Extraction method of torreya grandis crude polysaccharide and application of torreya grandis crude polysaccharide in neuronal differentiation

Abstract

The invention relates to the technical field of natural product extraction, in particular to a torreya grandis crude polysaccharide extraction method and application of the torreya grandis crude polysaccharide in neuronal differentiation.The torreya grandis crude polysaccharide is extracted through an ultrasonic wave-microwave-enzyme method combined auxiliary extraction technology, the polysaccharide content is detected to be 67.92% through a phenol-sulfuric acid method, the relative molecular mass is 3000 kDa, and the content of the polysaccharide in the torreya grandis crude polysaccharide is 87.92%. A novel method is provided for a torreya grandis polysaccharide extraction process; besides, activity research is carried out on the extracted torreya grandis crude polysaccharide, the torreya grandis crude polysaccharide can promote bone marrow-derived mesenchymal stem cells (BMSCs) to differentiate into neuronal cells, mRNA expression of neuron specific markers Netin, MAP2 and VEGF-A in the BMSCs is increased, the protein level of neuronal differentiation related genes beta-tubin III, Thy-1 and Enolase is up-regulated, and the effect of promoting the differentiation of the neuronal cells is achieved. A good nerve repairing effect is embodied, and the material is expected to be applied to clinical treatment of nerve diseases.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to a method for extracting crude polysaccharides from Torreya grandis seeds and its application in neuronal differentiation. Background Technology

[0002] Neurological diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), are a class of diseases characterized primarily by neurodegenerative changes, posing a serious threat to human health, especially among the elderly. These diseases are often accompanied by structural and functional damage to neurons, sometimes leading to irreversible neuronal death. For example, Alzheimer's patients experience a significant loss of neurons in their brains, resulting in cognitive decline and memory impairment; while Parkinson's patients suffer from motor dysfunction due to the degeneration of dopaminergic neurons in the midbrain. Because neurons in the central nervous system of adult mammals have extremely limited regenerative capacity, once damaged or dead, it is difficult for them to regain function through their own repair mechanisms. Therefore, the treatment of neurological diseases remains a major challenge in the medical field.

[0003] Currently, treatments for neurological diseases primarily focus on symptom relief and slowing disease progression, such as dopamine replacement therapy for Parkinson's disease or cholinesterase inhibitors to improve cognitive function in Alzheimer's patients. However, these treatments do not fundamentally address neuronal damage or death, nor do they effectively promote neuronal regeneration and functional recovery. Therefore, developing novel therapeutic strategies that promote neuronal differentiation and regeneration has become a key focus of current research.

[0004] In recent years, the role of plant polysaccharides in enhancing neurological and cognitive function has received increasing attention, especially those derived from edible and medicinal plants, such as Ganoderma lucidum polysaccharide, Ganoderma lucidum polysaccharide, and Ganoderma lucidum polysaccharide. These compounds exert neuromodulatory effects by regulating neurotransmitter expression and receptor activity, inflammatory responses, the HPA axis, and trophic factors. For example, Ganoderma lucidum polysaccharides can promote the proliferation of neural progenitor cells in transgenic mice with Alzheimer's disease, promote neurogenesis, and alleviate cognitive deficits. Therefore, compounds derived from natural products have potential value in the treatment of neurological diseases.

[0005] Torreya grandis, a gymnosperm belonging to the Taxaceae family (Taxaceae), is an evergreen tree with a long history of cultivation in my country, mainly distributed in Shengzhou, Dongyang, and Zhuji in Zhejiang Province. Torreya grandis seeds, also known as Yushan fruit, Torreya wood bark, red fruit, Yu Torreya, Zhen Torreya, and Xi Torreya, consist of an outer seed coat, middle seed coat, inner seed coat, and kernel. As a plant used for both food and medicine, Torreya grandis seeds are rich in various bioactive substances, including polysaccharides, flavonoids, and terpenes. Studies have shown that Torreya grandis seed extracts possess various biological activities such as antioxidant, anti-inflammatory, and immunomodulatory effects, providing a good foundation for the application of Torreya grandis active ingredients in the field of nerve repair. Currently, the incidence of neurodegenerative diseases continues to rise, increasing the clinical demand for drugs that can promote neuronal regeneration. Against this backdrop, exploring whether and how the crude polysaccharides in Torreya grandis seeds can promote neuronal differentiation and regeneration is particularly important and necessary. However, systematic research on the extraction methods of Torreya grandis polysaccharides and their specific mechanisms of action in neuronal differentiation is currently lacking. Therefore, conducting systematic research on Torreya grandis polysaccharides will not only contribute to the development of novel neuroreparative drugs, but also provide a scientific basis for the in-depth development and utilization of traditional medicinal and edible resources. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a method for extracting crude polysaccharides from Torreya grandis seeds and its application in neuronal differentiation.

