Gastrodia elata polysaccharide and application thereof

Abstract

The invention belongs to the field of new application of medicines, and particularly relates to gastrodia elata polysaccharide and application thereof. The gastrodia elata polysaccharide component GEP-2 is separated from crude gastrodia elata polysaccharide, structural characterization detection is conducted on the GEP-2, the result shows that the gastrodia elata polysaccharide GEP-2 is prepared from, by mass, 89.08% of glucose, 2.40% of galactose, 7.12% of galacturonic acid, 0.86% of arabinose and 0.54% of rhamnose, and the animal experiment result shows that the GEP-2 relieves the symptoms of mouse colitis, relieves weight loss and shortens the colon length; the traditional Chinese medicine composition has the advantages of reducing serum TNF-alpha, IL-1beta and IL-6 proinflammatory cytokine levels, improving the intestinal barrier function, reducing inflammatory cell infiltration, regulating the intestinal flora composition structure of mice and relieving ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of new drug applications, and more specifically, relates to a Gastrodia elata polysaccharide and an application thereof. Background Art

[0002] Ulcerative colitis (UC) is a difficult-to-treat intestinal disease characterized by chronic inflammation of the colorectal mucosa. Clinical manifestations include bloody and mucus-purulent stools, abdominal pain and diarrhea, and weight loss. Lesions often affect the rectum and sigmoid colon. Global epidemiological data show that the incidence of UC continues to rise, with 5 million new cases expected in 2023. The disease has a high recurrence rate and a low cure rate. The standardized incidence ratio (SIR) for colorectal cancer in patients is significantly increased by 1.7 times, and the risk of cancer increases to 4.5 times in patients with a disease course of more than 20 years. The pathogenesis of UC is not fully understood and may be related to an imbalanced immune response, oxidative stress, epithelial barrier defects, intestinal microbiome dysbiosis, genetic susceptibility, and the combined effects of environmental factors. Currently, the main clinical treatments for UC include 5-aminosalicylic acid drugs, steroid drugs, and immunosuppressants. These drugs can provide short-term symptom relief, but long-term use can easily lead to adverse reactions such as drug dependence and toxicity, infection risks, and complications, resulting in reduced treatment compliance. Existing therapies have a significant conflict between controlling disease recurrence and reducing adverse reactions, and there is an urgent need to develop safer and more effective therapeutic drugs to improve the current treatment status of UC. Traditional Chinese medicine can control the progression of UC by regulating immune inflammatory responses, reducing oxidative stress, and improving the intestinal mucosal barrier. Compounds that can be used to treat UC have been found in herbs such as Coptis chinensis and Atractylodes macrocephala, confirming the feasibility of natural medicines for treating UC. However, research on its therapeutic mechanisms and efficacy evaluation still requires support from modern scientific research.

[0003] Plant Gastrodia elata ( Gastrodiaelata Blume, G.elataGastrodia elata (Gastrodia elata) is a fully branched, heterotrophic orchid with a wide geographical distribution in China, Japan, Korea, Bhutan, and India. The traditional Chinese medicine (TCM) Gastrodia elata (Gastrodia elata) is derived from the dried tubers of this plant. Due to its outstanding medicinal value, it has been used as a herbal medicine for over 2,000 years, primarily for treating febrile seizures, tetanus, quadriplegia, limb numbness, and rheumatic joint pain in children, as well as for relieving epileptic seizures, headaches, and dizziness. Modern pharmacological research has revealed that Gastrodia elata contains a variety of bioactive components, such as flavonoids, steroidal saponins, and polysaccharides, among which polysaccharides have been shown to possess diverse biological activities. Gastrodia elata polysaccharides are a class of substances discovered in recent years that are abundant in Gastrodia elata and exhibit a wide range of biological activities. Research on their chemical composition, content determination, and pharmacological activities is increasing. Gastrodia elata polysaccharides have been shown to exhibit anti-vertigo, anti-aging, and antihypertensive effects. However, due to the numerous molecular weight fragments of Gastrodia elata polysaccharides, their specific composition and structure remain unclear, and their specific mechanisms of action remain elusive. Summary of the Invention

[0004] The purpose of the present invention is to provide a Gastrodia elata polysaccharide and its application to solve the above technical problems.

