Fresh Pu'er tea leaf polysaccharide, preparation method thereof, self-assembled nano material and application

Abstract

The invention provides Pu'er tea fresh leaf polysaccharide, a preparation method thereof, a self-assembled nano material and application, and belongs to the technical field of biological medicine. The Pu'er tea fresh leaf polysaccharide is homogeneous polysaccharide, and the repetitive sequence of the Pu'er tea fresh leaf polysaccharide is as follows: 1, 4-alpha-D-galacturonic acid, 1, 3, 6-beta-D-galactose and 1, 4, 6-beta-D-galactose are used as main chains, beta-D-glucuronic acid, alpha-L-arabinose and alpha-D-Rhap-(1-> 3)-alpha-L-Araf-(1-> 5)-alpha-L-Araf are used as side chains, and 1, 4, 6-beta-D-galactose and 1, 4, 6-beta-D-galactose are used as main chains. The Pu'er tea fresh leaf polysaccharide has methyl esterification in the presence of 2, 4-alpha-D-galacturonic acid, the methyl esterification degree is 32.08%, and the structure of the Pu'er tea fresh leaf polysaccharide is shown as a formula 1. The self-assembled nano material and the Pu'er tea fresh leaf polysaccharide have immunoregulatory activity, and the self-assembled nano material has better immunoregulatory activity than a single polysaccharide molecule or a single active small molecule compound.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a Pu'er tea fresh leaf polysaccharide and a preparation method thereof, a self-assembling nano material and an application thereof. Background Art

[0002] Tea is the most popular beverage in the world, and people have been drinking tea for the stimulation and desire of health since the cultivation of tea trees. Most researchers focus on low molecular weight compounds in tea, such as polyphenols, catechins, caffeine, and theanine. As a result, many significant advances have been made in understanding the biological activities of caffeine and polyphenols in tea. In recent studies, polysaccharides from tea have attracted more and more interest from researchers and revealed many biological activities, such as immune, antioxidant, anticancer, and anti-HIV activities. With the in-depth study of tea polysaccharides, more and more scholars have found that tea polysaccharides can usually bind to low molecular weight compounds and proteins through covalent and non-covalent bonds to form polysaccharide conjugates in tea.

[0003] At present, the research on the physicochemical properties and biological activities of tea polysaccharides is mostly focused on crude leaves and green tea, and there is little research on tea polysaccharides from other teas. Other types of tea, such as Pu'er tea polysaccharides in black tea, may have different physicochemical properties and biological activities due to the fermentation process. At present, the research on Pu'er tea polysaccharides only stays at the level of crude tea polysaccharides, and the research direction is mainly focused on extraction, separation and physicochemical property analysis. It is almost impossible to determine its structural characteristics and evaluate its exact efficacy. The molecular structure of Pu'er tea polysaccharides has not yet been reported. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a Pu'er tea fresh leaf polysaccharide and a preparation method thereof, a self-assembled nanomaterial and an application thereof, wherein the Pu'er tea fresh leaf polysaccharide and the self-assembled nanomaterial have an immunomodulatory effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a Pu'er tea fresh leaf polysaccharide. 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-α-D-galacturonic acid, 1,3,6-β-D-galactose and 1,4,6-β-D-galactose as the main chain, β-D-glucuronic acid, α-L-arabinose and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf as the side chain, 1,4-α-D-galacturonic acid is methyl esterified, and the methyl esterification degree is 32.08%. The Pu'er tea fresh leaf polysaccharide structure is: Formula 1.

[0006] Preferably, the molecular weight of the Pu'er tea fresh leaf polysaccharide is 31241 KDa; the monosaccharide composition of the Pu'er tea fresh leaf polysaccharide 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.

[0007] The present invention provides a method for preparing polysaccharides from fresh Pu'er tea leaves, comprising the following steps: (1) crushing and sieving the dried fresh Pu'er tea leaves to obtain tea powder; (2) soaking the tea powder in the first ethanol solution for degreasing to obtain defatted powder; (3) mixing the defatted powder with water for extraction, collecting the filtrate to obtain an extract, and concentrating the extract under reduced pressure to obtain a concentrated extract; (4) mixing the concentrated extract with the second ethanol solution for precipitation to obtain an alcohol-precipitated solution, centrifuging, collecting the precipitate, re-dissolving the precipitate, removing protein from the re-dissolved precipitate, collecting the supernatant to obtain a deproteinized extract, and drying to obtain Pu'er tea crude polysaccharides; (5) statically adsorbing the Pu'er tea crude polysaccharide with a macroporous adsorption resin suspension, shaking to obtain a decolorized suspension, filtering, washing, collecting the eluate a, and drying to obtain the Pu'er tea decolorized polysaccharide; (6) After the decolorized Pu'er tea polysaccharide is dissolved, the supernatant is collected by centrifugation and added to a chromatography column filled with DEAE cellulose DE-52, and eluted with water and 0.1M NaCl solution in sequence. The eluate after elution with 0.1M NaCl solution is collected, concentrated, dialyzed, and dried to obtain polysaccharide subfractions; (7) Purify the polysaccharide subcomponents using an propylene glycol gel S-400 gel column, elute, collect the homogeneous components, concentrate, perform a second dialysis, and dry to obtain Pu'er tea fresh leaf polysaccharides.

[0008] Preferably, the volume fraction of the first ethanol solution is 80-90%; the number of degreasing is 1-4 times, and the time of each degreasing is 3-4 hours; the mass volume ratio of the tea powder to the first ethanol solution is 1kg:3-4L.

[0009] Preferably, the mass 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° C., and the time for each extraction is 2-4 hours.

[0010] 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 re-dissolving the precipitate is 50-60° C. water.

[0011] Preferably, the macroporous adsorption resin is macroporous adsorption resin D101; the macroporous adsorption resin suspension is obtained by mixing macroporous adsorption resin and water according to 450-550 g:1000 mL; the shaking temperature is 40-60 ° C; the shaking time is 4-6 h; the shaking frequency is 110-130 r / min; the solvent for dissolving the bleached Pu'er tea polysaccharide is water, and the mass volume ratio of the bleached Pu'er tea polysaccharide to water is 15-25 mg:1 mL; in step (6), the elution flow rate is 0.5-1.5 mL / min, and the elution time with water and 0.1 M NaCl solution is 5-7 h respectively; the molecular weight cutoff of the first dialysis and the second dialysis is 3000-4000 Da; in step (7), the elution solvent is water, and the elution rate is 0.1-0.5 mL / min; The homogeneous component is an eluate obtained by elution detection by HPLC-RID, and a component with a retention time of 7.9 to 8.0 min in the HPLC-RID chromatogram; The HPLC-RID chromatographic conditions were as follows: chromatographic column: Thermo Acclaim 120 C18; mobile phase conditions: mobile phase A was pH 6.72 NaH 2 PO 4 / Na 2 HPO 4 Buffer, mobile phase B is acetonitrile; the NaH 2 PO 4 / Na 2 HPO 4 The buffer solution was prepared by mixing 1.6 g NaH 2 PO 4 , 0.8 g Na 2 HPO 4 Prepared by mixing with 100 mL of water; Elution program: 0~60min mobile phase A:mobile phase B volume ratio = 83:17; flow rate: 1mL / min; column temperature: 30℃.

