Sowthistle polysaccharide extract as well as extraction method and application thereof
The problem that existing drugs are not effective in all patients by extracting and refining polysaccharides from fresh stems and leaves of chicory is solved, which significantly reduces the symptoms and pathological changes of ulcerative colitis, and provides a new and effective treatment method.
Patent Information
- Application Number
- CN202311806522.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-26
- Publication Date
- 2025-03-11
AI Technical Summary
The existing drugs for treating ulcerative colitis are not effective for all patients, and long-term use has great side effects and financial burden.
The extract of chicory polysaccharide was extracted from the fresh stems and leaves of chicory, and the steps of boiling alcohol precipitation, Sevage method to remove impurity proteins, and DEAE-52 cellulose chromatography were used to obtain the chicory polysaccharide extract. This extract is used to treat ulcerative colitis.
Chicory polysaccharide extract significantly prevents weight loss and colon atrophy in mice, reduces DAI scores, reduces colon tissue structure disorders and inflammatory cell infiltration, reduces protein levels of iNOS, CD86 and IL-6, and reduces the immunohistochemical iNOS expression of colitis.
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Figure CN119591733A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of natural drug extraction and application, and specifically relates to a chicory polysaccharide extract and an extraction method and application thereof. Background Art
[0002] Ulcerative colitis is a common pathological type of inflammatory bowel disease. As a chronic nonspecific inflammatory bowel disease, the lesions mainly involve the rectal and colonic mucosa. Clinically, it is often manifested as abdominal pain, diarrhea, and bloody stools with mucus, pus, etc. The course of the disease is long, and it is protracted and recurrent, and may even become cancerous, which seriously affects the patient's quality of life. The patient population is very large. Its pathogenesis is complex and has not yet been fully elucidated, but the current research on disease-related factors has given scholars a certain understanding of its mechanism: intestinal flora disorders lead to intestinal mucosal barrier damage, which is an important pathogenic factor of chronic nonspecific inflammatory bowel disease.
[0003] As research deepens, new treatment methods are also increasing, such as fecal microbiota transplantation, leukocyte adsorption therapy, etc. For some severe patients, surgical treatment can be selected, but drug treatment is still the first choice. Existing mainstream treatment drugs include aminosalicylic acid preparations, glucocorticoids, probiotics, biological preparations and Chinese herbal prescriptions. Among them, aminosalicylic acid preparations are the first choice for maintenance medication for patients with mild to moderate active disease and patients in remission. Glucocorticoids can inhibit inflammation and immune response, and can be effective for treatment but cannot be used as maintenance medication. Immunosuppressants such as cyclosporine and azathioprine can be used for some glucocorticoid-resistant and dependent patients.
[0004] Although the above-mentioned traditional medicines can relieve symptoms of some patients and delay the course of the disease to a certain extent, the drugs currently used for treatment are not effective for all patients. Most patients cannot completely control their condition, the disease is lingering, and long-term use has significant side effects and economic burden. Summary of the invention
[0005] The purpose of the present invention is to overcome the above problems existing in the traditional technology and provide a chicory polysaccharide extract and an extraction method and application thereof.
[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:
[0007] The present invention provides a method for extracting cichorium polysaccharide extract, comprising the following steps:
[0008] 1) Using fresh stems and leaves of chicory as materials, crude polysaccharide precipitation was collected by boiling and alcohol precipitation;
[0009] 2) The crude polysaccharide is precipitated and then subjected to the sevage method to remove the impurity proteins contained therein;
[0010] 3) The obtained deproteinized polysaccharide is subjected to DEAE-52 cellulose chromatography again to remove the polysaccharide components with a lower content, and retain the main polysaccharide with a higher content;
[0011] 4) The main part of the polysaccharide is precipitated and purified to obtain the chicory polysaccharide extract.
[0012] Furthermore, in step 1), fresh stems and leaves of chicory are used as materials. After being crushed, they are boiled in 1:10-20 times water at normal pressure for 1-2 hours. The resulting solution is concentrated to 1 / 4 of the original volume, cooled, and then 3 times the volume of anhydrous ethanol is added for precipitation. After being refrigerated in a refrigerator overnight, the crude polysaccharide precipitate is collected by centrifugation.
[0013] Furthermore, in step 2), the mixed solution in the sevage method is composed of a crude polysaccharide solution, chloroform and n-butanol solution, and the volume ratio of the crude polysaccharide solution, chloroform and n-butanol solution is 1:1:3-5; the mixed solution is treated with a high-speed homogenizer for 2-4 minutes and then placed in a centrifuge for 4-6 minutes at a centrifugal speed of 5000-10000 rpm. After centrifugation, the white denatured protein part is removed, and the above process is repeated 3 times.
