CN112794923B - Ligusticum wallichii polysaccharide and preparation method, identification method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines and health-care foods, and particularly relates to ligusticum wallichii polysaccharide as well as a preparation method, an identification method and application thereof. The molecular weight range of the ligusticum wallichii polysaccharide provided by the invention is 1000-100000Da. The preparation method is simple, the reaction conditions are mild, and large-scale production can be realized; the invention identifies the chemical structure of the obtained high-purity ligusticum wallichii polysaccharide, defines the structure of the ligusticum wallichii polysaccharide and provides a structural basis for exploring the pharmacological activity mechanism of the ligusticum wallichii polysaccharide. Meanwhile, the pure product of the ligusticum wallichii polysaccharide obtained by the invention lays a foundation for ligusticum wallichii polysaccharide medicines, health-care foods and functional foods, as well as quality control and deep research on structure-activity relationship and action mechanism of the ligusticum wallichii polysaccharide medicines.

Description

Ligusticum wallichii polysaccharide and preparation method, identification method and application thereof

Technical Field

The invention belongs to the technical field of medicines and health-care foods, and particularly relates to ligusticum wallichii polysaccharide as well as a preparation method, an identification method and application thereof.

Background

The immune organs, immune cells and the like of the human body can protect the organism from being invaded by the outside and protect the health of the human body. These immune structures can effectively enhance immune cell activity, promote cytokine secretion of antibodies, and activate complement cells for immune regulation. The role of polysaccharides in immunomodulation is mainly 2, non-specific and specific. Wherein the specific immunity comprises cellular immunity and humoral immunity.

Polysaccharides, also known as polysaccharides, are a class of biological information macromolecules that are widely found in animals, plants, and microorganisms. The related research on polysaccharide has been in the past 70 years, especially in recent years, glycobiology becomes another research hotspot and difficulty after genomics and proteomics, and research has shown that polysaccharide has a plurality of biological activities such as immunoregulation, anti-inflammation, anti-tumor, blood sugar reduction, blood fat reduction and the like, wherein the immunoregulation function is one of the most important functions of polysaccharide substances. China also obtains a lot of achievements in the research, development and utilization aspects of traditional Chinese medicine polysaccharide in recent years, traditional Chinese medicine polysaccharide products such as ganoderma lucidum polysaccharide, astragalus polysaccharide, ginseng polysaccharide and the like are put on the market through analysis and identification, and polysaccharide health care products after deep processing also appear successively. Although the research on the immunoregulation effect of the traditional Chinese medicine polysaccharide is more, the research is not deep enough, and the research on the structure-activity relationship of the traditional Chinese medicine polysaccharide is less.

The rhizoma Ligustici Chuanxiong polysaccharide is one of dried rhizome of Ligusticum chuanxiong (Ligusticum chuanxiong Hort.) belonging to Umbelliferae. It is pungent in flavor and warm in nature, and has effects of promoting blood circulation, activating qi-flowing, dispelling pathogenic wind and relieving pain. The traditional Chinese medicine composition is clinically used for dispelling wind and eliminating dampness, dispelling exogenous wind, enriching and activating blood, promoting qi and activating blood and the like. The chemical components of the hemlock parsley mainly comprise volatile oil, alkaloid, phenolic substances, organic acid, phthalide lactone, polysaccharide and other components. Meanwhile, modern pharmacological research shows that the polysaccharide is one of the main active ingredients of the ligusticum wallichii, has wide pharmacological action and has various activities such as myocardial protection, vasodilation, anti-inflammation, anti-oxidation, anti-tumor and the like. At present, the research on the ligusticum wallichii polysaccharide is mainly focused on the research on the extraction process and the antioxidant activity of the ligusticum wallichii polysaccharide, the research on the structure of the ligusticum wallichii polysaccharide is not deep enough, and the reports on the immunoregulation action are less, so that a method for extraction, purification and structure identification is necessarily provided, and a foundation is laid for the quality control of the ligusticum wallichii polysaccharide and the deep research on the structure-effect relationship and action mechanism of the ligusticum wallichii polysaccharide.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides ligusticum wallichii polysaccharide and a preparation method, an identification method and application thereof. The preparation method is simple, the reaction conditions are mild, and large-scale production can be realized; the invention identifies the chemical structure of the obtained high-purity ligusticum wallichii polysaccharide, defines the components of the ligusticum wallichii polysaccharide and provides a structural basis for exploring the pharmacological activity mechanism of the ligusticum wallichii polysaccharide. Meanwhile, the pure product of the ligusticum wallichii polysaccharide obtained by the invention lays a foundation for the ligusticum wallichii polysaccharide medicine, quality control and deep research on the structure-activity relationship and action mechanism of the ligusticum wallichii polysaccharide medicine.

