Preparation method of scutellaria polysaccharide and its phosphorylated derivative, product and application thereof

Abstract

The application discloses a preparation method of a scutellaria polysaccharide and a phosphorylated derivative thereof, products and applications of the scutellaria polysaccharide and the phosphorylated derivative thereof; the scutellaria polysaccharide is extracted from dried roots of Scutellaria baicalensis Georgi; and the phosphorylated scutellaria polysaccharide is prepared by taking the scutellaria polysaccharide as raw material and taking sodium trimetaphosphate and sodium tripolyphosphate as phosphorylation reagents. The scutellaria polysaccharide and the phosphorylated scutellaria polysaccharide can effectively promote PC12 cell proliferation, reduce the content of active oxygen in H2O2-induced oxidative damage type PC12 cells, and further maintain the repair activity of the PC12 cells subjected to oxidative damage. Therefore, the scutellaria polysaccharide and the phosphorylated scutellaria polysaccharide are expected to be further developed into functional food, health products or medicines for preventing or treating neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to a method for preparing Scutellaria baicalensis polysaccharide and its phosphorylated derivatives, as well as the products and applications thereof, belonging to the field of application technology of Scutellaria baicalensis polysaccharide and its derivatives. Background Technology

[0002] Polysaccharides are long-chain macromolecules formed by monosaccharides linked by glycosidic bonds. They are widely found in animal cell membranes and the cell walls of plants and microorganisms, and are one of the four basic building blocks of life, possessing a variety of pharmacological activities. In recent years, plant-derived polysaccharides have become a research hotspot due to their advantages such as low toxicity, biocompatibility, and biodegradability.

[0003] Scutellaria baicalensis Georgi, also known as Scutellaria baicalensis root or Scutellaria baicalensis root, is a perennial herb belonging to the genus Scutellaria in the Lamiaceae family. It is native to Heilongjiang, Henan, Gansu, Shandong, and Sichuan provinces in my country and is used as both food and medicine. It is rich in flavonoids, saponins, polysaccharides, and terpenoids, among other active ingredients. The root is used medicinally and possesses antioxidant, antitumor, and immunomodulatory activities. Polysaccharides are one of the most important bioactive components of Scutellaria baicalensis. Currently, Scutellaria baicalensis polysaccharides have attracted much attention due to their antiviral, anti-inflammatory, antitumor, and immunomodulatory effects. However, despite the discovery of numerous pharmacological activities of Scutellaria baicalensis polysaccharides, research and application reports on their chemical modification and their effects on nerve cell protection and repair are relatively few. Summary of the Invention

[0004] Objective of the invention: The technical problem to be solved by the present invention is to provide a method for preparing Scutellaria baicalensis Georgi polysaccharide (SBGP) and / or phosphorylated Scutellaria baicalensis Georgi polysaccharide (P-SBGP) and its use in nerve cell protection and repair.

[0005] Technical Solution: To solve the above-mentioned technical problems, this invention provides a method for preparing Scutellaria baicalensis polysaccharide, the preparation method of which is as follows (Reference: Li Ping, Hu Jianran, Shi Baozhong, et al. Extraction of Scutellaria baicalensis polysaccharide and its antioxidant and antitumor activities [J]. Biotechnology Bulletin, 2021, 37(4), 155-163.):

[0006] (1) Weigh 50g of crushed Scutellaria baicalensis powder, add 200-500mL of petroleum ether, heat and reflux for 1-2 hours to remove fat-soluble components, filter, and evaporate the solvent from the filter residue.

[0007] (2) Add 500 mL of water to the filter residue, heat to boiling and maintain for 0.5–2 h, filter, repeat the extraction 2–4 times, remove the filter residue, and combine the filtrates. Distill the filtrate to remove some water, and obtain 100 mL of Scutellaria baicalensis aqueous extract solution, cool to room temperature (this distillation step is not mentioned in the references. This step helps to reduce the amount of ethanol used in the next experiment and reduce costs);

[0008] (3) Add 300-500 mL of anhydrous ethanol to precipitate the polysaccharide in the solution, let it stand overnight in a refrigerator at 4°C, collect the precipitate by centrifugation, wash it with 80% ethanol and acetone in sequence, filter it, dry it, and obtain crude polysaccharide.

