Active polysaccharide of swertia pseudochinensis, preparation method of active polysaccharide and application of active polysaccharide in preparation of antiepileptic drugs

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to active swertia pseudochinensis polysaccharide, a preparation method thereof and application of the active swertia pseudochinensis polysaccharide in preparation of antiepileptic drugs. The preparation method comprises the following steps: (1) crushing a dried swertia pseudochinensis sample, soaking with an alcoholic solution, and collecting residues; (2) carrying out water extraction treatment on the dried residues, and collecting an extracting solution; (4) concentrating the extracting solution, performing alcohol precipitation treatment, and collecting precipitate to obtain crude extraction polysaccharide; (3) dissolving the crude polysaccharide in water, deproteinizing, degreasing, decolorizing, dialyzing, concentrating, and freeze-drying to obtain crude polysaccharide; and (5) loading the crude polysaccharide solution to an anion exchange chromatographic column for purification, collecting eluent, concentrating, dialyzing, drying to obtain ion purified polysaccharide, loading the ion purified polysaccharide solution to a gel chromatographic column for further purification, collecting eluent, and drying to obtain the active polysaccharide of the swertia pseudochinensis. The swertia pseudochinensis polysaccharide D1N2 obtained from swertia pseudochinensis has the effects of preventing, relieving and / or treating rat epilepsy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug-active polysaccharide, its preparation method, and its application in the preparation of antiepileptic drugs. Background Technology

[0003] The medicinal herb is *Swertia*, a plant belonging to the genus *Swertia*. Swertia pseudochinensis The dried whole herb of *Swertia spp.* is distributed in Tibet, Yunnan, Sichuan, and other regions of my country. This medicinal material is used in the traditional medicine of several ethnic groups. The *Dian Yao Lu* records its whole herb as "Xu Bai Cai," primarily used to treat acute jaundice hepatitis. The *Mengzu Yaozhi Sanjuan* records preparations made from the whole herb of *Swertia spp.*, such as *Shan Huang Lian*, *Pu Di Qing*, and *Chou Cao*, primarily used to treat redness, swelling, pain, and damp-heat jaundice hepatitis. Its earliest medicinal record is found in *Inner Mongolia Traditional Chinese Medicine*, noting that its dried whole herb is widely distributed in northern provinces of my country, especially in North China, and has significant therapeutic effects, used to treat jaundice hepatitis, bacillary dysentery, and indigestion. The whole herb has the effects of clearing heat and dampness, strengthening the spleen and improving appetite, mainly used for damp-heat jaundice, hypochondriac pain, dysentery, abdominal pain, and loss of appetite. The herb is rich in chemical components, mainly containing iridoids, triterpenes, and flavonoids, with pharmacological effects primarily focused on hepatoprotection and choleretic effects. In recent years, polysaccharides extracted from traditional Chinese medicine (TCM) have attracted attention from the academic community due to their pharmacological activities such as antioxidant, free radical scavenging, anti-inflammatory, and antibacterial effects after purification and structural characterization. Related research has become a current hot topic, and they have been successfully applied in the food and pharmaceutical fields. However, research on polysaccharides from TCM remains relatively scarce, therefore, studying their structural characteristics and pharmacological effects is of great clinical significance. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a drug-active polysaccharide, its preparation method, and its application in the preparation of antiepileptic drugs.

[0005] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of this invention provides a method for preparing a pharmaceutically active polysaccharide, comprising the following steps: (1) After the dried medicine is pulverized, it is soaked in an alcohol solution. After soaking, the precipitate is collected and dried to obtain the dried residue. (2) The dried residue obtained in step (1) is mixed with water and then subjected to water extraction. After the water extraction is completed, the extract is collected. The temperature of the water extraction is 65-75℃. (3) The extract obtained in step (2) is concentrated to obtain a concentrated solution. The concentrated solution is subjected to alcohol precipitation. After the alcohol precipitation is completed, the precipitate is collected and dried to obtain crude polysaccharide. (4) Dissolve the crude polysaccharide prepared in step (3) in water to obtain a crude polysaccharide solution. The crude polysaccharide solution is subjected to deproteinization, defatting, decolorization, dialysis, concentration and freeze drying in sequence to obtain crude polysaccharide. (5) Dissolve the crude polysaccharide prepared in step (4) in water to obtain a crude polysaccharide solution; load the crude polysaccharide solution into an anion exchange chromatography column, elute the anion exchange chromatography column with an eluent, collect the eluent in segments, determine the total sugar content of the eluent in each collection tube using the sulfuric acid-phenol method, plot the ion purification elution curve with the tube number of the collected eluent in segments as the abscissa and the total sugar content of the eluent as the ordinate, combine the eluents corresponding to the main elution peak of the ion purification elution curve, and concentrate, dialyze and freeze dry the combined eluent to obtain purified polysaccharide; (6) Dissolve the purified polysaccharide prepared in step (5) in water to obtain a purified polysaccharide solution. Load the purified polysaccharide solution onto a gel chromatography column and elute the gel chromatography column with water. Collect the eluent in segments. Determine the total sugar content of the eluent in each collection tube using the sulfuric acid-phenol method. Plot the gel purification elution curve with the tube number of the collected eluent in segments as the abscissa and the total sugar content of the eluent as the ordinate. Combine the eluents corresponding to the main elution peak of the gel purification elution curve. The combined eluent is concentrated, dialyzed, and freeze-dried to obtain the active polysaccharide of the drug.

