Ligularifolium polysaccharide, preparation method thereof and application of ligularifolium polysaccharide in preparation of drugs for preventing and treating colonitis

Abstract

This invention discloses *Ligustrum lucidum* polysaccharide, its preparation method, and its application in the preparation of drugs for the prevention and treatment of colitis, belonging to the field of biomedical technology. Specifically, it relates to a method for preparing *Ligustrum lucidum* polysaccharide, comprising: pretreatment of *Ligustrum lucidum* leaves to obtain lyophilized *Ligustrum lucidum* leaf powder; mixing with petroleum ether, defatting, then mixing with deionized water, soaking and extracting to obtain *Ligustrum lucidum* extract; rotary evaporation to concentrate to 1 / 4 of the total volume to obtain a concentrated solution; mixing with anhydrous ethanol, allowing to stand and collect the precipitate; washing with an organic solvent to obtain crude *Ligustrum lucidum* polysaccharide; mixing with deionized water to obtain a crude *Ligustrum lucidum* polysaccharide solution; mixing with Sevag reagent, allowing to stand and separate into layers, collecting the upper aqueous phase; dialysis in deionized water to obtain a dialyzed polysaccharide solution; pre-freezing, vacuum freeze-drying to obtain *Ligustrum lucidum* polysaccharide. The *Ligustrum lucidum* polysaccharide prepared by this invention can prevent and / or treat ulcerative colitis by remodeling the intestinal flora structure and regulating intestinal metabolite levels.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to the polysaccharide of Ligusticum striatum leaf, its preparation method, and its application in the preparation of drugs for the prevention and treatment of colitis. Background Technology

[0002] Various chronic diseases caused by immune system disorders affect human health. Ulcerative colitis (UC) is an idiopathic disease characterized by intestinal mucosal damage and ulceration. Current drug treatments mostly have side effects. Modern medicine typically uses immunosuppressants, aminosalicylic acid drugs, antibiotics, and monoclonal antibodies to treat UC. However, long-term use of these drugs can lead to serious adverse reactions, poor clinical efficacy, and high relapse rates. Therefore, there is an urgent need to discover new therapies for treating UC patients.

[0003] The medicinal value of *Ligularia Fischeri* is one of the important research directions. The ethanol extract of its aerial parts has been shown to have significant anti-inflammatory effects. Studies have shown that this extract can effectively inhibit various inflammations, such as auricular swelling and adjuvant arthritis, and its mechanism of action may be related to inhibiting the synthesis or release of inflammatory mediators. The flavonoid extract of *Ligularia Fischeri* can effectively inhibit bacterial growth, prolong the bacterial lag phase, and has good stability. The minimum inhibitory concentrations against *E. coli*, *S. aureus*, *B. yeast*, and *A. niger* were 0.32 mg / mL, 0.64 mg / mL, 0.64 mg / mL, and 1.28 mg / mL, respectively. Meanwhile, a study systematically evaluated the in vitro antioxidant efficacy of *Ligularia Fischeri* polysaccharides (LFP), verifying its antioxidant properties through total antioxidant capacity determination, reducing power analysis, and DPPH radical, superoxide anion radical, and hydroxyl radical scavenging experiments. Experimental data show that this polysaccharide exhibits significant antioxidant activity, especially in scavenging DPPH free radicals, with a half-inhibitory concentration (IC50) of [missing value]. 50 The concentration was as low as 0.0351 mg / mL, indicating that this bioactive component has potential application value in the development of antioxidant functional foods and drugs. As a plant with ecological, edible, and medicinal value, the study of *Ligustrum lucidum* not only contributes to biodiversity conservation but also provides new ideas for the development of natural drugs. However, current research on the bioactivity of *Ligustrum lucidum*, both domestically and internationally, is still insufficient. In particular, the development and utilization of its abundant polysaccharides, flavonoids, and phenols remain in their early stages. This situation greatly restricts the development of *Ligustrum lucidum* in the field of deep processing and limits its widespread application in the functional food market. Therefore, studying the bioactive components and structure-activity relationships in *Ligustrum lucidum* is helpful for the development and utilization of its functional products and is of great significance for health promotion.

[0004] Currently, there is limited in-depth research on the biological activities of Ligustrum lucidum polysaccharides, as well as the structure and activity mechanisms of its components. For example, the structure of Ligustrum lucidum polysaccharides needs further characterization, and its regulatory effects on intestinal health require further investigation. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a polysaccharide from Ligusticum striatum, its preparation method, and its application in the preparation of drugs for the prevention and treatment of colitis. This invention provides a water extraction and alcohol precipitation method for preparing polysaccharide from Ligusticum striatum. It has been found that the prepared polysaccharide from Ligusticum striatum can regulate the abundance of intestinal flora, reshape the structure of intestinal flora, and prevent and / or treat ulcerative colitis by increasing the content of short-chain fatty acids and regulating the level of intestinal metabolites.

[0006] To achieve the above objectives, the present invention provides a method for preparing Ligustrum lucidum polysaccharide, comprising the following steps:

[0007] 1) Ligustrum lucidum leaves, quick-frozen, vacuum freeze-dried, pulverized and sieved to obtain freeze-dried powder of Ligustrum lucidum leaves;

[0008] 2) The freeze-dried powder of Ligustrum lucidum obtained in step 1) is mixed with petroleum ether, shaken to extract, allowed to stand and separate into layers, and the upper layer is discarded to obtain defatted Ligustrum lucidum powder.

[0009] 3) The defatted Ligustrum lucidum powder obtained in step 2) is mixed with deionized water and soaked and extracted 2 to 4 times. The extracts are combined to obtain Ligustrum lucidum extract.

[0010] 4) The Ligusticum striatum extract obtained in step 3) is concentrated by rotary evaporation to 1 / 4 of the total volume of the Ligusticum striatum extract to obtain a concentrated solution;

[0011] 5) Mix the concentrated solution obtained in step 4) with anhydrous ethanol, let it stand, and collect the precipitate;

[0012] 6) The precipitate obtained in step 5) was washed successively with anhydrous ethanol, acetone and anhydrous diethyl ether to obtain the washed precipitate, which was dried to constant weight to obtain crude polysaccharide of Ligusticum striatum.

[0013] 7) The crude polysaccharide of Ligustrum lucidum obtained in step 6) is mixed with deionized water to obtain a crude polysaccharide solution of Ligustrum lucidum; the crude polysaccharide solution of Ligustrum lucidum is mixed with Sevag reagent, shaken on a shaker, allowed to stand for separation, and the upper aqueous phase is taken.

[0014] 8) The upper aqueous phase obtained in step 7) is placed in a dialysis bag and dialyzed in deionized water to obtain a dialysis polysaccharide solution;

[0015] 9) The polysaccharide solution obtained after dialysis in step 8) is pre-frozen and then freeze-dried under vacuum to obtain Ligustrum lucidum polysaccharide.

[0016] Preferably, the quick-freezing temperature in step 1) is -70 to -90°C, and the quick-freezing time is 10 to 14 hours; the vacuum freeze-drying time in step 1) is 36 to 60 hours, the vacuum freeze-drying temperature is -40 to -60°C, and the vacuum degree of the vacuum freeze-drying is ≤0.1 mbar; the particle size of the sieve in step 1) is 70 to 90 mesh.

[0017] Preferably, in step 2), the ratio of freeze-dried Ligustrum lucidum leaf powder to petroleum ether is 1g:4-6mL; the shaking extraction speed in step 2) is 150-250rpm, and the shaking extraction time is 4-6h; the settling time in step 2) is 20-40min; in step 3), the ratio of defatted Ligustrum lucidum powder to deionized water is 1g:15-25mL; the soaking extraction temperature in step 3) is 70-90℃, the number of soaking extractions is 2-4 times, and the soaking extraction time is 1-3h each time.

[0018] Preferably, the rotary evaporation concentration temperature in step 4) is 70-90°C, and the vacuum degree of the rotary evaporation concentration is 0.08-0.09 MPa; the volume ratio of the concentrate to anhydrous ethanol in step 5) is 1:3-5, the standing temperature in step 5) is 4°C, and the standing time is ≥12h.

[0019] Preferably, the drying temperature in step 6) is 35-45℃; the volume ratio of the crude polysaccharide solution of Ligusticum striatum and the Sevag reagent in step 7) is 1:3-5, wherein the Sevag reagent is obtained by mixing chloroform and n-butanol in a volume ratio of 4:1; the shaking time in step 7) is 15-25 min, the shaking speed is 150-250 rpm; and the settling time in step 7) is ≥30 min.

[0020] Preferably, the molecular weight of the dialysis bag in step 8) is 8000-14000 Da, the dialysis treatment temperature in step 8) is 4°C, and the deionized water is replaced every 6-8 hours during the dialysis treatment; the pre-freezing temperature in step 9) is -70 to -90°C, the pre-freezing time is 10-14 hours, the vacuum freeze-drying time in step 9) is 36-60 hours, the vacuum freeze-drying temperature is -40 to -60°C, and the vacuum degree of the vacuum freeze-drying is ≤0.1 mbar.

[0021] The present invention also provides the preparation method described above for the preparation of Ligustrum lucidum polysaccharide.

[0022] The present invention also provides the application of the prepared Ligusticum striatum polysaccharide in the preparation of drugs for the prevention and / or treatment of colitis.

[0023] Preferably, the colitis is ulcerative colitis.

[0024] Preferably, the polysaccharide of Ligusticum striatum can prevent and / or treat ulcerative colitis by regulating the abundance of intestinal flora, reshaping the intestinal flora structure, and increasing the content of short-chain fatty acids (SCFAs) to regulate the level of intestinal metabolites.

