Pu'er tea fresh leaf polysaccharide and preparation method thereof, self-assembled nanomaterial and application

Abstract

The application provides a Pu'er tea fresh leaf polysaccharide and a preparation method thereof, a self-assembled nanomaterial and application, and belongs to the technical field of biological medicines. The Pu'er tea fresh leaf polysaccharide is a uniform polysaccharide, the repeating sequence of the Pu'er tea fresh leaf polysaccharide is 1,4-alpha-D-galacturonic acid, 1,3,6-beta-D-galactose and 1,4,6-beta-D-galactose as a main chain, beta-D-glucuronic acid, alpha-L-arabinose and alpha-D-Rhap-(1→3)-alpha-L-Araf-(1→5)-alpha-L-Araf as side chains, 1,4-alpha-D-galacturonic acid exists in a methyl esterification state, the degree of methyl esterification is 32.08%, and the structure of the Pu'er tea fresh leaf polysaccharide is shown as formula 1. The self-assembled nanomaterial and the Pu'er tea fresh leaf polysaccharide have immunoregulatory activity, and the self-assembled nanomaterial has better immunoregulatory activity than a single polysaccharide molecule or a single active small molecule compound.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a polysaccharide from fresh Pu-erh tea leaves, its preparation method, self-assembled nanomaterials, and their applications. Background Technology

[0002] Tea is the world's most popular beverage, and since the cultivation of tea trees, people have drunk it for health benefits and to satisfy their desires. Most researchers focus on low-molecular-weight compounds in tea, such as polyphenols, catechins, caffeine, and theanine. Therefore, significant progress has been made in understanding the bioactivity of caffeine and polyphenols in tea. In recent studies, polysaccharides from tea have attracted increasing interest from researchers, revealing numerous bioactivities, such as immune-enhancing, antioxidant, anticancer, and anti-HIV activities. With in-depth research on tea polysaccharides, more and more scholars have discovered that tea polysaccharides can often bind to low-molecular-weight compounds and proteins through covalent and non-covalent bonds, forming polysaccharide conjugates in tea.

[0003] Current research on the physicochemical properties and bioactivity of tea polysaccharides mainly focuses on coarse-leaf and green tea, with limited research on polysaccharides from other teas. Furthermore, polysaccharides from Pu-erh tea, such as those found in dark tea, may exhibit different physicochemical properties and bioactivity due to their fermentation process. Current research on Pu-erh tea polysaccharides is limited to coarse-leaf polysaccharides, primarily focusing on extraction, separation, and physicochemical property analysis. It is difficult to determine their structural characteristics or evaluate their precise efficacy, and the molecular structure of Pu-erh tea polysaccharides has not yet been reported. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a polysaccharide from fresh Pu-erh tea leaves, its preparation method, self-assembled nanomaterials, and their applications. The polysaccharide from fresh Pu-erh tea leaves and the self-assembled nanomaterials have immunomodulatory effects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a polysaccharide from fresh Pu-erh tea leaves. The polysaccharide is a homogeneous polysaccharide. Its repeating sequence consists of 1,4-α-D-galacturonic acid, 1,3,6-β-D-galactose, and 1,4,6-β-D-galactose as the main chain, and β-D-glucuronic acid, α-L-arabinose, and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf as the side chains. The 1,4-α-D-galacturonic acid is methylated to a degree of 32.08%. The structure of the Pu-erh tea leaf polysaccharide is as follows:

[0007] Formula 1.

[0008] Preferably, the molecular weight of the polysaccharide from fresh Pu-erh tea leaves is 31241 kDa; the monosaccharide composition of the polysaccharide from fresh Pu-erh tea leaves is rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose; the molar ratio of rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose is 1.55:2.47:35.06:25.63:35.28.

[0009] This invention provides a method for preparing polysaccharides from fresh Pu-erh tea leaves, comprising the following steps:

[0010] (1) Pulverize the dried Pu-erh tea leaves and sieve them to obtain tea powder;

[0011] (2) The tea powder was soaked in the first ethanol solution to remove the fat, and the defatted powder was obtained;

[0012] (3) Mix the defatted powder with water and extract, collect the filtrate to obtain the extract, concentrate the extract under reduced pressure to obtain the concentrated extract;

[0013] (4) The concentrated extract is mixed with the second ethanol solution to precipitate, and the precipitate is obtained by centrifugation, collection of precipitate, redissolution of precipitate, removal of protein in redissolution precipitate, collection of supernatant to obtain protein-free extract, drying to obtain Pu-erh tea crude polysaccharide;

[0014] (5) Static adsorption of crude polysaccharide of Pu'er tea with macroporous adsorption resin suspension, shaken to obtain decolorized suspension, filtered, washed, collected eluent a, dried to obtain decolorized polysaccharide of Pu'er tea;

[0015] (6) After the polysaccharide of Pu-erh tea is dissolved, the supernatant is collected by centrifugation and added to a chromatography column filled with DEAE cellulose DE-52. The column is eluted with water and 0.1M NaCl solution in sequence. The eluent after elution with 0.1M NaCl solution is collected, concentrated, dialyzed for the first time, and dried to obtain the polysaccharide subfraction.

[0016] (7) The polysaccharide subfractions were purified by using an S-400 propylene dextran gel column, eluted, and the homogeneous fractions were collected, concentrated, subjected to a second dialysis, and dried to obtain polysaccharides from fresh Pu-erh tea leaves.

[0017] Preferably, the volume fraction of the first ethanol solution is 80-90%; the degreasing is performed 1-4 times, with each degreasing session lasting 3-4 hours; and the mass-to-volume ratio of the tea powder to the first ethanol solution is 1 kg: 3-4 L.

[0018] Preferably, the mass-to-volume ratio of the defatted powder to water is 1:10~20; the number of extractions is 1~4 times; the extraction temperature is 90~100℃; and the extraction time for each extraction is 2~4 hours.

[0019] Preferably, the volume ratio of the concentrated extract to the second ethanol solution is 1:2~4; the volume percentage of the second ethanol solution is 80~97%; and the solvent for the reconstituted precipitate is water at 50~60℃.

[0020] Preferably, the macroporous adsorption resin is macroporous adsorption resin D101; the macroporous adsorption resin suspension is obtained by mixing macroporous adsorption resin and water at a ratio of 450~550g:1000mL; the shaking temperature is 40~60℃; the shaking time is 4~6h; the shaking frequency is 110~130r / min; the solvent for dissolving the Pu-erh tea decolorizing polysaccharide is water, and the mass-volume ratio of the Pu-erh tea decolorizing polysaccharide to water is 15~25mg:1mL; in step (6), the elution flow rate is 0.5~1.5mL / min, and the elution time with water and 0.1M NaCl solution is 5~7h; the molecular weight cutoff of the first dialysis and the second dialysis is 3000~4000Da; in step (7), the elution solvent is water, and the elution rate is 0.1~0.5mL / min;

[0021] The eluent obtained by elution was analyzed by HPLC-RID, and the components with a retention time of 7.9~8.0 min in the HPLC-RID chromatogram were identified.

[0022] The HPLC-RID chromatographic conditions were as follows: column: Thermo Acclaim 120 C18; mobile phase conditions: mobile phase A was a pH 6.72 NaH2PO4 / Na2HPO4 buffer solution, and mobile phase B was acetonitrile; the NaH2PO4 / Na2HPO4 buffer solution was prepared by mixing 1.6 g NaH2PO4, 0.8 g Na2HPO4, and 100 mL of water.

[0023] Elution program: 0-60 min; mobile phase A: mobile phase B volume ratio = 83:17; flow rate: 1 mL / min; column temperature: 30℃.

