A pharmaceutical composition for preventing and / or treating Alzheimer's disease and its use

Abstract

The present invention provides a pharmaceutical composition for preventing and / or treating Alzheimer's disease and the use of the active ingredient combination therein in preparing a drug for preventing and / or treating Alzheimer's disease. The pharmaceutical composition of the present invention comprises two Chinese medicine monomers (i.e., (-)-donkey herbivorous phenol or a pharmaceutically acceptable salt thereof and caryophyllene or a pharmaceutically acceptable salt thereof) as active ingredients, and the two Chinese medicine monomers have been confirmed to have obvious synergistic effects in improving both the pathology of AD disease and the symptoms of AD disease, and because they may affect multiple different pathological processes, the pharmaceutical composition of the present invention is expected to become an effective multi-target, multi-pathway therapeutic drug for Alzheimer's disease, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a pharmaceutical composition for preventing and / or treating Alzheimer's disease and use of the active ingredient combination therein in preparing a drug for preventing and / or treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory impairment and functional decline, which eventually leads to the loss of self-care ability. This disease not only has a serious impact on the patients themselves, but also poses a huge threat to global health and socioeconomic development. At present, the types of drugs approved by the US Food and Drug Administration (FDA) for the treatment of AD are limited. Although these drugs can improve patients' cognitive status and daily living ability, they cannot stop or reverse the course of AD. Since memantine was launched in 2003, the only FDA-approved drugs for the treatment of AD in the past 21 years are aducanumab and lencanumab, which can clear β-amyloid (Aβ) deposits in the brain to a certain extent, but are accompanied by side effects such as cerebral hemorrhage and brain edema, and are expensive. In recent years, the research direction of AD drugs has shifted from traditional single-target drugs to multi-target and multi-pathway combination drug treatment strategies.

[0003] As a natural drug extract, Chinese medicine monomers can exert neuroprotective effects through multiple mechanisms and have become an important area in AD drug research and development. Research in recent years has further revealed that there may be synergistic effects between different Chinese medicine monomers, and combined use may produce stronger therapeutic effects. For example, some Chinese medicine monomers have limited inhibitory effects on Aβ formation when used alone, but when used in combination with other Chinese medicine monomers, they may enhance their efficacy through interactions, thereby more effectively improving the pathological process of AD. In addition, the combination of Chinese medicine monomers may provide a new strategy for the comprehensive treatment of AD by simultaneously affecting multiple pathological processes, such as anti-inflammation, anti-oxidation, and regulating neurotransmitter systems. Therefore, exploring the strategy of combining Chinese medicine monomers is of great significance for discovering more effective AD treatment methods. Summary of the invention

[0004] Purpose of the Invention

[0005] In response to the needs existing in the above-mentioned prior art, the object of the present invention is to provide a pharmaceutical composition for preventing and / or treating Alzheimer's disease, comprising at least two Chinese medicine monomers as active ingredients, and the use of a combination of these two Chinese medicine monomers in the preparation of a drug for preventing and / or treating Alzheimer's disease.

[0006] The inventors have proved through a large number of monomer drug screening and animal experiments that the two traditional Chinese medicine monomers contained in the present invention have obvious synergistic effects in improving the symptoms of Alzheimer's disease and / or in treating Alzheimer's disease.

[0007] Solution

[0008] To achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a pharmaceutical composition for preventing and / or treating Alzheimer's disease, the pharmaceutical composition comprising:

[0010] (-)-Herba sphaerocarp or a pharmaceutically acceptable salt thereof;

[0011] Carnosolone or a pharmaceutically acceptable salt thereof;

[0012] and optionally pharmaceutically acceptable excipients.

[0013] In the above-mentioned uses, (-)-donkey phytophenol and carnosol ketone are both Chinese medicine monomers. 16 H 16 O4, CAS: 35878-41-2 is a small molecular weight natural isoflavanoid compound extracted from African rosewood, Indian rosewood and other plants, which has antibacterial, anti-inflammatory, antioxidant, anti-tumor and other therapeutic potentials; carnosinol ketone (C 18 H 20 O2, CAS: 119400-86-1) is a small molecular weight natural bis-norditerpenoid compound extracted from plants such as Salvia miltiorrhiza, which has therapeutic potentials such as anti-inflammatory, anti-oxidative stress, and anti-tumor.