[0025] Compared with the prior art, the present invention has the following advantages and technical effects:

[0026] This invention proposes a polysaccharide from *Ligustrum lucidum* (also known as 'Hoof Ligustrum'), its preparation method, and its application in the preparation of drugs for the prevention and treatment of colitis. The invention provides a water extraction and alcohol precipitation method for preparing *Ligustrum lucidum* polysaccharide. It was found that the prepared *Ligustrum lucidum* polysaccharide regulates intestinal flora abundance, reshapes intestinal flora structure, and prevents and / or treats ulcerative colitis by increasing short-chain fatty acid content and regulating intestinal metabolite levels. Specifically, the *Ligustrum lucidum* polysaccharide prepared in this invention alleviated weight loss in UC mice, reduced DAI scores and organ indices in UC mice, and improved colonic shortening and colonic tissue damage caused by colitis. The *Ligustrum lucidum* polysaccharide prepared in this invention improved the α-diversity index and β-diversity of the intestinal flora in UC mice, promoting the restoration of flora species richness and community structural heterogeneity to normal levels. The treatment significantly alleviated the dysbiosis of the gut microbiota. At the phylum level, the abnormal proliferation of Bacteroides / Firmicutes and Proteobacteria in the *Ligustrum lucidum* polysaccharide intervention group was effectively reversed. At the genus level, the abundance of *Bacteroides*, *Odoribacter*, *Cryptobacteroides*, *Prevotella*, and *Limosilactobacillus* all returned to normal levels. It also increased the content of SCFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid) in the cecal contents. The prepared *Ligustrum lucidum* polysaccharide activated the short-chain fatty acid receptor GPR41, regulated epithelial cell apoptosis-related factors, upregulated the expression levels of Bcl-2 and inhibited Bax mRNA and protein, reduced the expression of NLRP3 inflammasome signaling pathway mRNA and protein, and alleviated inflammatory damage, providing an effective intervention pathway for the relief and treatment of ulcerative colitis (UC). Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The figure shows the elution curves of LFP prepared in Example 1 using a DEAE-52 cellulose chromatography column. In the figure, LFP-W, LFP-1, LFP-2, LFP-3, LFP-4 and LFP-5 represent different components of LFP.

[0029] Figure 2 Elution curves of Sephadex G-100 dextran gel chromatography column are shown. In the figure, A is fraction LFP-W-1 separated by LFP-W, B is fractions LFP-1-1 and LFP-1-2 separated by LFP-1, C is fraction LFP-2-1 separated by LFP-2, and D is fraction LFP-3-1 separated by LFP-3.

[0030] Figure 3 Ion chromatograms of the components of LFP prepared as a monosaccharide standard and in Example 1;

[0031] Figure 4 The figures show the weight and DAI scores of mice in each group of colitis. In the figure, A is the weight statistics and B is the DAI score. In the figure, N group represents the normal group, M group represents the model group, P group represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=12. Different letters indicate significant differences between groups, P<0.05.

[0032] Figure 5 The statistical chart shows the regulation of organ indices in each group of colitis mice. In the figure, group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=12, different letters indicate significant differences between groups, P<0.05.

[0033] Figure 6 The graphs show the rectal bleeding and colon length of mice in each group of colitis. In the graphs, A represents rectal bleeding, B is a schematic diagram of colon length, and C is a statistical graph of colon length. In the graphs, N represents the normal group, M represents the model group, P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=12, different letters indicate significant differences between groups, P<0.05;

[0034] Figure 7 The following are statistical charts showing the pathological damage of the colon tissue of mice in each group. In the chart, A is the morphological observation chart, B is the damage score statistical chart, N represents the normal group, M represents the model group, P represents the positive control group, LFPL represents the low-dose LFP group, LFPH represents the high-dose LFP group, M±SD, n=12, different letters indicate significant differences between groups, P<0.05.

[0035] Figure 8 For the analysis of α-diversity of gut microbiota in mice in each group, where A is the species sparse curve, B is the Chao1 index, C is the Simpson index, D is the Shannon index, N represents the normal group, M represents the model group, P represents the positive control group, LFPL represents the low-dose LFP group, LFPH represents the high-dose LFP group, M±SD, n=5, * indicates P<0.05, ** indicates P<0.01;

[0036] Figure 9 The analysis of β-diversity of gut microbiota in mice in each group is shown in Figure A, where A is the principal coordinate analysis and B is the non-metric multidimensional scaling analysis. In the figure, N represents the normal group, M represents the model group, P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group.

[0037] Figure 10 The Venn plot shows the ASV levels between the groups. In the plot, group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group.

[0038] Figure 11 The species composition analysis at the phylum level for each group of mice is shown in Figure A, where A is a bar chart of relative abundance of species at the phylum level, B is the relative abundance ratio of Firmicutes to Bacteroidetes, N represents the normal group, M represents the model group, P represents the positive control group, LFPL represents the low-dose LFP group, LFPH represents the high-dose LFP group, M±SD, n=5, different letters indicate significant differences between groups, P<0.05;

[0039] Figure 12 The bar chart shows the relative abundance of mouse species at the genus level for each group. In the figure, group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group.

[0040] Figure 13 This is a genus-level difference diagram of mice in each group. In the diagram, group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group.

[0041] Figure 14 The diagram shows the differential classification units between groups of mice in the gut microbiota based on the classification hierarchy tree. In the diagram, group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group.

[0042] Figure 15The LDA scores of key genera in the gut microbiota of mice in each group are shown in the figure. Group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group.

[0043] Figure 16 The graph shows the predicted metabolic function of intestinal flora in mice in each group. In the graph, group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, LFPH represents the high-dose LFP group, M±SD, n=5, * indicates P<0.05, ** indicates P<0.01.

[0044] Figure 17 Pearson correlation heatmap analysis of gut microbiota abundance and SCFA content in mice at the genus level was performed for each group of mice. In the figure, group N represents the normal group, group M represents the model group, group P represents the positive control group, LFPL represents the low-dose LFP group, LFPH represents the high-dose LFP group, M±SD, n=5, * indicates P<0.05, ** indicates P<0.01;

[0045] Figure 18 The expression levels of SCFAs receptor GPR41 mRNA in each group of mice were statistically analyzed. In the figure, N group represents the normal group, M group represents the model group, P group represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=3, different letters indicate significant differences between groups, P<0.05.

[0046] Figure 19 The expression levels of GPR41 protein, the receptor for SCFAs, in each group of mice were statistically analyzed. In the figure, A is the immunoblot band image of the protein, B is the expression level of GPR41 protein, N represents the normal group, M represents the model group, P represents the positive control group, LFPL represents the low-dose LFP group, LFPH represents the high-dose LFP group, M±SD, n=3, different letters indicate significant differences between groups, P<0.05;

[0047] Figure 20 The expression levels of Bcl-2 and Bax mRNA in mice of each group were statistically analyzed. In the figure, A represents the expression level of Bcl-2 mRNA, B represents the expression level of Bax mRNA, N represents the normal group, M represents the model group, P represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=3, different letters indicate significant differences between groups, P<0.05;

[0048] Figure 21The expression levels of Bcl-2 and Bax proteins in mice in each group were statistically analyzed. In the figure, A is the immunoblot band image, B is the expression level of Bcl-2 protein, and C is the expression level of Bax protein. In the figure, N group represents the normal group, M group represents the model group, P group represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=3, different letters indicate significant differences between groups, P<0.05;

[0049] Figure 22 The expression of NLRP3 signaling pathway mRNA in the colon tissue of mice in each group was statistically analyzed. In the figure, A represents the expression of NLRP3 mRNA, B represents the expression of Caspase-1 mRNA, and C represents the expression of ASC mRNA. In the figure, N group represents the normal group, M group represents the model group, P group represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=3, different letters indicate significant differences between groups, P<0.05;

[0050] Figure 23 The expression of NLRP3 signaling pathway proteins in the colon tissue of mice in each group was statistically analyzed. In the figure, A is the protein immunoblot band, B is the expression of NLRP3 protein, C is the expression of Caspase-1 protein, and D is the expression of ASC protein. In the figure, N group represents the normal group, M group represents the model group, P group represents the positive control group, LFPL represents the low-dose LFP group, and LFPH represents the high-dose LFP group. M±SD, n=3, different letters indicate significant differences between groups, P<0.05;

[0051] Figure 24 Spearman correlation analysis plots were generated for the top 20 abundant bacterial species and SCFAs in each group of mice, and the detection indicators of GPR41, Bcl-2, and Bax in colon tissue. Red indicates a positive correlation, blue indicates a negative correlation, and darker colors indicate stronger correlations. M±SD, n=5, * indicates P<0.05, ** indicates P<0.01. Detailed Implementation

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0054] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0055] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0056] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0057] The source of the materials used in this invention: fresh Ligustrum lucidum leaves were purchased from Yanji West Market. GPR41 primary antibody was purchased from ABclonal; NLRP3, ASC, and Caspase-1 primary antibodies were purchased from Affinity Biosciences; Bcl-2 and Bax primary antibodies were purchased from CST; SDS-PAGE gel preparation kit was purchased from Beijing Dingguo; Immobilon Western Chemiluminescent HRP Substrate was purchased from MILLIPORE; 0.45 μM Immobilon-P Transfer Menbranes PVDF membrane was purchased from Immobilon; three-color prestained protein markers (10-180 kDa) were purchased from SMOBIO; specific amplification primers were purchased from Jilin Kumei Biotechnology; SuperReal fluorescence quantitative premixed reagent enhanced version was purchased from TIANGEN; FastKing one-step genomic cDNA first-strand synthesis premixed reagent was purchased from TIANGEN; ECOPLATE48 qPCR reaction plate was purchased from PCRmaxECO; ECOSEAL48 plate sealing film was purchased from PCRmaxECO; materials without specified sources were all commercially available.