[0009] (4) Dissolve the crude polysaccharide in 100 mL of hot water. Add Sevege reagent to the dissolved liquid at a volume ratio of 3:1 to remove protein. Repeat the above process until no protein is precipitated.

[0010] (5) After thoroughly mixing the dissolved liquid with Sevage reagent, filter the solution and place it into a 1kd dialysis bag. Dialyze the solution in tap water for 1-3 days, then in ultrapure water for 1-2 days. Finally, dry the dialysis solution in a 60°C oven to obtain Scutellaria baicalensis polysaccharide (SBGP) solid. (In the reference, an 8kd dialysis bag was used for dialysis for 1 day. In this step, a 1kd dialysis bag is used, which helps to retain polysaccharides with lower molecular weights. Extending the dialysis time helps to effectively remove low molecular weight impurities.)

[0011] Preferably, in step (1), the addition of 300 mL of petroleum ether and the extraction by heating and reflux for 2 hours...

[0012] Preferably, in step (2), the heating and boiling is maintained for 1 hour, filtered, and the extraction is repeated 3 times.

[0013] Preferably, in step (3), the volume of anhydrous ethanol added to precipitate the polysaccharide in the solution is 400 mL.

[0014] Preferably, in step (5), the dialysis bag containing polysaccharides is dialyzed in tap water for 2 days, and then dialyzed in ultrapure water for 1 day.

[0015] The Scutellaria baicalensis provided in this invention is the root of Scutellaria baicalensis Georgi, a herbaceous plant belonging to the genus Scutellaria in the family Lamiaceae.

[0016] The present invention also provides a Scutellaria baicalensis polysaccharide prepared by the method described above.

[0017] This invention also provides a method for preparing phosphorylated Scutellaria baicalensis polysaccharide, the preparation method of which is as follows:

[0018] (1) Dissolve 6g of sodium tripolyphosphate and 1g of sodium triphosphate in 80-120mL of ultrapure water, and then add 0.5g of Scutellaria baicalensis polysaccharide and 2.5g of sodium sulfate while stirring.

[0019] (2) After dissolving the Scutellaria baicalensis polysaccharide, the pH was adjusted to 8.7 with NaOH (20%, m / V). Then, a phosphorylation reaction was carried out at 75℃ for 2–6 hours.

[0020] (3) After the reaction, anhydrous ethanol was added to make the volume of the solution five times the original volume, and then the mixture was allowed to settle statically at room temperature for 24 hours. The precipitate was collected and then redissolved in 50 mL of ultrapure water to obtain a solution. The solution was placed in a 1 kDa dialysis bag and dialyzed in tap water for 2 days, and then dialyzed in ultrapure water for 1 day. Finally, the dialysate was dried in a 60 °C oven to obtain phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP).

[0021] Preferably, in step (1), the amount of ultrapure water used is 100 mL.

[0022] Preferably, in step (2), the phosphorylation reaction is carried out for 4 hours.

[0023] Preferably, in step (3), the dialysis bag containing phosphorylated polysaccharides is dialyzed in tap water for 2 days, and then dialyzed in ultrapure water for 1 day.

[0024] The present invention also provides a phosphorylated Scutellaria baicalensis polysaccharide prepared by the method described above.

[0025] The present invention also provides the application of the Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide in the preparation of drugs that promote cell proliferation, wherein the Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide can effectively promote the proliferation of PC12 cells.

[0026] The concentration of the Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide is 10–100 μg / mL.

[0027] The present invention also provides the application of the Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide in the preparation of drugs for treating and / or inhibiting cellular oxidative damage, wherein the Scutellaria baicalensis polysaccharide has a repairing effect on H2O2-induced oxidative damage to PC12 cells.

[0028] The phosphorylated Scutellaria baicalensis polysaccharide can effectively reduce the content of reactive oxygen species in PC12 cells induced by H2O2 oxidation damage and has a repair effect on H2O2-induced oxidative damage to PC12 cells.

[0029] The present invention also provides the use of the aforementioned Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide in the preparation of a drug for protecting cells from oxidative damage.

[0030] The present invention also provides the use of the aforementioned Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases.

[0031] The concentration of the Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide is 30–105 μg / mL.

