Arabic glucan as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation and application of natural product polysaccharide, and particularly relates to arabinoglucan as well as a preparation method and application thereof. The molecular weight of the arabinoglucan is 9.7 * 10 < 4 > Da; the repeating unit of the arabinoglucan is alpha-T-Araf-> 6)-alpha-D-Glcp-(1-> 6)-alpha-D-Glcp-(1-> 6)-alpha-D-Glcp-(1-> 6)-alpha-D-Glcp-(1-> 6)-alpha-D-Glcp-(1-> 3)-alpha-L-Araf-(1-> 5)-alpha-L-Araf-(1-> 6)-alpha-D-Glcp-(1-> 6)-alpha-D-Glcp-(1-> 6)-alpha-D-Glcp The arabinoglucan is composed of glucose Glc and arabinose Ara, and the molar ratio of the glucose Glc to the arabinose Ara is 3.55: 1.06. The Arabic glucan HMPS-3a is a homogeneous polysaccharide, the preparation process is simple, and factory application is easy to realize; the arabinoglucan HMPS-3a can significantly inhibit the release of inflammatory factors and NO in THP-1 cells induced by radiation, and is an active polysaccharide with anti-inflammatory and immunoregulation effects; the high-dose treatment of the Arabic glucan HMPS-3a can significantly maintain the integrity of the intestinal form, and the protection rate reaches 82.3%; meanwhile, the inflammatory response is remarkably inhibited through treatment, neutrophil infiltration is reduced, the goblet cell density is recovered, and the continuous integrity rate of mucous membranes is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of the preparation and application of natural product polysaccharides, and particularly relates to an arabinoglucan, a preparation method thereof and an application thereof. Background Art

[0002] With the extensive application of nuclear technology in the fields of military, industry and medical treatment, etc., the continuous increase of military and civilian nuclear facilities, the risks of nuclear terrorist attacks, nuclear power plant leaks and nuclear radiation accidents caused by improper use or storage are increasing day by day. The small intestine is highly sensitive to ionizing radiation. High-dose whole-body irradiation or abdominal and pelvic tumor radiotherapy can damage the crypt-villus structure of the small intestinal epithelium, affect its absorption and barrier functions, and cause radiation-induced intestinal injury (RII). RII is a key factor for death and treatment failure of systemic radiation injury, but its pathogenesis has not been fully elucidated, and there are lack of effective diagnosis and prevention and treatment measures. Finding new therapeutic drugs and targets is an urgent need faced by the treatment of RII.

[0003] Traditional Chinese medicine believes that the cause of radiation disease belongs to the exogenous "fiery toxin", the disease location is in the intestine, the pathological factors are mainly "dampness, heat and toxin", the pathological nature is deficiency in origin and excess in superficiality, with deficiency and excess intermingled, with spleen deficiency as the origin and dampness-heat as the superficiality. For this pathogenesis, "clearing heat and promoting diuresis, cooling blood and stopping bleeding" is the core of the traditional Chinese medicine treatment method for RII. Chinese herbal medicines for clearing heat and cooling blood have been proven to have significant therapeutic effects on RII. For example, compound kushen injection has obvious preventive and therapeutic effects on acute radiation hemorrhagic enteritis in animal experiments.

[0004] Tibetan medicine is an important part of the treasure house of traditional Chinese medicine in China. Tibetan medicine has unique efficacy, a long history, and great potential for development and research. Tibetan angelica, scientific name Heracleum millefolium Diels, is a plant of the genus Heracleum in the Umbelliferae family. It grows in alpine meadows, shrubs or gravel areas at an altitude of 2700-4800m and is one of the main varieties of Tibetan medicine. According to "Chinese Herbal Medicine", Tibetan medicine Heracleum millefolium Diels has a pungent, slightly sweet taste and a neutral nature, and has the effects of cooling blood and stopping bleeding, removing dampness and detoxifying, and is often used to treat rheumatism, epistaxis, gingival bleeding, skin ecchymosis, etc. Tibetan angelica has been proven to be one of the mainstream drugs for preventing and treating acute altitude sickness characterized by hypoxia and strong ultraviolet rays. As an important water-soluble component in Tibetan angelica, the research on its separation and purification method and pharmacological effects is still blank and urgently needs to be systematically and deeply explored. Summary of the Invention

[0005] The purpose of the invention is to provide an arabinoglucan, a preparation method thereof, and the application of the arabinoglucan in the preparation of drugs for anti-radiation intestinal injury.

[0006] The implementation process of the invention is as follows:

[0007] An arabinoglucan, the molecular weight of the arabinoglucan being 9.7×10 4 Da; the structural formula of the repeating unit of the arabinoglucan is as follows:

[0008]

[0009] wherein, A is an α-T-L-arabinose residue; B is an α-1,6-D-glucose residue; C is an α-1,3-L-arabinose residue; D is an α-1,5-L-arabinose residue.

[0010] The repeating unit of the arabinoglucan is α-T-Araf→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-L-Araf-(1→5)-α-L-Araf-(1→.

[0011] Furthermore, the arabinoglucan is composed of glucose (Glc) and arabinose (Ara), and the molar ratio of glucose (Glc) to arabinose (Ara) is 3.55:1.06.

[0012] The preparation method of the above-mentioned arabinoglucan comprises the following steps:

[0013] (1) Raw material pretreatment

[0014] Take the dried rhizome tissue of Heracleum millefolium, obtain Heracleum millefolium powder through an ultrafine pulverizer, add ethanol, carry out ultrasonic degreasing treatment and then centrifuge to discard the supernatant, and dry the residue in vacuo to constant weight;

[0015] (2) Preparation of crude polysaccharide HMPS

[0016] Mix the dried and degreased powder obtained in step (1) with ultrapure water, carry out dynamic extraction using a high-pressure thermal reflux extraction system, concentrate the extract by rotary evaporation, add precooled absolute ethanol until the mass percentage of ethanol is more than 80%, let it stand, collect the precipitate by centrifugation, and then use absolute ethanol and acetone, and freeze-dry to obtain crude polysaccharide HMPS;

[0017] (3) Deproteinization treatment

[0018] Dissolve the crude polysaccharide HMPS in Tris-HCl buffer solution, then add Sevage reagent, vortex and centrifuge to remove the protein layer, repeat the operation until there is no white floc in the interface; after combining the aqueous phases, add pepsin for enzymatic hydrolysis, inactivate by boiling water bath, and obtain a deproteinized solution;

[0019] (4) Decoloration and purification

[0020] The deproteinized solution was dynamically adsorbed through a D301 macroporous adsorption resin column, and the breakthrough solution was collected and filtered through a filter membrane. The molecular weight cut-off of the ultrafiltration system was 10 kDa. After concentration, it was freeze-dried to obtain the total polysaccharide;

[0021] (5) DEAE-52 cellulose column chromatography

[0022] The total polysaccharide was dissolved in distilled water and loaded onto a pre-equilibrated DEAE-52 anion exchange column. It was eluted successively with 0, 0.5, 1 mol·L -1 NaCl gradients. The main peak was detected by the phenol-sulfuric acid method and collected in the 1 mol·L -1 NaCl elution fraction. After dialysis with a MWCO of 3500 Da to desalt and then freeze-drying, the salt-washed component HMPS-3 was obtained;

[0023] (6) Sephadex G-100 gel chromatography

[0024] The salt-washed component HMPS-3 was dissolved in 0.1 mol·L -1 NH4HCO3 buffer solution and loaded onto a Sephadex G-100 column. It was eluted with 0.1 mol·L -1 NaCl. The sugar content was determined by the phenol-sulfuric acid method, and the sugar-containing fraction with a molecular weight of 9.7×10 4 Da was collected. After ultrafiltration and concentration, it was freeze-dried to obtain the homogeneous target product arabinoglucan HMPS-3a.

