Use of xiaoyao powder in preparation of drugs for treating alzheimer's disease

Abstract

The application belongs to the technical field of biological medicine, and provides an application of Xiaoyao San in preparation of an Alzheimer's disease treatment drug, wherein the Xiaoyao San comprises the following raw materials in parts by weight: 1 part of Radix Bupleuri, 1 part of Radix Angelicae Sinensis, 1 part of Radix Paeoniae Alba, 1 part of fried Rhizoma Atractylodis Macrocephalae, 1 part of Poria cocos, and 0.5 part of fried Radix Glycyrrhizae; the Xiaoyao San reduces A beta load in the brain, promotes A beta degradation, inhibits the NLRP3 inflammasome pathway, reduces IL-1 beta and IL-18 inflammatory factor levels, and improves histopathological damage of the hippocampus and the medial prefrontal cortex; the Xiaoyao San inhibits A beta1-42 fiber aggregation, reduces ROS and MDA, increases SOD activity, reduces TUNEL positive cells, and inhibits neural cell apoptosis. The application defines a new use of Xiaoyao San for Alzheimer's disease treatment, and provides a new traditional Chinese medicine drug selection for AD treatment; compared with a single target drug, the Xiaoyao San has a multi-target synergistic effect feature, and can simultaneously act on multiple pathological links such as blood-brain barrier penetration, A beta aggregation, neural inflammation, oxidative stress and cell apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of Xiaoyao San in the preparation of drugs for the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that commonly affects the elderly. Its core pathological features include senile plaques formed by the aggregation of β-amyloid protein (Aβ), neurofibrillary tangles formed by abnormal phosphorylation of tau protein, and neuronal damage, activated inflammatory responses, and oxidative stress imbalance. Clinically, AD patients mainly exhibit progressive cognitive decline, learning and memory impairment, and mood abnormalities, severely impacting their quality of life and placing a heavy burden on families and society.

[0003] Currently, treatments for Alzheimer's disease still have significant limitations, including at least the following shortcomings:

[0004] First, existing treatments are mostly symptomatic or single-target interventions, which are difficult to fully block or delay the complex pathological process of Alzheimer's disease.

[0005] Second, some drugs have problems such as limited efficacy, insufficient safety or poor patient compliance during long-term use, making it difficult to meet the needs of long-term management of chronic diseases.

[0006] Third, existing technologies are insufficient for the redevelopment and expansion of indications of classic Chinese medicine formulas in Alzheimer's disease, especially lacking systematic verification from multiple levels such as blood-brain barrier penetration, Aβ aggregation, inflammasomes, oxidative stress and apoptosis pathways.

[0007] Xiaoyao San is a classic traditional Chinese medicine formula, mainly composed of Bupleurum, Angelica sinensis, Paeonia lactiflora, Atractylodes macrocephala, Poria cocos, and Glycyrrhiza uralensis. Traditionally, it is believed to have effects such as soothing the liver and strengthening the spleen, nourishing blood and regulating menstruation. Modern pharmacological research suggests that Xiaoyao San may act on the central nervous system, improving mood, regulating nerve function, and potentially protecting cognition. Although Xiaoyao San is a classic formula with a long history of clinical application, its technical approach, preparation methods, key mechanisms, and dosage form protection range for the treatment of Alzheimer's disease have not yet been systematically proposed.

[0008] Therefore, finding drugs with high safety, suitable for long-term administration, and capable of intervening in the pathological process of Alzheimer's disease at multiple targets is an important problem that urgently needs to be solved in this field. Summary of the Invention

[0009] The purpose of this invention is to provide the application of Xiaoyao San in the preparation of Alzheimer's disease treatment drugs, aiming to solve the problems mentioned in the background art.

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

[0011] The application of Xiaoyao San in the preparation of drugs for treating Alzheimer's disease, wherein Xiaoyao San comprises the following raw materials in parts by weight: 1 part Bupleurum, 1 part Angelica sinensis, 1 part Paeonia lactiflora, 1 part stir-fried Atractylodes macrocephala, 1 part Poria cocos, and 0.5 parts prepared Glycyrrhiza uralensis.

[0012] The Xiaoyao San improves pathological damage to the hippocampus and medial prefrontal cortex by reducing the Aβ load in the brain, promoting Aβ degradation, inhibiting the NLRP3 inflammasome pathway, reducing the levels of IL-1β and IL-18 inflammatory factors.