[0032] The concentration of the Scutellaria baicalensis polysaccharide is 45–105 μg / mL.

[0033] The concentration of the phosphorylated Scutellaria baicalensis polysaccharide is 75–105 μg / mL.

[0034] The concentration of the Scutellaria baicalensis polysaccharide is 90–105 μg / mL.

[0035] Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides a method for preparing Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide. Both the prepared Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide can effectively promote the proliferation of PC12 cells, effectively reduce the content of reactive oxygen species in H2O2-induced oxidative damage PC12 cells, and show good repair effects on H2O2-induced oxidative damage PC12 cells. Therefore, it is expected to be further developed into functional foods, health products or medicines for the prevention or treatment of neurodegenerative diseases. Attached Figure Description

[0037] Figure 1 Infrared spectra (IR) of Scutellaria baicalensis polysaccharide (SBGP) and phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP);

[0038] Figure 2 The 1H NMR spectra of Scutellaria baicalensis polysaccharide (SBGP) and phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP) are shown. 1 ¹H NMR): a: Scutellaria baicalensis polysaccharide (SBGP); b: phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP);

[0039] Figure 3 Phosphoric NMR spectrum of phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP) 31 P NMR);

[0040] Figure 4 Bar graph showing the effects of Scutellaria baicalensis polysaccharide (SBGP) and phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP) on the proliferation activity of PC12 cells;

[0041] Figure 5Figure 1 shows the effect of Scutellaria baicalensis polysaccharide (SBGP) and phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP) on the reactive oxygen species content induced by H2O2 in PC12 cells: a: blank group; b: H2O2 group; c: Scutellaria baicalensis polysaccharide group; d: phosphorylated Scutellaria baicalensis polysaccharide.

[0042] Figure 6 The bar graph shows the effects of Scutellaria baicalensis polysaccharide (SBGP) and phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP) on the viability of H2O2-induced oxidative damage-induced PC12 cells. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0044] Example 1: Preparation of Scutellaria baicalensis polysaccharide solids

[0045] (1) Weigh 50g of crushed Scutellaria baicalensis powder, add 300mL of petroleum ether, heat and reflux for 2h to remove fat-soluble components, filter, and evaporate the solvent from the filter residue.

[0046] (2) Add 500 mL of water to the filter residue, heat to boiling and maintain for 1 hour, filter, repeat the extraction 3 times, remove the filter residue, and combine the filtrates. Distill the filtrate to remove some water, and obtain 100 mL of Scutellaria baicalensis water extract solution, and cool to room temperature;

[0047] (3) Add 400 mL of anhydrous ethanol to the above concentrated Scutellaria baicalensis water extract solution to precipitate the polysaccharide in the solution, let it stand overnight, filter, and dry to obtain crude polysaccharide.

[0048] (4) Dissolve the obtained crude polysaccharide in 100 mL of hot water. Add Sevege reagent (chloroform: n-butanol = 4:1) to the dissolved liquid at a volume ratio of crude polysaccharide to Sevege reagent of 3:1 for protein removal treatment. Repeat the above process until no protein is precipitated.

[0049] (5) The above mixed liquid was filtered and then dried in an oven at 60°C to obtain 4.32g of Scutellaria baicalensis polysaccharide (SBGP).

[0050] Example 2: Preparation of phosphorylated Scutellaria baicalensis polysaccharide

[0051] (1) Dissolve 6g of sodium tripolyphosphate and 1g of sodium triphosphate in 100mL of ultrapure water, and then add 0.5g of Scutellaria baicalensis polysaccharide and 2.5g of sodium sulfate while stirring;

[0052] (2) After dissolving the Scutellaria baicalensis polysaccharide, the pH was adjusted to 8.7 with NaOH (20%, m / V). The mixture was then reacted at 75℃ for 4 hours.

[0053] (3) After the reaction was complete, anhydrous ethanol was added to make the solution volume 5 times the original volume, and then the mixture was allowed to settle statically at room temperature for 24 hours. The precipitate was collected and then redissolved in 50 mL of ultrapure water to obtain a solution. The solution was placed in a 1 kDa dialysis bag and dialyzed in tap water for 2 days, and then dialyzed in ultrapure water for 1 day. Finally, the dialysate was dried in a 60 °C oven to obtain phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP).

