Methoxylated glabridin analogs and preparation methods and applications thereof

Abstract

The present invention discloses a methoxylated glabridin analogue, a preparation method and an application thereof. Experiments have shown that the methoxylated glabridin analogue MG01 has a dual-target agonist effect on ERβ / PGC-1α. In addition, MG01 has a good effect in treating Aβ 1‑42 In the constructed mouse hippocampal neuron cell HT-22 model, the cell activity damage can be significantly reduced. Methoxylated glabridin analogue MG01 inhibits Aβ 1‑42 MG01 can reverse the Aβ-induced decrease in mitochondrial membrane potential, increase in reactive oxygen species, and mitochondrial damage. 1‑42 The protein expression levels of induced mitochondrial biogenesis-related proteins PGC‑1α, NRF2, and TFAM indicate that MG01 activates ERβ / PGC‑1α, improves reactive oxygen species and mitochondrial function in cell models, exerts a neuroprotective effect, and thus prevents and treats Alzheimer's disease in women.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and in particular relates to a methoxylated glabridin analogue and a preparation method and application thereof. Background Art

[0002] The "China Alzheimer's Disease Report 2024" report shows that the prevalence of Alzheimer's disease (AD) in women in my country is twice that of men [1]. Due to the special physiological characteristics of women, estrogen deficiency caused by a sudden drop in estrogen levels in postmenopausal women has been considered an important risk factor for AD [2]. However, the specific mechanism by which a sudden drop in estrogen levels induces AD is still unclear. Although single estrogen therapy and estrogen replacement therapy have certain therapeutic effects, they can increase the risk of ovarian, endometrial and breast cancer, susceptibility to thrombosis, and susceptibility to cholecystitis [3]. Therefore, the development of targeted new drugs is still urgent.

[0003] The intracellular signal of estrogen is mainly the estrogen receptor (ER). ER is a ligand-dependent transcriptional regulator and a member of the nuclear receptor superfamily [4]. It can be divided into two categories: one is the classical nuclear receptor, which is located in the cell nucleus and exerts gene regulation effects by regulating the transcription of specific target genes, mainly including estrogen receptor α (ERα) and estrogen receptor β (ERβ); the second is membrane receptors, which indirectly exert transcriptional regulation through second messengers. ER is widely distributed in the brain. ERα is mainly expressed in the amygdala and hypothalamus, and ERβ is mainly expressed in the cortex, hippocampus, thalamus and cerebellum. ERβ is mainly related to cognition and other functions [5]. In addition, ERβ is relatively special. It exists both in the cell nucleus and in the cell membrane and mitochondria [6]. Therefore, regulating ERβ may be a key breakthrough in the treatment of female AD patients.

[0004] Mitochondria play a central role in regulating cell death[7]. Mitochondrial homeostasis imbalance is also one of the prominent early features of AD. Mitochondrial homeostasis imbalance and ferroptosis interact with each other and jointly promote the development of AD. Studies have shown that mitochondrial dysfunction promotes lipid peroxidation and ferroptosis. Mitochondria are an important source of intracellular ROS. Excessive ROS production not only causes mitochondrial damage, but also promotes further aggravation of lipid peroxidation[8]. Mitochondrial homeostasis is mainly controlled by three processes: mitochondrial biogenesis, mitochondrial dynamics, and mitochondrial autophagy. Among them, mitochondrial biogenesis is to maintain the number of mitochondria by producing new mitochondria, and plays an important role in maintaining mitochondrial health and function[9]. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) exists in healthy mitochondria. It is a co-transcription factor that regulates mitochondrial biogenesis by activating the transcription factor NRF1 / 2, thereby increasing the expression of the mitochondrial transcription factor mtTFA, promoting the replication of mtDNA, and maintaining the content of mitochondria

[10] . Mitochondria are also an important target of ERβ. Studies have shown that in the brains of female AD patients, the level of ERβ in the mitochondria of hippocampal neurons shows a significant decrease. Stimulating or overexpressing ERβ can significantly improve learning and memory impairment in AD model animals. Its mechanism of action is related to activating PGC-1α to promote mitochondrial development [11, 12].

