A polysaccharide extracted from pu'er tea and application thereof in alleviating side effects of chemotherapy

Abstract

The application provides a polysaccharide extracted from Pu'er tea and application thereof in relieving side effects of chemotherapy, first, removing water-soluble polysaccharides and macromolecular substances such as proteins in the Pu'er tea; second, further treating non-water-soluble residues with a certain amount of amylase to remove starch; then, gradient extraction is adopted to extract acidic polysaccharides; when the different Pu'er tea polysaccharides extracted by ethylenediaminetetraacetic acid solution are tested for immunomodulatory activity, the tea polysaccharides extracted by the ethylenediaminetetraacetic acid solution show a very strong promoting effect on the release of G-CSF factors from cells, the Pu'er tea polysaccharides have a significant effect of relieving granulocytopenia caused by chemotherapy drugs, the effect of relieving granulocytopenia caused by chemotherapy drugs is obvious, and the tea polysaccharides show a significant up-regulation of the expression of SOCS1 protein in the immunoblotting experiment, and the protein can negatively feedback regulate the abnormally activated JAK / STAT signal pathway in diseases such as tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a polysaccharide extracted from Pu-erh tea and its application in alleviating the side effects of chemotherapy. Background Technology

[0002] Chemotherapy, along with surgery and radiotherapy, is considered one of the three major treatments for cancer and is one of the most effective methods for treating cancer. Cyclophosphamide is a typical chemotherapy drug. The goals of chemotherapy include: inhibiting cancer cell growth, killing cancer cells, and alleviating cancer symptoms. Other treatment methods may also be used in conjunction with chemotherapy depending on the situation. Studies show that chemotherapy complications are increasingly becoming a concern. Because chemotherapy drugs primarily suppress bone marrow hematopoiesis, the most common complication during chemotherapy is the destruction of bone marrow hematopoietic function, with granulocytopenia being particularly prominent. Granulocytopenia manifests as fever due to granulocytopenia, a fever symptom that occurs when there is a persistent and severe decrease in granulocytes.

[0003] With the continuous improvement of people's living standards, various health foods with natural products as the main ingredients have quietly emerged. Plant polysaccharides are natural high-molecular complexes widely found in higher plants. Studies have shown that polysaccharides are not only important substances that constitute cells, but many polysaccharides also have good biological activity functions. Tea polysaccharides (TPS) are a large class of plant polysaccharides and a very important macromolecular active substance in tea. In nature, tea polysaccharides mainly exist in the form of glycoconjugates, that is, after various monosaccharides polymerize, they combine with proteins, nucleic acids, uronic acids, inorganic elements, etc. to form large complexes. Like polysaccharides in a broader sense, tea polysaccharides have also been proven to have antioxidant, immune-enhancing, blood sugar-lowering, anti-cancer, and antibacterial biological activities. Acidic polysaccharides with strong immune activity have been found to exist in large quantities in pectin, which is mainly found in the primary cell walls and intercellular spaces of plants and is a matrix polysaccharide of the cell wall.

[0004] Pu-erh tea, as a type of tea, is highly sought after by the public due to its unique flavor and taste as well as its health benefits. Therefore, research on the function of cell wall polysaccharides in Pu-erh tea has received widespread attention in this field. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the existing technology for extracting tea polysaccharides has complex processes such as purification and separation, and is not a cell wall polysaccharide, so it cannot be applied to granulocytopenia. The existing technology for extracting Pu-erh tea polysaccharides often focuses on water-alcohol extraction, which is relatively complex and has complex processes such as separation and purification. Furthermore, the content of acidic polysaccharides in Pu-erh tea polysaccharides extracted using the existing technology is relatively low. This invention provides a polysaccharide extracted from Pu-erh tea and its application in alleviating the side effects of chemotherapy. The tea polysaccharide extracted with ethylenediaminetetraacetic acid solution showed a strong promoting effect on the release of G-CSF factor from cells. In further studies, it was found that the Pu-erh tea polysaccharide has a significant effect in alleviating granulocytopenia induced by chemotherapy. When the dosage was 50 mg / kg, the number of neutrophils in mice with granulocytopenia was significantly increased, and the effect of alleviating granulocytopenia induced by chemotherapy was obvious. Moreover, the tea polysaccharide showed a significant upregulation of SOCS1 protein expression in immunoblotting experiments. This protein can negatively feedback regulate the abnormally activated JAK / STAT signaling pathway in diseases such as tumors.

