Preparation method of phosphorylated honeysuckle polysaccharide, product and application thereof

Abstract

The application discloses a preparation method of phosphorylated honeysuckle polysaccharide and the product and application thereof, the honeysuckle polysaccharide is extracted from the flowers of Lonicera japonica Thunb.; and the phosphorylated honeysuckle polysaccharide is prepared by taking the honeysuckle polysaccharide as raw material and taking sodium trimetaphosphate and sodium tripolyphosphate as phosphorylation reagents. The phosphorylated honeysuckle polysaccharide has potential application value in the preparation of functional food, health care products and medicines for treating neurodegenerative diseases, and belongs to the technical field of chemical modification and application of polysaccharide; the phosphorylated honeysuckle polysaccharide provided by the application can effectively promote the proliferation of PC-12 cells, and has good protection and repair effects on PC-12 cells with oxidative damage induced by H2O2, and thus is expected to be further developed into functional food, health care products or medicines for preventing or treating neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to a method for preparing phosphorylated honeysuckle polysaccharide, its products and applications, and belongs to the application field of honeysuckle polysaccharide. Background Technology

[0002] Neurodegenerative diseases, also known as neurodegenerative disorders, are a group of disease states caused by the gradual loss of neurons in the brain and spinal cord. The most common neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), which are widely recognized as highly dangerous neurological disorders. To date, no effective treatments have been found for these diseases; treatment options are limited to symptomatic relief and often have significant side effects, failing to achieve optimal prevention and treatment outcomes. Therefore, how to slow down or reverse the pathological progression of neurodegenerative diseases is a crucial problem that the medical community urgently needs to solve.

[0003] Honeysuckle (Lonicera japonica Thunb.) is the dried flower bud or newly opened flower of the honeysuckle plant (Lonicera japonica), belonging to the Caprifoliaceae family. It possesses various biological activities, including antioxidant, anti-inflammatory, anti-diabetic, and antifungal properties. Polysaccharides are one of the main active components of honeysuckle, exhibiting antiviral, antioxidant, anti-inflammatory, hypoglycemic, and antitumor activities. Recent studies have found that honeysuckle polysaccharide (Lonicera japonica Thunb. polysaccharide, LJTP) can promote PC12 cell differentiation and neurite regeneration, but the activity is relatively low.

[0004] Numerous studies have shown that the bioactivity of polysaccharides is closely related to their molecular properties, such as monosaccharide composition, molecular weight, chain conformation, and water solubility. Furthermore, molecular modification of polysaccharides provides an effective method for altering their molecular properties and bioactivity. Currently, research on honeysuckle polysaccharides mainly focuses on their isolation, purification, and bioactivity; however, there are few studies and reports on the preparation and bioactivity of honeysuckle polysaccharide derivatives, both domestically and internationally. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing phosphorylated honeysuckle polysaccharide, its product and application. The prepared phosphorylated honeysuckle polysaccharide can effectively promote the proliferation of PC-12 cells and shows a good repair effect on H2O2-induced oxidative damage PC-12 cells.

[0006] Technical Solution: To solve the above technical problems, this invention provides a method for preparing phosphorylated honeysuckle polysaccharide, comprising the following steps:

[0007] (1) Add anhydrous ethanol to the honeysuckle water extract solution to precipitate the polysaccharide in the solution and prepare crude polysaccharide;

[0008] (2) Dissolve the crude polysaccharide described in step (1), add Sevage reagent to remove protein, repeat the above process until no protein is precipitated; add Sevage reagent, filter, dry, and obtain honeysuckle polysaccharide.

[0009] (3) Add a mixed solution of sodium tripolyphosphate and sodium trimephosphate and sodium sulfate to the honeysuckle polysaccharide described in step (2); adjust the pH to alkaline with NaOH solution and then carry out the reaction.

[0010] (4) After the reaction was completed, anhydrous ethanol was added and static sedimentation was carried out. The precipitate was collected, redissolved in ultrapure water, dialyzed, and dried to obtain phosphorylated honeysuckle polysaccharide.

[0011] The present invention also provides a phosphorylated honeysuckle polysaccharide prepared by the aforementioned preparation method.

[0012] The present invention also provides the use of the phosphorylated honeysuckle polysaccharide in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases.

[0013] Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0014] The present invention also provides the use of the phosphorylated honeysuckle polysaccharide in the preparation of medicaments for treating and / or inhibiting cellular oxidative damage.

[0015] The present invention also provides the use of the phosphorylated honeysuckle polysaccharide in the preparation of a drug that protects cells from oxidative damage.

