A morindae officinalis polysaccharide, a preparation method thereof and a medicinal use of the polysaccharide for treating inflammation

Abstract

The application belongs to the technical field of plant polysaccharides, and specifically discloses a morindae polysaccharide, a preparation method thereof and a pharmaceutical use of the morindae polysaccharide in treating inflammation. Monosaccharides in the morindae polysaccharide include mannose, glucuronic acid, galacturonic acid, glucose and arabinose, and the molar ratio is 0.5-0.6:0.3-0.5:1-2:1-2:0.5-1.5. The morindae polysaccharide provided by the application is a new type of structural uniform polysaccharide which is first isolated from morindae stems, has a slightly acidic pH value, clear monosaccharide composition, determined molecular weight and clear glycosidic bond linkage mode, has an anti-inflammatory effect and is expected to be developed into a new anti-inflammatory drug. The preparation process is mild, simple to operate and environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of plant polysaccharide technology, and specifically discloses a chicken blood vine polysaccharide, its preparation method, and its use as a drug for treating inflammation. Background Technology

[0002] Chicken Blood Vine ( Spatholobus suberectus As a classic traditional Chinese medicine for treating "Bi syndrome" (including rheumatic diseases such as RA in modern medicine), *Spatholobus suberectus* (also known as chicken blood vine) was recorded as having the effects of "invigorating blood and nourishing blood, and clearing the meridians" as early as the Qing Dynasty in the *Compendium of Materia Medica Supplement*. It has been widely used by physicians and in folk clinical practice throughout history to treat rheumatic pain such as rheumatoid arthritis. Domestic and international research on the chemical components of *Spatholobus suberectus* has largely focused on small-molecule active substances such as flavonoids and coumarins, while research on its polysaccharide components is relatively scarce. For example, Liao Jiawei et al. reviewed the chemical components and pharmacological effects of *Spatholobus suberectus*, and predicted the quality markers of *Spatholobus suberectus* based on the specificity of chemical components, medicinal properties, efficacy, blood-entry components, and component measurability. They showed that *Spatholobus suberectus* has complex components, mainly including flavonoids, phenylpropanoids, and phenolic acids, and possesses activities such as regulating the blood system, antioxidation, antitumor, and antiviral activity (Liao Jiawei et al., Research progress on chemical components and pharmacological effects of *Spatholobus suberectus* and prediction of its quality markers (Q-Marker) [J]. Chinese Traditional and Herbal Drugs, 2023, 54(20):6866-6877.); Qin Weiqi et al., through transcriptome analysis of different organs of *Spatholobus suberectus*, explored the regulation of catechin biosynthesis in *Spatholobus suberectus* (Qin Weiqi et al., Transcriptome analysis of different organs of *Spatholobus suberectus* and research on catechin biosynthesis genes [J / OL]. Chinese Journal of Traditional and Herbal Drugs, 1-11 [2025-06-12]). Zhang Shengjie et al. studied the true reasons for the phenotypic characteristics of *Spatholobus suberectus* slices from different levels through literature review, market research, origin investigation, original plant identification, medicinal material phenotypic identification, and ITS2 sequence molecular identification (Zhang Shengjie et al., Origin Investigation and Identification of Two Types of Commercially Available *Spatholobus suberectus* Slices [J / OL]. Chinese Medicinal Herbs, 2025, (02): 327-331 [2025-06-12].); Xiao Xiaoji et al. used a combination of HPLC fingerprinting, chemical pattern recognition, and multi-index component content determination to conduct a comprehensive quality study on *Spatholobus suberectus* medicinal materials from 15 populations in 5 source areas (Xiao Xiaoji et al., Comprehensive Evaluation of *Spatholobus suberectus* Germplasm Resource Quality Based on HPLC Fingerprinting Combined with Chemical Pattern Recognition and Multi-Indicator Component Content Determination [J]. Chinese Pharmaceutical Journal, 2023, 58(12): 1123-1132.).

