A Yellow Tea Acidic Active Polysaccharide, Its Preparation Method and Application

Abstract

This invention relates to the field of tea active ingredient extraction technology, specifically disclosing an acidic active polysaccharide from *Tea macrophylla*, its preparation method, and its applications. The acidic active polysaccharide is a heteropolysaccharide composed of mannose, galactose, arabinose, glucose, rhamnose, and galacturonic acid, with a relative molecular weight of 722 kDa. The backbone of the acidic active polysaccharide consists of 1,2-α-L-Rhap, 1,4-α-D-Galp, and 1,6-α-D-Glcp residues, while the side chains include 1,4-β-D-GalpA, 1,6-β-D-GalpA, 1,3,6-β-D-Manp, 1,2,4-α-L-Rhap, T-β-Manp, and T-β-Galp residues. The acidic active polysaccharide L2B1 from *Tea macrophylla* described in this invention can regulate the intestinal microecological environment and effectively improve the symptoms of type 2 diabetes.

Description

Technical Field

[0001] This invention relates to the field of tea active ingredient extraction technology, and in particular to an acidic active polysaccharide from Huangda tea, its preparation method, and its application. Background Technology

[0002] With socioeconomic development and improved living standards, type 2 diabetes mellitus (T2DM) has become the most prevalent metabolic disease, primarily characterized by glucose intolerance and postprandial hyperglycemia. To slow the progression of T2DM and prevent complications, clinical treatments utilize drugs such as alpha-glucosidase inhibitors, insulin secretagogues, biguanides, and SGLT2 inhibitors to delay or reduce the digestion and absorption of dietary carbohydrates, thereby lowering blood glucose levels. However, long-term use of these drugs can cause gastrointestinal side effects, such as diarrhea, bloating, and liver disease. In recent years, hypoglycemic active ingredients from plants have become a hot topic in research and development. These naturally derived "green" active substances not only have stable efficacy and few toxic side effects but also effectively prevent and treat complications.

[0003] Huoshan Huangda tea is a major product among yellow teas, characterized by its "coarse branches and large leaves, bronze color, and crispy aroma." Previous research by the applicant's team has revealed that the coarse, mature leaves of Huangda tea have a high polysaccharide content and exhibit significant effects in liver protection, lipid reduction, and weight loss. Current research reports on Huangda tea polysaccharides primarily focus on crude polysaccharides; the main active components of Huangda tea polysaccharides remain unclear, and their active material basis and efficacy need further clarification. Therefore, the applicant has proposed an invention patent for "an acidic active polysaccharide from Huangda tea, its preparation method, and its application." Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention has applied for a patent for "An Acidic Active Polysaccharide from Huangda Tea, Its Preparation Method and Application". This invention isolates a homogeneous acidic active polysaccharide from Huangda tea, which can remodel the intestinal microenvironment in mice and effectively improve type 2 diabetes symptoms, making it an ideal candidate for hypoglycemic agents.

[0005] The monosaccharide composition of the Huangda Tea acidic active polysaccharide is mannose, arabinose, galactose, glucose, galacturonic acid, and rhamnose, with a molar ratio of 1:0.2:4.34:2.14:1.10:2.28.

[0006] The main chain of the acidic active polysaccharide of *Tea macrantha* includes 1,2-α-L-Rhap, 1,4-α-D-Galp, and 1,6-α-D-Glcp residues, and the side chains include 1,4-β-D-GalpA, 1,6-β-D-GalpA, 1,3,6-β-D-Manp, 1,2,4-α-L-Rhap, T-β-Manp, and T-β-Galp residues.

[0007] A method for preparing acidic active polysaccharides from Huangda tea includes the following steps:

[0008] Step 1: Grind Huangda tea into powder, sieve, add ethanol solution at a material-to-liquid ratio of 1:10 (g / mL), extract until the supernatant is colorless, centrifuge, concentrate and dry to obtain pretreated Huangda tea powder;

[0009] Step 2: Add the Huangda tea powder obtained in Step 1 to pure water, extract, filter, and combine the extracts;

[0010] Step 3: Concentrate the extract obtained in Step 2, add ethanol solution, stir thoroughly, let stand, and centrifuge to collect the precipitate;

[0011] Step 4: The precipitate obtained in Step 3 was subjected to protein removal by the Sevag method, dialysis, and freeze-drying to obtain Huangda Tea acidic crude polysaccharide.