[0005] The present invention provides Gastrodia elata polysaccharide GEP-2, which is derived from Gastrodia elata. The Gastrodia elata polysaccharide GEP-2 has a number average molecular weight of 2.54 kDa, a weight average molecular weight of 9.24 kDa, a z-average molecular weight of 37.92 kDa, and a polydispersity index of 3.64. The Gastrodia elata polysaccharide GEP-2 is composed of 89.11% glucose, 2.4% galactose, 7.13% galacturonic acid, 0.86% arabinose, and 0.5% rhamnose in percentages by mass.

[0006] The present invention isolated Gastrodia elata polysaccharide GEP-2 from crude Gastrodia elata polysaccharides. Structural elucidation using molecular weight measurement, monosaccharide composition determination, bond structure characterization, and NMR characterization revealed that GEP-2 is a polysaccharide with a (4)-α-D-glucose backbone, with branched polysaccharide fragments containing galacturonic acid, galactose (Gal), rhamnose, and arabinose substituents at positions 4 and 6. It exhibits a broad molecular weight distribution, with a number-average molecular weight of 2.54 kDa, a weight-average molecular weight of 9.24 kDa, a z-average molecular weight of 37.92 kDa, and a polydispersity index of 3.64. The monosaccharide composition is primarily glucose (89.08%), supplemented by galacturonic acid (7.12%), galactose (2.4%), rhamnose (0.54%), and arabinose (0.86%), connected by α-glycosidic bonds to form a complex branched structure.

[0007] In the activity study of GEP-2, it was confirmed that Gastrodia elata polysaccharide GEP-2 can significantly alleviate the symptoms of colitis, reduce inflammatory cell infiltration, reduce tissue edema and ulcers, and reduce the levels of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in colon tissue, while increasing the level of anti-inflammatory cytokine IL-10. It can regulate the composition structure of the intestinal flora of mice and increase the relative abundance of probiotics.

[0008] The present invention also provides the use of the Gastrodia elata polysaccharide GEP-2 in preparing a medicine for treating ulcerative colitis.

[0009] Furthermore, the Gastrodia elata polysaccharide GEP-2 is the only active ingredient in the medicine.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the auxiliary materials include any one or more of fillers, stabilizers, diluents, and adjuvants.

[0012] Furthermore, the diluent is any one of water and physiological saline.

[0013] The present invention also provides a medicine for treating ulcerative colitis. The medicine is prepared by mixing the Gastrodia elata polysaccharide GEP-2 and auxiliary materials. The content of the Gastrodia elata polysaccharide GEP-2 in the medicine is 0.1 wt% to 99 wt%.

[0014] Furthermore, the solid dosage forms include granules, tablets, capsules, pills, and dripping pills, and the solution dosage forms include oral liquid preparations, gavage preparations, and injection dosage forms.

[0015] Furthermore, the solution dosage form is a solution composed of water and the Gastrodia elata polysaccharide GEP-2, or a solution composed of physiological saline and the Gastrodia elata polysaccharide GEP-2.

[0016] Beneficial effects: The Gastrodia elata polysaccharide GEP-2 provided by the present invention can significantly alleviate the symptoms of colitis, such as weight loss and colon shortening. Histological examinations also show that Gastrodia elata polysaccharide GEP-2 can reduce inflammatory cell infiltration in colon tissue, alleviate tissue edema and ulcers. In addition, Gastrodia elata polysaccharide GEP-2 can also reduce the levels of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, etc. in colon tissue, while increasing the levels of anti-inflammatory cytokines such as IL-10, and can regulate the composition of the intestinal flora of mice and increase the relative abundance of probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electron microscopy characterization of Gastrodia elata polysaccharide component 2.

[0018] Figure 2 This is the nuclear magnetic resonance analysis diagram of Gastrodia elata polysaccharide component 2, where A is a one-dimensional proton nuclear magnetic resonance analysis diagram and B is a one-dimensional carbon nuclear magnetic resonance analysis diagram.

[0019] Figure 3 Figure 3 is a graph showing changes in mouse body weight.

[0020] Figure 4 Figure 2 is a graph of mouse colon length.

[0021] Figure 5 These are statistical graphs of mouse characterization data, where A is the statistical graph of mouse colon length and B is the statistical graph of DAI score.

[0022] Figure 6 This is a statistical diagram of mouse HE staining.