[0003] Although studies have shown that *Spatholobi caulis* extract exhibits significant activity in anti-inflammatory and immunomodulatory aspects, for example, in modern pharmacological research, Zhang Aiwen et al. systematically studied the preparation methods and anti-inflammatory and antioxidant activities of *Spatholobi caulis* extracts of different polarities, and evaluated their safety. The study found that the water extract of *Spatholobi caulis* showed better anti-inflammatory and antioxidant effects and higher safety than the alcohol extract and acetone extract (Zhang Aiwen et al., Preparation and Pharmacological and Toxicological Study of *Spatholobi caulis* Extract [J]. Chinese Veterinary Medicine Journal, 2011, 30(03):20-22.); Y Pan et al. summarized the pharmacological effects of *Spatholobi caulis* extract and pure components in vitro and in vivo, such as antitumor, hematopoietic, anti-inflammatory, antidiabetic, antioxidant, antiviral and antibacterial effects, as well as other activities (Pan Y, Luo X, Gong P. *Spatholobi caulis*: A systematic review of its traditional uses, chemical constituents, biological activities and clinical applications [J]. Journal of Ethnopharmacology, 2023, 317(000):14.); AS Ravipati et al. used the DPPH free radical scavenging method and yeast model to study the antioxidant activity of *Spatholobus suberectus* (Ravipati AS, et al. Antioxidant and anti-inflammatory activities of selected Chinese medicinal plants and their relation with antioxidant content[J]. BMC Complementary and Alternative Medicine, 2012.); Zhang Liangrong used mouse gouty arthritis model, rat knee osteoarthritis model and mouse knee osteoarthritis model to evaluate the anti-arthritis effect of *Spatholobus suberectus* extract. The results showed that the water extract was superior to the ethanol extract in relieving joint swelling and reducing matrix metalloproteinase (MMP) levels, further confirming its significant anti-inflammatory activity (Zhang Liangrong, Study on the anti-arthritis effect of *Spatholobus suberectus* [D]. Zhejiang University, 2015.). Lai Hongfang et al. improved the extraction efficiency by optimizing the extraction process of chicken blood vine polysaccharide (Lai Hongfang et al., Research on extraction process of chicken blood vine polysaccharide [J]. Shizhen Guoyi Guoyao, 2010, 21(08):1872-1874.).

[0004] However, existing research has not systematically elucidated the material basis of the biological functions of *Spatholobus suberectus*, such as the fine structural characteristics of its polysaccharides, including monosaccharide composition, glycosidic bond linkage, and spatial conformation. Furthermore, it lacks in-depth exploration of the intrinsic mechanism linking its structure and biological activity. This limitation severely restricts the in-depth development and utilization of *Spatholobus suberectus* polysaccharides, preventing the full realization of the medicinal material's potential in the field of anti-inflammatory drugs.

[0005] From a practical application perspective, with the increasing incidence of inflammatory diseases year by year, the development of safe and effective anti-inflammatory drugs has become an urgent need in the pharmaceutical field. Currently used anti-inflammatory drugs suffer from significant side effects and drug resistance, making research on anti-inflammatory components derived from natural drugs a focus of attention. Chicken blood vine, as a plant with both medicinal and edible uses, is abundant and highly safe. Clarifying the material basis of its biological functions will provide crucial experimental evidence for the research and development of novel anti-inflammatory drugs or functional foods using chicken blood vine as a raw material, promoting its transformation from a traditional Chinese medicine to a high-value-added health product. Simultaneously, it will provide technical reference for the development and utilization of polysaccharide components in other Chinese medicinal materials, contributing to the modernization and internationalization of the traditional Chinese medicine industry, and possessing significant economic and social value. Summary of the Invention

[0006] In view of the shortcomings of existing technologies, this invention provides a *Spatholobus suberectus* polysaccharide, its preparation method, and its use as a pharmaceutical ingredient for treating inflammation. This invention obtains crude *Spatholobus suberectus* polysaccharide from the stems of *Spatholobus suberectus* via water extraction and alcohol precipitation. After two-stage purification, and combined with MH7A cell activity evaluation, *Spatholobus suberectus* polysaccharide SSP-Ⅱa was obtained. A multi-dimensional characterization system, including ultraviolet spectroscopy, infrared spectroscopy, Congo red assay, and NMR, ensured the purity and structural uniformity of the product, and its primary structure was successfully resolved. It possesses anti-inflammatory effects and holds promise for development into a novel anti-inflammatory drug.

