Radix sophorae tonkinensis polysaccharide, and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of radix sophorae tonkinensis polysaccharide and its preparation method and application.The preparation method comprises the following steps: radix sophorae tonkinensis medicinal material is defatted, water extraction is concentrated, then sequentially subjected to step-by-step enzymolysis by pectinase and cellulase, deproteinization by Sevag method, alcohol precipitation, macroporous resin decolorization and ultrafiltration fractionation, collect the cut-off liquid with molecular weight of 8-300 kDa, and dry.The preparation method of the present application is stable, efficient and controllable, the radix sophorae tonkinensis polysaccharide prepared has high purity, good safety, clear anti-inflammatory and immunoregulatory activity, and wide application prospect, and can be used for preparing anti-inflammatory drugs or immunoregulators.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a Sophora tonkinensis polysaccharide, its preparation method, and its application. Background Technology

[0002] Inflammation is the body's defensive response to harmful stimuli. Imbalance in its regulation can trigger various acute and chronic inflammatory diseases, including inflammatory lung diseases, autoimmune inflammation, and intestinal inflammation. Its pathogenesis is closely related to abnormal immune cell activation, excessive secretion of inflammatory factors, and oxidative stress imbalance, leading to tissue damage and functional disorders, severely impacting patients' physical and mental health and posing significant challenges to clinical treatment. Currently, clinical treatment for inflammatory diseases primarily relies on glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs). While these drugs can rapidly relieve inflammatory symptoms, they suffer from poor targeting, significant systemic side effects (such as gastrointestinal damage, immunosuppression, and liver and kidney dysfunction), and the potential for drug resistance and dependence with long-term use. These limitations hinder precise regulation and long-term relief of inflammation, failing to meet the actual needs of clinical treatment.

[0003] Sophora tonkinensis, the dried root and rhizome of the legume Sophora japonica, is a traditional Chinese medicine. The *Kaibao Materia Medica* records its ability to "detoxify various poisons, relieve pain, and reduce swelling and boils." The *Chinese Pharmacopoeia* states that it enters the lung and stomach meridians, possessing the effects of clearing heat, detoxifying, reducing swelling, and relieving pain. Modern research confirms that its main active ingredients include alkaloids, polysaccharides, and flavonoids. Among them, Sophora tonkinensis polysaccharides, as one of the core active ingredients, have significant anti-inflammatory, antioxidant, and immunomodulatory biological activities. It exerts its anti-inflammatory effects by inhibiting the secretion of inflammatory factors such as TNF-α, IL-1β, and IL-6, increasing the survival rate of inflammatory cells, scavenging free radicals in the body, and regulating immune cell function. Sophora tonkinensis polysaccharides are of natural origin, have high biosafety, and low toxicity, offering unique advantages compared to chemical anti-inflammatory drugs, and have become one of the research hotspots in the field of natural anti-inflammatory drugs.

[0004] However, existing research on Sophora tonkinensis polysaccharides mainly focuses on the basic activity verification of crude extracts, which has shortcomings such as low extraction purity, unclear active substances, insufficient efficacy, and unstable process that makes it difficult to industrialize, thus limiting its progress towards clinical application and industrialization. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a Sophora tonkinensis polysaccharide, its preparation method, and its applications. This invention effectively overcomes the shortcomings of traditional crude extracts, such as low purity, unclear active substances, and difficulty in scaling up the process, through an integrated process encompassing defatting, water extraction, stepwise enzymatic hydrolysis with dual enzymes, Sevag deproteinization, alcohol precipitation, resin decolorization, and two-stage ultrafiltration purification.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing Sophora tonkinensis polysaccharide includes the following steps:

[0008] (1) Degreasing: Take dried Sophora tonkinensis root, soak it in ethanol to degrease it, separate the solid and liquid to obtain degreased residue;

[0009] (2) Water extraction: The defatted drug residue is heated and refluxed with water for extraction, filtered, and the extract is concentrated to obtain a concentrated solution;

[0010] (3) Enzymatic hydrolysis: Pectinase and cellulase are added to the concentrate for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymes are inactivated, centrifuged, and the supernatant is collected.

[0011] (4) Sevag method for deproteinization: The supernatant was treated with a mixture of chloroform and n-butanol, centrifuged, and the aqueous phase was collected;

[0012] (5) Alcohol precipitation: After concentrating the aqueous phase, add ethanol to the alcohol concentration to 70%~85% (volume fraction), let stand, and collect the precipitate by centrifugation;

[0013] (6) Resin decolorization: Dissolve the precipitate in water, add AB-8 macroporous adsorption resin for adsorption and decolorization, filter, and collect the filtrate;

[0014] (7) Ultrafiltration purification: The filtrate is fractionated by ultrafiltration membrane, and the retentate with a molecular weight greater than 8 kDa and less than 300 kDa is collected and dried to obtain the Sophora tonkinensis polysaccharide.

[0015] Further, in step (1), the ethanol is 95% ethanol by volume, and the soaking and degreasing time is 20-28 hours.

[0016] Further, in step (2), the heating and reflux extraction is performed 1 to 3 times, each time for 1 to 3 hours; the concentration is performed under reduced pressure to a relative density of 1.05 to 1.15 at 60°C.

[0017] Further, in step (3), the enzymatic hydrolysis specifically involves: first adjusting the pH of the concentrated solution to 6.0~7.0, adding pectinase, and hydrolyzing at 45~55℃ for 2~5 hours; then adding cellulase and continuing hydrolysis at 45~55℃ for 1~3 hours; the amount of pectinase added is 0.1%~0.5% of the weight of the dried Sophora tonkinensis root material, and the amount of cellulase added is 0.2%~1.0% of the weight of the dried Sophora tonkinensis root material.

[0018] Further, in step (4), the volume ratio of chloroform to n-butanol in the mixture of chloroform and n-butanol is (3~5):1; the treatment includes shaking and centrifugation, and the operation is repeated until there is no obvious denatured protein layer at the interface.

