CN115232224B - Oroxyli polysaccharide and medical application thereof in preparation of medicines for treating ulcerative colitis - Google Patents

Oroxyli polysaccharide and medical application thereof in preparation of medicines for treating ulcerative colitis Download PDF

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CN115232224B
CN115232224B CN202210930962.1A CN202210930962A CN115232224B CN 115232224 B CN115232224 B CN 115232224B CN 202210930962 A CN202210930962 A CN 202210930962A CN 115232224 B CN115232224 B CN 115232224B
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polysaccharide
oroxylum indicum
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ulcerative colitis
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万瑾毅
姚海强
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Beijing University of Chinese Medicine
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Abstract

The invention discloses oroxylum indicum polysaccharide and medical application thereof in preparing a medicament for treating ulcerative colitis. The oroxylum indicum polysaccharide is extracted from oroxylum indicum seeds by a classical water extraction and alcohol precipitation method, and experiments prove that the oroxylum indicum polysaccharide has the activity of treating ulcerative colitis, so that the oroxylum indicum polysaccharide has an application prospect of being developed into a medicament for treating ulcerative colitis.

Description

Oroxyli polysaccharide and medical application thereof in preparation of medicines for treating ulcerative colitis
Technical Field
The invention belongs to the field of chemistry, relates to polysaccharide and application thereof, and in particular relates to oroxylum indicum polysaccharide and medical application thereof in preparing a medicament for treating ulcerative colitis.
Background
Ulcerative colitis (ulcerative colitis, UC) is a chronic nonspecific inflammation of the colorectal, formerly known as "intestinal mucosal segmental inflammation" in the early 20 th century, and lesions often start from the rectum and are continuously diffusely distributed, which can involve the entire colorectal. The main clinical manifestations of UC are recurrent or persistent diarrhea, abdominal pain, mucopurulent bloody stool, and also parenteral systemic symptoms such as scleritis, joint damage, primary sclerosing cholangitis, etc. (thought of ulcerative colitis treatment strategy from a surgical point of view, journal of chinese utility surgery, volume 33, 7, 2013, 7).
Oroxylum indicum (L.) Vent, also known as semen Oroxyli, semen Pisi Sativi paper, fructus Psoraleae, and fructus Psoraleae, is dry mature seed of semen Oroxyli of Bignoniaceae. Semen Oroxyli is used as a traditional Chinese medicine, and is distributed in many places in China, mainly produced in Yunnan, guangxi, guizhou and the like. Semen Oroxyli is bitter and sweet in taste, cool in nature, enters lung, liver and stomach meridians, has the effects of moistening lung, relieving sore throat, soothing liver and harmonizing stomach, and is mainly used for cough due to lung heat, hoarseness, sore throat, pain in liver and stomach qi and sores. Modern researches have found that oroxylum indicum has various pharmacological effects such as antibacterial, anti-inflammatory, antiallergic, antiviral, antioxidant, cancer inhibiting, tumor inhibiting, blood glucose reducing, etc. (oroxylum indicum pharmacological effect, clinical application research progress, traditional Chinese medicine journal, volume 49, 5 of 2021, 5 month).
Oroxylum indicum polysaccharide is a component of oroxylum indicum that is ignored by researchers, and has been rarely studied. The Shanghai pharmaceutical institute Ding Kan of China academy of sciences and the like provides oroxylum indicum uniform polysaccharide, and pharmacological experimental research shows that the oroxylum indicum uniform polysaccharide has the activities of resisting novel coronaviruses and inhibiting coronavirus key protease 3CLpro proteolytic enzyme and PLpro proteolytic enzyme, and can be developed into a medicament for resisting the novel coronaviruses (Chinese patent CN 113372459A).
The invention provides the invention for discovering other pharmacological activities and medical applications of oroxylum indicum polysaccharide.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides oroxylum indicum polysaccharide and a medical application thereof in preparing a medicament for treating ulcerative colitis.
The above object of the present invention is achieved by the following technical scheme:
an oroxylum indicum polysaccharide prepared by the following steps: soaking semen Oroxyli powder in hot water, extracting, filtering the extractive solution, concentrating, adding 4 times of 95% ethanol water solution, mixing, standing, centrifuging, and collecting precipitate; and (3) re-dissolving the precipitate with water, dialyzing, adding 4 times of 95% ethanol water solution, fully and uniformly mixing, standing, centrifugally collecting the precipitate, and drying to obtain the product.
Preferably, the extraction solid-to-liquid ratio is 1kg:10L.
Preferably, the hot water temperature is 80 ℃.
Preferably, the extracts are combined, 3 times for 2 hours each.
Preferably, the standing condition is 4℃for 24 hours.
Preferably, the dialysis bag format is 3000Da.