[0025] Furthermore, in step (1), the dried rhizome tissue of Heracleum millefolium was processed into Heracleum millefolium powder with a particle size ≤100 μm by an ultrafine pulverizer; the mass-volume ratio of Heracleum millefolium powder to ethanol was 1 g:(14 - 16) mL; the mass percentage of the ethanol was more than 90%; the temperature of the ultrasound was 40 - 50 °C.

[0026] Furthermore, in step (2), the mass-volume ratio of the dried defatted powder to ultrapure water was 1 g:(28 - 33) mL; the pressure of the high-pressure thermal reflux extraction system was 0.14 - 0.16 MPa, the temperature was 110 - 130 °C, and the extraction time was 1 - 3 h; the temperature of the pre-cooled absolute ethanol was 4 - 10 °C; the temperature of the static standing was 2 - 6 °C.

[0027] Furthermore, in step (3), the mass-volume ratio of the crude polysaccharide HMPS to Tris-HCl buffer solution was 50:1; the volume ratio of the solution after dissolving the crude polysaccharide HMPS to the Sevage reagent added each time was 1:(3 - 5).

[0028] Furthermore, in step (3), the concentration of the Tris-HCl buffer solution was 0.04 - 0.06 mol·L -1, the pH is 8.0; the concentration of pepsin after being added to the aqueous phase is 0.4 - 0.6 mg / mL; the temperature of enzymatic hydrolysis is 36 - 38 °C.

[0029] Further, in step (3), the Sevage reagent consists of chloroform and n-butanol, and the volume ratio of chloroform to n-butanol in the Sevage reagent is 4:1;

[0030] Further, in step (4), the pore size of the filter membrane is 0.22 μm; in step (5), the concentration after the total polysaccharide is dissolved is 1.8 - 2.2 mg / mL.

[0031] Use of the above arabinoglucan in the preparation of drugs for preventing and treating radiation-induced intestinal injury.

[0032] Positive effects of the present invention:

[0033] (1) The arabinoglucan HMPS-3a of the present invention is a homogeneous polysaccharide, and the preparation process is simple and easy to realize industrial application.

[0034] (2) The arabinoglucan HMPS-3a of the present invention significantly inhibits the release of inflammatory factors and NO in radiation-induced THP-1 cells, and is an active polysaccharide with anti-inflammatory and immunomodulatory effects.

[0035] (3) High-dose treatment with the arabinoglucan HMPS-3a of the present invention significantly maintains the intestinal morphological integrity, the intestinal length is restored to 34.1 ± 0.6 cm, and the protection rate reaches 82.3%, which is significantly better than 68.5% of the glutamine group; at the same time, the treatment significantly inhibits the inflammatory response, reduces neutrophil infiltration, restores the goblet cell density, and improves the mucosal continuity integrity rate. Description of the drawings

[0036] Figure 1 is the separation and purification curve of HMPS-3 by Sephadex G-100 column;

[0037] Figure 2 is the FT-IR spectrum of arabinoglucan HMPS-3a;

[0038] Figure 3 is the HPSEC spectrum of arabinoglucan HMPS-3a;

[0039] Figure 4 is the HPLC spectrum of arabinoglucan HMPS-3a;

[0040] Figure 5 is the NMR spectrum of arabinoglucan HMPS-3a, where A is 1 1H NMR; B is 13CNMR; C is HSQC; D is COSY; E is TOCSY; F is HMBC;

[0041] Figure 6 is the effect diagram of arabinoglucan HMPS-3a on the levels of inflammatory factors and NO in radiation-induced THP-1 cells;

[0042] Figure 7 is the diagram of arabinoglucan HMPS-3a inhibiting radiation-induced DNA damage in HIEC cells;

[0043] Figure 8 is that arabinoglucan HMPS-3a inhibits radiation-induced apoptosis in HIEC cells;

[0044] Figure 9 is the effect diagram of HMPS-3a on the body weight of RII mice;

[0045] Figure 10 is the effect diagram of HMPS-3a on the small intestine length of RII mice;

[0046] Figure 11 is the diagram of arabinoglucan HMPS-3a improving radiation-induced pathological damage of intestinal tissues in mice. Specific embodiments

[0047] The present invention will be further described below in conjunction with embodiments.

[0048] In a first aspect, the present invention provides an arabinoglucan, and the molecular weight of the arabinoglucan is 9.7×10 4 Da; the structural formula of the repeating unit of the arabinoglucan is as follows:

[0049]

[0050] wherein, A is an α-T-L-arabinose residue; B is an α-1,6-D-glucose residue; C is an α-1,3-L-arabinose residue; D is an α-1,5-L-arabinose residue.

[0051] The repeating unit of the arabinoglucan is α-T-Araf→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-L-Araf-(1→5)-α-L-Araf-(1→; the arabinoglucan is composed of glucose Glc and arabinose Ara, and the molar ratio of the glucose Glc to the arabinose Ara is 3.55:1.06.

[0052] Second aspect, the present invention provides a method for preparing arabinogalactan, comprising the following steps:

[0053] (1) Raw material pretreatment

[0054] Take the dried rhizome tissue of Heracleum millefolium, and obtain Heracleum millefolium powder with a particle size ≤ 100 μm through an ultrafine pulverizer. Add ethanol with a mass percentage of more than 90% in an amount of 1 g:(14 - 16) mL according to the mass-volume ratio of Heracleum millefolium powder to ethanol. After ultrasonic degreasing treatment at 40 - 50 °C, centrifuge to discard the supernatant, and vacuum dry the residue at 30 - 50 °C until constant weight; the number of times of the above ultrasonic degreasing is not limited. In the embodiments of the present invention, 3 times, 20 min / time is taken as an example.

