Pharmaceutical applications of 1,8-diacetyloxy anthraquinone and pharmaceutical compositions

Abstract

The application relates to the field of biological medicine, in particular to pharmaceutical application and a pharmaceutical composition of 1,8-diacetylrhefa. The application of 1.1,8-diacetylrhefa in the preparation of a drug for treating Alzheimer's disease mediated by mitochondrial autophagy; and a pharmaceutical composition, which is composed of a therapeutically effective amount of 1,8-diacetylrhefa and a pharmaceutically acceptable carrier. 1,8-diacetylrhefa can specifically activate the PINK1 / Parkin signal pathway, thereby promoting the removal of damaged mitochondria and restoring the organelle homeostasis in neurons. Experimental results show that in cell and animal models, it can effectively reduce the beta-amyloid level and improve the cognitive dysfunction of model animals, such as increasing the spontaneous alternation rate of Y maze, prolonging the passive avoidance test latency, thereby providing a candidate treatment scheme with disease modification potential for neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the pharmaceutical applications of 1,8-diacetylemodin and pharmaceutical compositions thereof, particularly the application of 1,8-diacetylemodin in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases and pharmaceutical compositions comprising 1,8-diacetylemodin. Background Technology

[0002] Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are a group of central nervous system disorders caused by progressive neuronal damage or death, leading to a decline in cognitive, motor, and other functions. Their complex pathological mechanisms typically involve the abnormal deposition of misfolded proteins (such as β-amyloid and α-synuclein), oxidative stress, and chronic neuroinflammation. Recent research has further revealed that mitochondrial dysfunction and its quality control mechanisms, particularly the dysregulation of mitophagy, are common and early key factors driving various neurodegenerative diseases. The accumulation of damaged mitochondria not only leads to cellular energy crises but also exacerbates oxidative damage and neuronal death.

[0003] Currently, clinical treatment drugs (such as cholinesterase inhibitors for Alzheimer's disease, donepezil, rivastigmine, and galantamine) mainly act on the neurotransmitter system. Although they can temporarily relieve some symptoms, they cannot reverse or delay the essential progression of the disease. Furthermore, long-term use has problems such as significant side effects and limited efficacy.

[0004] Natural active substances offer a significant safety advantage in treating neurodegenerative diseases, thus sparking extensive research. Discovering a naturally active substance with exceptional therapeutic efficacy would be of paramount importance.

[0005] Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone natural product extracted from the traditional Chinese medicine rhubarb. Studies have reported that it can improve cognitive impairment in animal models of Alzheimer's disease (AD). For example, the article "Yang Le et al., 'Study on the improvement of cognitive impairment, endoplasmic reticulum stress and neuroinflammation in Alzheimer's disease by emodin' [J]. Journal of Shanghai Jiaotong University (Medical Edition), 2025" revealed that the target of emodin is mainly astrocytes and their endoplasmic reticulum stress-inflammation pathway (such as inhibiting IRE1α, NF-κB p65 and p38 phosphorylation), which mainly confirmed the downregulation effect of emodin on inflammatory factors. However, emodin contains multiple hydroxyl groups. These highly polar groups, which are prone to forming hydrogen bonds, result in a smaller effective nonpolar surface area, leading to poor lipophilicity and transmembrane transport capacity, and insufficient blood-brain barrier penetration. At the same time, emodin has a weak focusing effect on the translocation of YFP-Parkin protein to mitochondria in YFP-Parkin HeLa cells (a marker of the initiation of mitophagy), and its therapeutic effect on neurodegenerative diseases is not obvious.

[0006] Therefore, the discovery of a natural active substance with a high nonpolar surface area, high blood-brain barrier permeability, and strong translocation focusing effect on YFP-Parkin protein in YFP-Parkin HeLa cells has promising prospects for the development of drugs for the treatment of neurodegenerative diseases. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of existing clinical drugs for the treatment of neurodegenerative diseases, such as obvious side effects, limited efficacy, insufficient blood-brain barrier penetration of lead compounds (such as emodin) discovered from natural products, and weak translocation focusing effect on YFP-Parkin protein in YFP-Parkin HeLa cells. This invention provides pharmaceutical applications of 1,8-diacetylemodin and pharmaceutical compositions.

[0008] The first aspect of this invention provides a novel pharmaceutical application technology for a drug active ingredient that promotes mitophagy, as detailed below.

[0009] Application of 1,8-diacetylemodin in the preparation of drugs for treating Alzheimer's disease mediated by mitophagy.

[0010] Application of 1,8-diacetylemodin in the preparation of mitochondrial autophagy inducer drugs.

[0011] Among them, 1,8-diacetylemodin modifies the two phenolic hydroxyl groups of the 1,8-substituents of emodin to acetoxy groups.

[0012]

[0013] The molecular formula of 1,8-diacetylemodin is shown above, and its molecular weight is 354.3.

[0014] Furthermore, the drug is used to improve diseases mediated by mitophagy.

[0015] Furthermore, the drug is a drug for improving cognitive impairment, or the drug is a drug for preventing or treating neurodegenerative diseases.

[0016] Furthermore, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS). Preferably, the neurodegenerative disease is Alzheimer's disease.

[0017] A second aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of 1,8-diacetylemodin and a pharmaceutically acceptable carrier.

[0018] Furthermore, the content of 1,8-diacetylemodin in the pharmaceutical composition is from 10 mg to 100 mg per unit dosage form.

[0019] Preferably, the content of 1,8-diacetylemodin in the pharmaceutical composition is 50 mg to 100 mg per unit dosage form.

[0020] Example 3 of this invention demonstrated in APP / PS1 Alzheimer's disease model mice that daily intervention with 1,8-diacetylemodin at doses of 10 mg / kg and 20 mg / kg significantly cleared Aβ deposits in the brain and improved cognitive function. Based on interspecies dose extrapolation using a recognized body surface area conversion method, this effective dose range corresponds to an estimated daily dose of approximately 50 mg to 100 mg for adults (based on a 60 kg body weight). For ease of clinical use, this total daily dose can be designed to be provided in one or more unit dosage forms. Therefore, the pharmaceutical compositions of this invention can be formulated into unit dosage forms, such as tablets or capsules, wherein each unit dosage form contains 10 mg, 25 mg, 50 mg, or 100 mg of 1,8-diacetylemodin. A typical dosing regimen is 1 to 2 unit dosage forms taken once or twice daily.

[0021] Furthermore, pharmaceutically acceptable carriers are selected from one or more of fillers, binders, disintegrants, lubricants, and solubilizers.

[0022] Furthermore, the dosage form of the pharmaceutical composition is one of oral, injectable, or implantable formulations.

[0023] Furthermore, the dosage form of the pharmaceutical composition is one of the following: capsules, tablets, powders, granules, oral solutions, suspensions, oral emulsions, syrups, elixirs, injections, powder for injection, and subcutaneous implants.

[0024] Furthermore, the dosage form of the pharmaceutical composition is either a controlled-release formulation or a sustained-release formulation.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. This invention provides the application of 1,8-diacetylemodin in the preparation of drugs for the prevention or treatment of neurodegenerative diseases. In a YFP-Parkin HeLa cell model, 1,8-diacetylemodin (DAE) was found to significantly promote the translocation of Parkin protein to mitochondria in a dose-dependent manner (10-80 μM), with a significantly stronger effect than its parent compound, emodin. Mechanistic studies showed that DAE dose-dependently upregulates PINK1 protein expression and activates mitophagy via the PINK1 / Parkin pathway, promoting autophagic flux and effectively clearing damaged mitochondria. Cellular experiments confirmed that DAE intervention (80 μM, 24 h) significantly reduced intracellular Aβ levels through the aforementioned autophagy pathway, while this clearance effect was completely blocked after PINK1 knockdown or the use of the autophagy inhibitor chloroquine. Animal experiments showed that continuous intragastric administration of DAE (10 and 20 mg / kg) for 14 days dose-dependently increased PINK1 protein expression in the brain, reduced Aβ deposition in the hippocampus and cortex, and significantly improved cognitive impairment: prolonging escape latency and reducing the number of errors in the passive avoidance test, increasing the spontaneous alternation rate in the Y-maze test, and pathological sections showed a significant reduction in neuronal damage. The experimental data demonstrate the mechanism and efficacy of 1,8-diacetylemodin in improving AD pathology and cognitive function by activating PINK1 / Parkin-dependent mitophagy and clearing Aβ, providing empirical evidence for its application in the treatment of neurodegenerative diseases.