[0024] This invention provides a self-assembled nanomaterial. The process involves dissolving the aforementioned Pu-erh tea fresh leaf polysaccharide in water to obtain an aqueous solution of Pu-erh tea fresh leaf polysaccharide; mixing an active small molecule compound with water to obtain an aqueous solution of the active small molecule compound; and then co-assembling the Pu-erh tea fresh leaf polysaccharide aqueous solution and the active small molecule compound aqueous solution to obtain the self-assembled nanomaterial. The active small molecule compound includes one of epigallocatechin gallate and caffeine.

[0025] Preferably, the mass-to-volume ratio of the Pu-erh tea fresh leaf polysaccharide to water is 3-5:1; the mass-to-volume ratio of the active small molecule compound to water is 0.5-1.5:1; and the volume ratio of the Pu-erh tea fresh leaf polysaccharide aqueous solution to the active small molecule compound aqueous solution is 1-3:10-15.

[0026] The present invention also provides an application of the above-mentioned Pu-erh tea fresh leaf polysaccharide or self-assembled nanomaterial in the preparation of immunomodulatory products.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention provides a polysaccharide from fresh Pu-erh tea leaves, its preparation method, self-assembled nanomaterials, and their applications. The invention identifies a novel natural polysaccharide (CTP1) from fresh Pu-erh tea leaves. This polysaccharide is a novel homogeneous polysaccharide with a molecular weight of 31241 kDa. Based on the physicochemical properties of this novel polysaccharide, a polysaccharide platform was established to construct various types of self-assembled nanomaterials, such as polysaccharide-active small molecule compound nanomaterials. The self-assembled nanomaterials and the Pu-erh tea leaf polysaccharide exhibit immunomodulatory effects by activating RAW264.7 mononuclear macrophages and enhancing their phagocytic capacity, as well as regulating NO secretion levels. Furthermore, the self-assembled nanomaterials demonstrate better immunomodulatory activity than individual polysaccharide molecules or single active small molecule compounds. This invention provides a new strategy for constructing powerful nanomaterials by co-assembling Pu-erh tea leaf polysaccharides with various active small molecule compounds. Attached Figure Description

[0029] Figure 1 The high-performance liquid chromatogram of CTP1 is shown below.

[0030] Figure 2 High-performance liquid chromatogram of the monosaccharide composition of CTP1;

[0031] Figure 3 The infrared spectrum of CTP1;

[0032] Figure 4 The ultraviolet spectrum of CTP1;

[0033] Figure 5 A in the image is the nuclear magnetic resonance image of CTP1. 1 H NMR spectrum; B is CTP1. 13 C10 NMR spectrum; C is the DEPT-90 NMR spectrum of CTP1; D is the DEPT-135 NMR spectrum of CTP1;

[0034] Figure 6 The nuclear magnetic resonance TOCSY spectrum of CTP1;

[0035] Figure 7The nuclear magnetic resonance HSQC spectrum of CTP1;

[0036] Figure 8 The HMBC spectrum of the nuclear magnetic resonance labeled end-group isomer region of CTP1;

[0037] Figure 9 The HMBC spectrum of the aliphatic region of CTP1 is obtained from nuclear magnetic resonance labeling.

[0038] Figure 10 The structure is CTP1;

[0039] Figure 11 Transmission electron microscopy image of CTP1-EGCG;

[0040] Figure 12 Transmission electron microscopy image of CTP1-CAF;

[0041] Figure 13 The results show the comparison of the phagocytic capacity of CTP1, EGCG, CAF, CTP1-EGCG, and CTP1-CAF.

[0042] Figure 14 Results comparing NO secretion levels of CTP1, EGCG, CAF, CTP1-EGCG, and CTP1-CAF. Detailed Implementation

[0043] This invention provides a polysaccharide from fresh Pu-erh tea leaves. The polysaccharide is a homogeneous polysaccharide. Its repeating sequence consists of 1,4-α-D-galacturonic acid, 1,3,6-β-D-galactose, and 1,4,6-β-D-galactose as the main chain, and β-D-glucuronic acid, α-L-arabinose, and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf as the side chains. The 1,4-α-D-galacturonic acid is methylated to a degree of 32.08%. The structure of the Pu-erh tea leaf polysaccharide is as follows:

[0044] Formula 1.

[0045] In this invention, the molecular weight of the polysaccharide from fresh Pu-erh tea leaves is 31241 kDa; the monosaccharide composition of the polysaccharide from fresh Pu-erh tea leaves is rhamnose, glucuronic acid, galacturonic acid, galactose, and arabinose; the molar ratio of rhamnose, glucuronic acid, galacturonic acid, galactose, and arabinose is 1.55:2.47:35.06:25.63:35.28.

[0046] This invention has found that polysaccharides from fresh Pu-erh tea leaves can activate macrophages and enhance their phagocytic capacity, regulate NO secretion levels, and have immunomodulatory activity.

[0047] This invention provides a method for preparing polysaccharides from fresh Pu-erh tea leaves, comprising the following steps:

[0048] (1) Pulverize the dried Pu-erh tea leaves and sieve them to obtain tea powder;

[0049] (2) The tea powder was soaked in the first ethanol solution to remove the fat, and the defatted powder was obtained;

[0050] (3) Mix the defatted powder with water and extract, collect the filtrate to obtain the extract, concentrate the extract under reduced pressure to obtain the concentrated extract;

[0051] (4) The concentrated extract is mixed with the second ethanol solution to precipitate, and the precipitate is obtained by centrifugation, collection of precipitate, redissolution of precipitate, removal of protein in redissolution precipitate, collection of supernatant to obtain protein-free extract, drying to obtain Pu-erh tea crude polysaccharide;

[0052] (5) Static adsorption of the crude polysaccharide solution of Pu'er tea with macroporous adsorption resin, shaking, obtaining suspension, filtering, washing, collecting filtrate, drying, and obtaining decolorized polysaccharide of Pu'er tea;

[0053] (6) After the polysaccharide of Pu-erh tea is dissolved, the supernatant is collected by centrifugation and added to a chromatography column filled with DEAE cellulose DE-52 (purchased from Beijing Solarbio Technology Co., Ltd., item number C8930). The column is eluted with water and 0.1M NaCl solution in sequence. The eluent after elution with 0.1M NaCl solution is collected, concentrated, dialyzed for the first time, and dried to obtain the polysaccharide subfraction.

[0054] (7) The polysaccharide subfractions were purified by using an propylene dextran gel S-400 (purchased from Sitopan Biotechnology Co., Ltd., item number 17060901) gel column, eluted, collected, concentrated, subjected to a second dialysis, and dried to obtain polysaccharides from fresh Pu'er tea leaves.

[0055] In this invention, dried Pu-erh tea leaves are pulverized and sieved to obtain tea powder. The drying method can be air-drying, oven-drying, or freeze-drying. The mesh size of the sieve is preferably 50-70 mesh, more preferably 55-65 mesh, and even more preferably 60 mesh.

[0056] In this invention, to remove lipids from tea leaves and thus improve the purity of polysaccharides in fresh Pu-erh tea leaves, tea powder is obtained and then soaked in a first ethanol solution for degreasing to obtain degreased powder. The volume fraction of the first ethanol solution is preferably 80-90%, more preferably 83-87%, and even more preferably 85%; the number of degreasing cycles is preferably 1-4 times, more preferably 2-3 times, and even more preferably 3 times; the degreasing time for each cycle is preferably 3-4 hours; and the mass-to-volume ratio of tea powder to the first ethanol solution is preferably 1 kg: 3-4 L.

