A natural product-derived acetylcholinesterase inhibitor and use thereof

Abstract

This invention relates to the field of natural product pharmaceuticals, specifically to a natural product-derived acetylcholinesterase inhibitor and its application. Currently used chemically synthesized acetylcholinesterase inhibitors in clinical treatment, while achieving certain therapeutic effects, still suffer from side effects and drug resistance. Therefore, this invention provides an acetylcholinesterase inhibitor, which is a single-drug or dual-drug composition derived from a natural product. The single drug is behenin isoflavone (TI) or isoeugenol acetate (IA), and the dual-drug composition is TI and IA mixed in a specific ratio. The acetylcholinesterase inhibitor of this invention exhibits good AChE inhibitory activity, anti-Aβ aggregation efficacy, antioxidant activity, and good neuroprotective effects, indicating that TI and / or IA can be used as novel acetylcholinesterase inhibitors in the development and use of drugs for the clinical treatment and / or prevention of Alzheimer's disease and related diseases.

Description

Technical Field

[0001] This invention relates to the field of natural product pharmaceuticals, and more specifically to an acetylcholinesterase inhibitor derived from a natural product and its application. Background Technology

[0002] Alzheimer's disease (AD), also known as senile dementia, is a common neurodegenerative disease affecting the central nervous system in old age. Its clinical manifestations include memory impairment, aphasia, apraxia, agnosia, impairment of abstract thinking and calculation abilities, as well as personality and behavioral changes. With the accelerating aging of the global population, the incidence of Alzheimer's disease is rising year by year, becoming a significant factor affecting the quality of life and social burden of the elderly. Currently, the pathogenesis of this disease is not fully understood, but mainstream hypotheses include the β-amyloid (Aβ) cascade hypothesis, the tau protein hyperphosphorylation hypothesis, and the neuroinflammatory hypothesis.

[0003] Acetylcholinesterase inhibitors (AChEIs) are currently a major class of drugs used clinically to treat Alzheimer's disease. They work by inhibiting the activity of acetylcholinesterase (AChE) in the body, reducing the hydrolysis of acetylcholine, thereby increasing the level of acetylcholine in the brain and improving patients' cognitive function and daily living abilities. These drugs are mainly used to treat patients with mild to moderate Alzheimer's disease and have a certain alleviating effect on early symptoms. However, existing chemically synthesized acetylcholinesterase inhibitors, such as donepezil and rivastigmine, while achieving some efficacy, still have problems such as side effects and drug resistance.

[0004] In recent years, compounds derived from natural products have received continuous attention in the biomedical field. Studies have found that they have advantages over most clinical drugs, such as wide availability, structural diversity, and fewer side effects, providing a new direction for finding new acetylcholinesterase inhibitors with low toxicity and high efficacy. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a natural product-derived acetylcholinesterase inhibitor and its application.

[0006] This invention provides a natural product-derived acetylcholinesterase inhibitor containing an effective dose of gynostemma pentaphyllum isoflavones and / or isoeugenol acetate.

[0007] Furthermore, the structural formula of the toxicarol isoflavone (TI) is as shown in Formula I:

[0008]

[0009] Formula I

[0010] Furthermore, the isoeugenyl acetate (IA) has the structural formula shown in Formula II:

[0011]

[0012] Formula II

[0013] The CAS number of the isoeugenol acetate is 93-29-8. The propenyl side chain (-CH=CH-CH3) in its structure has cis-trans (Z / E) isomerism, and it is a cis-trans mixture of isoeugenol acetate.

[0014] The concentration of the gray leaf isoflavone is 1-4 μM, and the concentration of the isoeugenol acetate is 1-4 μM;

[0015] Further, the molar concentration ratio of gynostemma pentaphyllum isoflavone to isoeugenol acetate in the gynostemma pentaphyllum is (1-2):(1-2).

[0016] Furthermore, the molar concentration ratio is 1:2.

[0017] Furthermore, the optimal concentration of physcion isoflavone-isoeugenol acetate composition is 2 μM, and the optimal concentration of isoeugenol acetate is 4 μM.

