A cell wall polysaccharide extracted from pu'er tea and its extraction method and application

Abstract

The present application provides a kind of cell wall polysaccharide extracted from Pu'er tea and its extraction method and application.The first aspect of the present application provides a kind of method for extracting cell wall polysaccharide from Pu'er tea, first remove water-soluble polysaccharide and macromolecular substances such as protein;Second, a certain amount of amylase is used to further process the non-water-soluble residue to remove starch;Then, ethylenediaminetetraacetic acid combined with alkali solution is used to extract acidic polysaccharide, and it is found through test that the cell wall polysaccharide extracted by the method has immunoregulatory effect, and the activity is higher than that of polysaccharide extracted by traditional method.Therefore, by the method provided by the present application, the cell wall polysaccharide in Pu'er tea is developed and utilized, which helps to improve the industrial added value, broaden the product category, has higher application prospect and potential value.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of cell wall polysaccharide extracted from Pu'er tea and its extraction method and application, relate to the technical field of tea polysaccharide. BACKGROUND

[0002] Polysaccharide is a kind of natural macromolecular complex widely existing in animals, higher plants and microorganisms, and is one of important substances constituting life. Since people found that fungal polysaccharide has anticancer effect in the 1950s, the scientific community has carried out in-depth research on polysaccharide, including separation and extraction, physical and chemical properties, biological activity, molecular structure and the like. Studies have shown that polysaccharide is not only an important substance constituting cells, but also has good biological activity function, including immune regulation, antioxidant, antitumor, blood sugar reduction, intestinal flora regulation and the like. Tea polysaccharide (TPS) is a large class of plant polysaccharide, and is a very important macromolecular active substance in tea. In a natural state, tea polysaccharide mainly exists in the form of glycoconjugate, that is, various monosaccharides are polymerized and then combined with protein, nucleic acid, uronic acid, inorganic element and the like to form a large complex. Tea polysaccharide, like polysaccharide in a broad sense, has also been confirmed to have biological activities such as antioxidant, immune enhancement, blood sugar reduction, anticancer and antibacterial.

[0003] Pectin mainly exists in the primary cell wall and between cells of plants, and is a matrix polysaccharide of cell wall. From the structural point of view, pectin includes two kinds of acidic polysaccharides: polygalacturonic acid and polymuramic acid, and the number, type, connection mode and presence of other substituents of residues in the side chain of each polysaccharide vary considerably depending on the plant source, tissue and development stage.

[0004] Pu'er tea, as a kind of tea, has effects of antioxidant, anti-mutation, antibacterial, antiviral, antitumor, cholesterol reduction, obesity prevention, blood sugar reduction and anti-allergy. Studies have shown that the cell wall of Pu'er tea contains a variety of acidic polysaccharides with immunological activity. Conventional water extraction process is difficult to extract pectin bound by cellulose in the cell wall, and therefore the water extraction residue is usually used as fertilizer or animal feed without realizing its value. Therefore, how to extract acidic polysaccharides between the cell walls of Pu'er tea has attracted widespread attention in the field. SUMMARY

[0005] The present application provides an extraction method for extracting cell wall polysaccharide from Pu'er tea, which is used for extracting acidic polysaccharides between the cell walls of Pu'er tea.

[0006] The present application also provides the cell wall polysaccharide extracted from Pu'er tea as described above and its application in improving immunological activity.

[0007] The first aspect of the present application provides a method for extracting cell wall polysaccharide from Pu'er tea, comprising the following steps:

[0008] Step 1, crushing Pu'er tea to obtain Pu'er tea dry powder;

[0009] Step 2, sequentially mixing the Pu'er tea dry powder with water and an alcohol solution to remove water-soluble polysaccharides and proteins in the Pu'er tea dry powder, filtering and collecting to obtain a first filter residue;

[0010] Step 3, mixing the first filter residue with amylase to remove starch in the first filter residue, filtering and collecting to obtain a second filter residue;

[0011] Step 4, mixing the second filter residue with an ethylenediaminetetraacetic acid solution under the condition that the pH is 6-7, and assisting with ultrasonic treatment during the mixing process to change the cross-linking structure of the cell wall in the Pu'er tea cells, filtering and collecting to obtain a third filter residue and a first filtrate;

[0012] Step 5, mixing the third filter residue with an alkaline solution, and assisting with ultrasonic treatment during the mixing process, filtering and collecting to obtain a fourth filter residue and a second filtrate;

[0013] Step 6, mixing the first filtrate and the second filtrate and performing dialysis, collecting the dialysate and freeze-drying to obtain the cell wall polysaccharide.