[0057] In this invention, after obtaining the defatted powder, the defatted powder is mixed with water for extraction, the filtrate is collected to obtain an extract, and the extract is concentrated under reduced pressure to obtain a concentrated extract. The preferred mass-to-volume ratio of the defatted powder to water is 1:10-20, more preferably 1:12-18, and even more preferably 1:15. The preferred number of extractions is 1-4 times, more preferably 2-3 times, and even more preferably 3 times. The preferred extraction temperature is 90-100℃, more preferably 92-97℃, and even more preferably 95℃. The preferred extraction time is 2-4 hours, more preferably 2.5-3.5 hours, and even more preferably 3 hours. The preferred temperature for reduced pressure concentration is 50-60℃, more preferably 52-58℃, and even more preferably 55℃. The preferred pressure for reduced pressure concentration is 0.05-0.08 MPa, and even more preferably 0.07 MPa. This invention, through extraction by mixing the defatted powder with water, can effectively increase the polysaccharide content and improve the yield of polysaccharides from fresh Pu-erh tea leaves.

[0058] In this invention, after obtaining the concentrated extract, the concentrated extract is mixed with a second ethanol solution to precipitate, resulting in an alcohol-precipitated solution. The precipitate is then collected by centrifugation. The volume ratio of the concentrated extract to the second ethanol solution is preferably 1:2-4, more preferably 1:3; the volume percentage of the second ethanol solution is 80-97%, more preferably 85-95%, and even more preferably 95%. The centrifugation method can be 3500-4500 rpm for 10-20 minutes, such as 4000 rpm for 15 minutes. This invention effectively removes small-molecule polyphenols and pigments through alcohol precipitation, improving the yield and purity of polysaccharides from fresh Pu-erh tea leaves.

[0059] In this invention, after collecting the precipitate, the precipitate is redissolved to remove proteins from the redissolved precipitate. The supernatant is collected to obtain a protein-free extract, which is then dried to obtain crude polysaccharide from Pu-erh tea. The solvent for redissolving the precipitate is preferably water at 50-60°C, more preferably water at 60°C. The method for removing proteins from the redissolved precipitate can use a mixture of chloroform and n-butanol at a volume ratio of 4:1. The collection method can be centrifugation, preferably at 0°C and 3500-4500 r / min for 10-20 min, such as at 0°C and 4000 r / min for 15 min. The drying method can be lyophilization. This invention does not specifically limit the lyophilization method; conventional lyophilization methods known in the art are acceptable.

[0060] In this invention, after obtaining the crude polysaccharide from Pu-erh tea, the crude polysaccharide is statically adsorbed with a macroporous adsorption resin suspension, shaken, and the resulting suspension is filtered, washed, and the filtrate is collected and dried to obtain the decolorized polysaccharide from Pu-erh tea. The macroporous adsorption resin is preferably macroporous adsorption resin D101. The macroporous adsorption resin suspension is obtained by mixing macroporous adsorption resin and water at a ratio of 450-550g:1000mL. The macroporous adsorption resin is a pretreated macroporous adsorption resin. The pretreated macroporous adsorption resin is prepared by soaking macroporous adsorption resin D101 in a 95% (v / v) ethanol solution for 12 hours, washing with distilled water until the supernatant is clear, filtering with gauze, and then soaking in a 4% (v / v) NaOH solution for 4 hours, followed by washing with distilled water until neutral, resulting in a 5% (v / v) NaOH solution. The material is soaked in HCl solution for 4 hours, washed with distilled water until neutral, filtered through gauze, and then dried to obtain the pretreated macroporous adsorption resin. The shaking temperature is preferably 40-60℃, more preferably 45-55℃, and even more preferably 50℃. The shaking time is preferably 4-6 hours, more preferably 4.5-5.5 hours, and even more preferably 5 hours. The shaking frequency is preferably 110-130 r / min, more preferably 115-125 r / min, and even more preferably 120 r / min. The filtration method can be gauze filtration, the washing solvent is water, and the washing volume is 1-4 L, more preferably 2 L. The drying method can be freeze-drying. This invention uses static adsorption for decolorization, which can effectively remove phenolic pigments and improve the yield of polysaccharides.

[0061] In this invention, after obtaining the decolorized polysaccharide from Pu-erh tea, the decolorized polysaccharide is dissolved, the supernatant is collected by centrifugation, and added to a chromatography column filled with DEAE cellulose DE-52 (purchased from Beijing Solarbio Technology Co., Ltd., catalog number C8930). The column is eluted sequentially with water and 0.1M NaCl solution. The eluent after elution with 0.1M NaCl solution is collected, concentrated, subjected to a first dialysis, and dried to obtain the polysaccharide subfraction. The solvent for dissolving the Pu-erh tea decolorizing polysaccharide is water, and the preferred mass-to-volume ratio of the dissolved Pu-erh tea decolorizing polysaccharide to water is 15-25 mg:1 mL, more preferably 20 mg:1 mL. The preferred elution flow rate is 0.5-1.5 mL / min, more preferably 1 mL / min. The elution time with water and 0.1 M NaCl solution is 5-7 h each, i.e., elution with water for 5-7 h, followed by elution with 0.1 M NaCl solution for 5-7 h. The molecular weight cutoff for the first dialysis is 3000-4000 Da, more preferably 3500 Da. The drying method can be lyophilization. This invention does not have a specific limitation on the lyophilization method; any conventional lyophilization method known in the art can be used. The eluent obtained by elution with water and 0.1 M NaCl solution is collected using an automatic collector, and the carbohydrate content in each tube of eluent is monitored using the phenol-sulfuric acid method. The results show that the eluent eluted with 0.1 M NaCl solution contains the component with the highest carbohydrate content.

[0062] In this invention, after obtaining the polysaccharide subfractions, the subfractions are purified using an propylene dextran gel column S-400 (purchased from Sitopan Biotechnology Co., Ltd., catalog number 17060901). The elution is performed, and the homogenized fraction is collected, concentrated, subjected to a second dialysis, and dried to obtain Pu-erh tea leaf polysaccharides. The elution solvent is water, and the elution rate is preferably 0.1~0.5 mL / min, more preferably 0.2~0.4 mL / min, and even more preferably 0.3 mL / min. This invention does not specifically limit the concentration method; conventional methods in the art, such as vacuum concentration, are acceptable. The molecular weight cutoff for the second dialysis is 3000~4000 Da, more preferably 3500 Da. The drying method can be lyophilization; this invention does not specifically limit the lyophilization method, and conventional lyophilization methods known in the art are acceptable.

[0063] In this invention, the eluent obtained by elution is detected by HPLC-RID, and the fraction with a retention time of 7.9~8.0 min in the HPLC-RID chromatogram is a fraction, such as a fraction with a retention time of 7.957 min. The HPLC-RID chromatographic conditions are as follows: column: Thermo Acclaim 120 C18; mobile phase conditions: mobile phase A is pH 6.72 NaH2PO4 / Na2HPO4 buffer, and mobile phase B is acetonitrile; the NaH2PO4 / Na2HPO4 buffer is prepared by mixing 1.6 g NaH2PO4, 0.8 g Na2HPO4 and 100 mL of water; elution program: 0~60 min, mobile phase A:mobile phase B volume ratio = 83:17; flow rate: 1 mL / min; column temperature: 30℃. The detection wavelength of the HPLC-RID is 250 nm; the injection volume is 10 μL.

[0064] In the preparation of the NaH2PO4 / Na2HPO4 buffer solution, the mixing method can be stirring to completely dissolve NaH2PO4 and Na2HPO4 and mix evenly.

[0065] The polysaccharide prepared from fresh Pu-erh tea leaves using the above method has high purity, reaching 98%.

