Preparation method of physalis pubescens root polysaccharide with antioxidant and nerve cell protection activity

Abstract

This invention relates to a method for preparing polysaccharides from *Phyllostachys edulis* root with antioxidant and neuroprotective activities, and is a method for preparing natural drugs. The invention involves reflux extraction of pulverized *Phyllostachys edulis* root with ethanol to remove small molecules, followed by extraction with hot water reflux to obtain crude polysaccharides. The crude polysaccharides are then purified through a series of separation and purification processes, including protein removal, depigmentation, and column chromatography, to obtain four new natural polysaccharides: PPLR-W, PPLR-1, PPLR-2, and PPLR-3. The natural polysaccharides prepared by this invention lay the foundation for the development of antioxidant-related drugs and clinical research on the prevention or treatment of neurodegenerative diseases. The four new *Phyllostachys edulis* root polysaccharides possess DPPH and ABTS free radical scavenging abilities and exhibit significant neuroprotective effects against hydrogen peroxide-induced SH-SY5Y cell damage models.

Description

Technical Field

[0001] This invention relates to a method for preparing natural medicines, and in particular to a method for preparing polysaccharides from *Phyllostachys edulis* root with antioxidant and neuroprotective activities. Background Technology

[0002] Physalis peruviana ( Physalis pubescens Physalis alkekengi (also known as "Groundhog" or "Foreign Girl") is an annual herbaceous plant belonging to the genus Physalis in the Solanaceae family. It is reportedly native to Latin America and is now widely cultivated in Northeast China and Inner Mongolia. Its dual medicinal and edible properties, along with its rich nutritional content, have attracted considerable attention from scholars in the pharmaceutical and food industries. Physalis alkekengi is not only rich in nutrients but also a popular seasonal fruit, which can be processed into juice, canned goods, and other foods, giving it high industrial value. It is also a traditional folk remedy, used in prescriptions to treat symptoms such as coughs due to lung heat, acute bronchitis, hoarseness, and sore throat.

[0003] Currently, chemical components isolated from *Physalis* plants include steroids, flavonoids, sterols, alkaloids, volatile oils, abundant inorganic elements, and polysaccharides. Polysaccharides are natural high-molecular-weight polymers composed of monosaccharides linked by α- or β-glycosidic bonds. They are one of the basic substances constituting life activities, characterized by their large molecular weight and complex structure. As a major component of *Physalis* plants, polysaccharides possess rich biological activities. Modern pharmacological studies have shown that *Physalis* polysaccharides exhibit pharmacological activities such as immunomodulation, hepatoprotection, antioxidant activity, anti-hyperglycemia, and anti-hyperlipidemia.

[0004] Studies have shown that the higher-order spatial structure, monosaccharide composition, molecular weight, and specific functional groups of polysaccharides closely affect their biological activity and physicochemical properties. This invention investigated the antioxidant activity of purified *Phyllostachys edulis* root polysaccharides. The results showed that the polysaccharides exhibited scavenging activity against both types of free radicals, with the scavenging rate increasing with increasing sample concentration. Furthermore, the protective effect of the aforementioned *Phyllostachys edulis* root polysaccharides against H2O2-induced SH-SY5Y cell damage was detected using the MTT assay. The experimental results showed that the polysaccharides possessed certain neuroprotective activity, exhibiting a dose-dependent effect. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing polysaccharides from *Phyllostachys edulis* root with antioxidant and neuroprotective activities. This method involves extracting, separating, and purifying four natural polysaccharides from *Phyllostachys edulis* root. The prepared *Phyllostachys edulis* root polysaccharides possess antioxidant and neuroprotective activities, laying the foundation for the development of antioxidant-related drugs and for future clinical applications in the prevention or treatment of neurodegenerative diseases.

[0006] This invention is achieved through the following technical solution:

[0007] Preparation and purification of polysaccharides from *Phyllostachys pubescens* roots:

[0008] (1) Take the dried roots of *Physalis alkekengi*, grind them with a pulverizer, reflux them with ethanol to remove small molecule compounds, then reflux and condense them with distilled water 2-6 times, filter them, concentrate them under reduced pressure, and combine the filtrates.

