Pharmaceutical composition for treating Alzheimer's disease and preparation method and application thereof

Abstract

The invention relates to the technical field of pharmaceutical compositions, and particularly discloses a pharmaceutical composition for treating Alzheimer's disease and a preparation method and application thereof. The pharmaceutical composition provided by the invention consists of the following components in percentage by weight: 60-80% of human umbilical cord mesenchymal stem cells and 20-40% of a traditional Chinese medicine extract, the traditional Chinese medicine extract is prepared by mixing a cortex moutan aqueous extract and a caragana sinica rehd alcohol extract in a weight ratio of (1-5): (5-15). The invention further provides a preparation method and application of the pharmaceutical composition. The pharmaceutical composition prepared by the invention can effectively relieve the symptoms of the Alzheimer's disease and prolong the life of a patient.

Description

Technical Field

[0001] This application relates to the technical field of pharmaceutical compositions, specifically to a pharmaceutical composition for treating Alzheimer's disease, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease is the most common neurodegenerative disease among the elderly and the leading cause of dementia. Its core characteristics are progressive cognitive decline (such as memory loss, language impairment, and impaired judgment) and behavioral abnormalities, which ultimately severely affect the patient's daily living abilities and place a heavy burden on families and society.

[0003] Currently, there is no cure for Alzheimer's disease. Therefore, research on drug compositions for treating Alzheimer's disease is an urgent medical need and of social significance. Summary of the Invention

[0004] To address the aforementioned technical problems, this application provides a pharmaceutical composition for treating Alzheimer's disease, its preparation method, and its application.

[0005] In a first aspect, this application provides a pharmaceutical composition comprising the following components by weight percentage: 60-80% human umbilical cord mesenchymal stem cells and 20-40% traditional Chinese medicine extract; wherein the traditional Chinese medicine extract is composed of a mixture of peony bark water extract and schizonepeta root alcohol extract in a weight ratio of 1-5:6-15.

[0006] The human umbilical cord mesenchymal stem cells in the pharmaceutical composition provided in this application can secrete a variety of anti-inflammatory factors (such as IL-10, TGF-β, etc.), inhibit the excessive activation of microglia, and transform them from the harmful M1 phenotype to the protective M2 phenotype, thereby alleviating the inflammatory environment and creating favorable conditions for neuronal survival. Human umbilical cord mesenchymal stem cells can also secrete a series of neurotrophic factors (BDNF, NGF, GDNF, etc.), promoting the survival of existing neurons, enhancing synaptic plasticity, improving learning and memory functions, and potentially stimulating endogenous neurogenesis. In addition, human umbilical cord mesenchymal stem cells have immunomodulatory functions, which can regulate peripheral and central immune cells, reduce the attack of immune cells on the brain, and indirectly protect neurons; they can also activate microglia through paracrine effects, enhancing their ability to phagocytose and clear Aβ plaques, thus effectively alleviating the symptoms of Alzheimer's disease.

[0007] The aqueous extract of Paeonia suffruticosa is rich in effective components such as paeonol, which can significantly inhibit the activation of microglia, downregulate the expression of pro-inflammatory factors such as TNF-α and IL-1β, thereby alleviating neuroinflammation in AD models. It also possesses strong free radical scavenging capabilities, directly reducing oxidative stress damage. Simultaneously, it interferes with the aggregation process of Aβ peptides, reducing the formation of fibrillary Aβ and decreasing the toxicity of existing Aβ to neurons. Furthermore, it helps maintain the function of the blood-brain barrier, reducing the entry of harmful substances into the brain and improving brain homeostasis. The alcoholic extract of Zygophyllum truncatum has powerful antioxidant properties and activates antioxidant pathways, initiating the expression of a series of endogenous antioxidant and detoxification enzymes, fundamentally enhancing the neuronal resistance to oxidative stress. It can inhibit pro-inflammatory signaling pathways such as NF-κB, reducing the production of inflammatory factors; and reduce the production of Aβ and promote its clearance through multiple pathways, while also potentially inhibiting the hyperphosphorylation of Tau protein.

[0008] The pharmaceutical composition provided in this application utilizes a combination of human umbilical cord mesenchymal stem cells, peony root water extract, and schizonepeta root alcohol extract. With human umbilical cord mesenchymal stem cells as the core, it leverages their powerful paracrine and immunomodulatory functions to improve the brain microenvironment, nourish neurons, and reduce inflammation. Simultaneously, the peony root water extract and schizonepeta root alcohol extract, as highly effective small-molecule natural compounds, provide support from multiple levels, including anti-inflammatory, antioxidant, and Aβ inhibition, creating favorable conditions for stem cell survival and function. The synergistic effect of these three components constitutes a comprehensive therapeutic system targeting the multiple pathological mechanisms of Alzheimer's disease (AD), thus demonstrating significant efficacy in animal models.