[0005] Therefore, based on the above research background, it is concluded that ERβ and PGC-1α are two key targets for improving female AD. The present invention screened through the TCMSP Chinese System Pharmacology Platform and other methods to obtain a Chinese medicine monomer active ingredient, Glabridin, which can simultaneously activate the dual targets of ERβ / PGC-1α. Glabridin is an isoflavone compound extracted from the root of the plant of the genus Glycyrrhiza in the Leguminosae family

[13] . It has antioxidant, neuroprotective, free radical scavenging, anti-tumor, anti-inflammatory, anti-osteoporosis, and cardiovascular protection effects

[14] . However, Glabridin still has the disadvantages of low bioavailability and low blood-brain barrier permeability

[15] . Therefore, the present invention structurally modified and synthesized Glabridin to obtain the compound MG01. Therefore, as an ERβ / PGC-1α dual-target agonist, it has a good effect on Aβ 1-42 The neuroprotective effect of the glycyrrhizin-modified compound MG01 was investigated in an induced cell model, providing new effective compounds for the treatment of female AD patients.

[0006] References

[0007] [1] Mo Wei. New breakthrough in genetic basis research on Alzheimer's disease: epigenetic mechanism analysis of Alzheimer's disease based on single-cell multi-omics[J]. Science China: Life Sciences, 2024, 54(07): 1307-1309.

[0008] [2] Barth C, Crestol A, de Lange AG, et al. Sex steroids and the female brain across the lifespan: insights into risk of depression and Alzheimer's disease[J]. Lancet Diabetes Endocrinol. 2023;11(12):926-941.

[0009] [3] Zhang Tingting, Gang Jun, Fang Xiaoling. Advantages and disadvantages of estrogen replacement therapy [J]. Medicine and Philosophy (Clinical Decision Forum Edition), 2006, (01): 46-48.

[0010] [4] Zheng Xianghong, Xu Ying. Research progress on estrogen receptors and estrogen-related diseases[J]. Strait Pharmacy, 2016, 28(10): 1-5.

[0011] [5] Arao Y, Korach KS. The physiological role of estrogen receptorfunctional domains. Essays Biochem[J]. 2021;65(6):867-875.

[0012] [6] Pettersson K, Gustafsson JA. Role of estrogen receptor beta inestrogen action. Annu Rev Physiol[J]. 2001;63:165-192.

[0013] [7] Gao M, Yi J, Zhu J, et al. Role of Mitochondria in Ferroptosis.Mol Cell[J]. 2019;73(2):354-363.e3.

[0014] [8] Tadokoro T, Ikeda M, Ide T, et al. Mitochondria-dependentferroptosis plays a pivotal role in doxorubicin cardiotoxicity[J]. JCIInsight. 2023 Mar 22;8(6):e169756.

[0015] [9] Collier JJ, Oláhová M, McWilliams TG, et al. Mitochondrialsignalling and homeostasis: from cell biology to neurological disease. TrendsNeurosci[J]. 2023;46(2):137-152.

[0016]

[10] Abid H, Cartier D, Hamieh A, et al. AMPK Activation of PGC-1α / NRF-1-Dependent SELENOT Gene Transcription Promotes PACAP-InducedNeuroendocrine Cell Differentiation Through Tolerance to Oxidative Stress.Mol Neurobiol[J]. 2019;56(6):4086-4101.

[0017]

[11] Qian Yuanyuan, Liu Jinying, Sun Linlin, et al. Relationship between estrogen receptor β and Alzheimer's disease in women[J]. Journal of Stroke and Neurological Diseases, 2019, 36(11): 1046-1048.