[0006] Preferably, in step (5), the anion exchange column is a DEAE seplife FF weak anion exchange column, and after loading the sample, it is sequentially eluted with pure water and 0.1, 0.2, and 0.3 M NaCl solutions in a gradient.

[0007] More preferably, in step (5), the elution flow rate of the anion exchange column is 3 to 5 mL / min.

[0008] More preferably, the concentration of the crude polysaccharide solution in step (5) is 0.02 to 0.03 g / mL.

[0009] Preferably, in step (5), the eluent is collected in segments of 15 mL per tube.

[0010] Preferably, the gel chromatography column in step (6) is a Sephacryl S-400 HR column.

[0011] More preferably, the flow rate of the gel chromatography column elution in step (6) is 0.5 to 2.0 mL / min.

[0012] More preferably, the concentration of the purified polysaccharide in step (6) is 0.025–0.075 g / mL.

[0013] Preferably, in step (6), the eluent is collected in segments of 10 mL per tube.

[0014] Preferably, step (4) uses the Sevag method for protein removal; uses an organic solvent for defatting, wherein the organic solvent is petroleum ether; and uses AB-8 macroporous resin.

[0015] More preferably, in step (4) Sevag deproteinization, after adding Sevag reagent, centrifuge, mix thoroughly, and collect the upper aqueous phase; the Sevag reagent is composed of chloroform and n-butanol in a volume ratio of (2-6):1.

[0016] More preferably, the boiling range of the petroleum ether reagent is 30–60°C.

[0017] Preferably, dialysis is performed in steps (4), (5) and (6) using a dialysis bag with a molecular weight cutoff of 3000 Da.

[0018] More preferably, the dialysis time for steps (4), (5) and (6) is 48 to 72 hours.

[0019] Preferably, in step (2), the mass ratio of the dried residue to the volume ratio of water is 1:5 to 1:20, and the water extraction time is 3 to 5 hours.

[0020] Preferably, the mass ratio of crude polysaccharide to water in step (4) is 1:10 to 1:30.

[0021] Preferably, the alcohol precipitation process in step (3) is as follows: anhydrous ethanol is added to the concentrate, and the solution is allowed to stand at 4°C for 8 to 16 hours. The volume of anhydrous ethanol is 2 to 6 times the volume of the concentrate. The volume of the concentrate in step (3) is 1 / 5 to 1 / 20 of the volume of the extract.

[0022] Preferably, in step (1), the alcohol solution is anhydrous ethanol solution, the soaking temperature is 20-30℃, and the soaking time is 8-16h.

[0023] Preferably, the drying temperature in steps (1) and (3) is 40-60°C and the drying time is 40-55 h.

[0024] Preferably, the freeze-drying temperature in steps (4), (5), and (6) is -25 to -15°C, and the time is 40 to 55 hours.

[0025] A second aspect of the present invention provides a drug-active polysaccharide prepared using any of the preparation methods described in the first aspect.

[0026] The third invention provides the use of the pharmacologically active polysaccharide described in the second aspect in the preparation of drugs for the prevention, relief and / or treatment of epilepsy.

[0027] The fourth aspect of the present invention provides a medicament for preventing, alleviating and / or treating epilepsy, wherein the active ingredient of the medicament is the active polysaccharide described in the second aspect of the present invention.

[0028] Preferably, the drug can reduce the MDA content in hippocampal tissue and increase the SOD and CAT content.

[0029] Compared with the prior art, the present invention has the following advantages: This invention prepares *Dangyao* polysaccharide D1N2 from the natural drug *Dangyao*, and investigates its protective effect against acute seizures in rats through animal experiments. Proteomics analysis was used to detect the expression of differentially expressed proteins in the hippocampus of epileptic rats, and the results were validated by Western blot. The study found that *Dangyao* polysaccharide D1N2 significantly prolonged the seizure latency in epileptic rats, reduced MDA content in the hippocampus, increased SOD and CAT levels, improved hippocampal pathological morphology, and inhibited the expression levels of key proteins in the complement and coagulation cascade pathways (ITGB2 and SERPINE1), the IL-17 signaling pathway (PTGS2), the AGE-RAGE signaling pathway (STAT3), the sphingolipid signaling pathway (SGPL1), and the herpes simplex virus type 1 infection pathway (Jun). The protective effect of *Dangyao* polysaccharide D1N2 against rat seizures prepared in this invention can be used to prepare polysaccharide-based antiepileptic drugs and functional foods, possessing significant economic and market value. Attached Figure Description

