Use of Xinshubao tablets in preparing medicines for preventing and / or treating senile dementia

Abstract

The present invention discloses the use of Xinshubao tablets in preparing a drug for preventing and / or treating senile dementia. The present invention uses an AD animal model experiment constructed with scopolamine to determine that Xinshubao tablets improve the spatial learning and memory function of senile dementia mice by improving cortical and hippocampal tissue pathological damage, hippocampal neuron ultrastructure damage, and the cholinergic nervous system in the brain, and improving the neurotrophic state and synaptic function in the brain. The application of Xinshubao tablets in preventing and treating senile dementia has broad application prospects.

Description

Technical Field

[0001] The present invention specifically relates to the use of Xinshubao tablets in preparing medicines for preventing and / or treating senile dementia. Background Art

[0002] Dementia refers to a group of syndromes with cognitive impairment as the main clinical manifestation caused by various reasons such as neurodegeneration, cerebrovascular disease, infection, trauma, tumor, nutritional metabolism disorder, etc., which are usually more common in the elderly. It often manifests as multiple cognitive impairments such as orientation, memory, learning, language comprehension, and thinking. Most dementia patients also show abnormal behavior. This leads to a decline or loss of occupational and social functions. Dementia has a high prevalence, a long course of disease, a high disability and mortality rate, and a heavy social burden. The most common types of dementia are senile dementia (AD) and vascular dementia (VaD). The treatment methods of the two are different due to different causes. It is generally believed that vascular dementia can be treated through early active and effective treatment to achieve the treatment goal, while senile dementia, also known as Alzheimer's disease, has an important pathogenesis of abnormal cholinergic system function in the hippocampus. Therefore, up to now, there is no particularly effective treatment for it.

[0003] Xinshubao tablets are composed of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn. Among them, salvia miltiorrhiza can promote blood circulation and remove blood stasis, clear the heart and relieve restlessness, curcuma can promote blood circulation and relieve pain, promote qi and relieve depression, white peony root can nourish blood and restrain yin, soften the liver and relieve pain, acanthopanax senticosus can invigorate qi and strengthen spleen, invigorate kidney and calm the mind, and hawthorn can promote qi and disperse blood stasis, remove turbidity and reduce fat. The combination of all the medicines can play the effects of promoting blood circulation and removing blood stasis, invigorating qi and relieving pain. At present, it is mainly used clinically to treat coronary heart disease, chest tightness caused by qi deficiency and blood stasis, angina pectoris, and hypertension, hyperlipidemia and arteriosclerosis. There is no application research on the prevention or treatment of dementia, especially senile dementia, on Xinshubao tablets. Summary of the invention

[0004] To solve the above problems, the present invention provides a composition for use in preparing a drug for preventing and / or treating senile dementia, wherein the composition is a preparation prepared with salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn as raw materials and pharmaceutically acceptable excipients.

[0005] Furthermore, the drug is a drug for improving spatial learning and memory functions.

[0006] Furthermore, the drug is a drug that improves pathological damage of cortical and hippocampal tissues.

[0007] Furthermore, the drug is a drug for improving ultrastructural damage of hippocampal neurons.

[0008] Furthermore, the drug is a drug that improves the function of the cholinergic nervous system in the brain.

[0009] Furthermore, the drug is a drug that improves the neurotrophic state and synaptic function in the brain.

[0010] Furthermore, the mass ratio of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn in the composition is 150-250: 1500-2500: 200-400: 700-1300: 1500-2500.

[0011] Furthermore, the mass ratio of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn in the composition is 200:2000:300:1000:2000.

[0012] Furthermore, the preparation is Xinshubao tablets.

[0013] Scopolamine is a non-selective muscarinic acetylcholine receptor antagonist that causes central cholinergic dysfunction, thereby impairing learning ability and short-term memory. In addition, scopolamine can also cause central nervous system neurotrophic defects, oxidative stress, neuroinflammatory reactions, and aggravate Aβ protein accumulation, which can better simulate the pathological characteristics of AD patients.