[0018]

[12] Hou Y, Zhao W, Yu H, et al. Biochanin A alleviates cognitiveimpairment and hippocampal mitochondrial damage in ovariectomized APP / PS1mice. Phytomedicine[J]. 2022;100:154056.

[0019]

[13] Urban CF, Ermert D, Schmid M, et al. Neutrophil extracellulartraps contain calprotectin, a cytosolic protein complex involved in hostdefense against Candida albicans[J]. PLo SPathog, 2009, 5(10): e1000639.

[0020]

[14] PASTORINO G, CORNARA L, SOARES S, et al. Liquorice (Glycyrrhizaglabra): a phytochemical and pharmacological review[J]. Phytother Res, 2018,32(12): 2323-2339.

[0021]

[15] Wu Shaojuan. Study on biosafety evaluation and toxicity mechanism of glabridin[D]. Guangdong University of Technology, 2022. Summary of the invention

[0022] The present invention provides a methoxylated glabridin analogue and a preparation method and application thereof, which overcomes the defects and shortcomings of the existing multi-target drugs for treating Alzheimer's disease, that is, the few available types, and provides a methoxylated glabridin analogue with significant activation of ERβ / PGC-1α dual targets and the ability to inhibit Aβ 1-42 Induced cell damage and amelioration of mitochondrial damage by the methoxylated glabridin analog MG01.

[0023] The object of the present invention is to provide a methoxylated glabridin analog, wherein the methoxylated glabridin analog has a structure of formula (I):

[0024]

[0025] The purpose of the present invention is to provide a method for preparing a methoxylated glabridin analogue, and the synthetic route is as follows:

[0026] The specific steps include:

[0027]

[0028] S1, subjecting the compound of formula (II) to a substitution reaction with a methylating agent in an alkaline environment to obtain a methoxylated glabridin analogue of formula (I);

[0029] The mass ratio of the substrate formula (II), the inorganic base and the methylating agent is 176:105:75.5-85.

[0030] Another object of the present invention is to provide the use of the methoxylated glabridin analog in treating Alzheimer's disease in women.

[0031] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0032] Experiments have shown that the methoxylated glabridin analogue MG01 has dual-target ERβ / PGC-1α agonist effects. In addition, MG01 has a significant role in the treatment of Aβ 1-42 In the constructed mouse hippocampal neuron cell HT-22 model, MG01 can significantly reduce cell activity. 1-42 MG01 can reverse the Aβ-induced decrease in mitochondrial membrane potential, increase in reactive oxygen species, and mitochondrial damage. 1-42 The protein expression levels of induced mitochondrial biogenesis-related proteins PGC-1α, NRF2, and TFAM indicate that MG01 activates ERβ / PGC-1α, improves reactive oxygen species and mitochondrial function in cell models, exerts a neuroprotective effect, and thus prevents and treats Alzheimer's disease in women. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A The Western Blot provided in the embodiment of the present invention detects the expression levels of ERβ, PGC-1α, and ERα (Note: # P<0.05, ## P<0.01 vs. Control; * P<0.05, ** P<0.01 vs. the Aβ 1-42 );

[0034] Figure 1B is a graph showing the expression level of ERβ by MG01 provided in an embodiment of the present invention;

[0035] Figure 1C is a graph showing the expression level of PGC-1α by MG01 provided in an embodiment of the present invention;

[0036] Figure 1D is a graph showing the expression level of ERα by MG01 provided in an embodiment of the present invention;

[0037] Figure 2A is the CCK8 detection result provided by the embodiment of the present invention (Note: # P<0.05, ## P < 0.01 vs. theControl);

[0038] Figure 2B is the CCK8 detection result provided by the embodiment of the present invention (Note: # P<0.05, ## P < 0.01 vs. theControl);

[0039] Figure 2C is the CCK8 detection result provided by the embodiment of the present invention (Note: # P<0.05 vs. the Control; * P<0.05 vs. the Aβ 1-42 );

[0040] Figure 3A This is a staining result diagram of ROS provided by an embodiment of the present invention;

[0041] Figure 3B is the data experimental result of ROS provided by the embodiment of the present invention ( ## P<0.01 vs. Control; ** P<0.01, *** P<0.001 vs. the Aβ 1-42 );

[0042] Figure 3C This is a staining result diagram of JC-1 provided in an embodiment of the present invention.

