Traditional Chinese medicine composition and application thereof in improvement of Alzheimer disease

Abstract

The invention belongs to the field of marine traditional Chinese medicines, and relates to a traditional Chinese medicine composition and application thereof in improvement of Alzheimer's disease, the traditional Chinese medicine composition comprises the following raw materials by weight: 2-7 parts of hippocampus, 5-16 parts of Chinese angelica, and 2-7 parts of acorus tatarinowii. The traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method is used for preparing medicines for treating and / or preventing Alzheimer's disease. The traditional Chinese medicine composition disclosed by the invention is extremely simple in medicine taste, free of excessive complex components and relatively mild in medicine property; compared with a compound with more medicinal components, the traditional Chinese medicine composition is safer and more reliable in the use process due to the simple formula, the possibility of interaction with other medicines possibly taken by a patient at the same time is relatively small, and the risk of possible adverse reaction is greatly reduced. The traditional Chinese medicine composition is suitable for being taken for a long time, and can bring continuous benefits to the human body when being used for daily life cultivation and health preservation or adjuvant therapy for specific diseases.

Description

Technical Field

[0001] The invention belongs to the field of marine traditional Chinese medicine, and specifically relates to a traditional Chinese medicine composition and application thereof in improving Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is the most common type of dementia, mainly characterized by gradually worsening memory loss and cognitive dysfunction. The pathogenesis of AD is complex, including amyloid β-protein (Aβ)-related neurodegeneration, neurofibrillary tangles (NFT)-related neurodegeneration, synaptic dysfunction, neurotransmitter imbalance, neuroinflammation, intestinal flora imbalance, oxidative stress, cell autophagy, lipid droplet accumulation, and genetic factors. The complexity of its pathogenesis has greatly limited the development of new clinical drugs.

[0003] Currently, the Alzheimer's disease drugs marketed worldwide are mainly divided into three types: acetylcholinesterase inhibitors (donepezil, rivastigmine, galantamine), N-methyl-D-aspartate receptor antagonists (memantine) and anti-Aβ monoclonal antibodies (adunatumab, lencanezumab). Among them, donepezil-memantine compound preparation is a combination of Western medicines used to treat Alzheimer's disease. Donepezil is a cholinesterase inhibitor that increases the concentration of acetylcholine in the brain by inhibiting the hydrolysis of acetylcholine, thereby improving cognitive function. Memantine is an N-methyl-D-aspartate (NMDA) receptor antagonist that can regulate glutamatergic neurotransmission, reduce excitotoxicity, and protect neurons. However, the above-mentioned drugs are often accompanied by side effects such as gastrointestinal and nervous system, and their safety cannot be guaranteed for long-term use. Therefore, finding a safe and effective therapeutic drug is an urgent problem to be solved.

[0004] AD can be classified as "forgetfulness" or "dementia" in traditional Chinese medicine. Kidney deficiency is its basic pathogenesis. As the disease progresses, pathological products such as blood stasis and phlegm will appear, and the symptoms are manifested as mixed deficiency and excess. The treatment is mainly based on kidney tonification, supplemented by phlegm-removing and blood stasis-removing methods. In traditional Chinese medicine compound prescriptions such as Liuwei Dihuang Wan Jiajian, the traditional Liuwei Dihuang Wan is mainly composed of Rehmannia glutinosa, Cornus officinalis, Chinese yam, Alisma orientalis, Paeonia suffruticosa, and Poria cocos, which are used to nourish kidney yin. In some studies on Alzheimer's disease, Liuwei Dihuang Wan is used as the basis for additions and subtractions, but the number of drugs in the prescription is still relatively large.