[0014] Further, step 2 specifically comprises the following steps:

[0015] Step 2.1, mixing the Pu'er tea dry powder with distilled water at 36-37℃ for 1-1.5 hours, filtering and collecting to obtain a water extraction filter residue;

[0016] Step 2.2, mixing the water extraction filter residue with a 70% ethanol solution for 12-16 hours, filtering and collecting an alcohol extraction filter residue;

[0017] Step 2.3, washing and drying the alcohol extraction filter residue using anhydrous ethanol to obtain the first filter residue.

[0018] Further, step 3 specifically comprises the following steps:

[0019] Dissolving amylase in a Tris-maleic acid buffer, mixing the first filter residue obtained in step 2 with the Tris-maleic acid buffer in which the amylase is dissolved, and reacting at 37-40℃ for 2-4h, after the reaction is completed, filtering and collecting to obtain a second filter residue.

[0020] Further, the mass ratio of the second filter residue to the ethylenediaminetetraacetic acid solution is 1: (20-25).

[0021] Further, the mixing time of the second residue and the ethylenediamine tetraacetic acid solution is 10-14 hours, and the mixture is placed in an ultrasonic water bath for ultrasonic treatment for 30-40 minutes every 1 hour during the mixing.

[0022] Further, the mass ratio of the third residue and the alkaline solution is 1: (30-35).

[0023] Further, the mixing time of the third residue and the alkaline solution is 10-14 hours, and the mixture is placed in an ultrasonic water bath for ultrasonic treatment for 30 minutes every 1 hour during the mixing.

[0024] Further, step 6 specifically comprises the following steps:

[0025] Step 6.1, the first filtrate and the second filtrate are dialyzed using a dialysis membrane with a molecular weight of 8000 kDa for 44-48 hours, and the permeate is collected;

[0026] Step 6.2, the permeate is frozen at-20℃~-35℃ for 2-6 hours, and then the frozen permeate is placed in a vacuum freeze-drying chamber, the cold trap temperature is-40℃±2℃, and the vacuum degree is-30KPa~-60KPa, to obtain the cell wall polysaccharide.

[0027] The second aspect of the present application provides the cell wall polysaccharide prepared by the extraction method.

[0028] The third aspect of the present application provides the application of the cell wall polysaccharide in the preparation of a medicine with immunological activity.

[0029] The method provided by the present application first removes water-soluble polysaccharides and macromolecular substances such as proteins, improves the purity of the acidic polysaccharides, and avoids a complex purification process; secondly, a certain amount of amylase is used to further treat the non-water-soluble residue to remove starch, effectively avoiding the interference of starch in the extraction of polysaccharides and ensuring the purity of the active polysaccharides; then, ethylenediamine tetraacetic acid and an alkaline solution are used to extract the acidic polysaccharides, the ethylenediamine tetraacetic acid solution can chelate metal ions in the cell wall structure, change the cross-linking structure of the cell wall, extract the active polysaccharides in the cell wall without destroying the original structure of the polysaccharides, and the alkaline solution (KOH) can effectively destroy the H bonds between the heteroxylan and the cellulose microfibers.

