Acetylcholin esterase inhibitor from natural product and application of acetylcholin esterase inhibitor

Abstract

The invention relates to the field of natural product pharmacy, in particular to an acetylcholin esterase inhibitor sourced from a natural product and application of the acetylcholin esterase inhibitor. Although a chemical synthetic acetylcholin esterase inhibitor used in clinical treatment at present achieves a certain curative effect, the chemical synthetic acetylcholin esterase inhibitor still has the problems of side effects, drug resistance and the like. Therefore, the invention provides the acetylcholin esterase inhibitor, the inhibitor is a single-drug or double-drug composition derived from natural products, the single drug is grifolin isoflavone (TI) or isoeugenol acetate (IA), and the double drug is prepared from TI and IA in proportion. The acetylcholin esterase inhibitor disclosed by the invention has good AChE inhibitory activity, anti-Abeta aggregation effect, antioxidant activity and good neuroprotection effect, and the TI and / or IA can be used as a novel acetylcholin esterase inhibitor to be applied to the development and use of medicines for clinically treating and / or preventing related diseases such as Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of natural product pharmacy, and in particular to a natural product-derived acetylcholinesterase inhibitor and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD), also known as senile dementia, is a common central nervous system degenerative disease in the elderly. Its clinical manifestations include memory impairment, aphasia, apraxia, agnosia, impairment of abstract thinking and calculation ability, as well as personality and behavior changes, etc. With the acceleration of global aging process, the incidence of Alzheimer's disease is increasing year by year, which has become an important factor affecting the quality of life of the elderly and social burden. At present, the pathogenesis of the disease has not been fully elucidated, but the mainstream hypotheses include the β-amyloid (Aβ) cascade hypothesis, the tau protein hyperphosphorylation hypothesis and the neuroinflammation hypothesis, etc.

[0003] Acetylcholinesterase inhibitors (AChEI) are a major class of drugs currently used in the clinical treatment of Alzheimer's disease. By inhibiting the activity of acetylcholinesterase (AChE) in the body, it reduces the hydrolysis of acetylcholine, thereby increasing the level of acetylcholine in the brain and improving the cognitive function and daily living ability of patients. This class of drugs is mainly used for the treatment of mild to moderate Alzheimer's disease patients, and has certain remission effect on early symptoms. However, existing chemically synthesized acetylcholinesterase inhibitors, such as Donepezil, Rivastigmine, etc., although have achieved certain efficacy, still have problems such as side effects and drug resistance.

[0004] In recent years, natural product-derived compounds have been continuously concerned in the field of biological medicine. Research has found that they have advantages such as wide source, diverse structure, and small side effects compared to most clinical drugs, providing a new direction for finding new acetylcholinesterase inhibitors with low toxicity and high efficacy. SUMMARY

[0005] The present application aims to solve the above technical problems, and provides a natural product-derived acetylcholinesterase inhibitor and application thereof.

[0006] The present application provides a natural product-derived acetylcholinesterase inhibitor containing an effective dose of Toxicarol Isoflavone and / or Acetylacetate Isoeugenol ester;

[0007] Further, the structure of the Toxicarol Isoflavone (TI) is shown in Formula I:

[0008]

[0009] Formula I

[0010] Further, the structure of the isoeugenyl acetate (IA) is shown in Formula II:

[0011]

[0012] Formula II

[0013] The CAS number of the isoeugenyl acetate is 93-29-8, and the propenyl side chain (-CH=CH-CH3) in the structure thereof exists as a cis-trans (Z / E) isomer, which is a cis-trans mixture of isoeugenyl acetate.

[0014] The concentration of the formononetin isoflavone is 1-4 μM, and the concentration of the isoeugenyl acetate is 1-4 μM;

[0015] Further, the molar concentration ratio of the formononetin isoflavone to the isoeugenyl acetate in the formononetin isoflavone-isoeugenyl acetate composition is (1-2):(1-2);

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

[0017] Further, the optimal concentration of the formononetin isoflavone in the formononetin isoflavone-isoeugenyl acetate composition is 2 μM, and the optimal concentration of the isoeugenyl acetate is 4 μM.

[0018] The present application provides a natural product-derived acetylcholinesterase inhibitor for:

[0019] a. preparing a drug for preventing and / or treating an AChE-related disease;

[0020] b. preparing an anti-β-amyloid peptide (Aβ) aggregation drug;

[0021] c. preparing a drug for preventing and / or treating an Aβ aggregation-related disease;

[0022] The present application has the advantages that:

[0023] The natural product-derived compound TI and / or IA provided by the present application has good AChE inhibitory activity, moderate anti-Aβ aggregation efficacy, moderate antioxidant activity, and good neuroprotective effect, and has no obvious cytotoxicity to human neuroblastoma cells SH-SY5Y cells, and has good biological safety, indicating that TI and / or IA can be used as a new acetylcholinesterase inhibitor for clinical treatment and development of drugs for preventing and / or treating AD and other related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the data graph of AChE inhibition activity of TI and IA composition.

