A polysaccharide L2-1 from Huangda tea, its preparation method and uses

Abstract

This invention discloses a method for preparing Huangda tea polysaccharide L2-1 and its use in preventing and improving Alzheimer's disease. Huangda tea polysaccharide L2-1 has a total sugar content of 91.89%, a protein content of 1.19%, and a uronic acid content of 13.18%. The monosaccharide composition and its molar ratio are arabinose:rhamnose:mannose:glucose:galacturonic acid = 0.169:0.256:0.464:1:1.396. The Huangda tea polysaccharide L2-1 prepared by this invention can significantly improve the survival rate of L-Glu-induced PC12 cells and restore cell morphology, enhance the learning and memory abilities of APP / PS1 transgenic mice, inhibit the excessive activation of astrocytes in mouse brain tissue, and reduce neuroinflammation in the mouse brain. Huangda tea polysaccharide L2-1 has the function of improving / treating Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to a Huangda tea polysaccharide, its preparation method, and its uses. Background Technology

[0002] In recent years, with the diversification of tea consumption demands and people's pursuit of a healthy lifestyle, Huangda tea has become increasingly popular among consumers due to its unique flavor and multiple health benefits. Huangda tea is mainly produced in Jinzhai and Huoshan counties in western Anhui province. It has large leaves, long stems, a yellow liquor, and a strong, roasted aroma (commonly known as "crispy rice aroma"). It is rich in various active ingredients, such as catechins, theanine, and tea polysaccharides. Tea polysaccharides, as one of the important active components of tea, have been confirmed by relevant studies to have good antioxidant, anti-tumor, immune-enhancing, and blood sugar and lipid-lowering effects. Recent research in our group has found that crude polysaccharides from Huangda tea can alleviate cognitive impairment in Alzheimer's disease model mice by inhibiting oxidative stress, but the active components responsible for this effect are still unclear. The active components in Huangda tea that improve Alzheimer's disease, their physicochemical properties, and their relationship with efficacy urgently need to be clarified. Summary of the Invention

[0003] This invention provides a Huangda tea polysaccharide, its preparation method, and its uses. The Huangda tea polysaccharide with uniform components is obtained through separation and purification and applied to improve Alzheimer's disease.

[0004] This invention provides an active polysaccharide from Huangda tea, abbreviated as L2-1, which has a total sugar content of 91.89%, a protein content of 1.19%, a uronic acid content of 13.18%, and is free of phenolic and starchy substances.

[0005] The molecular weight of the Huangda tea polysaccharide L2-1 is 1.02 × 10⁻⁶. 4 The monosaccharide composition and its molar ratio are arabinose:rhamnose:mannose:glucose:galacturonic acid = 0.169:0.256:0.464:1:1.396.

[0006] The preparation method of Huangda tea polysaccharide L2-1 of the present invention includes the following steps:

[0007] Step 1: Grind Huangda tea into powder, pass it through a 200-mesh sieve to obtain Huangda tea powder, and pre-treat the Huangda tea powder by decolorization and degreasing; specifically, mix Huangda tea powder with anhydrous ethanol, extract at 70℃, repeat 3 times, collect the precipitate, dry at 37℃ to obtain pre-treated Huangda tea powder.

[0008] Step 2: Add distilled water to the pretreated Huangda tea powder obtained in Step 1, extract at 95℃, repeat 3 times, collect the supernatant, concentrate and add four times the volume of anhydrous ethanol, let stand at 4℃ for 8-12 hours, remove protein and dialyze to obtain Huangda tea crude polysaccharide.

[0009] Step 3: The crude polysaccharide of Huangda tea obtained in Step 2 was separated by ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 10kDa and 100kDa, and then concentrated and freeze-dried to obtain component L2.

[0010] Step 4: Pass the L2 fraction obtained in Step 3 through a DEAE-Sepharose Fast Flow anion exchange column. The loading concentration is 20 mg / mL, and the loading volume does not exceed 5% of the column volume. The mobile phases are distilled water (200 mL) and sodium chloride solution (0.1, 0.3, 0.5 M, pH 7, 600 mL), respectively. The elution rate is 2 mL / min. The elution curve is tracked and plotted using the phenol-sulfuric acid method. The main polysaccharide fraction is collected.