[0007] In a first aspect, the present invention provides a *Spatholobus suberectus* polysaccharide, wherein the monosaccharides in the *Spatholobus suberectus* polysaccharide include mannose, glucuronic acid, galacturonic acid, glucose and arabinose, and the molar ratio of the monosaccharides is 0.5~0.6:0.3~0.5:1~2:1~2:0.5~1.5.

[0008] In some implementations, the weight-average molecular weight of chicken blood vine polysaccharide is 300-400 kDa.

[0009] In some embodiments, the *Spatholobus suberectus* polysaccharide comprises a structure as shown in formula (I):

[0010] Equation (Ⅰ).

[0011] Secondly, the present invention provides a method for preparing the aforementioned *Spatholobus suberectus* polysaccharide, comprising: extracting the stem of *Spatholobus suberectus* with hot water to obtain an extract; subjecting the extract to defatting, deproteinization, and alcohol precipitation sequentially, and collecting the precipitate obtained by alcohol precipitation; freeze-drying the precipitate to obtain crude polysaccharide; reconstitute the crude polysaccharide with water and then subjecting it to ion exchange chromatography purification and elution sequentially, collecting the first eluent; freeze-drying the first eluent to obtain a primary purified product; reconstitute the primary purified product with water and then subjecting it to molecular sieve chromatography purification and elution, collecting the second eluent; freeze-drying the second eluent to obtain *Spatholobus suberectus* polysaccharide.

[0012] In some implementation schemes, the temperature of the hot water is 90~100℃, the ratio of chicken blood vine stem to hot water is 1g:3~5mL, the hot water extraction time is 50~70min, and the extraction is performed 1~3 times.

[0013] In some implementation schemes, petroleum ether is used for degreasing.

[0014] In some implementations, the sevage method is used for protein removal.

[0015] In some implementation schemes, 95% v / v ethanol is used for precipitation, the mixture is allowed to stand overnight at 4 °C, the supernatant is discarded, and the precipitate is dissolved in pure water.

[0016] In some embodiments, the packing material for ion exchange chromatography is cellulose, preferably DEAE cellulose, and more preferably cellulose DE-52. The eluent for ion exchange chromatography is sodium chloride solution, the sample concentration is 8-12 mg / mL, and the elution flow rate is 1-3 mL / min.

[0017] In some embodiments, the packing material for molecular sieve chromatography is agarose gel, preferably Sepharose CL-6B. The eluent for molecular sieve chromatography is water, with a sample concentration of 8-12 mg / mL and an elution flow rate of 0.1-0.3 mL / min.

[0018] Thirdly, the present invention provides a composition comprising the aforementioned *Spatholobus suberectus* polysaccharide.

[0019] Fourthly, the present invention provides the use of the aforementioned chicken blood vine polysaccharide or composition in the preparation of a medicament for treating inflammation.

[0020] The beneficial effects of this invention are as follows: The *Spatholobus suberectus* polysaccharide provided by this invention is a novel, homogeneous polysaccharide with a slightly acidic pH, clearly defined monosaccharide composition, definite molecular weight, and clear glycosidic bond linkage, isolated for the first time from the stem of *Spatholobus suberectus*. It possesses anti-inflammatory effects and holds promise for development into a novel anti-inflammatory drug. Its preparation process is mild, simple, and environmentally friendly. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of SSP-Ⅱa polysaccharide from *Spatholobus suberectus* in Example 1 of the present invention. Figure 2 The results of screening inflammatory cell activity of the eluents from different concentrations of NaCl solution in Example 1 of this invention; Figure 3 The results of total sugar content determination of SSP-Ⅱa polysaccharide in *Spatholobus suberectus* in Example 1 of this invention; Figure 4 The results of the determination of uronic acid content of SSP-Ⅱa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention; Figure 5 The image shows the ultraviolet spectrum of SSP-Ⅱa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention. Figure 6 The above is the HPGPC spectrum of SSP-IIa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention. Figure 7 The results of the triple helix structure analysis of SSP-Ⅱa, a polysaccharide from *Spatholobus suberectus*, in Example 1 of this invention; Figure 8A The results of particle size distribution determination of SSP-Ⅱa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention; Figure 8B The results of the potential distribution measurement of SSP-Ⅱa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention; Figure 9 The infrared spectrum of SSP-Ⅱa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention; Figure 10 The results of monosaccharide composition analysis of SSP-IIa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention are as follows: Peak 1: mannose; Peak 2: glucuronic acid; Peak 3: galacturonic acid; Peak 4: rhamnose; Peak 5: glucose; Peak 6: galactose; Peak 7: arabinose. Figure 11A The SSP-IIa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention. 1 H NMR spectrum; Figure 11B The SSP-IIa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention. 13 C NMR spectrum; Figure 11C The HSQC spectrum of SSP-Ⅱa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention is shown below. Figure 11D The SSP-IIa polysaccharide from *Spatholobus suberectus* in Example 1 of this invention. 1 H- 1 H COSY spectrum; Figure 11E The HMBC spectrum of SSP-Ⅱa, a polysaccharide from *Spatholobus suberectus*, in Example 1 of this invention is shown below. Figure 12 This describes the linkage of the seven sugar residues in SSP-Ⅱa, a polysaccharide from the chicken blood vine, in Example 1 of this invention. Detailed Implementation