[0066] This invention provides a self-assembled nanomaterial. The process involves dissolving the aforementioned Pu-erh tea fresh leaf polysaccharide in water to obtain an aqueous solution of Pu-erh tea fresh leaf polysaccharide; mixing an active small molecule compound with water to obtain an aqueous solution of the active small molecule compound; and then co-assembling the Pu-erh tea fresh leaf polysaccharide aqueous solution and the active small molecule compound aqueous solution to obtain the self-assembled nanomaterial. The active small molecule compound includes one of epigallocatechin gallate and caffeine.

[0067] In this invention, the mass-to-volume ratio of the polysaccharide from fresh Pu-erh tea leaves to water is 3-5 mg:1 mL; the mass-to-volume ratio of the active small molecule compound to water is 0.5-1.5 mg:1 mL; and the volume ratio of the aqueous solution of the polysaccharide from fresh Pu-erh tea leaves to the aqueous solution of the active small molecule compound is 1-3:10-15.

[0068] The present invention also provides an application of the above-mentioned Pu-erh tea fresh leaf polysaccharide or self-assembled nanomaterial in the preparation of immunomodulatory products.

[0069] In this invention, the product can be a drug, reagent, health product, or food. The Pu-erh tea leaf polysaccharide of this invention activates mouse mononuclear macrophages RAW264.7 and enhances their phagocytic capacity, regulates NO secretion levels, thereby exhibiting immunomodulatory activity. Furthermore, this self-assembled nanomaterial possesses better immunomodulatory activity than Pu-erh tea leaf polysaccharide or any single active small molecule compound. This invention can use Pu-erh tea leaf polysaccharide or self-assembled nanomaterial as the active ingredient of the above-mentioned product in combination with other products possessing immunomodulatory effects to achieve the purpose of immunomodulation. Alternatively, Pu-erh tea leaf polysaccharide or self-assembled nanomaterial can be used as the sole active ingredient of the above-mentioned product for immunomodulation, thereby enhancing immunity. The dosage form of the product includes tablets, syrups, injections, capsules, or granules. The Pu-erh tea leaf polysaccharide or self-assembled nanomaterial accounts for 20-90% of the product weight.

[0070] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0071] Example 1: Extraction, isolation, purification, and structural identification of polysaccharide (CTP1) from fresh Pu-erh tea leaves.

[0072] 1. Extraction, separation and purification of polysaccharide (CTP1) from fresh Pu-erh tea leaves

[0073] (1) Fresh Pu-erh tea leaves are air-dried, crushed, and passed through a 60-mesh sieve to obtain tea powder;

[0074] (2) Soak 2 kg of tea powder in 4 L of 85% ethanol solution for 4 h to remove fat, and collect the degreased powder a; add the degreased powder a to 4 L of 85% ethanol solution for 4 h to remove fat, and obtain the degreased powder b; add the degreased powder b to 4 L of 85% ethanol solution for 4 h to remove fat, and obtain the degreased powder c;

[0075] (3) 1 kg of defatted powder c was extracted with 15 L of distilled water at 95 °C for 3 h each time, for a total of 3 times. The filtrates after each extraction were collected and combined to obtain the extract. The extract was concentrated under reduced pressure at 55 °C using a rotary evaporator to 1 / 9 of the original extract volume to obtain the concentrated extract. The concentrated extract was precipitated overnight with 3 times the volume of 95% ethanol solution at room temperature to obtain the ethanol-precipitated solution. The ethanol-precipitated solution was centrifuged at 4000 rpm for 15 min to collect the precipitate. The precipitate was reconstituted with 60 °C hot water and then the protein in the precipitate was removed by Sevage (the Sevage is composed of chloroform and n-butanol, and the volume ratio of chloroform to n-butanol is 4:1). The supernatant was collected by centrifugation at 0 °C and 4000 r / min for 15 min to obtain the protein-free extract. The protein-free extract was freeze-dried to obtain 66 g of Pu-erh tea crude polysaccharide.

[0076] (4) After soaking macroporous adsorption resin D101 in 95% ethanol solution for 12 hours, wash it with distilled water until the supernatant is clear. After filtering with gauze, soak it in 4% NaOH solution for 4 hours, wash it with distilled water until neutral, soak it in 5% HCl solution for 4 hours, wash it with distilled water until neutral, filter it with gauze and dry the material. Take 500g of the dried material and add 1L of water to obtain a 0.5g / mL pretreated macroporous adsorption resin D101 suspension. Take 500mL of the pretreated macroporous adsorption resin D101 suspension and add 1g of the crude polysaccharide of Pu'er tea prepared in step (3). Shake at 50℃ for 5 hours with a shaking frequency of 120r / min to obtain a decolorized suspension. After shaking, filter it with gauze and rinse the solid phase with 2L of water. Collect the liquid phase, concentrate the liquid phase under reduced pressure and freeze dry to obtain the decolorized polysaccharide of Pu'er tea.

[0077] (5) Dissolve 400 mg of decolorized polysaccharide from Pu-erh tea in 20 mL of distilled water, centrifuge at 3000 rpm for 5 min, collect the supernatant, and load it onto a 2.9 × 60 cm chromatography column packed with DEAE cellulose DE-52 (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number C8930). Perform linear gradient elution with water (i.e., 0 M NaCl solution), 0.1 M, 0.3 M, and 0.5 M NaCl solutions at a flow rate of 1 mL / min, respectively, for 6 h at each concentration. Collect the eluents after elution with water, 0.1 M, 0.3 M, and 0.5 M NaCl solutions, respectively. Concentrate the eluents after elution with water, 0.1 M, 0.3 M, and 0.5 M NaCl solutions, dialyze at 3500 Da, and freeze dry to obtain four polysaccharide subfractions: DEAE-0, DEAE-1, DEAE-2, and DEAE-3, respectively.

[0078] (6) The fraction with the highest carbohydrate content (DEAE-1) was selected for further purification. Specifically, the DEAE-1 polysaccharide subfraction was purified by gel permeation chromatography on an S-400 propylene dextran gel column (purchased from Sitopan Biotechnology Co., Ltd., catalog number 17060901) (1.9×110cm). The elution solvent was water, the flow rate was 0.3mL / min, and the eluent b was collected in 2mL / tubes by an automatic collector. The polysaccharides in the eluent b were monitored by high performance liquid chromatography-refractive index detection (HPLC-RID). The fraction with the peak time of 7.957min was collected according to the HPLC-RID chromatogram. Then, the fraction was concentrated, dialyzed at 3500Da, and lyophilized to obtain Pu-erh tea fresh leaf polysaccharide (CTP1).

[0079] The chromatographic conditions for the above HPLC-RID were as follows: Column: Thermo Acclaim 120°C 18(4.6mm×250mm, 5µm); Mobile phase conditions: Mobile phase A is pH 6.72 NaH2PO4 / Na2HPO4 buffer, mobile phase B is acetonitrile; Elution program: 0~60min Mobile phase A:Mobile phase B = 83:17 (v / v); Column temperature: 30℃; Flow rate: 1mL / min; Detection wavelength: 250nm; Injection volume: 10μL.

[0080] The NaH2PO4 / Na2HPO4 buffer solution is prepared by mixing 1.6g NaH2PO4, 0.8g NaHPO4 and 100mL of water evenly.