[0012] Step 5: Prepare a solution of the crude acidic polysaccharide of *Tea macrantha* obtained in Step 4 with pure water, separate and purify it by passing it through a DEAE-Sepharose Fast Flow ion exchange chromatography column, and obtain the *Tea macrantha* acidic active polysaccharide by elution, collection, concentration and freeze drying.

[0013] A method for preparing acidic active polysaccharides from Huangda tea, wherein step 1 further includes: adding a 95% ethanol solution, an extraction temperature of 80℃, and an extraction time of 1.5h.

[0014] A method for preparing acidic active polysaccharides from Huangda tea, wherein step 2 further includes: an extraction temperature of 90℃, an extraction time of 1.5h, repeated extraction three times, and filtration using gauze with eight layers.

[0015] A method for preparing acidic active polysaccharides from Huangda tea, step 3 of which further includes: adding 95% ethanol solution to a final ethanol solution concentration of 80%, stirring thoroughly, and then standing in a refrigerator at 4°C for 12 hours.

[0016] A method for preparing acidic active polysaccharides from Huangda tea, step 5 further includes: preparing a 25 mg / mL solution of crude acidic polysaccharides from Huangda tea with pure water, eluting with NaCl (0.1 mol / L) at a flow rate of 1.2 mL / min, and freeze-drying to obtain a homogeneous component of acidic active polysaccharides from Huangda tea.

[0017] A drug for treating type 2 diabetes includes the above-mentioned Huangda Tea acidic active polysaccharide.

[0018] Application of Huangda Tea Acidic Active Polysaccharide in the Preparation of Drugs for the Treatment of Type 2 Diabetes.

[0019] The beneficial effects of this invention are reflected in:

[0020] 1. The present invention provides a separation and purification method for crude polysaccharides from Huangda tea using a DEAE-Sepharose Fast Flow chromatographic column, which can rapidly prepare a homogeneous component of acidic active polysaccharides from Huangda tea with a molecular weight of 722 kDa and a monosaccharide composition of mannose, arabinose, galactose, glucose, galacturonic acid, and rhamnose.

[0021] 2. This invention elucidates the primary structure of the homogeneous component of the acidic active polysaccharide of Huangda tea, further clarifying the active material basis of the hypoglycemic effect of L2B1. Attached Figure Description

[0022] Figure 1 The chromatograms are of the physicochemical characteristics of L2B1, a homogeneous component of acidic active polysaccharides from Huangda tea. Among them, A is the molecular weight standard curve, B is the high performance liquid chromatogram of L2B1, and C is the chromatogram of the composition of standard monosaccharides and L2B1 monosaccharides (*-solvent peak, 1-mannose, 2-rhamnose, 3-glucuronic acid, 4-galacturonic acid, 5-glucose, 6-galactose, 7-arabinose).

[0023] Figure 2 This is the nuclear magnetic resonance spectrum of L2B1, a homogeneous component of acidic active polysaccharides from Huangda tea; where A represents... 1 H NMR spectrum, B is 13 C10 NMR spectrum, C is COSY spectrum, D is HSQC spectrum, E is HMBC spectrum;

[0024] Figure 3 This is a schematic diagram of the predicted chemical structure of L2B1;

[0025] Figure 4 The effect of L2B1, a homogeneous component of acidic active polysaccharide from Huangda tea, on insulin levels and insulin resistance in T2DM mice;

[0026] Figure 5 The effect of L2B1, a homogeneous component of acidic active polysaccharide from Huangda tea, on the abundance of gut microbiota at the phylum level in T2DM mice. Detailed Implementation

[0027] The preparation and hypoglycemic activity of the present invention are described below with reference to specific embodiments and accompanying drawings. The listed embodiments are only for explaining the present invention and are not intended to limit the scope of protection of the present invention. The Huangda tea in the following embodiments is produced by Hengda Tea Co., Ltd. in Huoshan County, Anhui Province. The acidic active polysaccharide of Huangda tea, abbreviated as L2B1, has a total sugar content of 88.1% and a relative molecular weight of 722kDa.

[0028] Example 1: Preparation of L2B1, a homogeneous component of acidic active polysaccharides from Huangda tea.

[0029] Step 1: Grind Huangda tea into powder, pass it through an 80-mesh sieve, add 95% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL), extract at 80℃ for 1.5 hours until the supernatant is colorless, centrifuge, concentrate and dry to obtain pretreated Huangda tea powder.