[0055] (2) Preparation of crude polysaccharide HMPS

[0056] Mix the dried and degreased powder obtained in step (1) with ultrapure water (the mass-volume ratio of the dried and degreased powder to ultrapure water is 1 g:(28 - 33) mL), and perform dynamic extraction for 1 - 3 h using a high-pressure thermal reflux extraction system (pressure is 0.14 - 0.16 MPa, temperature is 110 - 130 °C). After the extract is concentrated by rotary evaporation to 1 / 5 of the original volume, add 3 times the volume of precooled absolute ethanol (4 - 10 °C) until the mass percentage of ethanol is more than 80%. Let it stand at 2 - 6 °C for 10 - 15 h, collect the precipitate by centrifugation, wash it with absolute ethanol and acetone 3 - 5 times, and then freeze-dry to obtain crude polysaccharide HMPS;

[0057] (3) Deproteinization treatment

[0058] Dissolve the crude polysaccharide HMPS in Tris-HCl buffer solution with a concentration of 0.04 - 0.06 mol·L -1 and pH of 8.0 (the mass-volume ratio of the crude polysaccharide HMPS to Tris-HCl buffer solution is 50:1). Then, add Sevage reagent in an amount of 1:(3 - 5) according to the volume ratio of the solution after dissolving the crude polysaccharide HMPS to the Sevage reagent added each time. After vortex oscillation for 8 - 15 min, centrifuge to remove the protein layer, and repeat the above operation until there is no white floc at the interface; after combining the aqueous phases, add pepsin (the concentration of pepsin in the aqueous phase is 0.4 - 0.6 mg / mL) and perform enzymatic hydrolysis at 36 - 38 °C for 1.5 - 2 h, and inactivate it in a boiling water bath for 10 min to obtain a deproteinized solution; wherein, the Sevage reagent is composed of chloroform and n-butanol, and the volume ratio of chloroform to n-butanol in the Sevage reagent is 4:1.

[0059] (4) Decolorization and purification

[0060] In an embodiment of the present invention, taking a flow rate of 1.5 BV / h as an example, the protein-depleted solution was dynamically adsorbed through a D301 macroporous adsorption resin column (column volume 200 mL) 3 to 4 times, and the breakthrough solution was collected, filtered through a 0.22 μm filter membrane, ultrafiltered with a molecular weight cut-off of 10 kDa, concentrated, and freeze-dried to obtain the total polysaccharide;

[0061] (5) DEAE-52 cellulose column chromatography

[0062] The total polysaccharide was dissolved in distilled water to a concentration of 1.8 - 2.2 mg / mL after dissolution, and was loaded onto a pre-equilibrated DEAE-52 anion exchange column (2.6 × 40 cm). Taking a flow rate of 1 mL / min as an example, 0, 0.5, and 1 mol·L - 1 NaCl gradient elution (flow rate 1 mL / min) was carried out, and the main peak was detected by the phenol-sulfuric acid method in the 1.0 mol·L -1 NaCl elution fraction. After dialysis with a MWCO of 3500 Da for desalting and then freeze-drying, the salt-washed component HMPS-3 was obtained;

[0063] (6) Sephadex G-100 gel chromatography

[0064] The salt-washed component HMPS-3 was dissolved in 0.1 mol·L -1 NH4HCO3 buffer solution, loaded onto a Sephadex G-100 column, eluted with 0.1 mol·L -1 NaCl, and the sugar content was determined by the phenol-sulfuric acid method. The sugar-containing fraction with a molecular weight of 9.7 × 10 4 Da was collected, ultrafiltered and concentrated, and then freeze-dried to obtain the homogeneous target product arabinoglucan HMPS-3a.

[0065] In a third aspect, the present invention provides the application of the arabinoglucan in the preparation of a drug for preventing and treating radiation-induced intestinal injury. Specifically, the arabinoglucan HMPS-3a of the present invention significantly inhibits the release of inflammatory factors and NO in radiation-induced THP-1 cells, and is an active polysaccharide with anti-inflammatory and immunomodulatory effects; high-dose treatment with the arabinoglucan HMPS-3a significantly maintains the integrity of the intestinal morphology, the intestinal length is restored to 34.1 ± 0.6 cm, and the protection rate reaches 82.3%, which is significantly better than 68.5% of the glutamine group; at the same time, the treatment significantly inhibits the inflammatory response, the infiltration of neutrophils is reduced, the density of goblet cells is restored, and the mucosal continuity integrity rate is improved.

[0066] Example 1

[0067] The preparation method of the arabinoglucan described in this example includes the following steps:

[0068] (1) Raw material pretreatment

[0069] Take the dry rhizome tissue of Heracleum millefolium, and obtain Heracleum millefolium powder with a particle size ≤ 100 μm through an ultrafine pulverizer. Add ethanol with a mass percentage of 95% in an amount according to the mass-volume ratio of Heracleum millefolium powder to ethanol of 1 g:15 mL. Perform ultrasonic degreasing treatment at 45°C for 3 times, 20 min each time, then centrifuge and discard the supernatant. The residue is vacuum dried at 40°C to constant weight;

[0070] (2) Preparation of crude polysaccharide HMPS

[0071] Mix the dry degreased powder obtained in step (1) with ultrapure water (the mass-volume ratio of the dry degreased powder to ultrapure water is 1 g:30 mL), and perform dynamic extraction for 2 h using a high-pressure thermal reflux extraction system (pressure is 0.15 MPa, temperature is 120°C). After the extract is concentrated by rotary evaporation to 1 / 5 of the original volume, add 3 volumes of pre-cooled absolute ethanol at 7°C until the mass percentage of ethanol is 80%. Let it stand at 4°C for 12 h, centrifuge to collect the precipitate, wash it 3 times with absolute ethanol and acetone, and then freeze-dry to obtain crude polysaccharide HMPS;

[0072] (3) Deproteinization treatment

[0073] Dissolve the crude polysaccharide HMPS in Tris-HCl buffer solution with a concentration of 0.05 mol·L -1 and pH of 8.0 (the mass-volume ratio of the crude polysaccharide HMPS to Tris-HCl buffer solution is 50:1). Then add Sevage reagent in an amount according to the volume ratio of the solution after dissolving the crude polysaccharide HMPS to the Sevage reagent added each time of 1:4. After vortex oscillation for 10 min, centrifuge to remove the protein layer, and repeat the above operation until there is no white floc at the interface; after combining the aqueous phases, add pepsin (the concentration after adding pepsin to the aqueous phase is 0.5 mg / mL) and perform enzymatic hydrolysis at 36 - 38°C for 2 h, and inactivate it in a boiling water bath for 10 min to obtain a deproteinized solution; among them, the Sevage reagent is composed of chloroform and n-butanol, and the volume ratio of chloroform to n-butanol in the Sevage reagent is 4:1.