[0012] The invention provides a self-assembling nano material. The above-mentioned Pu'er tea fresh leaf polysaccharide is dissolved in water to obtain a Pu'er tea fresh leaf polysaccharide aqueous solution; an active small molecule compound is mixed with water to obtain an active small molecule compound aqueous solution; and then the Pu'er tea fresh leaf polysaccharide aqueous solution and the active small molecule compound aqueous solution are co-assembled to obtain the self-assembling nano material; the active small molecule compound comprises one of epigallocatechin gallate and caffeine.

[0013] Preferably, the mass volume ratio of the Pu'er tea fresh leaf polysaccharide to water is 3~5:1; the mass volume ratio of the active small molecule compound to water is 0.5~1.5:1; the volume ratio of the Pu'er tea fresh leaf polysaccharide aqueous solution to the active small molecule compound aqueous solution is 1~3:10~15.

[0014] The present invention also provides an application of the above Pu'er tea fresh leaf polysaccharide or self-assembled nanomaterial in preparing an immunomodulatory product.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a Pu'er tea fresh leaf polysaccharide and a preparation method thereof, a self-assembled nanomaterial and an application thereof. The present invention identifies a new natural polysaccharide (CTP1) from Pu'er tea fresh leaves. The Pu'er tea fresh leaf polysaccharide is a new uniform polysaccharide with a molecular weight of 31241 KDa. Based on the physicochemical properties of the new polysaccharide, a polysaccharide platform is established to construct various types of self-assembled nanomaterials, such as polysaccharide-active small molecule compound nanomaterials. The self-assembled nanomaterial and Pu'er tea fresh leaf polysaccharide activate mononuclear macrophage RAW264.7 and improve its phagocytic ability, thereby regulating the NO secretion level, thereby having an immunoregulatory effect. And the self-assembled nanomaterial has better immunoregulatory activity than a single polysaccharide molecule or a single active small molecule compound. The present invention co-assembles Pu'er tea fresh leaf polysaccharide with a variety of active small molecule compounds, providing a new strategy for constructing powerful nanomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the HPLC chromatogram of CTP1; Figure 2 is the HPLC chromatogram of the monosaccharide composition of CTP1; Figure 3 is the infrared spectrum of CTP1; Figure 4 is the UV spectrum of CTP1; Figure 5 A in the figure is the NMR of CTP1 1 H NMR spectrum; B is that of CTP1 13 C is the DEPT-90 NMR spectrum of CTP1; D is the DEPT-135 NMR spectrum of CTP1; Figure 6 is the NMR TOCSY spectrum of CTP1; Figure 7 is the NMR HSQC spectrum of CTP1; Figure 8 This is the HMBC spectrum of the NMR labeled anomeric region of CTP1; Fig. 9This is the HMBC spectrum of the NMR labeled aliphatic region of CTP1; Fig.10 is the structure of CTP1; Fig.11 is the transmission electron microscopy image of CTP1-EGCG; Fig.12 is the transmission electron microscopy image of CTP1-CAF; Fig.13 The results are the comparison of the phagocytic ability of CTP1, EGCG, CAF, CTP1-EGCG and CTP1-CAF; Fig.14 The results are a comparison of the NO secretion levels of CTP1, EGCG, CAF, CTP1-EGCG and CTP1-CAF. DETAILED DESCRIPTION

[0017] The invention provides a Pu'er tea fresh leaf polysaccharide. 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-α-D-galacturonic acid, 1,3,6-β-D-galactose and 1,4,6-β-D-galactose as the main chain, β-D-glucuronic acid, α-L-arabinose and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf as the side chain, 1,4-α-D-galacturonic acid is methyl esterified, and the methyl esterification degree is 32.08%. The Pu'er tea fresh leaf polysaccharide structure is: Formula 1.

[0018] In the present invention, the molecular weight of the Pu'er tea fresh leaf polysaccharide is 31241KDa; the monosaccharide composition of the Pu'er tea fresh leaf polysaccharide 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.

[0019] The present invention has found that the polysaccharide from fresh leaves of Pu'er tea can activate macrophages and improve their phagocytic ability, regulate the NO secretion level, and has immunomodulatory activity.

[0020] The present invention provides a method for preparing polysaccharides from fresh Pu'er tea leaves, comprising the following steps: (1) crushing and sieving the dried fresh Pu'er tea leaves to obtain tea powder; (2) soaking the tea powder in the first ethanol solution for degreasing to obtain defatted powder; (3) mixing the defatted powder with water for extraction, collecting the filtrate to obtain an extract, and concentrating the extract under reduced pressure to obtain a concentrated extract; (4) mixing the concentrated extract with the second ethanol solution for precipitation to obtain an alcohol-precipitated solution, centrifuging, collecting the precipitate, re-dissolving the precipitate, removing protein from the re-dissolved precipitate, collecting the supernatant to obtain a deproteinized extract, and drying to obtain Pu'er tea crude polysaccharides; (5) statically adsorbing the Pu'er tea crude polysaccharide solution with a macroporous adsorption resin, shaking to obtain a suspension, filtering, washing, collecting the filtrate, and drying to obtain Pu'er tea decolorized polysaccharide; (6) After the decolorized polysaccharide of Pu'er tea is dissolved, the supernatant is collected by centrifugation and added to a chromatography column filled with DEAE cellulose DE-52 (purchased from Beijing Solebow Technology Co., Ltd., catalog number C8930), and eluted with water and 0.1 M NaCl solution in sequence. The eluate after elution with 0.1 M NaCl solution is collected, concentrated, dialyzed, and dried to obtain polysaccharide subfractions; (7) Purify the polysaccharide subfractions using an propylene glycol gel S-400 (purchased from Stefan Biotechnology Co., Ltd., catalog number 17060901) gel column, elute, collect the fractions, concentrate, perform a second dialysis, and dry to obtain Pu'er tea fresh leaf polysaccharides.

[0021] In the present invention, the dried fresh Pu'er tea leaves are crushed and sieved to obtain tea powder. The drying method can be air-dried, oven-dried or freeze-dried. The mesh number of the sieve is preferably 50-70 mesh, more preferably 55-65 mesh, and more preferably 60 mesh.

[0022] In the present invention, in order to remove lipid substances in tea leaves, thereby improving the purity of Pu'er tea fresh leaf polysaccharides. After obtaining tea powder, the tea powder is soaked in a first ethanol solution for degreasing to obtain a defatted powder. The volume fraction of the first ethanol solution is preferably 80-90%, more preferably 83-87%, and more preferably 85%; the number of degreasing is preferably 1-4 times, more preferably 2-3 times, and more preferably 3 times; the time for each degreasing is preferably 3-4 hours; the mass volume ratio of the tea powder to the first ethanol solution is preferably 1kg:3-4L.