The invention provides a ligusticum wallichii polysaccharide LC2-1-2A. The inventor obtains the chuanxiong polysaccharide LC2-1-2A by purifying and separating crude chuanxiong polysaccharide, and obtains the molecular weight range of 1000-100000Da by structural analysis.

Further, the structure of the chuanxiong polysaccharide LC2-1-2A is shown in figure 8, wherein x + y is more than or equal to 1 and less than or equal to 10.

Meanwhile, the invention also provides a preparation method of the ligusticum wallichii polysaccharide, which comprises the following steps:

s1, shearing: cutting rhizoma Ligustici Chuanxiong dry root into small segments, cleaning with water, and air drying to obtain rhizoma Ligustici Chuanxiong segments;

s2, water extraction: adding the ligusticum wallichii segments obtained in the step S1 into water, heating and extracting, and filtering to obtain an extracting solution and medicine residues;

s3, grading alcohol precipitation:

concentrating the extracting solution obtained in the step S2 under reduced pressure to obtain a concentrated solution 1; adding ethanol into the concentrated solution 1 until the volume concentration of the ethanol is a%, standing, and collecting precipitate and supernatant to obtain crude polysaccharide LC1 and supernatant 1;

concentrating the supernatant 1 under reduced pressure to obtain a concentrated solution 2; adding ethanol into the concentrated solution 2 until the volume concentration of the ethanol is b%, standing, and collecting precipitate and supernatant to obtain crude polysaccharide LC2 and supernatant 2;

concentrating the supernatant 2 under reduced pressure to obtain a concentrated solution 3; adding ethanol into the concentrated solution 3 until the volume concentration of the ethanol is c%, standing, and collecting precipitate to obtain crude polysaccharide LC3;

wherein a is more than or equal to 10 and more than b and more than c and less than 100;

s4, purification:

primary purification:

removing protein from the crude polysaccharide LC2 obtained in the step S3, dialyzing, and freeze-drying to obtain ligusticum wallichii polysaccharide;

and (3) secondary purification:

performing ion exchange column chromatography on the polysaccharide LC2 subjected to primary purification, performing gradient elution by using 0-2M NaCl, tracking an elution curve by using a phenol-sulfuric acid method, collecting sugar parts according to the elution curve respectively, concentrating, and freeze-drying; dissolving with water, centrifuging, and collecting supernatant;

and performing molecular sieve gel column chromatography on the supernatant, eluting with water, detecting an elution curve by using a phenol-sulfuric acid method, collecting a sugar part according to the elution curve, concentrating, and freeze-drying to obtain LC2-1-2A.

The inventor finds that the preferable length of the ligusticum wallichii section is 1-8 cm through research; meanwhile, the invention combines water extraction and a fractional alcohol precipitation method, the alcohol concentration is subjected to fractional alcohol precipitation from low to high, the ligusticum wallichii polysaccharide is primarily separated, and the polysaccharide with high polarity and good water solubility can be separated from the polysaccharide with low polarity and poor water solubility by high-concentration alcohol, so that the problem of complex and difficult separation in the later stage caused by extracting the polysaccharide by the traditional water boiling method is solved.

Further, the adding amount of water in the step S2 is 5-15 times of the weight of the ligusticum wallichii section, the heating temperature is 60-100 ℃, and the extraction time is 1-10 h.

Further, the reduced pressure concentration temperature in the step S3 is 40-70 ℃, and the standing time is 10-28 h.

In addition, the invention also provides an identification method of the ligusticum wallichii polysaccharide, which comprises the following steps:

(1) Completely hydrolyzing a ligusticum wallichii polysaccharide sample with acid, performing liquid chromatography detection after a hydrolysate PMP derivatization;

(2) Taking a ligusticum wallichii polysaccharide sample, performing complete acid hydrolysis, and performing D/L configuration analysis on hydrolysate derivatization;

(3) Taking a ligusticum polysaccharide sample, drying, tabletting and detecting by infrared spectroscopy;

(4) Taking a ligusticum wallichii polysaccharide sample, carrying out methylation, hydrolysis, reduction and acetylation, and carrying out GC-MS detection;

(5) Dissolving rhizoma Ligustici Chuanxiong polysaccharide sample in D ₂ And O, performing nuclear magnetic resonance analysis.

