Use of polygonatum multiflorum polysaccharide in preparation of medicine for treating ulcerative colitis

Abstract

The application discloses a polygonatum multiflorum polysaccharide, a preparation method and application thereof in preparation of a medicine for treating ulcerative colitis. The polygonatum multiflorum polysaccharide is obtained by extraction, deproteinization and column chromatography of polygonatum multiflorum. The anti-ulcerative colitis effect of the polygonatum multiflorum polysaccharide is evaluated by establishing a dextran sodium sulfate (DSS)-induced ulcerative colitis mouse model, and it is proved that the polygonatum multiflorum polysaccharide can significantly increase the body weight of the ulcerative colitis model mice, significantly reduce the blood stool index and loose stool index, increase the colon length, effectively improve the richness of intestinal flora, effectively improve the pathological damage of the colon tissue of the ulcerative colitis model mice, and achieve the purpose of treating ulcerative colitis. Therefore, it is firstly proved that the polygonatum multiflorum polysaccharide has a significant anti-ulcerative colitis effect, and important theoretical basis is provided for development of an anti-ulcerative colitis medicine.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine polysaccharide technology, specifically relating to the application of Polygonum multiflorum polysaccharide in the preparation of drugs for treating ulcerative colitis. Background Technology

[0002] Polygonum multiflorum, or He Shou Wu, is a perennial twining herbaceous plant belonging to the Polygonaceae family. It is slightly warm in nature and has a bitter, sweet, and astringent taste. The entire plant can be used medicinally, with the root being considered the best. Its tuberous roots are slender with enlarged ends, and are reddish or dark brown in color. It is a valuable traditional Chinese medicine and a good choice for dietary therapy and health maintenance. Polygonum multiflorum contains polysaccharide active ingredients, which are its main active components. These polysaccharides possess various biological activities, including antioxidant, anti-fatigue, anti-aging, anti-Alzheimer's disease, immunomodulatory, and lipid-lowering effects. These biological activities are closely related to its molecular structure (such as branching degree and functional group modification). Studies have shown that the extraction methods (such as water extraction and alcohol precipitation, ultrasound-assisted extraction) and purification processes (such as column chromatography) of Polygonum multiflorum polysaccharides significantly affect their yield and activity. Further development of extraction methods for Polygonum multiflorum polysaccharides and research into their potential medicinal value are of great significance.

[0003] Ulcerative colitis is a chronic, nonspecific disease of the colon and rectum with an unclear etiology. The lesions are limited to the colonic mucosa and submucosa, primarily involving inflammation and ulceration of the mucosa and submucosa of the colon and rectum. This includes distorted and shortened crypt structures, increased lymphocytes and plasma cells in the lamina propria, and decreased mucin. Clinically, it mainly manifests as diarrhea, abdominal pain, and bloody, mucoid stools. This disease is difficult to cure and carries a risk of malignant transformation, severely impacting patients' health and quality of life. While the exact cause of ulcerative colitis is unclear, it may be related to multiple factors such as genetics, immunity, environment, and infection. It is more common in individuals with a family history of the disease, long-term smokers, and those with a high-fat diet. The mortality rate is relatively low.

[0004] Ulcerative colitis (UC) is characterized by immune-mediated inflammation, oxidation, and gut microbiota dysbiosis. Current treatments, both domestically and internationally, primarily include drug therapy and surgery. Commonly used drugs include aminosalicylic acid, corticosteroids, immunosuppressants, and biologics. Because the pathogenesis of UC is not fully understood, most commercially available medications only alleviate symptoms. Furthermore, some patients experience poor response and severe adverse reactions after repeated administration, ultimately requiring resection of the affected area or even the entire colon. Therefore, the research and development of drugs for treating ulcerative colitis has long been of great importance, as it plays a crucial role in alleviating patient suffering and improving their quality of life. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing the use of Polygonum multiflorum polysaccharide in the preparation of drugs for treating ulcerative colitis.

