Paeoniflorin small-molecule active probe as well as preparation method and application thereof

Abstract

The invention discloses a paeoniflorin small-molecule active probe as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The paeoniflorin small-molecule active probe has a chemical structure shown in the specification. The paeoniflorin small molecule active probe is used for determining a direct action target of paeoniflorin. Experimental results show that the synthesized paeoniflorin small-molecule probe has bioactivity similar to that of paeoniflorin and can be specifically combined with an action target of paeoniflorin, it is proved that the small-molecule probe serves as a tool to replace paeoniflorin to conduct combination research on related protein targets, the blank in the prior art is filled, and a foundation is provided for deep research on the mechanism effect of paeoniflorin.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a paeoniflorin small molecule active probe, its preparation method, and its application. Background Technology

[0002] Peoniflorin, a pinane monoterpenoid bitter glycoside isolated from the dried root of peony, possesses various pharmacological effects, including anti-inflammatory, anticancer, vasodilatory, and neuroprotective properties. Previous studies have shown that paeoniflorin significantly improves motor dysfunction in Parkinson's disease (PD) mice by reducing damage and death of dopaminergic neurons, making it a potential drug for the prevention and treatment of PD.

[0003] Identification of drug target proteins helps in understanding the mechanisms of drug action, revealing the interactions between drugs and organisms, providing a theoretical basis for drug optimization and improvement, and offering clear molecular targets for drug design. This is of great significance to the modern pharmaceutical field. While paeoniflorin has been reported to possess anti-Parkinson's activity, its direct targets have not been reported. Identification and validation of the direct targets of paeoniflorin are crucial for developing paeoniflorin-based drugs and for drug discovery based on paeoniflorin targets. Summary of the Invention

[0004] The purpose of this invention is to provide a paeoniflorin small molecule active probe, its preparation method, and its application in order to solve the above-mentioned technical problems.

[0005] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, a paeoniflorin small molecule active probe, the chemical structure of which is as follows: .

[0006] As a second aspect of the present invention, a method for preparing the paeoniflorin small molecule active probe as described above is also provided, comprising the following steps: (1) Dissolve 5 mg of 1-hydroxybenzotriazole, 4 μL of 5-hexyneic acid and 6 μL of 1,3-diisopropylcarbodiimide in 100 μL of organic solvent and stir for 10 min to obtain a mixture. (2) Add 10 mg of paeoniflorin to the mixture obtained in step (1), stir and react at room temperature for 12 h to obtain the reaction solution; (3) The reaction solution obtained in step (2) is separated and purified to obtain the paeoniflorin small molecule active probe, which is a light yellow solid.

[0007] As a further optimization of the present invention, in step (1), the organic solvent is pyridine.

[0008] As a further optimization of the present invention, in step (3), separation and purification are carried out using the preparative liquid phase, wherein the mobile phase of the preparative liquid phase is a mixture of water and acetonitrile in a volume ratio of 7:3.

[0009] As a third aspect of the present invention, an application of the paeoniflorin small molecule active probe as described above in paeoniflorin target fishing is also provided.

[0010] As a fourth aspect of the present invention, the application of the paeoniflorin small molecule active probe as described above in the preparation of paeoniflorin-targeted drugs is also provided.

[0011] As a further optimization of the present invention, the drug is a drug for the prevention and treatment of Parkinson's disease.

[0012] The beneficial effects of this invention are as follows: This invention is the first to synthesize a bioactive small molecule probe containing paeoniflorin. Experimental results show that, firstly, the paeoniflorin small molecule probe has no cytotoxic effect on cells and significantly improves MPP. + The induced PD cell survival rate is similar to that of paeoniflorin, and the bioactivity is similar. Furthermore, the paeoniflorin small molecule active probe specifically binds to the target of paeoniflorin, enabling it to be used as a research tool for paeoniflorin target fishing and its mechanism of action, such as the study of the anti-Parkinson's disease mechanism of action. Attached Figure Description

