Gastrodin polycitrate as well as preparation method and application thereof

Abstract

The invention provides gastrodin poly-citrate, which belongs to the technical field of biochemistry and biomedical engineering, and the structural general formula of the gastrodin poly-citrate is as shown in the formula I. According to the invention, the gastrodin polyol and citric acid are subjected to high-temperature polymerization reaction by utilizing the property of the gastrodin polyol, the generated macromolecular gastrodin polycitrate is named as PGC, the PGC can improve the activity and bioavailability of gastrodin, and degradation product components play a medicinal synergistic effect, so that the gastrodin can be effectively degraded, and the bioavailability of the gastrodin can be improved. The composition has the effects of protecting cells, promoting cell proliferation or regeneration and resisting inflammation and oxidation. The invention further provides a preparation method and application of the gastrodin polycitrate.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biochemistry and biomedical engineering, and particularly relates to gastrodin polycitrate, a preparation method thereof, and applications thereof. Background Art

[0002] As a traditional Chinese herbal medicine, gastrodia elata was first recorded in "Shennong Ben Cao Jing" more than two thousand years ago. The main active ingredient of gastrodia elata, gastrodin (GAS), is a small molecule active compound of phenolic glycosides. Research shows that gastrodin has significant neuroprotective effects and can treat or improve epilepsy, Alzheimer's disease, Parkinson's disease, mood disorders, cognitive disorders, and neuropathic pain. Luo Yun et al. disclosed a drug combination for preventing and treating senile dementia in the patent CN102793708A in 2012, and the main ingredient contains gastrodin. In addition, gastrodin can also improve cardiovascular system diseases such as myocardial hypertrophy, hypertension, and myocardial ischemia-reperfusion injury. During the process of improving the above diseases, the antioxidant stress, anti-inflammatory, and anti-apoptotic effects of gastrodin can be observed, such as activating the Nrf2 signaling pathway, inhibiting the NF-κB and MAPK signaling pathways, increasing the expression of GSH-Px, SOD, and HO-1, reducing the expression of COX-2, TNF-α, and IL-1β, and regulating the mitochondrial cascade reaction. The extensive pharmacological effects of gastrodin and the advantages of low toxicity and few adverse reactions have attracted scientists to continuously explore its pharmacological mechanism and develop gastrodin-related preparations and new uses of gastrodin, such as gastrodin capsules (patent publication number: CN112426489A), injection solutions (patent publication number: CN112190545A), derivatives (patent publication number: CN113980062A), etc. preparations, and new uses for treating amyotrophic lateral sclerosis (patent publication number: CN115813941A), regulating macrophage polarization (patent publication number: CN113855687A), preventing and treating osteoporosis (patent publication number: CN104127425A), etc.

[0003] Research shows that after oral administration of gastrodin capsules (200 mg) to humans, gastrodin is rapidly absorbed, with an absorption half-life of only 0.18 hours and a time to reach the maximum plasma concentration of only 0.81 hours. However, after gastrodin enters the systemic circulation, it is rapidly and widely distributed, is quickly metabolized and cleared in the body, and most of the gastrodin is excreted through urine without being absorbed. The excretion also increases when the dose is increased, and it is not easy to accumulate in the body. Due to the short action time of gastrodin in the body, multiple-dose administration is required, which brings a bad experience to patients, and there are also disadvantages such as generally low bioavailability and weak functional activity, so its clinical application is limited. Therefore, taking gastrodin as a lead compound, further in-depth research is still needed to carry out structural modification and optimization on it to screen out drugs with better activity, smaller toxic and side effects, and longer action time in the body. Summary of the Invention

[0004] In order to solve the problems of low bioavailability, low activity and easy metabolism of gastrodin, the present invention provides a gastrodin polycitrate. Utilizing the properties of gastrodin polyol, it undergoes a high-temperature polymerization reaction with citric acid to generate a macromolecular gastrodin polycitrate named PGC. PGC can improve the activity and bioavailability of gastrodin, and the degradation product components play a synergistic medicinal role, having the effects of protecting cells, promoting cell proliferation or regeneration, and anti-inflammatory and antioxidant effects.

[0005] The present invention also provides a preparation method and application of gastrodin polycitrate.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a gastrodin polycitrate, and the structural general formula of the gastrodin polycitrate is shown in Formula I:

[0008]

[0009] n is an integer greater than 1.

