Schisandra polysaccharide, and preparation method and application thereof

Abstract

This invention discloses a Schisandra chinensis polysaccharide, its preparation method, and its application. The preparation method of the Schisandra chinensis polysaccharide includes the following steps: S1. Take Schisandra chinensis fruits, crush and soak them, then centrifuge to collect the precipitate, obtaining Schisandra chinensis powder; S2. Add the Schisandra chinensis powder to a low-temperature eutectic solvent, extract at 75-85℃ for 2-5 hours, centrifuge, and collect the supernatant to obtain the extract; the low-temperature eutectic solvent is an aqueous solution of 1,3-butanediol containing choline chloride, the molar ratio of choline chloride to 1,3-butanediol is 1:(3-5), and the volume ratio of 1,3-butanediol to water is (2-4):1; S3. Concentrate the extract, add an ethanol solution, precipitate with alcohol, centrifuge, collect the precipitate, and then wash and dry to obtain the Schisandra chinensis polysaccharide. The Schisandra chinensis polysaccharide provided by this invention can effectively activate the Nrf2 signaling pathway, increase the Nrf2 protein expression level, and inhibit Keap1 protein expression, thereby effectively alleviating APAP-induced liver damage.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically, to a Schisandra chinensis polysaccharide, its preparation method, and its application. Background Technology

[0002] Acetaminophen (APAP) is a widely used over-the-counter drug for antipyretic analgesia, also known as paracetamol. When taken at a safe dose, APAP can relieve pain. However, if patients take APAP for a long time or in large quantities for antipyretic analgesia, it can induce serious liver disease, which can lead to liver failure and even death.

[0003] The Keap1-Nrf2 signaling pathway plays a crucial role in cellular anti-oxidative stress responses, and its regulatory mechanism has become a research hotspot in the biomedical field. Activating the Nrf2 signaling pathway can enhance the expression of intracellular antioxidant enzymes, thereby effectively reducing oxidative stress-induced cellular damage. In recent years, the development of biopharmaceuticals based on natural products to regulate the Keap1-Nrf2 signaling pathway has gradually become a research direction. These biopharmaceuticals are characterized by their wide availability, high safety profile, and minimal side effects, demonstrating promising application prospects.

[0004] Patent CN103816347A discloses a stomach-nourishing biological preparation composed of traditional Chinese medicine ingredients such as pearl powder, cinnamon twig, processed arisaema, processed aconite, Paris polyphylla, Panax notoginseng, Notopterygium incisum, Stephania tetrandra, raw rhubarb, Bupleurum chinense, Citrus aurantium, Paeonia lactiflora, and Eupolyphaga sinensis. It aims to achieve detoxification, dampness removal, cold dispelling, blood stasis removal, tissue regeneration, and pain relief through the combination of traditional Chinese medicine ingredients. While this patent attempts to improve the body's health from multiple target perspectives through its compound traditional Chinese medicine design, it does not address the specific regulation of the Keap1-Nrf2 signaling pathway, nor does it explicitly mention the application of Schisandra chinensis polysaccharide and its mechanism of action in anti-oxidative stress.

[0005] Patent CN103798792A discloses a health-promoting biological preparation. This preparation uses millet, pine needles, holly, Paris polyphylla, Panax notoginseng, Notopterygium incisum, Acanthopanax senticosus, Stephania tetrandra, Clematis chinensis, raw rhubarb, and Eupolyphaga sinensis thrombolytic enzyme as its main raw materials, further adding processed Aconitum carmichaelii. Its aim is to achieve the effects of clearing heat and detoxifying, removing dampness, dispelling cold, promoting blood circulation and removing blood stasis, unblocking meridians and collaterals, and clearing blood vessels. Although this preparation emphasizes the synergistic effect of multiple traditional Chinese medicine components, its main objective is to improve the rehabilitation of brain tissue injury, brain atrophy, and vascular diseases. It lacks the ability to directly intervene in the Keap1-Nrf2 signaling pathway and does not include Schisandra chinensis polysaccharide as a core component, thus failing to fully utilize its potential in anti-oxidative stress.

