阅读说明：本技术 一种人参多糖组合物及其制备方法和应用 (Ginseng polysaccharide composition and preparation method and application thereof ) 是由 焦丽丽 吴巍 李慧 李波 陈长宝 刘淑莹 李珺铭 于 2020-05-14 设计创作，主要内容包括：本发明公开了一种具有免疫激活活性的人参多糖组合物及其制备方法。所述人参多糖组合物以摩尔百分比计,由3.0～3.6%鼠李糖、3.5～4.0%半乳糖醛酸、65.0%～70.0%葡萄糖、13.0%～13.5%半乳糖和9.5%～10.0%阿拉伯糖组成。所述人参多糖组合物的分子量大于3000Da且小于5000Da。在所述人参多糖组合物的制备方法中,使用20%、40%、60%、80%不同乙醇浓度的醇沉法。所述人参多糖体内能够促进淋巴细胞转化,增强巨噬细胞的吞噬作用及增强NK细胞的杀伤作用。本发明涉及的人参多糖组合物可用于制备增强机体免疫调节功能保健食品或药品。(The invention discloses a ginseng polysaccharide composition with immune activation activity and a preparation method thereof. The ginseng polysaccharide composition comprises, by mole, 3.0-3.6% of rhamnose, 3.5-4.0% of galacturonic acid, 65.0-70.0% of glucose, 13.0-13.5% of galactose and 9.5-10.0% of arabinose. The ginseng polysaccharide composition has a molecular weight greater than 3000Da and less than 5000 Da. In the preparation method of the ginseng polysaccharide composition, alcohol precipitation methods with different ethanol concentrations of 20%, 40%, 60% and 80% are used. The panaxan can promote lymphocyte transformation, enhance phagocytosis of macrophage and enhance killing effect of NK cell. The ginseng polysaccharide composition can be used for preparing health-care food or medicine for enhancing the immunoregulation function of organisms.)

1. A ginseng polysaccharide composition is characterized by comprising, by mole, 3.0-3.6% of rhamnose, 3.5-4.0% of galacturonic acid, 65.0-70.0% of glucose, 13.0-13.5% of galactose and 9.5-10.0% of arabinose, and the molecular weight of the ginseng polysaccharide composition is larger than 3000Da and smaller than 5000 Da.

2. The ginseng polysaccharide composition of claim 1, wherein rhamnose is 3.55%, galacturonic acid is 3.61%, glucose is 69.69%, galactose is 13.26%, arabinose is 9.89%, and the molecular weight is 3160 Da.

3. A method of preparing a ginseng polysaccharide composition according to any one of claims 1-2, comprising the steps of:

a, grinding ginseng to obtain ginseng powder;

b, extracting the ginseng powder with water, and then filtering to obtain filtrate;

c, concentrating the filtrate to obtain a ginseng polysaccharide extracting solution;

d, precipitating the ginseng polysaccharide extracting solution by using 20-80% ethanol, and centrifuging to obtain residues;

and E, dissolving the residue in water, and performing gel chromatography to obtain the ginseng polysaccharide composition.

4. A method according to claim 3, characterized in that said step D is carried out as follows:

adding 95% ethanol into the ginseng polysaccharide extracting solution to make the final concentration of the ethanol be 20%, and collecting precipitate and supernatant;

continuously adding 95% ethanol into the supernatant to prepare 40% ethanol concentration, and respectively collecting the supernatants;

continuously adding 95% ethanol into the supernatant to prepare 60% ethanol concentration, and respectively collecting the supernatants;

and continuously adding 95% ethanol into the supernatant to prepare 80% ethanol, collecting precipitate, redissolving in water, and freeze-drying.

5. Use of a ginseng polysaccharide composition according to any one of claims 1-2 in the preparation of a medicament having immune enhancing activity.

6. The use according to claim 5, wherein the immune enhancing activity is promoting proliferation of lymphocytes in mice, enhancing phagocytic function of macrophages and enhancing killing ability of NK cells.

Technical Field

The invention relates to a ginseng polysaccharide composition, a preparation method and application thereof.

