Compound capable of inhibiting activity of acetylcholin esterase and preparation method thereof

Abstract

The invention discloses a compound capable of inhibiting the activity of acetylcholin esterase and a preparation method thereof, and belongs to the field of research and development of natural medicines. The acetylcholin esterase inhibitor is obtained through antrodia camphorata microbial fermentation, product extraction, separation and purification. The inhibitor can be used for developing medicines for treating Alzheimer's disease or Alzheimer's disease, or can be used for developing anti-aging health-care foods and anti-oxidation products. The antrodia camphorata microbial fermentation preparation method is simple, the raw material source is wide, and industrial production is easy to realize.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine research and development, and specifically relates to the application of natural products fermented from Antrodia cinnamomea as a new type of acetylcholinesterase inhibitor and a preparation method thereof. Background Art

[0002] The pathogenesis of Alzheimer's disease (AD) remains unclear. Besides acetylcholine deficiency (cholinergic neuron degeneration), multiple factors are also involved, including β-amyloid deposition, tau hyperphosphorylation, oxidative stress, and inflammation. Acetylcholinesterase (AChE) is a serine hydrolase that catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) to produce acetic acid and choline. Acetylcholine plays a key role in normal cognitive function. Studies have shown that AChE levels in the brains of patients with Alzheimer's disease (AD) are significantly decreased, leading to a range of symptoms including cognitive impairment and decreased mobility. Therefore, inhibiting AChE activity may help increase ACh levels, thereby alleviating cognitive impairment and memory loss in the elderly. Currently available AChE inhibitors, while able to improve cognitive and behavioral impairments to some extent, suffer from limited efficacy, slow onset, short duration of action, poor efficacy in severe cases, and varying degrees of adverse reactions. The first-generation acetylcholinesterase inhibitor, tacrine, was withdrawn from the market due to significant hepatotoxicity. The second-generation inhibitor, donepezil, has a short half-life and requires daily dosing. Galantamine is extracted from plants, limiting raw materials. The third-generation inhibitor, huperzine A, is derived from natural plants, but has a bioavailability of less than 10% and is expensive to synthesize. With over 55 million people living with Alzheimer's disease worldwide, existing acetylcholinesterase inhibitors can only delay symptoms for 6-12 months. There is an urgent need for a new generation of inhibitors with both disease-modifying effects and long-term efficacy.

[0003] Natural products have advantages in natural drug screening due to their diverse chemical structures. Antrodia camphorata, also known as Antrodia camphorata and Antrodia camphorata, has been identified as a promising candidate for the screening of natural drugs. Summary of the Invention

[0004] The present invention provides a novel class of acetylcholinesterase inhibitors and a method for their preparation. These novel acetylcholinesterase inhibitors are obtained through microbial fermentation of Antrodia cinnamomea, product extraction, and separation and purification. Using microbial fermentation technology, the culture medium is readily available and inexpensive, and the preparation method is simple, making industrial production readily feasible.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing a compound capable of inhibiting acetylcholinesterase activity comprises taking Antrodia cinnamomea as a strain, and obtaining the compound capable of inhibiting acetylcholinesterase activity through microbial fermentation, product extraction and purification.

[0006] The compound having the ability to inhibit acetylcholinesterase activity includes any one of the following four compounds: .

[0007] Furthermore, the preparation method comprises the following steps: (1) Microbial fermentation: using Antrodia cinnamomea as the strain for solid or liquid fermentation; (2) Product extraction: The fermentation product obtained in step (1) is leached or extracted with a solvent, and the solvent is removed to obtain an extract containing the target compound; (3) Isolation and purification: The extract containing the target compound obtained in step (2) is purified by chromatography to obtain a compound having acetylcholinesterase inhibitory activity.

[0008] Preferably, the solvent in step (2) is one or more of ethanol, methanol, ethyl acetate, n-butanol, and chloroform.

[0009] Preferably, the chromatographic technique in step (3) comprises a combination of reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography.

[0010] Preferably, the compound prepared by the preparation method has the ability to inhibit acetylcholinesterase activity.

[0011] Application: Application of the compound capable of inhibiting acetylcholinesterase activity in the preparation of acetylcholinesterase inhibitors.

