阅读说明：本技术 川芎均一性多糖及其提取方法与应用 (Ligusticum wallichii homogeneous polysaccharide and extraction method and application thereof ) 是由 鲍依稀 钟诚 刘子靖 张旭裕 蒲有为 于 2020-08-17 设计创作，主要内容包括：本发明提供一种川芎多糖及其提取方法与应用,具体步骤如下：以川芎为原材料,经过热水提取步骤、乙醇沉淀步骤、去除蛋白步骤、透析浓缩冷冻干燥步骤,制得川芎粗多糖；对川芎粗多糖,采用DEAE Sepharose<Sup>TM</Sup>Fast Flow柱层析技术提取川芎多糖；对川芎多糖,采用Sephacryl<Sup>TM</Sup>S-300High Resolution柱层析技术提取川芎得到两种均一性多糖LCXP-1a、LCXP-3a,对比两种川芎均一性多糖在肝癌免疫调节的实验表明川芎均一性多糖具有免疫调节抗肿瘤作用。(The invention provides a ligusticum wallichii polysaccharide and an extraction method and application thereof, and the specific steps are as follows: taking ligusticum wallichii as a raw material, and preparing crude ligusticum wallichii polysaccharide through a hot water extraction step, an ethanol precipitation step, a protein removal step, a dialysis concentration and freeze drying step; for crude polysaccharide of rhizoma Ligustici Chuanxiong, DEAE Sepharose is used TM Extracting rhizoma Ligustici Chuanxiong polysaccharide by Fast Flow column chromatography; for rhizoma Ligustici Chuanxiong polysaccharide, Sephacryl is used TM S-300High Resolution column chromatography technology to extract rhizoma Ligustici Chuanxiong to obtain two homogeneous polysaccharides LCXP-1a and LCXP-3a, and experiment comparing the two homogeneous polysaccharides in liver cancer immunoregulation shows that Chuan XiongThe homogeneous polysaccharide of rhizoma Ligustici Chuanxiong has effects of regulating immunity and resisting tumor.)

1. A method for extracting rhizoma ligustici wallichii homogeneous polysaccharide is characterized by comprising the following specific steps:

1) crude extraction of crude polysaccharide of rhizoma Ligustici Chuanxiong;

2) for crude polysaccharide extract of rhizoma Ligustici Chuanxiong, DEAE Sepharose is used^TMExtracting rhizoma Ligustici Chuanxiong polysaccharides LCXP-1 and LCXP-3 by Fast Flow column chromatography;

3) sephacryl is used for rhizoma Ligustici Chuanxiong polysaccharides LCXP-1 and LCXP-3^TMExtracting with S-300High Resolution column chromatography to obtain homogeneous polysaccharides LCXP-1a and LCXP-3a of rhizoma Ligustici Chuanxiong.

2. The method for extracting Chuanxiong rhizome polysaccharide of claim 1, wherein the step 1) comprises the following steps:

1-1) hot water extraction step: pulverizing rhizoma Ligustici Chuanxiong in a pulverizer to obtain rhizoma Ligustici Chuanxiong powder; adding ligusticum wallichii powder into a hot water extraction reaction cup, adding distilled water I, placing the mixture in a water bath at the temperature of 75-85 ℃, stirring for 1.5-2.5 hours, filtering and collecting an extracting solution I; adding distilled water II into the residue, placing the mixture in a water bath at the temperature of 75-85 ℃, stirring the mixture for 1.5-2.5 hours, filtering and collecting an extracting solution II; mixing extractive solutions I and II, centrifuging to remove precipitate, and concentrating under reduced pressure on rotary evaporator to obtain rhizoma Ligustici Chuanxiong water soluble crude extract; the weight portion ratio of the ligusticum wallichii powder to the distilled water I to the distilled water II is as follows: 150-250 parts of ligusticum wallichii powder, 1500-2500 parts of distilled water I and 1200-2000 parts of distilled water II;

1-2) ethanol precipitation step: adding 4 times of anhydrous ethanol into the cooled crude water-soluble extract of the ligusticum wallichii while continuously stirring, placing the mixture in an environment at 4 ℃, precipitating for 16-24 hours, and centrifuging to obtain a precipitate I;

1-3) protein removal step: dissolving the precipitate I with distilled water III, adding sevag reagent with one fifth of the volume of the precipitate I, electrically and rapidly stirring for 8-12 min, and centrifuging at 4000rpm for 3-5 min; taking the supernatant I, repeatedly adding a sevag reagent with one fifth of the volume of the supernatant I, stirring and centrifuging until protein is completely removed to obtain a supernatant II; the weight part ratio of the precipitate I to the distilled water III is as follows: 10-20 parts of precipitate I and 25-50 parts of distilled water III;

1-4) dialysis concentration freeze drying: taking the supernatant II, selecting a dialysis bag with the molecular weight cutoff of 3000 Da-4000 Da, and dialyzing in pure water for 36-48 h; concentrating under reduced pressure on a rotary evaporator, and freeze drying in a freeze dryer to obtain rhizoma Ligustici Chuanxiong crude polysaccharide.