[0019] Further, in step (5), the alcohol concentration is 80%, and the standing is overnight at 4°C; in step (6), the volume of the AB-8 macroporous adsorption resin added is 1 / 10 to 1 / 5 of the volume of the solution after the precipitate is dissolved, and the adsorption conditions are room temperature shaking adsorption for 8 to 16 hours.

[0020] Further, in step (7), the ultrafiltration membrane grading specifically involves: firstly, the filtrate is subjected to a first-stage ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 300 kDa, and the permeate is collected; then, the permeate is subjected to a second-stage ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 8 kDa, and the retained liquid is collected; the drying is freeze drying.

[0021] A polysaccharide from Sophora tonkinensis prepared by the above method, wherein the molecular weight of the polysaccharide is distributed between 8 kDa and 300 kDa.

[0022] This invention provides the application of the aforementioned Sophora tonkinensis polysaccharide in the preparation of anti-inflammatory drugs or immunomodulators.

[0023] In this invention, the anti-inflammatory drug is a drug for treating inflammatory lung diseases; the immunomodulator is an immunomodulator that regulates macrophage polarization.

[0024] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] (1) This invention adopts a synergistic enzymatic hydrolysis strategy of stepwise feeding of pectinase and cellulase. First, pectinase is used to hydrolyze the outer pectin matrix to reduce viscosity and expose the cellulose skeleton. Then, cellulase is used to efficiently hydrolyze and break down the cell wall, which significantly promotes the release of intracellular active polysaccharides and greatly increases the yield of polysaccharides in the target molecular weight range. Then, the protein is removed by Sevag method and the product is decolorized by AB-8 macroporous resin to deeply remove impurities such as proteins and pigments. The purity of the finished polysaccharide can reach 85.6%. By using ultrafiltration membranes with molecular weight cutoffs of 300 kDa and 8 kDa for two-stage fractionation, the active components with molecular weights of 8 to 300 kDa are accurately enriched, and inactive or low-activity impurities such as oligosaccharides, monosaccharides, salts, and macromolecular nucleic acids and proteoglycans are eliminated, making the pharmacodynamic material basis clear, the product composition clear, and easy to control for industrialization.

[0026] (2) The present invention has high biocompatibility. Within the concentration range of 1.56 to 200 μg / mL, the Sophora tonkinensis polysaccharide of the present invention has no significant cytotoxicity to mouse alveolar macrophages MH-S, does not abnormally interfere with normal nitric oxide (NO) metabolism, and has no risk of inducing excessive NO production. Its safety is superior to that of glucocorticoids and nonsteroidal anti-inflammatory drugs with significant side effects from long-term use.

[0027] (3) The present invention exhibits significant anti-inflammatory and immunomodulatory activities. In the LPS-induced MH-S cell inflammation model, the Sophora tonkinensis polysaccharide of the present invention can dose-dependently inhibit excessive NO production, downregulate the expression of the pro-inflammatory factor TNF-α protein, and simultaneously regulate the level of the anti-inflammatory factor Arg-1 in a concentration-dependent manner, demonstrating a bidirectional regulatory effect and restoring inflammatory homeostasis. Transcriptome analysis further revealed that it can induce systemic changes in the gene expression profile of MH-S cells, significantly regulate chemotaxis, chemokine-mediated signaling pathways, and core inflammatory signaling pathways such as NF-κB, TLR4, IL-17, and TNF, and restore key protective genes such as Ccl7, Cxcl3, and IL11. Through multi-pathway and multi-target synergistic effects, it exerts anti-inflammatory and immunomodulatory functions, providing a strong scientific basis for its development into an anti-pulmonary inflammatory disease and immunomodulator.

[0028] In summary, the preparation method of this invention is stable, efficient and controllable, and the resulting Sophora tonkinensis polysaccharide has high purity, good safety, and clear anti-inflammatory and immunomodulatory activities, showing broad application prospects. Attached Figure Description

[0029] Figure 1 This is a figure showing the effect of different concentrations of Sophora tonkinensis polysaccharide on the activity of mouse alveolar macrophages MH-S cells in the embodiments of the present invention;

[0030] Figure 2 This is a figure showing the effect of Sophora tonkinensis polysaccharide on the survival rate of LPS-induced MH-S cells in an embodiment of the present invention;

[0031] Figure 3 This is a graph showing the detection effect of Sophora tonkinensis polysaccharide on LPS-induced NO production in MH-S cells in an embodiment of the present invention.

[0032] Figure 4 This is a graph showing the effect of Sophora tonkinensis polysaccharide on NO production in normal MH-S cells in an embodiment of the present invention.

[0033] Figure 5 This is a graph showing the effect of Sophora tonkinensis polysaccharide on the expression of the pro-inflammatory factor TNF-α in LPS-induced MH-S cells in an embodiment of the present invention.

[0034] Figure 6 This is a bar chart illustrating the effect of Sophora tonkinensis polysaccharide on LPS-induced Arg-1 protein content in MH-S cells in an embodiment of the present invention.

[0035] Figure 7 This is a PCA analysis diagram of Sophora tonkinensis polysaccharide acting on MH-S cells in an embodiment of the present invention;

[0036] Figure 8 This is a volcano diagram of Sophora tonkinensis polysaccharide acting on MH-S cells in an embodiment of the present invention;

[0037] Figure 9 This is a thermogram of Sophora tonkinensis polysaccharide acting on MH-S cells in an embodiment of the present invention;

[0038] Figure 10 This is a bar chart showing the GO functional enrichment analysis of differentially expressed genes between the LPS group and the control group, and between the Sophora tonkinensis polysaccharide + LPS group and the LPS group in the embodiments of the present invention.

[0039] Figure 11 This is a bubble chart showing the KEGG pathway enrichment analysis of differentially expressed genes between the LPS group and the control group, and between the Sophora tonkinensis polysaccharide + LPS group and the LPS group in the embodiments of the present invention.

[0040] Figure 12 This is a Venn diagram showing the differentially expressed genes between the LPS group and the control group, and between the Sophora tonkinensis polysaccharide + LPS group and the LPS group in the embodiments of the present invention, as well as the verification results of the expression of key differentially expressed genes. Detailed Implementation

[0041] Example 1: Preparation of Sophora tonkinensis polysaccharide

[0042] 1. Raw materials and reagents

[0043] Raw material: Dried Sophora tonkinensis root (commercially available, 10 kg).