[0013] The Xiaoyao San inhibits Aβ1-42 fiber aggregation, reduces ROS and MDA, increases SOD activity, reduces TUNEL-positive cells, and inhibits nerve cell apoptosis.

[0014] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0015] This invention clarifies a new use of Xiaoyao San in the treatment of Alzheimer's disease, expands the clinical indications of classic Chinese medicine formulas, and provides new Chinese medicine drug options for AD treatment;

[0016] Compared to single-target drugs, Xiaoyao San has the characteristic of multi-target synergistic effect, and can act on multiple pathological links such as blood-brain barrier penetration, Aβ aggregation, neuroinflammation, oxidative stress and cell apoptosis at the same time.

[0017] In vivo animal experiments have confirmed that Xiaoyao San can improve the learning and memory abilities, cognitive functions and emotional state of AD model mice, and the improvement effect is more obvious in the medium-dose group and the high-dose group.

[0018] Pathological and molecular tests have confirmed that Xiaoyao San can reduce the Aβ load in the brain, promote Aβ degradation, inhibit the NLRP3 inflammasome pathway, reduce the levels of inflammatory factors such as IL-1β and IL-18, and improve the pathological damage of hippocampal and medial prefrontal cortex tissues.

[0019] In vitro experiments have confirmed that Xiaoyao San has low cytotoxicity and can inhibit Aβ1-42 fiber aggregation, reduce ROS and MDA, increase SOD activity, reduce TUNEL positive cells, and inhibit nerve cell apoptosis.

[0020] Meanwhile, Xiaoyao San is a classic Chinese medicine compound with a wide range of medicinal materials, mature preparation technology, and relatively low cost. It is suitable for development into various dosage forms such as decoction, capsule, tablet, granule or oral liquid, and has good prospects for industrialization and clinical transformation. Attached Figure Description

[0021] Figure 1The effect of Xiaoyao San intervention on Aβ42 expression in mouse brain tissue provided in this embodiment of the invention; A: Representative images of Aβ42 in the CA1 region of the hippocampus in different groups, where A: blank group, B. model group, C. MCC950 group, DF. high, medium and low dose groups of Xiaoyao San; B: Quantitative analysis of Aβ42 expression level in the CA1 region of the hippocampus in different groups; **p < 0.01;

[0022] Figure 2 The effects of Xiaoyao San provided in this embodiment of the invention on Aβ1-40, Aβ1-42 and NEP in the hippocampus of AD mice; A: ELISA detection of Aβ1-40 level in mouse hippocampus; B: ELISA detection of Aβ1-42 level in mouse hippocampus; C: ELISA detection of NEP level in mouse hippocampus; **p < 0.01;

[0023] Figure 3 The effect of Xiaoyao San provided in this embodiment of the invention on the level of inflammatory factors in the hippocampus of AD mice; A: ELISA detection of IL-18 level in mouse hippocampus; B: ELISA detection of IL-1β level in mouse hippocampus; **p < 0.01;

[0024] Figure 4 The effect of Xiaoyao San provided in this embodiment of the invention on the expression of NLRP3 inflammasome-related protein in the hippocampus of AD mice; A: representative immunoblot image; BF: quantitative analysis results of the expression of NLRP3, Caspase-1, iNOS, COX2 and ASC proteins in the mouse hippocampus; **p < 0.01;

[0025] Figure 5 The effect of Xiaoyao San provided in this embodiment of the invention on the expression of NLRP3 inflammasome-related mRNA in mouse hippocampus; A: NLRP3 mRNA expression level in mouse hippocampus; B: ASC mRNA expression level in mouse hippocampus; C: Caspase-1 mRNA expression level in mouse hippocampus; **p < 0.01;

[0026] Figure 6 The effect of Xiaoyao San provided in this embodiment of the invention on the level of oxidative stress induced by Aβ1-42 in Bend.3 cells; A: MDA level in Bend.3 cells; B: ROS level in Bend.3 cells; C: SOD activity in Bend.3 cells; **p < 0.01;

[0027] Figure 7The effect of Xiaoyao San provided in this embodiment of the invention on the expression of apoptosis-related proteins induced by Aβ1-42 in Bend.3 cells; AD: Western blot detection of the expression levels of apoptosis-related proteins Bcl-2, Bax and Caspase-3 in Bend.3 cells; **p < 0.01. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] The Xiaoyao San used in this embodiment of the invention is prepared according to the following preparation method:

