Separation and extraction of neutral water-soluble polysaccharides from auricularia auricula and its new application

Abstract

This invention discloses the isolation, extraction, and novel application of a neutral, water-soluble polysaccharide from Sophora japonica extract. The invention extracts a water-soluble, neutral polysaccharide with a molecular weight between 20-22 kDa. This invention uses the Sophora japonica extract to treat renal interstitial fibrosis. Multiple studies, including cellular and animal studies, have demonstrated that the traditional Chinese medicine Sophora japonica can inhibit renal interstitial fibrosis, making it a potential drug for clinical application and bringing greater benefits to patients with kidney disease.

Description

Technical Field

[0001] This invention relates to the field of protein extraction technology, specifically to the separation, extraction, and new applications of neutral water-soluble polysaccharides from Sophora japonica extract. Background Technology

[0002] Chronic kidney disease (CKD) refers to chronic structural and functional impairment of the kidneys caused by various factors, including pathological damage with normal or abnormal glomerular filtration rate, abnormal blood or urine composition, abnormal imaging findings, or an unexplained decline in glomerular filtration rate (GFR) for more than 3 months. CKD has an irreversible, chronic, and progressive course, eventually leading to end-stage renal disease (ESRD). ESRD patients require lifelong renal replacement therapy (including maintenance hemodialysis, peritoneal dialysis, or kidney transplantation), which not only severely impacts their quality of life and places a heavy psychological and economic burden on patients and their families, but also consumes enormous social medical resources. Renal interstitial fibrosis (RIF) is a common pathological process in the progression of various chronic kidney diseases to end-stage renal failure. Its pathological feature is the replacement of normal renal interstitial and tubular structures with a large amount of accumulated extracellular matrix (ECM), such as type I, III, and IV collagen, fibronectin (FN), and laminin (LN). Therefore, inhibiting the progression of renal interstitial fibrosis is an important way to delay the development of end-stage renal disease (ESRD) in patients with chronic kidney disease.

[0003] Huaier, the fruiting body of *Trametes robiniophila* Murr. (family Polyporaceae), is a fungus belonging to the genus *Trametes*. Huaier extract is a hot water extract obtained after fermentation of the fungus. The main active components of its crude extract are polysaccharides and proteins. Huaier granules have long been shown to have good clinical efficacy in the adjuvant treatment of various cancers or post-cancer surgery. However, the preparation of refined polysaccharides from Huaier and their related applications in renal interstitial fibrosis have not yet been reported. Summary of the Invention

[0004] Based on the above background, the purpose of this invention is to provide a neutral water-soluble polysaccharide extracted from Sophora japonica extract, its preparation method and application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The first aspect of this invention provides a method for separating and extracting neutral water-soluble polysaccharides, the method comprising the following steps:

[0007] (1) Remove protein, pigment and small molecules from the aqueous solution of Sophora japonica extract to obtain total polysaccharide; (2) Dissolve the total polysaccharide obtained in (1) in water, purify and elute, perform tracking detection by phenol-sulfuric acid method, collect according to peak shape, concentrate, dialyze and freeze dry to obtain polysaccharide component; (3) Dissolve the polysaccharide component obtained in (2) in water, purify by chromatography column, collect symmetrical peaks, concentrate the collected liquid by rotary evaporator, and freeze dry to obtain purified polysaccharide.

[0008] In this invention, the term "polysaccharide" refers to a polymeric carbohydrate composed of monosaccharide units linked by glycosidic bonds. The structure of a polysaccharide typically consists of multiple monosaccharide units, such as glyceraldehyde (a triose); erythrose and threose (a tetraose); arabinose, ribose, xylose, and lyseose (a pentose); and glucose, mannose, fructose, and galactose (a hexose). Polysaccharides composed of the same monosaccharides are called homopolysaccharides, while those composed of different monosaccharides are called heteropolysaccharides.

[0009] The term "neutral polysaccharide" refers to a polymer composed of two or more different glycosyl monomers that do not contain organic acids. Acidic polysaccharides are more complex, containing not only two or more glycosyl monomers but also one or more uronic acid monomers. Most polysaccharides found in natural products are neutral or acidic, while only chitin / chitosan is considered a basic polysaccharide. The term "water-soluble polysaccharide" refers to a class of polysaccharide substances that can dissolve in water, typically possessing a high molecular weight and complex chemical structure. Water-soluble polysaccharides are widely distributed in nature, including in plants, algae, fungi, bacteria, and animal tissues.

[0010] In this invention, the method for removing proteins can be the Sevag method, centrifugal ultrafiltration, enzymatic digestion, polyacrylamide gel electrophoresis, guanidine chloride method, cell membrane disruption, etc. In some embodiments, the method for removing proteins is the Sevag method, which refers to a method for removing protein impurities from a mixed system by repeatedly shaking the mixture containing protein impurities with a chloroform solution containing a small amount of isoamyl alcohol or octanol, causing the proteins to denature and precipitate and be removed.

[0011] Furthermore, the specific steps for removing proteins are as follows: precipitation with four times the solution volume of anhydrous ethanol at 4°C for 24 hours, dialyzing with a 1000Da dialysis bag until the dialysate is clear, and deproteinization with Sevag reagent.

[0012] In this invention, the methods for removing pigments can include column chromatography, adsorption, oxidation, etc. Methods for removing small molecules can include ultrafiltration, dialysis, etc. In some embodiments, adsorption is used to remove pigments, and dialysis is used to remove small molecules.

[0013] Furthermore, the specific steps for removing pigments and small molecules are as follows: dialysis for one week using a 1000Da dialysis bag, depigmentation using AB-8 macroporous resin, dialysis using a 1kD pore size dialysis bag, and freeze-drying to obtain total polysaccharides.

[0014] In this invention, the "purification" method includes stepwise precipitation, salting out, metal complexation, cellulose column chromatography, quaternary ammonium salt precipitation, cellulose anion exchanger column chromatography, gel column chromatography, and preparative zone electrophoresis. In some embodiments, the purification method is gel column chromatography. The "elution" can be elution with a constant concentration of eluent or elution with linearly distributed eluents of different concentrations. In some embodiments, elution is performed using linearly distributed eluents of different concentrations.

