CN114524887A

CN114524887A - Method for separating and characterizing ginseng polysaccharide with pharmaceutical activity function

Info

Publication number: CN114524887A
Application number: CN202210324382.8A
Authority: CN
Inventors: 周鸿立; 曹冬雪; 赵晶丽
Original assignee: Jilin Institute of Chemical Technology
Current assignee: Kampo Extract Biotechnology Hainan Co ltd; Shenzhen Lizhuan Technology Transfer Center Co ltd
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2022-05-24
Anticipated expiration: 2040-07-01
Also published as: CN112409502B; CN112409502A; CN114524887B

Abstract

The invention relates to the field of drug development functional foods, in particular to a characterization method of ginseng polysaccharide with immunoregulation and anti-tumor activity. The invention extracts ginseng polysaccharide GSP by a microwave extraction method, adopts cotton-shaped DEAE for purification, and further separates by DEAE-52 column chromatography to obtain ginseng monomer polysaccharide. The different components of the ginseng polysaccharide are characterized by using instruments such as UV, IR, HPLC, NMR and the like. Congo red experiments prove that the four components have a triple-helix structure for the first time. GSP-2 contains a characteristic absorption peak of alpha-type shown by infrared; second derivative analysis polysaccharides are linked by alpha-type and beta-type glycosidic linkages. The immune regulation and anti-tumor effects of the components are studied in vitro, and the GSP-2 component with good immune activity is obtained, and is expected to be used as a potential immune regulator for the development of functional foods or medicines.

Description

Method for separating and characterizing ginseng polysaccharide with pharmaceutical activity function

Application No. 202010626447.5, entitled method for isolation and characterization of ginseng polysaccharides with immunomodulatory and antitumor activities, filing date 2020.07.01.

Technical Field

The invention relates to the field of drug development functional foods, in particular to a separation and characterization method of ginseng polysaccharide with a drug activity function.

Background

Research on polysaccharides shows that various biological activities of polysaccharides have close relations with the structure, spatial conformation and other aspects of the polysaccharides. The immunological activity of polysaccharides extracted from different natural sources depends on their monosaccharide composition, molecular weight, chain conformation, etc. For example, the ratio of arabinose, glucuronic acid and galactose in monosaccharide composition of polysaccharides has been reported to be related to their macrophage stimulating activity [ immunological activity and structure of polysaccharide from Streptococcus equi].International Journal of Biological Macromolecules,2013,57:218-225.]. It has been reported in the literature that ginseng polysaccharides are mainly composed of galactose, arabinose, rhamnose, glucose and galacturonic acid [ Structural and anti-inflammatory catalysis of a novel polysaccharide from North American ginseng (J quinquefolius) [ J ] polysaccharide].International Journal of Biological Macromolecules,2015.]。[Panax ginseng polysaccharide induces apoptosis by targeting Twist/AKR1C2/NF-1pathway in human gastric cancer[J].Carbohydrate Polymers,2014,102(Complete):103-109.]Isolating Ginseng radix antitumor polysaccharide (PGP2a), and analyzing by gas chromatography with galactose, arabinose, glucose and galacturonic acid content ratio of 3.7:1.6:0.5: 5.4. [ Purification, characterization and anti-activity of a polysaccharide from Panax gineng [ J ]].International Journal of Biological Macromolecules,2012,51(5):968---973.]The ginseng homogeneous polysaccharide (PGPW1) was isolated and analyzed to give a molar ratio of glucose (Glu), galactose (Gal), mannose (Man) and arabinose (Arab) of 3.3:1.2:0.5: 1.1.The reported molecular weight of ginseng polysaccharide is 1 × 10³～1×10⁶Better biological activity between Da [ systematic analysis of ginseng polysaccharide and immune activity research [ D ]]Northeast university, 2009.]. The glycosidic bond of the polysaccharide can be detected by nuclear magnetic resonance spectroscopy, and the monosaccharide types and some special groups can be determined according to some carbon signals appearing at special positions. [ Structural characterization of alkali-soluble polysaccharides from Panax ginseng C.A. Meyer [ J].Royal Society Open Science,2018,5(3):171644.]It is found by NMR technology that the RG-I structural domain of ginseng polysaccharide (NGP-1a, NGP-2a) may have high branching and beta-14-galactan alpha-1, 5/1,3, 5-arabinans and II type arabinogalactan side chain. According to the related literature, Congo red experiments can explore the triple-helix space structure of polysaccharide, and the triple-helix space structure is beneficial to the improvement of the bioactivity of polysaccharide [ the chain structure and the function of triple-helix polysaccharide research progress [ J]The term "functional polymer journal" (stage 2) "134-.]However, Congo red experiments on ginseng polysaccharides have not been reported.

The panaxan has good immunoregulatory activity. The ginseng polysaccharide GMP is obtained by separating [ Immunomodulating Activities of Polysaccharides Isolated from Panax generating [ J ]. Journal of Medicinal Food,2004,7(1):1-6 ], and the GMP under 100mg/ml is obtained by MTT experiment to improve the activity of macrophage; the fact that GMP has obvious phagocytosis effect under 100mg/ml is obtained through a neutral red experiment; the experiment of NO secretion amount shows that the phagocytosis effect of GMP is also obvious under 100 mg/ml. The polysaccharide GPN and GPNE-II of American ginseng are separated and obtained [ The core structure characterization and The engineering of nuclear polysaccharide with The immunological-enhancing activity ] [ J ]. International Journal of Biological Macromolecules,2018 ], and The GPN and GPNE-II have promotion effect on lymphocyte proliferation, thus showing application potential of immunoregulation. [ anticancer immunological activity of polysaccharides from Panax japonica C.A.Mey: rolls of the infection on CD4+ T cells and tumor associated macrophages [ J ]. International Journal of Biological Macromolecules Structure Function & Interactions,2018 ] American ginseng polysaccharide (PSPJ) was isolated and used for the study of the Antitumor activity of H22 hepatoma cells in mice, and it was found that PSPJ inhibited the growth of H22 tumors in vivo and had no detectable toxic effect on mice. [ Polysaccharides from viral sources in human tissues virus a macroporous and NK cell activation [ J ] International Journal of Biological Macromolecules Structure & Interactions,2017 ] isolation of the polysaccharide fraction of ginseng leaf (GS-P) in mice inhibited the proliferation of colon tumor 26-M3.1 in a dose-dependent manner, significantly enhancing cytotoxicity to tumor cells. The immunological and antitumor activities of the ginseng polysaccharides can be measured and compared by these several experimental methods.