[0018] This invention provides a natural product-derived acetylcholinesterase inhibitor for:

[0019] a. To prepare medicines for the prevention and / or treatment of AChE-related diseases;

[0020] b. Preparation of drugs to inhibit β-amyloid peptide (Aβ) aggregation;

[0021] c. To prepare drugs for the prevention and / or treatment of diseases related to Aβ aggregation;

[0022] The advantages of this invention are:

[0023] The present invention provides a natural product-derived compound TI and / or IA that exhibits good AChE inhibitory activity, moderate anti-Aβ aggregation efficacy, moderate antioxidant activity, and good neuroprotective effects. Furthermore, it shows no significant cytotoxicity to human neuroblastoma cells SH-SY5Y and demonstrates good biocompatibility. This indicates that TI and / or IA can be used as novel acetylcholinesterase inhibitors in the development and use of drugs for the clinical treatment and / or prevention of AD and other related diseases. Attached Figure Description

[0024] Figure 1 This is a graph showing the inhibitory activity data of the TI and IA combination against AChE.

[0025] Figure 2 Here is a graph showing the survival rate data of the compound on SH-SY5Y cells: Figure 2 In this context, A represents the individual effects of TI and IA on the survival rate of SH-SY5Y cells. Figure 2 In the figure, B represents the effect of the TI and IA combination on the survival rate of SH-SY5Y cells.

[0026] Figure 3 This is a graph showing the effect of the TI and IA combination on Aβ peptide (25–35)-induced cell death in the SH-SY5Y cell model in vitro. Detailed Implementation

[0027] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0028] Example 1: Assay of AChE inhibitory activity of TI and IA monotherapy drugs

[0029] Add 40 μL of phosphate buffer (pH = 8.0) to each well of a 96-well plate. Then, add 10 μL of the test compound (TI / IA) solution (0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25, 50, and 100 μM) or control solution to the corresponding well. Next, add 10 μL of AChE and incubate at 37°C with shaking for 5 min. Add 20 μL of DTNB solution and incubate at 37°C with shaking for another 5 min. Then, add 10 μL of substrate ATC and incubate at 37°C with shaking for 3 min. Measure the absorbance at 412 nm using a microplate reader and calculate the inhibition rate of the test compound against AChE. Calculate the IC50 of the compound based on the inhibition curve. 50 The value (inhibitor concentration when enzyme activity is 50%) is shown in Table 1.

[0030] Table 1

[0031]

[0032] Example 2: Determination of AChE inhibitory activity of the TI and IA combination

[0033] To investigate the optimal combination concentration of the two compounds, this invention is based on the IC50 values ​​of the two compounds. 50The values ​​were set as grouped in Table 2. The AChE inhibitory activity assay method described in Example 1 was used to calculate the inhibition rate of AChE in each group and draw a statistical analysis chart.

[0034] Table 2

[0035]

[0036] Experimental results: The experimental results are as follows Figure 1 As shown, donepezil, the positive control drug, exhibited an inhibition rate close to 100%, demonstrating excellent AChE inhibitory activity, indicating that the experimental procedures were rigorous and error-free. Next, the AChE inhibition rates of the various groups prepared with TI and IA in different proportions were observed. It can be seen that the AChE inhibition rates of TI and IA varied at different molar ratios and concentrations, with the overall trend being that the AChE inhibition rates of the low-concentration groups (a, b, c) were lower than those of the high-concentration groups (d, e, f). Furthermore, this invention also found that regardless of the concentration group, the composition prepared with a TI to IA molar ratio of 1:2 showed the highest AChE inhibition rate. Specifically, the AChE inhibition rate of group b was higher than that of group a / c, and the AChE inhibition rate of group e was higher than that of group d / f. This may be due to the stronger AChE inhibitory activity of IA compared to TI, indicating that IA plays a dominant role in the AChE inhibitory activity of the composition. Finally, the AChE inhibition rate of group e was higher than that of group b, which is due to the increased molar concentrations of TI and IA compounds. The above results indicate that the composition prepared by TI and IA at a molar ratio of 1:2 has the highest inhibition rate against AChE, and in this application, the optimal concentration of the TI and IA composition is 2 μM TI and 4 μM IA.

