Thesium chinense polysaccharide as well as preparation method and application thereof

Abstract

The invention provides thesium chinense polysaccharide as well as a preparation method and application thereof, compared with the prior art, neutral polysaccharide TP1 is efficiently extracted from thesium chinense by adopting a hot water extraction method, the neutral polysaccharide TP1 mainly comprises fructose, glucose and galactose according to a molar ratio of 44.7: 33.8: 17.9, and the molecular weight Mw of the TP1 measured by adopting a GPC-RI-MALS chromatographic method is 5377Da. The invention also researches the protective effect of thesium chinense polysaccharide on DSS-induced colitis. The TP1 reduces the loss of intestinal tight junction proteins (Claudin-1, Occludin and ZO-1), regulates the abundance of bacteria in the intestinal tract and enriches bacteria (ruminococcus, lactobacillus and bifidobacterium) capable of generating SCFAs, in addition, the TP1 increases the expression of GPR41 and GPR43 receptors in colon tissues, and the symptoms of colitis are relieved by reducing oxidative stress and inflammatory cytokine levels through the GPR41 and GPR43 receptors mediated by the SCFAs. The thesium chinense polysaccharide TP1 prepared by the invention can be used for preparing medicines for preventing and treating colitis and also can be used for preparing food.

Description

Technical Field

[0001] The present invention belongs to the fields of polysaccharides and medicine, and particularly relates to the polysaccharide of Thesium chinense Turcz. and its preparation method and application. Background Art

[0002] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic and complex inflammation marked by gastrointestinal lesions, characterized by diffuse mucosal inflammation, bloody stools, diarrhea, and abdominal pain, which seriously affects the quality of life of patients. At present, the exact cause and pathogenesis of UC are not fully understood. More and more studies have shown that UC may be related to factors such as genetic variation, intestinal flora imbalance, immune dysfunction, diet, and living environment. In the past decade, the incidence of UC has increased rapidly. If not treated in time, it may develop into colon cancer.

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

[0004] As a dietary resource, active polysaccharides derived from natural products have received extensive attention worldwide due to their good tolerance and various pharmacological activities such as anti-inflammatory, antibacterial, and anti-tumor effects. More and more studies have shown that polysaccharides have great potential in the prevention and treatment of UC. For example, the polysaccharide of Sagittaria trifolia L. alleviates colitis symptoms by repairing the intestinal barrier, promoting microbial metabolism, and regulating the intestinal microbiota-MAPK / NF-κB signaling pathway. The polysaccharide of Abelmoschus manihot (L.) Medic. relieves intestinal inflammation by regulating the abundance of intestinal bacteria and enhancing the intestinal mucus barrier. It is worth noting that some indigestible polysaccharides can undergo fermentation reactions under the metabolic action of the colonic microbiota and play anti-inflammatory, intestinal mucosa protection, and intestinal immunity regulation roles by affecting the diversity of the intestinal flora and the metabolites of the flora. At present, natural polysaccharides have been widely used in the health food and pharmaceutical industries. It is of great significance to develop active polysaccharides from natural plants and explore their beneficial properties. Summary of the Invention

[0005] The purpose of the present invention is to provide the polysaccharide of Thesium chinense Turcz. and its preparation method, and a neutral polysaccharide TP1 is efficiently extracted from Thesium chinense Turcz. by hot water extraction method. FT-IR and 1D / 2D-NMR analyses confirm that TP1 is mainly composed of fructose, glucose, and galactose, with a molar ratio of 44.7:33.8:17.9. The molecular weight Mw of TP1 measured by GPC-RI-MALS chromatography method is 5377 Da.

[0006] Another object of the present invention is to provide the application of Thesium chinense polysaccharide. The Thesium chinense polysaccharide TP1 prepared by the present invention can be used for preventing and treating colitis drugs, and 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 bacterial genera in the intestine, and enriches bacterial genera that can produce SCFAs (Ruminococcus, Lactobacillus and Prevotellaceae). In addition, TP1 increases the expression of GPR41 and GPR43 receptors in colon tissues, and reduces oxidative stress and inflammatory cytokine levels through SCFAs-mediated GPR41 and GPR43 receptors.

[0007] The application of Thesium chinense polysaccharide provided by the present invention can also be used for preparing food.

[0008] The specific technical solution of the present invention is as follows:

[0009] A Thesium chinense polysaccharide provided by the present invention is named TP1, which is mainly composed of fructose, glucose and galactose, and the molar ratio is 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.

[0010] A preparation method of Thesium chinense polysaccharide provided by the present invention includes the following steps:

[0011] 1) Fresh Thesium chinense is crushed, extracted by hot water distillation, concentrated, centrifuged to remove the precipitate, and then precipitated with anhydrous ethanol, and the precipitate is collected;

[0012] 2) The precipitate is redissolved with distilled water, decolorized, the protein is removed with Sevage reagent, and after dialysis, crude polysaccharide is obtained;

[0013] 3) After the crude polysaccharide is dissolved and filtered, it is eluted with distilled water for separation and purification, and the eluate is freeze-dried;

[0014] 4) The product solution in step 3) is separated on a gel column with distilled water, and the obtained eluate is freeze-dried to obtain the product.

[0015] The hot water distillation extraction described in step 1) is specifically: extracting 2 times with distilled water at 80-90 °C, the solid-liquid ratio = 1:20, g / mL; 2 h each time, and the extracts are combined;

[0016] The concentration described in step 1) refers to low-temperature rotary evaporation at 60 °C;

[0017] The centrifuging to remove the precipitate described in step 1) refers to centrifuging at 5000 r / min for 8 min to remove the precipitate;

[0018] The precipitation with absolute ethanol described in step 1) means: at 4°C, precipitate with 4 volumes of absolute ethanol for 12 h, centrifuge for 10 min, and collect the precipitate.

[0019] In step 2), decolorize through a D101 macroporous adsorption resin column, and then remove proteins with Sevage reagent (volume ratio of n-butanol: chloroform = 1:3);

[0020] In step 2), for the dialysis, use a 1000KDa dialysis bag, dialyze in deionized water for 48 h, change the water every 4 h, and concentrate and freeze-dry to obtain crude polysaccharide (TPs).

[0021] In step 3), prepare the crude polysaccharide TPs into a 20 mg / mL solution, filter using a 0.22 μm filter membrane, and load it onto a DEAE-52 (2.5×40 cm) column for separation and purification.