[0006] To achieve the above objectives, the present invention provides a method for extracting cell wall polysaccharides from Pu-erh tea, comprising the following steps:

[0007] Step 1: Grind the Pu-erh tea into powder to obtain dry Pu-erh tea powder;

[0008] Step 2: Mix the dried Pu-erh tea powder with water and alcohol solution in sequence to remove water-soluble polysaccharides and proteins from the dried Pu-erh tea powder, filter and collect the first filter residue;

[0009] Step 3: Mix the first filter residue with amylase to remove the starch from the first filter residue, filter and collect to obtain the second filter residue;

[0010] Step 4: Under pH 6-7 conditions, the second filter residue is mixed with ethylenediaminetetraacetic acid solution to change the cross-linking structure of the cell wall in Pu-erh tea cells, and the first filtrate and the third filter residue are obtained by filtration and collection.

[0011] Step 5: Mix the third filter residue with a saturated sodium carbonate solution, shake to extract and mix thoroughly, then filter to collect the fourth filter residue and the second filtrate;

[0012] Step 6: Mix the fourth filter residue with 1M KOH alkaline solution, shake to extract and mix thoroughly, filter and collect the fifth filter residue and the third filtrate;

[0013] Step 7: Mix the fifth filter residue with 4M KOH alkaline solution, shake to extract and mix thoroughly, filter and collect the sixth filter residue and the fourth filtrate;

[0014] Step 8: Dialyze the first, second, third and fourth filtrates, collect the permeate and freeze-dry it to obtain Pu-erh tea cell wall polysaccharides.

[0015] Further, step 2, mixing the Pu-erh tea powder with water and alcohol solution sequentially to remove water-soluble polysaccharides and proteins from the Pu-erh tea powder, filtering and collecting the first filter residue, includes the following steps:

[0016] Step 2.1: Mix the dried Pu-erh tea powder with distilled water at 36-37℃ for 1-1.5 hours, filter and collect the water extraction residue;

[0017] Step 2.2: Mix the water extraction residue with a 70% ethanol solution for 12-16 hours, filter and collect the ethanol extraction residue;

[0018] Step 2.3: Wash and dry the alcohol extraction filter residue with anhydrous ethanol to obtain the first filter residue.

[0019] Further, step 3, mixing the first filter residue with amylase to remove the starch from the first filter residue, filtering and collecting the second filter residue, specifically includes:

[0020] Dissolve the amylase in Tris-maleic acid buffer, mix the first filter residue obtained in step 2 with the Tris-maleic acid buffer containing dissolved amylase, and react at 37-40℃ for 2-4 hours. After the reaction is completed, filter and collect the second filter residue.

[0021] Furthermore, the mass ratio of the second filter residue to the ethylenediaminetetraacetic acid solution is 1:(20-25); the mass ratio of the third filter residue to the saturated sodium carbonate solution is 1:(20-25); the mass ratio of the fourth filter residue to the 1M KOH alkaline solution is 1:(20-25); and the mass ratio of the fifth filter residue to the 4M KOH alkaline solution is 1:(20-25).

[0022] Furthermore, the mixing time of the second filter residue with the ethylenediaminetetraacetic acid solution is 10-14 hours; the mixing time of the third filter residue with the saturated sodium carbonate solution is 10-14 hours; the mixing time of the fourth filter residue with the 1M KOH alkaline solution is 10-14 hours; and the mixing time of the fifth filter residue with the 4M KOH alkaline solution is 10-14 hours.

[0023] Further, the first, second, third, and fourth filtrates were dialyzed, and the permeate was collected and freeze-dried to obtain Pu-erh tea cell wall polysaccharides, specifically including the following steps:

[0024] Step 8.1: Dialyze the first, second, third and fourth filtrates for 44-48 hours. The molecular weight of the dialysis membrane is 8000kDa. Collect the permeate.