[0081] 2. Structural identification of CTP1

[0082] (1) Determination of the molecular weight of the above CTP1 by high performance gel permeation chromatography: The homogeneity and molecular weight distribution of the sample were analyzed by high performance gel permeation chromatography on an HPLC system equipped with a differential refractive index detector. Chromatographic column: Shodex SUGARKS-805 gel chromatography column (8×300mm, 10µm); column temperature: 45℃; detector temperature: 40℃; mobile phase: ultrapure water; flow rate: 0.8mL / min; injection volume: 10µL; sample concentration: 2mg / mL. The homogeneity of the sample was analyzed by peak area normalization on an HPLC chemistry workstation. A standard curve of molecular weight distribution was established with the logarithm of the relative molecular mass (Lg Mw) of a series of dextran standards with different molecular weights (5, 12, 25, 50, 410 and 670kDa) as the ordinate and the retention time (t) of the chromatographic peak as the abscissa. The molecular weight distribution of the sample was calculated according to the equation of the standard curve.

[0083] See results Figure 1 The results showed that the CTP1 prepared in this invention was a homogeneous polysaccharide, according to the established linear regression equation (Lg Mw =-0.6178t+12.453, R 2 Based on the retention time of CTP1 (t=0.9909) and CTP1 (t=7.957), the molecular weight of CTP1 was calculated to be 31241 kDa.

[0084] (2) Monosaccharide composition analysis of CTP1: The monosaccharide composition of CTP1 was analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization-reverse high performance liquid chromatography.

[0085] Acid hydrolysis of the sample: Accurately weigh 5 mg CTP1 and 2 mL 4 M trifluoroacetic acid and stir at 110 °C for 11 h. Dissolve the reaction product in methanol, concentrate under reduced pressure at 50 °C to remove residual TFA, repeat 5 times until TFA is completely removed, and obtain the hydrolysis product.

[0086] Derivatization of samples: The dried hydrolysis product was dissolved in 1 mL of distilled water to obtain a hydrolysate. Then, 50 μL of the 5 mg / mL hydrolysate, 50 μL of 0.3 M NaOH, and 100 μL of 0.5 M 1-phenyl-3-methyl-5-pyrazolone were mixed and reacted at 70 °C in the dark for 30 min. After adjusting the pH to 7.0 with 0.3 M HCl, 1 mL of chloroform was added and the extraction was repeated three times. The chloroform at the bottom was removed, and the aqueous phase at the top was filtered through a 0.22 μm microporous membrane as the test solution for liquid chromatography analysis.

[0087] Derivatization of standards: Weigh appropriate amounts of 10 monosaccharide reference standards (Man, Rib, Rhamnose, Glucuronic Acid, Galuronic Acid, Glucose, Gal, Xylose, Araose, and Fucose) dried to constant weight, dissolve in ultrapure water to prepare a 1 mg / mL solution. Accurately measure 50 μL and follow the derivatization procedure described above for the samples. Collect the top aqueous phase and filter it through a 0.22 μm microporous membrane as the reference solution for liquid chromatography analysis.

[0088] Chromatographic conditions: Column: Thermo Acclaim 120°C 18 (4.6mm×250mm, 5µm); Mobile phase conditions: Mobile phase A is pH 6.72 NaH2PO4 / Na2HPO4 buffer, mobile phase B is acetonitrile; Elution program: 0~60min Mobile phase A:Mobile phase B = 83:17 (v / v); Column temperature: 30℃; Flow rate: 1mL / min; Detection wavelength: 250nm; Injection volume: 10μL.

[0089] Figure 2 The results showed that the monosaccharide composition of CTP1 was rhamnose, glucuronic acid, galacturonic acid, galactose, and arabinose in a molar ratio of 1.55:2.47:35.06:25.63:35.28.

[0090] (3) Infrared spectral analysis: The infrared spectrum of CTP1 was measured using a Nicolet iS10 Fourier transform infrared spectrophotometer. After the sample was processed with KBr pellets, background samples were collected before sampling, and the spectra were recorded for 32 scans at 4.00 cm⁻¹. -1 Resolution, wavenumber range 4000~400cm -1 .

[0091] See results Figure 3 As can be seen, the infrared spectral characteristics of CTP1 show that at 3397 cm⁻¹... -1 and 2932cm -1The strong absorption bands appearing at 1613 cm⁻¹ are attributed to the stretching vibrations of the OH, methyl, and methylene groups, respectively. -1 and 1742cm -1 Stretching vibrations originating from carboxyl and ester carbonyl groups were observed at 1413 cm⁻¹. -1 The absorption in the vicinity is caused by the bending vibration of C-OH; 1260 cm⁻¹ -1 and 1237cm -1 The absorption bands at 1330 cm⁻¹ are attributed to the asymmetric stretching vibrations of CO in the esterified and unesterified carboxyl groups, respectively; -1 The absorption band at 1143 cm⁻¹ belongs to the in-plane bending vibration of CH within the sugar ring; -1 1098cm -1 and 1018cm -1 Pyranose absorption was observed at 957 cm⁻¹. -1 Absorption at this site belongs to furanose; in the range of 920~840cm -1 Multiple absorption bands were observed in the region, indicating that both α and β configurations exist simultaneously.

[0092] (4) Ultraviolet spectroscopy analysis: The ultraviolet spectrum of 1 mg / mL CTP1 was determined using a Shimadzu UV-2700 UV-Vis spectrophotometer in the wavelength range of 190–500 nm. Results are shown below. Figure 4 .

[0093] Figure 4 The results showed that CTP1 did not have obvious absorption peaks of protein and nucleic acid in the wavelength range of 260~280nm, indicating that CTP1 does not contain protein and nucleic acid.

[0094] (5) Nuclear Magnetic Resonance (NMR) Analysis: An appropriate amount of CTP1 sample was pre-dissolved in deuterated water (D2O, 99.9%), freeze-dried, and the D2O exchange was repeated three times. After freeze-drying, the sample was dissolved in 0.5 mL of D2O in a 3 mm NMR tube. The 1D and 2D NMR spectra were measured at 25 °C using a Bruker Avance III 800 MHz NMR spectrometer. The results are shown in […]. Figures 5-9 .

[0095] In CTP1 1 In the H NMR spectrum (see) Figure 5 A in δ H A large number of polysaccharide characteristic proton signals appeared in the signal range of 3.00–5.50 ppm. In the δ... H 4.30~4.95ppm and δ H The absorption peaks in the 4.95–5.50 ppm region confirm the presence of α and β glycosidic bonds in CTP1, consistent with the results of infrared spectroscopy. At δ... HEleven anomeric proton signals appeared in the low-field region at 5.23, 5.18, 5.14, 5.05, 4.99, 4.91, 4.64, 4.44, 4.41, 4.39, and 4.29 ppm. Based on the monosaccharide composition and methylation analysis of CTP1, these were labeled as residues AK. Among them, those at δ... H The anomeric proton signal at 4.64 ppm was compared with the proton signal (δ) of D2O. H The signal was masked by 4.69 ppm. Non-anomalous proton signals were all concentrated in the delta range. H In the 3.10–4.30 ppm region, some signals, due to severe overlap, require further analysis using the TOCSY spectrum. Figure 6 ) and HSQC spectrum ( Figure 7 The proton chemical shifts of each residue were assigned separately.