[0044] Reagents: Pectinase (30,000 U / g, Maclean), Cellulase (50 U / mg, Yuanye), AB-8 macroporous adsorption resin (analytical grade), Chloroform (China National Pharmaceutical Group Co., Ltd., analytical grade), n-Butanol (China National Pharmaceutical Group Co., Ltd., analytical grade), 95% ethanol (analytical grade), anhydrous ethanol, sodium dihydrogen phosphate (analytical grade), disodium hydrogen phosphate (analytical grade), sulfuric acid (analytical grade), and purified water.

[0045] 2. Preparation steps

[0046] (1) Degreasing treatment

[0047] Take 10 kg of dried Sophora tonkinensis root, crush it, place it in an extraction tank, add 40 L of 95% ethanol (volume fraction), soak at room temperature for 24 hours to defatting, filter, and evaporate the solvent from the residue.

[0048] (2) Water extraction and concentration

[0049] The defatted drug residue was transferred to an extraction tank, 80 L of pure water was added, and the mixture was heated under reflux for 2 hours. After filtration, another 60 L of pure water was added to the residue, and the mixture was heated under reflux for 2 hours. The two extracts were combined and filtered through 200-mesh gauze. The filtrate was concentrated under reduced pressure until the relative density at 60°C was 1.10, yielding approximately 14 L of concentrated solution.

[0050] (3) Dual-enzyme targeted enzymatic hydrolysis

[0051] Take all the concentrated solution obtained in step (2), add an appropriate amount of 0.05 mol / L phosphate buffer (pH 6.5), adjust the pH of the system to 6.5, and stir thoroughly for 2 hours;

[0052] Add pectinase to the above system at a rate of 0.2% based on the weight of Sophora tonkinensis root, and shake in a 50°C constant temperature water bath for 3 hours; then add cellulase at a rate of 0.5% based on the weight of Sophora tonkinensis root, mix well, and continue shaking at 50°C for 2 hours.

[0053] After enzymatic hydrolysis, the enzyme was inactivated by boiling in a water bath for 15 minutes, cooled, and centrifuged (8000 r / min, 15 min). The precipitate was discarded and the supernatant was collected.

[0054] (4) Sevag method for deproteinization

[0055] Add 1 / 4 of the volume of chloroform / n-butanol mixture (volume ratio 4:1) to the supernatant, shake vigorously for 30 minutes, centrifuge to separate the layers, and collect the upper aqueous phase; repeat the operation 3 to 4 times until there is no obvious white denatured protein layer at the interface between the aqueous phase and the organic phase, to obtain the purified aqueous phase (about 13 L).

[0056] (5) Alcohol precipitation and refining

[0057] The purified aqueous phase was concentrated under reduced pressure to 1 / 3 to 1 / 2 of its original volume to reduce the amount of ethanol used subsequently. While stirring, 95% ethanol was added until the final ethanol volume fraction reached 80%. The mixture was then transferred to 4°C and allowed to stand overnight. The precipitate was collected by centrifugation and washed twice with an appropriate amount of anhydrous ethanol to obtain the crude polysaccharide precipitate.

[0058] (6) Resin decolorization

[0059] The crude polysaccharide precipitate was reconstituted with an appropriate amount of pure water to about 5 L, and the pretreated AB-8 macroporous adsorption resin (the resin volume is about 1 / 10 to 1 / 5 of the solution volume) was added. The mixture was shaken at 200 r / min for 12 hours at room temperature. The resin was removed by filtration and the filtrate was collected.

[0060] (7) Purification

[0061] Dilute the filtrate to an appropriate concentration (approximately 1–2 mg / mL) and fractionate it using a two-stage ultrafiltration system.

[0062] First-stage ultrafiltration: Use a PES ultrafiltration membrane with a molecular weight cutoff of 300 kDa, operating pressure 0.1~0.3 MPa, temperature 25±2℃, circulate and concentrate until the volume of the retentate is 1 / 5~1 / 10 of the original solution, collect the permeate (molecular weight <300 kDa), and discard the retentate.

[0063] Second-stage ultrafiltration: The permeate from the first stage is transferred to another ultrafiltration system. A PES ultrafiltration membrane with a molecular weight cutoff of 8 kDa is used. The membrane is concentrated and repeatedly washed under the same operating conditions (deionized water is added for constant volume washing 3-5 times) to remove small molecule oligosaccharides, salts and pigments. Finally, the retentate (molecular weight > 8 kDa) is collected and the permeate is discarded.

[0064] (8) Freeze-drying

[0065] The collected retentate was concentrated to a small volume under reduced pressure, pre-frozen, and then freeze-dried to obtain freeze-dried Sophora tonkinensis polysaccharide powder.

[0066] 3. Test Results

[0067] The purity of the Sophora tonkinensis polysaccharide obtained in this embodiment was 85.6% as tested.

[0068] In this embodiment, the anthrone-sulfuric acid colorimetric method was used to detect the purity of polysaccharides. The principle is that polysaccharides react with anthrone in the presence of concentrated sulfuric acid to produce a blue-green compound. The absorbance was measured at a wavelength of 575 nm. The absorbance value is directly proportional to the polysaccharide content. The sugar content was calculated based on the regression equation obtained from the standard working curve. Glucose standard was accurately weighed, and glucose standard solutions of different concentrations were prepared with pure water. Anthrone-sulfuric acid reagent was added, and the reaction was carried out in a boiling water bath. After cooling to room temperature, the absorbance was measured at a wavelength of 575 nm using a UV-Vis spectrophotometer. A glucose standard curve was plotted, and the regression equation was obtained. The freeze-dried powder of Sophora tonkinensis polysaccharides prepared above was dissolved in pure water, and the absorbance was measured according to the above method. The polysaccharide content was calculated based on the regression equation. Polysaccharide content (mg / g) = C*V / M, where C is the result calculated from the standard curve; M is the actual weighed mass; and V is the total volume of the extract.

