Application of compound cistanche deserticola intelligence-promoting capsule in treatment of Alzheimer disease

Abstract

The invention discloses application of a compound cistanche deserticola intelligence-promoting capsule in treatment of Alzheimer's disease. Polygonum multiflorum, lotus leaves, cistanche deserticola, lumbricus and uniflower swisscentaury root in the compound cistanche deserticola intelligence-promoting capsule are applied in treatment of Alzheimer's disease. In this way, the compound cistanche deserticola intelligence promoting capsule can play a role in improving the Alzheimer's disease.

Description

[0001] This application claims priority to Chinese patent application No. 2025105586417, filed on April 29, 2025, entitled “Application of Compound Cistanche deserticola Capsules in the Treatment of Alzheimer’s Disease”, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present application relates to the technical field of Alzheimer's disease, and in particular to the application of compound Cistanche deserticola capsule in the treatment of Alzheimer's disease. Background Art

[0003] Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by progressive cognitive impairment, memory impairment, loss of functional independence, and behavioral changes. It is the primary clinical subtype of dementia, accounting for 50-70% of all cases. According to the Global Burden of Disease Study, AD ranks first among non-communicable diseases in terms of years of life lost and its rate of change worldwide, and is the fourth leading cause of all-cause mortality worldwide.

[0004] Compound Cistanche deserticola capsule is a traditional Chinese medicine preparation widely used in the long-term and collaborative treatment of mild vascular cognitive impairment, mild and moderate vascular dementia. It is composed of processed Polygonum multiflorum, lotus leaves, Cistanche deserticola, earthworms, and Rhizoma Cistanches. Its core therapeutic effects are to improve intelligence and nourish the liver, promote blood circulation and remove turbidity, and strengthen the brain and enhance intelligence.

[0005] However, the core goal of the initial development of the Compound Cistanche Alzheimer's Capsule was to improve vascular dementia, and previous clinical and basic research on this preparation still focused on vascular dementia, and there is still a lack of evidence to support its application value in the treatment of Alzheimer's disease. Summary of the Invention

[0006] The main technical problem solved by this application is to provide the application of compound cistanche and yizhi capsule in the treatment of Alzheimer's disease. The compound cistanche and yizhi capsule can improve Alzheimer's disease.

[0007] In order to solve the above technical problems, a technical solution adopted in this application is: to provide the use of compound Cistanche deserticola capsule in the treatment of Alzheimer's disease, and the use of Polygonum multiflorum, lotus leaf, Cistanche deserticola, earthworm and Rhizoma Cistanches in the compound Cistanche deserticola capsule in the treatment of Alzheimer's disease.

[0008] Among them, the compound Cistanche deserticola capsule is used to improve the target indicators of patients with Alzheimer's disease, and the target indicators include at least one of the animal's physical condition, behavioral ability, brain tissue condition, hippocampal tissue condition, and serum condition.

[0009] Among them, the target indicators include the state of the animal body, which includes at least one of the following: skin state, diet state, reaction state, grasping state, resistance state, mental state, weight state, and organ state; and / or, the target indicators include behavioral abilities, which include at least one of the following: motor coordination ability, balance ability, spatial memory ability, and learning ability; and / or, the target indicators include brain tissue conditions, which include NEFL conditions in brain tissue; and / or, the target indicators include hippocampal tissue conditions, which include at least one of the following: the structural condition of the hippocampal tissue, the condition of Nissl bodies in the hippocampal tissue, the Aβ deposition condition in the hippocampal tissue, and the Aβ 1-42 situation, PSD95 protein expression in hippocampal tissue, SYP protein expression in hippocampal tissue, TLR4 protein expression in hippocampal tissue, NF-κB protein expression in hippocampal tissue, MYD88 protein expression in hippocampal tissue, Bax protein expression in hippocampal tissue, Bc12 protein expression in hippocampal tissue, Caspase3 protein expression in hippocampal tissue; and / or, the target indicators include serum conditions, and the serum conditions include at least one of the following: Aβ1-42 situation in serum, NEFL situation in serum, 1L-6 situation in serum, 1L-1β situation in serum, and TNF-α situation in serum.

[0010] Among them, the efficacy of Compound Cistanche and Yizhi Capsules in treating Alzheimer's disease was verified by the following method, which includes: constructing a first control group comprising a normal mice; and, constructing a second control group comprising b Alzheimer's mice; and, constructing a first treatment group comprising c Alzheimer's mice; and, constructing a second treatment group comprising d Alzheimer's mice; injecting double distilled water into the first control group and the second control group; and, administering a Compound Cistanche and Yizhi Capsules aqueous solution to the first treatment group; and, administering a donepezil aqueous solution to the second treatment group; collecting at least one target indicator of each group of mice; and using the differences between the target indicators corresponding to each group to evaluate the efficacy of Compound Cistanche and Yizhi Capsules in treating Alzheimer's disease.

[0011] Among them, the target indicators include behavioral abilities, which include spatial memory abilities and learning abilities; the target indicators are collected by the following method, which includes: for each group of mice, performing a Morris water maze test on the group of mice for the first preset number of consecutive days, wherein the last day is the spatial exploration experiment stage, the first second preset days are the pre-training stage, and the remaining days are the positioning navigation experiment stage, and the second preset days are less than the first preset days; analyzing the test data of the spatial exploration experiment stage to obtain the spatial memory abilities and learning abilities of the group of mice.

[0012] Among them, the target indicators include behavioral abilities, and behavioral abilities include motor coordination abilities; the target indicators are collected by the following method, which includes: for each group of mice, performing a pole climbing experiment on the group of mice for the third consecutive preset number of days, wherein the last day is the first test period and the remaining days are the first training period; analyzing the test data of the first test period to obtain the motor coordination abilities of the group of mice.

[0013] Among them, the target indicators include behavioral abilities, which include motor coordination ability and balance ability; the target indicators are collected by the following method, which includes: for each group of mice, performing a rotating rod fatigue test on the group of mice for the fourth consecutive preset number of days, wherein the last day is the second test period and the remaining days are the second training period; the test data of the second test period is analyzed to obtain the motor coordination ability and balance ability of the group of mice.

[0014] Among them, the target indicators include brain tissue conditions, which include NEFL conditions in brain tissue; the target indicators include serum conditions, which include NEFL conditions in serum, Aβ1-42 conditions in serum, 1L-6 conditions in serum, 1L-1β conditions in serum and TNF-α conditions in serum; the target indicators include hippocampal tissue conditions, which include Aβ deposition conditions in hippocampal tissue and Aβ1-42 conditions in hippocampal tissue; the target indicators are collected by the following method, which includes: for each group of mice, collecting the first brain tissue sample, the first serum sample and the first hippocampal tissue sample of the group of mice; performing enzyme-linked immunosorbent assay experiments on the first brain tissue sample, the first serum sample and the first hippocampal tissue sample respectively to obtain the NEFL conditions in brain tissue, the NEFL conditions in serum, Aβ1-42 conditions in serum, 1L-6 conditions in serum, 1L-1β conditions in serum, TNF-α conditions in serum, Aβ1-42 conditions in hippocampal tissue and Aβ deposition conditions in hippocampal tissue.

[0015] Among them, the target indicators include hippocampal tissue conditions, including PSD95 protein expression in hippocampal tissue, SYP protein expression in hippocampal tissue, TLR4 protein expression in hippocampal tissue, NF-κB protein expression in hippocampal tissue, MYD88 protein expression in hippocampal tissue, Bax protein expression in hippocampal tissue, Bc12 protein expression in hippocampal tissue and Caspase3 protein expression in hippocampal tissue; the target indicators are collected by the following method, which includes: for each group of mice, collecting the second hippocampal tissue sample of each group of mice; performing protein immunoblotting experiment on the second hippocampal tissue sample to obtain PSD95 protein expression in hippocampal tissue, SYP protein expression in hippocampal tissue, TLR4 protein expression in hippocampal tissue, NF-κB protein expression in hippocampal tissue, MYD88 protein expression in hippocampal tissue, Bax protein expression in hippocampal tissue, Bc12 protein expression in hippocampal tissue and Caspase3 protein expression in hippocampal tissue.

[0016] Among them, the target indicators include the condition of hippocampal tissue, which includes the structural condition of the hippocampal tissue and the condition of Nissl bodies in the hippocampal tissue; the target indicators are collected by the following method, which includes: for each group of mice, collecting the third hippocampal tissue sample of the group of mice; performing HE staining on the third hippocampal tissue sample to obtain the structural condition of the hippocampal tissue; and performing Nissl staining on the third hippocampal tissue sample to obtain the condition of Nissl bodies in the hippocampal tissue.

[0017] Among them, the target indicators include the body state of the animal, and the body state of the animal includes the weight state; the target indicators are collected by the following method, which includes: for each group of mice, weighing the group of mice at every preset time interval to obtain the first weight of the mice as the weight state of the group of mice; and / or, the target indicators include the body state of the animal, and the body state of the animal includes the organ state; the target indicators are collected by the following method, which includes: for each group of mice, obtaining at least one organ of the group of mice; and, obtaining the first weight of the group of mice; for each organ, weighing each organ to obtain the second weight of the organ; obtaining the ratio of the second weight to the first weight, and obtaining the organ index corresponding to the organ as the organ state corresponding to the organ.

[0018] According to the above technical solution, the compound Cistanche deserticola capsule includes processed Polygonum multiflorum, lotus leaf, Cistanche deserticola, earthworm and Rhizoma Cistanches, and the compound Cistanche deserticola capsule can improve Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of an embodiment of a method for verifying the efficacy of Compound Cistanche and Yizhi Capsules in treating Alzheimer's disease provided by the present application;

[0020] Figure 2 Schematic diagram of the changes in body weight of mice in each group provided in this application;

[0021] Figure 3 This is a schematic diagram of the organ index of different organs of each group of mice provided in this application;

[0022] Figure 4 This is a schematic diagram of the trajectories of each group of mice during the pre-training phase of the water maze experiment provided in this application;

[0023] Figure 5 This is a schematic diagram of the escape latency of each group of mice in the positioning navigation test phase of the water maze experiment provided in this application;

[0024] Figure 6 This is a schematic diagram of the trajectory of each group of mice in the water maze experimental space exploration phase provided by this application;

[0025] Figure 7 Schematic diagram of the experimental results of each group of mice in the water maze experimental space exploration phase provided by this application;

[0026] Figure 8 This is a schematic diagram of the time it takes for each group of mice to climb a pole provided by this application;

[0027] Figure 9 This is a schematic diagram of the rotarod time of each group of mice provided in this application;

[0028] Figure 10 is a schematic diagram of the NEFL content in the serum and brain tissue of each group of mice provided in this application;

[0029] Figure 11 Schematic diagram of the Aβ1-42 levels in the serum and hippocampal tissues of each group of mice provided in this application;

[0030] Figure 12 Schematic diagram of the IL-6, IL-1β and TNF-α levels in the serum of each group of mice provided in this application;