[0014] The structural formulas of (-)-carnosinol and carnosin ketone are shown below:

[0015]

[0016] In a feasible embodiment, the salt of (-)-herbivorous alcohol is a salt type of (-)-herbivorous alcohol selected from the following salts: hydrochloride, nitrate, sulfate, phosphate, bromate, hydrobromide, citrate, formate, acetate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzoate, phthalate, malonate, maleate, perchlorate, fumarate, succinate, tartrate, lactate, gluconate, pamoate, aspartate or glutamate.

[0017] In a feasible embodiment, the salt of carnosol ketone is a salt type of carnosol ketone selected from the following salts: hydrochloride, nitrate, sulfate, phosphate, bromate, hydrobromide, citrate, formate, acetate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzoate, phthalate, malonate, maleate, perchlorate, fumarate, succinate, tartrate, lactate, gluconate, pamoate, aspartate or glutamate.

[0018] Preferably, the mass ratio of the (-)-caryophyllene phenol or a pharmaceutically acceptable salt thereof to the caryophyllene phenol or a pharmaceutically acceptable salt thereof is 1:(0.8-1.2), and more preferably 1:1.

[0019] Preferably, the pharmaceutically acceptable excipients include one or more of a carrier, an excipient, and a diluent; some examples suitable as carriers, excipients, and diluents include: one or more of lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water syrup, methylcellulose, methyl and propyl paraffin, talc, magnesium stearate, and liquid paraffin.

[0020] In some feasible implementations, pharmaceutically acceptable excipients also include: lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweeteners or flavoring agents and other auxiliary agents.

[0021] In some feasible implementations, the pharmaceutical composition is administered by one or more of the following: oral administration, injection, implantation, spraying and / or inhalation.

[0022] In some feasible implementations, the pharmaceutical composition is in the form of a preparation selected from the following: injection, oral solution, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol or suspension.

[0023] In a second aspect, the present invention provides a use of a combination of (-)-caryophyllene or a pharmaceutically acceptable salt thereof and caryophyllene or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating Alzheimer's disease.

[0024] For the above purposes:

[0025] Preferably, the salt of (-)-herbivorous alcohol is a salt type of (-)-herbivorous alcohol selected from the following: hydrochloride, nitrate, sulfate, phosphate, bromate, hydrobromide, citrate, formate, acetate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzoate, phthalate, malonate, maleate, perchlorate, fumarate, succinate, tartrate, lactate, gluconate, pamoate, aspartate or glutamate.

[0026] Preferably, the salt of carnosol ketone is a salt type of carnosol ketone selected from the following: hydrochloride, nitrate, sulfate, phosphate, bromate, hydrobromide, citrate, formate, acetate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzoate, phthalate, malonate, maleate, perchlorate, fumarate, succinate, tartrate, lactate, gluconate, pamoate, aspartate or glutamate.

[0027] Preferably, in the combination, the mass ratio of (-)-caryophyllene or a pharmaceutically acceptable salt thereof to caryophyllene or a pharmaceutically acceptable salt thereof is 1:(0.8-1.2), more preferably 1:1.

[0028] Preferably, the medicament comprises a preventively and / or therapeutically effective amount of a combination of the (-)-carnosin or a pharmaceutically acceptable salt thereof and the carnosin ketone or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0029] In some feasible implementations, the drug is administered by one or more of the following methods: oral administration, injection, implantation, spraying and / or inhalation.

[0030] In some feasible implementations, the drug is in the form of a preparation selected from the following: injection, oral solution, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol or suspension.