[0011] Step 5: The polysaccharide fraction of Huangda tea collected in Step 4 was further purified by Superdex 75 gel chromatography. The sample concentration was 100 mg / mL, the mobile phase was water, the flow rate was 0.3 mL / min, and the volume was 3 mL per tube. The phenol-sulfuric acid method was used for tracking. The same polysaccharide was combined by HPLC peak time and lyophilized to obtain fraction L2-1.

[0012] The Huangda tea polysaccharide L2-1 prepared by this invention is an acidic polysaccharide that has neuroprotective effects and can improve Alzheimer's disease.

[0013] The purpose of this invention, using Huangda tea polysaccharide L2-1, is to prepare pharmaceutical preparations for improving or treating Alzheimer's disease. These preparations significantly improve the survival and morphology of L-Glu-induced PC12 cells and have preventative and ameliorative effects on cognitive impairment in APP / PS1 mice.

[0014] Cellular experiments of this invention showed that Huangda tea polysaccharide could significantly restore the morphology of L-Glu-induced PC12 cells. Compared with the model group, the survival rate after L2-1 intervention increased from 49.39% to 81.96%. Animal experiments showed that L2-1 significantly increased the number of times APP / PS1 mice crossed the platform, the time spent in the platform quadrant, and shortened the escape latency of mice, with effects comparable to the positive control drug (donepezil). At the same time, it could alleviate the overactivation of GFAP in mouse brain tissue, reducing its expression by 62.7%, and significantly improving cognitive dysfunction in Alzheimer's mice.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention provides a homogeneous component of Huangda tea polysaccharide L2-1, which has good neuroprotective effects, can reduce neuroinflammation, alleviate neuronal apoptosis, and improve cognitive dysfunction. It can be used to prepare effective components for improving / treating Alzheimer's disease. Attached Figure Description

[0017] Figure 1 Elution curve of crude polysaccharide DEAE-Sepharose from Huangda tea.

[0018] Figure 2 HPLC molecular weight distribution chromatogram.

[0019] Figure 3 Monosaccharide composition analysis: A is the GC chromatogram of standard monosaccharides; B is the GC chromatogram of L2-1 monosaccharide composition.

[0020] Figure 4 Analysis of the neuroprotective effect of Huangda tea polysaccharide L2-1 on L-Glu-induced PC12 cells: A is the effect of L2-1 on the survival rate of L-Glu-induced PC12 cells; B is the effect of L2-1 on the cell morphology of L-Glu-induced PC12 cells.

[0021] Figure 5 The movement trajectory of mice in the water maze experiment.

[0022] Figure 6 : Latency period of mouse escape.

[0023] Figure 7 Number of times the mouse crossed the platform.

[0024] Figure 8 : Time spent in the plateau quadrant of the mouse.

[0025] Figure 9 Immunofluorescence dual localization map of GFAP and Aβ in mouse brain tissue.

[0026] Figure 10 : This refers to the expression of IL-1β in mouse brain tissue. Detailed Implementation

[0027] The preparation of Huangda tea polysaccharide and its neuroprotective and Alzheimer's disease-improving effects are further illustrated below with specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0028] Example 1: Preparation of Huangda Tea Polysaccharide

[0029] Step 1: Pulverize the dried Huangda tea and pass it through a 200-mesh sieve. Mix the resulting Huangda tea powder with anhydrous ethanol at a ratio of 1g:5mL, and heat in a 70℃ water bath for 2 hours. Repeat this process three times. Take the precipitate and dry it at 37℃ to obtain the pretreated Huangda tea powder.

[0030] Step 2: Mix the Huangda tea powder obtained in Step 1 with distilled water at a ratio of 1g:20mL, extract with water at 95℃, repeat 3 times, 2h each time, stirring intermittently, combine the supernatants, concentrate and add 4 times the volume of anhydrous ethanol, let stand at 4℃ for 12h, the final concentration of anhydrous ethanol is 80%, collect the precipitate after alcohol precipitation, remove the alcohol by rotary evaporation; mix the Huangda tea alcohol precipitate solution with 3% trichloroacetic acid 1:1, let stand at 4℃ for 12h, remove protein, repeat 2 times, dialyze with running water for 48h using a dialysis bag with a molecular weight cutoff of 3.5kDa, dialyze with still water for 24h, and freeze dry under vacuum to obtain Huangda tea crude polysaccharide.