[0031] Figure 13 Schematic diagram of Aβ immunohistochemical staining of the hippocampal CA1 region of each group of mice provided in this application;

[0032] Figure 14 This is a schematic diagram of the quantitative analysis results of Aβ in the hippocampal CA1 region of each group of mice provided in this application;

[0033] Figure 15 Schematic diagram of representative protein bands of PSD95 protein and SYP protein in the hippocampal tissue of each group of mice provided in this application;

[0034] Figure 16This is a quantitative schematic diagram of the band intensity of PSD95 protein relative to β-Actin in the hippocampal tissue of each group of mice provided in the present application;

[0035] Figure 17 This is a quantitative schematic diagram of the band intensity of SYP protein relative to GAPDH in the hippocampal tissue of each group of mice provided in the present application;

[0036] Figure 18 Schematic diagram of representative protein bands of TLR4 protein, NF-κB protein and MYD88 protein in the hippocampal tissue of each group of mice provided in this application;

[0037] Figure 19 This is a quantitative schematic diagram of the band intensity of TLR4 protein relative to GAPDH in the hippocampal tissue of each group of mice provided in the present application;

[0038] Figure 20 This is a quantitative schematic diagram of the band intensity of NF-κB protein relative to β-Actin in the hippocampal tissue of each group of mice provided in the present application;

[0039] Figure 21 This is a quantitative schematic diagram of the band intensity of MYD88 protein relative to β-Actin in the hippocampal tissue of each group of mice provided in the present application;

[0040] Figure 22 Schematic diagram of representative protein bands of Bax protein, Bcl2 protein and Caspase3 protein in the hippocampal tissue of each group of mice provided in this application;

[0041] Figure 23 This is a quantitative schematic diagram of the band intensity of Bax protein relative to GAPDH in the hippocampal tissue of each group of mice provided in the present application;

[0042] Figure 24 This is a quantitative schematic diagram of the band intensity of Bcl2 protein relative to GAPDH in the hippocampal tissue of each group of mice provided in this application;

[0043] Figure 25 This is a quantitative schematic diagram of the band intensity of Caspase 3 protein relative to β-Actin in the hippocampal tissue of each group of mice provided in the present application;

[0044] Figure 26 Schematic diagram of HE staining results of hippocampal tissue of each group of mice provided in this application;

[0045] Figure 27 Schematic diagram of Nissl body staining results of hippocampal tissues of each group of mice provided in this application;

[0046] Figure 28This is a schematic diagram of the number of Nissl bodies in the hippocampal tissue of each group of mice provided in this application. DETAILED DESCRIPTION

[0047] The following describes the embodiments of the present application in detail with reference to the accompanying drawings.

[0048] In the following description, for the purpose of explanation rather than limitation, specific details such as specific system structures, interfaces, and technologies are provided to facilitate a thorough understanding of the present application.

[0049] The term "and / or" in this article is simply a description of the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects are in an "or" relationship. In addition, "many" in this article means two or more than two. In addition, the term "at least one" in this article means any combination of at least two of any one or more of a plurality of. For example, including at least one of A, B, and C can mean including any one or more elements selected from the set consisting of A, B, and C.

[0050] The present application provides the use of compound Cistanche deserticola capsule (Cistanche deserticola formula, CRYZF) in the treatment of Alzheimer's disease (AD), and the use of processed Polygonum multiflorum, lotus leaf, Cistanche deserticola, earthworm and Rhizoma Cyperi in the compound Cistanche deserticola capsule in the treatment of Alzheimer's disease.

[0051] Alzheimer's disease is the most common neurodegenerative disease, with progressive cognitive dysfunction, memory impairment, loss of functional independence and behavioral changes as its core clinical manifestations.

[0052] Compound Cistanche deserticola capsule is a traditional Chinese medicine preparation widely used in the long-term and collaborative treatment of mild vascular cognitive impairment, mild and moderate vascular dementia. It is composed of processed Polygonum multiflorum, lotus leaves, Cistanche deserticola, earthworms and Rhizoma Cistanches, and its core therapeutic effects are to improve intelligence and nourish the liver, promote blood circulation and remove turbidity, and strengthen the brain and enhance intelligence. Compound Cistanche Alpiniae Capsules can improve memory ability by inhibiting neuronal apoptosis in the brain and enhancing synaptic plasticity; in addition, Compound Cistanche Alpiniae Capsules can exert an anti-thrombotic effect by inhibiting the maximum platelet aggregation rate; in addition, Compound Cistanche Alpiniae Capsules can increase brain tissue blood flow, maintain the stability of tissue cell energy metabolism, and protect the brain from cerebral ischemia-reperfusion-mediated brain damage; in addition, Compound Cistanche Alpiniae Capsules are beneficial in inhibiting oxidative stress in brain tissue and exerting a protective effect on mitochondrial function; in addition, Compound Cistanche Alpiniae Capsules combined with vascular dementia treatment drugs can effectively slow down the disease progression of patients with mild and moderate vascular dementia, and are beneficial to the improvement of corresponding mental and behavioral symptoms.

[0053] While Alzheimer's disease and vascular dementia differ in their modern medical pathological mechanisms, both fall within the categories of "forgetfulness," "dementia," and "dementia" within traditional medical theory, sharing similarities and connections in their etiology, pathogenesis, and treatment principles. Compound Cistanche Yizhi Capsules are composed of processed Polygonum multiflorum, lotus leaf, Cistanche deserticola, earthworm, and Rhizoma Cyperi. This traditional Chinese medicine combination, with processed Polygonum multiflorum as the monarch ingredient and the remaining four herbs as ministers, utilizes a "monarch-four-minister" Chinese medicine formula, maximizing its primary therapeutic effects of nourishing the liver and brain, promoting blood circulation and dissolving turbidity, and strengthening the brain and enhancing intelligence. Comprehensively considering the overall formula, lotus leaf, earthworm, and Rhizoma Cyperi contribute to ascending the clear and descending the turbid, dissolving dampness and promoting qi, and dispersing blood stasis, while Polygonum multiflorum and Cistanche deserticola focus on warming and nourishing the liver and kidneys, regulating mental function, and strengthening the brain and enhancing intelligence. The combined effect of these five herbs attacks pathogenic factors without harming vital energy, and nourishes without causing greasy sensations. This combination achieves a dual-action approach, dispelling pathogenic factors and supporting vital energy, addressing both the symptoms and the root cause.

[0054] In one embodiment, the compound Cistanche deserticola capsule is used to improve target indicators of patients with Alzheimer's disease, wherein the target indicator includes at least one of the following: physical condition, behavioral ability, brain tissue condition, hippocampal tissue condition, and serum condition. In other words, the compound Cistanche deserticola capsule can improve the physical condition, behavioral ability, brain tissue condition, hippocampal tissue condition, and serum condition of patients with Alzheimer's disease.

[0055] In a specific embodiment, the target indicator includes the animal's body state, and the animal's body state includes at least one of the following: skin state, eating state, reaction state, grasping state, resistance state, mental state, weight state, and organ state.

[0056] In a specific embodiment, the target indicator includes behavioral ability, and the behavioral ability includes at least one of the following: motor coordination ability, balance ability, spatial memory ability, and learning ability.

[0057] In one embodiment, the target indicator includes a brain tissue condition, and the brain tissue condition includes a NEFL condition in the brain tissue.

[0058] In a specific embodiment, the target indicator includes the condition of hippocampal tissue, and the condition of hippocampal tissue includes at least one of the following: the structure of the hippocampal tissue, the condition of Nissl bodies in the hippocampal tissue, the condition of Aβ deposition in the hippocampal tissue, the condition of Aβ1-42 in the hippocampal tissue, the expression of PSD95 protein in the hippocampal tissue, the expression of SYP protein in the hippocampal tissue, the expression of TLR4 protein in the hippocampal tissue, the expression of NF-κB protein in the hippocampal tissue, the expression of MYD88 protein in the hippocampal tissue, the expression of Bax protein in the hippocampal tissue, the expression of Bc12 protein in the hippocampal tissue, and the expression of Caspase3 protein in the hippocampal tissue.

[0059] In one embodiment, the target indicator includes serum levels, and the serum levels include at least one of the following: serum Aβ1-42 levels, serum NEFL levels, serum 1L-6 levels, serum 1L-1β levels, and serum TNF-α levels.

[0060] In one embodiment, if Figure 1 As shown, Figure 1 : This is a schematic diagram of an embodiment of a method for verifying the efficacy of Compound Cistanche and Yizhi Capsules in treating Alzheimer's disease provided by this application. The efficacy of Compound Cistanche and Yizhi Capsules in treating Alzheimer's disease is verified by the following method, which specifically includes the following steps:

[0061] Step S1: constructing a first control group comprising a normal mice; and constructing a second control group comprising b Alzheimer's disease mice; and constructing a first treatment group comprising c Alzheimer's disease mice; and constructing a second treatment group comprising d Alzheimer's disease mice.

[0062] In this embodiment, a first control group is constructed comprising a normal mice; a second control group is constructed comprising b Alzheimer's disease mice; a first treatment group is constructed comprising c Alzheimer's disease mice; and a second treatment group is constructed comprising d Alzheimer's disease mice. The number of mice in the first control group, the second control group, the first treatment group, and the second treatment group is not limited; that is, the sizes of a, b, c, and d are not limited and can be set according to actual needs.

[0063] Step S2: injecting double distilled water into the first control group and the second control group; and administering the Compound Cistanche Yizhi Capsule aqueous solution to the first treatment group; and administering the Donepezil aqueous solution to the second treatment group.

[0064] In this embodiment, double distilled water is injected into the first and second control groups; and the first treatment group is administered with an aqueous solution of Compound Cistanche and Yizhi Capsules; and the second treatment group is administered with an aqueous solution of Donepezil.

[0065] Step S3: Collect at least one target indicator from each group of mice.

[0066] In this embodiment, at least one target indicator is collected from each group of mice.

[0067] It should be noted that different target indicators of mice correspond to different collection methods.

[0068] Step S4: Using the differences between the target indicators corresponding to the groups, the efficacy of the compound Cistanche deserticola capsule in treating Alzheimer's disease was evaluated.

[0069] In this embodiment, the differences between the target indicators corresponding to the groups are used to evaluate the efficacy of the compound Cistanche deserticola capsule in treating Alzheimer's disease.

[0070] The following will illustrate and explain the method for verifying the efficacy of Compound Cistanche and Yizhi Capsules in treating Alzheimer's disease through specific experimental examples, but should not be used to limit the scope of this application.

[0071] A first control group, a second control group, a first treatment group and a second treatment group were constructed.

[0072] Specifically, the experimental animals were prepared by selecting 10 C57BL / 6J female mice aged 4-5 months and weighing (21±2) g; and 30 APP / PSI female Alzheimer's disease mice aged 4-5 months and weighing (21±2) g. The selected mice were then housed in an SPF-grade feeding room (room temperature (22.5±2.5)°C, relative humidity (50±5)%, 5 mice / cage, and a 12h / 12h light / dark cycle). During the feeding period, the mice had free access to water and food, and the bedding and cages were changed regularly.