[0022] In step 4), the separation of the product solution from step 3) on a gel column with distilled water means further separation on a Sephadex G-100 (1.6×60 cm) column with distilled water at a flow rate of 0.5 mL / min.

[0023] Through the above preparation method of the present invention, after hot water distillation extraction and ethanol precipitation, decolorization, protein removal, dialysis to remove small molecules, and then removal of charged substances and heterogeneous polysaccharides are carried out in sequence, impurities can be removed, the purity of polysaccharides can be improved, and the natural conformation of polysaccharides is maintained by using freeze-drying and neutral conditions (distilled water elution). The present invention comprehensively uses multiple mechanisms such as physical retention, chemical precipitation, and charge adsorption to cooperate synergistically to form a gradient purification system, and finally realizes the efficient separation and purification of polysaccharides. Each step independently removes specific impurities and jointly constructs a cross-validation purification network, and finally obtains a polysaccharide mainly composed of fructose, glucose, and galactose.

[0024] The present invention provides the application of the polysaccharide from Thesium chinense Turcz, which is used for drugs for preventing and treating colitis, or for preparing food.

[0025] The traditional Chinese medicine Thesium chinense Turcz is a plant of the family Santalaceae, a perennial parasitic herb, first recorded in "Illustrated Classic of Materia Medica", with aliases such as Bai Ru Cao, Di Shi Liu, Xiao Cao, Feng Ya Hao, Xi Xu Cao, etc., and is widely distributed in the north and south of China. In traditional Chinese medicine, Thesium chinense Turcz is called "plant antibiotic" and can be used to treat diseases such as pneumonia, acute tonsillitis, acute mastitis, pharyngitis, bronchitis, etc., and has a good inhibitory effect on a variety of pathogenic bacteria. These characteristics make Thesium chinense Turcz have high research value and application potential in the field of traditional Chinese medicine, but currently, there is less research on the polysaccharide components in Thesium chinense Turcz at home and abroad.

[0026] The present invention uses the whole plant of Thesium chinense Turcz. as raw material, from which a water-soluble polysaccharide is isolated and purified, and its structure is characterized by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, methylation analysis, monosaccharide composition analysis, etc. In addition, a UC mouse model is induced by DSS to evaluate its beneficial effects on DSS-induced colitis mice, laying a foundation for the in-depth study of Thesium chinense Turcz. polysaccharide and accumulating data for the development of related polysaccharide foods, new drugs or health products.

[0027] Compared with the prior art, the present invention efficiently extracts a neutral polysaccharide TP1 from Thesium chinense Turcz. by hot water extraction method. FT-IR and 1D / 2D-NMR analyses confirm that TP1 is mainly composed of fructose, glucose and galactose, and the molar ratio is 44.7:33.8:17.9. The molecular weight Mw of TP1 measured by GPC-RI-MALS chromatographic method is 5377 Da. The present invention also studies the protective effect of Thesium chinense Turcz. polysaccharide on DSS-induced colitis. TP1 reduces the loss of intestinal tight junction proteins (Claudin-1, Occludin and ZO-1), regulates the abundance of bacterial genera in the intestine, and enriches bacterial genera that can produce SCFAs (Ruminococcus, Lactobacillus and Prevotellaceae). In addition, TP1 increases the expression of GPR41 and GPR43 receptors in colon tissues, and reduces the levels of oxidative stress and inflammatory cytokines through SCFAs-mediated GPR41 and GPR43 receptors. The Thesium chinense Turcz. polysaccharide TP1 prepared by the present invention can be used for preventing and treating colitis drugs; it can also be used for preparing foods. Description of the Drawings

[0028] Figure 1 Detection diagrams at different stages in polysaccharide preparation; wherein (A) elution curve of DEAE-52 cellulose column, (B) elution curve of Sephadex G-100 gel chromatography column. (C) ultraviolet spectrum diagram, (D) monosaccharide composition analysis, a: standard monosaccharide, b: hydrolysis of TP1 at 60 °C, c: hydrolysis of TP1 at 121 °C, (E) infrared spectrum diagram of TP1, (F) molecular weight distribution map of TP1, (G) HPGPC map of TP1;

[0029] Figure 2A For the NMR of TP1 1 1H spectrum diagram;

[0030] Figure 2B For the NMR of TP1 13 13C spectrum diagram;

[0031] Figure 2C For the DEPT-135 spectrum diagram of TP1;

[0032] Figure 2D For the COSY spectrum diagram of TP1;

[0033] Figure 2E HSQC spectrum of TP1;

[0034] Figure 2F HMBC spectrum of TP1;

[0035] Figure 3 Main repetitive sugar chain structure of TP1;

[0036] Figure 4 Record of various indicators of mice after administration of DSS, (A) Administration scheme diagram; (B) Hematochezia condition; (C) Body weight change; (D) DAI score; (E) Colon length; (F) Spleen status;

[0037] Figure 5 Results of colon tissue staining and inflammatory factor detection; among them, (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;

[0038] Figure 6 Immunofluorescence chemistry analysis and WB analysis, (A) Immunofluorescence chemistry analysis of Occludin, Claudin-1, ZO-1, (B) WB analysis of Occludin, Claudin-1, ZO-1;

[0039] Figure 7 Analysis diagram of flora composition, among which (A) Abundance rank curve, (B) Veen diagram, (C) NMDS, (D) PCoA, (E) Chao1, Shannon, Simpson and observed_species indices, (F) F / B ratio, (G) Desulfovibrio, (H) Actinomycetes, (I) Helicobacter, (J) Ruminococcus, (K) Prevotellaceae_NK3B31_group, (L) Lactobacillus;

[0040] Figure 8A Heat map analysis at the genus level;

[0041] Figure 8B LDA histogram;

[0042] Figure 8C LEfSe evolutionary cladogram from phylum to genus;

[0043] Figure 8D Contents of acetic acid, propionic acid and butyric acid;

[0044] Figure 8E Analysis of GPR41 and GPR43 mRNA expression. Detailed implementation methods

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in 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 part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0046] The test materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0047] For those not specifying specific technologies or conditions in the embodiments, they can all be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions.