[0010] Based on the same inventive concept, the present invention provides a preparation method of gastrodin polycitrate, and the preparation method includes:

[0011] Mix citric acid and gastrodin, and then add an organic solvent to obtain a mixture;

[0012] Heat the mixture, and continue to react for 6 - 72 h after the mixture is dissolved to obtain a polymer;

[0013] Dissolve the polymer in water and adjust the pH to neutral to obtain a polymer solution;

[0014] The polymer solution is dialyzed and freeze-dried to obtain gastrodin polycitrate;

[0015] Among them, the molar ratio of citric acid to gastrodin in the mixture is (1 - 2):(1 - 2).

[0016] Further, the step of mixing citric acid and gastrodin, and then adding an organic solvent to obtain a mixture specifically includes:

[0017] Mix citric acid and gastrodin, and then add the organic solvent N,N-dimethylformamide to obtain a mixture;

[0018] Among them, the molar percentage of N,N-dimethylformamide in the mixture is 50 - 80 mol%.

[0019] Further, heating the mixture and continuously reacting for 6 - 72 h after the mixture is dissolved to obtain a polymer, specifically including:

[0020] Under an inert atmosphere, heating the mixture to 100 - 160 °C, starting timing after the mixture is dissolved, and continuously reacting at 100 - 160 °C for 6 - 72 h to obtain a polymer.

[0021] Further, dissolving the polymer in water and adjusting the pH to neutral (to completely dissolve it) to obtain a polymer solution, specifically including:

[0022] Adding water to the polymer, then adding sodium hydroxide to adjust the pH to neutral, and sonicating until dissolved to obtain a polymer solution.

[0023] Further, subjecting the polymer solution to dialysis and freeze - drying to obtain gastrodin polycitrate, specifically including:

[0024] Placing the polymer solution in a dialysis bag with a molecular weight cut - off of 400 Da and dialyzing for 48 h, changing the solution 3 - 6 times;

[0025] The liquid obtained by dialysis is freeze - dried to obtain gastrodin polycitrate.

[0026] Based on the same inventive concept, the present invention provides an application of gastrodin polycitrate in the preparation of a drug for preventing and treating nervous system diseases, where the nervous system diseases include at least one of nerve injury, epilepsy, Alzheimer's disease, Parkinson's disease, mood disorder, cognitive disorder, and neuropathic pain.

[0027] Based on the same inventive concept, the present invention provides an application of gastrodin polycitrate in the preparation of a drug for preventing and treating cardiovascular diseases, where the cardiovascular diseases include at least one of vascular injury, myocardial fibrosis, hypertension, and myocardial ischemia - reperfusion injury.

[0028] Based on the same inventive concept, the present invention provides an application of gastrodin polycitrate in the preparation of a drug for preventing and treating musculoskeletal system diseases, where the musculoskeletal system diseases include at least one of bone defect, osteoarthritis, and rheumatoid arthritis.

[0029] Based on the same inventive concept, the present invention provides an application of gastrodin polycitrate in the preparation of a biomedical material with a geometric shape or microstructure, where the biomedical material with a geometric shape or microstructure includes at least one of a catheter, hydrogel, biphasic scaffold, triphasic scaffold, nanoparticles, and microparticles.

[0030] Based on the same inventive concept, the present invention provides a drug, the active ingredient of which comprises the above-mentioned gastrodin polycitrate, and the drug includes drugs for preventing and treating central nervous system diseases, drugs for preventing and treating cardiovascular diseases, or drugs for preventing and treating musculoskeletal system diseases.

[0031] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0032] 1. For the gastrodin polycitrate of the present invention, a high-temperature polymerization esterification reaction is carried out between the hydroxyl group on gastrodin and the carboxyl group on citric acid to generate gastrodin polycitrate named PGC. PGC is a high-molecular polymer, which can solve the problems that gastrodin, as a small-molecule drug, has low bioavailability and is easily metabolized. At the same time, it also solves the problems of low medicinal activity and single function of traditional polycitrate. The PGC polymer of the present invention is a structural unit formed by connecting one molecule of citric acid and one molecule of gastrodin through an ester bond as its main repeating unit, and the molecular structure of this repeating unit is another medicinal active component in gastrodia elata - parishin E. Therefore, under the hydrolysis of the ester bond by esterase in vivo, the PGC polymer can degrade and release functional components such as parishin and gastrodin, and synergistically exert its medicinal value. PGC belongs to polymer molecules. Compared with small-molecule gastrodin, the polymer has a longer in vivo circulation time, which is beneficial to improving its bioavailability, and has the effects of protecting cells, promoting cell proliferation or regeneration, and anti-inflammatory and antioxidant, and will be very effective in treating diseases of the nervous system, cardiovascular system and musculoskeletal system.