[0023] Figure 7 These are statistical graphs of mouse serum ELISA detection, where A is the statistical graph of IL-1β expression, B is the statistical graph of TNF-α expression, C is the statistical graph of IL-6 expression, D is the statistical graph of IL-10 expression, E is the statistical graph of D-lactic acid expression, and F is the statistical graph of LPS expression.

[0024] Figure 8 This is a Venn diagram of the mouse intestinal flora.

[0025] Figure 9 The α-diversity analysis diagram of the mouse intestinal flora, where A is the Chao1 index statistical diagram, B is the Shannon index statistical diagram, C is the ACE index statistical diagram, and D is the Simpson index statistical diagram.

[0026] Figure 10 This is a β-diversity analysis diagram of mouse intestinal flora.

[0027] Figure 11 This is a statistical diagram of the relative abundance of mouse intestinal flora at the phylum level.

[0028] Figure 12 These are statistical graphs of the relative abundance of Bacteroidetes, Firmicutes, and Proteobacteria in the mouse intestine, where A is the relative abundance statistical graph of Bacteroidetes, B is the relative abundance statistical graph of Firmicutes, and C is the relative abundance statistical graph of Proteobacteria.

[0029] Figure 13 This is a heat map of the genus level of mouse intestinal flora.

[0030] Figure 14 is the relative abundance statistical diagram of the main bacterial genus in the mouse intestine, where A is Faecalibaculum Relative abundance statistics, B is unclassified_ Clostridia _UCG_014 relative abundance statistics, C is ParasutterellaRelative abundance statistics, D is Alistipes Relative abundance statistics, E is Dubosiella Relative abundance statistics. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] Example 1 1. Drugs and reagents.

[0033] Gastrodia elata (black red Gastrodia elata from Yunyang County, Chongqing) was purchased from Yunhai Pharmaceutical and was found to be in compliance with the quality standards of the Chinese Pharmacopoeia.

[0034] Analytical-grade n-butanol, anhydrous ethanol, and chloroform were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd., analytical-grade n-hexane, Adamas reagent, and filter paper were purchased from Shanghai Leigu Instrument Co., Ltd., 0.22 μm and 0.45 μm microporous filter membranes were purchased from Tianjin Jinteng Experimental Equipment Co., Ltd., ultrafiltration membranes and ultrafiltration cups were purchased from Shanghai Mosu Scientific Instrument Co., Ltd., dialysis bags were purchased from Solebo, and a rotary evaporator was purchased from Shanghai Rongya Biochemical Instrument Factory.

[0035] Dextrose sulfate sodium salt (DSS) was purchased from MP Biomedicals, USA (lot number: 0216011080), paraformaldehyde fixative was purchased from Wuhan Seville Biotechnology Co., Ltd. (lot number G1101), hematoxylin-eosin (HE) staining kit was purchased from Wuhan Seville Company, enzyme-linked immunosorbent assay (ELISA) kits (interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α)) were purchased from Jiangsu Enzyme Immunity Company, and mesalazine (5-aminosalicylic acid, 5-AS) was purchased from Sigma, USA.

[0036] 2. Extraction and identification of Gastrodia elata polysaccharides.

[0037] 1. Extraction of Gastrodia elata crude polysaccharides (GEPs): (1) Raw material pretreatment: Gastrodia elata tubers were mechanically crushed to a particle size of <1 cm³, mixed with ultrapure water at a volume ratio of 1:20, and incubated at a constant temperature of 25°C for 2 h to allow the tissue to fully hydrate and expand.

[0038] (2) Hot water extraction: After soaking, transfer to an 80°C constant temperature water bath extraction system and stir continuously for 3 hours. After initial filtration through double-layer gauze, the residue is extracted three times using the same steps, and the three filtrates are combined to obtain a composite filtrate.

[0039] (3) Concentration and purification: The composite filtrate was filtered twice through a 300-mesh filter cloth and then concentrated to a slightly viscous state using a vacuum rotary evaporator at 50°C to obtain a concentrated solution.

[0040] (4) Gradient alcohol precipitation: After the concentrate was cooled to 25°C, a 95% ethanol solution was slowly added using a gradient method, with continuous stirring until the ethanol volume fraction reached 70%. After continuous stirring for 30 minutes, the solution was allowed to settle for 12 hours. A clear phase interface was formed between the crude polysaccharide clumps of Gastrodia elata and the Gastrodia elata colloid complex phase.

[0041] (5) Washing and purification: After removing the supernatant by siphoning, the precipitate was redispersed and washed three times with a 70% ethanol solution. Finally, the precipitate was stored in a fresh 70% ethanol solution at 4°C for later use.

