CN112794923A - Ligusticum chuanxiong polysaccharide and its preparation method, identification method and application - Google Patents
Ligusticum chuanxiong polysaccharide and its preparation method, identification method and application Download PDFInfo
- Publication number
- CN112794923A CN112794923A CN202011459453.2A CN202011459453A CN112794923A CN 112794923 A CN112794923 A CN 112794923A CN 202011459453 A CN202011459453 A CN 202011459453A CN 112794923 A CN112794923 A CN 112794923A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- ligusticum chuanxiong
- supernatant
- chuanxiong
- ligusticum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
本发明属于医药及保健食品技术领域，具体涉及一种川芎多糖及其制备方法、鉴定方法和应用。本发明提供的川芎多糖的分子量范围为1000‑100000Da。本发明制备方法简单，反应条件温和，可大规模生产；且本发明对得到的高纯度川芎多糖化学结构进行了鉴定，明确了其结构，为探究其药理活性机制提供结构依据。同时，本发明得到的川芎多糖纯品为川芎多糖药物、保健食品、功能食品，及其质量控制和深入研究其构效关系、作用机制奠定基础。
The invention belongs to the technical field of medicine and health food, and in particular relates to a Ligusticum chuanxiong polysaccharide and a preparation method, identification method and application thereof. The molecular weight range of the Ligusticum chuanxiong polysaccharide provided by the invention is 1000-100000 Da. The preparation method of the invention is simple, the reaction conditions are mild, and the large-scale production is possible; and the chemical structure of the obtained high-purity chuanxiong polysaccharide is identified, the structure is clarified, and the structure basis is provided for exploring its pharmacological activity mechanism. At the same time, the pure Ligusticum chuanxiong polysaccharide obtained by the present invention lays the foundation for Ligusticum chuanxiong polysaccharide medicine, health food, functional food, and its quality control and in-depth study of its structure-activity relationship and action mechanism.
Description
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 bioinformatic 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 plurality 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 components of dried rhizome of Ligusticum chuanxiong (Ligusticum chuanxiong Hort) of Umbelliferae. It is pungent in flavor and warm in nature, enters liver, gallbladder and pericardium channels, 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 of myocardial protection, vasodilation, inflammation resistance, oxidation resistance, tumor resistance 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 LC 2-1-2A. The inventor obtains the chuanxiongpolysaccharide LC2-1-2A by purifying and separating crude chuanxiongpolysaccharide, and obtains the molecular weight range of 1000-100000Da by structural analysis.
Further, the structure of the ligusticum wallichii 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 and alcohol precipitating:
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 LC 3;
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;
subjecting the supernatant to molecular sieve gel column chromatography, eluting with water, detecting elution curve by phenol-sulfuric acid method, collecting sugar part according to the elution curve, concentrating, and freeze drying to obtain LC 2-1-2A.
The inventor finds out through research that the optimal length of the ligusticum wallichii section is 1-8 cm; 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 amount of water added 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 hours.
Further, in the step S3, the reduced pressure concentration temperature 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) taking a ligusticum wallichii polysaccharide sample, performing complete acid hydrolysis, derivatizing a hydrolysate PMP, and detecting by liquid chromatography;
(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 D2And 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, a main chain consists of → 5) -alpha-L-Araf- (1 → 2,3,5) -alpha-L-Araf- (1 → and → 3,5) -alpha-L-Araf- (1 → a branched chain consists of alpha-L-Araf- (1 → a branched chain.
The invention also relates to the application of the ligusticum wallichii polysaccharide in preparing immunoregulation medicaments or health-care 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, the preparation method is simple, the reaction condition is mild, and the large-scale production can be realized;
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 monosaccharide composition of LC 2-1-2A;
FIG. 2 is a high performance liquid chromatogram of the D/L structure of LC2-1-2A monosaccharide;
FIG. 3 is an infrared spectrum of LC 2-1-2A;
FIG. 4 shows LC2-1-2A1H NMR spectrum;
FIG. 5 shows LC2-1-2A13A C NMR spectrum;
FIG. 6 is the HSQC spectrum of LC 2-1-2A;
FIG. 7 is an HMBC map of LC 2-1-2A;
FIG. 8 is the primary structure of LC 2-1-2A;
FIG. 9 shows the immunomodulatory effect 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 of 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 10 times the weight of rhizoma Ligustici Chuanxiong segments obtained in step S1, heating to 80 deg.C, extracting for 3 hr, collecting extractive solution, and air drying the residue to obtain extractive solution and residue;
s3 grading and alcohol precipitating:
concentrating the extractive solution obtained in step S2 at 60 deg.C under reduced pressure, adding ethanol until the volume concentration of ethanol is 50%, standing at room temperature for 24 hr, 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 LC 3;
s4 purification: removing protein from the LC2 crude polysaccharide obtained in the step S3 by Sevag method, dialyzing the crude polysaccharide with dialysis bag (molecular weight cutoff is 3000Da) after removing protein, and lyophilizing to obtain rhizoma Ligustici Chuanxiong polysaccharide LC 2.