[0014] Furthermore, in step 3), the quercetin is dissolved and placed on a DEAE-52 cellulose chromatography column, and gradient elution is performed using 0.2-0.6% sodium chloride; the collected eluate is tested with a sulfuric acid phenol solution, and the collected liquid with a coffee color or dark brown color is collected and numbered, and the collected liquid with a significantly increased polysaccharide concentration is named SOP, and after precipitation with four times the volume of anhydrous ethanol, the purified quercetin is obtained.
[0015] The invention also provides a chicory polysaccharide extract obtained by the above extraction method.
[0016] The present invention also provides an application of the chicory polysaccharide extract in preparing a drug for treating small intestinal ulcer.
[0017] Furthermore, the drug can significantly prevent weight loss and colon atrophy in mice, and reduce the DAI scores of mice.
[0018] Furthermore, the drug can reduce colon tissue structural disorder, significantly reduce inflammatory cell infiltration, and lower histological scores.
[0019] Furthermore, the drug was able to reduce the protein levels of iNOS, CD86, and IL-6 in colon tissue.
[0020] Furthermore, the drug can reduce the expression of iNOS in the colon tissue of colitis mice by immunohistochemistry.
[0021] The beneficial effects of the present invention are:
[0022] The ciscargot polysaccharide extract provided by the present invention is extracted and refined from the stems and leaves of the perennial herbaceous plant ciscargot, specifically, crude polysaccharides are extracted by boiling and alcohol precipitation in sequence, the crude extract is subjected to the sevage method to remove the impurity protein contained therein, the obtained deproteinized polysaccharide is subjected to DEAE-52 cellulose chromatography again to remove the polysaccharide components with less content, retain the main part of the polysaccharide with large content, and then precipitate and purify to obtain; the ciscargot polysaccharide extract is the main part of ciscargot polysaccharide, which is the second elution peak part with the largest content eluted from the DEAE-52 cellulose chromatography column. The ciscargot polysaccharide prepared by the present invention can be used to treat ulcerative colitis.
[0023] Of course, any product implementing the present invention does not necessarily need to achieve all of the above advantages at the same time. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.
[0025] Figure 1 is a schematic diagram of chicory and its polysaccharides;
[0026] Figure 2 This is a schematic diagram of the composition of refined chicory polysaccharide;
[0027] Figure 3 Schematic diagram of the antioxidant activity of quercetin;
[0028] Figure 4 Schematic diagram of the effect of quercetin polysaccharide on the body weight of mice with DSS-induced colitis;
[0029] Among them, * indicates p < 0.05, # indicates p < 0.05;
[0030] Figure 5 Schematic diagram of the effect of quercetin polysaccharide on the disease activity index (DAI) score of DSS-induced colitis mice;
[0031] Among them, * indicates p<0.05, and # indicates p<0.05.
[0032] Figure 6 Schematic diagram of the effect of chicory polysaccharide on the length of the colon and rectum in mice with DSS-induced colitis;
[0033] Among them, ** indicates p < 0.01, and *** indicates p < 0.001.
[0034] Figure 7Schematic diagram of the effect of citronellol polysaccharide on the pathological changes of colon tissue HE staining in mice with DSS-induced colitis (16×);
[0035] Among them, * indicates p < 0.05, *** indicates p < 0.001;
[0036] Figure 8 Schematic diagram of the effect of quercetin on the expression of iNOS, CD86 and IL-6 inflammatory proteins in colon tissue of mice with DSS-induced colitis;
[0037] Among them, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001;
[0038] Fig. 9 Schematic diagram of the effect of quercetin polysaccharide on the expression of iNOS in the colon of DSS-induced colitis mice by immunohistochemical staining (20×);
[0039] Among them, *** indicates p<0.001. DETAILED DESCRIPTION
[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0041] The specific embodiments of the present invention are as follows:
[0042] Example 1
[0043] Purification of cichorium polysaccharide
[0044] 1) Fresh stems and leaves of chicory were used as materials. After being crushed, they were boiled in 1:15 times water at normal pressure for 1.5 hours. The resulting solution was concentrated to 1 / 4 of the original volume. After cooling, 3 times the volume of anhydrous ethanol was added for precipitation. After being refrigerated overnight in a refrigerator, the crude polysaccharide precipitate was collected by centrifugation.
[0045] 2) The crude chicory polysaccharide precipitate was used to remove the impurities by the modified Sevage method. The volume ratio of the crude polysaccharide solution, chloroform and n-butanol solution was 1:1:4. The mixed solution was treated with a high-speed homogenizer for 3 minutes and then centrifuged in a centrifuge at a speed of 8000 rpm. After centrifugation, the white denatured protein was removed. The above process was repeated 3 times.