[0006] Existing technologies have limitations in regulating the Keap1-Nrf2 signaling pathway, particularly in failing to fully utilize the advantages of Schisandra chinensis polysaccharide, an important natural active ingredient. Furthermore, existing formulations often focus on the comprehensive regulatory effects of traditional Chinese medicine formulas, while paying insufficient attention to the precise regulation of specific signaling pathways. This presents a significant opportunity for innovation in developing a biological agent based on Schisandra chinensis polysaccharide to regulate the Keap1-Nrf2 signaling pathway. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a Schisandra chinensis polysaccharide, its preparation method, and its applications.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides the application of Schisandra chinensis polysaccharide in regulating the Keap1-Nrf2 signaling pathway.

[0009] A second aspect of the present invention provides a method for preparing Schisandra chinensis polysaccharide, comprising the following steps: S1. Take Schisandra chinensis fruit, crush it, soak it, centrifuge it, and take the precipitate to obtain Schisandra chinensis powder; S2. Add Schisandra chinensis powder to a low-temperature eutectic solvent, extract at 75-85℃ for 2-5 hours, centrifuge, and collect the supernatant to obtain the extract; the low-temperature eutectic solvent is an aqueous solution of 1,3-butanediol containing choline chloride, the molar ratio of choline chloride to 1,3-butanediol is 1:(3-5), and the volume ratio of 1,3-butanediol to water is (2-4):1; S3. After concentrating the extract, add an ethanol solution, precipitate with alcohol, centrifuge, collect the precipitate, and then wash and dry it to obtain Schisandra polysaccharide.

[0010] In the preferred embodiment, the soaking conditions are: soaking at 30-40℃ for 12-24 hours; and pulverizing the Schisandra chinensis fruit to a particle size of 50-100 mesh.

[0011] In the preferred embodiment, in step S2, the ratio of Schisandra chinensis powder to low-temperature eutectic solvent is 1:(10~20).

[0012] In the preferred embodiment, in steps S1 to S3, the centrifugation conditions are centrifugation at a speed of 3000 to 5000 rpm for 5 to 20 minutes.

[0013] In the preferred embodiment, in step S3, the alcohol precipitation time is 24~48h, and the volume ratio of the extract before and after concentration is 10:1.

[0014] In the preferred embodiment, in step S3, after the extract is concentrated, 95% ethanol solution is added to make the final ethanol concentration 75%.

[0015] In a third aspect, the present invention provides a Schisandra chinensis polysaccharide prepared by any of the above methods.

[0016] In a fourth aspect, the present invention provides a biological agent containing the above-mentioned Schisandra chinensis polysaccharide.

[0017] In a preferred embodiment, the biological agent further includes excipients, such as mannitol, lactose, or microcrystalline cellulose, wherein the excipients account for 1% to 5% of the mass of the biological agent; and the dosage form of the biological agent is an oral liquid, capsule, or granules.

[0018] In a fifth aspect, the present invention provides the use of the above-mentioned Schisandra chinensis polysaccharide in the preparation of drugs for the prevention / treatment of drug-induced liver injury and / or health products.

[0019] Beneficial effects of the present invention The Schisandra chinensis polysaccharide provided by this invention can effectively activate the Nrf2 signaling pathway, increase Nrf2 protein expression levels, and inhibit Keap1 protein expression, thereby effectively alleviating APAP-induced liver injury. Animal experimental results show that, compared with Lycium barbarum polysaccharide and Schisandra chinensis polysaccharide extracted using choline chloride-ethylene glycol as a cryogenic eutectic solvent, the Schisandra chinensis polysaccharide extracted using choline chloride-1,3-butanediol as a cryogenic eutectic solvent in this invention has a significantly higher protective effect against APAP-induced liver injury. Attached Figure Description

[0020] Figure 1 These are H&E staining images of mouse liver tissue pathological sections; among them, A. CON group, B. MOD group, C. positive control group, D. SCP1 low-dose group, E. SCP1 medium-dose group, and F. SCP1 high-dose group. Figure 2 These are the results of Western blot analysis of mouse liver tissue. Detailed Implementation

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

[0022] Unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, the experimental methods used in the examples are conventional methods. Where specific conditions are not specified in the examples, they should be performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] Example 1