[0073] Preparation of experimental drugs: Prepare compound Cistanche deserticola capsules and Donepezil. Compound Cistanche deserticola capsules include five Chinese medicinal materials: processed Polygonum multiflorum, lotus leaves, Cistanche deserticola, earthworms and Rhizoma Cyperi.

[0074] Experimental groups: The selected mice were adaptively fed for one week and then divided into experimental groups. Ten C57BL / 6J female mice aged 4-5 months and weighing (21±2) g were used as the first control group (normal control group). Thirty female APP / PSI Alzheimer's disease mice aged 4-5 months and weighing (21±2) g were randomly divided into the second control group, the first treatment group, and the second treatment group according to the random number table method. Among them, the second control group, the first treatment group, and the second treatment group each contained 10 female APP / PSI Alzheimer's disease mice aged 4-5 months and weighing (21±2) g.

[0075] The mice in the first control group, the second control group, the first treatment group and the second treatment group were intervened.

[0076] Specifically, mice in the first and second control groups were injected with double-distilled water; mice in the first treatment group were gavage-administered an equal volume of a 468 mg / g aqueous solution of Compound Cistanche Yizhi Capsules; and mice in the second treatment group were administered an equal volume of a 0.65 mg / kg aqueous solution of Donepezil. The intervention was continued for 8 weeks in the first, second, first, and second treatment groups.

[0077] It should be noted that the dosage of donepezil is based on the drug instructions. The daily dose for a 70kg adult is 5mg / day. Therefore, based on the human-mouse equivalent dose conversion, the dosage for a 20g mouse is 0.013mg, and the equivalent dose of 0.65mg / kg is used as the intervention dose. The dosage of Compound Cistanche Yizhi Capsules is based on the body surface area conversion method in the "Pharmacological Experimental Methodology." The body size coefficient of a 20g mouse to a 70kg adult is 6.0. Body surface area = body size coefficient × (body weight) 2 / 3 / 10000. The daily dose for an adult is 3.6g. Therefore, based on the human-mouse equivalent dose conversion, the dosage for a 20g mouse is 9.36mg, and the equivalent dose of 468mg / kg is used as the intervention dose.

[0078] The target indicators of mice in the first control group, the second control group, the first treatment group and the second treatment group were collected. And evaluate the efficacy of compound Rongrong Yizhi capsule in the treatment of Alzheimer's disease.

[0079] The target indicator includes the animal's physical condition, which includes weight. The weight status is collected by weighing each group of mice at predetermined intervals to obtain a first weight, which serves as the weight status of the group of mice. The length of the predetermined interval is not limited and can be set based on actual needs.

[0080] Specifically, the experimental procedure involved injecting double-distilled water into the mice in the first and second control groups; gavage administering an equal volume of a 468mg / kg aqueous solution of Compound Cistanche Yizhi Capsules to the first treatment group; and administering an equal volume of a 0.65mg / kg aqueous solution of Donepezil to the mice in the second treatment group. The intervention was continued for eight weeks in the first, second, first, and second treatment groups.

[0081] During the experiment, the mice in the first control group, the second control group, the first treatment group and the second treatment group were weighed every week to obtain the first weight of the mice in the first control group, the second control group, the first treatment group and the second treatment group, which was used as the weight status of the mice in the first control group, the second control group, the first treatment group and the second treatment group. In addition, after the mice in the first control group and the second control group were injected with double distilled water for the last time, the mice in the first control group and the second control group were fasted for 12 hours, and 12 hours later, the mice in the first control group and the second control group were weighed to obtain the first weight of the mice in the first control group and the second control group, which was used as the weight status of the mice in the first control group and the second control group; and, after the mice in the first treatment group were administered the aqueous solution of Compound Cistanche Yizhi Capsules for the last time, the mice in the first treatment group were fasted for 12 hours, and 12 hours later, the mice in the first treatment group were weighed to obtain the first weight of the mice in the first treatment group, which was used as the weight status of the mice in the first treatment group; and, after the mice in the second treatment group were administered the aqueous solution of donepezil for the last time, the mice in the second treatment group were fasted for 12 hours, and 12 hours later, the mice in the second treatment group were weighed to obtain the first weight of the mice in the second treatment group, which was used as the weight status of the mice in the second treatment group.

[0082] As shown in Table 1 and Figure 2 As shown, Figure 2 : This is a schematic diagram of the changes in weight status of each group of mice provided in this application (Note: 0-8 weeks, compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05, n=9). The weight status changes of the mice in the first control group, the second control group, the first treatment group, and the second treatment group were consistent. Compared with the first control group, the weight status of the mice in the second control group, the first treatment group, and the second treatment group decreased. Compared with the second control group, the weight status of the mice in the first treatment group and the second treatment group further decreased.

[0083] Table 1 Body weight status of mice in each group ( n=9)

[0084]

[0085] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0086] The target indicators include the animal's physical condition, which includes fur condition, behavioral status, and mental state. The method for collecting fur condition, behavioral status, and mental state includes observing and recording the fur condition, behavioral status, and mental state of each group of mice at predetermined time intervals. The length of the predetermined time interval is not limited and can be set according to actual needs.

[0087] Specifically, during the experiment, the fur condition, behavioral condition and mental state of the mice in the first control group, the second control group, the first treatment group and the second treatment group were observed.

[0088] The mice in the first control group had smooth, shiny fur, intact skin, a good diet, quick reflexes, quick dodging when being grabbed, strong resistance, and a good mental state. Compared with the first control group, the mice in the second control group had loose or balding fur, a poor diet, slightly sluggish reflexes, low resistance when being grabbed, and a poor mental state. Compared with the second control group, the mice in the first and second treatment groups showed improvement in these symptoms. Their fur was smooth, shiny, and intact, their diet was acceptable, their reflexes were quick, their resistance was strong when being grabbed, and their mental state was acceptable. During the experiment, one mouse died in each group due to fighting caused by the mice's habit.

[0089] The target indicators include the state of the animal body, which includes the state of the organs. The method for collecting the state of the organs includes the following steps:

[0090] Step a: for each group of mice, obtaining at least one organ of each group of mice; and obtaining a first weight of each group of mice.

[0091] Step b: weighing each organ to obtain a second weight of the organ.

[0092] Specifically, the chest cavity and abdominal cavity of the mice in the first control group, the second control group, the first treatment group and the second treatment group were exposed, the liver organs, heart organs, spleen organs, lung organs and kidney organs of the mice in the first control group, the second control group, the first treatment group and the second treatment group were taken out, and the liver organs, heart organs, spleen organs, lung organs and kidney organs of the mice in the first control group, the second control group, the first treatment group and the second treatment group were weighed to obtain the second weight of the liver organs, heart organs, spleen organs, lung organs and kidney organs of the mice in the first control group, the second control group, the first treatment group and the second treatment group.

[0093] Step c: Obtaining a ratio of the second weight to the first weight to obtain an organ index corresponding to the organ as the organ status corresponding to the organ.

[0094] Specifically, based on the ratio of the second weight of the liver organs, heart organs, spleen organs, lung organs and kidney organs of the mice in the first control group, the second control group, the first treatment group and the second treatment group to the first weight of the corresponding mice, the organ index of the liver organs, heart organs, spleen organs, lung organs and kidney organs of the mice in the first control group, the second control group, the first treatment group and the second treatment group was calculated as the organ status corresponding to the liver organs, heart organs, spleen organs, lung organs and kidney organs of the mice in the first control group, the second control group, the first treatment group and the second treatment group.

[0095] As shown in Table 2 and Figure 3 As shown, Figure 3 Schematic diagram of the organ index of different organs of each group of mice provided in this application (Note: compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05, n=9). Compared with the first control group, the liver organ index, lung organ index and kidney organ index of the mice in the second control group all changed to varying degrees, but no statistical difference was found (P>0.05). Compared with the second control group, the liver organ index, heart organ index, spleen organ index, lung organ index and kidney organ index of the mice in the first treatment group and the second treatment group all changed to varying degrees. Except for the relative decrease in the liver organ index of the mice in the second treatment group (P<0.05), no statistical difference was found (P>0.05).

[0096] Table 2 Organ index of different organs of mice in each group ( n=9)

[0097] Group liver Heart spleen lung kidney The first control group 4.89±0.34 0.58±0.07 0.32±0.05 0.72±0.08 1.15±0.07 The second control group 5.19±0.22 0.50±0.09 0.29±0.05 0.72±0.05 1.16±0.09 The second treatment group <![CDATA[4.65±0.58 * ]]> 0.57±0.09 0.29±0.04 0.79±0.12 1.20±0.15 The first treatment group 4.83±0.30 0.53±0.06 0.31±0.05 0.72±0.15 1.19±0.05

[0098] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0099] Therefore, by analyzing the organ status of each organ of mice in the first control group, the second control group, the first treatment group and the second treatment group, it was determined that Compound Cistanche Yizhi Capsule and Donepezil had no obvious side effects on the main organs of mice in the short term.

[0100] The target indicators include behavioral abilities, which include spatial memory and learning abilities. The collection method of spatial memory and learning abilities includes the following steps:

[0101] Step a: For each group of mice, perform a Morris water maze test on the mice for a first predetermined number of consecutive days, wherein the last day is a spatial exploration phase, the first second predetermined number of days is a pre-training phase, and the remaining days are a positioning and navigation phase, wherein the second predetermined number of days is less than the first predetermined number of days. The first and second predetermined number of days are not limited and can be set according to actual needs.

[0102] The Morris water maze test is a behavioral experiment widely used to assess spatial memory and learning abilities in rodents. The experimental apparatus primarily consists of a circular pool (120 cm in diameter and 40 cm in height), an escape platform (8 cm in diameter and 20-35 cm in height), and a trajectory recorder. The Morris water maze test requires a quiet, dimly lit environment, a constant pool temperature (23-25°C), and an escape platform positioned in the fourth quadrant, 1.5 cm below the water level.

[0103] Specifically, the mice in the first control group, the second control group, the first treatment group and the second treatment group were subjected to Morris water maze (MWM) experimental detection for 7 consecutive days, with 3 measurements per day, and each measurement interval was no less than 60 minutes; among them, the Morris water maze experimental detection of the mice in the first control group, the second control group, the first treatment group and the second treatment group on the first day was the pre-training stage, the Morris water maze experimental detection of the mice in the first control group, the second control group, the first treatment group and the second treatment group on the 2nd to 6th days was the positioning navigation experimental stage, and the Morris water maze experimental detection of the mice in the first control group, the second control group, the first treatment group and the second treatment group on the 7th day was the spatial exploration experimental stage.

[0104] Pre-training stage: First, the mice in the first control group, the second control group, the first treatment group, and the second treatment group were placed on the escape platform and stayed for 10 seconds to adapt to the platform environment; then, the mice in the first control group, the second control group, the first treatment group, and the second treatment group were slowly placed in the water facing the edge of the second quadrant pool wall. If the mice failed to find the escape platform independently for more than 60 seconds, they were guided to the escape platform with a guide rod and stayed for another 10 seconds to consolidate their memory. After the training was completed, they were wiped dry and returned to the cage.