[0048] The materials used in the embodiments of the present invention:

[0049] Sodium dextran sulfate (DSS, molecular weight: 36 - 50 kDa) was provided by MP Biomedicals (Santa Ana, CA, USA). D101 macroporous adsorption resin, DEAE-52 cellulose, and Sephadex G-100 resin were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and myeloperoxidase (MPO) kits were all provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA kits for IL-6, TNF-α, IL-17, and IL-1β were purchased from Jiangsu Jingmei Biotechnology Co., Ltd. Antibodies Claudin-1, Occludin, ZO-1, and β-Actin were purchased from Affinity Biosciences (Changzhou, China). Dialysis bags with a molecular weight cut-off of 1000 kDa were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., China; The Chinese herbal medicine Thesium chinense Turcz. was purchased from Anhui Tuoyuantang Pharmaceutical Co., Ltd. and identified by Professor Li Chunyan (Wannan Medical College, Wuhu City). Except as otherwise specified, all other reagents were used as analytical grade.

[0050] Example 1

[0051] A preparation method of Thesium chinense Turcz. polysaccharide, specifically:

[0052] 1) Wash the fresh Thesium chinense Turcz. thoroughly, dry it, and powder it. Weigh 200 g of Thesium chinense Turcz. powder and extract it continuously twice with distilled water at 80 °C (solid-liquid ratio = 1:20, g / mL), each time for 2 h. Combine the extracts and concentrate them by rotary evaporation at 60 °C to 1 / 10 of the original volume of the combined extracts. Centrifuge at 5000 r / min for 8 min to remove the precipitate. At 4 °C, precipitate with 4 volumes of absolute ethanol for 12 h, centrifuge for 10 min, and collect the precipitate. The precipitate is fully dissolved in 1000 mL of distilled water at 60 °C, centrifuged again, and the supernatant is decolorized through a D101 macroporous adsorption resin column. Remove proteins with Sevage reagent (n-butanol: chloroform = 1:3 v / v). Subsequently, use a 1000 KDa dialysis bag and dialyze in deionized water for 48 h, changing the water every 4 h. Freeze-dry at -80 °C using a freeze-dryer to obtain crude polysaccharide (TPs). Dissolve 200 mg of TPs in 10 mL of distilled water, filter through a 0.22 μm filter membrane, and load it onto a DEAE-52 (2.5 × 40 cm) column for purification. Elute stepwise with distilled water, 0.1, 0.3, and 0.5 mol / L NaCl gradients. Collect 50 tubes (4 mL / tube) of eluate for each gradient, and determine the total sugar content using the anthrone-sulfuric acid method. Freeze-dry the collected distilled water eluate at -80 °C. The freeze-dried product is formulated into a 6 mg / mL aqueous solution, then loaded onto a Sephadex G-100 (1.6 × 60 cm) column, with each sample loading of 5 mL, and further separated with distilled water at a flow rate of 0.5 mL / min. Freeze-dry the obtained eluate and collect the purified polysaccharide, denoted as TP1.

[0053] After Thesium chinense Turcz. was extracted with hot water, precipitated with ethanol, decolorized by a D101 macroporous adsorption resin column, treated with Sevage reagent, dialyzed, and then freeze-dried to obtain TPs. Calculate the yield of TPs to be 0.79%. After TPs were separated by a DEAE-52 cellulose column, the water elution fraction and 0.1 M and 0.3 M NaCl elution fractions ( Figure 1 in A) were obtained and named TP1, TP2, and TP3 respectively. After collecting TP1 and freeze-drying it, it was purified using a Sephadex G-100 gel chromatography column ( Figure 1 in B). After elution with deionized water and freeze-drying with a freeze-dryer, the purified TP1 fraction was obtained. In addition, the HPGPC chromatogram showed that TP1 was a single-peak symmetry, indicating that the purity of TP1 was 95.25% ( Figure 1 in G). Using the anthrone-sulfuric acid method, the total sugar content in TP1 was 94.72%.

[0054] The structural characterization of TP1 prepared in Example 1 is as follows:

[0055] 1. UV and FT-IR analysis

[0056] Weigh 1 mg of TP1 and dissolve it in 1 mL of distilled water, and scan it from 200 nm to 400 nm on a NanoDrop one Ultra-micro spectrophotometer (Thermo, USA). The functional groups of TP1 were scanned and analyzed using a Nicolet iZ-10 Fourier transform infrared spectrometer with an instrument resolution of 4.00 cm -1 , mix TP1 (2 mg) with KBr (200 mg) and compress it into a 1 mm tablet, and scan it in the range of 4000 - 400 cm -1 .

[0057] The ultraviolet-visible spectrum of TP1 ( Figure 1 C) showed no obvious absorption peaks at 260 nm and 280 nm, indicating that TP1 does not contain nucleic acids and proteins.

[0058] Fourier transform infrared spectroscopy is widely used for the qualitative analysis of polysaccharide functional groups. As Figure 1 shown in E, the absorption peaks at 3276 cm -1 and 2929 cm -1 were assigned to O-H and C-H stretching vibrations respectively. These two infrared absorption peaks (3276 and 2929 cm -1 ) are characteristic peaks of polysaccharides. The infrared absorption peak at 1646 cm -1 was caused by the stretching vibration of C=O; 1430 - 1328 cm -1 corresponded to the deformation vibration of the H-O-H bond and the bending vibration of -CH(O-CH2) respectively. The absorption peak at 1020 cm -1 was due to the stretching vibration of the C-O-C glycosidic bond. The absorption peaks at 934 cm -1 and 818 cm -1 corresponded to the symmetric stretching vibration of the furan ring and the deformation vibration of the carbon-hydrogen bond in the furan ring respectively.

[0059] 2. Monosaccharide composition analysis

[0060] Weigh 5 mg of the TP1 sample, add 1 ml of 2 M trifluoroacetic acid (TFA) solution, hydrolyze it at 121 °C for 2 h. Dry it with nitrogen. Repeat the washing with methanol 2 - 3 times and dry it with nitrogen. Add sterile water to dissolve it and transfer it to a chromatographic vial for measurement. Another method condition was hydrolysis at 60 °C for 1 h. Use a Thermo ICS 5000+ ion chromatography system (ICS 5000 + , Thermo Fisher Scientific), equipped with Dionex TM CarboPac TMPA20 (150×3.0 mm, 10 μm) liquid chromatography column; The single sugar components of TP1 were analyzed and detected using an electrochemical detector, with an injection volume of 5 μl. Mobile phase A solution is H2O (water), mobile phase B solution is 0.1 M NaOH solution, mobile phase C solution is 0.1 M NaOH solution and 0.2 M NaAc, and the flow rate is 0.5 ml / min; The column temperature is 30 °C.