[0013] Figure 1 The synthesis reaction equation for the paeoniflorin small molecule active probe provided by this invention; Figure 2 The mass spectrum of the paeoniflorin small molecule active probe structure provided by this invention; Figure 3 The proton NMR spectrum of the paeoniflorin small molecule active probe structure provided by this invention; Figure 4 The carbon spectrum of the paeoniflorin small molecule active probe structure provided by this invention; Figure 5 The diagram shows the cytotoxicity results of the paeoniflorin small molecule active probe provided by this invention. Figure 6 The paeoniflorin small molecule active probe provided by this invention enhances MPP. + The survival rate of induced PD cells is shown in the figure. Figure 7 The paeoniflorin small molecule active probe provided by this invention reduces MPP. + The result of induced lactate dehydrogenase release in PD cells; Figure 8 The paeoniflorin small molecule active probe provided by this invention in MPP + The labeling results in induced PD cells (A: Fluorescent labeling; B: Coomassie Brilliant Blue). Detailed Implementation

[0014] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0015] Example 1: Synthesis and preparation method of paeoniflorin small molecule active probe The synthesis reaction equation for the paeoniflorin small molecule active probe is as follows: Figure 1 As shown, the specific synthesis and preparation method is as follows: (1) Accurately weigh 5 mg of 1-hydroxybenzotriazole and place it in a round-bottom flask. Add 100 μL of pyridine, 4 μL of 5-hexynic acid, and 6 μL of 1,3-diisopropylcarbodiimide to obtain a mixture. Stir the mixture at room temperature for 10 minutes. Then, add 10 mg of paeoniflorin to the reaction system and stir at room temperature for 12 hours to obtain the reaction solution.

[0016] (2) After the reaction solution was dried by nitrogen, it was redissolved in methanol to a 10 mg / ml solution. The solution was purified and separated by the preparative liquid phase. The mobile phase was a mixture of water and acetonitrile with a volume ratio of 7:3. The purified solution was a pale yellow solid, namely paeoniflorin small molecule active probe, with a yield of 3.6 mg and a yield of 36%.

[0017] The characterization data of the paeoniflorin small molecule active probe obtained above are as follows: See Figure 2 Mass spectrometry results, paeoniflorin probe MS (ES) m / z: calculated value C 29 H 33 O 12 [MH] - 573.1971, mass spectrometry value 573.1956.

[0018] See Figure 31H NMR results for paeoniflorin probe (600 MHz, methanol-d4): δ 8.05 (q, 2H), 7.61 (m, 1H), 7.49 (t, 2H), 5.43 (s, 1H), 4.74 (d, J=3.0 Hz, 2H), 4.55 (d, J=7.7 Hz, 1H), 4.48 (dd, J=11.8, 2.1 Hz, 1H), 4.13 (dd, J=11.8, 6.6 Hz, 1H), 3.43 (m, 1H), 3.35 (s, 1H), 3.33 (s, 1H), 3.27 (d, 1H), 3.23 (m, 1H), 2.60 (dd, J=6.8, 1.7 Hz, 1H), 2.48 (td, J=7.3, 1.1 Hz, 2H), 2.27 (t, J=2.6 Hz, 1H), 2.24 (td, J=6.9, 2.7 Hz, 2H), 2.13 (d, J=12.6Hz, 1H), 1.88 (d, J=10.9 Hz, 1H), 1.82 (m, 3H), 1.32 (s, 3H).

[0019] See Figure 4 Carbon spectroscopy results, paeoniflorin probe 13 C NMR (600 MHz, Methanol-d4)δ 174.4, 168.0, 134.4, 131.2, 130.7, 129.6, 106.3, 102.3, 100.0, 89.3, 87.1, 84.0, 77. 8, 75.2, 74.9, 72.1, 71.7, 70.5, 64.6, 61.6, 44.6, 44.0, 33.7, 25.0, 23.3, 19.6, 18.4.