[0096] and 1 Compared to the H NMR spectrum, CTP1 in 13 C NMR spectrum ( Figure 5 The signal distribution in (B) is wider. In the low-field region, δ C The coexistence of signals at 170.89 and 175.10 ppm confirms partial methyl esterification of uronic acid in CTP1, consistent with methylation analysis results. In CTP1... 1 H NMR and 13 The strongest signal in the 13C NMR spectrum (δ 3.70 / 52.85 ppm) originates from the methoxy group, while the signal in the high-field region at δ 1.15 / 16.52 ppm is attributed to the methyl group of Rha. The 13C NMR spectrum of CTP1 (… Figure 5 B) in δ C Eleven anodic carbon signals were observed at 109.24, 107.44, 107.09, 103.35, 103.26, 103.06, 102.69, 100.71, 99.43, 99.35, and 99.17 ppm. Combined with HSQC spectroscopy (… Figure 7 The cross-peaks in the anodic signal region determined the anodic signals of residues AB and DK to be δ 5.23 / 99.17, 5.18 / 99.43, 5.05 / 107.09, 4.99 / 107.44, 4.91 / 99.35, 4.64 / 100.71, 4.44 / 103.06, 4.41 / 102.69, 4.39 / 103.35, and 4.29 / 103.26 ppm, respectively. Due to the signal loss in the anodic region in the HSQC spectrum, the anodic signal of residue C is mainly determined by the HMBC spectrum ( Figure 8The combined 1H NMR and 13C NMR spectra of the end-group isomer region of the sample were determined to be δ 5.14 / 109.24 ppm. From the perspective of HMBC spectroscopy experiments, it is necessary to set a specific filter (145 Hz) to minimize (or filter out) the reactions generated by direct correlation, i.e., proton / carbon pairs generated by single bond coupling. 1 J C However, this setup may not completely eliminate the magnetization produced by the isomeric carbons, resulting in the isomeric carbon densities being detected in the HMBC spectrum. They retain coupling with their own protons, thus exhibiting two densities along the F2 dimension, with the central position matching the position of the isomeric proton.

[0097] HMBC spectrum of CTP1 (see) Figure 8 Two cross peaks, δ 5.25 / 109.24 ppm and δ 5.03 / 109.24 ppm, were observed in the anodic signal region. Therefore, the center position of the two cross peaks along the F2 dimension was determined to be the anodic signal of residue C. Meanwhile, as... Figure 7 As shown, the HSQC spectrum also shows a mapping of residue C anomaly signal (δ 5.14 / 109.24ppm) at δ 5.14 / 9.23ppm, which fully demonstrates the reliability of the residue C anomaly signal attribution.

[0098] For residues A, F, and G, TOCSY ( Figure 6 The H1 / H2 ratio was determined by the cross peaks at δ 5.23 / 4.07, 4.91 / 3.74, and 4.64 / 3.84 ppm in the TOCSY spectrum. Figure 6 The H3-H5 of residues A, F, and G were determined. All δH and δC values ​​in residues A, F, and G were assigned based on cross-peaks in the HSQC spectra, and HMBC spectroscopy was used. Figure 9 The results of the methylation analysis were verified. Based on the methylation analysis, residues A, F, and G were identified as →1)-α-D-GalpA-(4→). HMBC spectrum ( Figure 9 The cross peak at δ 3.77 / 175.10 ppm in the spectrum shows the correlation between H5 / C6 of residue A, indicating that residue A contains an unmethylated uronic acid group. Furthermore, based on the correlation between the cross peaks at δ 3.70 / 175.10 and 3.70 / 170.89 ppm in the HMBC spectrum, it is speculated that partial methylation may have occurred at the C6 position of residues F and G.

[0099] For residue B, H1 / H2 and H3 / H2 were determined based on the cross peaks at δ 5.18 / 4.06 ppm and δ 3.67 / 4.06 ppm in the TOCSY and COSY spectra, respectively. Furthermore, the TOCSY spectrum ( Figure 6 The cross-peak at δ 3.67 / 1.15 (H3 / H6) ppm suggests that residue B belongs to the rhamnose unit. HSQC spectrum ( Figure 7 The cross peak at δ 1.15 / 16.47 (H6 / C6) ppm in the TOCSY spectrum also indicates that residue B belongs to the rhamnose unit. Figure 6 The cross peaks at δ 3.93 / 1.15 ppm and δ 3.33 / 1.15 ppm were identified as H5 / H6 and H4 / H6 of residue B, respectively. In the HSQC spectrum, the δ H1-δ H6 of residue B were assigned based on the δ H1-δ H6 of residue B. C1 -δ C6 HMBC spectrum ( Figure 9 The cross peaks at δ 3.33 / 69.13 (H4 / C5), 3.33 / 70.00 (H4 / C3), 3.92 / 70.00 (H4 / C5), 4.06 / 70.00 (H2 / C3), 3.33 / 16.47 (H4 / C6), 1.15 / 69.13 (H4 / C5), and 1.15 / 71.89 (H4 / C5) ppm in the sample confirmed this, and combined with the results of methylation analysis, residue B was identified as α-D-Rhap-(1→).

[0100] For residues C, D, and E, as previously mentioned, the anodic signals of residues C, D, and E were identified as δ 5.14 / 109.24, 5.05 / 107.09, and 4.99 / 107.44 ppm, respectively. Their anodic carbon signals are significantly higher than those of other residues in CTP1, which is typical for furan-configured sugar residues. Combined with the results of methylation analysis, residues C, D, and E are presumed to belong to furan-configured arabinoses. According to TOCSY (… Figure 6 The cross peaks at δ 5.15 / 4.12, 5.05 / 4.04, and 4.99 / 4.02 ppm in the TOCSY spectrum indicated the H1 / H2 ratios of residues C, D, and E, respectively. Figure 6 The δ values ​​of residues C, D, and E were shown. H2 -δ H6 Signal, and based on HSQC spectrum ( Figure 7 ) δ assigned to residues C, D, and E C2 -δ C6 For residue C, HMBC spectrum ( Figure 9 ) in δ 3.61 / 76.60 (H 5aThe cross-peaks at ppm of 3.84 / 61.20 (H3 / C5), 3.84 / 81.27 (H3 / C2), 4.20 / 81.27 (H4 / C2), 5.14 / 76.60 (H1 / C3), and 5.14 / 81.27 (H1 / C2) confirm the reliability of the assignment. Similarly, residues D and E can also be confirmed in the HMBC spectrum. The difference is that, relative to residue C, the δ of residue D is higher. C3 The residue E shows a significant shift of 6-7 ppm to the lower field region. C5 A shift of 5–6 ppm to the low-field region indicates that residues D and E may contain substitution branches at C3 and C5, respectively. Residues C, D, and E were identified as α-L-Araf-(1→, →1)-α-L-Araf-(3→ and →1)-α-L-Araf-(5→), and the intensity of the anodic signal in 1D NMR for residues C, D, and E was consistent with the results of quantitative methylation analysis.

[0101] For residue H, the TOCSY spectrum ( Figure 6 The cross-peaks at δ 4.44 / 3.57, 4.44 / 3.85, 4.44 / 3.65, and 4.44 / 4.05 ppm in the HSQC spectrum indicate the H2-H5 of residue H. Figure 7 In ), based on residue H, δ H1 -δ H6 δ of residue H was allocated C1 -δ C6 HMBC spectrum ( Figure 9 The cross peaks at δ 4.05 / 80.11 (H5 / C4), 3.57 / 80.11 (H2 / C4), and 3.65 / 71.89 (H4 / C3) ppm in the sample confirmed this, and the residue H was identified as →1)-β-D-GlcpA-(4→) based on the results of methylation analysis.