[0016] The concentration of the phosphorylated honeysuckle polysaccharide is 30–105 μg / mL.

[0017] The concentration of the phosphorylated honeysuckle polysaccharide is 60–105 μg / mL.

[0018] The concentration of the phosphorylated honeysuckle polysaccharide is 75–105 μg / mL.

[0019] The concentration of the phosphorylated honeysuckle polysaccharide is 90–105 μg / mL.

[0020] The present invention also provides the application of the phosphorylated honeysuckle polysaccharide in the preparation of a drug that promotes cell proliferation.

[0021] The concentration of the phosphorylated honeysuckle polysaccharide is 30–100 μg / mL.

[0022] The concentration of the phosphorylated honeysuckle polysaccharide is 60–100 μg / mL.

[0023] The concentration of the phosphorylated honeysuckle polysaccharide is 70–100 μg / mL.

[0024] Based on the above technical solution, the phosphorylated honeysuckle polysaccharide can effectively promote the proliferation of PC-12 cells.

[0025] Based on the above technical solution, the phosphorylated honeysuckle polysaccharide has a repairing effect on H2O2-induced oxidative damage to PC-12 cells.

[0026] The principle of this invention: Cell surfaces possess a biomembrane composed of a phospholipid bilayer intercalated with embedded proteins and glycocalyxes. After damage, PC-12 cells require nutrients such as phospholipids, glycoproteins, glycolipids, and proteins for repair. Phosphorylated honeysuckle polysaccharides can provide damaged cells with their unique polysaccharide components, phosphate groups, and a small amount of glycoproteins, and possess certain antioxidant activity and good biocompatibility. Therefore, it can effectively promote the self-repair of damaged PC-12 cells and promote their growth and proliferation.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides a method for preparing phosphorylated honeysuckle polysaccharide. The prepared phosphorylated honeysuckle polysaccharide can effectively promote the proliferation of PC-12 cells and shows a good repair effect on H2O2-induced oxidative damage PC-12 cells. Therefore, it is expected to be further developed into a functional food, health product or medicine for the prevention or treatment of neurodegenerative diseases. Attached Figure Description

[0028] Figure 1 Infrared spectra of honeysuckle polysaccharide (LJTP) and phosphorylated honeysuckle polysaccharide (P-LJTP);

[0029] Figure 2 The 1H NMR spectra of honeysuckle polysaccharide (LJTP) and phosphorylated honeysuckle polysaccharide (P-LJTP) are shown (A: LJTP; B: P-LJTP).

[0030] Figure 3 Phosphorus NMR spectrum of phosphorylated honeysuckle polysaccharide (P-LJTP);

[0031] Figure 4 The effects of honeysuckle polysaccharide (LJTP) and phosphorylated honeysuckle polysaccharide (P-LJTP) on the viability of PC-12 cells;

[0032] Figure 5 The effects of honeysuckle polysaccharide (LJTP) and phosphorylated honeysuckle polysaccharide (P-LJTP) on the viability of PC-12 cells induced by H2O2 damage;

[0033] Figure 6The effect of different concentrations of P-LJTP on H2O2-induced PC-12 cell morphology (A: 0 μg / mL; B: 30 μg / mL; C: 60 μg / mL; D: 90 μg / mL). Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] Example 1

[0036] Preparation of phosphorylated honeysuckle polysaccharide solids

[0037] (1) Weigh 50g of honeysuckle whole herb, cut it into pieces, put it into a 500mL beaker, add 200mL of water, heat, boil for 1 hour, repeat the extraction 3 times, filter, remove the filter residue, combine the filtrates, distill off part of the water, and obtain 100mL of honeysuckle water extract solution, and cool to room temperature.

[0038] (2) Add 400 mL of anhydrous ethanol to the above concentrated honeysuckle water extract solution to precipitate the polysaccharide in the solution, let it stand overnight, filter, and dry to obtain crude polysaccharide.

[0039] (3) 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.

[0040] (4) The above mixed liquid was filtered and the resulting filter cake was dried in an oven at 60°C to obtain 3.29g of honeysuckle polysaccharide (LJTP).

[0041] (5) Dissolve 6g sodium tripolyphosphate and 1g sodium trimetaphosphate in 100mL of ultrapure water, and then add 0.5g honeysuckle polysaccharide and 2.5g sodium sulfate while stirring.

[0042] (6) After the honeysuckle polysaccharide was dissolved, the pH was adjusted to 8.7 with NaOH solution (20%, m / V). Then the reaction was carried out at 75℃ for 4 hours.