[0042] (6) Secondary purification: The precipitate and ultrapure water were mixed and dissolved in a volume ratio of 1:15, and steps (4) and (5) were repeated for secondary alcohol precipitation to effectively remove small molecular interferences such as phenols and obtain crude Gastrodia elata polysaccharides (GEPs).

[0043] The product obtained through the above process is Gastrodia elata polysaccharide (GEPs). Because the polysaccharide structure contains reducing end groups, it should be sealed and stored at low temperature in a 70% ethanol solution, or freeze-dried to produce a lyophilized powder, which should be stored at low temperature and protected from light until used.

[0044] 2. Purification of Gastrodia elata crude polysaccharides (GEPs): (1) Degreasing: After removing residual ethanol from the crude polysaccharide of Gastrodia elata by high-speed centrifugation, the product was transferred to a 500 mL standard ground-mouth round-bottom flask. Petroleum ether was added at a solid-liquid volume ratio of 1:5, and a condenser reflux tube was connected for heating and reflux (60°C, 400 rpm, 3 h). Based on the principle of polarity difference, this process can effectively remove fat-soluble impurities without losing polysaccharides. After the reaction system was naturally cooled to 25°C, the solid and liquid phases were separated by filtration using a Buchner funnel. The solid phase was dried in a vacuum oven (40°C, 6 h) to remove the residual solvent and obtain a solid precipitate.

[0045] (2) Improved Sevage protein treatment: The solid phase precipitate was mixed and dissolved with water in a volume ratio of 1:100, and then transferred into a separatory funnel. A chloroform-n-butanol mixed solvent was added in an equal volume ratio of aqueous phase to organic phase (the volume ratio of chloroform to n-butanol in the mixture was 4:1). The mixture was thoroughly mixed by back and forth oscillation. After standing and stratification, the lower organic phase and the interfacial denatured protein flocs were separated. The operation was repeated until no flocs were observed. The aqueous phase was the purified product, the crude Gastrodia elata polysaccharide aqueous solution. The purified Gastrodia elata polysaccharide was obtained by freeze-drying and stored at low temperature for future use.

[0046] 3. Obtaining Gastrodia elata polysaccharide fragments: A 50 mg / mL solution of crude Gastrodia elata polysaccharide was prepared. The solution was then filtered through a 0.22 μm filter membrane to remove water-insoluble macromolecules. The solution was then separated and purified using ultrafiltration membranes with molecular cutoffs of 100 kDa and 3 kDa, respectively. After alcohol precipitation and drying, Gastrodia elata polysaccharide fraction 1 (GEP-1) with a molecular weight <3 kDa and Gastrodia elata polysaccharide fraction 2 (GEP-2) with a molecular weight between 3 and 100 kDa were obtained.

[0047] 3. Identification and detection of Gastrodia elata polysaccharide component 2 (GEP-2).

[0048] 1. Structural characterization test: The structure of Gastrodia elata polysaccharide component 2 (GEP-2) was characterized by scanning electron microscopy. The molecular weights of the polysaccharides in GEP-2 were analyzed using gel permeation chromatography (GPC) coupled with a multi-angle laser light scattering (MALLS) detector and a differential refractive index detector (dRI). GPC works by eluting molecules sequentially in a solvent based on molecular weight or size, with larger molecules eluting first and smaller molecules eluting later, thus achieving size separation. After separation, a differential detector was used to determine the sample's concentration based on its refractive intensity, while a MALLS detector was used to measure light scattering information for the larger molecules. The corresponding molecular weights were then fitted using the Mark-Houwink equation.

[0049] 2. Monosaccharide composition detection: GEP-2 was completely hydrolyzed using trifluoroacetic acid. Using ion chromatography with an electrochemical detector, the different monosaccharide components in the polysaccharide were qualitatively analyzed based on retention times. Subsequently, quantitative analysis was performed using a standard curve method using monosaccharide standards. The detection principle is as follows: Under specific chromatographic conditions, different monosaccharides exhibit different retention capacities on the analytical column, resulting in significant differences in elution times. By observing the chromatogram of the polysaccharide hydrolyzate on the analytical column and eliminating interference from impurities, the monosaccharides in the structure can be qualitatively identified based on the peak elution time of each monosaccharide.