[0030] Figure 1 Figure 1 shows the characterization results of the active polysaccharide of *Dangyao*. Figure A is the ion purification elution curve of the eluent after desalting the polysaccharide using a DEAE seplife FF weak anion exchange column. Figure B shows the results of antioxidant activity determination, from left to right: ABTS free radical, total antioxidant capacity, DPPH free radical, and superoxide anion scavenging rate. Figure C is the gel purification elution curve of the eluent after passing through a Sephacryl S-400 HR column. Figure D is the UV-Vis spectrum of *Dangyao* polysaccharide D1N2. Figure E is the Fourier transform infrared spectrum of *Dangyao* polysaccharide D1N2. Figure F is the X-ray diffraction analysis spectrum of *Dangyao* polysaccharide D1N2. Figure G is the scanning electron microscope image of *Dangyao* polysaccharide D1N2, from left to right: 200×, 1000×, and 10000× magnification. Figure 2 Figure 1 shows the nuclear magnetic resonance (NMR) spectrum of the drug polysaccharide D1N2; Figure A is a one-dimensional proton NMR spectrum. 1 Figure B shows the H NMR spectrum; Figure B is the one-dimensional carbon spectrum. 13C NMR spectrum; Figure C is the COSY H-H correlation spectrum; Figure D is the HSQC C-H correlation spectrum; Figure E is the HMBC C-H correlation spectrum; Figure F is the NOESY H-H correlation spectrum; Figure G is the structural analysis of the D1N2 polysaccharide from the medicinal herb. Figure 3 The image shows the results of the monosaccharide composition analysis of the polysaccharide D1N2 from *Dendrobium nobile*. Figure 4 The total ion chromatogram of D1N2, a polysaccharide from the medicinal herb; Figure 5 Images of hippocampal tissue from rats in each group; A shows the biochemical test results, from left to right: malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels; B shows the H&E staining results; C shows the Nissl staining results. Figure 6 Figure 1 shows the proteomics characteristics of rat hippocampal tissue; Figure A is the peptide-protein diagram; Figure B is the peptide length distribution diagram; Figure C is the summary diagram of peptide and protein counts in the model group and the high-dose D1N2 polysaccharide group; Figure D is the protein identification overlap analysis diagram between the model group and the high-dose D1N2 polysaccharide group; Figure E is the differentially expressed protein diagram between the model group and the high-dose D1N2 polysaccharide group; Figure F is the PCA analysis diagram of samples from the model group and the high-dose D1N2 polysaccharide group; Figure G is the heatmap of paired quantitative correlation coefficients of samples; Figure H is the distribution diagram of logarithmic transformation fold change; Figure I is the histogram of logarithmic transformation fold change.

[0031] Figure 7 Subcellular localization map of differentially expressed proteins in rat hippocampus; Figure 8 The graph shows the GO analysis results of differentially expressed proteins; Figure 9 The image shows the results of KEGG analysis of differentially expressed proteins. Figure 10 The image shows the COG analysis results for differentially expressed proteins. Figure 11 Western blot was used to detect the expression of proteins ITGB2, SERPINE1, PTGS2, STAT3, SGPL1, and Jun in the hippocampus of rats in each group. Detailed Implementation

[0032] The embodiments described herein are merely illustrative of the technical content of the invention and are not intended to limit the scope of protection of the invention. The invention can be implemented in many forms and should not be construed as limited to the specific examples listed below.

[0033] Example 1: Preparation of active polysaccharides for pharmaceutical use A method for preparing a drug-active polysaccharide, the specific steps of which are as follows: (1) After the dried medicine is crushed, the medicine powder is obtained. Anhydrous ethanol is added to the medicine powder according to the mass ratio of the medicine powder to the volume ratio of anhydrous ethanol of 1:10. The medicine powder is soaked at 25°C for 12 h to degrease and decolorize. After soaking, the medicine powder is centrifuged and the precipitate is collected. The precipitate is dried at 50°C for 48 h to obtain the dried residue. (2) Add water to the dried residue obtained in step (1) according to the mass ratio of dried residue to water volume of 1:10, extract in a 60℃ water bath for 4 h, centrifuge to collect the supernatant, repeat the extraction twice, and combine the supernatants to obtain the extract. (3) The extract from step (2) was concentrated at 50°C for 2 hours to 1 / 10 of the extract volume using a rotary evaporator to obtain a concentrated solution. Anhydrous ethanol, four times the volume of the concentrated solution, was added to the concentrated solution. The solution was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation and dried at 50°C for 48 hours to obtain crude polysaccharide. (4) Dissolve the crude polysaccharide prepared in step (3) in water according to the mass ratio of crude polysaccharide to water of 1:20 to obtain crude polysaccharide solution. Add Sevag reagent composed of chloroform and n-butanol in a volume ratio of 4:1 to crude polysaccharide solution, centrifuge, mix thoroughly, collect the upper aqueous phase, defatt the upper aqueous phase with petroleum ether at a boiling range of 60℃, decolorize with AB-8 macroporous resin, dialyze with a 3000 Da dialysis bag for 48h, concentrate with a rotary evaporator, freeze dry at -20℃ for 48h to obtain crude polysaccharide. (5) Dissolve the crude polysaccharide prepared in step (4) in water to prepare a crude polysaccharide solution with a concentration of 0.025 g / mL. Centrifuge at 10000g for 10 min. Take the supernatant and load it into a DEAE Seplife FF weak anion exchange column (26 mm × 400 mm). Elute sequentially with distilled water and 0.1, 0.2, and 0.3 M NaCl solutions at a flow rate of 4 mL / min. Collect one tube of eluent every 15 mL. Collect the eluent in segments. Measure the total sugar content of the eluent in each collection tube at a wavelength of 490 nm using the phenol-sulfuric acid method. Plot the ion purification elution curve with the tube number of the collected eluent as the x-axis and the total sugar content of the eluent as the y-axis. Combine the eluents corresponding to the same main elution peak in the ion purification elution curve. Concentrate the combined eluent to 1 / 5 of the eluent volume using a rotary evaporator at 50℃. Then place it in a 3000 Da dialysis bag and dialyze against distilled water for 72 minutes. After desalting, two polysaccharide solutions, D1 and D2, were obtained. The solutions were then freeze-dried at -20°C for 48 hours to obtain two polysaccharides, D1 and D2. (6) Dissolve the D1 polysaccharide prepared in step (5) in water to prepare a purified polysaccharide solution with a concentration of 0.05 g / mL. Centrifuge at 10000g for 10 min. Take the supernatant and load it onto a Sephacryl S-400 HR column (26 mm × 1000 mm). Elute with distilled water at a flow rate of 1.0 mL / min. Elute with pure water for 1.5 column volumes. Collect one tube of eluent every 10 mL. Collect the eluent in segments. Measure the total sugar content of the eluent in each collection tube at a wavelength of 490 nm using the sulfuric acid-phenol method. Plot the gel purification elution curve with the tube number of the collected eluent as the x-axis and the total sugar content of the eluent as the y-axis. Combine the eluents corresponding to the same main elution peak in the gel purification elution curve. Concentrate the combined eluent to 1 / 5 of the eluent volume using a rotary evaporator at 50℃. Then place it in a 3000 Da dialysis bag and dialyze against distilled water for 72 minutes. After desalting, two components, D1N1 and D1N2, were obtained. They were then freeze-dried at -20℃ for 48 hours to obtain the active polysaccharide of the drug.