[0058] All instruments used in this invention are commercially available.

[0059] Example 1

[0060] 1) Ligustrum lucidum leaves were quick-frozen at -80℃ for 12 hours, then freeze-dried at -50℃ with a vacuum degree ≤0.1mbar for 48 hours, and pulverized through an 80-mesh sieve to obtain freeze-dried powder of Ligustrum lucidum leaves;

[0061] 2) Mix 100g of freeze-dried Ligustrum lucidum leaf powder with petroleum ether (boiling range 60-90℃) at a ratio of 1g:5mL, and extract by shaking at room temperature for 5h at 200rpm. Let stand for 30min to separate the layers, and carefully pour off the upper petroleum ether to obtain defatted Ligustrum lucidum powder.

[0062] 3) Mix defatted Ligustrum lucidum powder with deionized water at a ratio of 1g:20mL, soak and extract three times at 80℃, each time for 2 hours. After each soak and extraction, centrifuge at 4000rpm at room temperature for 10 minutes, collect the supernatant and combine them to obtain the extract, which is Ligustrum lucidum extract.

[0063] 4) The extract of Ligustrum lucidum was concentrated by rotary evaporation at a water bath temperature of 80℃ and a vacuum degree of 0.08-0.09MPa to 1 / 4 of the total volume of the extract.

[0064] 5) Mix the concentrate with anhydrous ethanol at a volume ratio of 1:4 (stir continuously and slowly add anhydrous ethanol), cover with plastic wrap, let stand at 4°C for 12 hours, carefully pour off the supernatant, centrifuge the precipitate at 4000 rpm at 4°C for 15 minutes, discard the supernatant, and take the precipitate.

[0065] 6) The precipitate was mixed with anhydrous ethanol at a volume ratio of 1:4, vortexed, centrifuged at 4000 rpm and 4°C, and the anhydrous ethanol used for washing was discarded. The washing with anhydrous ethanol was repeated 3 times. Then, the precipitate after washing with anhydrous ethanol was mixed with acetone at a volume ratio of 1:4, vortexed, centrifuged at 4000 rpm and 4°C, and the acetone used for washing was discarded. The washing with acetone was repeated 3 times. Then, the precipitate after washing with acetone was mixed with anhydrous diethyl ether at a volume ratio of 1:4, vortexed, centrifuged at 4000 rpm and 4°C, and the anhydrous diethyl ether used for washing was discarded. The washing with anhydrous diethyl ether was repeated 3 times. The washed precipitate was dried at 40°C and the organic solvent was evaporated to constant weight to obtain crude polysaccharide of Ligustrum lucidum.

[0066] 7) Mix the crude polysaccharide of Ligustrum lucidum with an appropriate amount of deionized water (the amount of deionized water should be sufficient to dissolve the crude polysaccharide in water) to obtain a crude polysaccharide solution. Mix the crude polysaccharide solution with Sevag reagent (chloroform and n-butanol mixed at a volume ratio of 4:1) at a volume ratio of 1:4. Shake at 200 rpm for 20 minutes, let stand for 30 minutes to separate into three distinct layers (upper aqueous phase containing polysaccharide, middle layer denatured protein gel, and lower organic phase). Carefully release and discard the lower organic phase and the middle denatured protein layer. Repeat the operation after mixing with Sevag reagent until no white flocculent denatured protein layer appears at the separation interface. Take the upper aqueous phase.

[0067] 8) The upper aqueous phase was placed in a dialysis bag with a molecular weight in the range of 8000 to 14000 Da, the two ends were tied tightly, and the bag was placed in a beaker filled with deionized water. The bag was dialyzed at 4°C for 48 hours. The deionized water in the beaker was replaced every 7 hours to obtain the polysaccharide solution after dialysis.

[0068] 9) After dialysis, the polysaccharide solution was pre-frozen at -80℃ for 12 hours, and then freeze-dried under vacuum at -50℃ and a vacuum degree of ≤0.1mbar for 48 hours to obtain Ligustrum lucidum polysaccharide (LFP).

[0069] Example 2

[0070] 1) Ligustrum lucidum leaves were quick-frozen at -70℃ for 10 hours, then at -40℃ with a vacuum degree of ≤0.1mbar, and freeze-dried under vacuum for 36 hours. The dried leaves were then pulverized and passed through a 70-mesh sieve to obtain freeze-dried powder of Ligustrum lucidum leaves.

[0071] 2) Mix 100g of freeze-dried Ligustrum lucidum leaf powder with petroleum ether (boiling range 60-90℃) at a ratio of 1g:4mL, and extract by shaking at room temperature for 4h at 150rpm. Let stand for 20min to separate the layers, and carefully pour off the upper petroleum ether to obtain defatted Ligustrum lucidum powder.

[0072] 3) Mix defatted Ligustrum lucidum powder with deionized water at a ratio of 1g:15mL, soak and extract twice at 70℃, each time for 1 hour. After each soak and extraction, centrifuge at 4000rpm at room temperature for 10 minutes, collect the supernatant and combine them to obtain the extract, which is Ligustrum lucidum extract.

[0073] 4) The extract of Ligustrum lucidum was concentrated by rotary evaporation at a water bath temperature of 70℃ and a vacuum degree of 0.08-0.09MPa to 1 / 4 of the total volume of the extract.

[0074] 5) Mix the concentrate with anhydrous ethanol at a volume ratio of 1:3 (stir continuously and slowly add anhydrous ethanol), cover with plastic wrap, let stand at 4°C for 14 hours, carefully pour off the supernatant, centrifuge the precipitate at 4000 rpm at 4°C for 15 minutes, discard the supernatant, and take the precipitate.

[0075] 6) The precipitate was mixed with anhydrous ethanol at a volume ratio of 1:3, vortexed, centrifuged at 4000 rpm and 4°C, and the anhydrous ethanol used for washing was discarded. The washing with anhydrous ethanol was repeated twice. Then, the precipitate after washing with anhydrous ethanol was mixed with acetone at a volume ratio of 1:3, vortexed, centrifuged at 4000 rpm and 4°C, and the acetone used for washing was discarded. The washing with acetone was repeated twice. Then, the precipitate after washing with acetone was mixed with anhydrous diethyl ether at a volume ratio of 1:3, vortexed, centrifuged at 4000 rpm and 4°C, and the anhydrous diethyl ether used for washing was discarded. The washing with anhydrous diethyl ether was repeated twice. The washed precipitate was dried at 35°C and the organic solvent was evaporated to constant weight to obtain crude polysaccharide of Ligustrum lucidum.

[0076] 7) Mix the crude polysaccharide of Ligustrum lucidum with an appropriate amount of deionized water (the amount of deionized water should be sufficient to dissolve the crude polysaccharide in water) to obtain a crude polysaccharide solution. Mix the crude polysaccharide solution with Sevag reagent (chloroform and n-butanol mixed at a volume ratio of 4:1) at a volume ratio of 1:3. Shake at 150 rpm for 15 min, let stand for 40 min to separate into three distinct layers (upper aqueous phase containing polysaccharide, middle layer denatured protein gel, and lower organic phase). Carefully release and discard the lower organic phase and the middle denatured protein layer. Repeat the operation after mixing with Sevag reagent until no white flocculent denatured protein layer appears at the separation interface. Take the upper aqueous phase.

[0077] 8) The upper aqueous phase was placed in a dialysis bag with a molecular weight in the range of 8000 to 14000 Da, the two ends were tied tightly, and the bag was placed in a beaker filled with deionized water. The bag was dialyzed at 4°C for 36 hours. The deionized water in the beaker was replaced every 6 hours to obtain the polysaccharide solution after dialysis.

[0078] 9) After dialysis, the polysaccharide solution was pre-frozen at -70℃ for 10 hours, and then freeze-dried under vacuum at -40℃ and a vacuum degree of ≤0.1mbar for 36 hours to obtain Ligustrum lucidum polysaccharide.

[0079] Example 3

[0080] 1) Ligustrum lucidum leaves were quick-frozen at -90℃ for 14 hours, then at -60℃ with a vacuum degree of ≤0.1mbar, and freeze-dried under vacuum for 60 hours. The dried leaves were then pulverized and passed through a 90-mesh sieve to obtain freeze-dried powder of Ligustrum lucidum leaves.

[0081] 2) Mix 100g of freeze-dried Ligustrum lucidum leaf powder with petroleum ether (boiling range 60-90℃) at a ratio of 1g:6mL, and extract by shaking at room temperature for 6h at 250rpm. Let stand for 40min to separate the layers, and carefully pour off the upper petroleum ether to obtain defatted Ligustrum lucidum powder.

[0082] 3) Mix defatted Ligustrum lucidum powder with deionized water at a ratio of 1g:25mL, soak and extract 4 times at 90℃, each time for 3 hours. After each soak and extraction, centrifuge at 4000rpm at room temperature for 10 minutes, collect the supernatant and combine them to obtain the extract, which is Ligustrum lucidum extract.

[0083] 4) The extract of Ligustrum lucidum was concentrated by rotary evaporation at a water bath temperature of 90℃ and a vacuum degree of 0.08-0.09MPa to 1 / 4 of the total volume of the extract.

[0084] 5) Mix the concentrate with anhydrous ethanol at a volume ratio of 1:5 (stir continuously and add anhydrous ethanol slowly), cover with plastic wrap, let stand at 4°C for 16 hours, carefully pour off the supernatant, centrifuge the precipitate at 4000 rpm at 4°C for 15 minutes, discard the supernatant, and take the precipitate.