[0054] Phosphorylated Scutellaria baicalensis polysaccharide (P-SBGP) was analyzed using infrared spectroscopy and nuclear magnetic resonance spectroscopy, and compared with unmodified Scutellaria baicalensis polysaccharide (SBGP). The results are shown in the appendix. Figures 1-3 .

[0055] Depend on Figure 1 (Infrared spectra) show that the waveform of phosphorylated Scutellaria baicalensis polysaccharide is generally similar to that of Scutellaria baicalensis polysaccharide. In the infrared spectra of both samples, the broad peak of Scutellaria baicalensis polysaccharide is at 3413 cm⁻¹. -1 The broad peak of phosphorylated Scutellaria baicalensis polysaccharide at 3393 cm⁻¹ -1 This is caused by OH bonds; in the range of 3000-2800 cm⁻¹ -1 At this location, tensile vibrations of –CH2 can be observed; 2027cm -1 The absorption peak at 1600 cm⁻¹ indicates that the polysaccharide contains an unsaturated carbon-carbon bond structure; the absorption peak at 1600 cm⁻¹ indicates that the polysaccharide contains an unsaturated carbon-carbon bond structure. -1 Nearby, a characteristic peak for the C=O bond of carbohydrates appears. (1354 cm⁻¹) -1 The peak at 1300-1000 cm⁻¹ is attributed to the in-plane bending vibration of the hydroxyl group (OH). -1 The peaks within the range are absorption peaks due to CO stretching vibrations. Specifically, phosphorylated Scutellaria baicalensis polysaccharide shows an absorption peak at 1101 cm⁻¹. -1 There is a strong absorption peak at this point, which is a characteristic peak of the P=O double bond of the phosphate group, indicating that the phosphate group has been incorporated into the structure of Scutellaria baicalensis polysaccharide.

[0056] Figure 2 a-b represent Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide, respectively. 1 The two graphs show the 1H NMR spectra. As can be seen from the two graphs, the peak at 4.72 ppm is the labeled peak of the residual solvent (D₂O), and the peak at 3.71-3.78 ppm represents the chemical shift of protons in the sugar ring. The absorption peak at 6.5-7.5 ppm indicates that the Scutellaria baicalensis polysaccharide structure contains an aromatic ring. Figure 2 As shown in Figure a, the peak around 1.56-2.70 ppm represents the proton signal attached to the -OH group of the Scutellaria baicalensis polysaccharide ring, as... Figure 2 As shown in b, no obvious -OH absorption peak was observed at this location for phosphorylated Scutellaria baicalensis polysaccharide, which means that the -OH on the sugar ring of Scutellaria baicalensis polysaccharide has been phosphorylated and modified to form a new group.

[0057] Figure 3 Phosphorylated Scutellaria baicalensis polysaccharide 31 The NMR spectrum shows two strong absorption peaks at -6.87 and 2.07 ppm, characteristic of phosphate groups, further confirming the successful phosphorylation of Scutellaria baicalensis polysaccharides.

[0058] Example 3: Effects of Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide on PC12 cell viability

[0059] 1. Preparation of cell viability test solution

[0060] A reference solution for PC12 cell viability testing was obtained by dissolving the Scutellaria baicalensis polysaccharide solid obtained in Example 1 in ultrapure water. The concentration of Scutellaria baicalensis polysaccharide in the obtained solution was 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg / mL.

[0061] A solution for PC12 cell viability testing was obtained by dissolving phosphorylated Scutellaria baicalensis polysaccharide solids obtained in Example 1 in ultrapure water. The concentration of phosphorylated Scutellaria baicalensis polysaccharide in the obtained solution was 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg / mL.

[0062] 2. Determination of PC12 cell viability

[0063] PC12 cells in logarithmic growth phase were regulated to 4 × 10⁶ cells using DMEM medium containing 10% fetal bovine serum (FBS). 4 Cells were seeded at a concentration of 100 μL / well in a 96-well plate and incubated at 37°C for 6 hours in a 5% CO2 incubator. After removing the culture supernatant, 200 μL of DMEM medium containing a certain concentration of Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL) was added to each well and incubated for 24 hours. Then, 10 μL of CCK-8 was added to each well and incubated at 37°C for 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader. Cell- and polysaccharide-free medium was used as a blank sample, and polysaccharide-free ultrapure water was used as a control. Cell viability was calculated using the following formula:

[0064] Cell viability (%) = (A 样品 -A 空白 ) / (A 对照 -A 空白 )

[0065] Where A 样品 A 空白 and A 对照 These are the absorbance values ​​of the sample, blank sample, and control sample, respectively.