[0069] In this embodiment, the temperature and time of the enzymatic hydrolysis reaction must be strictly controlled to avoid reducing enzyme activity and affecting the protein removal effect; the purity detection must be carried out in accordance with the operation specifications to ensure the accuracy and reliability of the detection data.

[0070] Example 2: Detection of the in vitro anti-inflammatory activity of Sophora tonkinensis polysaccharides

[0071] This embodiment verifies the in vitro anti-inflammatory effect of Sophora tonkinensis polysaccharide on LPS-induced mouse alveolar macrophages (MH-S), including cell resuscitation, passage, seeding, cytotoxicity detection, NO production regulation, inflammatory factor expression regulation, and transcriptome analysis.

[0072] (I) Experimental materials: Mouse alveolar macrophage cell line MH-S; MH-S cell culture medium (Zhongqiao Xinzhou), fetal bovine serum (FBS), penicillin and streptomycin antibiotics; LPS (lipopolysaccharide); Sophora tonkinensis polysaccharide prepared in Example 1; CCK-8 kit; RNA extraction kit (Novizan); NO detection kit; ELISA kit (Arg-1, TNF-α); microplate reader, transcriptome sequencer.

[0073] (II) Experimental Methods:

[0074] 1. Cell Culture

[0075] The routine procedures, including cell resuscitation, passage, and seeding, are briefly described below: Preheat the complete culture medium containing 10% fetal bovine serum (FBS). After removing the cryovials from the -80°C freezer or liquid nitrogen tank, thaw them rapidly by shaking in a 37°C water bath. Transfer the cell suspension to a centrifuge tube containing an equal volume of complete culture medium and centrifuge at 1200 rpm for 3 min. Discard the supernatant, add 5 mL of complete culture medium to resuspend the cells, transfer to a culture flask, and culture at 37°C with 5% CO2. When the cells have covered approximately 80% of the bottom of the flask, wash with PBS, trypsinize, and passage at an appropriate ratio. During experiments, take cells in the logarithmic growth phase, digest and centrifuge, count them, adjust to the required concentration, and seed them into the corresponding wells of a plate. All operations must be performed strictly according to aseptic principles.

[0076] 2. CCK-8 cell viability assay

[0077] To determine the safe and non-toxic concentration of Sophora tonkinensis polysaccharide, MH-S cells were seeded at a density of 5000 cells per well in 96-well plates with 6 replicates and 100 μL per well. After 24 h of culture, the old culture medium was discarded, and fresh culture medium containing different concentrations of Sophora tonkinensis polysaccharide (final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg / mL) was added. A LPS-induced drug administration group was also established: 24 h after cell seeding, the old culture medium was discarded, and the cells were pretreated with fresh culture medium containing 1 μg / mL LPS for 1 h, followed by administration of the aforementioned final concentration of the drug. After drug administration, incubation continued for 24 h, and 10 μL of CCK-8 solution was added to each well. After incubation for 1–4 h, the absorbance was measured at 450 nm using a microplate reader, and the relative cell viability was calculated.

[0078] 3. NO generation detection

[0079] MH-S cells were seeded at a density of 150,000 cells per well in 24-well plates, with a volume of 300 μL per well and four replicates per group. After 24 h of culture, the old culture medium was discarded. The drug-treated groups were pretreated for 1 h with 297 μL of fresh culture medium containing 1 μg / mL LPS, followed by the addition of 3 μL of different concentrations of *Sophora tonkinensis* polysaccharide stock solution, resulting in final drug concentrations of 100, 50, 25, 12.5, and 6.25 μg / mL. A blank control group was also included. After drug addition, incubation continued for 24 h, and 50 μL of the supernatant was collected by centrifugation. The NO content was determined according to the NO detection kit instructions. A separate detection group without LPS induction was also included. After 24 h of cell seeding, 300 μL of fresh culture medium containing different concentrations of *Sophora tonkinensis* polysaccharide (200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg / mL) was added directly. After 24 h of incubation, NO production was measured.

[0080] 4. Detection of inflammatory factor protein levels

[0081] Cell seeding and LPS induction procedures were the same as those for the LPS co-treatment group under the "NO Production Detection" section. The final concentrations of *Sophora tonkinensis* polysaccharide were set at 100, 50, and 10 μg / mL. After drug administration, the cells were incubated for another 24 h. 50 μL of cell supernatant was collected, and the protein levels of the pro-inflammatory factor TNF-α and the anti-inflammatory factor Arg-1 were detected strictly according to the ELISA kit instructions.

[0082] 5. Transcriptome analysis

[0083] MH-S cells were seeded at a density of 800,000 cells per well in 6-well plates, with a volume of 2 mL per well and 3 replicates per group. After 24 h of culture, the old culture medium was discarded. The treatment group was pretreated with 1.96 mL of fresh culture medium containing 1 μg / mL LPS for 1 h, followed by the addition of 40 μL of Sophora tonkinensis polysaccharide stock solution to achieve a final drug concentration of 100 μg / mL. After incubation for another 24 h, the supernatant was discarded, and the cells were washed twice with pre-chilled PBS. 1 mL of RNA extraction buffer was added to each well to lyse the cells, and the lysate was collected, frozen at -80°C, and then stored on dry ice for transtranscriptome sequencing.

[0084] (III) Experimental Results and Analysis:

[0085] 1. Cytotoxicity evaluation

[0086] Figure 1The figure shows the effect of different concentrations of Sophora tonkinensis polysaccharide on the activity of mouse alveolar macrophages MH-S cells in the embodiments of the present invention; the horizontal axis is the concentration of Sophora tonkinensis polysaccharide (μg / mL), and the vertical axis is the relative cell viability. The results show that Sophora tonkinensis polysaccharide has no obvious toxicity to MH-S cells at a concentration of ≤200μg / mL, and can play a safe role at a concentration of 100μg / mL (MH-S cell seeding density is 80W cells / well).

