Traditional Chinese medicine composition as gsk-3beta inhibitor and preparation and application thereof

Abstract

The present application relates to the field of biological medical technology, in particular to a traditional Chinese medicine composition as GSK-3beta inhibitor and preparation and application thereof. The traditional Chinese medicine composition as GSK-3beta inhibitor comprises gastrodin and ganoderma triterpene, and the mass ratio of gastrodin and ganoderma triterpene is (1-4):1. The traditional Chinese medicine composition provided by the present application can inhibit the overexpression of GSK-3beta in AD model, thereby inhibiting the overphosphorylation of Tau protein and playing a protective role on Alzheimer's disease. In addition, the traditional Chinese medicine composition provided by the present application contains gastrodin and ganoderma triterpene, which are homoeopathic traditional Chinese medicine efficacy components, greatly improving the safety of the drug, and providing a train of thought for the application of the traditional Chinese medicine composition as a functional food.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a traditional Chinese medicine composition as a GSK-3β inhibitor and its preparation and application. Background Technology

[0002] Currently, there is no cure for Alzheimer's disease. Commonly used clinical medications are mainly single-target drugs for symptomatic treatment, such as tacrine, donepezil hydrochloride, rivastigmine, and galantamine. These drugs have many adverse reactions and limitations. The cause of AD is still unclear, its pathogenesis is very complex, and there is currently no cure. It is considered one of the leading causes of death among the elderly. AD often has a long, irreversible course, high rates of disability and mortality, and multiple complications requiring long-term care, placing a heavy economic burden on society and families. In recent years, the National Health Commission of China has called for a nationwide campaign to promote the prevention and treatment of Alzheimer's disease. Early detection, early drug and non-drug interventions, and early control of risk factors can significantly reduce the risk of Alzheimer's disease and improve prognosis.

[0003] Several hypotheses exist regarding the pathogenesis of Alzheimer's disease (AD), including the cholinergic hypothesis, the Tau hypothesis, the amyloid hypothesis, the glutamate excitotoxicity hypothesis, vitamin B5 deficiency, and mitochondrial cascades. Among these, the amyloid hypothesis and the Tau hypothesis are the most studied. These hypotheses primarily involve the deposition of amyloid plaques in the brain due to the aggregation of insoluble β-amyloid protein (Aβ) with age, and the hyperphosphorylation of microtubule-associated protein tau at multiple sites leading to the aggregation of helical filaments, ultimately forming neurofibrillary tangles (NFTs). NFTs originate in the entorhinal cortex and hippocampus and then gradually spread to the cortex, representing a key characteristic of AD lesions.

[0004] Glycogen synthase kinase-3 (GSK-3) is a universally expressed serine-threonine kinase with constitutive activity, involved in the regulation of many key cellular biological pathways, some of which are related to neurodegeneration. GSK-3 is divided into two subtypes: GSK-3α and GSK-3β. GSK-3β kinase has a high content in the central nervous system, and its expression level increases with age. GSK-3β was first confirmed to have Tau protein kinase activity in 1993 by Koichi Ishiguro et al. (Ishiguro K, Shiratsuchi A, Sato S, et al. Glycogen synthase kinase 3beta is identical to tau protein kinase I generating several epitopes of paired helical filaments[J]. Febs Letters, 1993, 325(3):167-172). Tau protein has a large number of phosphorylation sites that can be catalyzed by GSK-3β. The prevailing view is that Aβ accumulates gradually with age, and GSK-3β activity increases with Aβ accumulation, ultimately leading to hyperphosphorylation of Tau protein. Numerous studies have shown that GSK-3β is a crucial participant in different aspects and pathways of AD pathogenesis and development; dysregulation of this kinase affects Tau phosphorylation, Aβ production, neurofibrillary tangles formation, memory, and synaptic dysfunction.

[0005] Research has found that when tau ceases to stably influence the neuronal cytoskeleton, loss of function occurs, and similarly, it can lead to defects in axonal transport. Higher levels of tau phosphorylation have also been shown to inhibit vesicle and organelle transport, including vesicles carrying amyloid precursor protein (APP), and increase oxidative stress levels. Tau can induce neurotoxicity through loss of function, gain of function, or mislocalization.

[0006] Tau protein phosphorylation is regulated by multiple kinases, including GSK-3β and extracellular Aβ-activated cyclin-dependent kinase 5 (CDK5). Kinase-phosphatase imbalances in the brain can affect Tau protein phosphorylation levels. Increased protein kinase activity or decreased phosphatase activity is one cause of hyperphosphorylation. In previous studies, GSK-3β was identified as a key kinase potentially involved in the development of Tau pathology in Alzheimer's disease (AD), and decreased GSK-3β activity can reduce Tau protein phosphorylation. Tobias Engel et al. (Engel T, Oliver P, Lucas JJ, et al. Chronic lithium administration to FTDP-17tau and GSK-3β overexpressing mice prevents tau hyperphosphorylation and neurofibrillary tangle formation, but pre-formed neurofibrillary tangles do not revert[J]. Journal of Neurochemistry, 2006, 99(6):1445-55.) A GSK-3β overexpression model was constructed in transgenic mice, and it was found that tau hyperphosphorylation was present in hippocampal neurons, accompanied by neurofibrillary tangles. Inhibition of GSK-3β overexpression did not eliminate the previously formed tangles, but it prevented abnormal tau accumulation caused by overexpression. Abnormal signaling of GSK-3β in the pathway is also one of the inducing factors of AD. In the brains of patients with mild cognitive impairment (MCI) and AD, GSK-3β activity was significantly increased compared to normal human brains. For example, in mice overexpressing GSK-3β, the phosphorylation level of Tau protein in the brain was significantly increased, and these mice also exhibited a series of behavioral defects such as lethargy. Hu et al. (Hu S, Begum AN, Jones MR, et al. GSK3 inhibitors show benefits in an Alzheimer's disease (AD) model of neurodegeneration but adverse effects in control animals[J]. Neurobiol Dis, 2009, 33(2): 193-206.) found that intracerebral injection of the GSK3s-specific inhibitor SB216763 (SB) into AD model mice improved the pathological manifestations of the AD mouse model by inhibiting the activity of GSK-3β. Hernandez et al. (Hernandez F, Lucas JJ, Avila J. GSK3 and Tau: Two Convergence Points in Alzheimer's Disease[J]. Journal of Alzheimers Disease, 2013, 33(Suppl 1.)) demonstrated that chronic treatment with lithium could alleviate AD by specifically inhibiting GSK-3β and improving Tau protein hyperphosphorylation.