[0031] Step 3: The crude polysaccharide obtained in Step 2 was separated by ultrafiltration using 10kDa and 100kDa molecular weight cutoff ultrafiltration membranes. The 10kDa to 100kDa polysaccharide fraction was collected, concentrated, and freeze-dried to obtain the L2 fraction.

[0032] Step 4: Load the L2-type DEAE-Sepharose Fast Flow anion exchange column prepared in Step 3 with a sample concentration of 20 mg / mL and a sample volume not exceeding 5% of the column volume. The mobile phases are distilled water (200 mL) and sodium chloride solution (0.1, 0.3, 0.5 M, pH 7, 600 mL), respectively. The elution rate is 2 mL / min, and the elution curve is plotted using the phenol-sulfuric acid method. Collect the main polysaccharide components.

[0033] Step 5: The polysaccharide fraction obtained in Step 4 was separated and purified by Superdex 75 gel chromatography. The sample concentration was 100 mg / mL, the mobile phase was water, the flow rate was 0.3 mL / min, and the volume was 3 mL per tube. The phenol-sulfuric acid method was used for tracking. The same polysaccharide was combined by HPLC peak time and lyophilized to obtain fraction L2-1.

[0034] Step 6: Physicochemical property analysis of L2-1. The total sugar content of the polysaccharide was determined using the phenol-sulfuric acid method. Glucose was used as a standard, and glucose solutions of different concentrations were prepared according to a gradient. A 100 μg / mL L2-1 polysaccharide solution was also prepared, with 1 mL of 6% phenol solution, 5 mL of sulfuric acid, and 2 mL of standard solutions and sample solutions of different concentrations added sequentially. After incubation at room temperature for 30 min, the absorbance was measured at 490 nm. The protein content of the polysaccharide was determined using the Coomassie brilliant blue method, with bovine serum albumin as a standard. Similar to the above method, 2.5 mL of Coomassie brilliant blue solution and 1 mL of standard solutions and sample solutions of different concentrations were added sequentially. After incubation at room temperature for 20 min, the absorbance was measured at 595 nm. The uronic acid content of the polysaccharide was determined using the carbazole-sulfuric acid method, with galacturonic acid as a standard, and the absorbance of each group was measured at 520 nm. The presence of starch in the sample was determined using iodine-potassium iodide. The total phenol content of the polysaccharide was determined using gallic acid as a standard. The results are shown in Table 1. The total sugar content of L2-1 was 91.89%, the protein content was 1.19%, and the uronic acid content was 13.18%.

[0035] Table 1 Physicochemical properties of L2-1

[0036] sample Total sugar protein Glucuronic acid Total phenols starch L2-1 91.89％ 1.19％ 13.18％ 0％ 0％

[0037] Example 2: Determination of molecular weight and monosaccharide composition of Huangda tea polysaccharide L2-1

[0038] Step 1: Detection of L2-1 using an Agilent 1260 high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). Prepare 1 mL of 3 mg / mL L2-1 and 1 mL of T5, T12, T41, T100, and T200 dextran standards, filter through a 0.22 μm aqueous membrane, and use a TSKGel G6000 PWXL column (300 × 7.0 mm, 13 μm), with N2 as the carrier gas at a flow rate of 2.5 L / min, an injection volume of 10 μL at a flow rate of 0.6 mL / min, and water as the mobile phase. A standard curve was plotted using the logarithm of the relative molecular mass (LgMw) and retention time (Rt) of the standards to determine the molecular weight of L2-1.

[0039] Step 2: L2-1 monosaccharide composition analysis employed an acid hydrolysis-pre-column derivatization method for sample processing, followed by gas chromatography analysis. 10 mg of polysaccharide was weighed into a 30 mL reaction flask, dissolved in 2.5 M trifluoroacetic acid, sealed with nitrogen, and incubated in an oil bath at 121 °C for 4 h to ensure complete hydrolysis. The solution was then evaporated to dryness under reduced pressure at 50 °C. Trifluoroacetic acid was removed by repeated additions of methanol until neutral, and the solution was then dried under N2. 2 mg of each standard monosaccharide was weighed for later use.