[0033] 2. For the gastrodin polycitrate of the present invention, the in vivo metabolism, pharmacokinetics, etc. of the macromolecular polymer PGC are different from those of small-molecule gastrodin. The PGC polymer can effectively increase the in vivo circulation time and improve its bioavailability. Therefore, multi-dose administration is not required, and it is particularly suitable for preparing drugs with high safety requirements and long-term medication. In addition, PGC can not only be used alone as a macromolecular drug for direct injection or oral administration, but also as a prepolymer to utilize its remaining available carboxyl and hydroxyl functional groups to continue polymerize itself or incorporate other monomers, and be processed into biomedical materials such as catheters, hydrogels, biphasic or triphasic scaffolds, and nano- or microparticles with geometric shapes and microstructures, solving the problem of single function of traditional POC (polyethylene glycol citrate), and enabling it to be more widely applied in the biomedical field.

[0034] 3. For the preparation method of the gastrodin polycitrate of the present invention, the method uses gastrodin and citric acid as the main raw materials, and a thermal polymerization reaction is carried out between the hydroxyl group on gastrodin and the carboxyl group on citric acid to generate gastrodin polycitrate. The preparation method of PGC of the present invention has the advantages of being simple and easy to operate, green and pollution-free, and low cost, is easy to realize industrial large-scale production, and has potential economic, social value and application prospects. Description of the Drawings

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0036] Figure 1 It is the Fourier transform infrared spectroscopy detection result of gastrodin polycitrate prepared in Examples 1 - 3.

[0037] Figure 2 is the nuclear magnetic resonance hydrogen spectrum ( Figure 2-1 ) and carbon spectrum ( Figure 2-2 ) results of gastrodin polycitrate prepared in Example 1.

[0038] Figure 3 is the ESI - MS mass spectrometry analysis results of gastrodin polycitrate prepared in Examples 1 - 3: Mass spectrum of Example 1 ( Figure 3-1 ), mass spectrum of Example 2 ( Figure 3-2 ), mass spectrum of Example 3 ( Figure 3-3 ).

[0039] Figure 4 It is the CCK8 result graph for evaluating the proliferation effect of gastrodin polycitrate prepared in Example 1 on PC12 cells.

[0040] Figure 5 It is the WB result of the polarization effect of gastrodin polycitrate prepared in Example 1 on macrophages.

[0041] Figure 6 It is the result of the migration effect of the gastrodin polycitrate polymer prepared in Example 1 on rat Schwann cells (RSC96 cells). Detailed implementation manners

[0042] The following will specifically elaborate on the present invention in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0043] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0044] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0045] The overall idea of the present invention is as follows:

[0046] Since gastrodin has a short action time in the body and requires multiple-dose administration, which brings an unpleasant experience to patients, combined with its generally low bioavailability and other disadvantages, the clinical application of gastrodin is restricted. Currently, the developed gastrodin-related drugs and their new uses have not been truly widely used in clinical diseases due to their low activity, easy metabolism and other disadvantages. Therefore, taking gastrodin as a lead compound, further structural modification and optimization are needed, and drugs with better activity, smaller toxic and side effects, and longer action time in the body still need to be further studied deeply.

[0047] Citric acid (CA) exists in plant and animal tissues, such as blood, bones and muscles. Citric acid is an intermediate in the tricarboxylic acid cycle and a non-toxic metabolite. Citric acid has functions such as whitening and skin care, improving the taste of food, anti-corrosion, enhancing immunity, promoting digestion and antioxidant, making it widely used in food, pharmaceuticals, environmental protection and biomedicine. In 2004, Professor Yang Jian et al. pioneered the biodegradable elastomer poly(1,8-octanediol-co-citrate) (POC) formed by the thermal polymerization of citric acid and 1,8-octanediol, creating a new family of citrate-based biomaterials. Currently, the FDA has approved three orthopedic implant devices based on POC.