[0009] Preferably, the pharmaceutical composition comprises the following components by weight percentage: 65-75% human umbilical cord mesenchymal stem cells and 25-35% traditional Chinese medicine extract.

[0010] Preferably, the pharmaceutical composition comprises the following components by weight percentage: 67-73% human umbilical cord mesenchymal stem cells and 27-33% traditional Chinese medicine extract.

[0011] In one specific embodiment, the pharmaceutical composition comprises the following components in weight percentages: 60% human umbilical cord mesenchymal stem cells and 40% traditional Chinese medicine extract; 65% human umbilical cord mesenchymal stem cells and 35% traditional Chinese medicine extract; 70% human umbilical cord mesenchymal stem cells and 30% traditional Chinese medicine extract; 75% human umbilical cord mesenchymal stem cells and 25% traditional Chinese medicine extract; and 80% human umbilical cord mesenchymal stem cells and 20% traditional Chinese medicine extract.

[0012] Experimental analysis shows that the drug composition composed of 60-80 wt% human umbilical cord mesenchymal stem cells and 20-40 wt% traditional Chinese medicine extracts in this application further improves the application effect of the drug composition.

[0013] Preferably, the number of cells in the human umbilical cord mesenchymal stem cells is 6-10 × 10⁻⁶. 8 The cell / mL count was 0.5-1.5 × 10⁻⁶ cells / mL, and the CD34 cell count was 0.5-1.5 × 10⁻⁶ cells / mL. 7 cell / mL.

[0014] Preferably, the herbal extract is composed of a mixture of peony root water extract and schizonepeta root alcohol extract in a weight ratio of 2-4:7-13.

[0015] Preferably, the herbal extract is composed of a mixture of peony root water extract and schizonepeta root alcohol extract in a weight ratio of 2.5-3.5:8-12.

[0016] In one specific implementation, the weight ratio of the aqueous extract of Paeonia suffruticosa and the alcoholic extract of Zygophyllum molybdites in the herbal extract can be 1:6, 2:6, 3:6, 4:6, 5:6, 1:7, 2:7, 3:7, 4:7, 5:7, 1:10, 2:10, 3:10, 4:10, 1:13, 2:13, 3:13, 4:13, 5:13, 1:15, 2:15, 3:15, or 4:15.

[0017] Experimental analysis shows that the present application utilizes a mixture of peony root water extract and strychnine root alcohol extract in the above weight ratio to form a traditional Chinese medicine extract, which further improves the application effect of the drug composition.

[0018] Preferably, the method for preparing the peony bark water extract is as follows: pulverize the peony bark into powder, add water and decoct 3-5 times, each decoction time is 1.5-3 hours; combine the filtrates, concentrate to a relative density of 1.1-1.16, add water to make up the volume, filter, sterilize, and the product is obtained.

[0019] Preferably, the preparation method of the ethanol extract of the root of the broom is as follows: the root of the broom is crushed into powder, and 50-80% ethanol extract is added and extracted under reflux for 3-5 times, with each extraction time being 1-2 hours; the filtrates are combined, concentrated to a relative density of 1.1-1.16, diluted with water to a fixed volume, filtered, and sterilized to obtain the extract.

[0020] Secondly, this application also provides the use of the pharmaceutical composition in the preparation of medicaments for the prevention or treatment of Alzheimer's disease.

[0021] Thirdly, this application also provides a medicament for the prevention or treatment of Alzheimer's disease, prepared using the aforementioned pharmaceutical composition.

[0022] In summary, the technical solution of this application has the following effects: This application Detailed Implementation

[0023] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application. Example

[0024] Example 1 Example 1 provides a pharmaceutical composition for treating Alzheimer's disease, its preparation method and application, and the preparation method thereof.

[0025] The preparation method of the pharmaceutical composition in Example 1 is as follows: (1) The preparation method of human umbilical cord mesenchymal stem cells is the same as the preparation method described in Example 1 of the patent application number 201610993092.7 (applied by the applicant on November 11, 2016). The specific preparation method is as follows.

[0026] Cell diluent: Prepare 2g of cell protectant consisting of 1.68g amino acids, 0.28g phospholipids, 0.02g adenine C, and 0.02g peptide growth factors; take 4.5g sodium chloride and the above cell protectant, add to water, dilute to 500ml, mix well, and set aside.