[0027] 2. The pharmaceutical composition provided by this invention lays a crucial foundation for transforming the unique neuroprotective mechanism of 1,8-diacetylemodin, based on PINK1 / Parkin's mitochondrial autophagy, into a stable and controllable clinical administration form. This composition can be flexibly prepared into different dosage forms, such as oral or injectable formulations, according to treatment needs, ensuring compliance and feasibility of administration. Attached Figure Description

[0028] Figure 1 Figure showing the effects of emodin and 1,8-diacetylemodin on the generation of fluorescent dots in YFP-Parkin HeLa cells.

[0029] Figure 2 This is a simulation diagram comparing the molecular electrostatic potentials of emodin and 1,8-diacetylemodin.

[0030] Figure 3 Figure 1 shows the experimental results of the effects of intervention time and concentration of 1,8-diacetylemodin on the aggregation and migration of YFP-Parkin protein.

[0031] Figure 4 The effect of DAE on the activity of YFP-Parkin HeLa cells (A) and N2a cells (B).

[0032] Figure 5 Figure 1 shows the experimental results related to the effect of DAE on Parkin-dependent mitophagy in YFP-Parkin HeLa cells and N2a cells.

[0033] Figure 6 The figure shows the experimental results of DAE maintaining mitochondrial homeostasis by clearing damaged mitochondria.

[0034] Figure 7 Figure showing experimental results related to DAE-dependent activation of mitophagy via the PINK1 / Parkin pathway and promotion of autophagic flux.

[0035] Figure 8 Figure showing experimental results related to DAE promoting Aβ clearance in N2a-Swed APP cells via autophagy.

[0036] Figure 9 Figure showing experimental results related to knocking down PINK1 to block the clearance of Aβ by DAE in N2a-Swed APP cells.

[0037] Figure 10 Figure 1 shows the experimental results related to DAE reducing Aβ deposition in the brains of APP / PS1 mice by activating PINK1-dependent mitophagy.

[0038] Figure 11 Figure showing experimental results related to DAE improving neuronal damage and restoring cognitive function in APP / PS1 mice. Detailed Implementation

[0039] Application of 1,8-diacetylemodin in the preparation of mitochondrial autophagy inducer drugs.

[0040] Alternatively, 1,8-diacetylemodin can be used as a mitophagy inducer or enhancer.

[0041] Alternatively, 1,8-diacetylemodin can be used as a drug to promote mitophagy. Utilizing 1,8-diacetylemodin to promote mitophagy can therapeutically address mitophagy deficiencies, achieving entirely new pharmaceutical applications. The drug's mechanism of action is completely different from traditional drugs used to treat such diseases, providing patients with a novel treatment option.

[0042] Application of 1,8-diacetylemodin in drug preparation.

[0043] Furthermore, the drug is a mitophagy inducer. Furthermore, the drug is a drug for improving diseases mediated by mitophagy.

[0044] Furthermore, the drug is a drug for improving cognitive impairment, or the drug is a drug for preventing or treating neurodegenerative diseases.

[0045] Furthermore, the drug is a treatment for at least one of neurodegenerative diseases, hereditary or primary mitochondrial diseases, cardiovascular diseases, metabolic diseases, or sarcopenia.

[0046] Furthermore, the aforementioned mitochondrial autophagy deficiency is one of the following: neurodegenerative diseases, hereditary or primary mitochondrial diseases, cardiovascular diseases, metabolic diseases, or sarcopenia. Specifically, 1,8-diacetylemodin can be used in the preparation of drugs for the prevention or treatment of neurodegenerative diseases.

[0047] Furthermore, the neurodegenerative disease mentioned is one of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia. Alzheimer's disease, Parkinson's disease, Huntington's disease, or ALS are all related to defects in mitochondrial autophagy. Promoting mitochondrial autophagy can exert a therapeutic effect; therefore, 1,8-diacetylemodin can be used in drug development for these diseases.

[0048] Furthermore, the drug works by at least one of the following: inducing or promoting PINK1 / Parkin-dependent mitophagy; Reduce or clear β-amyloid protein in the brain; Neuroprotection and / or improvement of cognitive impairment associated with neurodegenerative diseases.

[0049] The use of 1,8-diacetylemodin in the preparation of drugs for the prevention or treatment of neurodegenerative diseases.

[0050] In the molecular structure of 1,8-diacetylemodin, the acetylation of the polar phenolic hydroxyl group results in a larger effective nonpolar surface area. In the experimental investigation, the molecular electrostatic potential analysis showed that 1,8-diacetylemodin has a larger nonpolar surface area, stronger lipophilicity and transmembrane ability, which is conducive to penetrating the blood-brain barrier.

[0051] Since the acetyl group is an electron-withdrawing group, altering the electron cloud distribution of the anthraquinone nucleus may affect the binding affinity to the electrostatic interaction enzyme active sites of target proteins (such as PINK1 / Parkin pathway-related proteins). Experimental studies have found that both emodin and 1,8-diacetylemodin promote the translocation and focusing of YFP-Parkin protein in YFP-Parkin HeLa cells. Under the same drug intervention dose, the effect of 1,8-diacetylemodin is significantly better than that of emodin.

[0052] Experimental studies have shown that 1,8-diacetylemodin, compared with emodin, exhibits significantly enhanced PINK1 / Parkin pathway activation, mitophagy induction activity, and / or Aβ clearance. 1,8-diacetylemodin is significantly more effective than its parent compound, emodin, in the prevention or treatment of neurodegenerative diseases. Furthermore, in cell and animal models, 1,8-diacetylemodin effectively reduces β-amyloid protein levels and improves cognitive dysfunction in model animals (such as increasing the spontaneous alternation rate in the Y-maze and prolonging the latency of the passive avoidance test), thus providing a new research direction for neurodegenerative diseases.

[0053] 1,8-Diacetylemodin specifically activates the PINK1 / Parkin signaling pathway, thereby promoting the clearance of damaged mitochondria (i.e., mitophagy) and restoring organelle homeostasis within neurons. Experimental results show that in cell and animal models, it effectively reduces β-amyloid protein levels and improves cognitive dysfunction in model animals (e.g., increasing the spontaneous alternation rate in the Y-maze and prolonging the latency of the passive avoidance test), thus providing a potential therapeutic candidate for neurodegenerative diseases. Simultaneously, 1,8-Diacetylemodin promotes mitophagy, forming a fundamental protective pathway, and is a novel candidate drug capable of clearing pathogenic proteins and maintaining neuronal homeostasis. This has significant scientific and clinical translational value for overcoming the current treatment challenges of neurodegenerative diseases.

[0054] Furthermore, the drug works by promoting PINK1 / Parkin-dependent mitophagy.

[0055] Furthermore, the Alzheimer's disease described is the disease exhibited in APP / PS1 transgenic Alzheimer's disease model animals.

[0056] Furthermore, the drug is used to reduce or eliminate β-amyloid protein in the brain.

[0057] Furthermore, the drug is used to improve cognitive impairment associated with neurodegenerative diseases.

[0058] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0059] The following are the definitions of some of the English abbreviations used in this invention: APBS: Adaptive Poisson-Boltzmann Solver; MEP: Molecular electrostatic potential; DAE: 1,8-Diacetylemodin; Ac-1: 1,8-Diacetylemodin (this abbreviation is used in some figures); Emodin: emodin; DHS: emodin (this abbreviation is used in some figures); Kruskal-Wallis H Analysis: Kruskal-Wallis test; mtDNA: mitochondrial DNA; puncta: dotted structure; CCCP: carbonyl cyano-3-chlorophenylhydrazone / carbonyl cyano-chlorophenylhydrazone; CQ: chloroquine; 3-MA: 3-Methyladenine; siRNA: small interfering RNA; PEG: polyethylene glycol; PVP: polyvinylpyrrolidone; FBS: fetal bovine serum; PBS: phosphate buffered saline solution; CMC-Na: sodium carboxymethyl cellulose.

[0060] The YFP-Parkin HeLa cells used in the experiment are a genetically engineered special HeLa cell line. The basic cells are HeLa cells, which have a long history of widespread use in biological and medical research. Parkin is an E3 ubiquitin ligase, and mutations in its gene (PARK2) are one of the most common causes of autosomal recessive Parkinson's disease. Parkin protein plays a central role in mitophagy, marking damaged mitochondria and initiating their clearance by autophagosomes. YFP is an abbreviation for Yellow Fluorescent Protein. By fusing the YFP gene with the Parkin gene and introducing it into HeLa cells, a YFP-Parkin fusion protein was obtained that continuously expresses itself, emitting yellow fluorescence under a fluorescence microscope.