[0048] Traditional POC is synthesized from citric acid and a diol compound (such as 1,8-octanediol) through a simple, catalyst-free one-pot high-temperature polycondensation reaction to form a prepolymer. During the synthesis of the prepolymer, side carboxyl and hydroxyl functional groups can be partially retained, which not only helps the further cross-linking of the polymer network after polymerization, but also provides active sites for introducing functional groups. Therefore, its surface energy can be easily modified, thus having properties such as antioxidant, antibacterial, adhesive and fluorescence, and has been used in diseases of the cardiovascular system, musculoskeletal system, nervous system, renal system, gastrointestinal tissue, dermal tissue, etc. However, traditional POC has problems such as poor mechanical properties, fast degradation, local acidity during degradation and single function, which limit its wide application in the biomedical field. Moreover, the monomer diol reacting with citric acid has no medicinal function, so it is often necessary to introduce a third monomer to regulate the properties of POC.

[0049] For example, Li Xinrong disclosed a photocurable bioactive polycitrate-based polyurethane medical adhesive and its preparation method in the patent CN117224730A in 2023. Using polycitric acid diol ester prepolymer, diisocyanate, and vinyl monomer as the main raw materials, a double-bond-terminated polycitrate-based polyurethane prepolymer was prepared through stepwise reactions. Then, it was subjected to a graft reaction with an activator and N-hydroxysuccinimide to obtain a double-bond-terminated polycitrate-based polyurethane prepolymer containing NHS active ester. Finally, it was blended with a hydrophobic polymer and a photoinitiator was added and mixed evenly to obtain the photocurable bioactive polycitrate-based polyurethane medical adhesive.

[0050] Xu Jiajing disclosed a preparation method of a polycitrate porous scaffold in the patent CN112608457A in 2020. Citric acid, 1,8-octanediol, and 4-hydroxy-2-butenoic acid were placed in a container, heated and stirred in an oil bath at 160 - 165°C under nitrogen protection. After the mixture melted, the temperature was lowered to 135 - 140°C, and 2-aminoethanol-1-phosphate was added to obtain a prepolymer. a. Take the above prepolymer, dissolve it fully in acetone, pour it into a polytetrafluoroethylene mold, add NaCl particles and guanethidine sulfate, and stir evenly. b. Take out the sample, soak it in deionized water, wash away all the NaCl and guanethidine sulfate in the scaffold, and dry it to obtain the polycitrate porous scaffold.

[0051] To solve the problems of poor bioavailability, low activity, and easy metabolism of gastrodin as a small molecule drug, as well as the problems of single function of traditional POC and the need to introduce a third monomer to modify its properties, the present invention provides a gastrodin polycitrate polymer and its preparation method. Utilizing the properties of gastrodin polyol, an esterification reaction was carried out with citric acid to generate a macromolecular polymer: gastrodin polycitrate polymer, named PGC.

[0052] The in vivo metabolism, pharmacokinetics, cell uptake, etc. of this macromolecular polymer are different from those of small molecule gastrodin, and it will solve the problems of poor bioavailability, low activity, and easy metabolism of gastrodin. Different from traditional POC with a single function, PGC simultaneously has the functions of citric acid, gastrodin, and balisone glycoside, fully exerting the synergistic effect of each component, without containing components useless or harmful to the human body. Its degradation products, gastrodin and balisone glycoside, can also continue to exert their medicinal value. Compared with traditional POC, PGC does not need to introduce a third monomer to modify its properties. Since PGC can increase the in vivo circulation time and improve its bioavailability, multi-dose administration is not required, and it is particularly suitable for preparing drugs with high safety requirements and long-term medication needs.

[0053] In addition, PGC can not only be used alone as a macromolecular drug for direct injection or oral administration, but also as a prepolymer to utilize its remaining available carboxyl and hydroxyl functional groups to continue polymerizing itself or incorporating other monomers, and processed into biomedical materials such as catheters, hydrogels, biphasic or triphasic scaffolds, and nano- or microparticles with geometric shapes and microstructures, enabling it to be more widely applied in the biomedical field.