[0007] Alzheimer's disease (AD) is a chronic illness that initially presents with only mild cognitive impairment and no obvious symptoms, but its progression can take 15-25 years. With increasing life expectancy and a rapidly aging population, the number of AD patients is rapidly increasing, with a high proportion in the 65-year-old population. Studies have confirmed that GSK-3β expression increases with age, leading to increased Tau phosphorylation and promoting the development of AD. Therefore, with increasing age, such as after 65, regulating GSK-3β may be an effective strategy for preventing AD, suggesting the feasibility of using GSK-3β as an inhibitory target to prevent excessive Tau protein phosphorylation and thus prevent the occurrence and exacerbation of AD.

[0008] Therefore, the development of GSK-3β inhibitors could be a major breakthrough in the prevention and treatment of Alzheimer's disease. Summary of the Invention

[0009] This invention discloses a traditional Chinese medicine composition as a GSK-3β inhibitor, its preparation and application. It uses gastrodin (GAS) and Ganoderma lucidum triterpenoids (GLT), the main components of Gastrodia elata and Ganoderma lucidum, which are both medicinal and edible herbs, as raw materials. When combined in a specific ratio, it can significantly inhibit the overexpression of GSK-3β, thereby regulating the hyperphosphorylation of Tau protein and significantly improving Alzheimer's disease.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] The first objective of this invention is to provide a traditional Chinese medicine composition as a GSK-3β inhibitor, comprising gastrodin and ganoderic triterpenes, wherein the mass ratio of gastrodin to ganoderic triterpenes is (1-4):1.

[0012] The traditional Chinese medicine composition provided by this invention breaks through the previous method of using single components. It not only reduces the amount of each single component used, but also achieves a better synergistic effect by adjusting the mass ratio of the two traditional Chinese medicine components. At this mass ratio, the performance of the composition is significantly improved compared to any single component. This traditional Chinese medicine composition effectively improves Alzheimer's disease by inhibiting the expression of the key AD-related protein GSK-3β, thereby regulating the hyperphosphorylation of Tau protein. Therefore, the most important inventive point of this invention is the development of a traditional Chinese medicine composition that prevents and treats Alzheimer's disease by inhibiting GSK-3β protein expression.

[0013] To verify the practical effects of the above-mentioned invention, the applicant designed AD cell models and AD mouse models, and observed the results of cellular oxidative stress indicators (MDA, T-AOC, SOD), GSK-3β expression levels, and Tau protein phosphorylation levels in AD cell models and AD mouse models. Animal behavioral experiments in AD mouse models also showed that the traditional Chinese medicine composition prepared in this invention has a good protective effect against cellular oxidative stress damage and can help restore the oxidative stress damage state of AD cells and AD mice. Simultaneously, this traditional Chinese medicine composition can significantly inhibit the expression of GSK-3β protein in the AD model and significantly reduce phosphorylation at the Tau (Ser396) and Tau (Thr181) sites. More importantly, in the AD cell model, the use of gastrodin and Ganoderma triterpenes alone did not change the GSK-3β expression level; however, when the traditional Chinese medicine composition containing gastrodin and Ganoderma triterpenes was used, GSK-3β was significantly downregulated at both mRNA and protein levels.

[0014] GSK-3β is a key enzyme in the hyperphosphorylation of Tau protein, and its enhanced activity is closely related to the development of Alzheimer's disease (AD) because it promotes abnormal phosphorylation of Tau protein, leading to neurofibrillary tangles, a pathological feature of AD. No significant changes in GSK-3β expression levels were observed when gastrodin or ganoderic triterpenes were used alone, indicating that a single component may not be sufficient to effectively regulate this key pathological pathway. However, when used in combination, GSK-3β was significantly downregulated at both the mRNA and protein levels. This suggests that they intervene in the GSK-3β signaling pathway through different mechanisms of action or by enhancing each other's effects, thereby more effectively reducing Tau protein hyperphosphorylation and slowing the progression of neurodegenerative diseases. Specifically, gastrodin mainly works by inhibiting excessive excitation of nerve cells and providing neuroprotection; while ganoderic triterpenes exert their effects through their anti-inflammatory, antioxidant, and immunomodulatory properties, as well as by directly or indirectly affecting GSK-3β activity. The combined use of both can jointly combat the complex pathological process of AD through a multi-pathway, multi-target mechanism of action.

[0015] Furthermore, the interaction between gastrodin and ganoderic triterpenes enhances the bioavailability and / or stability and activity of their respective components in vivo, enabling them to exert their biological effects more effectively upon reaching the therapeutic site. This synergistic effect may not only slow disease progression but also promote neuronal protection and repair, improve cognitive function, and provide a new strategy for the treatment of Alzheimer's disease (AD). Therefore, the traditional Chinese medicine composition provided by this invention can effectively improve the symptoms of AD and holds promise for application in the prevention and treatment of AD.