Background

The Ginseng radix is Panax ginseng C.A.Meyer of AraliaceaePanax ginsengC.a. meyer) has since ancient been considered as a good medical plant to supplement both medical and food. Has effects in invigorating primordial qi, relieving depletion, promoting fluid production, and tranquilizing mind. A large number of researches show that the panaxan has good immunoregulation activity, and can induce lymphocyte proliferation and T lymphocyte activity.

In the past 60 s, the polysaccharides in ginseng have been classified by soviet scientists into ginseng starch and ginseng pectin. A plurality of researches later prove that the ginseng neutral sugar has remarkable effects of resisting oxidation and tumors and enhancing immunity. The structure of polysaccharides is closely related to their biological activity. The molecular weight, linkage type, branch chain and branch group, substituent site, number, etc. of the polysaccharide directly influence its biological function. Among these, molecular weight is an important factor affecting the activity of polysaccharides. In addition, different extraction or fractionation modes have certain influence on the activity of the polysaccharide. And it is considered that polysaccharides, like proteins, have active centers necessary for activity. The immune activity is one of the main activities of the ginseng polysaccharide, and the research on the essential structural unit of the ginseng polysaccharide for exerting the immune enhancement activity is important for the research on the structure-activity relationship of the ginseng polysaccharide. Meanwhile, a theoretical basis is provided for scientific development and utilization of the ginseng polysaccharide.

Disclosure of Invention

Problems to be solved by the invention

As mentioned above, polysaccharides are a class of biomacromolecules with complex structures, and molecular weight has important influence on biological functions of the biomacromolecules.

The invention aims to provide a preparation method of a purified sample of ginseng polysaccharide with immune enhancing activity, which is simple, simple and convenient to operate and easy to implement.

Means for solving the problems

The object of the present invention is achieved by the following embodiments.

1. A ginseng polysaccharide composition is characterized by comprising, by mole, 3.0-3.6% of rhamnose, 3.5-4.0% of galacturonic acid, 65.0-70.0% of glucose, 13.0-13.5% of galactose and 9.5-10.0% of arabinose, and the molecular weight of the ginseng polysaccharide composition is larger than 3000Da and smaller than 5000 Da.

2. The ginseng polysaccharide composition of claim 1, wherein rhamnose is 3.55%, galacturonic acid is 3.61%, glucose is 69.69%, galactose is 13.26%, arabinose is 9.89%, and the molecular weight is 3160 Da.

3. A method of preparing a ginseng polysaccharide composition according to any one of claims 1-2, comprising the steps of:

a, grinding ginseng to obtain ginseng powder;

b, extracting the ginseng powder with water, and then filtering to obtain filtrate;

c, concentrating the filtrate to obtain a ginseng polysaccharide extracting solution;

d, precipitating the ginseng polysaccharide extracting solution by using 20-80% ethanol, and centrifuging to obtain residues;

and E, dissolving the residue in water, and performing gel chromatography to obtain the ginseng polysaccharide composition.

4. A method according to claim 3, characterized in that said step D is carried out as follows:

adding 95% ethanol into the ginseng polysaccharide extracting solution to make the final concentration of the ethanol be 20%, and collecting precipitate and supernatant;

continuously adding 95% ethanol into the supernatant to prepare 40% ethanol concentration, and respectively collecting the supernatants;

continuously adding 95% ethanol into the supernatant to prepare 60% ethanol concentration, and respectively collecting the supernatants;

and continuously adding 95% ethanol into the supernatant to prepare 80% ethanol, collecting precipitate, redissolving in water, and freeze-drying.

5. Use of a ginseng polysaccharide composition according to any one of claims 1-2 in the preparation of a medicament having immune enhancing activity.

6. The use according to claim 7, wherein the immune enhancing activity is promoting proliferation of lymphocytes in mice, enhancing phagocytic function of macrophages and enhancing killing ability of NK cells.

The ginseng polysaccharide composition of the present invention can be prepared by the following specific method.

1. Extracting the ginseng crude polysaccharide: pulverizing Ginseng radix to 40-60 mesh, soaking in 10 times (w/w) of distilled water, placing on a heating jacket, and installing a reflux unit. Heating, timing for 2 hr when boiling, filtering the extractive solution in round bottom flask with 120 mesh nylon cloth after the first heating extraction, placing the residue in round bottom flask, storing the filtrate for use, extracting for three times, mixing the three Ginseng radix extractive solutions, and concentrating in 80 deg.C water bath.