[0040] Step 3: Add 1 mL of pyridine, 0.4 mL of hexamethyldisilazane, and 0.2 mL of trimethylchlorosilane sequentially to the above hydrolysis product, monosaccharide standard, and mixed standard. Mix thoroughly and let stand at room temperature for 5 min. Then filter the product through a 0.22 μm organic phase filter membrane. Then perform gas chromatography. Select an Agilent HP-5 capillary column (30 m × 0.25 mm × 0.25 μm), nitrogen as carrier gas, and column flow rate of 1 mL / min. Equipped with a flame ionization detector (FID) at 250 °C, hydrogen, air, and make-up gas flow rates of 40, 400, and 30 mL / min, respectively. Injector temperature is 250 °C, injection volume is 0.5 μL, and split ratio is 30:1. Column temperature program is 100 °C for 5 min, then increase to 150 °C at a rate of 5 °C / min, hold for 5 min, then increase to 240 °C at a rate of 5 °C / min and hold for 5 min.

[0041] The liquid chromatogram of L2-1 is as follows: Figure 3 As shown in Figure A, based on the standard curves of dextran with different molecular weights, the molecular weight of L2-1 was calculated to be 1.02 × 10⁻⁶. 4 Da. Monosaccharide composition and molar ratio Figure 3 As shown in B, the ratio of arabinose:rhamnose:mannose:glucose:galacturonic acid is 0.169:0.256:0.464:1:1.396.

[0042] Table 2. L2-1 Monosaccharide Composition Analysis

[0043] Monosaccharide composition Arabic sugar Rhamnose Mannose glucose Galacturonic acid L2-1 (mol%) 0.169 0.256 0.464 1 1.396

[0044] Example 3: Effect of L2-1 on L-Glu-induced PC12 cells

[0045] Step 1: PC12 cells were cultured in 1640 complete medium to the logarithmic growth phase and seeded into 96-well plates. They were cultured in a CO2 cell incubator for 24 hours until the cells adhered. The blank control group and the model group were given blank medium. The positive control group was given blank medium at a concentration of 10 μg / mL. The experimental groups were given blank medium at concentrations of 100, 200, and 400 μg / mL, respectively. After pretreatment of the cells for 3 hours, they were incubated with 20 mM L-Glu for 12 hours, followed by incubation with 0.5 mg / mL MTT for 24 hours. The live cells were stained purple. Finally, DMSO was added and the cells were shaken. The absorbance was measured at 490 nm.

[0046] L2-1-induced L-Glu-induced PC12 cell survival rate as follows Figure 4 As shown in Figure A, the high-dose L2-1 group increased the survival rate of PC12 cells after induction from 49% to 81.96%.

[0047] Table 3 Effect of L2-1 on L-Glu-induced PC12 cell viability

[0048] Blank group 100％ Model group (20 ml - Glu) <![CDATA[49.38%±3.33% e ]]> Donepezi (10 μg / mL) <![CDATA[73.75%±0.69% c ]]> L2-1 (100 mg / mL) <![CDATA[59.35%±3.99% d ]]> L2-1 (200 mg / mL) <![CDATA[75.27%±2.21% c ]]> L2-1 (400 mg / mL) <![CDATA[81.96%±2.4% b ]]>

[0049] Different letters represented significant differences compared to the model group (p < 0.05).

[0050] Step 2: After PC12 cells reach the logarithmic growth phase, they are seeded into 6-well plates and cultured until adherent. Following Step 1, different concentrations of polysaccharides are incubated for 3 hours, followed by co-incubation with 20 mM L-Glu for 12 hours. PC12 cell morphology is then observed using an inverted fluorescence microscope. Figure 4 As shown in B, the number of cells in the model group was significantly reduced, synapses were atrophied, and cell morphology was severely damaged. After administration of L2-1, as the dose increased, the synapses of PC12 cells recovered, the morphology tended to be normal, and the number of cells increased.

[0051] Example 4: Effects of Huangda Tea Polysaccharide L2-1 on Cognitive Function in APP / PS1 Mice

[0052] Step 1: Thirty 20-week-old APP / PS1 transgenic mice were divided into a model group, donepezi, and low, medium, and high-dose L2-1 groups. Wild-type C57BL / 6 mice from the same litter served as a control group. Each group consisted of six mice. The control and model groups were administered the same volume of physiological saline by gavage. The positive control group was administered 1 mg / kg / day of donepezi by gavage, and the polysaccharide groups were administered 100, 200, and 400 μg / mL L2-1 by gavage. This gavage treatment lasted for 10 weeks.