[0012] The beneficial effects of the present invention are as follows: the novel acetylcholinesterase inhibitor prepared by the present invention has strong acetylcholinesterase inhibitory activity, can be used to develop drugs or health products for treating Alzheimer's disease and other senile dementias and anti-aging, has great market prospects, and is prepared by liquid fermentation of Antrodia cinnamomea, has a wide range of culture medium raw materials, is simple in preparation method, and is easy to achieve industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound 1.

[0014] Figure 2 is the carbon NMR spectrum of the compound 1.

[0015] Figure 3 This is a high-resolution mass spectrometry analysis of compound 1.

[0016] Figure 4 is the chemical structure of compound 1.

[0017] Figure 5 is the hydrogen nuclear magnetic resonance spectrum of the compound 2.

[0018] Figure 6 is the carbon NMR spectrum of the compound 2.

[0019] Figure 7 is the chemical structure of compound 2.

[0020] Figure 8 is the hydrogen nuclear magnetic resonance spectrum of the compound 3.

[0021] Figure 9 is the carbon NMR spectrum of the compound 3.

[0022] Figure 10 is the chemical structure of compound 3.

[0023] Figure 11 is the hydrogen nuclear magnetic resonance spectrum of the compound 4.

[0024] Figure 12 is the carbon NMR spectrum of the compound 4.

[0025] Figure 13 is the chemical structure of compound 4.

[0026] Figure 14 This is the dose effect of compound 1 in inhibiting acetylcholinesterase activity. DETAILED DESCRIPTION

[0027] A novel class of acetylcholinesterase inhibitors and their preparation method. Using Antrodia cinnamomea as the strain, a novel class of acetylcholinesterase inhibitors was obtained through microbial fermentation, product extraction, and purification. The chemical structural characteristics of the novel class of acetylcholinesterase inhibitors are as follows: The novel acetylcholinesterase inhibitor and its preparation method comprise the following steps: Step 1: Add Antrodia cinnamomea ( Antrodiacamphorata ) After the strain is activated, solid-state fermentation or liquid fermentation is carried out under appropriate culture conditions using a culture medium suitable for fungal growth; Step 2: The fermentation product obtained by culturing in step 1 is extracted with an organic solvent, and solids are removed by filtration, centrifugation or membrane filtration. The solvent is concentrated to obtain a crude extract; Step 3: The crude extract obtained in step 2 is dissolved in a solvent such as methanol or ethanol, and then subjected to purification methods such as reverse phase silica gel column chromatography, gel column chromatography, and normal phase silica gel column chromatography. The mixture is analyzed by high performance liquid chromatography or thin layer chromatography to obtain the target component. Step 4: The novel acetylcholinesterase inhibitor obtained in step 3 is subjected to nuclear magnetic resonance spectroscopy and mass spectrometry detection.

[0028] Among them, the microbial fermentation described in step 1 can be solid-state fermentation using rice as the culture medium; the liquid fermentation can use a culture medium suitable for fungal growth, including a culture medium formula composed of: 5-25wt% potato, 1-5wt% glucose, and the balance water; the suitable culture conditions are that the culture medium has a natural pH, is sterilized at 0.1MPa, 100-140°C for 15-40min, is shaken at a temperature of 25-33°C, has a rotation speed of 150-250rpm, and is cultured for 5-30 days.

[0029] Wherein, the organic solvent described in step 2 is one or more of ethanol or methanol, ethyl acetate, n-butanol, and chloroform with different concentrations.

[0030] In the reversed-phase silica gel column chromatography described in step 3, the chromatographic medium is reversed-phase silica gel (RP-C18), and the chromatography parameters are as follows: using methanol water, ethanol water or acetone water of different concentrations as the eluent, the flow rate is 5-50 mL / min, collecting 5-200 mL in each tube, sampling each test tube for thin layer chromatography: the developing solvent is chloroform: methanol = 10:1, the color developer is 10% ethanolic sulfuric acid, and similar components are combined.

[0031] Wherein, the gel column chromatography described in step 3, the chromatography medium is gel Sephadex LH-20, the chromatography parameters are: methanol, ethanol or acetone as the eluent, the flow rate is 5-30s / drop, 3-20mL is collected in each tube, and each test tube is sampled for thin layer chromatography: the developing solvent is chloroform: methanol = 10:1, the color developer is 10% ethanolic sulfuric acid, and similar components are combined.