[0030] The ingredients are prepared according to the following proportions: Bupleurum chinense (1 part), Angelica sinensis (1 part), Paeonia lactiflora (1 part), Atractylodes macrocephala (fried) (1 part), Poria cocos (1 part), and Glycyrrhiza uralensis (processed) (0.5 parts by weight). First, the raw materials are selected and briefly rinsed with clean water to remove impurities and sand. After draining, Bupleurum chinense, Angelica sinensis, Paeonia lactiflora, Atractylodes macrocephala (fried), and Poria cocos are cut into 2-5mm segments or thick slices. Glycyrrhiza uralensis is used directly. Then, the herbs are air-dried in a clean, hot air environment at 40-50℃ until the moisture content is ≤12%. The pre-treated herbs are then placed in a multi-functional extraction tank, and 8-10 times their weight of purified water is added. The mixture is then soaked in a sealed container at room temperature for 30-60 minutes. After simmering at a gentle boil for 1.5–2 hours, the decoction is filtered through a 100-mesh screen. The residue is then simmered again with 6–8 times its weight of purified water for 1–1.5 hours and filtered once more. The two extracts are combined to obtain the combined extract of Xiaoyao San. The extract is transferred to a vacuum concentration tank and concentrated to a relative density of 1.15–1.25 at 60°C under a vacuum of -0.06–0.08 MPa and 60–70°C, yielding Xiaoyao San clear extract. The clear extract can be prepared into a dry powder using two methods: one is vacuum drying: the clear extract is spread into a layer ≤2 cm thick and dried under a vacuum of -0.06–0.08 MPa and 60–70°C. Drying can be done by: 1) Drying at 0.07-0.09 MPa and 55-65℃ until the moisture content of the dry extract is ≤5%, then pulverizing and passing through an 80-100 mesh sieve; 2) Spray drying: After adjusting the consistency of the clear extract, it is fed into a spray dryer, with the inlet air temperature controlled at 160-180℃, the outlet air temperature at 80-90℃, and the atomization pressure at 0.2-0.3 MPa. The powder is then collected and passed through an 80-mesh sieve. Various dosage forms can be prepared using the obtained dry extract powder or clear extract as raw materials. For decoctions, the dry extract powder can be dissolved in purified water and dispensed, or the combined extract can be directly sterilized and filled. For capsules, the dry extract powder is mixed with starch and microcrystalline cellulose, ethanol is added to make a soft mass, which is then granulated through a 16-mesh sieve, dried at 50℃, sized, and filled into hollow hard capsules, which are then polished and packaged in aluminum-plastic packaging. Tablets are prepared by granulating and drying the dry extract powder with starch, magnesium stearate, and sodium carboxymethyl starch, then pressing it into tablets using a rotary tablet press, and finally coating it with film as needed before packaging. Granules are prepared by mixing the dry extract powder with sucrose and dextrin, granulating it with ethanol as a wetting agent, drying it at 45–55°C, sizing it through a 14-mesh sieve, and quantitatively dispensing it into composite film bags. Oral liquids are prepared by mixing Xiaoyao San extract with purified water, stevioside or honey, and potassium sorbate, adjusting the pH to 4.0–6.0 with citric acid, filtering through a 0.22μm precision filter, sterilizing with flowing steam at 105°C for 15 minutes, and then aseptically filling and sealing. Experimental solutions are prepared by accurately weighing Xiaoyao San dry extract powder, dissolving it with sterile physiological saline for animal experiments using ultrasonication, and preparing 20... For cell experiments, drug solutions of g / kg, 10 g / kg, and 5 g / kg concentrations were dissolved and filtered for sterilization using cell-grade sterile culture medium or buffer, and then serially diluted to obtain working solutions of different concentrations. All solutions were prepared and used immediately.

[0031] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1: Experiment on the effect of Xiaoyao San on cognitive function in AD model mice:

[0033] 1. Experimental materials: APP / PS1 double transgenic AD model mice, 6-8 weeks old, male, weighing 20-25 g; normal wild-type mice were used as blank control group; Xiaoyao San was dissolved in physiological saline to prepare drug solutions of different concentrations; the experimental apparatus included Morris water maze, Y maze, Barnes maze, new object recognition experimental apparatus and open field experimental apparatus.