[0053] Step 2: After 8 weeks of gavage, mice underwent a water maze behavioral test. The water maze was a circular pool divided into four quadrants, with a platform placed 1 cm underwater. It was equipped with a temperature control system, a camera, and the Xeye Aba animal behavior video analysis system connected to a computer. The experiment lasted 6 days, consisting of 5 days of orientation and navigation tests and 1 day of spatial exploration tests. In the orientation and navigation test, the circular platform was placed in the third quadrant. Each mouse was placed into the maze facing the pool wall from one of the four quadrants in turn. The test lasted 120 seconds. If the mouse found the platform within 120 seconds, the time was recorded as the escape latency. If the mouse could not find the platform within 120 seconds, it was guided to stand on the platform for 20 seconds. This was repeated for 5 days. On the sixth day, the platform was removed, and the mice were placed into the maze facing the pool wall from the quadrant opposite the platform. They were allowed to swim freely for 60 seconds. The animal behavior video analysis system recorded the mice's movement trajectory, escape latency, and the number of times they crossed the platform. Figure 5 As shown, the water maze test revealed that the movement trajectories of the model group mice were scattered and mostly concentrated in the platform quadrant, while after L2-1 treatment, the movement trajectories of the mice gradually concentrated in the platform quadrant. Figure 6 The escape latency of mice was significantly reduced after L2-1 treatment, as shown. Figure 7 The number of times mice crossed the platform after L2-1 treatment was significantly increased compared to APP / PS1 mice; Figure 8 As shown, the time mice spent in the target quadrant increased significantly after L2-1 treatment.

[0054] Table 4. Escape latency and number of platform crossings in mice

[0055] Grouping Incubation period (s) Number of times the platform was traversed (times) Blank group <![CDATA[7.583±0.022 c ]]> <![CDATA[3.75±1.25 a ]]> APP / PS1 <![CDATA[50.028±8.009 a ]]> <![CDATA[0.667±0.33 c ]]> Donepezi (1 mg / kg / day) <![CDATA[9.01±1.377 c ]]> <![CDATA[3.667±0.33 a ]]> L2-1 (100 mg / mL) <![CDATA[29.536±1.005 b ]]> <![CDATA[2.25±0.75 b ]]> L2-1 (200 mg / mL) <![CDATA[17.18±2.645 bc ]]> <![CDATA[2.5±0.5 ab ]]> L2-1 (400 mg / mL) <![CDATA[10.423±3.453 c ]]> <![CDATA[3.33±0.667 ab ]]>

[0056] Different letters represented significant differences compared to the model group (p < 0.05).

[0057] Step 3: After 10 weeks of gavage, the mice were fasted for one night and then euthanized with sodium barbital. Blood was collected, and right brain tissue was obtained and fixed with paraformaldehyde for 24 hours. Immunofluorescence staining was then performed: paraffin sections were descaled into water for antigen retrieval. A circular motion was drawn around the tissue with a histochemical pen to prevent antibody migration. Endogenous peroxidase was blocked with hydrogen peroxide, followed by BSA for 30 minutes. Primary antibody (GFAP) was added and the mice were incubated overnight at 4°C. Slides were then placed in PBS (pH 7.4) and washed three times for 5 minutes each time on a decolorizing shaker. After slightly drying the slides, add HRP-labeled secondary antibody corresponding to the species of the primary antibody to the inner circle and cover the tissue. Incubate at room temperature for 50 min, destain the slides three times for 5 min each time, then add TSA, incubate in the dark for 10 min, wash three times for 5 min each time, and then microwave to bind the primary and secondary antibodies to the tissue. Add the second primary antibody (Aβ), incubate overnight at 4°C, then add the corresponding secondary antibody, incubate in the dark for 50 min, add DAPI staining solution, incubate in the dark for 10 min, wash three times for 5 min each time, dry, add autofluorescence quencher for 5 min, wash with running water for 10 min, dry, mount with antifluorescence quenching mounting medium, and finally observe and acquire images under a fluorescence microscope. The dual localization map of GFAP and Aβ immunofluorescence in mouse brain tissue is shown below. Figure 9 As shown, GFAPs are arranged in a star-like pattern around the Aβ plate, and L2-1 can significantly reduce the activation of GFAPs.

