A neuroprotective agent and its preparation method and application

Abstract

The present application relates to natural product extraction and functional product technical field, specifically a kind of neuroprotective agent and its preparation method and application, adopt water extraction alcohol precipitation combined with ultrafiltration subsection technology, schisandra chinensis powder is added water according to material liquid ratio 1:15, water extraction is carried out under the condition of 85 DEG C, after filtration concentration, alcohol precipitation is carried out, the obtained precipitate is redissolved, and the polysulfone ultrafiltration membrane with the molecular weight cut-off of 10kDa is used Ultrafiltration purification, collect cut-off liquid and dry, the total polysaccharide content of 59.2%, the glycuronidase content of 6.6%, the protein content of 3.6% schisandra chinensis polysaccharide enrichment is prepared;The enrichment has significant neuroprotective activity, can effectively antagonize corticosterone-induced PC12 cell damage, reduce intracellular reactive oxygen species level, inhibit inflammatory factor TNF-α, IL-1β secretion and inhibit cell apoptosis, polysaccharide enrichment degree is high, process is simple, and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and functional product technology, specifically a neuroprotective agent (SCP-EF) and its preparation method and neuroprotective applications. Background Technology

[0002] In recent years, with the accelerated pace of modern life, prolonged stress can activate the hypothalamic-pituitary-adrenal axis, increasing the release of glucocorticoids and leading to neuropathological damage, cognitive impairment, mental illness, and neurodegenerative diseases. Currently, clinically used neuroprotective drugs mainly include antioxidants, anti-inflammatory drugs, and neurotrophic factors; however, long-term use has limitations in efficacy and significant side effects, making it difficult to meet the growing demand.

[0003] Polysaccharides derived from natural plants have garnered increasing attention in the field of neuroprotection due to their high safety, good biocompatibility, and diverse biological activities. They exert neuroprotective effects through multiple pathways, including scavenging free radicals, enhancing antioxidant enzyme activity, inhibiting neuroinflammation, and promoting the expression of neurotrophic factors. Schisandra chinensis, a plant belonging to the genus Schisandra in the family Magnoliaceae, has the effects of invigorating qi and promoting body fluid production, tonifying the kidneys and calming the mind. It contains various active ingredients such as polysaccharides, lignans, and volatile oils. However, traditional high-concentration alcohol extraction methods result in significant polysaccharide loss, while water extraction, although effective, fails to yield fat-soluble active ingredients. Furthermore, existing water extraction and alcohol precipitation processes produce products with a wide molecular weight distribution and high impurity content, lacking effective molecular weight fractionation methods, which hinders the development and application of subsequent functional products. Therefore, there is an urgent need to develop a neuroprotective agent, its preparation method, and its applications to overcome the shortcomings in current practical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a neuroprotective agent, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A neuroprotective agent, wherein the neuroprotective agent is a polysaccharide enrichment of Schisandra chinensis, which is prepared by water extraction, filtration and concentration, alcohol precipitation, precipitation and redissolution of dried mature Schisandra chinensis fruit of the Magnoliaceae family, followed by ultrafiltration fractionation using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and drying of the collected retentate. The neuroprotective agent contains 59.2% total polysaccharides, 6.6% uronic acid, and 3.6% protein.

[0006] As a further aspect of the present invention: the material-to-liquid ratio of the water extraction is 1:15 (g / mL), the extraction temperature is 85℃, the extraction is performed twice, and the extraction time for each extraction is 2 hours.

[0007] As a further aspect of the present invention: in the alcohol precipitation step, the extract is concentrated to a relative density of 1.10 at 60°C, ethanol is added to make the final volume concentration of ethanol reach 70%, and the precipitate is collected by centrifugation after standing at room temperature of 20°C to 30°C for 12 hours.

[0008] A method for preparing the above-mentioned neuroprotective agent includes the following steps: extracting Schisandra chinensis fruit raw material with water, filtering and concentrating it, then precipitating it with alcohol, redissolving the precipitate, and then purifying it by ultrafiltration fractional purification using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10 kDa, collecting the retentate and drying it to obtain Schisandra chinensis polysaccharide enrichment.