[0024] A method for preparing Schisandra chinensis polysaccharide includes the following steps: S1. Take Schisandra chinensis fruit, crush it, soak it at 35℃ for 16 hours, then centrifuge it at 4500 rpm for 15 minutes, take the precipitate, and obtain Schisandra chinensis powder. S2. Schisandra chinensis powder was added to a cryogenic eutectic solvent at a material-to-liquid ratio of 1:15. After extraction at 80°C for 3 hours, the mixture was centrifuged at 4500 rpm for 15 minutes. The supernatant was collected to obtain the extract. The cryogenic eutectic solvent was an aqueous solution of 1,3-butanediol containing choline chloride, with a molar ratio of choline chloride to 1,3-butanediol of 1:4 and a volume ratio of 1,3-butanediol to water of 3:1. S3. The extract was concentrated to 1 / 10 of its original volume, and 95% ethanol solution was added to bring the final ethanol concentration to 75%. After ethanol precipitation for 36 hours, the precipitate was centrifuged at 4500 rpm for 15 minutes, and the precipitate was collected. It was washed twice with 95% ethanol solution and anhydrous ethanol, and then evaporated to dryness in a water bath at 60°C to obtain Schisandra polysaccharide, designated as SCP1.

[0025] Comparative Example 1 A method for preparing Schisandra chinensis polysaccharide includes the following steps: S1. Take Schisandra chinensis fruit, crush it, soak it at 35℃ for 16 hours, then centrifuge it at 4500 rpm for 15 minutes, take the precipitate, and obtain Schisandra chinensis powder. S2. Schisandra chinensis powder was added to a cryogenic eutectic solvent at a material-to-liquid ratio of 1:15. After extraction at 80°C for 3 hours, the mixture was centrifuged at 4500 rpm for 15 minutes. The supernatant was collected to obtain the extract. The cryogenic eutectic solvent was an aqueous solution of ethylene glycol containing choline chloride, with a molar ratio of choline chloride to ethylene glycol of 1:4 and a volume ratio of ethylene glycol to water of 3:1. S3. The extract was concentrated to 1 / 10 of its original volume, and 95% ethanol solution was added to bring the final ethanol concentration to 75%. After ethanol precipitation for 36 hours, the precipitate was centrifuged at 4500 rpm for 15 minutes, and the precipitate was collected. It was washed twice with 95% ethanol solution and anhydrous ethanol, and then evaporated to dryness in a water bath at 60°C to obtain Schisandra polysaccharide, designated as SCP2.

[0026] Comparative Example 2 A method for preparing Lycium barbarum polysaccharide includes the following steps: S1. Take wolfberries, crush them, soak them at 35℃ for 16 hours, then centrifuge them at 4500 rpm for 15 minutes, take the precipitate, and obtain wolfberry powder; S2. Add wolfberry powder to a cryogenic eutectic solvent at a material-to-liquid ratio of 1:15, extract at 80℃ for 3 hours, then centrifuge at 4500 rpm for 15 minutes, and collect the supernatant to obtain the extract; the cryogenic eutectic solvent is an aqueous solution of 1,3-butanediol containing choline chloride, with a molar ratio of choline chloride to 1,3-butanediol of 1:4 and a volume ratio of 1,3-butanediol to water of 3:1; S3. The extract was concentrated to 1 / 10 of its original volume, and 95% ethanol solution was added to make the final ethanol concentration reach 75%. After ethanol precipitation for 36 hours, the precipitate was centrifuged at 4500 rpm for 15 minutes, and the precipitate was collected. It was washed twice with 95% ethanol solution and anhydrous ethanol, respectively, and then evaporated to dryness in a water bath at 60℃ to obtain Lycium barbarum polysaccharide, denoted as LBP.

[0027] animal experiments Eighty 6-week-old male ICR mice weighing 20±2g were selected and, after one week of acclimatization, randomly divided into 8 groups of 10 mice each. The experimental groups were: low-dose SCP1 group, medium-dose SCP1 group, high-dose SCP1 group, medium-dose SCP2 group, medium-dose LBP group, positive control group, blank control group (CON), and acetaminophen (APAP)-induced liver injury model group (MOD). The drug administration details for each group are shown in Table 1. The mice were administered the drugs by gavage for 2 weeks, once daily. One hour after the last administration, mice in the low-, medium-, and high-dose SCP1 groups, the medium-dose SCP2 group, the medium-dose LBP group, the positive control group, and the MOD group were given a single intraperitoneal injection of APAP (250 mg / kg). The CON group was given saline in the same manner.