[0085] 6) The precipitate was mixed with anhydrous ethanol at a volume ratio of 1:5, vortexed, centrifuged at 4000 rpm and 4°C, and the anhydrous ethanol used for washing was discarded. The washing with anhydrous ethanol was repeated 3 times. Then, the precipitate after washing with anhydrous ethanol was mixed with acetone at a volume ratio of 1:5, vortexed, centrifuged at 4000 rpm and 4°C, and the acetone used for washing was discarded. The washing with acetone was repeated 3 times. Then, the precipitate after washing with acetone was mixed with anhydrous diethyl ether at a volume ratio of 1:5, vortexed, centrifuged at 4000 rpm and 4°C, and the anhydrous diethyl ether used for washing was discarded. The washing with anhydrous diethyl ether was repeated 3 times. The washed precipitate was dried at 45°C and the organic solvent was evaporated to constant weight to obtain crude polysaccharide of Ligustrum lucidum.

[0086] 7) Mix the crude polysaccharide of Ligustrum lucidum with an appropriate amount of deionized water (the amount of deionized water should be sufficient to dissolve the crude polysaccharide in water) to obtain a crude polysaccharide solution. Mix the crude polysaccharide solution with Sevag reagent (chloroform and n-butanol mixed at a volume ratio of 4:1) at a volume ratio of 1:5. Shake at 250 rpm for 25 min, let stand for 40 min to separate into three distinct layers (upper aqueous phase containing polysaccharide, middle layer denatured protein gel, and lower organic phase). Carefully release and discard the lower organic phase and the middle denatured protein layer. Repeat the operation after mixing with Sevag reagent until no white flocculent denatured protein layer appears at the separation interface. Take the upper aqueous phase.

[0087] 8) The upper aqueous phase was placed in a dialysis bag with a molecular weight in the range of 8000 to 14000 Da, the two ends were tied tightly, and the bag was placed in a beaker filled with deionized water. The bag was dialyzed at 4°C for 60 h, and the deionized water in the beaker was replaced every 8 h to obtain the polysaccharide solution after dialysis.

[0088] 9) After dialysis, the polysaccharide solution was pre-frozen at -90℃ for 14 hours, and then freeze-dried under vacuum at -60℃ and a vacuum degree of ≤0.1mbar for 60 hours to obtain Ligustrum lucidum polysaccharide.

[0089] Experimental Example 1

[0090] I. Purification of Polysaccharides from Ligusticum striatum.

[0091] 1. DEAE-52 cellulose column chromatography.

[0092] (1) Pretreatment of DEAE-52 cellulose filler: Weigh an appropriate amount of DEAE-52 filler and soak it in distilled water for 12 hours to allow it to fully swell. Change the water several times during this period to remove suspended impurities. Then, vacuum filter it through double-layer filter paper until dry. Soak the dried filler in 0.5M NaOH solution for 0.5 hours, rinse it with deionized water until neutral, soak it in 0.5M HCl solution for 0.5 hours, and then rinse it with water until neutral. Set aside for use.

[0093] (2) Column packing: After cleaning the glass chromatography column (1.7cm×70.0cm), fix it vertically and fill it with distilled water to 1 / 3 of the column height. The well-stirred packing material is slowly added to the chromatography column through a glass rod. After contacting the liquid surface, start the constant flow pump to allow it to settle naturally. After the packing material height stabilizes, turn off the pump and check that there are no air bubbles in the column bed and that the upper surface is flat.

[0094] (3) Sample loading and gradient elution: Equilibrate the chromatography column with 3 column volumes of deionized water (1.0 mL / min), and close the valve when the liquid level drops to the column inlet. Prepare a 10 mg / mL solution of the LFP sample prepared in Example 1, vortex and centrifuge (8000 r / min, 10 min) to remove the precipitate, filter through a 0.22 μm filter membrane, and slowly load the sample along the column wall. After standing for 10 min, start the pump to ensure the sample is completely in the column bed. Elute with a gradient of 0–0.5 M NaCl solution (flow rate 1.0 mL / min, volume 1000 mL, 20 mL per tube) until the eluent is colorless.

[0095] (4) Elution curve plotting and component collection: The eluent was analyzed by the phenol-sulfuric acid method, and the absorbance was measured at a wavelength of 490 nm. A curve was plotted with absorbance as the ordinate and the number of tubes as the abscissa. The purified components were divided according to the peak values. The main peak solutions were combined and concentrated. After dialyzing for 48 h, the solutions were freeze-dried under vacuum to obtain the preliminary purified products.

[0096] 2. Dextran gel G-100 column chromatography.

[0097] (1) Sephadex G-100 pretreatment: Weigh an appropriate amount of Sephadex G-100 dry powder, add distilled water and stir until it settles naturally. Rinse repeatedly with deionized water to remove surface impurities and broken gel. Then soak in distilled water for 24 hours, stirring continuously until the volume expands to above the minimum swelling volume specified in the instructions.

[0098] (2) Degassing of packing: Drain excess water from the pretreated Sephadex G-100 packing, transfer it to a vacuum filtration flask, seal it with a wash ball and degas it under vacuum until no more bubbles escape.

[0099] (3) Column packing: Inject a small amount of distilled water into the chromatography column (1.7cm×70.0cm), start the constant flow pump to backwash and remove air bubbles, and adjust to forward flow (flow rate 1mL / min). Use a glass rod to guide the flow and slowly and evenly pour the Sephadex G-100 packing into the column. After the packing has settled freely and stabilized on the column bed, seal the chromatography column.

[0100] (4) Sample loading and elution: Prepare 10 mg / mL solutions of each group separated by DEAE-52 column chromatography, slowly add them to the chromatography column until they completely permeate the packing material, add eluent to the column inlet, close the inlet, and start the constant flow pump to collect the eluent. The eluent is deionized water, the flow rate is 1 mL / min, the elution volume is 150 mL, and 5 mL is collected from each tube.

[0101] (5) Elution curve plotting and component collection: Same as described in DEAE-52 cellulose column chromatography.

[0102] Table 1. Composition of the Polysaccharide from Ligusticum striatum prepared in Example 1

[0103] name content(%) Total sugar 74.51±0.36 protein 0.82±0.07 Glucuronic acid 31.28±0.17

[0104] like Figure 1 As shown, the Ligusticum striatum polysaccharide (LFP) prepared in Example 1 was subjected to gradient elution with deionized water and different concentrations of NaCl using a DEAE-52 cellulose ion exchange column. Six elution peaks were detected by the phenol-sulfuric acid method and named LFP-W, LFP-1, LFP-2, LFP-3, LFP-4, and LFP-5, respectively. The yields of LFP-4 and LFP-5 were low, so they were discarded.

[0105] like Figure 2 China A Figure 2 B, Figure 2 C and Figure 2 As shown in Figure D, the four fractions with relatively high yields, LFP-W, LFP-1, LFP-2, and LFP-3, were further separated by a Sephadex G-100 chromatography column and eluted with deionized water to obtain five fractions: LFP-W-1, LFP-1-1, LFP-1-2, LFP-2-1, and LFP-3-1. The distribution of the elution curves and the size of the peak areas indicate that the LFP-1-2 fraction has a low polysaccharide content, and the amount of lyophilized powder obtained after collection is not large.

[0106] II. Determination of polysaccharide, protein and uronic acid content of Ligusticum striatum polysaccharide.

[0107] Polysaccharide content was determined using the phenol-sulfuric acid method: 1 mL of sample solution (pre-dissolved in distilled water) was added, followed by 1 mL of 5% phenol reagent, and then 5 mL of concentrated sulfuric acid. After thorough mixing, the mixture was reacted in a 40℃ water bath for 15 min. After cooling to room temperature, the absorbance was measured at 490 nm to calculate the polysaccharide content. A standard curve was plotted using glucose as the standard substance, with glucose concentration on the x-axis and absorbance at 490 nm on the y-axis. The fitted equation was y = 0.0037x + 0.0573(R²). 2 =0.9955).

[0108] Protein content was determined using the Coomassie Brilliant Blue method: the sample was dissolved in distilled water to prepare a sample solution. 0.2 mL of the sample solution was added to 1 mL of Coomassie Brilliant Blue, mixed well, and allowed to stand for 5 min. The absorbance was measured at 595 nm to calculate the protein content. A standard curve was constructed using bovine serum albumin as a standard. The protein concentration was plotted on the x-axis, and the absorbance at 595 nm on the y-axis. The fitted equation was y = 0.7123x + 1.2312, R0. 2 =0.9967.

[0109] The m-hydroxybiphenyl method was used to determine the uronic acid content. The specific steps are as follows: Weigh 0.15 g of m-hydroxybiphenyl, dissolve it in 5 mg / mL NaOH solution, and dilute to 100 mL. Weigh 3.9 g of aminosulfonic acid, add 5 mL of distilled water, heat and stir magnetically while continuously adding saturated KOH solution until completely dissolved. Dissolve the sample in distilled water. Take 0.4 mL of the polysaccharide solution, add 40 μL of aminosulfonic acid solution and shake gently. Then add 2.5 mL of concentrated H2SO4 and mix well. Heat to boiling for 20 min, cool, add 40 μL of m-hydroxybiphenyl solution at room temperature, mix well, let stand for 15 min, and measure the absorbance at 525 nm. A standard curve was plotted using D-galacturonic acid as a standard. The fitted equation was y = 2.1177x + 0.0649, R0. 2 =0.9950, the horizontal axis represents uronic acid content, and the vertical axis represents absorbance value.

[0110] Table 2. Chemical composition of each component after purification of the *Ligustrum lucidum* polysaccharide prepared in Example 1.

[0111] Components Polysaccharide content (%) Protein content (%) Glucuronic acid content (%) LFP-W-1 98.9±0.35 0.08±0.03 12.48±0.61 LFP-1-1 92.61±1.08 0.9±0.11 47.47±0.55 LFP-2-1 95.35±0.92 nd 55.38±0.82 LFP-3-1 93.88±0.47 0.4±0.19 88.25±0.93

[0112] As shown in Table 2, the polysaccharide, protein, and uronic acid contents of the purified Ligusticum striatum polysaccharide components LFP-W-1, LFP-1-1, LFP-2-1, and LFP-3-1 were determined by the phenol-sulfuric acid method, the Coomassie brilliant blue method, and the m-hydroxybiphenyl method, respectively.