[0061] The N2a cells used in the experiment were mouse-derived neuroblastoma cell lines.

[0062] The QuantityOne software used in this invention was developed by Bio-Rad and is mainly used in the field of molecular biology. It focuses on the analysis and processing of one-dimensional electrophoresis gels, dot blots, and narrow-line blots, and provides colony counting and molecular weight determination functions.

[0063] Example 1 1. Experimental Objective Based on the Parkin translocation screening, natural compounds that induce mitophagy were identified, and natural promoters of mitophagy were determined.

[0064] 2. Experimental Materials 2.1 Experimental Drugs Forty-two natural compounds were obtained from the Institute of Integrated Transformation of TCM Brain Disease Drugs, Chengdu University of Traditional Chinese Medicine. 1,8-Dacetylemodin was provided by Professor Han Bo of Chengdu University of Traditional Chinese Medicine (code name Ac-1). The compound codes are shown in Table 1. The purity of all compounds was >98%. (In some embodiments, the 1,8-Dacetylemodin can be prepared from emodin using an acetylation method, or it can be purchased through commercial channels. This is well-known technology in the field and will not be described further.) All compounds were dissolved in DMSO to prepare the corresponding concentrations, except for evodiamine (2.5, 1.25, 0.625 μM), anemarrhenasaponin A3 (0.5, 0.25, 0.125 μM), anemarrhenasaponin B2 (20, 10, 5 μM), total ginsenosides (20 ug / mL, 10 ug / mL), total astragalussaponins (20 ug / mL, 10 ug / mL), and CCCP (commercial positive control, full name Carbonyl cyanide m-chlorophenyl). Apart from hydrazone (20 μM), the cell-administered concentrations of the other compounds were 80, 40, and 20 μM or 50, 25, and 12.5 μM.

[0065] Table 1. List of test compound numbers No. name molecular weight Dosage concentration No. name molecular weight Dosage concentration 1 chlorogenic acid 354.31 80 / 40 / 20 μM 22 Ginsenosides 610.52 50 / 25 / 12.5 μM 2 Evodiamine 303.35 2.5 / 1.25 / 0.625 μM 23 Ginsenoside PK1 767.00 50 / 25 / 12.5 μM 3 ferulic acid 194.18 80 / 40 / 20 μM 24 3,6,7-Trimethylquercetin 360.32 50 / 25 / 12.5 μM 4 Ligustrazine 136.19 80 / 40 / 20 μM 25 Vitex flavonoids 374.34 50 / 25 / 12.5 μM 5 ligustilide 190.24 80 / 40 / 20 μM 26 4-Hydroxy-3-methoxycinnamon 178.18 50 / 25 / 12.5 μM 6 Angelica lactone 98.10 80 / 40 / 20 μM 27 1,8-Diaacetylemodin 354.07 80 / 40 / 20 μM 7 Sichuan pepper oil 196.20 80 / 40 / 20 μM 28 p-Hydroxybenzaldehyde 122.12 50 / 25 / 12.5 μM 8 Heptamethoxycoumarin 176.17 80 / 40 / 20 μM 29 Gastrodin 286.28 50 / 25 / 12.5 μM 9 Sichuan pepper toxin alcohol 202.16 80 / 40 / 20 μM 30 m-hydroxybenzoic acid 138.12 50 / 25 / 12.5 μM 10 Anemarrhena saponin B2 921.08 20 / 10 / 5μM 31 p-Hydroxybenzyl alcohol 124.14 50 / 25 / 12.5 μM 11 Anemarrhena saponin A3 740.92 0.5 / 0.25 / 0.125 μM 32 Clomid 624.69 50 / 25 / 12.5 μM 12 Paeonol 166.17 80 / 40 / 20 μM 33 Citronellol 154.25 50 / 25 / 12.5 μM 13 Luteolin 286.24 80 / 40 / 20 μM 34 p-hydroxybenzylamine 123.15 50 / 25 / 12.5 μM 14 Quercetin 302.24 50 / 25 / 12.5 μM 35 Bat puerarin 624.77 50 / 25 / 12.5 μM 15 Hypericin 464.38 80 / 40 / 20 μM 36 Zichun 362.33 50 / 25 / 12.5 μM 16 mangiferin 422.34 80 / 40 / 20 μM 37 Baicalein 270.24 80 / 40 / 20 μM 17 Myristic acid 228.37 80 / 40 / 20 μM 38 Anemarrhena saponin 416.65 80 / 40 / 20 μM 18 Vincristine 909.06 80 / 40 / 20 μM 39 baicalin 446.37 80 / 40 / 20 μM 19 Rutin 610.52 50 / 25 / 12.5 μM 40 Ginsenosides \ 20 / 10ug / mL 20 Ginsenoside Ro 957.11 50 / 25 / 12.5 μM 41 Astragalus total saponins \ 20 / 10ug / mL 21 Ginsenoside F1 638.87 50 / 25 / 12.5 μM 42 emodin 270.23 80 / 40 / 20 μM 2.1 Experimental Cells Stable HeLa cells expressing YFP-Parkin (YFP-Parkin HeLa cells) (In this example, the YFP-Parkin HeLa cells were kindly provided by Professor Shen Hanming of the National University of Singapore. Those skilled in the art can purchase the same HeLa cells from international / domestic standard cell banks (such as ATCC, Chinese Academy of Sciences Cell Bank) for the same experiments. HeLa cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin in an incubator at 37°C, 5% CO2 and saturated humidity.

[0066] 2.3 Experimental Reagents Table 2 Experimental Reagents Experimental reagents Item number batch number factory fetal bovine serum 11011-8611 19110505 Zhejiang Tianhang Biotechnology Co., Ltd. DMEM high glucose medium C11995500BT 8120428 Gibco, USA Penicillin and streptomycin mixture 15140-122 2199841 Gibco, USA Phosphate buffer solution C20012500BT 8120150 Gibco, USA 0.25% trypsin PYG0067 15L18B67 China's Boster Biotech Carbonyl cyano-3-chlorophenylhydrazone / carbonyl cyanide chlorophenylhydrazone C2759 MKBW0481V Sigma-Aldrich 2.4 Experimental Apparatus Table 3 Experimental Instruments instrument model factory Electronic constant temperature water bath DZKW-4 Beijing Zhongxing Weiye Instrument Co., Ltd. centrifuge L600 Hunan Xiangyi Laboratory Instrument Development Co., Ltd. Clean bench SW-QJ-2FD Suzhou Antai Air Technology Co., Ltd. Cell incubator 3111 Thermo Fisher Scientific, Inc. Electronic balance FA1204L Shanghai Yueping Scientific Instruments Co., Ltd. Inverted microscope XD-202 Nanjing Jiangnan Yongxin Optics Co., Ltd. Inverted fluorescence microscope DMI3000 Leica, Germany 3. Experimental Methods 3.1 YFP-Parkin HeLa cell culture and drug administration intervention YFP-Parkin HeLa cells in logarithmic growth phase were washed with PBS, digested with trypsin, and then the digestion was terminated with complete culture medium. Cells were collected, centrifuged, resuspended, and seeded at 200 μL per well into 24-well plates. The cells were then cultured at 37°C in a 5% CO2 incubator. For drug intervention, 300 μL / well of a 2.1 concentration was added for initial screening and treated for 6 hours. For time-gradient experiments, a concentration range of 0-80 μM was used, with treatments for 6, 8, 10, and 12 hours to observe the dynamic changes in YFP-Parkin protein translocation and fluorescent spot formation. All experiments included corresponding concentrations of DMSO as a blank control and a 20 μM CCCP positive control.

[0067] 3.2 Fluorescence microscopy observation of YFP-Parkin protein aggregation and translocation After YFP-Parkin HeLa cells were treated with the appropriate drugs, the drug-containing culture medium in each well was discarded. After washing with pre-cooled PBS, 100 μL of pre-cooled paraformaldehyde was added to each well for fixation for 15 min. 100 μL of PBS was added to each well. The cell plate was placed under an inverted fluorescence microscope, the GFP fluorescence channel was selected, and YFP-Parkin translocation and aggregation and fluorescence signals were observed and recorded.