[0028] Table 1. Dosage distribution in experimental groups CON group physiological saline MOD Group physiological saline Positive control group 37.8 mg / kg NAC saline solution SCP1 low-dose group 25 mg / kg SCP1 saline solution SCP1 medium dose group 50 mg / kg SCP1 saline solution SCP1 high-dose group 100 mg / kg SCP1 saline solution SCP2 medium dose group 50 mg / kg SCP2 saline solution LBP medium dose group 50 mg / kg LBP saline solution 1. Detection of serum biochemical markers ALT and AST levels and liver tissue oxidative stress markers GSH and MDA levels. After the experiment, all mice were fasted but allowed free access to water. 24 hours later, the mice were euthanized by medullary amputation, and blood was collected from the eyeballs. Blood samples were centrifuged at 3500 rpm and 4°C for 15 minutes. Serum was separated, and ALT and AST levels were measured using an automated biochemical analyzer. 100 mg of liver tissue was added to 9 volumes of physiological saline, homogenized in an ice-water bath, centrifuged, and the supernatant was collected. GSH and MDA levels were measured using the dithionitrobenzene method and the thiobarbituric acid method, respectively. All assays were performed according to the kit instructions, and the results are shown in Table 2. A portion of the remaining liver tissue was fixed in formalin, and the other portion was stored at -80°C for subsequent experiments.

[0029] Table 2. Results of ALT, AST, GSH, and MDA levels in mice. CON group 45 52 12.3 3.2 MOD Group 280 320 4.5 12.8 Positive control group 170 218 7.4 8.6 SCP1 low-dose group 200 220 6.8 9.5 SCP1 medium dose group 150 180 8.5 7.2 SCP1 high-dose group 100 120 10.2 5.8 SCP2 medium dose group 185 215 7.1 8.5 LBP medium dose group 220 260 5.8 10.5 Compared to the CON group, the MOD group showed significantly increased serum ALT and AST levels and significantly decreased liver MDA levels, indicating a successful establishment of the APAP-induced liver injury model. Compared to the model group, different doses of SCP1, SCP2, LBP, and NAC significantly reduced ALT, AST, and MDA levels and increased GSH levels in mice, with the high-dose SCP1 group showing the best protective effect. Compared to the medium-dose SCP2 group, the medium-dose SCP1 group showed significantly increased ALT, AST, and MDA levels and significantly decreased GSH levels, indicating that the Schisandra chinensis polysaccharide extracted using choline chloride-1,3-butanediol as a cryogenic eutectic solvent had a significantly higher protective effect against drug-induced liver injury than the Schisandra chinensis polysaccharide extracted using choline chloride-ethylene glycol as a cryogenic eutectic solvent. Compared with the medium-dose LBP group, mice in the low, medium and high-dose SCP1 groups showed significantly increased ALT, AST and MDA levels and significantly decreased GSH levels, indicating that Schisandra chinensis polysaccharide has a significantly higher protective effect against drug-induced liver injury than Lycium barbarum polysaccharide.

[0030] 2. Observation of pathological changes in liver tissue Formalin-fixed mouse liver tissues from the CON group, MOD group, positive control group, and low, medium, and high dose SCP1 groups were collected, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). The pathological morphological changes of hepatocytes were observed under a microscope (400x magnification). The results are as follows: Figure 1 As shown.

[0031] Figure 1 A shows the H&E staining results of liver tissue from mice in the CON group. Figure 1 A shows that the mouse liver lobules have a clear and intact structure, and the hepatocytes are morphologically intact and arranged in a neat radial pattern. Figure 1 B shows the H&E staining results of liver tissue from MOD group mice. Figure 1 B shows that the mouse hepatocytes are arranged in a disordered manner, with incomplete morphology, missing cell nuclei, and inflammatory infiltration near the central vein, indicating that APAP injection leads to liver damage, further demonstrating the successful establishment of the APAP-induced liver injury model. Figure 1 C~1F show the H&E staining results of liver tissue from mice in the positive control group, and the low, medium, and high dose groups of SCP1, respectively. Compared with the H&E staining results of liver tissue from mice in the MOD group, the liver cells of mice in the positive control group, and the low, medium, and high dose groups of SCP1 recovered completely. Among them, the protective effect of the medium and high dose groups of SCP1 on liver injury in mice was better than that of the positive control group, while the protective effect of the low dose group of SCP1 on liver injury in mice was slightly lower than that of the positive control group.