[0054] The PGC of the present invention has the effects of protecting cells, promoting cell proliferation or regeneration, and anti-inflammatory and antioxidant, and is effective in treating diseases in the nervous system, cardiovascular system, and musculoskeletal system. In addition, the preparation method of the PGC of the present invention has the advantages of being simple and easy to operate, green and pollution-free, and low cost, being easy to realize industrial production, and having potential economic, social value and application prospects.

[0055] Specifically, for a preparation method of gastrodin polycitrate of the present invention, the preparation method includes:

[0056] S1. Mix citric acid and gastrodin, and then add an organic solvent N,N-dimethylformamide to obtain a mixture, wherein the molar ratio of citric acid to gastrodin in the mixture is (1 - 2):(1 - 2);

[0057] S2. Heat the mixture to 100 - 160°C under an inert atmosphere, start timing after the mixture is dissolved, and keep reacting at 100 - 160°C for 6 - 72 h to obtain a polymer;

[0058] S3. Add water to the polymer, and then add sodium hydroxide to adjust the pH to neutral, and ultrasonicate until dissolved to obtain a polymer solution;

[0059] S4. Place the polymer solution in a dialysis bag with a cut-off molecular weight of 400 Da and dialyze for 48 h, changing the solution 3 - 6 times; the liquid obtained by dialysis is freeze-dried to obtain gastrodin polycitrate.

[0060] In step S1 of the present invention, the advantage of the molar ratio of citric acid to gastrodin in the mixture being (1 - 2):(1 - 2) is that the ratio of citric acid to gastrodin in the polymer can be regulated.

[0061] In step S2 of the present invention, the advantage of the reaction temperature of the mixture being 100 - 160°C is that citric acid and gastrodin can be fully dissolved and reacted.

[0062] In step S3 of the present invention, the purpose of adjusting the pH of the polymer solution to neutral is to neutralize the carboxyl groups on the polymer into carboxylate anions, making the product soluble in water and reducing the acidity of the product.

[0063] Next, the present application for a gastrodin polycitrate, its preparation method and application will be described in detail in conjunction with examples and experimental data.

[0064] Example 1

[0065] The preparation method of gastrodin polycitrate in this example includes:

[0066] (1) Weigh 10 mmol of citric acid and 10 mmol of gastrodin and put them into a 50 ml flask. Add N,N-dimethylformamide solvent with a molar fraction of 60% to promote dissolution. Place it in an oil bath at 100 °C under nitrogen protection and stir. Start timing after the mixture is dissolved and react for 12 h to obtain a polymer.

[0067] (2) Add an appropriate amount of deionized water to the polymer, adjust its pH to neutral with sodium hydroxide, and dissolve it by ultrasonic treatment to obtain a polymer solution.

[0068] (3) Place the polymer solution in a dialysis bag with a cut-off molecular weight of 400 Da and dialyze for 48 h, changing the solution 4 times.

[0069] (4) The dialyzed solution is freeze-dried to obtain gastrodin polycitrate.

[0070] Example 2

[0071] The preparation method of gastrodin polycitrate in this example includes:

[0072] (1) Put 10 mmol of citric acid and 10 mmol of gastrodin into a 50 ml flask. Add N,N-dimethylformamide solvent with a molar fraction of 60% to promote dissolution. Place it in an oil bath at 100 °C under nitrogen protection and stir. Start timing after the mixture is dissolved and react for 24 h to obtain a polymer.

[0073] (2) Add an appropriate amount of deionized water to the polymer, adjust its pH to neutral with sodium hydroxide, and dissolve it by ultrasonic treatment to obtain a polymer solution.

[0074] (3) Place the polymer solution in a dialysis bag with a cut-off molecular weight of 400 Da and dialyze for 48 h, changing the solution 4 times.

[0075] (4) The dialyzed solution is freeze-dried to obtain gastrodin polycitrate.

[0076] Example 3

[0077] The preparation method of gastrodin polycitrate in this example includes:

[0078] (1) Weigh 10 mmol of citric acid and 10 mmol of gastrodin and put them into a 50 ml flask. Add N,N-dimethylformamide solvent with a molar fraction of 60% to promote dissolution. Place it in an oil bath at 100 °C under nitrogen protection and stir. Start timing after the mixture is dissolved and react for 36 h to obtain a polymer.

