Polysaccharide from bletilla striata and preparation method and application thereof

Abstract

This invention provides *Centella asiatica* polysaccharide, its preparation method, and its applications. Compared with existing technologies, this invention uses hot water extraction to efficiently extract a neutral polysaccharide TP1 from *Centella asiatica*, mainly composed of fructose, glucose, and galactose in a molar ratio of 44.7:33.8:17.9. The molecular weight of TP1, Mw = 5377 Da, was determined by GPC-RI-MALS chromatography. This invention also investigates the protective effect of *Centella asiatica* polysaccharide against DSS-induced colitis. TP1 reduces the loss of intestinal tight junction proteins (Claudin-1, Occludin, and ZO-1), regulates the abundance of intestinal flora, and enriches flora that can produce SCFAs (Ruminococcus, Lactobacillus, and Bifidobacterium). In addition, TP1 increases the expression of GPR41 and GPR43 receptors in colonic tissue, thereby alleviating colitis symptoms by reducing oxidative stress and inflammatory cytokine levels through SCFA-mediated GPR41 and GPR43 receptor reduction. The polysaccharide TP1 prepared by this invention can be used as a drug for the prevention and treatment of colitis, and can also be used to prepare food.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharides and medicine, specifically relating to Centella asiatica polysaccharide, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic, complex inflammatory disease characterized by gastrointestinal lesions, diffuse mucosal inflammation, rectal bleeding, diarrhea, and abdominal pain, severely impacting patients' quality of life. Currently, the exact etiology and pathogenesis of UC are not fully understood, but increasing research suggests that UC may be related to genetic variations, gut microbiota dysbiosis, immune dysfunction, diet, and environmental factors. In the past decade, the incidence of UC has increased rapidly, and if left untreated, it may develop into colorectal cancer.

[0003] Currently used drugs for treating UC include aminosalicylic acid, corticosteroids, immunomodulators, and antibiotics. However, these drugs usually have problems such as long treatment cycles and many side effects. For example, corticosteroids can cause allergic reactions such as fever, nausea, and rash.

[0004] As a dietary resource, bioactive polysaccharides derived from natural products have garnered widespread attention worldwide due to their good tolerability and various pharmacological activities, including anti-inflammatory, antibacterial, and antitumor effects. Increasing research indicates that polysaccharides possess significant potential for the prevention and treatment of colitis (UC). For example, arrowhead polysaccharides alleviate colitis symptoms by repairing the intestinal barrier, promoting microbial metabolism, and regulating the gut microbiota-MAPK / NF-κB signaling pathway. Okra polysaccharides alleviate intestinal inflammation by regulating the abundance of gut microbiota and enhancing the intestinal mucus barrier. Notably, some recalcitrant polysaccharides can undergo fermentation under the metabolic action of the colonic microbiota, exerting anti-inflammatory, intestinal mucosal protective, and intestinal immune-regulating effects by influencing gut microbiota diversity and its metabolites. Currently, natural polysaccharides are widely used in the health food and pharmaceutical industries, making the development of bioactive polysaccharides from natural plants and the exploration of their beneficial properties of bioactive polysaccharides of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a polysaccharide from *Centella asiatica* and its preparation method. A neutral polysaccharide, TP1, is efficiently extracted from *Centella asiatica* using hot water extraction. FT-IR and 1D / 2D-NMR analyses confirmed that TP1 is mainly composed of fructose, glucose, and galactose in a molar ratio of 44.7:33.8:17.9. The molecular weight of TP1, Mw = 5377 Da, was determined by GPC-RI-MALS chromatography.

[0006] Another objective of this invention is to provide applications of *Lysimachia christinae* polysaccharide. The *Lysimachia christinae* polysaccharide TP1 prepared by this invention can be used as a drug for the prevention and treatment of colitis. Its mechanism of action is as follows: TP1 reduces the loss of intestinal tight junction proteins (Claudin-1, Occludin, and ZO-1), regulates the abundance of intestinal flora, and enriches flora that can produce SCFAs (Ruminococcus, Lactobacillus, and Prevotella). In addition, TP1 increases the expression of GPR41 and GPR43 receptors in colonic tissue, and reduces the levels of oxidative stress and inflammatory cytokines through SCFAs-mediated GPR41 and GPR43 receptors.

[0007] The specific technical solution of this invention is as follows:

[0008] The present invention provides a polysaccharide of Centella asiatica, named TP1, which is mainly composed of fructose, glucose and galactose in a molar ratio of fructose: glucose: galactose = 44.7 : 33.8 : 17.9. Its peak molecular weight (Mp) is 4589 Da, weight-average molecular weight (Mw) is 5377 Da and number-average molar mass (Mn) is 4797 Da.

[0009] The present invention provides a method for preparing polysaccharides from Centella asiatica, comprising the following steps:

[0010] 1) Fresh centrifuge is crushed, extracted by hot water distillation, concentrated, centrifuged to remove precipitate, and then precipitated with anhydrous ethanol and the precipitate is collected.

[0011] 2) The precipitate was dissolved in distilled water, decolorized, and the protein was removed by Sevage reagent. After dialysis, crude polysaccharide was obtained.

[0012] 3) After dissolving and filtering the crude polysaccharide, it was separated and purified by elution with distilled water, and the eluent was freeze-dried.

[0013] 4) The product solution from step 3) is separated on a gel column with distilled water, and the resulting eluent is freeze-dried to obtain the final product.

[0014] The hot water distillation extraction described in step 1) specifically involves: extracting twice with distilled water at 80-90℃, with a material-to-liquid ratio of 1:20, g / mL; each extraction lasts for 2 hours, and the extracts are then combined.

[0015] The concentration mentioned in step 1) refers to rotary evaporation at a low temperature of 60°C;

[0016] The centrifugation to remove precipitate mentioned in step 1) refers to centrifugation at 5000 r / min for 8 min to remove precipitate;

[0017] The precipitation with anhydrous ethanol mentioned in step 1) refers to: precipitating with 4 times the volume of anhydrous ethanol for 12 hours at 4℃, centrifuging for 10 minutes, and collecting the precipitate.

[0018] In step 2), the protein is removed by decolorizing with a D101 macroporous adsorption resin column and then by using Sevage reagent (volume ratio of n-butanol: chloroform = 1:3).

[0019] In step 2), the dialysis is performed using a 1000 kDa dialysis bag in deionized water for 48 hours, with the water changed every 4 hours, and the crude polysaccharide (TPs) is obtained by lyophilization after concentration.

[0020] In step 3), the crude polysaccharide TPs was prepared into a 20 mg / mL solution, filtered using a 0.22 μm filter membrane, and then packed into a DEAE-52 (2.5 × 40 cm) column for separation and purification.

[0021] In step 4), separating the product solution from step 3) on a gel column with distilled water refers to separating it on a Sephadex G-100 (1.6 × 60 cm) column with distilled water at a flow rate of 0.5 mL / min. The flow rates are further separated.

