Application of Polysaccharide from Rhizoma Cyperi in Preparation of Medicine for Preventing or Treating Inflammatory Bowel Disease

Abstract

The present invention discloses the use of schizonepeta polysaccharide in the preparation of drugs for preventing or treating inflammatory bowel disease. By establishing an IBD mouse model induced by dextran sodium sulfate (DSS), the results show that schizonepeta polysaccharide has a significant therapeutic effect on DSS-induced colitis and can significantly improve IBD symptoms, providing a theoretical basis and clinical foundation for new drugs for the clinical prevention and treatment of IBD.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to application of radix serrata polysaccharides in preparing medicines for preventing or treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a group of chronic nonspecific intestinal inflammatory diseases whose etiology is not yet clear. It poses a serious threat to people's health and quality of life, including ulcerative colitis (UC) and Crohn's disease (CD). The main clinical manifestations are abdominal pain, diarrhea, bloody stools, weight loss, etc. The occurrence and development of IBD are accompanied by a variety of serious complications, such as colon cancer, colon fibrosis, depression, anxiety, and metabolic disorders. The pathogenesis of IBD is still unclear. In recent years, it is believed that it may be related to factors such as the external environment, intestinal microorganisms, mucosal immunity, and genetics. At present, the treatment of IBD in clinical practice mostly adopts drug therapy. Common drugs include aminosalicylic acids, glucocorticoids, immunomodulators, and biological agents. However, these drugs have a variety of adverse drug reactions, which significantly reduce patient compliance and significantly weaken the clinical treatment effect.

[0003] The fruit of the Myrtaceae plant Myrtle, the Myrtaceae, is widely used as a traditional Chinese medicinal material for its rich nutrients. The fruit contains a variety of active ingredients such as flavonoids, phenolic components, amino acids and polysaccharides, which have the effects of nourishing blood and stopping bleeding, astringing the intestines and consolidating essence, promoting body fluid and quenching thirst, and improving immunity. Studies have shown that the flavonoids contained in the fruit can promote vasodilation, lower blood pressure and cholesterol levels, and prevent the occurrence of thrombosis and tumors. However, there is no literature report on the use of polysaccharides from the fruit of the Myrtaceae in the preparation of drugs for the prevention or treatment of inflammatory bowel disease. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a new application of Polysaccharide from Haworthia scoparia in the preparation of drugs for preventing or treating inflammatory bowel disease, in order to provide a theoretical basis and clinical foundation for the research of new drugs for preventing or treating inflammatory bowel disease.

[0005] The method for preparing the polysaccharide of the present invention comprises the following steps:

[0006] (1) Using water as an extraction solvent, extracting the medicinal material of the genus Cyperus rotundus by heating, combining the extracts and concentrating under reduced pressure, centrifuging the concentrate, taking the supernatant and adding anhydrous ethanol to a final ethanol concentration of 30%, precipitating with alcohol and standing to separate the precipitate 1 and the precipitate supernatant 1, collecting the precipitate 1 to obtain the genus Cyperus rotundus crude polysaccharide 30;

[0007] (2) taking the alcohol precipitation supernatant 1 and continuing to add anhydrous ethanol until the final ethanol concentration is 60%, letting it stand after alcohol precipitation, separating the precipitate 2 and the alcohol precipitation supernatant 2, collecting the precipitate 2 to obtain the crude polysaccharide 60 of the schizonepeta tenuifolia;

[0008] (3) Take the alcohol precipitation supernatant 2 and continue to add anhydrous ethanol to a final concentration of 90%. After alcohol precipitation, let it stand and separate

[0009] The precipitate 3 and the alcohol precipitation supernatant 3 were obtained, and the precipitate 3 was collected to obtain the crude polysaccharide 90 of the schisandra chinensis.

[0010] (4) Dissolve 30% crude polysaccharide of Psoralea corylifolia, 60% crude polysaccharide of Psoralea corylifolia and 90% crude polysaccharide of Psoralea corylifolia in deionized water

[0011] The mixture was centrifuged in water, and Sevage reagent was added to the supernatant to remove protein to obtain a polysaccharide solution after protein removal, which was concentrated, dialyzed with running water and then dried to obtain the schisandra chinensis polysaccharide 30, schisandra chinensis polysaccharide 60 and schisandra chinensis polysaccharide 90 respectively.

[0012] The mass content of the polysaccharide in the schisandra chinensis polysaccharide 30 is 40-50%, the average molecular weight of the polysaccharide is 200542Da, and the mass content of the protein is 15-20%.

