Application of salidroside derivative SHPL-49 in preparation of medicine for preventing / treating Alzheimer disease

Abstract

The invention provides application of a salidroside derivative SHPL-49 in preparation of a medicine for preventing / treating Alzheimer's disease (AD), and belongs to the technical field of biological medicines. The invention provides an application of a salidroside derivative SHPL-49 in preparation of a medicine for preventing / treating AD (Alzheimer's disease). According to the invention, the curative effect of the SHPL-49 on AD is evaluated by adopting two modes of preventive administration and therapeutic administration. The result shows that the SHPL-49 can effectively improve the learning and memory ability of an AD model and improve the cognitive function, and meanwhile, the brain injury is repaired. The SHPL-49 provided by the invention not only can effectively achieve the drug effect of preventing and / or treating the Alzheimer's disease, but also has good drug safety, and provides a new choice for the development of drugs for clinically treating the Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to the application of rhodioloside derivative SHPL-49 in the preparation of drugs for the prevention / treatment of Alzheimer's disease. Background Technology

[0002] Dementia is a syndrome that can be caused by a variety of diseases that damage nerve cells and impair the brain over time, often leading to a decline in cognitive function (the ability to process thoughts) that exceeds the generally expected consequences of biological aging. Increased age, genetic predisposition, and systemic vascular disease are major risk factors for developing dementia. Alzheimer's disease (AD) is the main type of dementia. The pathological mechanisms of AD are not fully understood, but current mainstream hypotheses include: abnormal deposition of Aβ protein outside nerve cells to form senile plaques (SP); abnormal phosphorylation of Tau protein to form neurofibrillary tangles (NFTs); and defects in neurotransmitters in the brain leading to damage to cholinergic neurons.

[0003] Currently, treatment for Alzheimer's disease (AD) primarily focuses on symptom relief, with no cure yet available. Many acetylcholinesterase (AChE) inhibitors, including donepezil and galantamine, have been developed to restore normal cholinergic function in the central nervous system of AD patients. These drugs are moderately effective in relieving AD symptoms but have numerous side effects, including gastrointestinal discomfort, bradycardia, headaches, and insomnia. Other medications used to treat AD, such as the anti-glutaminergic drug memantine, attempt to balance the cholinergic pathway by weakening the function of N-methyl-D-aspartate (NMDA) receptors. Like AChE inhibitors, memantine only provides moderate relief of AD symptoms and is accompanied by side effects such as constipation and drowsiness. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of the rhodioloside derivative SHPL-49 in the preparation of drugs for the prevention / treatment of Alzheimer's disease, providing a new option for the clinical treatment of AD.

[0005] This invention provides the application of the rhodioloside derivative SHPL-49 in the preparation of drugs for the prevention / treatment of Alzheimer's disease.

[0006] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0007] Preferably, the pharmaceutically acceptable excipients include at least one of diluents, wetting agents, binders, lubricants, colorants, and coating agents.

[0008] Preferably, the drug comprises at least one of the following dosage forms: tablets, capsules, granules, pills, injections, suspensions, dispersants, syrups, transdermal preparations, enteric-coated tablets, sprays, and lozenges.

[0009] Preferably, the Alzheimer's disease includes the following lesions: decreased learning and memory abilities, cognitive impairment, and brain damage.

[0010] Preferably, the inducing factors of Alzheimer's disease include at least one of the following: scopolamine induction, Aβ oligomer induction, and transAβ and / or Tau gene formation.

[0011] This invention provides the application of the rhodioloside derivative SHPL-49 in the preparation of drugs for the prevention / treatment of Alzheimer's disease. The embodiments of this invention are based on a scopolamine (SCOP)-induced Alzheimer's disease mouse model. Depending on the timing of administration (prevention and treatment), behavioral tests such as the Morris water maze, open field, and novel object recognition were used to evaluate the effects of SHPL-49 on Alzheimer's disease in mice. Results showed that in the Morris water maze test, SHPL-49 significantly reduced the escape latency and escape distance in the mouse model, while significantly increased the time spent in the target quadrant and the number of times the mouse crossed platforms, exhibiting a more pronounced exploratory tendency, indicating that SHPL-49 can significantly improve and enhance the learning and memory abilities of mice. The open field test results showed that SHPL-49 can increase the total distance traveled in the open field and the time spent in the central area, increase the frequency of mice entering the central area, and increase the number of squares crossed, demonstrating that SHPL-49 can significantly increase the autonomous exploratory activities of mice. Novel object recognition experiments showed that SHPL-49 effectively improved the preference for novel objects in mouse models and alleviated cognitive impairment. This indicates that both preventative and therapeutic administration of SHPL-49 can effectively reverse SCOP-induced memory and cognitive impairment in mice, and its effect on improving learning and memory is comparable to that of positive control drugs. The examples section of this invention also evaluates the effects of SHPL-49 on Alzheimer's disease in mice using a mouse model induced by Aβ oligomer intraventricular injection, through behavioral experiments such as novel object recognition and the Morris water maze. The results showed that novel object recognition results indicated that SHPL-49 effectively improved the preference for novel objects in mouse models and alleviated cognitive impairment. The Morris water maze test showed that during the navigation phase, SHPL-49 significantly reduced the escape latency and escape distance in mouse models, indicating that SHPL-49 can significantly improve and enhance the learning and memory abilities of Alzheimer's disease mice. This invention also uses transgenic *Caenorhabditis elegans* as a model organism to explore the therapeutic effect of SHPL-49 on Alzheimer's disease through nematode paralysis and lifespan experiments. The results showed that treatment with different concentrations of SHPL-49 significantly shifted the paralysis time curve and lifespan curve of the nematodes to the right, indicating that SHPL-49 may play a role in delaying the onset and progression of AD. Furthermore, the experiments showed that SHPL-49 did not damage the body weight, organs, or reproductive capacity of AD nematodes. Therefore, the SHPL-49 provided by this invention not only effectively prevents and / or treats Alzheimer's disease but also has good drug safety, providing a new option for drug development in the clinical treatment of Alzheimer's disease. Attached Figure Description

[0012] Figure 1The flowchart shows the experiment on the prevention of SCOP-induced Alzheimer's disease in a mouse model using SHPL-49.