[0022] This invention, through the above-described preparation method, involves hot water distillation and ethanol precipitation, followed by decolorization, protein removal, dialysis to remove small molecules, and then removal of charged substances and heterogeneous polysaccharides. This process effectively removes impurities and improves polysaccharide purity. Furthermore, freeze-drying and neutral conditions (elution with distilled water) maintain the native conformation of the polysaccharides. This invention comprehensively utilizes multiple mechanisms, including physical retention, chemical precipitation, and charge adsorption, to form a gradient purification system, ultimately achieving highly efficient separation and purification of polysaccharides. Each step independently removes specific impurities while simultaneously constructing a cross-validated purification network, ultimately yielding polysaccharides primarily composed of fructose, glucose, and galactose.

[0023] This invention provides the application of adding *Centella asiatica* polysaccharide for the prevention and treatment of colitis.

[0024] Thesium chinense Turcz, a perennial parasitic herb belonging to the Santalaceae family, is first recorded in the "Illustrated Materia Medica." It is also known as Bairu Cao, Di Shiliu, Xiaocao, Fengyahao, and Xixucao, and is widely distributed throughout northern and southern my country. In Traditional Chinese Medicine (TCM), it is referred to as a "plant antibiotic" and is used to treat pneumonia, acute tonsillitis, acute mastitis, pharyngitis, bronchitis, and other ailments. It also exhibits good inhibitory effects against various pathogenic bacteria. These properties give it high research value and application potential in the field of TCM. However, current research on the polysaccharide components of the herb is limited both domestically and internationally.

[0025] This invention uses the whole plant of *Hedyotis diffusa* as raw material to isolate and purify a water-soluble polysaccharide, and characterizes its structure using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, methylation analysis, and monosaccharide composition analysis. Furthermore, a UC mouse model was induced using DSS to evaluate its beneficial effects on DSS-induced colitis mice, laying the foundation for in-depth research on *Hedyotis diffusa* polysaccharides and accumulating data for the development of related polysaccharide-based new drugs or health products.

[0026] Compared with existing technologies, this invention uses hot water extraction to efficiently extract a neutral polysaccharide TP1 from *Hedyotis diffusa*. FT-IR and 1D / 2D-NMR analyses confirmed that TP1 is mainly composed of fructose, glucose, and galactose in a molar ratio of 44.7:33.8:17.9. The molecular weight of TP1, Mw = 5377 Da, was determined by GPC-RI-MALS chromatography. This invention also investigated the protective effect of *Hedyotis diffusa* polysaccharide against DSS-induced colitis. TP1 reduces the loss of intestinal tight junction proteins (Claudin-1, Occludin, and ZO-1), regulates the abundance of intestinal flora, and enriches bacteria that can produce SCFAs (Ruminococcus, Lactobacillus, and Prevotella). In addition, TP1 increases the expression of GPR41 and GPR43 receptors in colonic tissue, and reduces oxidative stress and inflammatory cytokine levels through SCFA-mediated GPR41 and GPR43 receptor expression. The polysaccharide TP1 prepared by this invention can be used as a drug for the prevention and treatment of colitis. Attached Figure Description

[0027] Figure 1 The images show the detection results at different stages of polysaccharide preparation; (A) elution curve of DEAE-52 cellulose column, (B) elution curve of Sephadex G-100 gel chromatography column, (C) UV spectrum, (D) monosaccharide composition analysis, a: standard monosaccharide, b: TP1 hydrolysis at 60℃, c: TP1 hydrolysis at 121℃, (E) infrared spectrum of TP1, (F) molecular weight distribution map of TP1, and (G) HPGPC spectrum of TP1.

[0028] Figure 2A NMR of TP1 1 H spectrum;

[0029] Figure 2B NMR of TP1 13 C spectrum;

[0030] Figure 2C The DEPT-135 spectrum of TP1;

[0031] Figure 2D The COSY spectrum of TP1;

[0032] Figure 2E (The HSQC spectrum of TP1)

[0033] Figure 2F The HMBC spectrum of TP1;

[0034] Figure 3 This is the main repeating sugar chain structure of TP1;

[0035] Figure 4 To record various indicators of mice after DSS administration, (A) Dosing regimen diagram; (B) Rectal hemorrhage; (C) Weight change; (D) DAI score; (E) Colon length; (F) Spleen status;

[0036] Figure 5 The results are for colon tissue staining and inflammatory factor detection; (A) HE staining of colon tissue, (B) AB-PAS staining of colon tissue, (C) IL-6, TNF-α, IL-17, IL-1β, (D) CAT, MDA, SOD, MPO;

[0037] Figure 6 For immunofluorescence chemical analysis and Western blot analysis, (A) immunofluorescence chemical analysis of Occludin, Claudin-1, and ZO-1, (B) Western blot analysis of Occludin, Claudin-1, and ZO-1;

[0038] Figure 7 The diagram shows the composition of the microbial community, including (A) abundance ranking curve, (B) Veen plot, (C) NMDS, (D) PCoA, (E) Chao1, Shannon, Simpson and observed_species indices, (F) F / B ratio, (G) Desulfovibrio, (H) Actinomyces, (I) Helicobacter, (J) Ruminococcus, (K) Prevotella, and (L) Lactobacillus.

[0039] Figure 8A This is a horizontal heatmap analysis;

[0040] Figure 8B For LDA histogram;

[0041] Figure 8C This is a phylogenetic branch diagram of LEfSe from phylum to genus;

[0042] Figure 8D Contents of acetic acid, propionic acid, and butyric acid;

[0043] Figure 8E Analysis of GPR41 and GPR43 mRNA expression. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0046] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0047] Materials used in the embodiments of this invention:

[0048] Sodium dextran sulfate (DSS, molecular weight: 36-50 kDa) was provided by MP Biomedical (Santa Ana, CA, USA). D101 macroporous adsorption resin, DEAE-52 cellulose, and dextran gel G-100 resin were purchased from Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and myeloperoxidase (MPO) kits were provided by Nanjing Jiancheng Biotechnology Institute (Nanjing, China). IL-6, TNF-α, IL-17, and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Jiangsu Jingmei Biotechnology Co., Ltd. Antibodies Claudin-1, Occludin, ZO-1, and β-Actin were purchased from Affinity Biosciences (Changzhou, China). 1000 kDa dialysis bags were purchased from Shanghai Yuanye Biotechnology Co., Ltd., China; the Chinese herbal medicine Bai Rui Cao was purchased from Anhui Tuoyuantang Pharmaceutical Co., Ltd., and was identified by Professor Liu Chunyan (Wannan Medical College, Wuhu City). Unless otherwise specified, all other reagents were of analytical grade.