[0006] To address the aforementioned technical problems, this invention first provides the use of Polygonum multiflorum polysaccharide in the preparation of drugs for treating ulcerative colitis.

[0007] The Polygonum multiflorum polysaccharide of this invention is obtained from Polygonum multiflorum through water extraction-alcohol precipitation-protein removal-column chromatography-dialysis and drying. The specific preparation method includes the following steps: Extraction: The Polygonum multiflorum herb was subjected to water extraction. The water extraction conditions included: temperature: 85~95℃, preferably 90℃; time: 2h; number of extractions: 2~3; material-to-liquid ratio: 1:20, to obtain the water extract. Alcohol precipitation: The obtained aqueous extract is mixed with ethanol to obtain a diluted solution, wherein the ethanol is anhydrous ethanol and the volume fraction of ethanol in the diluted solution is 70% to 95%. The solution is allowed to stand at 4°C for 12 hours or more. The diluted solution is then subjected to standing and centrifugation to obtain polysaccharide precipitate, and the precipitate is collected. Protein removal: The resulting precipitate is deproteinized using the Sevag method, and the protein removal is performed 8-10 times. Column chromatography: The polysaccharide was eluted using a DEAE-52 column with water, 1M sodium chloride solution, and 2M sodium chloride solution as eluents sequentially. The elution flow rate was 1-2 ml / min. The elution process was as follows: 100 ml of water, 100 ml of 1M sodium chloride solution, and 100 ml of 2M sodium chloride solution were used as eluents sequentially. One tube of eluent was collected every 8 mL. The eluents from tubes 20-30 were collected and combined to obtain the polysaccharide solution. Dialysis: The obtained polysaccharide solution is dialyzed, and the dialysis bag used for dialysis has a molecular weight cutoff of 3500D; Drying: The polysaccharide solution obtained by dialysis was subjected to rotary evaporation and freeze-drying to obtain the Polygonum multiflorum polysaccharide.

[0008] In a specific embodiment of the present invention, the obtained Polygonum multiflorum polysaccharide has Mw and Mn values ​​of 110-120 kDa and 55-60 kDa, respectively. The polydispersity (Mw / Mn) is 1.9-2.1.

[0009] This invention establishes a mouse model of ulcerative colitis using DSS (sodium dextran sulfate), observes the effects of Polygonum multiflorum polysaccharide on mice, and monitors changes in body weight, DAI index, colonic shortening, and pathological conditions after modeling. Mesalazine was used as a positive control. The results demonstrate that Polygonum multiflorum polysaccharide has a significant anti-ulcerative colitis effect, and compared to the control group, it significantly reduces inflammatory infiltration in the colonic tissue, slows colonic atrophy caused by ulcerative colitis, and delays disease progression. Its mechanism of action is related to improving intestinal flora and enhancing the body's immunity. The Polygonum multiflorum polysaccharide provided by this invention has significant efficacy, is widely available, and has high safety, making it suitable for preparing drugs for treating ulcerative colitis.

[0010] This invention also provides a traditional Chinese medicine preparation for treating ulcerative colitis, comprising the aforementioned Polygonum multiflorum polysaccharide. Polygonum multiflorum polysaccharide is the sole active ingredient in this preparation.

[0011] In some embodiments, the dosage form of the drug of the present invention may be capsules, pills, powders, syrups, tablets, granules, plasters, mixtures, drop pills, suppositories, aerosols, ointments, or injections.

[0012] The above-mentioned traditional Chinese medicine preparations are used in the preparation of drugs for the prevention and / or treatment of ulcerative colitis.

[0013] In a specific embodiment of the present invention, the dosage of the drug is 100mg / kg-200mg / kg.