[0030] Step 2: Add the pretreated Huangda tea powder to pure water at a liquid-to-solid ratio of 1:20 (g / mL), and extract in a 90℃ water bath for 1.5 hours. Repeat the extraction three times, then filter through 8 layers of gauze and combine the extracts. Concentrate the obtained extract to 1 / 20 of its original volume, add 95% ethanol solution until the final ethanol concentration is 80%, stir thoroughly, and let stand in a refrigerator at 4℃ for 12 hours. Centrifuge to collect the precipitate.

[0031] Step 3: Take 30g of Huangda Tea acidic active polysaccharide, i.e. the precipitate of Step 2, and prepare a polysaccharide solution of 20mg / mL with pure water. Add an equal volume of Sevag reagent (n-butanol: chloroform = 1:4), shake vigorously for 15min, centrifuge and collect the supernatant. Repeat until the protein is completely removed. Concentrate the polysaccharide solution, dialyze, and freeze dry to obtain Huangda Tea acidic crude polysaccharide.

[0032] Step 4: Prepare a 25 mg / mL solution of Huangda tea acidic crude polysaccharide with pure water, purify it using a DEAE-Sepharose FastFlow ion exchange chromatography column, elute with NaCl (0.1 mol / L) at a flow rate of 1.2 mL / min, collect the solutions in separate tubes, concentrate them, and freeze-dry them to obtain the homogeneous component L2B1 of Huangda tea acidic active polysaccharide. The total sugar content was determined to be 88.1% by the phenol-sulfuric acid method.

[0033] Example 2: Chemical structural characterization of L2B1, a homogeneous component of acidic active polysaccharides from Huangda tea.

[0034] Step 1: Prepare 2 mg / mL solutions of L2B1 and dextran standards (T10, T50, T500, T1000, T2000) with pure water. Analyze these solutions using an Agilent 1260 high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The chromatographic column was a TSKGel G6000PWXL, the mobile phase was deionized water, the carrier gas was N2, the gas flow rate was 2.5 L / min, and the injection volume was 10 μL. A standard curve was plotted using the logarithm of the relative molecular mass (Lg Mw) and retention time (Rt) of the standards. The results are shown below. Figure 1 As shown. Based on the standard curves of dextran with different molecular weights (Lg Mw=-0.2409x+8.7788, R…),… 2 =0.9989), the molecular weight of L2B1 was measured to be 722 kDa.

[0035] Step 2: The monosaccharide composition of the L2B1 acid hydrolysis-pre-column PMP (1-phenyl-3-methyl-5-pyrazolone) derivatized sample was determined using an Agilent 1260 HPLC system with a diode array detector (DAD). 10 mg of L2B1 was dissolved in 5 mL of 2 mol / L trifluoroacetic acid, sealed with nitrogen, and heated in an oil bath at 110 °C for 8 h. Methanol was added repeatedly and the solution was rotary evaporated until pH 7. 1 mL of deionized water was added for later use. 1 mL each of a 5 mg / mL standard monosaccharide solution and an acid-hydrolyzed L2B1 solution were added to each solution, along with 0.5 mL of NaOH (0.3 mol / L) and 0.5 mL of PMP working solution (0.5 mol / L), respectively. The mixture was shaken and reacted in a water bath at 70 °C for 1 h for PMP pre-column derivatization. The solution was adjusted to neutral with 0.5 mL of HCl (0.3 mol / L), and extracted three times with chloroform to remove excess PMP. The aqueous phase was filtered through a 0.22 μm filter and analyzed by HPLC-DAD.

[0036] HPLC-DAD conditions: Zorbox Eclipse XDB-C18 column, column temperature 30℃, wavelength 245nm; mobile phase A was acetonitrile, mobile phase B was 0.05mol / L phosphate buffer solution, gradient elution 0-60min, initial setting mobile phase A:mobile phase B = 17:83 (%), final elution ratio was mobile phase A:mobile phase B = 20:80 (%), injection volume was 10μL.

[0037] Figure 1 D indicates that L2B1 is composed of 6 monosaccharides, and the monosaccharide composition and molar percentage are mannose:arabinose:galactose:glucose:galacturonic acid:rhamnose = 1:0.2:4.34:2.14:1.10:2.28.