[0049] Example 1

[0050] A method for preparing polysaccharides from Centella asiatica, specifically as follows:

[0051] 1) Fresh *Centella asiatica* was washed, dried, and pulverized. 200g of the powder was extracted twice with distilled water at 80℃ (solid-to-liquid ratio = 1:20, g / mL), 2 hours each time. The extracts were combined and concentrated by rotary evaporation at 60℃ to 1 / 10 of the original volume. The precipitate was removed by centrifugation at 5000 rpm for 8 minutes. The precipitate was then precipitated with 4 times its volume of anhydrous ethanol at 4℃ for 12 hours, centrifuged for 10 minutes, and the precipitate was collected. The precipitate was fully dissolved in 1000mL of distilled water at 60℃, centrifuged again, and the supernatant was decolorized using a D101 macroporous adsorption resin column. Proteins were removed using Sevage reagent (n-butanol: chloroform = 1:3 v / v). Subsequently, the mixture was dialyzed in deionized water using a 1000 kDa dialysis bag for 48 hours, with water changes every 4 hours. The crude polysaccharide (TPs) was obtained by freeze-drying at -80℃. 200 mg TPs were dissolved in 10 mL of distilled water, filtered through a 0.22 μm filter membrane, and purified using a DEAE-52 (2.5 × 40 cm) column. Elution was performed stepwise with distilled water, 0.1, 0.3, and 0.5 mol / L NaCl. 50 tubes (4 mL / tube) of eluent were collected from each gradient, and the total sugar content was determined using the anthrone-sulfuric acid method. The collected distilled water eluent was freeze-dried at -80 °C. The freeze-dried product was prepared into a 6 mg / mL aqueous solution and packed into a Sephadex G-100 (1.6 × 60 cm) column, 5 mL at a time. Further separation was performed with distilled water at a flow rate of 0.5 mL / min. The resulting eluent was freeze-dried, and the purified polysaccharide was collected and designated TP1.

[0052] Centella asiatica was extracted with hot water and precipitated with ethanol. After decolorization using a D101 macroporous adsorption resin column, treatment with Sevage reagent, dialysis, and freeze-drying, total phosphoric acid (TPs) were obtained. The calculated TPs yield was 0.79%. The TPs were then separated using a DEAE-52 cellulose column to obtain water-eluted fractions and 0.1M and 0.3M NaCl-eluted fractions. Figure 1 The samples (A) were named TP1, TP2, and TP3, respectively. TP1 was collected, lyophilized, and then purified using a Sephadex G-100 gel chromatography column. Figure 1 (B) was eluted with deionized water and then freeze-dried to obtain purified TP1 fraction. Furthermore, the HPGPC spectrum showed that TP1 was a single, symmetrical peak, indicating a purity of 95.25% (B). Figure 1 (G). The total sugar content in TP1 was determined to be 94.72% using the anthrone sulfuric acid method.

[0053] The structure of TP1 prepared in Example 1 is characterized as follows:

[0054] 1. UV and FT-IR analysis

[0055] 1 mg of TP1 was dissolved in 1 mL of distilled water and scanned from 200 nm to 400 nm using a NanoDrop one Ultra-micro spectrophotometer (Thermo, USA). The functional groups of TP1 were analyzed using a Nicolet iZ-10 Fourier transform infrared spectrometer with a resolution of 4.00 cm⁻¹. -1 TP1 (2 mg) and KBr (200 mg) were mixed and compressed into 1 mm tablets, then heated at 4000-400 cm⁻¹. -1 Scan within the range.

[0056] UV-Vis spectrum of TP1 ( Figure 1 (C) No obvious absorption peaks were observed at 260 nm and 280 nm, indicating that TP1 does not contain nucleic acids and proteins.

[0057] Fourier transform infrared spectroscopy is widely used for the qualitative analysis of functional groups in polysaccharides. For example... Figure 1 As shown in E, 3276cm -1 and 2929cm -1 The absorption peaks were assigned to the stretching vibrations of OH and CH, respectively, and these two infrared absorption peaks (3276 and 2929 cm⁻¹) were respectively. −1 The peak at 1646 cm⁻¹ is a characteristic peak of polysaccharides. −1 The infrared absorption peak at 1430~1328 cm⁻¹ is caused by the stretching vibration of C=O; −1 These correspond to the HOH bond deformation and -CH(O-CH2) bending vibrations, respectively. 1020 cm -1 The absorption peak at 934 cm⁻¹ is due to the stretching vibration of the COC glycosidic bond. −1 and 818cm −1 The absorption peaks at these locations correspond to the symmetric stretching vibration of the furan ring and the deformation vibration of the carbon-hydrogen bonds in the furan ring, respectively.

[0058] 2. Monosaccharide composition analysis

[0059] Weigh 5 mg of TP1 sample and add 1 ml of 2 M trifluoroacetic acid (TFA) solution. Hydrolyze at 121 °C for 2 h. Dry under nitrogen. Wash 2-3 times with methanol and dry under nitrogen. Dissolve in sterile water and transfer to a chromatographic vial for analysis. Alternatively, hydrolyze at 60 °C for 1 h. Use a Thermo ICS 5000+ ion chromatography system (ICS 5000). +Thermo Fisher Scientific (TP1) was used, equipped with a Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm) HPLC column. TP1 monosaccharide components were analyzed using an electrochemical detector, with an injection volume of 5 μl. Mobile phase A consisted of H₂O (water), mobile phase B consisted of 0.1 M NaOH solution, and mobile phase C consisted of 0.1 M NaOH solution and 0.2 M NaAc. The flow rate was 0.5 mL / min, and the column temperature was 30 °C.

[0060] Using monosaccharides as the standard, by comparing standard monosaccharides ( Figure 1 (a line of D in the middle), hydrolysis at 60℃ ( Figure 1 (the b line of D in the middle) and hydrolysis at 121℃ ( Figure 1 The GC-MS ion chromatogram of the c-line of D is shown. The results show that TP1 is mainly composed of fructose, glucose and galactose, with a molar ratio of 44.7:33.8:17.9. Compared with the disclosed composition of *Cynanchum paniculatum* polysaccharide, the *Cynanchum paniculatum* polysaccharide (TP1) obtained by the method of this invention has a higher fructose content.

[0061] 3. Determination of molecular weight

[0062] Preparation of standard solution: Accurately weigh 5 mg of each PEG standard, dissolve it in 0.5 ml of 0.1 M NaNO3 mobile phase solution to prepare a 10 mg / ml solution, and transfer it to a 1.8 ml vial.

[0063] Sample solution preparation: Accurately weigh 10 mg of sample and dissolve it in 1 ml of 0.1 M NaNO3 mobile phase solution to prepare a 10 mg / ml solution. Vortex until the sample is dissolved, and centrifuge at 12000 rpm for 10 min. Pipette the supernatant, filter it through a 0.22 μm aqueous microporous membrane, and then transfer it to a 1.8 ml vial.

[0064] Chromatographic method: Mobile phase: 0.1M sodium nitrate aqueous solution; Column: Shodex SB 805-803 tandem gel column (8×300mm); Flow rate: 1ml / min; Column temperature: 40ºC; Injection volume: 50μL; Detector: Optilab T-rEX+ DawnHELEOSⅡ; Analysis time: 25min.