[0043] Figure 3D is the data experimental result of JC-1 provided in the embodiment of the present invention ( ## P<0.01 vs. Control; * P<0.05 vs. the Aβ 1-42 );

[0044] Figure 4A This is a graph of the expression levels of PGC-1α, NRF2, and TFAM detected by Western Blot provided in an embodiment of the present invention;

[0045] Figure 4B This is a graph showing the expression level of PGC-1α by MG01 provided in the embodiment of the present invention (Note: # P<0.05; * P<0.05vs. the Aβ 1-42 );

[0046] Figure 4C This is a graph showing the expression level of NRF2 by MG01 provided in the embodiment of the present invention (Note: ## P<0.01; * P<0.05vs. the Aβ1-42 );

[0047] Figure 4D This is a graph showing the expression level of TFAM by MG01 provided in the embodiment of the present invention (Note: ## P<0.01; * P<0.05vs. the Aβ 1-42 );

[0048] Figure 5 is the structural formula of the methoxylated glabridin analog MG01 provided in an embodiment of the present invention;

[0049] Figure 6 is the carbon spectrum of the methoxylated glabridin analog MG01 provided in the embodiment of the present invention;

[0050] Figure 7 It is the hydrogen spectrum of the methoxylated glabridin analog MG01 provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0052] Embodiment 1:

[0053] Weigh 3.52 g (10 mmol) of the substrate formula (II), dissolve it in 30 mL of DMF, add 2.1 g (20 mmol) of sodium carbonate, cool it in an ice bath, slowly drop 1.70 g (12 mmol) of methyl iodide, react at room temperature, and detect the end of the reaction by TLC. Pour the reaction solution into water, filter, and wash to obtain the product.

[0054] Example 2

[0055] Weigh 3.52 g (10 mmol) of the substrate formula (II), dissolve it in 30 mL of DMF, add 2.1 g (20 mmol) of sodium hydroxide, cool it in an ice bath, slowly drop 1.70 g (12 mmol) of methyl iodide, react at room temperature, and detect the end of the reaction by TLC. Pour the reaction solution into water, filter, and wash to obtain the product.

[0056] Example 3

[0057] Weigh 3.52 g (10 mmol) of the substrate formula (II), dissolve it in 30 mL of DMF, add 2.1 g (20 mmol) of sodium carbonate, cool it in an ice bath, slowly drop 1.51 g (12 mmol) of dimethyl sulfate, react at room temperature, and detect the end of the reaction by TLC. Pour the reaction solution into water, filter, and wash to obtain the product.

[0058] Example 4

[0059] Weigh 3.52 g (10 mmol) of the substrate formula (II), dissolve it in 30 mL of dichloromethane, add 2.1 g (20 mmol) of sodium carbonate, cool in an ice bath, slowly drop 1.51 g (12 mmol) of dimethyl sulfate, react at room temperature, and detect the end of the reaction by TLC. Pour the reaction solution into water, filter, and wash to obtain the product.

[0060] In the embodiment of the present invention, the inorganic base can be common inorganic bases such as potassium hydroxide, lithium hydroxide, potassium carbonate, etc. The inorganic base includes sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, and potassium carbonate.

[0061] In an embodiment of the present invention, the methylating agent includes: methyl iodide or dimethyl sulfate.