[0023] In the present 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 mass volume ratio of the defatted powder to water is preferably 1:10-20, more preferably 1:12-18, and more preferably 1:15; the number of extractions is preferably 1-4 times, more preferably 2-3 times, and more preferably 3 times. The extraction temperature is preferably 90-100°C, more preferably 92-97°C, and more preferably 95°C. The time for each extraction is preferably 2-4h, more preferably 2.5-3.5h, and more preferably 3h. The temperature of the reduced pressure concentration is preferably 50-60°C, more preferably 52-58°C, and more preferably 55°C. The pressure of the reduced pressure concentration is preferably 0.05-0.08MPa, and more preferably 0.07MPa. The present invention can effectively increase the polysaccharide content and the yield of fresh Pu'er tea leaf polysaccharides by mixing the defatted powder with water for extraction.

[0024] In the present invention, after obtaining the concentrated extract, the concentrated extract is mixed with the second ethanol solution for precipitation to obtain an alcohol-precipitated solution, centrifuged, and the precipitate is collected. 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% ethanol solution, more preferably 95% ethanol solution. The centrifugation method can be 3500-4500rpm centrifugal alcohol-precipitated solution for 10-20min, such as 4000rpm centrifugal alcohol-precipitated solution for 15min. The present invention can effectively remove small molecular polyphenols and pigment components by alcohol precipitation, and improve the yield and purity of Pu'er tea fresh leaf polysaccharides.

[0025] In the present invention, after collecting the precipitate, the precipitate is redissolved, the protein in the redissolved precipitate is removed, the supernatant is collected, a deproteinized extract is obtained, and the extract is dried to obtain Pu'er tea crude polysaccharide. The solvent for the redissolved precipitate is preferably 50-60°C water, and more preferably 60°C water. The method for removing protein from the redissolved precipitate can be a mixed reagent of chloroform: n-butanol in a volume ratio of 4:1. The collection method can be centrifugation, and the centrifugal method is preferably centrifugation at 0°C and 3500-4500r / min for 10-20min, such as centrifugation at 0°C and 4000r / min for 15min. The drying can be selected as freeze-drying. The present invention does not specifically limit the specific method of freeze-drying, and conventional freeze-drying methods known in the art can be used.

[0026] In the present invention, after obtaining the crude polysaccharide of Pu'er tea, the crude polysaccharide of Pu'er tea is subjected to static adsorption with a macroporous adsorption resin suspension, shaken to obtain a suspension, filtered, washed, the filtrate is collected, and dried to obtain decolorized polysaccharide of Pu'er tea. The macroporous adsorption resin is preferably macroporous adsorption resin D101. The macroporous adsorption resin suspension is obtained by mixing a macroporous adsorption resin with water at a ratio of 450-550 g:1000 mL, and the macroporous adsorption resin is a pretreated macroporous adsorption resin; the preparation method of the pretreated macroporous adsorption resin is as follows: soaking the macroporous adsorption resin D101 in a 95% by volume ethanol solution for 12 hours, washing with distilled water until the supernatant is free of turbidity, filtering with gauze, and then soaking in a 4% by mass NaOH solution for 4 hours, washing with distilled water until neutral, and washing with a mass fraction of 5%. Soak in HCl solution for 4 hours, wash with distilled water until neutral, filter with gauze and dry the material to obtain the pretreated macroporous adsorption resin; the shaking temperature is preferably 40-60°C, more preferably 45-55°C, and more preferably 50°C; the shaking time is preferably 4-6 hours, more preferably 4.5-5.5 hours, and more preferably 5 hours; the shaking frequency is preferably 110-130 r / min, further preselected as 115-125 r / min, and more preferably 120 r / min. The filtering method can be selected as gauze filtering, the washing solvent is water, and the washing amount is 1-4 L, and more preferably 2 L. The drying method can be selected as freeze-drying. The present invention adopts a static adsorption method for decolorization, which can effectively remove pigments such as phenols and improve the yield of polysaccharides.

[0027] In the present invention, after obtaining the decolorized Pu'er tea polysaccharide, the decolorized Pu'er tea polysaccharide is dissolved, the supernatant is collected by centrifugation, and added to a chromatography column filled with DEAE cellulose DE-52 (purchased from Beijing Solebow Technology Co., Ltd., item number C8930), eluted with water and 0.1M NaCl solution in sequence, and the eluate after elution with 0.1M NaCl solution is collected, concentrated, dialyzed for the first time, and dried to obtain polysaccharide subcomponents. The solvent for dissolving the decolorized polysaccharide of Pu'er tea is water, and the mass volume ratio of the decolorized polysaccharide of Pu'er tea to water is preferably 15~25mg:1mL, and more preferably 20mg:1mL; the flow rate of the elution is preferably 0.5~1.5mL / min, and more preferably 1mL / min; the elution time with water and 0.1M NaCl solution is 5~7h, that is, the elution time with water is 5~7h first, and then the elution time with 0.1M NaCl solution is 5~7h; the molecular weight cutoff of the first dialysis is 3000~4000Da, and more preferably 3500Da. The drying method can be selected as freeze-drying. The present invention does not specifically limit the specific method of freeze-drying, and conventional freeze-drying methods known in the art can be used. The present invention collects the eluate obtained by eluting water and 0.1M NaCl solution respectively using an automatic collector, and uses the phenol-sulfuric acid method to monitor the carbohydrate content in each tube of the eluate. The results show that the eluate after elution with 0.1MNaCl solution contains the component with the highest carbohydrate content.

[0028] In the present invention, after obtaining the polysaccharide subcomponents, the polysaccharide subcomponents are purified using a polypropylene polydextrose gel S-400 (purchased from Si Tuofan Biotechnology Co., Ltd., item number 17060901) gel column, eluted, and the uniform components are collected, concentrated, dialyzed for the second time, and dried to obtain Pu'er tea fresh leaf polysaccharides. The elution solvent is water, and the elution rate is preferably 0.1~0.5mL / min, more preferably 0.2~0.4mL / min, and more preferably 0.3mL / min. The present invention does not specifically limit the specific method of concentration, and conventional methods in the art can be used, such as reduced pressure concentration. The molecular weight cutoff of the second dialysis is 3000~4000Da, and more preferably 3500Da. The drying method can be selected as freeze-drying. The present invention does not specifically limit the specific method of freeze-drying, and conventional freeze-drying methods known in the art can be used.

[0029] In the present invention, the homogeneous component is an eluate obtained by eluting with HPLC-RID, and the component with a retention time of 7.9 to 8.0 min in the HPLC-RID chromatogram, such as a component with a retention time of 7.957 min; the chromatographic conditions of the HPLC-RID are: chromatographic column: Thermo Acclaim 120 C18; mobile phase conditions: mobile phase A is pH 6.72 NaH 2 PO 4 / Na 2 HPO 4 Buffer, mobile phase B is acetonitrile; the NaH 2 PO 4 / Na 2 HPO 4 The buffer solution was prepared by mixing 1.6 g NaH 2 PO 4 , 0.8 g Na 2 HPO 4 and 100 mL of water; elution procedure: 0-60 min mobile phase A: mobile phase B volume ratio = 83:17; flow rate: 1 mL / min; column temperature: 30° C. The detection wavelength of the HPLC-RID: 250 nm; injection volume: 10 μL.

