Application of polygonatum kingianum polysaccharide in preparation of medicine for treating ulcerative colitis

Abstract

The invention discloses application of polygonatum kingianum polysaccharide in preparation of medicine for treating ulcerative colitis, and relates to the field of biomedicine.The preparation method of the polygonatum kingianum polysaccharide comprises the following steps that S1, polygonatum kingianum roots are taken, smashed and then screened, and polygonatum kingianum powder is prepared; s2, the polygonatum kingianum powder obtained in the step S1 is soaked in an ethanol solution, and polygonatum kingianum powder residues are obtained; s3, performing water extraction on the polygonatum kingianum powder residues obtained in the step S2 to obtain an extracting solution; s4, concentrating the extracting solution obtained in the step S3, adding ethanol into the concentrated solution to precipitate polysaccharide, and centrifugally separating to obtain crude polysaccharide; the polygonatum kingianum polysaccharide disclosed by the invention is high in purity, the effectiveness of the polygonatum kingianum polysaccharide in treatment of ulcerative colitis is verified, and a new solution is provided for relieving the problem of shortage of current colitis treatment medicines.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to the application of Polygonatum cyrtonema polysaccharide in preparing a medicine for treating ulcerative colitis. Background Art

[0002] Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease (CD). The incidence has gradually increased in recent years and has become a complex and refractory digestive system disease that is prevalent worldwide. Although its etiology and pathogenesis have not yet been fully clarified, existing studies have shown that the disease may be related to multiple factors, including genetic susceptibility, immune system dysfunction, external environment, and intestinal microbial imbalance. The lesions usually manifest as diffuse inflammation, erosion, and fibrous scar formation in the colon mucosa and submucosa, and impaired intestinal mucosal barrier and immune imbalance are considered to be its important pathogenic mechanisms. Dextran sulfate sodium (DSS) is often used to induce animal models in research because it can significantly increase intestinal permeability, destroy intestinal mucosal barrier, upregulate inflammatory factors, activate inflammatory pathways, and lead to intestinal flora imbalance.

[0003] At present, there are various types of drugs for the clinical treatment of IBD, including 5-aminosalicylic acid, glucocorticoids, immunosuppressants and biological agents. However, these drugs are usually accompanied by serious side effects, such as causing ecological imbalance of intestinal microbiota and aggravating flora disorder. Long-term use may also cause serious adverse reactions. Therefore, it is urgent to develop a safe and effective new drug for the treatment of IBD.

[0004] Polysaccharides are natural polymers formed by aldose or ketose connected by glycosidic bonds. They are widely found in plants, marine organisms, fungi and other sources. As an important type of bioactive natural product, polysaccharides exhibit multiple activities such as anti-tumor, immunomodulatory, anticoagulant, hypoglycemic and antiviral. In recent years, Chinese herbal polysaccharides have attracted widespread attention due to their potential as the main active ingredients of Chinese medicine. Polygonatum spp. is a traditional medicinal and edible plant. The polygonatum polysaccharide contained in it has anti-tumor, antiviral, antibacterial, anti-aging and hypoglycemic effects. Studies have shown that polygonatum polysaccharides can also play an important role in the treatment of colitis through mechanisms such as anti-inflammatory, regulating excessive immune response and improving intestinal microecological imbalance. According to the 2020 edition of the Chinese Pharmacopoeia, Polygonatum mainly includes Polygonatum sibiricum Red., Polygonatum cyrtonema Hua and Polygonatum kingianum Coll. et Hemsl. Polygonatum sibiricum is a perennial herbaceous plant of the Liliaceae family that grows in Guizhou, Yunnan, Guangxi and other regions of China. Its main active ingredients include Polygonatum sibiricum polysaccharide, steroidal saponin, flavonoids, alkaloids, etc. Modern pharmacological studies have found that Polygonatum sibiricum polysaccharide has multiple pharmacological activities such as immunomodulation, antioxidant, anti-tumor and memory improvement. Despite this, the potential of Polygonatum sibiricum polysaccharide in the treatment of colitis has not been fully studied and developed.