[0009] (2) Prepare a polysaccharide solution, add a neutral protease to the polysaccharide solution, stir, and after reacting for a period of time, centrifuge and collect the supernatant; add 1 / 2-1 / 4 volume of Sevage reagent (1:3-1:5, v / v) prepared by n-butanol and chloroform to the supernatant, stir for 20-40 min, centrifuge, collect the supernatant, and remove the protein 2-5 times using the Sevage reagent method until no protein layer is precipitated.

[0010] (3) The supernatant obtained above is concentrated, decolorized using AB-8, D101, D301, and D307 resin columns, eluted with distilled water, and concentrated under reduced pressure to obtain the decolorized polysaccharide.

[0011] (4) The deproteinized and depigmented polysaccharides were separated using a DEAE-52 cellulose column and eluted with distilled water and NaCl solutions of different concentrations. The content was determined by the phenol-sulfuric acid method. The four components with higher sugar content were collected by Sephadex G-200 dextran gel purification, concentrated and dried under reduced pressure to obtain four new natural polysaccharides: PPLR-W, PPLR-1, PPLR-2 and PPLR-3.

[0012] (5) Structural analysis of four new *Phyllostachys pubescens* root polysaccharides: PPLR-W, PPLR-1, PPLR-2, and PPLR-3:

[0013] (6) The molecular weight range of natural polysaccharide 1PPLR-W is 20-23 kDa. The monosaccharide composition and molar ratio range is rhamnose: arabinose: xylose: mannose: glucose: galactose = (13.21-15.50): (1.70-2.13): (4.76-7.15): (15.58-17.99): (24.52-27.22): (19.88-22.40). The skeletal structure is shown below:

[0014] →6)- α -D-Gal p -(1→、→3)- α -D-Rha p -(1→、→3,5)- α -L-Ara f -(1→、→3)- α -D-Glc p-(1→、→6)- α -D-Man p -(1→and→2,4)- β -D-Xyl p -(1→).

[0015] The molecular weight range of natural polysaccharide 2PPLR-1 is 14-16 kDa. Its monosaccharide composition and molar ratio range are: rhamnose: arabinose: xylose: galacturonic acid: mannose: glucose: galactose = (10.95-13.35): (3.25-5.75): (4.74-7.20): (0.20-2.50): (11.37-13.78): (21.82-24.26): (22.90-25.41). The skeletal structure is shown below:

[0016] →3)- α -D-Gal p A-(1→、→6)- α -D-Gal p -(1→、→3)- α -D-Rha p -(1→、 α -L-Ara f -(1→、→3)- α -D-Glc p -(1→、→6)- α -D-Man p -(1→and→2,4)- β -D-Xyl p -(1→).

[0017] The molecular weight range of natural polysaccharide 3PPLR-2 is 20-24 kDa. Its monosaccharide composition and molar ratio range are: rhamnose: arabinose: xylose: galacturonic acid: mannose: glucose: galactose = (15.73-17.23): (2.78-5.17): (4.31-7.14): (0.19-2.51): (12.28-14.69): (22.42-24.82): (20.43-22.83). The skeletal structure is shown below:

[0018] →3)- α -D-Gal p A-(1→、→6)- α -D-Gal p -(1→、→4)- α -D-Rha p -(1→、 α -L-Ara f -(1→、 α -D-Glcp -(1→、→4)- α -D-Man p -(1→and→2,4)- β -D-Xyl p -(1→).

[0019] The molecular weight range of the natural polysaccharide 4PPLR-3 is 16-18 kDa. Its monosaccharide composition and molar ratio range are: rhamnose: arabinose: xylose: galacturonic acid: mannose: glucose: galactose = (13.09-15.49): (2.13-4.55): (4.15-6.56): (0.10-2.49): (12.59-14.99): (23.98-26.51): (20.07-22.47). The skeletal structure is shown below:

[0020] →3)- α -D-Gal p A-(1→、→6)- α -D-Gal p -(1→、→3)- α -D-Rha p -(1→、 α -L-Ara f -(1→、 α -D-Glc p -(1→、→6)- α -D-Man p -(1→and→2,4)- β -D-Xyl p -(1→).

