Preparation method and application of seabuckthorn acidic polysaccharide with high rhamnose content

Abstract

This invention belongs to the field of biomedical technology and discloses a method for preparing sea buckthorn acidic polysaccharide with high rhamnose content and its application. The sea buckthorn acidic polysaccharide is a homogeneous acidic heteropolysaccharide with a weight average molecular weight of 1.00 × 10⁻⁶. 6 The total sugar content is 90.7%, with rhamnose accounting for 53.83 mol%. Its preparation method includes raw material pretreatment, hot water reflux extraction, ethanol precipitation, deproteinization and decolorization, and purification using a DEAE-52 anion exchange column and a Sephadex G-200 gel column. This invention provides a mild and reproducible preparation process, yielding a well-defined sea buckthorn acidic polysaccharide with a triple helix conformation. Furthermore, it can significantly alleviate ulcerative colitis damage, downregulate the expression levels of pro-inflammatory factors TNF-α and IL-6, and regulate intestinal flora homeostasis. It is suitable for preparing drugs, functional foods, and dietary supplements for the prevention and treatment of colitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for preparing sea buckthorn acidic polysaccharide with high rhamnose content and its application. Background Technology

[0002] Ulcerative colitis (UC) is a core type of inflammatory bowel disease (IBD). Its main clinical manifestations include persistent inflammation of the colonic mucosa, recurrent diarrhea, mucus and bloody stools, and progressive weight loss. It is characterized by chronicity, relapse, and progressive exacerbation, which seriously reduces patients' quality of life. Furthermore, long-term illness significantly increases the risk of colorectal cancer.

[0003] Currently, clinical treatment for ulcerative colitis mainly relies on aminosalicylic acid drugs, glucocorticoids, immunosuppressants, and biologics. While these can alleviate inflammatory symptoms in the short term, they suffer from drawbacks such as the development of drug resistance with long-term use, high relapse rates after discontinuation, and significant systemic toxicity, failing to meet the clinical need for long-term safe medication. Therefore, developing natural active ingredients that are safe in origin, have a well-defined structure, a clear mechanism of action, and low toxicity has become a research hotspot in the prevention and treatment of ulcerative colitis.

[0004] Sea buckthorn is commonly used in traditional Chinese medicine for its spleen-strengthening, digestion-aiding, blood-activating, stasis-removing, cough-relieving, and phlegm-reducing properties. Studies have confirmed that it contains abundant bioactive components such as flavonoids, polyphenols, and polysaccharides. Among these, polysaccharides are considered to have immunomodulatory and anti-inflammatory potential. However, existing research has largely focused on crude polysaccharides or simple physicochemical characterization, lacking systematic studies on the structural confirmation of refined components and their relationship with anti-colitis effects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for preparing high-rhamnose-content sea buckthorn acidic polysaccharide and its application. This process is mild, reproducible, and scalable, and can isolate high-rhamnose-content sea buckthorn acidic polysaccharide SP-A with a clear source, distinct structural characteristics, stable composition, and good homogeneity. Simultaneously, the application and mechanism of action of sea buckthorn acidic polysaccharide SP-A in the prevention and treatment of ulcerative colitis are clarified, providing theoretical and experimental basis for its industrial development.

[0006] To achieve the above objectives, this invention provides a high-rhamnose-content sea buckthorn acidic polysaccharide, which is an acidic heteropolysaccharide. The monosaccharide composition of the sea buckthorn acidic polysaccharide includes rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, mannose, and fucose. The molar ratio of the monosaccharides is mannose:rhamnose:glucuronic acid:galacturonic acid:glucose:galactose:arabinose:fucose = 1.73:53.83:23.44:2.99:6.59:8.17:1.52:1.73.

[0007] A method for preparing sea buckthorn acidic polysaccharide with high rhamnose content is also provided, comprising the following steps: S1. Raw material pretreatment: The sea buckthorn fruit is dried and pulverized to obtain sea buckthorn dry powder, which is then degreased with petroleum ether and treated with ethanol to remove small molecule impurities, resulting in sea buckthorn filter residue. S2. Extraction of crude polysaccharides: Deionized water was added to the sea buckthorn filter residue, and the mixture was refluxed and centrifuged. The extract was concentrated, precipitated with ethanol, washed, and dried to obtain crude sea buckthorn polysaccharides. S3. Refining of crude polysaccharides: The crude polysaccharides of sea buckthorn are subjected to deproteinization and decolorization treatments in sequence to obtain refined crude polysaccharides; S4. Anion exchange column separation: The purified crude polysaccharide was prepared into an aqueous solution, centrifuged to remove impurities, and loaded onto an anion exchange column. NaCl solution was used for elution, the eluted fraction was collected, and after dialysis to remove salt and freeze-drying, SP-A fraction was obtained. S5. Gel column purification: SP-A group was prepared into an aqueous solution, loaded onto a gel column, eluted with distilled water as the mobile phase, the eluted fractions were combined, and freeze-dried to obtain the high rhamnose content sea buckthorn acidic polysaccharide.

[0008] Preferably, in step S1, drying is performed at 80°C, pulverization is performed by pulverizing through a 40-mesh sieve, and petroleum ether defatting is specifically performed by adding petroleum ether to the sea buckthorn dry powder, refluxing at 60°C for 1 hour, removing the petroleum ether by filtration, and repeating twice. Ethanol de-small molecule extraction is specifically performed by adding 95% ethanol solution, refluxing at 70°C for extraction, and repeating twice.