[0009] As a further aspect of the present invention: the material-to-liquid ratio of the water extraction is 1:15 (g / mL), the extraction temperature is 85℃, the extraction is performed twice, and the extraction time for each extraction is 2 hours.

[0010] As a further aspect of the present invention: in the alcohol precipitation step, the extract is concentrated to a relative density of 1.10 at 60°C, ethanol is added to make the final volume concentration of ethanol reach 70%, and the precipitate is collected by centrifugation after standing at room temperature of 20°C to 30°C for 12 hours.

[0011] As a further aspect of the present invention: the operating pressure of the ultrafiltration is 0.15 MPa, and the operating temperature is 20℃~30℃.

[0012] The application of the above-described neuroprotective agent in the preparation of neuroprotective drugs.

[0013] As a further aspect of the present invention, the neuroprotection specifically involves antagonizing corticosterone-induced neuronal damage.

[0014] As a further aspect of the present invention: the neuroprotection is achieved by reducing intracellular reactive oxygen species levels, inhibiting the secretion of inflammatory factors, and / or inhibiting cell apoptosis; The inflammatory factors include tumor necrosis factor-α and interleukin-1β.

[0015] Compared with the prior art, the beneficial effects of the present invention are: By employing ultrafiltration segmentation technology, polysaccharide enrichments with well-defined molecular weight distributions can be obtained. Appropriate molecular weight ranges can be screened according to the target application, thereby improving the product's targeting and efficacy predictability. The ultrafiltration process simultaneously removes small molecule impurities such as pigments, monosaccharides, and inorganic salts, improving product purity, enhancing the safety and compatibility of the ingredients, and making it more suitable as a raw material for neuroprotective drugs. The combined use of water extraction and alcohol precipitation with ultrafiltration fractional purification achieves preliminary purification during extraction, reduces subsequent purification steps, lowers production costs, and is suitable for industrial-scale production. This enrichment can significantly reduce corticosterone-induced PC12 cell damage, enhance antioxidant capacity, and inhibit cell apoptosis, providing a scientific basis for the development of neuroprotective products. The enriched compounds of this invention exert neuroprotective effects through multiple pathways, including anti-oxidation, anti-inflammation, and direct inhibition of apoptosis, exhibiting multi-target effects. Attached Figure Description

[0016] Figure 1 This is a graph showing the effect of neuroprotective agents on the survival rate of PC12 cells damaged by corticosterone in an embodiment of the present invention.

[0017] Figure 2 This figure shows the effect of neuroprotective agents on the level of reactive oxygen species in PC12 cells damaged by corticosterone, as described in this embodiment of the invention.

[0018] Figure 3 This is a diagram illustrating the effect of neuroprotective agents on the secretion of inflammatory factors in PC12 cells damaged by corticosterone, as described in this embodiment of the invention. In this table, A represents the result of tumor necrosis factor-α concentration detection, and B represents the result of interleukin-1β concentration detection.

[0019] Figure 4 This is a graph showing the effect of neuroprotective agents on the apoptosis rate of PC12 cells damaged by corticosterone in an embodiment of the present invention.

[0020] Figure 5 This is a scatter plot of flow cytometry results showing the effect of Schisandra chinensis polysaccharide enrichment on corticosterone-induced apoptosis in PC12 cells in an embodiment of the present invention. Among them, A is the control group, B is the corticosterone model group, C is the fluoxetine positive control group, D is the 5 μg / mL Schisandra polysaccharide enrichment treatment group, E is the 50 μg / mL Schisandra polysaccharide enrichment treatment group, and F is the 100 μg / mL Schisandra polysaccharide enrichment treatment group. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Please see Figures 1-5 The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0023] Unless otherwise specified, the experimental methods involved in the embodiments of this invention are all conventional experimental methods in the art; the raw materials, reagents, instruments and equipment used can all be obtained through legitimate commercial channels unless otherwise specified.