[0087] Figure 2 The figure shows the effect of Sophora tonkinensis polysaccharide on the survival rate of LPS-induced MH-S cells in this embodiment of the invention. The horizontal axis represents the concentration of Sophora tonkinensis polysaccharide (μg / mL), and the vertical axis represents the relative cell viability (%). The results show that after LPS induction, the cell viability of the model group decreased compared with that of the control group. After treatment with different concentrations of Sophora tonkinensis polysaccharide (6.25~100 μg / mL), the cell viability was maintained above 90%. Among them, the cell viability of the 50, 25, and 12.5 μg / mL concentration groups was close to or slightly higher than that of the LPS model group. This suggests that under LPS co-treatment conditions, Sophora tonkinensis polysaccharide can effectively inhibit the LPS-induced decrease in cell viability and improve cell survival rate, and has a good improving effect on LPS-damaged MH-S cells within the experimental concentration range.

[0088] CCK-8 assay results showed that treatment of MH-S cells with Sophora tonkinensis polysaccharide within the concentration range of 1.56–200 μg / mL did not significantly alter the relative cell viability compared to the control group, indicating that Sophora tonkinensis polysaccharide had no significant cytotoxicity to MH-S cells within this concentration range. Furthermore, a stable MH-S cell inflammation model was successfully established at 1 μg / mL LPS, and Sophora tonkinensis polysaccharide could safely exert its efficacy at this concentration. Therefore, subsequent experiments were conducted within the safe concentration range of ≤200 μg / mL.

[0089] 2. Regulatory effect on NO generation

[0090] Figure 3 This is a graph showing the effect of Sophora tonkinensis polysaccharide on LPS-induced NO production in MH-S cells in an embodiment of the present invention; the horizontal axis represents the concentration of Sophora tonkinensis polysaccharide (μg / mL), and the vertical axis represents the NO content in the cell supernatant (μmol / L). It visually presents the regulatory effect of different concentrations of Sophora tonkinensis polysaccharide on LPS-induced NO production in MH-S cells, and clarifies that it can dose-dependently inhibit LPS-induced excessive NO production in MH-S cells.

[0091] Figure 4The figure shows the effect of Sophora tonkinensis polysaccharide on NO production in normal MH-S cells in this embodiment of the invention. The horizontal axis represents the concentration of Sophora tonkinensis polysaccharide (μg / mL), and the vertical axis represents the amount of NO produced (μM). The results show that in normal MH-S cells without LPS induction, different concentrations (1.56~200 μg / mL) of Sophora tonkinensis polysaccharide had no significant effect on NO production. There was no significant difference between each concentration group and the control group, suggesting that Sophora tonkinensis polysaccharide does not pose a risk of inducing excessive NO production in normal cells within the experimental concentration range and has good biosafety.

[0092] NO detection results showed that LPS stimulation led to a sharp increase in NO content in the supernatant of MH-S cells, indicating the successful establishment of the inflammation model. In contrast, all doses of Sophora tonkinensis polysaccharide reduced NO production in a significant dose-dependent manner, with the most significant inhibitory effect observed at 100 μg / mL. This indicates that Sophora tonkinensis polysaccharide can effectively inhibit LPS-induced excessive NO production. Meanwhile, in normal MH-S cells without LPS stimulation, the NO production in all Sophora tonkinensis polysaccharide treatment groups showed no significant change compared to the blank control group, suggesting that the polysaccharide itself does not induce abnormal NO increases in normal cells and has good biocompatibility.

[0093] 3. Effects on the expression of inflammatory cytokine proteins

[0094] Figure 5 This is a graph showing the effect of Sophora tonkinensis polysaccharide on the expression of the pro-inflammatory factor TNF-α in LPS-induced MH-S cells in an embodiment of the present invention; the horizontal axis represents the concentration of Sophora tonkinensis polysaccharide (μg / mL), and the vertical axis represents the protein expression level. The results show that Sophora tonkinensis polysaccharide can downregulate TNF-α expression in a concentration-dependent manner.

[0095] Figure 6 This is a bar chart illustrating the effect of Sophora tonkinensis polysaccharide on LPS-induced Arg-1 protein content in MH-S cells in this embodiment of the invention. The horizontal axis represents different treatment groups (blank control group C, LPS model group, Sophora tonkinensis polysaccharide 10 μg / mL group, 50 μg / mL group, 100 μg / mL group), and the vertical axis represents Arg-1 protein content (μg / mL). The results show that LPS stimulation can significantly upregulate the expression level of Arg-1 in MH-S cells. After treatment with Sophora tonkinensis polysaccharide, the Arg-1 content in the 10 μg / mL and 50 μg / mL groups remained at a high level, while the Arg-1 content in the 100 μg / mL group was significantly lower than that in the LPS model group (*P<0.05). This suggests that Sophora tonkinensis polysaccharide can regulate LPS-induced Arg-1 expression in a concentration-dependent manner, and can maintain the expression of M2 macrophage markers within a certain concentration range, thus exerting anti-inflammatory and immunomodulatory effects.

[0096] ELISA results showed that LPS stimulation significantly upregulated the protein level of the pro-inflammatory cytokine TNF-α, while treatment with Sophora tonkinensis polysaccharide reversed this trend in a dose-dependent manner, significantly decreasing TNF-α levels. Conversely, for the anti-inflammatory cytokine Arg-1, Sophora tonkinensis polysaccharide treatment exhibited concentration-dependent regulation: the low and medium dose groups (10 and 50 μg / mL) maintained high Arg-1 levels, while the high dose group (100 μg / mL) showed a significant decrease in Arg-1 levels compared to the LPS model group (*P<0.05). Analysis indicates that Sophora tonkinensis polysaccharide can bidirectionally regulate the protein expression of inflammation-related factors, inhibiting the secretion of pro-inflammatory factors while regulating Arg-1 expression to normal levels, thereby restoring inflammatory homeostasis.

[0097] 4. Mechanism of action at the transcriptomics level

[0098] Figure 7 This is a PCA analysis diagram of Sophora tonkinensis polysaccharide applied to MH-S cells in an embodiment of the present invention. It can intuitively present the differences in gene expression among cells in the LPS-treated group, the blank control group, and the Sophora tonkinensis polysaccharide-treated group, and clearly distinguish the clustering of the three groups of samples, providing a basis for subsequent transcriptome analysis.