3. The method for extracting Chuanxiong rhizome polysaccharide of claim 2,

in the step 1-1), the water bath temperature is 80 ℃, the stirring time is 2 hours, the ligusticum wallichii powder is 200 parts, the distilled water I is 2000 parts, and the distilled water II is 1600 parts;

the precipitation time in the step 1-2) is 24 hours;

stirring for 10min, centrifuging for 3min, precipitating with I10 parts, and distilled water III 25 parts in step 1-3);

the molecular weight cut-off in the step 1-4) is 3500Da, and the dialysis time is 48 h.

4. The method for extracting Chuanxiong rhizome homogeneous polysaccharide of claim 1, wherein DEAE Sepharose is used in step 2)^TMThe specific steps of extracting the ligusticum wallichii polysaccharide by Fast Flow column chromatography technology are as follows:

2-1) pretreatment: 5ml of distilled water for preventing air bubbles was added to the column tube, and DEAE Sepharose was added^TMAdding Fast Flow into the column tube, and standing for 18-24 h; washing 1 column volume by using 0.4-0.6 mol/L HCl at the speed of 6-8 mL/min for 30-50 min; balancing 3-5 column volumes by using ultrapure water, wherein the speed is 6-8 mL/Min, and the time is 90-130 Min;

2-2) dissolving crude ligusticum wallichii polysaccharide in distilled water IV, loading at the speed of 3-4 mL/min, eluting 40 tubes, 8mL tubes and 6-8 mL/min by using water, 0.15mol/L and 0.3mol/L NaCl respectively to obtain an eluent LCXP-1, an eluent LCXP-2 and an eluent LCXP-3 respectively; the weight parts of the crude polysaccharide of the ligusticum wallichii and the distilled water IV are respectively as follows: 0.3-0.4 part of crude ligusticum wallichii polysaccharide and 25-30 parts of distilled water IV;

2-3) detecting the absorbance of the eluent LCXP-1, the eluent LCXP-2 and the eluent LCXP-3 by using a phenol-sulfuric acid method, and selecting the eluent LCXP-1 and the eluent LCXP-3 with the absorbance being a single peak.

2-4) selecting a dialysis bag with the molecular weight cutoff of 7500 Da-8000 Da for the eluent LCXP-1 and the eluent LCXP-3, dialyzing in pure water for 36-48h, and changing water once every 4 h; collecting the liquid in the dialysis bag, concentrating, lyophilizing, and storing at-20 deg.C to obtain rhizoma Ligustici Chuanxiong polysaccharide LCXP-1 and LCXP-3 respectively.

5. The method for extracting Chuanxiong rhizome polysaccharide of claim 4,

standing for 24 hours in the step 2-1), washing at the HCl concentration of 0.5mol/L, HCl at the speed of 6mL/min, and washing at the HCl for 35 min;

in the step 2-2), the sampling speed is 4mL/min, the elution speed is 6mL/min, the crude polysaccharide of the hemlock parsley is 0.3 part, and the distilled water is 25 parts;

the cut-off molecular weight in the step 2-4) is 8000Da, and the dialysis time is 48 h.

6. The method for extracting Chuanxiong rhizome polysaccharide of claim 1, wherein Sephacryl is used in step 3)^TMThe S-300High Resolution column chromatography technology for extracting the ligusticum wallichii homogeneity polysaccharide comprises the following specific steps:

3-1) pretreatment: 5ml of distilled water for preventing air bubbles was added to the column tube, and Sephacryl was added^TMAdding S-300Highresolution into a column tube, and standing for 18-24 h; washing 1-2 column volumes with PBS balance solution at a speed of 0.3-0.4 mL/min for 200-500 min.