The medical application of any one of the oroxylum indicum polysaccharides in preparing a medicament for treating ulcerative colitis.
The beneficial effects are that:
the oroxylum indicum polysaccharide is extracted from oroxylum indicum seeds by a classical water extraction and alcohol precipitation method, and pharmacological experiments prove that the oroxylum indicum polysaccharide has the activity of treating ulcerative colitis, so that the oroxylum indicum polysaccharide has an application prospect of being developed into a medicament for treating ulcerative colitis.
Drawings
FIG. 1 is a DAI score for each group of mice;
FIG. 2 shows inflammatory cytokine levels in intestinal tissues of mice in each group;
FIG.3showstheexpressionlevelsofzonaltightjunctionproteinsZO-1,Occludin,JAM-Aineachgroupofmice;
a, B in FIG. 4 shows the levels of MUC2 protein expressed in intestinal cup cells of mice of each group.
Detailed Description
The following describes the essential aspects of the present invention in detail with reference to examples, but is not intended to limit the scope of the present invention.
1. Experimental materials
1.1 materials and reagents
The modeling agent DSS (MW 36000-50000) was purchased from MP Biomedicals, USA, and the remaining reagents were purchased from the national drug group. Semen Oroxyli is purchased from Beijing Tongren Tang Co., ltd (origin: guangxi), and the voucher sample is stored in Beijing university of traditional Chinese medicine. Rabbit polyclonal antibodies (ZO-1, occidin, JAM-A, MUC-2) were purchased from Abcam, USA, DNA extraction kit was purchased from Life Technologies, USA, and ELISA kit was purchased from Wohan cloning technologies Co.
1.2 laboratory animals
30 healthy C57BL/6 male mice, 6-8 weeks old, weighing 20-25g, were purchased from Beijing Vitolith laboratory animal technologies Co. The method is characterized in that the method is fed to experimental animal centers of Chinese medical institute of Beijing university, SPF environment is kept at 20-25 ℃ all the time, relative humidity is controlled at 50-60%, light and darkness alternate every day, and the alternation time is 12 hours. After 1 week of pre-adaptation, the mice were randomly divided into three groups: control, model and oroxylum seed groups.
2. Experimental method
2.1 preparation of Oroxyli polysaccharide
Drying semen Oroxyli, pulverizing, and sieving with 60 mesh sieve to obtain semen Oroxyli dry powder. Firstly, soaking dry semen oroxyli powder in about 10 times of 80 ℃ hot water for 2 hours, carrying out vacuum filtration on the supernatant extract, circularly extracting for 3 times, and combining the 3 times of extract. Concentrating under reduced pressure to 1/4 of the original volume, slowly adding 95% ethanol with volume of 4 times, mixing, standing in refrigerator at 4deg.C for precipitation for 24 hr, and centrifuging to obtain precipitate. Dissolving in water, loading into 3000Da dialysis bag, dialyzing with flowing purified water for 48 hr, precipitating with 95% ethanol, and drying the precipitate to obtain semen Oroxyli polysaccharide.
2.2 establishment and administration of the UC model in mice
Model group mice were free to drink 3% dextran sodium sulfate aqueous solution for 7 days to induce UC model, and control group mice were free to drink distilled water. During the molding, 300mg/kg of oroxylum indicum polysaccharide aqueous solution (freshly prepared by adding distilled water into dry powder) is added to the oroxylum indicum group mice except for molding, and the other groups are filled with equal volume of distilled water in parallel. Once daily for 7 days.
2.3 assessment of mouse State and colon Condition
Mice were evaluated for their state performance such as mental state, hair, body weight, and stool characteristics, recorded daily. Wherein the quantitative evaluation is performed using disease activity index (Disease activity index, DAI). Dai= (weight loss fraction + fecal trait fraction + degree of occult blood)/3. Wherein the weight reduction rate is 0,1-5 is 1, 5-10 is 2, 10-15 is 3, and more than 15 is 4; the fecal character fraction is normally 0, the semithin stool is 2, and the thin stool is 4; the score of cryptotaemia was normally 0 score, cryptotaemia positive was 2 score, and dominant bleeding was 4 score. By day 7 of the experiment, 24 hours fasted, mice were sacrificed by cervical dislocation, anus was dissected to ileocecum colon, and length was measured.
2.4 determination of inflammatory cytokines in colon tissue of mice
Taking part of colon tissue of a mouse, adding PBS for fully homogenizing, centrifuging, taking supernatant, and detecting the inflammatory cytokines IL-6, IL-1 beta, TNF-alpha and IFN-gamma levels in the tissue by adopting an ELISA method.
2.5 determination of mouse colon tissue Tight-junction protein Westernblot
The colon tissue of the mouse is taken, about 20mg is weighed after thawing on ice, put into a precooled tissue homogenizer, 200 μl of precooled RIPA solution containing protease/phosphatase inhibitor is rapidly added, and homogenized fully on ice. After lysis, the samples were centrifuged at 13000 rpm at 4℃for 10 minutes and the supernatants were assayed for protein concentration by BCA. And after adjusting the concentration of each group of proteins to be equal, adding a loading buffer solution with a corresponding volume, uniformly mixing, boiling for 5 minutes by using a water bath, and preserving at the temperature of minus 20 ℃. theproteinextractistransferredtoPVDFmembraneafterpolyacrylamidegelelectrophoresis,incubatedwithZO-1,Occludin,JAM-Aprimaryantibodyovernight,andthenincubatedwithHRP-labeledsecondaryantibody. Development was performed using an enhanced chemiluminescent detection solution and the band density was analyzed using ImageJ software.