[0009] Preferably, in step S2, the reflux extraction is performed at 80°C for 1 hour; centrifugation is performed at 2000 r / min for 2 minutes; deionized water is added, and the reflux extraction and centrifugation steps are repeated 3 times; concentration is performed by rotary evaporation at 65°C; ethanol precipitation is performed by adding 4 times the volume of anhydrous ethanol, stirring to form a precipitate, then letting it stand at 4°C for 12 hours, and centrifuging at 5000 r / min for 5 minutes to separate the precipitate; washing is performed by washing with anhydrous ethanol.

[0010] Preferably, in step S3, the deproteinization is performed by the Sevag method; the decolorization is performed by AB-8 macroporous resin decolorization.

[0011] Preferably, in step S4, the aqueous solution concentration is 10 mg / mL; the centrifugation is 8000 r / min for 10 min; the anion exchange column is a DEAE-52 cellulose anion exchange column; the NaCl solution concentration is 0.2 mol / L; the elution flow rate is 2 mL / min; and the dialysis desalination time is 48 h.

[0012] Preferably, in step S5, the aqueous solution concentration is 5 mg / mL; the gel column is a Sephadex G-200 gel column; and when distilled water is used as the mobile phase for elution, the flow rate is 1.0 mL / min.

[0013] It also provides the application of high-rhamnose-content sea buckthorn acidic polysaccharide in the preparation of products for the prevention and treatment of colitis.

[0014] Preferably, sea buckthorn acidic polysaccharide reduces the expression levels of pro-inflammatory factors TNF-α and IL-6, thereby alleviating pathological damage to colonic tissue and improving the core symptoms of ulcerative colitis.

[0015] Preferably, sea buckthorn acidic polysaccharide plays a role in the prevention and treatment of colitis by improving the structure of intestinal flora, reducing the relative abundance of Proteobacteria, and increasing the relative abundance of Firmicutes, Bacteroidetes, and Verrucous Microbes, thereby regulating intestinal flora homeostasis.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: 1) The SP-A obtained by the directional separation of this invention is a homogeneous polysaccharide component with a total sugar content of 90.7%, a uronic acid content of 37.6%, and a rhamnose molar ratio of up to 53.83 mol%. This breaks through the technical bottleneck of complex composition, poor homogeneity, and unclear active ingredients of sea buckthorn crude polysaccharide in the prior art. The component has good batch stability and can achieve precise quality control.

[0017] 2) This invention systematically resolved the fine structure and spatial conformation of polysaccharides. Through techniques such as methylation-GC-MS and 600MHz two-dimensional nuclear magnetic resonance spectroscopy, the glycosidic bond linkage type, main chain backbone, branching sites and side chain structure of SP-A were clarified for the first time, confirming that it has a triple helix spatial conformation. The structure-activity relationship between polysaccharide structure and biological activity was established, laying a structural foundation for its activity mechanism research and industrial application.

[0018] 3) This invention not only verified the significant protective effect of SP-A against ulcerative colitis, but also clarified that it exerts its preventive and therapeutic effects through a dual mechanism of "inhibiting the overexpression of inflammatory factors + regulating intestinal flora homeostasis", providing sufficient experimental evidence for its clinical translation.

[0019] 4) SP-A of the present invention is derived from sea buckthorn, a plant that is both a food and a medicine. It has high biosafety and can be widely used in the development of drugs, functional foods, dietary supplements and other products related to the prevention and treatment of ulcerative colitis. It has extremely high market application value.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the extraction and purification process of sea buckthorn polysaccharide SP-A according to the present invention. Figure 2 Elution curve of sea buckthorn polysaccharide SP-A of the present invention on DEAE-52 anion exchange column; Figure 3 Elution curve of sea buckthorn polysaccharide SP-A of the present invention on a Sephadex G-200 gel column; Figure 4 This is the ultraviolet spectrum of sea buckthorn polysaccharide SP-A of the present invention; Figure 5 This is the infrared spectrum of sea buckthorn polysaccharide SP-A of the present invention; Figure 6 This is a high-performance gel permeation chromatogram of sea buckthorn polysaccharide SP-A of the present invention; Figure 7 The figure shows the monosaccharide composition of sea buckthorn polysaccharide SP-A of the present invention. In the figure, a is the HPLC chromatogram of the monosaccharide mixed standard derivative and b is the HPLC chromatogram of the monosaccharide composition of SP-A. Figure 8 This is a diagram showing the Congo Red test results of sea buckthorn polysaccharide SP-A of the present invention; Figure 9 The sea buckthorn polysaccharide SP-A of this invention 1 HNMR spectrum; Figure 10 The sea buckthorn polysaccharide SP-A of this invention 13 CNMR spectrum; Figure 11 This is the DEPT-135° spectrum of sea buckthorn polysaccharide SP-A of the present invention; Figure 12 This is the COSY spectrum of sea buckthorn polysaccharide SP-A of the present invention; Figure 13 This is the HSQC spectrum of sea buckthorn polysaccharide SP-A of the present invention; Figure 14 This is the HMBC spectrum of sea buckthorn polysaccharide SP-A of the present invention; Figure 15 This is the NOESY spectrum of sea buckthorn polysaccharide SP-A of the present invention; Figure 16 This is the TOCSY pattern of sea buckthorn polysaccharide SP-A of the present invention; Figure 17 This is a graph showing the effect of sea buckthorn polysaccharide SP-A on body weight changes in DSS-induced colitis mice. The sea buckthorn polysaccharide treatment group, compared to the blank control group, [shows a change in body weight]. represent represent represent represent P <0.0001; compared with the model group: # represent P <0.05, ## represent P <0.01, ### represent P <0.001, #### represent P <0.0001; Figure 18 This is a graph showing the effect of sea buckthorn polysaccharide SP-A of the present invention on the disease activity index (DAI) of DSS-induced colitis in mice. The sea buckthorn polysaccharide treatment group, compared with the blank control group, represent represent represent represent P <0.0001; compared with the model group: # represent P <0.05, ## represent P <0.01, ### represent P <0.001, #### represent P <0.0001; Figure 19 This is a comparison chart of colon length in mice of different groups (n=10). In the chart, a is a statistical analysis chart of colon length in each group of mice, and b is a representative physical image of colon in each group of mice. represent represent P <0.01; Figure 20 The images show H&E staining pathological images of colon tissue from mice in each group of the present invention (magnification: ×40, scale bar: 50 μm). In the figure, a is the H&E staining pathological image of different regions of colon tissue from mice in the Con group, b is the H&E staining pathological image of different regions of colon tissue from mice in the DSS group, and c is the H&E staining pathological image of different regions of colon tissue from mice in the SP-A group. Figure 21 This is a graph showing the detection levels of serum inflammatory factors TNF-α and IL-6 in mice of various groups according to the present invention. In the graph, a represents the serum IL-6 level of mice in each group, and b represents the serum TNF-α level of mice in each group. represent represent represent P <0.001; Figure 22 This is a graph showing the α-diversity analysis of the gut microbiota in mice of each group in this invention. In the graph, a is the statistical analysis graph of the ACE index of each group of mice, b is the statistical analysis graph of the Chao1 index of each group of mice, c is the statistical analysis graph of the Shannon index of each group of mice, and d is the statistical analysis graph of the Simpson index of each group of mice. represent represent represent represent P <0.0001; Figure 23 This is a PCoA analysis diagram of the intestinal flora structure of mice in each group of the present invention; Figure 24 This is a bar chart showing the relative abundance of gut microbiota at the phylum level in each group of mice in this invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0025] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0026] Example 1 like Figure 1 As shown, the preparation of sea buckthorn acidic polysaccharide SP-A is described.

