Angelica divaricata acidic polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a heracleum millefolium acidic polysaccharide as well as a preparation method and application thereof. According to the acid polysaccharide of the heracleum millefolium diels, the molecular weight of the acid polysaccharide HMPS-1b of the heracleum millefolium diels is 1.3 * 10 < 4 > Da, the acid polysaccharide HMPS-1b of the heracleum millefolium diels is composed of galacturonic acid GalA, glucose Glc, galactose Gal and arabinose Ara, and the molar ratio of the galacturonic acid GalA to the glucose Glc to the galactose Gal to the arabinose Ara is 2.2: 1.0: 1.1: 5.5. The heracleum millefolium acidic polysaccharide HMPS-1b obtained by the invention is a homogeneous polysaccharide, the preparation process is simple, and industrial application is easy to realize. The heracleum millefolium acidic polysaccharide HMPS-1b obtained by the invention can prolong the survival time of an AOM / DSS induced colon cancer model mouse; the weight loss of the AOM / DSS induced colon cancer model mouse can be relieved; the total number of tumors can be reduced, the tumor size is reduced, inflammatory injury is relieved, and the obvious effect on resisting colon cancer is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an acidic polysaccharide from Heracleum millefolium Diels, a preparation method thereof, and applications thereof. Background Art

[0002] Colorectal cancer is the most common malignant tumor of the digestive tract and is also one of the three major malignant tumors globally. Surgery is the main treatment method for colorectal cancer. For early-stage colorectal cancer, the 5-year survival rate after surgery can reach 90%. However, due to the insidious onset of colorectal cancer, approximately 83% of patients are already in the middle and late stages when they are diagnosed. The effect of surgery combined with radiotherapy and chemotherapy is not good, and it is extremely easy to relapse and metastasize. The survival time of patients does not increase significantly, and the prognosis is poor. 5-Fluorouracil, oxaliplatin, and irinotecan are currently commonly used chemotherapy drugs for colorectal cancer in clinical practice. Although they have significant curative effects, their adverse reactions are serious, and drug resistance is likely to occur. In recent years, targeted drugs and immunotherapy have brought hope for the treatment of colorectal cancer. However, their treatment cycles are short, they are prone to inducing drug resistance in tumor cells, and their adverse reactions are serious. Therefore, the need to find new drugs and targets for the treatment of colorectal cancer is still extremely urgent.

[0003] Tibetan Angelica, scientifically named Heracleum millefolium Diels, is a plant of the genus Heracleum in the family Apiaceae. It grows in alpine meadows, shrubs, or gravel areas at an altitude of 2,700 - 4,800 m and is one of the main varieties of Tibetan medicine. Tibetan Angelica has a long medicinal history in the Qinghai-Tibet region. Tibetan medicine uses the whole herb as medicine, and it is commonly used to treat rheumatism, disperse swelling, and break up mass and nodule. According to "Chinese Herbal Medicine", the Tibetan medicine Heracleum millefolium Diels is pungent, slightly sweet, and has a neutral nature, with the effects of cooling blood for hemostasis, expelling wind and detoxifying, and is commonly used to treat rheumatism, epistaxis, gingival bleeding, skin ecchymosis, leprosy, etc. Tibetan Angelica is one of the representative drugs of Tibetan medicine, and the research on its effective substances is an essential task that needs to be carried out urgently in the modernization research of Tibetan medicine. The reported chemical components in Tibetan Angelica include coumarins, volatile oils, organic acids, etc. Furanocoumarins have photosensitivity and can damage the DNA replication of bacteria and fungi under ultraviolet irradiation, inhibiting their growth and proliferation. The coumarin osthole can inhibit the growth of breast cancer cells by regulating the signal transducer and activator of transcription (STAT) signaling pathway and inhibit the release of related cytokines and regulate immunity through the NF-κB and Nrf2 signaling pathways. [3] The terpene compound α-pinene in Tibetan Angelica regulates the production of TNF-α, IL-6, and NO through the NF-κB signaling pathway, inhibits chondrocyte inflammation, and protects cartilage from inflammatory damage. In addition, the volatile oil components in Tibetan Angelica have significant bactericidal and bacteriostatic effects on various Gram-positive bacteria such as Streptococcus pneumoniae, and also have inhibitory effects on yeast-like fungi and Candida albicans. The volatile oil component γ-terpinene has significant in vitro antitumor effects and can effectively inhibit the proliferation of mouse melanoma B16 cells. However, as an important water-soluble component in Tibetan Angelica, the research on its separation and purification methods and pharmacological effects is still blank and urgently needs to be systematically and deeply explored. Summary of the Invention

[0004] The object of the present invention is to provide an acidic polysaccharide from Heracleum millefolium Diels and its preparation method and application.

[0005] The implementation process of the present invention is as follows:

[0006] An acidic polysaccharide from Heracleum millefolium Diels, the molecular weight of the acidic polysaccharide from Heracleum millefolium Diels is 1.3×10 4 Da, the main chain repeating unit of the acidic polysaccharide from Heracleum millefolium Diels is: →3)-α-L-Araf-(1→5)-α-L-Araf-(1→3,5)-α-L-Araf-(1→6)-α-D-Glcp-(1→4)-α-D-Ga lpA-(1→4)-α-D-GalpA-(1→3)-β-D-Galp-(1→, and the side chain is →5)-α-L-Araf-(1→3)-α-L-Araf-(1→;

[0007] The repeating unit of the structural formula of the acidic polysaccharide from Heracleum millefolium Diels is shown as follows:

[0008]

[0009] Among them, A is 1,3-α-L-arabinofuranosyl; B is 1,5-α-L-arabinofuranosyl; C is 1,3,5-α-L-arabinofuranosyl; D is 1,6-α-D-glucosyl; E is 1,4-α-D-glucuronic acid group; F is 1,3-β-D-galactosyl.

[0010] Furthermore, the acidic polysaccharide from Heracleum millefolium Diels is composed of galacturonic acid GalA, glucose Glc, galactose Gal and arabinose Ara, and the molar ratio of galacturonic acid GalA, glucose Glc, galactose Gal and arabinose Ara is 2.2:1.0:1.1:5.5.