[0034] Example 2: Activity determination of the drug polysaccharide solution 1. The activity of the two polysaccharide solutions D1 and D2 obtained in step (5) of Example 1 was determined. (1) Purity determination In Example 1, step (5), the total sugar content and purity of each eluent tube were determined using the phenol-sulfuric acid method. Then, an ion purification elution curve was plotted with the collection tube number as the X-axis and the absorbance value (representing the total sugar content) as the Y-axis. The specific procedure for determining the total sugar content was as follows: 100 μL of a 10-fold diluted total polysaccharide eluent was taken, and 550 μL of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:10 (V / V)) was added. The mixture was stirred, allowed to stand in the dark for 10 min, and the absorbance was measured at 490 mm. The ion purification elution curve is shown below. Figure 1 As shown in Figure A, tubes 7-16 contain polysaccharide D1, and tubes 28-40 contain polysaccharide D2. The specific procedure for determining the concentration of polysaccharides after ion purification using the phenol-sulfuric acid method was as follows: 25 mg of lyophilized samples of polysaccharides D1 and D2 were weighed, dissolved and diluted in 2.5 mL of water to prepare a 0.1 mg / mL polysaccharide solution. 100 μL of the polysaccharide supernatant was taken, and 550 μL of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:10 (V / V)) was added and mixed thoroughly. The mixture was reacted in the dark for 10 min, and the absorbance was measured at 490 mm. A standard curve was plotted based on the absorbance values ​​of D-anhydrous glucose reference solutions of different concentrations to determine the polysaccharide content of each component. Compared with the sample volume, the purity of component D1 was calculated to be 90.9%, and the purity of component D2 was 89.2%.

[0035] (2) Antioxidant activity assay The antioxidant activities of D1 and D2 polysaccharides were comprehensively analyzed by determining their scavenging abilities against DPPH radicals, ABTS radicals, and superoxide anions, as well as their total antioxidant capacity. Vitamin C at 0.1 mg / mL was used as a positive control, and double-distilled water as a negative control. Polysaccharide samples were tested at four concentrations: 0.5, 1, 1.5, and 2 mg / mL. Each sample group had three replicates. Experimental procedures and calculations were strictly performed according to the kit instructions. The DPPH radical scavenging ability assay kit (catalog number BC4750), ABTS radical scavenging ability assay kit (catalog number BC4770), superoxide anion scavenging ability assay kit (catalog number BC1410), and total antioxidant capacity (T-AOC) assay kit (catalog number 1310) were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0036] Experimental results are as follows Figure 1 As shown in B, Figure 1 B, from left to right, shows the detection results of ABTS free radicals, total antioxidant capacity, DPPH free radicals, and superoxide anion scavenging rate. The results indicate that the antioxidant capacity of component D1 is stronger than that of component D2.

[0037] 2. The purity of components D1N1 and D1N2 in step (6) of Example 1 was determined. After passing D1 polysaccharide through a Sephacryl S-400 HR column, the total sugar content and purity of each eluent tube were determined using the phenol-sulfuric acid method, as described above. The gel purification elution curves are shown below. Figure 1 As shown in C. The gel purification elution curves showed two main peaks, and the corresponding components were named D1N1 (tubes 21-31, purity 92.9%, weight 4.64 mg) and D1N2 (tubes 35-43, purity 94.4%, weight 4.63 mg), respectively.

[0038] Example 3: Structural characterization of the drug polysaccharide D1N2 Because the purity of D1N2 polysaccharide is higher, the structure of D1N2 polysaccharide component was characterized.

[0039] 1. Ultraviolet-Visible Spectroscopy Analysis The UV-Vis spectroscopy results showed that no obvious absorption peaks were observed in the 200-400 nm wavelength range, indicating that the sample did not contain impurities such as pigments, proteins, and nucleic acids. Figure 1 D).