[0013] The mass content of the polysaccharide in the schisandra chinensis polysaccharide 60 is 60-65%, the average molecular weight of the polysaccharide is 2374Da, and the mass content of the protein is 4-6%.

[0014] The mass content of polysaccharide in the schisandra chinensis polysaccharide 90 is 80-90%, the average molecular weight of the polysaccharide is 997 Da, and the mass content of protein is 3-5%.

[0015] The inflammatory bowel disease (IBD) described in the present invention includes ulcerative colitis and Crohn's disease.

[0016] The present invention uses C57BL / 6 mice as experimental animals, and induces an IBD mouse model by dextran sulfate sodium (DSS). The results show that 30% of Polysaccharide from the Mountain Twister, 60% of Polysaccharide from the Mountain Twister, and 90% of Polysaccharide from the Mountain Twister all have a significant reversal effect on weight loss caused by inflammatory bowel disease. By observing the fecal viscosity and fecal bleeding of mice every day, the DAI is calculated, and it is found that the DAI score of the Polysaccharide from the Mountain Twister group is significantly reduced; the length of the mouse colon is measured, and it is found that Polysaccharide from the Mountain Twister can significantly prolong the colon length of IBD mice. The above results show that 30% of Polysaccharide from the Mountain Twister, 60% of Polysaccharide from the Mountain Twister, and 90% of Polysaccharide from the Mountain Twister can significantly improve the symptoms of IBD mice and have a significant therapeutic effect on IBD.

[0017] The drug of the present invention comprises an effective amount of schisandra chinensis polysaccharide and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from pharmaceutically acceptable water extracts. Thus, the drug can be prepared and formed into a form suitable for administration of a clinical drug preparation.

[0018] Preferably, the dosage form of the drug is an aqueous extract.

[0019] Compared with the prior art, the present invention has the following excellent effects:

[0020] The present invention provides a new use of Polysaccharide from Haworthia scoparia in preparing a drug for preventing or treating inflammatory bowel disease. By establishing an IBD mouse model induced by dextran sodium sulfate (DSS), the results show that Polysaccharide from Haworthia scoparia has a significant therapeutic effect on DSS-induced colitis and can significantly improve IBD symptoms, thus providing a theoretical basis and clinical foundation for new drugs for the clinical prevention and treatment of IBD. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The weight average molecular weight test result of SNZ30;

[0022] Figure 2 is the test result of SNZ60 weight average molecular weight;

[0023] Figure 3 is the test result of SNZ90 weight average molecular weight;

[0024] Figure 4 . Effect of SNZ on body weight of colitis mice, ***p<0.001 (model group VS blank group); ##P<0.01, ###P<0.001 (SNZ group VS model group).

[0025] Figure 5 . Effect of SNZ on the disease activity index of colitis mice, ***p<0.001 (model group VS blank group); ##P<0.01, ###P<0.001 (SNZ group VS model group).

[0026] Figure 6 Effect of SNZ on colon length of colitis mice, ***p<0.001 (model group VS blank group); ###P<0.001 (SNZ, 5-ASA group VS model group). DETAILED DESCRIPTION

[0027] The present invention is further described below by means of specific implementation methods. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0028] Example 1: Therapeutic effect of Polysaccharide from Rhizoma Cyperi on IBD in mice

[0029] 1. Materials and Methods

[0030] 1.1 Drugs

[0031] The polysaccharide of the schisandra chinensis was prepared by water extraction and alcohol precipitation. 7 kg of the schisandra chinensis medicinal material was extracted at 80 ° C for 3 hours, with a solid-liquid ratio of 1:10. The extraction was performed 3 times, the extracts were combined, concentrated to 1 / 8 of the original volume, centrifuged at 4000 rpm for 10 minutes, the supernatant was added with anhydrous ethanol for alcohol precipitation, and the final concentration of the alcohol precipitation was 30%. After the alcohol precipitation, it was allowed to stand at 4 ° C for 48 hours, and the precipitate and the supernatant of the alcohol precipitation were separated. The alcohol precipitation was SNZ30. The supernatant of the alcohol precipitation was taken and anhydrous ethanol was continued to be added to the final concentration of ethanol of 60%, and the precipitate and the supernatant of the alcohol precipitation were separated. The alcohol precipitation was SNZ60. The supernatant of 60% of the alcohol precipitation was taken and anhydrous ethanol was continued to be added to the final concentration of 90%, and the precipitate and the supernatant of the alcohol precipitation were separated. The alcohol precipitation was SNZ90. Take the alcohol precipitate (SNZ30, SNZ60, SNZ90) and dissolve it in deionized water, centrifuge it at 4000rpm for 10min, take the supernatant and Sevage reagent (n-butanol: chloroform = 1:4) in a volume ratio of 1:4 to remove protein, and remove protein 4 times. Then take the deproteinized solution and concentrate it, dialyze it with running water for 48h, and then freeze-dry it. After freeze-drying, the sample obtained the dry mountain twisting nut crude polysaccharides SNZ30, SNZ60 and SNZ90. The polysaccharide content was detected by concentrated sulfuric acid-phenol method, the protein content was detected by Coomassie brilliant blue method, and the polysaccharide purity and average molecular weight were analyzed by HPGPC method. The determination conditions are as follows: chromatographic column: TSK G-3000PW XL ; Mobile phase: 0.1 mol / L K2PO4 solution; Flow rate: 0.5 mL / min; Injection volume: 20 μL.