[0011] The preparation method of the above-mentioned acidic polysaccharide from Heracleum millefolium Diels includes the following steps:

[0012] (1) Weigh the Tibetan medicine Heracleum millefolium Diels medicinal materials, add anhydrous ethanol and reflux to remove lipid impurities; evaporate to remove ethanol, add deionized water and decoct, combine the decoction, concentrate, centrifuge to take the supernatant, add anhydrous ethanol to precipitate, stand overnight at low temperature, centrifuge to collect the precipitate, and redissolve it in water to obtain the crude polysaccharide of Heracleum millefolium Diels;

[0013] (2) Add Sevag reagent to the crude polysaccharide of Heracleum millefolium Diels to remove protein impurities;

[0014] (3) Mix the Heracleum millefolium Diels polysaccharide, water, and hydrogen peroxide, heat for oxidative decolorization, concentrate, centrifuge to collect the supernatant, and dialyze to remove small molecular impurities of pigments. Collect the liquid in the bag to obtain the total Heracleum millefolium Diels polysaccharide HMPS;

[0015] (4) Pass the total Heracleum millefolium Diels polysaccharide HMPS through a DEAE Sephadex A-25 ion exchange column and elute with distilled water to obtain the water-washed fraction HMPS-1;

[0016] (5) Pass the water-washed fraction HMPS-1 through a Sephadex G-100 dextran gel column, elute with an NaCl solution, collect in fractions with an automatic collector, and determine the carbohydrate content of each tube by the phenol-sulfuric acid method. Plot a standard curve with elution volume-absorbance, and collect the fraction corresponding to the elution peak with a molecular weight of 1.3×10 4 Da to obtain the target product, Heracleum millefolium Diels acidic polysaccharide.

[0017] Further, in step (1), the decocting temperature is 75-85°C; the temperature of the low temperature is 3-5°C.

[0018] Further, in step (2), the volume ratio of the Heracleum millefolium Diels crude polysaccharide to the Sevag reagent is (3-5):1.

[0019] Further, in step (2), the volume ratio of chloroform to n-butanol in the Sevag reagent is 5:1.

[0020] Further, in step (3), the molar ratio of the Heracleum millefolium Diels crude polysaccharide, water, and hydrogen peroxide is 10:1:0.5.

[0021] Further, in step (3), the heating temperature is 65-75°C.

[0022] Further, in step (5), the concentration of the NaCl solution is 0.08-0.12 mol·L -1 .

[0023] Application of the above Heracleum millefolium Diels acidic polysaccharide in the preparation of anti-colorectal cancer drugs. The Heracleum millefolium Diels acidic polysaccharide HMPS-1b can prolong the survival time of mice in the AOM / DSS-induced colorectal cancer model; the Heracleum millefolium Diels acidic polysaccharide HMPS-1b can relieve the weight loss of mice in the AOM / DSS-induced colorectal cancer model.

[0024] Further, the Heracleum millefolium Diels acidic polysaccharide HMPS-1b can reduce the total number of tumors, shrink the tumor volume, and alleviate inflammatory damage.

[0025] Positive effects of the present invention:

[0026] (1) The acidic polysaccharide HMPS-1b from Heracleum millefolium var. dissectum obtained in the present invention is a homogeneous polysaccharide, and the preparation process is simple and easy to realize industrial application.

[0027] (2) The acidic polysaccharide HMPS-1b from Heracleum millefolium var. dissectum obtained in the present invention can prolong the survival time of mice in the AOM / DSS-induced colon cancer model; and can relieve the weight loss of mice in the AOM / DSS-induced colon cancer model.

[0028] (3) The acidic polysaccharide HMPS-1b from Heracleum millefolium var. dissectum obtained in the present invention can reduce the total number of tumors, shrink the tumor volume, and relieve inflammatory damage, showing obvious effects against colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the separation and purification curve of HMPS-1 by Sephadex G-100 column;

[0030] Figure 2 is the HPSEC chromatogram of HMPS-1a and HMPS-1b;

[0031] Figure 3 is the FT-IR spectra of HMPS-1a and HMPS-1b;

[0032] Figure 4 is the HPLC chromatogram of HMPS-1a and HMPS-1b;

[0033] Figure 5 is the 1D and 2D NMR spectra of HMPS-1b obtained in Example 1, (A) 1H NMR; (B) 13C NMR; (C) HSQC; (D) COSY; (E) TOCSY; (F) HMBC;

[0034] Figure 6 is the effect of HMPS-1a and HMPS-1b on the proliferation of colon cancer cells;

[0035] Figure 7 is the effect of HMPS-1b on the apoptosis of HCT116 cells;

[0036] Figure 8 is the effect of HMPS-1b on the proliferation cycle of HCT116 cells;

[0037] Figure 9 is the effect of HMPS-1b on the survival time of mice in the AOM / DSS-induced colon cancer model;

[0038] Figure 10 is the effect of HMPS-1b on the tumor growth of mice in the AOM / DSS-induced colon cancer model;

[0039] Figure 11 Effect of HMPS-1b on the body weight of mice in the AOM / DSS-induced colon cancer model;

[0040] Figure 12 Effect of HMPS-1b on the DAI score of the colon in mice with the AOM / DSS-induced colon cancer model;

[0041] Figure 13 Observation of the morphological and structural changes of the colon tissue in mice with the colon cancer model by HE staining. Specific implementation mode

[0042] The present invention will be further described below in conjunction with embodiments.

[0043] In the first aspect, the present invention provides an acidic polysaccharide HMPS-1b from Heracleum millefolium Diels, and the molecular weight of the acidic polysaccharide from Heracleum millefolium Diels is 1.3×10 4 Da. The main chain repeating unit of the acidic polysaccharide from Heracleum millefolium Diels is: →3)-α-L-Araf-(1→5)-α-L-Araf-(1→3,5)-α-L-Araf-(1→6)-α-D-Glcp-(1→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→3)-β-D-Galp-(1→, and the side chain is →5)-α-L-Araf-(1→3)-α-L-Araf-(1→;

[0044] The repeating unit of the structural formula of the acidic polysaccharide from Heracleum millefolium Diels is shown as follows:

[0045]

[0046] Among them, A is 1,3-α-L-arabinofuranosyl; B is 1,5-α-L-arabinofuranosyl; C is 1,3,5-α-L-arabinofuranosyl; D is 1,6-α-D-glucosyl; E is 1,4-α-D-glucuronic acid group; F is 1,3-β-D-galactosyl.

[0047] The acidic polysaccharide from Heracleum millefolium Diels is composed of galacturonic acid GalA, glucose Glc, galactose Gal and arabinose Ara, and the molar ratio of galacturonic acid GalA, glucose Glc, galactose Gal and arabinose Ara is 2.2:1.0:1.1:5.5.