[0031] Preferably, the subject is a mammal, preferably selected from humans, mice, rats, dogs, rabbits, pigs, monkeys, and more preferably humans. When the subject is a human, the effective dose of the combination of (-)-donkey herbivorous phenol or a pharmaceutically acceptable salt thereof and carnosol ketone or a pharmaceutically acceptable salt thereof in the prepared medicament is 3-8 mg / kg / d, preferably 5 mg / kg / d.

[0032] In the above use, the treatment of Alzheimer's disease includes one or more of the following:

[0033] (1) Slow down the progression of Alzheimer's disease;

[0034] (2) Improving symptoms caused by Alzheimer's disease; preferably, improving cognitive dysfunction, neuropsychological symptoms, psychiatric symptoms, behavioral abnormalities, and sleep disorders; in a specific embodiment, the improvement of neuropsychological symptoms is to improve anxiety and / or depression.

[0035] Preferably, the treatment of Alzheimer's disease is to improve cognitive dysfunction caused by Alzheimer's disease.

[0036] In the above use, the prevention and / or treatment of Alzheimer's disease is achieved by a mechanism selected from the following:

[0037] i) reducing the production or aggregation of β-amyloid protein, or clearing β-amyloid protein;

[0038] ii) reducing the production or aggregation of phosphorylated tau protein, or clearing phosphorylated tau protein;

[0039] iii) improve neuronal apoptosis;

[0040] iv) Improve synaptic damage;

[0041] v) Improve mitochondrial energy metabolism disorders;

[0042] vi) Inhibit oxidative stress damage, neuroinflammatory response or immune response, and reduce nerve damage and death;

[0043] vii) Promote the cell autophagy process to remove abnormal proteins and metabolites;

[0044] viii) Improve cerebrovascular function and promote cerebral blood circulation;

[0045] ix) Improve blood-brain barrier function;

[0046] x) Promote the synthesis or release of neurotrophic factors, increase nerve cell activity or promote nerve regeneration;

[0047] xi) Regulate the balance of neurotransmitters such as acetylcholine, glutamate and dopamine, and improve nerve conduction.

[0048] Preferably, the mechanism of preventing and / or treating Alzheimer's disease includes: reducing the deposition of Aβ plaques in the brain and reducing the content of toxic Aβ in the brain; further preferably, the toxic Aβ includes soluble Aβ 1-40 , soluble Aβ 1-42 and Aβ oligomers.

[0049] In a third aspect, the present invention also provides a method for preventing and / or treating Alzheimer's disease, comprising the following steps: administering to a subject in need thereof a preventive and / or therapeutically effective amount of the combination of the (-)-donkey herbivorous phenol or a pharmaceutically acceptable salt thereof and the caryophyllene ketone or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in the second aspect above.

[0050] Preferably, the treatment of Alzheimer's disease comprises one or more of the following:

[0051] (1) Slow down the progression of Alzheimer's disease;

[0052] (2) Improving symptoms caused by Alzheimer's disease; preferably, improving cognitive dysfunction, neuropsychological symptoms, psychiatric symptoms, behavioral abnormalities, and sleep disorders; in a specific embodiment, the improvement of neuropsychological symptoms is to improve anxiety and / or depression.

[0053] Further preferably, the treatment of Alzheimer's disease is to improve cognitive dysfunction caused by Alzheimer's disease.

[0054] Possibly, the prevention and / or treatment of Alzheimer's disease is achieved by a mechanism selected from the group consisting of:

[0055] i) reducing the production or aggregation of β-amyloid protein, or clearing β-amyloid protein;

[0056] ii) reducing the production or aggregation of phosphorylated tau protein, or clearing phosphorylated tau protein;

[0057] iii) improve neuronal apoptosis;

[0058] iv) Improve synaptic damage;

[0059] v) Improve mitochondrial energy metabolism disorders;

[0060] vi) Inhibit oxidative stress damage, neuroinflammatory response or immune response, and reduce nerve damage and death;

[0061] vii) Promote the cell autophagy process to remove abnormal proteins and metabolites;

[0062] viii) Improve cerebrovascular function and promote cerebral blood circulation;

[0063] ix) Improve blood-brain barrier function;

[0064] x) Promote the synthesis or release of neurotrophic factors, increase nerve cell activity or promote nerve regeneration;

[0065] xi) Regulate the balance of neurotransmitters such as acetylcholine, glutamate and dopamine, and improve nerve conduction.