[0021] The advantages and effects of this invention are:

[0022] 1. The four novel *Phyllostachys pubescens* root polysaccharides of this invention were tested for their scavenging effects on DPPH and ABTS free radicals. The natural *Phyllostachys pubescens* root polysaccharides exhibited scavenging activity against both of these free radicals. The MTT assay was used to detect the effect of *Phyllostachys pubescens* root polysaccharides on H2O2-induced SH-SY5Y cell damage. The results showed that *Phyllostachys pubescens* root polysaccharides significantly restored cell viability in a dose-dependent manner, with PPLR-1 showing the most significant protective effect against H2O2-induced SH-SY5Y cell damage.

[0023] 2. The four novel natural polysaccharides isolated and extracted from the roots of *Phyllostachys edulis* in this invention possess antioxidant and neuroprotective activities, laying the foundation for the development of antioxidant-related drugs and their clinical application in the prevention or treatment of neurodegenerative diseases. Attached Figure Description

[0024] Figure 1It is PPLR-W 1 H-NMR spectrum;

[0025] Figure 2 It is PPLR-W 13 C-NMR spectrum;

[0026] Figure 3 This is the HSQC spectrum of PPLR-W;

[0027] Figure 4 This is the HMBC map of PPLR-W;

[0028] Figure 5 It is PPLR-W 1 H- 1 H-COSY spectrum;

[0029] Figure 6 It is PPLR-1 1 H-NMR spectrum

[0030] Figure 7 It is PPLR-1 13 C-NMR spectrum;

[0031] Figure 8 This is the HSQC spectrum of PPLR-1;

[0032] Figure 9 This is the HMBC spectrum of PPLR-1;

[0033] Figure 10 It is PPLR-1 1 H- 1 H-COSY spectrum;

[0034] Figure 11 It is PPLR-2. 1 H-NMR spectrum;

[0035] Figure 12 It is PPLR-2. 13 C-NMR spectrum;

[0036] Figure 13 This is the HSQC spectrum of PPLR-2;

[0037] Figure 14 This is the HMBC spectrum of PPLR-2;

[0038] Figure 15 It is PPLR-2. 1 H- 1 H-COSY spectrum;

[0039] Figure 16 It is PPLR-3.1 H-NMR spectrum;

[0040] Figure 17 It is PPLR-3. 13 C-NMR spectrum;

[0041] Figure 18 This is the HSQC spectrum of PPLR-3;

[0042] Figure 19 This is the HMBC spectrum of PPLR-3;

[0043] Figure 20 It is PPLR-3. 1 H- 1 H-COSY spectrum;

[0044] Figure 21 This is a monosaccharide composition spectrum of Physalis alkekengi root polysaccharide;

[0045] Figure 22 This is a graph showing the ability of Physalis alkekengi root polysaccharides (PPLR-W, PPLR-1, PPLR-2, PPLR-3) to scavenge DPPH free radicals;

[0046] Figure 23 This is a graph showing the ability of Phyllanthus urinaria root polysaccharides (PPLR-W, PPLR-1, PPLR-2, PPLR-3) to scavenge ABTS free radicals;

[0047] Figure 24 This is a graph showing the neuroprotective activity of Phyllostachys edulis polysaccharides (PPLR-W, PPLR-1, PPLR-2, PPLR-3) against H2O2-induced SH-SY5Y cells. Implementation

[0048] The present invention will be described below through specific embodiments, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and biological materials are all of analytical or chromatographic purity and, unless otherwise specified, can be obtained commercially.

[0049] Example 1: Extraction, separation and purification of polysaccharides from *Phyllostachys pubescens* roots

[0050] The dried roots of *Physalis alkekengi* were ground using a pulverizer and refluxed twice with 3 times their volume of 80% ethanol for 3 hours each time. The mixture was filtered, and the residue was allowed to air dry naturally. Subsequently, it was extracted four times by reflux with distilled water, followed by filtration, vacuum concentration, and drying. A polysaccharide solution was prepared, and a neutral protease was added. After stirring and reacting for a period of time, the supernatant was collected by centrifugation. Sevage reagent (1:3-1:5, v / v) prepared from n-butanol and chloroform was added to the supernatant, and the mixture was stirred for 20-40 minutes. The supernatant was then collected by centrifugation, and the protein was removed four times using the Sevage method until no protein precipitate was observed. The obtained supernatant was concentrated and decolorized using an AB-8 resin column. Each time, 15 g of the deproteinized polysaccharide was dissolved and loaded onto the column, and deionized water was used as the mobile phase for elution. After concentration, decolorized *Physalis alkekengi* root polysaccharide was obtained.