[0105] Positioning and navigation experiment phase: First, an appropriate amount of titanium dioxide was added to the water to ensure water turbidity, allowing the track recorder to record the mouse's behavioral trajectory. Then, mice from the first control group, the second control group, the first treatment group, and the second treatment group were slowly lowered into the water facing the edge of the second quadrant. If the mouse independently found the escape platform within 60 seconds and remained there for at least 10 seconds, the escape latency was recorded. If the mouse failed to independently find the escape platform within 60 seconds, the escape latency was recorded as 60 seconds. A guide stick was used to guide the mouse to the escape platform, and the mouse remained there for another 20 seconds to consolidate its memory. After completion, the mouse was dried and returned to its cage. The mouse's escape latency was then recorded, which represented its learning performance for that day. Mice with shorter escape latencies demonstrated better learning and memory abilities.

[0106] During the spatial exploration phase, the escape platform was removed from the pool. Then, maintaining the same time and location as in the training phase, mice from the first control group, second control group, first treatment group, and second treatment group were placed into the pool. The mice's time spent in the target quadrant and the number of times they crossed the platform within 60 seconds were recorded. After the test, the mice were dried and returned to their cages. Mice that spent longer in the target quadrant and crossed the platform more frequently demonstrated better memory retention.

[0107] Step b: Analyze the detection data of the spatial exploration experiment phase to obtain the spatial memory ability and learning ability of the mice in each group.

[0108] Specifically, the Morris water maze test can be used to evaluate the spatial memory and learning abilities of rodents. Mice that spend a longer time in the target quadrant and cross the platform more times have better spatial memory and learning abilities.

[0109] like Figure 4 As shown, Figure 4 This is a schematic diagram of the trajectories of each group of mice in the pre-training phase of the water maze experiment provided by the present application. The mice in the first control group are normal mice, and the mice in the first control group can quickly find the hidden platform in the water. The mice in the second control group are Alzheimer's disease mice. The movement trajectories of the mice in the second control group are chaotic and cannot find the hidden platform in the water within the set time. The mice in the first treatment group are Alzheimer's disease mice. The mice in the first treatment group are administered with an aqueous solution of Compound Cistanche and Yizhi Capsules. After the intervention of Compound Cistanche and Yizhi Capsules, the mice in the first treatment group can find the hidden platform in the water more smoothly; the mice in the second treatment group are Alzheimer's disease mice. The mice in the second treatment group are administered with an aqueous solution of Donepezil. After the intervention of Donepezil, the mice in the second treatment group can find the hidden platform in the water more smoothly.

[0110] Positioning navigation experiment phase: as shown in Table 3 and Figure 5 As shown, Figure 5This is a schematic diagram of the escape latency of each group of mice in the water maze experiment positioning navigation test phase provided by the present application (Note: Compared with the first control group, ##P<0.01, #P<0.05; Compared with the second control group, **P<0.01, *P<0.05, n=9). The overall trend of the escape latency of mice in the first control group, the second control group, the first treatment group, and the second treatment group is the same. The escape latency gradually shortens with the increase in the number of training days. Compared with the first control group, the escape latency of mice in the second control group from day 2 to day 5 was prolonged to a certain extent, but no statistical difference was found (P>0.05); compared with the second control group, the escape latency of mice in the first treatment group and the second treatment group from day 1 to day 5 was shortened to a certain extent, but no statistical difference was found (P>0.05).

[0111] Table 3 Escape latency of mice in each group during the navigation experiment ( n=9)

[0112]

[0113]

[0114] Note: Compared with the first control group, ##P<U.U1, #P<0.05; compared with the second control group, **P<0.U1, *P<0.05.

[0115] Space exploration experiment phase: as shown in Table 4, Table 5, Figure 6 and Figure 7 As shown, Figure 6 This is a schematic diagram of the trajectory of each group of mice in the water maze experimental space exploration phase provided by this application. Figure 7 This is a schematic diagram of the experimental results of each group of mice in the water maze experimental space exploration experiment phase provided in the present application (Note: compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05, n=9). Compared with the first control group, the number of times the mice in the second control group crossed the platform and the time they stayed in the target quadrant were significantly reduced (P<0.01); compared with the second control group, the number of times the mice in the first treatment group and the second treatment group crossed the platform and the time they stayed in the target quadrant were significantly increased (P<0.01).

[0116] Table 4 The time that mice in each group stayed in the target quadrant ( n=9)

[0117]

[0118] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0119] Table 5 Number of times mice in each group crossed the platform (median (25%, 75%), n=9)

[0120] Group Number of platforms crossed The first control group 3.0(4.0，2.0) The second control group <![CDATA[1.0(1.0,0.0) ## ]]> The second treatment group <![CDATA[2.0(3.5.1.5) ** ]]> The first treatment group <![CDATA[2.0(4.0.2.0) ** ]]>

[0121] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0122] Therefore, by conducting Morris water maze test on mice in the first control group, the second control group, the first treatment group and the second treatment group, and analyzing the Morris water maze test data, it can be determined that Compound Cistanche Yizhi Capsule and Donepezil can improve the spatial memory and learning ability of Alzheimer's disease mice.

[0123] The target indicators include behavioral abilities, which include motor coordination abilities. The method for collecting motor coordination abilities includes the following steps:

[0124] Step a: For each group of mice, perform the pole climbing test on the mice for a third consecutive predetermined number of days, wherein the last day is the first test period and the remaining days are the first training period. The third predetermined number of days is not limited and can be set according to actual needs.

[0125] The pole climbing test is a behavioral test widely used to assess the motor coordination ability of rodents. The experimental apparatus consists of a base and a long cylindrical pole (gauze wrapped, 1 cm in diameter, 60 cm long). In addition, the pole climbing test requires mice to grasp and manipulate the pole to descend to the bottom of the pole. During the pole climbing test, the mouse is placed head-up on the top of the pole. The mouse will usually naturally descend and descend along the pole uninterruptedly to the base in order to return to its home cage.

[0126] Specifically, the mice in the first control group, the second control group, the first treatment group, and the second treatment group were subjected to a pole climbing test for three consecutive days, with three tests per day and an interval of no less than 60 minutes between each test. The first two days were the training period for the mice in the first control group, the second control group, the first treatment group, and the second treatment group, and the third day was the testing period for the mice in the first control group, the second control group, the first treatment group, and the second treatment group. During the training period, the time it took for the mice to crawl to the base until all four limbs touched the ground was recorded. If the time exceeded 60 seconds, a guide rod was used to moderately guide the mice to crawl. During the testing period, the time it took for the mice to crawl to the base until all four limbs touched the ground was recorded to evaluate the motor coordination ability of each group of mice. The mean of the three experimental times during the testing period was taken for statistical analysis. Mice that were able to complete the pole climbing test in a shorter time showed better motor coordination ability (Note: The experimental time was limited to 60 seconds. If the time exceeded 60 seconds, the timer was stopped, the mice were guided, and the time was uniformly recorded as 60 seconds).

[0127] Step b: Analyze the test data of the first test period to obtain the motor coordination ability of the group of mice.

[0128] Specifically, the pole climbing test can be used to evaluate the motor coordination ability of rodents. The shorter the pole climbing time, the better the coordination ability of the mouse.

[0129] As shown in Table 6 and Figure 8 As shown, Figure 8 This is a schematic diagram of the pole climbing time of each group of mice provided in the present application (Note: the experimental time is limited to 60 s. When the time exceeds 60 s, the timing is stopped, the mice are guided and the record is uniformly recorded in 60 s; compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=9). Compared with the first control group, the pole climbing time of the mice in the second control group was significantly increased (P<0.01); compared with the second control group, the pole climbing time of the mice in the first treatment group and the second treatment group was significantly reduced (P<0.01).

[0130] Table 6 Statistical results of climbing time of mice in each group ( n=9)

[0131]

[0132]

[0133] Note: The experimental time limit is 60 s. If the time exceeds 60 s, stop the timer, guide the mice, and record the results in 60 s. Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0134] Therefore, by conducting pole climbing test on mice in the first control group, the second control group, the first treatment group and the second treatment group, and analyzing the pole climbing test data, it can be determined that both Compound Cistanche Alpiniae Capsules and Donepezil effectively improve the pole climbing time and motor coordination ability of Alzheimer's disease mice, and Compound Cistanche Alpiniae Capsules are better than donepezil in improving the pole climbing time and motor coordination ability of Alzheimer's disease mice.

[0135] The target indicators include behavioral abilities, which include motor coordination and balance abilities. The collection method of motor coordination and balance abilities includes the following steps:

[0136] Step a: For each group of mice, perform a rotarod fatigue test on the mice for a fourth consecutive predetermined number of days, wherein the last day constitutes a second testing period, and the remaining days constitute a second training period. The fourth predetermined number of days is not limited and can be set based on actual needs.

[0137] The rotarod fatigue test is a behavioral experiment widely used to assess the motor coordination and balance abilities of rodents. The experimental apparatus consists of a rotating rod (approximately 3 cm in diameter), a drive motor, a gear transmission system, and five channels (approximately 6 cm wide). Furthermore, during the rotarod fatigue test, the mouse must be placed unidirectionally and stably on the rotarod in each channel. The spindle speed of the rotarod tester is set to accelerate at a constant rate of 0.12 rpm / s, gradually accelerating from 4 rpm / min to 40 rpm / min. A single test lasts for 300 seconds. The test is considered complete if the mouse falls from the rotarod or grasps the rotarod and rotates it continuously for three revolutions without crawling involuntarily.

[0138] Specifically, the mice in the first control group, the second control group, the first treatment group, and the second treatment group were subjected to a rotating rod fatigue test for three consecutive days, with the test performed three times a day, and the interval between each test was no less than 60 minutes; among them, the first two days were the training period for the mice in the first control group, the second control group, the first treatment group, and the second treatment group, and the third day was the testing period for the mice in the first control group, the second control group, the first treatment group, and the second treatment group. The experimental time of the mice was recorded during the testing period to systematically evaluate the motor coordination and balance abilities of the mice in each group. The mean of the three experimental times during the testing period was taken for statistical analysis. Mice with longer experimental times showed that their motor coordination and balance abilities were better (Note: the experimental time was limited to 300 seconds. When the time exceeded 300 seconds, the experiment was stopped and recorded as 300 seconds).

[0139] Step b: Analyze the test data of the second test period to obtain the motor coordination ability and balance ability of the group of mice.

[0140] Specifically, the rotarod fatigue test can be used to evaluate the motor coordination and balance abilities of rodents. The longer the rotarod test time, the better the motor coordination and balance abilities of the mice.