[0066] The experimental results on the effects of Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide on PC12 cell viability are shown in the figure. Figure 4 ,Depend on Figure 4 It can be seen that both Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide have strong proliferative effects on PC12 cells, and the higher the polysaccharide concentration, the more obvious the proliferative effect. When the polysaccharide concentration is 10 μg / mL, the survival rates of PC12 cells treated with Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide reach 171% and 147%, respectively; when the polysaccharide concentration is 100 μg / mL, the survival rates of PC12 cells treated with Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide are 209% and 178%, respectively. The figure also shows that the proliferative effect of Scutellaria baicalensis polysaccharide on PC12 cells is stronger than that of phosphorylated Scutellaria baicalensis polysaccharide.

[0067] Example 4: Measurement of Intracellular Reactive Oxygen Species (ROS) Levels

[0068] PC12 cells in logarithmic growth phase were regulated to a size of 1.2 × 10⁻⁶ using DMEM medium containing 10% fetal bovine serum (FBS). 6 Cells were seeded at a density of 1 mL / mL and then seeded into 6-well plates. After culturing for 6 hours at 37°C in a 5% CO2 incubator, each well was pretreated with hydrogen peroxide (1200 μmol / mL, 100 μL) for 2 hours. The culture medium was then discarded, and the cells were immediately incubated for 6 hours in fresh DMEM medium (1 mL) containing 10% fetal bovine serum (FBS) and either Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide solution (100 μg / mL). The culture medium was then removed, and the cells were washed with PBS to remove any residual medium. Fresh DMEM medium (1 mL) containing 10 μM DCFH-DA (Beyotime S0033S) and 10% FBS was added, and the cells were incubated at 37°C for 30 minutes. The cells were washed twice with PBS buffer to remove any DCFH-DA fluorescent probes that had not entered the cells. The fluorescence intensity of each group of cells was then observed using a confocal laser microscope to indicate the level of ROS generated. Cells that were not treated with hydrogen peroxide and polysaccharides served as the blank group, while the control group was treated with H2O2 and incubated with PBS instead of polysaccharide solution.

[0069] Figure 5 a-d are fluorescence images of PC12 cells in the blank group, H2O2 group, Scutellaria baicalensis polysaccharide group, and phosphorylated Scutellaria baicalensis polysaccharide group, respectively. Figure 5 As can be seen from a, the low fluorescence intensity in the blank group indicates a low content of reactive oxygen species in the cells; Figure 5 b shows that the H2O2 group has a high fluorescence intensity, indicating a higher content of reactive oxygen species in the cells; Figure 5 c shows that the reactive oxygen species content in cells treated with H2O2 decreased after the addition of Scutellaria baicalensis polysaccharide; Figure 5As can be seen from Figure d, after H2O2 treatment, the reactive oxygen species (ROS) content in cells treated with phosphorylated Scutellaria baicalensis polysaccharide was significantly reduced, approaching the level of the control group. In summary, the above experimental results indicate that Scutellaria baicalensis polysaccharide can reduce the ROS content in H2O2-induced oxidative damage PC12 cells to a certain extent; phosphorylated Scutellaria baicalensis polysaccharide has a more significant effect on reducing the ROS content in H2O2-induced oxidative damage PC12 cells than Scutellaria baicalensis polysaccharide alone.

[0070] Example 5: Effects of Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide on the viability of H2O2-induced oxidatively damaged PC12 cells.