[0020] Example 2: Paeoniflorin probe against MPP + Induced protective effect of PD cells SH-SY5Y cells (Shanghai Cell Bank, Chinese Academy of Sciences, CBP60913) were rapidly thawed and cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibodies. The cells were gently blown until they were evenly distributed in a cell culture incubator (37 ℃, 5% CO2) for subsequent experiments.

[0021] 2.1 Paeoniflorin probe has no cytotoxic effect. When SH-SY5Y cells reached approximately 90% confluence after 24 h of culture, they were seeded evenly in 96-well plates. After 24 h, the cells were administered the drug. A blank control group and paeoniflorin probe groups (1, 2.5, 5, 10, 25, 50, 100, 200 μM) were set up. Cell viability was detected by CCK-8 assay after 24 h.

[0022] See Figure 5 The results showed that the cell viability of the paeoniflorin probe groups at concentrations of 1-200 μM was 99.96%, 97.71%, 95.32%, 97.56%, 91.91%, 91.62%, 86.65%, and 86.80% respectively compared with the blank control group. This indicates that the paeoniflorin probe concentrations of 1-50 μM had no significant effect on the cells and were not significantly different from the blank control group, suggesting that the paeoniflorin probe at concentrations of 1-50 μM had no significant cytotoxicity to the cells.

[0023] 2.2 Paeoniflorin probe significantly increased MPP + induced PD cell survival rate When SH-SY5Y cells reached approximately 90% confluence after 24 h of culture, they were seeded evenly in 96-well plates. After 24 h, drug administration and model establishment were performed, including a blank control group, a model group, a paeoniflorin group (40 μM), and a paeoniflorin probe group (40 μM). MPP was applied 2 h after pre-drug administration. + (1.0 mM) was used to induce an in vitro Parkinson's disease model, and cell viability was detected by CCK-8 assay 24 h later.

[0024] See Figure 6 As a result, compared with the blank control group, the cell survival rate of 40 μM paeoniflorin was 77.98% of that of the blank control group, and the cell survival rate of 40 μM paeoniflorin probe was 74.94% of that of the blank control group. The results showed that the paeoniflorin probe significantly improved the survival rate of MPP+ induced PD cells, and its activity was close to that of paeoniflorin (40 μM), indicating that the paeoniflorin probe has certain biological activity.

[0025] 2.3. Paeoniflorin probe significantly reduced MPP. + Induced release of lactate dehydrogenase from PD cells Cells were seeded uniformly in 96-well plates. After 24 h, drug administration and model establishment were performed. The following groups were set up: a maximum enzyme activity control group, a blank control group, a model group, a paeoniflorin group (40 μM), and a paeoniflorin probe group (40 μM). An equal volume of dimethyl sulfoxide (DMSO) was added to the blank control group and the model group. Two hours after pre-drug administration, an in vitro Parkinson's disease model was induced using 1-methyl-4-phenylpyridine ions (MPP+, 1.0 mM). After 24 hours of culture, lactate dehydrogenase releasing agent was added to the maximum enzyme activity control group, and cells were repeatedly pipetted. After further culture in a cell culture incubator for 1 hour, the wells were centrifuged at 1000 rpm for 5 minutes. 60 μL of the supernatant was collected, and the absorbance was measured at 490 nm using a microplate reader according to the instructions. The lactate dehydrogenase release rate was calculated. The formula for the lactate dehydrogenase release rate is as follows: Lactate dehydrogenase release rate = (sample absorbance value - blank solvent absorbance value) / (maximum enzyme activity absorbance value - background blank control well absorbance value) × 100%.

[0026] See Figure 7 The results showed that a higher lactate dehydrogenase (LDH) release rate indicated more severe cell damage. The LDH release rate was 11.14% in the blank control group, 41.57% in the model group, 20.36% in the paeoniflorin group, and 21.72% in the paeoniflorin probe group. These results indicate that the paeoniflorin probe significantly reduced MPP (molecular phosphatase) levels. + The induced release of lactate dehydrogenase in PD cells was achieved, and the paeoniflorin probe at a concentration of 40 μM had a similar effect to paeoniflorin.