[0051] The deproteinized and depigmented polysaccharides were separated using a DEAE-52 cellulose column. The elution flow rate was set at 1.0 mL / min, with a collection interval of 10 min per tube. Elution was performed sequentially with distilled water and NaCl solutions of different concentrations. The absorbance of the polysaccharide solutions in the tubes was measured using the phenol-sulfuric acid method at 490 nm. Eluents with symmetrical peak shapes were collected and combined, concentrated, and dialyzed. Further purification of the obtained polysaccharide fractions was achieved using a Sephadex G-200 column. Four fractions with higher sugar content were collected, concentrated under reduced pressure, dialyzed, and dried to obtain polysaccharides PPLR-W, PPLR-1, PPLR-2, and PPLR-3.

[0052] Example 2: Nuclear Magnetic Spectroscopic Analysis of Physalis Root Polysaccharide

[0053] The primary structures of polysaccharides PPLR-W, PPLR-1, PPLR-2, and PPLR-3 from the roots of Physalis alkekengi were analyzed using NMR technology. The NMR spectra (600 MHz, D2O) are shown in the figure. 1 H-NMR and combined 13 C-NMR and HSQC analyses indicate that the PPLR-W spectrum at a shift value of 5.17 / 100.6 ppm indicates the presence of →6)- α -D-Gal p -(1→fraction, indicating presence at 5.11 / 98.1 ppm →3)- α -D-Rha p -(1→fraction, indicating presence at 5.06 / 101.1 ppm →3,5)- α -L-Ara f -(1→fraction, indicating presence at 5.02 / 96.2 ppm →3)- α -D-Glc p-(1→fraction, indicating presence at 4.91 / 98.0 ppm →6)- α -D-Man p -(1→fraction, indicating the presence of →2,4 at 4.42 / 102.7 ppm)- β -D-Xyl p -(1→fragment; the PPLR-1 spectrum at a shift value of 5.27 / 99.8 ppm indicates the presence of →3)- α -D-Gal p A-(1→fraction, indicating presence at 5.18 / 101.3ppm→6)- α -D-Gal p -(1→fraction, indicating presence at 5.11 / 97.9 ppm →3)- α -D-Rha p -(1→fraction, indicating presence at 5.06 / 100.7 ppm) α -L-Ara f -(1→fraction, indicating presence at 5.01 / 98.6 ppm →3)- α -D-Glc p -(1→fraction, indicating presence at 4.85 / 99.3ppm →6)- α -D-Man p -(1→fraction, indicating the presence of →2,4 at 4.43 / 102.8 ppm)- β -D-Xyl p -(1→fragment; the PPLR-2 spectrum at a chemical shift of 5.25 / 97.8 ppm indicates the presence of →3)- α -D-Gal p A-(1→fraction, indicating presence at 5.14 / 100.5 ppm →6)- α -D-Gal p -(1→fraction, indicating presence at 5.12 / 97.7 ppm →4)- α -D-Rha p -(1→fraction, indicating presence at 5.07 / 106.3 ppm) α -L-Ara f -(1→fraction, indicating presence at 5.02 / 100.4 ppm) α -D-Glc p -(1→fraction, indicating the presence of →4 at 4.99 / 98.3 ppm)- α -D-Man p -(1→fraction, indicating the presence of →2,4 at 4.44 / 103.6 ppm)- β-D-Xyl p -(1→fragment; the PPLR-3 spectrum at a chemical shift of 5.25 / 98.7 ppm indicates the presence of →3)- α -D-Gal p A-(1→fraction, indicating presence at 5.19 / 97.9 ppm →6)- α -D-Gal p -(1→fraction, indicating presence at 5.12 / 97.2 ppm →3)- α -D-Rha p -(1→fraction, indicating presence at 5.08 / 106.1 ppm) α -L-Ara f -(1→fraction, indicating presence at 5.02 / 100.2 ppm-) α -D-Glc p -(1→fragment, indicating the presence of →6 at 4.85 / 99.5)- α -D-Man p -(1→fraction, indicating the presence of →2,4 at 4.44 / 103.1 ppm)- β -D-Xyl p -(1→segment).