[0025] Step 8.2: Freeze the permeate at -70℃ to -80℃ for 6-12 hours, and then put the frozen permeate into a vacuum freeze-drying chamber. The cold trap temperature is -40℃±2℃ and the vacuum degree is -30KPa to -60KPa to obtain Pu-erh tea cell wall polysaccharides.

[0026] This invention also provides the application of cell wall polysaccharide in alleviating chemotherapy-induced granulocytopenia, characterized in that it has a relieving effect on granulocytopenia caused by chemotherapy and related inflammations such as fever and infection.

[0027] Furthermore, the chemotherapy drug is cyclophosphamide.

[0028] Further, the method includes the following steps: Step 1: Using the extracted Pu-erh tea cell wall polysaccharide as a sample, stimulate RAW264.7 cells and detect the release of immune active factors;

[0029] Step 2: Using the extracted Pu-erh tea cell wall polysaccharide as a sample, RAW264.7 cells were stimulated and total cell protein was extracted. The expression level of SOCS1 protein was detected by Western blotting.

[0030] Step 3: Using the extracted Pu-erh tea cell wall polysaccharides as a sample, treat BALB / c mice with granulocytopenia and evaluate the recovery of the model mice.

[0031] Furthermore, the concentration of cell wall polysaccharides in Pu-erh tea was 50 mg / kg.

[0032] Technical effect

[0033] This invention discloses a polysaccharide extracted from Pu-erh tea and its application in alleviating chemotherapy side effects. First, water-soluble polysaccharides and proteins, along with other macromolecules, are removed from the Pu-erh tea. Second, insoluble residues are further treated with a certain amount of amylase to remove starch. Next, acidic polysaccharides are extracted using gradient extraction with different solutions. When testing the immunomodulatory activity of different extracted Pu-erh tea polysaccharides, the tea polysaccharide extracted with ethylenediaminetetraacetic acid (EDTA) solution showed a strong promoting effect on the release of G-CSF factor from cells. This Pu-erh tea polysaccharide significantly alleviates granulocytopenia induced by chemotherapy. At a concentration of 50 mg / kg, the number of neutrophils in mice with granulocytopenia significantly increased, demonstrating a significant effect in alleviating chemotherapy-induced granulocytopenia. Furthermore, the tea polysaccharide showed a significant upregulation of SOCS1 protein expression in Western blotting experiments. This protein can negatively regulate the abnormally activated JAK / STAT signaling pathway in diseases such as tumors. This invention can be used to develop and prepare health care products to alleviate chemotherapy-induced granulocytopenia, which are expected to improve the quality of life of cancer patients and assist in cancer treatment.

[0034] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0035] Figure 1 The effect of tea polysaccharides extracted in Example 1 on G-CSF factor released by RAW264.7 cells was tested using the method in Example 2.

[0036] Figure 2 The effect of tea polysaccharides extracted with ethylenediaminetetraacetic acid in Example 1 on SOCS1 protein expression in RAW264.7 cells was tested using the method in Example 3.

[0037] Figure 3 The tea polysaccharide extracted with ethylenediaminetetraacetic acid in Example 1 was tested for its therapeutic effect on spleen tissue in a granulocytopenia model using the method in Example 4.

[0038] Figure 4 The tea polysaccharide extracted with ethylenediaminetetraacetic acid in Example 1 was tested using the method in Example 4 to treat the reduction in the number of neutrophils in a granulocytopenia model. Detailed Implementation

[0039] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0040] In the following description, specific details, such as particular internal procedures and techniques, are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will appreciate that the invention may be practiced in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods have been omitted so as not to obscure the description of the invention with unnecessary detail.

[0041] Example 1

[0042] This embodiment provides a method for extracting cell wall polysaccharides from Pu-erh tea, including the following steps:

[0043] Step 1: Pre-treatment of Pu-erh tea leaves: Crush the Pu-erh tea leaves, sift them, make Pu-erh tea powder, and seal and dry it for storage;

[0044] Step 2: Mix the Pu-erh tea powder with water and alcohol solution sequentially to remove water-soluble polysaccharides and proteins from the Pu-erh tea powder, filter and collect the first filter residue; specifically including:

[0045] Step 2.1: Dissolve 20g of the above tea powder in 100-200mL of distilled water, place on a shaker at 37℃ and shake for 1 hour, filter with double gauze and double filter paper, discard the filtrate and take the filter residue.