[0074] (4) Decolorization and purification

[0075] Pass the deproteinized solution through a D301 macroporous adsorption resin column (column volume 200 mL) at a flow rate of 1.5 BV / h for dynamic adsorption 3 times, collect the breakthrough liquid, filter it through a 0.22 μm filter membrane, and use an ultrafiltration system with a cut-off molecular weight of 10 kDa, concentrate it, and then freeze-dry to obtain the total polysaccharide;

[0076] (5) DEAE-52 cellulose column chromatography

[0077] The total polysaccharide was dissolved in distilled water to obtain a concentration of 2 mg / mL after dissolution. It was loaded onto a pre-equilibrated DEAE-52 anion exchange column (2.6×40 cm). Taking a flow rate of 1 mL / min as an example, it was eluted successively with 0, 0.5, 1 mol·L -1 NaCl gradients (flow rate 1 mL / min), and the main peak was detected by the phenol-sulfuric acid method. The 1.0 mol·L -1 NaCl elution fraction was collected, desalted by dialysis with MWCO 3500 Da, and then freeze-dried to obtain the salt-washed component HMPS-3;

[0078] (6) Sephadex G-100 gel chromatography

[0079] The salt-washed component HMPS-3 was dissolved in 0.1 mol·L -1 NH4HCO3 buffer solution, loaded onto a Sephadex G-100 column (1.6×60 cm). Taking a flow rate of 0.5 mL / min as an example, it was eluted with 0.1 mol·L -1 NaCl, and the sugar content was determined by the phenol-sulfuric acid method. The sugar-containing fraction with a molecular weight of 9.7×10 4 Da was collected, ultrafiltered and concentrated, and then freeze-dried to obtain the homogeneous target product arabinoglucan HMPS-3a, with a yield of 25.3%.

[0080] Example 2

[0081] The preparation method of the arabinoglucan described in this example includes the following steps:

[0082] (1) Raw material pretreatment

[0083] Take the dried rhizome tissue of Heracleum millefolium, and obtain Heracleum millefolium powder with a particle size ≤100 μm through an ultrafine pulverizer. Add ethanol with a mass percentage of 90% according to the mass-volume ratio of Heracleum millefolium powder to ethanol of 1 g:16 mL, perform ultrasonic degreasing treatment at 50°C for 3 times, 20 min each time, then centrifuge to discard the supernatant, and vacuum-dry the residue at 50°C to constant weight;

[0084] (2) Preparation of crude polysaccharide HMPS

[0085] Mix the dried degreased powder obtained in step (1) with ultrapure water (the mass-volume ratio of the dried degreased powder to ultrapure water is 1 g:28 mL), and perform dynamic extraction for 3 h using a high-pressure thermal reflux extraction system (pressure 0.14 MPa, temperature 110°C). After the extract is concentrated by rotary evaporation to 1 / 5 of the original volume, add 3 volumes of pre-cooled absolute ethanol at 10°C until the mass percentage of ethanol is 85%, let it stand at 6°C for 10 h, collect the precipitate by centrifugation, wash it 4 times with absolute ethanol and acetone, and then freeze-dry to obtain crude polysaccharide HMPS;

[0086] (3) Deproteinization treatment

[0087] Dissolve the crude polysaccharide HMPS in Tris-HCl buffer solution with a concentration of 0.06 mol·L -1 and a pH of 8.0 (the mass-volume ratio of the crude polysaccharide HMPS to the Tris-HCl buffer solution is 50:1). Then, add Sevage reagent according to the volume ratio of the solution after dissolving the crude polysaccharide HMPS to the Sevage reagent added each time of 1:5. After vortexing for 15 min, centrifuge to remove the protein layer, and repeat the above operation until there is no white floc at the interface; after combining the aqueous phases, add pepsin (the concentration of pepsin in the aqueous phase is 0.6 mg / mL) and carry out enzymatic hydrolysis at 38 °C for 1.5 h, and inactivate by boiling water bath for 10 min to obtain the deproteinized solution; wherein, the Sevage reagent is composed of chloroform and n-butanol, and the volume ratio of chloroform to n-butanol in the Sevage reagent is 4:1.

[0088] (4) Decoloration and purification

[0089] Pass the deproteinized solution through a D301 macroporous adsorption resin column (column volume 200 mL) at a flow rate of 1.5 BV / h for dynamic adsorption 4 times. Collect the breakthrough solution, filter it through a 0.22 μm filter membrane, and use an ultrafiltration system with a cut-off molecular weight of 10 kDa. After concentration, freeze-dry to obtain the total polysaccharide;

[0090] (5) DEAE-52 cellulose column chromatography

[0091] Dissolve the total polysaccharide in distilled water to obtain a concentration of 2.2 mg / mL after dissolution of the total polysaccharide. Load it onto a pre-equilibrated DEAE-52 anion exchange column (2.6×40 cm). Taking a flow rate of 1 mL / min as an example, elute successively with 0, 0.5, 1 mol·L -1 NaCl gradient (flow rate 1 mL / min), and detect and collect the main peak with the phenol-sulfuric acid method in the 0.3 mol·L -1 NaCl elution section. After desalting by dialysis with MWCO 3500 Da and then freeze-drying, obtain the salt-washed component HMPS-3;

[0092] (6) Sephadex G-100 gel chromatography

[0093] Dissolve the salt-washed component HMPS-3 in 0.1 mol·L -1 NH4HCO3 buffer solution, load it onto a Sephadex G-100 column (1.6×60 cm). Taking a flow rate of 0.5 mL / min as an example, elute with 0.1 mol·L -1 NaCl, determine the sugar content by the phenol-sulfuric acid method, and collect the fraction with a molecular weight of 9.7×10 4The sugar-containing components of Da were ultrafiltered and concentrated, and then freeze-dried to obtain the homogeneous target product arabinoglucan HMPS-3a with a yield of 25.1%.

[0094] Example 3

[0095] The preparation method of the arabinoglucan described in this example includes the following steps:

[0096] (1) Raw material pretreatment

[0097] Take the dried rhizome tissue of Heracleum millefolium, and obtain Heracleum millefolium powder with a particle size ≤ 100 μm through an ultrafine pulverizer. Add ethanol with a mass percentage of 98% according to the mass-volume ratio of Heracleum millefolium powder to ethanol of 1 g:14 mL, perform ultrasonic degreasing treatment at 40 °C for 3 times, 20 min each time, then centrifuge to discard the supernatant, and vacuum-dry the residue at 30 °C to constant weight;

[0098] (2) Preparation of crude polysaccharide HMPS

[0099] Mix the dried degreased powder obtained in step (1) with ultrapure water (the mass-volume ratio of the dried degreased powder to ultrapure water is 1 g:33 mL), and perform dynamic extraction for 1 h using a high-pressure thermal reflux extraction system (pressure is 0.16 MPa, temperature is 130 °C). After the extract is concentrated to 1 / 5 of the original volume by rotary evaporation, add 3 times the volume of anhydrous ethanol precooled at 4 °C until the mass percentage of ethanol is 90%, let stand at 2 °C for 15 h, centrifuge to collect the precipitate, wash it 5 times with anhydrous ethanol and acetone, and then freeze-dry to obtain crude polysaccharide HMPS;