[0022] 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. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] See Example 1 Figure 1 The preparation steps of Spatholobus suberectus polysaccharide SSP-Ⅱa are as follows: (1) Weigh 100 g of *Spatholobus suberectus* stems precisely at a material-to-liquid ratio of 1:4 and add 400 mL of distilled water. Boil in a pot for 1 hour and collect the decoction. Repeat this operation 3 times, boiling for 45 minutes on the last time. Collect the extract and combine the three extracts. Concentrate the extract to 50 mL using rotary evaporation. Add petroleum ether to the concentrated extract for extraction and defatting, and collect the aqueous phase. Repeat this step 3 times and combine the defatted aqueous phases. Mix the aqueous phase with Sevage reagent (chloroform: n-butanol = 5:1) at a ratio of 1:4. After complete mixing, let stand for 1 hour and separate the liquid to remove proteins. Repeat this step 3 times. The processed extract was rotary evaporated at 70℃, and then the concentrated extract was mixed with anhydrous ethanol at a volume ratio of 1:4. After ethanol precipitation at 4℃ for 24 hours, the mixture was centrifuged at 4750 rpm for 15 min, filtered, and the precipitate was completely dissolved in pure water and then freeze-dried to obtain the freeze-dried powder of chicken blood vine crude polysaccharide.

[0024] (2) The freeze-dried powder of chicken blood vine crude polysaccharide was dissolved in pure water to form a 10 mg / mL crude polysaccharide solution, and then separated and purified by DEAE-52 anion exchange column chromatography (3.5 × 30 cm). Gradient elution was performed with 0, 0.1, 0.2 and 0.3 mol / L NaCl solutions at a flow rate of 2.0 mL / min. The polysaccharide content in the eluent was detected by phenol-sulfuric acid colorimetric method. The eluents of different concentrations of NaCl solutions were freeze-dried to obtain four components, namely SSP-Ⅰ eluted with 0 mol / L NaCl solution, SSP-Ⅱ eluted with 0.1 mol / L NaCl solution, SSP-Ⅲ eluted with 0.2 mol / L NaCl solution and SSP-Ⅳ eluted with 0.3 mol / L NaCl solution.

[0025] (3) The four fractions (SSP-Ⅰ, SSP-Ⅱ, SSP-Ⅲ, and SSP-Ⅳ) obtained after DEAE-52 anion exchange column chromatography were subjected to in vitro MH7A cell experiments to clarify their anti-inflammatory activity. This experiment included a Normal group, a Control group, and a drug-treated group (SSP-Ⅰ eluted with 0 mol / L NaCl solution, SSP-Ⅱ eluted with 0.1 mol / L NaCl solution, SSP-Ⅲ eluted with 0.2 mol / L NaCl solution, and SSP-Ⅳ eluted with 0.3 mol / L NaCl solution). Cells were maintained in a CO2 (5%) environment using fetal bovine serum (10%), penicillin, and streptomycin added to high-glucose medium (DMEM). It should be noted that the Normal group consisted of MH7A cells and 10% DMEM; the Control group consisted of MH7A cells, 10% DMEM and TNF-α (10 ng / mL); and the drug administration group consisted of MH7A cells, 10% DMEM, TNF-α (10 ng / mL) and a four-component chicken blood vine polysaccharide solution.