[0046] 3) After the gesicornia polysaccharide is dissolved in distilled water, it is placed on a DEAE-52 cellulose chromatography column and gradient eluted with 0.4% sodium chloride. The collected eluate is tested with a sulfuric acid phenol solution, and the collected liquid with a coffee color or dark brown is collected and numbered. The collected liquid with a significantly increased polysaccharide concentration is named SOP, and after precipitation, it is the refined pure gesicornia polysaccharide.
[0047] The refined quercetin SOP part is shown in Tables 1 and 2, and meets the following monosaccharide composition and structural characteristics:
[0048] Table 1 Monosaccharide composition
[0049] composition content Galacturonicacid 72.13 Rhamnose 4.23 Arabinose 9.51 Galactose 10.57 Glucose 2.11 Mannose 0.40 Glucuronic acid 1.06
[0050] Table 2 SOP structural characteristics
[0051] Structural features Proportion % Rha / GalA 0.059 (Gal+Ara) / Rha 4.75 HG(mol%) 67.9 RG-I (mol%) 28.53 DE(%)* 23.81±1.34
[0052] Example 2
[0053] Application of refined chicory polysaccharide in the treatment of small intestinal ulcer
[0054] 1. Experimental Materials
[0055] Experimental animals and drugs: 30 C57BL / 6 male mice, weighing 18-20 g, were purchased from Huachuang Xinnuo (Taizhou, China), animal certificate number: SYXK (Su) 2023-0016. The experimental animals were kept in the SPF animal room of the Experimental Animal Research Center of Yijishan Hospital. The experimental animals were kept in separate cages, and the bedding was changed every 2 days. The animals were adaptively fed for 7 days and had free access to water and food during the experiment. The temperature of the breeding environment was 25±2℃, the relative humidity was 60%, and there was a 12-hour light and 12-hour dark cycle every day.
[0056] The chicory polysaccharide is the refined polysaccharide extracted from the fresh stems and leaves of chicory in Example 1.
[0057] Experimental reagents and chemical reagents: DSS (dextran sulfate sodium, 36-50 kDa; DR0597, HARVEYBIO, Beijing, China), 5-aminosalicylic acid (Aladdin, A129982, China), PBS buffer, 4% paraformaldehyde solution, 0.9% sodium chloride, Avertin mouse-specific anesthetic.
[0058] Kits: Total protein extraction kit (Keygen Biotechnology, KGP2100, Nanjing, China), BCA kit (White Shark, BL521S, Nanjing, China).
[0059] Experimental instruments: high-speed low-temperature centrifuge (Thermo Fisher, Pico17 microcentrifuge, Germany), fully automatic chemiluminescence image analyzer (Tianneng, 5200, Shanghai, China), Eppendorf pipette, analytical balance.
[0060] Experimental instruments: ophthalmic scissors, elbow forceps, measuring cylinder, 200ml beaker, 1.5mL, 15mL, 50mL centrifuge tubes.
[0061] 2. Drug Configuration
[0062] Preparation of 3% DSS solution: Weigh 30 g of DSS powder, pour into a 1000 mL beaker, add double distilled water to 1000 mL, stir until completely dissolved, and prepare a DSS solution with a final concentration of 3%. Each group of mice drinks 250 ml of DSS solution. Re-prepare every two days and replace the animals with new solution.
[0063] Preparation of ciscarotene gavage solution: Use an analytical balance to weigh 1g of ciscarotene powder and pour it into a 15ml centrifuge tube. Add double distilled water to make up to 10mL. Vortex to dissolve into a working solution with a final concentration of 100mg / mL. Dilute to 200ul solution according to the dose of 50 and 200mg / kg per mouse, respectively, and gavage. Prepare and use it daily.
[0064] Preparation of 5-aminosalicylic acid: Use an analytical balance to weigh 0.1 g of 5-aminosalicylic acid powder and pour it into a 15 ml centrifuge tube. Add double distilled water to make up to 10 ml. Vortex to dissolve into a working solution with a final concentration of 10 mg / ml. Dilute to 200 ul solution at a dose of 100 mg / kg per mouse and gavage. Prepare and use freshly prepared daily.
[0065] 3. Experimental Methods
[0066] Animal grouping and model establishment: 30 C57BL / 6 mice were randomly divided into 5 groups: control group, DSS group, DSS+SOP50mg / kg group, DSS+SOP200mg / kg group, and DSS+5-ASA100mg / kg group. Except for the control group (clean drinking water), the other 4 groups of mice drank drinking water containing 3% DSS, which was changed to normal clean drinking water after 7 days. From day 0 to day 9, SOP or 5-ASA (positive control) was given by gavage every day, and the control group was given double distilled water by gavage.