[0015] Furthermore, the specific steps of the purification and elution are as follows: heating, swirling, centrifugation at 12000 rpm, loading the supernatant onto a DEAE Sepharose Fast Flow purification column, and eluting with distilled water at a flow rate of 15 ml / min. The eluent is water and 0.2 M to 1.0 M NaCl aqueous solution.

[0016] Furthermore, the eluent is water, 0.2M NaCl aqueous solution, 0.5M NaCl aqueous solution, or 1.0M NaCl aqueous solution.

[0017] In this invention, the chromatography column can be a gel chromatography column or an ion exchange chromatography column. The gel chromatography column can be an agarose gel column, a polyacrylamide gel column, or a dextran gel column.

[0018] Furthermore, the chromatography column is a Sephadex G-200 dextran gel column.

[0019] A second aspect of the present invention provides a neutral water-soluble polysaccharide obtained by the method described in the first aspect of the present invention.

[0020] Furthermore, the monosaccharide composition of the neutral water-soluble polysaccharide includes arabinose, galactose, glucose, and mannose, with a molar ratio of 11.4:5.8:10.5:22.3.

[0021] Furthermore, the molecular formula of the neutral water-soluble polysaccharide is:

[0022]

[0023] Furthermore, the weight-average molecular weight of the neutral water-soluble polysaccharide is 2.0 × 10⁻⁶. 4 ~2.2×10 4 Da.

[0024] Furthermore, the weight-average molecular weight of the neutral water-soluble polysaccharide is 2.17 × 10⁻⁶.4 Da.

[0025] In this invention, the chemical formula of arabinose is C5H. 10 O5, originally extracted from a colloid secreted by the araffinose tree through complex chemical and physical methods, is a levorotatory monosaccharide with eight isomers, such as β-D-arabinose and β-L-arabinose. Galactose has the molecular formula C6H2O. 12 O6, commonly found in dairy products and beets, is often present in the brain and nerve tissue in the form of D-galactoside. The molecular formula of glucose is C6H2O. 12 In aqueous solution, glucose exists to a small extent in an open-chain form, primarily as α- or β-pyranose, and these two forms can interconvert. Three known forms can crystallize from aqueous solution: α-glucopyranose, β-glucopyranose, and α-glucopyranose monohydrate. The molecular formula for mannose is C6H₂O. 12 O6, also known as D-mannose, can be prepared by hydrolyzing polysaccharides rich in D-mannose (such as ivory palm seed mannan and yeast mannan). It can also be synthesized from D-mannitol (a byproduct of the iodine production industry from kelp) using hydrogen peroxide in the presence of ferrous ions. Furthermore, it can be prepared by epimerization of D-glucose or by extending the carbon chain of D-arabinose.

[0026] A third aspect of the present invention provides the application of the neutral water-soluble polysaccharide described in the second aspect of the present invention, the application comprising any one of the following:

[0027] 1) Application in the preparation of drugs for treating or delaying renal interstitial fibrosis.

[0028] 2) Application in the preparation of drugs for treating chronic kidney disease.

[0029] 3) Application in the study of the mechanism of renal interstitial fibrosis.

[0030] 4) Application in inhibiting macrophage activity.

[0031] 5) Application in inhibiting morphological changes in renal tubular epithelial cells.

[0032] 6) Application in inhibiting macrophage migration.

[0033] 7) Application in the treatment of renal tubular injury.

[0034] 8) Application in altering the levels of epigenetic regulatory factors in renal fibrosis.

[0035] In this invention, renal interstitial fibrosis (RIF) frequently occurs in both inflammatory and non-inflammatory kidney diseases and is associated with decreased renal excretory function. Fibroblasts, which occupy the renal interstitium, are primarily involved in RIF formation not only through the production of extracellular matrix but also through regulatory processes. They respond to a variety of cytokines released by different cell types. In some embodiments, the neutral water-soluble polysaccharide provided in the first aspect of this invention can be used as a tool to study the physiological and pathological mechanisms of RIF.

[0036] Furthermore, the renal interstitial fibrosis includes renal interstitial fibrosis caused by arterial nephrosclerosis, renal interstitial fibrosis caused by benign nephrosclerosis, renal interstitial fibrosis caused by malignant nephrosclerosis, folic acid-induced mouse renal interstitial fibrosis, adenine-induced mouse renal interstitial fibrosis, unilateral ureteral ligation mouse renal interstitial fibrosis, Tgfb1-induced primary mouse renal tubular epithelial cell damage and transdifferentiation, and primary mouse macrophage polarization and transdifferentiation caused by primary mouse renal tubular epithelial cell damage.

[0037] Furthermore, the chronic kidney disease includes chronic kidney disease caused by primary glomerulonephritis, hypertensive nephrotic arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial nephropathy, ischemic nephropathy, hereditary nephropathy, and other causes.

[0038] Furthermore, the renal tubulointerstitial lesions include chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, and drug-induced nephropathy.

[0039] Furthermore, the hereditary kidney diseases include polycystic kidney disease and hereditary nephritis.

[0040] Furthermore, the morphological changes in the renal tubular epithelial cells were caused by Tgfb1 stimulation.

[0041] Furthermore, the macrophage migration is caused by damage to renal tubular epithelial cells;

[0042] Furthermore, the renal tubular injury was caused by folic acid, adenine, and / or unilateral ureteral ligation.

[0043] Furthermore, the fibrosis epigenetic regulatory factors include one or more of α-sma, COL-1, COL-3, and E-cadherin.

[0044] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0045] In this invention, a "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity, including non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulation materials, or any type of formulation aid. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may comprise fluids such as water, saline, glycerol, and ethanol. Such carriers may also contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0046] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or drug carrier without toxic side effects.

[0047] Furthermore, the dosage forms of the drug include drops, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder sprays, patches, solutions, ointments, or creams.