In order to further develop the valuable resource of the ligusticum wallichii, the inventor discovers a new medicine source and obtains the invention through a large number of experimental researches: the method comprises the steps of taking dry roots of ligusticum wallichii as a raw material, separating by a water extraction and alcohol precipitation method to obtain crude polysaccharide, deproteinizing the extracted crude polysaccharide, purifying the crude polysaccharide of ligusticum wallichii by ion exchange chromatography and a gel molecular sieve column chromatography method, and preparing a purified product of the ligusticum wallichii polysaccharide for the first time, and carrying out systematic analysis and identification on physicochemical properties, molecular weight, monosaccharide composition and the like of the purified product of the polysaccharide to successfully obtain the structural information of the ligusticum wallichii polysaccharide, wherein the ligusticum wallichii polysaccharide LC2-1-2A is araban arabinoside, a main chain of the ligusticum wallichii polysaccharide consists of → 5) -alpha-L-Araf- (1 → → 2,3,5) -alpha-L-Araf- (1 → and → 3,5) -alpha-L-Araf- (1 → and a branch chain of the ligusticum wallichii polysaccharide consists of the alpha-L-Araf- (1 →.

The invention also relates to the application of the chuanxiong rhizome polysaccharide in preparing immunoregulation medicines or health products. And the immunoregulatory activity of the compound is evaluated through cell experiments and zebra fish experimental researches.

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

1. the invention adopts a water extraction and alcohol precipitation method to carry out preliminary separation on the ligusticum wallichii polysaccharide, the effect is obvious, and the preparation method is simple, the reaction condition is mild, and the large-scale production can be carried out;

2. the invention carries out secondary separation and purification on crude polysaccharide of ligusticum wallichii by column chromatography, has obvious effect and prepares a pure ligusticum wallichii polysaccharide for the first time;

3. the invention identifies the structure of the purified polysaccharide pure product, defines the physicochemical properties and the structures of all polysaccharide components, and provides a structural basis for exploring the pharmacological activity mechanism of the polysaccharide;

4. the pure product of the ligusticum wallichii polysaccharide obtained by the invention has the advantages of well-preserved components, definite structure and controllable quality, can enhance the phagocytic capacity of RAW264.7 macrophages, promote the release of molecules such as Nitric Oxide (NO), interleukin (IL) -6, IL-1 beta, tumor Necrosis Factor (TNF) -alpha and inflammatory factors, simultaneously improve the release of molecules such as NO and active oxygen (ROS) in zebra fish embryos, and provide a basis for the application of the ligusticum wallichii polysaccharide in the fields of medicines, health care products and the like.

5. Meanwhile, the invention lays a foundation for the medicine and quality control of the ligusticum wallichii polysaccharide and the deep research of the structure-activity relationship and the action mechanism of the ligusticum wallichii polysaccharide.

Drawings

FIG. 1 is a high performance liquid chromatogram of the LC2-1-2A monosaccharide composition;

FIG. 2 is a high performance liquid chromatogram of the D/L structure of LC2-1-2A monosaccharide;

FIG. 3 is an IR spectrum of LC2-1-2A;

FIG. 4 shows LC2-1-2A ¹ H NMR spectrum;

FIG. 5 shows LC2-1-2A ¹³ A C NMR spectrum;

FIG. 6 is HSQC spectrum of LC2-1-2A;

FIG. 7 is an HMBC profile of LC2-1-2A;

FIG. 8 is the primary structure of LC2-1-2A;

FIG. 9 shows the immunomodulatory effects of LC2-1-2A on RAW264.7 cells;

FIG. 10 is a graph of the effect of LC2-1-2A on ROS release in zebrafish embryos;

FIG. 11 is a graph showing the effect of LC2-1-2A on NO release from zebrafish embryos.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to be purely exemplary of the claimed technical solution and are not intended to be limiting. The scope of the present invention is defined by the appended claims.