[0005] At present, the extraction methods of polygonatum polysaccharide mainly include hot water extraction, microwave extraction, ultrasonic extraction and enzyme extraction. Among them, the ultrasonic-assisted extraction method is not suitable for large-scale industrial production due to the special equipment conditions; the microwave-assisted extraction method has problems such as limited raw material processing capacity and complex operation; although the enzyme extraction method is efficient, the amount of enzyme used is large and the subsequent removal is difficult. Taking all factors into consideration, the hot water extraction method is more suitable for large-scale production because of its simple operation and low cost. However, the existing extraction methods generally have problems such as high cost, low extraction rate, low purity and complex process, and they are urgently needed to be improved to improve the extraction efficiency and application value of polygonatum polysaccharide. Summary of the invention

[0006] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide the use of Polygonatum cyrtonema polysaccharide in the preparation of a medicine for treating ulcerative colitis.

[0007] The technical solution of the present invention is as follows:

[0008] Application of Polygonatum yunnanensis polysaccharide in preparing medicine for treating ulcerative colitis.

[0009] Furthermore, the preparation method of the Polygonatum cyrtonema polysaccharide comprises the following steps:

[0010] S1: taking the root of Polygonatum cyrtonema, crushing it and then sieving it to obtain Polygonatum cyrtonema powder;

[0011] S2: Soaking the polygonatum cyrtonema powder obtained in step S1 in an ethanol solution to obtain a polygonatum cyrtonema powder residue;

[0012] S3: extracting the powder residue of Polygonatum cyrtonema obtained in step S2 with water to obtain an extract;

[0013] S4: After the extract obtained in step S3 is concentrated, ethanol is added to the concentrate to precipitate polysaccharides, and crude polysaccharides are obtained by centrifugation; the crude polysaccharides are then defatted, purified, and dried to obtain Polygonatum cyrtonema polysaccharide.

[0014] Furthermore, in step S1, the particle size of the polygonatum odoratum powder is controlled at 20-40 meshes.

[0015] Further, in step S2, the volume concentration of the ethanol solution is 60%, the volume mass ratio of the ethanol solution to the Polygonatum odoratum powder is 10:1 mL / g, the soaking temperature is 60±2°C, each soaking time is 20-40 minutes, and a total of 2-4 soaking times.

[0016] Further, in step S3, pure water is added in a ratio of 10:1 mL / g of water to the residue of polygonatum odoratum powder, and reflux extraction is performed at 100±2° C. for 1-3 hours.

[0017] Furthermore, in step S4, pure ethanol is added to the concentrated solution to make the ethanol volume fraction of the solution reach 80wt%, the precipitation is fully stirred, and the crude polysaccharide is obtained by centrifugation after standing at room temperature (20-30°C) for 12 hours; the crude polysaccharide is defatted by a 2:1 volume chloroform-methanol mixture and then dried to finally obtain Yunnan Polygonatum cyrtonema polysaccharide.

[0018] Furthermore, the Polygonatum cyrtonema polysaccharide includes 91.28wt% fructose, 6.86wt% glucose, 0.89wt% mannose, 0.49wt% galacturonic acid, 0.23wt% galactose, 0.09wt% xylose and 0.16wt% arabinose.

[0019] The invention also discloses a medicine for treating ulcerative colitis, comprising Polygonatum cyrtonema polysaccharide and pharmaceutically acceptable pharmaceutical excipients.

[0020] The beneficial effects of the present invention are:

[0021] (1) The polygonatum cyrtonema polysaccharide extracted from the root of polygonatum cyrtonema of the present invention has high purity and adopts a high-efficiency extraction method, while reducing the cost of raw materials and simplifying the process flow, effectively solving the problem of low quality of existing extraction methods.