[0032] Wherein, the normal phase silica gel column chromatography described in step 3, the chromatography medium is silica gel, and the chromatography parameters are: using chloroform, dichloromethane, ethyl acetate, petroleum ether, acetone, and methanol in different volume ratios as eluents, wherein the volume ratio of dichloromethane or petroleum ether to ethyl acetate or acetone is 500:1~50, and the volume ratio of chloroform to methanol is 300:1~30.

[0033] Among them, the purification methods such as reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography described in step 3 can be adjusted in the order of using these methods; Wherein, the nuclear magnetic resonance spectrum detection described in step 4 is mainly to detect the hydrogen spectrum ( 1 H-NMR) and carbon spectroscopy ( 13 C-NMR), mass spectrometry can be high-resolution mass spectrometry or low-resolution mass spectrometry.

[0034] According to the results of the acetylcholinesterase inhibition assay of the compound, it was found that the compound had strong acetylcholinesterase inhibition activity (Example 3).

[0035] The present invention is further illustrated by the following examples, but the protection scope of the present invention is not limited to the following examples.

[0036] Example 1 The present invention provides a method for preparing a novel acetylcholinesterase inhibitor, comprising the following steps: Antrodia cinnamomea was inoculated into a potato glucose liquid medium (prepared as follows: 200 g of peeled and sliced potatoes were added to 1000 ml of water and boiled for 30 minutes. The mixture was then filtered through gauze. 20 g of glucose was added to the filtrate, stirred to dissolve, and the volume was adjusted to 1000 ml with water). Fermentation was carried out at 28°C and 220 rpm for 21 days. After fermentation, the fermentation broth was extracted with ethyl acetate, and the organic phase was concentrated, evaporated, and weighed to yield 8.97 g of a crude extract. The crude extract was initially separated on an RP-18 reverse-phase silica gel column (packing weight: 200 g) using a methanol-water mobile phase with a concentration gradient from pure water to 100% methanol (0%, 30%, 50%, 100%). Each gradient elution volume was 1.5 L, and 150 mL fractions were collected and concentrated. According to thin-layer chromatography analysis, fractions Fr.D (1.3 g) and Fr.E (1.5 g) containing the target compound were obtained by elution with 30% methanol in water. Fraction Fr.D (1.3 g) was further separated on a Sephadex LH-20 gel chromatography column (packing weight: 120 g) using methanol as the elution solvent at a rate of 15-20 seconds per drop. The eluate was collected every 60 minutes. Similar fractions were combined based on thin-layer chromatography analysis to obtain fractions Fr.D3 (708.3 mg), Fr.D5 (45.5 mg), and Fr.D6 (276.8 mg) containing the target compound. Fraction Fr.D3 was further isolated using an RP-C18 reverse-phase silica gel column (packing weight: 150 g) with pure water as the eluent, with a gradient elution of 1 L per column, and 100 mL per bottle was collected. Similar fractions were combined based on thin-layer chromatography analysis to obtain Fr.D3-3 (140 mg) containing the target compound. 55 mg of fraction Fr.D3-3 was chromatographed on a normal-phase silica gel column (packing weight: 2.0 g) using petroleum ether:ethyl acetate in varying volume ratios. When the elution gradient was 200:1, fraction Fr.D3-3-1 (32.6 mg) containing the target product was obtained. Finally, compound 4 (11.5 mg) was prepared by high-performance liquid chromatography. Fraction Fr.D5 (45.5 mg) was further separated by thin-layer chromatography on an RP-C18 reverse-phase silica gel column (packing weight: 40 g) using water, 20% methanol, and 30% methanol as eluents, sequentially. Based on the TLC analysis, similar fractions were combined and eluted with 30% methanol in water to obtain fraction Fr.D53 (17.5 mg) containing the target compound. Fraction Fr.D53 was purified by preparative HPLC and reverse-phase chromatography to obtain compound 3 (1.4 mg). A 50 mg portion of fraction Fr.D6 was chromatographed on a normal-phase silica gel column (packing weight: 2 g) using varying volume ratios of petroleum ether:ethyl acetate to afford compound 1 (23.7 mg) at an 8:1 gradient.