[0048] Advantages and beneficial effects of the present invention: 1. The present invention uses polysaccharide extract of Sophora japonica, a traditional Chinese medicine, to treat renal interstitial fibrosis. It has been confirmed at multiple levels, including cellular and animal studies, that Sophora japonica can inhibit renal interstitial fibrosis. It is a potential drug that can be translated into clinical applications, bringing more benefits to patients with kidney disease.

[0049] 2. At the cellular level, this drug significantly inhibited Tgfb1-induced damage and transdifferentiation of primary mouse renal tubular epithelial cells, as well as the migration, polarization, and transdifferentiation of primary mouse macrophages caused by primary mouse renal tubular epithelial cell damage. At the animal level, compared with folic acid, adenine, and unilateral ureteral ligation (UUO) mouse models, this drug significantly reduced kidney damage and fibrosis in the model mice. Attached Figure Description

[0050] Figure 1 Symmetrical peak diagram of polysaccharide extraction from Sophora japonica extract using gel column chromatography.

[0051] Figure 2 This is a peak diagram of neutral polysaccharide A (WHPA) sample.

[0052] Figure 3 This is a peak diagram of neutral polysaccharide B (WHPB) sample.

[0053] Figure 4The image shows the infrared results of the neutral polysaccharide B (WHPB) sample.

[0054] Figure 5 The ion chromatogram (A) and molar ratio diagram (B) of the monosaccharide components in neutral polysaccharide B (WHPB) are shown.

[0055] Figure 6 This image shows the cell activity assay of total polysaccharide (THP) from Sophora japonica on primary mouse renal tubular epithelial cells and primary mouse macrophages.

[0056] Figure 7 This is a graph showing the cell activity assay of primary mouse macrophages by total polysaccharides (THP), neutral polysaccharides (WHP), neutral polysaccharide A (WHPA), and neutral polysaccharide B (WHPB).

[0057] Figure 8 The effect of total polysaccharides (THP) / neutral polysaccharides (WHP) on inhibiting Tgfb1-induced primary renal tubular epithelial cell damage is shown in the figure.

[0058] Figure 9 The effect of total polysaccharides (THP) / neutral polysaccharides (WHP) on inhibiting macrophage migration induced by primary renal tubular epithelial cell damage is shown in the figure.

[0059] Figure 10 The image shows the pathological findings (PAS, Masson) of renal tubular injury and interstitial fibrosis in a mouse model of folic acid inhibition by total polysaccharide (THP) / neutral polysaccharide (WHP).

[0060] Figure 11 The images show the pathological findings (PAS, Masson) of renal tubular injury and interstitial fibrosis in a mouse model of adenine inhibition by total polysaccharide (THP) / neutral polysaccharide (WHP).

[0061] Figure 12 The image shows the pathological findings (PAS, Masson) of renal tubular injury and interstitial fibrosis in mice with a unilateral ureteral ligation (UUO) model induced by total polysaccharide (THP) / neutral polysaccharide (WHP).

[0062] Figure 13 Immunohistochemical staining of regulatory factors related to the inhibition of folic acid-induced renal fibrosis in mice by neutral polysaccharide (WHP).

[0063] Figure 14 Immunohistochemical staining of regulatory factors related to renal fibrosis in mice induced by the inhibition of adenine by neutral polysaccharide (WHP).

[0064] Figure 15 Immunohistochemical staining of regulatory factors related to renal fibrosis in mice induced by neutral polysaccharide (WHP) inhibition of unilateral ureteral ligation (UUO). Detailed Implementation

[0065] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0066] Example 1: Isolation, extraction and identification of total polysaccharides and water-soluble polysaccharides from Sophora japonica

[0067] I. Experimental Materials:

[0068] Table 1. Experimental Instruments

[0069] Instrument Name factory model Dextran gel GE (USA) Superdex-200 GPC-Autopurify Fully Automated Gel Purification System Borui Sugar Bio SHODEX Differential Refractive Detector Japan Shodex RID-10A centrifuge ThermoFisher D-37520 Electronic balance Sartorius CPA225D Automatic collector Borui BSZ-100 High Performance Liquid Chromatography SHIMADZU RID-10A FRC-10A Ion Chromatograph ThermoFisher ICS5000 Electric constant temperature drying oven Lichen Technology 101-1BS Nitrogen blowing device Lichen Technology UGC-24M

[0070] Table 2. Experimental Reagent List

[0071] reagents factory batch number Item number level Trifluoroacetic acid ACROS A0356762 139725000 AR 50% sodium hydroxide solution Alfa Aesar Z21E036 33382 GR Sodium acetate ThermoFisher 191126 059326 GR

[0072] Table 3. Standard Reagent Table

[0073]

[0074]

[0075] II. Experimental Methods

[0076] 1. Extraction: Sophora japonica extract (gifted by Qidong Gaitianli Pharmaceutical Co., Ltd.) was dissolved in water, precipitated with four times the volume of anhydrous ethanol at 4°C for 24 hours, dialyzed through a 1KD dialysis bag until the dialysate was clear, deproteinized with Sevag reagent, dialyzed through a 1KD dialysis bag for one week, depigmented with AB-8 macroporous resin, dialyzed through a 1KD dialysis bag, and freeze-dried to obtain total polysaccharides (THP). The total polysaccharides (THP) were dissolved in distilled water, heated, vortexed, and centrifuged at 12000 rpm. The supernatant was loaded onto a DEAE Sepharose Fast Flow purification column, and eluted with distilled water at a flow rate of 15 ml / min using three solvents: three column volumes of water, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl. The phenol-sulfuric acid method was used for elution. Detection was performed at 490 nm using a microplate reader. A scatter plot was plotted. Based on the peak shape, the samples were collected, concentrated, dialyzed through a 1000 Da dialysis bag, and freeze-dried. The obtained components were named WHP (aqueous neutral polysaccharide), HP-0.2, HP-0.5, and HP-1.0, respectively. The main purpose was to enrich neutral and acidic sugars, preparing for later experiments.