[0054] Example 3: Monosaccharide composition analysis of Physalis alkekengi polysaccharide

[0055] Take appropriate amounts of PPLR-W, PPLR-1, PPLR-2, and PPLR-3 samples, dissolve them in trifluoroacetic acid of a certain concentration, and then heat at 110 °C. o The reaction was carried out at C for 6 h. After hydrolysis, the mixture was cooled to room temperature and a small amount of chromatographic methanol was added. The remaining trifluoroacetic acid was removed in small amounts several times until there was no sour taste. After the sour taste was removed, the mixture was evaporated to dryness to obtain the hydrolyzed polysaccharide sample.

[0056] Accurately weigh appropriate amounts of each monosaccharide standard (rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid) and an appropriate amount of hydrolyzed polysaccharide sample into test tubes. Then add 500 μL of pyridine solution and 10 mg of hydroxylamine hydrochloride to each test tube. React in a 90℃ water bath for 1 h, and then cool to room temperature. Add 500 μL of acetic anhydride solution to each test tube, heat at 90℃ for 30 min, remove and let stand at room temperature. Then filter each portion of the solution through a 0.22 μm microporous membrane for later use.

[0057] Chromatographic conditions: Gas chromatography-mass spectrometry (GC-MS) was performed using an Agilent HP-5 capillary column and an FID detector. Helium was used as the carrier gas, with a split ratio of 110:1 and a flow rate of 1 mL / min. A programmed temperature ramp was used: the initial temperature was set at 160℃, and after 2 min, the temperature was increased to 250℃ at a rate of 2℃ per minute. The mass spectrometry conditions were set to a mass scan range of 50-350 amu, a solvent delay of 2.5 min, and a sample injection volume of 1 μL.

[0058] Chromatogram (see) Figure 21 Monosaccharide composition spectrum of Prunus cerasifera root polysaccharide (in the monosaccharide standard spectrum, the elution order of each monosaccharide is as follows: 1: rhamnose, 2: arabinose, 3: xylose, 4: glucuronic acid, 5: galacturonic acid, 6: mannose, 7: glucose, 8: galactose)

[0059] PPLR-W is composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose, with a molar ratio of 14.31:1.93:5.95:16.78:25.92:21.18. PPLR-1, PPLR-2, and PPLR-3 are composed of rhamnose, arabinose, xylose, galacturonic acid, mannose, glucose, and galactose, with monosaccharide molar ratios of 12.15:4.45:5.94:1.40:12.57:23.02:24.20; 16.03:3.97:5.51:1.41:13.48:23.62:21.63; and 14.29:3.33:5.35:1.30:13.79:25.30:21.27, respectively.

[0060] Example 4: Determination of molecular weight of polysaccharides from *Phyllostachys pubescens* roots

[0061] Weigh out a certain amount of dextran standards with molecular weights of T4 (4000 Da), T10 (10000 Da), T20 (20000 Da), T40 (40000 Da), T200 (200000 Da), and T500 (500000 Da), as well as PPLR-W, PPLR-1, PPLR-2, and PPLR-3. Dissolve in 1 mL of Wahaha purified water, mix thoroughly to obtain the standard solution, and filter through a 0.22 μm filter membrane for later use.

[0062] Chromatographic conditions: LC-20AR high performance liquid chromatograph;

[0063] Detector: RID-20A differential refractive index detector;

[0064] Chromatographic column: TSK Gel G5000 PW column (ID=7.8 mm, L=300 mm);

[0065] Mobile phase: 0.02 mol / L KH₂PO₄;

[0066] Column temperature: room temperature;

[0067] Flow rate: 0.6 mL / min;

[0068] Injection volume: 50 μL;

[0069] A standard curve was obtained based on the logarithm of the molecular weight and the elution time of the standard dextran. The molecular weights of PPLR-W, PPLR-1, PPLR-2 and PPLR-3 were 21.57 kDa, 15.31 kDa, 20.64 kDa and 16.53 kDa, respectively, as determined by the elution time of the polysaccharides.