[0027] Cell precipitation agent: 6 wt% hydroxyethyl starch aqueous solution.

[0028] Layering agent: An aqueous solution with a density of 1.1 g / ml prepared from sucrose and meglumine diatrizoate.

[0029] The above-mentioned cell diluent, cell precipitant, and stratifying agent were sterilized at 115°C for 20 minutes, and their endotoxin content was tested to be ≤0.5 EU / ml before being bottled.

[0030] Procedure: Add 50ml of umbilical cord blood containing sodium citrate anticoagulant to 200ml of cell diluent, then add 100ml of cell precipitant, mix and shake well for 10min, let stand for 2h, and after separation, aspirate the upper cell liquid, dispense into centrifuge tubes, and centrifuge at 1500 rpm for 20min.

[0031] The lower layer of cell sap was collected, diluted with physiological saline, and evenly spread on a layering agent. The mixture was then centrifuged at 1500 rpm for 20 minutes. The middle, cloudy stem cell layer was collected, washed three times with physiological saline, and then diluted to 200 ml with physiological saline to obtain human umbilical cord mesenchymal stem cells with a cell count of 8 × 10⁶ cells. 8 cell / mL, CD34 cell count was 1×10⁻⁶ 7 cell / mL.

[0032] (2) The preparation method of the water extract of peony bark is as follows: Peony bark (peony bark sourced from Anguo Xuancao Trading Co., Ltd.) is pulverized into powder, and decocted 3 times with 8 times the amount of water, each time for 2 hours. The filtrates from the three decoctions are collected and combined, and the dregs are discarded. The combined filtrate is concentrated under vacuum at 65℃ to a relative density of 1.12 (measured at 60℃), cooled to room temperature, and water is added to make up to 1000ml. The solution is filtered and sterilized to obtain the final product.

[0033] (3) The preparation method of the ethanol extract of the root of the golden sparrow is as follows: the root of the golden sparrow (the root of the golden sparrow comes from Anguo Xuancao Trading Co., Ltd.) is crushed into powder, and 50-80% ethanol extract is added and refluxed for 3-5 times. Then, 8 times the amount of 70wt% ethanol aqueous solution is added and refluxed for 3 times, and the extraction time is 1 hour each time. The filtrates from the 3 extractions are collected and combined, and the residue is discarded. The combined filtrate is concentrated in vacuum at 65℃ to a relative density of 1.12 (measured at 60℃), cooled to room temperature, and water is added to make up to 1000ml. The solution is filtered and sterilized to obtain the extract.

[0034] (4) The pharmaceutical composition consists of the following components by weight percentage: 70wt% human umbilical cord mesenchymal stem cells and 30wt% traditional Chinese medicine extract; weigh 70g of human umbilical cord mesenchymal stem cells and 30g of traditional Chinese medicine extract (composed of a mixture of peony bark water extract and strychnine root alcohol extract in a weight ratio of 3:10), add them to sterile water, mix evenly, and the pharmaceutical composition is obtained.

[0035] Examples 2-7 Examples 2-7 provide a pharmaceutical composition for treating Alzheimer's disease, its preparation method, its application, and its preparation method.

[0036] The specific differences between the above embodiments and Embodiment 1 are as follows:

[0037] In Example 2, the pharmaceutical composition consisted of 60 wt% human umbilical cord mesenchymal stem cells and 40 wt% traditional Chinese medicine extract.

[0038] In Example 3, the pharmaceutical composition consisted of 80 wt% human umbilical cord mesenchymal stem cells and 20 wt% traditional Chinese medicine extract.

[0039] In Example 4: The herbal extract is composed of a mixture of Paeonia suffruticosa water extract and Sophora flavescens root alcohol extract in a weight ratio of 1:15.

[0040] In Example 5: The herbal extract is composed of a mixture of Paeonia suffruticosa water extract and Sophora flavescens root alcohol extract in a weight ratio of 5:6.

[0041] In Example 6: The herbal extract is composed of a mixture of Paeonia suffruticosa water extract and Sophora flavescens root alcohol extract in a weight ratio of 2:13.

[0042] In Example 7: The herbal extract is composed of a mixture of Paeonia suffruticosa water extract and Sophora flavescens root alcohol extract in a weight ratio of 4:7.

[0043] All other process parameters in the above embodiments are the same as those in Embodiment 1.

[0044] Comparative Example Comparative Examples 1-4 Comparative Examples 1-4 each provide a pharmaceutical composition, its preparation method, and its application, as well as the preparation method thereof.

[0045] The difference between the above comparative example and Example 1 is as follows.