[0027] S1. Pretreatment of sea buckthorn: Spread the Chinese subspecies of sea buckthorn fruit evenly on the iron plate in a vacuum drying oven, dry at 80℃ to constant weight, and place in a ventilated place for 24 hours to ensure complete removal of moisture; put the dried sea buckthorn fruit into a multi-functional pulverizer to crush, and sieve using a 40-mesh stainless steel sieve to collect sea buckthorn powder with a particle size of less than 40 mesh, and store it in a sealed container in a 4℃ refrigerator dry environment.

[0028] S2. Extraction of crude polysaccharides from sea buckthorn: Weigh 100g of sea buckthorn powder after constant weight, add 500mL of petroleum ether, reflux at 60℃ for 1h to defatt it, filter to remove the petroleum ether extract, and repeat the defatting step twice to fully remove lipid compounds; add 500mL of 95% ethanol solution to the defatted sea buckthorn powder, reflux at 70℃ for 1h, and repeat the extraction twice to fully remove small molecule impurities such as lipids, glycosides, phenols and water-soluble monosaccharides that are soluble in organic solvents.

[0029] The treated sea buckthorn residue was added to deionized water at a material-to-liquid ratio of 1:10 (g / mL), and extracted by reflux at 80°C for 1 hour in an oil bath. The extract was then divided into fractions and centrifuged at 2000 rpm for 2 minutes to separate the aqueous phase. The solid fraction was then subjected to the same water extraction process three times. All water extracts were combined and concentrated to less than 100 mL by rotary evaporation at 65°C until the liquid became viscous. Four times the volume of anhydrous ethanol was slowly added to the concentrate while stirring constantly to induce precipitation. The mixture was then allowed to stand at 4°C for 12 hours and centrifuged at 5000 rpm for 5 minutes. The precipitate was separated and repeatedly washed with anhydrous ethanol until the elution solution was colorless. After preliminary drying, the product was freeze-dried to obtain crude sea buckthorn polysaccharide, which was named SP.

[0030] S3. Isolation and purification of crude polysaccharides from sea buckthorn: The crude polysaccharide SP was deproteinized five times using the Sevag method. The deproteinized solution was then decolorized using an AB-8 macroporous resin column. A 10 mg / mL polysaccharide solution was then prepared. Insoluble matter was removed before centrifugation, followed by centrifugation at 8000 rpm for 10 min. The supernatant was loaded onto a DEAE-52 cellulose anion exchange column (2.6 cm inner diameter × 40 cm height). A gradient elution was performed sequentially using deionized water, 0.1, 0.2, 0.3, 0.5, and 1.0 mol / L NaCl solutions at a flow rate of 2 mL / min. 5 mL of eluent was collected from each tube. The eluent was monitored at 490 nm using the phenol-sulfuric acid method, and an elution curve was plotted (e.g., ...). Figure 2As shown in the figure, the elution peak liquids were collected separately to obtain six polysaccharide components: SP-0.0, SP-0.1, SP-0.2, SP-0.3, SP-0.5, and SP-1.0. Among them, the components eluted with NaCl were dialyzed and desalted for 48 hours, then concentrated and freeze-dried for later use. The remaining polysaccharides were freeze-dried for later use.

[0031] The main active component of sea buckthorn polysaccharide, SP-0.2, was further purified. SP-0.2 was dissolved in distilled water to prepare a 5 mg / mL aqueous solution, which was then loaded onto a Sephadex G-200 gel column (1.6 cm × 100 cm). Distilled water was used as the mobile phase, and the flow rate was controlled at 1.0 mL / min. One tube was collected for every 5 mL, for a total of 50 tubes. The sugar content of each tube was determined by the phenol-sulfuric acid method, and elution curves were plotted (e.g., ...). Figure 3 (As shown), the elution fractions from tubes 10-24 were combined and freeze-dried to obtain sea buckthorn fruit acidic polysaccharide SP-A.