[0005] Western medicines currently used to treat AD have many side effects, and Chinese patent medicines used to treat AD have many medicinal flavors and complex compositions, making them unsuitable for long-term use. Therefore, developing a safe, effective, and simple Chinese medicine combination is of great significance for the long-term treatment of AD. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes a Chinese medicine composition and its application in improving Alzheimer's disease. The Chinese medicine composition has a simple composition, consisting of only three medicines, reducing the risk of drug interaction, and all of them are natural ingredients. It is very safe even for long-term medication, and has less toxicity and side effects than Western medicine. As a new therapeutic drug, it has great potential and application value in the treatment of AD.

[0007] The technical solution of the present invention is:

[0008] The invention provides a traditional Chinese medicine composition. The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 2 to 7 parts of seahorses, 5 to 16 parts of angelica sinensis and 2 to 7 parts of calamus tatarinowii.

[0009] The Chinese medicine composition is composed of three Chinese medicines: seahorse, angelica and calamus. Among them, seahorse, as a kidney-tonifying Chinese medicine, has the effects of tonifying the kidney and strengthening yang, and its peptides, minerals and other ingredients have been proven to have neuroprotective effects; angelica, as a Chinese medicine with a long history and significant efficacy, has been widely used in the clinical treatment of AD in ancient and modern times. It contains a variety of chemical components, among which volatile oil components such as ligustilide, organic acids such as ferulic acid and polysaccharides are the main active ingredients for its treatment of AD; calamus has the effects of opening the mind and resolving phlegm, refreshing the mind and improving intelligence, and can be used in combination with a variety of drugs to treat forgetfulness. The combination of the three drugs can comprehensively, multi-facetedly and multi-pathway regulate the human body, thereby exerting a systemic therapeutic effect; at the same time, the Chinese medicine composition has small toxic and side effects, good safety and tolerance, and can be taken for a long time.

[0010] Furthermore, the Chinese medicine composition is composed of the following raw materials in parts by weight: 4 parts of seahorse, 8 parts of angelica, and 4 parts of calamus.

[0011] The present invention also provides a method for preparing any of the above-mentioned Chinese medicine compositions, comprising the following steps:

[0012] Weigh seahorse, angelica and calamus according to the weight of each raw material, crush them and mix them, add ethanol in a ratio of 1:8-12 between the raw material mixture and the ethanol liquid, soak for 20-50 minutes, heat and reflux for extraction, and filter to obtain a filtrate;

[0013] The filtrates were combined, concentrated, and freeze-dried to obtain a powder.

[0014] Furthermore, the concentration of ethanol used is 50% to 90%. In the preparation process, ethanol with a concentration range of 50% to 90% is used as a solvent, and the Chinese medicine composition is extracted and prepared by heating and reflux. This preparation method has undergone rigorous scientific research and practical verification, and can effectively extract the active ingredients in hippocampus and angelica. The ethanol solvent in the above concentration range can better dissolve the active substances in Chinese medicinal materials, and the heating and reflux method can ensure the sufficiency and efficiency of the extraction process, so that the final Chinese medicine extract has higher purity and stronger efficacy.

[0015] Furthermore, the heating reflux temperature is 80-110° C., the time is 2-3 hours, and the reflux times are 2-5 times.

[0016] The present invention also provides use of any of the above-mentioned Chinese medicine compositions or the Chinese medicine compositions prepared by any of the above-mentioned preparation methods in preparing drugs for treating and / or preventing Alzheimer's disease.

[0017] The present invention further provides a traditional Chinese medicine preparation for improving Alzheimer's disease, wherein the effective ingredient is any one of the traditional Chinese medicine compositions described above or a traditional Chinese medicine composition prepared by any one of the preparation methods described above.

[0018] Furthermore, the Chinese medicine preparations include tablets, powders, decoctions, capsules, pills and granules.

[0019] Beneficial effects of the present invention:

[0020] (1) The Chinese medicine composition of the present invention has a very simple taste, does not have too many complex ingredients, and has a relatively mild medicinal property. Compared with compound prescriptions with more medicinal ingredients, this simple formula makes it safer and more reliable during use, and the possibility of interaction with other drugs that the patient may take at the same time is relatively small, greatly reducing the risk of possible adverse reactions.