[0007] This invention employs a combined ultrasonic-microwave-enzymatic extraction process to extract crude polysaccharide (TMSP) from Torreya grandis seeds, and the TMSP content is detected by the phenol-sulfuric acid method. Furthermore, the activity of the extracted TMSP was studied. TMSP can promote the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into neurons, increase the mRNA expression of neuron-specific markers Nestin, MAP2, and VEGF-A in BMSCs, and upregulate the protein levels of neuron differentiation-related genes β-tublin III, Thy-1, and Enolase.

[0008] This invention provides a method for preparing crude polysaccharides from Torreya grandis seeds using a combined ultrasonic-microwave-enzymatic extraction process, specifically comprising the following steps:

[0009] S1: Wash the Torreya grandis seeds, dry them at 40℃, crush them, pass them through a 100-mesh sieve, and remove fat and pigment with petroleum ether.

[0010] S2: Add phosphate buffer to the sample obtained in S1 at a ratio of 1g:30mL, and extract to TMSP extract using a combination of ultrasonic-microwave-enzymatic method.

[0011] S3: Centrifuge the TMSP extract obtained in S2 at 5000 r / min for 15 min to obtain the supernatant, concentrate by rotation, precipitate the polysaccharide with ethanol, and freeze-dry to obtain the crude TMSP extract.

[0012] S4: Dissolve the crude TMSP extract obtained in S3 in distilled water to prepare a polysaccharide concentrate of 2 mg / mL. Add an organic solvent prepared by chloroform and n-butanol at a volume ratio of 5:1 to 1 / 5 of the concentrate volume. Stir the mixture on a magnetic stirrer for 15 min (1000 r / min). Centrifuge (5000 r / min, 5 min), retain the supernatant, remove the precipitate, and repeat the previous steps until no protein precipitation occurs.

[0013] Furthermore, the conditions for removing fats and pigments with petroleum ether as described in S1 are: reflux at 50°C for 10 hours according to the ratio of Torreya grandis powder to petroleum ether = 1g: 20mL.

[0014] Furthermore, the ultrasound-microwave-enzyme hydrolysis combined method described in S2 is as follows: add the enzymatic hydrolysis reactant, adjust the pH to 4.5, place it in a 55℃ water bath shaker, and hydrolyze for 2 hours. After the enzymatic hydrolysis is completed, heat the mixture to 90℃ and maintain it for 10 minutes to inactivate the enzyme. Next, perform ultrasound-microwave extraction with the following conditions: ultrasound power 360W, microwave power 500W, extraction time 80min, and ultrasound temperature 75℃.

[0015] Furthermore, the amount of the enzymatic hydrolysis reactant added is 2% of the mass of Torreya grandis powder;

[0016] Furthermore, the enzymatic hydrolysis reactants are prepared by mixing Trichoderma cellulase and Penicillium pectinase at a mass ratio of 1:1.

[0017] Furthermore, the method for precipitating polysaccharides with ethanol described in S3 is as follows: add 4 times the volume of 95% ethanol solution to make the final ethanol volume fraction above 85%, incubate at 4°C overnight, and centrifuge at 5000 r / min for 15 min to obtain the precipitate.

[0018] This invention also provides an application of crude polysaccharide from Torreya grandis in the preparation of nerve repair drugs.

[0019] Furthermore, the neural repair drug is a drug that promotes the differentiation of bone marrow-derived mesenchymal stem cells into neurons.

[0020] The effective concentration of crude polysaccharide from Torreya grandis in the drug that promotes the differentiation of bone marrow-derived mesenchymal stem cells into neurons is 100 μg / mL.

[0021] Furthermore, the neural repair drug is a drug that promotes the mRNA expression of neuron-specific markers Nestin, MAP2, and VEGF-A.

[0022] Furthermore, the neural repair drug is a drug that upregulates the protein levels of neuronal differentiation-related genes β-tublin III, Thy-1, and Enolase.