[0014] The use of Xinshubao tablets of the present invention in preparing a drug for preventing and / or treating senile dementia, and an AD animal model experiment constructed with scopolamine, determined that Xinshubao tablets improve the spatial learning and memory function of senile dementia mice by improving cortical and hippocampal tissue pathological damage, hippocampal neuron ultrastructure damage, and the cholinergic nervous system in the brain, and improving the neurotrophic state and synaptic function in the brain. The application of Xinshubao tablets in preventing and treating senile dementia has broad application prospects.

[0015] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0016] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Xinshubao tablets improved the cognitive function of model mice (A: Schematic diagram of novel object recognition experiment; B: Movement trajectory of mice in novel object recognition experiment; C: Statistical graph of recognition index of each group of mice in novel object recognition experiment; D: Schematic diagram of Y maze experiment; E: Movement trajectory of mice in Y maze experiment; F: Statistical graph of spontaneous alternation times of each group of mice in Y maze experiment; G: Typical graph of swimming trajectory of mice in each group, H: Statistical graph of escape latency of mice in each group; I: Statistical graph of the number of times mice in each group crossed the platform; J: Statistical graph of the percentage of target quadrant residence time of mice in each group. #, ## compared with blank control group p<0.05, p<0.01; *, * compared with model control group p<0.05, p<0.01, n=12)

[0018] Figure 2 H&E staining and Nissl staining results (A: typical HE staining picture; B: typical Nissl staining picture, C: Nissl body statistical picture; #, ## compared with blank control group p<0.05, p<0.01; *, * compared with model control group p<0.05, p<0.01, n=3)

[0019] Figure 3 Ultrastructure of CA1 neurons in mouse hippocampus (A: typical image of mitochondria, B: typical image of synapses, C: statistical image of the number of damaged mitochondria, D: statistical image of the number of synapses; #, ## compared with blank control group p<0.05, p<0.01; *, * compared with model control group p<0.05, p<0.01, n=3)

[0020] Figure 4 Xinshubao tablets improved the cholinergic nervous system in the brain of model mice (A: statistical graph of Ach levels in the brains of mice in each group; B: statistical graph of AchE activity in the brains of mice in each group; C: typical graph of CHAT and CHT1 protein blots in the brains of mice in each group; D: statistical graph of CHAT and CHT1 protein expression levels in the brains of mice in each group. #, ## compared with blank control group p<0.05, p<0.01; *, * compared with model control group p<0.05, p<0.01)

[0021] Figure 5 Xinshubao tablets promoted the expression of BDNF and synaptic proteins in the brains of model mice (A: Western blot typical images of the expression of BDNF, SYN and PSD95 proteins in the brains of each group of mice; B: Statistical graph of relative expression of BDNF protein; C: Statistical graph of relative expression of SYN protein; D: Statistical graph of relative expression of PSD95 protein. ##, ### compared with the blank control group p<0.01, p<0.001; *, * compared with the model control group p<0.05, p<0.01). DETAILED DESCRIPTION

[0022] The raw materials, equipment and reagents used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products. Among them, Xinshubao tablets are provided by Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd., which are prepared from Salvia miltiorrhiza, White Peony Root, Curcuma Radix, Acanthopanax Senticosus and Crataegus Pinellia with a mass ratio of 200:2000:300:1000:2000 as raw materials.

[0023] Example 1 Study on the application of Xinshubao tablets in treating senile dementia

[0024] 1. Experimental Methods

[0025] 1.1 Experimental animals

[0026] Male C57BL / 6J mice, 6-8 weeks old, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. (certificate number: SCXK (Beijing) 2019-0010). The animal room temperature was 20-25°C, the relative humidity was 40%-60%, and the light was maintained for 12 hours per day, with a day and night cycle, and free access to food and water. After one week of adaptive feeding, the experiment began. The experiment was approved by the Experimental Animal Welfare Ethics Committee of the Medical Experiment Center of the China Academy of Chinese Medical Sciences (ERCCACMS21-2201-01).