[0013] Figure 2 The flowchart shows the SHPL-49 treatment of a SCOP-induced Alzheimer's disease mouse model for 28 days.

[0014] Figure 3 The flowchart shows the 14-day treatment experiment of SHPL-49 on a mouse model of SCOP-induced Alzheimer's disease.

[0015] Figure 4 Flowchart of an experiment involving intraventricular injection of Alzheimer's disease Aβ oligomers into a mouse model;

[0016] Figure 5 A graph showing the weight change in a mouse model during the experiment;

[0017] Figure 6 To prevent the statistical results of escape latency in mouse models during experiments;

[0018] Figure 7 To prevent the statistical results of escape latency in mouse models during experiments;

[0019] Figure 8 To prevent the statistical results of the percentage of time spent in the original platform quadrant in the mouse model during the experiment;

[0020] Figure 9 To prevent the statistical results of the number of times the mouse model crossed the platform in the experiment;

[0021] Figure 10 To prevent spatial exploration trajectory mapping in mouse models during experiments;

[0022] Figure 11 To prevent discriminant index results in mouse models during experiments;

[0023] Figure 12 To prevent the mouse model from exploring object trajectory diagrams in experiments;

[0024] Figure 13 H&E staining of the brain region of a mouse model was used to prevent H&E staining in the experiment, where the scale bar = 50 μm;

[0025] Figure 14 To prevent adverse reactions in the Nissl staining results of the mouse model brain region, the scale bar was set to 50 μm.

[0026] Figure 15 A graph showing the weight change of a mouse model during a 28-day treatment experiment.

[0027] Figure 16 The organ index results of a mouse model during a 28-day treatment experiment;

[0028] Figure 17 The results of escape latency statistics for mouse models during the 28-day treatment experiment;

[0029] Figure 18 The escape latency of a mouse model during a 28-day treatment experiment was statistically analyzed.

[0030] Figure 19 This is a statistical result of the percentage of time spent in the original plateau quadrant in the mouse model during the 28-day treatment experiment;

[0031] Figure 20 This is a statistical result of the number of times the mouse model crossed the platform during the 28-day treatment experiment;

[0032] Figure 21 Spatial exploration trajectory diagram of a mouse model during a 28-day treatment experiment;

[0033] Figure 22 This is a statistical result of the total distance traveled in an open field by a mouse model during a 28-day treatment experiment;

[0034] Figure 23 This is a statistical result of the number of grid crossings in the mouse model during the 28-day treatment experiment;

[0035] Figure 24 Results of the frequency of mouse models entering the central region during the 28-day treatment experiment;

[0036] Figure 25 This is a statistical result of the time the mouse model spent in the central region during the 28-day treatment experiment;

[0037] Figure 26 A trajectory diagram of a mouse model exploring within an open field device during a 28-day treatment experiment;

[0038] Figure 27 H&E staining of the brain region of a mouse model after 28 days of treatment, with scale bar = 50 μm;

[0039] Figure 28 Nissl staining results of the brain region of the model animals during the 28-day treatment experiment, where the scale bar = 50 μm;

[0040] Figure 29 The results of weight changes in a mouse model during a 14-day treatment experiment;

[0041] Figure 30 The results of escape latency statistics for mouse models during a 14-day treatment experiment;

[0042] Figure 31 The escape latency of a mouse model during a 14-day treatment experiment was statistically analyzed.

[0043] Figure 32 This is a statistical result of the percentage of time spent in the original platform quadrant in the mouse model during the 14-day treatment experiment;

[0044] Figure 33 This is a statistical result of the number of times the mouse model crossed the platform during the 14-day treatment experiment;

[0045] Figure 34 Spatial exploration trajectory diagram of a mouse model during a 14-day treatment experiment;

[0046] Figure 35 The results of the discrimination index in the mouse model during the 14-day treatment experiment;

[0047] Figure 36 A trajectory diagram of an object exploration in a mouse model during a 14-day treatment experiment;

[0048] Figure 37 H&E staining of the brain region of a mouse model after 14 days of treatment, with scale bar = 50 μm;

[0049] Figure 38 Nissl staining results of the brain region of a mouse model during the 14-day treatment experiment, where the scale bar = 50 μm;

[0050] Figure 39 Discrimination index results for a mouse model of Aβ oligomer intraventricular injection;

[0051] Figure 40 A mouse model of object exploration trajectory after Aβ oligomer injection into the lateral ventricle;

[0052] Figure 41 Statistical results of escape latency distance in a mouse model of Aβ oligomer intraventricular injection;

[0053] Figure 42 Statistical results of escape latency distance in a mouse model of Aβ oligomer intraventricular injection;

[0054] Figure 43 The paralysis time curve of the nematode model;

[0055] Figure 44 The lifespan variation curve of the nematode model;

[0056] Figure 45 The results show the statistical results of the reproductive capacity of the nematode model. Detailed Implementation

[0057] This invention provides the application of the rhodioloside derivative SHPL-49 in the preparation of drugs for the prevention / treatment of Alzheimer's disease.

[0058] In this invention, the rhodioloside derivative SHPL-49 belongs to the glycoside class of compounds with a molecular weight of 342.39 g / mol. Its specific structural formula is shown in Formula I. In this embodiment, SHPL-49 was purchased from Shanghai Hutchison Pharmaceuticals Co., Ltd.