[0030] It is found through tests that the cell wall polysaccharide extracted by the method has an immunomodulatory effect, and has higher activity than polysaccharide extracted by a traditional method; finally, the liquid polysaccharide is concentrated into a solid by using a freeze-drying technology, the stability of the polysaccharide is increased, storage and transportation are facilitated, and the polysaccharide is added to various dosage forms, so that practical application is more convenient. In summary, the method provided by the application develops and utilizes the cell wall polysaccharide in the cell wall of Pu'er tea, helps to improve the industrial added value, broaden the product types, has a high application prospect and potential value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Fig. 1 Effect of tea polysaccharide extracted for example 1 and comparative examples 1-4 on G-CSF factor released by RAW264.7 cells;

[0032] Fig. 2 Effect of tea polysaccharide extracted for example 1 and comparative examples 1-4 on IL-1α factor released by RAW264.7 cells;

[0033] Fig. 3 Effect of tea polysaccharide extracted for example 1 and comparative examples 1-4 on NO released by RAW264.7 cells. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0035] Example 1

[0036] The present embodiment provides a method for extracting cell wall polysaccharide from Pu'er tea, comprising the following steps:

[0037] Step 1, Pu'er tea leaf pretreatment: crushing Pu'er tea leaves in three periods of fermentation, sieving, and preparing Pu'er tea dry powder, and sealing and dry storing;

[0038] Step 2.1, 20 g of tea powder in each of the three periods is dissolved in 100-200 mL of distilled water, and placed on a shaking bed at 37℃ for 1 hour of shaking and shaking, and then filtered with double-layer gauze and double-layer filter paper, and the filtrate is discarded and the filter residue is taken;

[0039] Step 2.2, 100-200 mL of 70% ethanol was added to the residue of the above three samples, respectively, and shaken on a shaking table at 37°C overnight. The next day, the residue was filtered and washed with anhydrous ethanol for 4-5 times. After filtration, the residue was dried in a fume hood until there was no alcohol smell.

[0040] Step 3, 10 g of the above dried sample was added with 20 mg of amylase and reacted in 20 mM Tris-maleic acid buffer at 37°C for 2-4 hours to fully dissolve the starch. The residue after reaction was washed several times with distilled water to ensure that the starch was completely removed. After filtration, the residue was dried at room temperature. After drying, the weight change after the removal of starch was measured.

[0041] Step 4, the residue obtained after drying after the removal of starch was mixed with ethylenediaminetetraacetic acid solution with pH at 6-7 at a mass ratio of 1:20, and shaken on a shaking table for 10 hours to ensure that the solution was fully chelated to change the cross-linking structure of the cell wall. During the shaking, it was placed in an ultrasonic water bath every 1 hour for 30 minutes. After the treatment was completed, it was filtered with double-layer gauze and double-layer filter paper, and the residue was dried at room temperature. The filtrate was dialyzed for 48 hours with a dialysis membrane with a molecular weight of 8000 kDa.

[0042] Step 5, the dried residue obtained above was mixed with 1M KOH solution at a mass ratio of 1:30, and shaken on a shaking table for 10 hours to ensure that the alkali solution (KOH) could effectively destroy the H bonds between the xylan and the cellulose microfibers, and was used for efficient extraction of xyloglucan and glucosamine hydrochloride. During the shaking, it was placed in an ultrasonic water bath every 1 hour for 30 minutes. After the treatment was completed, it was filtered with double-layer gauze and double-layer filter paper, and the residue was dried at room temperature. The filtrate was dialyzed for 48 hours with a dialysis membrane with a molecular weight of 8000 kDa.

[0043] Step 6, the concentrated liquid obtained after dialysis in steps 4-5 was placed in a quick-freezing freezer at -20°C to -35°C and frozen for 2 hours. The frozen concentrate was placed in a vacuum freeze-drying chamber with a cold trap temperature of -40°C±2°C and a vacuum degree of -30KPa to obtain dried flocculent material, which was a cell wall polysaccharide with biological immune activity.

[0044] Comparative Example 1

[0045] This comparative example provides a method for extracting polysaccharides from Pu'er tea, comprising the following steps:

[0046] Step 1, Pu'er tea pretreatment: The Pu'er tea leaves of the three periods before, during and after fermentation were crushed, sieved and made into Pu'er tea dry powder, which was sealed and dried for storage.