[0100] (3) Deproteinization treatment

[0101] Dissolve the crude polysaccharide HMPS in Tris-HCl buffer solution with a concentration of 0.04 mol·L -1 and pH of 8.0 (the mass-volume ratio of the crude polysaccharide HMPS to Tris-HCl buffer solution is 50:1), then add Sevage reagent according to the volume ratio of the solution after dissolving the crude polysaccharide HMPS to the Sevage reagent added each time of 1:3, vortex and oscillate for 8 min, then centrifuge to remove the protein layer, and repeat the above operation until there is no white floc at the interface; after combining the aqueous phases, add pepsin (the concentration of pepsin in the aqueous phase is 0.4 mg / mL) and perform enzymatic hydrolysis at 36 °C for 2 h, inactivate at boiling water bath for 10 min to obtain a deproteinized solution; among them, the Sevage reagent is composed of chloroform and n-butanol, and the volume ratio of chloroform to n-butanol in the Sevage reagent is 4:1.

[0102] (4) Decolorization and purification

[0103] The deproteinized solution was passed through a D301 macroporous adsorption resin column (column volume 200 mL) at a flow rate of 1.5 BV / h for dynamic adsorption three times. The breakthrough solution was collected, filtered through a 0.22-μm filter membrane, concentrated by an ultrafiltration system with a molecular weight cut-off of 10 kDa, and freeze-dried to obtain the total polysaccharide;

[0104] (5) DEAE-52 cellulose column chromatography

[0105] The total polysaccharide was dissolved in distilled water to a concentration of 1.8 mg / mL after dissolution. It was loaded onto a pre-equilibrated DEAE-52 anion exchange column (2.6 × 40 cm). Taking a flow rate of 1 mL / min as an example, it was eluted successively with 0, 0.5, 1 mol·L -1 NaCl gradients (flow rate 1 mL / min), and the main peak was detected by the phenol-sulfuric acid method. The fraction eluted with 1.0 mol·L -1 NaCl was collected, desalted by dialysis with an MWCO of 3500 Da, and freeze-dried to obtain the salt-washed fraction HMPS-3;

[0106] (6) Sephadex G-100 gel chromatography

[0107] The salt-washed fraction HMPS-3 was dissolved in 0.1 mol·L -1 NH4HCO3 buffer solution and loaded onto a Sephadex G-100 column (1.6 × 60 cm). Taking a flow rate of 0.7 mL / min as an example, it was eluted with 0.1 mol·L -1 NaCl, and the sugar content was determined by the phenol-sulfuric acid method. The sugar-containing fraction with a molecular weight of 9.7 × 10 4 Da was collected, ultrafiltered and concentrated, and then freeze-dried to obtain the homogeneous target product arabinoglucan HMPS-3a, with a yield of 25.5%.

[0108] Performance test:

[0109] (I) Structural characterization analysis of arabinoglucan HMPS-3a

[0110] (1) Isolation and purification of arabinoglucan HMPS-3a

[0111] In step (6) of Example 1, the salt-washed fraction HMPS-3 was separated and purified by a Sephadex G-100 column ( Figure 1 ), with a flow rate of 0.5 mL / min, eluted with 0.1 mol·L -1 NaCl, and the sugar content was determined by the phenol-sulfuric acid method. The sugar-containing fraction with a molecular weight of 9.7 × 10 4 Da was collected, ultrafiltered and concentrated, and then freeze-dried to obtain the homogeneous target product arabinoglucan HMPS-3a, with a yield of 25.3%.

[0112] (2) Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0113] Test procedure: Accurately weigh 2.0 mg of the arabinoglucan HMPS-3a sample prepared in Freeze-dried Example 1, mix it with dry potassium bromide powder (200 mg, activated at 110 °C for 4 h), grind it until uniform, and press it into a transparent thin film (pressure 10 MPa, holding pressure for 30 s). Scan and detect using a Nicolet iS50 Fourier transform infrared spectrometer, with parameter settings: scanning range 4000 - 400 cm -1 , resolution 4 cm -1 , accumulate scans 32 times, and deduct background CO2 / H2O interference in real time. Analyze characteristic absorption peaks through OMNIC software, and focus on analyzing sugar ring characteristic peaks (such as 3400 cm -1 O-H stretching vibration, 1070 cm -1 C-O-C pyran ring vibration) and arabinose characteristic absorption (890 cm -1 β-configuration C-H vibration).

[0114] Experimental results: See Figure 2 . Among them, the signal near 3402 cm -1 is the O-H stretching vibration, the signals near 2936 cm -1 and 1418 cm -1 are the C-H stretching vibration and bending vibration, the signal near 1503 cm -1 is the polysaccharide ring-external C-O stretching vibration, and the signal near 1620 cm -1 is the O-H deformation vibration generated by polysaccharide containing bound water. In addition, the signals near 1026 cm -1 and 1143 cm -1 are the C-O bending vibrations of pyranose.

[0115] (3) High Performance Liquid Chromatography (HPLC) Analysis for Molecular Weight Determination

[0116] Test procedure: Use a TSK-Gel G4000PWxl chromatographic column (7.8×300 mm), with the mobile phase being 0.1 M Na2SO4 (containing 0.02% NaN3), flow rate 0.6 mL / min, and column temperature 35 °C. Establish a standard curve (lgMw = -0.267t + 10.58, R 2 = 0.998) using dextran standard products (T series, molecular weight 1 - 1000 kDa). Inject 20 μL of the sample solution (5 mg / mL, filtered through 0.22 μm), collect data with a differential refractive index detector (RID-20A), and calculate the molecular weight distribution of arabinoglucan HMPS-3a through the retention time.

[0117] The HPSEC determination results showed that the HPSEC chromatogram peak of the araboglucan HMPS-3a obtained in Example 1 was single and symmetric, indicating a homogeneous polysaccharide ( Figure 3 ), and the relative molecular weights were calculated by the standard curve method to be 9.7×10 4 Da.

[0118] (4) Determination of sugar content, uronic acid content, and impurity protein content

[0119] 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. The sugar content, uronic acid content, and impurity protein content of the araboglucan HMPS-3a in Example 1 were 96.3%, 1.5%, and 0.9%, respectively.

[0120] (5) High-performance liquid chromatography (HPLC) analysis of monosaccharide composition

[0121] Test procedure: 10 mg of the araboglucan HMPS-3a obtained in Example 1 was hermetically hydrolyzed with 2 M trifluoroacetic acid (TFA) at a ratio of 1:10 (w / v) (110 °C, 4 h), and after removing TFA by nitrogen blowing, it was redissolved in ultrapure water. 100 μL of the hydrolyzate was taken, 100 μL of 0.5 M PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution and 50 μL of 0.3 M NaOH were added, and derivatization was carried out at 70 °C for 30 min. After neutralization with glacial acetic acid, HPLC analysis was performed. Chromatographic conditions: Agilent ZORBAX SB-C18 column (4.6×250 mm), mobile phase A (0.1 M phosphate buffer, pH 6.8)-B (acetonitrile) gradient elution (0-20 min, 83%→77% A), flow rate 1.0 mL / min, detection wavelength 245 nm. The retention times of the control monosaccharide standards (glucose, arabinose, etc.) were used for qualitative analysis, and the external standard method was used for quantitative analysis.