[0024] In this invention, the total polysaccharide content was determined by the phenol-sulfuric acid method, with glucose as the standard; the uronic acid content was determined by the m-hydroxybiphenyl method, with galacturonic acid as the standard; and the protein content was determined by the Coomassie brilliant blue method, with bovine serum albumin as the standard.

[0025] I. Preparation of Schisandra chinensis polysaccharide concentrate (neuroprotective agent) Example 1: Preparation of Schisandra chinensis polysaccharide concentrate using a preferred process (1) Raw material pretreatment: Take 10 kg of dried Schisandra chinensis fruit, crush it and pass it through a 40-mesh sieve to obtain Schisandra chinensis powder; (2) Water extraction: Add the Schisandra chinensis powder to 150L of pure water at a material-to-liquid ratio of 1:15 (g / mL), and extract twice at 85℃ for 2 hours each time. Combine the two extracts. (3) Alcohol precipitation: Filter the extract to remove the residue, concentrate the filtrate under reduced pressure to a relative density of 1.10 at 60°C; add 95% ethanol aqueous solution to the concentrate to make the final volume concentration of ethanol reach 70%, let it stand at room temperature of 20°C to 30°C for 12 hours, centrifuge at 4000 rpm for 15 minutes, and collect the precipitate. (4) Ultrafiltration segmentation: The obtained precipitate was redissolved in 20L of pure water and ultrafiltration was performed using a polyethersulfone (PES) ultrafiltration membrane with a molecular weight cutoff of 10kDa. The operating pressure was 0.15MPa and the operating temperature was 20℃~30℃ room temperature. The retentate was collected. (5) Drying: The retentate was spray-dried to obtain approximately 0.72 kg of Schisandra polysaccharide concentrate.

[0026] Preferably, the process parameters for spray drying in step (5) are: inlet air temperature 130℃~180℃ (preferably 150℃~160℃), outlet air temperature 60℃~90℃ (preferably 75℃~85℃), and spray pressure 0.15~0.35Mpa.

[0027] The Schisandra chinensis polysaccharide enrichment prepared in this embodiment was found to contain 59.2% total polysaccharide, 6.6% uronic acid, and 3.6% protein, which are within the range of component contents defined in this invention.

[0028] Example 2: Preparation of Schisandra chinensis polysaccharide concentrate using a low-intensity process (1) Raw material pretreatment: Take 10 kg of dried Schisandra chinensis fruit, crush it and pass it through a 20-mesh sieve to obtain Schisandra chinensis powder; (2) Water extraction: Add the Schisandra chinensis powder to 80L of pure water at a material-to-liquid ratio of 1:8 (g / mL), and extract three times at 60℃ for three hours each time. Combine the three extracts. (3) Alcohol precipitation: Filter the extract to remove the residue, concentrate the filtrate under reduced pressure to a relative density of 1.05 at 60°C; add an aqueous ethanol solution to the concentrate to make the final volume concentration of ethanol reach 60%, let it stand at room temperature of 20°C to 30°C for 24 hours, and collect the precipitate by centrifugation. (4) Ultrafiltration segmentation: The obtained precipitate was redissolved in pure water and ultrafiltration was performed using a regenerated cellulose ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The operating pressure was 0.1 MPa and the operating temperature was 20℃~30℃ room temperature. The retentate was collected. (5) Drying: The retentate was freeze-dried to obtain approximately 0.48 kg of Schisandra polysaccharide concentrate.

[0029] The freeze-drying process parameters are as follows: pre-freezing temperature -30℃ to -80℃, vacuum degree ≤50Pa, cold trap temperature ≤-40℃, and main drying temperature -30℃ to 0℃.

[0030] The Schisandra chinensis polysaccharide concentrate prepared in this embodiment was found to contain 43.5% total polysaccharide, 4.4% uronic acid, and 2.8% protein, which are within the range of component contents defined in this invention.