[0040] 2. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis Fourier transform infrared spectroscopy revealed that the sample was within the range of 3600-3200 cm⁻¹. -1The absorption band within this range is attributed to the stretching vibration absorption peak of the OH group, which is a characteristic absorption peak of carbohydrates. Specifically: 3296.63 cm⁻¹ -1 The absorption peak at 2918.88 cm⁻¹ corresponds to the stretching vibration of the OH group in carbohydrates. -1 The absorption peak at 1023.89 cm⁻¹ is attributed to the CH stretching vibration, while the peak at 1023.89 cm⁻¹ is attributed to the CH stretching vibration. -1 The absorption peak at that point corresponds to the CO stretching vibration ( Figure 1 E).

[0041] 3. X-ray diffraction (XRD) analysis X-ray diffraction patterns can reflect the crystalline or amorphous properties of polysaccharides: crystalline substances exhibit a series of sharp diffraction peaks, while amorphous substances show broad, diffuse peaks. Consistent with FT-IR analysis results, D1N2 exhibits similar characteristics in XRD patterns at almost all diffraction angles. Figure 1 F).

[0042] 4. Scanning electron microscopy analysis Scanning electron microscopy revealed that the surface morphology of D1N2 polysaccharide exhibited an irregular, uneven structure with several spherical aggregates attached. Figure 1 G, from left to right, represents 200×, 1000×, and 10000× times respectively.

[0043] 5. Nuclear magnetic resonance structural analysis Nuclear magnetic resonance results showed that the one-dimensional hydrogen spectrum 1 H NMR such as Figure 2 A, One-dimensional carbon spectrum 13 C NMR such as Figure 2 B, COSY hydrogen-hydrogen correlation spectrum as follows Figure 2 C, HSQC carbon-hydrogen correlation spectrum as follows Figure 2 D, HMBC C-H correlation spectrum as follows Figure 2 E, NOESY hydrogen-hydrogen correlation spectrum as follows Figure 2 F. The results showed that the 1H NMR signal of the sample was mainly concentrated between δ 3.0 and 5.5 ppm. Multiple coupled signal peaks were identified in the anodic signal region of δ 4.3-5.4 ppm, indicating the presence of various sugar residues in the sample. The corresponding chemical shifts of the anodic hydrogens were δ 4.38, 4.43, 4.54, 4.65, 4.75, 4.99, 5.09, and 5.14, respectively. The non-anodic hydrogen signals were mainly concentrated in the δ 3.1-4.2 ppm region. Due to severe overlap, some signals required further analysis using COSY and HSQC spectra to assign the H2-H6 chemical shifts of each sugar residue. The strong signal peak near δ 4.71 ppm was a solvent peak.

[0044] Multiple signal peaks were identified in the anodic carbon region of the sample, combined with13 The cross-peaks in the anodic regions of the C NMR and HSQC spectra identified the anodic signals present in the sample as follows: δ 4.43 / 103.18, 4.38 / 103.47, 4.65 / 100.13, 5.14 / 109.21, 4.99 / 107.58, 5.09 / 100.69, 4.54 / 104.28, and 4.75 / 99.7 ppm, which were denoted as sugar residues A, B, C, D, E, F, G, and H, respectively. Based on the sample's bonding structure (methylation) information, anodic signals, and comprehensive literature reports, it is inferred that sugar residue A is →4)-β-D-Galp-(1→, sugar residue B is →3,6)-β-D-Galp-(1→, sugar residue C is →4)-β-D-Manp-(1→, sugar residue D is →5)-α-L-Araf-(1→, sugar residue E is α-L-Araf-(1→, sugar residue F is →3)-α-D-Glcp-(1→, sugar residue G is →6)-β-D-Glcp-(1→, sugar residue H is α-L-Rhap-(1→, as shown in the original text) Figure 2 G).

[0045] Example 4: Monosaccharide composition analysis of the drug polysaccharide D1N2 (1) Sample extraction Take a clean chromatographic vial, weigh 20 mg of the polysaccharide sample, add 1 mL of 2 M TFA acid solution, and heat at 121 °C for 2 h. Purge with nitrogen and dry. Wash with 99.99% methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water, transfer to a chromatographic vial for analysis.

[0046] (2) Preparation of standard products After accurately weighing the required standards for this project, add water to prepare a 10 mg / mL standard solution stock solution. Then, take an appropriate amount of the stock solution stock solution and mix them to prepare a standard mixed standard with a maximum index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL. Prepare the series of standards required for the instrument according to the concentration gradient in Table 1.

[0047] Table 1. Monosaccharide mixed standard gradient concentration information (3) Chromatographic analysis The chromatographic system used was a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), which utilized an electrochemical detector to analyze and detect monosaccharide components. A Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A / B / C (95:5:0, V / V), 26 min A / B / C (85:5:10, V / V), 42 min A / B / C (85:5:10, V / V), 42.1 min A / B / C (60:0:40, V / V), 52 min A / B / C (60:40:0, V / V), 52.1 min A / B / C (95:5:0, V / V), 60 min A / B / C (95:5:0, V / V).

[0048] (4) Results of monosaccharide composition analysis The monosaccharide composition analysis results of D1N2 polysaccharide are as follows: Figure 3 As shown, the D1N2 polysaccharide is mainly composed of Fuc, Ara, Rha, Gal, Glc, Xyl, Man, Gal-UA, and Glc-UA, with molar percentages of 3.03%, 29.09%, 9.11%, 28.17%, 17.44%, 4.24%, 7.04%, 1.09%, and 0.77%, respectively.