[0014] The advantages of this invention compared to existing technologies are as follows: This invention provides the application of Polygonum multiflorum polysaccharide in the preparation of drugs for treating ulcerative colitis. This invention, through recording changes in mouse body weight, colon length, and stool blood, and using methods such as HE staining and immunohistochemistry, comprehensively confirmed the intestinal pathological structure and the improvement after drug treatment. It shows that Polygonum multiflorum polysaccharide can significantly improve inflammatory damage in the colon, reduce weight loss, and shorten the colon; it also promotes the repair of DSS-induced mucosal damage in mice with ulcerative colitis, and can be used to treat ulcerative colitis. Attached Figure Description

[0015] Figure 1 This is an absolute molecular weight analysis diagram of Polygonum multiflorum polysaccharide according to an embodiment of the present invention; Figure 2 This is the weight of mice according to one embodiment of the present invention (n=6). Figure 3 This is the mouse DAI score (n=6) according to one embodiment of the present invention. Figure 4 This is a mouse colon image (n=6) according to an embodiment of the present invention. Figure 5 This is the length of the mouse colon (n=6) according to one embodiment of the present invention. Figure 6 This is the weight of a mouse spleen (n=6) according to one embodiment of the present invention. Figure 7 These are mouse pathological sections (n=6) according to one embodiment of the present invention. Figure 8 This is a Venn diagram of different groups of operational classification units (OTUs) and overlapping OTUs according to one embodiment of the present invention; Figure 9 It is the Simpson index of gut microbiota according to one embodiment of the present invention. Detailed Implementation

[0016] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0017] Example 1: Preparation of Polygonum multiflorum polysaccharide Step 1 Extraction: The Polygonum multiflorum herb was subjected to water extraction. The water extraction process included: temperature: 90℃; time: 2 hours; number of extractions: 2; material-to-liquid ratio: 1:20, to obtain the water extract. Step 2: Alcohol precipitation: Mix the aqueous extract obtained in Step 1 with anhydrous ethanol. The volume fraction of ethanol in the resulting diluted solution is 75%. Let it stand at 4°C for 12 hours. Then, let the diluted solution stand and centrifuge sequentially to obtain polysaccharide precipitate. Collect the precipitate. Step 3: Protein Removal: The precipitate obtained in Step 2 was subjected to protein removal using the Sevag method, repeated 10 times. 200 mg of dried crude polysaccharide was weighed and dissolved in 8 ml of distilled water. 2 ml (1 / 4 volume) of Sevag reagent (chloroform:n-butanol = 4:1 / V:V) was added. The mixture was vortexed for 30 min at room temperature, followed by centrifugation (9000 rpm, 15 min). Denatured proteins were then observed between the aqueous and organic layers. The upper aqueous phase was collected and purified again. This process was repeated 10 times.

[0018] Step 4: Column chromatography: The polysaccharide obtained in Step 3 was eluted using a DEAE-52 column. The Polygonum multiflorum polysaccharide solution was eluted using a DEAE-52 column at a flow rate of 1–2 ml / min. The elution process was as follows: water, 1M sodium chloride solution, and 2M sodium chloride solution were used as eluents sequentially. The volume of water, 1M sodium chloride solution, and 2M sodium chloride solution was 100 ml. One tube of eluent was collected every 8 mL. The eluent from the 20th to 30th tubes was collected. Step 5 Dialysis: Dialyze the polysaccharide solution obtained in Step 4, changing the water every four hours for four times. The dialysis bag used for dialysis has a molecular weight cutoff of 3500D. Step 6 Drying: The polysaccharide solution obtained in Step 5 is subjected to rotary evaporation at 50°C and freeze-drying to obtain the Polygonum multiflorum polysaccharide.

[0019] The polysaccharide content was determined using the phenol-sulfuric acid method. In each tube, 1 mL of the sample was added to 1 mL of freshly prepared 5% phenol solution, shaken well, and then 5.0 mL of concentrated sulfuric acid was quickly added and immediately shaken well again. The volume was then brought to 10 mL with deionized water and incubated in a 70°C water bath for 30 min. The tubes were then placed in an ice-water bath for approximately 5 min. The absorbance of each solution was measured at 490 nm. The calculated polysaccharide content was 90.2%.