[0077] 2. Separation: Weigh 100 mg WHP, dissolve in 3 mL of distilled water, centrifuge at 12000 rpm for 10 min, load the supernatant onto a sample, and purify using a polysaccharide gel purification system specially designed by Borui Sugar Biotechnology Co., Ltd., combined with online detection and collection using a differential detector (RI-502SHODEX). Collect symmetrical peaks. Concentrate the collected solution using a rotary evaporator, freeze-dry, and obtain the gel column purified polysaccharide.

[0078] 3. Measurement:

[0079] 3.1 Determination of polysaccharide molecular weight and purity by HPGPC. Experimental procedure: Reagent preparation: 0.05M NaCl solution: accurately prepared, filtered through a 0.45μm filter membrane, and sonicated for 10 min; Sample and standard solution preparation: accurately weigh the sample and standard, prepare a 5 mg / ml solution for the sample, centrifuge at 12000 rpm for 10 min, filter the supernatant through a 0.22μm microporous membrane, and then transfer the sample to a 1.8 ml vial; Chromatographic method: Column: BRT105-104-102 tandem gel column (8×300 mm); Mobile phase: 0.05M NaCl solution; Flow rate: 0.6 ml / min; Column temperature: 40℃; Injection volume: 20 μl; Detector: Differential detector RD-10A.

[0080] 3.2 Analysis of functional groups of polysaccharides by FT-IR. Experimental procedure: Accurately weigh 2 mg of sample and 200 mg of potassium bromide, compress them into tablets, and use potassium bromide powder tablets as blank control. Scan and record the results using a Fourier transform infrared spectrometer (FT-IR650, Tianjin Gangdong Technology Development Co., Ltd.).

[0081] 3.3 Determination of Monosaccharide Composition Using Ion Chromatography. This method utilizes the electrochemical activity of carbohydrate molecules and their ionization in strongly alkaline solutions. Carbohydrates are weak acids with pKa > 11, and in high-pH eluents, they exist partially or entirely as anions. Based on the differences in ion exchange caused by the pKa of different carbohydrates and the varying hydrophobic interactions between certain carbohydrates and anion exchange resins, efficient anion exchange separation of carbohydrates is achieved. The current generated by the oxidation of hydroxyl groups in the carbohydrate molecule on the gold electrode surface is then detected. Reagent Preparation: 2.4 g of 15 mM NaOH solution, 2 L of 50% NaOH solution, 1.2 g of 5 mM NaOH & 100 mM NaOAC solution, 8.2 g of 50% NaOH solution, 1 L of NaOAC; Preparation and Calculation of Standard Solutions: Prepare approximately 10 mg / ml standard solutions using 16 monosaccharide standards (as shown in Table 3). Prepare 5 mg / L gradient concentration standards for each monosaccharide as Standards. Determine the mass of different monosaccharides using an absolute quantification method, and calculate the molar ratio based on the molar mass of the monosaccharides. Sample preparation: Accurately weigh 10 mg of sample into an ampoule, add 10 ml of 3M TFA, and hydrolyze at 120℃ for 3 h. Accurately pipette the acid-hydrolyzed solution into a tube, blow dry under nitrogen, add 5 ml of water, vortex mix, pipette 100 μL, add 900 μL of deionized water, and centrifuge at 12000 rpm for 5 min. Inject the supernatant into an IC50 analyzer. Chromatographic method: Column: Dionex Carbopac™ PA20 (3*150); Mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH & 100 mM NaOAC; Flow rate: 0.3 ml / min; Injection volume: 5 μL; Column temperature: 30℃; Detector: Electrochemical detector.

[0082] 3.4 NMR Determination of Molecular Structure. Experimental Materials: Heavy water (D2O, 99.9%), deuterated acetone as internal control; freeze dryer; Bruker 600M NMR spectrometer. Experimental Procedure: Weigh 50 mg of the polysaccharide sample WHPB, dissolve it in 0.5 ml of heavy water, and freeze-dry. Then, dissolve the freeze-dried powder again in 0.5 ml of heavy water and continue freeze-drying. Repeat this process to ensure sufficient exchange of active hydrogens. Then, dissolve the sample in 0.5 ml of heavy water and measure the 1H NMR spectrum, 13C NMR spectrum, DEPT135 one-dimensional spectrum, and two-dimensional spectrum at 600 MHz at room temperature (25 °C).

[0083] III. Experimental Results

[0084] 1. According to Figure 1The peak chromatogram is shown. The symmetrical portion of the first peak (100-110 min) is collected as component A. This is concentrated using a rotary evaporator, dialyzed, and freeze-dried to obtain the gel column purified polysaccharide WHPA. The symmetrical portion of the second peak (125-135 min) is collected as component B. This is concentrated using a rotary evaporator, dialyzed, and freeze-dried to obtain the gel column purified polysaccharide WHPB, with yields of 7.8% and 12.4%, respectively.

[0085] 2. According to Figure 2 , Figure 3 As shown, calibration curves for lgMp-RT (peak molecular weight), lgMw-RT (weight-average molecular weight), and lgMn-RT (number-average molecular weight) were obtained. Based on the standard curves, calculation formulas were derived to calculate the molecular weight of each sample.

[0086] The equation for the lgMp-RT correction curve is: y = -0.195x + 12.375, R 2 =0.9913;

[0087] The equation for the lgMw-RT correction curve is: y = -0.2078x + 12.968, R 2 =0.993;

[0088] The equation for the lgMn-RT correction curve is: y = -0.181x + 11.734, R 2 =0.9972.

[0089] Table 4. WHPA Sample Parameters

[0090] Sample ID RT(min) lgMp lgMw lgMn Mp Mw Mn WHPA 41.535 4.3 4.3 4.2 18866 21728 16450

[0091] Table 5. Parameters of WHPB Samples

[0092] Sample ID RT(min) lgMp lgMw lgMn Mp Mw Mn WHPB 41.640 4.3 4.3 4.2 17997 20664 15746

[0093] 3. For example Figure 4 The image shows the infrared (IR) results for the WHPB polysaccharide component. The absorption band is located at 3600-3200 cm⁻¹. -1 This is the absorption peak of the stretching vibration of -OH, and the absorption peak in this region is a characteristic peak of carbohydrates. Specifically: 3372 cm⁻¹ -1 This is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. At 2927 cm⁻¹ -1 There is an absorption peak at 1650 cm⁻¹, attributed to the CH stretching vibration. -1 The absorption peak at 1411 cm⁻¹ is attributed to the C=O stretching vibration. -1 An absorption peak is observed at 1241 cm⁻¹, which is attributed to the CO stretching vibration. -1 and 1027cm -1The absorption peak at that point is attributed to the variable-angle vibration of OH.