[0061] Using monosaccharides as standards, by comparing the GC-MS ion chromatograms of the standard monosaccharides ( Figure 1 line a of D in Figure 1 ), hydrolysis at 60 °C ( Figure 1 line b of D in

[0062] 3. Determination of molecular weight

[0063] Preparation of standard solution: Accurately weigh 5 mg of each standard PEG, 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 injection vial.

[0064] Preparation of sample solution: Accurately weigh 10 mg of the sample, dissolve it in 1 ml of 0.1 M NaNO3 mobile phase solution to prepare a 10 mg / ml solution. After vortexing until the sample is dissolved, 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 injection vial.

[0065] Chromatographic method: Mobile phase: 0.1 M aqueous sodium nitrate solution; Chromatographic column: Shodex SB 805-803 tandem gel column (8×300 mm); Flow rate: 1 ml / min; Column temperature: 40 °C; Injection volume: 50 μL; Detector: Optilab T-rEX+Dawn HELEOSⅡ; Analysis time: 25 min.

[0066] Place the standard in the injection tray and analyze it using the GPC-RI-MALS chromatographic method. After the instrument is normalized by PEG21600 and 22000, the collected data is analyzed by Astra 6.1 software.

[0067] The method of the present invention uses the GPC-RI-MALS chromatographic method to determine the average molecular weight and uniformity 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.

[0068] In addition, as Figure 1 shown in F in

[0069] 4. Methylation analysis

[0070] TP1 was fully dissolved in DMSO, and the sample solution was mixed with NaOH and CH3I for methylation reaction. 1 mL of water and 2 mL of dichloromethane were added, vortex-mixed, centrifuged, and the aqueous phase was discarded. The washing was repeated 3 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 and evaporated to dryness at 30 °C. 50 μL of 2M ammonia water 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, dried with nitrogen, washed twice with 250 μL of methanol, and dried with nitrogen. 250 μL of acetic anhydride was added, vortex-mixed, and the reaction was carried out at 100 °C for 2.5 h. 1 mL of water was added and allowed to stand for 10 min, then 500 μl of dichloromethane was added, vortex-mixed, centrifuged, and the aqueous phase was discarded. The washing was repeated 3 times. Finally, the lower dichloromethane phase was removed and tested on the machine. Another treatment method is to add 100 μL of 1M TFA and react at 70 °C for 3 h.

[0071] The analytical instrument for this experiment is the 7890A-5977B gas chromatography-mass spectrometry instrument of Agilent Technologies Inc. (CA, USA).

[0072] The chromatographic system used is the Agilent gas chromatographic system (Agilent 7890A; Agilent Technologies, USA), an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), the carrier gas is high-purity helium (purity not less than 99.999%), the flow rate is 1.0 mL / min, and the temperature of the injection port is 260 °C. The injection volume is 1 μL, split injection, the split ratio is 10:1, and the solvent delay is 2.2 min.

[0073] The mass spectrometry system used was a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent, USA, equipped with an electron impact ion source (EI) and a MassHunter workstation. The electron impact ion source (EI), the injection port temperature was 230°C, the quadrupole temperature was 150°C, and the electron energy was 70 eV. The scanning mode was full scan mode (SCAN), and the mass scanning range (m / z): 30-600.

[0074] The hydroxyl groups of TP1 polysaccharide were methylated, hydrolyzed and reduced, and acetylated to sugar alcohol acetate. The prepared samples and methyl glycoside types were determined by gas chromatography-mass spectrometry and combined with mass spectrometry database analysis to obtain the types and molar ratios of methyl glycosides, and 7 connection modes were inferred. The results are shown in Table 1.

[0075] Table 1 Results of methylation analysis of TP1

[0076]

[0077] 5. NMR analysis

[0078] 50 mg of dried TP1 sample was taken and 0.6 mL (99.9%) of D2O was added to fully dissolve it. 1D-NMR ( 1 H and 13 C) and 2D-NMR spectra ( 1 H– 1 H COSY, NOESY, HSQC and HMBC), and data analysis was performed using MestReNova (6.1.0-6224) software.

[0079] 1 H NMR spectrum shows that the proton signals are concentrated in the range of δ3.3-5.4 ( Figure 2A ), 13 The signals of C NMR spectrum are concentrated in the region of δ55-110, and there is no peak signal between δ150-220, indicating that TP1 does not contain uronic acid ( Figure 2B ). There are multiple anomeric hydrogen signals in the δ4.3-5.4 region and multiple anomeric carbon signals in the δ99.5-104.5 region, indicating that TP1 has both α- and β-configurations, which is consistent with the results of infrared analysis. 1 H. 13 C.DEPT-135, 1 H- 1 H COSY, HSQC and HMBC NMR spectra of residues A, B, C, E, F, G1 The 13 H and C chemical shift assignments are shown in Table 2.

[0080] Four strong signals at δ 103.31, 103.76, 103.90, and 104.27 were found in the anomeric carbon region but disappeared in the DEPT-135 spectrum ( Figure 2C ), indicating that all four anomeric carbons are quaternary carbons. In addition, there are no hydrogen-related signals for them in the HSQC spectrum ( Figure 2E ). Based on the monosaccharide composition, combined with the methylation results and relevant literature reports, it can be inferred that the anomeric carbon signals δ 103.76, 103.90, 103.31, and 104.27 belong to the C-2 signals of the β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →1)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→ residues, and are labeled as residues A, B, C, and D in sequence. At the same time, the signals δ 60.48 and 62.53 are inverted peaks in the DEPT-135 spectrum, determined to be the C-1 and C-6 signals of fructose. Due to the HSQC correlation of C-1 with 3.80 ppm, it is considered that 3.80 ppm is the H-1 signal, and due to the HSQC correlation of C-6 with 3.61 ppm, it is considered that 3.61 ppm is the H-6 signal. From the COSY ( Figure 2D ) correlation of H-6 to 3.75 ppm, it is considered that 3.75 ppm is the H-5 signal. From the HMBC ( Figure 2F ) correlation of H-6 to 74.53 ppm, and the COSY correlation from 3.75 ppm to 4.01 ppm and the HSQC correlation from 4.01 ppm to 74.53 ppm indicate that 74.53 ppm and 4.01 ppm are the 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 the 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.