Example 1 preparation of Ligusticum chuanxiong polysaccharide

The preparation method of the ligusticum wallichii polysaccharide comprises the following steps:

s1, shearing: cutting 10kg of dry root of the ligusticum wallichii into 1-3 cm by using scissors, quickly cleaning by using cold water, and drying in the air to obtain ligusticum wallichii sections;

s2, water extraction: adding water with the weight being 10 times of that of the ligusticum wallichii segments obtained in the step S1, heating to 80 ℃, extracting for 3 hours, collecting an extracting solution, and airing dregs of a decoction to obtain an extracting solution and dregs of a decoction;

s3, grading alcohol precipitation:

concentrating the extracting solution obtained in the step S2 at 60 ℃ under reduced pressure, adding ethanol until the volume concentration of the ethanol is 50%, standing at room temperature for 24h, centrifuging, and collecting precipitate and supernatant to obtain crude polysaccharide LC1 and supernatant 1;

concentrating the supernatant 1 at 60 deg.C under reduced pressure, adding ethanol until ethanol volume concentration is 70%, standing at room temperature for 24 hr, centrifuging, and collecting precipitate and supernatant to obtain crude polysaccharide LC2 and supernatant 2;

concentrating the supernatant 2 at 60 deg.C under reduced pressure, adding ethanol until the volume concentration of ethanol is 90%, standing at room temperature for 24 hr, centrifuging, and collecting precipitate and supernatant to obtain crude polysaccharide LC3;

s4, purification: and (4) removing proteins of the LC2 crude polysaccharide obtained in the step (S3) by using a Sevag method, dialyzing the crude polysaccharide after removing the proteins by using a dialysis bag (the molecular weight cutoff is 3000 Da), and freeze-drying to obtain the ligusticum wallichii polysaccharide LC2.

Example 2 Ligusticum chuanxiong polysaccharide LC2-1-2A

The chuanxiong polysaccharide LC2-1-2A is obtained by secondary purification of chuanxiong polysaccharide LC2 obtained in example 1, and the specific method is as follows:

1) Ion exchange column chromatography: dissolving 200mg of ligusticum wallichii polysaccharide LC2 obtained in example 1 in 5mL of deionized water, loading the mixture on a DEAE-FF column, and generating 2 peaks under the conditions of eluents with different salt concentrations, wherein the elution peak is a 0.1M NaCl elution part (a phenol-sulfuric acid method is used for tracking an elution curve in an elution process, sugar parts are respectively collected according to the elution curve), and concentrating and freeze-drying the obtained eluents respectively to obtain polysaccharide LC2;

2) Molecular sieve gel chromatography: dissolving the lyophilized polysaccharide sample with water, centrifuging, collecting supernatant, separating with Sephadex G-75 column, eluting with water, tracking elution curve with phenol-sulfuric acid method to obtain a single symmetric peak, collecting main peak, concentrating, and freeze drying to obtain rhizoma Ligustici Chuanxiong polysaccharide LC2-1-2A.

Example 3 structural analysis of Ligusticum chuanxiong polysaccharide LC2-1-2A

Test materials (one): ligusticum wallichii polysaccharide LC2-1-2A.

(II) test method:

1. monosaccharide composition analysis

Sample treatment:

a sample of cnidium officinale polysaccharide (4.0 mg each) was accurately weighed into a stoppered test tube, 2.0mL of 2M trifluoroacetic acid (TFA) was added, and the mixture was put in an oil bath pan and hydrolyzed in an oil bath at 120 ℃ for 6 hours. Cooling to room temperature, repeatedly adding methanol for spin-drying, removing TFA, dissolving with deionized water to 1mL, centrifuging, absorbing 100 μ L of sample solution respectively, adding 100 μ L of 0.3M NaOH solution, adding 100 μ L of 0.5M PMP methanol solution, mixing, reacting at 70 deg.C for 30min, cooling, adding 105 μ L of 0.3M HCL solution for neutralization, adding deionized water to 1mL, adding equal volume of chloroform solution, shaking vigorously, centrifuging, removing chloroform phase, repeating for 2 times, filtering the water phase with 0.45 μ M filter membrane, and analyzing by HPLC sample injection.

Chromatographic conditions are as follows:

a chromatographic column: kromasil 100-5-C18, 4.6X 250mm,5 μm; mobile phase: 0.1M phosphate (pH = 6.9) buffer-acetonitrile (83 v/v; detection wavelength: 250nm; flow rate: 0.8mL/min; sample injection volume: 20 μ L.

2. Monosaccharide D/L conformation analysis

LC2-1-2A (5 mg) was reacted with trifluoroacetic acid (2M, 2mL) at 120 ℃ in an oil bath for 4h. After the reaction is finished, adding equal volume of chloroform for extraction twice, discarding the chloroform layer, and spin-drying the water layer.