[0043] (7) 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 honeysuckle polysaccharide (P-LJTP).

[0044] Phosphorylated honeysuckle polysaccharide (P-LJTP) was analyzed using infrared spectroscopy and nuclear magnetic resonance spectroscopy, and compared with unmodified honeysuckle polysaccharide (LJTP). The results are shown in the appendix. Figures 1-3 .

[0045] Depend on Figure 1 Infrared spectra show that the overall waveform of phosphorylated honeysuckle polysaccharide is similar to that of honeysuckle polysaccharide. In the spectra of both samples, the waveform is similar in the 3000-2800 cm⁻¹ range. -1 At this location, tensile vibrations of -CH2 can be observed; 2026 cm -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 Characteristic peaks of C=O bonds in carbohydrates appear nearby; 1300-1000 cm⁻¹ -1 The peak within the range is the CO stretching vibration absorption peak, 1353 cm⁻¹. -1 The peak at 3425 cm⁻¹ is attributed to the in-plane bending vibration of the hydroxyl group (OH). The broad peak of honeysuckle polysaccharide is at 3425 cm⁻¹. -1 The broad peak of phosphorylated honeysuckle polysaccharide at 3433 cm⁻¹ -1 This is caused by OH bonds. Specifically, phosphorylated honeysuckle polysaccharides at 1102 cm⁻¹ -1 There is a 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 honeysuckle polysaccharide.

[0046] Figure 2 A through B show honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide, respectively. 1 The two graphs show the 1H NMR spectra. As can be seen from the two graphs, 4.72 ppm is the labeled peak of the residual solvent (D₂O), and the peak near 3.71 ppm represents the chemical shift of protons in the sugar ring. Figure 2 As shown in Figure A, the peak value around 1.43-2.70 ppm represents the proton signal attached to the -OH group of the honeysuckle polysaccharide ring. Figure 2 As shown in Figure B, no obvious absorption peak was observed at this location for phosphorylated honeysuckle polysaccharide, which means that the -OH group on the sugar ring of honeysuckle polysaccharide has been phosphorylated and a new group has been formed.

[0047] Figure 3 Phosphorylated honeysuckle polysaccharide 31 The NMR spectrum shows two strong absorption peaks at -7.19 and 1.77 ppm, characteristic of phosphate groups, further confirming the successful phosphorylation of honeysuckle polysaccharides.

[0048] Example 2: Effects of honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide on PC-12 cell viability

[0049] 1. Preparation of cell viability test solution

[0050] A reference solution for PC-12 cell viability testing was obtained by dissolving the honeysuckle polysaccharide solid obtained in Example 1 in ultrapure water. The concentration of honeysuckle polysaccharide in the obtained solution was 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg / mL.

[0051] A solution for PC-12 cell viability testing was obtained by dissolving phosphorylated honeysuckle polysaccharide solids obtained in Example 1 in ultrapure water. The concentration of phosphorylated honeysuckle polysaccharide in the obtained solution was 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg / mL.

[0052] 2. Determination of PC-12 cell viability

[0053] PC-12 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 density of 100 μL / well in 96-well plates and incubated at 37°C for 6 hours in a 5% CO2 incubator. After removing the culture supernatant, 200 μL of DMEM medium containing specific concentrations of honeysuckle polysaccharide or phosphorylated honeysuckle 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:

[0054] Cell viability (%) = (A 样品 -A 空白 ) / (A 对照 -A 空白 )

[0055] Among them, A 样品 A 空白 and A 对照 These are the absorbance values ​​of the sample, blank sample, and control sample, respectively.

[0056] The experimental results on the effects of honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide on PC-12 cell viability are shown in the figure. Figure 4 ,Depend on Figure 4 It can be seen that both honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide have strong proliferative effects on PC-12 cells. The higher the polysaccharide concentration, the more obvious the proliferative effect. When the concentrations of honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide are 100 μg / mL, the survival rates of PC-12 cells reach 292% and 370%, respectively. Figure 4 It can also be seen that phosphorylated honeysuckle polysaccharide has a stronger proliferative effect than honeysuckle polysaccharide.

[0057] Example 3: Effects of honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide on the viability of H2O2-induced oxidatively damaged PC-12 cells.