[0032] Sulfasalazine (lot: J2212276) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dextran sulfate sodium was purchased from MP Biomedicals (lot: S7980).

[0033] 1.2 Instruments

[0034] MTB1000D electronic balance (Shenzhen Meifu Electronics Co., Ltd.).

[0035] 1.3 Animals

[0036] C57BL / 6 male mice (purchased from Guangdong Medical Experimental Animal Center, license number SCXK (Yue) 2022-0002 Yuejianzhengzi 2006A018), weighing 19-21 g. Mice were housed in the Experimental Animal Center of Guangdong Medical University under 12 h light / darkness and free access to food.

[0037] 1.4 Methods

[0038] After 7 days of adaptive feeding with standard diet and water (24-25°C, humidity 70-75%, 12 hours of light / darkness), the mice were randomly divided into 6 groups, 6 mice in each group, including control group, model group, 5-ASA group (200 mg / kg), SNZ30 group (200 mg / kg, po), SNZ60 group (200 mg / kg, po), SNZ90 group (200 mg / kg, po). SNZ30, SNZ60, and SNZ90 were orally administered for 5 days, the model group was orally administered with purified water for 5 consecutive days (from day 0 to day 5), and on day 5, the mice were free to drink 2.5% DSS and continued to be administered for 7 days (from day 5 to day 12). The blank group was free to drink purified water. The weight, fecal viscosity, and fecal bleeding of the mice were observed and recorded every day. On day 12, the anesthetized mice were killed after blood was taken. The length of the mouse colon tissue was measured.

[0039] Evaluation by disease activity index (DAI): The disease activity index score was determined according to the weight loss (no loss was recorded as 0 points, loss of 1% to 5% was recorded as 1 point, loss of 5% to 10% was recorded as 2 points, loss of 10% to 5% was recorded as 3 points, and loss greater than 15% was recorded as 4 points), stool viscosity (normal stool was recorded as 0 points, soft stool was recorded as 1 point, loose and wet stool was recorded as 2 points, diarrhea and dry stool was recorded as 3 points, diarrhea and wet stool was recorded as 4 points), and fecal bleeding of IBD mice (no blood was recorded as 0 points, slight blood was recorded as 1 point, occult blood was recorded as 2 points, little bleeding was recorded as 3 points, and heavy bleeding was recorded as 4 points). DAI calculation: DAI = (weight loss score + stool viscosity score + stool blood degree score) / 3

[0040] 1.5 Statistical methods

[0041] The results were analyzed using Graphpad Prism 7.0 statistical analysis software. The comparison between samples was performed using t-test.

[0042] 2. Results

[0043] 2.1 Determination of sugar content, protein content, polysaccharide purity and average molecular weight of SNZ polysaccharide

[0044] The SNA polysaccharide and protein contents are shown in Table 1 , and the polysaccharide purity and average molecular weight were determined as shown in Figure 1 , 2 , as shown in Figure 3.

[0045] Table 1. Analysis of polysaccharide content and protein content of SNZ

[0046]

[0047] Effect of SNZ on the body weight of IBD mice

[0048] The experimental results showed that SNZ had an inhibitory effect on weight loss caused by IBD, such as Figure 4 As shown, there was a significant difference in body weight between SNZ30 and the model group (p < 0.001), there was a significant difference in body weight between SNZ60 and the model group (P < 0.01), there was a significant difference in body weight between SNZ90 and the model group (P < 0.01), and there was a significant difference in body weight between the model group and the blank group (P < 0.001). The results are shown in Table 2.

[0049] Table 2. Effect of TSXL on body weight of colitis mice (x - ±SD N=6)

[0050]

[0051]

[0052] ***p<0.001 (model group VS blank group); ##P<0.01, ###P<0.001 (SNZ group VS model group).