[0023] The advantages of this invention are:

[0024] 1. This invention is the first to utilize a combined ultrasonic-microwave-enzymatic extraction process. In the enzymatic process, Trichoderma cellulase hydrolyzes cellulose and some hemicellulose to disrupt the cell wall skeleton, while penicillinase further disrupts the cell wall network structure by degrading pectin-like substances. The combination of these two processes greatly enhances the release of intracellular polysaccharides. TMSP is then obtained through deproteinization, dialysis, and vacuum freeze-drying. The polysaccharide content, as determined by the sulfuric acid-phenol method, is 67.92%, with a relative molecular mass of 3000 kDa. This provides a new method for the extraction of polysaccharides from Torreya grandis seeds.

[0025] 2. The TMSP of this invention can promote the differentiation of BMSCs into neurons, upregulate the mRNA expression of neuron-specific markers Nestin, MAP2 and VEGF-A and the protein levels of neuron differentiation-related genes β-tublin III, Thy-1 and Enolase, providing a theoretical basis for the clinical application of Torreya grandis polysaccharides. Attached Figure Description

[0026] Figure 1 This is a graph showing the effect of TMSP prepared in this invention on the growth of BMSCs.

[0027] Figure 2 This is a graph showing the effect of TMSP prepared in this invention on the differentiation of BMSCs into neuronal cells: Figure 2 -A is a graph showing the effect of Giemsa staining in identifying neuronal differentiation; Figure 2 -B is Figure 2 -Quantitative analysis diagram of the maximum diameter of cells in A; Figure 2 -C is Figure 2 -Quantitative analysis diagram of the longest synapse length in cells A; Figure 2 -D is Figure 2 -Quantitative analysis of cell contact numbers in A.

[0028] Figure 3 The following is a diagram showing the effect of the TMSP prepared in this invention on the mRNA expression of specific markers for BMSC differentiation into neuronal cells: Figure 3 -A represents the relative mRNA expression level of Nestin in the cell; Figure 3 -B represents the relative mRNA expression level of MAP2 in cells; Figure 3 -C represents the relative expression level of VEGF-A mRNA in cells.

[0029] Figure 4This is a graph showing the effect of the TMSP prepared in this invention on the protein expression of genes related to the differentiation of BMSCs into neuronal cells: Figure 4 -A shows the protein expression bands of neuronal cell differentiation-related genes β-tublin III, Thy-1, and Enolase. Figure 4 BD is a quantitative analysis of the protein expression of β-tublin III, Thy-1, and Enolase. Detailed Implementation

[0030] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0031] Example 1: Combined preparation of TMSP using ultrasound-microwave-enzyme method

[0032] S1: Wash the Torreya grandis seeds, dry them at 40℃, pulverize them, and pass them through a 100-mesh sieve. Then, according to the ratio of Torreya grandis powder to petroleum ether = 1g: 20mL, reflux with petroleum ether at 50℃ for 10h to remove fat and pigment.

[0033] S2: The sample obtained in S1 was added to phosphate buffer at a ratio of 1g:30mL. An ultrasonic-microwave-enzymatic extraction method was used. 2% (by weight) of the enzymatic hydrolysis product (Trichoderma cellulase: Penicillium pectinase = 1:1 by weight) of Torreya grandis powder was added. The pH was adjusted to 4.5, and the mixture was placed in a 55℃ water bath shaker for 2 hours for enzymatic hydrolysis. After hydrolysis, the mixture was heated to 90℃ and held for 10 minutes to inactivate the enzyme. Next, ultrasonic-microwave extraction was performed under the following conditions: ultrasonic power 360W, microwave power 500W, extraction time 80min, and ultrasonic temperature 75℃. Finally, the TMSP extract was obtained.

[0034] S3: Centrifuge the TMSP extract obtained in S2 at 5000 r / min for 15 min to obtain the supernatant, concentrate by rotation, add 4 times the volume of 95% ethanol solution to make the final ethanol volume fraction above 85%, incubate at 4℃ overnight, centrifuge at 5000 r / min for 15 min to obtain the precipitate, and freeze-dry to obtain the crude TMSP extract.