[0099] Figure 8 This is a volcano diagram of Sophora tonkinensis polysaccharide applied to MH-S cells in an embodiment of the present invention; it is used to show the differential gene distribution between the LPS_3.5Kd group and the LPS group, and between the LPS group and the Cont group, to visually present the expression fold and significance of differential genes, and to clarify the regulatory role of Sophora tonkinensis polysaccharide on LPS-induced gene expression in MH-S cells.

[0100] Figure 9 This is a heatmap of Sophora tonkinensis polysaccharide applied to MH-S cells in an embodiment of the present invention; it clearly shows the expression patterns of differentially expressed genes among different treatment groups, and intuitively demonstrates the effect of Sophora tonkinensis polysaccharide on the gene expression profile of MH-S cells.

[0101] Figure 10 This is a bar chart showing the GO functional enrichment analysis of differentially expressed genes between the LPS group and the control group, and between the Sophora tonkinensis polysaccharide + LPS group and the LPS group in the embodiments of this invention. The results show that LPS stimulation can significantly enrich chemokine-mediated signaling pathways, chemotaxis, and other immune-inflammatory biological processes. After treatment with Sophora tonkinensis polysaccharide, chemotaxis is still a significantly enriched biological process, suggesting that Sophora tonkinensis polysaccharide can exert anti-inflammatory and immunomodulatory effects by regulating chemotaxis-related biological processes.

[0102] Figure 11This is a bubble chart showing the KEGG pathway enrichment analysis of differentially expressed genes between the LPS group and the control group, and between the Sophora tonkinensis polysaccharide + LPS group and the LPS group in the embodiments of the present invention. The results show that LPS stimulation can significantly enrich inflammation-related pathways such as Cytokine-cytokine receptor interaction, IL-17 signaling pathway, and TNF signaling pathway. After treatment with Sophora tonkinensis polysaccharide, the above-mentioned inflammation-related pathways are still significantly enriched, but the degree of enrichment is changed, suggesting that Sophora tonkinensis polysaccharide can exert anti-inflammatory effects by regulating these core inflammatory pathways.

[0103] Figure 12 This is a Venn diagram of differentially expressed genes between the LPS group and the control group, and between the Sophora tonkinensis polysaccharide + LPS group and the LPS group in the embodiments of the present invention, as well as a diagram of the expression verification results of key differentially expressed genes. The results show that a total of 60 common differentially expressed genes were screened between the LPS stimulation group and the control group, and between the Sophora tonkinensis polysaccharide + LPS group and the LPS group. These genes are dependent on LPS expression. Among them, the expression of chemokines Ccl7 and Cxcl3 and anti-inflammatory factor IL11 was significantly downregulated after LPS stimulation and significantly upregulated after Sophora tonkinensis polysaccharide treatment, suggesting that Sophora tonkinensis polysaccharide can exert anti-inflammatory and immunomodulatory effects by regulating these key genes.

[0104] PCA analysis showed that the gene expression profiles of the blank control group, LPS model group, and Sophora tonkinensis polysaccharide treatment group were clearly distinguishable, indicating that Sophora tonkinensis polysaccharide treatment induced systemic changes in MH-S cells at the transcriptome level. Differential gene screening identified 60 LPS-dependent common differentially expressed genes between the LPS group and the blank control group, and between the Sophora tonkinensis polysaccharide group and the LPS group. Venn diagrams and volcano diagrams visually displayed the distribution and fold changes of these genes. Further GO functional enrichment analysis showed that the differentially expressed genes were significantly enriched in immune-inflammatory biological processes such as chemotaxis. KEGG pathway enrichment analysis revealed that Sophora tonkinensis polysaccharide can significantly regulate key inflammatory signaling pathways such as NF-κB, TLR4, and IL-17. Further differential gene trend analysis indicated that Sophora tonkinensis polysaccharide can stimulate the secretion of chemokines CCL7 and CXCL3. Comprehensive analysis suggests that Sophora tonkinensis polysaccharides may exert their anti-inflammatory and immunomodulatory effects through multiple pathways and targets, including regulating inflammatory signal transduction, inhibiting the expression of pro-inflammatory factors, and promoting immune cell chemotaxis and pathogen clearance.

[0105] Based on the above experimental results, the *Sophora tonkinensis* polysaccharide of this invention showed no significant cytotoxicity to MH-S cells within a concentration range of 1.56–200 μg / mL, and exhibited good protective effects against LPS-induced cell damage, demonstrating high biocompatibility. Functionally, the *Sophora tonkinensis* polysaccharide dose-dependently inhibited LPS-induced excessive NO production without affecting the NO levels in normal cells. At the protein expression level, the polysaccharide bidirectionally regulated inflammatory factors, downregulating the expression of the pro-inflammatory factor TNF-α while flexibly regulating the expression of the anti-inflammatory factor Arg-1 at different concentrations, thus restoring overall inflammatory homeostasis. At the transcriptomic level, PCA analysis confirmed that the *Sophora tonkinensis* polysaccharide induced systemic changes in the gene expression profile of MH-S cells, identifying 60 LPS-dependent common differentially expressed genes. GO and KEGG enrichment analyses further revealed that its mechanism of action involves chemotaxis, chemokine-mediated signaling pathways, and core inflammatory signaling pathways such as NF-κB, TLR4, IL-17, and TNF, and can significantly revert the expression of key genes such as Ccl7, Cxcl3, and IL11.

[0106] In summary, Sophora tonkinensis polysaccharides exert significant anti-inflammatory and immunomodulatory activities through multi-pathway and multi-target synergistic effects, and have the potential to be developed into anti-inflammatory drugs or immunomodulators.

[0107] To verify the necessity of the key steps and parameter ranges in the preparation method of the present invention, the inventors have set up the following comparative examples.

[0108] Comparative Example 1: Omitted the two-enzyme directed enzymatic hydrolysis step

[0109] The procedure was carried out in accordance with Example 1, except that in step (3), the water extract concentrate was not subjected to enzymatic hydrolysis by pectinase and cellulase, but was directly boiled in a boiling water bath for 15 minutes, cooled and centrifuged, and then proceeded to step (4) for deproteinization using the Sevag method. The remaining steps and parameters were exactly the same as in Example 1.