[0027] Example 3: Target Fishing Experiment SH-SY5Y cells were cultured in cell culture dishes, and experiments were conducted when the cells reached approximately 90% confluence after 24 hours. Groups were set up including a blank control group (equal volume of DMSO), a probe group (40 μM paeoniflorin probe), and a competition group (40 μM paeoniflorin + 40 μM paeoniflorin probe). MPP was applied 2 hours after pre-drug administration. + (1.0 mM) induced PD cell model.

[0028] After 24 hours, the cell culture medium was discarded, and the cells were washed twice with pre-cooled PBS. Cells were collected by centrifugation (1500 rpm, 4 ℃, 5 min), the supernatant was discarded, and cell lysis buffer was added. Cells were then lysed thoroughly for 30 min, centrifuged again (15000 rpm, 4 ℃, 15 min), and the supernatant was collected. Protein concentration was determined using a BCA kit, and the protein concentration for each group was adjusted to 1 mg / mL. The rhodamine fluorescent group was linked to the paeoniflorin probe using click chemistry. Specifically, 100 μM Cy3-azide (Cy3-N3), 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), 1 mM tricarboxyethylphosphine (TCEP), and 1 mM anhydrous copper sulfate (CuSO4) were added sequentially to each group. The mixture was thoroughly mixed and incubated at 25 ℃ in the dark for 2 h. After the reaction, pre-cooled acetone (-20℃) was added to precipitate the protein. The protein was then collected by centrifugation at 15000 rpm for 10 minutes at 4℃. 200 μL of cell lysis buffer was added, and the precipitated protein was sonicated to dissolve it. Finally, the supernatant was collected by centrifugation (15000 rpm, 4℃, 15 min). 5× Loading Butter was added, and the reaction was stopped by boiling. SDS-PAGE electrophoresis was performed, and the gel was observed using a gel imaging system. After the reaction, the gel was stained with Coomassie Brilliant Blue and photographed.

[0029] See Figure 8 As shown in Figure A, the probe group clearly shows a fluorescent band at 40-55 kDa on the gel, while the band in the competition group is significantly weakened. This indicates that the paeoniflorin probe can specifically bind to the protein bound by paeoniflorin and can be used as a research tool for studying the target and mechanism of action of paeoniflorin.

[0030] See Figure 8 The results in Figure B show that the Coomassie Brilliant Blue staining intensity of the blank control group, probe group, and competition group is basically the same, indicating that the paeoniflorin probe can specifically bind to the protein bound by paeoniflorin and can be used as a research tool for paeoniflorin's target and mechanism of action.

[0031] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A small molecule active probe for paeoniflorin, characterized in that, The chemical structure of the paeoniflorin small molecule active probe is as follows: 。 2. A method for preparing the paeoniflorin small molecule active probe as described in claim 1, characterized in that, Includes the following steps: 5 mg of 1-hydroxybenzotriazole, 4 μL of 5-hexyneic acid, and 6 μL of 1,3-diisopropylcarbodiimide were dissolved in 100 μL of organic solvent and stirred for 10 min to obtain a mixture. Add 10 mg of paeoniflorin to the mixture obtained in step (1), stir and react at room temperature for 12 h to obtain the reaction solution; The reaction solution obtained in step (2) was separated and purified to obtain the paeoniflorin small molecule active probe.

3. The method for preparing a paeoniflorin small molecule active probe according to claim 2, characterized in that, In step (1), the organic solvent is pyridine.

4. The method for preparing a paeoniflorin small molecule active probe according to claim 2, characterized in that, In step (3), separation and purification are performed using the preparative liquid phase, wherein the mobile phase of the preparative liquid phase is a mixture of water and acetonitrile in a volume ratio of 7:

3.

5. The application of the paeoniflorin small molecule active probe as described in claim 1 in paeoniflorin target fishing.

6. The use of the paeoniflorin small molecule active probe as described in claim 1 in the preparation of paeoniflorin-targeted drugs.

7. The application according to claim 6, characterized in that, The drug in question is for the prevention and treatment of Parkinson's disease.

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