[0066] Preferably, the mechanism of preventing and / or treating Alzheimer's disease includes: reducing the deposition of Aβ plaques in the brain and reducing the content of toxic Aβ in the brain; further preferably, the toxic Aβ includes soluble Aβ 1-40 , soluble Aβ 1-42 and Aβ oligomers.

[0067] Preferably, the subject is a mammal, preferably selected from humans, mice, rats, dogs, rabbits, pigs, monkeys, and more preferably humans.

[0068] The term "effective amount" as used in the present invention refers to the amount or dosage of the active ingredient that provides the expected effect to the patient being diagnosed or treated by single or multiple administration to the patient. The effective amount can be determined by the participating diagnostician as a person skilled in the art through known techniques and observations obtained under similar circumstances. In determining the effective amount or dosage of the active ingredient to be administered, the participating diagnostician should consider a variety of factors, including but not limited to: the species of the mammal; size, age and general health; the specific disease involved; the degree of involvement or severity of the disease; the response of the individual patient; the specific compound administered; the mode of administration; the bioavailability properties of the administered formulation; the selected dosage regimen; the use of concomitant drug therapy; and other relevant circumstances.

[0069] Beneficial Effects

[0070] In order to develop a multi-target, multi-pathway combined drug treatment for Alzheimer's disease, the inventors of the present invention have discovered two Chinese medicine monomers with significant synergistic effects in improving the pathology of Alzheimer's disease and / or treating Alzheimer's disease through a large number of experimental screenings and animal experimental verifications: (-)-donkey herbivorous phenol and carnosol ketone; the inventors found that when the two were combined and applied to AD disease animal models in a reasonable ratio, the cognitive function of AD disease animals was significantly improved, and its efficacy was comparable to that of donepezil hydrochloride, the current mainstream drug for AD, and therefore it is expected to become one of the mainstream therapeutic drugs for AD disease; in addition, the Aβ plaque deposition, toxic Aβ (including soluble Aβ) and 1-40 , soluble Aβ 1-42 and Aβ oligomers) contents were also significantly reduced, and the improvements in these Aβ pathologies in the combination group were significantly better than those in any of the Chinese medicine monomer groups at the same dose. It can be seen that the combined use of (-)-caryophyllene and caryophyllene has a significant synergistic effect in improving AD disease pathology, showing unexpected technical effects.

[0071] Moreover, since (-)-herbivorous phenol and carnosolone may affect multiple different pathological processes, the pharmaceutical composition provided by the present invention, which includes two traditional Chinese medicine monomers, (-)-herbivorous phenol and carnosolone, as active ingredients, is expected to become an effective multi-target, multi-pathway therapeutic drug for Alzheimer's disease, and has good clinical application prospects.

[0072] In addition, the two active ingredients (-)-caryophyllene and caryophyllene ketone contained in the pharmaceutical composition of the present invention are both natural compounds, have the advantages of wide sources, small side effects, etc., and have extremely high cost-effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0074] Figure 1 The spontaneous alternation rates of mice in different drug administration groups in the Y-maze experiment described in Example 1 of the present invention are shown; wherein the horizontal axis shows the group, and the vertical axis shows the spontaneous alternation rate (%); ***: P<0.001 compared with the wild-type group; #: P<0.05 compared with the solvent control group; ###: P<0.001 compared with the solvent control group.

[0075] Figure 2 The figure shows the escape latency of mice in different drug administration groups during the training phase in the Morris water maze experiment described in Example 2 of the present invention; wherein the horizontal axis shows the group, and the vertical axis shows the escape latency (s); *: On the 5th day of training, the wild-type group, donepezil hydrochloride group and combination group were compared with the solvent control group by P<0.05.

