for analyzing an effective component of a ginseng polysaccharide according to claim 1, The method according to claim 1,
Wherein the mobile phase of the HPLC is a mixture of potassium dihydrogen phosphate (ACN) in a ratio of 55:45 to 85:15. The method according to claim 1,
Wherein the allose is diluted in purified water in the above HPLC analysis and used as an internal standard solution and quantitatively analyzed through a calibration curve. delete (I) selectively extracting ginseng polysaccharide contained in ginseng by extraction, filtration, concentration, precipitation and removal;
(Ii) preparing a sample by powdering the extracted ginseng polysaccharide at low temperature;
(Iii) taking a manufactured sample to a subject to be tested and performing a human body reaction test; And
(Iv) evaluating immune enhancement efficacy and safety according to glucose, galactose, arabinose, glucuronic acid, and galacturonic acid as effective ingredients of the ginseng polysaccharide using the human body reaction analysis data; Lt; / RTI >
The steps (i) and (ii)
(1) a step of transferring white ginseng or water-washed ginseng to an extraction tank, (2) a step of extracting at 70 to 80 ° C using 4 to 8 times of purified water relative to the mass of ginseng or white ginseng, (3) (4) a step of secondly filtering the cooled extract through a 0.6-μm filter (5) a step (5) of filtering the extracted second extract (6) adding a concentrate to any one of ethanol, propanol, butanol, pentanol, and acetone to precipitate the ginseng polysaccharide material, and separating and removing the saponin layer of the supernatant, (7) adding a purified water to the ginseng polysaccharide from which the saponin layer has been removed, dissolving in water and concentrating to 50 Brix, and (8) low temperature aging the concentrate at 10 ° C or less. (9) Stabilizing the concentrate by stirring, Through Phase, 10 human response way analysis of ginseng polysaccharides characterized in that the concentrate by freeze drying or hot air drying a spray of the sample prepared in powder form to kill. delete The method according to claim 6,
Wherein the analysis of human ginseng polysaccharide body application is performed by evaluating at least one selected from NK cell activity, phagocytic activity, monocyte-derived mediators, gastrointestinal infections and safety factors.
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