2. Alcohol precipitation and grading of ginseng polysaccharide: precipitating the obtained Ginseng radix extractive solution with 20%, 40%, 60%, and 80% ethanol to obtain Ginseng radix polysaccharide compositions with different concentrations.

3. And (3) grading and purifying the ginseng polysaccharide: taking 0.5 g of the ginseng polysaccharide composition, putting the ginseng polysaccharide composition into a centrifuge tube, adding 10mL of water, centrifuging for 5 minutes at 8000 rpm, taking supernate by using a rubber head dropper, adding the supernate into a prepared Sepharose CL-6B preparation column, taking physiological saline as a mobile phase, collecting by using an automatic collector, measuring the sugar content of the eluent by using a phenol-sulfuric acid method, drawing a corresponding curve graph of elution volume and sugar content by using software, collecting the elution volume corresponding to a peak in the curve graph, dialyzing for 24 hours by using flowing tap water of a dialysis bag and distilled water for 24 hours, pouring the eluent into a freeze-drying container, placing the freeze-drying container in an ultra-low temperature refrigerator for freezing, putting the frozen sample into a freeze dryer for freeze-drying, and obtaining powder which is a purified sample.

Effects of the invention

The ginseng polysaccharide composition can promote the mouse lymphocyte proliferation in vivo, has an activating effect on macrophages, and has a remarkable effect of enhancing NK cell toxicity, so that the ginseng polysaccharide composition has an immune activating effect.

Drawings

FIG. 1 is a high performance gel permeation chromatogram of a ginseng polysaccharide composition.

FIG. 2 is a monosaccharide composition analysis diagram of the ginseng polysaccharide composition.

Detailed Description

The apparatus used in the present invention is: liquid chromatography system Ultimate3000 from Thermo Fisher Scientific, Eishi Spectroscopy column, UV-VIS DAD Detector, Infinite M200 PRO microplate reader from TECAN.

Example 1

Accurately weighing 500 g of pulverized Ginseng radix, placing into a 10L round bottom flask, adding 5L of water, placing on a heating jacket, and installing a reflux device. Heating, timing for 2 hr when boiling, filtering the extractive solution in round bottom flask with 120 mesh nylon cloth after the first heating extraction, placing the residue in round bottom flask, storing the filtrate for use, extracting for three times, mixing the three Ginseng radix extractive solutions, and concentrating in 80 deg.C water bath.

Adding 95% ethanol into the Ginseng radix extractive solution to make ethanol concentration 20%, standing for 24 hr, centrifuging at 8000 rpm for 10 min, taking out supernatant, adding 95% ethanol to make ethanol concentration 40%, standing for 24 hr, centrifuging, and collecting supernatant; adding 95% ethanol into the supernatant to reach a final concentration of 80%, standing for 24 hr, centrifuging at 8000 rpm for 10 min to obtain precipitate, dissolving in water, and lyophilizing to obtain panaxan fraction.

Dissolving 500 mg of the above panaxan in distilled water (10 ml), centrifuging at 10000 rpm for 5 min, collecting supernatant, purifying the obtained supernatant with Sepharose CL-6B gel column (3.0 cm × 90 cm), using distilled water as eluent, flow rate of 0.5 ml/min, 20 min/tube, performing phenol-sulfuric acid method tracking detection, collecting corresponding polysaccharide fraction, and freeze drying to obtain panaxan composition with uniform molecular weight.

The ginseng polysaccharide composition obtained by the invention is analyzed by HPLC under the following measurement conditions to obtain a single narrow symmetrical elution peak, the elution time is 16.557 minutes, therefore, the ginseng polysaccharide composition has uniform molecular weight, and the molecular weight of the ginseng polysaccharide composition is calculated to be 3160Da according to a molecular weight curve drawn by glucan with known molecular weight

The HPLC measurement conditions were as follows:

a chromatographic column: TSK-G3000 PWXL (7.8 mm ID X30.0 cm L)

A detector: RID-10A

Mobile phase: deionized water

Flow rate: 1.0 mL/min

Sample introduction amount: 10 μ L

The ginseng polysaccharide composition obtained by the invention is subjected to complete acid hydrolysis and PMP derivatization, and is analyzed by HPLC under the following determination conditions, according to the relative retention time of standard monosaccharides, the monosaccharides contained in the ginseng polysaccharide consist of rhamnose, galacturonic, glucose, galactose and arabinose, and the molar percentage contents of the monosaccharides are respectively 3.55%, 3.61%, 69.69%, 13.26% and 9.89%.