[0141] As shown in Table 7 and Figure 9 As shown, Figure 9 This is a schematic diagram of the rotarod time of each group of mice provided in this application (Note: The experimental time is limited to 300 seconds. When the time exceeds 300 seconds, the experiment is stopped and the time is uniformly recorded as 300 seconds; compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=9). Compared with the first control group, the rotarod time of mice in the second control group was significantly reduced (P<0.01). Compared with the second control group, the rotarod time of mice in the first treatment group and the second treatment group was significantly increased (P<0.01).

[0142] Table 7 Statistical results of the rotarod time of mice in each group (x±s, n=9)

[0143]

[0144] Note: The experimental time is limited to 300 s. If the time exceeds 300 s, the experiment is stopped and the time is recorded as 300 s. Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0145] Therefore, by conducting rotarod fatigue test on mice in the first control group, the second control group, the first treatment group and the second treatment group, and analyzing the rotarod fatigue test data, it can be determined that both Compound Cistanche Alpiniae Capsules and Donepezil can effectively improve the rotarod time, motor coordination and balance ability of Alzheimer's disease mice, and the effect of Compound Cistanche Alpiniae Capsules on improving the rotarod time, motor coordination and balance ability of Alzheimer's disease mice is better than donepezil.

[0146] The target indicators include brain tissue conditions, including NEFL levels in brain tissue; serum conditions, including NEFL levels, Aβ1-42 levels, 1L-6 levels, 1L-1β levels, and TNF-α levels; and hippocampal tissue conditions, including Aβ deposition and Aβ1-42 levels in hippocampal tissue. The method for collecting brain tissue NEFL levels, serum NEFL levels, serum Aβ1-42 levels, serum 1L-6 levels, serum 1L-1β levels, serum TNF-α levels, hippocampal Aβ deposition, and hippocampal Aβ1-42 levels includes the following steps:

[0147] Step a: For each group of mice, a first brain tissue sample, a first serum sample, and a first hippocampus tissue sample are collected from the mice in the group.

[0148] Specifically, the experimental procedure involved injecting double-distilled water into the mice in the first and second control groups; gavage-administering an equal volume of a 468 mg / kg aqueous solution of Compound Cistanche Yizhi Capsules to the first treatment group; and administering an equal volume of a 0.65 mg / kg aqueous solution of donepezil to the mice in the second treatment group. The intervention continued for eight weeks in the second, third, first, and second treatment groups.

[0149] Collection of the first serum sample: After the last injection of double-distilled water into the mice in the first and second control groups, the mice in the first and second control groups were fasted for 12 hours. 12 hours later, the mice in the first and second control groups were anesthetized by intraperitoneal injection of tribromoethanol; then, blood was collected from the inner canthus of the eyes of the mice in the first and second control groups, and the blood was allowed to stand at 4°C for 30 minutes and then centrifuged at 3000 rpm / min for 15 minutes; then, the upper serum was collected and packaged, and stored in a -80°C refrigerator for later use, thereby collecting the first serum samples of the mice in the first and second control groups.

[0150] After the last administration of the aqueous solution of Compound Cistanche Yizhi Capsules to the first treatment group, the mice in the first treatment group were fasted for 12 hours. Twelve hours later, the mice in the first treatment group were anesthetized by intraperitoneal injection of tribromoethanol; then, blood was collected from the inner canthus of the eyes of the mice in the first treatment group, and the blood was allowed to stand at 4°C for 30 minutes and then centrifuged at 3000 rpm / min for 15 minutes; then, the upper serum was collected and packaged, and stored in a -80°C refrigerator for later use, thereby collecting the first serum samples of the mice in the first treatment group.

[0151] After the last administration of donepezil aqueous solution to the second treatment group, the mice in the second treatment group were fasted for 12 hours. Twelve hours later, the mice in the second treatment group were anesthetized with tribromoethanol intraperitoneally. Then, blood was collected from the inner canthus of the mice in the second treatment group. The blood was allowed to stand at 4°C for 30 minutes and then centrifuged at 3000 rpm / min for 15 minutes. Then, the upper serum was collected and packaged, and stored in a -80°C refrigerator for later use, thereby obtaining the first serum samples of the mice in the second treatment group.

[0152] Collection of the first brain tissue sample: After the last injection of double-distilled water into the mice in the first and second control groups, the mice in the first and second control groups were fasted for 12 hours. 12 hours later, the mice in the first and second control groups were anesthetized by intraperitoneal injection of tribromoethanol; then, blood was collected from the inner canthus of the eyes of the mice in the first and second control groups; then, after blood collection, the mice in the first and second control groups were killed by cervical dislocation, and brain tissue samples were collected, thereby collecting the first brain tissue samples of the mice in the first and second control groups.

[0153] After the last administration of the aqueous solution of Compound Cistanche deserticola Capsules to the mice in the first treatment group, the mice in the first treatment group were fasted for 12 hours. 12 hours later, the mice in the first treatment group were anesthetized by intraperitoneal injection of tribromoethanol; then, blood was collected from the inner canthus of the eyes of the mice in the first treatment group; then, after the blood was collected, the mice in the first treatment group were killed by cervical dislocation, and brain tissue samples were collected, thereby obtaining the first brain tissue samples of the mice in the first treatment group.

[0154] After the last administration of the donepezil aqueous solution to the mice in the second treatment group, the mice in the second treatment group were fasted for 12 hours. 12 hours later, the mice in the second treatment group were anesthetized with tribromoethanol intraperitoneally; then, blood was collected from the inner canthus of the eyes of the mice in the second treatment group; then, after the blood was collected, the mice in the second treatment group were killed by cervical dislocation, and brain tissue samples were collected, thereby obtaining the first brain tissue samples of the mice in the second treatment group.

[0155] Collection of the first hippocampal tissue sample: After the first brain tissue sample of the first control group was collected, the brain tissue was divided into two parts along the midline. The left side of the brain tissue was fixed in formalin solution for more than 48 hours for subsequent histological staining observation. The hippocampal tissue on the right side of the brain tissue was separated and stored in a -80°C refrigerator until use, thereby collecting the first hippocampal tissue sample of the mice in the first control group.

[0156] After obtaining the first brain tissue sample of the second control group, the brain tissue was divided into two along the midline, and the left side of the brain tissue was fixed in formalin solution for more than 48 hours for subsequent histological staining observation. The hippocampal tissue was isolated from the right side of the brain tissue and stored in a -80°C refrigerator for later use, thereby obtaining the first hippocampal tissue sample of the mice in the second control group.

[0157] After collecting the first brain tissue sample from the first treatment group, the brain tissue was divided into two along the midline, and the left side of the brain tissue was fixed in formalin solution for more than 48 hours for subsequent histological staining observation. The hippocampal tissue was isolated from the right side of the brain tissue and stored in a -80°C refrigerator for later use, thereby collecting the first hippocampal tissue samples of mice in the first treatment group.

[0158] After collecting the first brain tissue sample from the second treatment group, the brain tissue was divided into two along the midline, and the left side of the brain tissue was fixed in formalin solution for more than 48 hours for subsequent histological staining observation. The hippocampal tissue was isolated from the right side of the brain tissue and stored in a -80°C refrigerator for later use, thereby collecting the first hippocampal tissue samples of mice in the second treatment group.

[0159] Step b: performing enzyme-linked immunosorbent assay on the first brain tissue sample, the first serum sample, and the first hippocampal tissue sample, respectively, to obtain the NEFL level in the brain tissue, the NEFL level in the serum, the Aβ1-42 level in the serum, the 1L-6 level in the serum, the 1L-1β level in the serum, the TNF-α level in the serum, the Aβ1-42 level in the hippocampal tissue, and the Aβ deposition level in the hippocampal tissue.

[0160] Specifically, enzyme-linked immunosorbent assay (ELISA) experiments are performed on the first brain tissue sample, the first serum sample, and the first hippocampal tissue sample, respectively, to obtain NEFL levels in the brain tissue, NEFL levels in the serum, Aβ1-42 levels in the serum, 1L-6 levels in the serum, 1L-1β levels in the serum, TNF-α levels in the serum, Aβ1-42 levels in the hippocampal tissue, and Aβ deposition levels in the hippocampal tissue, specifically comprising the following steps:

[0161] a) Dilution and loading of standard samples: Set up 10 standard sample wells on the enzyme-labeled coated plate, and dilute and load the plate.

[0162] b) Sample loading: Set up blank wells and test sample wells on the enzyme-labeled plate; dilute the first brain tissue sample, the first serum sample, and the first hippocampal tissue sample 5-fold. Then, load 10 μL of the diluted first brain tissue sample, the first serum sample, and the first hippocampal tissue sample into each well.

[0163] c) Add enzyme: Add 50 μL of enzyme labeling reagent to the sample wells and standard wells of the enzyme labeling coated plate.

[0164] d) Incubation: Seal the plate with a sealing film and incubate in a 37°C incubator for 30 min.

[0165] e) Washing: Remove the sealing film, discard the liquid and pat dry. Fill each well with washing solution, let it stand for 30 seconds, and then discard. Repeat 5 times.

[0166] f) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop the color at 37°C in the dark for 10 min.

[0167] g) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0168] h) Assay: Zero the well with a blank and measure the absorbance of each well at 450 nm for 15 minutes. The absorbance value correlates with the concentration of the target substance in the sample. By measuring the absorbance and comparing it with a standard curve, the levels of NEFL in brain tissue, NEFL in serum, Aβ1-42 in serum, IL-6 in serum, IL-1β in serum, TNF-α in serum, Aβ1-42 in hippocampus, and Aβ deposition in hippocampus can be calculated.

[0169] Neurofilament light chain polypeptide (NEFL) is a key component of neuronal skeleton formation. Neuronal damage will lead to an increase in NEFL content. Figure 10 As shown, Figure 10 Schematic diagram of NEFL levels in the serum and brain tissue of each group of mice provided in this application (Note: Compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=6). Compared with the first control group, the NEFL levels in the serum and brain tissue of the mice in the second control group were significantly increased (P<0.01). Compared with the second control group, the NEFL levels in the serum and brain tissue of the mice in the first and second treatment groups were significantly decreased (P<0.01, P<0.01, P<0.05, P<0.01).

[0170] Table 8 NEFL content in serum and brain tissue of mice in each group ( n=6)

[0171]

[0172] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0173] Therefore, by conducting enzyme-linked immunosorbent assay experiments on mice in the first control group, the second control group, the first treatment group, and the second treatment group, it was determined that both Compound Cistanche Yizhi Capsule and Donepezil can reduce the NEFL content in the serum and brain tissue of Alzheimer's disease mice, and can both improve neuronal damage in Alzheimer's disease mice.

[0174] As shown in Table 9 and Figure 11 As shown, Figure 11Schematic diagram of the Aβ1-42 levels in the serum and hippocampal tissues of each group of mice provided in this application (Note: Compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=6). Compared with the first control group, the Aβ1-42 levels in the serum and hippocampal tissues of the mice in the second control group were significantly increased (P<0.01). Compared with the second control group, the Aβ1-42 levels in the serum and hippocampal tissues of the mice in the first treatment group and the second treatment group were significantly decreased (P<0.01, P<0.05, P<0.05, P<0.05).