[0030] In the above preparation of NaH 2 PO 4 / Na 2 HPO 4 When the buffer solution is used, the mixing method can be stirring to mix the NaH 2 PO 4 and Na 2 HPO 4 Dissolve completely and mix well.

[0031] The Pu'er tea fresh leaf polysaccharide prepared by the above preparation method has a high purity of 98%.

[0032] The invention provides a self-assembling nano material. The above-mentioned Pu'er tea fresh leaf polysaccharide is dissolved in water to obtain a Pu'er tea fresh leaf polysaccharide aqueous solution; an active small molecule compound is mixed with water to obtain an active small molecule compound aqueous solution; and then the Pu'er tea fresh leaf polysaccharide aqueous solution and the active small molecule compound aqueous solution are co-assembled to obtain the self-assembling nano material; the active small molecule compound comprises one of epigallocatechin gallate and caffeine.

[0033] In the present invention, the mass volume ratio of the Pu'er tea fresh leaf polysaccharide to water is 3~5mg:1mL; the mass volume ratio of the active small molecule compound to water is 0.5~1.5mg:1mL; the volume ratio of the Pu'er tea fresh leaf polysaccharide aqueous solution to the active small molecule compound aqueous solution is 1~3:10~15.

[0034] The present invention also provides an application of the above Pu'er tea fresh leaf polysaccharide or self-assembled nanomaterial in preparing an immunomodulatory product.

[0035] In the present invention, the product can be a medicine, a reagent, a health product or a food. The Pu'er tea fresh leaf polysaccharide of the present invention activates mouse mononuclear macrophage RAW264.7 and improves its phagocytic ability, regulates the NO secretion level, and thus has immunomodulatory activity, and the self-assembled nanomaterial has better immunomodulatory activity than Pu'er tea fresh leaf polysaccharide or a single active small molecule compound. The present invention can use Pu'er tea fresh leaf polysaccharide or self-assembled nanomaterial as the active ingredient of the above-mentioned product in combination with other products with immunomodulatory effects to achieve the purpose of immunomodulation, and can also use Pu'er tea fresh leaf polysaccharide or self-assembled nanomaterial as the only active ingredient of the above-mentioned product for immunomodulation, thereby improving immune ability. The dosage form of the product includes tablets, syrups, injections, capsules or granules. The Pu'er tea fresh leaf polysaccharide or self-assembled nanomaterial accounts for 20-90% of the weight of the product.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1 Extraction, separation, purification and structural identification of Pu'er tea fresh leaf polysaccharide (CTP1) 1. Extraction, separation and purification of Pu'er tea fresh leaf polysaccharide (CTP1) (1) Fresh Pu'er tea leaves are air-dried and crushed, and passed through a 60-mesh sieve to obtain tea powder; (2) 2 kg of tea powder was soaked in 4 L of 85% ethanol solution for 4 h to degrease, and the defatted powder a was collected; the defatted powder a was added to 4 L of 85% ethanol solution for 4 h to degrease, and the defatted powder b was obtained; the defatted powder b was added to 4 L of 85% ethanol solution for 4 h to degrease, and the defatted powder c was obtained; (3) 1 kg of defatted powder c was extracted with 15 L of distilled water at 95° C. for 3 hours each time, for a total of 3 times, and the filtrate after each extraction was collected and combined to obtain an extract; the extract was concentrated to 1 / 9 of the original extract volume by a rotary evaporator at 55° C. under reduced pressure to obtain a concentrated extract; the concentrated extract was added with 3 times the volume of 95% ethanol solution at room temperature for precipitation overnight to obtain an alcohol-precipitated solution, and the alcohol-precipitated solution was centrifuged at 4000 rpm for 15 minutes to collect the precipitate; the precipitate was re-dissolved in 60° C. hot water and then Sevage (the Sevage is composed of chloroform and n-butanol, and the volume ratio of chloroform:n-butanol is 4:1) was used to remove the protein in the precipitate, and the supernatant was collected by centrifugation at 0° C. and 4000 r / min for 15 minutes to obtain a deproteinized extract; the deproteinized extract was freeze-dried to obtain 66 g of Pu'er tea crude polysaccharide; (4) After soaking the macroporous adsorption resin D101 in 95% by volume ethanol solution for 12 h, wash it with distilled water until the supernatant is free of turbidity, filter it with gauze, soak it in 4% by mass NaOH solution for 4 h, wash it with distilled water until it is neutral, soak it in 5% by mass HCl solution for 4 h, wash it with distilled water until it is neutral, filter it with gauze, and dry the material. Take 500 g of the dried material and add 1 L of water to obtain a 0.5 g / mL pretreated macroporous adsorption resin D101 suspension. Take 500 mL of the pretreated macroporous adsorption resin D101 suspension, add 1 g of the Pu'er tea crude polysaccharide prepared in step (3), shake it at a constant temperature of 50°C for 5 h, and shake it at a frequency of 120 r / min to obtain a decolorized suspension. After the shaking, filter it with gauze and rinse the solid phase with 2 L of water. Collect the liquid phase, concentrate the liquid phase under reduced pressure, and then freeze-dry it to obtain a decolorized Pu'er tea polysaccharide.

[0038] (5) 400 mg of Pu'er tea decolorized polysaccharide was dissolved in 20 mL of distilled water, centrifuged at 3000 rpm for 5 min, and the supernatant was collected and loaded onto a chromatography column (2.9×60 cm) filled with DEAE cellulose DE-52 (purchased from Beijing Solebow Technology Co., Ltd., product number C8930). Linear gradient elution was performed 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, and each concentration was eluted for 6 h. The eluates after elution with water, 0.1 M, 0.3 M, and 0.5 M NaCl solutions were collected respectively; the eluates after elution with water, 0.1 M, 0.3 M, and 0.5 M NaCl solutions were concentrated, dialyzed at 3500 Da, and freeze-dried to obtain four polysaccharide subfractions, DEAE-0, DEAE-1, DEAE-2, and DEAE-3, respectively; (6) The component with the highest carbohydrate content (DEAE-1) was selected for further purification, that is, the DEAE-1 polysaccharide subcomponent was purified by gel permeation chromatography on a polyacrylamide gel S-400 (purchased from Si Tuofan Biotechnology Co., Ltd., item number 17060901) gel column (1.9×110 cm). The elution solvent was water, the flow rate was 0.3 mL / min, and the eluent b was collected by an automatic collector at 2 mL / tube. The polysaccharides in the eluent b were monitored by high performance liquid chromatography-differential refractive index detector (HPLC-RID). The component with a peak time of 7.957 min in the HPLC-RID chromatogram was collected. Then, it was concentrated, dialyzed at 3500 Da, and freeze-dried to obtain Pu'er tea fresh leaf polysaccharide (CTP1).