The HPLC measurement conditions were as follows:

a chromatographic column: DIKMA Inertsil ODS-3 (4.6 mm. times.150 mm)

A detector: UV-VIS DAD

Detection wavelength: 245 nm

Mobile phase: PBS (0.1M, p hr 7.0): acetonitrile =82:18 (v/v), p h 7.0

Flow rate: 1.0 mL/min

Sample introduction amount: 10 μ L

Test of drug efficacy

Immunity assay Using the panaxan composition prepared in example 1

1. In vivo immunocompetence study of ginseng polysaccharide compositions

1.1 Effect of the Ginseng polysaccharide composition on the body weight and immune organs of mice

Male mouse ICR mice were randomly divided into a normal group, a normal saline control group, and a ginseng polysaccharide composition administration group (50 mg/kg), and 3 groups were formed, each group consisting of only 12 mice. The negative control group was administered with 0.2 ml/tube of physiological saline for 30 days. After the experiment is finished, the mice are killed by cervical dislocation, weighed, the body weight is recorded, the mice are moved into an ultra-clean bench after being sterilized by alcohol, the spleen and the thymus of the mice are taken out under the aseptic condition and weighed, and the spleen index and the thymus index are calculated.

TABLE 1 Effect of Ginseng polysaccharide compositions on mouse body weight, spleen index and thymus index

Note: ^*indicates that p is the ratio of the experimental group to the negative control group<0.05

The results showed that the weight of the mice in the ginseng polysaccharide composition 50mg/kg administration group was increased compared to the negative control group, but did not show significant difference; the spleen index and the thymus index of mice in the administration group are obviously improved compared with those in the negative control group, and have obvious difference (P < 0.05). And the appetite, vitality and fur gloss of the mice in the group to which the ginseng polysaccharide composition is administered are better than those in the negative control group.

1.2. Effect of Ginseng polysaccharide composition on mouse lymphocyte transformation

Preparation of single cell suspension of spleen lymphocytes: administering the ginseng polysaccharide composition to normal mice by gavage for 30 consecutive days. The day after the last administration, the mice were sacrificed by decapitation, the spleen was aseptically dissected, the connective tissue and fat were removed and placed in a dish containing an ice-bath D-Hanks balanced salt solution, the spleen was ground with forceps, and the cell suspension was filtered through a 200 mesh sieve to remove tissue mass, transferred into a centrifuge tube, and centrifuged at 1000 rpm for 5 minutes. The lymphocyte separation medium was slowly added to remove the erythrocytes. And washed twice with Hanks balanced salt solution. Cells were resuspended in DMEM medium. Trypan blue staining counts viable cells greater than 95%. The cells were diluted to 1X 106 cells/mL with DMEM medium to prepare a single cell suspension. The results show that in this experiment, when the panaxan composition is used alone (table 2), lymphocyte transformation can be stimulated remarkably, and the ratio to negative contrast can reach remarkable difference (P < 0.01); in addition, the panaxan composition can induce ConA to induce T lymphocyte proliferation at 50mg/kg (Table 2), and compared with the negative control group, the action effect is very significant (P < 0.01).

TABLE 2 Effect of the Ginseng polysaccharide compositions on lymphocyte proliferation

Note: ^*indicates that p is the ratio of the experimental group to the negative control group<0.05, ^**Indicates that p is the ratio of the experimental group to the negative control group<0.01

1.3 Effect of the Ginseng polysaccharide composition on stimulating phagocytic function of macrophages in the abdominal cavity

Preparing abdominal cavity macrophages: the ginseng polysaccharide composition (50 mg/kg) was administered to normal mice by gavage for 30 consecutive days. The day after the last dose, mice were sacrificed by cervical dislocation, the abdominal cavity was washed aseptically with pre-cooled PBS (10 mL), and the abdominal cavity fluid was collected in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 minutes at 4 ℃. Centrifugation was repeated twice, and the supernatant was discarded and the cells were resuspended in DMEM medium. The adjusted cell concentration was adjusted to 5X 106 cells/mL and added to a 12-well plate at 100. mu.l/well. Culturing the culture plate in an incubator with 5% CO2 at 37 ℃ for 3 hours, washing the culture plate with PBS (phosphate buffer solution) pre-warmed at 37 ℃ for 2-3 times, and removing nonadherent cells to obtain the purified adherent macrophages.