[0037] 479 mg of fraction Fr.E was further separated using a Sephadex LH-20 gel chromatography column with methanol as the eluent. Based on the results of thin-layer chromatography analysis of each fraction, similar fractions were combined to obtain fraction Fr.E2 (63.6 mg) containing the target compound. Fraction Fr.E2 was eluted using a normal-phase silica gel column (packing weight: 2.1 g) with varying ratios of petroleum ether:ethyl acetate. At 200:1, fraction Fr.E2-1 (12 mg) containing the target product was obtained. Finally, preparative HPLC was used to obtain compound 2 (4.0 mg).

[0038] Example 2 The pure compound was subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR and 13 C-NMR) determination, and the structure of the compound can be determined based on the nuclear magnetic spectrum data.

[0039] Compound 1 is a white substance, soluble in methanol, acetone and chloroform, and can be detected by nuclear magnetic resonance spectroscopy ( 1 H-NMR, 13 C-NMR, HSQC, HMBC and DEPT) (Table 1 and Figure 1-Figure 2 ), it was found that compound 1 contains 6 hydrogen signals and 8 carbon signals, of which 10 carbon atoms include 1 primary carbon, 1 secondary carbon, 1 tertiary carbon and 5 quaternary carbons. From the HMBC spectrum, it can be seen that there are carbon-hydrogen long-range correlations between H-2 and C-1, C-3 and C-4, H-5 and C-6 and C-8, and H-8 and C-5, C-6 and C-7. According to the chemical shift values of C-1, C-3, C-4, C-5, C-6 and C-7, which are δ137.0, δ134.1, δ134.7, δ112.6, δ133.0 and δ120.7 respectively, it is judged that C-1, C-3, C-4, C-5, C-6 and C-7 are carbon atoms on the benzene ring. 1 H-5 in H-NMR is a doublet with a coupling constant of J =0.9 Hz, indicating that there is no strong coupling effect near the hydrogen, and C-5 is a tertiary carbon, it is judged that H-7 is the hydrogen in the unsubstituted position on the benzene ring; according to the chemical shift value of C-2, which is δ101.8 and is a secondary carbon, combined with 1 H-2 in H-NMR can be judged to be methylenedioxy, connected to the C-1 and C-3 positions of the benzene ring; according to the chemical shift value of C-8, which is δ15.8 and is a primary carbon, combined with 1H-8 in H-NMR can be judged to be a methyl group connected to C-6; since C-4 and C-7 are quaternary carbons, it can be judged that they are each connected to a hydroxyl group. The compound was further subjected to high-resolution mass spectrometry detection, and the molecular formula of the compound was determined to be C8H8O4 by HRQ-TOF MS, and the mass spectrometry signal of the compound can be detected. m / z [M + H] + 169.0496 (theoretical value is C8H9O4, 169.0501) and m / z [M + Na] + 191.0319 (theoretical value is C8H8NaO4, 199.0320) Figure 3 ). Combined 1 H-NMR and 13 Based on the chemical shift values of C-NMR and the literature, 1 was resolved as 5-methyl-benzo[1,3]dioxole-4,7-diol, with the molecular formula C8H8O4 ( Figure 4 ).

[0040] Table 1 NMR data of compound 1 (H spectrum 600 MHz, C spectrum 151 MHz, deuterated acetone) Compound 2 is an orange-red needle-like substance that is easily soluble in chloroform. 1 H-NMR, 13 C-NMR, data are shown in Table 2 and Figure 5-Figure 6 ) determination, compound 2 contains 9 hydrogen signals and 9 carbon signals, of which the 9 carbon atoms include 3 primary carbons, 1 tertiary carbon and 5 quaternary carbons. According to the chemical shift values of C-2, C-3, C-5 and C-6, which are δ145.2, δ145.0, δ144.2 and δ131.4 respectively, it is judged that they are aromatic carbons, and C-2 and C-3, C-5 and C-6 are connected by carbon-carbon double bonds; according to the chemical shift values of C-1 and C-4, which are δ184.4 and δ184.6 respectively, it is judged that C-1 and C-4 are 2 carbonyl groups, and C-1 to C-5 are quaternary carbons, and C-6 is tertiary carbon. 1 H-6 in the H-NMR spectrum indicates that C-1 to C-6 form an aromatic ring, and C-6 is an unsubstituted position on the aromatic ring; according to the chemical shift values of C-8 and C-9, which are primary carbons, 1 H-8 and H-9 in H-NMR can be judged that C-8 and C-9 are two methoxy groups, connected to the C-2 and C-3 positions of the aromatic ring respectively; according to the chemical shift value of C-7, which is δ15.6 and is a primary carbon, combined with 1H-7 in H-NMR can be judged that C-7 is a methyl group connected to C-5. Based on the above data analysis, 2 is interpreted as 2,3-dimethoxy-5-methyl-p-benzoquinone, with a molecular formula of C9H9O4 ( Figure 7 ).