[0032] 3. Western blot detection 100 mg of frozen liver tissue was added to 1 mL of lysis buffer and lysed on ice for 1 h. The tissue was then centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected as the target protein sample. The protein concentration of the target protein sample was determined using the BCA method. The target protein was separated on a 10% SDS-polyacrylamide gel under constant voltage (80 V–120 V) and transferred to a PVDF membrane for 2 h. It was then blocked for 1 h with blocking buffer (TBST buffer containing 5% skim milk powder). After incubation at room temperature, the blocking buffer was discarded, and Keap1 and Nrf2 primary antibodies (1:1000) were added and incubated overnight at 4 °C. The next day, the sample was washed with TBST buffer 3 × 10 min, and incubated with HRP-labeled secondary antibody (1:5000) at room temperature for 1 h, followed by washing with TBST buffer 3 × 10 min. Finally, ECL developing solution was added, and the grayscale value was quantified using a gel imaging and analysis system on a chemiluminescence instrument. The results are shown below. Figure 2 As shown.

[0033] like Figure 2 As shown, compared to the CON group mice, the MOD group mice showed a significant increase in liver Nrf2 protein content. Compared to the MOD group mice, the low- and high-dose SCP1 groups mice also showed a significant increase in liver Nrf2 protein content. Compared to the CON group mice, the MOD group mice showed no significant change in liver Keap1 protein content. Compared to the MOD group mice, the low- and high-dose SCP1 groups mice showed a decrease in liver Keap1 protein content, with the high-dose SCP1 group showing a significant decrease. This indicates that SCP1 can effectively activate the Nrf2 signaling pathway, increase Nrf2 protein expression levels, and inhibit Keap1 protein expression, thereby effectively reducing oxidative stress-induced cell damage.

[0034] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Application of a Schisandra chinensis polysaccharide in regulating the Keap1-Nrf2 signaling pathway.

2. A method for preparing Schisandra chinensis polysaccharide, characterized in that, Includes the following steps: S1. Take Schisandra chinensis fruit, crush it, soak it, centrifuge it, and take the precipitate to obtain Schisandra chinensis powder; S2. Add Schisandra chinensis powder to a low-temperature eutectic solvent, extract at 75-85℃ for 2-5 hours, centrifuge, and collect the supernatant to obtain the extract; the low-temperature eutectic solvent is an aqueous solution of 1,3-butanediol containing choline chloride, the molar ratio of choline chloride to 1,3-butanediol is 1:(3-5), and the volume ratio of 1,3-butanediol to water is (2-4):1; S3. After concentrating the extract, add an ethanol solution, precipitate with alcohol, centrifuge, collect the precipitate, and then wash and dry it to obtain Schisandra polysaccharide.

3. The method for preparing Schisandra chinensis polysaccharide according to claim 2, characterized in that, In step S1, the soaking conditions are 30-40℃ for 12-24 hours; the Schisandra chinensis fruit is crushed to a particle size of 50-100 mesh.

4. The method for preparing Schisandra chinensis polysaccharide according to claim 2, characterized in that, In step S2, the ratio of Schisandra chinensis powder to low-temperature eutectic solvent is 1:(10~20).

5. The method for preparing Schisandra chinensis polysaccharide according to claim 2, characterized in that, In steps S1 to S3, the centrifugation conditions are centrifugation at a speed of 3000 to 5000 rpm for 5 to 20 minutes.

6. The method for preparing Schisandra chinensis polysaccharide according to claim 2, characterized in that, In step S3, the alcohol precipitation time is 24-48 hours, and the volume ratio of the extract before and after concentration is 10:1; after the extract is concentrated, 95% ethanol solution is added to make the final ethanol concentration 75%.

7. A Schisandra chinensis polysaccharide prepared by the method according to any one of claims 2 to 6.

8. A biological agent containing the Schisandra chinensis polysaccharide as described in claim 7.

9. The biological agent of Schisandra chinensis polysaccharide according to claim 8, characterized in that, The biological agent also includes excipients, such as mannitol, lactose, or microcrystalline cellulose, wherein the excipients account for 1% to 5% of the mass of the biological agent; the dosage form of the biological agent is an oral liquid, capsule, or granules.

10. The use of the Schisandra chinensis polysaccharide according to claim 7 in the preparation of drugs for the prevention / treatment of drug-induced liver injury and / or health products.

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