[0048] In the second aspect, the present invention provides a preparation method of the above-mentioned acidic polysaccharide HMPS-1b from Heracleum millefolium Diels, including the following steps:

[0049] (1) Weigh the Tibetan medicine Heracleum millefolium Diels, add anhydrous ethanol and reflux to remove lipid impurities; volatilize the ethanol, add deionized water and decoct at 75 - 85 °C, combine the decoction liquids, concentrate, centrifuge to obtain the supernatant, add anhydrous ethanol for precipitation, let it stand overnight at 3 - 5 °C, centrifuge to collect the precipitate, and redissolve it in water to obtain the crude polysaccharide of Heracleum millefolium Diels. In step (1), the present invention does not limit the quality of the medicinal material and the volume of anhydrous ethanol, as long as it can remove the lipid impurities in the medicinal material through multiple anhydrous ethanol refluxes. The dosage in the following examples is only for illustration. The present invention does not limit the dosage of deionized water, the number of decoction times and the decoction time each time, as long as it can achieve sufficient decoction and extract as many active ingredients as possible. The dosage in the following examples is only for illustration. The present invention does not limit the dosage of anhydrous ethanol added after the decoction is completed, as long as as much precipitation as possible can occur. The dosage in the following examples is only for illustration. The present invention does not limit the amount of water used to redissolve the precipitate, as long as it can be completely dissolved.

[0050] (2) Add Sevag reagent (the volume ratio of chloroform to n-butanol is 5:1) to the crude polysaccharide of Heracleum millefolium Diels to remove protein impurities. The volume ratio of the crude polysaccharide of Heracleum millefolium Diels to Sevag reagent is (3 - 5):1. In step (2), it is necessary to repeatedly add Sevag reagent to the crude polysaccharide of Heracleum millefolium Diels and freeze-thaw to remove protein impurities. The present invention does not limit the number of times of adding Sevag reagent, until no precipitation or turbidity appears. However, the amount of Sevag reagent added each time is limited. The volume ratio of the crude polysaccharide of Heracleum millefolium Diels to Sevag reagent is (3 - 5):1.

[0051] (3) According to the molar ratio of the crude polysaccharide of Heracleum millefolium Diels, water and hydrogen peroxide of 10:1:0.5, mix the crude polysaccharide of Heracleum millefolium Diels, water and hydrogen peroxide, heat at 65 - 75 °C for oxidative decolorization, concentrate, centrifuge to collect the supernatant, and dialyze to remove small molecular impurities of pigments. Collect the liquid in the bag, which is the total polysaccharide HMPS of Heracleum millefolium Diels. In step (3), the present invention does not limit the time of oxidative decolorization, as long as decolorization can be achieved. The present invention also does not limit the dialysis time, as long as as much total polysaccharide HMPS of Heracleum millefolium Diels as possible can be dialyzed out.

[0052] (4) Pass the total polysaccharide HMPS of Heracleum millefolium Diels through a DEAE Sephadex A-25 ion exchange column and elute with distilled water to obtain the eluted fraction HMPS-1. In step (4), the present invention does not limit the dosage of distilled water, based on eluting the eluted fraction HMPS-1.

[0053] (5) Pass the eluted fraction HMPS-1 through a Sephadex G-100 dextran gel column, 0.08 - 0.12 mol·L - 1Elute with NaCl solution, collect in separate tubes using an automatic collector, and determine the carbohydrate content of each tube by the phenol-sulfuric acid method. Plot a standard curve with elution volume - absorbance, and collect the component corresponding to the elution peak with a molecular weight of 1.3×10 4 Da to obtain the target product, Heracleum millefolium acidic polysaccharide HMPS-1b. In step (5), the present invention does not limit the amount of NaCl solution used, and the criterion is to elute the target product, Heracleum millefolium acidic polysaccharide HMPS-1b.

[0054] Thirdly, the present invention provides the application of the above Heracleum millefolium acidic polysaccharide in anti-colorectal cancer drugs.

[0055] The Heracleum millefolium acidic polysaccharide HMPS-1b can prolong the survival time of mice in the AOM / DSS-induced colorectal cancer model; the Heracleum millefolium acidic polysaccharide HMPS-1b can alleviate the weight loss of mice in the AOM / DSS-induced colorectal cancer model. The Heracleum millefolium acidic polysaccharide HMPS-1b can reduce the total number of tumors, shrink the tumor volume, and alleviate inflammatory damage.

[0056] Example 1

[0057] The preparation method of the Heracleum millefolium acidic polysaccharide HMPS-1b described in this example includes the following steps:

[0058] (1) The medicinal material of Angelica tibetica was identified by Professor Hubenxiang of Shaanxi University of Chinese Medicine as the dried whole herb of Heracleum millefolium. Weigh 1 kg of the medicinal material, reflux it 3 times with 4 times the volume of anhydrous ethanol to remove lipid impurities; evaporate the ethanol, add deionized water to 6 times the volume, wait for the medicinal slices to absorb water completely, decoct at 80 °C for 2 times, 1 h each time; combine the decoction liquids, concentrate, centrifuge to obtain the supernatant, add 4 times the volume of anhydrous ethanol for precipitation, let it stand overnight at 4 °C, centrifuge to collect the precipitate, and redissolve it in water to obtain the crude polysaccharide of Heracleum millefolium.

[0059] (2) Repeatedly add Sevag reagent (the volume ratio of chloroform to n-butanol is 5:1) to the crude polysaccharide of Heracleum millefolium and freeze-thaw to remove protein impurities. The volume ratio of the crude polysaccharide of Heracleum millefolium to the Sevag reagent added each time is 4:1;

[0060] (3) According to the molar ratio of the crude polysaccharide of Heracleum millefolium, water and hydrogen peroxide of 10:1:0.5, mix the crude polysaccharide of Heracleum millefolium, water and hydrogen peroxide evenly, adjust the pH to 7, heat at 70 °C for 4 h for oxidative decolorization, concentrate, centrifuge to collect the supernatant, and dialyze for 48 h to remove small molecule impurities of pigments. Collect the liquid inside the dialysis bag, which is the total polysaccharide HMPS of Heracleum millefolium;

[0061] (4) Pass the total polysaccharide HMPS of Heracleum millefolium through a DEAE Sephadex A-25 ion exchange column and elute with distilled water to obtain the water-washed fraction HMPS-1;

[0062] (5) Pass the water-washed component HMPS-1 through a Sephadex G-100 dextran gel column and elute with 0.1 mol·L -1 NaCl solution. The automatic collector collects 5 mL per tube, and the carbohydrate content of each tube is determined by the phenol-sulfuric acid method. A standard curve is plotted with elution volume-absorbance. Collect the component corresponding to the elution peak with a molecular weight of 1.3×10 4 Da to obtain the target product Heracleum millefolium acidic polysaccharide HMPS-1b; through a Sephadex G-75 column and elute with deionized water to desalt and purify the Heracleum millefolium acidic polysaccharide HMPS-1b.