3-2) dissolving the ligusticum wallichii polysaccharide LCXP-1 in distilled water V, and loading the sample at the rate of 0.3-0.4 mL/min; eluting 40 tubes with PBS (phosphate buffer solution) balance solution at a speed of 0.3mL/min for 3 mL/tube to obtain an eluent LCXP-1 a; the weight parts of the ligusticum wallichii polysaccharide LCXP-1 and the distilled water V are respectively as follows: 0.025-0.035 parts of ligusticum wallichii polysaccharide LCXP-1 and 1-1.5 parts of distilled water V;

dissolving ligusticum wallichii polysaccharide LCXP-3 in distilled water VI, and sampling at the rate of 0.3-0.4 mL/min; eluting 40 tubes with PBS (phosphate buffer solution) balance solution at a speed of 0.3mL/min for 3 mL/tube to obtain an eluent LCXP-3 a; the weight parts of the ligusticum wallichii polysaccharide LCXP-3 and the distilled water VI are respectively as follows: 0.025-0.035 parts of ligusticum wallichii polysaccharide LCXP-3 and 1-1.5 parts of distilled water VI.

3-3) detecting the absorbance of the eluates LCXP-1a and LCXP-3a, and selecting the eluates LCXP-1a and LCXP-3a with single peak absorbance.

3-4) selecting dialysis bags with the molecular weight cutoff of 7500 Da-8000 Da for the LCXP-1a and LCXP-3a eluents with the single peak absorbance, respectively dialyzing in pure water for 36-48h, and changing water once every 4 h; collecting the liquid in the dialysis bag, rotary evaporating for concentration, lyophilizing, and storing at-20 deg.C to obtain homogeneous polysaccharides LCXP-1a and LCXP-3a of rhizoma Ligustici Chuanxiong with two elution fragments.

7. The method for extracting Chuanxiong rhizome polysaccharide of claim 6,

standing for 24h in the step 3-1), washing 1 column volume by using PBS (phosphate buffer solution) equilibrium solution, keeping the equilibrium speed of the PBS equilibrium solution at 0.3mL/min, and keeping the equilibrium time of the PBS equilibrium solution at 260 min;

in the step 3-2), the sample loading speed is 0.3mL/min, the Ligusticum wallichii polysaccharide LCXP-10.03 parts, the distilled water V1 parts, the Ligusticum wallichii polysaccharide LCXP-30.03 parts and the distilled water VI 1 parts;

the cut-off molecular weight in the step 3-4) is 8000Da, and the dialysis time is 48 h.

8. The Chuanxiong rhizome homogeneous polysaccharide extracted by the method for extracting Chuanxiong rhizome homogeneous polysaccharide according to claims 1 to 7, wherein the composition of the Chuanxiong rhizome polysaccharide LCXP-1a is as follows:

the molecular weight is: 11159 Da;

the total polysaccharide content is: 99.68 percent;

the uronic acid content is: 4.05 percent;

the monosaccharide composition is: mannose, ribose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose.

9. The Chuanxiong rhizome homogeneous polysaccharide extracted by the method for extracting Chuanxiong rhizome homogeneous polysaccharide according to claims 1 to 7, wherein the composition of the Chuanxiong rhizome polysaccharide LCXP-3a is as follows:

the molecular weight is: 203486 Da;

the total polysaccharide content is: 90.04 percent;

the uronic acid content is: 63.36 percent;

the monosaccharide composition is: mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose.

10. The use of the chuanxiong rhizome homopolysaccharide of claim 8 or 9 for immunomodulation and anti-tumor of liver cancer.

Technical Field

The invention relates to the technical field of plant extraction, in particular to a ligusticum wallichii homogeneous polysaccharide and an extraction method and application thereof.

Background

Ligusticum wallichii is a traditional Chinese medicine with a long history, and in recent years, various biological activities of Ligusticum wallichii are widely researched, and mainly comprise anti-inflammatory activity, cardiovascular effect, cell aging slowing, oxidation resistance and in-vitro anti-tumor activity. At present, more than 170 components are separated from ligusticum wallichii, including phthalate, terpenes and enol thereof, alkaloids, polysaccharides, organic acids and esters thereof and the like. Ligusticum wallichii polysaccharide (LCXP) is one of the main biological active substances of Ligusticum wallichii, and the research on LCXP is increasing, and generally comprises the research on the extraction, separation and purification and biological activity of LCXP. Studies have shown that many immune-based polysaccharides can exert immunomodulatory effects by activating immune cells, particularly antigen presenting cells. The surface of the antigen presenting cell has a large number of Toll-like receptors (TLRs), can recognize antigens and activate intracellular signal pathways, is important in innate and adaptive immune response, and participates in the antitumor activity of some traditional Chinese medicine polysaccharides.