[0070] Example 5: DPPH free radical scavenging ability

[0071] The samples were prepared into a series of aqueous solutions with different concentrations. 100 μL of each concentration was added to 100 μL of 0.2 mmol / L DPPH stock solution (prepared from anhydrous ethanol and DPPH powder). The solutions were reacted at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm. Vitamin C (Vc) was used as a positive control. The scavenging capacity was calculated using the formula R% = [A0 - (A1 - A2)] / A0 × 100%. A0 was 100 μL of 0.2 mmol / L DPPH stock solution and 100 μL of ethanol, representing the blank group; A1 was 100 μL of ethanol and 100 μL of samples at different concentrations, representing the control group; A2 was 100 μL of DPPH stock solution and 100 μL of samples at different concentrations, representing the sample group. The ability of *Physalis alkekengi* root polysaccharide to scavenge DPPH free radicals was dose-dependent. When the concentration reached 5 mg / mL, the DPPH free radical scavenging effect was PPLR-1>PPLR-3>PPLR-2>PPLR-W, with scavenging rates of 79.46%, 77.74%, 71.08%, and 65.95%, respectively.

[0072] Example 6: ABTS free radical scavenging ability

[0073] Samples were prepared into a series of aqueous solutions with different concentrations. 10 μL of each concentration was added to 190 μL of ABTS stock solution, and the mixture was incubated in the dark for 10 min before being placed in a microplate reader for detection. The absorbance was measured at 734 nm (A1). Vitamin C (Vc) was used as a positive control. The blank control (A0) consisted of 100 μL of ABTS and 100 μL of ethanol. 100 μL of each concentration of sample and 100 μL of ABTS constituted the background group (A2). The scavenging capacity was calculated using the formula R% = [A0 - (A1 - A2)] / A0 × 100%.

[0074] Depend on Figure 23 It was found that the ability of *Phyllostachys pubescens* polysaccharide to scavenge ABTS free radicals was dose-dependent. When the concentration reached 5 mg / mL, the scavenging effect on DPPH free radicals was PPLR-1>PPLR-3>PPLR-2>PPLR-W, with scavenging rates of 55.94%, 50.55%, 48.23%, and 47.32%, respectively.

[0075] Example 7: Neuroprotective activity of Physalis alkekengi polysaccharide against H2O2-induced SH-SY5Y cells

[0076] After routine culture of SH-SY5Y cells, they were seeded in 96-well plates and incubated at 37°C. o Cells were statically cultured at C under 5% CO2 for 24 h until adherence. Samples at concentrations of 31.25, 62.5, 125, 250, 500 μg / mL, and 50 μL / well were added to 96-well plates and incubated for 24 h. Then, 800 μmol / L H2O2 was added for reaction, and cell viability was measured by MTT assay. A blank control group was established without cell seeding, receiving no H2O2 or drug treatment; all other experimental procedures were identical to the drug-treated group.

[0077] Survival rate (%) = [A 490 (Drug administration group)-A 490 (Blank control)] / [A 490 (Negative control)-A 490 (Blank control) × 100%

[0078] In the experiment using the MTT assay to detect the effect of Physalis alkekengi polysaccharide on H2O2-induced damage to SH-SY5Y cells, the results showed that, compared with the control group, pretreatment of cells with different concentrations of polysaccharide compounds significantly restored cell viability in a dose-dependent manner. The highest cell viability was observed at 500 μg / mL, and the optimal activities of PPLR-W, PPLR-1, PPLR-2, and PPLR-3 were 75.13%, 80.12%, 75.31%, and 77.98%, respectively.