2.6 staining of the colonic tissue mucus layer AB-PAS in mice
A portion of the colon tissue of the mice was taken, fixed in 4% paraformaldehyde, and then sectioned by paraffin embedding. Slice dewaxing and water washing, sequentially dip-dyeing with alisxin blue dye liquor, oxidizing with 0.5% periodate aqueous solution, dip-dyeing with Schiff's reagent in dark place, counterstaining with hematoxylin, differentiating with ethanol hydrochloride, returning blue with ammonia water, sealing with neutral gum after xylene transparency, and microscopic examination.
2.7 colon tissue mucin MUC2 immunohistochemistry
After dewaxing and washing colon sections of mice, antigen retrieval is carried out by using sodium citrate solution, peroxidase blocking agent is dripped, the primary antibody and the secondary antibody of MUC2 are incubated, DAB color development is carried out, hematoxylin counterstain is carried out, and the colon sections are dehydrated, transparent, neutral gum sealed and microscopic examination is carried out conventionally.
2.8 statistical analysis
The data were analyzed using SPSS 23.0 and GraphPad Prism 9 software, the experimental results were expressed as mean ± standard deviation, the comparison between groups was statistically significant using single factor analysis of variance, P < 0.05, P < 0.01 as the difference.
3. Experimental results
3.1 Effect of Oroxyle polysaccharide on the status manifestations of UC model mice
Experimental results show that the mice in the control group have good overall state, luster of hair, weight gain and normal fecal characteristics; the other two groups of mice have the phenomena of listlessness, dull hair, weight reduction, loose stool, purulent blood and stool with different degrees, but the overall performance of the oroxylum indicum group is better than that of the model group. The DAI scores for each group of mice are shown in figure 1. The oroxylum indicum polysaccharide can significantly improve the state performance of mice compared with the model group.
3.2 Effect of Oroxyli polysaccharide on the colon and Length of UC model mice
The colon condition of the mice is observed, and the conditions that the colon mucous membrane of the mice in the control group is smooth, the color of the intestinal wall is normal, the colon surface of the mice in the model group is hyperemia or ulceration and the like with different degrees are found, and the whole colon intestinal wall of the mice in the oroxylum indicum group is slightly hyperemia and has less ulceration. Furthermore, as can be seen from the colon length of each group of mice in table 1, oroxylin significantly counteracted the phenomenon of colonic atrophy caused by inflammation compared to the model group.
TABLE 1 results of colon length in mice
Figure SMS_1
3.3 Effect of Oroxyle polysaccharide on levels of intestinal inflammatory factors in mice with UC model
As can be seen from fig. 2, the level of inflammatory cytokines in intestinal tissues of the UC model mice was significantly increased. Compared with the model group, the oroxylin can obviously reduce the level of inflammatory cytokines IL-6, IL-1 beta, TNF-alpha and IFN-gamma in intestinal tissues, and can play an anti-inflammatory role on UC mice.
3.4 Effect of Oroxyle polysaccharide on the expression level of the intestinal canal tight junction protein of UC model mice
The physical barrier of the intestinal tract is a histological barrier formed by intestinal epithelial cells and tight connection among cells, wherein the tight connection structure of the intestinal tract is formed by various proteins, including transmembrane proteins Occludin, cytoplasmic protein ZOs family, connection adhesion molecule JAM family and the like. ascanbeseenfromfig.3,theexpressionleveloftheintestinalcanalcompactjunctionproteinoftheUCmodelmiceissignificantlyreduced,andtheoroxylumindicumpolysaccharidecansignificantlypromotetheexpressionoftheintestinalcanalcompactjunctionproteinsZO-1,Occludin,JAM-a,andenhancetheintestinalcanalphysicalbarrierfunctionoftheUCmice,comparedwiththemodelgroup.
3.5 Effect of Oroxyle polysaccharide on the intestinal mucus layer and MUC2 mucin expression level of UC model mice
The mucus layer serves as the primary structure of the intestinal chemical barrier, and its mucus is composed of a highly glycosylated polymeric network of mucins secreted by goblet cells, such as MUC proteins. From fig. 4 a, it can be seen that the mucus layer of UC model mice is significantly thinner, and oroxylin significantly increases intestinal mucus layer thickness relative to the model group. The immunohistochemical results of B in FIG. 4 show that the MUC2 protein staining in the intestinal goblet cells of the oroxylum indicum mice is obviously enhanced, and further shows that the oroxylum indicum polysaccharide obviously improves the expression level of the MUC2 mucin in the intestinal tracts of the mice, and has a certain repairing effect on the intestinal chemical barrier of UC mice.
The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.