[0027] 1.2 Grouping and Dosing

[0028] The experiment was divided into the following 6 groups: blank control group (Control), model control group (Model), three dose groups of Xinshubao tablets (low (XSB-L), medium (XSB-M), and high (XSB-H), and positive control group (Donepezil), with 12 mice in each group. Mice in the blank control group were intraperitoneally injected with normal saline, and mice in the other groups were intraperitoneally injected with 3 mg / kg scopolamine hydrobromide solution once a day for 3 consecutive weeks to cause cognitive dysfunction. At the same time as modeling, the Xinshubao tablets group was gavaged with 3.75 g / kg, 7.5 g / kg, and 15 g / kg of Xinshubao tablets (calculated according to the amount of crude drug) every day, the blank control group and the model control group were gavaged with an equal volume of CMC-Na, and the positive control group was gavaged with 0.65 mg / kg of Donepezil hydrochloride CMC-Na suspension.

[0029] 1.3 Behavioral experiments to detect cognitive function of mice in each group

[0030] Starting from the 21st day of drug administration, novel object recognition test, Y maze test and Morris water maze test were performed to detect the cognitive function of mice in each group:

[0031] (1) New object recognition experiment: The experiment was divided into three stages: adaptation period, training period and test period. Adaptation period: Each mouse was placed in a new object recognition box and allowed to move freely for 5 min to adapt to the environment. After 24 h, the training period experiment was conducted. Training period: Two identical objects were placed in the middle of the bottom of the box. Each mouse was allowed to explore freely in the box for 5 min. After 1 h, the test period experiment was conducted. Training period: One object in the training period was replaced with an object of different shape and similar size. Each mouse was placed in the box and allowed to explore freely for 5 min. The mouse activities were recorded, and the ANY-Maze software was used to analyze the time each mouse spent exploring the new object T1 and the time each mouse spent exploring the old object T2. The cognitive index RI of each mouse was calculated as T1 / (T1+T2).

[0032] (2) Self-alternation Y-maze test: After the novel object recognition test, a Y-maze spontaneous alternation test was conducted to evaluate the working memory of each group of mice. The mice were placed at the end of one arm and allowed to explore freely for 5 minutes. The behavior trajectory of the mice was recorded using a camera system, and the spontaneous alternation rate of the mice was analyzed using ANY-Maze software.

[0033] (3) Morris water maze experiment: After the Y-maze experiment, the Morris water maze experiment was performed to detect the spatial learning and memory abilities of mice in each group. ① Spatial exploration experiment: The escape platform was 1 cm below the water surface, and the water temperature was 20-21°C. Each of the four quadrants was trained once a day. During the training, the mice were gently placed in the water from a 1 / 2 arc in any quadrant, facing the pool wall. If the mice could not find the platform within 60 seconds, they were led to the platform and stayed there for 20 seconds. The training was continued for 5 days. The escape latency of each group of mice before going on stage was recorded to evaluate the spatial learning ability of the mice; ② Positioning navigation experiment: On the 6th day, the platform was removed, and the mice were placed in the water from the quadrant opposite to the original platform, facing the pool wall. The number of times the mice crossed the platform within 60 seconds and the percentage of time they stayed in the quadrant where the platform was located were detected and analyzed to evaluate the spatial memory ability of the mice.

[0034] 1.4 Preparation of paraffin sections

[0035] After the water maze experiment, three mice were taken from each group and anesthetized with 50 mg / kg sodium pentobarbital. The heart was exposed and 20 mL of normal saline and 4% paraformaldehyde solution were perfused through the left ventricle in sequence. The brain was taken out and fixed with 4% paraformaldehyde for 8 h. The brain was dehydrated, immersed in wax, and embedded in paraffin to prepare paraffin tissue blocks. The brain was cut into 3 μm sections using a paraffin slicer and stored in a refrigerator at 4°C.