[0065] The standard was placed in the injection tray and analyzed using the GPC-RI-MALS chromatographic method. After normalization by PEG21600 and 22000, the collected data were analyzed using Astra 6.1 software.

[0066] The present invention uses GPC-RI-MALS chromatography to determine the average molecular weight and homogeneity of TP1. The peak molecular weight (Mp) of TP1 is 4589 Da, the weight-average molecular weight (Mw) is 5377 Da, and the number-average molar mass (Mn) is 4797 Da. Correspondingly, the polydispersity index (Mw / Mn) of TP1 is 1.121, clearly indicating its homogeneity.

[0067] In addition, such as Figure 1 As shown in Figure F, both the light scattering LS and RI detectors detected only a single peak, indicating that there is no obvious TP1 aggregation in the aqueous solution.

[0068] 4. Methylation analysis

[0069] TP1 was thoroughly dissolved in DMSO. The sample solution was then mixed with NaOH and CH3I for methylation. 1 mL of water and 2 mL of dichloromethane were added, vortexed, centrifuged, and the aqueous phase was discarded. This washing process was repeated three times. The lower dichloromethane phase was aspirated and evaporated to dryness. Then, 100 μL of 2M TFA was added, and the reaction was carried out at 121 °C for 2 h, followed by evaporation to dryness at 30 °C. 50 μL of 2M ammonia and 50 μL of 1M NaBD4 solution were added and mixed. After reacting at room temperature for 2.5 h, 20 μL of acetic acid was added to stop the reaction. The mixture was dried under nitrogen, and then washed twice with 250 μL of methanol, followed by drying under nitrogen. 250 μL of acetic anhydride was added, vortexed, and reacted at 100 °C for 2.5 h. 1 mL of water was added, and the mixture was allowed to stand for 10 min. Then, 500 μL of dichloromethane was added, vortexed, centrifuged, and the aqueous phase was discarded. This washing process was repeated three times. Finally, the lower dichloromethane phase was removed, and the sample was tested on an instrument. Another treatment method is to add 100 μL of 1M TFA and react at 70°C for 3 hours.

[0070] The analytical instrument used in this experiment was an Agilent Technologies Inc. CA, UAS 7890A-5977B gas chromatography-mass spectrometry (GC-MS) system.

[0071] The chromatographic system used was an Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), with an HP-5MS capillary column (30m × 0.25mm × 0.25μm, Agilent J&W Scientific, Folsom, CA, USA). High-purity helium (purity not less than 99.999%) was used as the carrier gas. The flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, using split injection at a split ratio of 10:1, with a solvent delay of 2.2 min.

[0072] The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometer from Agilent Technologies, USA, equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source had an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode was full scan (SCAN), with a mass scan range (m / z) of 30-600.

[0073] The hydroxyl groups of TP1 polysaccharide were methylated, hydrolyzed and reduced, and acetylated to sugar alcohol acetate. Gas chromatography-mass spectrometry (GC-MS) was used to determine the types of methyl glycosides in the prepared samples, and the binding ion signals were analyzed using a mass spectrometry database to obtain the types and molar ratios of methyl glycosides, and seven linkage modes were deduced. The results are shown in Table 1.

[0074] Table 1. Methylation analysis results of TP1

[0075]

[0076] 5. Nuclear magnetic resonance analysis

[0077] Take 50 mg of dried TP1 sample, add 0.6 mL (99.9%) of D2O to dissolve it completely, and record 1D-NMR at 25 °C using a JNM-ECZ600R / S1 spectrometer (600 MHz, JEOL, Japan). 1 H and 13 C) and 2D-NMR spectra ( 1 H– 1 H COSY, NOESY, HSQC, and HMBC), data analysis was performed using MestReNova (6.1.0-6224) software.

[0078] 1 The 1H NMR spectrum indicates that the proton signal is concentrated in the range of δ 3.3-5.4. Figure 2A ), 13 The C NMR spectrum signal is concentrated in the δ55-110 region, and there is no peak signal between δ150-220, indicating that TP1 does not contain uronic acid. Figure 2B Multiple anomeric hydrogen signals were observed in the δ 4.3–5.4 region, and multiple anomeric carbon signals were observed in the δ 99.5–104.5 region, indicating that TP1 possesses both α- and β-configurations, consistent with the infrared analysis results. This finding, combined with relevant literature and… 1 H, 13 C. DEPT-135 1 H- 1H COSY, HSQC, and HMBC NMR spectra were used to analyze residues A, B, C, E, F, and G. 1 H and 13 The chemical shifts of C are assigned in Table 2.

[0079] Four strong signals at δ 103.31, 103.76, 103.90, and 104.27 were found in the anomeric carbon region, but they disappeared in the DEPT-135 spectrum. Figure 2C This indicates that all four anomers are quaternary carbons. Furthermore, no hydrogen-related signal was found in the HSQC spectrum. Figure 2E Based on the monosaccharide composition, combined with methylation results and relevant literature reports, the anomeric carbon signals δ103.76, 103.90, 103.31, and 104.27 can be inferred to be the C-2 signals of β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→) residues, and are respectively labeled as residues A, B, C, and D. Meanwhile, in the DEPT-135 spectrum, signals δ60.48 and 62.53 are inverted peaks, identified as the C-1 and C-6 signals of fructose. Since C-1 is correlated with the HSQC at 3.80 ppm, 3.80 ppm is considered the H-1 signal; and since C-6 is correlated with the HSQC at 3.61 ppm, 3.61 ppm is considered the H-6 signal. From H-6 to 3.75 ppm COSY (… Figure 2D The correlation was analyzed, and 3.75 ppm was considered to be the H-5 signal. The HMBC (H-6) ranged from 74.53 ppm. Figure 2F The correlation between 4.08 ppm and 74.53 ppm, along with the COSY correlation from 3.75 ppm to 4.01 ppm and the HSQC correlation from 4.01 ppm to 74.53 ppm, indicates that 74.53 ppm and 4.01 ppm are C-4 and H-4 signals, respectively. 4.08 ppm shows a COSY correlation with H-4 and an HMBC correlation with C-1, indicating that 4.08 ppm is an H-3 signal. Therefore, based on the HSQC correlation between 4.08 ppm and 77.19 ppm, 77.19 ppm is the C-3 signal.

[0080] In addition, significant correlation signals were observed in the HSQC spectrum, including δ 4.46 / 103.67, 4.55 / 104.40, and 5.3 / 99.88. Combining the HMBC and HSQC spectra, it can be inferred that δ 4.46 / 103.67, 4.55 / 104.40, and 5.3 / 99.88 are the H-1 / C-1 correlation signals of the residues β-D-Galp-(1→, →3,6)-β-D-Galp-(1→, →4)-α-D-Glcp-(1→), and are respectively labeled as residues E, F, and G.