[0047] Step 2, 20 g of tea powder of each of the three periods was dissolved in 100-200 mL of distilled water, and placed in a shaking bed at 37°C for shaking for 1 hour, then placed in a boiling water bath at 95-100°C for extraction for 2 hours. During the treatment, the sample was placed in an ultrasonic water bath for ultrasonic treatment for 30 minutes every 1 hour. After the treatment, the filtrate was collected after filtration with double-layer gauze and double-layer filter paper;

[0048] Step 3, the filtrate was dialyzed for 48 hours, and the concentrated liquid obtained after dialysis was placed in a quick-freezing library at -20°C to -35°C for freezing for 2 hours. The frozen concentrated liquid was placed in a vacuum freeze-drying chamber, the cold trap temperature was -40°C±2°C, and the vacuum degree was -30KPa to -60KPa, to obtain the hot water extraction product of Pu'er tea.

[0049] Comparative Example 2

[0050] The present comparative example provides a method for extracting polysaccharides from Pu'er tea, comprising the following steps:

[0051] Step 1, Pu'er tea leaf pretreatment: Pu'er tea leaves of three periods before, during and after fermentation were crushed, sieved, and made into Pu'er tea dry powder, which was sealed and dried for storage;

[0052] Step 2, 20 g of tea powder of each of the three periods was dissolved in 100-200 mL of distilled water, and placed in a shaking bed at 37°C for shaking for 1 hour, then placed in a boiling water bath at 95-100°C for extraction for 2 hours. During the treatment, the sample was placed in an ultrasonic water bath for ultrasonic treatment for 30 minutes every 1 hour. After the treatment, the filtrate was collected after filtration with double-layer gauze and double-layer filter paper;

[0053] Step 3, the filtrate was dialyzed for 48 hours, and the concentrated liquid obtained after dialysis was placed in a quick-freezing library at -20°C to -35°C for freezing for 2 hours. The frozen concentrated liquid was placed in a vacuum freeze-drying chamber, the cold trap temperature was -40°C±2°C, and the vacuum degree was -30KPa to -60KPa, to obtain the hot water extraction product of Pu'er tea.

[0054] Comparative Example 3

[0055] The present comparative example provides a method for extracting polysaccharides from Pu'er tea, comprising the following steps:

[0056] Step 1, Pu'er tea leaf pretreatment: Pu'er tea leaves of three periods before, during and after fermentation were crushed, sieved, and made into Pu'er tea dry powder, which was sealed and dried for storage;

[0057] Step 2, 20 g of tea powder of each of the three periods was dissolved in 100-200 mL of distilled water, and placed in a shaking bed at 37°C for shaking for 1 hour, then placed in a boiling water bath at 95-100°C for extraction for 2 hours. During the treatment, the sample was placed in an ultrasonic water bath for ultrasonic treatment for 30 minutes every 1 hour. After the treatment, the filtrate was collected after filtration with double-layer gauze and double-layer filter paper;

[0058] Step 3, the filtrate is dialyzed for 48 hours, and the concentrated liquid obtained after dialysis is placed in a quick freezer at -20℃ to -35℃ for 2 hours, and the frozen concentrated liquid is placed in a vacuum freeze-drying chamber with a cold trap temperature of -40℃±2℃ and a vacuum degree of -30KPa to -60KPa to obtain the ultrasonic extraction product of Pu'er tea.

[0059] Comparative Example 4

[0060] Step 1, Pu'er tea leaf pretreatment: the Pu'er tea leaves of the three periods before, during and after fermentation are crushed, sieved and made into Pu'er tea dry powder, which is sealed and dried for storage;

[0061] Step 2, 20 g of tea powder of each of the three periods is dissolved in 100-200 mL of distilled water, and placed in a 37℃ shaking bed for 1 hour of shaking. After filtering with double-layer gauze and double-layer filter paper, the filtrate is discarded and the residue is dried in a fume hood.

[0062] Step 3, the dried residue obtained above is mixed with 1M KOH solution at a mass ratio of 1:30, and placed in a shaking bed for 10 hours of shaking. After treatment, it is filtered with double-layer gauze and double-layer filter paper, and the residue is dried at room temperature. The filtrate is dialyzed for 48 hours with a dialysis membrane with a molecular weight of 8000kDa;

[0063] Step 4, the concentrated liquid obtained after dialysis is placed in a quick freezer at -20℃ to -35℃ for 2 hours, and the frozen concentrated liquid is placed in a vacuum freeze-drying chamber with a cold trap temperature of -40℃±2℃ and a vacuum degree of -30KPa to -60KPa to obtain the alkaline solution extraction product of Pu'er tea.