[0031] Example 3: Preparation of Schisandra chinensis polysaccharide concentrate using a high-intensity process (1) Raw material pretreatment: Take 10 kg of dried Schisandra chinensis fruit, crush it and pass it through a 60-mesh sieve to obtain Schisandra chinensis powder; (2) Water extraction: Add the Schisandra chinensis powder to 200L of pure water at a material-to-liquid ratio of 1:20 (g / mL), and extract twice at 100℃ for 1 hour each time. Combine the two extracts. (3) Alcohol precipitation: Filter the extract to remove the residue, concentrate the filtrate under reduced pressure to a relative density of 1.20 at 60°C; add an aqueous ethanol solution to the concentrate to make the final volume concentration of ethanol reach 80%, let it stand at room temperature of 20°C to 30°C for 8 hours, and collect the precipitate by centrifugation. (4) Ultrafiltration segmentation: The obtained precipitate was redissolved in pure water and ultrafiltration was performed using a polyethersulfone (PES) ultrafiltration membrane with a molecular weight cutoff of 50 kDa. The operating pressure was 0.3 MPa and the operating temperature was 20℃~30℃ room temperature. The retentate was collected. (5) Drying: The retentate was spray-dried to obtain approximately 0.85 kg of Schisandra polysaccharide concentrate.

[0032] Upon testing, the Schisandra chinensis polysaccharide enrichment (SCP-EF) prepared in this embodiment contained a total polysaccharide content of 61.5%, a uronic acid content of 5.6%, and a protein content of 5.3%, which are within the component content range defined by this invention.

[0033] It should be noted that Examples 2 and 3 above are only used to illustrate the feasibility of the preparation scheme under different process parameters. The verification of neuroprotective activity and effect described in this invention is based on the preferred Example 1.

[0034] II. Verification of the neuroprotective activity of Schisandra chinensis polysaccharide-rich extracts The following activity verification experiments all used the Schisandra chinensis polysaccharide enrichment (SCP-EF) prepared in Example 1 as the sample, which has balanced process parameters and strong representativeness.

[0035] All cell experiments below were conducted using high-glucose DMEM complete medium containing 10% fetal bovine serum, and the culture environment was 37°C, 5% CO2, and saturated humidity.

[0036] Example 4: Effect of Schisandra chinensis polysaccharide concentrate on the survival rate of PC12 cells damaged by corticosterone To verify the neuroprotective activity of the Schisandra chinensis polysaccharide enrichment of the present invention, a corticosterone-induced PC12 cell injury model was used, and cell viability was detected by the Cell Counting Kit-8 (CCK-8) method to evaluate the protective effect of the enrichment on nerve cell injury.

[0037] 1. Cell seeding: PC12 cells in logarithmic growth phase were digested with 0.25% trypsin and the cell concentration was adjusted to 1×10⁻⁶. 4 Cells were seeded per well in a 96-well cell culture plate with 200 μL of cell suspension added to each well. The plates were then incubated overnight in a cell culture incubator to allow the cells to adhere to the plate.

[0038] 2. Drug Treatment: After cell adhesion, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS buffer. Fresh culture medium containing different concentrations of Schisandra chinensis polysaccharide concentrate (5 μg / mL, 50 μg / mL, and 100 μg / mL) was added to each well (100 μL). Simultaneously, blank control group (culture medium only, no cells), normal control group (cells and culture medium only, no corticosterone or drug treatment), corticosterone model group (corticosterone added after 24 hours of culture, no drug treatment), and positive drug control group (culture medium containing 10 μM fluoxetine for 24 hours) were set up, with 4 parallel wells in each group. The edge wells of the 96-well plate were filled with sterile PBS buffer to eliminate the influence of edge effects on the experimental results. The plates were then incubated in a cell culture incubator for 24 hours.

[0039] 3. Corticosterone modeling: After pretreatment with the positive control and test drugs for 24 hours, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS buffer. Fresh culture medium containing 500 μM corticosterone was added to the corticosterone model group and the groups treated with different concentrations of drugs, while fresh culture medium without corticosterone was added to the normal control group, 100 μL per well. The cells were then incubated in a cell culture incubator for 24 hours.