[0113] III. Monosaccharide composition analysis.

[0114] Monosaccharide composition was determined using polysaccharide hydrolysis and PMP derivatization. Sixteen standard monosaccharides were analyzed as standard samples. Chromatographic conditions were as follows: Dionex Carbopac™ PA20 (3×150 mm) column; mobile phase A: H2O, B: 15 mM NaOH, C: 15 mM NaOH + 100 mM NaAc; flow rate 0.3 mL / min; injection volume 25 μL; column temperature 30 °C; and electrochemical detector. Finally, the mass of each monosaccharide was determined by absolute quantification using the external standard method (based on the standard mixture), and the molar ratio was calculated based on the molar mass.

[0115] Table 3. Proportion of monosaccharide components in each part of the purified Ligusticum striatum polysaccharide prepared in Example 1.

[0116]

[0117]

[0118] As shown in Table 3 and Figure 3 The ion chromatograms of LFP components shown indicate that the monosaccharide compositions of different polysaccharide components vary. Among them, LFP-W-1 is a neutral polysaccharide, with fructose as the main monosaccharide, accounting for 82.9% of the total. LFP-1-1, LFP-2-1, and LFP-3-1 are acidic polysaccharides, with relatively high contents of galacturonic acid, accounting for 55.8%, 71.5%, and 64.6% of the total, respectively. Figure 3 In the study, the peak of sodium hydroxide (solvent) was observed at 2.0 min, and the peak of sodium acetate (solvent) was observed at 40.5 min. The peak times and peak areas of each component of LFP and monosaccharide standards were measured and calculated. The monosaccharide components and their proportions were analyzed, and the results are shown in Table 3. Comparison with the mixed standard curve of 16 monosaccharides revealed that LFP is mainly composed of nine monosaccharides: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, galacturonic acid (GalA), and glucuronic acid (GlcA). The study showed that fructose and galactose can specifically promote the production of butyrate and acetate in the intestine. The galacturonic acid domain can effectively maintain the balance of colonic collagen metabolism and significantly promote wound healing in mouse colonic inflammation.

[0119] Experiment Example 2

[0120] The effect of Ligusticum striatum polysaccharide on colonic inflammation in mice was determined.

[0121] I. Induction of UC mouse model.

[0122] Seventy-two SPF-grade female Balb / c mice (weighing 18–20 g) were acclimatized for 7 days in an IVC system (temperature 20±4℃, relative humidity 50±10%, 12h light-dark cycle) and then randomly divided into five groups: normal group (N group), model group (M group), positive control group (P group), low-dose LFP group (LFPL), and high-dose LFP group (LFPH). All mice had free access to water and food. The administration regimens were as follows: the normal group and model group were administered physiological saline by gavage (days 1–14); the positive control group was administered physiological saline by gavage from days 1–7 and 200 mg / kg SASP enteric-coated tablets by gavage from days 8–14; the LFPL and LFPH groups were administered 200 mg / kg and 800 mg / kg LFP prepared in Example 1 by gavage, respectively (days 1–14). Except for the normal group, the other four groups were given 3% dextran sulfate sodium (DSS) via drinking water to induce ulcerative colitis (UC) from days 8–14. The animal testing protocol was approved by the Ethics Committee of Yanbian University, and the experimental procedures were conducted in accordance with the Ethics Committee's guidelines, providing humane care according to the 3R principle. Testing License Number: NO.SCXK(Liaoning)2020-0001.

[0123] II. Biological sample collection.

[0124] Immediately after euthanizing the mice, colon, liver, kidney, and spleen tissue samples were collected on ice for morphological comparison, weighing, and photographic recording. The length of the colon tissue was precisely measured, and the organ mass coefficient (organ weight / mouse body weight × 100%) was calculated. Colon samples used for histological analysis (uniformly taken from the distal colon) were immediately fixed in 4% paraformaldehyde solution to ensure complete preservation of tissue morphology.

[0125] III. DAI score calculation.

[0126] Record the mouse weight and fecal characteristics during the experiment, observe the mice for bloody stools, and calculate the DAI score according to Table 4 below.

[0127] Table 4 DAI Scoring Table

[0128]

[0129]

[0130] IV. Pathological observation of H&E staining.

[0131] Distal colon tissue was rinsed with physiological saline, blotted dry with filter paper, and immediately fixed in 4% paraformaldehyde (w / v). The process included: rinsing the fixed tissue with running water to remove residual fixative, followed by graded ethanol dehydration, xylene clearing, and then immersion in soft and hard paraffin wax for 1 hour each. The fully paraffin-impregnated tissue was vertically embedded, and 4μm sections were cut, spread in a 42℃ water bath, dewaxed, stained with H&E, dehydrated and cleared, and then mounted with neutral resin. Finally, the sections were observed under an optical microscope at 100× and 200×, and colonic pathological damage was scored according to Table 5 below. The results are expressed as the sum of the scores of the two parameters.

[0132] Table 5 Histological Pathological Injury Scoring Table

[0133]

[0134] V. Data Statistics and Analysis.

[0135] Experimental data are presented based on the mean and corresponding standard deviation (Mean ± SD) of three independent trials. One-way ANOVA and Waller-Duncan multiple comparisons were performed using SPSS 22.0 statistical software, with P < 0.05 considered statistically significant. GraphPadPrism 10 software was used for analysis and graphing.

[0136] VI. Results.

[0137] 1. Effects of LFP prepared in Example 1 on body weight and DAI score in mice with colitis.

[0138] DSS can induce clinical manifestations in mice similar to inflammatory bowel disease (IBD), such as bloody stools, weight loss, and diarrhea. Figure 4 As shown in Figure A, starting from day 11, except for the normal group, mice treated with 3% DSS exhibited varying degrees of disheveled fur, lethargy, and weight loss, consistent with the pathogenesis of this disease. Compared to the model group, the positive control group, low-dose LFP group, and high-dose LFP group showed recovery of mouse weight and reduced diarrhea and bloody stools. Figure 4 As shown in Figure B, the severity of colitis in mice was assessed by the DAI score. Except for the normal group, the DAI scores of other groups continued to increase, indicating successful modeling. In addition, compared with the model group, the DAI levels of the positive control group, the low-dose LFP group and the high-dose LFP group were significantly reduced (P<0.05), indicating that LFP intake can improve the trend of weight loss in mice to a certain extent and alleviate diarrhea and bloody stools caused by colitis.

[0139] 2. Effects of LFP prepared in Example 1 on organ indices in mice with colonic inflammation.

[0140] Internal organs are the carriers of an animal's physiological functions, participating in metabolism and maintaining normal life activities. Organ indices are important biological characteristics in experiments, reflecting the animal's physiological condition and the degree of inflammation within the body. Increased organ indices indicate visceral edema, congestion, or hypertrophy, while decreased indices indicate visceral atrophy. Indices for the kidneys, spleen, thymus, and liver are as follows: Figure 5 As shown, the organ indices of the model group mice were significantly higher than those of the normal group mice (P<0.05), which may be due to pathological enlargement of organs caused by inflammatory damage in the mice. Under LFP intervention, the liver and kidney indices of mice in the low-dose LFP group and the high-dose LFP group were significantly lower than those in the model group (P<0.05), and there was no significant difference from the normal group. The effect on the spleen index was not significant.

[0141] 3. Effects of LFP prepared in Example 1 on fecal bleeding and colon length in mice with colitis.

[0142] like Figure 6 China A Figure 6 China B and Figure 6 As shown in Figure C, compared with the normal group, the colon length of mice in the model group was significantly shortened and the rectal bleeding was more severe. After SASP intervention, the colon length of mice in the positive control group and the low-dose LFP group and the high-dose LFP group was significantly restored (P<0.05), and the rectal bleeding was significantly improved, indicating that LFP has an ameliorative effect on UC.

[0143] 4. Effects of LFP prepared in Example 1 on colonic pathological damage in mice with colonic inflammation.

[0144] Studies have shown that DSS has a toxic effect on the colonic epithelium, inducing erosion and ultimately damaging the integrity of the intestinal barrier. This leads to increased colonic epithelial permeability and further allows pro-inflammatory intestinal contents to diffuse into the underlying tissues, disrupting the balance between innate immunity and the gut microbiota. To observe the ameliorative effect of LFP on colonic pathological damage in UC mice, the extent of colonic damage in mice was assessed using H&E staining images and histopathological damage scores.

[0145] like Figure 7 China A and Figure 7As shown in Figure B, the normal group showed intact intestinal mucosal structure, regular arrangement of epithelial cells, and intact goblet cells. In the model group, the intestinal glands and tissue cells of the mucosal layer were severely damaged after DSS intervention, with missing crypt structures, disrupted intestinal lumen structure, and extensive inflammatory cell infiltration that had invaded the submucosa and muscularis propria. Compared with the model group, the positive control group, after SASP intervention, and the low-dose LFP group and high-dose LFP group, showed some recovery of colonic damage, with more intact colonic structure, a greater number of goblet cells, reduced inflammatory cell infiltration, and relatively more intact crypt structure (P<0.05). These results indicate that LFP protects the colon and alleviates DSS-induced colonic pathological changes.

[0146] In summary, after DSS induction, the LFP intervention prepared in Example 1 alleviated the weight loss in UC mice, reduced the DAI score and organ index in UC mice, and improved the shortening of colon length and colon tissue damage caused by colon inflammation.