[0064] Test Example 1

[0065] The components and contents of the cell wall polysaccharides extracted from Pu'er tea are tested, and the test results are shown in Table 1:

[0066] Table 1 Composition and content of tea polysaccharides prepared in Example 1 and Comparative Examples 1-4

[0067]

[0068] In Table 1, Rha represents rhamnose, Ara represents arabinose, GlcN represents glucosamine hydrochloride, Gal represents galactose, Glc represents glucose, Xyl represents xylose, Man represents mannose, GalA represents galacturonic acid, and ManA represents mannuronic acid. According to the results shown in Table 1, the cell wall polysaccharides extracted by the method of Example 1 contain a large amount of acidic polysaccharides with immunological activity, which is significantly improved compared with the water-soluble tea polysaccharides extracted by conventional hot water extraction.

[0069] Test Example 2

[0070] The cell wall polysaccharide obtained in Example 1 was subjected to an immune activity analysis experiment, including the following steps:

[0071] Step 1, 10% fetal bovine serum (FBS) and 1% penicillin (100 μg / mL) and streptomycin (100 μg / mL) were added to DMEM high-sugar medium as the culture medium for culturing RAW264.7 mouse mononuclear macrophages;

[0072] Step 2, RAW264.7 cells were cultured in the medium in step 1, and after the cell density was sufficient, the cells were collected to 1*10 5 The density of cells per well was 1*10

[0073] Step 3, the tea polysaccharides extracted from Example 1 and Comparative Example 1 were added to the culture medium at a concentration of 20 μg / mL or 100 μg / mL for culture. ConA (concanavalin, 2 μg / mL) was used as a negative control, and lipopolysaccharide (1 μg / mL) was used as a positive control. After 24 hours of culture, the culture supernatant was collected for determination of granulocyte colony-stimulating factor (G-CSF), interleukin-1 (IL-1), and nitric oxide (NO) release.

[0074] Granulocyte colony-stimulating factor (G-CSF) is the first myeloid growth factor approved by the FDA. Its main role is to support post-chemotherapy treatment, reduce the degree and duration of chemotherapy-induced granulocytopenia, and reduce the incidence of granulocytopenia fever. In vivo and in vitro experiments have confirmed that granulocyte colony-stimulating factor (G-CSF) can activate granulocyte function, activate neutrophil respiratory burst, enhance its chemotaxis, and improve its phagocytic function.

[0075] Interleukin-1 (IL-1) is a cytokine of the chemokine family. IL-1 family ligands include IL-1α, IL-1β, and IL-Ra. Interleukin-1 mainly plays an immune regulatory role, and its effects include ① synergistic effect with antigen, which can activate CD4+ T cells and express IL-2R; ② promotes B cell growth and differentiation, and can increase the number of hemolytic plaque (PFC) of spleen cells by 100 ③ promotes the antigen presentation ability of mononuclear-macrophage APC; ④ synergizes with IL-2 or interferon to enhance NK cell activity; ⑤ attracts neutrophils and causes the release of inflammatory mediators.

[0076] Nitric oxide (NO) is an autocrine and paracrine signaling molecule that can diffuse into biological membranes. NO participates in processes such as the immune defense system, neurotransmission, and angiogenesis. Downstream targets of NO include guanylate cyclase and NF-κB; the former can increase cGMP levels, while the latter is an important transcription factor expressed in the iNOS gene. Dysregulation of NO levels and signaling pathways is common in certain disease states. Diabetic patients have lower than global average NO levels, and atherosclerosis often leads to impaired NO signaling pathways; therefore, NO levels are of great significance for research on NO signaling pathways.