After the sample was spun dry, L-cysteine methyl ester hydrochloride (2.5 mg) and anhydrous pyridine (1 mL) were added, and the mixture was reacted in a water bath at 60 ℃ for 1 hour. O-phenyl isothiocyanate (5. Mu.L) was added thereto, and the reaction was continued in a water bath at 60 ℃ for 1 hour. After the reaction, the sample is subjected to liquid phase analysis after passing through a 0.45 mu m microporous filter membrane.

Chromatographic conditions are as follows:

mobile phase: 25% of CH ₃ CN-H ₂ O (0.1% acetic acid); flow rate: 0.8mL/min; sample introduction amount: 10 mu L of the solution; detection wavelength: UV 250nm; detection time: and (5) 60min.

3. Infrared spectroscopy detection

Grinding dried test material 2.0mg with KBr, tabletting, and subjecting to Perkin EImer FT/IR-100 at 4000-400cm ^-1 Scanning is performed over the range.

4. methylation/GC-MS analysis

Weighing 8.0mg of a dried test material into a reaction bottle, adding 8mL of anhydrous DMSO, adding 800mg of dried sodium hydroxide, performing ultrasonic treatment for 30min, adding 3.0mL of iodomethane in a dark place under an ice bath condition, performing ultrasonic treatment for 30min in three times in each ice bath, adding 2mL of distilled water after the reaction is finished to decompose residual iodomethane, adding 1mL of chloroform for extraction, and centrifuging to obtain a chloroform layer.

After the methylation is completed, the sample is put into a test tube with a plug, oil bath is carried out for hydrolysis for 6h at the constant temperature of 120 ℃ by using 2mol/L TFA, reduced pressure is evaporated to dryness, methanol is repeatedly added for spin-drying until the pH is neutral, and then 20mg of NaBH is added ₄ The reaction was carried out at 40 ℃ for 30min to reduce the hydrolysate. The reaction was stopped with 100. Mu.L of glacial acetic acid and the sample was spun dry under reduced pressureThen 2mL of acetic anhydride and pyridine were added for acetylation. The reaction was stirred magnetically for 2h at 95 ℃. Then methanol was added repeatedly 3 times, spin-dried, dissolved with 1mL chloroform, washed with distilled water of the same volume for 3 times, the aqueous layer was removed, finally the chloroform layer was dried with anhydrous sodium sulfate, filtered to remove the sodium sulfate solid, concentrated under reduced pressure and evaporated to dryness for GC-MS analysis.

5. Nuclear magnetic resonance analysis

Freeze drying rhizoma Ligustici Chuanxiong polysaccharide sample LC2-1-2A repeatedly, dissolving 60mg in 0.6mL D ₂ O, placing in a nuclear magnetic tube, and recording by using a 400MHz nuclear magnetic resonance instrument Bruker AV-400 ¹ H NMR， ¹³ C NMR, HSQC, HMBC, etc.

(III) test results:

1. LC2-1-2A structural analysis of rhizoma Ligustici Chuanxiong polysaccharide

(1) Monosaccharide composition analysis

As shown in FIG. 1, LC2-1-2A contained only arabinose as seen from the HPLC profile. ( Chromatographic peak sequence: 1: mannose, 2: rhamnose, 3: glucuronic acid, 4: galacturonic acid, 5: glucose, 6: galactose, 7: xylose, 8: arabinose, 9: fucose sugar )

(2) Monosaccharide D/L conformation analysis

As shown in FIG. 2, the configuration of arabinose in LC2-1-2A was L configuration as seen from HPLC.

(2) Infrared spectroscopic analysis

As shown in FIG. 3, the LC2-1-2A infrared spectrum shows that the Chuan-xiong rhizome LC2-1-2A contains the characteristic infrared absorption peak of polysaccharide.

(3) methylation/GC-MS analysis

After the methylation analysis of LC2-1-2A, hydrolysis, reduction acetylation and GC-MS detection, the GC-MS spectrum shows that LC2-1-2A contains → 5) -alpha-L-Araf- (1 →, → 2,3,5) -alpha-L-Araf- (1 →, → 3,5) -alpha-L-Araf- (1 → and alpha-L-Araf- (1 → iso-sugar residues.

(4) Nuclear magnetic resonance analysis

This test passes ¹ H NMR、 ¹³ C NMR and HSQC were assigned to the chemical shifts of the carbon and hydrogen atoms of the sugar residue of LC2-1-2A and then confirmed with HMBCThe connection order is recognized. FIGS. 4-7 are LC2-1-2A ¹ H NMR、 ¹³ C NMR, HSQC and HMBC spectra.