[0081] In addition, there are obvious correlation signals 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 β-D-Galp-(1→, →3,6)-β-D-Galp-(1→, →4)-α-D-Glcp-(1→ residues, and are labeled as residues E, F, and G in sequence.

[0082] β-D-Galp-(1→(residue E): The correlation signals of H-1 and 3.61 ppm are shown in the COSY spectrum, indicating that 3.61 ppm belongs to H-2. Correspondingly, because of the HSQC correlation from H-2 to 74.54 ppm, 74.54 ppm is considered as the C-2 signal. The HMBC correlation of H-1 to 68.56 ppm, the COSY correlation of 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, the COSY correlation of 3.78 ppm with H-3, and the HMBC correlation between 3.78 ppm and C-2 indicate that 3.78 ppm is the H-4 signal. Therefore, according to the HSQC correlation between 3.78 ppm and 72.54 ppm, 72.54 ppm is the C-4 signal. In addition, 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 as H-5 and C-5 signals. At the same time, H-4 shows an HMBC correlation with 62.58 ppm, indicating that 62.58 ppm is the C-6 signal. H-5 and 3.74 ppm show a good correlation, indicating that 3.74 ppm is considered as the H-6 signal.

[0083] →3,6)-β-D-Galp-(1→(residue F): The correlation signals of H-1 and 3.68 ppm were shown in the COSY spectrum, indicating that 3.68 ppm belongs to H-2. The HSQC correlation between H-2 and 73.87 ppm confirmed that 73.87 ppm is the C-2 signal. The HMBC correlation between H-1 and 77.64 ppm, the COSY correlation between H-2 and 4.08 ppm, and the HSQC correlation between 4.08 ppm and 77.64 ppm indicated that 4.08 ppm and 77.64 ppm are the H-3 and C-3 signals respectively. In addition, the COSY correlation between 4.02 ppm and H-3, and the HMBC correlation between 4.02 ppm and C-2 indicated that 4.02 ppm is the H-4 signal. Therefore, according to the HSQC correlation between 4.02 ppm and 75.86 ppm, 75.86 ppm is the C-4 signal. Based on the COSY correlation between H4 and 3.77 ppm, the HMBC correlation between 3.77 ppm and C-3, and the HSQC correlation between 3.77 ppm and 72.54 ppm, 3.77 ppm and 72.54 ppm were considered as the H-5 and C-5 signals. Meanwhile, the HMBC correlation between H-4 and 63.24 ppm indicated that 63.24 ppm is the C-6 signal. The good COSY correlation between H-5 and 3.89 ppm indicated that 3.89 ppm is considered as the H-6 signal.

[0084] →4)-α-D-Glcp-(1→(residue G): H-1 and 3.48 ppm are correlated in the COSY spectrum, indicating that 3.48 ppm is attributed 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 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, and 3.84 ppm is correlated with C-2 in the HMBC spectrum, indicating that 3.84 ppm is the H-4 signal. 72.54 ppm is found as the C-4 signal in the HSQC. In addition, 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. Meanwhile, H-4 is correlated with 62.13 ppm in the HMBC spectrum, and the signal δ62.13 is an inverted peak in the DEPT-135 spectrum, indicating that 62.13 ppm is the C-6 signal. H-5 and 3.75 ppm show good COSY correlation, indicating that 3.75 ppm is the H-6 signal.

[0085] The HMBC spectrum can provide long-range correlation signals between carbon atoms and hydrogen atoms, which are helpful for inferring the linkage order of each residue in the polysaccharide molecule. The HMBC spectrum of TP1 shows that the correlation signal δ3.61 / 103.76 (A C-2 / DH-6) indicates that C-2 of residue A is linked to H-6 of residue D; meanwhile, the correlation signal δ3.65 / 103.76 (A C-2 / C H-1) indicates that C-2 of residue A is linked to H-1 of residue C, and the correlation signal δ3.78 / 103.31 (C H-1 / C C-2) indicates that H1 of residue C is linked to C2 of residue C; the correlation signal δ3.60 / 104.27 (B H-6 / D C-2) indicates that H-6 of residue B is linked to C-2 of residue D. The correlation signal at δ3.84 / 103.31 (G H-4 / C C-2) indicates that H-4 of residue G is linked to C-2 of residue C. The correlation signal at δ5.30 / 71.65 (G H-1 / G C-4) indicates that H-1 of residue G is linked to C-4 of residue G. The correlation signal at δ5.30 / 77.64 (G H-1 / F C-3) indicates that H-1 of residue G is linked to C-3 of residue F, and the correlation signal at δ4.55 / 77.64 (F H-1 / FC-3) indicates that H-1 of residue F is linked to C-3 of residue F, and the correlation signal at δ3.46 / 103.67 (F H-6 / EC-1) indicates that H-6 of residue F is linked to C-1 of residue E.

[0086] Since fructose is easily decomposed under high-temperature conditions, the monosaccharide composition and methylation analysis cannot accurately reflect the proportion of monosaccharides in the sugar chain. Therefore, by means of 1 H and 13 C spectra, the proportion of each monosaccharide in TP1 was judged. In the 13 C 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 the proportion of residues A, B, C, and D in TP1 is approximately 2:2:8:2; the integral ratio of the C-1 signal peaks of residues E and F is approximately 1:1, indicating that the proportion of residues E and F in TP1 is approximately 1:1. In the 1 H spectrum, the integral ratio of the H1 signal peaks of E, F, and G to 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 total proportion of fructose is approximately 3:3:10:14. Therefore, it can be inferred that the proportion of residues A, B, C, D, E, F, and G in TP1 is approximately 2:2:8:2:3:3:10. The possible sugar chain structure of TP1 is as shown in Figure 3 shown.

[0087] Table 2 1 H and 13 C chemical shift δ (ppm)

[0088]

[0089] Example 2

[0090] Application of Thesium chinense polysaccharide, and the specific process is as follows:

[0091] The animal experiment procedures were approved by the Animal Ethics Committee Laboratory of Wannan Medical College (WNMC-AWE-2024301) and were carried out strictly in accordance with the regulations of the China Laboratory Animal Administration. C57BL / 6J mice (male, 8 weeks old, body weight 21±2 g) were purchased from Qinglongshan Laboratory Animal Center (Nanjing, Jiangsu, China) (license number: SCXK(Zhe)2024-0002). All SPF-grade mice were placed in an environment with a humidity of 50-55% and a temperature of 22-25°C, with a 12-h light-dark cycle, and had free access to food and pure water. All mice were humanely treated.