[0094] 4. For example Figure 5 As shown, the monosaccharide composition of WHPB includes arabinose, galactose, glucose, and mannose, with a molar ratio of 11.4:5.8:10.5:22.3.

[0095] 5. The molecular formula of WHPB determined by NMR is as follows:

[0096]

[0097] Example 2: Comparison of the therapeutic effects of total polysaccharides from Sophora japonica and neutral water-soluble polysaccharides on protecting renal tubules.

[0098] I. Experimental Materials: Total polysaccharide (THP) and neutral water-soluble polysaccharide (WHP) extracted and isolated from Sophora japonica in Example 1, purified neutral water-soluble polysaccharides WHPA and WHPB, primary mouse renal tubular epithelial cells, primary mouse macrophages, C57 / BL-6 mice, and ICR mice.

[0099] II. Experimental Methods

[0100] 1. The activity of total polysaccharide (THP) from Sophora japonica was detected at the level of primary mouse renal tubular cells using the CCK8 assay.

[0101] The procedure is as follows: Primary mouse renal tubular epithelial cells were cultured in DMEM medium (containing 10% fetal bovine serum + 1% penicillin-streptomycin mixture). Cell growth was observed, the supernatant was discarded, and the cells were digested with trypsin, centrifuged at 800 rpm for 5 min, and then mixed with complete culture medium to prepare a single-cell suspension. The number of cells was counted using a cell counting chamber, and 15,000 cells / well were seeded into 96-well plates, with 3 replicates per group. The cells were incubated overnight in a constant temperature incubator (37℃, 5% CO2). After 24 hours of starvation, serum-free culture media with different drug concentrations were prepared (concentration gradient set to 0, 0, 0.

[0102] Add culture medium (200 μL / well) to each well (100, 200, 400, 800, 1600 μg / mL), and set up 3 replicates per group. After culturing for 24 h, replace with CCK8 solution, incubate for 1-2 h, and then measure the absorbance at 450 nm using an ELISA reader and calculate cell viability.

[0103] 2. The activity of different refined polysaccharide components from the traditional Chinese medicine Sophora japonica was detected at the level of primary mouse macrophages using the CCK8 assay.

[0104] The procedure is as follows: Primary mouse renal tubular epithelial cells were cultured in DMEM medium (containing 10% fetal bovine serum + 15% L929 cell culture supernatant + 1% penicillin-streptomycin mixture). Cell growth was observed, the supernatant was discarded, and the cells were digested with trypsin, centrifuged at 800 rpm for 5 min, and then mixed with complete culture medium to prepare a single-cell suspension. The number of cells was counted using a cell counting chamber, and 15,000 cells / well were seeded into 96-well plates, with 3 replicates per group. Incubate overnight in a constant temperature incubator (37℃, 5% CO2). After starvation for 24 hours, prepare culture media with different drug concentrations (concentration gradients set to 0, 100, 200, 400, 800, 1600 μg / mL) and add culture media (200 μL / well), with 3 replicates per group. After culturing for 24 hours, replace with CCK8 solution and incubate for 1-2 hours. Measure absorbance at 450 nm using a microplate reader and calculate cell viability.

[0105] 3. Primary mouse renal tubular epithelial cells were divided into four groups: PBS control group, PBS+THP / WHP control group, Tgfb1 stimulation group, and THP / WHP+Tgfb1 group. Their morphology was photographed under a light microscope.

[0106] 4. Primary mouse renal tubular epithelial cells and primary mouse macrophages were co-cultured. Primary mouse renal tubular epithelial cells were seeded in the lower layer, and primary mouse macrophages were seeded in the upper layer. The primary mouse renal tubular epithelial cells in the lower layer were stimulated with Tgfb1, and the migration of cells in the upper layer was observed. The cells were divided into four groups: PBS control group, PBS+WHP control group, Tgfb1 stimulation group, and WHP+Tgfb1 group. The migration of primary mouse macrophages was photographed under an inverted microscope.

[0107] 5. In mice (C57 / BL-6), the effects of PAS and Masson staining on the inhibition of folic acid-induced renal tubular damage and interstitial fibrosis by the neutral polysaccharide WHP from Sophora japonica were detected.

[0108] The procedure was as follows: 20 mice were randomly divided into 4 groups of 5 mice each. The groups were: sodium bicarbonate solution intraperitoneal injection group, folic acid model group, and WHP intraperitoneal injection folic acid model group. One week later, the mice were euthanized by cervical dislocation after anesthesia, fixed in 10% formalin, sectioned in paraffin at 4 pm, and stained with PAS and Masson. PAS staining: Dewaxing twice in xylene, 10 min each time; hydration twice in anhydrous ethanol, 1 min each time; rinsing with tap water for 2-3 min; oxidation with periodic acid for 20 min, then rinsing with water; staining with hexamine silver solution at 60℃ for 30-50 min, until the basement membrane appears black under the microscope; (Hexamine silver solution preparation: 22 ml distilled water + 6 ml 15% hexamethylenetetramine + 4.5 ml 5% borax + 3 drops 4% boric acid, prepared before staining, placed in a 60℃ oven, and after 20 min, add 1 ml 5% silver nitrate); add a few drops of 2% gold chloride to the tissue, wash with 5% sodium thiosulfate; stain the nucleus with hematoxylin for 1 min, then rinse with water; differentiate with 1% hydrochloric acid ethanol for 2-3 s, then rinse with water; blue with ammonia for 2-3 s, then rinse with water; stain with eosin for 1 min, then rinse with water; dehydrate with graded alcohols, clear with xylene, and mount with resin. Masson staining: Dewaxing twice in xylene for 10 min each time; hydration twice in anhydrous ethanol for 1 min each time; rinsing with tap water for 2-3 min; oxidation with potassium dichromate for 20 min, then rinsing with water; staining the nucleus with hematoxylin for 1 min, then rinsing with water; differentiation with 1% hydrochloric acid ethanol for 2-3 s, then rinsing with water; blueing with ammonia for 2-3 s, then rinsing with water; staining with Masson's solution for 20 min, then rinsing with water; dehydration with graded alcohols, clearing with xylene, and mounting with resin.