[0021] At the same time, precisely because of its simple medicinal taste, it is very suitable for long-term use. Whether it is used for daily health care or as an auxiliary treatment for specific diseases, long-term use of this Chinese medicine composition can bring lasting benefits to the human body and maintain a good physical condition.

[0022] (2) The three Chinese medicines in the Chinese medicine composition synergistically improve symptoms, following the holistic concept of traditional Chinese medicine. They not only focus on a local symptom or a single target of the disease, but also improve the symptoms of AD patients through synergistic effects, through multiple pathways and multiple targets, and can regulate the body's qi and blood, viscera functions, etc. as a whole;

[0023] Because the composition contains the marine Chinese medicine seahorse, the human body tends to tolerate it better; compared with chemically synthesized drugs, the two Chinese medicines, seahorse and angelica, are already accepted by people in their daily diet and are safe even for long-term use; with the correct usage and dosage, there will be no obvious side effects, and it is highly safe, especially with less toxicity and side effects than Western medicine.

[0024] (3) Through a series of experiments and studies, it was found that the Chinese herbal extract obtained by the present invention has significant efficacy, can effectively improve PC12 cell damage, and provide strong support for cell repair and regeneration; at the same time, in the zebrafish AD model, the Chinese herbal composition can also significantly improve motor dysfunction; in the AD mouse model, it shows the potential for improving learning and memory disorders. This discovery provides new ideas and methods for the treatment of related diseases, and further proves the huge potential and value of the Chinese herbal composition.

[0025] (4) The three Chinese medicines proposed in the present invention have a long history of application in my country's traditional Chinese medicine culture. People have a certain cultural identity and trust in them. Compared with some newly developed Western medicines or unfamiliar foreign drugs, the public is more likely to accept Chinese medicine compositions prepared based on the concept of traditional Chinese medicine. This cultural familiarity is also conducive to their promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To investigate the effects of hippocampus, angelica and calamus alone or in combination on the survival rate of PC12 cells after injury.

[0027] Figure 2 To investigate the effects of the combination of hippocampus, angelica sinensis, and calamus tatarinowii, and the combination of the three herbs on the total movement distance and speed of the AD zebrafish model;

[0028] Figure 3 To study the effects of the combination of hippocampus, calamus and angelica on the body weight, kidney deficiency syndrome score and total distance in the open field of AD kidney deficiency mouse model;

[0029] Figure 4 Effects of hippocampus, calamus, angelica and kidney deficiency syndrome on water maze test, object recognition test and serum Aβ in AD mice model 1-42 The influence of content. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0032] Example 1

[0033] The Chinese medicine composition of this embodiment is prepared from the following raw materials in parts by weight: 4 parts of hippocampus, 8 parts of angelica, and 4 parts of calamus.

[0034] The preparation method of the Chinese medicine composition of this embodiment comprises the following steps:

[0035] Prepare the materials according to the above weight proportions, weigh 4g of seahorse, 8g of angelica, and 4g of calamus, respectively, with a weighing accuracy of ±0.01g. Crush the weighed seahorse, angelica, and calamus to a powder state with a particle size of less than 0.5mm, place them in a clean large beaker, and use a glass rod to gently stir the powder to fully mix them. Calculate the volume of 90% ethanol required according to the 10-fold amount of the solid-liquid ratio, accurately measure it, and slowly add it to the mixed medicinal powder, stirring with a glass rod while adding, so that the ethanol is in full contact with the medicinal powder. Seal the mixed medicinal ethanol solution with plastic wrap and soak it at room temperature for 35 minutes. During the soaking process, gently shake the container once every 10 minutes to allow the medicinal materials to fully contact with the ethanol and promote the dissolution of the medicinal ingredients.