[0076] Figure 3 The graph shows the percentage of exploration time in the target quadrant of mice in different drug administration groups in the probe test in the Morris water maze experiment described in Example 2 of the present invention; wherein the horizontal axis shows the group, and the vertical axis shows the percentage of time in the target quadrant; **: P<0.01 compared with the wild-type group; #: P<0.05 compared with the solvent control group.

[0077] Figure 4 The figure shows the percentage of exploration distance of mice in different drug administration groups in the target quadrant in the probe test in the Morris water maze experiment described in Example 2 of the present invention; wherein the horizontal axis shows the group, and the vertical axis shows the percentage of target quadrant distance; **: P<0.01 compared with the wild-type group; #: P<0.05 compared with the solvent control group.

[0078] Figure 5The figure shows the average swimming speed of mice in different drug administration groups in the probe test in the Morris water maze experiment described in Example 2 of the present invention; wherein the abscissa shows the group, and the ordinate shows the average swimming speed (cm / s).

[0079] Figure 6 The representative route map of mice in different drug administration groups in the probe test in the Morris water maze experiment described in Example 2 of the present invention is shown.

[0080] Figure 7 The percentage of Aβ plaque area in the cerebral cortex of mice in different drug administration groups described in Example 3 of the present invention is shown; wherein, the horizontal axis shows the group, and the vertical axis shows the percentage of Aβ plaque area in the cerebral cortex; *: P<0.05 compared with the solvent control group; ****: P<0.0001 compared with the solvent control group; #: P<0.05 compared with the combination group.

[0081] Figure 8 The graph shows the percentage of Aβ plaque area in the hippocampus of mice in different drug administration groups described in Example 3 of the present invention; wherein, the horizontal axis shows the group, and the vertical axis shows the percentage of Aβ plaque area in the hippocampus; **: P<0.01 compared with the solvent control group; ****: P<0.0001 compared with the solvent control group; #: P<0.05 compared with the combination group; ####: P<0.0001 compared with the combination group.

[0082] Fig. 9 Representative histological sections of Aβ plaques in the cerebral cortex and hippocampus of mice in different drug administration groups described in Example 3 of the present invention are shown.

[0083] Fig.10 The Aβ in the cerebral cortex of mice in the different drug administration groups described in Example 4 of the present invention is shown. 1-40 The horizontal axis shows the group, and the vertical axis shows the Aβ content in the cerebral cortex. 1-40 content; *: compared with the solvent control group, P<0.05; **: compared with the solvent control group, P<0.01; ****: compared with the solvent control group, P<0.0001; ##: compared with the combination group, P<0.01; ###: compared with the combination group, P<0.001.

[0084] Fig.11 The Aβ in the hippocampus of mice in the different drug administration groups described in Example 4 of the present invention is shown. 1-40 The horizontal axis shows the groups, and the vertical axis shows the Aβ content in the hippocampus 1-40 content; **: compared with the solvent control group, P<0.01; ****: compared with the solvent control group, P<0.0001; #: compared with the combination group, P<0.05.

[0085] Fig.12The Aβ in the cerebral cortex of mice in the different drug administration groups described in Example 4 of the present invention is shown. 1-42 The horizontal axis shows the group, and the vertical axis shows the Aβ content in the cerebral cortex. 1-42 content; *: compared with the solvent control group, P<0.05; **: compared with the solvent control group, P<0.01; ****: compared with the solvent control group, P<0.0001; #: compared with the combination group, P<0.05.

[0086] Fig.13 The Aβ in the hippocampus of mice in the different drug administration groups described in Example 4 of the present invention is shown. 1-42 The horizontal axis shows the groups, and the vertical axis shows the Aβ content in the hippocampus 1-42 content; *: compared with the solvent control group, P<0.05; **: compared with the solvent control group, P<0.01; ***: compared with the solvent control group, P<0.001; ****: compared with the solvent control group, P<0.0001; #: compared with the combination group, P<0.05.