[0109] 6. In mice (ICR), PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting adenine-induced renal tubular injury and interstitial fibrosis.

[0110] The procedure was as follows: 20 mice were randomly divided into 4 groups of 5 mice each. The groups were: sodium carboxymethyl cellulose gavage group, adenine gavage model group, and adenine model group treated with WHP intraperitoneal injection. After 4 weeks, the mice were euthanized by cervical dislocation under anesthesia, fixed in 10% formalin, sectioned in paraffin at 4 pm, and stained with PAS and Masson. PAS staining: Dewaxing twice in xylene, 10 min each time; hydration twice in anhydrous ethanol, 1 min each time; rinsing with tap water for 2-3 min; oxidation with periodic acid for 20 min, then rinsing with water; staining with hexamine silver solution at 60℃ for 30-50 min, until the basement membrane appears black under the microscope; (Hexamine silver solution preparation: 22 ml distilled water + 6 ml 15% hexamethylenetetramine + 4.5 ml 5% borax + 3 drops 4% boric acid, prepared before staining, placed in a 60℃ oven, and after 20 min, add 1 ml 5% silver nitrate); add a few drops of 2% gold chloride to the tissue, wash with 5% sodium thiosulfate; stain the nucleus with hematoxylin for 1 min, then rinse with water; differentiate with 1% hydrochloric acid ethanol for 2-3 s, then rinse with water; blue with ammonia for 2-3 s, then rinse with water; stain with eosin for 1 min, then rinse with water; dehydrate with graded alcohols, clear with xylene, and mount with resin. Masson staining: Dewaxing twice in xylene for 10 min each time; hydration twice in anhydrous ethanol for 1 min each time; rinsing with tap water for 2-3 min; oxidation with potassium dichromate for 20 min, then rinsing with water; staining the nucleus with hematoxylin for 1 min, then rinsing with water; differentiation with 1% hydrochloric acid ethanol for 2-3 s, then rinsing with water; blueing with ammonia for 2-3 s, then rinsing with water; staining with Masson's solution for 20 min, then rinsing with water; dehydration with graded alcohols, clearing with xylene, and mounting with resin.

[0111] 7. In mice (C57 / BL-6), PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting renal tubular damage and interstitial fibrosis induced by UUO.

[0112] The procedure was as follows: 20 mice were randomly divided into 4 groups of 5 mice each. The groups were: sham operation group, unilateral ureteral ligation (UUO) model group, and unilateral ureteral ligation (UUO) model group treated with Huai Er Qing Gao (6g / kg / d) by gavage. One week later, the mice were euthanized by cervical dislocation after anesthesia, fixed in 10% formalin, sectioned in paraffin at 4pm, and stained with PAS and Masson. PAS staining: Dewaxing twice in xylene, 10 min each time; hydration twice in anhydrous ethanol, 1 min each time; rinsing with tap water for 2-3 min; oxidation with periodic acid for 20 min, then rinsing with water; staining with hexamine silver solution at 60℃ for 30-50 min, until the basement membrane appears black under the microscope; (Hexamine silver solution preparation: 22 ml distilled water + 6 ml 15% hexamethylenetetramine + 4.5 ml 5% borax + 3 drops 4% boric acid, prepared before staining, placed in a 60℃ oven, and after 20 min, add 1 ml 5% silver nitrate); add a few drops of 2% gold chloride to the tissue, wash with 5% sodium thiosulfate; stain the nucleus with hematoxylin for 1 min, then rinse with water; differentiate with 1% hydrochloric acid ethanol for 2-3 s, then rinse with water; blue with ammonia for 2-3 s, then rinse with water; stain with eosin for 1 min, then rinse with water; dehydrate with graded alcohols, clear with xylene, and mount with resin. Masson staining: Dewaxing twice in xylene for 10 min each time; hydration twice in anhydrous ethanol for 1 min each time; rinsing with tap water for 2-3 min; oxidation with potassium dichromate for 20 min, then rinsing with water; staining the nucleus with hematoxylin for 1 min, then rinsing with water; differentiation with 1% hydrochloric acid ethanol for 2-3 s, then rinsing with water; blueing with ammonia for 2-3 s, then rinsing with water; staining with Masson's solution for 20 min, then rinsing with water; dehydration with graded alcohols, clearing with xylene, and mounting with resin.

[0113] 8. In mice (C57 / BL-6), immunohistochemical staining was used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting folic acid-induced renal tubular damage and interstitial fibrosis.