[0059]

[0060] In this invention, the Alzheimer's disease preferably includes the following lesions: decreased learning and memory ability, cognitive impairment, and brain damage. In embodiments of this invention, the Alzheimer's disease animal model is preferably induced by scopolamine, Aβ oligomer intraventricular injection, or Aβ and / or Tau gene conversion. In the prevention experiment, the SCOP induction dose was 3 mg / kg, and the induction time was 14 days; in Example 2, the 28-day treatment experiment, the SCOP induction dose was 2 mg / kg, and the induction time was 28 days; in Example 3, the 14-day treatment experiment, the SCOP induction dose was 2 mg / kg, and the induction time was 14 days; in the Aβ oligomer intraventricular injection experiment, the Aβ oligomer concentration was 1 mg / mL, the injection volume was 5 μL, and a single injection induced formation. In embodiments of the invention, behavioral differences in the mouse model were assessed using the Morris water maze test, open field test, and novel object recognition test, respectively, and pathological changes in brain tissue sections were assessed using brain slice staining. Experiments showed that successful SCOP induction induced learning, memory, and cognitive impairment in mice. This was primarily manifested in a significant increase in escape latency distance and time, indicating a decline in learning ability. The mice also exhibited a significant reduction in time spent in the original platform quadrant and the number of platform crossings, and their movement trajectories did not show a clear tendency to explore the target quadrant. This indicated a decline in cognitive and memory retention abilities. Furthermore, the discrimination index showed a significant downward trend during the identification of new objects, suggesting significant cognitive impairment. Pathological staining results showed increased interneuronal gaps, uneven staining, and a reduced number of neurons in the hippocampus of the mouse model, with visible necrotic neurons and pyknosis of cell nuclei, indicating brain tissue damage. Intraventricular injection of Aβ oligomers induced learning and cognitive impairment in Alzheimer's disease mice. This was mainly manifested in a significant downward trend in the discrimination index during the identification of new objects, indicating cognitive deficits. In the Morris water maze navigation experiment, the escape latency distance and time were significantly prolonged, indicating a decline in learning ability.

[0061] In this invention, to evaluate the efficacy of SHPL-49, a SCOP-induced Alzheimer's disease mouse model was used. Two administration methods were employed: pre-induction prophylaxis and post-induction administration. Behavioral tests, including the Morris water maze, open field, and novel object recognition, were used to evaluate the effect of SHPL-49 on Alzheimer's disease in mice. A new object recognition and Morris water maze tests were also used to evaluate the effect of SHPL-49 on Alzheimer's disease in mice using an Aβ oligomer intraventricular injection model. Simultaneously, using transgenic *Caenorhabditis elegans* (CL4176 and BR5270) as model organisms, nematode paralysis and lifespan experiments were conducted to explore the therapeutic effect of SHPL-49 against Alzheimer's disease.

[0062] In one embodiment of the present invention, a preventative dosing experiment was conducted. SHPL-49 was administered at a dose of 180 mg / kg for 42 consecutive days. Starting on day 29 of dosing, SCOP was injected to induce Alzheimer's disease in mice. Donepezil (DNP) at a dose of 3 mg / kg served as a positive control group. The experiment showed that after administration of SHPL-49 and DNP, the escape latency distance and escape latency time in the mouse model were significantly shortened, indicating that SHPL-49 and DNP significantly improved the learning ability of mice. SHPL-49 administration prolonged the time the mouse model spent in the target quadrant and increased the number of times the mice crossed the platform, exhibiting a more pronounced exploratory tendency, indicating that both SHPL-49 and DNP significantly improved the memory retention ability of mice, with no significant difference between the two. In the assessment of cognitive and memory retention abilities in mice, administration of both SHPL-49 and DNP significantly increased the mouse model's preference for new objects, indicating that these two drugs can improve memory impairment in AD mice, with no significant difference between SHPL-49 and DNP. In pathological examination of the slides, the SHPL-49-treated group showed more uniform staining, more regular neuronal cell morphology, and increased cell number, indicating that SHPL-49 can alleviate brain damage in mice. Furthermore, preventative experiments showed that SHPL-49 had no significant effect on the body weight of the mouse model.

[0063] In another embodiment of the invention, SCOP-induced mouse model experiments were conducted after 14 days and 28 days of drug treatment, with dosages of 130 mg / kg and 180 mg / kg body weight, respectively. The experimental evaluation methods were the same as those used in the preventative drug administration experiment and will not be repeated here. Morris water maze test results showed that the escape latency distance and escape latency time of SHPL-49-treated mice were significantly shortened. SHPL-49 administration resulted in a more pronounced exploratory tendency in the mouse model, and the time spent searching for the target quadrant was significantly reduced. This indicates that a SHPL-49 dosage of 180 mg / kg can significantly improve the memory ability of Alzheimer's disease mice. Simultaneously, both dosages of SHPL-49 significantly increased the total distance traveled by mice in the open field and the time spent in the central area of ​​the open field. The SHPL-49 dosage of 180 mg / kg increased the frequency of mice entering the center of the open field and increased the number of squares crossed, demonstrating that SHPL-49 can significantly increase the autonomous exploratory activity of mice. Brain tissue section experiments showed that the SHPL-49 group had more uniform staining, more regular neuronal cell morphology, and an increased number of neurons. This indicates that SHPL-49 can effectively reduce brain damage in mice.

[0064] In another embodiment of the invention, a mouse model induced by intraventricular injection of Aβ oligomers was established for 16 days. SHPL-49 was administered at a dose of 180 mg / kg body weight, with DNP at a dose of 10 mg / kg serving as a positive control. The experimental evaluation methods were the same as in the prophylactic administration experiment and will not be repeated here. The experiment showed that, in the assessment of cognitive and memory retention abilities in mice, SHPL-49 administration significantly improved the mouse model's preference for novel objects, indicating that SHPL-49 can improve memory impairment in AD mice. SHPL-49 administration significantly shortened the escape latency distance and escape latency time in the mouse model, demonstrating that SHPL-49 has a significant effect on improving the learning ability of mice.

[0065] In another embodiment of the present invention, the effects of SHPL-49 on the paralysis phenotype were investigated using the transgenic Aβ nematode CL4176 as the experimental subject, and the effects of SHPL-49 on the lifespan and reproductive capacity of the transgenic Tau nematode BR5270 were investigated using the experimental subject. The results showed that SHPL-49 administration could delay the paralysis time of the nematodes and prolong their lifespan, but had no effect on their reproductive capacity, indicating that SHPL-49 administration has no toxic effect on the nematodes.