Disclosure of Invention

The invention adopts a microwave extraction method to extract ginseng polysaccharide, firstly separates and purifies the ginseng monomer polysaccharide through DEAE-52 cellulose to obtain ginseng monomer polysaccharide, and then measures ultraviolet, infrared, monosaccharide composition, molecular weight, helical structure and immunoregulation activity, thereby laying a theoretical foundation for further developing ginseng resources.

The invention realizes the aim through the following technical scheme, and the separation method of the ginseng monomer polysaccharide with the immunity and the anti-tumor activity comprises the following steps:

(1) extracting Ginseng radix polysaccharide with water as extractant by microwave extraction method, and measuring the content and extraction rate of Ginseng radix polysaccharide by phenol-sulfuric acid method.

(2) Purifying the crude ginseng polysaccharide by adopting the pretreated DEAE cellulose to obtain purified ginseng polysaccharide;

the method comprises the following specific steps: the adsorption capacity of DEAE-cellulose to ginseng polysaccharide is determined by a method of gradually increasing the sample amount, and the grading effect of the polysaccharide is ensured. DEAE-cellulose ion exchange chromatography can not only classify the polysaccharide according to the ionic strength, but also achieve the purpose of decolorization. The DEAE cellulose pretreatment comprises the following steps: firstly, soaking DEAE cellulose dry powder in distilled water, soaking in 0.5mol/L HCl aqueous solution for 1-2 h, then washing with distilled water until the pH value of the filtrate is neutral, then, soaking the drained DEAE cellulose in 0.5mol/L NaOH solution for 1-2 h, then washing the pH value of the filtrate to be neutral with distilled water, and then, packing the filtrate into a column.

The ginseng polysaccharide is purified by cotton-shaped DEAE cellulose: the sample concentration was 10.0mg/mL, the sample volume was 50mL, the flow rate was 1.0mL/min, and the eluates were distilled water and 0.3mol/L aqueous NaCl. Eluting with distilled water to obtain neutral sugar fraction, and eluting with 0.3mol/L NaCl water solution to obtain acidic sugar fraction; collecting the eluent after passing through the column, and respectively carrying out rotary evaporation and concentration; and then putting the polysaccharide solution into a dialysis bag, dialyzing with distilled water for 24h, changing water for many times, concentrating the polysaccharide solution, and freeze-drying to obtain the purified ginseng polysaccharide.

(3) Separating purified panaxan with DEAE-52 cellulose, sequentially eluting with distilled water, 0.1mol/L, 0.2mol/L, and 0.3mol/L NaCl aqueous solution at flow rate of 1mL/min to obtain panaxan.

Preferably, the microwave extraction method comprises the following specific steps: taking 5g of ginseng crude polysaccharide powder, and performing microwave extraction at the temperature of 70 ℃, wherein the material-liquid ratio is 1: 30(mg/ml), and the extraction time is 6min under the condition of the power of 550W, and then the ginseng crude polysaccharide is obtained after concentration and drying.

Preferably, the DEAE-52 cellulose is used for separating the purified ginseng polysaccharide by the following specific steps: weighing purified panaxan, dissolving with 5mL distilled water, and slowly adding along DEAE-52 cellulose column wall during loading to prevent uneven loading caused by filler flying. Eluting the neutral sugar fraction with distilled water; sequentially eluting the acidic sugar fraction with 0.1mol/L, 0.2mol/L and 0.3mol/L NaCl aqueous solution at the flow rate of 1mL/min, and collecting by bottles; distilled water is eluted and collected to obtain monomeric polysaccharide GSP-0, 0.1mol/L NaCl aqueous solution is eluted and collected to obtain monomeric polysaccharide GSP-1, 0.2mol/L NaCl aqueous solution is eluted and collected to obtain monomeric polysaccharide GSP-2, and 0.3mol/L NaCl aqueous solution is eluted and collected to obtain monomeric polysaccharide GSP-3.

Preferably, collecting 0.2mol/L NaCl water solution to obtain ginseng monomer polysaccharide, which is defined as GSP-2; the average molecular weight of the GSP-2 is 1.76 multiplied by 10⁵Da, in terms of molar ratios, mannose in GSP-2: ribose: rhamnose: glucose furfural acid: galacturonic acid: glucose: galactose: arabinose 0.56: 0.62: 1:1.45:0.67:2.31:2.46:3.30. GSP-2 may haveEffectively promoting the increment capability and phagocytosis of macrophages and promoting the secretion of NO; can also effectively promote the inhibition effect on the proliferation of 5 colorectal cancer cells (HCT-116, HCT-8, HT-29, SW48 or Caco2), has better immunity and anti-tumor activity, and can be used for preparing anti-tumor drugs.

According to another aspect of the present invention, there is provided a method for characterizing monomeric polysaccharides of ginseng having immunological and anti-tumor activities, comprising the steps of:

(1) and (3) ultraviolet absorption spectrum detection: dissolving 2.00mg of the separated ginseng polysaccharide in 2ml of distilled water, scanning by using an ultraviolet spectrophotometer within the range of 200-800nm, and recording an ultraviolet absorption spectrum to detect whether protein and nucleic acid exist in the ginseng monomer polysaccharide.

(2) Infrared spectrum detection:

precisely weighing purified and separated ginseng monomer polysaccharide 2mg and dried potassium bromide 100mg, mixing, grinding with agate mortar, tabletting, and measuring 4000cm in infrared spectrometer^-1～400cm^-1Scanning, continuously scanning for 32 times, determining the absorption peak of the sample, and scanning the sample to remove air peak polysaccharide so as to measure the functional group and glycosidic bond linkage mode of ginseng monomer polysaccharide.

(3) Carrying out high performance liquid chromatography analysis on ginseng monomer polysaccharide by adopting a 1-phenyl-3-methyl-5-pyrazolone derivatization method:

accurately weighing 2-3 mg of separated ginseng monomer polysaccharide sample, placing the ginseng monomer polysaccharide sample in a reaction kettle, adding 2mL of 2mol/L trifluoroacetic acid aqueous solution, sealing the reaction kettle, placing the reaction kettle in a 110 ℃ oven for hydrolysis for 5 hours, taking supernatant, evaporating to dryness, repeatedly washing with methanol for 3 times, and evaporating to dryness to obtain a hydrolysate; adding 0.2mL of 0.5mol/L methanol solution of 1-phenyl-3-methyl-5-pyrazolone and 0.2mL of 0.3mol/L NaOH aqueous solution into a water bath at 70 ℃ for heating for 60min, cooling, adding 0.2mL of 0.3mol/L HCl aqueous solution for neutralizing, adding 1mL of chloroform for extraction, centrifuging to remove supernatant, repeating for 3 times, filtering through a 0.45 mu L filter membrane to obtain a derivatized solution, filtering, performing high performance liquid chromatography analysis, and calculating the amount of each monosaccharide.