[0092] After 1 week of adaptive feeding, 12 mice were randomly selected as the normal control group and were fed with normal drinking water, serving as the Control group. Mice in other groups were continuously given water containing 3% DSS (w / v) for 5 days to induce acute colitis. From the 8th day, the body weight, fecal characteristics, and fecal blood level of the mice were recorded every day, and the disease activity index (DAI) was scored to determine whether the modeling was successful. The successfully modeled mice were randomly divided into 4 groups (n = 12), namely the DSS group, the TP1-L (100 mg / kg) group, the TP1-H (400 mg / kg) group, and the 5-aminosalicylic acid (5-ASA) (200 mg / kg) group ( Figure 4 in A). From the 13th day, mice in the Control group and the DSS group were given normal saline to drink freely, while the TPl-L group, the TP1-H group, and the 5-ASA group were given intragastric administration for 7 days simultaneously. On the 21st day of the experiment, the mice were anesthetized and euthanized, blood was taken from the orbital cavity, and mouse serum was extracted by centrifugation. The viscera, cecal contents, and colon tissues of all mice were collected and stored at -80°C. 1 cm of the distal colon was fixed in 4% paraformaldehyde.

[0093] 1. Evaluation of the severity of UC based on the disease activity index (DAI) score

[0094] In animal models, the DAI score has been widely used to evaluate the severity of UC. The body weight, fecal consistency, and fecal occult blood status of the mice were recorded every day, and these three parameters were scored according to the DAI scoring criteria. The DAI score is the combination of the scores of all the above parameters, and the disease activity index of each mouse was calculated.

[0095] The intervention effect of TP1 was revealed by recording the body weight of mice during modeling, observing fecal traits, calculating the DAI index, measuring the length of the colon and the weight of the spleen. The results showed that DSS administration caused continuous weight loss in mice of each group, which was consistent with the disease pathogenesis ( Figure 4 as shown in A), at the end of modeling, the DAI scores of all groups except the normal group (Control group) increased ( Figure 4 as shown in D), indicating that the UC mouse model was successfully established. The mice in the DSS group had severe fecal blood loss ( Figure 4 as shown in B), continuous weight loss ( Figure 4 as shown in C), and significantly increased DAI scores. After administration of TP1 and 5-ASA, the fecal blood loss and weight changes of mice were significantly improved. Especially in the TP1-H administration group, the weight loss was significantly reduced. The colon length of mice in the DSS group was significantly shortened ( Figure 4 as shown in E), and the spleen was significantly swollen ( Figure 4 as shown in F). After administration of TP1, the shortening of the colon length and the swelling of the spleen were significantly inhibited (P<0.05). It was shown that TP1 played a protective role in the colon inflammation of mice, TP1 could improve DSS-induced colitis, and could be used for the preparation of drugs for the treatment of colitis.

[0096] 2. ELISA and Oxidative Stress Assay

[0097] An enzyme-linked immunosorbent assay (ELISA) kit was used to detect the levels of inflammatory cytokines IL-6, IL-1β, TNF-α, and IL-17 in serum. Detection kits were used to detect the levels of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and myeloperoxidase (MPO), and the measurements were performed according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China).

[0098] Inflammatory factors play a central role in the pathogenesis of UC. The levels of these cytokines were measured by enzyme-linked immunosorbent assay. It was found that the levels of TNF-α, IL-17, IL-6, and IL-1β in mice with DSS-induced colitis were significantly increased ( Figure 5 as shown in C). The pro-inflammatory cytokines IL-6, TNF-α, and IL-1β have been shown to have a significant impact on the pathogenesis and exacerbation of UC. IL-17 inhibits the expression of intestinal epithelial tight junction proteins, increases intestinal permeability, promotes bacterial translocation and inflammation spread. Administration of TP1 to colitis mice effectively inhibited the increase in the levels of TNF-α, IL-17, IL-6, and IL-1β, but the effect of TP1 on the TNF-α level was almost not dose-dependent, which may be related to individual differences in mice. Generally speaking, these results indicate that TP1 has a significant anti-inflammatory effect and can regulate the inflammatory response of UC mice.

[0099] The severity of oxidative stress can be reflected by the activities of CAT and SOD and the contents of MPO and MDA. MPO mainly exists in neutrophils and can exacerbate mucosal damage. MPO is considered a biomarker of intestinal tissue injury and neutrophil infiltration. In this study, compared with the control group, the contents of MDA and the activity of MPO in the colon tissue of the DSS group were significantly increased, while the contents of CAT and the activity of SOD were significantly decreased. After administration of TP1, the increases in the activities of MDA and MPO and the decreases in the activities of CAT and SOD induced by DSS were significantly improved( Figure 5 in D), especially the results of the TP1-H group were better than those of the TP1-L group. Therefore, TP1 can reduce the abnormal secretion of inflammatory cytokines and oxidative stress response induced by DSS, thereby alleviating the symptoms of UC mice.

[0100] In summary, TP1 inhibits colonic oxidative stress and the levels of pro-inflammatory cytokines in DSS-induced colitis mice.

[0101] 3. Histopathological H&E and AB-PAS staining

[0102] Take 1.0 cm of distal colon tissue, fix it with 4% paraformaldehyde, and embed it in paraffin. Cut the colon into 5-μm sections and perform hematoxylin & eosin (H&E) and Alcian blue (AB) and Periodic acid Schiff (PAS) stain (AB-PAS) according to the standard procedure. Observe the differences between groups with an optical microscope (NI-U, NIKON).

[0103] HE staining was used to further evaluate colonic pathological damage( Figure 5 in A). The intestinal epithelium of mice in the normal group was intact and continuous, the goblet cells were arranged neatly, and there was no inflammatory cell infiltration. In the DSS group, the colonic epithelial cells were damaged (blue arrow), the mucosa was damaged (green arrow), the 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 alleviated, the epithelium was more intact, and the histological score was significantly reduced.

[0104] 4. Histopathological immunofluorescence staining

[0105] For immunofluorescence evaluation, the sections were dewaxed, fixed, and incubated with primary antibodies Claudin-1, ZO-1, and Occludin (1:500) at 4°C overnight. The sections were incubated with CY3-labeled goat anti-mouse (1:300) at room temperature in the dark for 50 min, and then counterstained with DAPI for nuclei and incubated at room temperature in the dark for 10 min. The stained sections were observed using a fluorescence microscope (NI-U, NIKO), and the average density of Claudin-1, Occludin, and ZO-1 was analyzed using ImageJ software.