[0079] (2) An appropriate amount of deionized water was added to the polymer, and its pH was adjusted to neutral with sodium hydroxide and then dissolved by ultrasonic treatment to obtain a polymer solution.

[0080] (3) The polymer solution was placed in a dialysis bag with a molecular weight cut-off of 400 Da and dialyzed for 48 h with 4 buffer exchanges.

[0081] (4) The dialyzed solution was freeze-dried to obtain gastrodin polycitrate.

[0082] Example 4

[0083] The gastrodin polycitrate prepared in Examples 1-3 was characterized in this example, and the results are as Figure 1 shown in -3.

[0084] Figure 1 are the Fourier transform infrared spectroscopy detection results of the gastrodin polycitrate prepared in Examples 1-3. Figure 1 Among them, characteristic peaks of C=O and C-O-C ester bonds appeared at 1720 cm -1 and 1230 cm -1 respectively. Characteristic peaks of C-O-C (pyran ring) and C=C (benzene ring) appeared at 1020 cm -1 and 1510 cm -1 respectively, and a characteristic peak of para-disubstituted benzene ring appeared at 830 cm -1 , indicating that citric acid and gastrodin were successfully polymerized.

[0085] Figure 2 shows the 1H nuclear magnetic resonance spectrum ( Figure 2-1 ) and 13C nuclear magnetic resonance spectrum ( Figure 2-2 ) results of the gastrodin polycitrate prepared in Example 1. According to the analysis of the 1H and 13C nuclear magnetic resonance spectrum results, signals of both gastrodin and citric acid were contained in the polymer. When gastrodin and citric acid were reacted with a molar ratio of 1:1, a polymer with a ratio of about 1:1 of the two was obtained, and its chemical reaction formula is as follows:

[0086]

[0087] Figure 3 shows the mass spectrometry analysis results of the gastrodin polycitrate prepared in Examples 1, 2, and 3. From the analysis of the results in Figure 3, it can be obtained that the molecular weight of the synthesized polymer conforms to the structure shown in the following figure, and its basic repeating unit is the structural general formula I. Figure 3-1 is the mass spectrometry analysis result of the polymer of gastrodin polycitrate prepared in Example 1, and its molecular weight distribution ranges from 728 to 1786 Da; Figure 3-2 is the mass spectrometry analysis result of the polymer of gastrodin polycitrate prepared in Example 2, and its molecular weight distribution ranges from 633 to 2228 Da; Figure 3-3The mass spectrometry analysis results of the gastrodin polycitrate polymer prepared in Example 3, and its molecular weight distribution ranges from 633 to 5054 Da. The reaction durations of Examples 1, 2, and 3 are 12 h, 24 h, and 36 h respectively. The above results prove that as the reaction time extends, the molecular weight of the obtained polymer also increases.

[0088] Combined with the NMR and mass spectrometry results, it can be analyzed that the basic repeating units of Examples 1, 2, and 3 are all the structural general formula I.

[0089] Example 5

[0090] In this example, various properties of the prepared gastrodin polycitrate were tested.

[0091] 1. Evaluation of the effect of the gastrodin polycitrate polymer prepared in Example 1 on cell proliferation:

[0092] (1) Prepare the PGC and GAS mother liquors: Weigh 0.03 g of PGC and GAS respectively and add them to 10 ml of PBS to dissolve them completely, obtaining a mother liquor with a concentration of 3 mg / ml. After filtering with a 0.22 μm filter head, it is reserved for use.

[0093] (2) Prepare PGC and GAS solutions with different concentrations: Take 1 ml of the mother liquor and add it to 9 ml of DMEM complete medium containing 10% serum and 1% double antibody to obtain a solution with a concentration of 300 μg / ml; take 0.5 ml of the solution with a concentration of 300 μg / ml and add it to 1 ml of DMEM complete medium containing 10% serum and 1% double antibody to obtain a solution with a concentration of 200 μg / ml; dilute them successively according to this method to obtain solutions with concentrations of 100, 50, 20, 10, and 5 μg / ml respectively, and store them at -20 °C for later use.

[0094] (3) Resuscitate PC12 cells (nerve cell line). After trypsinizing the cells cultured to the third generation, centrifuge and discard the supernatant, and resuspend and perform cell counting after resuspension.