(4) Measuring the molecular weight of ginseng monomer polysaccharide by adopting a high performance liquid phase size exclusion method and a differential refraction detector under the following conditions;

20 μ L of 2.0mg/mL isolated ginseng monomer polysaccharide sample aqueous solution was injected through a 0.45 μm filter into Shodex Sugar KS-804(8.0 mm. times.300 mm2) column, sample injection and analysis were performed under chromatographic conditions, peak areas were recorded, and the relative molecular mass of each component was calculated by substituting into the regression equation. The standard curve linear regression equation is:

log(Mw)＝13.87440-5.01843x+0.96087x²-0.07993x³

mw weight-average molecular weight x: retention time

(5) Analyzing ginseng polysaccharide by adopting a Congo red experiment:

mixing 1mg/mL of ginseng monomeric polysaccharide sample aqueous solution with 2mL of 80 mu mol/L Congo red solution, and then gradually adding the mixture into 1mol/L of NaOH aqueous solution to form NaOH mixed solution with final concentrations of 0mol/L, 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L and 0.5 mol/L; maintaining the mixture at the dark room temperature for 5 min; and (3) carrying out ultraviolet-visible absorption spectrum full-wavelength scanning within the range of 200-800nm, and recording the maximum absorption wavelength.

Preferably, the conditions of the high performance liquid chromatography are as follows:

the separation column is an ODS 2C 18 column (250X 4.6 mm)²5mm) mobile phase of acetonitrile and 0.05mol/mL pH 6.8 phosphate buffer, wherein the volume ratio of acetonitrile to phosphate buffer is 18:82, flow rate: 0.8mL/min, column temperature: 30 ℃, an ultraviolet detector, a detection wavelength of 245nm and a sample injection amount of 20 mu L.

The activity test is carried out on the separated ginseng monomer polysaccharide with the immunocompetence, and the method comprises the following steps:

(A) the effect of ginseng monomeric polysaccharides on the proliferative capacity of RAW264.7 cells was evaluated by MTT assay. The absorbance was measured at 490nm using a microplate reader. The cell viability equation is calculated as follows:

phagocytic activity of RAW264.7 cells was measured by a method using neutral red. The absorbance was measured at 490nm with a microplate reader. The phagocytosis index is calculated as follows:

nitric Oxide (NO) production was determined by measuring nitrite concentration in the culture supernatant of RAW264.7 cells cultured using Griess reaction. The absorbance at 490nm was measured with a microplate reader to calculate the nitrite concentration. By comparison with NO standards.

(B) And comparing the in vitro immune activity through partial characterization to obtain the most effective part of GSP-2, and further performing an anti-tumor activity test. The cell survival rate is detected by a CCK-8 method, and the absorbance at 450nm is detected by an enzyme-labeling instrument. Calculating the cell survival rate and inhibition rate under the action of the medicine:

survival rate ═ OD (addition group)/OD (blank group)) × 100%;

inhibition rate ═ 1-OD (addition group)/OD (blank group)) × 100%.

IC for calculating drug effect₅₀Values were plotted as a function of drug, inhibition and drug action concentration using GraphPad Prism.

(C) Through partial characterization, the in vitro immune and antitumor activities are compared to obtain the most effective part of GSP-2, and the structure is further characterized. Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker DRX-500NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) using D₂O is used as a solvent. Respectively at 128k and 32k¹H-NMR and¹³C-NMR spectrum.

The invention provides a systematic characterization method by purifying and separating ginseng polysaccharide, and performing characterization analysis such as UV, IR, HPLC, NMR and Congo red experiments on different components. RAW264.7 macrophage is used as an immunological experimental object, and the inhibition effect of the proliferation of 5 colorectal cancer cells is detected by adopting a CCK-8 method, so that the immunoregulation and the anti-tumor effect of the ginseng polysaccharide are comprehensively and comprehensively clarified. The structure-activity relationship of the ginseng polysaccharide is deeply discussed, and a theoretical basis is provided for the development and utilization of the ginseng polysaccharide.

Compared with the prior art, the invention has the following advantages:

1) the invention provides a systematic characterization method for the ginseng polysaccharide with immune and anti-tumor activity for the first time.

2) The invention adopts cotton DEAE cellulose to purify the ginseng polysaccharide for the first time, and the ginseng polysaccharide GSP-2 with immunity and anti-tumor activity is obtained. GSP-2 can effectively promote the increment capability and phagocytosis of macrophages, promote the secretion of NO, effectively promote the inhibition effect on the proliferation of 5 colorectal cancer cells, and has better immunity and anti-tumor activity.

3) The invention carries out Congo red experiment on the ginseng polysaccharide for the first time and proves that the ginseng polysaccharide has a triple-helix space structure.

Drawings

FIG. 1 is a DEAE-52 column separation elution curve;

FIG. 2 is a UV full wavelength scan of GSP-0, GSP-1, GSP-2, GSP-3;

FIG. 3 is an IR scan of GSP-0, GSP-1, GSP-2 and GSP-3;

FIG. 4 is an IR second derivative spectrogram of GSP-0, GSP-1, GSP-2 and GSP-3;

FIG. 5 is a triple helix constellation diagram for GSP-0, GSP-1, GSP-2, GSP-3;

FIG. 6 is a graph showing the effect of GSP-0, GSP-1, GSP-2 and GSP-3 on the proliferation potency of RAW264.7 cells;

FIG. 7 is a graph of the effect of GSP-0, GSP-1, GSP-2 and GSP-3 on the phagocytic capacity of RAW264.7 cells;

FIG. 8 is a graph showing the effect of GSP-0, GSP-1, GSP-2 and GSP-3 on the secretion of RAW264.7 cells;

FIG. 9 is a graph of the inhibition of cell proliferation of 5 colorectal cancers by GSP-2;

FIG. 10 is of GSP-2¹H-NMR spectrum;

FIG. 11 is of GSP-2¹³C-NMR spectrum.

Detailed Description

Example 1 extraction, purification and content determination of crude Ginseng polysaccharide

The raw material is rhizome of Changbai mountain ginseng in Jilin province, which is cleaned, dried and crushed into powder by a crusher for standby. Extracting 5g of ginseng powder with water as extractant by microwave extraction; the microwave extraction time is 6min, the temperature is 70 ℃, and the material-liquid ratio is 1: 30(mg/ml) under the condition of power of 550W, concentrating and drying to obtain the ginseng crude polysaccharide, named GSP.