[0049] Example 5: Methylation analysis of the drug polysaccharide D1N2 (1) Sample pretreatment Weigh 2 mg of polysaccharide sample and dissolve it in 500 μL of DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times. Take the lower dichloromethane phase and dry it under nitrogen. Add 100 μL of 2M TFA and react at 121℃ for 90 min. Evaporate to dryness at 30℃. Add 50 μL of 2M ammonia and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL of acetic acid to terminate the reaction, dry under nitrogen, wash twice with 250 μL of methanol, and dry under nitrogen. Add 250 μL of acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h. Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water three times. Take the lower dichloromethane phase and analyze it by GC-MS.

[0050] (2) Mass spectrometry detection An Agilent Technologies Inc. (CA, USA) 6890A-5977B gas chromatography-mass spectrometry (GC-MS) system with an autosampler model G4567A was used. The chromatographic system employed was an Agilent GC system (6890A; Agilent Technologies, USA), with a BPX70 column (30 m × 0.25 mm × 0.25 µm, SGE, Australia). The injection volume was 1 μL, the split ratio was 10:1, the carrier gas was high-purity helium, and the flow rate was 1.5 mL / min. The column oven was initially set at 140 °C and held for 2.0 min, then programmed to reach 230 °C at a rate of 3 °C / min and held for 3 min. The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. Electron impact ionization (EI) was used, and the analytes were detected in full scan (SCAN) mode with a mass scan range (m / z) of 50-350.

[0051] (3) Test results When the total ion chromatogram of the drug polysaccharide D1N2 is as follows Figure 4 The main glycosidic bonds of the polysaccharide D1N2 are four types: t-Gal(p), t-Glc(p), t-Ara(f), and 4-Man(p), with molar ratios of 37.53%, 25.09%, 20.06%, and 11.00%, respectively.

[0052] Example 6: Pharmacological verification of the drug polysaccharide D1N2 1. Animal model construction and dosing regimen SD rats (weighing 180-220 g) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed in the SPF-grade animal room of Henan Shuangyun Biotechnology Co., Ltd. The environmental conditions were 22 ± 2°C, 50% humidity, 12 h light-dark cycle, and free access to food and water.

[0053] Thirty rats were randomly divided into five groups: control group, model group, carbamazepine (CBZ) group, high-dose D1N2 polysaccharide group, and low-dose D1N2 polysaccharide group, with six rats in each group. After 7 days of acclimatization, the control group and model group were administered physiological saline by gavage daily at a dose of 5 mL / kg; the carbamazepine group was administered carbamazepine by gavage daily at a dose of 0.125 g / kg; the high-dose D1N2 polysaccharide group was administered D1N2 polysaccharide by gavage daily at a dose of 10 g / kg; and the low-dose D1N2 polysaccharide group was administered D1N2 polysaccharide by gavage daily at a dose of 5 g / kg. This treatment was continued for 14 days. On day 15, except for the control group, the other four groups were induced with epilepsy models by intraperitoneal injection of 3 mmol / kg lithium chloride, followed by intraperitoneal injection of 35.3 mg / kg pilocarpine 20 h later. Seizures occurred in rats 15-35 minutes after induction, and the intensity of seizures within 30 minutes was recorded using the modified Racine scale. One hour after the onset of seizures, intraperitoneal injection of 10% chloral hydrate saline solution terminated the seizures. After modeling, rats continued to eat freely and were administered the same drug regimen by gavage for 7 days before being sacrificed.

[0054] 2. Effects of drugs on the latency and frequency of epileptic seizures in rats Table 2 shows the latency and frequency of seizures in four groups of rats induced by lithium chloride-pilocarpine. As shown in Table 2, compared with the model group, the CBZ group, the high-dose group of D1N2 polysaccharide, and the low-dose group of D1N2 polysaccharide significantly prolonged the latency of seizures and reduced the frequency of seizures. The effect of the high-dose group of D1N2 polysaccharide was similar to that of the CBZ group, indicating that D1N2 polysaccharide has a good preventive effect on rat epilepsy.

[0055] Table 2. Effects of D1N2 polysaccharide on latency and frequency of epileptic seizures in rats. 3. Effects of drugs on biochemical indicators and tissue morphology of hippocampal tissue in epileptic rats The hippocampal tissues of rats in each group were collected for the detection of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels. The results are as follows: Figure 5As shown in Figure A, compared with the control group, the MDA content in the hippocampus of epileptic rats was significantly increased, while the SOD and CAT contents were significantly decreased. Compared with the model group, administration of carbamazepine and D1N2 polysaccharide significantly reduced the MDA content and increased the SOD and CAT contents in the hippocampus of rats.

[0056] H&E staining results are as follows Figure 5 As shown in Figure B, the hippocampal CA1 region cells in the control group showed normal morphology, orderly cell arrangement, abundant cytoplasm, and large, round nuclei. In contrast, the model group showed a significant decrease in cell count, with loose and disordered cell arrangement. The cytoplasm exhibited deep staining, and nuclear dissolution and fragmentation were evident, accompanied by vacuolation. Compared to the model group, both the carbamazepine group and the D1N2 group showed improved hippocampal cell characteristics, increased cell density, gradual restoration of normal cell morphology, more orderly cell arrangement, and a significant reduction in the pathological manifestations of nuclear fragmentation and pyknosis.