[0068] 3.3 Calculation of Molecular Electrostatic Potential (MEP) Molecular electrostatic potential (MEP) is a parameter describing the electrostatic interactions between molecules, providing an intuitive method for understanding the relative polarity of molecules. To compare the interaction strength differences between emodin and Ac-1 (i.e., 1,8-diacetyl emodin) from the perspective of molecular polarity, their molecular electrostatic potentials were calculated. Partial charge of the molecules was distributed in Chimera using the AM1-BBC method, and the molecular electrostatic potential values ​​were calculated using an adaptive Poisson-Boltzmann solver.

[0069] 3.4 Data Statistics and Analysis All measurement data are expressed as mean ± standard deviation. If the data follow a normal distribution, one-way ANOVA is used; otherwise, a nonparametric Kruskal-Wallis test is performed. p <0.05 indicates a statistically significant difference.

[0070] 4. Experimental Results 4.1 1,8-Dacetylemodin promotes YFP-Parkin translocation more strongly than emodin. like Figure 1 As shown, the effects of emodin and 1,8-diacetylemodin on the generation of fluorescent dots in YFP-Parkin HeLa cells are as follows: Figure 1 In the formula A, we have the structural formula of emodin (DHS). Figure 1 B is 1,8-diacetylemodin (Ac-1). Figure 1 In the middle C, there is a fluorescence image of YFP-Parkin fluorescent dots induced by emodin and Ac-1. Figure 1 The middle section (D) shows the statistical distribution of YFP-Parkin fluorescent dots induced by emodin and Ac-1. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the DHS 40μM group, △p<0.05, △△p<0.01, △△△p<0.001; compared with the DHS 80μM group, #p<0.05, ##p<0.01, ###p<0.001.

[0071] Of the 42 natural compounds, only emodin and 1,8-diacetylemodin significantly promoted the translocation and focusing of YFP-Parkin protein in YFP-Parkin HeLa cells. Under the same intervention dose, the effect of 1,8-diacetylemodin was significantly better than that of emodin (see...). Figure 1 ,like Figure 1 The D-value in the figure shows that the number of YFP-Parkin puncta was low in the control group (Ctrl) (mean 0.09), while the positive control CCCP significantly induced the formation of puncta structures (mean 11.43). At the same concentrations (20, 40, and 80 μM), the number of puncta in the 1,8-diacetylemodin treatment group was significantly higher than that in the emodin (marked DHS in the figure) treatment group. Particularly noteworthy is that at a concentration of 80 μM, the number of puncta in the 1,8-diacetylemodin group (mean 9.11) was close to 80% of the CCCP level, while the emodin group was only about half that of CCCP (mean 5.29), suggesting that 1,8-diacetylemodin is a Parkin-dependent inducer of mitophagy.

[0072] 4.2 Comparison of the molecular electrostatic potentials of emodin and 1,8-diacetylemodin To explain why 1,8-diacetylemodin induces stronger mitophagy activity than emodin from the perspective of molecular polarity, the molecular electrostatic potentials of the two were calculated and compared, such as... Figure 2The simulated molecular electrostatic potential diagram shows that the electron-rich region (blue) and electron-deficient region (red) of emodin are larger than those of 1,8-diacetylemodin, while the neutral region (green) is the opposite, suggesting that 1,8-diacetylemodin has a higher molecular nonpolar surface area and stronger receptor affinity than emodin.

[0073] 4.3 Effect of 1,8-Diacetylemodin on the generation of fluorescent dots in YFP-Parkin HeLa cells like Figure 3 As shown, after intervention with four concentration gradients (10, 20, 40, 80 μM) and four administration time points (6, 8, 10, 12 h) of 1,8-diacetylemodin, the results showed that the number of fluorescent dots formed by YFP-Parkin translocation was significantly enhanced with the increase of the intervention dose of 1,8-diacetylemodin. However, at the same dosage, different intervention times had no significant effect on the formation of fluorescent dots, suggesting that the promotion of YFP-Parkin translocation by 1,8-diacetylemodin is dose-dependent rather than time-dependent.

[0074] 5. Experimental Conclusions Of the 42 candidate natural compounds, only emodin and 1,8-diacetylemodin promoted the formation of fluorescent dots in YFP-Parkin translocation (a marker of initiation of mitophagy) in YFP-Parkin HeLa cells, with 1,8-diacetylemodin showing a significantly stronger effect than emodin. Molecular electrostatic potential results suggest that 1,8-diacetylemodin has a higher nonpolar surface area than emodin, which may enhance its lipophilicity and transmembrane transport capacity. 1,8-diacetylemodin is a concentration-dependent rather than time-dependent compound that promotes YFP-Parkin translocation.

[0075] Example 2 1. Experimental Objective To investigate the effects of 1,8-diacetylemodin on PINK1 / Parkin-mediated mitophagy and mitochondria.

[0076] 2. Experimental Materials 2.1 Drugs and Reagents 1,8-Diaacetylemodin was provided by Professor Han Bo of Chengdu University of Traditional Chinese Medicine; the main reagents are shown in Table 4.

[0077] Table 4 Main Experimental Reagents Experimental reagents Item number batch number factory Mitochondrial red fluorescent probe M4067 HM0722 Suzhou Yuheng Biotechnology Co., Ltd., China H2DCFDA fluorescent probe D1002 DD0323 Suzhou Yuheng Biotechnology Co., Ltd. Ad-mCherry-GFP-LC3B C3011 070519191024 Biyuntian Biotechnology Co., Ltd. Hochest 33258 G1011 CR2012063 Wuhan Saiweier Biotechnology Carbonyl cyano-3-chlorophenylhydrazone / carbonyl cyanide chlorophenylhydrazone C2759 MKBW0481V Sigma-Aldrich Chloroquine C843545 C11407224 McLean Biotechnology 3-MA HY-19312 / CS-5207 40214 MCE Sodium pentobarbital 69020100 69020100 Merck Group, Germany Cell mitochondrial isolation kit C3601 110317171213 Biyuntian Biotechnology RIPA pyrolysis fluid G2002 HJ202804 Wuhan Saiweier Biotechnology Supersensitive ECL chemiluminescent substrate 4AW011-200 4AL242017F Beijing Sizhengbai Biotechnology Co., Ltd. Opti-MEM medium 31985-062 2177708 Gibco, USA RNAFit RNA-specific transfection reagent HB-RF-50 20200819 Hanheng Biotechnology Co., Ltd. Total RNA Isolation Kit Cat.No.RE-03113 Lot No. R201001 Chengdu Fuji Biotechnology Co., Ltd. cDNA Reverse Transcription Kit G3330-50 YL203301 Wuhan Saiweier Biotechnology Co., Ltd. Real-time PCR assay kit G3320-05 LT202201 Wuhan Saiweier Biotechnology Goat serum AR0009 15K04AD09 Boster Biologics Anti-fluorescence quenching sealing tablets G1401 HJ202501 Wuhan Saiweier Biotechnology G-418 G8160 7160033 Beijing Solarbio Technology LC3 primary antibody 14600-1-AP 00085064 Wuhan Sanying Company p62 primary antibody 5114S 6 CST Parkin primary antibody 4211S 7 CST GAPDH primary antibody Mab5456 A90545 Wuhan Saiweier Biotechnology Co., Ltd. COX IV primary antibody 11242-1-AP 00081701 Wuhan Sanying Company PINK1 primary antibody 6946T 11 CST Aβ antibody 15126S 1 CST HRP-marked goat anti-rabbit GB23303 YH201001 Wuhan Saiweier Biotechnology Co., Ltd. HRP-tagged goat anti-mouse GAM007 A90545 Linko Biotechnology Co., Ltd. Cy3-labeled goat anti-mouse GB21301 LY195118 Wuhan Saiweier Biotechnology <![CDATA[Aβ 1-40 ELISA kit E-EL-H0542c 66571QLM6X Elabscience <![CDATA[Aβ 1-42 ELISA kit E-EL-H0543c BPXHDEJDWH Elabscience 2.2 Experimental Cells YFP-Parkin HeLa cells were donated by Professor Shen Hanming of the National University of Singapore; N2a cells were donated by Professor Li Min of Hong Kong Baptist University; and HeLa cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Those skilled in the art can obtain the same experimental cells for the same experiments from international / domestic standard cell banks (such as ATCC and the Cell Bank of the Chinese Academy of Sciences).