The traditional polysaccharide separation and extraction methods comprise a water extraction and alcohol precipitation method, an enzyme extraction method, an ultrasonic-assisted extraction method, a supercritical fluid extraction method, an ultrahigh-pressure extraction technology, a microwave-assisted extraction method, a two-aqueous-phase extraction and other new technologies, and the separation principle, the treatment mode and the effect are different.

In the invention patent with publication number CN105273094, entitled "a method for rapidly separating polysaccharide from Ligusticum wallichii", rhizoma Ligustici Chuanxiong root is dried, pulverized and degreased, then mixed with ethanol-inorganic salt aqueous two-phase extract, microwave extracted, centrifuged, separated alcohol, precipitated, dried to obtain crude polysaccharide; in the process research of [ journal articles ] Pare Hati. Ehrzel Hemaiti, Saide Aihemai Hemai, Xu Shansan, Jiergli, Wuqiang, Pare Hati. Aihemai maiti, Saide Aihemai maiti, Xu Shannhan, Ji' er Gel i, Wu Qiang- "scientific friend 2011 18-stage Ligusticum chuanxiong polysaccharide separation and extraction, Ligusticum chuanxiong polysaccharide is obtained by degreasing rhizoma chuanxiong root with petroleum ether, extracting with hot water, precipitating with ethanol, removing protein, washing with anhydrous ethanol, acetone and ether, and freeze-drying. The defects of the method are that column chromatography is not used for further separation and purification, the extracted ligusticum wallichii polysaccharide is not homogeneous polysaccharide, the purity is low, the molecular weight and monosaccharide composition cannot be detected, and the biological activity is not explored.

Disclosure of Invention

The first purpose of the invention is to provide a method for extracting ligusticum wallichii homogeneous polysaccharide.

The second purpose of the invention is to provide two kinds of chuanxiong rhizome homogeneous polysaccharide.

The third purpose of the invention is to provide the application of two chuanxiong rhizome homogeneous polysaccharides in the aspect of liver cancer immunoregulation and anti-tumor.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting rhizoma Ligustici Chuanxiong homogeneous polysaccharide comprises the following steps:

1) crude extraction of crude polysaccharide of rhizoma Ligustici Chuanxiong;

2) for crude polysaccharide extract of rhizoma Ligustici Chuanxiong, DEAE Sepharose is used^TMExtracting rhizoma Ligustici Chuanxiong polysaccharides LCXP-1 and LCXP-3 by Fast Flow column chromatography;

3) sephacryl is used for rhizoma Ligustici Chuanxiong polysaccharides LCXP-1 and LCXP-3^TMExtracting with S-300High resolution ion column chromatography to obtain homogeneous polysaccharides LCXP-1a and LCXP-3a of rhizoma Ligustici Chuanxiong.

Further, the specific method for roughly extracting the crude polysaccharide of the ligusticum wallichii in the step 1) comprises the following steps:

1-1) hot water extraction step: pulverizing rhizoma Ligustici Chuanxiong in a pulverizer to obtain rhizoma Ligustici Chuanxiong powder; adding ligusticum wallichii powder into a hot water extraction reaction cup, adding distilled water I, placing the mixture in a water bath at the temperature of 75-85 ℃, stirring for 1.5-2.5 hours, and filtering to collect an extracting solution I; adding distilled water II into the residue, placing the residue in a water bath at the temperature of 75-85 ℃, stirring the mixture for 1.5-2.5 hours, filtering and collecting an extracting solution II; mixing the extractive solutions I and II, centrifuging to remove precipitate, and concentrating under reduced pressure on a rotary evaporator to obtain rhizoma Ligustici Chuanxiong water-soluble crude extract; the weight portion ratio of the ligusticum wallichii powder, the distilled water I and the distilled water II is as follows: 150-250 parts of ligusticum wallichii powder, 1500-2500 parts of distilled water I and 1200-2000 parts of distilled water II;

1-2) ethanol precipitation step: adding 4 times of anhydrous ethanol into the cooled crude water-soluble extract of the ligusticum wallichii while continuously stirring, placing the mixture in an environment at 4 ℃, precipitating for 16-24 hours, and centrifuging to obtain a precipitate I;