[0026] The specific experimental procedure was as follows: First, MH7A cells in the logarithmic growth phase were harvested, and the cell concentration was adjusted. 100 μL of cells were seeded into each well of a 96-well plate, with three replicates per group. The plates were incubated at 37°C with 5% CO2 for 3 hours until cell attachment. Then, TNF-α (10 ng / mL) was used to stimulate the cells, followed by the addition of *Spatholobus suberectus* polysaccharide solution. After culturing for another 24 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours. The absorbance was measured at 450 nm using a microplate reader. A lower absorbance value indicated lower cell viability and a stronger anti-inflammatory effect.

[0027] The results showed that the survival rate of MH7A cells was significantly reduced after administration of all four components, suggesting that *Spatholobus suberectus* polysaccharide can inhibit the growth of inflammatory cells. Meanwhile, the component with the best anti-inflammatory activity was SSP-II, as this component showed the most significant inhibitory effect on MH7A inflammatory cells. (See also...) Figure 2 The anti-inflammatory activity of the four components was screened using MH7A cells, and SSP-II was found to have the best anti-inflammatory activity.

[0028] (4) Dissolve SSP-II in pure water to a 10 mg / mL solution, then separate and purify it using an agarose CL-6B gel column (16×1000 mm), elute with pure water at a flow rate of 0.2 mL / min, freeze dry to obtain SSP-IIa.

[0029] I. Physicochemical Properties Analysis of SSP-IIa Polysaccharide from Chicken Blood Vine (1) The total sugar content of the chicken blood vine polysaccharide (SSP-Ⅱa) obtained in Example 1 was determined by the phenol-sulfuric acid method. Accurately weigh 10 mg of D-anhydrous glucose and place it in a 100 mL volumetric flask. Dissolve and dilute to volume with distilled water to obtain a glucose reference solution with a concentration of 0.1 mg / mL. Subsequently, pipette 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, and 1.0 mL of the prepared standard solution into 25 mL stoppered glass test tubes, respectively. Add distilled water to each tube to a final volume of 2.0 mL, then add 1.0 mL of 5% phenol solution and 5.0 mL of concentrated sulfuric acid solution. Mix well and heat in a boiling water bath (80 ℃) for 15 min. Remove and cool to room temperature. Using water as a blank control, measure the absorbance at a wavelength of 490 nm. Perform three parallel measurements. Plot a standard curve with glucose concentration (C) on the x-axis and absorbance (A) on the y-axis to obtain the regression equation and calculate the correlation coefficient (R).

[0030] Accurately weigh 5 mg of crude polysaccharide from *Spatholobus suberectus*, add distilled water to a 50 mL volumetric flask, accurately pipette 1 mL of the sample into a stoppered glass test tube, add water to make up to 2 mL, and measure the absorbance of the sample at 490 nm according to the standard curve plotting and determination method.

[0031] Establish the standard curve of total sugar from chicken blood vine as follows: Figure 3 The given value is y = 0.6497x + 0.1130, R. 2 =0.9992. Substituting the absorbance value of the crude polysaccharide from *Spatholobus suberectus* at 490 nm into the equation of the standard curve, the total sugar content of SSP-Ⅱa was calculated to be 63.62% (w / w).