[0122] The monosaccharide composition of the araboglucan HMPS-3a was determined by HPLC, and the results are as Figure 4 shown. The araboglucan HMPS-3a was composed of Glc and Ara with a molar ratio of 3.55:1.06, indicating that HMPS-3a is an araboglucan.

[0123] (6) Nuclear magnetic resonance (NMR) analysis

[0124] Test procedure:

[0125] (6.1) Sample pretreatment

[0126] 50 mg of the arabinoglucan HMPS-3a prepared in Example 1 was dissolved in 0.5 mL of 99.9% D2O. After ultrasonic dissolution, it was centrifuged (12,000×g, 10 min), and freeze-thaw was repeated 3 times to replace protons. The final solution was transferred to a 5 mm NMR tube.

[0127] (6.2) One-dimensional spectrum (1D NMR)

[0128] Using a Bruker Avance III HD 500 MHz nuclear magnetic resonance spectrometer, the probe temperature was 298 K. 1 1H NMR parameters: spectral width 12 ppm, relaxation delay 2 s, number of scans 64 times; 13 13C NMR parameters: spectral width 240 ppm, relaxation delay 1.5 s, number of scans 1024 times. Characteristic signals were identified by chemical shifts (such as the anomeric hydrogen region at δ 5.1 - 5.3 ppm and the carbon skeleton region at δ 60 - 110 ppm).

[0129] (6.3) Two-dimensional spectrum (2D NMR)

[0130] HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple bond correlation), and COSY (homonuclear chemical shift correlation) experiments were performed:

[0131] ·HSQC: 1H detection, evolution time 0.1 s, number of scans 128×1024, JCH = 145 Hz

[0132] ·HMBC: long-range coupling constant J = 8 Hz, delay time 60 ms

[0133] ·COSY: spectral width 10 ppm, number of scans 8×2048

[0134] The sugar ring linkage mode was analyzed by cross peaks (such as the β-arabinose C1-H1 correlation indicated by δH 4.45 / δC 104.5), and the glycosidic bond linkage site was determined by combining HMBC long-range coupling.

[0135] 1D and 2D NMR were used to analyze the sugar residue composition and linkage mode of arabinoglucan HMPS-3a. The results showed that, see Figure 5, Araboglucan HMPS-3a is composed of 1 kind of glucose residue α-1,6-Glcp and 3 kinds of arabinose residues α-1,3-Araf, α-1,5-Araf and α-T-Araf. The repeating unit of araboglucan HMPS-3a was obtained by analyzing HMBC and NOESY spectra: α-T-Araf→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-L-Araf-(1→5)-α-L-Araf-(1→.

[0136] The structural formula of the repeating unit is:

[0137]

[0138] Structure analysis showed that HMPS-3a is an araboglucan, and its repeating unit is: α-T-Araf→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-L-Araf-(1→5)-α-L-Araf-(1→.

[0139] (2) Evaluation of the in vitro anti-RII effect of Heracleum millefolium polysaccharide

[0140] (1) Establishment of cell culture system

[0141] (1.1) Culture of THP-1 monocytes

[0142] The human monocyte cell line THP-1 purchased from ATCC was cultured in suspension in RPMI-1640 medium (containing 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 0.05 mM β-mercaptoethanol) at 37 °C and 5% CO2. Passage was performed at a ratio of 1:3 every 2 days, and the cell viability was ≥95% (verified by trypan blue staining).

[0143] (1.2) Culture of HIEC intestinal epithelial cells

[0144] Normal human intestinal epithelial cells HIEC (ScienCell #2950) were seeded on collagen-coated culture dishes and cultured adherently in a special intestinal epithelial cell medium (containing 10 ng / mL EGF and 5 μg / mL insulin) at 37 °C and 5% CO2. The medium was changed every 3 days, and when the confluence reached 80%, digestion and passage were performed with 0.25% trypsin-EDTA.

[0145] (2) Effects of HMPS-3a on radiation-induced inflammatory factors and NO in THP-1

[0146] The density of THP-1 cells was adjusted to 2×10 5 cells / mL. After pre-incubating with arabinoglucan HMPS-3a (25, 50, 100 μg / mL) or dexamethasone (10 μM, positive control) obtained in Example 1 for 6 h, the cells were irradiated with 6 Gy of X-rays (dose rate 1 Gy / min, filter plate 2 mm Al). The supernatant was collected 24 h after irradiation, and cell debris was removed by centrifugation (300×g, 5 min), and stored at -80 °C for further measurement. ELISA assay: According to the instructions of the kit (R&D Systems), the concentrations of TNF-α, IL-6, and IL-1β were quantified, and the standard curve R 2 > 0.99. NO assay: Griess reagent method (Sigma). Take 100 μL of the supernatant + an equal volume of Griess reagent, and measure the absorbance at 540 nm, and calculate the concentration using the NaNO2 standard curve.

[0147] As Figure 6 shown, arabinoglucan HMPS-3a significantly inhibited the release of inflammatory factors and NO in radiation-induced THP-1 cells, and it is an active polysaccharide with anti-inflammatory and immunomodulatory effects.

[0148] (3) Effect of HMPS-3a on radiation-induced DNA damage in HIEC

[0149] HIEC cells were seeded in 6-well plates (5×10 4 cells / well), and divided into a low-dose group (HMPS-3a L) and a high-dose group (HMPS-3a H). The low-dose group was added with arabinoglucan HMPS-3a 50 μg / mL obtained in Example 1, and the high-dose group was added with arabinoglucan HMPS-3a 100 μg / mL obtained in Example 1. The blank group (Control) had 3 wells and was only added with culture medium. The model group (Model) had 3 wells and was seeded with HIEC cells (5×10 4 cells / well) and culture medium. After 24 h, the cells were irradiated with 8 Gy of local abdominal simulated irradiation (Varian Clinac iX, 6 MV photon beam, SSD = 100 cm). γ-H2AX immunofluorescence: The cells were fixed 2 h after irradiation (4% paraformaldehyde, 20 min), permeabilized with 0.5% Triton X-100, blocked with 5% BSA for 1 h, incubated with anti-γ-H2AX primary antibody (1:500, CST#9718) overnight at 4 °C, incubated with AlexaFluor 488 secondary antibody (1:1000) for 1 h in the dark, and counterstained with DAPI for nuclei. The number of foci was counted using a confocal microscope (Zeiss LSM 900) (≥30 cells / group).