[0032] Example 2 Structural identification of sea buckthorn acidic polysaccharide SP-A.

[0033] 1) Ultraviolet spectroscopy analysis.

[0034] A 0.1 mg / mL polysaccharide aqueous solution was prepared from SP-A sample, and the UV-Vis absorption spectrum was measured using a UV-Vis spectrophotometer in the scanning range of 200 nm–600 nm. The results are as follows: Figure 4 As shown, SP-A has no characteristic absorption peaks at 260 nm and 280 nm, indicating that the polysaccharide does not contain nucleic acid and protein impurities, which meets the purity requirements for subsequent structural analysis.

[0035] 2) Infrared spectroscopy analysis.

[0036] Take 2 mg of dried SP-A sample and grind it thoroughly with an appropriate amount of dried KBr, press it into a transparent thin film, and use a Fourier transform mid- / far-infrared spectrometer at 4000-400 cm⁻¹. -1 Infrared spectra were measured within the scanning interval. Results are as follows: Figure 5 As shown, 3200-3600cm -1 The strong absorption peak at 2920 cm⁻¹ is attributed to the stretching vibration of the OH groups within the polysaccharide molecule. -1 The absorption peak at 1400-1600 cm⁻¹ is attributed to the stretching vibration of the CH bond in polysaccharides, while the other two are characteristic absorption peaks of carbohydrates; -1 The strong absorption peak at that point is related to the carboxyl group (-COO). - The correlation with stretching vibrations confirms that SP-A is an acidic polysaccharide; 1000-1200cm -1The strong absorption peaks between the two peaks indicate that the polysaccharide backbone is linked by glycosidic bonds. Infrared spectroscopy results show that SP-A has the basic structure and acidic functional group characteristics of a polysaccharide.

[0037] 3) Molecular weight determination.

[0038] The molecular weight (Mw) distribution of SP-A samples was determined by high-performance gel permeation chromatography (HPLC) using an Agilent-1200 HPLC system (Agilent-1200 HPLC system, purchased from Agilent Technologies, USA), equipped with a TSK gel G-4000 PWxL column (7.8 mm × 300 mm) and a refractive index detector. 3 mg each of SP-A sample and dextran standards (T-10, T-40, T-70, T-110, T-500, T-2000) were dissolved in 1 mL of ultrapure water, filtered through a 0.22 μm filter membrane, and then analyzed. Ultrapure water was used as the mobile phase, with a flow rate of 0.6 mL / min and a column temperature of 30 °C.

[0039] A standard curve was plotted based on the peak times and molecular weights of the standards. The molecular weight of the SP-A sample was calculated, and the average molecular weight of sea buckthorn polysaccharide SP-A was determined by high-performance gel permeation chromatography (HPGPC). Figure 6 As shown), according to the standard curve log 10 MW = -0.339X + 9.1997, R 2 =0.9918; Calculations show that the weight-average molecular weight of SP-A is approximately 1.00 × 10⁻⁶. 6 Da.

[0040] 4) Monosaccharide composition analysis.

[0041] Acid hydrolysis of polysaccharides: Accurately weigh 5 mg of SP-A sample and add it to a reaction flask. Add 4 mL of 4M trifluoroacetic acid (TFA) under nitrogen atmosphere and seal the flask. Hydrolyze in an oven at 110 °C for 6 h. After hydrolysis, evaporate the solvent under vacuum, add methanol and evaporate to dryness. Repeat the operation 3 times to ensure complete removal of TFA. Add 0.5 mL of deionized water to prepare a 10 mg / mL polysaccharide hydrolysis solution.

[0042] Derivatization of hydrolyzed polysaccharides: Take 200 μL of polysaccharide hydrolysate, add 200 μL of 0.3M NaOH solution and 200 μL of 0.5M PMP methanol solution in sequence, mix well, and derivatize in a water bath at 70℃ for 2 h; after cooling to room temperature, add 200 μL of 0.3M HCl solution, then add an equal volume of chloroform, vortex for 2 min, centrifuge at 10000 r / min for 10 min, collect the upper aqueous phase, and repeat the extraction 3 times; take the upper aqueous phase, dilute 20 times, filter through a 0.45 μm filter membrane, and analyze by instrument.

[0043] Preparation and derivatization of monosaccharide standards: Prepare monosaccharide solutions and mixed monosaccharide standard solutions. Accurately weigh 2 mg of each monosaccharide (D-mannose, D-ribose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-glucose, D-galactose, D-arabinose, L-fucose monosaccharide) and dissolve it in 1 mL of deionized water. Take 200 μL of the monosaccharide solution and mixed monosaccharide standard solution, and perform derivatization treatment using the same method as for SP-A polysaccharide, followed by instrumental analysis.

[0044] Chromatographic conditions: An Essentia LC-16 high-performance liquid chromatography system (purchased from Shimadzu Corporation, Japan) was used for detection. A 10 μL solution was injected into an Extend-C18 column (4.6 mm × 250 mm, 5 μm, purchased from Agilent Technologies, USA) using a refractive index detector (RID). The mobile phase was phosphate buffer (0.1 mol / L, pH 6.8) and acetonitrile, with a gradient ratio of 83:17. The flow rate was 1.0 mL / min; the column temperature was 25 °C; the detector wavelength was 250 nm; and the injection volume was 10 μL. Based on the peak times of each monosaccharide in the mixed standard, the types of monosaccharides after polysaccharide hydrolysis were evaluated, and the molar ratio of each monosaccharide was calculated based on the peak area integral.

[0045] The results are as follows Figure 7 As shown, the elution order of the nine monosaccharide standards is D-mannose, D-ribose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-glucose, D-galactose, L-arabinose, and L-fucose.