[0078] 2.3 Experimental Apparatus Table 5 Experimental Instruments instrument model factory Electronic constant temperature water bath DZKW-4 Beijing Zhongxing Weiye Instrument Co., Ltd. centrifuge L600 Hunan Xiangyi Laboratory Instrument Development Co., Ltd. Clean bench SW-QJ-2FD Suzhou Antai Air Technology Co., Ltd. Cell incubator 3111 Thermo Fisher Scientific, Inc. Electronic balance FA1204L Shanghai Yueping Scientific Instruments Co., Ltd. Inverted microscope XD-202 Nanjing Jiangnan Yongxin Optics Co., Ltd. Inverted fluorescence microscope DMI3000 Leica, Germany Laser confocal microscope FV1200 Olympus 3. Experimental Methods 3.1 Cell culture, drug administration, and viability assay YFP-Parkin HeLa and N2a cells were digested, centrifuged, and resuspended, and then seeded at 5 × 10³ cells per well in 96-well plates. After culturing for 12 hours, the cells were treated with 5, 10, 20, 40, and 80 μM DAE for the corresponding time periods. 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated at 37°C for 4 hours. Subsequently, formazan crystals were dissolved in 100 μL DMSO, and the absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the solvent control group as a baseline. The results were the average of three replicates.

[0079] 3.2 Small interfering RNA transfection experiment Using RNA Fit transfection reagent (Hanheng Biotechnology, HB-RF-50), and following the manufacturer's instructions, scrambled siRNA double strands or PINK1-specific siRNA double strands were transfected into YFP-Parkin HeLa cells, N2a cells, and N2a-SwedAPP cells. The scrambled siRNA double strands served as a negative control, as their sequence was not targeted. The PINK1 siRNA sequence used in YFP-Parkin HeLa cells was 5′-CGCUGUUCCUCGUUAUGAATT-3′; the PINK1 siRNA sequence used in N2a cells was 5′-GCCCUGAAGAACCUGAAAUTT-3′.

[0080] 3.3 Confocal Microscopy Imaging and Image Analysis To analyze whether DAE can activate mitophagy, the formation of YFP-Parkin punctate structures and their co-localization with mitochondria were assessed. YFP-Parkin HeLa cells were seeded in 96-well plates (1 × 10⁶ cells per well). 4Cells were incubated overnight in either a single cell or a confocal culture dish. After 6 hours of DAE treatment, appropriate staining was performed: Cells in 96-well plates had their nuclei labeled with Hoechst 33258 and were imaged using an ImageXpress Micro confocal system with a 20x objective lens. The granularity of the YFP-Parkin dotted structures in each cell was calculated. Cells in confocal culture dishes were stained with MitoScene™ Red CMXRos dye at 37°C for 30 minutes to label mitochondria. Images were acquired using an Olympus FV1200 confocal microscope with a 60x oil immersion lens, and the colocalization and quantitative analysis of YFP-Parkin and mitochondrial fluorescence signals were performed using ImageJ software.

[0081] 3.4 Mitochondrial isolation and Western blot analysis To perform protein detection, mitochondria were isolated from treated YFP-Parkin HeLa and N2a cells: Cells were suspended in mitochondrial extraction buffer containing 1% PMSF, incubated on ice for 30 minutes, homogenized, and centrifuged sequentially at 600×g (10 min) and 12,000×g (10 min) at 4°C. The precipitate was collected as the mitochondrial fraction, and proteins were extracted using standard methods. For whole-cell protein extraction, cells were lysed using RIPA lysis buffer containing 1% PMSF, centrifuged, and the supernatant was collected. Protein concentration was determined by the BCA method. Equal volumes of protein samples were separated by SDS-PAGE and transferred to a membrane, then blocked with 5% BSA at room temperature for 90 minutes, and subsequently incubated overnight at 4°C with the corresponding primary antibody (GAPDH, 1:5000; LC3, COX II, COX IV, p62, Parkin, PINK1, BACE1, PS1, etc., were all 1:1000). After washing with TBST, the sample was incubated with secondary antibody (1:5000) at room temperature for 60 minutes, washed again, and developed with high-sensitivity ECL reagent. The relative expression level of the target band was analyzed using Quantity One software.

[0082] 4. Experimental Results 4.1 Effects of DAE on the viability of YFP-Parkin HeLa cells and N2a cells The results are shown in Table 6 and Figure 4 As shown, treatment of YFP-ParkinHeLa cells with DAE (5μM, 10μM, 20μM, 40μM, 80μM) for 6h and N2a cells for 24h had no significant effect on cell viability.

[0083] Table 6: Effects of DAE on the viability of YFP-Parkin HeLa and N2a cells

[0084] Note: Compared with the blank control group, * p <0.05, ** p <0.01, *** p <0.001.

[0085] 4.2 DAE promotes the translocation and expression of Parkin protein into mitochondria and upregulates mitophagy-related proteins. The experimental results related to the effect of DAE on Parkin-dependent mitophagy in YFP-Parkin HeLa cells and N2a cells are shown in the figure. Figure 5 As shown. Among them, Figure 5 In the image, A represents the fluorescence image of YFP-Parkin protein co-localization with mitochondria; Figure 5 In this context, B represents the expression of Parkin protein in the mitochondria of N2a cells; Figure 5 In the figure, C represents the expression of mitochondrial LC3 II / 1 and p62 proteins in YFP-Parkin HeLa cells. Figure 5 In the figure, D represents the expression of LC3 II / 1 and p62 proteins in the mitochondria of N2a cells. Compared with the Ctrl group, * p <0.05, ** p <0.01.

[0086] Figure 5 A in the figure shows that DAE (80 μM) treatment for 6 h can induce the formation of fluorescent spots co-localized with the mitochondrial probe (MitoScene™ Red CMXRos) in YFP-Parkin HeLa cells, indicating that it can promote the translocation of Parkin protein to mitochondria.

[0087] further Figure 5 Figure B shows that mitochondrial protein extraction and analysis in N2a cells revealed that DAE (20-80 μM) treatment for 24 h dose-dependently increased mitochondrial Parkin protein expression. These results confirm that DAE promotes Parkin localization and expression in mitochondria.

[0088] Given that Parkin activates ubiquitinated outer membrane proteins on the mitochondrial surface, thereby recruiting adaptor proteins such as p62 and binding LC3 to initiate mitophagy, this embodiment further examined the expression of autophagy marker proteins in mitochondria. Figure 5The results (C and D) showed that in YFP-Parkin HeLa and N2a cells, DAE dose-dependently increased the protein levels of p62 and LC3-II in mitochondria, suggesting that DAE can promote the enrichment of key autophagy proteins in mitochondria. These findings suggest that 1,8-diacetylemodin promotes Parkin-dependent mitophagy in YFP-Parkin HeLa and N2a cells.

[0089] 4.3 DAE maintains mitochondrial homeostasis by clearing damaged mitochondria. To verify whether DAE reduces the content of damaged mitochondria through mitophagy, the expression of mitochondrial DNA and marker proteins was detected. The final results are as follows: Figure 6 As shown.

[0090] in, Figure 6 In this context, A represents a dose-dependent decrease in mtDNA expression in YFP-Parkin HeLa cells; Figure 6 B in the equation represents the time-dependent decrease in mtDNA expression in N2a cells. Figure 6 C in the equation represents a dose-dependent decrease in mtDNA expression in N2a cells; Figure 6 In the D, the expression of COX IV and COX II proteins in YFP-Parkin HeLa cells was reduced in a dose-dependent manner. Figure 6 E in the figure represents a time-dependent decrease in the expression of COX IV and COX II proteins in N2a cells; Figure 6 In FG, the expression of COX IV and COX II proteins in N2a cells was reduced in a dose-dependent manner. Figure 6 The H in the text represents the dose-dependent decrease in mitochondrial fragmentation expression in YFP-Parkin HeLa cells; Figure 6 The I in the figure represents a time-dependent decrease in mitochondrial fragmentation expression in N2a cells; Figure 6 J in the figure represents a dose-dependent decrease in mitochondrial fragmentation expression in N2a cells. Compared to the Ctrl group, * p <0.05, ** p <0.01.

[0091] Figure 6 The AC results showed that DAE intervention dose-dependently reduced mtDNA levels in YFP-Parkin HeLa cells (6 h) and N2a cells (24 h), with a time-dependent decrease in N2a cells. Similarly, Figure 6The DG study showed that DAE could induce the degradation of mitochondrial proteins COX IV and COX II in YFP-Parkin HeLa cells in a dose-dependent manner, and exhibited dose- and time-dependent degradation in N2a cells.