[0036] 1.5 H&E staining experiment

[0037] Brain tissue paraffin sections were baked for 2 h, dewaxed conventionally, stained with hematoxylin (60°C) for 60 s, washed off with running water for 5-10 s, differentiated with 1% hydrochloric acid ethanol for 3 s, washed with water for 1-2 s, rinsed with running water for 10 min, stained with 0.5% eosin solution for 30-60 s, washed with distilled water for 1-2 s, 80% ethanol for 1-2 s, 95% ethanol for 1-2 s, anhydrous ethanol I for 1-2 s, anhydrous ethanol II for 1-2 s, xylene I for 5 min, xylene II for 5 min, sealed with neutral gum, observed under a microscope and photographed.

[0038] 1.6 Nissl staining experiment

[0039] Brain tissue paraffin sections were baked for 2 h, routinely dewaxed, stained with Nissl staining solution for 10 min (37°C, protected from light), washed with distilled water for 5 s, 95% ethanol for 2 min, xylene I for 5 min, xylene II for 5 min, sealed with neutral gum, observed under a microscope and photographed.

[0040] 1.7 Detection of Ach and AchE levels in brain tissue

[0041] After the water maze experiment, 6 mice were selected from each group. The brains were removed after deep anesthesia, and the cortex and hippocampus were retained. A 10% tissue homogenate was prepared and the protein concentration was determined by the BCA method. The Ach and AchE levels in the brain tissues were detected according to the method in the kit instructions.

[0042] 1.8 Transmission electron microscopy to detect ultrastructural damage of hippocampal neurons

[0043] After the water maze experiment, the mice were deeply anesthetized by intraperitoneal injection of an overdose of sodium pentobarbital, and the hippocampal tissue was quickly excised and fixed in 2% glutaraldehyde (pH = 7.2). The hippocampal CA1 region was selected and cut into 1.0 × 1.0 × 1.0 mm 3 The tissues were blocked and fixed with 1% osmium tetroxide for 2 h at room temperature. After dehydration, infiltration, embedding, and polymerization, the tissues were cut into 70 nm sections and stained with 2% uranyl acetate saturated alcohol solution and 2.6% lead citrate in sequence. Finally, the sections were observed and photographed under TEM (HT7700, Hitachi, Tokyo, Japan). Image J software was used to analyze the number of mitochondria and synapses.

[0044] 1.9 Western blot experiment

[0045] Total hippocampal protein was extracted, and the protein concentration was determined by BCA method. Loading buffer was added and boiled for 5 minutes. Proteins were separated by SDS-PAGE gel electrophoresis, and wet transfer was performed at 250 mA for 80 minutes. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, and the primary antibodies: CHAT / CHT1 / BDNF / SYN and PSD95 were incubated at 4°C overnight, and then the corresponding secondary antibodies were incubated. The gel imager was used to take pictures, and the grayscale value was analyzed by Image J. The protein expression of each group was compared after normalization with the internal reference (β-tubulin).

[0046] 1.10 Statistical analysis

[0047] Data were expressed as mean ± standard deviation (mean ± sd), and statistical analysis was performed using GraphPad Prism 9.0 software. The escape latency data in the water maze experiment were analyzed using repeated measures two-way ANOVA combined with Bonferroni post-hoc test; other data were compared between multiple groups using one-way ANOVA combined with Tukey's post-hoc test, and two-tailed Students' test was used for comparison between two groups. p < 0.05 indicated that the difference was statistically significant. Fluorescence intensity and gray value were semi-quantitatively analyzed using Image J software.

[0048] 2. Experimental results

[0049] 2.1 Xinshubao Tablets Improve Cognitive Function in Model Mice

[0050] The results of the novel object recognition experiment showed that compared with the blank control group, the recognition index of the model group mice was significantly reduced, indicating that the short-term memory function of the model mice was impaired; XSB-L, XSB-M and XSB-H could significantly improve the recognition index of the model mice, and the effect was equivalent to that of the positive drug donepezil hydrochloride ( Figure 1 AC), indicating that Xinshubao tablets can improve the short-term memory function of model mice.

[0051] The results of the Y maze showed that compared with the blank control group, the spontaneous alternation rate of the model group mice was significantly reduced, indicating that the working memory of the model mice was impaired; XSB-H could significantly improve the recognition index of the model mice, and its effect was equivalent to that of the positive drug donepezil ( Figure 1 DF), indicating that Xinshubao tablets can improve the working memory function of model mice.