[0147] Experimental Example 3

[0148] The effects of Ligusticum striatum polysaccharide on intestinal flora and short-chain fatty acid content in mice with colitis were determined.

[0149] I. Biological sample collection.

[0150] After the mice in Experiment 2 were euthanized, the contents of the cecum and feces were collected, stored in cryovials, labeled and temporarily stored in liquid nitrogen, and then transferred to a -80°C freezer for later use.

[0151] II. Intestinal flora determination.

[0152] Total genomic DNA of microorganisms was extracted from cecal contents using a rapid DNA spin extraction kit (MP Biomedicals, CA, USA, MagBeads FastDNA Kit for Soil 116564384). The V3–V4 region of the 16S rRNA gene was amplified using primers 338F and 806R. Sequencing libraries were prepared from the PCR amplification products and sequenced on the Illumina MiSeq platform. QIIME (v1.8.0) was used to identify and eliminate problematic sequences. Sequences were merged based on 97% sequence similarity, and operational taxonomic units (OTUs) were assigned. Colony composition analysis of the fecal microbiota was performed based on α-diversity and β-diversity, comparing the effects of LFP treatment on microorganisms at the phylum and genus levels.

[0153] III. Determination of SCFAs content in cecal contents.

[0154] Take an appropriate amount of sample into a 1.5 mL centrifuge tube, add 500 L of water, homogenize, and centrifuge at 12000 rpm for 10 min at 4 °C. Collect 200 μL of the supernatant. Add 100 μL of 15% phosphoric acid, 20 μL of 375 μg / mL internal standard (4-methylvaleric acid) solution, and 280 μL of diethyl ether to the supernatant. Homogenize again and centrifuge under the same conditions. Collect the supernatant for instrumental analysis. The instrumental analysis conditions are as follows:

[0155] Chromatographic system: Thermo Trace 1310 gas chromatograph (Thermo Fisher Scientific, USA), equipped with an Agilent HP-INNOWAX capillary column (30m × 0.25mm × 0.25μm). Split injection mode (split ratio 10:1) was used, with an injection volume of 1μL. Temperature program: initial 90℃, increased to 120℃ at 10℃ / min, increased to 150℃ at 5℃ / min, and finally increased to 250℃ at 25℃ / min and held for 2 min. Helium was used as the carrier gas at a flow rate of 1.0 mL / min.

[0156] Mass spectrometry system: Thermo ISQ LT mass spectrometer (Thermo Fisher Scientific, USA), equipped with an electron impact ionization (EI) source, using SIM scan mode, electron energy 70 eV.

[0157] The content of SCFAs was calculated by referring to the standard equation and using the peak area.

[0158] IV. Data Statistics and Analysis.

[0159] Experimental data are presented based on the mean and corresponding standard deviation (Mean ± SD) of three independent trials. One-way ANOVA and Waller-Duncan multiple comparisons were performed using SPSS 22.0 statistical software, with P < 0.05 considered statistically significant. GraphPadPrism 10 software was used for analysis and graphing. Bioinformatics analysis was performed using QIIME (v1.8.0).

[0160] V. Results.

[0161] 1. Effect of LFP prepared in Example 1 on α-diversity of gut microbiota in mice with colitis.

[0162] Alpha diversity analysis can be used to determine sample size and the diversity, richness, and evenness of a community. For example... Figure 8As shown in Figure A, the species sparsity curve results indicate that the number of species gradually levels off as sequencing depth increases, suggesting that the sequencing data is sufficiently reliable for further analysis. The sequencing depth of all samples was determined using Chao1, Simpson, and Shannon indices, as shown below. Figure 8 B in the middle, C in the diagram and Figure 8 As shown in Figure D, the community diversity of the LFPL group and the normal group was relatively high, and the difference from the model group was significant (P<0.05). The positive control group and the LFPH group were slightly improved, indicating that the richness of the gut microbiota was restored after the intervention of SASP and LFP. In particular, there was no significant difference in community richness between the LFPH group and the normal group (P>0.05).

[0163] 2. Effect of LFP prepared in Example 1 on the β-diversity of gut microbiota in mice with colitis.

[0164] β-diversity is an important ecological indicator characterizing the degree of difference among biological community samples. To analyze the differences in microbial community structure among samples, principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) were used to visualize β-diversity. Figure 9 China A and Figure 9 As shown in Figure B, PCoA and NMDS analyses revealed that the normal group samples exhibited significant spatial separation from other experimental groups in the two-dimensional ordination space (P<0.05), indicating that DSS induction significantly altered the overall composition of the gut microbiota (P<0.05). Although the clustering of samples from each intervention group partially overlapped with that of the model group, their spatial distribution showed a characteristic shift trend, suggesting that the low-dose LFP group and the high-dose LFP group, through LFP intervention, and the positive control group, through SASP intervention, promoted a remodeling process beneficial to the remission of ulcerative colitis through specific regulatory mechanisms.

[0165] 3. Effect of LFP prepared in Example 1 on the composition of intestinal microbiota in mice with colitis.

[0166] The Venn diagram at the level of amplicon sequence variants (ASVs) illustrates the number of common and specific species within the six groups. For example... Figure 10 The figure shows the Venn plot of ASVs levels among the five groups. According to the results of the Venn plot, different interventions have different effects on the gut microbiota. The five groups share 370 ASVs, the normal group has 1920 ASVs that are different from the other groups, the model group has 1099 ASVs that are unique, the positive control group has 1297 ASVs that are unique, the LFPL group has 1955 ASVs that are unique, and the LFPH group has 1364 ASVs that are unique.

[0167] like Figure 11 China A and Figure 11 As shown in Figure B, the analysis of the relative abundance of species at the phylum level (bar chart) indicates that the top 10 most abundant phyla essentially cover the entire gut microbiota structure. Among them, Bacteroidetes, Firmicutes, Desulfobacterota, Actinobacteria, Deferribacterota, and Proteobacteria are the dominant bacterial groups in the gut, accounting for over 95% of all phyla (e.g., ...). Figure 11 (A) Proteobacteria are pathogenic phyla in the gut microbiota, including Escherichia coli, Salmonella, Vibrio cholerae, and Helicobacter pylori. Studies have found that they are associated with metabolic disorders and colitis (UC). The main bacterial phyla in the gut are Firmicutes and Bacteroidetes, which play important roles in nutrient absorption and metabolism. After DSS induction, the abundance of Bacteroidetes and Proteobacteria significantly increased, while the abundance of Firmicutes significantly decreased. The decrease in the Firmicutes / Bacteroidetes ratio is a typical marker of colitis, and this ratio is considered to play an important role in maintaining the stability of the gut environment. The LFPL and LFPH groups were treated with LFP intervention, while the positive control group was treated with SASP intervention, which induced changes in the gut microbiota. In particular, the F / B ratio in the LFPH group was significantly higher than that in the model group, and the microbiota composition of the LFPH group was more similar to that of the normal group (e.g., Figure 11 (B)

[0168] like Figure 12 and Figure 13 As shown, at the genus level, the model group had a higher proportion of *Bacteroides* and *Odoribacter*, belonging to the phylum *Bacteroides*. After LFP intervention, the abundance of the gut microbiota was closer to the normal level compared to the model group. Specifically, the relative abundance of *Cryptobacteroides*, *Prevotella*, and *Limosilactobacillus* increased. Most of these bacteria can ferment polysaccharides in the gut to produce short-chain fatty acids, such as acetic acid, propionic acid, and butyric acid. In conclusion, LFP intervention can, to some extent, regulate the relative abundance of the gut microbiota to improve colonic inflammation.

[0169] like Figure 14 and Figure 15As shown, based on the linear discriminant analysis (LEfSe) effect size algorithm, the system identified significantly different gut microbiota biomarkers among different groups. The analysis results showed that the LDA values ​​of all groups were significantly higher than the preset threshold (LDAscore>3), with the normal group exhibiting the most significant perturbation of microbiota characteristics, identifying a total of 10 landmark taxonomic units. Further comparison revealed that the model group, positive control group, LFPL group, and LFPH group had 10, 7, 4, and 2 statistically significant specific response taxonomic units, respectively (LDA score>3, P<0.05). The number of biomarkers among the intervention groups showed a clear dose-dependent decreasing trend, providing important clues for elucidating the microbiota regulation mechanism of fructooligosaccharides in improving ulcerative colitis. At different taxonomic levels, the dominant bacteria in the normal group, model group, positive control group, LFPL group, and LFPH group were Lactobacillus, Bacteroidaceae, Catonella, UBA932, and Pararaprevotella, respectively. The dominant bacteria in each group could be distinguished by biomarkers.

[0170] 4. Intestinal flora function prediction analysis.

[0171] Functional prediction studies were conducted by integrating microbial community difference data from the LFPL, LFPH, and model groups, combined with the KEGG metabolic database. For example... Figure 16 As shown, the gut microbiota exhibits significant specificity in regulating host metabolic pathways, primarily involving carbohydrate and amino acid metabolism, as well as energy conversion, nucleotide synthesis, and coenzyme / vitamin metabolism. Under inflammatory pathological conditions, each metabolic pathway shows differential changes: carbohydrate metabolism is upregulated, amino acid metabolism is inhibited, while energy metabolism, nucleotide synthesis, and coenzyme / vitamin metabolism show an enhanced trend. Notably, after LFP intervention, the metabolic profiles of the LFPL and LFPH groups showed a trend towards regression to normal physiological states.

[0172] 5. SCFAs content analysis.

[0173] Polysaccharides, as biomolecules formed by monosaccharide subunits linked by glycosidic bonds, cannot be directly digested and absorbed by the human body. Gut microorganisms, by expressing diverse carbohydrate-active enzymes, hydrolyze and modify the glycosidic bonds of the polysaccharide backbone and side chains. Their synergistic multi-enzyme action degrades complex carbohydrate structures, generating signaling molecules that can be utilized by human cells, thereby regulating physiological and pathological processes. After being degraded into monosaccharides or oligosaccharides, polysaccharides enter different metabolic pathways, ultimately producing SCFAs, which help maintain the balance of the gut microbiota.