[0039] The chromatographic conditions of the above HPLC-RID are as follows: Chromatographic column: Thermo Acclaim 120 C 18(4.6 mm × 250 mm, 5 µm); mobile phase conditions: mobile phase A was pH 6.72 NaH 2 PO 4 / Na 2 HPO 4 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.

[0040] The NaH 2 PO 4 / Na 2 HPO 4 The buffer solution was prepared by mixing 1.6 g NaH 2 PO 4 , 0.8 g NaHPO 4 and 100 mL of water, stirring and mixing evenly.

[0041] 2. Structural identification of CTP1 (1) Determination of the molecular weight of the above CTP1 by high performance gel permeation chromatography: The uniformity 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°C; detector temperature: 40°C; mobile phase: ultrapure water; flow rate: 0.8mL / min; injection volume: 10µL; sample concentration: 2mg / mL. The uniformity of the sample was analyzed by peak area normalization method on an HPLC chemical workstation. The logarithmic value (Lg Mw) of the relative molecular mass of a series of different molecular weight dextran standards (5, 12, 25, 50, 410 and 670kDa) was used as the ordinate and the retention time (t) of the chromatographic peak was used as the abscissa to establish a molecular weight distribution standard curve, and the molecular weight distribution of the sample was calculated according to the standard curve equation.

[0042] Results Figure 1 The results show that the CTP1 prepared by the present invention is a homogeneous polysaccharide. According to the established linear regression equation (Lg Mw =-0.6178t+12.453, R 2 =0.9909) and the retention time of CTP1 (t=7.957), the molecular weight of CTP1 was calculated to be 31241 KDa.

[0043] (2) Analysis of monosaccharide composition of CTP1: The monosaccharide composition of CTP1 was analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization-reverse phase HPLC.

[0044] Acid hydrolysis of the sample: 5 mg of CTP1 was accurately weighed and mixed with 2 mL of 4 M trifluoroacetic acid and stirred at 110°C for 11 h. The reaction product was dissolved in methanol and concentrated under reduced pressure at 50°C to remove the residual TFA. This was repeated 5 times until the TFA was completely removed to obtain the hydrolyzed product.

[0045] Derivatization of samples: Dissolve the dried hydrolyzate in 1 mL of distilled water to obtain a hydrolyzate, then take 50 μL of 5 mg / mL hydrolyzate, 50 μL of 0.3 M NaOH and 100 μL of 0.5 M 1-phenyl-3-methyl-5-pyrazolone and mix them, and react at 70 ° C in the dark for 30 minutes. After adjusting the pH to 7.0 with 0.3 M HCl, add 1 mL of chloroform and repeat the extraction three times. Remove the chloroform at the bottom, collect the top aqueous phase and filter it through a 0.22 μm microporous membrane as the test solution for liquid phase analysis.

[0046] Derivatization of standard products: Weigh an appropriate amount of 10 monosaccharide reference products, including mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc), which have been dried to a constant weight, and dissolve them in ultrapure water to prepare a 1 mg / mL solution. Accurately measure 50 μL and follow the steps of the above sample derivatization treatment. Collect the top aqueous phase and filter it through a 0.22 μm microporous membrane as the reference solution for liquid phase analysis.

[0047] Chromatographic conditions: Chromatographic column: Thermo Acclaim 120 C 18 (4.6 mm × 250 mm, 5 µm); mobile phase conditions: mobile phase A was pH 6.72 NaH 2 PO 4 / Na 2 HPO 4 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.

[0048] 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.

[0049] (3) Infrared spectrum analysis: The infrared spectrum of CTP1 was measured using a Nicolet iS10 Fourier transform infrared spectrophotometer. After the sample was treated with KBr pellets, the background was collected before sampling, and the spectrum was recorded for 32 scans at 4.00 cm -1 Resolution, wave number range is 4000~400cm -1 .

[0050] Results Figure 3 It can be seen that the infrared spectrum of CTP1 shows that at 3397cm -1 and 2932cm -1 The strong absorption bands at 1613 cm-1 are attributed to the stretching vibrations of OH, methyl and methylene. -1 and 1742cm -1 Stretching vibrations from the carboxyl group and the ester carbonyl group were observed at 1413 cm -1 The absorption near 1260cm is caused by the bending vibration of C-OH; -1 and 1237cm -1 The absorption bands at 1330 cm-1 are attributed to the asymmetric stretching vibration of CO in the esterified and unesterified carboxyl groups, respectively; -1 The absorption band at 1143cm -1 、1098cm -1 and 1018cm -1 The absorption of pyranose was observed at 957 cm -1 The absorption at 920~840cm belongs to furanose; -1 Multiple absorption bands were observed in the region, indicating the coexistence of α and β configurations.

[0051] (4) UV spectral analysis: The UV spectrum of 1 mg / mL CTP1 was measured using a Shimadzu UV-2700 UV-visible spectrophotometer in the wavelength range of 190-500 nm. The results are shown in Figure 4 .

[0052] Figure 4 The results showed that CTP1 had no obvious absorption peaks of proteins and nucleic acids in the wavelength range of 260~280nm, indicating that CTP1 did not contain proteins and nucleic acids.

[0053] (5) Nuclear magnetic resonance (NMR) analysis: Predissolve an appropriate amount of CTP1 sample in deuterated water (D 2 O, 99.9%), freeze-dried, repeated three times. 2 O exchange, freeze-dried and washed with 0.5 mL D 2O was dissolved in a 3 mm NMR tube. 1D and 2D NMR spectra were measured at 25°C using a Bruker Avance III 800 MHz NMR spectrometer. Figure 5~Figure 9 .

[0054] In CTP1 1 H NMR spectroscopy (see Figure 5 A in δ H A large number of polysaccharide characteristic proton signals appeared in the signal range of 3.00~5.50ppm. H 4.30~4.95ppm and δ H The signal absorption peaks in the region of 4.95-5.50 ppm confirmed the existence of α- and β-configuration glycosidic bonds in CTP1, which was consistent with the results of infrared spectroscopy. H Eleven 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, which were labeled as residues AK based on the results of monosaccharide composition and methylation analysis of CTP1. H The anomeric proton signal at 4.64 ppm was replaced by D 2 The proton signal of O (δ H 4.69ppm). The non-anomeric proton signals are concentrated in δ H In the 3.10~4.30 ppm region, some individual signals overlap severely and need to be combined with TOCSY spectra ( Figure 6 ) and HSQC spectra ( Figure 7 ) were used to assign the proton chemical shifts of each residue.