[0035] S4: Dissolve the crude TMSP extract obtained in S3 in distilled water to prepare a polysaccharide concentrate of 2 mg / mL. Add an organic solvent (chloroform: n-butanol = 5:1) at 1 / 5 of the concentrate volume. Stir the mixture on a magnetic stirrer for 15 min (1000 r / min). Centrifuge (5000 r / min, 5 min), retain the supernatant, remove the precipitate, and repeat the previous steps until no protein precipitation occurs.

[0036] S5: Transfer the solution obtained in S4 into a dialysis bag (3000kDa) and dialyze for 24 hours at room temperature and under running water. Then dialyze for another 24 hours with distilled water. After dialysis, pour out the polysaccharide solution from the dialysis bag, pre-freeze it in a -20℃ freezer, and then freeze-dry it in a vacuum freeze dryer to obtain crude TMSP polysaccharide.

[0037] Example 2: Determination of polysaccharide content using the sulfuric acid-phenol method

[0038] The polysaccharide content in the extracted TMSP was determined by the sulfuric acid-phenol method. A standard curve of polysaccharide concentration and absorbance was plotted using glucose. The crude polysaccharide yield (%) was then calculated according to the calculation formula (1).

[0039]

[0040] In the formula, m is the mass of dried Torreya grandis powder (g); c is the mass concentration of crude Torreya grandis polysaccharide corresponding to the standard curve (mg / mL); V is the volume of the test liquid (mL); and D is the dilution factor.

[0041] The results showed that the polysaccharide content of the TMSP extracted in this experiment was 67.92%, and the relative molecular mass was 3000 kDa, proving that the present invention successfully extracted crude TMSP polysaccharide with a polysaccharide content of more than 60%.

[0042] Example 3: Evaluation of TMSP cytotoxicity against BMSCs using the MTT assay

[0043] Male Sprague-Dawley (SD) rats aged 4–6 weeks were euthanized by cervical dislocation and immersed in 75% ethanol for 15 minutes. The femur, tibia, and surface muscles were aseptically removed, and bone marrow mesenchymal stem cells (BMSCs) were flushed from the bone marrow cavity using a 1 mL syringe in DMEM medium. The cell suspension was then collected and cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. Non-adherent cells were removed after 24 hours of culture. BMSCs were passaged when they reached approximately 70%–80% confluence. BMSCs were seeded into 96-well plates at 5000 cells per well and cultured overnight at 37°C and 5% CO2 until cell adherence. Using a serial dilution method, TMSP was prepared into different concentrations of neural basal medium (DMEM / F12 containing 10% FBS, 1% P / S, 20 ng / mL basic fibroblast growth factor (bFGF), and 20 ng / mL epidermal growth factor (EGF)) containing a neural inducer (2% B27). 100 μL of the solution was added to each well of a 96-well plate to achieve final TMSP concentrations of 0, 25, 50, 100, and 200 μg / mL. After co-culturing with cells for 7 days, the medium was changed every two days. After treatment, the original medium in the 96-well plates was discarded, and 20 μL of sterile MTT at a concentration of 5 mg / mL was added to each well under light-protected conditions. The plates were incubated at 37°C for 4 hours under light-protected conditions. After 4 hours, the MTT was discarded, and 150 μL of DMSO was added to fully dissolve the formazan crystals in the wells. Cell viability was analyzed by measuring the absorbance at 490 nm using a microplate reader.

[0044] Experimental results: Statistical analysis results of MTT results are as follows Figure 1 TMSP showed significant growth-promoting effects on BMSCs at concentrations of 25, 50, and 100 μg / mL, but inhibited BMSCs growth at a concentration of 200 μg / mL. Therefore, 100 μg / mL TMSP was selected as the concentration for activity evaluation in subsequent examples.

[0045] Example 4: Giemsa staining experiment to observe the differentiation of BMSCs

[0046] BMSCs were dissociated using trypsin and then seeded on poly-L-lysine-coated glass coverslips containing 10% FBS and 1% penicillin-streptomycin in DMEM for one day. After cell attachment, the medium was replaced with neural basal medium containing a neuroinducer (2% B27) and 100 μg / mL TMSP, and cultured in an incubator at 37°C and 5% CO2. BMSCs cultured in DMEM (containing 10% FBS) were designated as the Control group; BMSCs cultured in DMEM / F12 medium containing 2% B27, 20 ng / mL bFGF, and 20 ng / mL EGF were designated as the Vehicle group; and BMSCs treated with 100 μg / mL TMSP were designated as the TMSP group. Neuron-like cells were identified using the Giemsa staining kit. Under an inverted fluorescence microscope, 10 non-overlapping fields of view (×40) were randomly selected for image capture. ImageJ was then used to calculate the maximum diameter, longest neurite length, and number of synapses of neuron-like positive cells, and statistical analysis was performed.