[0046] Step 2.2: Add 100-200 mL of 70% ethanol to the filter residue of the above samples, shake on a shaker at 37°C overnight, filter the next day, wash the filter residue thoroughly with anhydrous ethanol 4-5 times, filter again, and place the filter residue in a fume hood to dry until there is no alcohol odor.

[0047] Step 3: Take 10g of the dried sample above, add 20mg of amylase, and react in 20mM Tris-maleic acid buffer at 37℃ for 2-4 hours to fully dissolve the starch. Wash the filter residue after the reaction several times with distilled water to ensure that the starch is completely removed. After filtration, dry the filter residue at room temperature and weigh it to measure the weight change after removing the starch.

[0048] Step 4: Mix the filter residue obtained after starch removal and drying with an ethylenediaminetetraacetic acid solution with a pH of 6-7 at a mass ratio of 1:20, place it on a shaker and shake for 10 hours to ensure that the solution fully chelates and changes the cross-linking structure of the cell wall. After the treatment, filter it with double-layer gauze and double-layer filter paper. Dry the filter residue at room temperature. Dialyze the filtrate with a dialysis membrane with a molecular weight of 8000kDa for 48 hours.

[0049] Step 5: Mix the filter residue obtained after filtration in Step 4 with saturated sodium carbonate solution at a weight ratio of 1:20, place on a shaker and shake for 10 hours to ensure full extraction. After the treatment, filter with double-layer gauze and double-layer filter paper. Dry the filter residue at room temperature. Dialyze the filtrate with a dialysis membrane with a molecular weight of 8000kDa for 48 hours.

[0050] Step 6: Mix the filter residue obtained after filtration in Step 5 with 1M KOH solution at a weight ratio of 1:20, place on a shaker and shake for 10 hours to ensure full extraction. After the treatment, filter with double-layer gauze and double-layer filter paper. Dry the filter residue at room temperature. Dialyze the filtrate with a dialysis membrane with a molecular weight of 8000kDa for 48 hours.

[0051] Step 7: Mix the filter residue obtained after filtration in Step 6 with 4M KOH solution at a weight ratio of 1:20, place on a shaker and shake for 10 hours to ensure full extraction. After the treatment, filter with double-layer gauze and double-layer filter paper. Dry the filter residue at room temperature. Dialyze the filtrate with a dialysis membrane with a molecular weight of 8000kDa for 48 hours.

[0052] Step 8: Dialyze the first, second, third, and fourth filtrates for 44-48 hours. The molecular weight of the dialysis membrane is 8000 kDa. Collect the permeate. Place the concentrated liquid obtained after dialysis in steps 4, 5, 6, and 7 into an ultra-low temperature freezer and freeze it at -70℃ to -80℃ for 6 hours. Place the frozen concentrated liquid into a vacuum freeze-drying chamber with a cold trap temperature of -40℃ ± 2℃ and a vacuum degree of -30 kPa to -60 kPa to obtain a dry flocculent substance, which is a cell wall polysaccharide with biological immune activity.

[0053] Example 2:

[0054] An experiment was conducted to stimulate the release of cellular immune factors using the cell wall polysaccharide obtained in Example 1.

[0055] Step 1: Add 10% fetal bovine serum (FBS), 1% penicillin (100 μg / mL), and streptomycin (100 μg / mL) to DMEM high glucose medium as the culture medium for culturing RAW264.7 mouse mononuclear macrophages;

[0056] Step 2: Culture RAW264.7 cells in the culture medium from Step 1 and collect the cells at 9*103 after the cell density is sufficient. 4 Cell density per well: Cells were colonized in 96-well plates and adhered for 12 hours.

[0057] Step 3: When the cells in each well of the 96-well plate reach a density of 80%-90% in Step 2, discard the old culture medium and replace it with a new culture medium containing tea polysaccharides. The new culture medium containing tea polysaccharides is prepared as follows: the tea polysaccharides extracted in Example 1 are prepared with serum-free DMEM culture medium to a concentration of 20 μg / mL or 100 μg / mL. ConA (concanavalin A, 2 μg / mL) is used as a negative control, and lipopolysaccharide (1 μg / mL) is used as a positive control. After stimulating culture for 24 hours, the culture supernatant is collected for the determination of granulocyte colony-stimulating factor (G-CSF) release.