Example 2 Ligusticum wallichii 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, 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 the elution process, sugar parts are respectively collected according to the elution curve), and concentrating and freeze-drying the obtained eluents respectively to obtain polysaccharide LC 2;
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 LC 2-1-2A.
Example 3 structural analysis of Ligusticum wallichii polysaccharide LC2-1-2A
Test materials (one): rhizoma Ligustici Chuanxiong polysaccharide LC 2-1-2A.
(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: 17 v/v); detection wavelength: 250 nm; flow rate: 0.8 mL/min; sample introduction volume: 20 μ L.
2. Monosaccharide D/L conformation analysis
LC2-1-2A (5mg) was reacted with trifluoroacetic acid (2M, 2mL) in an oil bath at 120 ℃ for 4 h. 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 (1mL) 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% CH3CN-H2O (0.1% acetic acid); flow rate: 0.8 mL/min; sample introduction amount: 10 mu L of the solution; detection wavelength: UV 250 nm; detection time: and (5) 60 min.
3. Infrared spectroscopy detection
Will be dried2.0mg of the test material(s) was ground with KBr, tabletted, and examined with a Perkin EImer FT/IR-100 at 4000-400cm-1Scanning is performed over the range.
4. methylation/GC-MS analysis
Weighing 8.0mg of dry test material into a reaction bottle, adding 8mL of anhydrous DMSO (dimethylsulfoxide), adding 800mg of dry sodium hydroxide, performing ultrasonic treatment for 30min, adding 3.0mL of methyl iodide in a dark place under an ice bath condition, performing ultrasonic treatment for three times, performing ultrasonic treatment for 30min in each ice bath, adding 2mL of distilled water after the reaction is finished, decomposing residual methyl iodide, adding 1mL of chloroform, performing 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 added4The reaction was carried out at 40 ℃ for 30min to reduce the hydrolysate. The reaction was stopped with 100. mu.L of glacial acetic acid, the sample was spun dry under reduced pressure, and then 2mL of acetyl anhydride and pyridine were added for acetylation. The reaction was stirred for 2h with magnetic stirring 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 D2O, placing in a nuclear magnetic tube, and recording by using a 400MHz nuclear magnetic resonance instrument Bruker AV-4001H NMR，13C NMR, HSQC, HMBC, etc.
(III) test results:
1. structure analysis of Ligusticum wallichii polysaccharide LC2-1-2A
(1) Monosaccharide composition analysis
As shown in FIG. 1, LC2-1-2A contained only arabinose as seen from the HPLC profile. (chromatographic peak order: 1: mannose, 2: rhamnose, 3: glucuronic acid, 4: galacturonic acid, 5: glucose, 6: galactose, 7: xylose, 8: arabinose, 9: fucose)
(2) Monosaccharide D/L conformation analysis
As shown in FIG. 2, the configuration of arabinose in LC2-1-2A is L configuration as seen from HPLC chromatogram.
(2) Infrared spectroscopic analysis
As shown in FIG. 3, the infrared spectrum of LC2-1-2A shows that the Ligusticum wallichii LC2-1-2A contains the characteristic infrared absorption peak of polysaccharide.
(3) methylation/GC-MS analysis
LC2-1-2A methylation analysis, after hydrolysis, reduction acetylation, GC-MS detection, and 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 → etc. sugar residues.