[0063] Comparative Example 1

[0064] The method and parameters are the same as those in Example 1, except that: while obtaining the target product HMPS-1b, the component corresponding to the elution peak with a molecular weight of 1.7×10 5 Da can be collected to obtain Heracleum millefolium polysaccharide HMPS-1a. The differences and performance differences between the two polysaccharides are analyzed by structural feature comparison.

[0065] Example 2

[0066] The preparation method of the Heracleum millefolium acidic polysaccharide HMPS-1b described in this example includes the following steps:

[0067] (1) The medicinal material of Angelica tibetica was identified by Professor Hubenxiang of Shaanxi University of Chinese Medicine as the dried whole herb of Heracleum millefolium. Weigh 1 kg of the medicinal material, add 5 times the volume of anhydrous ethanol and reflux 4 times to remove lipid impurities; volatilize the ethanol, add deionized water to 5 times the volume, wait for the cut pieces to absorb water completely, decoct at 75°C for 3 times, 2 h each time; combine the decoction liquids, concentrate, centrifuge to take the supernatant, add 3 times the volume of anhydrous ethanol for precipitation, stand overnight at 5°C, centrifuge to collect the precipitate, and redissolve it in water to obtain the crude polysaccharide of Heracleum millefolium.

[0068] (2) Repeatedly add Sevag reagent (the volume ratio of chloroform to n-butanol is 5:1) to the crude polysaccharide of Heracleum millefolium and freeze-thaw to remove protein impurities. The volume ratio of the crude polysaccharide of Heracleum millefolium to the Sevag reagent added each time is 3:1;

[0069] (3) According to the molar ratio of the crude polysaccharide of Heracleum millefolium, water and hydrogen peroxide of 10:1:0.5, mix the crude polysaccharide of Heracleum millefolium, water and hydrogen peroxide evenly, pH = 7, heat at 75°C for 3 h for oxidative decolorization, concentrate, centrifuge to collect the supernatant, and dialyze for 36 h to remove small molecule impurities of pigments. Collect the liquid in the bag, which is the total polysaccharide HMPS of Heracleum millefolium;

[0070] (4) Pass the total polysaccharide HMPS of Heracleum millefolium through a DEAE Sephadex A-25 ion exchange column and elute with distilled water to obtain the water-washed fraction HMPS-1;

[0071] (5) Pass the water-washed fraction HMPS-1 through a Sephadex G-100 dextran gel column and elute with 0.12 mol·L -1 NaCl solution. Automatically collect 5 mL per tube with an automatic collector, and determine the carbohydrate content of each tube by the phenol-sulfuric acid method. Plot a standard curve with elution volume - absorbance, and collect the fraction corresponding to the elution peak with a molecular weight of 1.3×10 4 Da to obtain the target product, the acidic polysaccharide HMPS-1b of Heracleum millefolium; Pass through a Sephadex G-75 column and elute with deionized water to desalt and purify the acidic polysaccharide HMPS-1b of Heracleum millefolium.

[0072] Example 3

[0073] The preparation method of the acidic polysaccharide HMPS-1b of Heracleum millefolium described in this example includes the following steps:

[0074] (1) The medicinal material of Angelica tibetica was identified by Professor Hubenxiang of Shaanxi University of Chinese Medicine as the dried whole herb of Heracleum millefolium. Weigh 1 kg of the medicinal material, reflux twice with 6 times the volume of anhydrous ethanol to remove lipid impurities; volatilize the ethanol, add deionized water to 5 times the volume, wait for the cut pieces to absorb water completely, decoct at 85 °C for 3 times, 2 h each time; combine the decoction liquids, concentrate, centrifuge to take the supernatant, add 5 times the volume of anhydrous ethanol to precipitate, stand overnight at 3 °C, centrifuge to collect the precipitate, and redissolve it in water to obtain the crude polysaccharide of Heracleum millefolium.

[0075] (2) Repeatedly add Sevag reagent (the volume ratio of chloroform to n-butanol is 5:1) to the crude polysaccharide of Heracleum millefolium and freeze-thaw to remove protein impurities. The volume ratio of the crude polysaccharide of Heracleum millefolium to the Sevag reagent added each time is 5:1;

[0076] (3) According to the molar ratio of the crude polysaccharide of Heracleum millefolium, water and hydrogen peroxide of 10:1:0.5, mix the crude polysaccharide of Heracleum millefolium, water and hydrogen peroxide evenly, adjust the pH to 7, heat at 65 °C for 5 h for oxidative decolorization, concentrate, centrifuge to collect the supernatant, and dialyze for 40 h to remove small molecule impurities of pigments. Collect the liquid inside the bag, which is the total polysaccharide HMPS of Heracleum millefolium;

[0077] (4) Pass the total polysaccharide HMPS of Heracleum millefolium through a DEAE Sephadex A-25 ion exchange column and elute with distilled water to obtain the water-washed fraction HMPS-1;

[0078] (5) Pass the water-washed fraction HMPS-1 through a Sephadex G-100 dextran gel column and elute with 0.08 mol·L-1 Eluted with NaCl solution, and the automatic collector collected 5 mL per tube. The carbohydrate content of each tube was determined by the phenol-sulfuric acid method. A standard curve was plotted with elution volume - absorbance. The elution peak corresponding to the component with a molecular weight of 1.3×10 4 Da was collected to obtain the target product Heracleum millefolium acidic polysaccharide HMPS-1b; through a Sephadex G-75 column and eluted with deionized water, the Heracleum millefolium acidic polysaccharide HMPS-1b was desalted and purified.

[0079] Structure and property tests of Heracleum millefolium acidic polysaccharide HMPS-1b:

[0080] (I) Analysis of the structural characteristics of Heracleum millefolium polysaccharide

[0081] (1) Isolation and purification of HPMS-1b

[0082] As in Example 1, HMPS was separated by a DEAE-Sephadex A-25 ion exchange column and eluted with distilled water to obtain 1 sub-component HMPS-1. HMPS-1 was further separated and purified by a Sephadex G-100 dextran gel column ( Figure 1 ), eluted with 0.1 M NaCl, and the sugar content was determined by the phenol-sulfuric acid method. The sugar-containing components with different molecular weights were collected and named HMPS-1a and HMPS-1b respectively, with yields of 18.7% and 13.5% respectively.