[0022] (2) The high-purity Polygonatum cyrtonema polysaccharide prepared by the present invention can effectively treat colitis drugs and has potential applications in the drug, providing a new solution to alleviate the current shortage of drugs for the treatment of colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A in the figure represents the weight changes of mice in each group; Figure 1 B in the figure is the disease activity index (DAI) of each group of mice; Figure 1 C and D in the figure are the colon lengths of mice in each group; Figure 1 E in the figure is the spleen size of mice in each group; Figure 1 F in the figure is the spleen index of mice in each group;

[0024] Figure 2 H&E staining of colon, liver and kidney tissues of mice in each group

[0025] Figure 3 A in the figure shows the cells expressing the intestinal tight junction protein occludin, ZO-1 and the immune cell marker Ly6C in each group of mice; Figure 3 B in the figure is the CD45 in the colon of each group of mice + The proportion of cells in colon tissue; Figure 3 C is the proportion of macrophages in the colon tissue of mice in each group; Figure 3 D in the figure is the proportion of secondary granulocytes in the colon tissue of mice in each group;

[0026] Figure 4 is the protein expression level of HMOX1, KEAP1, and p-NF-κB in the colon tissues of mice in each group;

[0027] Figure 5 A in the figure is the expression level of inflammatory factor IL6 in the serum of mice in each group; Figure 5 B in the figure is the mRNA expression level of inflammatory factor IL6 in the colon tissue of mice in each group;

[0028] Figure 6 A in the figure is the abundance index of bacterial flora in the feces of mice in each group (chao index); Figure 6 B in the figure is the diversity index of bacterial flora in the feces of mice in each group (Shannon index); Figure 6 C in the table is the diversity index of bacterial flora in the feces of mice in each group (Simpson index); Figure 6 D in the figure is the proportion of each bacterial group in the feces of mice in each group; Figure 6 E in the table is the metabolite Myristic acid in the feces of mice in each group; Figure 6 F in the figure is the metabolite Desmosterol in the feces of mice in each group. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below with reference to specific embodiments.

[0030] The raw materials and equipment used in the specific implementation of the present invention are all known commodities and are obtained by purchasing commercially available products.

[0031] Among them, DSS: dextran sulfate sodium; SASP: sulfasalazine.

[0032] The animals used in the experiment were SPF-grade male C57BL / 6J mice (20±2g), a total of 50, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. [SCXK(Su)-2023-0009]. The mouse breeding environment was controlled at a temperature of 20-26°C, a relative humidity of 40%-60%, a daily light of 10-12 hours, and good indoor ventilation. Before the experiment, the mice were adaptively fed for 1 week.

[0033] The modeling drug used was dextran sulfate sodium (DSS) with a molecular weight of 36,000 to 50,000 Da, purchased from MP Company.

[0034] Example 1: Preparation of Polygonatum cyrtonema polysaccharide

[0035] Take 200g of the root of Polygonatum yunnanensis, crush it and pass it through a No. 5 sieve (80 mesh) to obtain Polygonatum yunnanensis powder. Then add 60% ethanol by volume concentration (the volume mass ratio of ethanol to powder is 10:1mL / g) to the powder, extract it at 60°C for 30 minutes, repeat 3 times and filter it, and retain the residue. Add pure water to the residue (the volume mass ratio of pure water to residue is 10:1mL / g), reflux extract at 100°C for 2 hours, collect the supernatant after filtration and concentrate it under vacuum to obtain a crude Polygonatum yunnanensis polysaccharide concentrate. Add pure ethanol to the concentrate to make the ethanol content of the system reach 80wt%, stir the precipitation and stand at room temperature for 12 hours and then centrifuge to obtain a crude Polygonatum yunnanensis polysaccharide precipitate. Finally, the crude Polygonatum yunnanensis polysaccharide precipitate is degreased by a chloroform-methanol mixture with a volume ratio of 2:1 and then dried to finally obtain Polygonatum yunnanensis polysaccharide powder, and finally obtain 35.92g of Polygonatum yunnanensis polysaccharide.

[0036] A standard curve was drawn by liquid chromatography using standard samples, and then the composition of Polygonatum cyrtonema polysaccharide was clarified by comparing with the standard samples: it consists of fructose (91.28%), glucose (6.86%) and mannose (0.89%), and contains a small amount of galacturonic acid (0.49%), galactose (0.23%), xylose (0.09%) and arabinose (0.16%).