[0057] Nissl staining results as follows Figure 5 As shown in Figure C, neurons in the control group were evenly arranged, with regular morphology and clearly visible Nissl bodies. Neurons in the treatment group were slightly disordered, with relatively regular cell morphology, but Nissl bodies stained slightly lighter and intercellular spaces increased. Neurons in the model group were significantly absent, disordered, and generally lightly stained.

[0058] 4. Effects of drugs on the proteomic characteristics of hippocampal tissue in epileptic rats To elucidate the mechanism by which D1N2 polysaccharide improves acute seizures in epileptic rats, we used proteomics analysis on the hippocampus of rats in the model group and the high-dose D1N2 polysaccharide group. The results showed that the proteomics data identified a total of 43,338 peptides and 6,336 proteins, including 15,431 unique peptides and 2,597 quantifiable proteins. Figure 6 A). The identified peptide lengths were mainly distributed between 7 and 21 amino acids, consistent with the expected distribution characteristics of peptides digested by trypsin. Figure 6 B). The number of peptides and proteins detected in each group is summarized in [the table below]. Figure 6 C. Protein identification overlap analysis showed that there were a total of 6,324 proteins (99.34%) in the two groups, while the model group and the D1N2 polysaccharide group specifically identified 6,333 and 6,357 proteins, respectively. Figure 6 D).

[0059] According to |FC|>1.5 and P Based on the criterion of <0.05, a total of 291 differentially expressed proteins were identified. Compared with the D1N2 polysaccharide group, 154 proteins in the model group were upregulated and 137 proteins were downregulated. Figure 6E). Principal component analysis (PCA) further revealed a clear separation between the two groups of samples, indicating significant differences in their proteomic expression profiles. Figure 6 F). Based on the quantitative data of each protein, the Pearson correlation coefficient (R) between each pair of samples was calculated to assess the quantitative repeatability between replicate samples and the quantitative correlation between different sample groups. A heatmap of paired quantitative correlation coefficients for all samples is shown below. Figure 6 As shown in G, red indicates a correlation coefficient close to 1, indicating good quantitative repeatability and correlation. The correlation among D1N2 polysaccharide groups is better than that among model groups, mainly because modeling treatment may cause more significant changes in the overall proteome, thereby increasing intra-group heterogeneity.

[0060] Comparative analysis of the log-transformation fold change (Log2FC) distribution showed that although the median ratios of each group were close to zero, the standard deviation of the model group / D1N2 polysaccharide group (SD = 0.67) was significantly higher than that of the D1N2 polysaccharide group / D1N2 polysaccharide group (SD = 0.31) and the model group / model group (SD = 0.37). Figure 6 H). This increased variability indicates that lithium chloride-pilocarpine treatment induced significant heterogeneity in protein expression. In the initial dataset, 4,308 proteins that were quantifiable in at least two replicates were retained for differential analysis. Of these, 154 proteins in the model group were significantly upregulated (FC ≥ 1.5, H). P <0.05), 146 proteins were significantly downregulated (FC ≤ −1.5, P <0.05)( Figure 6 I).

[0061] 5. Subcellular localization of differentially expressed proteins in hippocampal tissue Subcellular localization analysis of proteins helps to reveal their functional roles within cells. For example... Figure 7 As shown, we present the distribution and proportion of proteins in different cell compartments in the proteomic data. Differentially expressed proteins are mainly located in the protoplasm (227 proteins, accounting for 6.48%), followed by the cytoplasm (200 proteins, 5.71%) and nucleoplasm (138 proteins, 3.94%).

[0062] 6. GO analysis results of differentially expressed proteins GO functional enrichment analysis of differentially expressed proteins helps to systematically understand their biological processes, molecular functions, and cellular composition in organisms. Results are as follows: Figure 8As shown, differentially expressed proteins were enriched in 155 GO-BP entries, mainly involving the following metabolic and regulatory processes: biological process regulation, organic matter metabolism, primary metabolism, cellular metabolism, nitrogenous compound metabolism, cellular response to stimuli, anatomical development, cell component organization or biogenesis, multicellular development, and localization. 47 GO-MF entries were also enriched, primarily focusing on binding functions (protein binding, organic cyclic compound binding, heterocyclic compound binding, ion binding, enzyme regulatory activity, protein complex binding, transferase activity, catalytic activity, protein action, hydrolase activity, and carbohydrate derivative binding). Additionally, 134 GO-CC entries were enriched, mainly involving cellular structures such as organelles, intracellular organelles, cytoplasm, membrane-bound chambers, membranes, endometrial systems, cytosol, pericellular space, extracellular regions, and nucleoplasm.

[0063] 7. KEGG analysis results of differentially expressed proteins We performed KEGG pathway analysis on differentially expressed proteins. For example... Figure 9 As shown, the results indicate that these proteins are significantly enriched in multiple key pathways, including complement and coagulation cascades, IL-17 signaling pathway, AGE-RAGE signaling pathway, sphingolipid signaling pathway, and herpes simplex virus type 1 infection pathway.

[0064] 8. COG analysis results of differentially expressed proteins To screen for significantly enriched COG functional categories among differentially expressed proteins, we compared the COG annotation results of the differentially expressed protein list with the complete list of identified proteins. For example... Figure 10 As shown, COG enrichment analysis revealed that differentially expressed proteins were significantly enriched in the "extracellular structures" category.