The LC2-1-2A hydrocarbon assignments are shown in Table 1 below, based on the NMR spectra of FIGS. 4-7.

TABLE 1 LC2-1-2A NMR analysis results

^a Unresolved from other signals，nd：no detected.

In conclusion: LC2-1-2A is an arabinan composed of arabinose, which contains → 5) -alpha-L-Araf- (1 →, → 2,3,5) -alpha-L-Araf- (1 →, → 3,5) -alpha-L-Araf- (1 → and alpha-L-Araf- (1 → isosaccharide residues, from methylation analysis, the order of linkage between the different saccharide residues is derived from two-dimensional nuclear magnetic HMBC spectrum analysis, and the structure of LC2-1-2A derived from the above analysis is shown in FIG. 8, wherein 1. Ltoreq. X + y. Ltoreq.10.

Example 4 study of immunomodulatory effects of pure Ligusticum chuanxiong polysaccharide on macrophage

Test materials (one): ligusticum wallichii polysaccharide LC2-1-2A.

(II) test subjects: RAW264.7 cells (supplied by shanghai cell bank, chinese academy of sciences).

(III) test method:

1. experiment design and grouping:

the invention adopts a macrophage RAW264.7 model, and is provided with a blank group, a positive medicine group and a ligusticum wallichii polysaccharide administration group, wherein the concentration of the ligusticum wallichii polysaccharide LC2-1-2A is 50 mug/mL, 100 mug/mL and 200 mug/mL, the positive medicine is Lipopolysaccharide (LPS), and the concentration is 1 mug/mL.

2. Mother liquor preparation and cell counting

Dissolving LC2-1-2A20mg in PBS buffer salt 1ml, filtering the prepared mother liquor with 0.22 μm filter membrane, sterilizing by ultraviolet irradiation for 30min, and placing in refrigerator at-20 deg.C.

The flask was placed in a clean bench, the old medium was removed, and 5mL of PBS buffer was added to wash the cellsAfter the cells are washed, PBS buffer solution is removed, 600 mu L of trypsin is added into a culture flask, RAW264.7 cells are digested for 1min, then DMEM culture medium is added, and the cells are repeatedly blown to form uniform single cell suspension. Adding 20 μ L of the single cell suspension into 180 μ L DMEM medium, repeatedly blowing and beating, counting 10 μ L in cell counting plate, diluting the cells to 5 × 10 ⁴ Per mL, plating.

3. Administration of drugs

Inoculating RAW264.7 cells into a 96-well plate, culturing for 24h, adding a sample to be tested, respectively administering LC2-1-2A at 50 μ g/mL,100 μ g/mL and 200 μ g/mL, and placing RAW264.7 cells after administration in CO ₂ Culturing in an incubator for 24h.

4.MTT assay

The culture medium in the 96-well plate was removed, and 20. Mu.L of 5mg/mL thiazole blue (MTT) solution was added to each well in CO ₂ Culturing in an incubator for 4h. After 4h, the MTT solution was removed from the 96-well plate, and 150 μ L of dmso (analytical grade) was added to each well to dissolve formazan crystals, and absorbance (OD value) at 492nm was measured using a microplate reader. The proliferation promoting effect of the sample on RAW264.7 cells is calculated, and the experiment is repeated three times.

5. Neutral Red phagocytosis assay

RAW264.7 cells were treated by LC2-1-2A administration at different concentrations, washed three times with PBS, incubated for 1h with 100. Mu.L of neutral red solution (0.1%), the neutral red solution was discarded, washed three times with PBS, blotted, 150. Mu.L of cell lysate (glacial acetic acid: ethanol = 1:1) was added, incubated for 1h at room temperature, OD was measured at 540 nm with microplate reader, and the experiment was repeated three times.

NO Release test

RAW264.7 cells were seeded in 96-well plates and treated with different concentrations of LC2-1-2A (50. Mu.g/mL, 100. Mu.g/mL and 200. Mu.g/mL) and 1. Mu.g/mL LPS for 4h, after which the supernatants were collected and reacted with Griess reagent and OD was measured at 550nm with a microplate reader.

Effect of LC2-1-2A on RAW264.7 cytokines

RAW264.7 cells were inoculated into 24-well plates and incubated for 4h, LC2-1-2A (50. Mu.g/mL, 100. Mu.g/mL and 200. Mu.g/mL) and 1. Mu.g/mL LPS were treated for 24h, and then supernatants were collected and assayed for inflammatory factors such as TNF-. Alpha., IL-6 and IL-1. Beta. Using an ELISA kit.