[0092] Further morphological analysis revealed the following experimental results: Figure 6 As shown in HJ, DAE reduced mitochondrial fragmentation in YFP-Parkin HeLa cells in a dose-dependent manner, and reduced the number and degree of mitochondrial fragmentation in N2a cells in a dose- and time-dependent manner.

[0093] The above results indicate that DAE can effectively clear mitochondria and maintain mitochondrial network homeostasis.

[0094] 4.4 DAE activates mitophagy and promotes autophagic flux through the PINK1 / Parkin pathway The experimental results related to DAE's activation of mitophagy and promotion of autophagic flux via the PINK1 / Parkin pathway are shown in the figure below. Figure 7 As shown. Among them, Figure 7 In the figure, A represents the dose-dependent increase in PINK1 protein expression in YFP-Parkin HeLa cells by DAE. Figure 7 In the figure, B represents the time-dependent effect of DAE on PINK1 protein expression in N2a cells. Figure 7 C in the figure represents the dose-dependent increase in PINK1 protein expression in N2a cells by DAE. Figure 7 In the diagram, D represents the colocalization fluorescence of Parkin protein with mitochondria in YFP-Parkin HeLa cells with knocked-down PINK1. Figure 7 In the figure, E represents the expression of Parkin protein in mitochondria in N2a cells with knocked-down PINK1. Figure 7 F in the figure indicates that knocking down PINK1 in YFP-Parkin HeLa cells prevented the degradation of COX IV protein. Figure 7 G in the figure indicates that knocking down PINK1 in N2a cells prevented the degradation of COX IV protein. Figure 7 In the figure, H represents the expression of LC3 II / 1 and p62 proteins in YFP-ParkinHeLa cells after 6 hours of DAE intervention, as a function of the drug dosage. Figure 7 In the figure, I represents the expression of LC3 II / 1 and p62 proteins in N2a cells after treatment with DAE (80 μM) over time. Figure 7 J in the figure represents the expression of LC3 II / 1 and p62 proteins in N2a cells after 24 hours of DAE intervention, as a function of the drug dosage. Figure 7K represents the effect of DAE on LC3 II / I protein in YFP-Parkin HeLa cells after blocking autophagic flux. Figure 7 L in the figure represents the effect of DAE on LC3 II / I protein in N2a cells after blocking autophagic flux. Figure 7 In this context, M represents the effect of Ad-mCherry-GFP-LC3B adenovirus on DAE-induced autophagic flux. Compared with the Ctrl / 0h / siNC CCCP group, * p <0.05, ** p <0.01; compared with the DAE group / siPINK1 DAE group, # p <0.05, ## p <0.01. Compared with the siNC CCCP group, p <0.05, ΔΔ p <0.01.

[0095] Specifically, Figure 7 The results of the AC experiment showed that DAE could dose-dependently increase the expression of PINK1 protein in YFP-ParkinHeLa cells. In N2a cells, DAE (80 μM) treatment for 6 h significantly increased PINK1 protein levels, while its expression gradually decreased when the treatment time was extended to 18-24 h, suggesting that mitochondria may be gradually cleared with the extension of intervention time; and the 6 h intervention showed a dose-dependent effect.

[0096] To clarify the dependence of this pathway, PINK1 was knocked down using siRNA. In YFP-Parkin HeLa cells, PINK1 knockdown inhibited DAE-induced YFP-Parkin protein translocation to mitochondria and the positive control CCCP. The results are as follows: Figure 7 As shown in DE, the upregulation of mitochondrial Parkin protein was also blocked in N2a cells.

[0097] Furthermore, PINK1 knockdown also blocked the degradation of the mitochondrial marker protein COX IV induced by DAE and CCCP, such as Figure 7 As shown in FG. These results collectively indicate that DAE-induced mitophagy depends on the PINK1 / Parkin pathway.

[0098] Further examination of key autophagy proteins yielded the following experimental results: Figure 7 As shown in HJ, DAE was found to promote LC3 in a dose-dependent manner in both cell types. I to LC3 II transformation and reduction of p62 protein levels; also time-dependent in N2a cells.

[0099] As the experimental results Figure 7 As shown in KL, using early autophagy inhibitor 3 MA can inhibit DAE-induced LC3 II transformation; while treatment alone with the late autophagy inhibitor chloroquine (CQ) significantly increased LC3. II. Accumulation, and LC3 after being used in conjunction with DAE The further increase in Ⅱ suggests that DAE can both promote autophagosome generation and enhance autophagic flux.

[0100] Using Ad mCherry GFP Autophagic flux was directly observed using the LC3B adenovirus reporter system. Results showed that control group cells exhibited diffuse yellow fluorescence; orange-red spots appeared after 6 h of DAE treatment, indicating successful fusion of autophagosomes and lysosomes; 3 The combination of MA and DAE can inhibit spot formation; CQ treatment alone results in the accumulation of yellow spots (autophagosomes have not fused with lysosomes); and the combination of CQ and DAE further increases the number of yellow spots. Figure 7 M in (the middle part).

[0101] The above results confirm that DAE relies on the PINK1 / Parkin pathway to activate mitophagy and promote autophagic flux.

[0102] 5. Experimental Results 1,8-Diacetylemodin promotes autophagic flux to maintain cellular mitochondrial homeostasis through PINK1 / Parkin pathway-mediated mitophagy.

[0103] Example 3 1. Experimental Objective To investigate the effect of 1,8-diacetylemodin on PINK1 / Parkin-mediated mitophagy in promoting Aβ clearance and improving cognitive impairment in APP / PS1 mice.

[0104] 2. Experimental Materials 2.1 Drugs and Reagents The drugs and reagents are sourced from the same sources as in Example 2.

[0105] 2.2 Cells and Animals N2a cells and N2a-Swed APP cells were donated by Professor Li Min of Hong Kong Baptist University; HeLa cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Six-month-old APP / PS1 mice were purchased from Beijing Co., Ltd. (Certificate No.: 110322251103401058), and the same-month-old C57BL / 6J mice were purchased from Beijing Co., Ltd. (Certificate No.: 110322251103406024).

[0106] 2.3 Experimental Apparatus The main experimental instruments were sourced from the same source as in Example 2.

[0107] 3. Experimental Methods 3.1 Cell Culture and Viability Assay In the experiment, N2a-Swed APP cells were cultured in a 1:1 mixture of high-glucose DMEM and Opti-MEM medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin, and 200 μg / mL G418); other cells (including YFP-Parkin HeLa and N2a cells) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. All cells were incubated at 37°C in a 5% CO2 incubator. The effect of compound DAE on cell viability was determined using the MTT assay: cells were seeded at 5 × 10³ cells / well in 96-well plates and cultured for 12 hours. Then, cells were treated with different concentrations of DAE (5–80 μM) for corresponding time periods. 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated at 37°C for 4 hours. Subsequently, formazan crystals were dissolved in 100 μL of DMSO, and the absorbance was measured at 450 nm. Cell viability was calculated using the solvent control group as a baseline. All experimental data were averaged from three replicates.

[0108] 3.2 Cellular Immunofluorescence The Aβ content in DAE-treated N2a-Swed APP cells was detected using confocal microscopy with Aβ antibody. N2a-Swed APP cells were seeded on coverslips in six-well plates and treated with a specified concentration of DAE for 24 hours. The mitochondrial outer membrane marker TOM20 is commonly used to reflect intracellular mitochondrial content; YFP-Parkin HeLa cells and N2a cells were treated with the corresponding drug for detection. The culture medium was aspirated, cells were washed with PBS, and fixed with 4% paraformaldehyde at 4°C for 15 minutes. After washing three times with PBS, cells were blocked with 5% normal goat serum (containing 0.1% Triton X-100) at room temperature for 30 minutes. Cells were washed three more times with PBS, incubated overnight with primary antibodies (Aβ, TOM20) at 4°C, and then incubated with the corresponding secondary antibodies at room temperature for 1 hour. After washing three times with PBS, cells were stained with Hoechst 33258 at room temperature for 10 minutes. Images were acquired using a 20x objective lens under a confocal microscope, and the average fluorescence intensity was quantified using ImageJ software.