[0077] Test results are as follows Figs. 1-3 As shown, the cell wall polysaccharides extracted using EDTA combined with alkaline solution and ultrasonic treatment exhibited the greatest ability to modulate the immunity of RAW264.7 mouse mononuclear macrophages. Furthermore, the cell wall polysaccharides extracted using EDTA combined with alkaline solution and ultrasonic treatment yielded more acidic polysaccharides compared to conventional hot water extraction. These acidic polysaccharides possess immunomodulatory capabilities and can moderately activate macrophages. Our test results indicate that the cell wall polysaccharides extracted using EDTA combined with alkaline solution and ultrasonic treatment significantly promoted IL-1α secretion and G-CSF production, providing immunomodulatory functions in the innate immune response and subsequently protecting the host from pathogenic infectious agents.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of cell wall polysaccharides extracted from Pu'er tea in the preparation of medicines with immunocompetence, characterized in that, The extraction method of the cell wall polysaccharide comprises the following steps: Step 1, crushing Pu'er tea to obtain Pu'er tea dry powder; Step 2, sequentially mixing the Pu'er tea dry powder with water and an alcohol solution to remove water-soluble polysaccharides and proteins in the Pu'er tea dry powder, filtering and collecting to obtain first filter residue; Step 3, mixing the first filter residue with amylase to remove starch in the first filter residue, filtering and collecting to obtain second filter residue; Step 4, mixing the second filter residue with ethylenediaminetetraacetic acid solution under the condition that the pH is 6-7, and assisting with ultrasonic treatment during the mixing process to change the cross-linking structure of the cell wall in the Pu'er tea cells, filtering and collecting to obtain third filter residue and first filtrate; Step 5, mixing the third filter residue with an alkaline solution, and assisting with ultrasonic treatment during the mixing process, filtering and collecting to obtain fourth filter residue and second filtrate; Step 6, mixing the first filtrate and the second filtrate and performing dialysis, collecting the permeate and freeze-drying to obtain the cell wall polysaccharide; The mixing time of the second filter residue and the ethylenediaminetetraacetic acid solution is 10-14 hours, and the second filter residue is placed in an ultrasonic water bath for ultrasonic treatment for 30-40 minutes every 1 hour during the mixing process; The mixing time of the third filter residue and the alkaline solution is 10-14 hours, and the third filter residue is placed in an ultrasonic water bath for ultrasonic treatment for 30-40 minutes every 1 hour during the mixing process.

2. Use according to claim 1, characterized in that, Step 2 specifically comprises the following steps: Step 2.1, mixing the Pu'er tea dry powder with distilled water at 36-37 DEG C for 1-1.5 hours, filtering and collecting to obtain water extraction filter residue; Step 2.2, mixing the water extraction filter residue with a 70% ethanol solution for 12-16 hours, filtering and collecting alcohol extraction filter residue; Step 2.3, washing and drying the alcohol extraction filter residue using anhydrous ethanol to obtain first filter residue.

3. Use according to claim 1, characterized in that, Step 3 specifically comprises the following steps: Dissolving amylase in Tris-maleic acid buffer, mixing the first filter residue obtained in step 2 with the Tris-maleic acid buffer in which the amylase is dissolved, and reacting at 37-40 DEG C for 2-4 hours, after the reaction is completed, filtering and collecting to obtain second filter residue.

4. Use according to claim 1, characterized in that, The mass ratio of the second filter residue to the ethylenediaminetetraacetic acid solution is 1: (20-25).

5. The use according to claim 1, characterized in that, The mass ratio of the third filter residue to the alkaline solution is 1: (30-35).

6. The use according to claim 1, characterized in that, Step 6 specifically comprises the following steps: Step 6.1, dialyzing the first filtrate and the second filtrate using a dialysis membrane for 44-48 hours, the molecular weight of the dialysis membrane is 8000 kDa, and the permeate is collected; Step 6.2, freezing the permeate at -20 DEG C to -35 DEG C for 2-6 hours, then placing the frozen permeate into a vacuum freeze-drying chamber, the cold trap temperature is -40 DEG C ± 2 DEG C, and the vacuum degree is -30 KPa to -60 KPa, to obtain the cell wall polysaccharide.

Images