[0055] and 1 Compared with the H NMR spectrum, CTP1 13 C NMR spectrum ( Figure 5 The signal distribution in B) is broader. In the low field region δ C The co-existence of the signals at 170.89 and 175.10 ppm confirmed that the uronic acid in CTP1 was partially methylated, which was consistent with the results of methylation analysis. 1 H NMR and 13 The strongest signal in the C NMR spectrum (δ 3.70 / 52.85 ppm) comes from the methoxy group, and the signal at δ 1.15 / 16.52 ppm in the high-field region is attributed to the methyl group of Rha. Figure 5 B) in δ CEleven anomeric carbon signals were shown 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 the HSQC spectrum ( Figure 7 ) cross peaks in the anomeric signal region, the anomeric signals of residues AB and DK were determined 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 loss of the signal in the anomeric region in the HSQC spectrum, the anomeric signal of residue C was mainly determined by the HMBC spectrum ( Figure 8 ) was determined by combining 1H NMR and 13C NMR spectra to be δ 5.14 / 109.24 ppm. From the point of view of HMBC spectroscopy experiments, it is necessary to set a specific filter (145 Hz) to minimize (or filter out) reactions arising from direct correlation, i.e., proton / carbon pairs arising from single bond coupling ( 1 J C , H). However, in general, this setup may not completely eliminate the magnetization generated by the isomeric carbons, resulting in the detection of densities of the isomeric carbons in the HMBC spectrum. They retain the coupling with their own protons, so they appear as two densities along the F2 dimension, with the central position matching the position of the isomeric protons.

[0056] HMBC spectrum of CTP1 (see Figure 8 ) showed two cross peaks at δ 5.25 / 109.24 ppm and δ 5.03 / 109.24 ppm in the anomeric signal region, so the center positions of the two cross peaks along the F2 dimension were determined to be the anomeric signals of residue C. Figure 7 As shown, the mapping of the residue C anomer signal (δ 5.14 / 109.24ppm) also appeared at δ 5.14 / 9.23ppm in the HSQC spectrum, which fully demonstrated the reliability of the attribution of the residue C anomer signal.

[0057] For residues A, F, and G, TOCSY( Figure 6 The cross peaks at δ 5.23 / 4.07, 4.91 / 3.74 and 4.64 / 3.84 ppm in the spectrum were used to determine the H 1 / H 2 . From the TOCSY spectrum ( Figure 6 ) determined the H 3 -H 5All δH and δC in residues A, F, and G were assigned based on cross peaks in the HSQC spectrum and using the HMBC spectrum ( Fig. 9 ) was verified. Based on the results of methylation analysis, residues A, F, and G were identified as →1)-α-D-GalpA-(4→. HMBC spectrum ( Fig. 9 ) shows the correlation of H5 / C6 of residue A, indicating that residue A contains an unmethylated uronic acid group. In addition, based on the correlation of the cross peaks at δ 3.70 / 175.10 and 3.70 / 170.89 ppm in the HMBC spectrum, it is inferred that the C6 of residues F and G 6 Partial methyl esterification may have occurred at this position.

[0058] 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. Figure 6 )δ 3.67 / 1.15(H 3 / H 6 ) ppm cross peak suggests that residue B belongs to the rhamnose unit. HSQC spectrum ( Figure 7 )δ 1.15 / 16.47(H 6 / C 6 ) ppm also shows that residue B belongs to the rhamnose unit. TOCSY spectrum ( 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 C1 -δ C6 . HMBC spectrum ( Fig. 9 )δ 3.33 / 69.13(H 4 / C 5 )、3.33 / 70.00(H 4 / C 3 )、3.92 / 70.00(H 4 / C 5 )、4.06 / 70.00(H 2 / C 3 )、3.33 / 16.47(H 4 / C 6 )、1.15 / 69.13(H 4 / C 5 ) and 1.15 / 71.89 (H 4 / C 5This was confirmed by a cross peak at ppm, and combined with the results of the methylation analysis, residue B was identified as α-D-Rhap-(1→.

[0059] For residues C, D and E, as mentioned above, the anomeric signals of residues C, D and E were confirmed to be δ 5.14 / 109.24, 5.05 / 107.09 and 4.99 / 107.44 ppm, respectively. Their anomeric carbon signals were significantly higher than those of other residues in CTP1. Generally, the anomeric carbon signals of furanose residues are higher. Combined with the results of methylation analysis, it is speculated that residues C, D and E belong to furanose arabinose. 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 spectrum were determined for the H 1 / H 2 .TOCSY spectrum ( Figure 6 ) shows the δ values ​​of residues C, D, and E H2 -δ H6 signal, and according to the HSQC spectrum ( Figure 7 ) δ assigned to residues C, D, and E C2 -δ C6 For residue C, the HMBC spectrum ( Fig. 9 )δ 3.61 / 76.60(H 5a / C 3 )、3.84 / 61.20(H 3 / C 5 )、3.84 / 81.27(H 3 / C 2 )、4.20 / 81.27(H 4 / C 2 )、5.14 / 76.60(H 1 / C 3 ) and 5.14 / 81.27 (H 1 / C 2 ) ppm confirms the reliability of the assignment. Similarly, residues D and E can also be confirmed in the HMBC spectrum. The difference is that the δ C3 The δ of residue E is obviously shifted to the low field region by 6~7ppm. C5 The shift to the downfield region is 5-6 ppm, indicating that residues D and E may be located at C 3 and C 5 The residues C, D and E were identified as α-L-Araf-(1→, →1)-α-L-Araf-(3→ and →1)-α-L-Araf-(5→, respectively. The intensities of the anomeric signals of residues C, D and E in 1D NMR were consistent with the results of quantitative methylation analysis.

[0060] For residue H, TOCSY spectrum ( Figure 6 ) at δ 4.44 / 3.57, 4.44 / 3.85, 4.44 / 3.65, and 4.44 / 4.05 ppm show the H 2 -H 5 In the HSQC spectrum ( Figure 7 ), based on the δ of residue H H1 -δ H6 The δ of residue H is assigned C1 -δ C6 . HMBC spectrum ( Fig. 9 )δ 4.05 / 80.11(H 5 / C 4 )、3.57 / 80.11(H 2 / C 4 ) and 3.65 / 71.89 (H 4 / C 3 This was confirmed by a cross peak at 10 ppm and, combined with the results of the methylation analysis, residue H was identified as (→1)-β-D-GlcpA-(4→.

[0061] For residues I, J, and K, according to TOCSY ( Figure 6 ) and HSQC ( Figure 7 ) spectrum is assigned δ H2 -δ H6 and δ C2 -δ C6 , and using the HMBC spectrum ( Fig. 9 ) was verified. Based on the results of 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. Among them, residues I and J were substituted by -OMe at the O-4 position, respectively. HSQC spectrum ( Figure 7 ) at δ 3.13 / 54.12 and 3.38 / 59.98 ppm were detected by DEPT-135 spectrum ( Figure 5 D) confirmed as the proton / carbon signal of -OMe. Fig. 9 ) is found that the proton of -OMe binds to the C 4 (δ 3.39 / 82.07ppm) and C 5 The cross peak at δ 3.39 / 74.83 ppm in the HMBC spectrum also shows the cross peak between the proton of -OMe and the C of residue J.4 There is a strong correlation between them, suggesting that the O-4 position of residue J is substituted by -OMe.