[0032] (2) The uronic acid content of SSP-IIa polysaccharide obtained in Example 1 was determined by the m-hydroxybiphenyl method. The uronic acid content in *Spatholobus suberectus* polysaccharide was determined using the m-hydroxybiphenyl method. This method is simple, easy to perform, and highly specific, making it suitable for determining the uronic acid content in *Spatholobus suberectus* polysaccharide. Accurately weigh 5 mg of anhydrous galacturonic acid into a 10 mL volumetric flask, dissolve it in distilled water, and dilute to the mark to obtain a 0.5 mg / mL galacturonic acid standard solution. Accurately weigh 477 mg of sodium tetraborate into a 50 mL volumetric flask, dissolve it in concentrated sulfuric acid, and dilute to the mark. Accurately weigh 50 mg of solid sodium hydroxide into a 10 mL volumetric flask, dissolve it in distilled water, and dilute to the mark to obtain an aqueous sodium hydroxide solution. Weigh 15 mg of m-hydroxybiphenyl into a 10 mL volumetric flask, dissolve it in the freshly prepared sodium hydroxide solution, and dilute to the mark. Accurately measure 0 μL, 50 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, and 600 μL of 0.5 mg / mL galacturonic acid standard solution into 20 mL stoppered test tubes, add water to a final volume of 1 mL, add 5 mL of sodium tetraborate solution, shake well, heat in boiling water for 20 min, then remove and cool to room temperature. Add 50 μL of m-hydroxybiphenyl solution, shake well, and let stand at room temperature for 30 min. Measure the absorbance of the standard at 525 nm and plot the curve. Accurately weigh 5 mg of SSP-Ⅱa sample and place it in a 10 mL volumetric flask. Add distilled water to the mark and then pipette 400 μL into a 20 mL stoppered test tube. Add water to 1 mL and add 5 mL of sodium tetraborate solution. Shake well and heat in boiling water for 20 min. Then let it cool to room temperature. Add 100 μL of m-hydroxybiphenyl solution, mix well, and let it stand at room temperature for 30 min. Measure and record the absorbance of the sample at 525 nm.

[0033] Establish a standard curve for the sugar and acid content of chicken blood vine, as follows: Figure 4 The given value is y = 1.0048x + 0.0689, R. 2 =0.9996. Substituting the absorbance value measured at 525 nm for SSP-Ⅱa into the equation of the standard curve, the uronic acid content was calculated to be 36.33% (w / w). The instrument used was a SHIMADZU UV-2550 UV-Vis spectrophotometer.

[0034] (3) The UV absorption of the Spatholobus suberectus polysaccharide SSP-IIa obtained in Example 1 was determined by UV scanning. like Figure 5 As shown, the UV scan spectrum of the obtained chicken blood vine polysaccharide SSP-Ⅱa showed no absorption peaks of pigments, proteins, or nucleic acids. The instrument used was a SHIMADZU UV-2550 UV-Vis spectrophotometer with a scanning range of 200~800nm.

[0035] (4) The homogeneity and relative molecular weight of the SSP-IIa polysaccharide obtained in Example 1 were determined by HPGPC method. Test conditions: Agilent 1260 Infinity system; 0.05 mol / L phosphate (pH=6.7) buffer: acetonitrile = 79:21, column temperature 35℃; detection wavelength: 245 nm; injection volume 10 μL; flow rate 1.0 mL / min.

[0036] like Figure 6 As shown, the SSP-Ⅱa polysaccharide obtained in Example 1 was tested and found to be a homogeneous polysaccharide with a relative molecular mass of 324.74 kDa.

[0037] (5) The triple helix structure of the Spatholobus suberectus polysaccharide SSP-IIa obtained in Example 1 was determined by UV scanning. A 1 mg / mL solution of *Spatholobus suberectus* polysaccharide (SSP-Ⅱa) was prepared and mixed with Congo red solution (100 μM) at a 1:1 (v:v) ratio. Subsequently, NaOH solutions with concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5 mol / L were added, and the reaction solutions were scanned under UV light at a wavelength of 400–600 nm.

[0038] The triple-helix structure of SSP-IIa was characterized using the Congo red assay. Under alkaline conditions, polysaccharides with a triple-helix structure can complex with Congo red, and the maximum absorption wavelength of this complex exhibits a red shift compared to the Congo red solution. With increasing alkalinity, the maximum absorption wavelength of the polysaccharide-Congo red complex decreases as the triple-helix structure is disrupted. Figure 7 As shown, the maximum absorption wavelength of this complex is 509 nm, which shows a slight redshift trend compared to 500 nm in the Congo red solution, indicating that SSP-Ⅱa does not have a triple helix structure.

[0039] (6) The morphology of the surface ultrastructure of the chicken blood vine polysaccharide SSP-Ⅱa obtained in Example 1 was determined by scanning electron microscopy.

[0040] Take 2-3 mg of SSP-Ⅱa sample lyophilized powder and adhere it to the conductive adhesive on the sample stage. Place it in a vacuum coating instrument for gold sputtering, and then observe and record the image using a JSM.7001F thermal field emission scanning electron microscope.