Phagocytosis of neutral red by macrophages: the detached macrophages were collected by first incubating with EDTA for 30 min, adjusted to a cell concentration of 1X 106 cells/mL, added to a 96-well plate, and cultured for 24 hours, and then a neutral red solution (concentration of 0.075%, 100. mu.L/well) was added to each well and cultured for 1 hour. Finally, the plate was washed with PBS, and 100. mu.L of cell lysate (ethanol: acetic acid =1:1, v/v) was added thereto for lysis for 2 hours, followed by detection of OD value at 550 nm using a microplate reader.

TABLE 3 phagocytosis of macrophage by panaxan composition

Note: p <0.05 in the experimental group compared to the negative control group, and p <0.01 in the experimental group compared to the negative control group.

In order to observe the regulation effect of the ginseng polysaccharide on abdominal cavity macrophages, the phagocytic function of the ginseng polysaccharide composition on mouse macrophages is firstly detected. The result shows that the ginseng polysaccharide composition (50 mg/kg) can remarkably enhance the capability of macrophages in the abdominal cavity of a mouse to phagocytose neutral red. The ginseng polysaccharide composition is suggested to have remarkable immunostimulation activity on macrophages.

1.4 Effect of the Ginseng polysaccharide composition on the production of NO by peritoneal macrophages

Direct detection of NO production is difficult because NO is extremely unstable extracellularly and has a short half-life. The currently used method is to measure the nitrite concentration in the supernatant of macrophage culture broth using Griess reagent to indirectly reflect the level of NO produced by macrophages.

Peritoneal macrophages were prepared as above. Cells were added to 24-well plates at a concentration of 2X 106 cells/mL. Adding a sugar sample to be detected, culturing for 24 hours, sucking cell culture solution supernatant (100 mu l) and adding the cell culture solution supernatant into another culture plate, adding an equal volume of Griess reagent, detecting by using an enzyme-labeling instrument after 10 minutes, wherein the detection wavelength is 540 nm, drawing a standard curve by using NaNO2 solution, and calculating the NO yield after samples with different concentrations stimulate macrophages. The results show that the panaxan can promote macrophage to secrete NO, and further prove that the panaxan composition can activate macrophage.

3. Effect experiment of ginseng polysaccharide on NK cytotoxic Activity

NK cells, as another subset of lymphocytes, are capable of lysing or killing infected tumor cells. To investigate the effect of ginseng polysaccharide on NK cell killing ability in vivo, mice were gavaged with a ginseng polysaccharide composition (50 mg/kg) for 10 days. A negative control group and a blank control group were also set. After 30 days of continuous administration of the panaxan composition, lymphocytes were prepared according to the method 2.1, cultured in vitro for 48 hours, and viability was measured using YAC-1 as the target cell (effective-to-target ratio 20:1, respectively). As shown in Table 4, the ginseng polysaccharide compositions all have a certain regulation effect on the cytotoxic activity of NK cells. The ginseng polysaccharide composition is prompted to have a remarkable NK cell toxicity enhancing effect. YAC-1 cells were used as target cells for the detection of NK cytotoxic activity. The effective target ratio of 20:1 is added into a 96-well plate, namely 5 multiplied by 106 splenocytes/well and 2.5 multiplied by 105 YAC-1 cells respectively per well, after CO-culturing for 24 hours at 37 ℃ in a CO2 incubator, MTT solution (5 mg/ml) is added, the culture is continued for 4 hours, the supernatant is discarded, 100 mu l DMSO is added, and the light absorption value at 490 nm is detected by a microplate reader. The cytotoxic activity is calculated as follows:

NK cell viability (%) =

TABLE 4 Effect of ginseng polysaccharide compositions on NK cytotoxic Activity

	NK cell Activity (%)
		Blank group	11.978±5.65
Negative group	9.333±4.70
		Ginseng polysaccharide composition	20.88±10.42*

Note: denotes p <0.05 in the experimental group compared to the negative control group.