[0081] β-D-Galp-(1→(residue E): H-1 and 3.61 ppm show a correlation signal in the COSY spectrum, indicating that 3.61 ppm belongs to H-2. Accordingly, because of the correlation of HSQC from H-2 to 74.54 ppm, 74.54 ppm is considered to be the C-2 signal. The HMBC correlation from H-1 to 68.56 ppm, the COSY correlation from H-2 to 3.83, and the HSQC correlation from 3.83 ppm to 68.56 ppm indicate that 3.83 ppm and 68.56 ppm are H-3 and C-3 signals, respectively. In addition, 3.78 ppm is COSY correlated with H-3, while 3.78 ppm is HMBC correlated with C-2, indicating that 3.78 ppm is the C-2 signal. The H-4 signal is identified. Therefore, based on the HSQC correlation between 3.78 ppm and 72.54 ppm, 72.54 ppm is considered the C-4 signal. Furthermore, based on the COSY correlation between H4 and 3.47 ppm, the HMBC correlation between 3.47 ppm and C-3, and the HSQC correlation between 3.47 ppm and 71.88 ppm, 3.47 ppm and 71.88 ppm are considered the H-5 and C-5 signals, respectively. Simultaneously, H-4 shows an HMBC correlation with 62.58 ppm, indicating that 62.58 ppm is the C-6 signal. The strong correlation between H-5 and 3.74 ppm suggests that 3.74 ppm is considered the H-6 signal.

[0082] →3,6)-β-D-Galp-(1→(residue F): H-1 and 3.68 ppm show a correlation signal in the COSY spectrum, indicating that 3.68 ppm belongs to H-2. The correlation between H-2 and HSQC at 73.87 ppm confirms that 73.87 ppm is the C-2 signal. H-1 and HMBC at 77.64 ppm are correlated, H-2 and COSY at 4.08 ppm are correlated, and HSQC at 4.08 ppm and 77.64 ppm are correlated, indicating that 4.08 ppm and 77.64 ppm are the H-3 and C-3 signals, respectively. In addition, 4.02 ppm is correlated with COSY for H-3, and 4.02 ppm is correlated with HMBC for C-2, indicating that 4.02 ppm is the H-4 signal. Therefore, based on the correlation of HSQC between 4.02 ppm and 75.86 ppm, 75.86 ppm is the C-4 signal. Based on H4 and 3.77 ppm... Based on the COSY correlation between m, the HMBC correlation between 3.77ppm and C-3, and the HSQC correlation between 3.77ppm and 72.54ppm, 3.77ppm and 72.54ppm are considered to be H-5 and C-5 signals, respectively. Meanwhile, H-4 shows an HMBC correlation with 63.24ppm, indicating that 63.24ppm is a C-6 signal. H-5 and 3.89ppm exhibit a good COSY correlation, suggesting that 3.89ppm is considered the H-6 signal.

[0083] →4)-α-D-Glcp-(1→(residue G): H-1 and 3.48 ppm are correlated in the COSY spectrum, indicating that 3.48 ppm belongs to H-2. H-2 and 69.42 ppm are correlated in the HSQC spectrum, so 69.42 ppm is considered to be the C-2 signal. H-1 and 73.21 ppm are correlated in the HMBC spectrum, H-2 and 3.71 ppm are correlated in the COSY spectrum, and 3.71 ppm and 73.21 ppm are correlated in the HSQC spectrum, confirming that 3.71 ppm and 73.21 ppm are the H-3 and C-3 signals, respectively. In addition, 3.84 ppm is correlated with H-3 in the COSY spectrum, while 3.84 ppm is correlated with C-2 in the HMBC spectrum. The results indicate that 3.84 ppm is the H-4 signal. 72.54 ppm was found to be the C-4 signal in the HSQC spectrum. Furthermore, based on the correlation between H4 and 3.61 ppm in the COSY spectrum, the correlation between 3.61 ppm and C-3 in the HMBC spectrum, and the correlation between 3.61 ppm and 77.19 ppm in the HSQC spectrum, 3.61 ppm and 77.19 ppm are considered to be the H-5 and C-5 signals, respectively. Meanwhile, H-4 is correlated with 62.13 ppm in the HMBC spectrum, and the signal δ62.13 in the DEPT-135 spectrum shows an inverted peak, indicating that 62.13 ppm is the C-6 signal. H-5 and 3.75 ppm show a good COSY correlation, indicating that 3.75 ppm is the H-6 signal.

[0084] HMBC spectra provide long-range correlation signals between carbon and hydrogen atoms, which help infer the connection sequence of residues in a polysaccharide molecule. The HMBC spectrum of TP1 shows that the correlation signal δ 3.61 / 103.76 (A C-2 / D H-6) indicates that the C-2 of residue A is linked to the H-6 of residue D; simultaneously, the correlation signal δ 3.65 / 103.76 (A C-2 / CH-1) indicates that the C-2 of residue A is linked to the H-1 of residue C; the correlation signal δ 3.78 / 103.31 (CH-1 / C C-2) indicates that the H1 of residue C is linked to the C2 of residue C; and the correlation signal δ 3.60 / 104.27 (B H-6 / D C-2) indicates that the H-6 of residue B is linked to the C-2 of residue D. The correlation signal at δ 3.84 / 103.31 (G H-4 / C C-2) indicates that the H-4 of residue G is linked to the C-2 of residue C. The correlation signal at δ 5.30 / 71.65 (G H-1 / G C-4) indicates that the H-1 of residue G is linked to the C-4 of residue G. The correlation signal at δ 5.30 / 77.64 (G H-1 / F C-3) indicates that the H-1 of residue G is linked to the C-3 of residue F, the correlation signal at δ 4.55 / 77.64 (F H-1 / F C-3) indicates that the H-1 of residue F is linked to the C-3 of residue F, and the correlation signal at δ 3.46 / 103.67 (F H-6 / E C-1) indicates that the H-6 of residue F is linked to the C-1 of residue E.

[0085] Because fructose is easily decomposed under high temperatures, monosaccharide composition and methylation analysis cannot accurately reflect the proportion of monosaccharides in the sugar chain. Therefore, by using... 1 H and 13 C-chromatogram to determine the proportion of each monosaccharide in TP1. 13 In the C-1 spectrum, the integral ratio of the C-2 signal peaks of residues A, B, C, and D is approximately 2:2:8:2, indicating that residues A, B, C, and D account for approximately 2:2:8:2 in TP1; the integral ratio of the C-1 signal peaks of residues E and F is approximately 1:1, indicating that residues E and F account for approximately 1:1 in TP1. 1 In the H spectrum, the ratio of the integral of the H1 signal peaks of E, F, and G to the integral of the H3 signal peak of fructose is approximately 3:3:10:14, indicating that the ratio of the proportion of residues E, F, and G in TP1 to the proportion of total fructose is approximately 3:3:10:14. Therefore, it can be inferred that the ratio of residues A, B, C, D, E, F, and G in TP1 is approximately 2:2:8:2:3:3:10. The possible glycan structure of TP1 is as follows: Figure 3 As shown.