[0053] Effect of SNZ on the disease activity index of colitis mice

[0054] After SNZ treatment, the symptoms of diarrhea, rectal bleeding and weight loss in IBD mice were significantly alleviated, and the DAI score was significantly reduced. There was a significant difference in DAI between the SNZ30 group and the model group (P<0.01), between the SNZ60 group and the model group (P<0.001), between the SNZ90 group and the model group (P<0.01), and between the model group and the blank group (p<0.001). The results are shown in Table 3. Figure 5 .

[0055] Table 3. Effect of SNZ on the disease activity index of colitis mice (x - ±SD N=6)

[0056]

[0057] ***p<0.001 (model group VS blank group); ##P<0.01, ###P<0.001 (SNZ group VS model group).

[0058] Effect of SNZ treatment on colon length

[0059] Compared with the blank group, the colon length of the model group was significantly shortened (P<0.001), and after 12 days of SNZ treatment, the shortening of colon length was significantly reversed. There was a significant difference in colon length between the SNZ30 group and the model group (p<0.001), a significant difference in colon length between the SNZ60 group and the model group (p<0.001), and a significant difference in colon length between the SNZ90 group and the model group (p<0.001). The results are shown in Table 4. Figure 6 .

[0060] Table 4. Effect of SNZ on colon length in colitis mice (x - ±SD, N = 6)

[0061]

[0062] ***p<0.001 (model group VS blank group); ###P<0.001 (SNZ, 5-ASA group VS model group).

[0063] In summary, it can be seen that there are significant differences between the SNZ30, SNZ60, and SNZ90 groups and the model group in terms of body weight, DAI score, and colon length. It can be concluded that SZN has a significant therapeutic effect on DSS-induced colitis and can significantly improve IBD symptoms.

Claims

1. Use of a polysaccharide from Rhizoma Cibotii in the preparation of a drug for preventing or treating inflammatory bowel disease; the polysaccharide from Rhizoma Cibotii is Rhizoma Cibotii polysaccharide 30, Rhizoma Cibotii polysaccharide 60 or Rhizoma Cibotii polysaccharide 90; The mass content of polysaccharide in the schisandra chinensis polysaccharide 30 is 40-50%, the weight average molecular weight of the polysaccharide is 200542 Da, and the mass content of protein is 15-20%; The mass content of polysaccharide in the schisandra chinensis polysaccharide 60 is 60-65%, the average molecular weight of the polysaccharide is 2374 Da, and the mass content of protein is 4-6%; The mass content of polysaccharide in the schisandra chinensis polysaccharide 90 is 80-90%, the average molecular weight of the polysaccharide is 997Da, and the mass content of protein is 3-5%; The preparation method of the schisandra chinensis polysaccharide comprises the following steps: (1) Using water as the extraction solvent, the medicinal material of the Herba Cynoglossi was heated and extracted, the extracts were combined and concentrated under reduced pressure, the concentrate was centrifuged, and the supernatant was added with anhydrous ethanol to a final ethanol concentration of 30%. After alcohol precipitation, the mixture was allowed to stand to separate the precipitate 1 and the alcohol precipitation supernatant 1, and the precipitate 1 was collected to obtain the Herba Cynoglossi crude polysaccharide 30; (2) taking the alcohol precipitation supernatant 1 and continuing to add anhydrous ethanol until the final ethanol concentration is 60%, letting it stand after alcohol precipitation, separating the precipitate 2 and the alcohol precipitation supernatant 2, collecting the precipitate 2 to obtain the crude polysaccharide 60 of the schizonepeta tenuifolia; (3) Take the alcohol precipitation supernatant 2 and continue to add anhydrous ethanol until the final ethanol concentration is 90%. After alcohol precipitation, let it stand to separate the precipitate 3 and the alcohol precipitation supernatant 3. Collect the precipitate 3 to obtain the crude polysaccharide 90 of the schizonepeta tenuifolia. (4) Dissolve crude polysaccharide 30, crude polysaccharide 60 and crude polysaccharide 90 of Haworthia scoparia in deionized water, centrifuge and add Sevage reagent to the supernatant to remove protein to obtain a polysaccharide solution after protein removal. Concentrate the solution, dialyze with running water and then dry to obtain Haworthia scoparia polysaccharide 30, Haworthia scoparia polysaccharide 60 and Haworthia scoparia polysaccharide 90, respectively.

2. The use according to claim 1, characterized in that: The inflammatory bowel diseases are ulcerative colitis and Crohn's disease.

3. The use according to claim 1, characterized in that: The medicine comprises an effective amount of schisandra chinensis polysaccharide and a pharmaceutically acceptable carrier.

4. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, powder, granule or capsule.

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