DEAE-pretreatment of cellulose: firstly, DEAE-cellulose dry powder is soaked in distilled water, soaked in 0.5mol/L HCl solution for 1-2 h, then washed by distilled water until the pH value of filtrate is neutral, the DEAE-cellulose after being dried is soaked in 0.5mol/L NaOH solution for 1-2 h, washed by distilled water until the pH value of the filtrate is neutral, and then packed into a column.

Cotton-like DEAE loading and elution: the method comprises the steps of measuring pretreated cotton DEAE-cellulose, filling the cotton DEAE-cellulose into a chromatographic column by a wet method, wherein the sample loading concentration is 10.0mg/mL, the sample loading volume is 50mL, the flow rate is 1.0mL/min, and the eluent is distilled water and 0.3mol/L NaCl aqueous solution. Eluting with distilled water to obtain neutral sugar fraction, and eluting with 0.3mol/L NaCl water solution to obtain acidic sugar fraction; collecting the eluent after passing through the column, and respectively carrying out rotary evaporation and concentration. And then putting the polysaccharide solution into a dialysis bag, dialyzing with distilled water for 24h, changing water for many times, concentrating the polysaccharide solution, and freeze-drying to obtain the purified ginseng polysaccharide.

The content of the ginseng crude polysaccharide is determined by adopting a phenol-sulfuric acid method. The content of uronic acid is determined by adopting m-hydroxyl biphenyl method and galacturonic acid as standard substance. The content of protein is determined by adopting a Coomassie brilliant blue G-250 staining method and taking calf serum as a standard substance.

TABLE 1 extraction rate of crude polysaccharides, polysaccharide, uronic acid and protein content of ginseng before and after purification

Note: the purified polysaccharide herein is a mixture of a neutral sugar fraction and an acidic sugar fraction

The yield of ginseng polysaccharide obtained by microwave extraction is 69.34% +/-0.07 by phenol-sulfuric acid method. The crude ginseng polysaccharide is purified by cotton-shaped DEAE cellulose, so that the separation effect of the ginseng polysaccharide is improved. The purity of the ginseng polysaccharides before and after purification, and the contents of protein and uronic acid are shown in Table 1. The purity of the polysaccharide and the content of uronic acid are obviously improved, which shows that the cotton-shaped DEAE cellulose can effectively purify the ginseng polysaccharide. The protein content was increased, and it was presumed that a glycoprotein might be present.

Example 2 isolation of ginseng polysaccharides

DEAE-52 loading and elution. Specifically, the purified panaxan is weighed and dissolved by 5mL of distilled water, and is slowly added along the column wall during sample loading, so that the filler is prevented from flying, and the sample loading is not uniform. Eluting neutral sugar fraction with distilled water; the acidic sugar fractions were sequentially eluted with 0.1mol/L, 0.2mol/L, and 0.3mol/L aqueous NaCl at a flow rate of 1mL/min, collected in vials, and 5 mL/vial for each 5 min. The absorbance of each liquid was measured by the phenol-sulfuric acid method, and an elution curve was plotted with the vial number as the abscissa and the absorbance a as the ordinate, with the results shown in fig. 1.

As shown in FIG. 1, the elution profile obtained by DEAE-52 cellulose separation after the purification with cotton-like DEAE was 4, which was observed to exist and eluted as a single and symmetrical sharp peak in a cross-sectional view, indicating the uniformity and high purity of the sample. Respectively named as GSP-0, GSP-1, GSP-2 and GSP-3, and is subjected to concentration, dialysis and freeze drying for later use; wherein GSP-0 is obtained by eluting with distilled water solution, GSP-1 is obtained by eluting with 0.1mol/L NaCl water solution, GSP-2 is obtained by eluting with 0.2mol/L NaCl water solution, and GSP-3 is obtained by eluting with 0.3mol/L NaCl water solution.

Example 3 structural characterization of the ginseng polysaccharides

1. Ultraviolet spectrometry and analysis

Dissolving 2.00mg of the separated ginseng polysaccharide in 2ml of distilled water, scanning by using an ultraviolet spectrophotometer within the range of 200-800nm, and recording an ultraviolet absorption spectrum.

And (4) ultraviolet analysis: the scanning range is in the range of 200-800 nm. As can be seen from FIG. 2, GSP-0 and GSP-1 detected the absorption peaks of the proteins, indicating that these two polysaccharides may have glycoproteins, consistent with the rationale of the physicochemical properties at the purification stage. As can be seen in FIG. 2, the spectral curves for GSP-2 and GSP-3 are relatively smooth, indicating that protein and nucleic acid impurities in GSP-2 and GSP-3 have been minimized or absent.

2. Determination and analysis of infrared spectra and second derivative

Precisely weighing purified and separated 2mg of panaxan and 100mg of dried potassium bromide, mixing, grinding with agate mortar, tabletting, and measuring 4000cm in infrared spectrometer^-1～400cm^-1Scanning, continuously scanning 32 times, determining the absorption peak of the sample, and sweeping off the air peak polysaccharide before scanning the sample. The obtained spectrum was subjected to absorbance normalization, baseline correction and smoothing with Omnic software, and then processed with origin8.0 software.

Certain features of the polysaccharide structure can be inferred from the position and shape of various characteristic peaks in the infrared spectrum. As shown in figure 3, the OH stretching vibration of the GSP-0, GSP-1, GSP-2 and GSP-3 components is 3370-3450 cm^-1Shows broad and strong vibrational peaks, indicating the presence of intramolecular and intermolecular hydrogen bonds in the polysaccharide. At 2927cm^-1The absorption peak shown here is that of methylene group C-H in the polysaccharide. 1414cm^-1、1632cm^-1The peaks shown on the left and right are the symmetric and asymmetric shock absorption peaks of C ═ O. At 1000 to 1200cm^-1The middle region is the vibration absorption peak of bones C-O and C-C, and the contrast shows that GSP-0 and GSP-1 are 1150cm^-1There are characteristic absorption peaks of uronic acid around the peak. GSP-0, GSP-1 and GSP-2 are 855cm^–1、1026cm^–1The peak in (b) indicates that this fraction is linked by alpha-pyranoside linkages. It is presumed that GSP-0, GSP-1, GSP-2 and GSP-3 are formed by connecting alpha-glycosidic bonds and contain heteropolypyranose structures with different functional groups.