[0037] Example 2: Effect of Polygonatum cyrtonema polysaccharide on improving colitis

[0038] (1) Experimental process

[0039] Thirty SPF male C57BL / 6J mice were randomly divided into 6 groups according to body weight (n=5 in each group):

[0040] Blank control group (Ctrl): normal drinking water;

[0041] Model group (DSS): freely drink 2.5wt% DSS solution;

[0042] Low-dose group of Polygonatum cyrtonema polysaccharide (DSS+PKP-L): DSS+100 mg / kg Polygonatum cyrtonema polysaccharide solution;

[0043] DSS+PKP-M: DSS+200mg / kg DSS solution;

[0044] High-dose group of Polygonatum cyrtonema polysaccharide (DSS+PKP-H): DSS+400mg / kg Polygonatum cyrtonema polysaccharide solution;

[0045] Positive control group (DSS+SASP): DSS+200 mg / kg SASP solution.

[0046] Before the experiment, mice were adaptively fed for 1 week. The model group, Polygonatum cyrtonema polysaccharide group and positive control group were allowed to drink 2.5wt% DSS solution freely for 7 consecutive days, and fresh DSS solution was replaced every other day; Polygonatum cyrtonema polysaccharide group was given Polygonatum cyrtonema polysaccharide solution of different concentrations by gavage once a day from the first day of model construction; the positive control group was given SASP solution by gavage once a day; the blank control group was given normal water.

[0047] (2) Experimental observation and analysis

[0048] Starting from the first day of administration, the weight, stool morphology and blood in the stool of the mice were recorded daily. After 7 days of administration, the level of the inflammatory factor IL6 was assessed by ELISA through orbital blood sampling. Subsequently, the mice were killed by cervical dislocation, the colon and spleen were removed, the colon length was recorded, and the colon contents were cleaned with phosphate-buffered saline. Part of the colon tissue was fixed with 4.0wt% paraformaldehyde, embedded in paraffin and prepared into 4μm thick sections for staining and pathological observation under an optical microscope. Another part of the colon tissue was crushed and lysed to extract protein, and the expression level of oxidative stress-related proteins was detected by Western blot. In addition, the fecal samples were analyzed for flora by 16S rDNA sequencing, and combined with label-free metabolomics sequencing, the therapeutic effects and potential mechanisms of Polygonatum odoratum polysaccharide were systematically evaluated. The experimental results are shown in Figure 1-5 .

[0049] from Figure 1The experimental results show that PKP (polysaccharide from Yunnan Polygonatum) significantly improved the weight loss of mice on the 7th day (see Figure 1 Compared with the model group, PKP significantly reduced the disease activity index (DAI) score (see Figure 1 B) and significantly alleviated DSS-induced colon shortening in mice (see Figure 1 PKP can effectively reduce spleen swelling (see Figure 1 In conclusion, PKP significantly improved the symptoms of DSS-induced ulcerative colitis in mice.

[0050] from Figure 2 The experimental results show that PKP can significantly repair colon tissue damage. Compared with the model group, the colon tissue structure of mice treated with PKP was more complete, the glands were arranged more regularly, and the inflammatory cell infiltration was significantly reduced, showing a good colon protection effect (see Figure 2 In addition, PKP did not show obvious toxic side effects on liver and kidney tissues (see Figure 2 ). In summary, PKP alleviated DSS-induced colon tissue damage in mice.

[0051] from Figure 3 The experimental results show that PKP repairs the intestinal barrier function by upregulating the expression of proteins ZO-1 and Occludin (see Figure 3 DSS treatment led to increased macrophage infiltration in colon tissue, while PKP significantly reduced this infiltration phenomenon (see Figure 3 These results suggest that PKP can effectively repair intestinal barrier function by reducing inflammatory cell infiltration and upregulating the expression of multiple tight junction proteins.

[0052] from Figure 4 The experimental results show that PKP can inhibit the expression of oxidative stress-related protein HMOX1 in intestinal tissue and promote the expression of KEAP1 protein (see Figure 4 ). These results suggest that PKP plays a protective role by alleviating oxidative stress in intestinal tissues.