[0175] Table 9 Aβ1-42 levels in serum and hippocampus of mice in each group ( n=6)

[0176]

[0177] Note: Compared with the first control group, ##P<0.U1, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0178] Therefore, by performing enzyme-linked immunosorbent assay experiments on mice in the first control group, the second control group, the first treatment group, and the second treatment group, it was determined that both Compound Cistanche Yizhi Capsule and Donepezil can reduce the Aβ1-42 content in the serum and brain tissue of Alzheimer's disease mice, and can improve Aβ deposition.

[0179] As shown in Table 10 and Figure 12 As shown, Figure 12 Schematic diagram of the 1L-6, 1L-1β and TNF-α levels in the serum of each group of mice provided in the present application (Note: Compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=6). Compared with the first control group, the 1L-6 (interleukin, IL)-6), 1L-1β and TNF-α (tumor necrosis factor-α) levels in the serum of the mice in the second control group were significantly increased (P<0.01). Compared with the second control group, the 1L-6 levels in the serum of the mice in the first treatment group and the second treatment group decreased to a certain extent (P<0.01, P>0.05). Compared with the second control group, the 1L-1β levels in the serum of the mice in the first treatment group and the second treatment group were significantly decreased (P<0.01, P<0.01). Compared with the second control group, the TNF-α levels in the serum of the mice in the first treatment group and the second treatment group were significantly decreased (P<0.01, P<0.05).

[0180] Table 10 IL-6, IL-1β and TNF-α contents in the serum of mice in each group ( n=6)

[0181]

[0182] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0183] Therefore, by performing enzyme-linked immunosorbent assay experiments on mice in the first control group, the second control group, the first treatment group, and the second treatment group, it was determined that both Compound Cistanche Yizhi Capsule and Donepezil can regulate the levels of 1L-6, 1L-1β, and TNF-α in the serum of Alzheimer's disease mice to improve the inflammatory state.

[0184] Aβ deposition in hippocampal tissue plays an important role in the development of Alzheimer's disease and is an early adverse event in the pathogenesis of Alzheimer's disease. Figure 13 、 Figure 14 As shown, Figure 13 Schematic diagram of Aβ immunohistochemical staining of the hippocampal CA1 region of each group of mice provided in this application. Figure 14 Schematic diagram of the quantitative analysis results of Aβ in the CA1 region of the hippocampus of each group of mice provided in this application (Note: compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=4). Aβ is mainly expressed in the pyramidal neurons of the hippocampus of the mice in the second control group. Compared with the first control group, the deposition of Aβ in the neurons of the hippocampus of the mice in the second control group was significantly enhanced (P<0.01). Compared with the second control group, the deposition of Aβ in the neurons of the hippocampus of the mice in the first treatment group and the second treatment group was reduced (P<0.05).

[0185] Table 11 Quantification of Aβ in the hippocampal CA1 region of mice in each group ( n=4)

[0186]

[0187] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0188] Therefore, by performing enzyme-linked immunosorbent assay experiments on mice in the first control group, the second control group, the first treatment group, and the second treatment group, it was determined that both Compound Cistanche Yizhi Capsule and Donepezil could improve Aβ deposition in the hippocampus of mice with Alzheimer's disease.

[0189] The target indicators include hippocampal tissue conditions, including PSD95 protein expression, SYP protein expression, TLR4 protein expression, NF-κB protein expression, MYD88 protein expression, Bax protein expression, Bc12 protein expression, and Caspase3 protein expression in the hippocampus. The method for collecting PSD95 protein expression, SYP protein expression, TLR4 protein expression, NF-κB protein expression, MYD88 protein expression, Bax protein expression, Bc12 protein expression, and Caspase3 protein expression in the hippocampus includes the following steps:

[0190] Step a: For each group of mice, collect the second hippocampal tissue sample from the mice in the group.

[0191] Specifically, the experimental procedure involved injecting double-distilled water into the mice in the first and second control groups; gavage administering an equal volume of a 468mg / kg aqueous solution of Compound Cistanche Yizhi Capsules to the first treatment group; and administering an equal volume of a 0.65mg / kg aqueous solution of Donepezil to the mice in the second treatment group. The intervention was continued for eight weeks in the first, second, first, and second treatment groups.

[0192] After the last injection of double distilled water into the mice in the first and second control groups, the mice in the first and second control groups were fasted for 12 hours. 12 hours later, the mice in the first and second control groups were anesthetized with tribromoethanol intraperitoneally: then, blood was collected from the inner canthus of the eyes of the mice in the first and second control groups; then, after blood collection, the mice in the first and second control groups were killed by cervical dislocation, and brain tissue sampling was performed: then, the brain tissues of the mice in the first and second control groups were divided into two along the midline, and the hippocampus tissue was separated from the right side of the brain tissue and stored in a -80°C refrigerator for future use, thereby collecting the second hippocampus tissue samples of the mice in the first and second control groups.

[0193] After the last administration of the aqueous solution of Compound Cistanche Yizhi Capsules to the mice in the first treatment group, the mice in the first treatment group were fasted for 12 hours, and 12 hours later, the mice in the first treatment group were anesthetized by intraperitoneal injection of tribromoethanol; then, blood was collected from the inner canthus of the mice in the first treatment group; then, after blood collection, the mice in the first treatment group were killed by cervical dislocation, and brain tissue sampling was performed; then, the brain tissue of the mice in the first treatment group was divided into two along the midline, and the hippocampus tissue was separated from the right side of the brain tissue and stored in a -80°C refrigerator for future use, thereby collecting the second hippocampus tissue samples of the mice in the first treatment group.

[0194] After the last administration of donepezil aqueous solution to the mice in the second treatment group, the mice in the second treatment group were fasted for 12 hours. 12 hours later, the mice in the second treatment group were anesthetized with tribromoethanol intraperitoneally; then, blood was collected from the inner canthus of the mice in the second treatment group; then, after blood collection, the mice in the second treatment group were killed by cervical dislocation, and brain tissue sampling was performed; then, the brain tissue of the mice in the second treatment group was divided into two along the midline, and the hippocampus tissue was isolated from the right side of the brain tissue and stored in a -80°C refrigerator for future use, thereby collecting the second hippocampus tissue samples of the mice in the second treatment group.

[0195] Step b: Performing a protein immunoblotting experiment on the second hippocampal tissue sample to obtain the expression of PSD95 protein in the hippocampal tissue, the expression of SYP protein in the hippocampal tissue, the expression of TLR4 protein in the hippocampal tissue, the expression of NF-κB protein in the hippocampal tissue, the expression of MYD88 protein in the hippocampal tissue, the expression of Bax protein in the hippocampal tissue, the expression of Bc12 protein in the hippocampal tissue, and the expression of Caspase3 protein in the hippocampal tissue.

[0196] Specifically, a protein immunoblotting experiment was performed on the second hippocampal tissue sample to obtain the expression of postsynaptic density protein 95 (PSD95) protein in the hippocampal tissue, the expression of synaptophysin (SYP) protein in the hippocampal tissue, the expression of Toll-like receptor 4 (TLR4) protein in the hippocampal tissue, the expression of nuclear factor (NF-κB (65)) protein in the hippocampal tissue, the expression of myeloid differentiation primary response 88 (MYD88) protein in the hippocampal tissue, the expression of Bcl2 associated X (Bax) protein in the hippocampal tissue, the expression of B-cell lymphoma 2 (Bcl2) protein in the hippocampal tissue and the expression of Caspase3 protein in the hippocampal tissue, which specifically includes the following steps:

[0197] a) Tissue Protein Extraction: Weigh 20 mg of the second hippocampal tissue sample into a 1.5 mL enzyme-free EP tube, add 300 μL of tissue lysis buffer (1 mL RIPA; 10 μL PMSF), and homogenize and lyse the sample on ice using ultrasound. The homogenized sample was then placed on ice for 30 min, vortexing every 5 min. The sample was then centrifuged at 12,000 rpm / min for 5 min. The supernatant was aspirated and transferred to a new 1.5 mL enzyme-free EP tube and placed on ice for subsequent experiments.

[0198] b) BCA protein quantification: prepare BCA working solution, dilute standards, dilute samples, incubate, and detect.

[0199] c) Protein denaturation: Add the remaining second hippocampal tissue sample to 5× Loading Buffer at a ratio of 4:1, mix well, and denature in a 100°C water bath for 10 min. After the protein cools, aliquot and store at -20°C for subsequent experiments.

[0200] d) Rapid preparation and electrophoresis of PAGE gels.

[0201] e) Transfer

[0202] f) Blocking: After transfer, turn off the power and remove the transfer clip. Place the removed PVDF membrane in a protein incubation box containing 1×TBST solution and wash it three times with 1×TBST solution on a shaker, each time for 5 minutes. After washing, add rapid blocking solution and block on a shaker at room temperature for 15-30 minutes.

[0203] g) Primary Antibody Incubation: After blocking, recover the rapid blocking buffer and wash three times with 1× TBST on a shaker for 5 minutes each. Add the corresponding primary antibody solution based on the target protein and incubate on a shaker at room temperature for 10 minutes, then transfer to a 4°C refrigerator for overnight incubation.

[0204] h) Secondary Antibody Incubation: After the primary antibody incubation is complete, the primary antibody solution is recovered and washed three times with 1× TBST solution, each wash for 10 minutes. The corresponding secondary antibody solution is added according to the primary antibody protein, and the mixture is incubated on a shaker at room temperature for 1-2 hours.

[0205] i) Development: After the secondary antibody incubation is completed, the secondary antibody solution is recovered and washed three times with 1× TBST solution, each wash for 10 minutes. The developing equipment is turned on, and ECL developer is prepared in the dark. An appropriate amount of developer is pipetted onto the strip for exposure and development, and the developed image is saved.

[0206] PSD95 is a postsynaptic scaffold protein that plays a key role in protein assembly, synaptic development, and synaptic plasticity; SYP is a glycoprotein located on the synaptic vesicle membrane that plays a role in docking, fusion, and endocytosis during membrane transport. The expression of both proteins can be used to evaluate synaptic plasticity in mice. Figure 15-17 As shown, Figure 15 Schematic diagram of representative protein bands of PSD95 protein and SYP protein in the hippocampal tissue of each group of mice provided in this application, Figure 16 This is a quantitative schematic diagram of the band intensity of PSD95 protein relative to β-Actin in the hippocampal tissue of each group of mice provided in this application (Note: compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=6), Figure 17 : This application provides a quantitative schematic diagram of the band intensity of SYP protein for GAPDH in the hippocampus tissue of each group of mice (Note: Compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=6). Compared with the first control group, the expression of PSD95 protein and SYP protein in the hippocampus tissue of the mice in the second control group were significantly reduced (P<0.05). Compared with the second control group, the expression of PSD95 protein and SYP protein in the hippocampus tissue of the mice in the first treatment group and the second treatment group were significantly increased (P<0.01, P<0.05, P<0.05, P<0.01).