[0037] Example 3: Detection of Aβ self-aggregation activity of TI and IA composition

[0038] A total of 1 mg Aβ was dissolved in 1 mL of hexafluoroisopropanol and aliquoted into 50 μL tubes. Then, 20 μL of LDMSO was added to one tube of Aβ solution. Test compounds were prepared using PBS: group e (2 μM TI and 4 μM IA), and the positive control (donepezil, 4 μM). Then, 20 μL of the prepared test compounds and 20 μL of Aβ solution were added to a 96-well black plate, gently tapped to mix, capped tightly, and sealed with black tape to prevent solvent evaporation. The plate was then incubated at room temperature in the dark for 24 h. 160 μL of 5 μM THT solution was added, and fluorescence absorption was measured at an excitation wavelength of 445 nm and an emission wavelength of 490 nm.

[0039] Experimental Results: Aβ aggregation is one of the main histopathological markers of Alzheimer's disease (AD). Therefore, this invention uses thioflavin T assays to determine the preventive effect of a combination of TI and IA on Aβ aggregation. As shown in Table 3, composition e has a moderate inhibitory effect on Aβ aggregation, with an inhibition rate of 46.95% ± 2.92% for TI at 2 μM and IA at 4 μM. Although the inhibition rate of composition e on Aβ aggregation is higher than that of donepezil, the effects are relatively similar. The results indicate that the combination of compounds TI at 2 μM and IA at 4 μM exhibits moderate anti-Aβ aggregation efficacy.

[0040] Table 3

[0041]

[0042] Example 4: In vitro antioxidant activity assay of the TI and IA combination

[0043] The free radical scavenging activity of the TI and IA compositions was tested using 1,1-diphenyl-2-trinitrohydrazide (DPPH). To prepare a 0.1 mM DPPH stock solution, DPPH was dissolved in methanol. Test compounds were prepared with PBS: group e (2 μM TI and 4 μM IA), and a positive control (donepezil, 4 μM). In a 96-well plate, 10 μL of the test compound and 90 μL of DPPH solution were added to each well. After reacting at 25 °C for 30 min, the absorbance was measured at 545 nm.

[0044] Experimental Results: DPPH assay was used to evaluate the free radical scavenging ability of composition e, while donepezil was used as a positive control. As shown in Table 4, composition e exhibited moderate free radical scavenging activity (inhibition rate of 32.66% ± 2.69% at TI concentration of 2 μM and IA concentration of 4 μM), comparable to the positive control donepezil. The results indicate that the composition possesses moderate antioxidant activity at TI concentration of 2 μM and IA concentration of 4 μM.

[0045] Table 4

[0046]

[0047] Example 5: In vitro cytotoxicity detection

[0048] Human neuroblastoma cells SH-SY5Y were observed at 25 cm. 2Cells were cultured in fresh MEM / F12 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 g / mL streptomycin, and incubated at 37°C and 5% CO2 at a growth rate of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 10 cells / well in 96-well plates. After incubation for 24 hours, the culture medium was removed. The compounds were prepared in serum-free medium in the following groups: single-drug group: TI or IA (0, 1, 2, 4, 8 μM); two-drug group: group af in Table 2 of Example 2, and added to each well of the plate for 24 hours. After 24 hours of treatment, 10 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazole bromide was added to each well. After incubation at 37°C for 4 hours, 100 μL of DMSO was added to dissolve the formazan crystals, and the absorbance of the mixture was measured at 490 nm using a microplate reader.