[0016] Furthermore, the gastrodin and ganoderic triterpenoids selected in this invention are derived from Gastrodia elata and Ganoderma lucidum, respectively. Both Gastrodia elata and Ganoderma lucidum are medicinal and edible raw materials.

[0017] Therefore, the implicit inventive point of this invention is to prepare functional foods using a complex of medicinal and edible herbal active ingredients, providing a new health strategy for the prevention of chronic diseases. The use of a combination of gastrodin and Ganoderma lucidum triterpenes, both medicinal and edible active ingredients, in the preparation of functional foods to aid in memory improvement has a preventive and symptom-alleviating effect on Alzheimer's disease, and is of significant social importance in preventing and controlling the high incidence of Alzheimer's disease.

[0018] In summary, the traditional Chinese medicine composition provided by this invention has significant effects in preventing and treating Alzheimer's disease (AD); and the traditional Chinese medicine ingredients used are medicinal and edible medicinal components with high drug safety.

[0019] More preferably, the mass ratio of gastrodin to Ganoderma triterpenoids is 4:1.

[0020] Preferably, the extraction method of gastrodin includes the following steps: gastrodin powder is extracted with alcohol by ultrasonication, then filtered, centrifuged, evaporated and concentrated, then separated and purified, eluted with ethanol, concentrated and dried to obtain gastrodin.

[0021] Preferably, the method for extracting gastrodin includes the following steps:

[0022] S3.1. According to the ratio of ethanol solution to gastrodia powder liquid to material is 30:1 mL / g, gastrodia powder that has passed through a 60-mesh sieve is soaked in 70% ethanol solution for 12 hours, and then ultrasonically extracted for 60 minutes to obtain gastrodin extract.

[0023] S3.2. Gastrodin extract is obtained by filtration, centrifugation and evaporation to obtain crude gastrodin extract.

[0024] S3.3. The crude extract of gastrodin was loaded onto a macroporous adsorption resin, allowed to stand for adsorption, and then separated and purified. The eluent was obtained by elution with 70% ethanol solution. The eluent was then rotary evaporated, concentrated, and dried to obtain gastrodin. The separation conditions were a flow rate of 4 BV / h and a loading volume of 100 mL.

[0025] Preferably, the extraction method of Ganoderma triterpenes includes the following steps: ultrasonic extraction of Ganoderma powder with alcohol, followed by filtration, centrifugation and merging of the filtrates, followed by evaporation, separation and purification, elution, concentration and drying to obtain Ganoderma triterpenes.

[0026] Preferably, the extraction method of the Ganoderma lucidum triterpenes includes the following steps:

[0027] S4.1. Following the ratio of ethanol solution to Ganoderma lucidum powder liquid to 25:1 mL / g, Ganoderma lucidum powder that has passed through a 40-mesh sieve was subjected to two ultrasonic cycles at 80°C with 75% ethanol solution to obtain Ganoderma lucidum triterpenoid extract. One ultrasonic cycle time was 30 min.

[0028] S4.2 After filtration of the Ganoderma lucidum triterpenoid extract, the filtrate is retained. The filter residue is then centrifuged and the supernatant is retained. The filtrate and supernatant are combined to obtain a comprehensive filtrate. After evaporation to remove ethanol from the comprehensive filtrate, it is separated and purified by macroporous resin to obtain a crude Ganoderma lucidum triterpenoid extract. The separation conditions are pH=4, flow rate 2 BV / h, and loading volume 30 mL.

[0029] S4.3 The crude extract of Ganoderma lucidum triterpenes was eluted sequentially with 15% ethanol solution, 35% ethanol solution and 95% ethanol solution, and then evaporated, concentrated and dried with 95% ethanol solution to obtain Ganoderma lucidum triterpenes.

[0030] Preferably, the traditional Chinese medicine composition is formulated with pharmaceutically acceptable excipients to produce one of the following dosage forms: powder, tablet, granule, and liquid preparation.

[0031] To formulate traditional Chinese medicine compositions with pharmaceutically acceptable excipients into powders, tablets, granules, and liquid preparations, each dosage form has its own unique preparation process, as detailed below:

[0032] Powder: The traditional Chinese medicine composition and excipients (such as starch, lactose, etc. as fillers or glidants) are separately pulverized to the required fineness and then graded and screened through sieves of different mesh sizes to ensure uniform particle size. The powder is then mixed, divided into doses, and packaged to obtain the traditional Chinese medicine composition.

[0033] Tablets: The traditional Chinese medicine composition is mixed with a binder (starch paste, syrup, etc.), then wet granulated, dry granulated, or directly compressed into tablets, and finally compressed into tablets using a tableting machine to obtain tablets.

[0034] Granules: Similar to the wet granulation process for tablets, but granules are typically larger for easier administration. Granules are obtained by drying the granules after compression, eliminating the need for tableting.

[0035] Liquid preparation: The liquid preparation is obtained by combining the extracted gastrodin solution and Ganoderma triterpenoid solution.

[0036] In addition to the dosage forms mentioned above, any feasible dosage form falls within the application scope of the traditional Chinese medicine composition provided by this invention.

[0037] The second objective of this invention is to provide a method for preparing a traditional Chinese medicine composition as a GSK-3β inhibitor, wherein gastrodin and Ganoderma triterpenes are dissolved in solvents and refrigerated for later use; then the two solutions are mixed in a mass ratio of gastrodin to Ganoderma triterpenes of (1-4):1 to prepare the traditional Chinese medicine composition.

[0038] The solvent used here to dissolve gastrodin and Ganoderma triterpenes can be a commonly used solvent, that is, a reagent that can achieve complete dissolution and is safe and harmless, such as sterile water, deionized water, distilled water, anhydrous ethanol, etc.

[0039] Preferably, the method for preparing the traditional Chinese medicine composition as a GSK-3β inhibitor includes the following steps:

[0040] S6.1 Gastrodin solution was prepared by mixing gastrodin with sterile water and stored in a refrigerator at 4°C for later use.