[0046] In Comparative Example 1, the drug composition consisted of 30 wt% human umbilical cord mesenchymal stem cells and 70 wt% traditional Chinese medicine extract.

[0047] In Comparative Example 2: an equal amount of Paeonia lactiflora water extract was used instead of Paeonia suffruticosa water extract (Paeonia lactiflora was sourced from Anguo Xuancao Trading Co., Ltd.); the preparation method of Paeonia lactiflora water extract was the same as that of Paeonia suffruticosa water extract.

[0048] In Comparative Example 3, an equal amount of aqueous extract of *Broomcornu* root was used instead of alcoholic extract of *Broomcornu* root; the preparation method of aqueous extract of *Broomcornu* root was the same as that of aqueous extract of *Paeonia suffruticosa* root.

[0049] In Comparative Example 4: the Chinese herbal extract was composed of a mixture of peony bark water extract and sage root alcohol extract in a weight ratio of 10:3.

[0050] All other process parameters in the above comparative examples are the same as those in Example 1.

[0051] Performance testing This experiment, referencing information from "Pharmacological Experimental Methodology" and "Pharmacological Research Methodology of Traditional Chinese Medicine" and related literature, used the Morris water maze experiment to investigate the therapeutic effect of the drug compositions prepared in the examples or comparative examples on AD model mice.

[0052] Experimental animal groups: Normal control group: age-matched, strained wild-type mice (without AD pathological background) were given physiological saline; AD model group: AD model mice (APP / PS1 double transgenic mice) were given the same physiological saline as the NC group; Drug treatment group: AD model mice were given the test drug composition prepared in the examples or comparative examples.

[0053] Experimental equipment: Morris water maze apparatus: a circular pool with four equidistant visual markers (cards of different colors, used as references for the mouse's spatial localization) attached to the inner wall; the pool is filled with clean water at a temperature of 22-25℃, with the water level 2cm higher than the hidden platform (10cm in diameter and 2cm lower than the water level), so that the platform is completely hidden underwater.

[0054] Video tracking system: A camera installed directly above the pool is connected to a computer and behavior analysis software to automatically record the mouse's movement trajectory, swimming speed, and dwell time.

[0055] During the experiment, the laboratory environment was kept quiet and the lighting was stable (to avoid noise / light changes interfering with the mice's visual localization), and the position of the visual markers remained unchanged throughout the experiment.

[0056] (1) Phase 1: Positioning and navigation experiment to assess spatial learning ability Mice were trained to locate a hidden platform using visual markers. The difficulty they faced in finding the platform was recorded to assess their spatial learning ability. The pool was divided into four quadrants (A, B, C, and D). The platform was fixed in the center of quadrant A, and its position remained unchanged throughout the exercise. Each day, one entry point was randomly selected from the four quadrants (each entry point was approximately 10 cm from the pool wall), and the mouse was placed in the water with its face facing the pool wall, while video tracking was initiated.

[0057] Recording metrics: Escape latency: the time from when the mouse enters the water to when it finds and climbs onto the platform; Total distance traveled: the total distance traveled from when the mouse enters the water to when it finds and climbs onto the platform.

[0058] After each training session, allow the mice to remain on the platform for 20 seconds to reinforce their memory, then dry them with a towel and return them to their cages. Repeat this training for 7 consecutive days.

[0059] (2) Phase 2: Spatial exploration experiment to assess spatial memory retention ability To avoid interference from immediate memory, the experiment was conducted on the third day after the navigation experiment. After removing the hidden platform, the visual marker position remained unchanged, and the mice were placed into the water from the opposite quadrant C (the original platform was in quadrant A) of the original platform quadrant, and video tracking was initiated.

[0060] Record indicators: Number of times the mouse seeks a safe platform: Observe whether the mouse can remember the original platform location within 120 seconds and record the number of times it seeks a safe platform to reflect the long-term retention effect of memory; Percentage of time spent in the target quadrant: The proportion of time the mouse spends in quadrant A, where the original platform is located, out of the total exploration time of 120 seconds.

[0061] Test results are shown in Table 1.

[0062] Table 1. Performance test results of the pharmaceutical compositions in the examples and comparative examples. The test results in Table 1 above show that, compared with the normal control group, the AD model mice exhibited abnormal learning and memory. In the navigation test, they showed prolonged latency and total distance traveled; in the spatial exploration test, they showed a decrease in the number of times they sought safe platforms and a decrease in the percentage of time spent in the target quadrant, indicating that the mouse model was exhibiting symptoms of Alzheimer's disease. Compared with the AD model group, after treating AD model mice with the drug composition prepared in this application, the mice showed a shortened latency and a reduced total distance traveled in the navigation test; and an increase in the number of times they sought safe platforms and the percentage of time spent in the target quadrant in the spatial exploration test. This indicates that the mice's learning speed was accelerated, their memory retention was enhanced, and their behavioral indicators approached those of normal mice. This demonstrates that the drug composition prepared according to the technical solution of this application can effectively alleviate the symptoms of Alzheimer's disease model mice.