[0095] (4) Take a 96-well plate, seed the cells at a density of 10,000 cells / well, then place it in an incubator at 37 °C for 24 h, and then add the pre-warmed PGC and GAS solutions with different concentrations. Discard the medium at 24 h of culture. Add 100 μl of the mixture of medium and CCK8 (10:1) to each well, and measure the absorbance at a wavelength of 450 nm after culturing in an incubator at 37 °C for 2 h.

[0096] (5) Process the data according to the formula: Cell Viability = (absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well) * 100%.

[0097] The results are as Figure 4 shown. FromFigure 4 It can be seen that PGC is basically non-toxic to cells, and compared with the gastrodin group at the same concentration, the PGC group has a significant promoting effect on the proliferation of PC12 cells.

[0098] 2. Polarization effect test of the gastrodin polycitrate polymer prepared in Example 1 on mouse mononuclear macrophage cell line (Raw264.7 cells):

[0099] (1) Prepare the mother solutions of PGC and GAS: Weigh 14.3 mg of GAS and 23.9 mg of PGC and add them to 5 ml of PBS respectively to dissolve them completely, obtaining the mother solutions of GAS and PGC containing 10 mM of gastrodin. After filtering with a 0.22 μm filter head, they are reserved for use.

[0100] (2) Prepare PGC and GAS solutions with different concentrations: Take 50 μl of the mother solution and add it to 4.95 ml of DMEM complete medium containing 10% serum and 1% double antibody to obtain a solution with a concentration of 100 μM; Take 3 ml of the solution with a concentration of 100 μM and add it to 3 ml of DMEM complete medium containing 10% serum and 1% double antibody to obtain a solution with a concentration of 50 μM; Take 3 ml of the solution with a concentration of 50 μM and add it to 3 ml of DMEM complete medium containing 10% serum and 1% double antibody to obtain a solution with a concentration of 25 μM. Store at -20 °C for later use.

[0101] (3) Resuscitate Raw264.7 cells. Discard the culture medium of the cells cultured to the third generation, add PBS pre-warmed to 37 °C for washing, and then add fresh DMEM complete medium containing 10% serum and 1% double antibody. Pipette the cells 20 - 30 times, and take the cell suspension for counting.

[0102] (4) Take a 6-well plate and seed the cells at a density of 5×10^ 6 cells / well. Then place it in an incubator at 37 °C for 24 h, and then add different concentrations of PGC and GAS solutions containing 1 μg / ml LPS. Culture for 24 h, and collect the culture medium and store it at -20 °C for later use.

[0103] (5) Wash the cells 2 times with PBS and then extract the protein. Determine the protein concentration by the BCA method and perform Western Blot experiment. Measure the macrophage M1 polarization markers: α tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS); macrophage M2 polarization markers: arginase-1 (Arg-1), mannose receptor (CD206); internal reference: β-actin. M1 macrophages exhibit pro-inflammatory characteristics and are involved in mechanisms such as anti-microbial defense, tissue destruction, and anti-tumor drug resistance; M2 macrophages exhibit anti-inflammatory characteristics and are involved in processes such as wound repair, angiogenesis, anti-parasite, and tumor growth.

[0104] The results are as Figure 5 shown. It can be seen from Figure 5 that macrophages treated with PGC or GAS for LPS-induced inflammation can significantly inhibit the upregulation of M1 polarization markers TNF-α and iNOS, and promote the upregulation of M2 polarization markers Arg-1 and CD206, indicating that both PGC and GAS can inhibit macrophage M1 polarization and induce macrophage M2 polarization to a certain extent, reversing the downregulation of Arg-1 caused by LPS. The above results indicate that both PGC and GAS have significant anti-inflammatory effects.

[0105] 3. Experiment on the migration of gastrodin polycitrate polymer on rat Schwann cells (RSC96 cells):

[0106] (1) Resuscitate RSC96 cells. After trypsinizing the cells cultured to the third generation, centrifuge and discard the supernatant, and resuspend the cells for cell counting.

[0107] (2) Take a 6-well plate and seed the cells at a density of 5×10^ 6 cells / well. Then place it in an incubator at 37°C for 36 h and perform a scratch assay.