[0052] The results of Morris water maze showed that compared with the blank control group, the escape latency of the mice in the model group increased significantly, the number of crossing the platform decreased significantly, and the percentage of time spent in the target quadrant decreased significantly, indicating that the spatial learning and memory function of the model mice was impaired. The escape latency of the mice in the Xinshubao tablets group gradually shortened with the extension of training time. On the 4th and 5th days of training, there were significant differences between the XSB-L and XSB-H groups and the model group. In the spatial exploration experiment, the number of crossing the platform and the percentage of time spent in the target quadrant of the mice in the Xinshubao tablets group increased, and there were significant differences between the XSB-L and XSB-H groups and the model group ( Figure 1 HJ), which was equivalent to the positive control drug donepezil hydrochloride, indicating that Xinshubao tablets can improve the spatial learning and memory function of model mice.

[0053] 2.2 Xinshubao Tablets Improve Cortical and Hippocampal Tissue Structural Damage in Model Mice

[0054] After the water maze experiment, H&E staining and Nissl staining were used to detect the damage of the cortical and hippocampal tissues of mice in each group. The results of H&E staining showed that the neurons in the cortex, hippocampal CA1, CA3 and DG regions of the model group were loosely arranged, with shrunken and darkly stained nuclei, while the neurons in the XSB-L, XSB-M and XSB-H groups were neatly and tightly arranged, with round nuclei and uniform staining of the nucleus and cytoplasm ( Figure 2 A). Nissl staining results showed that the neurons in the cortex and hippocampal CA1, CA3 and DG regions of the model group were loosely arranged, the Nissl body staining was reduced, and the number was reduced. The neurons in the Xinshubao tablets group were compactly arranged, the number of Nissl bodies increased, and the staining was deepened. The number of Nissl bodies in the cortex and hippocampal CA1 and DG regions of the XSB-M and XSB-H groups was significantly different from that of the model group, and the effect was equivalent to that of the positive control drug donepezil hydrochloride ( Figure 2 BC). The above results show that Xinshubao tablets can effectively improve the pathological damage of the cortex and hippocampus tissues of model mice.

[0055] 2.3 Xinshubao Tablets Improve the Ultrastructure of Hippocampal Neurons in Model Mice

[0056] Transmission electron microscopy was used to observe the ultrastructure of hippocampal CA1 neurons in each group of mice. Figure 3 As shown in the figure, the double-layered nuclear membrane structure of neurons in the model group was unclear, mitochondrial cristae were missing, mitochondrial vacuolization was obvious, and the number of synapses decreased, indicating that continuous intraperitoneal injection of scopolamine damaged the ultrastructure of hippocampal neurons. After administration of Xinshubao tablets, the mitochondrial morphology returned to the typical long spindle shape, the mitochondrial cristae were clearly visible, the double-layered nuclear membrane structure was intact, and the number of synapses increased significantly, which was equivalent to the positive control drug donepezil hydrochloride ( Figure 3AD). The above results indicate that Xinshubao tablets can effectively improve the ultrastructural damage of hippocampal neurons in VaD model mice.

[0057] 2.4 Xinshubao Tablets Improve the Cholinergic Nervous System in the Brain of Model Mice

[0058] like Figure 4 As shown, compared with the blank control group, the Ach level in the brain of the model group mice was significantly reduced ( Figure 4 A), AchE activity increased significantly ( Figure 4 B), CHAT and CHT1 protein expressions were significantly decreased ( Figure 4 CD), indicating that the cholinergic nervous system of the cortex and hippocampus of the model mice was damaged; XSB-H could significantly increase the Ach level in the brain of the model mice and reduce the activity of AchE. At the same time, both XSB-M and XSB-H could increase the expression levels of CHAT and CHT1 proteins, indicating that Xinshubao tablets can effectively improve the cholinergic nervous system in the brain of the model mice.