Claims

1. A method for preparing polysaccharides from *Phyllostachys pubescens* root with antioxidant and neuroprotective activities, characterized in that, The method includes the following steps: (1) Take the dried roots of *Physalis alkekengi*, grind them with a pulverizer, reflux them twice with 3 times the volume of 80% ethanol for 3 hours each time, filter them, let the residue air dry naturally, then reflux and condense them with distilled water 4 times, filter them, concentrate them under reduced pressure, and dry them. Prepare a polysaccharide solution, add a neutral protease to the polysaccharide solution, stir, react for a period of time, and then centrifuge to collect the supernatant. (2) Add Sevage reagent prepared by n-butanol:chloroform = (0.5-2):(3-5), v / v to the above supernatant, stir for 20-40 min, centrifuge, collect the supernatant, and remove the protein 4 times by the Sevage method until no protein precipitates out. (3) The supernatant obtained above is concentrated and decolorized with AB-8 resin column. Each time, 15 g of protein-free polysaccharide is dissolved and loaded onto the sample. Deionized water is used as the mobile phase for elution. After concentration, decolorized Prunus cerasifera root polysaccharide is obtained. (4) The polysaccharides after deproteinization and decolorization were separated using a DEAE-52 cellulose column. The elution flow rate was set to 1.0 mL / min, and the time interval between collections was 10 min. The polysaccharides were eluted with distilled water and NaCl solutions of different concentrations in sequence. The absorbance of the polysaccharide solution in the test tube was measured by the phenol-sulfuric acid method. The eluents with symmetrical peak shapes were collected and combined, concentrated and dialyzed. The obtained polysaccharide components were further purified by elution using a Sephadex G-200 column. The four components with higher sugar content were collected, concentrated, dialyzed and dried under reduced pressure to obtain polysaccharides PPLR-W, PPLR-1, PPLR-2 and PPLR-3.

2. The method for preparing *Phyllostachys edulis* root polysaccharide with antioxidant and neuroprotective activities according to claim 1, characterized in that, The natural polysaccharide 1PPLR-W has a molecular weight range of 20-23 kDa, and its monosaccharide composition and molar ratio range is rhamnose:arabinose:xylose:mannose:glucose:galactose = (13.21-15.50):(1.70-2.13):(4.76-7.15):(15.58-17.99):(24.52-27.22):(19.88-22.40). Its skeletal structure is shown below: →6)- α -D-Gal p -(1→, →3)- α -D-Rha p -(1→, →3,5)- α -L-Ara f -(1→, →3)- α -D-Glc p -(1→, →6)- α -D-Man p -(1→ and →2,4)- β -D-Xyl p -(1→.

3. The method for preparing *Phyllostachys edulis* root polysaccharide with antioxidant and neuroprotective activities according to claim 1, characterized in that, The natural polysaccharide 2PPLR-1 has a molecular weight range of 14-16 kDa, and its monosaccharide composition and molar ratio range is rhamnose:arabinose:xylose:galacturonic acid:mannose:glucose:galactose = (10.95-13.35):(3.25-5.75):(4.74-7.20):(0.20-2.50):(11.37-13.78):(21.82-24.26):(22.90-25.41). Its skeletal structure is shown below: →3)- α -D-Gal p A-(1→,→6)- α -D-Gal p -(1→,→3)- α -D-Rha p -(1→, α -L-Ara f -(1→,→3)- α -D-Glc p -(1→,→6)- α -D-Man p -(1→ and →2,4)- β -D-Xyl p -(1→.

4. The method for preparing *Phyllostachys edulis* root polysaccharide with antioxidant and neuroprotective activities according to claim 1, characterized in that, The natural polysaccharide 3PPLR-2 has a molecular weight range of 20-24 kDa, and its monosaccharide composition and molar ratio range is rhamnose:arabinose:xylose:galacturonic acid:mannose:glucose:galactose = (15.73-17.23):(2.78-5.17):(4.31-7.14):(0.19-2.51):(12.28-14.69):(22.42-24.82):(20.43-22.83). Its skeletal structure is shown below: →3)- α -D-Gal p A-(1→, →6)- α -D-Gal p -(1→, →4)- α -D-Rha p -(1→, α -L-Ara f -(1→, α -D-Glc p -(1→, →4)- α -D-Man p -(1→ and →2,4)- β -D-Xyl p -(1→.

5. The method for preparing *Phyllostachys edulis* root polysaccharide with antioxidant and neuroprotective activities according to claim 1, characterized in that, The natural polysaccharide 4PPLR-3 has a molecular weight range of 16-18 kDa, and its monosaccharide composition and molar ratio range is rhamnose:arabinose:xylose:galacturonic acid:mannose:glucose:galactose = (13.09-15.49): (2.13-4.55): (4.15-6.56): (0.10-2.49): (12.59-14.99): (23.98-26.51): (20.07-22.47). Its skeletal structure is shown below: →3)- α -D-Gal p A-(1→,→6)- α -D-Gal p -(1→,→3)- α -D-Rha p -(1→, α -L-Ara f -(1→, α -D-Glc p -(1→,→6)- α -D-Man p -(1→ and →2,4)- β -D-Xyl p -(1→.

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