[0041] The results showed that the *Spatholobus suberectus* polysaccharide exhibited a mostly sheet-like morphology with a smooth surface and uneven size under scanning electron microscopy at 2 kV and 5 kV. Microscopic gaps between molecules were also observed, indicating that the polysaccharide was not completely aggregated.

[0042] (7) The particle size distribution and potential analysis of the *Spatholobus suberectus* polysaccharide SSP-Ⅱa obtained in Example 1 were determined using a nanoparticle analyzer, as shown below. Figure 8A , Figure 8B As shown.

[0043] (8) The characteristic groups of the Spatholobus suberectus polysaccharide SSP-IIa obtained in Example 1 were analyzed using an infrared spectrometer, such as... Figure 9 As shown.

[0044] Approximately 2.0 mg of dried polysaccharide sample was weighed, mixed with 200 mg of KBr powder, compressed into a pellet, and then analyzed on an L1600400Spectrum TWO FT-IR Fourier transform infrared spectrometer with a scanning range of 4000 cm⁻¹. -1 ~400cm -1 .

[0045] Testing revealed that the Spatholobus suberectus polysaccharide SSP-IIa obtained in Example 1 exhibited characteristic peaks for polysaccharides, appearing at 3310 cm⁻¹. -1 The absorption peak at 2890 cm⁻¹ is due to the stretching vibration of the OH group in SSP-IIa. -1 The absorption peak at 1644 cm⁻¹ corresponds to the stretching vibration of CH in the methyl group, while the bending vibration of -COOH appears at 1644 cm⁻¹. -1 and 1416 cm -1 At this location, the asymmetric stretching vibrations of the COC and COH groups in SSP-Ⅱa are located at 1152 cm⁻¹. -1 and 1015 cm -1 The absorption peak at that location.

[0046] II. Chemical Structure Identification of Chicken Blood Vine Polysaccharide (1) The monosaccharide composition of the *Spatholobus suberectus* polysaccharide SSP-IIa obtained in Example 1 was analyzed by pre-column derivatization using PMP reagent HPLC, such as... Figure 10 As shown.

[0047] Weigh 20 mg of *Spatholobus suberectus* polysaccharide and dissolve it in ultrapure water (3 mL). Place the solution in a Schlenk tube and add trifluoroacetic acid (3 mL, 4 M). Stir the mixture at 110 °C for 5 hours. Then, transfer the acid-hydrolyzed product sample to a flask, add methanol solution, and rotary evaporate. Repeat the above operation 3 times until the trifluoroacetic acid is completely removed. Then dissolve the product in ultrapure water (2 mL) and allow it to derivatize. Pipette 1 mL of the hydrolysis solution into a test tube, and then add sodium hydroxide solution (1 mL, 0.3 M) and PMP methanol solution (1 mL, 0.5 M) respectively. Stir the mixture at 70 °C for 1 hour. After natural cooling, add hydrochloric acid solution (1 mL, 0.3 M). Then extract the solution 3 times with dichloromethane solution. Take 100 μL of the upper aqueous layer, dilute and mix it, and filter it through a membrane for monitoring.

[0048] Upon testing, the SSP-IIa polysaccharide obtained in Example 1 was found to be composed of mannose, glucuronic acid, galacturonic acid, glucose, and arabinose, with the molar ratio of mannose, glucuronic acid, galacturonic acid, glucose, and arabinose being 0.55:0.40:1.35:1.55:1.

[0049] (2) The chemical structural characteristics of the Spatholobus suberectus polysaccharide SSP-IIa obtained in Example 1 were determined by NMR. 30 mg of Spatholobus suberectus polysaccharide SSP-Ⅱa was completely dissolved in 99.9% D2O, loaded into an NMR tube, and subjected to NMR analysis.

[0050] Detection conditions: VNMRS600 superconducting nuclear magnetic resonance spectrometer; One NMR probe (5mm); 1 The H-spectrum operates at a frequency of 599.81 MHz. 13 The C-spectrum operating frequency was 150.84 MHz; measurements were taken at 25℃. 1 H spectrum, measured at 25℃ 13 C, COSY, HSQC, HMBC spectra.