(IV) results of the experiment

As shown in FIG. 9, after treatment with different concentrations of Chuan Xiong polysaccharide LC2-1-2A, the activity of RAW264.7 cells was not significantly different (P > 0.05) compared with the blank group, indicating that Chuan Xiong polysaccharide LC2-1-2A is safe and non-cytotoxic at 50 μ g/mL-200 μ g/mL. Secondly, after the RAW264.7 cells are treated by LC2-1-2A with different concentrations, the phagocytic activity of the cells is remarkably increased (P <0.05 or P < 0.01) and the expression level of inflammatory factors such as NO, TNF-alpha, IL-6, IL-1 beta and the like is remarkably increased (P <0.05, P < -0.01 or P < 0.001), which indicates that the chuanxiongpolysaccharide LC2-1-2A can enhance the immune regulation capability of organisms by improving the phagocytic capability of macrophages of the organisms, increasing the release of NO and promoting the release of inflammatory factors such as TNF-alpha, IL-6, IL-1 beta and the like.

Example 5 research on immunoregulation of chuanxiong polysaccharide pure product LC2-1-2A on zebra fish embryo (I) experimental method

1. Zebra fish embryo collection and culture

Controlling the day and night rhythm of adult zebra fish, controlling the day and night time to be 14h, collecting fertilized eggs of the zebra fish, placing the fertilized eggs into a culture dish, adding a culture medium (0.2% of instant sea salt is dissolved in deionized water), and placing the fertilized eggs into a constant-temperature incubator for culture, wherein the culture temperature is 28.5 ℃.

2. Design and grouping of experiments

The invention adopts a zebra fish animal model to establish a fast and efficient immunocompetence screening model. The fertilized zebrafish embryos after 7-8 hours were placed in 12-well plates with 6 zebrafish larvae per well and incubated with different concentrations of LC2-1-2A (50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL) at 28.5 ℃ for 24h. After 24h, the culture medium containing LCP70-2A is removed, the regular fresh culture medium is replaced, and the culture is continued until 72h after the fertilization of the zebra fish juvenile fish, wherein the zebra fish is not fed in the culture process.

3. Zebra fish in-vivo fluorescence imaging observation

(1) Reactive Oxygen Species (ROS) release assay

The zebra fish juvenile fish are cultured according to the method until 72h after fertilization, and an ROS fluorescent probe DCF-DA (20 mug/mL) is added into the culture medium and the culture is continued for 1h in the dark. After the incubation was completed, the zebra fish larvae were repeatedly washed three times with water, and then anesthetized with a 0.02% tricaine solution and fixed with 3% methylcellulose. And observing the relative fluorescence intensity in the zebra fish juvenile fish under a laser confocal microscope and taking a picture. And (3) carrying out quantitative analysis on the relative fluorescence intensity in the zebra fish juvenile fish by using Image J software so as to detect the influence of LC2-1-2A on the ROS release amount in the zebra fish juvenile fish.

(2) Nitric Oxide (NO) release assay

The zebra fish juvenile fish were cultured as described above until 72h after fertilization, and the NO fluorescent probe DAF-FMDA (5. Mu.M) was added to the medium and incubation continued for 2h in the dark. After the incubation was completed, the zebrafish juvenile fish were repeatedly washed with water three times, followed by anesthetizing the zebrafish juvenile fish with a 0.02% tricaine solution and fixing with 3% methylcellulose. And observing the relative fluorescence intensity in the zebra fish juvenile fish under a laser confocal microscope and photographing. And (3) carrying out quantitative analysis on the relative fluorescence intensity in the zebra fish juvenile fish by using Image J software so as to detect the influence of LC2-1-2A on the NO release amount in the zebra fish juvenile fish.

(II) results of the experiment

As shown in fig. 10 and fig. 11, as the concentration of LC2-1-2A is increased (50 μ g/mL,100 μ g/mL,200 μ g/mL) compared to the control group, the fluorescence intensity of ROS in zebrafish is 1.59 times, 2.28 times and 3.16 times that of the blank group, and the fluorescence intensity of NO is 1.75 times, 2.41 times and 3.73 times, respectively, which indicates that LC2-1-2A has a significant enhancing effect on the ROS and NO contents in zebrafish larvae, and can enhance the in vivo immunomodulatory activity of zebrafish larvae.