Claims (6)

1. Use of oroxylum indicum polysaccharide for the manufacture of a medicament for the treatment of ulcerative colitis, the oroxylum indicum polysaccharide being prepared by the steps of: soaking semen Oroxyli powder in hot water, extracting, filtering the extractive solution, concentrating, adding 4 times of 95% ethanol water solution, mixing, standing, centrifuging, and collecting precipitate; and (3) re-dissolving the precipitate with water, dialyzing, adding 4 times of 95% ethanol water solution, fully and uniformly mixing, standing, centrifugally collecting the precipitate, and drying to obtain the product.
2. Use according to claim 1, characterized in that: the extraction solid-to-liquid ratio is 1kg:10L.
3. Use according to claim 1, characterized in that: the hot water temperature was 80 ℃.
4. Use according to claim 1, characterized in that: extracting for 3 times and 2 hours each time, and mixing the extracts.
5. Use according to claim 1, characterized in that: the standing condition is 4 ℃ for 24 hours.
6. Use according to claim 1, characterized in that: the dialysis bag had a specification of 3000Da.
CN202210930962.1A 2022-08-04 2022-08-04 Oroxyli polysaccharide and medical application thereof in preparation of medicines for treating ulcerative colitis Active CN115232224B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104892702A (en) * 2015-05-18 2015-09-09 徐州医学院 Method for extracting, separating and purifying two flavonoid glycosides from semen oroxyli
CN104958330A (en) * 2015-06-05 2015-10-07 辽宁中医药大学 Oroxylum indicum general flavone extraction and purification method and application thereof
CN113372459A (en) * 2021-05-07 2021-09-10 中国科学院上海药物研究所 Oroxylum indicum polysaccharide, preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104892702A (en) * 2015-05-18 2015-09-09 徐州医学院 Method for extracting, separating and purifying two flavonoid glycosides from semen oroxyli
CN104958330A (en) * 2015-06-05 2015-10-07 辽宁中医药大学 Oroxylum indicum general flavone extraction and purification method and application thereof
CN113372459A (en) * 2021-05-07 2021-09-10 中国科学院上海药物研究所 Oroxylum indicum polysaccharide, preparation method and application thereof

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