[0053] from Figure 5 The experimental results show that PKP significantly inhibited the protein level of inflammatory factor IL6 in the serum of mice induced by DSS ( Figure 5 A) and mRNA levels of inflammatory factor IL6 in colon tissue ( Figure 5 B).

[0054] from Figure 6 The experimental results show that PKP can significantly increase the abundance of intestinal microorganisms and increase the proportion of beneficial bacteria such as Firmicutes ( Figure 6In addition, PKP also improved the metabolite profile of intestinal microorganisms, significantly increasing the abundance of anti-inflammatory metabolites such as myristic acid and desmosterol ( Figure 6 E and F in ).

[0055] In summary, the purity of the Polygonatum yunnanensis polysaccharide prepared by the present invention is not less than 95%, and meets the pharmacopoeia standards. It is specifically used for the preparation of drugs for preventing and treating ulcerative colitis, and its therapeutic effect is mainly achieved by regulating intestinal microbial communities, inhibiting the release of inflammatory factors and repairing the intestinal mucosal barrier.

[0056] Under the premise that no conflict occurs, those skilled in the art may freely combine and superimpose the above-mentioned additional technical features.

[0057] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. The use of Polygonatum yunnanensis polysaccharide in the preparation of a drug for treating ulcerative colitis, characterized in that: The preparation method of polygonatum yunnanensis polysaccharide comprises the following steps: S1: taking the root of Polygonatum cyrtonema, crushing it and then sieving it to obtain Polygonatum cyrtonema powder; S2: Soaking the polygonatum cyrtonema powder obtained in step S1 in an ethanol solution to obtain a polygonatum cyrtonema powder residue; S3: extracting the powder residue of Polygonatum cyrtonema obtained in step S2 with water to obtain an extract; S4: After the extract obtained in step S3 is concentrated, ethanol is added to the concentrate to precipitate polysaccharides, and crude polysaccharides are obtained by centrifugation; the crude polysaccharides are then defatted, purified, and dried to obtain Polygonatum cyrtonema polysaccharide.

2. The use of Polygonatum yunnanensis polysaccharide according to claim 1 in the preparation of a drug for treating ulcerative colitis, characterized in that: In step S1, the particle size of the polygonatum odoratum powder is controlled at 20-40 meshes.

3. The use of Polygonatum yunnanensis polysaccharide according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that: In step S2, the volume concentration of the ethanol solution is 60%, the volume mass ratio of the ethanol solution to the Polygonatum odoratum powder is 10:1 mL / g, the soaking temperature is 60±2°C, each soaking time is 20-40 minutes, and the soaking is performed 2-4 times in total.

4. The use of Polygonatum yunnanensis polysaccharide according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that: In step S3, pure water is added in a ratio of 10:1 mL / g of water to the residue of polygonatum cyrtonema powder, and reflux extraction is performed at 98±2° C. for 1-3 hours.

5. The use of Polygonatum yunnanensis polysaccharide according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that: In step S4, pure ethanol is added to the concentrated solution to make the ethanol volume fraction of the solution reach 80wt%, the solution is fully stirred and precipitated, and the crude polysaccharide is obtained by centrifugation after standing at 20-30°C for 12 hours; the crude polysaccharide is defatted and purified by a 2:1 volume chloroform-methanol mixture and then dried to finally obtain Yunnan Polygonatum cyrtonema polysaccharide.

6. The use of Polygonatum yunnanensis polysaccharide according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that: The polygonatum yunnanensis polysaccharide includes 91.28wt% fructose, 6.86wt% glucose, 0.89wt% mannose, 0.49wt% galacturonic acid, 0.23wt% galactose, 0.09wt% xylose and 0.16wt% arabinose.

7. A drug for treating ulcerative colitis, characterized in that: It comprises the polygonatum yunnanensis polysaccharide as described in any one of claims 1 to 6 and pharmaceutically acceptable pharmaceutical excipients.

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