[0062] Furthermore, according to the δ H and δ C , combined with HMBC ( Figure 8 ) spectrum (anomeric region) to infer the sequence information of the sugar moiety. For the most abundant GalA-related residues, the HMBC spectrum ( Figure 8 ) shows that the cross peaks at δ 4.64 / 79.12 ppm in CTP1 contain HG domains connected by (1,4)-α-glycosidic bonds. HMBC spectrum ( Figure 8 ) shows H of residues I and J 1 C with residue J 6 , H of residue K 1 C with residue K 6 There are coupled signals respectively, suggesting that CTP1 contains a galactomannan fragment linearly connected by a (1,6)-β-glycosidic bond. HMBC spectrum ( Figure 8 ) shows that H1 of residue G is bound to C of residue I and residue J, respectively. 6 There are coupling signals, indicating that the HG domain is connected to the Gal part through a (1,6)-α-glycosidic bond, and CTP1 is a linear connection between the HG domain and galactan as the main chain. According to the HMBC spectrum ( Figure 8 ) 1 C with residue J 3 The coupling signals between residues H and C suggest that residues H and C are connected to the O-3 position of residue J as two separate branches. In addition, the HMBC spectrum ( Figure 8 ) 1 C with residue D 3 , H of residue E 5 C with residue D 1 , H of residue E 1 C with residue K 4 The coupling signals between them suggest that there is a branch chain with residue B as the terminal unit in CTP1, which is connected to the O-4 position of residue K. Based on all the above results, the primary structure of CTP1 is a linear connection of →1)-α-D-GalpA-(4→ and →1)-β-D-Galp-(6→ as the main chain, containing β-D-GlcpA-(1→, α-L-Araf-(1→ and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf-(1→) three branch chain fragments connected to the 3rd and 4th positions of the Gal residue, respectively, and the GalA and Gal fragments contain methyl esterification and methoxylation groups at positions 6 and 4, respectively. The structure of CTP1 is shown in Fig.10 .

[0063] Example 2 Preparation of CTP1-epigallocatechin gallate (EGCG) nanomaterials Take 5 mg of Pu'er tea fresh leaf polysaccharide (CTP1) prepared in Example 1 and dissolve it in 2 mL of pure water to prepare a polysaccharide aqueous solution with a concentration of 2.5 mg / mL, and filter it through a 0.45 μm filter membrane for standby use to obtain a polysaccharide aqueous solution. Take EGCG and prepare it into a 1 mg / mL EGCG aqueous solution with pure water, and filter it through a 0.45 μm filter membrane for standby use to obtain an EGCG aqueous solution. The polysaccharide aqueous solution prepared above and the EGCG aqueous solution are mixed in a volume ratio of 2:13 to form a CTP1 and EGCG self-assembled nanomaterial (referred to as CTP1-EGCG), which is prepared and used immediately.

[0064] Example 3 Preparation of CTP1-caffeine (CAF) nanomaterials Take 8 mg of Pu'er tea fresh leaf polysaccharide (CTP1) prepared in Example 1 and dissolve it in 2 mL of pure water to prepare a polysaccharide aqueous solution with a concentration of 4 mg / mL, and filter it through a 0.45 μm filter membrane for use to obtain a Pu'er tea fresh leaf polysaccharide aqueous solution. Take caffeine and add pure water to prepare a CAF aqueous solution with a concentration of 1 mg / mL, and filter it through a 0.45 μm filter membrane for use to obtain a CAF aqueous solution. The Pu'er tea fresh leaf polysaccharide aqueous solution prepared above is mixed with the CAF aqueous solution in a volume ratio of 2:13 to form a CTP1 and CAF self-assembled nanomaterial (abbreviated as CTP1-CAF), which is prepared and used immediately.

[0065] Example 4 Characterization of Nanomaterials 10 μL of each of the CTP1-EGCG prepared in Example 2 and the CTP1-CAF prepared in Example 3 was taken to observe the morphology of the nanomaterials using a transmission electron microscope (TEM).

[0066] Results Fig.11 and Fig.12 The results showed that CTP1 could self-assemble with EGCG and CAF to form spherical nanoparticles.

[0067] Example 5 Immunomodulatory activity of nanomaterials formed by polysaccharide CTP1, EGCG and CAF Specific methods for in vitro immunomodulatory activity: (1) The phagocytic activity of RAW264.7 macrophages was measured by the neutral red method. RAW264.7 macrophages were cultured at 2×10 4Each well of the cells was seeded in a 96-well plate and incubated with different concentrations (12.5, 25, and 50 μg / mL) of CTP1, different concentrations (12.5, 25, and 50 μg / mL) of EGCG, and different concentrations (12.5, 25, and 50 μg / mL) of CTP1-EGCG nanomaterials at 37°C and 5% CO. 2 Incubate in a humidified atmosphere for 24 h. Alternatively, RAW264.7 macrophages were cultured at 2×10 4 Each well of the cells was seeded in a 96-well plate and incubated with different concentrations (12.5, 25, and 50 μg / mL) of CAF, different concentrations (12.5, 25, and 50 μg / mL) of CTP1, and different concentrations (12.5, 25, and 50 μg / mL) of CTP1-CAF nanomaterials at 37°C and 5% CO. 2 The cells were co-incubated for 24 h in a humidified atmosphere. 1 μg / mL lipopolysaccharide (LPS) was used as the positive control group, and an equal volume of DMEM medium was used as the blank control. After the cell incubation, 150 μL of 0.075% neutral red per well was used to replace the drug treatment group and the cells were incubated at 37°C with 5% CO 2 Incubate in a humidified atmosphere for 1 h. Discard the neutral red that was not phagocytosed by the cells and rinse twice with PBS, add lysate (glacial acetic acid: ethanol = 1:1) and lyse at 25°C for 1 h. Measure the absorbance at 540 nm with an ELISA reader. Calculate the phagocytic rate of macrophages RAW264.7 according to the following formula: phagocytic rate (%) = (AS / A0) × 100%; A0 is the absorbance value of the control group; AS is the absorbance value in the treatment group.

[0068] Fig.13 The results showed that both CTP1-EGCG and CTP1-CAF nanomaterials can play an immunomodulatory role by activating mononuclear macrophages RAW264.7 and improving their phagocytic ability. In addition, the effect of CTP1-EGCG nanomaterials on improving the phagocytic ability of RAW264.7 cells was significantly higher than that of EGCG and CTP1 monomers (P<0.05, *), and the effect of CTP1-CAF nanomaterials on improving the phagocytic ability of RAW264.7 cells was significantly higher than that of CAF monomers (P<0.05, *). In summary, the self-assembled nanomaterials constructed based on CTP1 have the advantage of improving the phagocytic ability of macrophages in terms of immunomodulatory activity.