[0062] The raw materials used in the composition, use and product provided by the present invention can be purchased from the market. Mouse hippocampal neuron cells HT-22 (Jiangsu KeyGen Biotechnology Co., Ltd., China, catalog number: KGG1312-1), 10% fetal bovine serum (Sijiqing, China, catalog number: KGL3001-500), DMEM high glucose medium (Jiangsu KeyGen Biotechnology Co., Ltd., China, catalog number: KGL1206-500), Aβ 1-42 Oligomers (Gill Biochemical Co., Ltd., China, Catalog No.: 52487), CCK-8 detection kit (Beyotime Biotechnology Co., Ltd., China, Catalog No.: C0042), reactive oxygen species detection kit (Beyotime Biotechnology Co., Ltd., China, Catalog No.: S0033S), mitochondrial membrane potential detection kit (JC-1) (Beyotime Biotechnology Co., Ltd., China, Catalog No.: C2003S), ERα (Abcam, USA, Catalog No.: ab32063), ERβ (Santa, USA, Catalog No.: 1531), PGC-1α (Abcam, USA, Catalog No.: ab176328), TFAM (Cell Signaling, USA, Catalog No.: 8076S), NRF2 (CellSignaling, USA, Catalog No.: 12721S).

[0063] Example 1: Verification of the dual-target agonist effect of the compound MG01 of the present invention on ERβ / PGC-1α

[0064] 1. Methods

[0065] Cell culture, model construction and drug administration: Mouse hippocampal neuron cell line HT-22 was cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum in a 5% CO 2 The incubator was set at 37°C. The cells were digested and subcultured with trypsin containing EDTA, and the cells in the logarithmic growth phase were used for the experiment. All experiments were repeated 3 times. HT-22 cells were divided into control group, 20µmol / L Aβ 1-42 group and 20µmol / L Aβ 1-42 +1µmol / L MG01 group, drug intervention for 24h.

[0066] Western blotting was used to detect the expression levels of ERβ, PGC-1α, and β-actin proteins: cells were inoculated into 6-well cell culture plates in a 37°C incubator, and HT-22 cells were divided into control group, 20µmol / L Aβ 1-42 group and 20µmol / L Aβ 1-42 In the +1µmol / L MG01 group, cells were collected 24 hours after drug intervention, lysed with RIPA lysis buffer, and total cell protein was extracted. The protein concentration was measured by BCA method. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then electrotransferred to PVDF membrane, and the PVDF membrane was blocked with TBST buffer containing 5% skim milk powder at room temperature for 2 hours. Primary antibody was added and incubated at 4 °C overnight. The primary antibody incubation solution was discarded, and TBST buffer containing secondary antibody was added. After incubation at room temperature for 1 hour, the secondary antibody was washed away. The image lab chemiluminescence system was used for detection. The experiment was repeated 3 times independently.

[0067] Statistical analysis: Prism software was used to process and analyze the data. Measurement data were expressed as mean ± standard deviation. Independent sample t test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. To ensure the accuracy of the experiment, each group of experiments was repeated at least 3 times, and p < 0.05 was considered statistically significant.

[0068] 2. Results

[0069] The expression levels of ERβ and PGC-1α were detected by Western Blot to verify the agonistic effect of MG01 on the two targets. The results of Western Blot showed that in the model group, the expression of ERβ and PGC-1α was significantly downregulated, and both of them were significantly increased after the administration of MG01 (P < 0.05 or P < 0.01), while the expression of ERα did not change significantly (P > 0.05) ( Figure 1A , Figure 1B , Figure 1C , Figure 1D ). This indicates that MG01 has dual-target agonistic effects on ERβ / PGC-1α.

[0070] Example 2: Effect of the compound MG01 of the present invention on Aβ 1-42 Effects of induced HT22 cell activity in mouse hippocampal neurons

[0071] In order to study the efficacy of MG01 in the present invention in preventing and treating AD, the present invention uses Aβ 1-42 The AD cell model was constructed using induced mouse hippocampal neuronal cells HT22, and the effect of MG01 on cell activity was detected by CCK-8 method.