[0041] Table 2 NMR data of compound 2 (H spectrum 600 MHz, C spectrum 151 MHz, deuterated chloroform) Compound 3 is a yellow substance, easily soluble in methanol, and can be detected by NMR spectroscopy ( 1 H-NMR, 13 C-NMR, data are shown in Table 3 and Figure 8-Figure 9 ) determination, compound 3 contains 8 hydrogen signals and 8 carbon signals, of which the 8 carbon atoms include 2 primary carbons, 2 tertiary carbons and 4 quaternary carbons. According to the chemical shift values of C-1 and C-4, which are δ182.6 and δ187.6 respectively, and both are quaternary carbons, it is shown that C-1 and C-4 are 2 carbonyl groups; according to the chemical shift values of C-2 and C-3, which are δ159.0 and δ107.5, and the chemical shift values of C-5 and C-6, which are δ134.0 and δ143.8 respectively, it is judged that C-2, C-3, C-5 and C-6 are aromatic carbons, and C-2 and C-3, C-5 and C-6 are connected by carbon-carbon double bonds; according to the chemical shift value of C-7, which is δ15.7 and is a primary carbon, combined with 1 H-7 in H-NMR can be used to determine that C-7 is a methyl group; according to the chemical shift value of C-8, which is δ56.4 and is a primary carbon, combined with 1 H-8 in H-NMR can be used to determine that C-8 is a methoxy group; 1 H-3 and H-5 in H-NMR, and C-3 and C-5 are tertiary carbons, C-2 and C-6 are quaternary carbons, which can be judged that there is a methoxy group connected to C-2 and a methyl group connected to C-6 on the aromatic ring. Based on the above data analysis, 3 is interpreted as 2-Methoxy-6-methyl-1,4-benzoquinone, with a molecular formula of C8H8O3 ( Figure 10 ).

[0042] Table 3 NMR data of compound 3 (H spectrum 600 MHz, C spectrum 151 MHz, deuterated chloroform) Compound 4 is an oily substance, easily soluble in chloroform, and can be detected by nuclear magnetic resonance spectroscopy ( 1 H-NMR, 13 C-NMR, data are shown in Table 4 and Figure 11-12) determination, compound 4 contains 10 hydrogen signals and 9 carbon signals, of which the 9 carbon atoms include 3 primary carbons, 1 tertiary carbon and 5 quaternary carbons. According to the chemical shift values of C-1, C-2, C-3, C-4, C-5 and C-6, which are δ141.7, δ137.3, δ140.6, δ139.2 and δ119.5 respectively, it is judged that C-1 to C-6 form a benzene ring; according to 1 The chemical shift value of H-6 in H-NMR is δ6.48, and it is a single peak, indicating that there is no adjacent hydrogen around this hydrogen to couple with it. Combined with the fact that C-6 is a tertiary carbon, it is judged that H-6 is the hydrogen in the unsubstituted position on the benzene ring; according to the chemical shift values of C-7 and C-8, which are primary carbons, δ61.0 and δ60.9, respectively, combined with the fact that 1 H-7 and H-8 in H-NMR can be judged that C-7 and C-8 are two methoxy groups, connected to the C-2 and C-3 positions of the benzene ring respectively; according to the chemical shift value of C-9, which is δ15.5 and is a primary carbon, combined with 1 H-9 in H-NMR can be used to determine that C-9 is a methyl group connected to C-5. Based on the above data analysis, 4 is interpreted as 2,3-dimethoxy-5-methylbenzene-1,4-diol, with a molecular formula of C9H 12 O4 ( Figure 13 ).