[0066] In this invention, various dosage forms of SHPL-49 and all formulations containing SHPL-49 are used. The drug is administered orally, intravenously, topically, over-the-counter, by inhalation or spray, sublingually, transdermally, rectally, or otherwise. The drug preferably also includes pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients preferably include at least one of diluents, wetting agents, binders, lubricants, colorants, and coating agents. The drug includes at least one of the following dosage forms: tablets, capsules, granules, pills, injections, suspensions, dispersants, syrups, transdermal preparations, enteric-coated tablets, sprays, and lozenges. This invention does not impose any particular limitation on the preparation method of the drug; any drug preparation method well known in the art can be used.

[0067] The following examples illustrate the application of the rhodioloside derivative SHPL-49 provided by this invention in the preparation of drugs for the prevention / treatment of Alzheimer's disease. However, these examples should not be construed as limiting the scope of protection of this invention.

[0068] Example 1

[0069] The preventive effect of SHPL-49 on Alzheimer's disease:

[0070] I. Methods

[0071] Forty male SPF-grade C57BL / 6J mice (12 weeks) were randomly divided into four groups: a control group (Sham), a model group (Model), an SHPL-49 group (180 mg / kg), and a positive control group (donepezil 3 mg / kg), with 10 mice in each group. Mice were acclimatized for 7 days to adapt to the experimental environment and diet. The control and model groups were administered saline by gavage, the SHPL-49 group was administered 180 mg / kg SHPL-49 by gavage, and the positive control group was administered 3 mg / kg DNP by gavage, for 42 consecutive days. From day 29 of administration, the Sham group received 10 mL / kg of saline intraperitoneally daily. The other groups were induced with Alzheimer's disease by intraperitoneal injection of scopolamine (SCOP 3 mg / kg) 30 minutes after administration, with SCOP induction continuing for 14 days until day 42 of administration. The effects of the drug on a SCOP-induced Alzheimer's disease mouse model were evaluated by Morris water maze test on days 29-34 of drug administration and by new object recognition test on days 41 and 42 of drug administration.

[0072] The Morris water maze test is primarily used to assess the learning and memory abilities of mice. Significant differences in data between groups indicate successful model establishment. The method is briefly described below: The experimental setup consists of a circular gray pool with a diameter of 120 cm, divided into four quadrants. An entry point is marked on the wall of each quadrant. A circular platform with a diameter of 5 cm is placed in the center of the target quadrant, submerged 1–2 cm underwater, and its position remains unchanged throughout the experiment. The pool is heated to maintain a water temperature of 22–24°C. The entire experiment comprises a visible platform test (1 day, four tests per day), a hidden platform test (4 days, four tests per day), and a spatial exploration test (24 hours after the last hidden platform test, one test per day). Modeling and treatment drugs are administered 30 minutes before each experiment, allowing the mouse to acclimatize to the experimental environment beforehand. In each experiment, the experimenter gently placed the mouse face down into the pool and used a video monitoring system (SMART 3.0, Panlab Harvard Apparatus, Bioscience Company, Holliston, MA, USA) to record the mouse's swimming trajectory and various movement parameters. If the mouse found the platform within 60 seconds, the time required to search for and climb onto the platform was recorded as the latency. If the mouse failed to find the platform within 60 seconds, the experimenter guided it to the platform and kept it there for 15 seconds; the latency was recorded as 60 seconds. For the spatial exploration experiment, the platform was removed, and the quadrant furthest from the platform was selected as the entry point. The mouse was placed in the water and swam for 60 seconds. The percentage of time spent in the quadrant where the original platform was located was measured, along with the number of times the mouse crossed the original platform area and its search strategy.

[0073] The new object recognition experiment evaluates an animal's cognitive memory ability by comparing the number of times and the time taken for experimental animals to explore new and old objects. The experiment consists of several phases. Environmental adaptation period: Mice are placed in an empty box for 10 minutes to adapt. Familiarization period: Mice are placed in a test box at an equidistant distance with their backs to two identical objects (A, A'), and the number of times they touch the objects with their mouths or noses is recorded within 5 minutes. Testing period: One of the objects is replaced with an object B, which is different in shape and color, and the mouse is placed in the test box with its back to both objects for 5 minutes. Testing begins 1 hour after the familiarization period. The Recognition Index (RI) is calculated using Formula I.

[0074] RI = New object exploration time / (New object exploration time + Old object exploration time) × 100% Formula I.

[0075] During the familiarization period, the experimental animals spent roughly the same amount of time in contact with two identical objects. After introducing a new object, normal animals showed a significant increase in their exploration of the new object, with a cognitive coefficient higher than 0.5; however, when animals had memory impairment, the cognitive coefficient was lower than 0.5, meaning that the animals lost their preference for the new object.

[0076] Twenty-four hours after the behavioral experiment, three or four mice from each group were randomly selected. The mice were deeply anesthetized and their brains were harvested via cardiac perfusion. The brain tissue was fixed in 4% paraformaldehyde and then sequentially embedded in paraffin, sectioned, dewaxed, stained, and mounted. Finally, the pathological changes of each brain tissue section were observed under a microscope.

[0077] II. Results

[0078] Figure 5 The results show the changes in body weight of experimental animals during the drug prevention model. The results indicate that on day 35 of drug administration, the Model group showed a significant decrease in body weight compared to the Sham group, while there was no statistically significant difference between the drug-treated group and the Model group.

[0079] First, the mice were trained in adaptability and spatial memory using the Morris water maze test. The experiment began on day 29 after drug administration, and days 29-33 were the orientation and navigation experiment period, with the main goal of enabling the mice to find the escape platform located 1-2 cm underwater. Figures 6-7 The results showed that in the orientation navigation experiment, compared with the Sham group mice, the escape latency distance and escape latency time within 60 seconds were significantly prolonged in the Model group mice, indicating that SCOP induction successfully caused learning, memory, and cognitive impairment in the mice. Compared with the Model group, the escape latency distance and escape latency time of mice on days 29, 30, 32, and 33 after SHPL-49 administration were significantly shortened, indicating that SHPL-49 has a significant improving effect on the learning ability of mice. Day 34 was the spatial exploration experiment period, mainly recording the motor behavior of the mice. Figure 8 , Figure 9 and Figure 10 The results showed that after platform removal, compared with the Sham group mice, the Model group mice spent significantly less time in the original platform quadrant and crossed the platform more frequently, and their movement trajectories did not show a significant exploratory tendency in the target quadrant. Compared with the Model group mice, the SHPL-49 group and the positive drug DNP group mice spent significantly more time in the target quadrant and crossed the platform more frequently, and showed a more obvious exploratory tendency, indicating that both SHPL-49 and the positive drug DNP can significantly improve the memory retention ability of mice. Furthermore, there were no significant differences in these indicators between the SHPL-49 group and the positive drug group. This indicates that prophylactic administration of SHPL-49 can effectively reverse SCOP-induced memory and cognitive impairment in mice, and its effect on improving learning and memory ability is comparable to that of the positive drug.