[0063] In Comparative Example 1, the drug composition consisted of 30 wt% human umbilical cord mesenchymal stem cells and 70 wt% traditional Chinese medicine extract. In Comparative Example 2, an equal amount of white peony root water extract was used instead of peony bark water extract. In Comparative Example 3, an equal amount of sage root water extract was used instead of sage root alcohol extract. In Comparative Example 4, the traditional Chinese medicine extract was composed of a mixture of peony bark water extract and sage root alcohol extract in a weight ratio of 10:3. The prepared drug compositions showed low efficacy in alleviating the symptoms of Alzheimer's disease model mice.

[0064] In contrast, this application utilizes a pharmaceutical composition consisting of 60-80 wt% human umbilical cord mesenchymal stem cells and 20-40 wt% traditional Chinese medicine extracts, and a traditional Chinese medicine extract consisting of a mixture of Paeonia suffruticosa water extract and Sophora flavescens root alcohol extract in a weight ratio of 1-5:6-15. The resulting pharmaceutical composition can effectively alleviate the symptoms of Alzheimer's disease in mouse models.

[0065] By comparing the test results of Examples 1-3, it can be seen that the present application further improves the application effect of the drug composition by controlling the composition to consist of 65-75wt% human umbilical cord mesenchymal stem cells and 25-35wt% traditional Chinese medicine extract.

[0066] By comparing the test results of Examples 1 and 4-7, it can be seen that the present application utilizes a mixture of Paeonia suffruticosa water extract and Sophora flavescens alcohol extract in a weight ratio of 2-4:7-13 to form a traditional Chinese medicine extract, which further improves the application effect of the drug composition.

[0067] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It is composed of the following components by weight percentage: 60-80% human umbilical cord mesenchymal stem cells and 20-40% traditional Chinese medicine extract; the traditional Chinese medicine extract is composed of a mixture of peony bark water extract and sage root alcohol extract in a weight ratio of 1-5:6-15.

2. The pharmaceutical composition according to claim 1, characterized in that, It consists of the following components by weight percentage: 65-75% human umbilical cord mesenchymal stem cells and 25-35% traditional Chinese medicine extracts.

3. The pharmaceutical composition according to claim 1, characterized in that, It consists of the following components by weight percentage: 67-73% human umbilical cord mesenchymal stem cells and 27-33% traditional Chinese medicine extracts.

4. The pharmaceutical composition according to claim 1, characterized in that, The number of cells in the human umbilical cord mesenchymal stem cells is 6-10 × 10⁻⁶. 8 The cell / mL count was 0.5-1.5 × 10⁻⁶ cells / mL, and the CD34 cell count was 0.5-1.5 × 10⁻⁶ cells / mL. 7 cell / mL.

5. The pharmaceutical composition according to claim 1, characterized in that, The herbal extract is composed of a mixture of peony root water extract and strychnine root alcohol extract in a weight ratio of 2-4:7-13.

6. The pharmaceutical composition according to claim 1, characterized in that, The herbal extract is composed of a mixture of peony root water extract and strychnine root alcohol extract in a weight ratio of 2.5-3.5:8-12.

7. The pharmaceutical composition according to claim 1, characterized in that, The method for preparing the peony bark water extract is as follows: pulverize the peony bark into powder, add water and decoct 3-5 times, each decoction time is 1.5-3 hours; combine the filtrates, concentrate to a relative density of 1.1-1.16, add water to make up the volume, filter, sterilize, and the product is obtained.

8. The pharmaceutical composition according to claim 1, characterized in that, The preparation method of the ethanol extract of the root of the golden privet is as follows: the root of the golden privet is crushed into powder, and 50-80% ethanol extract is added and refluxed for 3-5 times, with each extraction time being 1-2 hours; the filtrates are combined, concentrated to a relative density of 1.1-1.16, water is added to make up the volume, filtered, and sterilized to obtain the extract.

9. Use of the pharmaceutical composition according to any one of claims 1-8 in the preparation of a medicament for the prevention or treatment of Alzheimer's disease.

10. A drug for the prevention or treatment of Alzheimer's disease, characterized in that, Prepared using the pharmaceutical composition according to any one of claims 1-8.

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