[0072] 1. Methods

[0073] Cell culture, model construction and drug administration: Mouse hippocampal neuron cell line HT-22 was cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum in a 5% CO 2 The incubator was set at 37°C. The cells were digested and subcultured with trypsin containing EDTA, and the cells in the logarithmic growth phase were used for the experiment. All experiments were repeated 3 times. 5µmol / L, 10µmol / L, 20µmol / L, and 40µmol / L Aβ 1-42 Oligomers were induced in HT22 cells for 24 h to screen for the best Aβ 1-42 Damage concentration. AD cells were treated with MG01 at gradient concentrations (0.1µmol / L, 1µmol / L, 5µmol / L, 10µmol / L) for 24 h to screen the optimal administration concentration.

[0074] Cell viability detection: According to the above grouping, cells were inoculated into 96-well culture plates and cultured in a 37°C incubator. After 24 hours, CCK-8 detection reagent was added to each well. After incubation for 1 hour, the absorbance value of each cell culture well at 450nm was detected using an ELISA reader.

[0075] Statistical analysis: Prism software was used to process and analyze the data. Measurement data were expressed as mean ± standard deviation. Independent sample t test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. To ensure the accuracy of the experiment, each group of experiments was repeated at least 3 times, and p < 0.05 was considered statistically significant.

[0076] 2. Results

[0077] In the CCK8 results, as shown in Table 1, different concentrations of Aβ 1-42 After that, the cell viability was damaged to varying degrees. 1-42The cell viability was 80% at a concentration of 20 μM, which was the optimal concentration for model construction. Subsequent experiments were modeled at this concentration (Table 1). Similarly, the CCK-8 results showed that, as shown in Table 2, after culturing for 24 hours with different concentrations of MG01 (0 μM, 0.1 μM, 1 μM, 5 μM, 10 μM), MG01 concentrations below 1 μM had no effect on cell viability (Table 2). 1-42 Induce cell damage, give different concentrations of MG01, and test after 24 hours of culture. Table 3 shows that the cell viability is improved to varying degrees when different concentrations of MG01 are given. The improvement is most significant when the concentration of MG01 is 1 μM (P < 0.05), so this concentration is selected as the optimal concentration for subsequent experiments (Table 3) ( Figure 2A , Figure 2B , Figure 2C ).

[0078] Table 1

[0079]

[0080] Table 2

[0081]

[0082] Table 3

[0083]

[0084] Example 3: Effect of the compound MG01 of the present invention on Aβ 1-42 Effects of induced mitochondrial homeostasis in mouse hippocampal neuronal cells HT22

[0085] Mitochondrial homeostasis imbalance is also one of the prominent early features of AD, and mitochondrial homeostasis imbalance promotes the development of AD. In order to study the efficacy of MG01 in the present invention in preventing and treating AD, the present invention uses Aβ 1-42 The AD cell model was constructed using induced mouse hippocampal neuronal cells HT22, and the effect of MG01 on mitochondrial homeostasis was detected using ROS kit, JC-1 kit and Western Blot.

[0086] 1. Methods

[0087] Cell model construction and drug administration: HT-22 cells were divided into control group, 20µmol / L Aβ 1-42 group and 20µmol / LAβ 1-42 +1µmol / L MG01 group, drug intervention for 24h.

[0088] ROS detection: divided into control group, reactive oxygen control group, 20µmol / L Aβ 1-42group and 20µmol / L Aβ 1-42 +1µmol / L MG01 group cells were inoculated into 6-well cell culture plates and cultured in a 37℃ incubator for 24h. The reactive oxygen species positive control was incubated in the incubator for 20 minutes before staining. After adding the staining reagent, the cells were incubated in the incubator for 20 minutes. The cells were washed three times with serum-free cell culture medium. The results were then observed using a fluorescence microscope with an excitation wavelength of 488nm and an emission wavelength of 525nm.