[0034] 2. Experimental grouping: Transgenic AD model mice were randomly divided into the model group, the high-dose Xiaoyao San group (20 g / kg), the medium-dose Xiaoyao San group (10 g / kg), the low-dose Xiaoyao San group (5 g / kg), and the positive control group (donepezil, 1 mg / kg), with 10 mice in each group; normal wild-type mice served as the blank control group, with 10 mice in each group.

[0035] 3. Administration method: Mice in all groups were administered the drug orally once daily for 12 consecutive weeks; the blank control group and the model group were given an equal volume of physiological saline.

[0036] 4. Detection methods: Morris water maze was used to record escape latency, number of platform crossings, and time spent in the target quadrant; Y maze was used to record the rate of spontaneous alternation behavior; Barnes maze was used to record escape latency and number of errors; new object recognition experiment was used to record the discrimination index; open field experiment was used to record the total movement distance and time spent in the central area.

[0037] 5. Experimental Results: As shown in Tables 1 and 2, compared with the model group, the escape latency of mice in each dose group of Xiaoyao San was significantly shortened, and the number of platform crossings, the time spent in the target quadrant, the rate of spontaneous alternation behavior, the discrimination index, and the time spent in the central region were all significantly increased, while the number of errors was significantly reduced. The improvement effects were more significant in the medium-dose group and the high-dose group, and were comparable to those in the positive control group. The results indicate that Xiaoyao San can significantly improve the learning and memory abilities and cognitive functions of AD model mice.

[0038] Table 1. Effects of Xiaoyao San on the total number of arm insertions and spontaneous alternation rate in mice of each group.

[0039] Blank group 36.60±4.92 0.64±0.21 Model group 33.70±6.72 0.49±0.15 Positive control group 36.60±7.99 0.57±0.11 High-dose group 34.60±7.45 0.62±0.12 medium dose group 37.50±6.58 0.59±0.07 low-dose group 37.60±7.22 0.64±0.13

[0040] Table 2. Escape latency and number of platforms crossed by mice in each group during the Morris water maze experiment.

[0041] Example 2: Effects of Xiaoyao San on intracranial pathological markers in AD model mice:

[0042] 1. Experimental materials: The animals and drugs in Example 1 were used, along with Aβ detection kit, inflammatory factor detection kit, Western blot reagents, real-time quantitative PCR reagents, and HE staining reagents;

[0043] 2. Experimental Methods: Mice were sacrificed 12 weeks after drug administration, and brain tissue was collected. The hippocampus and medial prefrontal cortex were isolated. ELISA was used to detect the expression levels of Aβ1-40, Aβ1-42, Aβ1-42 oligomers, NEP, IL-1β, and IL-18 in the brain. Western blot was used to detect the expression levels of iNOS, COX2, NLRP3, ASC, Pro-caspase-1, Caspase-1 p20, and apoptosis-related proteins Bcl2, Bax, caspase9, and caspase3. Real-time quantitative PCR was used to detect the mRNA expression levels of NLRP3, ASC, and Caspase-1. HE staining was used to observe the histopathological changes in the hippocampus and medial prefrontal cortex.

[0044] 3. Experimental results: such as Figures 1 to 5 As shown, compared with the model group, the levels of Aβ1-40, Aβ1-42, and Aβ1-42 oligomers in the brains of mice in each dose group of Xiaoyao San were significantly reduced, while the expression level of NEP was significantly increased; the expression levels of IL-1β and IL-18 were decreased, and the expression levels of iNOS and COX2 were downregulated; the expression levels of NLRP3, ASC, Pro-caspase-1, and Caspase-1 p20 proteins and their mRNA transcription levels were all reduced; the expression level of Bcl2 was increased, while the expression levels of Bax, caspase9, and caspase3 were decreased; and the pathological damage of the hippocampus and medial prefrontal cortex was improved. The results indicate that Xiaoyao San can improve the pathological state in the brains of AD model mice by regulating Aβ metabolism, inhibiting inflammatory responses, and promoting neuronal apoptosis.

[0045] Example 3: Blood-brain barrier penetration and cytotoxicity experiments of Xiaoyao San:

[0046] 1. Experimental Materials: A blood-brain barrier model was constructed using human brain microvascular endothelial cells (HBMEC), and a neural cell injury model was constructed using SH-SY5Y cells; the drug used was Xiaoyao San extract; experimental reagents included Transwell chambers, CCK-8 reagent, ThT reagent, ROS detection kit, SOD detection kit, MDA detection kit, TUNEL staining kit, and Western blot related reagents.