[0036] Pour the soaked medicinal material ethanol solution into the reaction bottle of the reflux device, turn on the condensation water, place the reaction bottle in the electric heating jacket, adjust the temperature of the electric heating jacket to 80°C, and control the reflux time to 2.5h. After the reflux is completed, stop heating, wait for the solution to cool to room temperature, pour the cooled reflux liquid into the Buchner funnel, use a suction bottle to filter, and collect the filtrate.

[0037] Reflux 3 times, filter to obtain the filtrate, pour the combined filtrate into a rotary evaporation bottle, set the temperature to 50°C and the speed to 80rpm, and stop concentrating when the filtrate volume is concentrated to about 1 / 3 of the original volume to obtain a concentrated solution. Pour the concentrated solution evenly into a freeze drying plate and place it in a -80 degree refrigerator for 2 hours, transfer it to a freeze dryer, control the vacuum degree to 10Pa, the drying temperature to -20°C, and the drying time to 48 hours. After the freeze drying is completed, gradually increase the temperature to about 30°C and continue drying for 2 hours to remove residual moisture. After drying, take out the freeze drying plate and collect the obtained powder, which is the prepared Chinese medicine composition powder.

[0038] Example 2

[0039] The Chinese medicine composition of this embodiment is prepared from the following raw materials in parts by weight: 2 parts of hippocampus, 5 parts of angelica, and 2 parts of calamus.

[0040] The preparation method of the Chinese medicine composition of this embodiment comprises the following steps:

[0041] Prepare the materials according to the above weight proportions, weigh 2g of seahorse, 5g of angelica, and 2g of calamus, respectively, with a weighing accuracy of ±0.01g. Crush the weighed seahorse, angelica, and calamus to a powder state with a particle size of less than 0.5mm, and mix them thoroughly. Calculate and accurately measure the required volume of 50% ethanol according to the 8-fold amount of the solid-liquid ratio, and slowly add it to the mixed medicinal powder, stirring with a glass rod while adding to allow ethanol to fully contact the medicinal powder. Seal the mixed medicinal ethanol solution with plastic wrap and soak it at room temperature for 20 minutes. During the soaking process, gently shake the container once every 10 minutes to allow the medicinal materials to fully contact the ethanol and promote the dissolution of the medicinal ingredients.

[0042] Pour the soaked medicinal material ethanol solution into the reaction bottle of the reflux device, turn on the condensation water, place the reaction bottle in the electric heating jacket, adjust the temperature of the electric heating jacket to 90°C, and control the reflux time to 2h. After the reflux is completed, stop heating, wait for the solution to cool to room temperature, pour the cooled reflux liquid into the Buchner funnel, use a suction bottle to filter, and collect the filtrate.

[0043] Reflux twice, filter to obtain the filtrate, pour the combined filtrate into a rotary evaporation bottle, set the temperature to 55°C and the speed to 90rpm, and stop concentrating when the filtrate volume is concentrated to about 1 / 4 of the original volume to obtain a concentrated solution. Pour the concentrated solution evenly into a freeze drying plate and place it in a -80 degree refrigerator for 2.5 hours, transfer it to a freeze dryer, control the vacuum degree to 20Pa, the drying temperature to -15°C, and the drying time to 48 hours. After the freeze drying is completed, gradually increase the temperature to about 30°C, and continue drying for 2.5 hours to remove residual moisture. After the drying is completed, take out the freeze drying plate and collect the obtained powder, which is the prepared Chinese medicine composition powder.

[0044] Example 3

[0045] The Chinese medicine composition of this embodiment is prepared from the following raw materials in parts by weight: 7 parts of hippocampus, 16 parts of angelica sinensis, and 7 parts of calamus tatarinowii.