[0041] S6.2 Dissolve Ganoderma lucidum triterpenes in food additive ethanol (GB 30610—2014) or DMSO solution, then dilute with sterile water to obtain Ganoderma lucidum triterpenes solution, and store in a refrigerator at 4℃ for later use.

[0042] S6.3 Mix gastrodin solution and Ganoderma triterpenoid solution so that the mass ratio of gastrodin to Ganoderma triterpenoid in the final prepared traditional Chinese medicine composition is (1-4):1.

[0043] A third objective of this invention is to provide the application of traditional Chinese medicine compositions in the preparation of GSK-3β inhibitors.

[0044] The fourth objective of this invention is to provide the application of traditional Chinese medicine compositions in the preparation of Tau protein inhibitors.

[0045] The fifth objective of this invention is to provide the application of traditional Chinese medicine compositions in the preparation of drugs for the prevention and treatment of Alzheimer's disease or health products for Alzheimer's disease.

[0046] The inventors designed AD cell and mouse models to verify the efficacy of the traditional Chinese medicine composition prepared in this invention in the prevention and treatment of AD. Observation of cellular oxidative stress indicators (MDA, T-AOC, SOD), GSK-3β expression levels, and Tau protein phosphorylation levels in AD cell and mouse models, as well as animal behavioral results in the AD mouse model, showed that the traditional Chinese medicine composition prepared in this invention has a good protective effect against cellular oxidative stress damage and can help restore the oxidative stress-damaged state of AD cells and AD mice. Oxidative stress is an important pathological mechanism in the development of AD, involving excessive free radical generation and imbalance of the antioxidant defense system, leading to neuronal damage and death. By regulating oxidative stress-related biomarkers (such as MDA, T-AOC, SOD, etc.), this traditional Chinese medicine composition can reduce neuroinflammation, protect neurons from damage, and potentially improve cognitive function. Simultaneously, this traditional Chinese medicine composition can significantly inhibit GSK-3β protein expression and significantly reduce phosphorylation at the Tau (Ser396) and Tau (Thr181) sites. Therefore, the traditional Chinese medicine composition containing gastrodin and Ganoderma triterpenes provided by this invention can effectively improve the symptoms of Alzheimer's disease (AD) and is expected to be applied in the prevention and treatment of AD. Furthermore, the medicinal and edible properties of gastrodin and Ganoderma triterpenes can be utilized to prepare functional health products that improve memory or safe drugs for the prevention and treatment of AD.

[0047] Therefore, the present invention has the following beneficial effects:

[0048] (1) The traditional Chinese medicine composition provided by the present invention can inhibit the overexpression of GSK-3β in AD model, thereby inhibiting the excessive phosphorylation of Tau protein and exerting a protective effect against AD.

[0049] (2) The traditional Chinese medicine composition provided by the present invention can be used as an inhibitor of GSK-3β and has a significant effect on improving Alzheimer's disease.

[0050] (3) The traditional Chinese medicine composition provided by the present invention contains gastrodin and Ganoderma triterpenoids, which are both medicinal and edible traditional Chinese medicine components, greatly improving drug safety and providing ideas for the application of traditional Chinese medicine composition as functional food. Attached Figure Description

[0051] Figure 1 The graph shows the changes in MDA level, SOD activity and T-AOC in cells (A: MDA level, B: SOD activity, C: T-AOC; *p<0.05, **p<0.01; compared with AD group).

[0052] Figure 2 Phosphorylation levels of Tau proteins (Thr181 and Ser396) in cells (*p<0.05, **p<0.01; compared with AD group).

[0053] Figure 3 Plot showing GSK-3β expression levels in cells (*p<0.05; compared with the AD group).

[0054] Figure 4 The graph shows the effect of different treatments on the number of times and time it takes for mice to traverse the platform in the water maze experiment.

[0055] Figure 5 The graph shows the changes in MDA level, SOD activity and T-AOC in the mouse brain (A: MDA level, B: SOD activity, C: T-AOC; *p<0.05, **p<0.01; compared with AD group).

[0056] Figure 6 Phosphorylation of Tau protein and (Thr181 and Ser396) in the mouse brain (*p<0.05, **p<0.01; compared with AD group).

[0057] Figure 7 Plot of GSK-3β expression levels in mouse brains (*p<0.05; compared with AD group). Detailed Implementation

[0058] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0059]

Example

[0060] Example 1

[0061] 1. Gastrodin Extract

[0062] S1. According to the ratio of ethanol solution to gastrodia powder liquid to material is 30:1mL / g, 5g of gastrodia powder after passing through a 60-mesh sieve is soaked in 150mL of 70% ethanol solution for 12h, and then ultrasonically extracted for 60min to obtain gastrodin extract.

[0063] S2. Gastrodin extract was filtered, centrifuged and then evaporated and concentrated to obtain crude gastrodin extract.

[0064] S3. The crude extract of gastrodin was loaded onto a macroporous adsorption resin, allowed to stand for adsorption, and then separated and purified. The eluent was obtained by elution with 70% ethanol solution. The eluent was then rotary evaporated, concentrated, and dried to obtain gastrodin. The separation conditions were a flow rate of 4 BV / h and a loading volume of 100 mL.

[0065] 2. Ganoderma lucidum triterpenoid extraction

[0066] S1. According to the ratio of ethanol solution to Ganoderma lucidum powder liquid to material is 25:1mL / g, 5g of Ganoderma lucidum powder after passing through a 40-mesh sieve is subjected to 75mL of 75% ethanol solution and ultrasonic cycles are repeated at 80℃ for two times to obtain Ganoderma lucidum triterpenoid extract. One ultrasonic cycle time is 30min.