[0080] The novel object recognition experiment primarily observes mice's preference for unfamiliar objects in a quiet environment without significant external interference, in order to evaluate their cognitive and memory retention abilities. The experiment began on day 41 of drug administration, and mice were tested after adaptation and familiarization training. Figure 11 Figures 1 and 12 show that, during the testing period, compared with the Sham group, the discrimination index of the Model group mice during the recognition of new objects showed a significant downward trend, suggesting that the Model group mice had significant memory impairment. The comparison shows that administration of SHPL-49 and DNP significantly improved the preference of AD mice for new objects, indicating that these two drugs can improve memory impairment in AD mice, and the effect of SHPL-49 is comparable to that of the positive control drug.

[0081] H&E staining ( Figure 13 The results showed that neurons in the Sham group were orderly arranged, uniformly stained, and had full morphology; compared with the Sham group, neurons in the Model group had larger gaps, uneven staining, and fewer neurons, with visible necrotic neurons and condensed nuclei; compared with the Model group, the SHPL-49 group had more uniform staining, more regular cell morphology, and increased cell number. Nissl staining ( Figure 14 The results showed that neurons in the Sham group had no obvious damage, and Nissl bodies were clearly visible. Compared with the Sham group, neurons in the Model group were shrunken, with Nissl bodies showing dissolution and fragmentation, a reduced number, and deeply stained cytoplasm. Compared with the Model group, neurons in the SHPL-49 group had regular morphology and increased number. This indicates that SHPL-49 can alleviate brain damage in mice.

[0082] Example 2

[0083] The therapeutic effects of SHPL-49 on Alzheimer's disease:

[0084] A 28-day drug-induced Alzheimer's disease model was established using 30 male SPF-grade C57BL / 6J mice (8 weeks old). Thirty mice were acclimatized for 7 days, with seven mice designated as the Sham group. The remaining mice were subcutaneously injected with D-galactose (300 mg / kg / day) for 42 consecutive days to induce aging. The Sham group received an equal volume of saline. The last day of injection was designated as day 0. These aging mice were randomly divided into three groups: a Model group (n=7), an SHPL-491 30 mg / kg administration group (n=8), and an SHPL-491 80 mg / kg administration group (n=8). All groups received the prescribed doses via gavage for 28 consecutive days, while simultaneously receiving intraperitoneal injections of SCOP (2 mg / kg) to induce Alzheimer's disease. The Sham group received an equal volume of saline. Behavioral experiments, including the Morris water maze and open field test, were conducted from day 21 to day 27 of drug administration. The specific methods were the same as described in Example 1, except that the spatial exploration experiment was conducted for 90 seconds.

[0085] Figure 15 The study presented the results of weight changes in a mouse model after 28 days of drug treatment. From the start of intraperitoneal injection of SCOP, the mouse weight in the Model group was significantly lower than that in the Sham group. There was no statistically significant difference between the drug-treated group and the Model group, suggesting that SCOP modeling may have some negative effects on the mice. Figure 16 The organ coefficients of each group were displayed, and no statistically significant differences were found. The results suggest that SHPL-49 does not significantly harm the health of mice.

[0086] Mice were trained in adaptability and spatial memory using the Morris water maze test. The experiment began on day 21 after drug administration, with days 21-25 being the orientation and navigation experiment period. The main objective was to enable mice to locate an escape platform located 1-2 cm underwater. Figure 17 and Figure 18 The results showed that in the navigation experiment, compared with the Sham group mice, the escape latency distance and escape latency time within 90 seconds were significantly prolonged in the Model group mice, proving the model's success and causing learning, memory, and cognitive impairment in the mice; compared with the Model group, the escape latency distance and escape latency time of the SHPL-49 treatment group mice were significantly shortened. Day 26 was the space exploration experiment, mainly recording the mice's motor behavior. Figures 19-21 The spatial exploration trajectory experiment results showed that after the platform was removed, compared with the Sham group mice, the Model group mice spent significantly less time in the original platform quadrant and crossed the platform more frequently, and their movement trajectories did not show a significant tendency to explore the target quadrant. Compared with the Model group mice, the SHPL-49 180 mg / kg treatment group mice showed a more significant tendency to explore, and the time spent finding the target quadrant was significantly reduced. This indicates that the SHPL-49 180 mg / kg dosage can significantly improve the memory retention ability of mice.

[0087] The open field test in Alzheimer's disease (AD) model primarily considers the mouse's motor activity and exploratory ability. Spontaneous motor ability is defined as average speed and number of squares crossed, while exploratory ability can be defined as the frequency of entering the central region and the time spent in the central region. The experimental procedure is as follows: Mice are placed in a quiet laboratory environment to allow them to acclimatize for a period of time, minimizing interference from the external environment. During the formal test, the mouse is gently placed in the center of the open field box (50×50×35cm) with its back to the experimenter, avoiding interference with its behavior. A video monitoring system (VisuTrack 2.0) is activated to record the mouse's movement trajectory, dwell time, distance, and other behavioral indicators in the open field. The experiment lasts for 5 minutes. Each mouse is tested once, during which the equipment is wiped with 75% alcohol to remove any odor or feces left by the previous mouse. The next mouse is placed in only after the alcohol has completely evaporated. The total distance traveled, average speed, number of squares crossed, and time spent in the central region are analyzed. To determine the effects of SHPL-49 on motor function and exploratory behavior, mice were tested using an open field apparatus, with the experiment commencing on day 27 of drug administration. Open field experiment ( Figures 22-26 The results showed that, compared with the Sham group mice, the Model group mice had significantly reduced total distance traveled and number of squares crossed within a 5-minute test period, significantly shorter frequency of entering the central area of ​​the open field, and significantly reduced time spent in the central area, indicating a significant decrease in spontaneous activity. Compared with the Model group mice, both SHPL-49 doses of 130 mg / kg and 180 mg / kg significantly increased the total distance traveled in the open field and the time spent in the central area, while SHPL-49 doses of 180 mg / kg increased the frequency of entering the central area and the number of squares crossed, demonstrating that SHPL-49 can significantly increase the autonomous exploration activity of mice.