[0046] The preparation method of the Chinese medicine composition of this embodiment comprises the following steps:

[0047] Prepare the materials according to the above weight proportions, weigh 7g of seahorse, 16g of angelica, and 7g of calamus, respectively, with a weighing accuracy of ±0.01g. Crush the weighed seahorse, angelica, and calamus to a powder state with a particle size of less than 0.5mm, and stir to fully mix them. Calculate and accurately measure the required volume of 70% ethanol according to the 12-fold amount of the solid-liquid ratio, and slowly add it to the mixed medicinal powder, stirring with a glass rod while adding to make the ethanol fully contact with the medicinal powder. Seal the mixed medicinal ethanol solution with plastic wrap and soak it at room temperature for 50 minutes. During the soaking process, gently shake the container once every 10 minutes to make the medicinal materials fully contact with the ethanol and promote the dissolution of the medicinal ingredients.

[0048] Pour the soaked medicinal material ethanol solution into the reaction bottle of the reflux device, turn on the condensation water, place the reaction bottle in the electric heating jacket, adjust the temperature of the electric heating jacket to 100°C, and control the reflux time to 3h. After the reflux is completed, stop heating, wait for the solution to cool to room temperature, pour the cooled reflux liquid into the Buchner funnel, use a suction bottle to filter, and collect the filtrate.

[0049] Reflux 5 times, filter to obtain the filtrate, pour the combined filtrate into a rotary evaporation bottle, set the temperature to 60°C and the speed to 100 rpm, and stop concentrating when the filtrate volume is concentrated to about 1 / 4 of the original volume to obtain a concentrated solution. Pour the concentrated solution evenly into a freeze drying plate and place it in a -80 degree refrigerator for 3 hours, transfer it to a freeze dryer, control the vacuum degree to 30Pa, the drying temperature to -10°C, and the drying time to 48 hours. After the freeze drying is completed, gradually increase the temperature to about 30°C, and continue drying for 3 hours to remove residual moisture. After drying, take out the freeze drying plate and collect the obtained powder, which is the prepared Chinese medicine composition powder.

[0050] Test Example 1

[0051] Study on the protective effect of Chinese herbal medicine combination on PC12 cell damage

[0052] The protective effects of different Chinese herbal medicine compositions on PC12 cell damage were evaluated by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) experiment:

[0053] PC12 cells in logarithmic growth phase were inoculated in 96-well plates, 5000 cells / 100 μL medium / well. They were divided into control group (C), model group (M), Angelica sinensis alone group (DG), Acorus calamus alone group (SCP), Hippocampus alone group (HM), Hippocampus Angelica sinensis combined group (HD), Hippocampus Acorus calamus combined group (HS), and Hippocampus Acorus calamus Angelica sinensis combined group (HSD). Three replicates were set in each group to ensure the reliability of the experimental data.

[0054] Each Chinese medicine group was given drug intervention 2 hours before modeling. After 2 hours of drug intervention, the control group was added with complete culture medium, the drug intervention group was added with complete culture medium containing each Chinese medicine composition and L-glutamic acid (L-Glu) (the concentration of each drug intervention was determined to be 400 μg / mL based on the previous preliminary experiment, and the concentration of L-Glu modeling was 160 μM), and the model group was added with complete culture medium containing 160 μM L-Glu. After 24 hours of drug co-incubation, 10 μL of MTT reagent was added to each well for incubation for 4 hours, the cell culture medium was discarded, 100 μL of dimethyl sulfoxide was added to each well, and the absorbance was detected at a wavelength of 490 nm using an enzyme marker.

[0055] By comparing the absorbance differences among different groups, the protective effect of each drug intervention group on PC12 cell damage was evaluated.

[0056] The formula for calculating cell survival rate is: cell survival rate (%) = (absorbance value of experimental group - absorbance value of blank well) / (absorbance value of control group - absorbance value of blank well) × 100%.

[0057] The above-mentioned combination group of seahorse, calamus and angelica is a Chinese medicine composition prepared in Example 1;

[0058] The drug treatment methods of the angelica single-use group (8g angelica), the Acorus calamus single-use group (4g Acorus calamus), the hippocampus single-use group (4g hippocampus), the hippocampus and angelica combined group (4g hippocampus, 8g angelica), and the hippocampus and Acorus calamus combined group (4g hippocampus, 4g Acorus calamus) are the same as those in Example 1.