[0038] Step 3: Add 20 mg L2B1 to 6 mL of anhydrous DMSO, seal with nitrogen, add 240 mg NaOH, seal with nitrogen, mix well, add 4 mL of iodomethane, seal with nitrogen, repeat the addition of iodomethane 3 times, add 6 mL of pure water to terminate the reaction, dialyze, and concentrate. Repeat the above methylation process until the methylated sample shows a high IR spectrum at 3400 cm⁻¹. -1 No absorption peak was observed nearby. The fully methylated L2B1 sample was dissolved in 2 mL of trifluoroacetic acid (2 mol / L), sealed with nitrogen, and incubated in an oil bath at 110 °C for 6 h. Methanol was added repeatedly and the mixture was rotary evaporated until pH 7. 3 mL of deionized water was added for later use. 60 mg of sodium borodeuteride was added, and the mixture was sealed with nitrogen and magnetically stirred for 24 h. Methanol was added repeatedly to obtain the reduced polysaccharide L2B1. 0.5 mL each of acetic anhydride and anhydrous pyridine were added, the mixture was sealed with nitrogen, incubated in an oil bath at 100 °C for 2 h, cooled, and evaporated to dryness. Extraction was performed with water and chloroform, and the sample was analyzed by GC-MS.

[0039] GC-MS conditions: RTX-5MS column, helium as carrier gas, inlet rate 1 mL / min; temperature program: initial temperature 50℃, 7 min; ramp to 320℃ at 10℃ / min, 14 min; ion source temperature 230℃, solvent separation for 12 min; mass spectrum in the range of mass-to-charge ratio (m / z) 30-580.

[0040] The methylation results are shown in Table 1 and Figure 3 As shown, L2B1 contains nine glycosidic bonds: 1,2,4-α-L-Rhap, 1,4-α-D-Galp, 1,6-α-D-Glcp, 1,3,6-β-D-Manp, 1-β-D-Galp, 1,4-β-D-GalpA, 1,6-β-D-GalpA, 1-β-D-Manp, and 1,2-β-L-Rhap, with corresponding molar percentages of [missing information]. The values ​​16.88:17.28:19.66:5.19:15.16:4.38:7.18:4.13:4.45 indicate that the L2B1 backbone is composed of 1,2-α-L-Rhap, 1,4-α-D-Galp, and 1,6-α-D-Glcp residues. The presence of 1,3,6-β-D-Manp and 1,2,4-α-L-Rhap residues indicates that L2B1 has side chains.

[0041] Table 1. Analysis of L2B1 methylated sugar alcohol acetyl ester (PMAA) results

[0042]

[0043] Step 4: Dissolve 80 mg of polysaccharide L2B1 in 1.5 mL of heavy water, centrifuge (5000 rpm, 5 min), collect the supernatant, freeze dry, replace twice, dissolve in 0.55 mL of heavy water, centrifuge (5000 rpm, 5 min), collect the supernatant into an NMR tube, and determine its one-dimensional (H1N and C1N) and two-dimensional (HSQC, HMBC, COSY) spectra using an NMR spectrometer (JNM-ECZ600R).

[0044] In L2B1 1 H and 13 C NMR spectrum ( Figure 2 A and Figure 2 In B), the proton signal between δ3.30-5.10 ppm and the carbon signal between δ60-110 ppm are characteristic regions of polysaccharides. 13 The signals at δ 174.81 and 179.61 ppm in the C NMR spectrum confirm the presence of uronic acid, and δ 16.65 and 16.88 ppm belong to... 13 The Rha C6 signal peak in the C10 NMR spectrum. Analysis 1 H- 1In the anodic regions of H COSY and HSQC spectra, the anodic proton / carbon chemical shifts of residue AH are 5.27 / 98.41, 5.04 / 97.77, 4.98 / 97.75, 4.74 / 100.78, 4.65 / 103.70, 4.63 / 103.70, 4.51 / 102.82, 4.47 / 103.71, and 4.71 / 103.71 ppm, respectively, indicating that residue AC is in the α configuration and residue DI is in the β configuration.

[0045] 1 H- 1 H COSY spectrum reflects the coupling relationship between adjacent hydrocarbons. Figure 2 C). The signals for residues A, H–2 to H–6 can be deduced as 4.12, 3.91, 3.51, 3.77, and 1.25 ppm. The chemical shifts from C-2 to C-6 can be obtained via HSQC spectroscopy (…). Figure 2 The CH correlations in residue D) were obtained as 70.21, 72.13, 69.43, 72.13, and 16.65 ppm. By comparison with literature, residue A was identified as 1,2,4-α-L-Rhap. Residues B1 and C1 were 1,4-α-D-Galp, 1,6-α-D-Glcp, 1,3,6-β-D-Manp, 1-β-D-Galp, 1,4-β-D-GalpA, 1,6-β-D-GalpA, 1-β-D-Manp, and 1,2-β-L-Rhap. All proton / carbon chemical shifts in residues A1 and C1 are detailed in Table 3.