[0067] Experimental process: The activity, food intake, status, body weight, feces and death of mice were observed and recorded daily. Two days after the model was established, the mice were killed after the last weighing, the cecum and colon were cut off, the intestinal tissue was carefully peeled off, the length of the colon was recorded and photographed for preservation, the cecum 1.5 cm away from the anus was discarded, and the distal colon of about 1 cm was cut and placed in 4% paraformaldehyde for HE staining and immunohistochemical staining, and the remaining colon was stored at -80°C for subsequent WB and Elisa experiments.
[0068] HE staining: The colon of mice fixed with 4% paraformaldehyde was dehydrated and embedded in paraffin, cut into 4 μm sections and then hydrated, stained with hematoxylin and eosin, dehydrated and sealed, and observed under a microscope.
[0069] Immunohistochemical staining: The mouse colon fixed with 4% paraformaldehyde was dehydrated and embedded in paraffin, cut into 4 μm sections and baked at 60°C, dewaxed and hydrated, followed by antigen retrieval, endogenous peroxidase blocking, and blocked with iNOS (18985-1-AP, 1:200, Wuhan Mitaka) primary antibody incubated at 4°C overnight. After bio-hormone labeling, horseradish enzyme was labeled with streptavidin, and the color was developed and counterstained with hematoxylin. Finally, the sections were dehydrated and the results were observed under a microscope.
[0070] WB detection of iNOS, CD86, and IL-6 proteins in intestinal tissue: The total protein of intestinal tissue was extracted using the Keygen total protein extraction kit and then subjected to protein electrophoresis using 4-20% precast gel, and then transferred to a PVDF membrane and blocked with skim milk for 1 hour. Subsequently, the membrane was incubated with primary antibodies for iNOS (18985-1-AP, 1:2000, Wuhan Tri-Tac), CD86 (13395-1-AP, 1:1000, Wuhan Tri-Tac), and IL-6 (DF6087, 1:1500, Affinity Biosciences, USA) at 4°C overnight, and developed after incubation with HRP-conjugated secondary antibodies for 1 hour.
[0071] Data statistics: The measurement data were expressed as mean ± standard deviation, the differences among the components were compared using one-way analysis of variance, and the pairwise comparisons were performed using LSD-t test.
[0072] IV. Experimental Results
[0073] 1. Effect of citronellol polysaccharide on body weight of mice with DSS-induced colitis
[0074] The research results are as follows Figure 4As shown in the figure, starting from the 5th day, the effect of DSS began to appear, and all mice drinking DSS began to show obvious weight loss. Overall, whether it is 50mg / kgSOP or 200mg / kgSOP or the positive control 100mg / kg5-ASA, they can alleviate the weight loss induced by DSS. The effect of 200mg / kg SOP is equivalent to that of 100mg / kg 5-ASA, and the effect of 50mg / kgSOP is slightly inferior to that of 200mg / kg SOP, but it is still significantly different from the DSS group.
[0075] 2. Effect of cichorium polysaccharide on DAI score of DSS-induced colitis mice
[0076] The research results are as follows Figure 5 As shown in the figure, starting from the 3rd day, the DAI score induced by DSS began to increase, and all mice drinking DSS began to show an increase in DSS score. Whether it is 50mg / kgSOP or 200mg / kgSOP or the positive control 100mg / kg5-ASA, they can reduce the increase in DAI induced by DSS. The effect of 200mg / kg SOP is equivalent to that of 100mg / kg 5-ASA, and the effect of 50mg / kgSOP is slightly inferior to that of 200mg / kg SOP, but it is still significantly different from the DSS group.
[0077] 3. Effect of cichorium polysaccharide on colorectal length in mice with DSS-induced colitis
[0078] The research results are as follows Figure 6 As shown in the figure, all mice drinking DSS had different degrees of colon shortening, and both 50mg / kg SOP, 200mg / kg SOP and the positive control 100mg / kg 5-ASA could alleviate DSS-induced colon shortening. The effect of 200mg / kg SOP was equivalent to that of 100mg / kg 5-ASA, and the effect of 50mg / kg SOP was slightly inferior to that of 200mg / kg SOP, but still significantly different from the DSS group.