[0046] Based on the retention time and peak areas of each peak in the sample, SP-A was found to be composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and fucose, with a molar ratio of 1.73:53.83:23.44:2.99:6.59:8.17:1.52:1.73. This indicates that polysaccharide SP-A is a heteropolysaccharide composed of multiple monosaccharides, with rhamnose as the main monosaccharide, while also containing a certain proportion of uronic acid monosaccharides, glucose, galactose, arabinose, and a small amount of fucose.

[0047] 5) Congo red staining test.

[0048] Prepare 80 mmol / L Congo red standard solution, 1 mg / mL SP-A solution, and NaOH solutions with concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. Take 1 mL of SP-A solution and 1 mL of Congo red solution, and add 1 mL of NaOH solution of different concentrations respectively. Mix well to achieve final NaOH concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L in the mixture. Replace the NaOH solution with deionized water as a blank control. After mixing, let stand at room temperature for 30 min, and measure the absorbance in the 400-600 nm range using a UV-Vis spectrophotometer. Plot a line graph of absorbance versus NaOH concentration.

[0049] The results are as follows Figure 8 As shown, when the NaOH concentration is in the range of 0-0.1 mol / L, the maximum absorption wavelength of SP-A gradually increases, reaching a peak at a NaOH concentration of 0.1 mol / L. As the NaOH concentration further increases, the triple helix structure is destroyed, and the maximum absorption wavelength gradually decreases, confirming that SP-A has a triple helix spatial conformation.

[0050] 6) Methylation-GC-MS analysis.

[0051] Weigh 5 mg of SP-A sample, dissolve it in 1 mL of distilled water, add 1 mL of 100 mg / mL CMC activator, and react for 2 h. Then add 1 mL of 2 M imidazole solution and 1 mL of 30 mg / mL NaBH4 solution, and react for 3 h. Finally, add 100 μL of glacial acetic acid to terminate the reaction. Dialyze the mixture and freeze-dry to obtain the carboxyl-reduced polysaccharide sample for subsequent experiments.

[0052] Take the reduced polysaccharide sample into a test tube, add 500 μL of DMSO to dissolve it; add 1 mg of NaOH powder and react for 30 min. Add 50 μL of CH3I and react in the dark for 1 h. Add 1 mL of distilled water and 2 mL of dichloromethane, vortex to mix, centrifuge to discard the aqueous phase, repeat the washing with water 3 times, collect the lower dichloromethane phase and blow dry with nitrogen.

[0053] Then add 100 μL of 2M TFA, react at 121℃ for 90 min, and evaporate to dryness at 30℃; add 50 μL of 2M ammonia and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, and blow dry with nitrogen; add 250 μL of acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h; add 1 mL of water and let stand for 10 min; add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the washing with water 3 times. Take the lower dichloromethane phase and analyze it.

[0054] GC-MS detection was performed with the following instrument parameters: injection port temperature 280℃, helium flow rate 1.0 mL / min, initial temperature 80℃, held for 2 min, then increased to 300℃ at a rate of 10℃ / min for 5 min, split ratio 10:1, and SH-Rxi-5Sil MS column used.

[0055] The methylation results of SP-A are shown in Table 1. SP-A polysaccharide is composed of sugar residues with six different linkages. SP-A is mainly composed of Glc p Rha p Gal p and GlcA p Composition. Among them, terminal Glc was detected. p 1→4-Gal p 1→2-Rha p and 1→4-Rha p Equal linear connection, with both →3,4)-GlcA present. p -(1→and→2,4)-Rha p -(1→ and other disubstituted residues indicate that SP-A is a heteropolysaccharide with a distinct branched structure.

[0056] Table 1. SP-A methylation results

[0057] 7) Nuclear magnetic resonance spectroscopy analysis.

[0058] 50 mg of pure polysaccharide SP-A was dissolved in 1 mL of D2O and lyophilized in a freeze dryer to exchange deuterium. The lyophilization was repeated twice, and the solution was then dissolved again in 0.5 mL of D2O and collected in an NMR tube. The polysaccharide solution was transferred to the NMR tube and detected using a 600 MHz NMR spectrometer at 25 °C to obtain a one-dimensional NMR spectrum. 1 H NMR, 13 C NMR (DEPT-135°) and two-dimensional NMR spectra (COSY, HSQC, HMBC, NOESY, and TOCSY).

[0059] 1 H NMR spectra show (e.g.) Figure 9 As shown), from 1In the ¹H NMR spectroscopy, multiple clear anodic signals (A1-F1) were observed in the anodic region (δH 4.9-5.3 ppm), with values ​​of 4.98, 5.21, 5.00, 4.89, 5.20, and 5.08 ppm, indicating that the polysaccharide contains at least six sugar residues. The chemical shifts of these anodic protons were generally around 5.0 ppm, and combined with their peak shape characteristics, this suggests that the polysaccharide is predominantly composed of α-glycosidic bonds, consistent with the common configuration characteristics of rhamnose and galactose in plant polysaccharides. Outside the anodic region, the signal was dense in the δH 3.2-4.5 ppm range, reflecting the overlap of numerous sugar ring protons (H-2 to H-6). This indicates that the differences in substitution sites between different monosaccharide residues lead to dispersed chemical shifts, a typical manifestation of multiple linkage modes.