[0059] The results are as follows Figure 1 As shown in A, the three drugs, Hippocampus (HM), Angelica sinensis (DG) and Acorus tatarinowii (SCP), all showed good safety without obvious toxic reactions.

[0060] The results are as follows Figure 1 As shown in B, compared with the normal control group (C), the cell survival rate of the model group (M) showed a significant downward trend, indicating that the cell injury model was successfully constructed. The DG group, SCP group, HM group, HD group, HS group and HSD group were able to significantly improve the cell survival rate, among which the synergistic effect of the three Chinese medicines in the HSD group was particularly prominent, and the effect on improving the cell survival rate was more significant, fully demonstrating the unique advantages and potential of the combined use of the three drugs in promoting cell survival and reducing damage.

[0061] Test Example 2

[0062] Protective effect of Chinese herbal medicine combination on motor function of AD zebrafish model

[0063] The protective effect of the Chinese medicine composition on the motor function of the AD zebrafish model was evaluated by the zebrafish light-dark alternation behavioral test:

[0064] The zebrafish larvae at 3 days after fertilization were randomly transferred to a 6-well cell culture plate, and the larvae in poor condition and deformed were eliminated, with 30 fish in each well. The zebrafish were randomly divided into a control group (C), a model group (M), a seahorse and angelica combination group (HD), a seahorse and Acorus calamus combination group (HS), a seahorse and Acorus calamus and angelica combination group (HSD, a traditional Chinese medicine composition prepared in Example 1) and a donepezil hydrochloride positive drug group (DPZ).

[0065] The control group was treated with experimental water only, the model group was treated with aluminum chloride (150 μM concentration), and the drug group was intervened with drugs and aluminum chloride (drug concentration was 50 μg / mL) for 3 days. The drugs were changed once a day and cultured in a light incubator. The movement distance and speed of zebrafish in each group were observed by alternating light and dark behavior 6 days after fertilization.

[0066] The results are as follows Figure 2 As shown in the figure, during the first light cycle of the light-dark alternating behavioral test, the total movement distance and speed of zebrafish in the model group showed a significant downward trend compared with the normal control group.

[0067] The total movement distance of zebrafish treated with the combination of seahorse and angelica sinensis, seahorse and calamus tatarinowii, and seahorse, calamus, and angelica sinensis was significantly increased (see Figure 2 A).

[0068] In addition, the zebrafish treated with the Chinese medicine composition prepared in Example 1, the combined use of Acorus calamus and Angelica sinensis and the donepezil hydrochloride positive drug group achieved significant improvements in both the total movement distance and movement speed (see Figure 2 A and B), indicating that these two treatments have more prominent effects in improving the locomotor ability of zebrafish.

[0069] Test Example 3

[0070] Study on the efficacy of Chinese herbal medicine combination in improving AD kidney deficiency mouse model

[0071] The improvement effect of the Chinese medicine composition on the AD kidney deficiency mouse model was evaluated by behavioral experiments and microscopic index detection:

[0072] The experiment used KM mice, which were kept in an environment with a temperature of 21-25°C, a relative humidity of 50%-60%, and a light / dark cycle of 12h. They were adaptively fed for 7 days and had free access to water and food. Before the experiment, the mice were grouped by body weight and open field test, and divided into 4 groups: normal group, model group, Chinese medicine composition group (540mg / mL) and donepezil hydrochloride intervention group (1.5mg / mL).