[0040] 4. Cell viability assay: After co-incubating for 48 hours, carefully aspirate the supernatant from each well and add 110 μL of fresh cell culture medium containing 10 μL of CCK-8 solution to each well. Continue incubation for another 2 hours. After incubation, measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Calculate cell viability using the following formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of normal control group - OD value of blank group) × 100%; The blank group consisted of wells containing only culture medium and CCK-8 solution, without cells.

[0041] 5. Experimental Results: The experimental results showed that after treatment with corticosterone, the survival rate of PC12 cells decreased to 71.5%, indicating that the corticosterone-induced PC12 cell damage model was successfully established. Compared with the model group, the cell viability of the positive control drug fluoxetine group was increased by 11.3%. The cell viability of the Schisandra chinensis polysaccharide enrichment treatment groups of 5 μg / mL and 50 μg / mL reached 92.0% and 121.1%, respectively, both significantly higher than the model group, with the 50 μg / mL group showing the most significant recovery of cell viability. The cell viability of the 100 μg / mL treatment group was 74.7%, and no significant protective effect was observed at this concentration. The above results indicate that the Schisandra chinensis polysaccharide enrichment prepared in this invention has a protective effect against corticosterone-damaged PC12 cells, and 50 μg / mL is the optimal protective concentration.

[0042] Example 5: Effects of Schisandra chinensis polysaccharide accumulation on intracellular reactive oxygen species levels in PC12 cells damaged by corticosterone To evaluate the antioxidant activity of Schisandra chinensis polysaccharide enrichment, a corticosterone-induced PC12 cell injury model was used, and the intracellular reactive oxygen species (ROS) level was detected for verification.

[0043] 1. Cell seeding: PC12 cells in logarithmic growth phase were digested with 0.25% trypsin and then seeded at a rate of 2 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of 1 cell per well in a 6-well cell culture plate. 2 mL of cell suspension was added to each well, and the plate was incubated overnight in a cell culture incubator to allow the cells to adhere.

[0044] 2. Drug Treatment: After cell adhesion, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS buffer. The drug-treated groups were treated with fresh culture medium containing 100 μg / mL, 200 μg / mL, and 300 μg / mL Schisandra chinensis polysaccharide enrichment, respectively. The positive control group was treated with fresh culture medium containing 10 μM fluoxetine, while the normal control group and the corticosterone model group were treated with an equal volume of fresh culture medium without the drug. Three parallel wells were set up for each group. The cells were incubated in a cell culture incubator for 24 hours.

[0045] Preliminary experimental results showed that low concentrations of Schisandra chinensis polysaccharide enrichment did not significantly inhibit the accumulation of reactive oxygen species induced by corticosterone in PC12 cells. Therefore, this experiment selected three concentration gradients of 100 μg / mL, 200 μg / mL, and 300 μg / mL for investigation.

[0046] 3. Corticosterone modeling: After 24 hours of drug pretreatment, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS buffer. Fresh culture medium containing 500 μM corticosterone was added to the corticosterone model group, positive drug control group, and drug-treated group, while fresh culture medium without corticosterone was added to the normal control group, 2 mL per well. The cells were then placed in a cell culture incubator and cultured for another 24 hours.

[0047] 4. Detection of Intracellular Reactive Oxygen Species (ROS) Levels: After 24 hours of corticosterone treatment, the culture supernatant was discarded, and cells were washed twice with sterile PBS. 1 mL of serum-free medium containing 10 μM DCFH-DA probe was added to each well, and the cells were incubated at 37°C in the dark for 30 minutes. After incubation, the probe working solution was discarded, and the cells were washed twice with pre-chilled PBS. Cells were collected by digestion with 0.25% trypsin, digestion was terminated by adding complete culture medium, and the cells were centrifuged at 300 × g for 5 minutes, discarding the supernatant. The cell pellet was resuspended in 500 μL of pre-chilled PBS, transferred to flow cytometry tubes, and immediately stored on ice in the dark. Flow cytometry was used for detection, with an excitation wavelength of 488 nm and an emission channel of 525 nm (FITC channel). 20,000 cells were collected from each tube, and the FITC-A Median value of each group was recorded to reflect the relative level of intracellular ROS.