[0060] 13 CNMR spectra show (e.g.) Figure 10 As shown), multiple sets of anomeric carbon signals appear in the δC range of 98-103 ppm, corresponding to the C-1 atoms of the six monosaccharide residues in the methylation at 100.5, 99.5, 101.7, 102.1, 102.8, and 98.2 ppm; the signal distribution is widespread in the δC region of 60-85 ppm, suggesting that the carbons in the sugar ring are substituted to varying degrees, and are related to →4)-Rha p -(1→、→2)-Rha p The structural characteristics of SP-A are consistent with those of 1→; the presence of uronic acid carbonyl characteristic signals in the δC range of 170-180ppm (located at 176.25, 175.62, and 171.06ppm) further confirms that SP-A is an acidic polysaccharide.

[0061] DEPT-135° chart (e.g.) Figure 11 In the spectrum shown, the positive phase peaks mainly correspond to CH and CH3 carbons, while the negative phase peaks correspond to CH2 carbons. Quaternary carbons without hydrogen are not displayed in this spectrum. Multiple distinct positive phase signals can be observed in the δC 98-103 ppm region. These signals belong to the anodic carbons of different monosaccharide residues, indicating that SP-A contains multiple sugar residues, and their anodic carbons are all CH-type. This is completely consistent with the hemiacetal carbon structure characteristics linked by glycosidic bonds in polysaccharides. Numerous positive phase peaks in the sugar ring region (δC 70-85 ppm) correspond to CH on the ring from C-2 to C-5. The large number and crowding of peaks reflect the complexity of substitution sites and residue types. The negative phase peaks (δC 60–66 ppm) generally correspond to the methylene-CH2 at the C-6 position of galactose or glucose. Rhamnose is a 6-deoxyhexose with CH3 at the C-6 position. The signal at δC 16.20 ppm belongs to the C-6 position of rhamnose. Carboxycarbon (δC 170-180ppm) is not displayed in DEPT-135.

[0062] Through HSQC (such as Figure 13 As shown), COSY (as shown) Figure 12 As shown), TOCSY (as shown) Figure 16 The carbon and hydrogen signals of each sugar residue in SP-A were fully assigned using the spectrum shown in Table 2. The results are shown in Table 2. HMBC (such as...) was used to... Figure 14 (as shown) and NOESY (as shown) Figure 15 Long-range correlation and spatial proximity correlation analysis of the spectrum (as shown) clarified the glycosidic bond linkage mode of the polysaccharide: the main chain backbone of SP-A is α-L-Rha p -(1→2) and α-L-Rha p -(1→4) is the main component, in →2,4)-α-L-Rha p -(1→ Residue sites undergo branching, and the side chain is mainly composed of α-D-GlcA p With α-D-Gal p The residues are composed of side chains that are linked to the main chain via (1→4) glycosidic bonds, forming a polysaccharide molecule with a branched structure.

[0063] Table 2 SP-A 1 H and 13 CNMR chemical shift assignment

[0064] Therefore, based on the above results, the SP-A structure is inferred to be: Example 3 Study on the alleviating effect of sea buckthorn polysaccharide SP-A on DSS-induced ulcerative colitis in mice.

[0065] 1) Experimental animals and model construction.

[0066] Thirty SPF-grade male C57BL / 6 mice, weighing 18-22g, were randomly divided into 3 groups after 7 days of acclimatization using a random number table. n=10 : Blank control group (Con group), 4% DSS model group (DSS group), polysaccharide intervention group (SP-A group).

[0067] Mice were acclimatized for 7 days before the experiment. After the acclimatization period, mice in the polysaccharide intervention group were given SP-A solution (0.1 mL / mouse) by gavage for 3 consecutive days to establish a polysaccharide pretreated intestinal protection model; mice in the blank control group and DSS model group were given an equal volume of physiological saline by gavage for 3 consecutive days during the same period.

[0068] After pretreatment, except for the blank control group, the drinking water of mice in all other groups was replaced with a solution containing 4% sodium dextran sulfate (DSS) to induce an acute colitis model. During the modeling period, mice in the polysaccharide intervention group were administered sea buckthorn polysaccharide SP-A solution by gavage daily, with the administration volume calculated at 0.005 mL / g body weight; mice in the blank control group and the DSS model group were administered an equal volume of physiological saline by gavage concurrently for 7 consecutive days.

[0069] During the experiment, the mice's mental state, activity level, and fur condition were observed and recorded at fixed times each day. Fecal characteristics and the presence of blood in the stool were also monitored, and body weight was measured daily. Based on changes in body weight, the severity of diarrhea, and blood in the stool, the Disease Activity Index (DAI) was calculated to comprehensively assess the severity of colitis. After the last administration, the mice were fasted and deprived of water for 12 hours, then sacrificed for sample collection and subsequent indicator testing.

[0070] 2) Effects of SP-A on the overall phenotype of colitis mice.

[0071] Weight changes and DAI score results as follows Figure 17 , Figure 18 As shown, compared with the Con group, the DSS model group mice experienced a significant decrease in body weight starting from day 3 of modeling, reaching their lowest body weight on day 7. P <0.0001 indicates that DSS successfully induced a significant disease state. Simultaneously, the DAI score in the DSS group continued to increase with the modeling time, and was significantly higher than that in the Con group after day 5. P <0.001, mice exhibited typical symptoms of ulcerative colitis such as diarrhea, bloody stools, and lethargy. Compared to the DSS model group, the SP-A group showed a significantly slower rate of weight loss, and its body weight was significantly higher than that of the DSS model group in the later stages of modeling. P <0.05, the DAI score was significantly lower than that of the DSS group ( P (<0.01), clinical symptoms were significantly improved, and the severity of the disease was reduced. The results indicate that SP-A has a significant protective effect against DSS-induced colitis in mice, can improve the general condition of mice at the overall level, reduce body weight loss, and effectively reduce the disease activity index.