[0049] Experimental Results: To evaluate the cytotoxicity of these two compounds at different concentrations, cell viability was tested in SH-SY5Y cells. The results of single-drug treatments were first observed as follows: Figure 2 As shown in Figure A, compared with the control group, SH-SY5Y cells showed cell proliferation at concentrations of 1-4 μM after the addition of TI or IA, and the cell number increased with increasing concentration. Although the proliferation of SH-SY5Y cells stopped when the concentration of TI or IA increased to 8 μM, the cell survival rate was higher than 80%. Therefore, TI or IA at concentrations of 1-8 μM did not have significant cytotoxicity. The results of the dual-drug treatment were then observed as follows... Figure 2 As shown in Figure B, compared with the control group, the cell survival rate of SH-SY5Y cells treated with donepezil and af in each group was around 100%, indicating that the composition of TI and IA prepared at a molar concentration ratio of 1-2:1-2 at 1-4 μM had no obvious cytotoxic effect on SH-SY5Y cells, demonstrating the good biosafety of the TI and IA composition.

[0050] Example 6: Effect of TI and IA composition on Aβ peptide (25–35)-induced cell death in vitro in SH-SY5Y cell model

[0051] SH-SY5Y cells (10,000 cells / well) were seeded and cultured for 24 hours in DMEM / F-12 medium containing 10% FBS at 37°C under a humid atmosphere and 5% CO2. After removing the medium, except for the control group, compounds were prepared in serum-free medium and grouped as follows: positive control: donepezil 4 μM; single-drug group: TI 2 μM, IA 4 μM; double-drug group: a combination of TI 2 μM and IA 4 μM, and added to each well of the plate for 24 hours. The medium was then removed, and 40 μg / mL Aβ peptide (25–35) (dissolved in PBS) was added to the medium, followed by 24 hours of culture. Cell viability was assessed using the MTT assay, and absorbance at 570 nm was measured.

[0052] Experimental results: Aβ peptide (25–35) is a fragment of β-amyloid peptide (Aβ), which is derived from the cleavage of the Aβ precursor protein (APP). It has neurotoxicity and can induce neuronal cell apoptosis. Figure 3 This study demonstrated the neuroprotective activity of TI, IA, and their combination against Aβ peptide (25-35)-induced cell death in the SH-SY5Y cell model. Treatment with 40 μg / mL Aβ peptide (25-35) reduced cell viability from 100% to approximately 60% (control group). Pretreatment with TI, IA, and their combination for 24 hours significantly increased cell viability compared to the control group, with the two-drug group showing higher cell viability than the single-drug group and approaching that of donepezil as a positive control. These results indicate that TI and IA possess good neuroprotective effects, and the combination of 2 μM TI and 4 μM IA exhibits the best neuroprotective effect.

[0053] In summary, the TI and / or IA composition of the present invention exhibits good AChE inhibitory activity, moderate anti-Aβ aggregation efficacy, moderate antioxidant activity, and good neuroprotective effects. Furthermore, it does not show significant cytotoxicity against human neuroblastoma cells SH-SY5Y, indicating that the TI and / or IA composition can be used as a novel acetylcholinesterase inhibitor for clinical treatment and / or the development of drugs for AD and other related diseases.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. An acetylcholinesterase inhibitor derived from a natural product, characterized in that, The acetylcholinesterase inhibitor contains an effective dose of a composition of physcion isoflavone and isoeugenol acetate, wherein the composition of physcion isoflavone and isoeugenol acetate is formulated at a molar concentration ratio of (1-2):(1-2).

2. The acetylcholinesterase inhibitor according to claim 1, characterized in that, The molar concentration of the gray leaf isoflavone is 1-4 μM, and the molar concentration of the isoeugenol acetate is 1-4 μM.

3. The acetylcholinesterase inhibitor according to claim 1, characterized in that, The molar concentration ratio is 1:

2.

4. The acetylcholinesterase inhibitor according to claim 1 or 2, characterized in that, The optimal concentration of gynostemma pentaphyllum is 2 μM and the optimal concentration of isoeugenol acetate is 4 μM in the gynostemma pentaphyllum isoflavone-isoeugenol acetate composition.

5. Use of a natural product-derived acetylcholinesterase inhibitor as described in any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease.

6. The use as described in claim 5, characterized in that, The drug exerts its therapeutic effect by inhibiting acetylcholinesterase activity and / or inhibiting β-amyloid peptide aggregation.

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