[0174] Table 6. SCFA content in mouse cecal contents

[0175]

[0176]

[0177] Note: (M±SD, n=5), different letters indicate significant differences between groups, P<0.05.

[0178] As shown in Table 6, the short-chain fatty acids (SCFAs) in the cecal contents of mice were mainly acetic acid, propionic acid, and butyric acid. Their biological effects included stimulating the proliferation of beneficial bacteria, activating regulatory T cells to inhibit the release of inflammatory mediators, and were significantly associated with increased oxygen consumption of colonic epithelial cells and enhanced intestinal barrier function. DSS led to a significant decrease in SCFA content in mice, and interventions in the LFPL and LFPH groups significantly reversed the decreasing trend of SCFAs (P<0.05), bringing them close to normal levels.

[0179] like Figure 17 As shown, Pearson correlation analysis also indicated that, among the dominant bacterial phyla in each group, the abundance of bacteria in the normal group was positively correlated with SCFA levels. Conversely, the number of dominant gut bacteria in the model group was negatively correlated with SCFA levels. However, in the positive control group under SASP intervention, and in the LFPL and LFPH groups under LFP intervention, the correlation trend between dominant bacterial abundance and SCFAs gradually shifted towards that of the normal group.

[0180] In summary, the LFP intervention prepared in Example 1 improved the α-diversity index and β-diversity of the gut microbiota in UC mice, promoting the restoration of microbial species richness and community structural heterogeneity to normal levels. It significantly alleviated the state of microbial dysbiosis. At the phylum level, the abnormal proliferation of Bacteroidetes / Firmicutes and Proteobacteria in the LFP intervention group was effectively reversed. At the genus level, the abundance of Bacteroides, Odoribacter, Cryptobacteroides, Prevotella, and Limosilactobacillus all returned to normal levels. It increased the content of SCFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid) in the cecal contents, and the gut microbiota and SCFAs in the LFPL and LFPH groups showed a trend towards normal levels.

[0181] Experiment Example 4

[0182] The regulatory effect of GPR-NLRP3 on colitis in mice was determined by examining the polysaccharide of Ligusticum striatum.

[0183] I. Biological sample collection.

[0184] Colon tissue was immediately harvested from mice euthanized in Experiment 2 and placed on ice. Tissues were classified and preserved according to different experiments: tissues for qRT-PCR were placed in DEPC-treated EP tubes; tissues for Western blot assays were stored in sterilized EP tubes. After labeling, the tissues were temporarily stored in liquid nitrogen and then transferred to a -80°C freezer for long-term storage.

[0185] II. qRT-PCR detection of the expression of related factor mRNAs.

[0186] 1. RNA extraction from tissues using a kit method.

[0187] Total RNA was extracted from colon tissue using a column purification method. 50 mg of fresh tissue sample was placed in a pre-chilled RNase-free grinding tube, 500 μL of lysis buffer was added, and the sample was cryogenically ground. The resulting lysis buffer was incubated at 4°C for 30 min. The supernatant was collected by centrifugation at 4°C, RNA dilution buffer was added, and the mixture was incubated for 3 min. The supernatant was collected by centrifugation again. Anhydrous ethanol was added, and the mixture was centrifuged at 4°C for 1 min, then the filtrate was discarded. RNA washing buffer and DNase I incubation buffer were then added sequentially (incubated at room temperature for 15 min). This centrifugation and filtrate discarding process was repeated twice. Finally, the purification column was transferred to a sterile collection tube, DEPC water was added, and the mixture was incubated at room temperature for 2 min before centrifugation to elute RNA. RNA purity was measured using Nanodrop (A). 260 / A 280 (Ratio 1.8–2.1), qualified samples were aliquoted and stored in an ultra-low temperature freezer at -80°C.

[0188] 2. cDNA synthesis.

[0189] Place the EP tube in an ice box, accurately measure the reagents according to Table 7 to prepare the reaction solution, mix thoroughly, and then place it in the PCR instrument. Set the reaction conditions: 42℃ for 15 min, 95℃ for 3 min.

[0190] Table 7 Reverse Transcription Procedure

[0191] reagents Dosage (μL) 5×FastKing-RTSuperMix 4.0 TotalRNA 50ng~2μg <![CDATA[RNaseFreedH2O]]> Supplement to 20

[0192] 3. Real-time quantitative PCR analysis.

[0193] The PCR reaction system was prepared on ice according to Table 8. Table 9 shows the primer sequences for the reaction. The amplification program was set as follows: 95℃ pre-denaturation for 15 min; 40 cycles (95℃ for 10 s, 60℃ for 30 s); melting curve analysis was performed using three-step temperature control (95℃, 60℃, and 95℃ for 15 s each). mRNA expression levels were measured using β-actin as an internal control, and a 2... -ΔΔCt The relative expression level is calculated using this method.

[0194] Table 8 PCR reaction solution preparation system

[0195] reagents Dosage (μL) 2×SuperRealPreMixPlus 10 PCRForwardPrimer (10μM) 0.6 PCRReversePrimer (10μM) 0.6 <![CDATA[50×ROXReferenceDye △ ]]> 0.4 cDNA template * <![CDATA[RNase-freeddH2O]]> Supplement to 20 Total 20

[0196] Table 9 Primer sequences

[0197]

[0198] III. Western Blot method for detecting the expression of related proteins.

[0199] 1. Extraction of total protein from tissues.

[0200] Colon tissue was added to a grinding tube along with an appropriate amount of lysis buffer prepared in the ratio (RIPA:PMSF:phosphatase inhibitor:protease inhibitor = 100:1:1:1). After being thoroughly ground in a cryogenic grinder, the mixture was allowed to stand at 4°C for 30 minutes. Subsequently, it was centrifuged at 4°C and the supernatant was collected for later use.

[0201] 2. Determination of protein concentration.

[0202] (1) Solution preparation: Accurately weigh bovine serum albumin (BSA) powder and prepare a 25 mg / mL stock solution with sterile PBS buffer. After sterilization by filtering through a 0.22 μm filter membrane, serially dilute to a working concentration of 0.5 mg / mL for the establishment of a standard curve. Mix reagent A and reagent B thoroughly by vortexing at a volume ratio of 50:1 (v / v) to prepare the colorimetric working solution. Store at 4°C protected from light for later use.

[0203] (2) Standard curve and sample determination: 20 μL of tissue protein solution and standard solutions of different concentrations were added sequentially to the reaction system, followed by 200 μL of working solution. After incubation at 37°C for 30 min, the absorbance was measured at 562 nm. A standard curve was plotted based on the obtained data, and the protein concentration was calculated. After balancing the sample concentrations, 5×SDS protein loading buffer was added, and the mixture was boiled for 10 min, aliquoted, and stored for subsequent use.

[0204] 3. SDS-polyacrylamide gel electrophoresis.

[0205] (1) Gel preparation: Prepare the separating gel and stacking gel sequentially according to the instructions in the kit. The optimal separation range for different concentrations of separating gel is shown in Table 10.

[0206] Table 10 Optimal separation range of separating gels at different concentrations

[0207] SDS-PAGE separating gel concentration (%) Optimal separation range kDa 6 57～212 8 36～94 10 20～80 12 12～60 15 10～43

[0208] (2) Electrophoresis: After the sample is denatured at 95℃ for 5 min, it is loaded and concentrated at a constant voltage of 80V for 20 min (to the boundary of the bromophenol blue gel). Then the voltage is adjusted to 120V and electrophoresis is continued for 60 min (until the indicator is 0.5 cm from the bottom of the gel).

[0209] (3) Membrane transfer (semi-dry method): Immerse filter paper and methanol-activated PVDF membrane in transfer solution and assemble them into a "sandwich" structure in the order of "filter paper-gel (face up)-PVDF membrane-filter paper". Set the transfer time according to the molecular weight of the target protein (<120kDa: transfer with a constant current of 0.2A for 45min; ≥120kDa: transfer for 60min).

[0210] (4) Blocking and hybridization: After the transfer was completed, the target band was cut according to the pre-labeled section and blocked with 5% BSA (dissolved in TBS-T) at room temperature for 2 h. After washing with TBS-T, the primary antibody diluted 1:1000 was added and incubated overnight at 4°C. The next day, after washing with TBS-T, it was incubated with HRP-labeled secondary antibody diluted 1:10000 at room temperature for 2 h.

[0211] (5) Development: Mix equal volumes of developer A and B, evenly cover the surface of the strip, incubate, and then expose and photograph.

[0212] IV. Data Statistics and Analysis.

[0213] Experimental data visualization and analysis were performed using the Origin 2021 platform. Western blotting band grayscale values ​​were quantitatively measured using the ImageJ image processing system. One-way ANOVA was performed using the SPSS 22.0 statistical analysis platform, and the Waller-Duncan method was used for multiple comparisons. The statistical significance threshold was P < 0.05.

[0214] V. Results.

[0215] 1. Effects on GPR41 mRNA expression.

[0216] Studies have shown that SCFAs can activate GPCRs on the surface of epithelial cells, thereby participating in the regulation of various physiological homeostasis, such as energy balance, metabolism, and inflammation. Simultaneously, SCFAs-GPRs signaling can maintain epithelial cell layer stability by regulating apoptosis-related pathways and influencing the expression of Bcl-2 and Bax.