[0114] The procedure was as follows: Twenty mice were randomly divided into four groups of five each: a sodium bicarbonate solution intraperitoneal injection group, a folic acid model group, and a WHP intraperitoneal injection folic acid model group. One week later, the mice were anesthetized and euthanized by cervical dislocation. The mice were fixed in 10% formalin, sectioned in paraffin at 4 pm, and subjected to immunohistochemical staining. Immunohistochemical staining included: dewaxing twice in xylene for 10 minutes each time; gradient alcohol dehydration: dehydration was performed in 100%, 95%, 80%, and 70% alcohol solutions for 1 minute each; rinsing in tap water for approximately 10 minutes; PBS washing: 3 minutes × 3 times each time; adding endogenous peroxidase inhibitor to block endogenous peroxidase, incubating in a humidified chamber at room temperature for 10 minutes; PBS washing: 3 minutes × 3 times each time; microwave antigen retrieval: the iron slide holder was replaced with a plastic slide holder, placed in a solution of sodium bicarbonate solution... In a glass staining jar containing sodium citrate buffer, the sections are fully submerged in the sodium citrate buffer, ensuring the tissue is not exposed outside the buffer. Microwave on medium speed for 4 minutes, 4 times. After microwave retrieval, remove the jar from the microwave and allow it to cool naturally at room temperature. Wash with PBS for 3 minutes each time, 3 times. Serum blocking: Remove the sections from the jar, wipe the tissue around the sections dry with tissue paper while keeping the tissue moist, place the sections in a humidified chamber, and add goat serum for blocking. Incubate at room temperature for 20-30 minutes. Discard the blocking serum, and while still moist, add the pre-prepared primary antibody. Incubate overnight at 4°C in a humidified chamber. The next day, remove the humidified chamber from the refrigerator, let it stand for 30 minutes, and then wash with PBS for 3 minutes each time, 3 times. Select the appropriate horseradish peroxidase-labeled secondary antibody according to the species of the primary antibody, prepare the secondary antibody with PBS at a 1:100 ratio, add the secondary antibody, and incubate at 37°C for 40 minutes. Wash with PBS: 3 minutes x 3 times each time; DAB staining: Prepare DAB staining solution, mix thoroughly, add DAB staining solution to the slide, observe under a microscope, and stop the reaction with tap water when the tissue color turns brown; counterstain with hematoxylin for 30 seconds, rinse with tap water; dehydrate with graded alcohols: dehydrate for 2 minutes each in 80%, 95%, and 100% alcohol solutions; clear with xylene: clear twice in xylene for 5 minutes each time; mount with neutral resin, observe and photograph under a microscope.

[0115] 9. In mice (ICR), PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting adenine-induced renal tubular injury and interstitial fibrosis.

[0116] The procedure was as follows: Twenty mice were randomly divided into four groups of five each: a sodium carboxymethyl cellulose gavage group, an adenine gavage model group, and an HP-W intraperitoneal injection adenine model group. Four weeks later, mice were euthanized by cervical dislocation after anesthesia, fixed in 10% formalin, sectioned in paraffin at 4 pm, and subjected to immunohistochemical staining. Immunohistochemical staining included: dewaxing twice in xylene for 10 minutes each time; gradient alcohol dehydration: dehydration in 100%, 95%, 80%, and 70% alcohol solutions for 1 minute each; rinsing in tap water for approximately 10 minutes; PBS washing: 3 minutes x 3 times; adding endogenous peroxidase inhibitor to block endogenous peroxidase, incubating in a humidified chamber at room temperature for 10 minutes; PBS washing: 3 minutes x 3 times; microwave antigen retrieval: replacing the iron slide holder with a plastic slide holder, and placing it in a solution of... In a glass staining jar containing sodium citrate buffer, the sections are fully submerged in the sodium citrate buffer, ensuring the tissue is not exposed outside the buffer. Microwave on medium speed for 4 minutes, 4 times. After microwave retrieval, remove the jar from the microwave and allow it to cool naturally at room temperature. Wash with PBS for 3 minutes each time, 3 times. Serum blocking: Remove the sections from the jar, wipe the tissue around the sections dry with tissue paper while keeping the tissue moist, place the sections in a humidified chamber, and add goat serum for blocking. Incubate at room temperature for 20-30 minutes. Discard the blocking serum, and while still moist, add the pre-prepared primary antibody. Incubate overnight at 4°C in a humidified chamber. The next day, remove the humidified chamber from the refrigerator, let it stand for 30 minutes, and then wash with PBS for 3 minutes each time, 3 times. Select the appropriate horseradish peroxidase-labeled secondary antibody according to the species of the primary antibody, prepare the secondary antibody with PBS at a 1:100 ratio, add the secondary antibody, and incubate at 37°C for 40 minutes. Wash with PBS: 3 minutes x 3 times each time; DAB staining: Prepare DAB staining solution, mix thoroughly, add DAB staining solution to the slide, observe under a microscope, and stop the reaction with tap water when the tissue color turns brown; counterstain with hematoxylin for 30 seconds, rinse with tap water; dehydrate with graded alcohols: dehydrate for 2 minutes each in 80%, 95%, and 100% alcohol solutions; clear with xylene: clear twice in xylene for 5 minutes each time; mount with neutral resin, observe and photograph under a microscope.

[0117] 10. In mice (C57 / BL-6), PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting UUO-induced renal tubular damage and interstitial fibrosis.

[0118] The procedure was as follows: Twenty mice were randomly divided into four groups of five mice each: a sham-operated group, a unilateral ureteral ligation (UUO) model group, and a WHP intraperitoneal injection-treated unilateral ureteral ligation (UUO) model group. One week later, mice were euthanized by cervical dislocation after anesthesia, fixed in 10% formalin, sectioned in paraffin at 4 pm, and subjected to immunohistochemical staining. Immunohistochemical staining: Dewaxing: dewaxing twice in xylene, 10 min each time; gradient alcohol dehydration: dehydration was performed in 100%, 95%, 80%, and 70% alcohol tanks, 1 minute each; rinsing in tap water for about 10 minutes; PBS washing: 3 minutes × 3 times each time; adding endogenous peroxidase inhibitor to block endogenous peroxidase, incubating in a humidified chamber at room temperature for 10 minutes; PBS washing: 3 minutes × 3 times each time; microwave antigen retrieval: the iron slide holder was replaced with a plastic slide holder, placed in a container of... In a glass staining jar containing sodium citrate buffer, the sections are fully submerged in the sodium citrate buffer, ensuring the tissue is not exposed outside the buffer. Microwave on medium speed for 4 minutes, 4 times. After microwave retrieval, remove the jar from the microwave and allow it to cool naturally at room temperature. Wash with PBS for 3 minutes each time, 3 times. Serum blocking: Remove the sections from the jar, wipe the tissue around the sections dry with tissue paper while keeping the tissue moist, place the sections in a humidified chamber, and add goat serum for blocking. Incubate at room temperature for 20-30 minutes. Discard the blocking serum, and while still moist, add the pre-prepared primary antibody. Incubate overnight at 4°C in a humidified chamber. The next day, remove the humidified chamber from the refrigerator, let it stand for 30 minutes, and then wash with PBS for 3 minutes each time, 3 times. Select the appropriate horseradish peroxidase-labeled secondary antibody according to the species of the primary antibody, prepare the secondary antibody with PBS at a 1:100 ratio, add the secondary antibody, and incubate at 37°C for 40 minutes. Wash with PBS: 3 minutes x 3 times each time; DAB staining: Prepare DAB staining solution, mix thoroughly, add DAB staining solution to the slide, observe under a microscope, and stop the reaction with tap water when the tissue color turns brown; counterstain with hematoxylin for 30 seconds, rinse with tap water; dehydrate with graded alcohols: dehydrate for 2 minutes each in 80%, 95%, and 100% alcohol solutions; clear with xylene: clear twice in xylene for 5 minutes each time; mount with neutral resin, observe and photograph under a microscope.