In conclusion, the pure chuanxiong polysaccharide product LC2-1-2A prepared by the invention can enhance the phagocytic capacity of RAW246.7 cells and promote the release of related inflammatory factors, and meanwhile, the LC2-1-2A can enhance the release of ROS and NO in zebra fish juvenile fish, thereby enhancing the immunoregulatory capacity of organisms.

Claims (6)

1. A rhizoma Ligustici Chuanxiong polysaccharide is characterized in that the rhizoma Ligustici Chuanxiong polysaccharide is LC2-1-2A;

the rhizoma Ligustici Chuanxiong polysaccharide LC2-1-2A is composed of arabinose, and its molecular weight range is 1000-100000Da;

the structural formula of LC2-1-2A is:

1≤x+y≤10。

2. the method for preparing cnidium officinale polysaccharide according to claim 1, comprising the steps of:

s1, shearing: cutting rhizoma Ligustici Chuanxiong dry root into small segments, cleaning with water, and air drying to obtain rhizoma Ligustici Chuanxiong segments;

s2, water extraction: adding the ligusticum wallichii segments obtained in the step S1 into water, heating and extracting, and filtering to obtain an extracting solution and dregs;

s3, grading alcohol precipitation:

concentrating the extracting solution obtained in the step S2 under reduced pressure to obtain a concentrated solution 1; adding ethanol into the concentrated solution 1 until the volume concentration of the ethanol is a%, standing, and collecting precipitate and supernatant to obtain crude polysaccharide LC1 and supernatant 1;

concentrating the supernatant 1 under reduced pressure to obtain a concentrated solution 2; adding ethanol into the concentrated solution 2 until the volume concentration of the ethanol is b%, standing, and collecting precipitate and supernatant to obtain crude polysaccharide LC2 and supernatant 2;

concentrating the supernatant 2 under reduced pressure to obtain a concentrated solution 3; adding ethanol into the concentrated solution 3 until the volume concentration of the ethanol is c%, standing, and collecting precipitate to obtain crude polysaccharide LC3;

wherein a is more than or equal to 10 and more than b and more than c and less than 100;

s4, purification:

primary purification:

removing proteins from the crude polysaccharides LC1, LC2 and LC3 obtained in the step S3, dialyzing, and freeze-drying to obtain the ligusticum wallichii polysaccharide;

and (3) secondary purification:

performing ion exchange column chromatography on the polysaccharide LC2 subjected to primary purification, wherein the ion exchange column is a DEAE-FF column, performing gradient elution by using 0-2M NaCl, tracking an elution curve by using a phenol-sulfuric acid method, respectively collecting sugar parts according to the elution curve, concentrating, and freeze-drying; dissolving with water, centrifuging, and collecting supernatant;

and performing molecular sieve gel column chromatography on the supernatant, wherein the molecular sieve gel column is a Sephadex G-75 column, eluting with water, detecting an elution curve by using a phenol-sulfuric acid method, collecting a sugar part according to the elution curve, concentrating, and freeze-drying to obtain LC2-1-2A.

3. The method for preparing Chuanxiong polysaccharide as claimed in claim 2, wherein the amount of water added in step S2 is 5-15 times of the weight of the said Chuanxiong blocks, the heating temperature is 60-100 deg.C, and the extraction time is 1-10 h.

4. The method for preparing Chuanxiong polysaccharide as claimed in claim 2, wherein the concentration under reduced pressure in step S3 is at 40-70 deg.C, and the standing time is 10-28 h.

5. The method for identifying cnidium officinale polysaccharide according to claim 1, comprising the steps of:

(1) Taking a ligusticum wallichii polysaccharide sample, performing complete acid hydrolysis, derivatizing a hydrolysate PMP, and detecting by liquid chromatography;

(2) Completely hydrolyzing a ligusticum wallichii polysaccharide sample with acid, performing D/L configuration detection after derivatization of a hydrolysate;

(3) Taking a ligusticum polysaccharide sample, drying, tabletting and detecting by infrared spectroscopy;

(4) Taking a ligusticum wallichii polysaccharide sample, carrying out methylation, hydrolysis, reduction and acetylation, and carrying out GC-MS detection;

(5) Dissolving rhizoma Ligustici Chuanxiong polysaccharide sample in D ₂ And O, performing nuclear magnetic resonance analysis.

6. The use of the cnidium officinale polysaccharide of claim 1 in preparing a medicament or health product for enhancing immunity.

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