[0110] The final freeze-dried product had a purity of 82.3%, but the yield of polysaccharides in the target molecular weight range (8-300 kDa) was significantly lower than that in Example 1. During ultrafiltration, the permeation flux of the first-stage 300 kDa ultrafiltration membrane decreased significantly, and the time required to process the same volume of liquid was significantly longer than that in Example 1.

[0111] In vitro activity comparison results showed that at a polysaccharide concentration of 100 μg / mL, the inhibitory rate of the product of Comparative Example 1 on LPS-induced NO production in MH-S cells was reduced by about one-third compared with that of the product of Example 1, and the downregulation effect on the pro-inflammatory factor TNF-α was also weakened. This indicates that the dual-enzyme targeted enzymatic hydrolysis in step (3) is not an optional auxiliary operation, but plays a key role in breaking down the cell wall structure, releasing intracellular active polysaccharides, reducing the viscosity of the extract, and ensuring the efficiency of subsequent ultrafiltration purification. Omitting this step will lead to a decrease in the yield of the target product, a deterioration in process operability, and a weakening of biological activity.

[0112] Comparative Example 2: Using the traditional water extraction and alcohol precipitation method

[0113] The procedure was performed according to Example 1, with the following difference: after completing step (2) water extraction and concentration, step (3) dual-enzyme targeted hydrolysis, step (4) Sevag deproteinization, step (6) resin decolorization, and step (7) ultrafiltration purification were not performed. Instead, the concentrate was directly added to 95% ethanol to a final concentration of 80% (volume fraction), allowed to stand overnight at 4°C, the precipitate was collected by centrifugation, washed with anhydrous ethanol, and then vacuum dried to obtain the traditional crude polysaccharide product. The remaining steps and parameters were the same as in Example 1 (i.e., only defatting and water extraction with alcohol precipitation were retained).

[0114] The final product, after testing, showed a polysaccharide purity of 38.6% and a protein content as high as 12.5%. It was dark brown in appearance, had poor resolubility, and the solution was turbid.

[0115] In vitro activity comparison results showed that, at the same total sugar dose (100 μg / mL), the inhibitory rate of the product of Comparative Example 2 on LPS-induced NO production in MH-S cells was much lower than that of the product of Example 1, and its downregulation effect on the pro-inflammatory factor TNF-α was not significant. This indicates that the combined purification process of "enzymatic hydrolysis, Sevag deproteinization, resin decolorization, and ultrafiltration fractionation" constructed in this invention is not a simple superposition of existing technologies, but rather achieves high enrichment of active polysaccharide components and efficient removal of impurities through multi-step synergistic action, ultimately achieving a significant improvement in efficacy at the same dosage.

[0116] Comparative Example 3: Ultrafiltration molecular weight fractionation step omitted

[0117] The procedure was carried out in accordance with Example 1, except that in step (7), the filtrate after resin decolorization was not subjected to two-stage ultrafiltration, but was directly concentrated to a small volume under reduced pressure, pre-frozen, and then freeze-dried to obtain a polysaccharide mixture with a full molecular weight range. The remaining steps and parameters were exactly the same as in Example 1.

[0118] The final freeze-dried product was found to have a total polysaccharide content of 52.4%, with a molecular weight distribution ranging from 3 kDa to over 500 kDa. It contained a large number of small molecule impurities such as oligosaccharides, monosaccharides, and salts with a molecular weight below 8 kDa, as well as large molecules such as nucleic acids and proteoglycans with a molecular weight above 300 kDa.

[0119] In vitro activity comparison results showed that, at the same total polysaccharide concentration (100 μg / mL), the inhibitory rate of Comparative Example 3 product on LPS-induced NO production in MH-S cells was significantly lower than that of Example 1 product, and its downregulation effect on the pro-inflammatory factor TNF-α was also significantly weaker than that of Example 1 product. When the dosage concentration was increased to 200 μg / mL, Comparative Example 3 product achieved an inhibitory effect similar to that of Example 1 product at 100 μg / mL, indicating that its potency was significantly lower than that of Example 1. This demonstrates that the present invention, through precise fractionation using two-stage ultrafiltration membranes with molecular weight cutoffs of 8 kDa and 300 kDa, selectively removes low-activity and inactive impurity fractions, achieving a high enrichment of active polysaccharides within the 8-300 kDa molecular weight window. This specific molecular weight range makes a decisive contribution to the potency improvement and component clarity of the final product.

[0120] Comparative Example 4: Enzymatic hydrolysis using only a single enzyme

[0121] The operation is carried out in accordance with Example 1, except that in step (3), only a single type of enzyme is used for enzymatic hydrolysis.

[0122] Comparative Example 4A (Pectinase Only): Only pectinase was added at a rate of 0.7% based on the weight of the Sophora tonkinensis root. The mixture was reacted with shaking in a 50°C water bath for 5 hours. Cellulase was not added. The remaining steps and parameters were exactly the same as in Example 1.

[0123] The yield of polysaccharides in the target molecular weight range (8-300 kDa) of the final lyophilized product was lower than that of Example 1; although the viscosity of the enzymatic hydrolysate was significantly lower than that of the unenzymatic hydrolysate group, it was still higher than that of Example 1 (dual enzyme compound group); in vitro activity assay showed that at a polysaccharide concentration of 100 μg / mL, its inhibition rate on LPS-induced NO production in MH-S cells was lower than that of the product of Example 1.

[0124] Comparative Example 4B (Cellulase Only): Only cellulase was added at a rate of 0.7% based on the weight of the Sophora tonkinensis root. The mixture was reacted with shaking in a 50°C water bath for 5 hours. Pectinase was not added. The remaining steps and parameters were exactly the same as in Example 1.

[0125] The yield of polysaccharides in the target molecular weight range (8-300 kDa) of the final lyophilized product was lower than that in Example 1; the viscosity of the enzymatic hydrolysate was not significantly improved and was significantly higher than that in Example 1, which led to a decrease in the efficiency of subsequent ultrafiltration purification; in vitro activity assay showed that at a polysaccharide concentration of 100 μg / mL, its inhibition rate on LPS-induced NO production in MH-S cells was lower than that of the product in Example 1.