[0083] (2) Determination of homogeneity and relative molecular weight

[0084] A series of standard dextrans (T-10, T-50, T-120, T-50, T-150, and T-410) and the samples were dissolved in 0.1 M NaNO3 before use, with a final concentration of 1 mg / mL. After high-speed centrifugation, 200 μL was taken for analysis by GPC-RI-MALS. Chromatographic columns: Ohpak SB-805HQ (300×8 mm), Ohpak SB-804HQ (300×8 mm), and Ohpak SB-803HQ (300×8 mm) in series; injection volume 10 μL, mobile phase 0.1 M NaNO3, flow rate 0.5 mL / min, column temperature 45 °C, isocratic elution for 100 min. The sample concentration was detected by the refractive index intensity provided by the differential detector, the light scattering of macromolecules was detected by a multi-angle laser light scattering instrument, and the corresponding molecular weight of the component was calculated according to the Mark-Houwink Equation.

[0085] The HPSEC determination results showed that the HPSEC chromatogram peaks of the two Heracleum millefolium polysaccharides HMPS-1b and HMPS-1a in Example 1 and Comparative Example 1 were both single and symmetrical, indicating homogeneous polysaccharides (Figure 2 ) The relative molecular masses were calculated by the standard curve method, and the relative molecular masses of HMPS-1a and HMPS-1b were 1.7×10 5 Da and 1.3×10 4 Da, respectively.

[0086] (3) Determination of sugar content, uronic acid content, and impurity protein content of HMPS-1a in Comparative Example 1 and HMPS-1b in Example 1

[0087] The sugar content, impurity protein content, and uronic acid content were determined by the phenol-sulfuric acid method, Coomassie brilliant blue method, and sulfuric acid-carbazole method, respectively. Standard curves were plotted and the mass percentages of sugar content, impurity protein content, and uronic acid content were obtained by calculation. The mass percentages of sugar content, uronic acid content, and impurity protein content of HMPS-1a were 95.8%, 1.1%, and 1.4%, respectively; the mass percentages of sugar content, uronic acid content, and impurity protein content of HMPS-1b were 96.4%, 1.3%, and 0.8%, respectively.

[0088] (4) FT-IR analysis

[0089] Weigh 2 mg of polysaccharide respectively, grind and mix it evenly with dry KBr, press it into tablets, and measure the IR from 4000 cm -1 to 400 cm -1 using an infrared spectrometer to determine the main functional groups in the sugar molecule and the glycosidic bond configuration of pyranose.

[0090] The characteristic functional groups of HMPS-1a in Comparative Example 1 and HMPS-1b in Example 1 were determined by FT-IR, and the results are shown in Figure 3 . Among them, the signal near 3393 cm -1 is the O-H stretching vibration, the signals near 2929 cm -1 and 1422 cm -1 are the C-H stretching vibration and bending vibration, the signal near 1356 cm -1 is the out-of-ring C-O stretching vibration of the polysaccharide ring, and the signal near 1652 cm -1 is the O-H deformation vibration produced by the polysaccharide containing bound water. In addition, the signals near 1015, 1279, and 1156 cm -1 are the C-O bending vibrations of pyranose.

[0091] (5) Determination of monosaccharide composition and proportion by high performance liquid chromatography

[0092] Dissolve 5 mg of polysaccharide sample in 1 mL of TFA (2 M), heat and hydrolyze it at 121 °C for 2 h, dry it under nitrogen, wash it with methanol 3 times, dissolve the sample with sterile water, and take 200 μL for testing. Chromatographic column: Dionex TM CarboPacTM PA20 (150 * 3.0 mm, 10 μm); Detector: Electrochemical detector; Injection volume: 5 μL; Column temperature: 30 °C. Mobile phase A (H2O), Mobile phase B (0.1 M NaOH), Mobile phase C (0.1 M NaOH, 0.2 M NaAc), Flow rate 0.5 mL / min; Column temperature: 30 °C; Gradient elution.

[0093] The monosaccharide composition of HMPS-1a in Comparative Example 1 and HMPS-1b in Example 1 was determined by HPLC method, and the results are as Figure 4 shown. HMPS-1a is mainly composed of Rha, GalA, Gal and Ara, with a molar ratio of 2.3:5.5:6.2:1.8; HMPS-1b is mainly composed of GalA, Glc, Gal and Ara, with a molar ratio of 2.2:1.0:1.1:5.5.

[0094] (6) 1D and 2D NMR analysis of the sugar residue types and linkages of the acidic polysaccharide HMPS-1b from Heracleum millefolium in Example 1

[0095] 1D and 2D NMR were used to further analyze the sugar residue composition and linkage sequence of HMPS-1b in Example 1: ① Each polysaccharide sample was deuterium-exchanged three times and then dissolved in 500 μL of deuterium oxide. ② 1D NMR (1H NMR, 13C NMR) and 2D NMR (1H-1HCOSY, HSQC, HMBC, NOESY) spectra were detected using a Bruker Avance 500 MHz spectrometer.

[0096] The results are as Figure 5 shown. The main chain repeating unit of HMPS-1b in Example 1 is: →3)-α-L-Araf-(1→5)-α-L-Araf-(1→3,5)-α-L-Araf-(1→6)-α-D-Glcp-(1→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→3)-β-D-Galp-(1→, and the side chain is →5)-α-L-Araf-(1→3)-α-L-Araf-(1→; The repeating unit of the structural formula of the acidic polysaccharide from Heracleum millefolium is as follows:

[0097]

[0098] Among them, A is 1,3-α-L-arabinofuranosyl; B is 1,5-α-L-arabinofuranosyl; C is 1,3,5-α-L-arabinofuranosyl; D is 1,6-α-D-glucopyranosyl; E is 1,4-α-D-glucuronic acid group; F is 1,3-β-D-galactopyranosyl.

[0099] (2) Evaluation of the in vitro anti-tumor activity of Heracleum millefolium polysaccharide

[0100] (1) Culture of human colon cancer cells and normal human intestinal epithelial cells

[0101] Human colon cancer cell lines HCT116 and SW480 were selected and cultured in DMEM or RPMI1640 culture medium containing 10% fetal bovine serum in an incubator at 37°C with 5% CO2.