[0050] 3. Bonding structure characterization: First, GEP-2 was treated with alkali and iodomethane to convert all non-glycosidically bound hydroxyl groups to methylated structures. Trifluoroacetic acid was then added for complete acid hydrolysis of the polyether compound. After all glycosidic bonds between monosaccharides were hydrolyzed to free monosaccharides, acetic anhydride was added for acetylation, converting the free hydroxyl groups to acetylated structures. The products were then analyzed by GC-MS. Due to their different chromatographic migration rates, the different products exhibited distinct chromatographic peaks at different retention times, as shown in the total ion current chromatogram. In electron impact ionization (EI) mass spectrometry, different monosaccharides exhibited distinct fragmentation peaks depending on their methylated and acetylated structures. Polysaccharides are composed of monosaccharide units linked by glycosidic bonds, with multiple hydroxyl groups attached to the sugar rings. Polysaccharide methylation analysis is a key method for exploring polysaccharide structure. Its core objective is to assess the extent of hydroxyl methylation on the sugar chain to infer the linkage pattern and location within the polysaccharide. The methylation process converts free hydroxyl groups not involved in glycosidic bond formation into methoxy groups (-OCH3). The methylated polysaccharide then undergoes hydrolysis, reduction, and acetylation to produce a series of partially methylated sugar alcohol acetate derivatives. Analysis of these derivatives using gas chromatography-mass spectrometry (GC-MS) can elucidate the linkage patterns and structural characteristics of each monosaccharide.

[0051] 4. NMR characterization and structural analysis: Nuclear Magnetic Resonance (NMR) analysis of polysaccharides involves recording the chemical shifts of protons and carbon atoms in a high-frequency magnetic field to determine information such as the polysaccharide's anomeric configuration, glycosidic bond pattern, and linkage sequence. Specifically, one-dimensional proton NMR (1H NMR) and one-dimensional carbon NMR (13C NMR) can be used to determine the chemical shifts of carbon and hydrogen atoms within sugar residues. Furthermore, due to the significant signal overlap between identical atoms, two-dimensional NMR techniques are employed to analyze the molecular structure of polysaccharides. These techniques include correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond coherence (HMBC).

[0052] 4. Verification of the efficacy of Gastrodia elata polysaccharide component 2 (GEP-2).

[0053] 1. Animal Experiment Design Forty SPF C57BL / 6J male mice (7 weeks old, weighing 18-22 g) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Changsha, China, Laboratory Animal Production License No. SCXK (Xiang) 2025-0004). All mice were housed in the mouse breeding room of the Laboratory Animal Research Institute of Chongqing Academy of Traditional Chinese Medicine with free access to water and a basic diet. The environmental conditions were 22°C, 65% relative humidity, and a 12 / 12 h light / dark cycle. The animal care and use procedures were approved by the Laboratory Animal Ethics Committee of Chongqing Academy of Traditional Chinese Medicine (Approval No. 2024-ZJS01). The experimental design involved randomly assigning mice to five groups (n = 8). After a 7-day acclimatization period, the mice were administered drugs for 7 days. Mice in the blank control group (Control), model group (Model), and mesalazine-positive drug group (MS) were fed a standard diet. Mice in the blank control group drank distilled water, while mice in the other experimental groups drank freely a 3% DSS solution (refreshed daily). Furthermore, mice in the positive drug group received daily oral administration of 100 mg / kg mesalazine, mice in the GEP-1 group received daily oral administration of 100 mg / kg GEP-1, and mice in the GEP-2 group received daily oral administration of 100 mg / kg GEP-2. The weight and general condition of the mice were observed daily. Body weight, diarrhea, and fecal bleeding were recorded daily. On day 8, blood was collected from the orbital vein after anesthesia. The mice were euthanized, and colon tissue was dissected and isolated, and colon length was measured.

[0054] 2. Disease Activity Index Score: The disease activity index (DAI) criteria used to evaluate DSS colitis are shown in Table 1 . The scores were scored based on the percentage of body weight loss, stool characteristics, and bleeding. These scores were added and divided by three to obtain a DAI score between 0 and 4.