[0067] S2. After filtration of Ganoderma lucidum triterpenoid extract, the filtrate is retained. The filter residue is then centrifuged and the supernatant is retained. The filtrate and supernatant are combined to obtain a comprehensive filtrate. After evaporation to remove ethanol from the comprehensive filtrate, it is separated and purified by macroporous resin to obtain crude Ganoderma lucidum triterpenoid extract. The separation conditions are pH=4, flow rate 2 BV / h, and sample loading volume 30 mL.

[0068] S3. The crude extract of Ganoderma lucidum triterpenes was eluted sequentially with 15% ethanol solution, 35% ethanol solution and 95% ethanol solution, and then evaporated, concentrated and dried with 95% ethanol solution to obtain Ganoderma lucidum triterpenes.

[0069] This invention employs a mixed method of vanillin-glacial acetic acid and perchloric acid for the qualitative analysis of Ganoderma lucidum triterpenes. This method primarily utilizes colorimetric reactions of different eluting components to identify and differentiate them. This method is particularly suitable for the analysis of compounds containing triterpenes, sterols, and saponins.

[0070] Vanillin, under acidic conditions (such as perchloric acid), can undergo a condensation reaction with compounds possessing appropriate functional groups (such as phenolic hydroxyl groups and carbonyl groups) to form products with specific colors. This colorimetric reaction is based on the oxidation of vanillin with the phenolic hydroxyl groups in the compound, forming a conjugated system, thus producing a color change. Perchloric acid is not only a strong acid but also has oxidizing properties; it can promote structural changes in certain compounds, such as oxidizing phenolic hydroxyl groups to carboxyl groups, increasing the conjugated system in the molecule, which is beneficial for the colorimetric reaction. Simultaneously, perchloric acid can accelerate the reaction rate and improve the stability and sensitivity of the colorimetric reaction. Glacial acetic acid acts as a solvent and auxiliary reagent here, aiding in the dissolution of reactants and potentially participating in the formation of stable colorimetric products. Ganoderma lucidum triterpenoids often exhibit a purple color during the determination using a mixed method of vanillin-glacial acetic acid and perchloric acid.

[0071] Therefore, a mixture of vanillin-glacial acetic acid and perchloric acid can be used to qualitatively analyze the presence and relative content of Ganoderma triterpenes. This allows for precise control of the mass ratio of gastrodin and Ganoderma triterpenes in the traditional Chinese medicine composition, resulting in a traditional Chinese medicine composition with better AD prevention and treatment effects.

[0072] 3. Preparation of Traditional Chinese Medicine Compositions

[0073] S1. The gastrodin extracted above was mixed with sterile water to prepare a 40 μg / mL gastrodin solution, which was then stored at 4℃ for later use.

[0074] S2. Dissolve the Ganoderma triterpenes obtained above with food additive ethanol (GB 30610—2014) or DMSO solution, then add sterile water to dilute and mix to prepare a 20 μg / mL Ganoderma triterpenes solution, and store it in a refrigerator at 4℃ for later use.

[0075] S3. Gastrodin solution and Ganoderma triterpenoid solution are mixed in a volume ratio of 1:1 to obtain traditional Chinese medicine composition A, and the mass ratio of gastrodin to Ganoderma triterpenoid in traditional Chinese medicine composition A is 2:1.

[0076] Example 2

[0077] This embodiment is basically the same as Embodiment 1, except that:

[0078] S3. Gastrodin solution and Ganoderma triterpenoid solution are mixed in a volume ratio of 1:2 to obtain traditional Chinese medicine composition B, and the mass ratio of gastrodin to Ganoderma triterpenoid in traditional Chinese medicine composition B is 1:1.

[0079] Example 3

[0080] This embodiment is basically the same as Embodiment 1, except that:

[0081] S3. Gastrodin solution and Ganoderma triterpenoid solution were mixed in a volume ratio of 2:1 to obtain traditional Chinese medicine composition C, and the mass ratio of gastrodin to Ganoderma triterpenoid in traditional Chinese medicine composition C was 4:1.

[0082] Comparative Example 1

[0083] 1. Gastrodin Extract

[0084] S1. According to the ratio of ethanol solution to gastrodia powder liquid to material is 30:1mL / g, 5g of gastrodia powder after passing through a 60-mesh sieve is soaked in 150mL of 70% ethanol solution for 12h, and then ultrasonically extracted for 60min to obtain gastrodin extract.

[0085] S2. Gastrodin extract was filtered, centrifuged and then evaporated and concentrated to obtain crude gastrodin extract.

[0086] S3. The crude extract of gastrodin was loaded onto a macroporous adsorption resin, allowed to stand for adsorption, and then separated and purified. The eluent was obtained by elution with 70% ethanol solution. The eluent was then rotary evaporated, concentrated, and dried to obtain gastrodin. The separation conditions were a flow rate of 4 BV / h and a loading volume of 100 mL.

[0087] 2. Preparation of Gastrodin Solution

[0088] The gastrodin extracted above was mixed with sterile water to prepare a 40 μg / mL gastrodin solution.

[0089] Comparative Example 2

[0090] 1. Ganoderma lucidum triterpenoid extract

[0091] S1. According to the ratio of ethanol solution to Ganoderma lucidum powder liquid to material is 25:1mL / g, 5g of Ganoderma lucidum powder after passing through a 40-mesh sieve is subjected to 75mL of 75% ethanol solution and ultrasonic cycles are repeated at 80℃ for two times to obtain Ganoderma lucidum triterpenoid extract. One ultrasonic cycle time is 30min.