[0059] 2.5 Xinshubao tablets promote the expression of BDNF and synaptic proteins in the brain of model mice

[0060] like Figure 5 As shown, compared with the blank control group, the expression levels of brain-derived neurotrophic factor BDNF, synaptic protein SYN and PSD95 in the brain of the model mice were significantly reduced. XSB-H can significantly increase the protein levels of BDNF, SYN and PSD95 in the brain of the model mice, and its effect is equivalent to that of the positive drug donepezil hydrochloride, indicating that Xinshubao tablets can effectively improve the neurotrophic status and synaptic function in the brain of the model mice.

[0061] 3. Discussion

[0062] The cholinergic system in the hippocampus plays an important role in memory formation and cognition. The cognitive impairment caused by its functional decline is considered to be one of the important pathogenesis of Alzheimer's disease (AD). Cholinergic neurons are mainly located in the basal nucleus and septal diagonal complex of the basal forebrain, providing the main source of cholinergic innervation for the hippocampus and cerebral cortex. Neurotransmitters transmit signals from neurons in the basal forebrain to neurons in the hippocampus. This process involves the synthesis, packaging, secretion and clearance of neurotransmitters. Choline and acetyl-CoA are synthesized into acetylcholine (ACh) under the catalysis of choline acetyltransferase (ChAT). The latter is transported from the cytoplasm to synaptic vesicles under the action of vesicular acetylcholine transporter (VAChT). The ACh stored in the synaptic vesicles is released into the synaptic cleft when the neuron discharges, completing the signal transduction. Next, some ACh is hydrolyzed by acetylcholinesterase (AChE) to produce choline and acetate, and choline is taken up by the high-affinity choline transporter (CHT) on the presynaptic plasma membrane and used again by ChAT for ACh synthesis. Therefore, any abnormality in these processes may lead to abnormal cholinergic function and cause cognitive dysfunction.

[0063] Scopolamine is a non-selective muscarinic acetylcholine receptor antagonist that causes central cholinergic dysfunction, thereby impairing learning ability and short-term memory. In addition, scopolamine can also cause central nervous system neurotrophic defects, oxidative stress reactions, neuroinflammatory reactions, and aggravate Aβ protein accumulation, which can better simulate the pathological characteristics of AD patients. Therefore, the scopolamine-induced cognitive dysfunction model (amnesia model) has been widely used to evaluate the anti-dementia effect of drugs. In this experiment, a dementia mouse model was prepared by intraperitoneal injection of scopolamine, and its cognitive function was detected using a variety of behavioral methods. It was fully confirmed that Xinshubao tablets can improve the short-term memory, working memory and spatial learning memory abilities of scopolamine-induced dementia model mice. The action pathway and characteristics of Xinshubao tablets in improving the cognitive function of dementia model mice were further clarified from the tissue pathology level and protein expression level. It was found that Xinshubao tablets can improve brain tissue pathological damage in dementia model mice, reduce neuronal ultrastructural damage, improve the cholinergic nervous system, and increase neurotrophic levels and synaptic protein levels. It is expected to be developed into a new drug for the treatment of AD.

Claims

1. Use of a composition in the preparation of a drug for preventing and / or treating senile dementia, characterized in that: The composition is a preparation prepared by using salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn as raw materials and adding pharmaceutically acceptable excipients; The mass ratio of salvia miltiorrhiza, white peony root, curcuma, acanthopanax senticosus and hawthorn in the composition is 200:2000:300:1000:2000.

2. The use according to claim 1, characterized in that: The drug is a drug for improving spatial learning and memory functions.

3. The use according to claim 2, characterized in that: The medicine is a medicine for improving pathological damage of cortical and hippocampal tissues.

4. The use according to claim 2, characterized in that: The medicine is a medicine for improving ultrastructural damage of hippocampal neurons.

5. The use according to claim 2, characterized in that: The drug is a drug that improves the function of the cholinergic nervous system in the brain.

6. The use according to claim 4, characterized in that: The drug is a drug that improves neurotrophic status and synaptic function in the brain.

7. The use according to claim 1, characterized in that: The preparation is Xinshubao tablets.

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