[0048] 5. Experimental Results: The experimental results showed that the FITC-A Median value of the model group was 2594. After pretreatment with different concentrations of Schisandra chinensis polysaccharide concentrate, the FITC-A Median value of the 100 μg / mL group decreased to 2151, and that of the 300 μg / mL group decreased to 1979, both significantly lower than that of the model group. This indicates that these two concentrations can effectively inhibit corticosterone-induced reactive oxygen species (ROS) accumulation. These results demonstrate that the Schisandra chinensis polysaccharide concentrate prepared in this invention can effectively inhibit corticosterone-induced excessive ROS accumulation in PC12 cells, exhibiting a significant anti-oxidative stress effect.

[0049] Example 6: Effects of Schisandra chinensis polysaccharide concentrate on the secretion of inflammatory factors in PC12 cells damaged by corticosterone To evaluate the anti-inflammatory activity of Schisandra chinensis polysaccharide enrichment, a corticosterone-induced PC12 cell injury model was used, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the cell supernatant were detected for verification.

[0050] 1. Cell seeding: PC12 cells in logarithmic growth phase were digested with 0.25% trypsin and the cell concentration was adjusted to 1×10⁻⁶. 4 Cells / well. Seed the cell suspension into 96-well cell culture plates, adding 200 μL to each well, and incubate overnight at 37°C in a 5% CO2 cell culture incubator to allow the cells to adhere.

[0051] 2. Drug Treatment: After cell adhesion, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS buffer. Fresh culture medium containing different concentrations of Schisandra chinensis polysaccharide concentrate (5 μg / mL, 50 μg / mL, and 100 μg / mL) was added to each well (100 μL). Simultaneously, blank control group (culture medium only, no cells), normal control group (cells and culture medium only, no corticosterone or drug treatment), corticosterone model group (corticosterone added after 24 hours of culture, no drug treatment), and positive drug control group (culture medium containing 10 μM fluoxetine for 24 hours) were set up, with 4 parallel wells in each group. The edge wells of the 96-well plate were filled with sterile PBS buffer to eliminate the influence of edge effects on the experimental results. The plates were then incubated in a cell culture incubator for 24 hours.

[0052] 3. Corticosterone modeling: After pretreatment with the positive control and test drugs for 24 hours, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS buffer. Fresh culture medium containing 500 μM corticosterone was added to the corticosterone model group and the groups treated with different concentrations of drugs, while fresh culture medium without corticosterone was added to the normal control group, 100 μL per well. The cells were then incubated in a cell culture incubator for 24 hours.

[0053] 4. Supernatant collection and ELISA detection: After 24 hours of treatment with corticosterone, the cell culture supernatant from each well was collected into a sterile centrifuge tube and centrifuged at 4°C and 5000 rpm for 5 minutes. The supernatant was then diluted with the sample diluent provided with the kit at a 1:1 volume ratio. After thorough mixing, the detection was performed strictly in accordance with the ELISA kit instructions.

[0054] 5. Experimental Results: The results showed that, compared with the normal control group, the levels of both TNF-α and IL-1β, two inflammatory factors, were increased in the culture supernatant of the corticosterone model group. For TNF-α, the 100 μg / mL Schisandra chinensis polysaccharide enrichment group showed the most significant decrease compared to the model group, followed by the 5 μg / mL group, while the 50 μg / mL group showed a smaller decrease. Overall, Schisandra chinensis polysaccharide enrichment had a certain inhibitory effect on TNF-α secretion, with a relatively significant effect at high concentrations. For IL-1β, both the 5 μg / mL and 100 μg / mL Schisandra chinensis polysaccharide enrichment groups showed strong inhibitory activity, while the 50 μg / mL group showed no significant inhibitory effect. This suggests that the inhibitory effect of Schisandra chinensis polysaccharide enrichment on corticosterone-induced IL-1β secretion exhibits a non-monotonic dose-response relationship, which may be related to changes in the spatial conformation of the polysaccharide at different concentrations or receptor saturation. The above results indicate that the Schisandra chinensis polysaccharide enrichment prepared in this invention can effectively inhibit the secretion of inflammatory factors in PC12 cells induced by corticosterone, exhibiting a significant anti-inflammatory effect.