[0092] S2. After filtration of Ganoderma lucidum triterpenoid extract, the filtrate is retained. The filter residue is then centrifuged and the supernatant is retained. The filtrate and supernatant are combined to obtain a comprehensive filtrate. After evaporation to remove ethanol from the comprehensive filtrate, it is separated and purified by macroporous resin to obtain crude Ganoderma lucidum triterpenoid extract. The separation conditions are pH=4, flow rate 2 BV / h, and sample loading volume 30 mL.

[0093] S3. The crude extract of Ganoderma lucidum triterpenes was eluted sequentially with 15% ethanol solution, 35% ethanol solution and 95% ethanol solution, and then evaporated, concentrated and dried with 95% ethanol solution to obtain Ganoderma lucidum triterpenes.

[0094] 2. Preparation of Ganoderma lucidum triterpenoid solution

[0095] The Ganoderma triterpenes obtained above were dissolved in food additive ethanol (GB 30610—2014) or DMSO solution, and then diluted with sterile water to prepare a 20 μg / mL Ganoderma triterpenes solution.

[0096] [Preparation before the test]

[0097] 1. Aβ 25-35 preparation

[0098] Aβ 25-35 The powder was dissolved using HFIP to remove impurities. Appropriate amounts of HFIP-dissolved Aβ were dispensed. 25-35 Pour into 1.5 mL EP tubes, air dry HFIP, and finally form a peptide membrane. Store at -80°C. Before use, dissolve the peptide membrane in sterile PBS with 10% DMSO using sonication in an ice bath, and incubate at 4°C for 14-30 days to allow Aβ to settle. 25-35 Oligopoly.

[0099] 2. Cell experiments and grouping

[0100] The cells were human neuroblastoma cells (SH-SY5Y), divided into 7 groups: NC blank group, AD model group, GAS group (comparative example 1), GLT group (comparative example 2), traditional Chinese medicine composition A (Example 1), traditional Chinese medicine composition B (Example 2), and traditional Chinese medicine composition C (Example 3). The drugs (i.e., gastrodin, ganoderic triterpenes, and gastrodin and ganoderic triterpenes) were incubated for 24 hours. 20 μM Aβ... 25-35 An AD cell model was constructed by incubating for 24 hours.

[0101] 3. Protein extraction, sample preparation, and Western blotting

[0102] (1) Take an appropriate amount of lysis buffer, add a mixture of protease inhibitor and phosphatase inhibitor before use, with a composition ratio of 100:1:1, mix well, and place the mixture on ice for later use.

[0103] (2) Take cell samples or fresh animal tissue and wash them 1-3 times with pre-cooled PBS.

[0104] (3) Add lysis buffer and lyse at 4°C for 30-60 min. The tissue needs to be homogenized 30-50 times in an ice bath until fully lysed using a grinding rod.

[0105] (4) Centrifuge at 12000g for 10 min at 4℃. Take the supernatant and add 5x loading buffer according to the ratio, mix well, and heat in a metal bath at 100℃ for 10 min to fully denature the protein.

[0106] (5) Perform SDS-PAGE protein gel electrophoresis. After the electrophoresis, remove the protein gel from the electrophoresis tank and transfer the protein onto a PVDF membrane using the wet transfer method.

[0107] (6) The membrane was sealed with 5% skim milk powder at room temperature for 2 hours. After sealing, the PVDF membrane was washed 4 times with 1x TBST for 5 minutes each time.

[0108] (7) Based on the protein to be detected, incubate with the corresponding primary antibody at 4°C overnight. The next day, wash 4 times with 1x TBST for 5 minutes each time. Continue incubating with the secondary antibody at 4°C for 1 hour. After the incubation, wash 4 times with 1x TBST for 5 minutes each time.

[0109] (8) Mix the ECL colorimetric solution (A:B = 1:1), add an appropriate amount of the mixture evenly on the membrane, and scan and image it using a Tanon 5500 ultrasensitive chemiluminescence imager.

[0110] 4. Cell RNA extraction

[0111] (1) After cell induction is completed, remove the cell culture plate and discard the old culture medium.

[0112] (2) Add 1 mL PBS to wash the cell surface, discard the PBS, repeat twice, and add 1 mL Trizol to lyse the cells. After standing at room temperature for 5 min, collect the cells into a 1.5 mL RNA-free EP tube.

[0113] (3) Add 200 μL of chloroform, mix well, and let stand at room temperature for 10 min.

[0114] (4) Centrifuge at 12,000 rpm for 15 min at 4℃.

[0115] (5) Take the upper aqueous phase containing RNA into a new 1.5 mL RNA-free EP tube, add isopropanol at a ratio of 1:1, mix by inversion, and let stand at room temperature for 10 min.

[0116] (6) Centrifuge at 12,000 rpm for 10 min at 4℃.

[0117] (7) Carefully discard the supernatant, add 1 mL of 75% DEPC ethanol along the tube wall, and invert to resuspend the RNA to wash the RNA precipitate.

[0118] (8) Centrifuge at 6000 rpm for 5 min at 4℃, and carefully discard 75% DEPC ethanol. Repeat steps (7)-(8) twice.

[0119] (9) Carefully aspirate the remaining small amount of 75% DEPC ethanol from the bottom of the EP tube and allow the RNA precipitate to dry at room temperature.

[0120] (10) Add an appropriate amount of DEPC water to dissolve the RNA precipitate according to the sample. Take 2 μL of RNA solution, zero the instrument with DEPC water, and use Nanodrop to detect the RNA concentration and purity. Store the remaining RNA at -80℃.

[0121] (11) For subsequent operations, please refer to the instructions for the AG reverse transcription kit and... qPCR GreenMaster Mix (No Rox) Kit Instruction Manual.

[0122] 5. Detection of oxidative stress-related indicators

[0123] The levels of SOD / MDA / T-AOC in cells and tissues were detected according to the manufacturer's instructions.

[0124] 6. Method for establishing a mouse AD model: D-galactose (150 mg / kg) was injected intraperitoneally, and aluminum trichloride (AlCl3, 15 mg / kg) was administered by gavage to induce AD.