[0058] Granulocyte colony-stimulating factor (G-CSF) was the first myeloid growth factor approved by the FDA. Its main function is as supportive therapy after chemotherapy, reducing the severity and duration of chemotherapy-induced granulocytopenia and decreasing the incidence of fever due to granulocytopenia. Both in vivo and in vitro experiments have confirmed that G-CSF can activate granulocyte function, activate the respiratory burst of neutrophils, enhance their chemotaxis, and improve their phagocytic function.

[0059] Test results as follows Figure 1As shown, the four types of Pu-erh tea cell wall polysaccharides extracted using different solutions all exhibited a promoting effect on the immunomodulation of mononuclear macrophages in RAW264.7 mice. Among them, the cell wall polysaccharides extracted using ethylenediaminetetraacetic acid solution showed the strongest ability, increasing the production of G-CSF by several hundred times compared with the control group. This provides immunomodulatory function in the innate immune response and subsequently protects the host from pathogenic infectious agents.

[0060] Example 3

[0061] Experiments were conducted to stimulate cell protein expression using the cell wall polysaccharide obtained in Example 1:

[0062] Step 1: The cell culture medium is the same as in Step 1 of Example 1;

[0063] Step 2: Culture RAW264.7 cells in the culture medium from Step 1 and collect cells at 1.5*102 after the cell density is sufficient. 6 Cell density per well: Cells were colonized in 6-well plates and adhered for 12 hours.

[0064] Step 3: When the cells in each well of the 6-well plate reach a density of 80%-90% in Step 2, discard the old culture medium and replace it with a new culture medium containing tea polysaccharides. The new culture medium containing tea polysaccharides is prepared by using the tea polysaccharides extracted in Example 1 to a concentration of 20 μg / mL or 100 μg / mL in serum-free DMEM culture medium. Lipopolysaccharide (1 μg / mL) is used as a positive control, and the cells are stimulated for 24 hours.

[0065] Step 4: After 24 hours, discard the old culture medium from Step 3. Add 150 μL of lysis buffer to each well of cells to lyse the cells and collect total cell protein. During this process, place the cells on ice for a total of 5-8 minutes for lysis. Then, collect the cells and transfer them into EP tubes containing a small amount of glass beads. Place the tubes on ice for another 25 minutes, vortexing 5 times for 20 seconds each time. After that, place the tubes in a refrigerated centrifuge and centrifuge at 12,000 rpm at 4°C for 25 minutes.

[0066] Step 5: After centrifugation, aspirate the supernatant into a new EP tube and use the kit to quantify the protein. Calculate the protein loading amount based on the absorbance. If the protein amount is X μL (generally around 30 μg), add 18-X μL of 1×PBS and 6 μL of solution A (4×SDS:DTT = 10:1). Mix the mixture thoroughly and denature at 95°C for 8 min.

[0067] Step 6: Add the protein loading solution prepared in step 5 to the pre-prepared protein gel and perform SDS-PAGE (protein electrophoresis). The electrophoresis conditions are 80V for about 30 minutes for the stacking gel. After running through the stacking gel, switch to 120V and wait for the 4XSDS to reach the bottom.

[0068] Step 7, Gel Cutting and Transfer: Cut the filter paper, sponge plate, and 0.2μm PVDF membrane specifically for transfer. Prepare a transfer buffer containing 20% ​​methanol. Remove the gel glass plate after electrophoresis and cut the gel in the region containing the target protein. On the semi-dry transfer apparatus, place each layer in the following order: sponge plate, filter paper, PVDF membrane, gel, filter paper, sponge plate. After each step, gently roll a glass tube dipped in transfer buffer to remove air bubbles. Blot up excess transfer buffer with a paper towel, close the semi-dry transfer apparatus lid, and set the transfer apparatus conditions to 0.3A, 20V, 60min.

[0069] Step 8, Milk Sealing: Wash the stained PVDF membrane with TBST until completely decolorized, at room temperature.