[0102] For residues I, J, and K, according to TOCSY ( Figure 6 ) and HSQC ( Figure 7 The spectrum was sequentially assigned δ H2 -δ H6 and δ C2 -δ C6 and using HMBC spectrum ( Figure 9 The results were verified. Based on methylation analysis, residues I, J, and K were identified as →1)-β-D-Galp-(4-OMe)-(6→), →1)-β-D-Galp-(4-OMe)-(3,6→), and →1)-β-D-Galp-(4,6→), respectively. Residues I and J were substituted with -OMe at the O-4 positions. HSQC spectra ( Figure 7The cross peaks at δ 3.13 / 54.12 and 3.38 / 59.98 ppm in the DEPT-135 spectrum were overridden. Figure 5 The D signal in the spectrum was confirmed as a -OMe proton / carbon signal. In the HMBC spectrum ( Figure 9 In the HMBC spectrum, a correlation was found between the proton of -OMe and the C4 (δ 3.39 / 82.07ppm) and C5 (δ 3.39 / 74.83ppm) positions of residue I, indicating that the O-4 position of residue I is substituted by -OMe. Similarly, the cross peak at δ 3.13 / 68.42ppm in the HMBC spectrum also showed a strong correlation between the proton of -OMe and the C4 position of residue J, suggesting that the O-4 position of residue J is substituted by -OMe.

[0103] Furthermore, based on the δ of each sugar residue in CTP1 H and δ C Combined with HMBC ( Figure 8 HMBC spectroscopy (terminal isomer regions) is used to infer the sequence information of glycosyl groups. For the most abundant GalA-related residues, HMBC spectroscopy ( Figure 8 The cross peaks at δ 4.64 / 79.12 ppm in the spectrum indicate that CTP1 contains HG domains linked by (1,4)-α-glycosidic bonds. HMBC spectrum ( Figure 8 The HMBC spectrum showed coupling signals between H1 of residues I and J and C6 of residue J, and between H1 of residue K and C6 of residue K, suggesting that CTP1 contains a galactan fragment linearly linked by a (1,6)-β-glycosidic bond. Figure 8 The results showed that H1 of residue G was coupled to C6 of residues I and J, respectively, indicating that the HG domain and the Gal moiety are linked by (1,6)-α-glycosidic bonds, and CTP1 has a main chain with the HG domain linearly linked to galactan. Based on the HMBC spectrum ( Figure 8 The coupling signal between H1 of residue C and C3 of residue J suggests that residues H and C are connected to the O-3 position of residue J as two separate branches. Furthermore, the HMBC spectrum ( Figure 8The coupling signals between H1 of residue B and C3 of residue D, H5 of residue E and C1 of residue D, and H1 of residue E and C4 of residue K suggest the existence of a branch chain in CTP1 with residue B as the terminal unit, which is connected to the O-4 position of residue K. Based on all the above results, the primary structure of CTP1 has a main chain with →1)-α-D-GalpA-(4→ and →1)-β-D-Galp-(6→) linearly linked, containing three branch chain segments β-D-GlcpA-(1→, α-L-Araf-(1→, and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf-(1→) connected to the 3 and 4 positions of the Gal residue, respectively. Furthermore, the GalA and Gal segments contain methyl esterification and methoxylation groups at the 6 and 4 positions, respectively. The structure of CTP1 is shown in [reference needed]. Figure 10 .

[0104] Example 2: Preparation of CTP1-epigallocatechin gallate (EGCG) nanomaterials

[0105] 5 mg of the Pu-erh tea leaf polysaccharide (CTP1) prepared in Example 1 was dissolved in 2 mL of pure water to prepare a polysaccharide aqueous solution with a concentration of 2.5 mg / mL. The solution was then filtered through a 0.45 μm filter membrane for later use. EGCG was dissolved in pure water to prepare an EGCG aqueous solution with a concentration of 1 mg / mL. This solution was then filtered through a 0.45 μm filter membrane for later use. The polysaccharide aqueous solution and the EGCG aqueous solution prepared above were mixed at a volume ratio of 2:13 to form a self-assembled CTP1-EGCG nanomaterial (referred to as CTP1-EGCG), which was prepared and used immediately.

[0106] Example 3: Preparation of CTP1-Caffeine (CAF) Nanomaterials

[0107] 8 mg of the Pu-erh tea fresh leaf polysaccharide (CTP1) prepared in Example 1 was dissolved in 2 mL of pure water to prepare a polysaccharide aqueous solution with a concentration of 4 mg / mL. The solution was then filtered through a 0.45 μm filter membrane for later use. Caffeine was dissolved in pure water to prepare a CAF aqueous solution with a concentration of 1 mg / mL. This solution was then filtered through a 0.45 μm filter membrane for later use. The Pu-erh tea fresh leaf polysaccharide aqueous solution and the CAF aqueous solution prepared above were mixed at a volume ratio of 2:13 to form a self-assembled nanomaterial of CTP1 and CAF (referred to as CTP1-CAF). This mixture was prepared and used immediately.

[0108] Example 4 Characterization of Nanomaterials

[0109] 10 μL of CTP1-EGCG prepared in Example 2 and CTP1-CAF prepared in Example 3 were taken respectively, and the morphology of the nanomaterials was observed using transmission electron microscopy (TEM).

[0110] See results Figure 11 and Figure 12 The results showed that CTP1 can self-assemble with EGCG and CAF to form spherical nanoparticles, respectively.

[0111] Example 5 Immunomodulatory activity of nanomaterials formed by polysaccharide CTP1 with EGCG and CAF, respectively

[0112] Specific methods for in vitro immunomodulatory activity:

[0113] (1) Phagocytosis of RAW264.7 macrophages was measured using the neutral red assay. RAW264.7 macrophages were subjected to a phagocytic activity of 2 × 10⁻⁶ cells / mL. 4 Cells were seeded at 2 × 10⁶ cells per well in 96-well plates and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO₂ with different concentrations (12.5, 25, and 50 μg / mL) of CTP1, EGCG, and CTP1-EGCG nanomaterials, respectively. Alternatively, RAW264.7 macrophages were seeded at 2 × 10⁶ cells per well. 4 Cells were seeded per well in 96-well plates and incubated for 24 h at 37 °C with different concentrations (12.5, 25, and 50 μg / mL) of CAF, CTP1, and CTP1-CAF nanomaterials. A positive control group was prepared with 1 μg / mL lipopolysaccharide (LPS), and an equal volume of DMEM medium was used as a blank control. After incubation, 150 μL of 0.075% neutral red was added to each well to replace the drug-treated groups, and the cells were incubated for 1 h at 37 °C with 5% CO2. Unphased neutral red was discarded, and the cells were washed twice with PBS. Lysis was performed at 25 °C for 1 h using lysis buffer (acetic acid:ethanol = 1:1). Absorbance was measured at 540 nm using a microplate reader. The phagocytic rate of RAW264.7 macrophages was calculated using the following formula: Phagocytic rate (%) = (AS / A0) × 100%; A0 is the absorbance value of the control group; AS is the absorbance value of the treatment group.

[0114] Figure 13The results showed that both CTP1-EGCG and CTP1-CAF nanomaterials exerted immunomodulatory effects by activating RAW264.7 monocytes and macrophages and enhancing their phagocytic capacity. Furthermore, the effect of CTP1-EGCG nanomaterials in enhancing the phagocytic capacity of RAW264.7 cells was significantly higher than that of EGCG and CTP1 monomers (P<0.05, *), and the effect of CTP1-CAF nanomaterials in enhancing the phagocytic capacity of RAW264.7 cells was significantly higher than that of CAF monomers (P<0.05, *). In summary, the self-assembled nanomaterials based on CTP1 exhibit superior immunomodulatory activity in enhancing macrophage phagocytic capacity.