[0047] Experimental Results: To investigate the effect of TMSP on BMSCs cell differentiation, this application treated BMSCs cells with 100 μg / mL TMSP and detected cell morphological changes 7 days after neuronal induction. Based on Giemsa staining results... Figure 2 As shown in Figure A, the cells in the Control group exhibited a spindle-shaped morphology, while the Vehicle and TMSP groups showed similar neuronal morphology after 7 days of induction. This was based on the maximum cell diameter (…). Figure 2 -B), the longest synaptic length of neuron-like cells ( Figure 2 -C) and the number of synapses of neuron-like cells ( Figure 2 -D) The differentiation differences between the two groups of cells were analyzed from three aspects. The cells in the TMSP group showed typical neuronal morphology, with more synapses and longer neurites than the cells in the Vehicle group, indicating that TMSP promotes the differentiation of BMSCs into neuron-like cells.

[0048] Example 5: Effects of TMSP on gene and protein expression in neuronal differentiation

[0049] (1) Real-time quantitative PCR (RT-qPCR): Cell treatment was performed as described in Example 4. After treatment, cells were collected, and total RNA was extracted from undifferentiated and differentiated BMSCs using an RNA isolation kit. The RNA was then reverse transcribed into cDNA using PrimeScript reverse transcriptase. qRT-PCR was performed using the SYBR Green PCR mixture in a Quant Studio6 Flex real-time quantitative PCR system to detect the mRNA expression of Nestin, MAP2, and VEGF-A in BMSCs.

[0050] (2) Western blot (WB) experiment: Cell treatment was as described in Example 4. After treatment, cells were collected, proteins were extracted by lysing BMSCs, and neuron-like cells were incubated with RIPA buffer supplemented with protease and phosphatase inhibitors at 4°C for 30 minutes. Next, the samples were centrifuged at 12,000g for 15 minutes to obtain the supernatant, and the protein concentration was measured using the Bradford assay. Then, the samples were loaded onto a protein electrophoresis apparatus for gel transfer. The membrane was blocked overnight with 5% milk powder solution prepared with PBS. The next day, after washing the membrane, antibody incubation solution for neuronal differentiation-related genes was added and incubated for 2 hours. After incubation, the membrane was washed again, and secondary antibody was added and incubated for 1 hour. After incubation, the membrane was developed.

[0051] Experimental Results: To observe the effect of TMSP on the expression levels of specific markers in neurons differentiated from BMSCs, this application used RT-qPCR and Western blotting to detect the mRNA and protein expression of neuron-specific markers. The RT-qPCR results are as follows: Figure 3 As shown, compared with the Control group, the Vehicle group and TMSP group showed a higher level of the neuron-specific biomarker Nestin after 7 days of induction. Figure 3 -A), MAP2 Figure 3 -B) and VEGF-A ( Figure 3 -C) mRNA expression in BMSCs was increased, and the promoting effect of TMSP was stronger in the TMSP group than in the Vehicle group, indicating that TMSP enhanced the differentiation of BMSCs into neurons. Furthermore, the protein levels of neuron-specific markers in cells were examined 7 days after TMSP induction, and the results were as follows: Figure 4 ,right Figure 4 The quantitative analysis results of each protein in A are as follows: Figure 4 BD, compared with the other two groups, the TMSP group had a higher concentration of the neuronal differentiation-related gene β-tublin III ( Figure 4 -D), Thy-1( Figure 4 -C) and Enolase ( Figure 4The protein level of -B was also significantly increased, which was largely consistent with mRNA expression. In summary, these results indicate that TMSP enhances neuronal differentiation in BMSCs.