[0070] The membrane was incubated in blocking buffer (1.25 g of skim milk powder dissolved in 25 mL of PBST) and blocked on a horizontal shaker at 60 rpm for 1 h.

[0071] Step 9, Primary antibody incubation: After blocking, wash the PVDF membrane with TBST until the liquid is clear, add the diluted primary antibody (primary antibody: PBST = 1:500), and incubate overnight at 4°C.

[0072] Step 10, Secondary antibody incubation: Recover the primary antibody, wash the PVDF membrane 5 times with TBST at 90 rpm for 5 min each time, and after washing, add the diluted secondary antibody (secondary antibody: PBST = 1:2000), 30 rpm, and incubate in the dark for 1 h.

[0073] Step 11, Colorimetric Imaging: ECL Luminescence: Recover the secondary antibody, wash the PVDF membrane 5 times with TBST at 90 rpm for 5 min each time. Add ECL staining agent (1.5 mL of solution A + 1.5 mL of solution B), react in the dark for 5 min, expose using a two-color laser imager, and analyze the grayscale using ImageJ.

[0074] Test results as follows Figure 1-2 As shown, the expression level of SOCS1 protein in RAW264.7 mouse monocytes and macrophages was significantly increased after the cell wall polysaccharide extracted by treatment with ethylenediaminetetraacetic acid solution was stimulated. Related studies have shown that the JAK-STAT signaling pathway is generally continuously activated in leukemia cells and tumor cells. The main negative feedback regulators of JAK-STAT are cytokine signaling inhibitors (SOCSs). The SOCS family is the main signaling molecule that weakens the JAK-STAT pathway, and SOCS1 protein plays an important role in this process.

[0075] Example 4

[0076] Experiments were conducted on mice with granulocytopenia using the cell wall polysaccharide obtained in Example 1:

[0077] In the experiment of Example 2, we previously found that treating the extracted Pu-erh tea cell wall polysaccharides with ethylenediaminetetraacetic acid solution significantly increased the release of G-CSF factor by several hundred times compared to the normal group. G-CSF plays a significant role in promoting granulocyte release. Therefore, we wondered if Pu-erh tea cell wall polysaccharides could alleviate granulocytopenia following chemotherapy. To verify this hypothesis, we treated mice with granulocytopenia using cell wall polysaccharides, specifically including the following steps:

[0078] Step 1: Place 8-week-old female BALB / c mice in a suitable temperature environment and provide them with sufficient water and food for one week to allow them to adapt to the environment.

[0079] Step 2: One week later, the mice from Step 1 were divided into four groups: normal group, model group, positive group, and polysaccharide group, with three mice in each group. Before the experiment, the mice were weighed to record their body weight, and the dosage was determined based on each mouse's weight. The dosage of cyclophosphamide was 100 mg / kg, levamisole was 25 mg / kg, and tea polysaccharide was 50 mg / kg. After determining the dosage, the mice in the model group, positive group, and polysaccharide group were intraperitoneally injected with 100 mg / kg of cyclophosphamide solution for three consecutive days to induce granulocytopenia. The normal group was injected with the same amount of physiological saline as a control.

[0080] Step 3: After successfully establishing the model in mice after three days of treatment, the normal group and the model group continued to be injected with physiological saline intraperitoneally for four days. The positive group was treated with levamisole by gavage at a dose of 25 mg / kg, and the polysaccharide group was treated with tea polysaccharide by intraperitoneal injection at a dose of 50 mg / kg.

[0081] Step 4: After 7 days, blood samples were collected from each group of experimental mice for testing. Mice were then euthanized by cervical dislocation, and their spleens and livers were harvested, weighed, embedded, sectioned, stained with hematoxylin and eosin (HE), and photographed to observe the tissue lesions.