[0106] The colonic mucus barrier is composed of colonic mucus mainly secreted by goblet cells and is the first line of defense for the host to protect the intestinal epithelium from invasion. AB-PAS staining can stain acidic mucins blue and mixed mucins blue-purple ( Figure 5 (B). The results showed that goblet cells in the DSS group were damaged and mucin secretion was significantly reduced compared with the normal group. Importantly, TP1 administration was able to attenuate DSS-induced goblet cell depletion, and the results of the TP1-H group were better than those of the TP1-L group. Then, the blue color in the images was quantified by ImageJ software. The blue intensity in the DSS group was significantly lower than that in the normal group, and the blue intensity increased significantly after TP1 administration, that is, TP1 treatment restored the number of goblet cells. These results indicate that TP1 has a protective effect on colon tissue and can alleviate tissue damage caused by UC.

[0107] 5. Western blot analysis

[0108] The colon tissue was washed with PBS, ground on ice, and then lysed in RIPA lysis buffer for 20 min. The lysate was centrifuged (13000 rpm) at 4°C for 20 min. The protein concentration of the supernatant was detected 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 with 5% skim milk powder in TBST at 25°C for 2 h, and the membrane was incubated with primary antibodies (Claudin-1, Occludin, ZO-1, and β-actin) at 4°C overnight, and then incubated with enzyme-labeled secondary antibodies at room temperature for 1 h. The protein blot imaging system (Amersham ImageQuant TM 800, Citiva) to analyze protein signals, and ImageJ software to quantify protein levels.

[0109] Immunofluorescence analysis results showed that the expression of Claudin-1, Occludin and ZO-1 was inhibited in the DSS group, indicating that DSS damaged the intestinal barrier (Figure 6 In A). The TP1 treatment group significantly maintained the colonic mucosal barrier by upregulating TJ expression to varying degrees. Among them, the TP1-H group had the largest positive expression areas 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, WB analysis was performed to verify the expression levels of Claudin-1, Occludin, and ZO-1 proteins again, so as to accurately evaluate the barrier function. Combining the results of three repetitions, it was found that compared with the control group, the DSS-treated mice showed a significant decrease in the expression of TJ proteins. However, after administration of TP1, the loss of Occludin, ZO-1, and Claudin-1 proteins was significantly reduced, and the effect was more significant in the TP1-H group( Figure 6 In B). This is consistent with the results obtained by immunofluorescence chemical analysis. At the same time, it is consistent with the result of rich goblet cells detected by H&E staining in the TP1-H group. In summary, these observations indicate that TP1 alleviates DSS-induced damage to the colonic tissue of mice and reduces the disruption of the intestinal barrier, thus preventing UC damage in mice.

[0110] 6. Analysis of intestinal flora composition

[0111] The analysis method of 16S rRNA is based on the inventors' previous research. The colonic contents of mice were collected, quickly frozen in liquid nitrogen, and then stored in a -80 °C refrigerator. Gene sequencing was detected by PANOMIX Biomedical Technology Co., Ltd. (Suzhou, China). The sequencing library was prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit, and 16S rRNA sequencing was performed on the Illumina MiSeq-PE250 platform (Illumina, USA).

[0112] In the alpha diversity analysis, for microbial community samples, the abundance rank curve can intuitively reflect the number of high-abundance and rare ASV / OTUs in the community( Figure 7 In A). According to the results of the Veen diagram, 3928, 2669, and 2935 independent OTUs were found in the Control group, DSS group, and TP1-H group respectively, indicating obvious changes in the intestinal microbiota composition among these groups( Figure 7 In B). To evaluate the effect of TP1 on the abundance and diversity of intestinal flora, the alpha diversity index was analyzed. The results showed that the DSS group decreased in the Chao1, Shannon, Simpson, and Observed_species indices Figure 7In (E), while TP1-H significantly restored these indices. Beta diversity analysis (PCoA and NMDS) was subsequently used to analyze the similarities and differences between the microbial communities. PCoA showed that the distribution directions of the DSS group and the TP1-H group were different and the distances were large, indicating that TP1 and DSS had significant effects on the intestinal microbiota of mice ( Figure 7 In (D), NMDS analysis further confirmed the above theory ( Figure 7 In (C). These results indicated that TP1 regulated the richness and diversity of the intestinal microbiota.

[0113] At the phylum level, the intestinal microbiota of mice was mainly composed of Firmicutes, Bacteroidetes, and Proteobacteria. Compared with the Control group, the ratio of Firmicutes to Bacteroidetes in the DSS group was significantly increased, indicating a certain degree of disorder in the intestinal microbiota of mice, but TP1 intervention could effectively regulate the F / B ratio ( Figure 7 In (F). At the genus level, according to the cluster heatmap analysis ( Figure 8A ), in the DSS group, the relative abundances of bacteria related to regulating immunity, protecting the intestinal mucosa, and promoting SCFA production, such as Ligilactobacillus, Alloprevotella, and Ruminococcus, were significantly lower than those in the Control group, while the relative abundances of Helicobacter, Adlercreutzia, and Desulfovibrio were significantly increased ( Figure 7 In (G-L)). After TP1 administration, the relative abundances of Ligilactobacillus and Alloprevotella were significantly increased, and the relative abundances of Helicobacter, Adlercreutzia, and Desulfovibrio were significantly decreased. Linear discriminant analysis effect size (LEfSe) was used to evaluate the hierarchy and abundance of the intestinal microbiota in each group, and the LDA score threshold was set at 4.0 ( Figure 8B 、 Figure 8C ). The results showed that DSS promoted the proliferation of some pathogenic bacteria, such as Adlercreutzia, Desulfovibrio, and Helicobacter. TP1-H enriched genera that could regulate immunity, protect the intestinal mucosa, and promote SCFA production, such as Ruminococcus, Ligilactobacillus, and Alloprevotella bacteria.

[0114] 7. Determination of short-chain fatty acids (SCFAs)

[0115] SCFAs in feces include acetate, propionate, butyrate, valerate, etc. The SCFA content was analyzed using a GC system (7890A, California, USA) equipped with a flame ionization detector and an HP-FFAP column (30m×0.32mm×0.25μm, Agilent Technologies, USA). The experiment was carried out using the following procedure: maintaining at 80°C for 0.5 min, heating from 80 - 150°C at a rate of 4°C / min, and then heating from 150 - 230°C at a rate of 20°C / min, and maintaining for 10 min. The signal was detected using a flame ionization detector at 300°C.