[0052] In summary, this invention utilizes a combined ultrasound-microwave-enzyme extraction process to extract TMSP, achieving a polysaccharide content of 67.92%. This process promotes the differentiation of BMSCs into neurons, upregulates the mRNA expression of neuron-specific markers Nestin, MAP2, and VEGF-A, and increases the protein levels of neuron differentiation-related genes β-tublin III, Thy-1, and Enolase, providing a new approach for the development and utilization of Torreya grandis seeds.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting crude polysaccharides from Torreya grandis seeds using a combined ultrasonic-microwave-enzymatic method, characterized in that, Specifically, the steps include the following: S1: Wash the Torreya grandis seeds, dry them at 40℃, crush them, pass them through a 100-mesh sieve, and remove fat and pigment with petroleum ether. S2: Add phosphate buffer to the sample obtained in S1 at a ratio of 1g:30mL, and extract to TMSP extract using a combination of ultrasonic-microwave-enzymatic method. S3: Centrifuge the TMSP extract obtained in S2 at 5000 r / min for 15 min to obtain the supernatant, concentrate by rotation, precipitate the polysaccharide with ethanol, and freeze-dry to obtain the crude TMSP extract. S4: Dissolve the crude TMSP extract obtained in S3 in distilled water to prepare a polysaccharide concentrate of 2 mg / mL. Add an organic solvent prepared by chloroform and n-butanol at a volume ratio of 5:1 to 1 / 5 of the concentrate volume. Stir the mixture on a magnetic stirrer for 15 min at 1000 r / min. After centrifugation at 5000 r / min for 5 min, retain the supernatant and remove the precipitate. Repeat the previous steps until no protein precipitation occurs.

2. The method for extracting crude polysaccharides from Torreya grandis seeds using a combined ultrasonic-microwave-enzymatic method according to claim 1, characterized in that, The conditions for removing fat and pigment with petroleum ether as described in S1 are: reflux at 50°C for 10 hours according to the ratio of Torreya grandis powder to petroleum ether = 1g: 20mL.

3. The method for extracting crude polysaccharides from Torreya grandis seeds using a combined ultrasonic-microwave-enzymatic method according to claim 1, characterized in that, The ultrasound-microwave-enzyme combined method described in S2 is as follows: add the enzymatic reaction mixture, adjust the pH to 4.5, place it in a 55℃ water bath shaker, and enzymatically hydrolyze for 2 hours. After the enzymatic hydrolysis is completed, heat the mixture to 90℃ and maintain it for 10 minutes to inactivate the enzyme. Next, perform ultrasound-microwave extraction with the following conditions: ultrasound power 360W, microwave power 500W, extraction time 80min, and ultrasound temperature 75℃.

4. A method for extracting crude polysaccharides from Torreya grandis seeds using a combined ultrasonic-microwave-enzymatic method as described in claim 3, characterized in that... The amount of the enzymatic hydrolysis reactant added is 2% of the mass of Torreya grandis powder.

5. A method for extracting crude polysaccharides from Torreya grandis seeds using a combined ultrasonic-microwave-enzymatic method as described in claim 3, characterized in that... The enzymatic hydrolysis reaction mixture is prepared by mixing Trichoderma cellulase and Penicillium pectinase at a mass ratio of 1:

1.

6. The method for extracting crude polysaccharides from Torreya grandis seeds using a combined ultrasonic-microwave-enzymatic method according to claim 1, characterized in that, The method for precipitating polysaccharides with ethanol described in S3 is as follows: add 4 times the volume of 95% ethanol solution to make the final ethanol volume fraction above 85%, incubate overnight at 4°C, and centrifuge at 5000 r / min for 15 min to obtain the precipitate.

7. Application of crude polysaccharide from Torreya grandis in the preparation of nerve repair drugs.

8. The application of the crude polysaccharide of Torreya grandis fruit according to claim 7 in the preparation of nerve repair drugs, characterized in that, The neurorepair drug is a drug that promotes the differentiation of bone marrow-derived mesenchymal stem cells into neurons.

9. The application of the crude polysaccharide from Torreya grandis seeds according to claim 7 in the preparation of nerve repair drugs, characterized in that, The neurorepair drug is a drug that promotes the mRNA expression of neuron-specific markers Nestin, MAP2, and VEGF-A and upregulates the protein levels of neuron differentiation-related genes β-tublin III, Thy-1, and Enolase.

10. The application of the crude polysaccharide of Torreya grandis according to claim 8 in the preparation of nerve repair drugs, characterized in that, The effective concentration of crude polysaccharide from Torreya grandis in the drug that promotes the differentiation of bone marrow-derived mesenchymal stem cells into neurons is 100 μg / mL.

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