[0055] Table 1: DAI scoring criteria 3. ELISA test: Blood was collected from the eye socket and allowed to stand at room temperature for 1 hour before centrifugation at 1500g for 10 minutes at 4°C. The supernatant was separated as serum and stored in an ultra-low temperature freezer until needed. According to the reagent manufacturer's instructions, 10 μL of serum from each group of mice was diluted and added to the reaction wells of a 96-well plate. Subsequently, 50 μL each of the standard and biotin-labeled antibody were added and incubated at 37°C for 1 hour. After washing three times, avidin-horseradish peroxidase conjugate was added and incubated again for 30 minutes, with the same washing steps as before. Finally, 50 μL each of substrates A and B were added and incubated for 10 minutes, protected from light. Stop solution was quickly added, and absorbance was measured using a multi-function microplate reader. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, LPS, and D-lactic acid were measured according to the kit instructions.

[0056] 4. Histopathological analysis: Mice were euthanized under anesthesia, and the cecum to rectum was rapidly removed. The length from the cecum to the terminal rectum was measured and photographed. Colonic tissue was then segmented. One portion was snap-frozen in liquid nitrogen and stored at −80°C for subsequent analysis. The remaining portion was fixed in 4% formaldehyde. Colonic tissue was fixed overnight in 4% paraformaldehyde, dehydrated with graded ethanol, embedded in paraffin, sectioned (5 μm thick), and dried. The tissue was then dewaxed in xylene, rehydrated with anhydrous ethanol and graded ethanol, stained with hematoxylin and eosin, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral gum. Pathological changes in colonic tissue were analyzed under a light microscope in different fields of view within each section.

[0057] 5. 16S rRNA sequencing: Colonic contents were collected and stored in liquid nitrogen until further use. Fecal genomic DNA was extracted using a magnetic bead-based bacterial / fungal DNA extraction kit. The extracted DNA was then synthesized into cDNA and amplified using paired-end sequencing on the Illumina NovaSeq platform for 16S rRNA sequencing. The raw data were processed for denoising and chimera filtering, and the resulting data tables were used to analyze the bacterial taxa present in each fecal sample. The 16S rRNA gene sequencing data were analyzed using the R packages hyloseq and DESeq2. Linear discriminant analysis (LEfSe) was used to identify bacterial taxa with the largest effect sizes and differential abundances at the genus level.

[0058] 6. Data Analysis GraphPad Prism 9.0 software (GraphPad Software, USA) was used for statistical analysis and statistical graphs. The experimental data were expressed as mean ± standard deviation (SD). One-way ANOVA and t-test were used for statistical analysis. The differences were considered statistically significant when P < 0.05.

[0059] 5. Experimental results.

[0060] 1. Structural characteristics of GEP-2: The SEM examination results of GEP-2 structure are as follows Figure 1 As shown, GEP-2 possesses a unique three-dimensional porous structure with pore sizes ranging from 5 to 20 μm. Fitting results indicate that GEP-2 has a number-average molecular weight of 2.54 kDa, a weight-average molecular weight of 9.24 kDa, a z-average molecular weight of 37.92 kDa, and a polydispersity index of 3.64. After normalization of the monosaccharide composition, as shown in Table 2, glucose (Glc) is the most abundant monosaccharide in Gastrodia elata polysaccharide fraction 2, accounting for approximately 89.08%. Galactose (Gal) and galacturonic acid (Gal-UA) contribute to 2.4% and 7.12%, respectively. Furthermore, small amounts of arabinose (Ara) and rhamnose (Rha) are present, accounting for approximately 0.86% and 0.54%, respectively. These five monosaccharides collectively constitute the composition of Gastrodia elata polysaccharide GEP-2.

[0061] Table 2: Monosaccharide composition analysis Based on the structural characterization of the bonds, rhamnose and galactose each exhibited a single linkage type, 2-Rha(p) and 3-Gal(p). Analysis was difficult due to the low content of arabinose. Galacturonic acid exhibited two binding modes: 4-Gal(p)-UA and 3,4-Gal(p)-UA, while glucose exhibited six linkage types, as shown in Table 3.

[0062] Table 3: Monosaccharide bond analysis results like Figure 2 As shown in the results in Table 4, NMR characterization and structural analysis combined the NMR results and analyzed the main structure of the polysaccharide as follows: GEP-2 is a branched polysaccharide fragment with →4)-α-D-glucose (Glc) as the main chain and galacturonic acid (Gal-UA), galactose (Gal), rhamnose (Rha) and arabinose (Ara) substituents at positions 4 and 6.