[0125] The dosage of the herbal composition administered via gavage was determined by converting the human dosage of gastrodin. The concentration ratio of herbal composition C in the low-dose group (Low), medium-dose group (Medium), and high-dose group (High) was 1:2:4. Animal experiments were grouped as follows:

[0126]

[0127] 7. Animal behavioral experiments

[0128] After feeding, the mice underwent the Morris water maze test. For the first three days, platforms were placed in the pool for acclimatization training. The pool was divided into four quadrants along the cardinal directions. Mice were released into the water at the midpoints of each quadrant, guided to locate the platforms, and developed some memory of their positions after three days of training. On the fourth day, the platforms were removed, and a spatial exploration experiment was conducted. Random entry points were selected, and the mice's exploration paths in the pool were recorded using an imaging system over 120 seconds. The number of times the mice crossed platform locations and the effective exploration time in the quadrants where the platforms were placed were statistically analyzed.

[0129] [Performance Testing]

[0130] 1. Study on the inhibitory effect of the composition on GSK-3β in an AD cell model

[0131] Cells were incubated with the traditional Chinese medicine compositions obtained in Examples 1-3, the single GAS solution obtained in Comparative Examples 1-2, and the single GLT solution, respectively, and Aβ was added. 25-35 Following stimulation, the inhibitory effect of the composition on GSK-3β was investigated using Western blotting and RT-qPCR. Further changes in Tau protein phosphorylation (Thr181 and Ser396 sites) were detected using Western blotting. Subsequently, oxidative stress indicators MDA, T-AOC, and SOD in AD model cells after treatment with the traditional Chinese medicine composition were measured.

[0132] 2. Animal experiments to investigate the inhibitory effect of the composition on GSK-3β in an AD mouse model.

[0133] Seven-month-old male C57 / BL mice were selected and divided into 6 groups (the same 6 groups as described in section 6, "Pre-test Preparation," section 6, "Mouse AD Model Construction Method"), and administered the drugs via gavage and intraperitoneal injection for 40 consecutive days. After feeding, behavioral experiments were conducted to assess the mice's learning and memory abilities. Subsequently, the mice were dissected, and brain tissue was collected. RT-qPCR was used to detect changes in GSK-3β mRNA expression in the brain tissue of AD model mice, and Western blotting was used to detect protein expression, exploring the inhibitory effect of the composition on GSK-3β in AD model mice. Western blotting was used to detect phosphorylation of Tau protein (Thr181 and Ser396 sites) in brain tissue, and changes in MDA / T-AOC / SOD indices were detected to further explore the protective effect of the composition on AD model mice.

[0134]

Experimental Results

[0135] 1. Effects of traditional Chinese medicine composition on oxidative stress-related indicators in an AD cell model

[0136] observe Figure 1 It was found that different mass ratios of GAS and GLT reduced the effective concentrations of each component, but the traditional Chinese medicine composition still exhibited a certain protective effect against oxidative stress damage in cells. Specifically, compared to the AD group, GAS group, and GLT group, the traditional Chinese medicine composition showed the following: when the mass ratio was 1:1, the MDA level was significantly reduced; after a mass ratio of 4:1, the cell SOD activity and MDA level recovered compared to the model group, and p < 0.05, indicating a statistically significant difference. This suggests that the traditional Chinese medicine composition C obtained at a mass ratio of 4:1 has a protective effect against oxidative stress damage in the AD cell model.

[0137] 2. Effects of traditional Chinese medicine composition on Tau protein phosphorylation level in AD cell model

[0138] like Figure 2 As shown, in combinations of GAS and GLT at different mass ratios, treatment with the traditional Chinese medicine (TCM) composition reduced the hyperphosphorylation of Tau protein at the Ser396 site. TCM composition C showed a significant difference compared to the AD group (p<0.01), indicating that the inhibitory effect of TCM composition C on Tau protein phosphorylation was significantly better than that of TCM compositions A and B. Furthermore, hyperphosphorylation of Tau protein at the Thr181 site was significantly downregulated only under the action of TCM composition C, with a p<0.05 effect compared to the AD group. These results indicate that in the AD cell model, TCM composition C has a significant regulatory effect on the phosphorylation level of Tau protein, reducing phosphorylation at both the Ser396 and Thr181 sites. This suggests that even at low concentrations of a single component, the combination of GAS and GLT can also have a significant protective effect on the AD cell model. Compared with the GAS and GLT groups, TCM composition C significantly inhibited the hyperphosphorylation of Tau protein at the Ser396 and Thr181 sites, and can better improve the pathological condition of AD.

[0139] 3. Effects of traditional Chinese medicine composition C on GSK-3β expression levels in an AD cell model

[0140] Based on the combined results of oxidative stress and Tau protein phosphorylation, the traditional Chinese medicine composition C has a significant effect on reducing oxidative stress damage and inhibiting excessive Tau protein phosphorylation. Therefore, further investigation is needed to determine whether the traditional Chinese medicine composition C downregulates GSK-3β protein and mRNA levels in the AD cell model.

[0141] The result is as follows Figure 3 As shown, observe Figure 3 It can be seen that in Aβ 25-35 After 24 hours of treatment, GSK-3β expression levels increased. GSK-3β levels in the GAS and GLT groups showed no significant change, while the traditional Chinese medicine composition C downregulated GSK-3β at both mRNA and protein levels compared to the AD group, showing a significant difference (p<0.05). This indicates that in the AD cell model, treatment with the traditional Chinese medicine composition downregulated GSK-3β, with a significantly better effect than the GAS and GLT groups, demonstrating a significant synergistic effect between GAS and GLT, which can help enhance the efficacy of single GAS or single GLT in the prevention and treatment of AD. Furthermore, the mechanism of action of the traditional Chinese medicine composition was verified to be: by inhibiting GSK-3β protein expression, thereby reducing Tau protein hyperphosphorylation.