(4) Nuclear magnetic resonance analysis
This test passes1H NMR、13C NMR and HSQC assigned chemical shifts of carbon and hydrogen atoms of sugar residues of LC2-1-2A, and then confirmed the order of attachment with HMBC. FIG. 4-7 shows LC2-1-2A1H NMR、13C NMR, HSQC and HMBC spectra.
According to the NMR spectra of FIGS. 4-7, the LC2-1-2A hydrocarbon assignments are shown in Table 1 below.
TABLE 1 LC2-1-2A NMR analysis results
a Unresolved from other signals，nd：no detected.
In summary, the following steps: LC2-1-2A is an arabinan composed of arabinose, which contains → 5) -alpha-L-Araf- (1 → 2,3,5) -alpha-L-Araf- (1 → and alpha-L-Araf- (1 → isosaccharide residues from methylation analysis, and the sequence of linkage between different saccharide residues is obtained from two-dimensional nuclear magnetic HMBC spectrum analysis, and the structure of LC2-1-2A obtained 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): rhizoma Ligustici Chuanxiong polysaccharide LC 2-1-2A.
(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 1ml PBS buffer salt, filtering the prepared mother liquor with 0.22 μm filter membrane, sterilizing by ultraviolet irradiation for 30min, and placing in a refrigerator at-20 deg.C for use.
Placing the culture bottle in a super clean bench, removing the old culture medium, adding 5mL PBS buffer solution to clean the cells, removing the PBS buffer solution after cleaning the cells, adding 600 mu L trypsin into the culture bottle, adding DMEM culture medium after digesting RAW264.7 cells for 1min, and repeatedly blowing to obtain 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 × 104Per 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 CO2Culturing in an incubator for 24 h.
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 CO2Culturing in an incubator for 4 h. After 4h, the MTT solution in the 96-well plate was removed, 150 μ L of dimethyl sulfoxide (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 with LC2-1-2A dosing at different concentrations, washed three times with PBS, incubated for 1h with 100 μ L of neutral red solution (0.1%), discarded, washed three times with PBS, blotted, 150 μ 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 a microplate reader, and the experiment was repeated three times.
NO Release test
RAW264.7 cells were seeded in 96-well plates and after 4h 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 24h, the supernatants were collected and reacted with Griess reagent and OD was determined with a microplate reader at 550 nm.
Effect of LC2-1-2A on RAW264.7 cytokines
RAW264.7 cells were inoculated in a 24-well plate and incubated for 4h, and then treated with LC2-1-2A (50. mu.g/mL, 100. mu.g/mL and 200. mu.g/mL) and 1. mu.g/mL LPS for 24h, and then the supernatant was collected and subjected to detection of 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 50-200 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 chuanxiong polysaccharide LC2-1-2A can enhance the immune regulation ability of organisms by improving the phagocytic ability of macrophages of the organisms, increasing the NO release 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 Zebra fish embryo by pure Ligusticum chuanxiong polysaccharide LC2-1-2A (one) 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 at 14h:10h, 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, 6 zebrafish larvae per well, and cultured with different concentrations of LC2-1-2A (50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL) at 28.5 ℃ for 24 hours. After 24h, the culture medium containing LCP70-2A was removed, replaced with fresh culture medium periodically, and the culture was continued until 72h after fertilization of the zebra fish larvae, during which time the zebra fish were not fed.
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. The influence of LC2-1-2A on the ROS release amount in the zebra fish body is detected by carrying out quantitative analysis on the relative fluorescence intensity in the zebra fish juvenile fish body by using Image J software.
(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 was increased (50 μ g/mL,100 μ g/mL,200 μ g/mL), the fluorescence intensity of ROS in zebrafish was 1.59 times, 2.28 times and 3.16 times that of the blank group, and the fluorescence intensity of NO was 1.75 times, 2.41 times and 3.73 times, respectively, compared to the control group, which indicates that LC2-1-2A has a significant enhancing effect on ROS and NO content in zebrafish juvenile fish, and can enhance the in vivo immunomodulatory activity of zebrafish juvenile fish.
In conclusion, the pure chuanxiong polysaccharide product LC2-1-2A prepared by the invention can enhance the phagocytosis capability of RAW246.7 cells and promote the release of related inflammatory factors, and meanwhile, LC2-1-2A can enhance the release of ROS and NO in zebra fish juvenile fish, thereby enhancing the immunoregulation capability of the body.
Claims (7)
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