[0071] PC12 cells in logarithmic growth phase were regulated to 4 × 10⁶ cells using DMEM medium containing 10% FBS. 4 Cells were seeded at a concentration of 100 μL / well in 96-well plates and cultured at 37°C for 6 hours in a 5% CO2 incubator. After removing the culture supernatant, the cells were pretreated with H2O2 (1200 μmol / L) for 4 hours, followed by 200 μL of DMEM medium containing either a specific concentration of Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide (0, 15, 30, 45, 60, 75, 90, 105 μg / mL) for 24 hours. Then, 10 μL of LCK-8 was added to each well and incubated at 37°C for 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader. Cell- and polysaccharide-free medium was used as a blank sample, and polysaccharide-free ultrapure water was used as a control. Cell viability was calculated using the following formula:

[0072] Cell viability (%) = (A 样品 -A 空白 ) / (A 对照 -A 空白 )

[0073] Where A 样品 A 空白 and A 对照 These are the absorbance values ​​of the sample, blank sample, and control sample, respectively.

[0074] Figure 6This study presents the effects of Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide on the viability of H2O2-induced oxidatively damaged PC12 cells. The figures show that the cell viability of the H2O2-treated model group was reduced. Both Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide exhibited good protective and repair effects on H2O2-induced oxidatively damaged PC12 cells, and these effects were dose-dependent. Compared with the model group, when the concentration of Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide was 60 μg / mL, the cell viability of the polysaccharide pretreatment group was significantly increased, reaching 60% and 73%, respectively. When the concentration of Scutellaria baicalensis polysaccharide and phosphorylated Scutellaria baicalensis polysaccharide was 105 μg / mL, the cell viability increased to 86% and 92%, respectively. The results also indicate that phosphorylated Scutellaria baicalensis polysaccharide was more effective than Scutellaria baicalensis polysaccharide in repairing H2O2-induced oxidative damage.

Claims

1. A method for preparing phosphorylated Scutellaria baicalensis polysaccharide, characterized in that, Includes the following steps: Sodium tripolyphosphate and sodium triphosphate were dissolved in ultrapure water, and then scutellaria baicalensis polysaccharide and sodium sulfate were added while stirring. The pH was adjusted to alkaline using NaOH solution, and a phosphorylation reaction was carried out. Anhydrous ethanol was added for static precipitation, the precipitate was collected, dissolved, dialyzed, and dried to obtain the phosphorylated Scutellaria baicalensis polysaccharide. The preparation method of the Scutellaria baicalensis polysaccharide includes the following steps: (1) Add petroleum ether to Scutellaria baicalensis powder, heat and reflux to extract, remove fat-soluble components, filter, evaporate the solvent to obtain filter residue; (2) Add water to the filter residue in step (1), heat to boiling, filter, extract repeatedly, remove the filter residue, combine the filtrates, and obtain Scutellaria baicalensis water extract solution; (3) Add anhydrous ethanol to the Scutellaria baicalensis aqueous extract solution described in step (2), let stand overnight, centrifuge to collect the precipitate, wash with 80% ethanol and acetone in sequence, filter, dry, and obtain crude polysaccharide; (4) Dissolve the crude polysaccharide obtained in step (3) in hot water, add Sevege reagent for protein removal treatment, repeat the above process until no protein is precipitated; filter, dialyze, dry to obtain Scutellaria baicalensis polysaccharide.

2. A phosphorylated Scutellaria baicalensis polysaccharide prepared by the method of claim 1.

3. The use of the Scutellaria baicalensis polysaccharide of claim 1 or the phosphorylated Scutellaria baicalensis polysaccharide of claim 2 in the preparation of a drug that promotes the proliferation of PC12 cells.

4. The application according to claim 3, characterized in that, The concentration of the Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide is 10~100 µg / mL.

5. The use of the Scutellaria baicalensis polysaccharide of claim 1 or the phosphorylated Scutellaria baicalensis polysaccharide of claim 2 in the preparation of a medicament for preventing and / or treating and / or inhibiting H2O2-induced oxidative damage to PC12 cells.

6. The use of the Scutellaria baicalensis polysaccharide of claim 1 or the phosphorylated Scutellaria baicalensis polysaccharide of claim 2 in the preparation of a medicament for protecting PC12 cells from H2O2-induced oxidative damage.

7. The use of the Scutellaria baicalensis polysaccharide of claim 1 or the phosphorylated Scutellaria baicalensis polysaccharide of claim 2 in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases.

8. The application according to any one of claims 5 to 7, characterized in that, The concentration of the Scutellaria baicalensis polysaccharide or phosphorylated Scutellaria baicalensis polysaccharide is 30~105 µg / mL.

Images