[0089] Mitochondrial membrane potential detection: cells were inoculated into 6-well cell culture plates in a 37°C incubator. HT-22 cells were divided into control group, 20µmol / L Aβ 1-42 group and 20µmol / L Aβ 1-42 +1µmol / L MG01 group, after 24h of culture, remove the culture medium, wash once with PBS, add 1ml JC-1 staining working solution, and mix thoroughly. Incubate at 37℃ for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4ml distilled water per 1ml JC-1 staining buffer (5X), and place it in an ice bath. After the incubation, remove the supernatant, wash twice with JC-1 staining buffer (1X). Observe under a fluorescence microscope.

[0090] Western blotting was used to detect the expression levels of PGC-1α, NRF2, and TFAM proteins: cells were inoculated into 6-well cell culture plates in a 37°C incubator, and HT-22 cells were divided into control group, 20µmol / L Aβ 1-42 group and 20µmol / L Aβ 1-42 In the +1µmol / LMG01 group, cells were collected 24 hours after drug intervention, lysed with RIPA lysis buffer, and total cell protein was extracted. The protein concentration was measured by BCA method. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electrotransferred to PVDF membranes. The PVDF membranes were blocked with TBST buffer containing 5% skim milk powder at room temperature for 2 hours. Primary antibody was added and incubated overnight at 4 °C. The primary antibody incubation solution was discarded, and TBST buffer containing secondary antibody was added. After incubation at room temperature for 1 hour, the secondary antibody was washed away. The images were detected with Image Lab chemiluminescence system. The experiment was repeated 3 times independently.

[0091] Statistical analysis: Prism software was used to process and analyze the data. Measurement data were expressed as mean ± standard deviation. Independent sample t test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. To ensure the accuracy of the experiment, each group of experiments was repeated at least 3 times, and p < 0.05 was considered statistically significant.

[0092] 2. Results

[0093] JC-1 results show ( Figure 3A, Figure 3B ), compared with the control group, Aβ 1-42 The mitochondrial membrane potential in the induced model group was significantly reduced (P<0.01), and the mitochondrial membrane potential was significantly restored after the administration of MG01 (P<0.05). Figure 3C , Figure 3D ), in showing that compared with the control group, Aβ 1-42 The ROS level in the induced cell model was significantly increased (P<0.01), while after administration of MG01, the ROS level was significantly inhibited (P<0.01).

[0094] Western Blot results showed that compared with the control group, the expression levels of PGC-1α, NRF2, and TFAM in the AD model were significantly reduced (P<0.05 or P<0.01). After administration of MG01, the expression levels of PGC-1α, NRF2, and TFAM in the cells were significantly improved (P<0.05) ( Figure 4A , Figure 4B , Figure 4C , Figure 4D ).

[0095] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a methoxylated glabridin analogue, the synthetic route is as follows: ； The specific steps include: S1, subjecting the compound of formula (II) to a substitution reaction with a methylating agent in an inorganic base to obtain a methoxylated glabridin analogue of formula (I); The mass ratio of the substrate formula (II), the inorganic base and the methylating agent is 176:105:75.5-85.

2. The method for preparing a methoxylated glabridin analogue according to claim 1, characterized in that: The methylating agent includes: one of methyl iodide and dimethyl sulfate.

3. The method for preparing a methoxylated glabridin analogue according to claim 1, characterized in that: The inorganic base includes one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate and potassium carbonate.

4. Use of the methoxylated glabridin analogue according to claim 1 in the preparation of a drug for treating Alzheimer's disease.

5. The use according to claim 4, characterized in that: The drug dosage form for treating Alzheimer's disease is capsule, tablet, microcapsule preparation, injection, suppository, spray or ointment.

6. The use according to any one of claims 4 to 5, characterized in that: The medicament is used to treat patients suffering from Alzheimer's disease.

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