[0046] According to the HMBC spectrum ( Figure 2E) The sequence of L2B1 glycosyl residues can be deduced. H-1 of 1,2,4-α-L-Rhap(A) is correlated with C-4 of 1,4-α-D-Galp(B). H-1 of 1,4-α-D-Galp(B) is correlated with C-6 of 1,6-α-D-Glcp(C). H-1 of 1,6-α-D-Glcp(C) is strongly correlated with C-2 of 1,2,4-α-L-Rhap(A) and C-6 of 1,3,6-β-D-Manp(D). H-1 of 1,3,6-β-D-Manp(D) is correlated with C-2 of 1,2,4-α-L-Rhap(A). H-1 of T-Galp(E) is correlated with C-4 of 1,4-α-D-Galp(B). The H-1 of 1,4-β-D-GalpA(F) is correlated with the C-2 of 1,2-β-L-Rhap(I), and the H-6 of 1,4-β-D-GalpA(F) is linked to an acetyl group (-OAc). The H-1 of 1,6-β-D-GalpA(G) and T-β-D-Manp(H) is correlated with the C-2 of 1,2,4-α-L-Rhap(A). The H-1 of 1,2-β-L-Rhap(I) is correlated with the C-3 of 1,3,6-β-D-Manp(D). Based on these correlations, it can be inferred that the L2B1 backbone consists of 1,2-α-L-Rhap, 1,4-α-D-Galp, and 1,6-α-D-Glcp, with a branch at C-4 of 1,2,4-α-L-Rhap.

[0047] In summary, it can be deduced that the main chain of L2B1 consists of 1,2-α-L-Rhap, 1,4-α-D-Galp, and 1,6-α-D-Glcp residues, while the side chains consist of 1,4-β-D-GalpA, 1,6-β-D-GalpA, 1,3,6-β-D-Manp, 1,2,4-α-L-Rhap, T-β-Manp, and T-β-Galp residues. Branches are connected to the main chain at the C-4 position. For the specific structure, see [link to structural details]. Figure 3 .

[0048] Table 2. L2B1 1 H and 13 C Chemical Shift Classification

[0049]

[0050] Example 3: Hypoglycemic effect of L2B1, a homogeneous component of acidic active polysaccharide from Huangda tea.

[0051] A type 2 diabetic mouse model was established by inducing streptozotocin (STZ) to evaluate the hypoglycemic efficacy of L2B1, a homogeneous component of the acidic active polysaccharide from Huangda tea.

[0052] Step 1: Forty-eight male C57BL / 6J mice were randomly divided into four groups after one week of acclimatization: normal group, model group, LL2B1 group (low dose), ML2B1 group (medium dose), HL2B1 group (high dose), and MET (metformin, positive control) group. Two weeks later, the mice were treated as follows: NC and DM groups were administered 0.9% NaCl solution by gavage daily; L2B1 groups were administered L2B1 (75, 150, and 300 mg / kg) by gavage daily; and MET groups were administered MET (150 mg / kg) daily. Except for the control group, all mice were fed a high-fat diet for 6 weeks, injected with STZ (40 mg / kg) for 5 days, and began gavage two weeks later. At week 12, mice were anesthetized and sacrificed, and blood, liver, and fresh fecal samples were collected and stored at -80°C. Data were analyzed for significance using SPSS 27.0 software.

[0053] Step 2: Mice were fasted overnight. Blood was collected from the tail vein of the mice using a needle prick method. The 0-minute blood glucose (BG0) was measured using a portable Roche blood glucose meter. Glucose was administered at a rate of 1 / 500, and the blood glucose values ​​at 15, 30, 60, 90, and 120 minutes were recorded as BG15, BG30, BG60, BG90, and BG120, respectively. The area under the OGTT curve (AUC) was calculated using the formula: AUC = 1 × (BG60 + BG120) / 2 + 0.5 × (BG30 + BG60) / 2 + 0.5 × (BG0 + BG30) / 2.

[0054] Step 3: Extraction of total microbial DNA, followed by purification and quantification using quantitative qRT-PCR. Then, paired-end sequencing was performed on the overlap sequence. The sequencing data was processed using DADA2, and an OTU-like table was constructed to obtain an ASV characteristic table and sequences for diversity analysis, species classification annotation, etc.

[0055] Table 3. Changes in fasting blood glucose levels (mmol / L) in mice after different intervention conditions

[0056]

[0057] Note: Numerical values ​​are expressed as Mean ± SD (n = 8). Compared to the NC group, ** P<0.01, highly significant; compared with the DM group, ## P<0.01, highly significant; # P<0.05, significant.