[0025] Figure 2 is the data graph of SH-SY5Y cell survival rate of the compound: Figure 2 A in is the effect of TI and IA alone on SH-SY5Y cell survival rate, Figure 2 B in is the effect of TI and IA composition on SH-SY5Y cell survival rate.

[0026] Figure 3 is the data graph of the effect of TI and IA composition on Aβ peptide (25-35) induced cell death in SH-SY5Y cell model in vitro. DETAILED DESCRIPTION

[0027] The technical solutions described in the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the embodiments described in the present application are only a part of the feasible technical solutions of the present application, and other embodiments obtained by those skilled in the art on the basis of the embodiments of the present application without any creative labor should be regarded as belonging to the protection scope of the present application.

[0028] Example 1: AChE inhibition activity determination of TI and IA single drug

[0029] Add 40 μL of phosphate buffer (pH = 8.0) to each well of a 96-well plate, then add 10 μL of 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25, 50 and 100 μM of the test compound (TI / IA) solution or the control group to the corresponding wells, then add 10 μL of AChE, and incubate at 37°C on a shaking table for 5 min. Add 20 μL of DTNB solution, and incubate at 37°C on a shaking table for 5 min, then add 10 μL of substrate ATC, and incubate at 37°C on a shaking table for 3 min, and then measure the absorbance at 412 nm by an enzyme label instrument to calculate the inhibition rate of the test compound on AChE. According to the inhibition curve, the IC 50 value (inhibitor concentration when the enzyme activity is inhibited by 50%) of the compound is obtained, and the data statistics are shown in Table 1.

[0030] Table 1

[0031] Example 2: AChE inhibition activity determination of TI and IA composition

[0032] In order to study the optimal compounding concentration of the two compounds, the present application determines the IC 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.

[0033] Table 2

[0034] 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.

[0035] Example 3: Detection of Aβ self-aggregation activity of TI and IA composition

[0036] 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.

[0037] Experimental results: Aggregation of Aβ is one of the main histopathological markers of AD. Therefore, the present application used thioflavin T to determine the prevention of Aβ aggregation by the TI and IA combination. As shown in Table 3, the composition e had a moderate inhibitory effect on Aβ aggregation, with an inhibition rate of 46.95% ± 2.92% for the composition of TI at 2 μM concentration and IA at 4 μM concentration; although the inhibitory effect of composition e on Aβ aggregation was higher than that of the positive drug donepezil, the effect was also relatively close. The results showed that the composition of TI at 2 μM concentration and IA at 4 μM concentration exhibited moderate anti-Aβ aggregation efficacy.

[0038] Table 3

[0039] Example 4: In vitro antioxidant activity determination of TI and IA combination

[0040] The radical scavenging activity of TI and IA combination was tested using 1,1-diphenyl-2- trinitro-phenylhydrazine (DPPH). To prepare a 0.1 mM concentration of DPPH stock solution, DPPH was dissolved in methanol. The test compounds were prepared with PBS solution: group e (2 μM TI and 4 μM IA), positive control (donepezil, 4 μM). In a 96-well plate, 10 μL of test compound and 90 μL of DPPH solution were added to each well. After 30 minutes of reaction at 25 °C, the absorbance was measured at 545 nm.

[0041] Experimental results: DPPH assay was used to evaluate the radical scavenging ability of composition e, with donepezil as a positive control. As shown in Table 4, composition e showed moderate radical scavenging activity (32.66% ± 2.69% inhibition rate for the composition of TI at 2 μM concentration and IA at 4 μM concentration), with an inhibitory effect comparable to that of the positive control donepezil. The results showed that the composition of TI at 2 μM concentration and IA at 4 μM concentration had moderate antioxidant activity.