[0020] like Figure 1 As shown in the figure, the absolute molecular weight analysis revealed that the Mw and Mn of Polygonum multiflorum polysaccharide were 1.18 × 10⁵ Da and 5.74 × 10⁴ Da, respectively. The polydispersity (Mw / Mn) was 2.065.

[0021] Example 2: Therapeutic effect of Polygonum multiflorum polysaccharide on mice with DSS-induced ulcerative colitis 1. Experimental Materials Animals: Seven-week-old male C57BL / 6 mice, weighing 20-22 g, were purchased from the Experimental Animal Center of Yangzhou University (SPF grade). The environment was clean-grade, with a temperature controlled at 23±2℃ and humidity maintained at 50%-60%. A 12-hour alternating light and dark cycle was maintained. Free access to food was provided, and drinking water was filtered and autoclaved before use.

[0022] Drugs and reagents: DSS sodium dextran sulfate (Shanghai Bid Pharmaceutical Technology Co., Ltd.); 5-aminosalicylic acid (Shanghai Bid Pharmaceutical Technology Co., Ltd.).

[0023] 2. Experimental Methods After the mice underwent acclimatization, they were randomly divided into 6 groups of 8 mice each based on their body weight. These groups were designated as a blank control group (Control), a model group (Model), a positive control group (5-aminosalicylic acid, 100 mg / kg / d), a low-dose polysaccharide group (100 mg / kg / d), and a high-dose polysaccharide group (200 mg / kg / d). Except for the blank control group, the other 5 groups were given 3% DSS in the drinking water freely for 7 days (with fresh 3% DSS solution replaced on days 1, 3, and 5) to establish a mouse model of ulcerative colitis.

[0024] Mouse body weight, water and food intake, stool consistency, and fecal occult blood were recorded daily. The Disease Activity Index (DAI) was scored according to the literature standard (Lu Huidong, Li Yanmei. Atractylodes macrocephala lactone III alleviates intestinal damage in a mouse model of ulcerative colitis by regulating the JAK2 / STAT3 signaling pathway [J]. Chinese Journal of Pathophysiology, 2023, 39(1): 142-149). After successful modeling, the mice were administered the drug via gavage. The blank control group and the model control group were given pure water via gavage, while the other groups were given the corresponding polysaccharide solution via gavage, once daily for 7 consecutive days. On day 8, the animals were euthanized, the spleen was weighed, and the colon tissue was removed. The length of the colon from the cecum to the anus was measured and recorded. Samples of the colon contents were collected in sterile centrifuge tubes and immediately frozen at -80 °C for intestinal microbiota analysis. The distal colon portion was fixed in 4% paraformaldehyde for HE staining and immunohistochemical analysis. The remaining colon tissue was stored at -80 °C for other assays.

[0025] 3. Experimental Results like Figure 2 As shown, the body weight of mice decreased significantly after DSS induction. However, the decrease in body weight was alleviated after the administration of positive control drug and polysaccharide treatment on the eighth day. From the tenth day onwards, the body weight of the treatment group showed an upward trend, indicating that Polygonum multiflorum polysaccharide treatment can alleviate the body weight loss caused by DSS. The body weight reduction rate (%) of mice was calculated as follows: (body weight on day 1 (g) - body weight on day n (g)) / (body weight on day 1 (g)) × 100%.

[0026] like Figure 3 As shown, the DAI score of the control group mice remained around 0. After DSS stimulation, the scores of all groups showed a steady increase. However, after the DSS stimulation was stopped, the DAI scores of all groups showed a decreasing trend, but the decrease in the drug-treated groups was much greater than that in the DSS group. This indicates that Polygonum multiflorum polysaccharide has an ameliorative effect on weight loss, bloody stools, and diarrhea in mice with colitis.

[0027] like Figure 4 and Figure 5 As shown, compared with the normal control group, the colon length of mice in the DSS group was significantly shortened, while the positive control drug and low and high doses of Polygonum multiflorum polysaccharide alleviated the shortening of colon length to varying degrees, indicating that Polygonum multiflorum polysaccharide can alleviate the colon shortening in DSS-induced ulcerative colitis in mice.