The second derivative spectrum can improve the resolution of the one-dimensional spectrum, overlapping peaks on some infrared spectrums are separated on the second derivative infrared spectrum, the positions of absorption peaks are more accurate, and more detailed comparison can be carried out, as shown in fig. 4, under the treatment of the second derivative, a plurality of masked peaks are processedThe absorption peak of the cap was revealed. GSP-1, GSP-2 and GSP-3 in the one-dimensional map are 1740cm^–1The peak of (A) is weaker, and 1740cm can be clearly seen in a two-dimensional map^–1The absorption peak of (1) was confirmed to contain an ester carbonyl group. At 1200-1000 cm ^–11081 is a characteristic absorption peak of polysaccharide belonging to a pyranose ring characteristic structure, and GSP-0, GSP-1, and GSP-2 at 762cm^–1Typical absorption around and shows the C-O-C symmetric oscillation peak of the d-pyranosyl ring, and it can be seen from the figure that GSP-3 does not contain the C-O-C symmetric oscillation peak, and GSP-0 and GSP-1 are at 923cm^–1Is a beta-type glycosidic bond. The combination of the first derivative and the second derivative conjectures that GSP-0 and GSP-1 have beta-type glycosidic bonds and alpha-glycosidic bonds.

Under multiple wavelengths, the first derivative and the second derivative map are combined to better reflect the difference of the 4 polysaccharides, and more accurate data and results can be deduced.

3. Monosaccharide composition analysis

Accurately weighing 2-3 mg of separated ginseng monomer polysaccharide sample, placing the ginseng monomer polysaccharide sample in a reaction kettle, adding 2mL of 2mol/L trifluoroacetic acid aqueous solution, sealing the reaction kettle, placing the reaction kettle in a 110 ℃ oven for hydrolysis for 5 hours, taking supernatant, evaporating to dryness, repeatedly washing with methanol for 3 times, and evaporating to dryness to obtain a hydrolysate; adding 0.2mL of 0.5mol/L methanol solution of 1-phenyl-3-methyl-5-pyrazolone and 0.2mL of 0.3mol/L NaOH aqueous solution into a water bath at 70 ℃ for heating for 60min, cooling, adding 0.2mL of 0.3mol/L HCl aqueous solution for neutralizing, adding 1mL of chloroform for extraction, centrifuging to remove supernatant, repeating for 3 times, filtering through a 0.45 mu L filter membrane to obtain a derivatized solution, filtering again, performing high performance liquid chromatography analysis, and calculating the amount of each monosaccharide. HPLC conditions: separation HPLC conditions: separating the column: ODS 2C 18 column (250X 4.6mm2, 5mm), mobile phase: acetonitrile-0.05 mol/mL phosphate buffered pH 6.8 (volume ratio 18:82), flow rate: 0.8mL/min, column temperature: 30 ℃, an ultraviolet detector, a detection wavelength of 245nm and a sample injection amount of 20 mu L. Mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose as standard. Monosaccharides and their molar ratios are obtained as in table 2.

TABLE 2 monosaccharide compositions and molar ratios of GSP-0, GSP-1, GSP-2, GSP-3

From table 2, monosaccharide compositions and molar ratios of four polysaccharides are shown, and the monosaccharide compositions are determined by high performance liquid chromatography, wherein GSP-0, GSP-1, GSP-2 and GSP-3 mainly comprise ribose, rhamnose, glucose, galactose and arabinose, and structurally, the monosaccharide compositions are similar, and contain glucuronic acid and galacturonic acid, which is consistent with infrared results. Previous studies on the composition of ginseng polysaccharides have focused on extraction and purification, among other things, which have a higher abundance of monosaccharide combinations, particularly with a higher proportion of glucose and arabinose, than other classes of ginseng polysaccharides.

4. Molecular weight analysis

The relative molecular weight is determined by high performance gel permeation chromatography, and the purity is detected by a differential detector. The standard curve linear regression equation is:

log(Mw)＝13.87440-5.01843x+0.96087x²-0.07993x³

mw weight-average molecular weight x: retention time

TABLE 3 molecular weights of GSP-0, GSP-1, GSP-2, GSP-3

Generally, the molecular weight distribution of the polysaccharide with biological activity is in a certain regularity, the molecular weights of the polysaccharide with different activity functions are in different ranges, and the structure and the biological activity of the polysaccharide are influenced to a certain extent when the molecular weights are too high or too low. As shown in Table 3, the weight average molecular weight distribution of GSP-0, GSP-1, GSP-2 and GSP-3 is 1.76-9.24 × 10⁵Da. The polydispersity index is the ratio of weight average to number average, and the closer the index is to 1, the narrower and uniformly symmetrical the molecular weight distribution. GSP-2 has a polydispersity index of 0.357 due to pointsThe biological activity of these high molecular weight polysaccharides is limited by their large molecular weight, poor apparent viscosity or water solubility or complex conformation. The search for low molecular weight bioactive polysaccharides is therefore a need for the search for new products to be developed.

5. Determination of ginseng polysaccharide helical configuration

And analyzing the interaction of Congo red and polysaccharide by using an ultraviolet-visible spectrophotometry to determine the triple helix configuration. 1mg/mL of the sample aqueous solution of each component was mixed with 2mL of Congo red solution (80. mu. mol/L), and then the mixture was gradually added to 1mol/L of NaOH solution to form NaOH mixed solutions having final concentrations of 0mol/L, 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, and 0.5 mol/L. The mixture was kept at the dark room temperature for 5 min. And (3) carrying out ultraviolet-visible absorption spectrum full-wavelength scanning within the range of 200-800nm, and recording the maximum absorption wavelength.

The conformation transition of polysaccharide under different sodium hydroxide concentrations is measured by congo red-polysaccharide complex colorimetry, and it is reported in literature that polysaccharide with triple helix structure undergoes helix-helix transition, shows red shift and blue shift with the increase of alkali concentration and the sequence of lambda max, and the triple helix structure can endow polysaccharide with higher bioactivity. In the present study, the complex of ginseng polysaccharide and congo red at different NaOH concentrations is shown in fig. 5, it can be seen that the NaOH concentration is increased from 0mol/L to 0.5mol/L, the maximum absorption wavelength of the 4-component ginseng polysaccharide and congo red complex is red-shifted, contrary to the blue-shift trend when congo red is used alone, therefore, the 4-component ginseng polysaccharide has triple helix conformation, indicating that the regular spatial conformation is closely related to the biological activity of the polysaccharide.

Example 4 Ginseng polysaccharide immunomodulatory Activity

1. Cell culture

The RAW264.7 cell line is a mouse macrophage cell line obtained from a type culture specimen of the chinese academy of sciences (shanghai). Cells were cultured in complete medium (Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% total bone serum (FBS), 1mmol/L sodium pyruvate, 100U/mL penicillin and 100. mu.g/mL streptomycin) and maintained at 37 ℃ moist 5% CO₂An incubator. Cells were cultured for 36-48h to log phase and then used for experiments.