[0088] H&E staining ( Figure 27 The results showed that neurons in the Sham group were tightly and orderly arranged, with uniform staining and full morphology; compared with the Sham group, neurons in the Model group had larger gaps, uneven staining, and fewer neurons, with visible necrotic neurons and condensed nuclei; compared with the Model group, the SHPL-49 group had more uniform staining, more regular cell morphology, and increased number of neurons, with the 180mg / kg group being superior to the 130mg / kg group. Nissl staining ( Figure 28 The results showed that neurons in the Sham group showed no significant damage, and Nissl bodies were clearly visible. Compared with the Sham group, neurons in the Model group were shrunken, vacuolated, and Nissl bodies were dissolved and broken, with a reduced number and deeply stained cytoplasm. Compared with the Model group, neurons in the SHPL-49 group had regular morphology and increased number, with the 180 mg / kg group being superior to the 130 mg / kg group. From the above, we can conclude that SHPL-49 can effectively reduce brain damage in mice.

[0089] Example 3

[0090] A 14-day drug-induced Alzheimer's disease model was established using 50 male SPF-grade C57BL / 6J mice (8 weeks old). Fifty mice were acclimatized for 7 days, and 10 mice were designated as the Sham group. The remaining mice were subcutaneously injected with D-galactose (300 mg / kg / day) for 42 consecutive days to induce aging. The Sham group received an equal volume of saline. The last day of injection was designated as day 0. These aging mice were randomly divided into four groups: Model group, SHPL-491 30 mg / kg administration group, SHPL-491 80 mg / kg administration group, and DNP 3 mg / kg administration group, with 10 mice in each group. All groups received the prescribed doses via gavage for 14 consecutive days, while simultaneously receiving intraperitoneal injection of SCOP (2 mg / kg) to induce Alzheimer's disease. The Sham group received an equal volume of saline. Behavioral experiments, including the Morris water maze and novel object recognition tests, were conducted from day 6 to day 13 of drug administration, following the methods described in Example 1.

[0091] Figure 29 The study presents the weight changes in a mouse model after 14 days of drug treatment. Starting with intraperitoneal injection of SCOP, the mice in the SHPL-49 180 mg / kg group showed a significant weight loss compared to the model. This suggests that the effect of simultaneous SCOP administration and model establishment on the mouse body is complex and requires further analysis.

[0092] First, the mice were trained in adaptability and spatial memory using the Morris water maze test. The experiment began on day 6 of drug administration, with days 6-10 being the orientation and navigation experiment period. The main goal was to enable the mice to find the escape platform located 1-2 cm underwater. Figures 30-31 The results showed that in the orientation navigation experiment, compared with the Sham group mice, the escape latency distance and escape latency time within 60 seconds were significantly prolonged in the Model group mice, proving the model's success and causing learning, memory, and cognitive impairment in the mice. Compared with the Model group, the escape latency distance and escape latency time of mice in the SHPL-49 administration group and the positive control group were significantly shortened. Regarding escape latency distance, there was a significant difference between the SHPL-49 130 mg / kg group and the positive control group on days 6 and 9 of administration; regarding escape latency time, there was a significant difference between the SHPL-49 130 mg / kg group and the positive control group on days 7 and 9 of administration. Day 11 was the space exploration experiment period, mainly recording the mice's motor behavior. Figures 32-34The spatial exploration trajectory experiment results showed that after the platform was removed, compared with the Sham group mice, the Model group mice spent significantly less time in the original platform quadrant and crossed the platform more frequently, and the movement trajectory did not show a significant exploratory tendency in the target quadrant. Compared with the Model group mice, the SHPL-49 administration group and the positive drug group mice showed a more significant exploratory tendency, spending significantly more time in the target quadrant and crossing the platform more frequently than the mouse model, indicating that both SHPL-49 and the positive drug DNP can significantly improve the memory retention ability of mice. Furthermore, there were no significant differences in these indicators between the SHPL-49 180 mg / kg group and the positive drug group. This indicates that SHPL-49 180 mg / kg administration can effectively treat SCOP-induced memory and cognitive impairment in mice, and its effect on improving learning and memory ability is comparable to that of the positive drug.

[0093] from Figure 35 Figures 3 and 36 show that, during the testing period, compared with the Sham group, the discrimination index of the Model group mice showed a significant downward trend during the recognition of new objects, suggesting that the Model group mice had significant memory impairment. The comparison also shows that administration of SHPL-49 and DNP significantly improved the preference of AD mice for new objects, indicating that these two drugs can improve memory impairment in AD mice.

[0094] H&E staining ( Figure 37 The results showed that neurons in the Sham group were tightly and orderly arranged, with uniform staining and full morphology; compared with the Sham group, neurons in the Model group had larger gaps, uneven staining, and fewer neurons, with visible necrotic neurons and condensed nuclei; compared with the Model group, the SHPL-49 group had more uniform staining, more regular cell morphology, and increased number of neurons, with the 180mg / kg group being superior to the 130mg / kg group. Nissl staining ( Figure 38 The results showed that neurons in the Sham group showed no significant damage, and Nissl bodies were clearly visible. Compared with the Sham group, neurons in the Model group were shrunken, vacuolated, and Nissl bodies were dissolved and broken, with a reduced number and deeply stained cytoplasm. Compared with the Model group, neurons in the SHPL-49 group had regular morphology and increased number, with the 180 mg / kg group being superior to the 130 mg / kg group. From the above, we can conclude that SHPL-49 can effectively reduce brain damage in mice.