[0150] As Figure 7As shown, arabinoglucan HMPS-3a significantly inhibits radiation-induced DNA damage in HIEC cells. The inhibition rates of the low-dose group (HMPS-3a L) and the high-dose group (HMPS-3a H) are 39.2% and 85.5% respectively.

[0151] (4) Effect of HMPS-3a on radiation-induced apoptosis of HIEC

[0152] After HIEC was irradiated with 10 Gy and cultured for another 48 h, the experimental groups were divided into a low-dose group (HMPS-3a L) and a high-dose group (HMPS-3a H). The low-dose group was added with 50 μg / mL of arabinoglucan HMPS-3a obtained in Example 1, and the high-dose group was added with 100 μg / mL of arabinoglucan HMPS-3a obtained in Example 1. The blank group (Control) had 3 wells and only added culture medium, and the model group (Model) had 3 wells, inoculating HIEC cells (5×10 4 cells / well) and culture medium. The cells were digested and collected, washed twice with PBS, resuspended in Binding Buffer, and then added with 5 μL of AnnexinV-FITC and 2 μL of PI (BD Pharmingen), and reacted in the dark for 15 min. Detection was performed using a flow cytometer (BD FACSCanto II): Fluorescence channels: FITC (530 / 30 nm), PI (670 nm LP). Gating strategy: AnnexinV+ / PI- is early apoptosis, and Annexin V+ / PI+ is late apoptosis.

[0153] As Figure 8 shown, arabinoglucan HMPS-3a effectively inhibits radiation-induced apoptosis of HIEC cells. The inhibition rates of the low-dose group (HMPS-3a L) and the high-dose group (HMPS-3a H) are 16.1% and 53.5% respectively.

[0154] (5) Evaluation of the anti-RII effect of HMPS-3a in vivo

[0155] Animal model: SPF-grade C57BL / 50 mice (male, 8 weeks old) were randomly divided into 5 groups (n = 10): blank group (Control), model group (Model), low-dose HMPS-3a (HMPS-3a L, 50 mg / kg), high-dose (HMPS-3a H, 100 mg / kg), and the positive control was the glutamine group (150 mg / kg).

[0156] Blank group (Control): Not irradiated and not given drugs.

[0157] Irradiation and drug administration process:

[0158] Model group, HMPS-3a low dose (HMPS-3a L, 50 mg / kg), high dose (HMPS-3a H, 100 mg / kg), the positive control was the glutamine group (150 mg / kg). After anesthesia, local abdominal irradiation was performed (15 Gy, with other organs shielded by lead).

[0159] HMPS-3a low dose (HMPS-3a L, 50 mg / kg), high dose (HMPS-3a H, 100 mg / kg), the positive control was the glutamine group (150 mg / kg). From 7 days before irradiation, gavage administration was given daily until the samples were taken 72 h after irradiation.

[0160] Sample processing: Retrieve the ileum tissue, fix it with 4% paraformaldehyde (for pathological sections) or freeze it quickly in liquid nitrogen (for Western blot), and store it at -80 °C.

[0161] The RII mouse model was used to evaluate the anti-RII effect of HMPS-3a in vivo. The mice in the model group showed typical characteristics of radiation-induced intestinal injury: The body weight decreased to the peak on the 7th day after irradiation (23.7 ± 2.1% vs the baseline before irradiation), which was significantly higher than that of the blank control group. After intervention with the high-dose group of HMPS-3a (HMPS-3a H, 100 mg / kg), the weight loss rate was significantly inhibited to 9.2 ± 1.6%, and the effect was better than that of the glutamine group ( Figure 9 ). Severe atrophic changes occurred in the ileum of the mice in the model group: The intestinal segment length shortened from normal (36.8 ± 0.7 cm) to 29.3 ± 0.9 cm. Treatment with the high-dose group of HMPS-3a (HMPS-3a H) significantly maintained the integrity of the intestinal morphology, and the intestinal length recovered to 34.1 ± 0.6 cm, with a protection rate of 82.3%, which was significantly better than 68.5% of the glutamine group ( Figure 10 ).

[0162] (6) HE staining

[0163] Take the intestinal tissues of the mice in the blank group (Control), model group (Model), low-dose group (HMPS-3a L, 50 mg / kg), high-dose group (HMPS-3a H, 100 mg / kg), and the positive control glutamine group (150 mg / kg) in experiment (5) for staining.

[0164] The specific process of HE staining is as follows:

[0165] Deparaffinization of 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, thus achieving 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 treatment with xylene, place the sections in absolute ethanol. The role of absolute ethanol is to remove xylene and further remove the residual paraffin. Place them 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 the organic solvents and complete the hydration process.

[0166] Antigen retrieval: Immerse the sections in a citrate antigen retrieval buffer (pH 6.0), and use a microwave oven to boil them on medium heat for 3 min for antigen retrieval. After standing at room temperature, turn to medium heat again and boil for 2 min, then cool to room temperature. Immerse the sections in PBS (pH 7.4), shake and wash for 5 min * 3 times, 10 min * 1 time. Then permeabilize them 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.

[0167] Blocking endogenous peroxidase: Place the sections in a 3% hydrogen peroxide aqueous solution, and let them stand in the dark at room temperature for 25 min. Then shake and wash them in PBS for 5 min * 3 times, 10 min * 1 time.

[0168] Serum blocking: Drop goat serum on the tissue and block it at room temperature for 50 min. (Use rabbit serum to block if the primary antibody is from goat, and use BSA to block for other sources) Add primary antibody: Gently shake off the blocking solution, drop the primary antibody on the section tissue, and incubate it in a wet box at room temperature for 2 h. Add secondary antibody: Wash it in PBS with shaking for 5 min * 3 times, 10 min * 1 time for the 4th time. Gently shake off the PBS, drop the secondary antibody on the tissue, incubate it at room temperature for 1 h, and wash it with PBS for 5 min * 3 times. Then incubate it with SABC at room temperature for 30 min, and wash it with PBS for 5 min * 3 times, 10 min * 1 time for the 4th time.

[0169] DAB color development: Gently shake off the PBS, drop the DAB color development solution (prepared freshly) on the tissue, control the color development time (about 10 s) under microscopic observation. The positive result is brownish yellow, and gently rinse the sections with running water for more than 10 min to terminate the color development.

[0170] Counterstaining the cell nucleus: Quickly drop hematoxylin on the tissue, counterstain for 2 s, shake off the staining solution and wash it with tap water for about 1 min.