[0119] III. Experimental Results

[0120] 1. By Figure 6 , Figure 7 It is evident that the total polysaccharide THP extracted from Sophora japonica extract did not inhibit the activity of normal mouse renal tubular epithelial cells. Figure 6 ), and inhibits macrophage activity ( Figure 7 (A) The extracted and isolated neutral polysaccharide WHP has twice the inhibitory efficiency against macrophage activity compared to THP. Figure 7 B in the text), and neutral polysaccharide WHPA ( Figure 7C) and WHPB ( Figure 7 D in the formula has the same inhibitory effect as WHP, and there is no difference between the two. Figure 7 (E in the example). Therefore, subsequent experiments will only select one of them as a representative for the experiment.

[0121] 2. By Figure 8 , Figure 9 It is evident that both THP and WHP can effectively inhibit the morphological changes in primary mouse renal tubular epithelial cells induced by Tgfb1 stimulation. Figure 8 ) and macrophage migration caused by damage to primary mouse renal tubular epithelial cells ( Figure 9 Furthermore, WHP (800 μg / mL) requires only half the dose of THP (1600 μg / mL) to achieve the same effect.

[0122] 3. By Figure 10-12 It is evident that both THP and WHP can effectively inhibit folic acid ( Figure 10 ), adenine ( Figure 11 ) and unilateral ureteral ligation ( Figure 12 WHP (0.085 g / kg / d) caused renal tubular damage and renal interstitial fibrosis in mice, and only half the dose of THP (0.17 g / kg / d) was required to achieve the same effect.

[0123] 4. By Figure 13-15 It is evident that WHP can effectively inhibit folic acid ( Figure 13 ), adenine ( Figure 14 ) and unilateral ureteral ligation ( Figure 15 This study induced an increase in α-sma, COL-1, and COL-3 levels and a decrease in E-cadherin in the kidneys of mice.

[0124] The above results indicate that the neutral water-soluble polysaccharides WHP, WHPA, and WHPB extracted from Sophora japonica extract using the extraction and separation method provided by this invention can treat renal interstitial fibrosis caused by various reasons, and the therapeutic effect is twice that of the crude extract of total polysaccharides from Sophora japonica.

[0125] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for separating and extracting neutral water-soluble polysaccharides, characterized in that, The steps of the method include: (1) precipitating the aqueous solution of Sophora japonica extract with four times the volume of anhydrous ethanol at 4°C for 24 hours, dialyzing with a 1000Da dialysis bag until the dialysate is clear, removing proteins, pigments and small molecules with Sevag reagent to obtain total polysaccharides; (2) dissolving the total polysaccharides obtained in (1) in water, purifying and eluting, tracking and detecting with phenol-sulfuric acid method, collecting according to peak shape, concentrating, dialyzing, and freeze-drying to obtain polysaccharide components; (3) dissolving the polysaccharide components obtained in (2) in water, purifying with a chromatography column, collecting symmetrical peaks, concentrating the collected liquid through a rotary evaporator, and freeze-drying to obtain purified polysaccharides.

2. The method according to claim 1, characterized in that, The removal of pigments and small molecules is achieved through dialysis. The specific steps of dialysis are as follows: dialysis for one week using a 1000Da dialysis bag, depigmentation using AB-8 macroporous resin, dialysis using a 1kD pore size dialysis bag, and freeze-drying to obtain total polysaccharides.

3. The method according to claim 1, characterized in that, The specific steps of the purification and elution are as follows: heating, swirling, centrifugation at 12000 rpm, loading the supernatant onto a DEAE Sepharose Fast Flow purification column, and eluting with distilled water at a flow rate of 15 ml / min. The eluent is water and 0.2 M to 1.0 M NaCl aqueous solution.

4. The method according to claim 3, characterized in that, The eluent is water, 0.2M NaCl aqueous solution, 0.5M NaCl aqueous solution, or 1.0M NaCl aqueous solution.

5. The method according to claim 1, characterized in that, The chromatography column was a Sephadex G-200 dextran gel column.

6. A neutral water-soluble polysaccharide obtained by the method according to any one of claims 1-5.

7. The neutral water-soluble polysaccharide according to claim 6, characterized in that, The neutral water-soluble polysaccharide has a monosaccharide composition including arabinose, galactose, glucose, and mannose, with a molar ratio of 11.4:5.8:10.5:22.

3.

8. The neutral water-soluble polysaccharide according to claim 6, characterized in that, The molecular formula of the neutral water-soluble polysaccharide is: .

9. The neutral water-soluble polysaccharide according to claim 6, characterized in that, The weight-average molecular weight of the neutral water-soluble polysaccharide is 2.0 × 10⁻⁶. 4 ~2.2×10 4 Da.

10. The neutral water-soluble polysaccharide according to claim 6, characterized in that, The weight-average molecular weight of the neutral water-soluble polysaccharide is 2.17 × 10⁻⁶. 4 Da.

11. The use of the neutral water-soluble polysaccharide according to any one of claims 6-10 in the preparation of a drug for treating or delaying renal interstitial fibrosis.

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