[0086] Table 2 TP1 1 H and 13C chemical shift δ (ppm)

[0087]

[0088] Example 2

[0089] The application of Centella asiatica polysaccharides is carried out in the following specific process:

[0090] Animal experimental procedures were approved by the Animal Ethics Committee Laboratory of Wannan Medical College (WNMC-AWE-2024301) and strictly followed the regulations of the State Administration for Laboratory Animal Science of China. C57BL / 6J mice (male, 8 weeks old, weighing 21±2g) were purchased from Qinglongshan Laboratory Animal Center (Nanjing, Jiangsu, China) (License No.: SCXK (Zhe) 2024-0002). All SPF-grade mice were kept in an environment with 50-55% humidity and 22-25℃, with a 12-hour light-dark cycle, and had free access to food and purified water. All mice underwent humane treatment.

[0091] After one week of acclimatization, 12 mice were randomly selected as the normal control group, fed with normal drinking water. The other groups of mice were given water containing 3% DSS (w / v) for 5 consecutive days to induce acute colitis. Starting from day 8, mouse weight, fecal characteristics, and degree of blood in the feces were recorded daily, and the Disease Activity Index (DAI) score was calculated to determine the success of the model. Mice with successful modeling were randomly divided into 4 groups (n=12): DSS group, TP1-L (100mg / kg) group, TP1-H (400mg / kg) group, and 5-aminosalicylic acid (5-ASA) (200mg / kg) group. Figure 4 (A). Starting from day 13, mice in the Control and DSS groups had free access to saline solution, while the TPL-L, TP1-H, and 5-ASA groups were administered the drugs via gavage for 7 days. On day 21, the mice were anesthetized and euthanized, and blood was collected from the orbital cavity. Serum was extracted by centrifugation. All organs, cecal contents, and colonic tissue were collected and stored at -80°C. A 1 cm segment of the distal colon was fixed in 4% paraformaldehyde.

[0092] 1. Assess UC severity based on Disease Activity Index (DAI) score

[0093] In animal models, the DAI score has been widely used to assess the severity of ulcerative colitis (UC). Mice's body weight, fecal consistency, and fecal occult blood status are recorded daily, and these three parameters are scored according to the DAI scoring criteria. The DAI score is a combination of the scores for all the above parameters, and the disease activity index is calculated for each mouse.

[0094] The intervention effect of TP1 was revealed by recording mouse body weight, observing fecal characteristics, calculating the DAI index, and measuring colon length and spleen weight during the modeling period. The results showed that DSS administration led to a sustained decrease in body weight in all groups of mice, consistent with the disease pathogenesis. Figure 4 In the middle A group, at the end of the modeling process, except for the control group, the DAI scores of all other groups increased. Figure 4 The DSS group mice showed severe fecal blood loss (D), indicating that the UC mouse model was successfully established. Figure 4 (Medium B), weight continues to decrease ( Figure 4 In the DSS group, the DAI score was significantly increased, and TP1 and 5-ASA administration significantly improved fecal blood loss and weight changes in mice. Especially in the TP1-H administration group, weight loss was significantly reduced. The colon length in the DSS group was significantly shortened. Figure 4 In the middle E), the spleen became significantly swollen ( Figure 4 (F). TP1 administration significantly inhibited the shortening of colon length and the swelling of spleen (P<0.05). This indicates that TP1 has a protective effect against colonic inflammation in mice and can improve DSS-induced colitis, and can be used in the preparation of drugs for the treatment of colitis.

[0095] 2. ELISA and oxidative stress assay

[0096] Serum levels of inflammatory cytokines IL-6, IL-1β, TNF-α, and IL-17 were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Levels of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and myeloperoxidase (MPO) were detected using a detection kit, according to the manufacturer's instructions (Nanjing Jiancheng Biotechnology Research Institute, China).

[0097] Inflammatory factors play a central role in the pathogenesis of ulcerative colitis (UC). The levels of these cytokines were measured using an enzyme-linked immunosorbent assay (ELISA). It was found that the levels of TNF-α, IL-17, IL-6, and IL-1β were significantly elevated in mice with DSS-induced colitis. Figure 5 (C). Pro-inflammatory cytokines IL-6, TNF-α, and IL-1β have been shown to significantly influence the pathogenesis and exacerbation of colitis (UC). IL-17 inhibits the expression of intestinal epithelial tight junction proteins, increases intestinal permeability, and promotes bacterial translocation and inflammatory spread. Administration of TP1 to colitis mice effectively inhibited the increase in TNF-α, IL-17, IL-6, and IL-1β levels, but the effect of TP1 on TNF-α levels was almost dose-dependent, which may be related to individual differences among mice. Overall, these results indicate that TP1 has a significant anti-inflammatory effect and can modulate the inflammatory response in UC mice.

[0098] The severity of oxidative stress can be reflected by the activities of CAT and SOD, and the contents of MPO and MDA. MPO is mainly found in neutrophils and can aggravate mucosal damage. MPO is considered a biomarker of intestinal tissue damage and neutrophil infiltration. In this study, compared with the control group, the DSS group showed significantly increased MDA content and MPO activity in colon tissue, while significantly decreased CAT content and SOD activity. TP1 administration significantly improved the DSS-induced increase in MDA and MPO activity and the decrease in CAT and SOD activity. Figure 5 The results in the TP1-H group were superior to those in the TP1-L group. Therefore, TP1 can alleviate the abnormal secretion of inflammatory cytokines and oxidative stress induced by DSS, thereby alleviating the symptoms in UC mice.

[0099] In summary, TP1 inhibits colonic oxidative stress and pro-inflammatory cytokine levels in DSS-induced colitis mice.

[0100] 3. Histopathological H&E and AB-PAS staining

[0101] A 1.0 cm section of distal colon tissue was harvested, fixed with 4% paraformaldehyde, and embedded in paraffin. The colon was sectioned into 5 μm pieces and stained with hematoxylin and eosin (H&E) and Alcian blue (AB) and Periodic acid Schiff (PAS) stain (AB-PAS) according to standard procedures. Differences between groups were observed using an optical microscope (NI-U, NIKON).

[0102] HE staining was used to further assess colonic pathological damage. Figure 5 (A) In the normal group of mice, the intestinal epithelium was intact and continuous, with goblet cells arranged neatly and no inflammatory cell infiltration. In the DSS group, the colonic epithelial cells were destroyed (blue arrow), the mucosa was damaged (green arrow), crypts and glands were lost (black arrow), and the number of goblet cells was significantly reduced (red arrow). After treatment with TP1 and 5-ASA, the inflammation of the colonic mucosa was relieved, the epithelium was more intact, and the histological score was significantly reduced.

[0103] 4. Histopathological immunofluorescence staining

[0104] For immunofluorescence evaluation, sections were dewaxed, repaired, and incubated overnight at 4°C with primary antibodies Claudin-1, ZO-1, and Occludin (1:500). Sections were then incubated with CY3-labeled goat anti-mouse (1:300) at room temperature in the dark for 50 min, followed by nuclei counterstaining with DAPI and incubation at room temperature in the dark for 10 min. The stained sections were observed using a fluorescence microscope (NI-U, NIKO), and the average densities of Claudin-1, Occludin, and ZO-1 were analyzed using ImageJ software.