[0079] 4. Effect of citronellol polysaccharide on pathological changes of colon tissue HE staining in mice with DSS-induced colitis
[0080] The research results are as follows Figure 7As shown, the inventors used HE staining to evaluate the pathological changes in the colon tissue of each group of mice, and found that the mucosal structure of the colon tissue of the control group mice was intact and there was no pathological damage. The structural integrity of the colon of the mice in the DSS group was completely destroyed, the submucosal edema was obvious, a large number of granulocytes and monocytes infiltrated, and the glandular structure basically disappeared. The pictures confirmed that whether it was 0mg / kgSOP or 200mg / kgSOP or the positive control 100mg / kg5-ASA, they could alleviate the pathological damage induced by DSS, among which 200mg / kg SOP and 100mg / kg 5-ASA had better recovery effects on the colon tissue structure than 50mg / kg SOP.
[0081] 5. Effect of cissin on the expression of iNOS, CD86 and IL-6 inflammatory proteins in colon tissue of mice with DSS-induced colitis
[0082] The research results are as follows Figure 8 As shown in the data, iNOS and IL-6, as inflammatory proteins, were significantly increased in the colon tissues intervened by DSS, and then significantly decreased after the treatment of SOP. Previous studies have confirmed that macrophages account for a high proportion of inflammatory cells in the colon tissues of colitis. CD86, as an M1 macrophage marker, was also significantly increased after DSS intervention, and was significantly decreased after both SOP and 5-ASA interventions, suggesting that SOP plays an important role in the phenotypic transformation of macrophages.
[0083] 6. Effect of citronellol polysaccharide on the expression of iNOS in colon immunohistochemical staining of DSS-induced colitis mice
[0084] The research results are as follows Fig. 9 As shown, iNOS is an important inflammatory factor. The inventors used intestinal tissue paraffin sections for immunohistochemical staining, and the results showed that both SOP and 5-ASA affected the expression of iNOS, which was the same as the WB results.
[0085] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.
Claims
1. A method for extracting quercetin polysaccharide extract, characterized in that: The steps include: 1) Using fresh stems and leaves of chicory as materials, crude polysaccharide precipitation was collected by boiling and alcohol precipitation; 2) The crude polysaccharide is precipitated and then subjected to the sevage method to remove the impurity proteins contained therein; 3) The obtained deproteinized polysaccharide is subjected to DEAE-52 cellulose chromatography again to remove the polysaccharide components with a lower content, and retain the main polysaccharide with a higher content; 4) The main part of the polysaccharide is precipitated and purified to obtain the chicory polysaccharide extract.
2. The extraction method according to claim 1, characterized in that: In step 1), fresh stems and leaves of chicory are used as materials. After being crushed, they are boiled in clean water at a ratio of 1:10 to 20 for 1 to 2 hours under normal pressure. The resulting solution is concentrated to 1 / 4 of the original volume, cooled, and then 3 times the volume of anhydrous ethanol is added for precipitation. After being refrigerated in a refrigerator overnight, the crude polysaccharide precipitate is collected by centrifugation.
3. The extraction method according to claim 2, characterized in that: In step 2), the mixed solution in the sevage method is composed of a crude polysaccharide solution, chloroform and n-butanol solution, and the volume ratio of the crude polysaccharide solution, chloroform and n-butanol solution is 1:1:3-5; the mixed solution is treated with a high-speed homogenizer for 2-4 minutes and then placed in a centrifuge for 4-6 minutes at a centrifugal speed of 5000-10000 rpm. After centrifugation, the white denatured protein part is removed, and the above process is repeated 3 times.
4. The extraction method according to claim 3, characterized in that: In step 3), the quercetin is dissolved and placed on a DEAE-52 cellulose chromatography column, and gradient elution is performed using 0.2-0.6% sodium chloride; the collected eluate is tested with a sulfuric acid phenol solution, and the collected liquid with a coffee color or dark brown color is collected and numbered, and the collected liquid with a significantly increased polysaccharide concentration is named SOP, and after precipitation with four times the volume of anhydrous ethanol, the purified quercetin is obtained.
5. A quercetin polysaccharide extract obtained by the extraction method according to any one of claims 1 to 4.
6. Use of the quercetin polysaccharide extract according to claim 5 in the preparation of a drug for treating small intestinal ulcer.
7. The use according to claim 6, characterized in that: The drug can significantly prevent weight loss and colon atrophy in mice, and reduce the DAI scores of mice.
8. The use according to claim 6, characterized in that: The drug can reduce the disorder of colon tissue structure, significantly reduce the infiltration of inflammatory cells, and lower the histological score.
9. The use according to claim 6, characterized in that: The drug can reduce the protein levels of iNOS, CD86 and IL-6 in colon tissue.
10. The use according to claim 6, characterized in that: The drug can reduce the expression of iNOS in the colon tissue of colitis mice by immunohistochemistry.