[0142] 4. Animal behavioral experiments

[0143] In the Morris water maze experiment, the AD group mice traversed platforms significantly less and spent significantly less time exploring than the NC group, indicating that the AD group did not memorize the platform locations during training, and their learning and memory abilities were significantly weaker than the NC group. The PC group, after modeling and administration of donepezil hydrochloride, showed an increase in the number of platform traversals and the effective exploration time. (Observation) Figure 4 It was found that the number of times mice crossed the platform was significantly increased in the medium and high dose groups compared to the AD group (p<0.05). In the effective exploration time statistics, gavage administration of the medium and high doses of the traditional Chinese medicine composition C also showed a prolonged effective exploration time, with the medium dose group showing a significantly greater increase in exploration time compared to the AD group (p<0.01). These results indicate that the traditional Chinese medicine composition C can improve the learning and memory abilities of AD mice and alleviate cognitive impairment in AD mice.

[0144] 5. Effects of Traditional Chinese Medicine Composition C on Oxidative Stress Indicators in the Brain of an AD Mouse Model

[0145] An appropriate amount of mouse brain tissue was collected to detect the oxidative stress indicators MDA / T-AOC / SOD, exploring the effect of the traditional Chinese medicine composition C on an AD mouse model. The results are as follows: Figure 5 As shown in the figure, compared with the AD group, with the increase of the concentration of the traditional Chinese medicine composition C administered by gavage, the MDA content in the mouse brain tissue decreased, while the levels of T-AOC and SOD increased. The oxidative stress level in the low-dose group showed no significant change compared with the AD group, while the oxidative stress damage in the medium- and high-dose groups showed some recovery. In the medium-dose group, the recovery effect of MDA and SOD was significant, showing a significant difference compared with the AD group (p<0.01). This indicates that the traditional Chinese medicine composition C can reduce oxidative stress damage at medium and high doses and has a significant protective effect.

[0146] 6. Effects of Traditional Chinese Medicine Composition C on Tau Protein Phosphorylation Levels in the Brain of an AD Mouse Model

[0147] The phosphorylation levels of Tau protein at Thr181 and Ser396 sites in mouse brain tissue were investigated, and the results are as follows: Figure 6 As shown in the figure, compared with the NC group, the combined use of D-gal and aluminum trichloride induced hyperphosphorylation of Tau protein at two sites in the mouse brain tissue, further demonstrating the successful establishment of the AD mouse model. After gavage administration of traditional Chinese medicine composition C, a certain concentration of composition C also inhibited the excessive activation of Tau protein after modeling. The medium-dose and high-dose groups significantly reduced the abnormal phosphorylation level of Tau protein in the brain tissue, showing significant differences compared with the AD group (p values ​​less than 0.05), indicating that a certain concentration of composition C has a neuroprotective effect on the AD mouse model, and composition C has a significant ameliorative effect on AD.

[0148] 7. Effects of traditional Chinese medicine composition C on GSK-3β levels in the brain of an AD mouse model

[0149] GSK-3β is widely expressed in tissues, and dysregulation of this kinase affects Tau protein phosphorylation. Based on the experimental results in [6. Effects of Traditional Chinese Medicine Composition C on Tau Protein Phosphorylation Levels in the Brain of an AD Mouse Model], a certain concentration of Traditional Chinese Medicine Composition C can inhibit the hyperphosphorylation of Tau protein. Therefore, the applicant further examined the level of GSK-3β in brain tissue. The results are as follows... Figure 7 As shown in the results, q-PCR and Western blotting revealed that GSK-3β levels decreased to varying degrees with increasing concentration of the traditional Chinese medicine composition C. The GSK-3β levels in the medium-dose and high-dose groups were significantly lower than those in the AD group (p<0.05). In conclusion, both medium- and high-dose traditional Chinese medicine composition C exhibit significant inhibitory effects on GSK-3β.

Claims

1. A traditional Chinese medicine composition for treating Alzheimer's disease, characterized in that, The components are gastrodin and ganoderic triterpenoids, with a mass ratio of gastrodin to ganoderic triterpenoids of 4:

1. The extraction method of gastrodin is as follows: gastrodin powder is ultrasonically extracted with 70% ethanol solution, then filtered, centrifuged, evaporated and concentrated, then separated and purified, eluted with 70% ethanol solution, concentrated and dried to obtain gastrodin. The extraction method of Ganoderma lucidum triterpenes is as follows: Ganoderma lucidum powder is ultrasonically extracted with 75% ethanol solution at 80℃, then filtered, centrifuged and combined, and the filtrates are evaporated, separated and purified, eluted, concentrated and dried to obtain Ganoderma lucidum triterpenes; wherein the elution method is: gradient elution with 15% ethanol solution, 35% ethanol solution and 95% ethanol solution in sequence.

2. The traditional Chinese medicine composition as described in claim 1, characterized in that, The traditional Chinese medicine composition is formulated with pharmaceutically acceptable excipients to produce one of the following dosage forms: powder, tablet, granule, and liquid preparation.

3. A method for preparing the traditional Chinese medicine composition as described in claim 1 or 2, characterized in that, Gastrodin was dissolved in sterile water, and Ganoderma triterpenes were dissolved in ethanol or DMSO solution and then diluted with sterile water and refrigerated for later use. Then, the two solutions were mixed at a mass ratio of 4:1 (gastrodin to Ganoderma triterpenes) to prepare the traditional Chinese medicine composition.

4. The use of the traditional Chinese medicine composition as described in claim 1 or 2 in the preparation of drugs for the prevention and treatment of Alzheimer's disease.

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