[0069] (2) The effect of RAW264.7 macrophages on NO secretion was determined by the Griess method. RAW264.7 macrophages were cultured at 2×10 4Each well of the cells was seeded in a 96-well plate and incubated with different concentrations (12.5, 25, and 50 μg / mL) of CTP1, different concentrations (12.5, 25, and 50 μg / mL) of EGCG, and different concentrations (12.5, 25, and 50 μg / mL) of CTP1-EGCG nanomaterials at 37°C and 5% CO. 2 Incubate in a humidified atmosphere for 24 h. Alternatively, RAW264.7 macrophages were cultured at 2×10 4 Each well of the cells was seeded in a 96-well plate and incubated with different concentrations (12.5, 25, and 50 μg / mL) of CTP1, different concentrations (12.5, 25, and 50 μg / mL) of CAF, and different concentrations (12.5, 25, and 50 μg / mL) of CTP1-CAF nanomaterials at 37°C and 5% CO. 2 The cells were incubated in a humidified atmosphere for 24 h. 1 μg / mL LPS was used as a positive control group, and an equal volume of DMEM medium was used as a blank control. The cell supernatant was mixed with the same volume of Griess reagent and incubated at room temperature for 10 min. The absorbance was read at 540 nm using an ELISA plate reader, and NaNO 2 The NO content was calculated based on the obtained standard curve.

[0070] Fig.14 The results showed that both CTP1-EGCG and CTP1-CAF nanomaterials can promote RAW264.7 cells to secrete NO to play an immunomodulatory role. Although the ability of CTP1-EGCG nanomaterials to promote NO secretion by RAW264.7 cells is lower than that of CTP1 monomers, it is significantly higher than EGCG monomers within the experimental concentration range (P<0.05, *). Similarly, CTP1-CAF nanomaterials have similar effects to CTP1-EGCG. In particular, at a low dose concentration (12.5µg / mL), the ability of CTP1-CAF nanomaterials to promote NO secretion by RAW264.7 cells is significantly higher than that of CTP1 and CAF monomers. In summary, self-assembled nanomaterials constructed based on CTP1 have the advantage of promoting macrophages to secrete NO and thus play an immunomodulatory role.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Pu'er tea fresh leaf polysaccharide, characterized in that: 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-α-D-galacturonic acid, 1,3,6-β-D-galactose and 1,4,6-β-D-galactose as the main chain, β-D-glucuronic acid, α-L-arabinose and α-D-Rhap-(1→3)-α-L-Araf-(1→5)-α-L-Araf as the side chain, 1,4-α-D-galacturonic acid is methyl esterified, and the degree of methyl esterification is 32.08%. The Pu'er tea fresh leaf polysaccharide structure is: Formula 1.

2. The Pu'er tea fresh leaf polysaccharide according to claim 1, characterized in that: The molecular weight of the Pu'er tea fresh leaf polysaccharide is 31241 KDa; the monosaccharide composition of the Pu'er tea fresh leaf polysaccharide 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.

3. A method for preparing Pu'er tea fresh leaf polysaccharide according to claim 1 or 2, characterized in that: The following steps are involved: (1) crushing and sieving the dried fresh Pu'er tea leaves to obtain tea powder; (2) soaking the tea powder in the first ethanol solution for degreasing to obtain defatted powder; (3) mixing the defatted powder with water for extraction, collecting the filtrate to obtain an extract, and concentrating the extract under reduced pressure to obtain a concentrated extract; (4) mixing the concentrated extract with the second ethanol solution for precipitation to obtain an alcohol-precipitated solution, centrifuging, collecting the precipitate, re-dissolving the precipitate, removing protein from the re-dissolved precipitate, collecting the supernatant to obtain a deproteinized extract, and drying to obtain Pu'er tea crude polysaccharides; (5) subjecting the crude Pu'er tea polysaccharide to static adsorption with a macroporous adsorption resin suspension, shaking to obtain a decolorized suspension, filtering, washing, collecting an eluate a, and drying to obtain a decolorized Pu'er tea polysaccharide; (6) After the decolorized Pu'er tea polysaccharide is dissolved, the supernatant is collected by centrifugation and added to a chromatography column filled with DEAE cellulose DE-52, and eluted with water and 0.1M NaCl solution in sequence. The eluate after elution with 0.1M NaCl solution is collected, concentrated, dialyzed, and dried to obtain polysaccharide subfractions; (7) Purify the polysaccharide subcomponents using an propylene glycol gel S-400 gel column, elute, collect the homogeneous components, concentrate, perform a second dialysis, and dry to obtain Pu'er tea fresh leaf polysaccharides.

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

5. The preparation method according to claim 3, characterized in that The mass 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°C, and the time of each extraction is 2-4 hours.

6. The preparation method according to claim 3, 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 re-dissolving the precipitate is 50-60° C. water.

7. The preparation method according to claim 3, 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 according to 450-550 g:1000 mL; the shaking temperature is 40-60 ° C; the shaking time is 4-6 h; the shaking frequency is 110-130 r / min; the solvent for dissolving the bleached Pu'er tea polysaccharide is water, and the mass volume ratio of the bleached Pu'er tea polysaccharide to water is 15-25 mg:1 mL; in step (6), the elution flow rate is 0.5-1.5 mL / min, and the elution time with water and 0.1 M NaCl solution is 5-7 h; the molecular weight cutoff of the first dialysis and the second dialysis is 3000-4000 Da; in step (7), the elution solvent is water, and the elution rate is 0.1-0.5 mL / min; The homogeneous component is an eluate obtained by elution detection by HPLC-RID, and a component with a retention time of 7.9 to 8.0 min in the HPLC-RID chromatogram; Chromatographic conditions of the HPLC-RID: Column: Thermo Acclaim 120 C 18 ; Mobile phase conditions: mobile phase A is pH 6.72 NaH2PO4 / Na2HPO4 buffer, and mobile phase B is acetonitrile; the preparation method of the NaH2PO4 / Na2HPO4 buffer comprises mixing 1.6 g NaH2PO4, 0.8 g Na2HPO4 and 100 mL water; Elution program: 0~60min mobile phase A:mobile phase B volume ratio = 83:17; flow rate: 1mL / min; column temperature: 30℃.

8. A self-assembled nanomaterial, characterized in that: The Pu'er tea fresh leaf polysaccharide described in claim 1 or 2 or the Pu'er tea fresh leaf polysaccharide prepared by the preparation method according to any one of claims 3 to 7 is dissolved in water to obtain a Pu'er tea fresh leaf polysaccharide aqueous solution; an active small molecule compound is mixed with water to obtain an active small molecule compound aqueous solution, and then the Pu'er 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 comprises one of epigallocatechin gallate and caffeine.

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

10. Use of the Pu'er tea fresh leaf polysaccharide according to claim 1 or 2, the Pu'er tea fresh leaf polysaccharide prepared by the preparation method according to any one of claims 3 to 7, or the self-assembled nanomaterial according to claim 8 or 9 in the preparation of immunomodulatory products.

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