[0055] Example 7: Effect of Schisandra chinensis polysaccharide concentrate on apoptosis in PC12 cells damaged by corticosterone To evaluate the anti-apoptotic effect of Schisandra chinensis polysaccharide enrichment, a corticosterone-induced PC12 cell injury model was used, and the cell apoptosis rate was detected by Annexin V-FITC / PI double staining flow cytometry for verification.

[0056] 1. Cell seeding and grouping: PC12 cells in logarithmic growth phase were digested with 0.25% trypsin and the cell concentration was adjusted to 1.5 × 10⁻⁶. 5 Cells were seeded per well in 12-well cell culture plates, with 1 mL of cell suspension added to each well. The plates were then incubated overnight to allow cell adhesion. Experimental setups included a normal control group (containing only cells and culture medium, without corticosterone or other drug treatments), a corticosterone model group (corticosterone added after 24 hours of culture, without drug treatment), a positive drug control group (culture medium containing 10 μM fluoxetine), and Schisandra chinensis polysaccharide enrichment treatment groups (5 μg / mL, 50 μg / mL, and 100 μg / mL), with three parallel wells in each group.

[0057] 2. Drug treatment: After cell adhesion, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS buffer. The drug-treated groups were added to fresh culture medium containing 5 μg / mL, 50 μg / mL, and 100 μg / mL Schisandra chinensis polysaccharide enrichment, respectively. The normal control group and the corticosterone model group were added to an equal volume of fresh culture medium without the drug. The positive control group was added to fresh culture medium containing 10 μM fluoxetine. All cells were incubated in a cell culture incubator for 24 hours.

[0058] 3. Corticosterone modeling: After 24 hours of drug pretreatment, the original culture medium was discarded, and the cells were washed twice with sterile PBS. Fresh culture medium containing 500 μM corticosterone was added to the corticosterone model group, positive drug control group, and drug-treated group, while fresh culture medium without corticosterone was added to the normal control group. The cells were then cultured in a cell culture incubator for another 24 hours.

[0059] 4. Flow Cytometry Detection of Apoptosis: After corticosterone treatment, carefully collect the culture supernatant from each well into the corresponding labeled centrifuge tubes. For adherent cells in 12 wells, wash once with PBS, then gently digest with 0.25% trypsin. Once the cells become rounded and detach, stop the digestion with the collected supernatant and collect the cells. Centrifuge at 2000 rpm for 5 minutes at 4°C to collect the cell pellet. Wash the cells twice with pre-cooled PBS. Discard the supernatant and add 200 μL of 1×Binding Buffer to each tube to resuspend the cells. Add 3 μL of Annexin V-FITC to each tube sequentially, mix well, and incubate at room temperature for 15 minutes in the dark. Add 3 μL of PI staining to each tube 5 minutes before flow cytometry. Add another 200 μL of 1×Binding Buffer to each tube before flow cytometry. Simultaneously set up unstained cell tubes, Annexin V-FITC single-stained tubes, and PI single-stained tubes for voltage adjustment, fluorescence compensation, and gating. Flow cytometry was used for detection. The excitation wavelength was 488 nm. FITC fluorescence was collected in the FL1 channel (525 nm), and PI fluorescence was collected in the FL2 channel (575 nm). 40,000 cells were collected per tube.

[0060] Results Analysis: A scatter plot was drawn with Annexin V-FITC as the x-axis and PI as the y-axis, dividing the cells into four quadrants: the lower left quadrant (Q3) represented viable cells (Annexin V-FITC). - / PI - The lower right quadrant (Q4) contains early apoptotic cells (Annexin V). + / PI - The upper right quadrant (Q2) contains late-stage apoptotic and necrotic cells (Annexin V). + / PI + The upper left quadrant (Q1) contains mechanically damaged or necrotic cells (Annexin V). - / PI + The total apoptosis rate (%) was calculated as the sum of the early apoptosis rate (Q4) and the late apoptosis rate (Q2).