[0095] Example 4

[0096] The therapeutic effect of SHPL-49 on Alzheimer's disease induced by intraventricular injection of Aβ oligomers

[0097] Mice were acclimatized for one week, with 5 mice designated as the control group and another 5 as the sham-operated group. The remaining mice were injected with Aβ oligomers into the right ventricle. The sham-operated group received an equal volume of PBS, while the control group received no treatment. The model mice were randomly divided into a model group and a drug treatment group. The drug treatment group consisted of SHPL-49 and DNP groups, with 6 mice in each group. SHPL-49 was administered at 180 mg / kg, and DNP at 10 mg / kg, respectively, via gavage. The other groups received an equal volume of saline via gavage. Drug administration began on the second day after surgery and continued for 16 days. Behavioral experiments began on the 11th day of drug administration.

[0098] from Figure 39 and Figure 40 It can be seen that, during the test period, compared with the Sham group, the discrimination index of the Model group mice showed a significant downward trend during the recognition of new objects, indicating that the Model group mice had significant memory impairment. The comparison shows that SHPL-49 administration significantly improved the preference of AD mice for new objects, indicating that the drug can improve memory impairment in AD mice. Figures 41-42 The results showed that, in the navigation experiment, compared with the Sham group mice, the escape latency distance and escape latency time of the Model group mice were significantly prolonged within 60 seconds, proving the success of the model and causing learning, memory and cognitive impairment in the mice. Compared with the Model group, the escape latency distance and escape latency time of the mice in the SHPL-49 administration group and the positive drug group were significantly shortened, indicating that SHPL-49 can improve the learning and memory abilities of AD mice.

[0099] Example 5

[0100] Experimental methods for detecting Caenorhabditis elegans in the treatment of Alzheimer's disease with SHPL-49.

[0101] 1) Nematode culture: CL4176 and CL802 are temperature-sensitive nematodes, inoculated onto NGM plates and cultured at 16°C. BR5270 and BR5271 were inoculated onto NGM plates and cultured at 20°C. E. coli OP50 was used as a food source.

[0102] 2) Grouping: control group (non-transgenic nematode CL802 / BR5271), model group (transgenic nematode CL4176 / BR5270, no drug treatment), drug treatment group (transgenic nematode CL4176 / BR5270, treated with different concentrations of drug), positive control group (transgenic nematode CL4176 / BR5270, treated with DNP).

[0103] 3) Preparation of drug reservoir and administration method

[0104] Accurately weigh 51.36 mg of SHPL-49 and dissolve it in 1 mL of DMSO to prepare a 150 mmol / L solution. Dilute this solution with E. coli OP50 bacterial suspension to concentrations of 15 mmol / L, 10 mmol / L, and 5 mmol / L. Then, add 500 μL of each of these solutions to 50 mL of E. coli OP50 to obtain SHPL-49 bacterial suspensions with final concentrations of 150 μmol / L, 100 μmol / L, and 50 μmol / L, respectively. Accurately weigh 10.40 mg of DNP and dissolve it in 1 mL of DMSO to prepare a 25 mmol / L solution. Dilute this solution with E. coli OP50 bacterial suspension to a concentration of 1 mmol / L. Then, add 1250 μL of this solution to 50 mL of E. coli OP50 to obtain a DNP bacterial suspension with a final concentration of 25 μmol / L. Add an appropriate amount of E. coli OP50 bacterial suspension to ordinary NGM medium, and add different drug-containing bacterial suspensions to NGM medium containing drugs.

[0105] 4) Preparation and coating of E. coli OP50

[0106] Select a single E. coli OP50 colony and incubate it overnight at 37°C and 220 rpm. Then, transfer 2 mL of the bacterial culture to 20 mL of fresh LB broth and continue to culture at 37°C and 220 rpm until the OD reaches the target value. 600 =0.8~1.0. Add 120μL of bacterial suspension to each NGM solid medium (120μL of bacterial suspension is recommended for a 60mm diameter petri dish). Then, evenly spread the bacterial suspension on the bottom of a sterile test tube, leaving the edge of the bacterial suspension 3-5mm away from the dish wall to prevent nematodes from burrowing into the medium from between the medium and the dish wall. After evenly spreading the bacterial suspension, let the NGM solid medium air dry at room temperature, then invert it and incubate at room temperature until a sufficiently thick bacterial layer is observed on the surface of the medium. It is then ready for use. If not used immediately, it can be stored upside down in a 4℃ refrigerator for later use.

[0107] 5) Cryopreservation of nematodes

[0108] Collect a large number of nematodes in the starvation (Dauer) stage, and wash the culture medium with 600 μL of M9 buffer in a clean bench. Repeat four times until all the nematodes on the surface of the culture medium are washed off. Aspirate all the M9 solution from the culture medium and dispense it into sterile cryovials, 600 μL per cryovial. Add another 600 μL of cryopreservation buffer to each cryovial and mix well. Place the cryovials in an ultra-low temperature freezer at -80°C for slow freezing.

[0109] 6) The resurgence of nematodes

[0110] Remove the required nematode cryovials from the -80℃ freezer and thaw them rapidly in a 37℃ water bath. In a clean bench, aspirate the cryopreservation solution from the cryovials and add 200–400 μL to the edge of a culture medium coated with E. coli OP50. Place the culture dishes upright in the desired culture temperature and incubate until all the liquid in the culture dishes has evaporated. Then, invert the dishes for further incubation and transfer the successfully revived and healthy nematodes to a new culture medium.