[0047] 2. Blood-brain barrier penetration experiment: A Transwell model of HBMEC monolayer cells was constructed. The extract of Xiaoyao San was added to the upper chamber of the Transwell chamber. After culturing for a certain period of time, the content of the characteristic components of Xiaoyao San in the lower chamber was detected by high performance liquid chromatography, and the penetration rate was calculated. The results showed that the characteristic components of Xiaoyao San could penetrate the HBMEC monolayer cell model, and the penetration rate gradually increased with the prolongation of time, indicating that Xiaoyao San has good blood-brain barrier penetration;

[0048] 3. Cytotoxicity and protection experiment: The CCK-8 assay was used to detect the toxicity of Xiaoyao San to SH-SY5Y cells. The results showed that within a certain concentration range, Xiaoyao San had no significant effect on the survival rate of SH-SY5Y cells, suggesting low cytotoxicity. An SH-SY5Y cell injury model induced by Aβ1-42 fibrils was established, and a normal control group, a model group, a low-concentration group of Xiaoyao San, a medium-concentration group of Xiaoyao San, and a high-concentration group of Xiaoyao San were set up. The aggregation of Aβ1-4 fibrils was observed by ThT staining. The results showed that Xiaoyao San could significantly inhibit the aggregation of Aβ1-42 fibrils.

[0049] 4. Detection of oxidative stress and apoptosis: Detection kits for ROS, SOD, and MDA were used to detect oxidative stress indicators. The results were as Figure 6 shown. Xiaoyao San could reduce the levels of ROS and MDA in the cells of the model group and increase the activity of SOD; TUNEL staining and Western blot were used to detect cell apoptosis. The results were as Figure 7 shown. Xiaoyao San could reduce the number of TUNEL-positive cells, up-regulate the expression of Bcl2, down-regulate the expressions of Bax, caspase9, and caspase3, and inhibit the apoptosis of nerve cells induced by Aβ1-42 fibrils.

[0050] Example 4. Preparation of a pharmaceutical preparation of Xiaoyao San for treating Alzheimer's disease:

[0051] Taking Xiaoyao San as the active ingredient and using the conventional process of traditional Chinese medicine preparations, it can be prepared into decoctions, capsules, tablets, granules or oral liquids. In the preparation process, pharmaceutically acceptable excipients, diluents, fillers, disintegrants, binders, flavoring agents or stabilizers can be added according to the specific dosage form. The preparation is preferably administered orally, and the dosage can be converted and adjusted according to the patient's age, weight, disease severity and the screening results of animal experiments;

[0052] This example shows that the pharmaceutical preparation of Xiaoyao San for treating Alzheimer's disease has the characteristics of flexible dosage form development, mature preparation process, wide raw material sources, and controllable production cost, and is suitable for further industrialization transformation.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Xiaoyao San in the preparation of drugs for the treatment of Alzheimer's disease.

2. The application of Xiaoyao San according to claim 1 in the preparation of Alzheimer's disease treatment drugs, characterized in that, The Xiaoyao Powder comprises the following ingredients in parts by weight: 1 part Bupleurum, 1 part Angelica sinensis, 1 part Paeonia lactiflora, 1 part stir-fried Atractylodes macrocephala, 1 part Poria cocos, and 0.5 parts prepared Glycyrrhiza uralensis.

3. The application of Xiaoyao San according to claim 1 in the preparation of Alzheimer's disease treatment drugs, characterized in that, The Xiaoyao San formula improves pathological damage in the hippocampus and medial prefrontal cortex by reducing the Aβ load in the brain, promoting Aβ degradation, inhibiting the NLRP3 inflammasome pathway, reducing the levels of IL-1β and IL-18 inflammatory factors.

4. The application of Xiaoyao San according to claim 1 in the preparation of Alzheimer's disease treatment drugs, characterized in that, The Xiaoyao San inhibits Aβ1-42 fiber aggregation, reduces ROS and MDA, increases SOD activity, reduces TUNEL-positive cells, and inhibits nerve cell apoptosis.

5. The application of Xiaoyao San according to claim 1 in the preparation of Alzheimer's disease treatment drugs, characterized in that, The dosage form of Xiaoyao San is one of the following: decoction, capsule, tablet, granule, or oral liquid.

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