[0061] 5. Experimental Results: The experimental results showed that the total apoptosis rate of the model group cells was significantly higher than that of the normal control group after corticosterone treatment, indicating that corticosterone successfully induced apoptosis in PC12 cells. Compared with the model group, the total apoptosis rate of the fluoxetine group was significantly reduced. The total apoptosis rate of cells treated with different concentrations of Schisandra chinensis polysaccharide enrichment decreased to varying degrees, with a significant decrease in the apoptosis rate in the 5 μg / mL group; the apoptosis rates of the 50 μg / mL group and the 100 μg / mL group decreased further, and the reduction in the 50 μg / mL group was relatively better. The above results indicate that the Schisandra chinensis polysaccharide enrichment prepared in this invention can significantly inhibit corticosterone-induced apoptosis in PC12 cells, exhibiting a clear anti-apoptotic neuroprotective effect.

[0062] In this invention, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A neuroprotective agent, characterized in that, The neuroprotective agent is a polysaccharide enrichment of Schisandra chinensis, which is prepared by water extraction, filtration and concentration, alcohol precipitation, precipitation and redissolution of dried mature Schisandra chinensis fruit of the Magnoliaceae family, followed by ultrafiltration fractionation using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and drying of the collected retentate. The neuroprotective agent contains 59.2% total polysaccharides, 6.6% uronic acid, and 3.6% protein.

2. The neuroprotective agent according to claim 1, characterized in that, The water extraction process involves a material-to-liquid ratio of 1:15 (g / mL), an extraction temperature of 85℃, two extractions, and an extraction time of 2 hours for each extraction.

3. The neuroprotective agent according to claim 1, characterized in that, In the alcohol precipitation step, the extract is concentrated to a relative density of 1.10 at 60°C, ethanol is added to make the final volume concentration of ethanol reach 70%, and the precipitate is collected by centrifugation after standing at room temperature of 20°C to 30°C for 12 hours.

4. A method for preparing the neuroprotective agent according to claim 1, characterized in that, The process includes the following steps: the raw material of Schisandra chinensis fruit is extracted with water, filtered and concentrated, and then precipitated with alcohol. The precipitate is redissolved and then purified by ultrafiltration using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The retentate is collected and dried to obtain the Schisandra chinensis polysaccharide concentrate.

5. The method for preparing the neuroprotective agent according to claim 4, characterized in that, The water extraction process involves a material-to-liquid ratio of 1:15 (g / mL), an extraction temperature of 85℃, two extractions, and an extraction time of 2 hours for each extraction.

6. The method for preparing the neuroprotective agent according to claim 5, characterized in that, In the alcohol precipitation step, the extract is concentrated to a relative density of 1.10 at 60°C, ethanol is added to make the final volume concentration of ethanol reach 70%, and the precipitate is collected by centrifugation after standing at room temperature of 20°C to 30°C for 12 hours.

7. The method for preparing the neuroprotective agent according to claim 4, characterized in that, The ultrafiltration system operates at a pressure of 0.15 MPa and a temperature of 20°C to 30°C.

8. The use of the neuroprotective agent according to claim 1 in the preparation of a neuroprotective drug.

9. The use of the neuroprotective agent according to claim 8 in the preparation of neuroprotective drugs, characterized in that, The neuroprotection specifically refers to antagonizing corticosterone-induced neuronal damage.

10. The use of the neuroprotective agent according to claim 9 in the preparation of neuroprotective drugs, characterized in that, The neuroprotection is achieved by reducing intracellular reactive oxygen species levels, inhibiting the secretion of inflammatory factors, and / or inhibiting cell apoptosis; The inflammatory factors include tumor necrosis factor-α and interleukin-1β.

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