[0207] Table 12 Quantification of PSD95 protein and SYP protein in the hippocampus tissue of mice in each group ( n=6)

[0208] Group PSD95 SYP The first control group 1.01±0.22 1.04±0.10 The second control group <![CDATA[0.77±0.05 # ]]> <![CDATA[0.84±0.14 # ]]> The second treatment group <![CDATA[1.08±0.13 ** ]]> <![CDATA[1.06±0.13 * ]]> The first treatment group <![CDATA[1.04±0.13 * ]]> <![CDATA[1.14±0.09 ** ]]>

[0209] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0210] Therefore, by performing protein immunoblotting experiments on mice in the first control group, the second control group, the first treatment group and the second treatment group, it was determined that both Compound Cistanche Yizhi Capsule and Donepezil can increase the expression of PSD95 protein and SYP protein in the hippocampal tissue of Alzheimer's disease mice and improve the synaptic plasticity of Alzheimer's disease mice.

[0211] TLR4 plays an important role in mediating the binding and phagocytosis of protofilament amyloid by microglia, and can effectively mediate the activation of the NF-κB / MYD88 inflammatory pathway. The expression of proteins in this pathway can be used to evaluate the level of neuroinflammation in mice. As shown in Table 13 and Figures 18-21 As shown, Figure 18 Schematic diagram of representative protein bands of TLR4 protein, NF-κB protein and MYD88 protein in the hippocampal tissue of each group of mice provided in this application, Figure 19 : This is a quantitative schematic diagram of the band intensity of TLR4 protein for GAPDH in the hippocampal tissue of each group of mice provided in the present application (Note: compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=5), Figure 20 This is a quantitative diagram of the band intensity of NF-κB protein relative to β-Actin in the hippocampal tissue of each group of mice provided in this application (Note: Compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=5), Figure 21: This is a schematic diagram of the quantitative intensity of the MYD88 protein for β-Actin in the hippocampal tissue of each group of mice provided in the present application (Note: Compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=5). Compared with the first control group, the TLR4 protein expression, NF-κB protein expression and MYD88 protein expression in the hippocampal tissue of the mice in the second control group were significantly increased (P<0.05, P<0.01, P<0.05). Compared with the second control group, the TLR4 protein expression in the hippocampal tissue of the mice in the first treatment group and the second treatment group was significantly reduced (P<0.01, P<0.01). Compared with the second control group, the NF-κB protein expression in the hippocampal tissue of the mice in the first treatment group and the second treatment group was reduced to varying degrees (P>0.05, P<0.05). Compared with the second control group, the expression of MYD88 protein in the hippocampus of mice in the first treatment group and the second treatment group was significantly decreased (P < 0.05, P < 0.05).

[0212] Table 13 Quantification of TLR4 protein, NF-κB protein and MYD88 protein in the hippocampus tissue of mice in each group ( n=5)

[0213] Group TLR4 NF-κB (p65) MYD88 The first control group 0.72±0.19 0.62±0.24 1.08±0.17 The second control group <![CDATA[1.06±0.03 # ]]> <![CDATA[1.00±0.04 ## ]]> <![CDATA[1.39±0.03 # ]]> The second treatment group <![CDATA[0.68±0.21 ** ]]> 0.77±0.11 <![CDATA[1.12±0.15 * ]]> The first treatment group <![CDATA[0.61±0.12 ** ]]> <![CDATA[0.72±0.15 * ]]> <![CDATA[1.12±0.15 * ]]>

[0214] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0215] Therefore, by performing protein immunoblotting experiments on mice in the first control group, the second control group, the first treatment group and the second treatment group, it was determined that Compound Cistanche Yizhi Capsule and Donepezil can reduce TLR4 protein expression, NF-κB protein expression and MYD88 protein expression in the hippocampus tissue of Alzheimer's disease mice to varying degrees, and improve the inflammation level of the hippocampus tissue of Alzheimer's disease mice.

[0216] Bax, Bcl2, and Caspase3 are key proteins in the process of endogenous and exogenous apoptosis and can be used to evaluate the apoptosis level of mouse hippocampal tissue. Figure 22-Figure 25 As shown, Figure 22 Schematic diagram of representative protein bands of Bax protein, Bcl2 protein and Caspase3 protein in the hippocampal tissue of each group of mice provided in this application, Figure 23: This is a quantitative diagram of the band intensity of Bax protein relative to GAPDH in the hippocampal tissue of each group of mice provided in the present application (Note: compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=3-5), Figure 24 : This is a quantitative diagram of the band intensity of Bcl2 protein relative to GAPDH in the hippocampal tissue of each group of mice provided in this application (Note: compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=3-5), Figure 25 : This is a schematic diagram of the quantitative intensity of the Caspase3 protein relative to β-Actin in the hippocampal tissue of each group of mice provided in this application (Note: Compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=3-5). Compared with the first control group, the expression of Bcl2 protein in the hippocampal tissue of the mice in the second control group was reduced (P>0.05), and the expression of Bax protein and Caspase3 protein was increased (P<0.05, P<0.01). Compared with the second control group, the expression of Bcl2 protein in the hippocampal tissue of the mice in the second treatment group was increased (P>0.05), and the expression of Bax protein and Caspase3 protein was decreased (P<0.05, P<0.05). Compared with the second control group, the expression of Bc12 protein in the hippocampus of mice in the first treatment group was increased (P < 0.05), and the expression of Bax protein and Caspase3 protein were decreased (P < 0.01, P < 0.05).

[0217] Table 14 Quantification of Bax protein, Bcl2 protein and Caspase3 protein in the hippocampus tissue of each group of mice ( n=3-5)

[0218] Group Bax Bcl2 Caspase3 The first control group 0.80±0.11 0.92±0.06 0.75±0.14 The second control group <![CDATA[1.04±0.01 # ]]> 0.72±0.02 <![CDATA[1.06±0.12 ## ]]> The second treatment group <![CDATA[0.80±0.09 * ]]> 0.89±0.11 <![CDATA[0.83±0.17 * <!-- 20 -->]]> The first treatment group <![CDATA[0.60±0.11 ** ]]> <![CDATA[1.06±0.27 * ]]> <![CDATA[0.82±0.13 * ]]>

[0219] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0220] Therefore, by performing protein immunoblotting experiments on mice in the first control group, the second control group, the first treatment group and the second treatment group, it was determined that Compound Cistanche Yizhi Capsule and Donepezil can increase the Bcl2 protein expression in the hippocampus tissue of Alzheimer's disease mice to varying degrees, reduce the Bax protein expression and Caspase3 protein expression in the hippocampus tissue of Alzheimer's disease mice, and improve the apoptosis level of the hippocampus tissue of Alzheimer's disease mice.

[0221] The target indicators include hippocampal tissue conditions, including the structural conditions of the hippocampal tissue and the Nissl bodies in the hippocampal tissue. The method for collecting the structural conditions of the hippocampal tissue and the Nissl bodies in the hippocampal tissue includes the following steps:

[0222] Step a: For each group of mice, collect the third hippocampal tissue sample from the mice in the group.

[0223] Specifically, the experimental procedure involved injecting double-distilled water into the mice in the first and second control groups; gavage-administering an equal volume of a 468 mg / kg aqueous solution of Compound Cistanche Yizhi Capsules to the first treatment group; and administering an equal volume of a 0.65 mg / kg aqueous solution of donepezil to the mice in the second treatment group. The intervention continued for eight weeks in the second, third, first, and second treatment groups.

[0224] a) Fixation: The brain tissues of the mice in the first control group, the second control group, the first treatment group, and the second treatment group were fixed with formalin solution for 48 hours before use in subsequent experiments.

[0225] b) Modification: The olfactory bulbs and cerebellum of the brain tissues of the mice in the first control group, the second control group, the first treatment group, and the second treatment group were removed.

[0226] c) Wax impregnation: follow the preparation process in sequence, including: 75% ethanol for 1 hour, 95% ethanol overnight, 100% ethanol I for 1 hour, 100% ethanol II for 4 hours, xylene I for 15 minutes, xylene II for 45 minutes, soft wax for 30 minutes, and hard wax for 1 hour.

[0227] d) Embedding: Pour liquid hard wax into the embedding frame, then place the tissue into the embedding frame and position the tissue. Cover the disposable embedding box and place it on the cooling table of the paraffin embedding machine. After the liquid paraffin solidifies, remove the embedding frame.

[0228] e) Sectioning: After the liquid paraffin solidifies, fix it on a desktop paraffin slicer, replace the blade and set the section thickness to 5 μm; when complete hippocampal tissue (including the CA1 area, CA3 area, and dentate gyrus) is observed, retain the slices and continuously section them. Place the tissue slices in 48°C water for expansion, segmentation, and retrieval; after sectioning, place the slices in a hot air circulating oven (65°C) for 4-6 hours, cool to room temperature, and store them, thereby collecting the third hippocampal tissue samples of mice in the first control group, the second control group, the first treatment group, and the second treatment group.

[0229] Step b: performing HE staining on the third hippocampal tissue sample to obtain the structural condition of the hippocampal tissue; and performing Nissl staining on the third hippocampal tissue sample to obtain the condition of Nissl bodies in the hippocampal tissue.

[0230] Specifically, HE staining and Nissl staining were performed on the third hippocampal tissue samples of the mice in the first control group, the second control group, the first treatment group, and the second treatment group.

[0231] HE staining was used to observe the changes in the hippocampal tissue structure of mice in each group to evaluate the effect of Compound Cistanche Yizhi Capsule on the pathological improvement of the hippocampal tissue of Alzheimer's disease mice. Figure 26 As shown, Figure 26 Schematic diagram of HE staining results of hippocampal tissue of each group of mice provided in the present application. The hippocampal tissue structure and cell morphology of the mice in the first control group are complete, and there are 2-3 layers of small pyramidal cells in the hippocampal CA1 region, with full nuclei, tightly arranged and neatly arranged, mostly round or oval, with darkly stained nucleoli, clear borders, and lightly stained cytoplasm. The hippocampal tissue structure and cell morphology of the mice in the second control group are significantly changed, and the small pyramidal cells in the hippocampal CA1 region are irregular in morphology, fuzzy in cell structure, loosely arranged, unclear in demarcation between nucleus and cytoplasm, partially visible nuclear condensation, and neuronal loss. Compared with the second control group, the hippocampal tissue structure and cell morphology of the mice in the first treatment group and the second treatment group are relatively complete, and the small pyramidal cells in the hippocampal CA1 region are complete in morphology, with a clearer structure and regular and neat arrangement, and no obvious neuronal loss is seen.

[0232] Therefore, by performing HE staining on the mice in the first control group, the second control group, the first treatment group and the second treatment group, it can be determined that Compound Cistanche Yizhi Capsule and Donepezil can effectively improve the hippocampal tissue structure and the morphological structure of the small pyramidal cells in Alzheimer's disease mice.