[0109] 3.3 Animal feeding, drug administration, and behavioral testing This protocol was approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (License No.: IBD2025001). Six-month-old APP / PS1 transgenic mice and age-matched C57BL / 6J wild-type mice were selected. After 3 days of acclimatization, the APP / PS1 mice were randomly divided into a model group, a low-dose (10 mg / kg) DAE group, and a high-dose (20 mg / kg) DAE group. Wild-type mice served as the control group. The DAE group was administered the corresponding dose of DAE suspension (0.5% CMC-Na solvent) by gavage for 14 consecutive days, while the control and model groups received the same volume of solvent. After the administration, the cognitive function of the mice was assessed sequentially using the Y-maze and passive avoidance tests: In the Y-maze test, the rate of spontaneous alternation during free exploration within 5 minutes was recorded to reflect working memory; the passive avoidance test was conducted 24 hours after training, measuring the escape latency and number of times the mice entered the dark room to assess their memory retention ability. The day after the behavioral test, the mice were anesthetized and their brains were harvested. The right hemisphere was cryopreserved for protein and ELISA analysis, while the left hemisphere was fixed in 4% paraformaldehyde for subsequent immunohistochemical studies.

[0110] 3.4 ELISA kit for detecting Aβ content According to the kit instructions, the levels of Aβ1-40 and Aβ1-42 in DAE-treated N2a-Swed APP cells or mouse brain tissue were determined by ELISA.

[0111] 3.5 H&E and Immunohistochemical Staining H&E staining was performed according to standard procedures; IHC staining was performed as follows: 4 µm brain tissue paraffin sections were baked, rehydrated, had antigen retrieval and peroxidase blocked, and then incubated overnight at 4°C with mouse anti-Aβ primary antibody (1:500, #15126, CST, USA). The sections were then incubated with HRP-labeled secondary antibody (1:300) at room temperature for 2 hours. Staining was performed using a DAB kit (#AR1022, Boster Biological, Wuhan, China), and the cell nuclei were counterstained with hematoxylin. Finally, the IHC sections were analyzed using ImageJ software.

[0112] 3.6 Statistical Analysis Data are expressed as mean ± standard error (x The mean ± standard deviation (SEM) is used. One-way ANOVA was used for samples that conformed to a normal distribution; otherwise, the Kruskal-Wallis test was used. A p-value < 0.05 was considered statistically significant.

[0113] 4. Experimental Results 4.1 DAE promotes Aβ clearance in N2a-Swed APP cells via autophagy. To clarify whether DAE clearance of Aβ depends on the autophagy pathway, chloroquine, an autophagy flux inhibitor, was used for intervention. Experimental results Figure 8 As shown in the figure, the experimental results related to DAE promoting the clearance of Aβ in N2a-Swed APP cells through autophagy are presented.

[0114] The results of immunofluorescence and ELISA assays were consistent, indicating that: like Figure 8 The immunofluorescence image of Aβ in N2a-Swed APP cells shown in Figure A shows that, compared with N2a cells, the content of Aβ (including Aβ1-40 and Aβ1-42) in N2a-Swed APP cells is significantly increased. Figure 8 The graph in Figure B shows the statistical changes in Aβ immunofluorescence intensity, indicating that DAE treatment for 24 h can dose-dependently reduce the level of Aβ in this cell model. Figure 8 In this context, C represents Aβ in N2a-Swed APP cells after DAE treatment. 1-40 The content of. Figure 8 In this context, D represents Aβ in N2a-Swed APP cells after DAE treatment. 1-42 The content of [the substance / method / etc.]. Compared to group N2a, [the figure shows / description / etc.]. * p <0.05, ** p <0.01; compared with the N2a APP group, # p <0.05, ## p<0.01. Treatment with CQ alone significantly increased the accumulation of intracellular Aβ, while treatment with CQ in combination with DAE completely blocked the scavenging effect of DAE on Aβ.

[0115] The above results confirm that the clearance of Aβ by DAE depends on a complete and unobstructed autophagic flow.

[0116] 4.2 Knockdown of PINK1 blocks DAE clearance of Aβ in N2a-Swed APP cells The results of immunofluorescence and ELISA assays consistently indicate that, Figure 9 As shown, where, Figure 9 In the image, A represents the Aβ immunofluorescence pattern. Figure 9 B in the graph represents the statistical graph of Aβ immunofluorescence intensity. Figure 9 In this context, C represents Aβ in N2a-Swed APP cells after DAE treatment. 1-40 The content of. Figure 9 In this context, D represents Aβ in N2a-Swed APP cells after DAE treatment. 1-42 The content of [the substance / method]. Compared with group N2a, * p <0.05, ** p <0.01; compared with the N2a APP group, # p <0.05, ## p <0.01.

[0117] Compared with N2a cells, N2a-Swed APP cells showed significantly increased levels of Aβ (including Aβ1-40 and Aβ1-42). DAE (80 μM) intervention for 24 h effectively reduced Aβ levels in this cell model. However, after knocking down PINK1 using siPINK1, Aβ levels in N2a-Swed APP cells further accumulated, and the Aβ-clearing effect of DAE was completely blocked. These results confirm that DAE-promoted Aβ clearance depends on the expression and function of the PINK1 protein.

[0118] 4.3 DAE reduces Aβ deposition in the brain of APP / PS1 mice by activating PINK1-dependent mitophagy Experimental results are as follows Figure 10 The figure shown illustrates experimental results regarding the reduction of Aβ deposition in the brains of APP / PS1 mice by DAE through activation of PINK1-dependent mitophagy. Figure 10 A in the figure represents the PINK1 band plot and quantitative analysis (n=6). Figure 10 In the figure, B represents the intracranial Aβ level of APP / PS1 after DAE administration. 1-40 The content of (n=6). Figure 10 C in the figure represents the intracranial Aβ level of APP / PS1 after DAE administration. 1-42 The content of (n=6). Figure 10 D in the figure represents the representative IHC of Aβ in the APP / PS1 intracerebral cortex and hippocampus after DAE administration (n=5). Figure 10 E represents the quantitative area of ​​cortical Aβ. F represents the quantitative area of ​​hippocampal Aβ. Compared with the C57BL / 6J group, ** p <0.01, compared with the APP / PS1 group, # p <0.05, ## p <0.01.

[0119] Animal experiments showed that compared with wild-type C57BL / 6J mice, APP / PS1 mice had significantly lower PINK1 protein expression levels in their brains, suggesting PINK1-dependent mitophagy dysfunction. After DAE intervention, PINK1 protein expression in the brains of APP / PS1 mice showed a dose-dependent recovery. Figure 10 The A in the figure indicates that DAE can improve the mitochondrial autophagy disorder in this model.

[0120] Further analysis of Aβ deposition revealed that the area and content of Aβ-positive cells in the hippocampus and cortex of APP / PS1 mice were significantly higher than those in the wild-type control group. DAE treatment resulted in a dose-dependent reduction in Aβ deposition in the mouse brain, a result confirmed by ELISA. Figure 10 BC in the middle) and immunohistochemistry ( Figure 10 This has been consistently confirmed in DF.

[0121] In summary, DAE can effectively promote the clearance of Aβ in the brain of APP / PS1 mice by activating the PINK1-dependent mitophagy pathway.

[0122] 4.4 DAE improves neuronal damage and restores cognitive function in APP / PS1 mice The experimental results related to DAE's improvement of neuronal damage and restoration of cognitive function in APP / PS1 mice are shown in the figure below. Figure 11 As shown.

[0123] in, Figure 11 A in the image represents HE staining of the cerebral cortex and hippocampus of a representative APP / PS1 mouse (red arrows indicate nuclear lysis, blue arrows indicate increased intercellular spaces, n=5). Figure 11 In the experiment, B represents the escape latency period (n=6) of the darkness avoidance experiment. Figure 11 In this context, C represents the number of errors in the dark avoidance experiment (n=6). Figure 11In this context, D represents the total number of arm advances in the Y-maze test (n=6). Figure 11 In this context, E represents the spontaneous alternation rate (n=6) of the Y-maze test. Compared to the C57BL / 6J group, * p <0.05, compared with the APP / PS1 group, # p <0.05.

[0124] Specifically, histopathological analysis (HE staining) showed that, compared with wild-type C57BL / 6J mice, APP / PS1 mice exhibited disordered and reduced neuronal arrangement in the CA1 and CA3 regions of the hippocampus, as well as damage characteristics such as nuclear condensation, nuclear dissolution, and increased intercellular spaces. Figure 11 As shown in Figure A, the aforementioned pathological changes were significantly improved after DAE treatment, and the cell arrangement and morphology tended to be normal, indicating that DAE can effectively reduce neuronal damage in APP / PS1 mice.