[0063] Table 4: NMR: Bonding analysis results 2. Comparison of DAI score, body weight and colon length of mice: Weight loss and changes in colon length are important indicators for assessing the severity of UC. This study found that the DSS-induced mouse model showed significant weight loss and shortened colon length. The mice in the blank group had shiny hair, normal diet and behavior, and their body weight gradually increased slightly; the mice in the model group showed mental depression, obvious diarrhea, blood in the stool, and weight loss. Figure 4 As shown in Figure 2, compared with the model group, the body weight of mice in the GEP-2 group was significantly increased. Figure 4 and Figure 5 As shown, analysis of the colon length and DAI scores of mice revealed that compared with the blank group, the DAI scores of mice in the model group were increased (P < 0.05), and the colons were significantly shortened (P < 0.001). This decreased DAI score indicates that the model was successfully established. Compared with the model group, the colons of mice in the mesalazine-positive group were enlarged, indicating that mesalazine can effectively slow the shortening of the colon in UC mice, a result that was consistent with expectations. Furthermore, the colons of mice in the GEP-2 group were significantly enlarged, especially on day 6 of modeling. Significant differences in body weight and DAI scores were observed, indicating that GEP-2 can effectively alleviate the symptoms of DSS-induced UC in mice, alleviating weight loss in mice with ulcerative colitis and alleviating the degree of colon shortening in mice with colitis.

[0064] 3. Histopathological status of mouse colon: like Figure 6 As shown, HE staining results further revealed that the colonic morphology and structure of the control mice were normal, with no pathological damage. The mucosal epithelium was intact, the intestinal glands were abundant, and the goblet cells were numerous. The cells were neatly arranged, and the crypt structure was clear. Compared with the blank group, the colonic tissue structure of the model and GEP-1 groups was severely damaged, with severe destruction of the crypt structure, accompanied by infiltration of inflammatory cells and lymphocytes, a significant decrease in goblet cells, and lesions involving the submucosa. Compared with the model group, the pathological damage of the colonic tissue of the mice in the mesalazine and GEP-2 groups was significantly improved. GEP-2 can effectively alleviate intestinal barrier damage in mice, reduce ulcer hyperplasia and inflammatory infiltration, partially preserve the crypt structure and intestinal gland structure, increase the number of goblet cells, and alleviate colonic tissue inflammation to varying degrees, indicating that GEP-2 can improve the pathological damage of the colon in UC mice to a certain extent.

[0065] 4. Changes in inflammatory-related cytokines in mouse serum: Inflammation plays an important role in the development of UC. There are a large number of pro-inflammatory cytokines in the intestines of UC patients, such as TNF-α, IL-1, IL-6, etc. These cytokines promote the continuation and amplification of inflammatory responses by activating signaling pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB. This study used ELISA to detect the levels of TNF-α, IL-1β, IL-6, and IL-10 in the small intestinal tissues of mice. Figure 7 As shown, the results showed that compared with the control group, the serum concentrations of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the model group mice were significantly increased, while the anti-inflammatory cytokine IL-10 was significantly decreased. Compared with the model group, the serum concentrations of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the mesalazine and GEP-2 groups were decreased. In addition, the expression of the anti-inflammatory cytokine IL-10 in the serum of the GEP-2 group mice was increased, suggesting that GEP-2 can effectively reduce the levels of inflammatory factors in UC mice and alleviate the occurrence of inflammation. In addition, when intestinal barrier function is impaired, LPS from the intestine can enter the blood through the damaged intestinal mucosa, which can mediate intestinal mucosal inflammation and aggravate intestinal barrier dysfunction. The results showed that the serum LPS and D-lactose levels in the GEP-2 group mice were significantly lower than those in the model group, indicating that GEP-2 can effectively reduce the level of inflammation in mice and improve intestinal barrier permeability.