[0065] 9. Verify the effect of the drug on differentially expressed proteins in the hippocampus of epileptic rats. Western blot analysis was used to validate differentially expressed proteins screened by proteomics. The results showed that, compared to the control group, the expression levels of key proteins in the complement and coagulation cascade pathways (ITGB2 and SERPINE1), the IL-17 signaling pathway (PTGS2), the AGE-RAGE signaling pathway (STAT3), the sphingolipid signaling pathway (SGPL1), and the herpes simplex virus type 1 infection pathway (Jun) were significantly increased in the hippocampus of the model group rats. The drug-derived polysaccharide D1N2 significantly reduced the expression levels of these proteins in a dose-dependent manner. These results indicate that the drug-derived polysaccharide D1N2 exerts its anti-epileptic effect by inhibiting the expression of these pathways.

[0066] The above embodiments are merely specific examples of the present invention and are intended to illustrate the invention, not to limit it. Any obvious modifications or equivalent alternatives guided by the core technical concept of the present invention fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a pharmaceutically active polysaccharide, characterized in that, Includes the following steps: (1) After the dried medicine is pulverized, it is soaked in an alcohol solution. After soaking, the precipitate is collected and dried to obtain the dried residue. (2) The dried residue obtained in step (1) is mixed with water and then subjected to water extraction. After the water extraction is completed, the extract is collected. The temperature of the water extraction is 65-75℃. (3) The extract obtained in step (2) is concentrated to obtain a concentrated solution. The concentrated solution is subjected to alcohol precipitation. After the alcohol precipitation is completed, the precipitate is collected and dried to obtain crude polysaccharide. (4) Dissolve the crude polysaccharide prepared in step (3) in water to obtain a crude polysaccharide solution. The crude polysaccharide solution is subjected to deproteinization, defatting, decolorization, dialysis, concentration and freeze drying in sequence to obtain crude polysaccharide. (5) Dissolve the crude polysaccharide prepared in step (4) in water to obtain a crude polysaccharide solution; load the crude polysaccharide solution into an anion exchange chromatography column, elute the anion exchange chromatography column with an eluent, collect the eluent in segments, determine the total sugar content of the eluent in each collection tube using the sulfuric acid-phenol method, plot the ion purification elution curve with the tube number of the collected eluent in segments as the abscissa and the total sugar content of the eluent as the ordinate, combine the eluents corresponding to the main elution peak of the ion purification elution curve, and concentrate, dialyze and freeze dry the combined eluent to obtain purified polysaccharide; (6) Dissolve the purified polysaccharide prepared in step (5) in water to obtain a purified polysaccharide solution. Load the purified polysaccharide solution onto a gel chromatography column and elute the gel chromatography column with water. Collect the eluent in segments. Determine the total sugar content of the eluent in each collection tube using the sulfuric acid-phenol method. Plot the gel purification elution curve with the tube number of the collected eluent in segments as the abscissa and the total sugar content of the eluent as the ordinate. Combine the eluents corresponding to the main elution peak of the gel purification elution curve. The combined eluent is concentrated, dialyzed, and freeze-dried to obtain the active polysaccharide of the drug.

2. The preparation method according to claim 1, characterized in that, Step (5) The anion exchange column is a DEAEseplife FF weak anion exchange column. After loading the sample, the column is eluted sequentially with water and 0.1, 0.2, and 0.3 M NaCl solutions.

3. The preparation method according to claim 2, characterized in that, Step (6) The gel chromatography column is a Sephacryl S-400 HR column.

4. The preparation method according to claim 1, characterized in that, Step (4) Deproteinize using the Sevag method; defatting using an organic solvent, wherein the organic solvent is petroleum ether reagent; decolorizing using a macroporous resin, wherein the macroporous resin is AB-8 macroporous resin; Step (5) elution using an anion exchange column at a flow rate of 3 to 5 mL / min; Step (6) elution using a gel chromatography column at a flow rate of 0.5 to 2.0 mL / min.

5. The preparation method according to claim 1, characterized in that, In steps (4), (5) and (6), dialysis is performed using dialysis bags with a molecular weight cutoff of 3000 Da; in step (2), the mass ratio of the dried residue to the volume of water is 1:5 to 1:20, and the water extraction time is 3 to 5 h; in step (4), the mass ratio of the crude polysaccharide to the volume of water is 1:10 to 1:

30.

6. The preparation method according to claim 5, characterized in that, Step (3) alcohol precipitation process is as follows: add anhydrous ethanol to the concentrate, let it stand at 4℃ for 8 to 16 hours, the volume of anhydrous ethanol is 2 to 6 times the volume of the concentrate; the volume of the concentrate in step (3) is 1 / 5 to 1 / 20 of the volume of the extract.

7. The preparation method according to claim 1, characterized in that, Step (1) The alcohol solution is anhydrous ethanol solution, the soaking temperature is 20-30℃, and the soaking time is 8-16 h.

8. The active polysaccharide prepared by any one of the preparation methods according to claims 1 to 7.

9. The use of the pharmacoactive polysaccharide of claim 8 in the preparation of a drug for the prevention, relief and / or treatment of epilepsy.

10. A drug for the prevention, relief, and / or treatment of epilepsy, characterized in that, The active ingredient of the drug is the active polysaccharide as described in claim 8.

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