[0042] Table 4

[0043] Example 5: In vitro cytotoxicity test

[0044] Human neuroblastoma cells SH-SY5Y cells were cultured in 25 cm 2T75 flasks were incubated at 37°C and 5% CO2 with 5 x 105 cells / flask in fresh MEM / F12 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 g / mL streptomycin. After 24 hours of incubation, the medium was removed. The compounds were prepared in serum-free medium and grouped as follows: single drug groups: TI or IA (0, 1, 2, 4, 8 μM); double drug groups: groups a-f of Table 2 in Example 2 were 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-tetrazolium bromide was added to each well. After 4 hours of incubation at 37°C, 100 μL of DMSO was added to dissolve the formazan crystals, and the absorbance of the mixture was measured at 490 nm on a microplate reader. 3

[0045] Experimental results: To evaluate the cytotoxicity of each concentration of the two compounds, cell viability was tested on SH-SY5Y cells. First, the experimental results of single drug treatment were observed as shown in A of Figure 2 Compared with the control group, after the addition of TI or IA, SH-SY5Y cells showed cell proliferation at a concentration of 1-4 μM, and the number of cells increased with the increase of the concentration, although the proliferation line of SH-SY5Y cells stopped when the concentration of TI or IA increased to 8 μM, but the cell survival rate was higher than 80%, so TI or IA had no obvious cytotoxicity at a concentration of 1-8 μM. Then, the experimental results of double drug treatment were observed as shown in B of Figure 2 Compared with the control group, after the treatment of positive drug donepezil and each group a-f on SH-SY5Y cells, the cell survival rate of each group was 100% up and down, which indicated that the composition of TI and IA at a molar concentration ratio of 1-2:1-2 at a concentration of 1-4 μM had no obvious cytotoxicity to SH-SY5Y cells, which reflected the good biological safety of the composition of TI and IA.

[0046] Example 6: Effect of TI and IA composition on Aβ peptide (25-35)-induced cell death in SH-SY5Y cell model in vitro

[0047] ​SH-SY5Y cells (10,000 cells / well) were seeded and cultured in DMEM / F-12 medium containing 10% FBS for 24 hours at 37°C under a humidified atmosphere and 5% CO2. After removing the medium, except for the control group, the compounds were prepared in serum-free medium as follows: positive drug: donepezil 4 μM; single drug group: TI 2 μM, IA 4 μM; double drug group: combination of TI 2 μM and IA 4 μM, and added to each well of the plate for 24 hours. Then the medium was removed, 40 μg / mL Aβ peptide (25-35) (dissolved in PBS) was added to the medium, and incubated for another 24 hours. Cell viability was evaluated using MTT, and the absorbance at 570 nm was measured.

[0048] Experimental results: Aβ peptide (25-35) is a fragment of β-amyloid peptide (Aβ) derived from the product of cleavage of the precursor protein of Aβ (APP), which has neurotoxicity and can induce neuronal apoptosis. Figure 3 The results show the neuroprotective activity of TI, IA and their combination against Aβ peptide (25-35)-induced cell death in the SH-SY5Y cell model. After treatment with 40 μg / mL Aβ peptide (25-35), the cell viability decreased from 100% to about 60% (control group). Pretreatment with TI, IA and their combination for 24 hours showed that the cell viability was significantly improved compared with the control group, and the cell viability of the double drug group was not only higher than that of the single drug group but also close to that of the positive drug donepezil. These results indicate that TI and IA have good neuroprotective effects, and the combination of TI 2 μM and IA 4 μM has the best neuroprotective effect.

[0049] In summary, the combination of TI and IA has good AChE inhibitory activity, moderate anti-Aβ aggregation efficacy, moderate antioxidant activity and good neuroprotective effect, and has no obvious cytotoxicity to human neuroblastoma cells SH-SY5Y cells, indicating that the combination of TI and IA can be used as a new acetylcholinesterase inhibitor for clinical treatment and / or development of drugs for AD and related diseases.

[0050] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and supplements without departing from the principles of the present application, and these improvements and supplements should also be considered within the protection scope of the present application.

Claims

1. A natural product-derived acetylcholinesterase inhibitor, characterized in that, The acetylcholinesterase inhibitor contains an effective dose of the genistein isoflavone and / or the eugenyl acetate.

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

3. The acetylcholinesterase inhibitor according to claim 1, characterized in that, The genistein isoflavone-eugenyl acetate composition is formulated according to a molar concentration ratio of the genistein isoflavone to the eugenyl acetate of (1-2):(1-2).

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

2.

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

6. Use of the natural product-derived acetylcholinesterase inhibitor of claim 1 in the preparation of a medicament for preventing and / or treating acetylcholinesterase-related diseases.

7. Use of the natural product-derived acetylcholinesterase inhibitor of claim 1 in the preparation of a medicament for resisting β-amyloid peptide aggregation.

8. Use of the natural product-derived acetylcholinesterase inhibitor of claim 1 in the preparation of a medicament for preventing and / or treating Aβ aggregation-related diseases.

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