[0058] Fasting blood glucose reflects the function of pancreatic β-cells and insulin secretion. Fasting blood glucose testing is a routine method for evaluating type 2 diabetes mellitus (T2DM). In the normal control group, blood glucose levels were relatively stable (5.03 mmol / L–5.84 mmol / L), while in the model group, blood glucose levels significantly increased, generally remaining above 20 mmol / L and continuing to rise (see Table 3). After four weeks of treatment with different concentrations of L2B1 and MET, blood glucose levels in diabetic mice decreased significantly over time. At week 12, the high-dose L2B1 intervention group showed a blood glucose level of 9.76 mmol / L, comparable to the effect of a positive control drug.

[0059] like Figure 4 As shown, compared with the NC group, the insulin resistance index (HOMA-IR) and insulin level (FINS) of the DM group were significantly increased (P<0.01), while after intervention with L2B1 and MET, the insulin level and insulin resistance index of diabetic mice were significantly decreased (P<0.01). Specifically, the insulin level and insulin resistance index of the ML2B1 group were similar to those of the MET group, while the insulin level of the HL2B1 group was lower than that of the MET group but similar to that of the NC group, and the insulin resistance index was similar to that of the MET group. These results indicate that after 4 weeks of L2B1 intervention, insulin sensitivity in mice was increased, excessive insulin secretion was inhibited, and insulin resistance was reduced in a dose-dependent manner.

[0060] Gut microbiota play a crucial role in the development of type 2 diabetes mellitus (T2DM). An increased Firmicutes / Bacteroidetes ratio and elevated Proteobacteria abundance are common characteristics of the gut microbiota in T2DM mice. For example... Figure 5 As shown, the abundance of Firmicutes in the model group mice increased and the abundance of Bacteroidetes decreased compared with normal mice. After high-dose L2B1 intervention, the abundance of Firmicutes in the model group mice decreased from 54.8% to 44.5%, and the abundance of Bacteroidetes increased from 11.8% to 31%, indicating that L2B1 can effectively improve the symptoms of T2DM by regulating the abundance of gut-specific flora and reshaping the gut microenvironment.

[0061] 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. Use of an acidic active polysaccharide of Camellia reticulata Lindl. in the preparation of a medicament for treating type 2 diabetes. The preparation method of the acidic active polysaccharide of Camellia reticulata Lindl. comprises the following steps: Step 1: grinding Camellia reticulata Lindl. into powder, sieving, adding ethanol solution at a solid-liquid ratio of 1 g:10 mL, extracting until the supernatant is colorless, centrifuging, concentrating and drying to obtain pretreated Camellia reticulata Lindl. powder; Step 2: adding the Camellia reticulata Lindl. powder obtained in step 1 to pure water, extracting, filtering, and combining the extract; Step 3: concentrating the extract obtained in step 2, adding ethanol solution, stirring thoroughly, then standing, centrifuging to collect the precipitate; Step 4: removing protein from the precipitate obtained in step 3 by the sevag method, dialyzing, and freeze-drying to obtain acidic crude polysaccharide of Camellia reticulata Lindl.; Step 5: preparing a solution of the acidic crude polysaccharide of Camellia reticulata Lindl. obtained in step 4 with pure water, separating and purifying by DEAE-Sepharose FastFlow ion exchange chromatography column, collecting and concentrating by elution, and freeze-drying to obtain the acidic active polysaccharide of Camellia reticulata Lindl. In step 5, the acidic crude polysaccharide of Camellia reticulata Lindl. is prepared into a 25 mg / mL solution with pure water, elution is performed using NaCl at a flow rate of 1.2 mL / min and a concentration of 0.1 mol / L, and the freeze-dried acidic active polysaccharide of Camellia reticulata Lindl. is obtained.

2. Use according to claim 1, characterized in that, In step 1, the added ethanol solution is 95% ethanol solution, the extraction temperature is 80℃, and the extraction time is 1.5 h.

3. Use according to claim 1, characterized in that, In step 2, the extraction temperature is 90℃, the extraction time is 1.5 h, the number of repeated extractions is 3, the filter cloth used for filtering is 8 layers.

4. Use according to claim 1, characterized in that, In step 3, 95% ethanol solution is added to a final concentration of 80% ethanol solution, the mixture is thoroughly stirred, then stood in a refrigerator at 4℃ for 12 h.

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