[0072] Colon length measurement results as follows Figure 19 As shown, compared with the Con group, the colon length of mice in the DSS model group was significantly shortened ( P <0.01 indicates that DSS successfully induced an acute colitis model, with significant inflammatory damage occurring in the colonic tissue; compared with the DSS model group, the colon length of mice in the SP-A group was significantly prolonged ( PThe value was <0.05, indicating that sea buckthorn polysaccharide SP-A can effectively alleviate colonic shortening caused by DSS. Macroscopic morphological observation of the colon showed that mice in the DSS group exhibited significant shortening and thickening, along with pathological features such as congestion and edema. In contrast, the colons of mice in the SP-A group showed intact morphology, with significantly restored length compared to the DSS group, and the degree of tissue damage was improved to some extent, confirming that SP-A has a significant protective effect on colonic tissue.

[0073] The pathological results of H&E staining of colon tissue are as follows: Figure 20 As shown, the colonic tissue of mice in the Con group was intact, with continuous intestinal mucosal epithelium, clear crypt structures, normal goblet cell distribution, and no obvious inflammatory cell infiltration in the lamina propria, exhibiting normal histological morphology. Compared with the Con group, the colonic tissue of mice in the DSS group showed obvious pathological damage, mainly manifested as severe destruction of the intestinal mucosal epithelium, disordered or even absent crypt structures, significant edema in the mucosal and submucosal layers, and a large number of inflammatory cell infiltrations, indicating that DSS successfully induced typical pathological changes of acute ulcerative colitis. After SP-A intervention, the degree of pathological damage in the colonic tissue of mice was significantly reduced. Compared with the DSS group, the colonic mucosal structure of the SP-A group was relatively intact, the crypt arrangement was more regular, the inflammatory cell infiltration was significantly reduced, and the degree of mucosal edema was reduced, indicating that SP-A had a significant ameliorative effect on DSS-induced colonic tissue damage.

[0074] 3) Effects of SP-A on serum inflammatory factors in mice with colitis.

[0075] The expression levels of pro-inflammatory factors IL-6 and TNF-α in mouse serum were detected by ELISA. The results are as follows: Figure 21 As shown. Compared with the Con group, the serum levels of IL-6 and TNF-α in the DSS model group mice were significantly increased ( P <0.01, P The value <0.001 indicates that DSS successfully induced an inflammatory response in the body. Compared with the DSS model group, the serum IL-6 level in the SP-A group mice was significantly reduced ( P <0.01), sea buckthorn polysaccharide SP-A can effectively inhibit the abnormal increase of inflammatory factors induced by DSS; TNF-α level is significantly reduced ( P The value was <0.05, confirming that SP-A can significantly inhibit the pro-inflammatory response in mice with colitis and exert anti-inflammatory activity.

[0076] Based on the results of changes in body weight, DAI score, colon length, and serum inflammatory factors (IL-6, TNF-α), it can be inferred that sea buckthorn polysaccharide SP-A may play a role in alleviating DSS-induced colitis by inhibiting inflammatory response, reducing excessive infiltration of immune cells, and protecting the intestinal mucosal barrier structure.

[0077] 4) The regulatory effect of SP-A on the intestinal flora of colitis mice.

[0078] Colonic contents were collected from mice to analyze changes in gut microbiota structure. Results are as follows: Figure 22 As shown, compared with the Con group, the ACE index and Chao1 index of mice in the DSS group were significantly reduced ( P <0.0001 indicates that DSS treatment significantly weakened the species richness of the gut microbiota. DSS-induced colitis led to severe disruption of the gut microbiota structure, a change consistent with the decreased microbiota richness observed in ulcerative colitis patients and related animal models. Regarding community diversity, the Shannon index in the DSS group mice was significantly decreased, while the Simpson index was significantly increased (…). P The value <0.0001 indicates that DSS treatment not only reduced the number of bacterial species but also exacerbated the enrichment of dominant bacterial groups, leading to a more homogeneous and unbalanced community structure. These results demonstrate that DSS-induced intestinal inflammation is accompanied by a significant decrease in gut microbiota diversity. After SP-A intervention, all alpha-diversity indices of the mouse gut microbiota showed significant improvement. Compared to the DSS group, the SP-A group showed significantly increased ACE and Chao1 indices (…). P <0.0001 indicates that sea buckthorn polysaccharide can effectively alleviate the decrease in bacterial richness caused by DSS. Meanwhile, the Shannon index of the SP-A group was significantly higher than that of the DSS group, while the Simpson index was significantly lower ( P <0.0001), indicating that SP-A not only increases the number of bacterial species, but also improves the uniformity of community structure and promotes the recovery of the gut microbiota to a healthy state.

[0079] PCoA analysis results are as follows: Figure 23 As shown, PCoA analysis (PC1 and PC2) explained 29% and 25% of the variation in gut microbiota structure, respectively. The Con group samples showed good aggregation, indicating a relatively stable gut microbiota structure. The DSS group samples were significantly separated from the Con group, suggesting that DSS treatment significantly altered the overall composition of the mouse gut microbiota. Compared to the DSS group, the SP-A treated group samples showed a significantly closer distribution to the control group, confirming that SP-A can improve DSS-induced gut microbiota dysbiosis and promote the restoration of the microbiota structure to a normal physiological state.