[0116] As the main products of gut microbiota fermentation, SCFAs are the main energy source of 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 are involved in maintaining intestinal homeostasis through GPR41 and GPR43 receptors. The q-PCR results showed that compared with the normal group, DSS significantly inhibited the expression of GPR41 and GPR43 genes in the colon tissue, and the administration of TP1 significantly increased the expression of GPR41 and GPR43 genes in the colon tissue ( Figure 8E ). This indicates that TP1 promotes the proliferation of some short-chain fatty acid-producing bacteria in the intestine to alleviate DSS-induced colitis.

[0117] 8. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR)

[0118] RT-PCR was used to detect the expression of GPR41 and GPR43 mRNA in the colon tissue. 50 mg of colon tissue from the blank 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 the ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan) and a gradient thermal cycler (Easy Cycle Gradient 96, AnalytikJena, Beijing, China). RT-PCR analysis was performed using a real-time fluorescence quantitative PCR instrument (Applied Biosystems QuantStudio 5, Shanghai, China). The primer sequences are shown in Table 3.

[0119] Table 3 Primer sequences for semi-quantitative RT-qPCR analysis

[0120]

[0121] 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 at *P<0.05 and **P<0.01, and ns indicated no significant difference.

[0122] In summary, a neutral polysaccharide was extracted and purified from Thesium chinense Turcz., and named TP1.

[0123] Structurally: Through monosaccharide composition analysis, methylation analysis, NMR nuclear magnetic resonance and other analyses, different from the polysaccharides of Thesium chinense Turcz. reported previously, the TP1 polysaccharide extracted and isolated in this study was found to be a neutral heteropolysaccharide with a narrow molecular weight distribution by molecular weight detection, and its molecular weight was 5377 Da. Monosaccharide composition analysis showed that it was mainly composed of fructose, glucose, and galactose, among which the fructose content was relatively high, and the molar ratio was 44.7:33.8:17.9. NMR nuclear magnetic resonance and other analyses found that 13 the signals of the C spectrum were mainly concentrated in the region of δ55-110, and there were no peak signals between δ150-220, indicating that TP1 did not contain uronic acid. It was verified by DEPT-135 spectrum analysis that TP1 did contain a relatively high proportion of fructan. The main chain of TP1 was 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→, and the side chain was mainly composed of β-D-Fruf-(2→6)-β-D-Fruf-(2→ connected to the C-6 position of →1,6)-β-D-Fruf-(2→ and β-D-Galp-(1→ connected to the C-6 position of →3,6)-β-D-Galp-(1→.

[0124] In terms of function: TP1, which is composed of different types of fructose, glucose, and galactose, has a complex structure and diverse biological activities. Studies have shown that TP1 can alleviate the symptoms of DSS-induced colitis in mice, including weight loss, increased DAI score, spleen swelling, colon shortening, etc. Compared with the prior art, TP1 protects the intestinal barrier by restoring the number of goblet cells, increasing the content of goblet cell glycoproteins and crypt mucins, and reducing the loss of tight junction proteins Claudin-1, Occludin, and ZO-1. In addition, 16sRNA sequencing analysis shows that TP1 promotes the production of SCFAs in the colon by improving the dysbiosis of the gut microbiota and enriching the genera of bacteria that can promote the production of SCFAs, such as Ruminococcus, Lactobacillus, and Bifidobacterium, and significantly increases the expression of GPR41 and GPR43 receptors in the colon tissue. TP1 reduces oxidative stress and the levels of inflammatory cytokines through the SCFAs-mediated GPR41 and GPR43 receptors to alleviate intestinal inflammation.

[0125] The description of the above embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A polysaccharide of Herba Lycopodii, characterized in that: The herbaceous lily polysaccharide is mainly composed of fructose, glucose and galactose, and the molar ratio of fructose: glucose: galactose is 44.7: 33.8: 17.

9.

2. The Herba Lysimachiae polysaccharide according to claim 1, characterized in that: Its weight-average molecular weight (Mw) is 5377 Da, and its number-average molar mass (Mn) is 4797 Da.

3. A method for preparing the Herba Lysimachiae polysaccharides according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: 1) crushing fresh hyssop, distilling and extracting with hot water, concentrating, centrifuging to remove precipitate, and then precipitating with anhydrous ethanol to collect the precipitate; 2) The precipitate was redissolved in distilled water, decolorized, and the protein was removed by Sevage reagent. After dialysis, crude polysaccharide was obtained; 3) After the crude polysaccharide is dissolved and filtered, it is then eluted with distilled water for separation and purification, and the eluate is freeze-dried; 4) The product solution of step 3) is separated on a gel column with distilled water, and the obtained eluate is freeze-dried to obtain the product.

4. The preparation method according to claim 3, characterized in that: The hot water distillation extraction in step 1) is specifically as follows: extracting twice with 80°C-90°C distilled water, with a solid-liquid ratio of 1:20, g / mL; each time for 2 hours, and combining the extracts.

5. The preparation method according to claim 3, characterized in that: The step 1) of precipitating with anhydrous ethanol means: precipitating with 4 times the volume of anhydrous ethanol at 4° C. for 12 hours, centrifuging for 10 minutes, and collecting the precipitate.

6. The preparation method according to claim 3, characterized in that: In step 2), the product was decolorized by D101 macroporous adsorption resin column, and the protein was removed by Sevage reagent (n-butanol: chloroform = 1:3 v / v); the product was dialyzed in deionized water for 48 h using a 1000 KDa dialysis bag, with the water changed every 4 h, and the product was concentrated and freeze-dried to obtain crude polysaccharide.

7. The preparation method according to claim 3, characterized in that: 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 loaded onto a DEAE-52 column for purification.

8. The preparation method according to claim 3, characterized in that: In step 4), separating the product solution of step 3) on a gel column using distilled water means separating on a Sephadex G-100 column using distilled water at a flow rate of 0.5 mL / min.

9. An application of the Herba Lysimachiae polysaccharides according to claim 1 or 2, characterized in that: Used for preparing medicines for preventing and treating colitis.

10. An application of the Herba Lysimachiae polysaccharides according to claim 1 or 2, characterized in that: For use in preparing food.

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