1.1 Effect of Ginseng polysaccharides on the proliferative Capacity of RAW264.7 cells

The effect of ginseng polysaccharides on the proliferative capacity of RAW264.7 cells was evaluated by MTT assay. The cells were suspended in the medium at 1X 10⁵cell/well density was seeded in 96-well plates and incubated (37 ℃, 5% CO)₂) And (4) 12 h. Polysaccharide samples were dissolved in culture medium at different concentrations (25, 50, 100, 200, 400, 800 μ g/mL) and applied to the cells. Lipopolysaccharide (LPS, 25. mu.g/mL) and intact medium without added polysaccharide were positive and blank controls, respectively. After 24h of incubation, 10. mu.L of MTT solution (5mg/mL) was added to each well. With 5% CO at 37 deg.C₂The cultivation was continued for 4 h. Subsequently, the supernatant was discarded, and the plate was shaken and left in the dark for 10min by adding 100 μ L of dimethyl sulfoxide to each well to ensure complete dissolution of the resulting purple crystals. Finally, the absorbance was measured at 490nm using a microplate reader. The cell viability equation is calculated as follows:

the results of using MTT method to detect the proliferation promoting ability of RAW264.7 cells by GSP are shown in fig. 6. From the figure, it can be seen that the cell activities of GSP-0, GSP-1 and GSP-2 are all larger than that of the blank control in the range of 25-400 mug/mL, which indicates that 3 components have a certain promotion effect on the proliferation of RAW264.7 cells. The proliferation indexes of GSP-0, GSP-1, GSP-2 and GSP-3 reach the highest values when the concentration is 25-50 mg/mL, and have significant difference compared with a blank control group. GSP-3 had no effect at 200 mg/mL-800 mg/mL, and it is presumed that a high concentration would have a slightly toxic effect on cells.

1.2 Effect of Panax ginseng polysaccharides on phagocytic Capacity of RAW264.7 cells

Phagocytic activity of RAW264.7 cells was measured by the neutral red method. RAW264.7 cells in the logarithmic growth phase in a 96-well plate were suspended in the culture medium and cultured for 24 hours, and then the supernatant was transferred to a new 96-well plate to determine the nitric oxide content. Neutral erythrophagocytosis assay of phagocytic potency of RAW264.7 cells. After removing the supernatant, the non-adherent cells were removed by washing twice with PBS, and 100. mu.L of neutral red solution (0.075%, g/g) was added to each well. Incubate for 1h, then discard the supernatant. To remove excess neutral red solution, cells were washed twice with PBS. After this time, 100 μ L of cell lysate (ethanol: 1.0mol/L acetic acid 1:1, v/v) was added and the plate was left at room temperature overnight. The absorbance was measured at 490nm with a microplate reader. The phagocytosis index is calculated as follows:

the neutral red test is one of means for testing the phagocytic capacity of the polysaccharide, the result of the neutral red test is shown in figure 7, and compared with a blank control, the four-component polysaccharide has a promoting effect on the phagocytic capacity of RAW264.7 cells within the concentration range of 25-400 mu g/mL, and has a remarkable phagocytic effect within the concentration range of 100-400 mu g/mL. It can be seen from FIG. 7 that the GSP-2 component-promoting effect is in an ascending trend, relatively good in activity and remarkable in phagocytosis.

1.3 Effect of Ginseng polysaccharides on NO secretion from RAW264.7 cells

Nitric Oxide (NO) production was determined by measuring nitrite concentration in the culture supernatant of RAW264.7 cells cultured using Griess reaction. The supernatant collected in a 96-well plate in the phagocytosis assay was mixed with an equal volume of Griess reagent (0.1% N-1-naphthylethylenediamine dihydrochloride in 5% phosphoric acid), kept at room temperature for 10min in the dark, and the nitrite concentration was calculated by measuring the absorbance at 490nm with a microplate reader. By comparison with NO standards.

It has been demonstrated that Nitric Oxide (NO) produced by macrophages is involved in the destruction of various intracellular pathogens as well as tumor cells and places the cells in a state of cell arrest. The result of NO secretion is shown in FIG. 8, compared with the blank control, GSP-0, GSP-1, GSP-2 and GSP-3 can stimulate RAW264.7 macrophage to secrete NO within the concentration range of 25-800 mug/mL, and the stimulation effect of the GSP-1, GSP-2 and GSP-3 components within the concentration range of 50-400 mug/mL tends to increase along with the increase of the concentration.

In the test results of GSP-0, GSP-1, GSP-2 and GSP-3 in FIGS. 6 to 8, the arrangement order of the histograms is the order in which Blank (Blank), LPS and concentration are increased in the order of 25 to 800. mu.g/mL.

Example 5 antitumor Activity of Ginseng polysaccharide GSP-2

1.1 cell culture

Human colorectal cancer cells HCT-116, HCT-8 and SW48, and culture conditions are as follows: using RPMI-1640 culture medium, 10% fetal bovine serum, at 37 deg.C and 5% CO₂And (5) culturing in an incubator at constant temperature. Human colorectal cancer cell Caco2, culture conditions: DMEM culture medium, 10% fetal calf serum, 5% CO at 37 deg.C₂And (5) culturing in an incubator at constant temperature. Human colorectal cancer cell HT-29, culture conditions: using McCoy's 5A culture solution, 10% fetal bovine serum, 5% CO at 37 deg.C₂And (5) culturing in an incubator at constant temperature.

1.2 drug formulation

Preparing a medicament: weighing appropriate amount of positive drug LPS and tested drug panaxan GSP-2, dissolving with PBS solution to obtain final concentration of 2mg/mL, subpackaging, and storing at 4 deg.C. LPS and panaxan are diluted to required drug concentration for relevant experiments.

1.3 the CCK-8 method is used for detecting the cell survival rate.

Each well-grown cell was resuspended in a culture medium containing 10% fetal bovine serum, and the cell concentration was adjusted to 4X 10⁴Per mL, 100. mu.L of cell suspension was added to each well, and 5% CO was added at 37 ℃₂Culturing in an incubator for 24 h. The total culture medium in each well was changed to 100. mu.L of culture medium containing different concentrations of panaxan (0. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL). The cells added with the ginseng polysaccharide were incubated in a cell incubator for 48 h. After 48h, 10. mu.L of CCK-8 solution was added to each well by a shotgun. After addition of CCK-8, incubate for 4h in the incubator. Before reading, shake for 1min to evenly shake the color of the liquid to prevent bubbles from affecting the microplate reader readings. Absorbance at 450nm was measured using a microplate reader. Calculating the cell survival rate and inhibition rate under the action of the medicine:

survival rate ═ OD (addition group)/OD (blank group)) × 100%;

inhibition rate ═ 1-OD (addition group)/OD (blank group)) × 100%. IC for calculating drug effect₅₀Values were plotted as a function of drug, inhibition and drug action concentration using GraphPad Prism.