1-3) protein removal step: dissolving the precipitate I with distilled water III, adding sevag reagent with one fifth of the volume of the precipitate I, electrically and rapidly stirring for 8-12 min, and centrifuging at 4000rpm for 3-5 min; taking the supernatant I, repeatedly adding a sevag reagent with one fifth of the volume of the supernatant I, stirring and centrifuging until protein is completely removed to obtain a supernatant II; the weight part ratio of the precipitate I to the distilled water III is as follows: 10-20 parts of precipitate I and 25-50 parts of distilled water III;

1-4) dialysis concentration freeze drying: taking the supernatant II, selecting a dialysis bag with the molecular weight cutoff of 3000 Da-4000 Da, and dialyzing in pure water for 36-48 h; concentrating under reduced pressure on a rotary evaporator, and freeze drying in a freeze dryer to obtain rhizoma Ligustici Chuanxiong crude polysaccharide.

Further, in the step 1-1), the water bath temperature is 80 ℃, the stirring time is 2 hours, the ligusticum wallichii powder is 200 parts, the distilled water I is 2000 parts, and the distilled water II is 1600 parts;

the precipitation time in the step 1-2) is 24 hours;

in the step 1-3), stirring for 10min, centrifuging for 3min, 10 parts of precipitate I and 25 parts of distilled water III;

the molecular weight cut-off in the step 1-4) is 3500Da, and the dialysis time is 48 h.

Further, the step 2) is performed by using DEAE Sepharose^TMThe specific steps of extracting the ligusticum wallichii polysaccharide by Fast Flow column chromatography technology are as follows:

2-1) pretreatment: 5ml of distilled water for preventing air bubbles was added to the column tube, and DEAE Sepharose was added^TMAdding FastFlow into a column tube, and standing for 18-24 h; washing 1 column volume by using 0.4-0.6 mol/L HCl at the speed of 6-8 mL/min for 30-50 min; balancing 3-5 column volumes by using ultrapure water, wherein the speed is 6-8 mL/Min, and the time is 90-130 Min;

2-2) dissolving crude ligusticum wallichii polysaccharide in distilled water IV, loading at the speed of 3-4 mL/min, and eluting 40 tubes, 8 mL/tube and 6-8 mL/min respectively with water, 0.15mol/L NaCl and 0.3mol/L NaCl to obtain an eluent LCXP-1, an eluent LCXP-2 and an eluent LCXP-3 respectively; the weight parts of the crude polysaccharide of the ligusticum wallichii and the distilled water IV are respectively as follows: 0.3-0.4 part of crude ligusticum wallichii polysaccharide and 25-30 parts of distilled water IV;

2-3) detecting the absorbance of the eluent LCXP-1, the eluent LCXP-2 and the eluent LCXP-3 by using a phenol-sulfuric acid method, and selecting the eluent LCXP-1 and the eluent LCXP-3 with the absorbance being a single peak.

2-4) selecting a dialysis bag with the molecular weight cutoff of 7500 Da-8000 Da for the eluent LCXP-1 and the eluent LCXP-3, dialyzing in pure water for 36-48h, and changing water once every 4 h; collecting the liquid in the dialysis bag, concentrating, lyophilizing, and storing at-20 deg.C to obtain rhizoma Ligustici Chuanxiong polysaccharide LCXP-1 and LCXP-3 respectively.

Further, in the step 2-1), the standing time is 24 hours, the HCl concentration is 0.5mol/L, HCl cleaning speed is 6mL/min, and the HCl cleaning time is 35 min;

in the step 2-2), the sampling speed is 4mL/min, the elution speed is 6mL/min, the crude polysaccharide of the ligusticum wallichii is 0.3 part, and the distilled water is IV 25 parts;

the cut-off molecular weight in the step 2-4) is 8000Da, and the dialysis time is 48 h.

Further, said step 3) uses Sephacryl^TMThe S-300High resolution ion column chromatography technology for extracting the ligusticum wallichii homogeneity polysaccharide comprises the following specific steps:

3-1) pretreatment: 5ml of distilled water for preventing air bubbles was added to the column tube, and Sephacryl was added^TMAdding S-300Highresolution into a column tube, and standing for 18-24 h; washing 1-2 column volumes with PBS balance solution at a speed of 0.3-0.4 mL/min for 200-500 min.