[0028] like Figure 6 As shown, DSS induction affects spleen weight. Compared with the blank group, the spleen weight of the model group mice increased, but after treatment with Polygonum multiflorum polysaccharide, it decreased compared with the model group, indicating that Polygonum multiflorum polysaccharide can alleviate DSS-induced splenomegaly and increased spleen index in mice.

[0029] like Figure 7 HE (×50) staining results showed that the colon in the control group had clear stratification, distinct crypt structures, and tightly and regularly arranged goblet cells. In the DSS-stimulated mice, the cells lacked stratification, and crypt cells, goblet cells, and glands were largely absent. In contrast, the colonic stratification in the low- and high-polysaccharide groups and the positive control group remained largely intact, with only a small number of crypt cells and goblet cells lost. This indicates that Polygonum multiflorum polysaccharide treatment can reduce inflammatory infiltration and gradually restore tissue damaged by colitis.

[0030] like Figure 8 The OTU species composition study revealed that the number of species decreased after ulcerative colitis modeling, but the species richness was significantly improved after treatment with high-dose Polygonum multiflorum polysaccharide compared to low-dose Polygonum multiflorum polysaccharide.

[0031] like Figure 9 The Simpson index for species diversity ranges from 0 to 1. A value closer to 1 indicates a greater variety and uniformity of species within the system, representing the highest richness and evenness of the environment. The Simpson index for species diversity in the gut microbiota of the model group mice was lower than that of the control group. However, compared to the model group, the species richness in the polysaccharide group increased with polysaccharide dosage. This suggests that Polygonum multiflorum polysaccharides can regulate gut microbiota diversity, enhance immune function, thereby slowing the progression of ulcerative colitis, restoring damaged colitis tissue, and exerting a therapeutic effect on ulcerative colitis.

[0032] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Polygonum multiflorum polysaccharide in the preparation of drugs for treating ulcerative colitis, characterized in that, The preparation method of the Polygonum multiflorum polysaccharide includes the following steps: extracting Polygonum multiflorum medicinal material with water to obtain an aqueous extract; diluting the aqueous extract with ethanol to obtain a diluted solution, wherein the volume fraction of ethanol in the diluted solution is 70-95%; allowing the diluted solution to stand and centrifuge sequentially to obtain a polysaccharide precipitate; removing protein from the polysaccharide precipitate using the Sevag method and eluting with a DEAE-52 column to obtain a polysaccharide solution; dialyzing, rotary evaporating, and freeze-drying the polysaccharide solution to obtain the Polygonum multiflorum polysaccharide; the dialysis bag used for dialysis has a molecular weight cutoff of 3500D.

2. The application according to claim 1, characterized in that, The polysaccharide content of the Polygonum multiflorum polysaccharide is ≥90%.

3. The application according to claim 1, characterized in that, The polysaccharide from Polygonum multiflorum has a Mw of 110-120 kDa and a Mn of 55-60 kDa.

4. The application according to claim 1, characterized in that, The water extraction temperature is 85~95℃, the extraction time is 2h, the extraction is performed 2~3 times, and the material-liquid ratio is 1:

20.

5. The application according to claim 1, characterized in that, The diluted solution should be allowed to stand at 4°C for at least 12 hours.

6. The application according to claim 1, characterized in that, The Sevag method is used to remove protein 8-10 times.

7. The application according to claim 1, characterized in that, The DEAE-52 column was used for elution at a flow rate of 1–2 mL / min. The elution process was as follows: water, 1 mol / L sodium chloride solution, and 2 mol / L sodium chloride solution were used as eluents in sequence, with each eluent volume being 100 mL. Every 8 mL of eluent was collected, and the eluents from the 20th to 30th tubes were combined to obtain the polysaccharide solution.

8. The application according to claim 1, characterized in that, The aforementioned drug for treating ulcerative colitis uses Polygonum multiflorum polysaccharide as its sole active ingredient.

Images