The cytotoxic effect of the ginseng polysaccharide GSP-2 on HCT-116, HCT-8, HT-29, SW48 and Caco2 cells is examined, and the action time of the ginseng polysaccharide GSP-2 is 48 h. The results showed that the panaxan GSP-2 had little cytotoxic effect on Caco2 and HCT-8, and showed growth inhibitory effect on HCT-116, HT-29 and SW48, with HCT-116 cells being most sensitive and HCT-116 being most cytotoxic at the maximum concentration of 400 mg/ml. The maximum drug concentration was about 40% on the cell, and the cytotoxicity was increased with the drug concentration, and the survival rate of each cell under the effect of different drug concentrations is shown in fig. 9.

FIG. 9 shows the cell proliferation inhibitory effect of GSP-2 on 5 colorectal cancer cells at 48 h. FIG. A, B, C, D, E, histogram of survival of HCT-116, HT-29, SW48, Caco2 and HCT-8 cells treated with LPS (50. mu.g/mL), vs Blank groups, NS, No signalicicane, P <0.05, P <0.01, P < 0.001.

Example 6 Nuclear magnetic characterization of the Panax ginseng polysaccharide GSP-2

Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker DRX-500NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) using D₂O is used as a solvent. Respectively at 128k and 32k¹H-NMR and¹³C-NMR spectrum.

As shown in fig. 10, there are 8 signals at δ 5.27ppm, δ 5.18ppm, δ 5.02ppm, δ 4.91ppm, δ 4.79ppm, δ 4.71ppm, δ 4.62.ppm, δ 4.61ppm, representing that GSP-2 has 8 monosaccharide species, which is consistent with the monosaccharide composition results; 5.0ppm is the critical value of proton signal for differentiating pyranose configuration, and if the proton shift on the first hydrogen of the sample is more than 5.0ppm, it is delta 0-type glycoside and less than beta-type glycoside, so GSP-2 is presumed to be alpha-type glycoside configuration. At 4.70ppm¹H signal belongs to D₂And O. In that¹The H proton region, where the chemical is transferred from 3.30ppm to 4.41ppm, is assigned to the proton of carbon-2 to the proton of carbon-6 of the sugar ring, and these signals are typical of the peak polysaccharide, 1.Signals around 98ppm indicate that GSP-2 has been acetylated, signals at 1.07 and 1.20ppm indicate the methyl group of rhamnose, which is consistent with the infrared results.

As shown in FIG. 11, under nuclear magnetism¹³In the C spectrum, the monosaccharide class and certain specific groups can be determined based on some carbon signals that appear at specific positions. In that¹³C NMR isofield regions, signals from 107.0 to 109.3ppm were assigned to Araf, and signals from 102.7 to 104.5ppm were assigned to Galp and Glcp. 16.10ppm → 2,4) - α -Rhap- (1 → ligation; 76.15ppm indicates a c-4 substitution, which is → 4) - α -GalpA- (1 → ligation; 78.69ppm indicates a c3 substitution, → 5) - α -Araf- (1 → ligation. The results show that GSP-2 is → 4) - α -GalpA- (1 → and → 5) - α -Araf- (1 → link and → 2,4) - α -Rhap- (1 → link, consistent with the monosaccharide composition results.

The results show that GSP-0, GSP-1, GSP-2 and GSP-3 have immune stimulation to RAW264.7 cells in a certain concentration range. Polysaccharides are reported to have immunological activity, which is related to their monosaccharide composition, molecular weight, triple helix structure or uronic acid content, and the molar ratio of GluA of GSP-2 in GSP-0, GSP-1, GSP-2, GSP-3 is 2.59: 0.39: 1.45:1.30, uronic acid, glucose, mannose receptor, abundant monosaccharide components, plays a key important role in macrophage activation. The literature reports show that the polysaccharide with relatively low or medium molecular weight and high uronic acid content has high immunoregulatory activity. By comparison of three experiments, the immunological activity of GSP-2 was the best. GSP-2 has a minimum molecular weight of 1.76 × 10 in four components⁵Da, which may be due to its smaller molecular weight. GSP-2 has triple-helix structure, which is beneficial to improving bioactivity. It is speculated that GSP is immunologically active, and one of the reasons for this may be that it has a triple helix structure. The ginseng polysaccharide GSP-2 shows growth inhibition on HCT-116, HT-29 and SW48, and is effective on three kinds of anti-tumor cells, of which HCT-116 cells are most sensitive and are presumed to be related to triple helix structure, uronic acid and molecular weight. In addition, the monosaccharide linkage pattern and higher configuration of polysaccharides also affect the immunomodulatory and antitumor activities of polysaccharides, and GSP-2 is an α -glycosidically linked polysaccharide consisting of → 4) - α -GalpA- (1 → and → 5) - α -Araf- (1 → link and → 2,4) - αRhap- (1 → ligation, therefore, its structure-function relationship is to be further studied.

To summarize:

in the invention, four components of polysaccharides GSP-0, GSP-1, GSP-2 and GSP-3 are obtained by microwave extraction, cotton-shaped DEAE cellulose purification and DEAE-52 column chromatography separation. When the structure-activity relationship between the structure of the ginseng polysaccharide and the immunity and the anti-tumor activity is researched, the influence of factors such as monosaccharide composition, molecular weight, functional groups, the connection mode of glycosidic bonds and the like on the immunity and the anti-tumor activity is comprehensively considered, and the fact that the ginseng polysaccharide has the spatial structure of a triple helix structure is proved for the first time, so that the immunity and the anti-tumor activity of the ginseng polysaccharide are greatly improved. The connection sequence of each glycosidic bond, the configuration of the branched chain and the like are studied next, and the structure, the biological activity and the structure-activity relationship of the panaxan are further studied deeply, so that theoretical support is provided for promoting the discovery and research of new panaxan drugs.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.

Claims

1. A method for separating ginseng monomer polysaccharide with immunological activity and anti-tumor effect comprises the following steps:

(1) extracting ginseng polysaccharide with water as extractant by microwave extraction method to obtain ginseng crude polysaccharide, and measuring the content and extraction rate of the ginseng crude polysaccharide by phenol-sulfuric acid method after extraction;

(2) purifying the crude ginseng polysaccharide by adopting pretreated cotton-shaped DEAE cellulose to obtain purified ginseng polysaccharide;

(3) separating purified ginseng polysaccharide with DEAE-52 cellulose, sequentially eluting with distilled water, 0.1mol/L, 0.2mol/L, and 0.3mol/L NaCl aqueous solution at flow rate of 1mL/min to obtain ginseng monomer polysaccharide GSP-2.