[0102] (2) Detection of cell proliferation by CCK-8

[0103] Logarithmic growth phase colon cancer HCT116 and SW480 cells were seeded into 96-well plates at 5×10 3 / well. After the cells adhered overnight, they were cultured with HMPS-1a in Comparative Example 1 or HMPS-1b in Example 1 at a final concentration of 0, 0.1 - 100 mg / L for 48 h. Each group had 8 replicates. 10 μL of CCK-8 solution was added to each well, and the mixture was incubated at 37°C for 2 h. The absorbance value (OD) was measured at 490 nm using an enzyme-linked immunosorbent assay reader. The experiment was repeated 3 times. The cell inhibition rate was calculated according to the following formula: Cell inhibition rate (%) = (ODcontrol group - ODexperimental group) / (ODcontrol group - ODblank group) × 100%. A proliferation inhibition curve was plotted with the logarithmic drug concentration as the abscissa and the inhibition rate as the ordinate.

[0104] The effects of HMPS-1a in Comparative Example 1 and HMPS-1b in Example 1 on the proliferation of two colon cancer cells, HCT116 and SW480, were determined by the CCK-8 method. The results are as Figure 6 shown. Both HMPS-1a in Comparative Example 1 and HMPS-1b in Example 1 significantly inhibited the proliferation of HCT116 and SW480, and the inhibitory effect of HMPS-1b in Example 1 was stronger. The inhibitory effect of HMPS-1b on HCT116 cells was the most prominent, with a maximum inhibition rate of 78.5%, and the maximum inhibition rate on SW480 was 75.2%. The maximum inhibition rates of HMPS-1a in Comparative Example 1 on HCT116 and SW480 were 48.4% and 55.4% respectively.

[0105] (3) Detection of cell cycle and apoptosis by flow cytometry

[0106] Logarithmic growth phase colon cancer cells were taken and seeded at 1×10 5Cells were inoculated into six-well plates at a density of / mL, 3 mL per well. After the cells adhered overnight, HMPS-1b from Example 1 with final concentrations of 50 and 100 mg / L were added respectively, denoted as HMPS-1b-L (low concentration) and HMPS-1b-H (high concentration). The control group was only added with culture medium, and each group was set with 4 replicate wells. After culturing for 48 h, the cells were collected and washed twice with pre-cooled PBS. The cells were resuspended with binding buffer, and the cell density was adjusted to 1 - 5×10 6 / mL. 100 μL of the cell suspension was taken respectively for cell cycle and apoptosis assays. Cell cycle: The cells were suspended with pre-cooled 95% ethanol and fixed at 4°C for 12 h. After washing twice with pre-cooled PBS, 10 μL of PI fluorescent dye was added and stained for 15 min in the dark, and then detected by flow cytometry. Apoptosis rate: 5 μL of Annexin V / FITC was added and incubated at room temperature in the dark for 5 min; 10 μL of PI staining solution and 400 μL of PBS were added, and flow cytometry was immediately performed.

[0107] As Figure 7 shown, compared with the normal group, after administration of HMPS-1b-L (low concentration) or HMPS-1b-H (high concentration), the apoptosis rates of HCT116 increased to 19.3% and 33.0% respectively. This result indicates that HMPS-1b can significantly induce apoptosis of HCT116 cells.

[0108] As Figure 8 shown, compared with the normal group, after administration of HMPS-1b-L (low concentration) or HMPS-1b-H (high concentration), the proportions of HCT116 cells in the S phase of the rapid proliferation period decreased to 25.92% and 20.41% respectively. This result indicates that HMPS-1b can significantly inhibit the rapid proliferation of HCT116 cells.

[0109] (III) Study on the anti-colon cancer effect of Heracleum millefolium polysaccharide in vivo

[0110] (1) Construction of the model, experimental process, recording and analysis

[0111] (1.1) Experimental animals

[0112] 100 C57BL / 6J mice (5 weeks old, male). Purchased from Vital River Laboratories in Beijing. Raised in the animal experiment center. The mice were experimentally treated after 1 week of adaptive feeding. The mice were experimentally treated after 1 week of adaptive feeding. The mice had normal food intake during the experiment.

[0113] (1.2) Reagents and consumables

[0114] Drugs: 13 g of HMPS-1b obtained in Example 1. Dextran sulfate sodium (DSS), Yisheng Company, 135 g is needed. Azoxymethane (AOM), sigma, 25 mg. 1 ml syringe; tin foil; 50 ml disposable centrifuge tube; plastic-sealed bag; 15 ml centrifuge tube; centrifuge tube rack, etc.

[0115] (1.3) Establishment of AOM / DSS mouse model, experimental grouping and administration

[0116] After one week of adaptive feeding, on the 8th day, after weighing, AOM was intraperitoneally injected at a dose of 10 mg / kg (a total of 92 mice); in the third week, the drinking water of the groups other than the normal group was changed to an aqueous DSS solution for one week, and normal drinking water was provided in the fourth and fifth weeks; in the sixth week, the drinking water of the groups other than the normal group was changed to an aqueous DSS solution for one week, and normal drinking water was provided in the seventh and eighth weeks; in the ninth week, the drinking water of the groups other than the normal group was changed to an aqueous DSS solution for one week, and normal drinking water was provided in the tenth, eleventh, and twelfth weeks, and the mice were sacrificed on the 85th day. After SPF environmental adaptation feeding, the mice were numbered and weighed, and they were randomly divided into 7 groups (20 cage positions were needed) by the random number table method: ① Normal control group (Control; n = 10); ② Model group (Model; n = 15); ③ Low-dose HMPS-1b group (HMPS-1b-L; n = 15); ④ High-dose HMPS-1b group (HMPS-1b-H; n = 15); After adaptive feeding, 10 mice in the normal control group were randomly selected as the blank control group. The remaining mice were intraperitoneally injected with AOM for modeling, and deaths occurred 2 - 3 days later. One week later, the surviving mice were grouped according to the above-mentioned planned numbers. Starting from the fourth week, gavage administration was carried out according to the above grouping. The low-dose and high-dose HMPS-1b groups were gavaged with the corresponding doses of the drug, and the Control group and the Model group were given an equal amount of normal saline once a day for 63 days.

[0117] (1.4) General condition observation

[0118] During the experiment, the general conditions of the mice in each group were observed and recorded weekly, including activity response, mental state, body hair color, food intake, water intake, body weight change, stool characteristics, and fecal conditions. ① Record the survival percentage. ② Record the body weight of the mice every week (weigh on Sunday), and draw a line graph of body weight change and a percentage graph of initial weight. ③ Conduct a disease activity index score DAI = (body weight loss score + stool character score + blood in stool score) / 3, and the scoring standard was formulated with reference to relevant research standards. The body weight loss score was based on the body weight of the mice on the first day of the intervention (the day of intraperitoneal injection of AOM). ④ Record the food consumption of the mice every week. Starting from the day of intraperitoneal injection of AOM, 200 g of food should be weighed well when feeding, and it should be recorded when adding food, 50 g each time. Weigh the remaining food amount on Sunday every week and start recording again.