[0082] Test results as follows Figure 4 As shown, the cell wall polysaccharides extracted by treatment with ethylenediaminetetraacetic acid solution exhibited a significant effect in treating granulocyte model mice. Pu-erh tea cell wall polysaccharides increased the number of neutrophils in mice with a cyclophosphamide-induced granulocytopenia, and also... Figure 3 The tissue sections also clearly show that, in Figure b, the spleen sections of the model group mice show an unclear boundary between the red and white pulp, while in Figure a, the spleen sections of the normal group mice show a clear boundary between the red and white pulp. At the same time, the spleen sections of the positive treatment group (Figure c) and the polysaccharide treatment group (Figure d) show obvious therapeutic effects, with the boundary between the red and white pulp becoming clear and obvious.

[0083] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of cell wall polysaccharides extracted from Pu-erh tea in the preparation of drugs to alleviate chemotherapy-induced granulocytopenia, characterized in that... The extraction method of cell wall polysaccharides from Pu-erh tea includes the following steps: Step 1: Grind the Pu-erh tea into powder to obtain dry Pu-erh tea powder; Step 2: Mix the dried Pu-erh tea powder with water and alcohol solution in sequence to remove water-soluble polysaccharides and proteins from the dried Pu-erh tea powder, filter and collect the first filter residue; Step 3: Mix the first filter residue with amylase to remove the starch from the first filter residue, filter and collect to obtain the second filter residue; Step 4: Under pH 6-7 conditions, the second filter residue is mixed with ethylenediaminetetraacetic acid solution to change the cross-linking structure of the cell wall in Pu-erh tea cells. The first filtrate and the third filter residue are then collected by filtration. Specifically, the second filter residue is mixed with ethylenediaminetetraacetic acid solution at pH 6-7 at a mass ratio of 1:20 and placed on a shaker for 10 hours to ensure that the solution is fully chelated and the cross-linking structure of the cell wall is changed. After the treatment, the mixture is filtered with double-layer gauze and double-layer filter paper. The filter residue is dried at room temperature, and the filtrate is dialyzed with a dialysis membrane with a molecular weight of 8000kDa for 48 hours. Step 5: Dialyze the first filtrate, freeze it at -70℃ to -80℃ for 6 hours, dry the frozen solid in a freeze dryer, and obtain the Pu-erh tea cell wall polysaccharide after freeze-drying.

2. The application of cell wall polysaccharides extracted from Pu-erh tea as described in claim 1 in the preparation of a drug for alleviating chemotherapy-induced granulocytopenia, characterized in that... Step 2: The dried Pu-erh tea powder is mixed sequentially with water and alcohol solution to remove water-soluble polysaccharides and proteins from the dried Pu-erh tea powder. The mixture is then filtered and the first filter residue is collected. This includes the following steps: Step 2.1: At 36-37℃, mix the dried Pu-erh tea powder with distilled water for 1-1.5 h, filter and collect the water extraction residue; Step 2.2: Mix the water extraction residue with a 70% ethanol solution for 12-16 h, filter and collect the ethanol extraction residue; Step 2.3: Wash and dry the alcohol extraction filter residue with anhydrous ethanol to obtain the first filter residue.

3. The application of cell wall polysaccharides extracted from Pu-erh tea as described in claim 1 in the preparation of a drug for alleviating chemotherapy-induced granulocytopenia, characterized in that... Step 3: Mix the first filter residue with amylase to remove the starch from the first filter residue, filter and collect the second filter residue, specifically including: Dissolve the amylase in Tris-maleic acid buffer, mix the first filter residue obtained in step 2 with the Tris-maleic acid buffer containing the dissolved amylase, and react at 37-40℃ for 2-4 h. After the reaction is completed, filter and collect the second filter residue.

4. The application of cell wall polysaccharides extracted from Pu-erh tea as described in claim 1 in the preparation of a drug for alleviating chemotherapy-induced granulocytopenia, characterized in that... The mass ratio of the second filter residue to the ethylenediaminetetraacetic acid solution is 1:(20-25).

5. The application of cell wall polysaccharides extracted from Pu-erh tea as described in claim 1 in the preparation of a drug for alleviating chemotherapy-induced granulocytopenia, characterized in that... The mixing time between the second filter residue and the ethylenediaminetetraacetic acid solution is 10-14 hours.

6. The application of the cell wall polysaccharide extracted from Pu-erh tea as described in claim 1 in the preparation of a drug for alleviating chemotherapy-induced granulocytopenia, characterized in that... The chemotherapy drug is cyclophosphamide.

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