[0043] Table 4 NMR data of compound 4 (H spectrum 600 MHz, C spectrum 151 MHz, deuterated chloroform) Example 3 The inhibitory activity of the compounds against acetylcholinesterase was determined by the Ellman method (spectrophotometry). Huperzine A was used as the positive control, and different reaction groups (sample group, sample blank group, positive control group, positive blank group, negative control group, and negative blank group) were set up. First, the sample to be tested was prepared into a 1 mg / mL solution using chromatographic grade methanol. First, 50 μL PBS buffer, 20 μL test sample, and 10 μL acetylcholinesterase were added to each experimental area of the 96-well plate in sequence, mixed thoroughly, and reacted at 4°C for 20 minutes. After the completion of the first stage of the reaction, the 96-well plate was quickly transferred to ice, and 100 μL DTNB (5,5'-dithiobis-2-nitrobenzoic acid) and 20 μL ATch (thioacetylcholine) were added respectively. After mixing again, the reaction was incubated at 37°C for 20 minutes. Finally, the plate was detected by a microplate reader at 412°C. The absorbance value at 400 nm was used. For the blank control, the acetylcholinesterase in the reaction system was replaced with PBS buffer; for the negative control, the test sample was replaced with methanol. To ensure data reliability, three parallel control groups were performed for each experiment. Based on the measured absorbance, the inhibition rate of each compound on acetylcholinesterase was calculated according to the following formula.

[0044] The inhibition rates of the compounds against acetylcholinesterase are shown in Table 5. At a final concentration of 0.1 mg / mL in the reaction system, compound 1 exhibited a strong inhibitory effect on acetylcholinesterase, with its inhibitory activity significantly superior to that of the other tested compounds, reaching an inhibition rate of 99.14%, comparable to that of the positive control, huperzine A (inhibition rate of 101.98%). Compounds 2, 4, and 3 also exhibited strong inhibitory effects, with inhibition rates of 90.68%, 71.23%, and 88.48%, respectively.

[0045] Table 5 Inhibition rate of compounds on acetylcholinesterase To further determine the inhibitory activity of compound 1 on acetylcholinesterase, it was prepared into 7 concentrations of 0.025 mg / mL, 0.05 mg / mL, 0.15 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1.0 mg / mL, and the same experimental method was used for detection, that is, the final concentrations of the samples in the experimental system (200 μL) were 2.5, 5.0, 15, 25, 50, 75, and 100 μg / mL, respectively. The inhibition rates of compound 1 on acetylcholinesterase at different concentrations are shown in Tables 6 and Figure 14 SPSS software analysis showed that the IC value of compound 1 for acetylcholinesterase inhibition was 50 The inhibitory activity was 5.97 μg / mL, and within the experimental concentration range, there was a significant positive correlation between the inhibitory activity and its concentration (P<0.01).

[0046] Table 6 Inhibition rate of compound 1 at different concentrations on acetylcholinesterase The results showed that compounds 1, 2, 3, and 4 had significant inhibitory effects on acetylcholinesterase, especially compound 1, whose inhibitory activity was comparable to that of the commercial drug huperzine A, and had great potential for development as an acetylcholinesterase inhibitor.

[0047] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing a compound having acetylcholinesterase inhibitory activity, characterized in that: Antrodia cinnamomea was used as the fungus, and a compound capable of inhibiting acetylcholinesterase activity was obtained through microbial fermentation, product extraction and purification.

2. The preparation method according to claim 1, characterized in that The compound having the ability to inhibit acetylcholinesterase activity includes any one of the following four compounds: 。 3. The preparation method according to claim 1, wherein: The following steps are involved: (1) Microbial fermentation: using Antrodia cinnamomea as the strain for solid or liquid fermentation; (2) Product extraction: The fermentation product obtained in step (1) is leached or extracted with a solvent, and the solvent is removed to obtain an extract containing the target compound; (3) Isolation and purification: The extract containing the target compound obtained in step (2) is purified by chromatography to obtain a compound having acetylcholinesterase inhibitory activity.

4. The preparation method according to claim 2, wherein: The solvent in step (2) is one or more of ethanol, methanol, ethyl acetate, n-butanol, and chloroform.

5. The preparation method according to claim 2, wherein: The chromatographic technique in step (3) includes a combination of reverse phase silica gel column chromatography, gel column chromatography and normal phase silica gel column chromatography.

6. A compound having acetylcholinesterase inhibitory activity obtained according to the preparation method according to any one of claims 1 to 5.

7. Use of the compound having acetylcholinesterase inhibitory activity according to claim 6 in the preparation of acetylcholinesterase inhibitors.

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