[0119] (1.5) Organ Index

[0120] Isolate the spleen, rinse it with normal saline, conduct gross observation and take photos for record, blot it dry with filter paper and weigh it, then calculate the spleen index. Spleen index = spleen mass (mg) / mouse body mass (g). Isolate the thymus, rinse it with normal saline, conduct gross observation and take photos for record, blot it dry with filter paper and weigh it, then calculate the thymus index. Thymus index = thymus mass (mg) / mouse body mass (g).

[0121] (1.6) HE Staining

[0122] Deparaffinizing the paraffin sections to water is a process of gradually removing paraffin and rehydrating the sections. The detailed steps are as follows: Place the paraffin sections in xylene. Xylene can dissolve paraffin to achieve deparaffinization. Generally, it needs to be placed in xylene for 15 - 20 minutes, and the specific time can be adjusted according to the actual situation. After xylene treatment, place the sections in absolute ethanol. The function of absolute ethanol is to remove xylene and further remove the residual paraffin. Place it in absolute ethanol for 5 minutes. Then, place the sections successively in 85% alcohol and 75% alcohol, each time for 5 minutes. This is to gradually reduce the content of organic solvents in the sections and prepare to enter the water environment. Finally, place the sections in distilled water to completely remove organic solvents and complete the hydration process.

[0123] Antigen retrieval: Immerse the sections in a citric acid antigen retrieval buffer (pH = 6.0), and perform antigen retrieval using a microwave oven at medium heat until boiling for 3 min, let it stand at room temperature and then re - boil at medium heat for 2 min, and cool to room temperature. Immerse the sections in PBS (pH = 7.4), shake and wash for 5 min * 3 times and 10 min * 1 time. Then permeabilize with Triton (0.3%) at room temperature for 20 min, and wash with PBS * 4 times; 5 min for the first 3 times and 10 min for the 4th time.

[0124] Blocking endogenous peroxidase: Place the sections in a 3% hydrogen peroxide aqueous solution, let it stand at room temperature in the dark for 25 min, then shake and wash in PBS for 5 min * 3 times and 10 min * 1 time.

[0125] Serum blocking: Drop goat serum on the tissue and block at room temperature for 50 min. (If the primary antibody is from goat, use rabbit serum to block; for other sources, use BSA to block) Add the primary antibody: Gently shake off the blocking solution, drop the primary antibody on the section tissue, and incubate at room temperature in a wet box for 2 h. Add the secondary antibody: Wash in PBS by shaking for 5 min * 3 times and 10 min * 1 time. Gently shake off the PBS, drop the secondary antibody on the tissue, incubate at room temperature for 1 h, and wash with PBS for 5 min * 3 times. Then incubate with SABC at room temperature for 30 min, and wash with PBS for 5 min * 3 times and 10 min * 1 time.

[0126] DAB Chromogenic Reaction: Gently flick off the PBS, add DAB chromogenic solution (prepared freshly before use) dropwise onto the tissue, control the chromogenic time under microscopic observation (about 10 s), positive reaction shows brownish-yellow color, and gently rinse the section with running water for more than 10 min to terminate the chromogenic reaction.

[0127] Nuclear Counterstaining: Quickly add hematoxylin dropwise onto the tissue, counterstain for 2 s, flick off the staining solution and wash with tap water for about 1 min.

[0128] The steps of dehydration and mounting are as follows: Place the tissue section on the section rack, after standing at room temperature for 60 min, immerse it in xylene for 10 min, then transfer it to another staining jar with more xylene and immerse for another 10 min; after dewaxing, place the tissue section in absolute ethanol for 5 min, then 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, 50% ethanol for 5 min; after hydration treatment, incubate the tissue section in pure water for 5 min, then transfer it to PBS buffer and incubate for 5 min; conversely, first incubate in pure water for 5 min, then place it in 50% ethanol for 5 min, then 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, absolute ethanol for 5 min, and finally, perform transparency treatment in xylene twice, 10 min each time; after dehydration is completed, wipe off the liquid around the section, drop a drop of neutral balsam, cover with a coverslip, and gently tap the coverslip with the blunt end of the forceps to remove air bubbles to complete the mounting.

[0129] Microscopic examination and image acquisition and analysis.

[0130] (2) Experimental Results:

[0131] (2.1) Effect of HMPS-1b in Example 1 on the survival time of mice in the AOM / DSS-induced colon cancer model

[0132] The in vivo anti-colon cancer effect of HMPS-1b in Example 1 was evaluated using an AOM / DSS-induced colon cancer mouse model. First, the effect of HMPS-1b on the survival time of model mice was evaluated. The results are as Figure 9 shown. The average survival time of mice in the model group was 66.3 ± 6.9 days; the average survival times of mice in the low-dose (100 mg / kg) and high-dose (200 mg / kg) HMPS-1b treatment groups were 71.2 ± 7.2 days and 81.0 ± 2.3 days, respectively; compared with the model group, HMPS-1b significantly prolonged the survival time of mice after AOM / DSS modeling.

[0133] (2.2) Effect of HMPS-1b in Example 1 on the tumor growth of mice in the AOM / DSS-induced colon cancer model

[0134] Observe the effects of HMPS-1b on the growth size and number of intestinal tumors in model mice in Example 1. The results are as Figure 10 shown. Tumors occurred in all three groups of mice treated with AOM / DSS. The average number of tumors in the colon of the model group mice was 3.3 ± 0.3, and the tumor burden was 12.5 ± 0.7 mm; the average number of tumors in the low-dose and high-dose HMPS-1b treatment group mice were 3.0 ± 0.6 and 1.0 ± 0.6 respectively, and the tumor burdens were 7.0 ± 0.8 mm and 2.5 ± 1.3 mm respectively. Compared with the model group, the total number of tumors in the two HMPS-1b treatment groups was significantly reduced, the tumor volume was significantly reduced, and it showed a dose-dependent change.