[0115] (2) The effect of RAW264.7 macrophages on NO secretion was determined by the Griess method. RAW264.7 macrophages were subjected to a concentration of 2 × 10⁻⁶ cells / mL. 4 Cells were seeded at 2 × 10⁶ cells per well in 96-well plates and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO₂ with different concentrations (12.5, 25, and 50 μg / mL) of CTP1, EGCG, and CTP1-EGCG nanomaterials, respectively. Alternatively, RAW264.7 macrophages were seeded at 2 × 10⁶ cells per well. 4 Cells were seeded into each well of a 96-well plate and incubated for 24 h at 37 °C under a humidified atmosphere of 5% CO2 with different concentrations (12.5, 25, and 50 μg / mL) of CTP1, CAF, and CTP1-CAF nanomaterials. 1 μg / mL LPS was used as a positive control, and an equal volume of DMEM medium was used as a blank control. The cell supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 min. The absorbance was read at 540 nm using a microplate reader, and the NO content was calculated using a standard curve obtained with NaNO2.

[0116] Figure 14The results showed that both CTP1-EGCG and CTP1-CAF nanomaterials could promote NO secretion from RAW264.7 cells to exert immunomodulatory effects. Although the ability of CTP1-EGCG nanomaterials to promote NO secretion from RAW264.7 cells was lower than that of CTP1 monomer, it was significantly higher than that of EGCG monomer within the experimental concentration range (P<0.05, *). Similarly, CTP1-CAF nanomaterials also had similar effects to CTP1-EGCG. In particular, at a low concentration (12.5 µg / mL), the ability of CTP1-CAF nanomaterials to promote NO secretion from RAW264.7 cells was significantly higher than that of CTP1 and CAF monomers. In conclusion, self-assembled nanomaterials based on CTP1 have the advantage of promoting NO secretion from macrophages to exert immunomodulatory activity.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polysaccharide from fresh Pu-erh tea leaves, characterized in that, The polysaccharide from fresh Pu-erh tea leaves is a homogeneous polysaccharide. The repeating sequence of this polysaccharide consists of 1,4-α-D-galacturonic acid, 1,3,6-β-D-galactose, and 1,4,6-β-D-galactose as the main chain, with β-D-glucuronic acid, α-L-arabinose, and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf as the side chains. 1,4-α-D-galacturonic acid is methylated, with a methylation degree of 32.08%. The structure of this Pu-erh tea leaf polysaccharide is as follows: Formula 1; The molecular weight of the polysaccharide from fresh Pu-erh tea leaves is 31241 kDa; the monosaccharide composition of the polysaccharide from fresh Pu-erh tea leaves is rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose; the molar ratio of rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose is 1.55:2.47:35.06:25.63:35.

28.

2. A method for preparing the polysaccharide from fresh Pu-erh tea leaves according to claim 1, characterized in that, Includes the following steps: (1) Pulverize the dried Pu-erh tea leaves and sieve them to obtain tea powder; (2) The tea powder was soaked in the first ethanol solution to remove the fat, and the defatted powder was obtained; (3) Mix the defatted powder with water and extract, collect the filtrate to obtain the extract, concentrate the extract under reduced pressure to obtain the concentrated extract; (4) The concentrated extract is mixed with the second ethanol solution to precipitate, and the precipitate is obtained by centrifugation, collection of precipitate, redissolution of precipitate, removal of protein in redissolution precipitate, collection of supernatant to obtain protein-free extract, drying to obtain Pu-erh tea crude polysaccharide; (5) Static adsorption of crude polysaccharide of Pu'er tea with macroporous adsorption resin suspension, shaken to obtain decolorized suspension, filtered, washed, collected eluent a, dried to obtain decolorized polysaccharide of Pu'er tea; (6) After the polysaccharide of Pu-erh tea is dissolved, the supernatant is collected by centrifugation and added to a chromatography column filled with DEAE cellulose DE-52. The column is eluted with water and 0.1M NaCl solution in sequence. The eluent after elution with 0.1M NaCl solution is collected, concentrated, dialyzed for the first time, and dried to obtain the polysaccharide subfraction. (7) The polysaccharide subfractions were purified by using an S-400 propylene dextran gel column, eluted, and the homogeneous fractions were collected, concentrated, subjected to a second dialysis, and dried to obtain polysaccharides from fresh Pu-erh tea leaves.

3. The preparation method according to claim 2, characterized in that, The volume fraction of the first ethanol solution is 80-90%; the degreasing is performed 1-4 times, with each degreasing session lasting 3-4 hours; the mass-to-volume ratio of the tea powder to the first ethanol solution is 1 kg: 3-4 L.

4. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of the defatted powder to water is 1:10~20; the extraction is performed 1~4 times, the extraction temperature is 90~100℃, and the extraction time for each extraction is 2~4 hours.

5. The preparation method according to claim 2, characterized in that, The volume ratio of the concentrated extract to the second ethanol solution is 1:2~4; the volume percentage of the second ethanol solution is 80~97%; and the solvent for the reconstituted precipitate is water at 50~60℃.

6. The preparation method according to claim 2, characterized in that, The macroporous adsorption resin is macroporous adsorption resin D101; the macroporous adsorption resin suspension is obtained by mixing macroporous adsorption resin and water at a ratio of 450~550g:1000mL; the shaking temperature is 40~60℃; the shaking time is 4~6h; the shaking frequency is 110~130r / min; the solvent for dissolving the Pu-erh tea decolorizing polysaccharide is water, and the mass-volume ratio of the Pu-erh tea decolorizing polysaccharide to water is 15~25mg:1mL; in step (6), the elution flow rate is 0.5~1.5mL / min, and the elution time with water and 0.1M NaCl solution is 5~7h; the molecular weight cutoff of the first dialysis and the second dialysis is 3000~4000Da; in step (7), the elution solvent is water, and the elution rate is 0.1~0.5mL / min. The aforementioned group consisted of components whose retention time in the HPLC-RID chromatogram was 7.9 to 8.0 min, obtained by elution using HPLC-RID detection. The HPLC-RID chromatographic conditions were as follows: Column: Thermo Acclaim 120 C 18 Mobile phase conditions: Mobile phase A is a pH 6.72 NaH2PO4 / Na2HPO4 buffer solution, and mobile phase B is acetonitrile; the preparation method of the NaH2PO4 / Na2HPO4 buffer solution includes mixing 1.6g NaH2PO4, 0.8g Na2HPO4 and 100mL water. Elution program: 0-60 min; mobile phase A: mobile phase B volume ratio = 83:17; flow rate: 1 mL / min; column temperature: 30℃.

7. A self-assembled nanomaterial, characterized in that, The Pu-erh tea fresh leaf polysaccharide of claim 1 or the Pu-erh tea fresh leaf polysaccharide prepared by any one of claims 2 to 6 is dissolved in water to obtain an aqueous solution of Pu-erh tea fresh leaf polysaccharide; the active small molecule compound is mixed with water to obtain an aqueous solution of the active small molecule compound, and then the Pu-erh tea fresh leaf polysaccharide aqueous solution and the active small molecule compound aqueous solution are co-assembled to obtain a self-assembled nanomaterial; the active small molecule compound includes one of epigallocatechin gallate and caffeine.

8. The self-assembled nanomaterial according to claim 7, characterized in that, The mass-to-volume ratio of the polysaccharide from fresh Pu-erh tea leaves to water is 3-5:1; the mass-to-volume ratio of the active small molecule compound to water is 0.5-1.5:1; and the volume ratio of the aqueous solution of the polysaccharide from fresh Pu-erh tea leaves to the aqueous solution of the active small molecule compound is 1-3:10-15.

9. The application of the Pu-erh tea fresh leaf polysaccharide according to claim 1, the Pu-erh tea fresh leaf polysaccharide prepared by the preparation method according to any one of claims 2 to 6, or the self-assembled nanomaterial according to claim 7 or 8 in the preparation of immunomodulatory products.

Images