[0126] A comprehensive comparison reveals that while pectinase alone can reduce the viscosity of the extract to some extent by hydrolyzing pectin, the release efficiency of intracellular polysaccharides is limited because the cellulose backbone remains intact. Similarly, while cellulase alone can partially break down the cell wall, the outer pectin layer restricts sufficient contact between the enzyme and substrate, and the high viscosity of the system affects mass transfer efficiency, resulting in unsatisfactory cell wall disruption and sugar release. This invention combines pectinase and cellulase, employing a stepwise feeding strategy of adding pectinase first, followed by cellulase. This allows pectinase to preferentially hydrolyze the outer pectin matrix to reduce viscosity and expose the cellulose backbone, followed by efficient hydrolysis of the exposed cellulose to achieve complete cell wall disintegration. The two enzymes exhibit a significant synergistic effect in substrate exposure and enzymatic hydrolysis timing, resulting in higher yields and bioactivity of the final target product compared to either single-enzyme treatment group.

[0127] Based on the above experimental results, the *Sophora tonkinensis* polysaccharide of this invention showed no significant cytotoxicity to MH-S cells within a concentration range of 1.56–200 μg / mL, and exhibited good protective effects against LPS-induced cell damage, demonstrating high biocompatibility. Functionally, the *Sophora tonkinensis* polysaccharide dose-dependently inhibited LPS-induced excessive NO production without affecting the NO levels in normal cells. At the protein expression level, the polysaccharide bidirectionally regulated inflammatory factors, downregulating the expression of the pro-inflammatory factor TNF-α while flexibly regulating the expression of the anti-inflammatory factor Arg-1 at different concentrations, thus restoring overall inflammatory homeostasis. At the transcriptomic level, PCA analysis confirmed that the *Sophora tonkinensis* polysaccharide induced systemic changes in the gene expression profile of MH-S cells, identifying 60 LPS-dependent common differentially expressed genes. GO and KEGG enrichment analyses further revealed that its mechanism of action involves chemotaxis, chemokine-mediated signaling pathways, and core inflammatory signaling pathways such as NF-κB, TLR4, IL-17, and TNF, and can significantly revert the expression of key genes such as Ccl7, Cxcl3, and IL11.

[0128] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing Sophora tonkinensis polysaccharide, characterized in that, Includes the following steps: (1) Degreasing: Take dried Sophora tonkinensis root, soak it in ethanol to degrease it, separate the solid and liquid to obtain degreased residue; (2) Water extraction: The defatted drug residue is heated and refluxed with water for extraction, filtered, and the extract is concentrated to obtain a concentrated solution; (3) Enzymatic hydrolysis: Pectinase and cellulase are added to the concentrate for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymes are inactivated, centrifuged, and the supernatant is collected. (4) Sevag method for deproteinization: The supernatant was treated with a mixture of chloroform and n-butanol, centrifuged, and the aqueous phase was collected; (5) Alcohol precipitation: After concentrating the aqueous phase, add ethanol to the alcohol concentration to 70%~85%, let stand, and collect the precipitate by centrifugation; (6) Resin decolorization: Dissolve the precipitate in water, add AB-8 macroporous adsorption resin for adsorption and decolorization, filter, and collect the filtrate; (7) Ultrafiltration purification: The filtrate is fractionated by ultrafiltration membrane, and the retentate with a molecular weight greater than 8 kDa and less than 300 kDa is collected and dried to obtain the Sophora tonkinensis polysaccharide.

2. The preparation method according to claim 1, characterized in that, In step (1), the ethanol is 95% ethanol by volume, and the soaking and degreasing time is 20-28 hours.

3. The preparation method according to claim 1, characterized in that, In step (2), the heating and reflux extraction is performed 1 to 3 times, each time for 1 to 3 hours; the concentration is performed under reduced pressure to a relative density of 1.05 to 1.15 at 60°C.

4. The preparation method according to claim 1, characterized in that, In step (3), the enzymatic hydrolysis specifically involves: first adjusting the pH of the concentrated solution to 6.0-7.0, adding pectinase, and hydrolyzing at 45-55°C for 2-5 hours; then adding cellulase and continuing hydrolysis at 45-55°C for 1-3 hours; the amount of pectinase added is 0.1%-0.5% of the weight of the dried Sophora tonkinensis root material, and the amount of cellulase added is 0.2%-1.0% of the weight of the dried Sophora tonkinensis root material.

5. The preparation method according to claim 1, characterized in that, In step (4), the volume ratio of chloroform to n-butanol in the mixture of chloroform and n-butanol is (3~5):1; the treatment includes shaking and centrifugation, and the operation is repeated until there is no obvious denatured protein layer at the interface.

6. The preparation method according to claim 1, characterized in that, In step (5), the alcohol concentration is 80%, and the standing is overnight at 4°C; in step (6), the volume of AB-8 macroporous adsorption resin added is 1 / 10 to 1 / 5 of the volume of the solution after the precipitate is dissolved, and the adsorption conditions are room temperature shaking adsorption for 8 to 16 hours.

7. The preparation method according to claim 1, characterized in that, In step (7), the ultrafiltration membrane grading specifically involves: firstly, the filtrate is subjected to a first-stage ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 300 kDa, and the permeate is collected; then, the permeate is subjected to a second-stage ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 8 kDa, and the retained liquid is collected; the drying is freeze drying.

8. A polysaccharide from Sophora tonkinensis prepared by the method according to any one of claims 1 to 7, characterized in that: The molecular weight of the Sophora tonkinensis polysaccharide ranges from 8 kDa to 300 kDa.

9. The use of the Sophora tonkinensis polysaccharide according to claim 8 in the preparation of anti-inflammatory drugs or immunomodulators.

10. The application according to claim 9, characterized in that: The anti-inflammatory drug is a drug for treating inflammatory diseases of the lungs; the immunomodulator is an immunomodulator that regulates macrophage polarization.

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