[0171] The steps of dehydrating and mounting the slides are as follows: Place the tissue sections in a slide rack. After standing at room temperature for 60 min, immerse them in xylene. After 10 min, transfer them to another staining jar with more xylene and soak for another 10 min. After dewaxing, place the tissue sections in absolute ethanol for 5 min, then 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and 50% ethanol for 5 min. After hydration treatment, place the tissue sections in pure water and incubate for 5 min, then transfer them to PBS buffer and incubate for 5 min. Conversely, first place them in pure water for 5 min, then 50% ethanol for 5 min, 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, and absolute ethanol for 5 min. Finally, place them in xylene for transparency treatment twice, 10 min each time. After dehydration is completed, wipe off the liquid around the sections, drop a drop of neutral balsam, cover with a coverslip, and gently tap the coverslip with the blunt end of forceps to remove air bubbles to complete the slide mounting.

[0172] Microscopic examination and image acquisition and analysis.

[0173] HE staining ( Figure 11 ) showed that the ileum of the mice in the model group presented typical pathological features of radiation-induced intestinal injury: the villus height decreased significantly from the normal level, the number of surviving crypts decreased, and the crypt depth atrophied. After intervention with the high-dose HMPS-3a group (HMPS-3a H, 100 mg / kg), the villus height recovered, and the number of surviving crypts increased, which was significantly better than that of the glutamine group. Moreover, extensive epithelial shedding was visible in the intestinal mucosa of the model group, with a large number of neutrophil infiltrations in the lamina propria and a decrease in the density of goblet cells. HMPS-3a treatment significantly inhibited the inflammatory response, with a reduction in neutrophil infiltration, a recovery in the density of goblet cells, and an increase in the mucosal integrity rate.

[0174] Those skilled in the art will readily conceive of other embodiments of the present application after considering the specification and the practice disclosed 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 the common general knowledge or conventional technical means in the technical field not disclosed in the present application. The specification and the embodiments are only regarded as exemplary, 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 arabinoglucan, characterized in that, The molecular weight of the arabinoglucan is 9.7×10 4 Da; The structural formula of the repeating unit of the arabinoglucan is as follows: Among them, A is an α-T-L-arabinose residue; B is an α-1,6-D-glucose residue; C is an α-1,3-L-arabinose residue; D is an α-1,5-L-arabinose residue.

2. The arabinoglucan according to claim 1, characterized in that: The arabinoglucan is composed of glucose (Glc) and arabinose (Ara), and the molar ratio of glucose (Glc) to arabinose (Ara) is 3.55:1.

06.

3. The preparation method of the araboglucan according to claim 1, characterized in that, It includes the following steps: (1) Pretreatment of raw materials Take the dried rhizome tissue of Heracleum millefolium, and obtain Heracleum millefolium powder through an ultrafine pulverizer. Add ethanol, perform ultrasonic degreasing treatment, then centrifuge to discard the supernatant, and dry the residue in vacuum until constant weight. (2) Preparation of crude polysaccharide HMPS Mix the dried and degreased powder obtained in step (1) with ultrapure water, and perform dynamic extraction using a high-pressure thermal reflux extraction system. After the extract is concentrated by rotary evaporation, add pre-cooled absolute ethanol until the mass percentage of ethanol is more than 80%. Let it stand, centrifuge to collect the precipitate, and then use absolute ethanol and acetone, and freeze-dry to obtain crude polysaccharide HMPS. (3) Deproteinization treatment Dissolve the crude polysaccharide HMPS in Tris-HCl buffer solution, then add Sevage reagent, vortex and centrifuge to remove the protein layer, and repeat the operation until there is no white floc at the interface; after combining the aqueous phases, add pepsin for enzymatic hydrolysis, and inactivate it in a boiling water bath to obtain a deproteinized solution. (4) Decolorization and purification Perform dynamic adsorption of the deproteinized solution through a D301 macroporous adsorption resin column, collect the breakthrough solution, filter it through a filter membrane, the cut-off molecular weight of the ultrafiltration system is 10 kDa, concentrate and then freeze-dry to obtain the total polysaccharide. (5) DEAE-52 cellulose column chromatography Dissolve the total polysaccharide in distilled water, load it onto a pre-equilibrated DEAE-52 anion exchange column, and elute it successively with 0, 0.5, and 1 mol·L -1 NaCl gradient elution, and detect the collected main peak by the phenol-sulfuric acid method in the 1 mol·L -1 NaCl elution section. After desalting by dialysis with MWCO 3500 Da and freeze-drying, the salt-washed component HMPS-3 is obtained; (6) Sephadex G-100 gel chromatography Dissolve the salt-washing component HMPS-3 in 0.1 mol·L -1 NH4HCO3 buffer solution, load it onto a Sephadex G-100 column, and elute with 0.1 mol·L -1 NaCl. Determine the sugar content by the phenol-sulfuric acid method, collect the sugar-containing component with a molecular weight of 9.7×10 4 Da, ultrafilter and concentrate it, and then freeze-dry it to obtain the homogeneous target product arabinoglucan HMPS-3a.

4. The preparation method of arabinoglucan according to claim 3, wherein: In step (1), the dried rhizome tissue of Heracleum millefolium is processed into Heracleum millefolium powder with a particle size ≤100 μm by an ultrafine pulverizer; the mass-volume ratio of Heracleum millefolium powder to ethanol is 1 g:(14 - 16) mL; the mass percentage of the ethanol is more than 90%; the temperature of the ultrasonic treatment is 40 - 50 °C.

5. The preparation method of the arabogalactan according to claim 3, wherein: In step (2), the mass-volume ratio of the dried and degreased powder to ultrapure water is 1 g:(28 - 33) mL; the pressure of the high-pressure thermal reflux extraction system is 0.14 - 0.16 MPa, the temperature is 110 - 130 °C, and the extraction time is 1 - 3 h; the temperature of the pre-cooled absolute ethanol is 4 - 10 °C; the temperature of the standing is 2 - 6 °C.

6. The preparation method of arabogalactan according to claim 3, characterized in that: In step (3), the mass-volume ratio of the crude polysaccharide HMPS to Tris-HCl buffer solution is 50:1; the volume ratio of the solution after dissolving the crude polysaccharide HMPS to the Sevage reagent added each time is 1:(3 - 5).

7. The preparation method of arabinoglucan according to claim 3, characterized in that: In step (3), the concentration of the Tris-HCl buffer solution is 0.04 to 0.06 mol·L -1 , the pH is 8.0; the concentration of the pepsin after being added to the aqueous phase is 0.4 to 0.6 mg / mL; the temperature of the enzymatic hydrolysis is 36 to 38 °C.

8. The preparation method of arabinoglucan according to claim 3, characterized in that: In step (3), the Sevage reagent is composed of chloroform and n-butanol, and the volume ratio of chloroform to n-butanol in the Sevage reagent is 4:

1.

9. The preparation method of the arabogalactan according to claim 3, characterized in that: In step (4), the pore size of the filter membrane is 0.22 μm; in step (5), the concentration of the total polysaccharide after dissolution is 1.8 - 2.2 mg / mL.

10. Use of the arabinoglucan according to claim 1 in the preparation of a drug for preventing radioactive intestinal injury.

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