[0135] (2.3) Effects of HMPS-1b on the body weight of AOM / DSS-induced colon cancer model mice in Example 1

[0136] During the entire study process, the body weight of the mice was recorded weekly, and the results are as Figure 11 shown. The body weight of the model group mice decreased rapidly during DSS treatment. When the DSS water was removed, although the body weight of the model group mice recovered somewhat, it was still lower than that of the normal group. At the last week (week 13), the average body weight ratio of the model group mice was 106.1% ± 1.8%; the average body weight ratios of the low-dose and high-dose HMPS-1b treatment group mice were 114.1% ± 2.3% and 122.7% ± 3.2% respectively. The body weight of the high-dose HMPS-1b treatment group mice was significantly higher than that of the model group (P < 0.01), indicating that HMPS-1b can alleviate AOM / DSS-induced weight loss.

[0137] (2.4) DAI score

[0138] The DAI score results of the mice are as Figure 12 shown. Compared with the normal group, the model group mice showed obvious clinical features related to colorectal cancer such as significant weight loss, diarrhea, and bloody stools, which was reflected in the stagewise sharp increase in the DAI score of the model group mice (P < 0.001). At the last week (week 13), the DAI score of the model group mice was 3.7 ± 0.3; the DAI scores of the low-dose and high-dose HMPS-1b treatment group mice were 1.5 ± 0.3 and 0.3 ± 0.2 respectively. HMPS-1b alleviated the symptoms of diarrhea, rectal bleeding, prolapse, and weight loss in the model group mice, and significantly reduced the DAI score (P < 0.001).

[0139] (2.5) HE staining

[0140] HE staining further confirmed the pathological manifestations of AOM / DSS-induced colorectal cancer in colon tissue and the improvement effect of HMPS-1b on colorectal cancer. As Figure 13As shown, the colonic crypts of the mice in the normal group were arranged neatly and compactly, and inflammatory cells were occasionally seen in the lamina propria and submucosa. In contrast, the glands in the colonic tissue of the mice in the model group were disorderly arranged, the crypts disappeared, the intestinal wall structure was damaged, the muscularis mucosa was discontinuous, and a large number of inflammatory cell infiltrations were visible in the submucosa. At the same time, abnormal crypt foci were observed in the colonic tissue of the mice in the model group, with enlarged and deeply stained cell nuclei, a significantly increased nuclear-cytoplasmic ratio, increased cellular atypia, and an elevated mitotic figure in local areas. In contrast, in the mice treated with HMPS-1b in Example 1, these inflammatory injuries were significantly alleviated and showed a dose-dependent change.

[0141] Other embodiments of the present application will be readily apparent to those skilled in the art upon consideration of the specification and practice of the disclosure herein. The present application is intended to cover any variations, uses, or adaptations of the present application that follow the general principles of the present application and include common general knowledge or conventional technical means in the technical field not disclosed herein. The specification and examples are only illustrative, and the present application is not limited to the precise structures described above and shown in the drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. An acidic polysaccharide from Heracleum millefolium, characterized in that, The molecular weight of the acidic polysaccharide from Heracleum millefolium is 1.3×10 4 Da, and the repeating unit of the structural formula is as follows: Among them, A is 1,3-α-L-arabinofuranosyl; B is 1,5-α-L-arabinofuranosyl; C is 1,3,5-α-L-arabinofuranosyl; D is 1,6-α-D-glucopyranosyl; E is 1,4-α-D-glucuronic acid; F is 1,3-β-D-galactosyl.

2. The acidic polysaccharide of Heracleum millefolium var. dissectum according to claim 1, characterized in that: The acidic polysaccharide from Heracleum millefolium is composed of galacturonic acid (GalA), glucose (Glc), galactose (Gal) and arabinose (Ara), and the molar ratio of galacturonic acid (GalA), glucose (Glc), galactose (Gal) and arabinose (Ara) is 2.2:1.0:1.1:5.

5.

3. The preparation method of the acidic polysaccharide of Heracleum millefolium Diels as claimed in claim 1, wherein, It includes the following steps: (1) Weigh the medicinal material Heracleum millefolium, add anhydrous ethanol and reflux to remove lipid impurities; volatilize to remove ethanol, add deionized water and decoct, combine the decoction, concentrate, centrifuge to obtain the supernatant, add anhydrous ethanol for precipitation, stand overnight at low temperature, centrifuge to collect the precipitate, redissolve it in water to obtain the crude polysaccharide from Heracleum millefolium. (2) Add Sevag reagent to the crude polysaccharide from Heracleum millefolium to remove protein impurities. (3) Mix the crude polysaccharide from Heracleum millefolium, water and hydrogen peroxide, heat for oxidative decolorization, concentrate, centrifuge to collect the supernatant, dialyze to remove small molecular impurities of pigments, and collect the liquid inside the bag, which is the total polysaccharide HMPS from Heracleum millefolium. (4) Pass the total polysaccharide HMPS from Heracleum millefolium through a DEAE Sephadex A-25 ion exchange column and elute with distilled water to obtain the eluted fraction HMPS-1. (5) Pass the water-washed component HMPS-1 through a Sephadex G-100 dextran gel column, elute with an NaCl solution, collect in separate tubes using an automatic collector, and determine the carbohydrate content of each tube by the phenol-sulfuric acid method. Plot a standard curve with elution volume-absorbance, and collect the component corresponding to the elution peak with a molecular weight of 1.3×10 4 Da to obtain the target product, Heracleum millefolium acidic polysaccharide.

4. The preparation method of the acidic polysaccharide of Heracleum millefolium var. dissectum according to claim 3, characterized in that: In step (1), the decocting temperature is 75-85 °C; the low temperature is 3-5 °C.

5. The preparation method of the acidic polysaccharide of Heracleum millefolium Diels according to claim 3, characterized in that: In step (2), the volume ratio of the crude polysaccharide from Heracleum millefolium to Sevag reagent is (3-5):

1.

6. The preparation method of the acidic polysaccharide of Heracleum millefolium var. dissectum according to claim 3, wherein: In step (2), the volume ratio of chloroform to n-butanol in the Sevag reagent is 5:

1.

7. The preparation method of the acidic polysaccharide of Heracleum millefolium var. dissectum according to claim 3, characterized in that: In step (3), the molar ratio of the crude polysaccharide from Heracleum millefolium, water and hydrogen peroxide is 10:1:0.

5.

8. The preparation method of the acidic polysaccharide of Heracleum millefolium Diels according to claim 3, wherein: In step (3), the heating temperature is 65-75 °C.

9. The preparation method of the acidic polysaccharide of Heracleum millefolium Diels according to claim 3, characterized in that: In step (5), the concentration of the NaCl solution is 0.08 to 0.12 mol·L -1 .

10. Use of the acidic polysaccharide from Heracleum millefolium according to claim 1 in the preparation of an anti-colorectal cancer drug.

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