[0105] The colonic mucus barrier, primarily composed of colonic mucus secreted by goblet cells, is the host's first line of defense against intestinal invasion. AB-PAS staining stained acidic mucin blue and mixed mucin blue-purple. Figure 5 (Middle B). The results showed that, compared with the normal group, the goblet cells in the DSS group were damaged, and mucin secretion was significantly reduced. Importantly, TP1 administration could attenuate DSS-induced goblet cell depletion, with the TP1-H group showing better results than the TP1-L group. Then, the blue intensity in the images was quantified using ImageJ software. The blue intensity in the DSS group was significantly lower than that in the normal group, while the blue intensity increased significantly after TP1 administration, indicating that TP1 treatment restored the number of goblet cells. These results suggest that TP1 has a protective effect on colonic tissue and can alleviate tissue damage caused by UC. 5. Western blot analysis

[0106] Colon tissue was washed with PBS, homogenized on ice, and then lysed in RIPA lysis buffer for 20 min. The lysate was centrifuged at 4°C (13000 rpm) for 20 min. The protein concentration in the supernatant was determined using a BCA protein assay kit. The same amount of protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane (Hybond, Sunnyvale, CA, USA) using a semi-dry transfer system (Bio-Rad, Hercules, CA, USA). The membrane was blocked in TBST with 5% skim milk at 25°C for 2 h, incubated overnight with primary antibodies (Claudin-1, Occludin, ZO-1, and β-actin) at 4°C, and then incubated with enzyme-labeled secondary antibody at room temperature for 1 h. Protein signals were analyzed using a Western blot imaging system (Amersham ImageQuant™ 800, Citiva), and protein levels were quantified using ImageJ software.

[0107] Immunofluorescence chemical analysis results showed that the expression of Claudin-1, Occludin, and ZO-1 was inhibited in the DSS group, indicating that DSS impairs the intestinal barrier. Figure 6(A) The TP1 treatment group significantly maintained the colonic mucosal barrier by upregulating TJ expression to varying degrees. Among them, the TP1-H group showed the largest positive expression area of ​​Claudin-1, Occludin, and ZO-1. Therefore, TP1 can maintain the integrity of the mucosal barrier by upregulating the expression of Claudin-1, Occludin, and ZO-1. In addition, Western blotting analysis was performed to further verify the expression levels of Claudin-1, Occludin, and ZO-1 proteins, thereby accurately assessing barrier function. Combining the results of three replicates, it was found that DSS-treated mice showed significantly reduced TJ protein expression compared with the control group. However, TP1 administration significantly reduced the loss of Occludin, ZO-1, and Claudin-1 proteins, and the effect was more significant in the TP1-H group. Figure 6 (Middle B). This is consistent with the results obtained from immunofluorescence chemical analysis. It also matches the results of H&E staining detecting abundant goblet cells in the TP1-H group. In summary, these observations suggest that TP1 alleviates DSS-induced damage to mouse colonic tissue and reduces intestinal barrier disruption, thereby preventing UC injury in mice.

[0108] 6. Analysis of gut microbiota composition

[0109] The 16S rRNA analysis method is based on the inventors' previous research. The colon contents of mice were collected, rapidly frozen in liquid nitrogen, and then stored in a freezer at -80°C. Gene sequencing was performed by PANOMIX Biomedical Technology Co., Ltd. (Suzhou, China). Sequencing libraries were prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit, and 16S rRNA sequencing was performed on the Illumina MiSeq-PE250 platform (Illumina, USA).

[0110] In alpha diversity analysis, for microbial community samples, the abundance rank curve can intuitively reflect the number of high-abundance and rare ASVs / OTUs in the community. Figure 7 According to the Veen plot results, 3928, 2669, and 2935 independent OTUs were found in the Control group, DSS group, and TP1-H group, respectively, indicating significant differences in gut microbiota composition among these groups. Figure 7 (Medium B). To assess the impact of TP1 on gut microbiota abundance and diversity, the alpha diversity index was analyzed. Results showed that the DSS group exhibited decreases in the Chao1, Shannon, Simpson, and Observed_species indices. Figure 7The DSS and TP1-H groups showed significantly improved gut microbiota, while TP1-H significantly restored these indices. Beta diversity analyses (PCoA and NMDS) were subsequently used to analyze the similarities and differences between the microbiota. PCoA showed that the DSS and TP1-H groups had different distribution directions and were far apart, indicating that TP1 and DSS had a significant impact on the mouse gut microbiota. Figure 7 (D), NMDS analysis further confirmed the above theory ( Figure 7 (C). These results indicate that TP1 regulates the richness and diversity of the gut microbiota.

[0111] At the phylum level, the gut microbiota of mice is mainly composed of Firmicutes, Bacteroidetes, and Proteobacteria. Compared with the Control group, the ratio of Firmicutes to Bacteroidetes was significantly increased in the DSS group, indicating a certain degree of dysbiosis in the mouse gut microbiota. However, TP1 intervention can effectively regulate the F / B ratio. Figure 7 (F). At the genus level, based on cluster heatmap analysis ( Figure 8A In the DSS group, the relative abundance of bacteria associated with immune regulation, intestinal mucosal protection, and SCFA production, such as *Ligilactobacillus*, *Alloprevotella*, and *Ruminococcus*, was significantly lower than in the Control group, while the relative abundance of *Helicobacter*, *Adlercreutzia*, and *Desulfovibrio* was significantly increased. Figure 7 (GL). After TP1 administration, the relative abundance of *Ligilactobacillus* and *Alloprevotella* significantly increased, while the relative abundance of *Helicobacter*, *Adlercreutzia*, and *Desulfovibrio* significantly decreased. Linear discriminant analysis (LDA) effect size (LEfSe) was used to assess the hierarchy and abundance of gut microbiota in each group, with the LDA score threshold set at 4.0 (Figure 8 B). Figure 8C The results showed that DSS promoted the proliferation of some pathogenic bacteria, such as *Adlerecreutzia*, *Desulfovibrio*, and *Helicobacter*. TP1-H enriched bacteria that could modulate immunity, protect the intestinal mucosa, and promote SCFA production, such as *Ruminococcus*, *Ligilactobacillus*, and *Alloprevotella*.

[0112] 7. Determination of Short-Chain Fatty Acids (SCFAs)

[0113] SCFAs in feces include acetate, propionate, butyrate, and valeric acid. SCFA content was analyzed using a GC system (7890A, California, USA) equipped with a flame ionization detector and an HP-FFAP column (30 m × 0.32 mm × 0.25 µm, Agilent Technologies, Inc., USA). The experiment was conducted using the following procedure: heating at 80 °C for 0.5 min, then heating at a rate of 4 °C / min from 80 to 150 °C, followed by heating at a rate of 20 °C / min from 150 to 230 °C, and holding for 10 min. The signal was detected at 300 °C using a flame ionization detector.