3-2) dissolving the ligusticum wallichii polysaccharide LCXP-1 in distilled water V, and loading the sample at the concentration of 0.3-0.4 mL/min; eluting 40 tubes with PBS (phosphate buffer solution) balance solution at a speed of 0.3mL/min for 3 mL/tube to obtain an eluent LCXP-1 a; the weight parts of the ligusticum wallichii polysaccharide LCXP-1 and the distilled water V are respectively as follows: 0.025-0.035 parts of ligusticum wallichii polysaccharide LCXP-1 and 1-1.5 parts of distilled water V;

dissolving ligusticum wallichii polysaccharide LCXP-3 in distilled water VI, and sampling at the rate of 0.3-0.4 mL/min; eluting 40 tubes with PBS (phosphate buffer solution) balance solution at a speed of 0.3mL/min for 3 mL/tube to obtain an eluent LCXP-3 a; the weight parts of the ligusticum wallichii polysaccharide LCXP-3 and the distilled water VI are respectively as follows: 0.025-0.035 parts of ligusticum wallichii polysaccharide LCXP-3 and 1-1.5 parts of distilled water VI.

3-3) detecting the absorbance of the eluates LCXP-1a and LCXP-3a, and selecting the eluates LCXP-1a and LCXP-3a with single peak absorbance.

3-4) selecting dialysis bags with the molecular weight cutoff of 7500 Da-8000 Da for the LCXP-1a and LCXP-3a eluents with the single peak absorbance, respectively dialyzing in pure water for 36-48h, and changing water once every 4 h; collecting the liquid in the dialysis bag, rotary evaporating for concentration, lyophilizing, and storing at-20 deg.C to obtain homogeneous polysaccharides LCXP-1a and LCXP-3a of rhizoma Ligustici Chuanxiong with two elution fragments.

Further, in the step 3-1), the standing time is 24 hours, the volume of 1 column is washed by PBS (phosphate buffer solution) equilibrium liquid, the equilibrium speed of the PBS equilibrium liquid is 0.3mL/min, and the equilibrium time of the PBS equilibrium liquid is 260 min;

in the step 3-2), the sample loading speed is 0.3mL/min, the Ligusticum wallichii polysaccharide LCXP-10.03 parts, the distilled water V1 parts, the Ligusticum wallichii polysaccharide LCXP-30.03 parts and the distilled water VI 1 parts;

the cut-off molecular weight in the step 3-4) is 8000Da, and the dialysis time is 48 h.

Further, the ligusticum wallichii polysaccharide LCXP-1a comprises the following components:

the molecular weight is: 11159 Da;

the total polysaccharide content is: 99.68 percent;

the uronic acid content is: 4.05 percent;

the monosaccharide composition is: mannose, ribose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose.

Further, the ligusticum wallichii polysaccharide LCXP-3a comprises the following components:

the molecular weight is: 203486 Da;

the total polysaccharide content is: 90.04 percent;

the uronic acid content is: 63.36 percent;

the monosaccharide composition is: mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose.

Further, the application of the ligusticum wallichii homogeneous polysaccharide in liver cancer immunoregulation and anti-tumor.

Due to the adoption of the technical scheme, the invention has the following advantages:

the invention firstly uses the chuanxiong rhizome as raw material, adopts the traditional water extraction and alcohol precipitation method to extract chuanxiong rhizome crude polysaccharide, and then passes the extracted chuanxiong rhizome crude polysaccharide through DEAE Sepharose^TMFast Flow column, Sephacryl^TMThe method comprises the steps of carrying out separation and purification on an S-300High Resolution column twice to obtain two fragments of ligusticum wallichii homogeneous polysaccharides LCXP-1a and LCXP-3a, and having strong immunocompetence, further carrying out separation and purification through a column chromatography method, extracting and purifying the obtained ligusticum wallichii polysaccharides from ligusticum wallichii, wherein the LCXP-1a and the LCXP-3a are single symmetrical peaks, so that the LCXP-1a and the LCXP-3a are homogeneous polysaccharides, the content of LCXP-1a total polysaccharides is 99.68%, the content of LCXP-3a total polysaccharides is 90.04%, the purity is High, the molecular weight of LCXP-1a is 59Da, the molecular weight of LCXP-3a is 203486Da, and monosaccharide components of the monosaccharides of the LCXP-3a are respectively mannose, ribose, glucuronic acid, glucose, galactose, xylose, arabinose and fucose, the content of the extracted polysaccharides of the LCXP-3a is High in the aspects of directly inhibiting the liver cancer cells, such as the proliferation of liver cancer cells, liver cancer cells.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof.

Drawings

Detailed Description

The invention is further illustrated by the following figures and examples.