[0051] like Figures 11A-11E As shown, the Spatholobus suberectus polysaccharide SSP-Ⅱa obtained in Example 1 was determined to be composed of →1)α-D-Man p -(4→,→4)-α-D-Glc p -(4→ / →4)-α-D-Glc p -(6→,→1) -α-D-Ara p， →4)-α-D-Glc p -(1→,→1)-α-D-GalA p -(4→,→1)-α-D-GlcA p -(4→,→4)-α-D-Man p -(1→ constitutes, where Figure 11A For the hydrogen spectrum ( 1 H NMR spectrum), Figure 11B For carbon spectrum ( 13 (C NMR spectrum) Figure 11C This is a heteronuclear single quantum relation spectrum (HSQC diagram). Figure 11D For homonuclear chemical shift correlation spectrum ( 1 H- 1 H COSY diagram). Figure 11E The HMBC diagram shows the carbon-hydrogen relationship of multiple bonds. The chemical shift analysis of the three sugar residues is shown in Table 1. The specific NMR spectral structure analysis is as follows: Table 1. NMR analysis of SSP-IIa polysaccharides from *Spatholobus suberectus*

[0052] Based on the above analysis, the following results can be obtained: The *Spatholobus suberectus* polysaccharide SSP-IIa has a uniform composition and a relative molecular mass of 324.74 kDa. Monosaccharide composition analysis shows that the *Spatholobus suberectus* polysaccharide SSP-IIa of this invention contains furan-type arabinose (Arap), pyran-type galacturonic acid (GalAp), pyran-type glucuronic acid (GlcAp), pyran-type glucose (Glcp), and pyran-type mannose (Manp). NMR analysis shows that the sugar residue configuration is α-type, and its primary structural unit contains →1)α-D-Man. p -(4→,→4)-α-D-Glc p -(4→ / →4)-α-D-Glc p -(6→,→1) -α-D-Ara p， →4)-α-D-Glc p -(1→,→1)-α-D-GalA p -(4→,→1)-α-D-GlcA p -(4→,→4)-α-D-Man p -(1→Seven sugar residues, the connection method of the seven sugar residues is as follows Figure 12 As shown.

[0053] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. The use of a *Spatholobus suberectus* polysaccharide or a composition containing *Spatholobus suberectus* polysaccharide in the preparation of a medicament for treating inflammation, characterized in that... The monosaccharides in the *Spatholobus suberectus* polysaccharide include mannose, glucuronic acid, galacturonic acid, glucose, and arabinose, with a molar ratio of 0.5~0.6:0.3~0.5:1~2:1~2:0.5~1.5; The weight-average molecular weight of the *Spatholobus suberectus* polysaccharide is 300-400 kDa; The *Spatholobus suberectus* polysaccharide comprises the structure shown in formula (Ⅰ): Equation (Ⅰ).

2. The application according to claim 1, characterized in that, The preparation method of the aforementioned chicken blood vine polysaccharide includes: The stems of *Spatholobus suberectus* were extracted with hot water to obtain an extract; the extract was then subjected to defatting, deproteinization, and alcohol precipitation, and the precipitate obtained from the alcohol precipitation was collected; the precipitate was then freeze-dried to obtain crude polysaccharide. The crude polysaccharide was reconstituted with water and then subjected to ion exchange chromatography for purification and elution. The first eluent was collected and then freeze-dried to obtain the first purified product. The purified product was reconstituted with water and then purified by molecular sieve chromatography and eluted. The second eluent was collected. The second eluent was then freeze-dried to obtain the chicken blood vine polysaccharide. The temperature of the hot water is 90~100℃, the ratio of the amount of chicken blood vine stem to hot water is 1g:3~5mL, the extraction time of the hot water is 50~70min, and the number of extractions is 1~3 times; The packing material for the ion exchange chromatography is DEAE cellulose, and the eluent is 0.1 mol / L sodium chloride solution; the packing material for the molecular sieve chromatography is Sepharose CL-6B, and the eluent is water.

3. The application according to claim 2, characterized in that, In the ion exchange chromatography, the sample concentration is 8-12 mg / mL, and the elution flow rate is 1-3 mL / min.

4. The application according to claim 2, characterized in that, In the molecular sieve chromatography, the sample concentration is 8~12 mg / mL, and the elution flow rate is 0.1~0.3 mL / min.

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