[0108] (3) After scratching the cells, replace them with the conditioned medium obtained in 2.(4) to create an inflammatory microenvironment after PGC treatment of macrophages.

[0109] (4) Use an inverted white light microscope to take scratch photos at 0, 12, 24, and 48 h respectively. Use ImageJ to process the scratch area and perform statistical analysis with GraphPad Prism 8.

[0110] The results are as Figure 6 shown. It can be seen from Figure 6 that the conditioned medium after PGC treatment can promote the migration of Schwann cells.

[0111] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0112] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0113] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A gastrodin polycitrate, characterized in that: The general structural formula of the gastrodin polycitrate is shown in Formula I: n is an integer >1.

2. The method for preparing gastrodin polycitrate according to claim 1, characterized in that: The preparation method comprises: Mixing citric acid and gastrodin, and then adding an organic solvent to obtain a mixture; heating the mixture, and continuing the reaction for 6 to 72 hours after the mixture is dissolved to obtain a polymer; Dissolving the polymer in water and adjusting the pH to neutral to obtain a polymer solution; The polymer solution is dialyzed and freeze-dried to obtain gastrodin polycitrate; Wherein, the molar ratio of citric acid to gastrodin in the mixture is (1-2): (1-2).

3. The method for preparing gastrodin polycitrate according to claim 2, characterized in that: The step of mixing citric acid and gastrodin and then adding an organic solvent to obtain a mixture specifically comprises: Mixing citric acid and gastrodin, and then adding an organic solvent, N,N-dimethylformamide, to obtain a mixture; Wherein, the molar percentage of N,N-dimethylformamide in the mixture is 50-80 mol%.

4. The method for preparing gastrodin polycitrate according to claim 2, characterized in that: The step of heating the mixture and continuing the reaction for 6 to 72 hours after the mixture is dissolved to obtain a polymer specifically comprises: The mixture is heated to 100-160° C. in an inert atmosphere, and timing is started after the mixture is dissolved. The temperature is maintained at 100-160° C. and the reaction is continued for 6-72 hours to obtain a polymer.

5. The method for preparing gastrodin polycitrate according to claim 2, characterized in that: The step of dissolving the polymer in water and adjusting the pH to neutral to obtain a polymer solution specifically comprises: Water is added to the polymer, and then sodium hydroxide is added to adjust the pH to neutral, and ultrasonic treatment is performed to dissolve the polymer to obtain a polymer solution.

6. The method for preparing gastrodin polycitrate according to claim 2, characterized in that: The polymer solution is dialyzed and freeze-dried to obtain gastrodin polycitrate, which specifically comprises: The polymer solution was placed in a dialysis bag with a molecular weight cut-off of 400 Da and dialyzed for 48 hours, with the solution being changed 3 to 6 times; The dialyzed liquid was freeze-dried to obtain gastrodin polycitrate.

7. Use of a gastrodin polycitrate according to claim 1 in the preparation of a drug for preventing and treating nervous system diseases, characterized in that: The nervous system disease includes at least one of nerve damage, epilepsy, Alzheimer's disease, Parkinson's disease, mood disorders, cognitive disorders and neuropathic pain.

8. The use of a gastrodin polycitrate according to claim 1 in the preparation of a drug for preventing and treating cardiovascular diseases, characterized in that: The cardiovascular disease includes at least one of vascular injury, myocardial fibrosis, hypertension and myocardial ischemia-reperfusion injury.

9. Use of a gastrodin polycitrate according to claim 1 in the preparation of a medicament for preventing and treating musculoskeletal system diseases, characterized in that: The musculoskeletal disease includes at least one of bone defects, osteoarthritis and rheumatoid arthritis.

10. Use of a gastrodin polycitrate according to claim 1 in preparing a biomedical material with a geometric shape or microstructure, characterized in that: The biomedical material with geometric shape or microstructure includes at least one of a catheter, a hydrogel, a two-phase scaffold, a three-phase scaffold, nanoparticles and microparticles.

11. A drug, characterized in that The active ingredient of the drug comprises the gastrodin polycitrate as claimed in claim 1, and the drug comprises a drug for preventing and treating central nervous system diseases, a drug for preventing and treating cardiovascular diseases, or a drug for preventing and treating musculoskeletal system diseases.

Images