[0073] The AD kidney deficiency model was prepared by injection of aluminum chloride (concentration of 100 mg / mL) and D-galactose (concentration of 120 mg / mL) for a total of 8 weeks. Drug intervention began in the 5th week, and behavioral tests such as open field test, object recognition test, and water maze test were performed in the last week, and kidney deficiency syndrome scores were performed (refer to the evaluation criteria of the literature "Establishment and Evaluation of Alzheimer's Disease Kidney Deficiency Rat Model Based on Fear of Kidney Injury"). After the experiment, serum was collected for pathological index detection.

[0074] (1) Behavioral indicators and kidney deficiency syndrome scores

[0075] Open field test

[0076] The experiment was conducted in an open field box with a length of 50 cm, a width of 50 cm, and a depth of 50 cm. The bottom of the open field box was divided into 9 squares, with the middle area being the central area and the remaining area being the peripheral area. The bottom and surrounding areas of the open field box were all white. At the beginning, the mice were placed in the center of the open field box and their activities were observed within 6 minutes. The total distance traveled by the mice, the distance traveled in the central area, and the number of times they entered the central area were recorded.

[0077] Object recognition experiment

[0078] On the first day, the mice were first placed in the test room for 1 hour of environmental adaptation and familiarization, and then placed in an open field box for 20 minutes of adaptation (without objects in the box).

[0079] On the second day, the mice were first placed in the test room for 1 hour of environmental adaptation and familiarization. Then, two yellow cylindrical bottles of exactly the same size and texture were placed on the left and right ends of the same side of the open field box as familiar objects. The mice were then placed in the box with their backs facing the objects, and the mice's exploration of the objects was recorded within 5 minutes.

[0080] After the 2-hour familiarization period, a yellow cylindrical bottle in the open field box was randomly replaced with a green conical bottle of the same size as a strange object, and then the mouse was placed in the box with its back facing the object. The exploration time of the mouse on the strange object and the exploration time on the familiar object were recorded within 5 minutes, and the recognition index was calculated.

[0081] Morris water maze test

[0082] The water maze experiment was divided into two parts, with a total of 5 consecutive days of testing. The first part was a positioning navigation experiment that tested mice for 4 days, and the second part was a spatial exploration experiment that tested for 1 day. The black vegetarian color powder was washed with warm water to hide the platform, and it was kept in sharp contrast with the color of the mice during video recording for easy tracking. The platform was located 0.8 cm below the water surface, 21 cm deep, and positioned in the NW quadrant. The water temperature in the water maze was kept constant at 22-24°C using a heating rod.

[0083] Positioning navigation experiment: Each mouse was trained 4 times / d, with an interval of 15 to 20 minutes between each training. The entry point was selected semi-randomly during the test. In each training, the mouse could find the platform within 60 seconds, and the stay time on the platform exceeded 2 seconds as a sign of successful platform search. The time taken by the mouse from entering the water to successfully finding the platform was recorded as the latency. If the mouse failed to find the platform within 60 seconds, the experimenter would guide it to the platform by hand, and the latency was recorded as 60 seconds. The average latency per day was calculated to evaluate the animal's ability to acquire spatial memory.

[0084] Spatial exploration experiment: The platform was removed and each mouse was tested once for 60 seconds. The spatial memory ability of the animal was evaluated by the number of times the animal crossed the original platform position within 60 seconds, the ratio of the original platform quadrant travel distance and time ratio (i.e. the ratio of the animal's original platform quadrant travel distance and time to the total travel distance and total time).

[0085] Table 1 Syndrome scores of mice

[0086]

[0087] (2) Pathological index detection

[0088] Elisa detection of Aβ in serum 1-42 content

[0089] After equilibration at room temperature for 10 minutes, the required panels were taken out from the aluminum bag, and the remaining panels were sealed in a ziplock bag and returned to 4°C.

[0090] Sample addition: Add 100 μL of samples or standards of different concentrations to the corresponding wells, add 100 μL of universal diluent to the blank wells, cover with sealing film and incubate at 37°C for 1 hour.

[0091] Add biotinylated antibody: Take out the ELISA plate, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well, cover with sealing film and incubate at 37℃ for 1 hour.