[0114] SCFAs, as the main products of gut microbiota fermentation, are a major energy source for intestinal cells and play an important role in maintaining intestinal barrier integrity and regulating host immune responses. The concentrations of acetic acid, propionic acid, and butyric acid in the feces of mice in the DSS group were significantly lower than those in the normal group, but their concentrations significantly recovered after TP1 intervention. Figure 8D Meanwhile, SCFAs participate in maintaining intestinal homeostasis through GPR41 and GPR43 receptors. q-PCR results showed that, compared with the normal group, DSS significantly inhibited the expression of GPR41 and GPR43 genes in colonic tissue, while TP1 administration significantly increased the expression of GPR41 and GPR43 genes in colonic tissue. Figure 8E This indicates that TP1 promotes the proliferation of some bacteria in the gut that produce short-chain fatty acids, thereby alleviating DSS-induced colitis.

[0115] 8. Real-time quantitative polymerase chain reaction (RT-qPCR)

[0116] RT-PCR was used to detect the expression of GPR41 and GPR43 mRNA in colon tissue. 50 mg of colon tissue from the control group, model group, and TP1-H mice was weighed, and total RNA was extracted according to the kit instructions (Fastagen, Shanghai, China). The extracted RNA was reverse transcribed into cDNA using a ReverTra AceqPCR RT kit (TOYOBO, Osaka, Japan) and gradient thermal cycling (Easy Cycle Gradient 96, AnalytikJena, Beijing, China). RT-PCR analysis was performed using a real-time quantitative PCR system (Applied Biosystems QuantStudio 5, Shanghai, China). Primer sequences are shown in Table 3.

[0117] Table 3 Primer sequences used for semi-quantitative RT-qPCR analysis

[0118]

[0119] In this invention, Graphpad 10.1.2 software was used for data difference analysis, and the mean ± standard deviation was used to represent the quantitative results of the experiment. When performing statistical comparisons of more than two groups, one-way ANOVA and Dunnett's test were used. Differences were considered statistically significant when *P < 0.05 and **P < 0.01, and ns indicated no significant difference.

[0120] In summary, this invention extracts and purifies a neutral polysaccharide from Centella asiatica, named TP1.

[0121] Structurally: Monosaccharide composition analysis, methylation analysis, and NMR analysis revealed that, unlike previously reported polysaccharides from *Gnaphalium affine*, the TP1 polysaccharide extracted and isolated in this study is a neutral heteropolysaccharide with a narrow molecular weight distribution, measuring 5377 Da. Monosaccharide composition analysis showed that it is mainly composed of fructose, glucose, and galactose, with fructose being the most abundant, in a molar ratio of 44.7:33.8:17.9. NMR analysis further revealed... 13 The C-spectral signal is mainly concentrated in the δ55-110 region, with no peak signal between δ150-220, indicating that TP1 does not contain uronic acid. DEPT-135 spectral analysis confirmed that TP1 does indeed contain a high proportion of fructans. The main chain of TP1 is mainly composed of →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →4)-α-D-Glcp-(1→ and →3,6)-β-D-Galp-(1→). The side chains are mainly composed of β-D-Fruf-(2→6)-β-D-Fruf-(2→) attached to the C-6 position of →1,6)-β-D-Fruf-(2→) and β-D-Galp-(1→) attached to the C-6 position of →3,6)-β-D-Galp-(1→).

[0122] Functionally, TP1, composed of different types of fructose, glucose, and galactose, possesses a complex structure and diverse biological activities. Studies have shown that TP1 can alleviate DSS-induced colitis symptoms in mice, including weight loss, elevated DAI scores, splenic swelling, and colonic shortening. Compared with existing technologies, TP1 protects the intestinal barrier by restoring goblet cell numbers, increasing goblet cell glycoprotein and crypt mucin content, and reducing the loss of tight junction proteins Claudin-1, Occludin, and ZO-1. Furthermore, 16sRNA sequencing analysis revealed that TP1 promotes SCFA production in the colon by improving gut microbiota dysbiosis, enriching genera that promote SCFA production, such as Ruminococcus, Lactobacillus, and Bifidobacterium, and significantly increasing the expression of GPR41 and GPR43 receptors in colonic tissue. This reduces intestinal inflammation through SCFA-mediated reduction of oxidative stress and inflammatory cytokine levels via GPR41 and GPR43 receptor mediated by SCFAs.

[0123] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A polysaccharide from Centella asiatica, characterized in that, The polysaccharide from *Centella asiatica* is mainly composed of fructose, glucose, and galactose, with a molar ratio of fructose: glucose: galactose = 44.7: 33.8: 17.

9. Its weight-average molecular weight (Mw) is 5377 Da, and its number-average molar mass (Mn) is 4797 Da.

2. A method for preparing the *Centella asiatica* polysaccharide according to claim 1, characterized in that, The preparation method includes the following steps: 1) Fresh centrifuge is crushed, extracted by hot water distillation, concentrated, centrifuged to remove precipitate, and then precipitated with anhydrous ethanol and the precipitate is collected. 2) The precipitate was dissolved in distilled water, decolorized, and the protein was removed by Sevage reagent. After dialysis, crude polysaccharide was obtained. 3) After dissolving and filtering the crude polysaccharide, it was separated and purified by elution with distilled water, and the eluent was freeze-dried. 4) The product solution from step 3) is separated on a gel column using distilled water, and the resulting eluent is freeze-dried to obtain the final product; The hot water distillation extraction described in step 1) specifically involves: extracting twice with 80℃ distilled water at a material-to-liquid ratio of 1:20, g / mL; each extraction lasts for 2 hours, and the extracts are then combined. In step 2), the protein was removed by decolorizing with a D101 macroporous adsorption resin column and by using Sevage reagent n-butanol:trichloromethane = 1:3 v / v to remove the protein. The protein was then dialyzed in deionized water for 48 hours using a 1000 kDa dialysis bag, with the water changed every 4 hours. The protein was then concentrated and freeze-dried to obtain crude polysaccharide. In step 3), the crude polysaccharide TPs was prepared into a 20 mg / mL solution, filtered using a 0.22 μm filter membrane, and the filtrate was then packed into a DEAE-52 column for purification. In step 4), separating the product solution from step 3) on a gel column with distilled water means separating it on a Sephadex G-100 column with distilled water at a flow rate of 0.5 mL / min.

3. The preparation method according to claim 2, characterized in that, The precipitation with anhydrous ethanol mentioned in step 1) refers to: precipitating with 4 times the volume of anhydrous ethanol for 12 hours at 4℃, centrifuging for 10 minutes, and collecting the precipitate.

4. An application of the *Centella asiatica* polysaccharide according to claim 1, characterized in that, Used to prepare drugs for the prevention and treatment of colitis.

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