[0217] like Figure 18As shown, the mRNA expression of GPR41 in the normal group, positive control group, LFPL group, and LFPH group was significantly different from that in the model group (P<0.05). Among them, the mRNA expression of GPR41 in the normal group was not significantly different from that in the two groups (LFPL group and LFPH group) treated with LFP (P>0.05), indicating that LFP can effectively upregulate the mRNA expression of GPR41.

[0218] 2. Effects on GPR41 protein expression.

[0219] like Figure 19 China A and Figure 19 As shown in Figure B, there were significant differences in protein expression between the normal group and the model group (P<0.05), indicating that DSS significantly inhibited the expression of GPR41 protein. GPR41 protein expression was restored in the positive control group, LFPL group, and LFPH group, consistent with the mRNA expression regulation results, suggesting that LFP can regulate the damaged intestinal epithelial structure of UC mice by activating GPCRs.

[0220] 3. Effects on Bcl-2 and Bax mRNA expression.

[0221] Disseminated cerebrospinal fluid (DSS) can disrupt the homeostasis of apoptosis regulation by downregulating the expression level of the anti-apoptotic protein Bcl-2 and upregulating the activity of the pro-apoptotic protein Bax, thereby triggering an abnormal increase in programmed cell death in colonic epithelial cells. This pathological change leads to the destruction of the integrity of the intestinal physical barrier, forming a trigger point for an inflammatory cascade, ultimately exacerbating or prolonging the inflammatory damage process of the intestinal mucosa.

[0222] like Figure 20 China A and Figure 20 As shown in Figure B, there were significant differences in Bcl-2 / Bax mRNA expression levels between the normal group and the model group (P<0.05). The LFPL and LFPH groups, which were treated with LFP, significantly reversed the abnormal expression patterns and brought the relevant indicators closer to the physiological levels of healthy mice (P<0.05), demonstrating better regulatory efficacy than the positive control group.

[0223] 4. Effects on the expression of Bcl-2 and Bax proteins.

[0224] like Figure 21 China A Figure 21 China B and Figure 21As shown in Figure C, the protein expression levels of Bcl-2 and Bax in the normal group were significantly different from those in the model group (P<0.05). The positive control group, LFPL group, and LFPH group upregulated Bcl-2 protein expression and downregulated Bax protein expression. In particular, the LFPH group had a significant regulatory effect on the protein expression of Bcl-2 and Bax (P<0.05), which is consistent with the trend of the above mRNA expression results, indicating that LFP can significantly reverse the inflammatory state of the colon tissue of UC mice.

[0225] 5. Effects on the expression of NLRP3, Caspase-1 and ASC mRNA.

[0226] The NLRP3 inflammasome is an important component of the innate immune system, consisting of NLRP3, ASC, and Caspase-1, and plays a key role in ulcerative colitis (UC).

[0227] like Figure 22 China A Figure 22 China B and Figure 22 As shown in Figure C, compared with the model group, the mRNA transcription levels of NLRP3, Caspase-1, and ASC in the normal group showed significant differences (P<0.05), indicating that DSS induction successfully activated the NLRP3 inflammasome pathway. In the intervention group, both the positive control group and the different doses of LFP intervention groups (LFPL / LFPH) showed significant inhibitory effects on NLRP3 and ASC gene expression (P<0.05).

[0228] 6. Effects on the expression of NLRP3, Caspase-1 and ASC proteins.

[0229] like Figure 23 China A Figure 23 B, Figure 23 C and Figure 23 As shown in Figure D, DSS upregulated the protein expression of NLRP3, Caspase-1, and ASC. In the LFPL and LFPH groups treated with LFP, the expression of NLRP3 and Caspase-1 was significantly reduced (P<0.05), and ASC protein expression was also downregulated to some extent. This indicates that LFP can improve inflammatory damage in UC by regulating the NLRP3 inflammasome signaling pathway.

[0230] 7. Correlation analysis.

[0231] Spearman correlation analysis was performed on the top 20 most abundant bacterial groups at the genus level, the content of SCFAs, and the detection indicators of GPR41, Bcl-2, and Bax in colon tissue. Figure 24 As shown, combined with Figure 17In the model group, the abundance of *Bacteroides* and *Odoribacter* was high, showing a negative correlation with cytokines such as GPR41 and Bcl-2, and a positive correlation with Bax and the NLRP3 signaling pathway components NLRP3, Caspase-1, and ASC. In the LFPL and LFPH groups, the abundance of *Limosilactobacillus* and *Ligilactobacillus* was improved, showing a positive correlation with cytokines such as GPR41 and Bcl-2, and a negative correlation with Bax and the NLRP3 signaling pathway components NLRP3, Caspase-1, and ASC, the opposite trend to the model group. This indicates that LFP improves colonic inflammation by regulating the gut microbiota structure, thereby modulating the expression of the GPR-NLPR3 signaling pathway and related factors.

[0232] In summary, LFP activates the short-chain fatty acid receptor GPR41, regulates epithelial cell apoptosis-related factors, upregulates the mRNA and protein expression levels of Bcl-2 and inhibits Bax, and reduces the mRNA and protein expression of the NLRP3 inflammasome signaling pathway, thus alleviating inflammatory damage and providing an effective intervention for the relief and treatment of UC. LF can regulate the structure of the gut microbiota in colitis-prone mice, promoting the production of SCFAs and modulating related cytokines and the NLRP3 signaling pathway, thereby preventing and improving colitis. This provides a reference for the development of polysaccharides and the intervention and relief of UC.

[0233] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a polysaccharide from Ligustrum lucidum leaves in the preparation of a medicament for the prevention and / or treatment of ulcerative colitis, characterized in that, The preparation method of the polysaccharide from Ligustrum lucidum includes the following steps: 1) Ligustrum lucidum leaves, quick-frozen, vacuum freeze-dried, pulverized and sieved to obtain freeze-dried powder of Ligustrum lucidum leaves; 2) The freeze-dried powder of Ligustrum lucidum obtained in step 1) is mixed with petroleum ether, shaken to extract, allowed to stand and separate into layers, and the upper layer is discarded to obtain defatted Ligustrum lucidum powder. 3) The defatted Ligustrum lucidum powder obtained in step 2) is mixed with deionized water and soaked and extracted 2-4 times. The extracts are combined to obtain Ligustrum lucidum extract. 4) The Ligusticum striatum extract obtained in step 3) is concentrated by rotary evaporation to 1 / 4 of the total volume of the Ligusticum striatum extract to obtain a concentrated solution; 5) Mix the concentrated solution obtained in step 4) with anhydrous ethanol, let it stand, and collect the precipitate; 6) The precipitate obtained in step 5) was washed successively with anhydrous ethanol, acetone and anhydrous diethyl ether to obtain the washed precipitate, which was dried to constant weight to obtain crude polysaccharide of Ligusticum striatum. 7) The crude polysaccharide of Ligustrum lucidum obtained in step 6) is mixed with deionized water to obtain a crude polysaccharide solution of Ligustrum lucidum; the crude polysaccharide solution of Ligustrum lucidum is mixed with Sevag reagent, shaken on a shaker, allowed to stand for separation, and the upper aqueous phase is taken. 8) The upper aqueous phase obtained in step 7) is placed in a dialysis bag and dialyzed in deionized water to obtain a dialysis polysaccharide solution; 9) The polysaccharide solution obtained after dialysis in step 8) is pre-frozen and then freeze-dried under vacuum to obtain Ligustrum lucidum polysaccharide; The quick-freezing temperature in step 1) is -70~-90℃, and the quick-freezing time is 10~14h; the vacuum freeze-drying time in step 1) is 36~60h, the vacuum freeze-drying temperature is -40~-60℃, and the vacuum degree of the vacuum freeze-drying is ≤0.1mbar; the particle size of the sieve in step 1) is 70~90 mesh. In step 3), the ratio of defatted Ligustrum lucidum powder to deionized water is 1g: 15~25mL; the soaking and extraction temperature in step 3) is 70~90℃, the number of soaking and extractions is 2~4 times, and the soaking and extraction time is 1~3h each time. The molecular weight of the dialysis bag mentioned in step 8) is 8000~14000 Da, the dialysis temperature mentioned in step 8) is 4℃, and the deionized water is replaced every 6~8 hours.

2. The application according to claim 1, characterized in that, In step 2), the ratio of freeze-dried Ligusticum striatum leaf powder to petroleum ether is 1g: 4~6mL; in step 2), the shaking extraction speed is 150~250rpm, and the shaking extraction time is 4~6h; in step 2), the standing layering time is 20~40min.

3. The application according to claim 1, characterized in that, The rotary evaporation concentration temperature in step 4) is 70~90℃, and the vacuum degree of the rotary evaporation concentration is 0.08~0.09MPa; the volume ratio of the concentrate to anhydrous ethanol in step 5) is 1:3~5, the standing temperature in step 5) is 4℃, and the standing time is ≥12h.

4. The application according to claim 1, characterized in that, The drying temperature in step 6) is 35~45℃; the volume ratio of the crude polysaccharide solution of Ligusticum striatum and the Sevag reagent in step 7) is 1:3~5, and the Sevag reagent is obtained by mixing chloroform and n-butanol in a volume ratio of 4:1; the shaking time in step 7) is 15~25min, and the shaking speed is 150~250rpm; the settling time in step 7) is ≥30min.

5. The application according to claim 1, characterized in that, The pre-freezing temperature in step 9) is -70~-90℃, the pre-freezing time is 10~14h, the vacuum freeze-drying time in step 9) is 36~60h, the vacuum freeze-drying temperature is -40~-60℃, and the vacuum degree of the vacuum freeze-drying is ≤0.1mbar.

6. The application according to claim 1, characterized in that, Ligusticum striatum polysaccharide can prevent and / or treat ulcerative colitis by regulating the abundance of gut microbiota, reshaping the gut microbiota structure, and increasing the content of short-chain fatty acids to regulate the level of gut metabolites.

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