[0080] The results of the relative abundance analysis at the phylum level are as follows: Figure 24 As shown, the gut microbiota of group Con mainly consists of the phylum Proteobacteria (… Proteobacteria Bacteroidetes ( Bacteroidetes ) and Firmicutes ( Firmicutes The composition of the gut microbiota is relatively stable, with the proportions of each major phylum being relatively stable, reflecting the balance of the microbiota structure under normal gut microecological conditions; DSS treatment significantly altered the phylum-level composition of the mouse gut microbiota. Compared to the Con group, the relative abundance of Proteobacteria was significantly increased, while the relative proportions of Bacteroidetes and Firmicutes changed markedly. Overall, this indicated abnormal proliferation of Proteobacteria and an imbalance in the dominant phylum structure, a pattern consistent with common gut microbiota dysbiosis in inflammatory bowel disease models. Previous studies have shown that excessive enrichment of Proteobacteria is closely related to enhanced intestinal inflammation, elevated oxidative stress, and disruption of the intestinal mucosal barrier. In the SP-A group, the phylum-level composition of the gut microbiota showed a clear regulatory trend. Compared with the DSS group, the relative abundance of Proteobacteria decreased significantly, while the proportions of Bacteroidetes and Firmicutes increased relatively. The overall microbiota structure recovered towards that of the Con group, confirming that SP-A can regulate the gut microbiota composition of colitis mice, inhibit the abnormal proliferation of inflammation-related pathogens, promote the growth of beneficial bacteria, and restore gut microecological homeostasis.

[0081] The above experimental results confirm that the sea buckthorn acidic polysaccharide SP-A prepared in this invention can exert a significant protective effect against DSS-induced ulcerative colitis in mice by inhibiting inflammatory responses and regulating intestinal flora homeostasis, and has excellent potential for the prevention and treatment of ulcerative colitis.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A high-rhamnose content acidic polysaccharide from sea buckthorn, characterized in that, Sea buckthorn acidic polysaccharide is an acidic heteropolysaccharide; its monosaccharide composition includes rhamnose, ribose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, mannose, and fucose; the molar ratio of monosaccharide composition is mannose:rhamnose:glucuronic acid:galacturonic acid:glucose:galactose:arabinose:fucose = 1.73:53.83:23.44:2.99:6.59:8.17:1.52:1.

73.

2. A method for preparing the high rhamnose content sea buckthorn acidic polysaccharide according to claim 1, characterized in that, Includes the following steps: S1. Raw material pretreatment: The sea buckthorn fruit is dried and pulverized to obtain sea buckthorn dry powder, which is then degreased with petroleum ether and treated with ethanol to remove small molecule impurities, resulting in sea buckthorn filter residue. S2. Extraction of crude polysaccharides: Deionized water was added to the sea buckthorn filter residue, and the mixture was refluxed and centrifuged. The extract was concentrated, precipitated with ethanol, washed, and dried to obtain crude sea buckthorn polysaccharides. S3. Refining of crude polysaccharides: The crude polysaccharides of sea buckthorn are subjected to deproteinization and decolorization treatments in sequence to obtain refined crude polysaccharides; S4. Anion exchange column separation: The purified crude polysaccharide was prepared into an aqueous solution, centrifuged to remove impurities, and loaded onto an anion exchange column. NaCl solution was used for elution, the eluted fraction was collected, and after dialysis to remove salt and freeze-drying, SP-A fraction was obtained. S5. Gel column purification: SP-A group was prepared into an aqueous solution, loaded onto a gel column, eluted with distilled water as the mobile phase, the eluted fractions were combined, and freeze-dried to obtain the high rhamnose content sea buckthorn acidic polysaccharide.

3. The preparation method according to claim 2, characterized in that, In step S1, drying is done at 80°C, and pulverization is done by passing the powder through a 40-mesh sieve. Petroleum ether defatting is specifically done by adding petroleum ether to the dried sea buckthorn powder, refluxing at 60°C for 1 hour, removing the petroleum ether by filtration, and repeating twice. Ethanol de-small molecule extraction is specifically done by adding 95% ethanol solution, refluxing at 70°C, and repeating twice.

4. The preparation method according to claim 2, characterized in that, In step S2, reflux extraction was performed at 80℃ for 1 hour; centrifugation was performed at 2000 r / min for 2 minutes; deionized water was added, and the reflux extraction and centrifugation steps were repeated 3 times; concentration was performed by rotary evaporation at 65℃; ethanol precipitation was performed by adding 4 times the volume of anhydrous ethanol, stirring to form a precipitate, then letting it stand at 4℃ for 12 hours, and centrifuging at 5000 r / min for 5 minutes to separate the precipitate; washing was performed with anhydrous ethanol.

5. The preparation method according to claim 2, characterized in that, In step S3, the deproteinization is performed using the Sevag method; the decolorization is performed using AB-8 macroporous resin.

6. The preparation method according to claim 3, characterized in that, In step S4, the aqueous solution concentration was 10 mg / mL; the centrifugation was performed at 8000 r / min for 10 min; the anion exchange column was a DEAE-52 cellulose anion exchange column; the NaCl solution concentration was 0.2 mol / L; the elution flow rate was 2 mL / min; and the dialysis desalination time was 48 h.

7. The preparation method according to claim 2, characterized in that, In step S5, the aqueous solution concentration was 5 mg / mL; the gel column was a Sephadex G-200 gel column; and the flow rate was 1.0 mL / min when using distilled water as the mobile phase for elution.

8. The application of the high rhamnose content sea buckthorn acidic polysaccharide according to claim 1 in the preparation of products for the prevention and treatment of colitis.

9. The application according to claim 8, characterized in that, Sea buckthorn acidic polysaccharide reduces the expression levels of pro-inflammatory factors TNF-α and IL-6, alleviates pathological damage to colon tissue, and improves the core symptoms of ulcerative colitis.

10. The application according to claim 8, characterized in that, Sea buckthorn acidic polysaccharides play a role in the prevention and treatment of colitis by improving the structure of the intestinal flora, reducing the relative abundance of Proteobacteria, and increasing the relative abundance of Firmicutes, Bacteroidetes, and Verrucous Microbes, thereby regulating the homeostasis of the intestinal flora.

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