2. The method of claim 1, wherein: the microwave extraction method comprises the following specific steps: taking 5g of ginseng crude polysaccharide powder, and performing microwave extraction at the temperature of 70 ℃, wherein the material-liquid ratio is 1: 30mg/ml, 550W extraction power, 6min extraction time, concentration after extraction, and drying to obtain ginseng crude polysaccharide.

3. The method of claim 1, wherein: the DEAE cellulose pretreatment comprises the following steps: firstly, soaking DEAE cellulose dry powder in distilled water, soaking in 0.5mol/L HCl aqueous solution for 1-2 h, then washing with distilled water until the pH value of the filtrate is neutral, washing the drained DEAE cellulose in 0.5mol/L NaOH solution for 1-2 h, then washing the pH value of the filtrate to be neutral with distilled water, and then filling the filtrate into a column;

the ginseng polysaccharide is purified by cotton-shaped DEAE cellulose: the sample loading concentration is 10.0mg/mL, the sample loading volume is 50mL, the flow rate is 1.0mL/min, and the eluent is distilled water and 0.3mol/L NaCl aqueous solution; eluting with distilled water to obtain neutral sugar fraction, and eluting with 0.3mol/L NaCl water solution to obtain acidic sugar fraction; collecting the eluent after passing through the column, and respectively carrying out rotary evaporation and concentration; and then putting the polysaccharide solution into a dialysis bag, dialyzing with distilled water for 24h, changing water during dialysis, concentrating the polysaccharide solution, and freeze-drying to obtain the purified ginseng polysaccharide.

4. The method of claim 3, wherein:

the DEAE-52 cellulose is used for separating the purified ginseng polysaccharide, and the specific steps are as follows: weighing the purified panaxan of neutral sugar fraction and acidic sugar fraction, dissolving with 5mL distilled water, and slowly adding along DEAE-52 cellulose column wall during loading to prevent uneven loading caused by filler flying; eluting neutral sugar fraction with distilled water; sequentially eluting the acidic sugar fraction with 0.1mol/L, 0.2mol/L and 0.3mol/L NaCl aqueous solution at the flow rate of 1mL/min, and collecting by bottles; eluting with 0.2mol/L NaCl water solution, and collecting monomer polysaccharide GSP-2.

5. The method of claim 4The method is characterized in that: the average molecular weight of the GSP-2 is 1.76 multiplied by 10⁵Da, in terms of molar ratios, mannose in GSP-2: ribose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose 0.56: 0.62: 1:1.45:0.67:2.31:2.46:3.30.

6. Use of GSP-2 obtained by the method of claim 4, characterized in that: the composition is used for preparing a medicament for treating human colorectal cancer, wherein the human colorectal cancer is any one or more of cancers corresponding to HCT-116, HT-29 and SW48 cells.

7. Use of GSP-2 obtained by the method of claim 6, characterized in that: is used for preparing a medicament for treating human colorectal cancer, which is cancer corresponding to three cells of HCT-116, HT-29 and SW 48.

8. Use according to claim 6, characterized in that: the cancer is colon cancer corresponding to HCT-116 cells.

9. A method for characterizing monomeric polysaccharides of ginseng having immunological and antitumor activities, which are obtained by the method of any one of claims 1 to 6, comprising the steps of:

(1) and (3) ultraviolet absorption spectrum detection: dissolving the separated ginseng monomer polysaccharide in distilled water, scanning by using an ultraviolet spectrophotometer within the range of 200-800nm, and recording an ultraviolet absorption spectrum so as to detect whether the ginseng monomer polysaccharide has protein and nucleic acid;

(2) infrared spectrum detection:

precisely weighing purified and separated ginseng monomer polysaccharide and dried potassium bromide, mixing, grinding with agate mortar, tabletting, and measuring to 4000cm in infrared spectrometer^-1～400cm^-1Scanning, namely measuring the absorption peak of the sample, and sweeping away air peak polysaccharide before scanning the sample so as to measure the functional group and glycosidic bond mode of ginseng monomer polysaccharide;

accurately weighing 2-3 mg of separated ginseng monomer polysaccharide sample, placing the ginseng monomer polysaccharide sample in a reaction kettle, adding 2mL of 2mol/L trifluoroacetic acid aqueous solution, sealing the reaction kettle, placing the reaction kettle in a 110 ℃ oven for hydrolysis for 5 hours, taking supernatant, evaporating to dryness, repeatedly washing with methanol for 3 times, and evaporating to dryness to obtain a hydrolysate; adding 0.2mL of 0.5mol/L methanol solution of 1-phenyl-3-methyl-5-pyrazolone and 0.2mL of 0.3mol/L NaOH aqueous solution into a water bath at 70 ℃ for heating for 60min, cooling, adding 0.2mL of 0.3mol/L HCl aqueous solution for neutralizing, adding 1mL of chloroform for extraction, centrifuging to remove supernatant, repeating for 3 times, filtering through a 0.45 mu L filter membrane to obtain a derivatized solution, filtering, performing high performance liquid chromatography analysis, and calculating the amount of each monosaccharide;

(4) measuring the molecular weight of ginseng monomer polysaccharide by adopting a high-efficiency liquid size exclusion method and a differential refraction detector under the following conditions;

collecting 20 μ L of 2.0mg/mL separated ginseng monomer polysaccharide sample water solution, filtering with 0.45 μm filter, and injecting into 8.0mm × 300mm²Feeding a sample into the Shodex Sugar KS-804 column under the chromatographic condition, analyzing, recording the peak area, and substituting into a regression equation to calculate the relative molecular mass of each component; the standard curve linear regression equation is:

log(Mw)＝13.87440-5.01843x+0.96087x²-0.07993x³

mw weight-average molecular weight x: retention time

(5) Analyzing ginseng polysaccharide by adopting a Congo red experiment:

10. The characterization method of claim 9, wherein: the conditions of the high performance liquid chromatography analysis are as follows: the separation column is an ODS 2C 18 column, the mobile phase is acetonitrile and 0.05mol/mL phosphate buffer with pH of 6.8, wherein the volume ratio of the acetonitrile to the phosphate buffer is 18:82, the flow rate: 0.8mL/min, column temperature: 30 ℃ and an ultraviolet detector, wherein the detection wavelength is 245nm, and the sample injection amount is 20 mu L.