[0058] PC-12 cells in logarithmic growth phase were regulated to 4 × 10⁶ cells using DMEM medium containing 10% FBS. 4 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated at 37°C with 5% CO2 for 6 hours. After removing the culture supernatant, the cells were pretreated with H2O2 (1200 μmol / L) for 4 hours, followed by incubation in 200 μL of DMEM medium containing specific concentrations of honeysuckle polysaccharide or phosphorylated honeysuckle polysaccharide (0, 15, 30, 45, 60, 75, 90, 105 μg / mL) 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:

[0059] Cell viability (%) = (A 样品 -A 空白 ) / (A 对照 -A 空白 )

[0060] Among them, A 样品 A 空白 and A 对照 These are the absorbance values ​​of the sample, blank sample, and control sample, respectively.

[0061] Figure 5 This figure shows the effect of phosphorylated honeysuckle polysaccharide on the viability of H2O2-induced oxidatively damaged PC-12 cells. As can be seen from the figure, the cell viability of the H2O2-treated model group was reduced. Both honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide had good protective and repair effects on H2O2-induced oxidatively damaged PC-12 cells, and both were dose-dependent. Compared with the model group, when the concentration of honeysuckle polysaccharide or phosphorylated honeysuckle polysaccharide reached 60 μg / mL, the cell viability of the polysaccharide pretreatment group was significantly improved. When the concentration of honeysuckle polysaccharide and phosphorylated honeysuckle polysaccharide reached 90 μg / mL, the cell viability increased to 69% and 96%, respectively. The experimental results also show that phosphorylated honeysuckle polysaccharide has a much better repair effect on H2O2-induced oxidatively damaged cells than honeysuckle polysaccharide.

[0062] The effects of different concentrations of phosphorylated honeysuckle polysaccharide on the morphology of PC-12 cells induced by H2O2 oxidative damage are shown in the figure. Figure 6 .like Figure 6 As shown, in the normal control group, PC-12 cells were slender and spindle-shaped, and grew well through static adhesion. Compared with the normal control group, the model group showed severe damage, mainly manifested as wrinkled, smaller, rounder, and clumped cell morphology. This indicates that the PC-12 cells treated with phosphorylated honeysuckle polysaccharide were effectively improved, similar to the normal control group, further demonstrating that phosphorylated honeysuckle polysaccharide can effectively protect PC-12 cells from H2O2-induced oxidative damage.

Claims

1. A method for preparing phosphorylated honeysuckle polysaccharide, characterized in that, Includes the following steps: (1) Add anhydrous ethanol to the aqueous extract of honeysuckle to precipitate the polysaccharides in the solution and prepare crude polysaccharides; (2) Dissolve the crude polysaccharide described in step (1), add Sevage reagent to remove protein, repeat the above process until no protein is precipitated; add Sevage reagent, filter, dry, and obtain honeysuckle polysaccharide; (3) Add a mixed solution of sodium tripolyphosphate and sodium trimetaphosphate and sodium sulfate to the honeysuckle polysaccharide described in step (2); adjust the pH to alkaline with NaOH solution before proceeding with the reaction; (4) After the reaction is complete, add anhydrous ethanol and allow it to settle statically; The precipitate was collected, redissolved in ultrapure water, dialyzed, and dried to obtain phosphorylated honeysuckle polysaccharide.

2. A phosphorylated honeysuckle polysaccharide prepared by the preparation method described in claim 1.

3. The use of the phosphorylated honeysuckle polysaccharide according to claim 2 in the preparation of a medicament for the prevention and / or treatment of neurodegenerative diseases.

4. The use of the phosphorylated honeysuckle polysaccharide according to claim 2 in the preparation of a medicament for treating and / or inhibiting H2O2-induced oxidative damage to PC-12 cells.

5. The use of the phosphorylated honeysuckle polysaccharide according to claim 2 in the preparation of a medicament for protecting PC-12 cells from H2O2-induced oxidative damage.

6. The application according to any one of claims 3 to 5, characterized in that, The concentration of the phosphorylated honeysuckle polysaccharide is 30~105 µg / mL.

7. The application according to any one of claims 3 to 5, characterized in that, The concentration of the phosphorylated honeysuckle polysaccharide was 60~105 µg / mL.

8. The use of the phosphorylated honeysuckle polysaccharide according to claim 2 in the preparation of a drug that promotes the proliferation of PC-12 cells.

9. The application according to claim 6, characterized in that, The concentration of the phosphorylated honeysuckle polysaccharide is 30~100 µg / mL.

10. The application according to claim 6, characterized in that, The concentration of the phosphorylated honeysuckle polysaccharide is 60~100µg / mL.

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