[0092] Wash the plate: discard the liquid, add 300μL 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 3 times.

[0093] Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0094] Wash the plate: discard the liquid, add 300 μL 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times.

[0095] Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes.

[0096] Add stop solution: Take out the ELISA plate, add 50 μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm.

[0097] The results are as follows Figure 3 As shown: The test results revealed that there was no significant difference in the body weight of each group of mice (see Figure 3 A), indicating that the experimental treatment had no significant effect on the growth and development of mice. In terms of kidney deficiency syndrome scores, compared with the normal control group, the scores of the AD kidney deficiency syndrome model group showed a significant increase, reflecting that the model was successfully constructed and the mice showed more obvious kidney deficiency symptoms. However, after the intervention of the Chinese medicine composition and the positive control drug donepezil hydrochloride, the kidney deficiency syndrome score was significantly reduced, indicating that both intervention measures can effectively alleviate the kidney deficiency symptoms of mice and improve their overall health (see Figure 3 B).

[0098] In the open field test, the total movement distance of the mice in the model group showed a downward trend compared with the normal control group, suggesting that the symptoms of kidney deficiency may have a negative impact on the activity of the mice. However, the total movement distance of the mice after intervention with the Chinese medicine composition and donepezil hydrochloride showed an upward trend, suggesting that these two intervention methods may have a positive effect on the behavioral activities of the mice by improving the kidney deficiency state of the mice, thereby enhancing their exercise ability and vitality (see Figure 3 C).

[0099] Test results such as Figure 4 As shown: In the water maze experiment, the latency of each group of mice to find the platform during the training period tended to decrease (see Figure 4 A), in the exploration period, compared with the normal group, the model group mice's residence time and distance in the target quadrant showed a downward trend, and after the intervention of the Chinese medicine composition and the positive drug donepezil hydrochloride, there was an upward trend (see Figure 4 B and C). In the object recognition experiment, compared with the normal group, the recognition index of the model group showed a downward trend, and after the intervention of the Chinese medicine composition and the positive drug donepezil hydrochloride, there was an upward trend (see Figure 4 D) In ​​serum Aβ 1-42 In the content detection experiment, compared with the normal group, the model group mice had Aβ 1-42 The content has a decreasing trend, and after the intervention of the Chinese medicine composition and the positive drug donepezil hydrochloride, it has an increasing trend (see Figure 4 E).

[0100] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 2-7 parts of seahorse, 5-16 parts of angelica, and 2-7 parts of calamus.

2. The Chinese medicine composition according to claim 1, characterized in that: The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 4 parts of seahorse, 8 parts of angelica and 4 parts of calamus.

3. The method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: The following steps are involved: Weigh seahorse, angelica and calamus according to the weight of each raw material, crush them and mix them, add ethanol in a ratio of 1:8-12 between the raw material mixture and the ethanol liquid, soak for 20-50 minutes, heat and reflux for extraction, and filter to obtain a filtrate; The filtrates were combined, concentrated, and freeze-dried to obtain a powder.

4. The preparation method according to claim 3, characterized in that: The concentration of ethanol used is 50% to 90%.

5. The preparation method according to claim 3, characterized in that: The heating reflux temperature is 80-110°C, the time is 2-3h, and the reflux times are 2-5 times.

6. Use of the Chinese medicine composition according to claim 1 or 2 or the Chinese medicine composition prepared by the preparation method according to any one of claims 3 to 5 in the preparation of drugs for treating and / or preventing Alzheimer's disease.

7. A Chinese medicine preparation for improving Alzheimer's disease, wherein the effective ingredient is the Chinese medicine composition according to claim 1 or 2 or the Chinese medicine composition prepared by the preparation method according to any one of claims 3 to 5.

8. The Chinese medicine preparation according to claim 7, characterized in that: Including tablets, powders, decoctions, capsules, pills and granules.

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