[0125] Figure 11 The BC data showed that APP / PS1 mice exhibited significant memory impairment in cognitive behavioral tests: an increased number of errors and a shortened escape latency in the passive avoidance test.

[0126] Figure 11 The DE data shows that the spontaneous alternation rate decreases in the Y maze (the total number of arm advances remains unchanged). Figure 11 The BE study showed that DAE intervention dose-dependently reduced the number of errors, prolonged the escape latency, and significantly increased the spontaneous alternation rate.

[0127] The above experimental results demonstrate that DAE can effectively improve fear memory and spatial working memory in APP / PS1 mice.

[0128] 5. Experimental Conclusions 1,8-Diacetylemodin promoted Aβ clearance through PINK1-dependent mitophagy, alleviated neuropathological damage in APP / PS1 mice, and thus improved cognitive dysfunction.

[0129] This study comprehensively utilized YFP-Parkin HeLa cells, N2a cells, N2a-Swed APP cells, and APP / PS1 mouse models to systematically explore the mechanism and effects of compound DAE. The main conclusions are as follows: DAE can promote mitophagy by inducing PINK1 protein accumulation and activating the PINK1 / Parkin pathway; this effect disappears after PINK1 is disrupted. Further research revealed that DAE can effectively clear Aβ in the brains of N2a-Swed APP cells and APP / PS1 mice via this pathway, and this clearance effect is inhibited by blocking autophagic flux or knocking down PINK1. These findings suggest that DAE is a PINK1-dependent mitophagy inducer that can promote Aβ clearance and maintain mitochondrial homeostasis through the PINK1 / Parkin-dependent mitophagy pathway, thereby alleviating cognitive impairment in APP / PS1 mice and providing experimental evidence for its use in intervention studies of Alzheimer's disease.

[0130] Example 4 The use of 1,8-diacetylemodin in the preparation of drugs for the prevention or treatment of neurodegenerative diseases.

[0131] In some specific implementations, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS). Preferably, the neurodegenerative disease is Alzheimer's disease.

[0132] A second aspect of this embodiment provides a pharmaceutical composition comprising a therapeutically effective amount of 1,8-diacetylemodin and a pharmaceutically acceptable carrier.

[0133] In some specific embodiments, the 1,8-diacetylemodin is present in the pharmaceutical composition at a concentration of 10 mg to 100 mg per unit dosage form. The pharmaceutical compositions of the present invention can be prepared in unit dosage forms, such as tablets or capsules, wherein each unit dosage form contains 10 mg, 25 mg, 50 mg, or 100 mg of 1,8-diacetylemodin.

[0134] In some specific implementation schemes, the pharmaceutically acceptable carrier is selected from one or more of fillers, binders, disintegrants, lubricants, and solubilizers.

[0135] In some specific implementations, the dosage form of the pharmaceutical composition is capsules, tablets, powders, or granules.

[0136] Specifically, fillers, also known as diluents, are the largest component of excipients in tablets or capsules. Their main function is to increase the weight and volume of the formulation, ensuring that each dose contains an accurate and uniform amount of active ingredient, thereby guaranteeing the precision of the dosage. Commonly used fillers include lactose, microcrystalline cellulose, pregelatinized starch, and inorganic materials such as calcium hydrogen phosphate. They have good chemical inertness and do not interact with the active pharmaceutical ingredient.

[0137] The role of binders is to provide adhesive force, binding powdered active pharmaceutical ingredients together with dry materials such as fillers. This imparts the mechanical strength required for direct compression of wet-granulated granules or powders, preventing breakage or wear during subsequent processing, packaging, and transportation. Common binders include povidone, hydroxypropyl methylcellulose, starch paste, and hydroxypropyl cellulose (HPC).

[0138] The core function of disintegrants is to promote the rapid rupture and dispersion of tablet or capsule contents into fine particles in gastrointestinal fluids, thereby greatly increasing the contact surface area between the drug and body fluids and accelerating the dissolution and absorption of active ingredients. Their mechanism of action is primarily strong swelling upon water absorption. Highly effective disintegrants include crospovidone, sodium carboxymethyl starch, sodium crospovidone carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose.

[0139] Lubricants are primarily used to reduce friction between granules or powder and the die punch, ensuring tablets are smoothly ejected from the compression mold and preventing material from adhering to the die surface. Additionally, they improve granule flowability, resulting in a more uniform and accurate filling process. The most widely used lubricant is magnesium stearate. Other options include micronized talc, hydrogenated vegetable oil, and sodium fumarate stearate.

[0140] Cosolvents (or solubilizers) are excipients that significantly improve the apparent solubility and dissolution rate of poorly soluble drugs in aqueous media by forming micelles, inclusion complexes, or altering solvent polarity. This is crucial for drugs with potentially poor water solubility, such as 1,8-diacetylemodin, as it aids in their absorption in vivo. Commonly used cosolvents include polyethylene glycol series, polyvinylpyrrolidone, and cyclodextrins and their derivatives (such as hydroxypropyl-β-cyclodextrin, which can solubilize through molecular inclusion). Surfactants such as polysorbate 80 (Tween 80) are also frequently used as cosolvents.

[0141] Example 5 Preparation of 1,8-diacetylemodin tablets Accurately weigh the following ingredients: 5g of 1,8-diacetylemodin, 7g of microcrystalline cellulose, 1g of hydroxypropyl methylcellulose, 1.5g of sodium carboxymethyl starch, and 0.5g of magnesium stearate.

[0142] First, thoroughly dry-mix 1,8-diacetylemodin, microcrystalline cellulose, and sodium carboxymethyl starch in a mixer to ensure uniform material distribution. Then, dissolve hydroxypropyl methylcellulose in an appropriate amount of purified water to prepare a binder solution, which is gradually added to the dry-mixed materials while stirring until uniform wet granules are formed. The wet granules are granulated through a sieve and dried in a fluidized bed dryer until the moisture content is below 2% to obtain dried granules. The granules are then sieved to remove lumps and ensure uniformity. Magnesium stearate, a lubricant, is added to the granulated granules, and the mixture is gently mixed in a mixer to avoid granule breakage and uneven lubricant distribution. The mixed granules are then compressed into tablets using a rotary tableting machine. The pressure and compression speed are adjusted to ensure that the weight, hardness, and disintegration time of each tablet meet pharmacopoeia standards. An ambient temperature of 20-25°C and a relative humidity below 50% are maintained to ensure stable tablet quality.

[0143] Example 6 Preparation of 1,8-diacetylemodin capsules Accurately weigh the following raw materials: 2 g of 1,8-diacetylemodin, 10 g of lactose, 6 g of corn starch, and 1.8 g of gelatin. Dissolve the gelatin in water to prepare a 10 wt% aqueous solution. Mix the 1,8-diacetylemodin, lactose, and corn starch evenly, add the prepared gelatin aqueous solution, granulate, pass through a 1 mm mesh sieve, and dry at 40°C. Then, fill the granules into hydroxypropyl methylcellulose (HPMC) empty capsules to obtain the capsule formulation.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. 1,8-Dacetylemodin in the preparation of drugs for treating Alzheimer's disease mediated by mitophagy. 2.1 Application of 8-diacetylemodin in the preparation of mitochondrial autophagy inducer drugs.

3. The application according to claim 2, characterized in that, The drug is used to improve diseases mediated by mitophagy.

4. The application according to claim 3, characterized in that, The drug is a drug used to improve cognitive impairment, or the drug is a drug for the prevention or treatment of neurodegenerative diseases.

5. The application according to claim 3, characterized in that, The neurodegenerative diseases mentioned are Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

6. A pharmaceutical composition, characterized in that, It consists of a therapeutically effective amount of 1,8-diacetylemodin and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The content of 1,8-diacetylemodin in the pharmaceutical composition is 10 mg to 100 mg per unit dosage form.

8. The pharmaceutical composition according to claim 7, characterized in that, The content of 1,8-diacetylemodin in the pharmaceutical composition is 50 mg to 100 mg per unit dosage form.

9. The pharmaceutical composition according to any one of claims 6-8, characterized in that, Pharmaceutically acceptable carriers are selected from one or more of fillers, binders, disintegrants, lubricants, and solubilizers.

10. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is one of oral, injectable, or implantable formulations.

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