[0233] Nissl bodies are a type of basophilic substance that is widely present in the cytoplasm of neurons. Their main function is to synthesize proteins required for neural activity. By observing the morphology and number of Nissl bodies, we can reflect the damage of neurons. Figure 27 、 Figure 28 As shown, Figure 27 Schematic diagram of Nissl body staining results of hippocampal tissues of each group of mice provided in this application, Figure 28Schematic diagram of the number of Nissl bodies in the hippocampal tissue of each group of mice provided in this application (Note: compared with the first control group, ##P<0.01, #P<0.05, compared with the second control group, **P<0.01, *P<0.05, n=3), the hippocampal neurons of the mice in the first control group are arranged neatly and densely, with regular morphology, and the Nissl bodies in the neurons are filled with tiger stripes or dots. The number of hippocampal neurons in the mice in the second control group is reduced, the arrangement is scattered, the gaps are larger, the Nissl bodies in the cytoplasm are reduced, and the boundaries are unclear (P<0.05). Compared with the second control group, the morphology of hippocampal neurons in the mice in the first treatment group and the second treatment group was improved, the arrangement was more neat, the number of Nissl bodies increased, and the staining was darker (P<0.05).

[0234] Table 15 The number of Nissl bodies in the hippocampus of mice in each group ( n=3)

[0235]

[0236] Note: Compared with the first control group, ##P<0.01, #P<0.05; compared with the second control group, **P<0.01, *P<0.05.

[0237] Therefore, by performing Nissl staining on the mice in the first control group, the second control group, the first treatment group and the second treatment group, it was determined that both Compound Cistanche Yizhi Capsule and Donepezil can improve the morphology and structure of hippocampal neurons and the morphology and number of Nissl bodies in Alzheimer's disease mice, and improve neuronal damage.

[0238] It should be noted that Image J analysis software was used to select immunohistochemically positive signal areas and calculate the proportion of positive areas. Image J analysis software was used to quantitatively analyze Western blot bands, and the grayscale value ratio of the target protein band to the GAPDH / β-Actin internal reference band was used as the relative expression level of its protein level. PRISM8 statistical software (Graph Pad software) was used for statistical analysis of experimental data. Quantitative data are expressed as mean ± standard deviation. . The t-test was used to analyze data between two groups, and one-way analysis of variance (ANOVA) was used to analyze data between multiple groups. If the data were normally distributed and the variances were equal, the least significant difference (LSD-t) method was used; if the variances were unequal, the Dunnett's T3 test was used. Rank data were expressed as quartiles, and the Wilcoxon rank-sum test was used to compare the two groups. P < 0.05 indicated a statistically significant difference, and P > 0.05 indicated no statistically significant difference. Compared with the Control group, ##P < 0.01, #P < 0.05; compared with the Model group, **P < 0.01, *P < 0.05.

[0239] If the technical solution of this application involves personal information, the product that applies the technical solution of this application has clearly informed the personal information processing rules and obtained the individual's voluntary consent before processing personal information. If the technical solution of this application involves sensitive personal information, the product that applies the technical solution of this application has obtained the individual's separate consent before processing sensitive personal information, and at the same time meets the "explicit consent" requirement. For example, on personal information collection devices such as cameras, a clear and prominent sign is set to inform that the personal information collection scope has been entered and personal information will be collected. If the individual voluntarily enters the collection scope, it is deemed that they agree to the collection of their personal information; or on the personal information processing device, when the personal information processing rules are notified by obvious signs / information, the individual's authorization is obtained through pop-up information or by asking the individual to upload their personal information; among which, the personal information processing rules may include information such as the personal information processor, the purpose of personal information processing, the processing method, and the type of personal information processed.

[0240] The above description is only an implementation method of the present application and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the description and drawings of this application, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. The use of compound Cistanche deserticola capsule in treating Alzheimer's disease is characterized by: The invention relates to an application of the processed Polygonum multiflorum, lotus leaf, Cistanche deserticola, earthworm and Rhizoma Cyperi in the compound Cistanche deserticola capsule in treating Alzheimer's disease.

2. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 1, characterized in that: The compound Cistanche deserticola capsule is used to improve target indicators of patients with Alzheimer's disease, and the target indicators include at least one of the animal's physical condition, behavioral ability, brain tissue condition, hippocampal tissue condition, and serum condition.

3. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 2, characterized in that: The target indicator includes the animal's body state, which includes at least one of the following: skin state, diet state, reaction state, grasping state, resistance state, mental state, weight state, and organ state; And / or, the target indicator includes the behavioral ability, and the behavioral ability includes at least one of the following: motor coordination ability, balance ability, spatial memory ability, and learning ability; And / or, the target indicator includes the brain tissue condition, and the brain tissue condition includes the NEFL condition in the brain tissue; And / or, the target indicator includes the condition of the hippocampal tissue, and the condition of the hippocampal tissue includes at least one of the following: the structure of the hippocampal tissue, the condition of Nissl bodies in the hippocampal tissue, the condition of Aβ deposition in the hippocampal tissue, the condition of Aβ1-42 in the hippocampal tissue, the expression of PSD95 protein in the hippocampal tissue, the expression of SYP protein in the hippocampal tissue, the expression of TLR4 protein in the hippocampal tissue, the expression of NF-κB protein in the hippocampal tissue, the expression of MYD88 protein in the hippocampal tissue, the expression of Bax protein in the hippocampal tissue, the expression of Bc12 protein in the hippocampal tissue, and the expression of Caspase3 protein in the hippocampal tissue; And / or, the target indicator includes the serum status, and the serum status includes at least one of the following: Aβ1-42 status in serum, NEFL status in serum, 1L-6 status in serum, 1L-1β status in serum, and TNF-α status in serum.

4. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 1, characterized in that: The efficacy of the compound Cistanche deserticola capsule in treating Alzheimer's disease was verified by the following method, which includes: A first control group including a normal mice was constructed; and, constructing a second control group comprising b Alzheimer's disease mice; and, constructing a first treatment group comprising c Alzheimer's disease mice; and, constructing a second treatment group comprising d Alzheimer's disease mice; injecting double distilled water into the first control group and the second control group; and administering the aqueous solution of Compound Cistanche and Yizhi Capsules to the first treatment group; and, administering an aqueous solution of donepezil to the second treatment group; Collect at least one target indicator from each group of mice; The efficacy of the compound Cistanche deserticola capsule in treating Alzheimer's disease was evaluated by utilizing the differences between the target indicators corresponding to each group.

5. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 4, characterized in that: The target indicators include behavioral abilities, which include spatial memory ability and learning ability. The target indicators are collected by the following method, which includes: For each group of mice, the Morris water maze test is performed on the mice for a first preset number of consecutive days, wherein the last day is a spatial exploration test phase, the first second preset number of days is a pre-training phase, and the remaining days are a positioning navigation test phase, wherein the second preset number of days is less than the first preset number of days; The detection data of the space exploration experiment phase are analyzed to obtain the spatial memory ability and the learning ability of the group of mice.

6. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 4, characterized in that: The target indicator includes behavioral ability, and the behavioral ability includes motor coordination ability; the target indicator is collected by the following method, which includes: For each group of mice, the mice were subjected to a pole climbing test for the third consecutive preset number of days, wherein the last day was the first test period and the remaining days were the first training period; The detection data of the first test period are analyzed to obtain the motor coordination ability of the group of mice.

7. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 4, characterized in that: The target indicators include behavioral abilities, which include motor coordination and balance abilities; The target indicators are collected by the following method, which includes: For each group of mice, the mice were subjected to a rotarod fatigue test for the fourth consecutive preset number of days, wherein the last day was the second test period and the remaining days were the second training period; The detection data of the second test period are analyzed to obtain the motor coordination ability and the balance ability of the group of mice.

8. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 4, characterized in that: The target indicators include brain tissue conditions, including NEFL conditions in the brain tissue; serum conditions, including NEFL conditions, Aβ1-42 conditions, 1L-6 conditions, 1L-1β conditions, and TNF-α conditions in the serum; and hippocampal tissue conditions, including Aβ deposition conditions and Aβ1-42 conditions in the hippocampal tissue. The target indicators are collected by the following method, which includes: For each group of mice, the first brain tissue sample, the first serum sample, and the first hippocampal tissue sample of the mice in the group were collected; Enzyme-linked immunosorbent assay experiments were performed on the first brain tissue sample, the first serum sample and the first hippocampal tissue sample, respectively, to obtain the NEFL level in the brain tissue, the NEFL level in the serum, the Aβ1-42 level in the serum, the 1L-6 level in the serum, the 1L-1β level in the serum, the TNF-α level in the serum, the Aβ1-42 level in the hippocampal tissue and the Aβ deposition level in the hippocampal tissue.

9. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 4, characterized in that: The target indicators include hippocampal tissue conditions, including PSD95 protein expression in hippocampal tissue, SYP protein expression in hippocampal tissue, TLR4 protein expression in hippocampal tissue, NF-κB protein expression in hippocampal tissue, MYD88 protein expression in hippocampal tissue, Bax protein expression in hippocampal tissue, Bc12 protein expression in hippocampal tissue, and Caspase3 protein expression in hippocampal tissue; the target indicators are collected by the following method, which includes: For each group of mice, a second hippocampal tissue sample was collected from the mice in that group; A protein immunoblotting experiment was performed on the second hippocampal tissue sample to obtain the expression of PSD95 protein in the hippocampal tissue, the expression of SYP protein in the hippocampal tissue, the expression of TLR4 protein in the hippocampal tissue, the expression of NF-κB protein in the hippocampal tissue, the expression of MYD88 protein in the hippocampal tissue, the expression of Bax protein in the hippocampal tissue, the expression of Bc12 protein in the hippocampal tissue, and the expression of Caspase3 protein in the hippocampal tissue.

10. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 4, characterized in that: The target index includes the condition of hippocampal tissue, including the structural condition of the hippocampal tissue and the condition of Nissl bodies in the hippocampal tissue; the target index is collected by the following method, which includes: For each group of mice, the third hippocampal tissue sample was collected from the mice in the group; Performing HE staining on the third hippocampal tissue sample to obtain the structural condition of the hippocampal tissue; Furthermore, Nissl staining is performed on the third hippocampal tissue sample to obtain the Nissl bodies in the hippocampal tissue.

11. The use of the compound Cistanche deserticola capsule in treating Alzheimer's disease according to claim 4, characterized in that: The target indicator includes the animal's body state, which includes the animal's weight state; the target indicator is collected by the following method, which includes: For each group of mice, weighing the mice in the group at intervals of a preset time period to obtain a first weight of the mice as the body weight status of the mice in the group; And / or, the target indicator includes the state of the animal body, and the state of the animal body includes the state of an organ; the target indicator is collected by the following method, and the method includes: For each group of mice, obtaining at least one organ of the mice in the group; and, obtaining a first weight of the group of mice; For each of the organs, weighing each of the organs to obtain a second weight of the organ; A ratio of the second weight to the first weight is obtained to obtain an organ index corresponding to the organ as the organ state corresponding to the organ.

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