[0066] 5. Changes in intestinal flora of mice: The intestinal microbiota is crucial for intestinal health, and its imbalance is closely associated with inflammatory bowel disease (IBD). In this study, 16S rRNA gene sequencing was used to investigate the effects of GEP-2 on the intestinal microbiota in mice with DSS-induced colitis. The composition, α-diversity, β-diversity, and differential bacterial communities were assessed to determine how GEP-2 may help restore microbial balance and alleviate UC. Figure 8 The results showed that there were 1044 unique OTUs in the control group, 772 unique OTUs in the model group, 637 unique OTUs in the mesalazine group, and 3127 unique OTUs in the GEP-2 group, indicating that there were certain structural differences in the intestinal flora of mice in each group. α-diversity is an indicator of species richness and diversity within a specific region or ecosystem, such as Figure 9 As shown in Figure 2, this study used Chao1, Shannon, ACE, and Simpson indices to analyze the α-diversity of the intestinal flora of mice. The results showed that the species richness and diversity of the GEP-2 group were significantly different from those of the control group, model group, and mesalazine group. The β-diversity analysis of the intestinal microorganisms in the colon contents of mice in each group was performed. Figure 10As shown in the figure, the control group, model group, mesalazine-positive drug group, and GEP-2 group were clearly separated, indicating that there were significant differences in the microbial community structure between different groups. The model group and mesalazine-positive drug group had a small overlap, and most areas were separated, while the GEP-2 group was clearly separated, indicating that GEP-2 had a significant effect on the intestinal flora of UC mice.

[0067] like Figure 11 As shown in the figure, the results of comparing the phylum level of the intestinal flora of mice in different groups show that GEP-2 has the effect of regulating the composition of the mouse intestinal flora. At the phylum level, Firmicutes, Bacteroidetes and Proteobacteria are the dominant bacteria in the intestinal microbiota of mice in all experimental groups, such as Figure 12 As shown in Figure 3, GEP-2 can specifically reduce the number of Bacteroidetes and increase the number of Firmicutes and Proteobacteria. Figure 13 As shown, the results showed that GEP-2 can significantly improve Faecalibaculum 、unclassified_ Clostridia _UCG_014, Parasutterella 、 Alistipes 、 Dubosiella Such as bacterial genus categories. Figure 14 As shown, it is noteworthy that GEP-2 can significantly improve Dubosiella Studies have found a new mouse symbiotic bacterium in this genus. Dubosiella newyorkensis , which has a probiotic immunomodulatory effect on DSS-induced colitis. In addition, Clostridium spp. ( Clostridia _UCG-014) can withstand gastric acid entering the intestine, promote the growth of beneficial bacteria, inhibit harmful growth in the intestine, and restore intestinal flora.

[0068] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0069] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0070] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Gastrodia elata polysaccharide GEP-2, characterized in that: Derived from Gastrodia elata, Gastrodia elata polysaccharide GEP-2 has a number average molecular weight of 2.54 kDa, a weight average molecular weight of 9.24 kDa, a z-average molecular weight of 37.92 kDa, and a polydispersity index of 3.

64. In the Gastrodia elata polysaccharide GEP-2, the proportion of each monosaccharide is as follows, calculated by mass percentage: 89.08% glucose, 2.40% galactose, 7.12% galacturonic acid, 0.86% arabinose and 0.54% rhamnose.

2. Use of the Gastrodia elata polysaccharide GEP-2 according to claim 1 in the preparation of a drug for treating ulcerative colitis.

3. The use of Gastrodia elata polysaccharide GEP-2 in the preparation of a drug for treating ulcerative colitis according to claim 1, characterized in that: In the medicine, Gastrodia elata polysaccharide GEP-2 is the only active ingredient.

4. The use of Gastrodia elata polysaccharide GEP-2 in the preparation of a drug for treating ulcerative colitis according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The use of Gastrodia elata polysaccharide GEP-2 in the preparation of a drug for treating ulcerative colitis according to claim 4, characterized in that: The auxiliary materials include any one or more of fillers, stabilizers, diluents, and adjuvants.

6. The use of Gastrodia elata polysaccharide GEP-2 in the preparation of a drug for treating ulcerative colitis according to claim 5, characterized in that: The diluent is any one of water and physiological saline.

7. A drug for treating ulcerative colitis, characterized in that: The medicine is prepared by mixing the Gastrodia elata polysaccharide GEP-2 described in claim 1 and excipients, and the content of Gastrodia elata polysaccharide GEP-2 in the medicine is 0.1 wt% to 99 wt%.

8. The drug according to claim 7, characterized in that The dosage form of the drug is a solid dosage form or a solution dosage form. The solid dosage form includes granules, tablets, capsules, pills, and dripping pills. The solution dosage form includes oral liquid preparations, gavages, and injection dosage forms.

9. The drug according to claim 8, characterized in that The solution dosage form is a solution consisting of water and the Gastrodia elata polysaccharide GEP-2, or a solution consisting of physiological saline and the Gastrodia elata polysaccharide GEP-2.

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