[0111] 7) Synchronization of nematodes

[0112] The experiment requires nematodes to be at the same growth and development stage, therefore, synchronization treatment should be performed before the experiment. There are two main synchronization methods based on the required nematode quantity: First, select an appropriate number of nematodes in the oviposition stage and place them on a suitable plate to allow them to lay eggs. Generally, the oviposition rate is 6-8 eggs per hour, with the specific number depending on the experimental needs. After 1-2 hours, remove the nematodes from the plate and wait for the eggs to hatch. Second, use the lysis method. Wash the plate with M9 buffer, collect a large number of egg-bearing nematodes and eggs into centrifuge tubes, add 5 mL of bleach lysis buffer (5% NaClO:10M NaOH:pure water = 4:1:15, volume ratio), shake vigorously for about 4 minutes, observe under a microscope that most of the nematodes have ruptured and the eggs have been released, then add about 10 mL of M9 solution to stop the lysis. Repeat centrifugation and washing 3-5 times, finally resuspend in about 5-10 mL of M9 solution, and incubate overnight in an incubator to obtain a large number of larvae (L1 stage).

[0113] 8) Culture and observation of paralysis phenotype of CL4176 nematode

[0114] Synchronized CL802 and CL4176 nematodes were inoculated onto ordinary NGM medium and drug-treated medium, respectively, with at least 100 eggs per plate. The plates were placed in a 16°C incubator and cultured for 48 hours. The temperature was then increased to 25°C for further induction of Aβ expression. Through gradient experiments with varying culture times after temperature increases, it was determined that the paralysis status of CL4176 nematodes should be observed starting 28 hours after temperature increase, with observations every 2 hours. Paralysis was defined as the CL4176 nematodes ceasing their curvilinear movements, and only slight head swaying when the tail was gently touched, with no further body movement upon stimulation. The paralyzed nematodes were then counted.

[0115] 9) Nematode lifespan experiment

[0116] Nematodes BR5271 and BR5270 were synchronized and inoculated onto ordinary NGM medium and drug-treated medium, respectively, and cultured in a 20℃ incubator. Each plate contained 30 nematodes, with 3 plates per group. After the synchronized eggs matured into L4 stage adults (day 0), to prevent the eggs and hatched larvae from affecting the experimental observation records, the nematodes were transferred to a new plate daily for the first 7 days, and then every 2 days thereafter until all nematodes died. The movement of the nematodes was observed daily, and the survival and mortality of the nematodes were recorded. The criteria for nematode mortality were: inability to move, no pharyngeal pump activity, and no response to touch. Nematodes that adhered to the culture wall, were lost, had worm bags, or experienced vaginal rupture were not included in the statistical data.

[0117] 10) Nematode reproductive capacity experiment

[0118] BR5271 and BR5270 nematodes were synchronized and inoculated onto ordinary NGM medium and drug-treated medium, respectively, and cultured in a constant temperature incubator at 20℃. After the synchronized eggs grew into L4 stage adults (marked as day 0), the oviposition-stage nematodes were transferred to corresponding new plates for culture, with one nematode per plate and 10 plates per group. Every 24 hours, the oviposition-stage nematodes were transferred to new corresponding plates. Plates containing eggs were cultured in the incubator for another 24 hours and then placed at 4℃ for 1 hour to stiffen the nematodes. The number of nematodes was then quickly recorded. The total number of progeny nematodes in each plate was the number of eggs laid by each nematode.

[0119] Figure 43 This study demonstrated the effect of SHPL-49 on the paralytic phenotype of the transgenic Aβ nematode CL4176 (Yong Y, Yan L, Wei J, et al. A novel ferroptosis inhibitor, Thonningianin A, improves Alzheimer's disease by activating GPX4. [J] Theranostics. 2024 Sep 23; 14(16): 6161-6184.). Nematodes were cultured with different concentrations of SHPL-49, and the number of paralyzed nematodes was recorded every 2 hours. The results showed that treatment with 50, 100, 150 μM SHPL-49, and 25 μM DNP significantly shifted the paralysis time curve of CL4176 nematodes to the right, suggesting that SHPL-49 may play a role in delaying the onset and progression of Alzheimer's disease (AD).

[0120] To investigate the effect of SHPL-49 on the lifespan of the transgenic Tau nematode BR5270 (Garzón-García L, Ayuda-Durán B, González-Manzano S. Neuroprotective Potential of the Flavonoids Quercetin and Epicatechin in a C. elegans Tauopathy Model. [J] Mol Nutr Food Res. 2025 May 12: e70108.), nematodes were cultured with different concentrations of SHPL-49, and the survival rate was recorded daily. Analysis of the experimental data showed that the median survival time of the nematodes in each group was 17 days, 13.5 days, 17 days, 18 days, 15 days, and 17 days, respectively. The results are as follows: Figure 44 As shown, the life curves of the model group nematode BR5270 were significantly shifted to the left compared to the control group nematode BR5271, with a 28.56% reduction in average lifespan. The life curves of the drug-treated groups were significantly shifted to the right compared to the model group. Treatment with 50, 100, and 150 μM SHPL-49, and 25 μM M NP, respectively, extended the average lifespan by 10.62%, 19.87%, 14.00%, and 21.45%, with 100 μM SHPL-49 showing the best effect. This indicates that SHPL-49 has the effect of prolonging the lifespan of the transgenic Tau nematode BR5270.

[0121] This embodiment also investigated the effect of SHPL-49 on the reproductive capacity of the transgenic Tau nematode BR5270. After culturing nematodes with different concentrations of SHPL-49, the total number of progeny nematodes in each group was counted. The results are as follows: Figure 45 As shown, there was no significant difference in the total number of nematode offspring among the groups. The results indicate that SHPL-49 had no significant effect on the reproductive capacity of BR5270 nematodes, suggesting it has no toxic effect.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of SHPL-49, a rhodioloside derivative, in the preparation of drugs for the prevention / treatment of Alzheimer's disease.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable excipients include at least one of diluents, wetting agents, binders, lubricants, colorants, and coating agents.

4. The application according to any one of claims 1 to 3, characterized in that, The drug includes at least one of the following dosage forms: tablets, capsules, granules, pills, suspensions, dispersants, syrups, transdermal preparations, enteric-coated tablets, injections, sprays, and lozenges.

5. The application according to claim 1, characterized in that, Alzheimer's disease includes the following conditions: decreased learning and memory abilities, cognitive impairment, and brain damage.

6. The application according to claim 1 or 5, characterized in that, The inducing factors of Alzheimer's disease include at least one of the following: scopolamine induction, Aβ oligomer induction, and transAβ and / or Tau gene formation.

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