[0058] Step 4: Obtain left brain tissue, rapidly freeze in liquid nitrogen, and then store at -80℃ for ELISA detection of relevant inflammatory factors: Weigh 10mg of tissue, add PBS at a 1:9 ratio, grind on ice, centrifuge to collect the supernatant, determine the total BCA protein concentration, and determine the IL-1β content according to the kit instructions. Mouse brain tissue IL-1β as... Figure 10 As shown, L2-1 can significantly reduce IL-1β expression in the brain tissue of APP / PS1 mice.

Claims

1.A polysaccharide L2-1 of Camellia reticulata Lindl, characterized in that: the polysaccharide L2-1 of Camellia reticulata Lindl has a total sugar content of 91.89%, a protein content of 1.19%, and an uronic acid content of 13.18%, and is free of phenolic and starch substances; and the polysaccharide L2-1 of Camellia reticulata Lindl is prepared by a method comprising the following steps: Step 1: crushing Camellia reticulata Lindl, passing the powder through a 200-mesh sieve to obtain Camellia reticulata Lindl powder, and performing decolorization and degreasing pretreatment on the Camellia reticulata Lindl powder; Step 2: sequentially subjecting the pretreated Camellia reticulata Lindl powder obtained in Step 1 to water extraction, alcohol precipitation, deproteinization, and dialysis to obtain Camellia reticulata Lindl crude polysaccharides; Step 3: subjecting the Camellia reticulata Lindl crude polysaccharides prepared in Step 2 to ultrafiltration separation using an ultrafiltration membrane with a molecular weight cut-off of 10 kDa and 100 kDa, and then concentrating and freeze-drying to obtain an L2 component; Step 4: passing the L2 component obtained in Step 3 through an anion exchange column, and collecting a main polysaccharide component; and Step 5: further purifying the polysaccharide component collected in Step 4 by passing it through a Superdex 75 gel chromatography column, combining the same polysaccharide by HPLC peak time, and freeze-drying to obtain an L2-1 component. 2.The polysaccharide L2-1 of Camellia reticulata Lindl according to claim 1, characterized in that: in Step 1, the decolorization and degreasing pretreatment is performed by mixing the Camellia reticulata Lindl powder with anhydrous ethanol, extracting at 70℃ for 3 times, taking the precipitate, and drying at 37℃ to obtain the pretreated Camellia reticulata Lindl powder. The molecular weight of the yellow large tea polysaccharide L2-1 is 1.02 x 10 4 Da, the monosaccharide composition and the molar ratio thereof are arabinose: rhamnose: mannose: glucose: galacturonic acid = 0.169: 0.256: 0.464: 1: 1.396; 3.The polysaccharide L2-1 of Camellia reticulata Lindl according to claim 1, characterized in that: in Step 2, the water extraction is performed at 95℃ for 2h, and the extraction is repeated 3 times, and the extraction solutions are combined. 4.The polysaccharide L2-1 of Camellia reticulata Lindl according to claim 1, characterized in that: in Step 2, the alcohol precipitation is performed by adding four times the volume of anhydrous ethanol, and the solution is allowed to stand at 4℃ for 8-12h. 5.The polysaccharide L2-1 of Camellia reticulata Lindl according to claim 1, characterized in that: in Step 4, the anion exchange column is a DEAE-Sepharose Fast Flow anion exchange column, the loading concentration is 20 mg / mL, the loading volume does not exceed 5% of the column volume, the mobile phase is a gradient elution of distilled water and sodium chloride solution, and the concentration of the sodium chloride solution is set to 0.1M, 0.3M, and 0.5M during the elution stage, and the elution speed is 2 mL / min. 6.The polysaccharide L2-1 of Camellia reticulata Lindl according to claim 1, characterized in that: in Step 5, when the Superdex 75 gel chromatography column is used for further purification, the loading concentration is 100 mg / mL, the mobile phase is water, and the flow rate is 0.3 mL / min. 7.Use of the polysaccharide L2-1 of Camellia reticulata Lindl according to any one of claims 1-6, characterized in that: the polysaccharide L2-1 of Camellia reticulata Lindl is used for preparing a pharmaceutical preparation for improving or treating Alzheimer's disease. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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