[0087] Fig.14 The figure shows the content of Aβ oligomers in the cerebral cortex of mice in different drug administration groups described in Example 4 of the present invention; wherein the horizontal axis shows the group and the vertical axis shows the content of Aβ oligomers in the cerebral cortex; *: P<0.05 compared with the solvent control group; **: P<0.01 compared with the solvent control group; ***: P<0.001 compared with the solvent control group; ****: P<0.0001 compared with the solvent control group; #: P<0.05 compared with the combination group.

[0088] Fig.15 The figure shows the Aβ oligomer content in the hippocampus of mice in the different drug administration groups described in Example 4 of the present invention; wherein the horizontal axis shows the group, and the vertical axis shows the Aβ oligomer content in the hippocampus; *: P<0.05 compared with the solvent control group; **: P<0.01 compared with the solvent control group; ****: P<0.0001 compared with the solvent control group; #: P<0.05 compared with the combination group. DETAILED DESCRIPTION

[0089] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described in conjunction with the embodiments below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0090] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0091] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0092] The present invention is further described in detail below by way of examples.

[0093] Example 1: (-)-Carnosine combined with carnosine significantly improves cognitive function (specifically, short-term working memory) in AD mice

[0094] APP / PS1 double transgenic mice are one of the most commonly used AD mouse models, expressing mutant human presenilin 1 (PS1-dE9) and human-mouse amyloid precursor protein (APPswe) fusions driven by the prion protein promoter. Morris water maze experiments found that the spatial memory and learning abilities of 9-month-old APP / PS1 double transgenic mice were significantly different from those of wild-type mice of the same age, and their learning and memory abilities were significantly reduced. The typical pathological characteristics of APP / PS1 double transgenic mice are the formation of Aβ plaques in the brain at 6 months of age, a large number of Aβ plaques in the cortex and hippocampus at 9 months of age, and the Aβ plaques continue to increase until about 12 months.

[0095] In this experiment, APP / PS1 male double transgenic mice and C57BL / 6J male wild mice were purchased from Jiangsu Lingfei Biotechnology Co., Ltd.; (-)-donkey herbivores were purchased from Yunnan Xili Biotechnology Co., Ltd.; carnosol was purchased from WuXi Lanbo (Wuhan) Chemical Technology Co., Ltd.; donepezil hydrochloride was purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd. Donepezil hydrochloride is a cholinesterase inhibitor, which can inhibit the activity of cholinesterase, slow down the decomposition of acetylcholine (ACh) in the synaptic cleft, thereby increasing the content of ACh and improving the cognitive function of patients with Alzheimer's disease (AD). It is currently one of the main therapeutic drugs for Alzheimer's disease.

[0096] In this example, 6-month-old male APP / PS1 double transgenic mice were used as AD model mice, and C57BL / 6J male mice of the same age were used as wild mouse controls to explore the effect of the combined use of (-)-donkey herbivorous phenol and carnosol ketone on the cognitive function of AD mice. In the experiment, a group of (-)-donkey herbivorous phenol and carnosol ketone alone, a donepezil hydrochloride positive drug control group, and a wild mouse control group were set up.

[0097] In this embodiment, AD model mice were randomly divided into 5 groups, namely: a solvent control group, a group treated with (-)-herbivorous phenol and carnosine alone, a group treated with (-)-herbivorous phenol and carnosine together, and a donepezil hydrochloride positive drug control group, plus a wild mouse control group, a total of 6 groups of mice, 8 mice in each group.

[0098] All drugs were dissolved into appropriate concentrations using 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline as solvents. Each group was intraperitoneally injected once a day according to the scheme in Table 1. The wild-type group and the solvent control group were intraperitoneally injected with the same amount of solvents for a total of 3 months.

[0099] Table 1. Mouse grouping and dosing regimen

[0100]

[0101] After 3 months of administration according to the above scheme, the Y maze experiment was first performed to test the short-term working memory and exploration ability of mice. The Y maze consists of three opaque arms, and the angle between any two arms is 120 degrees. The mouse was placed from the blind end of one of the arms and taken out after being allowed to freely explore the maze for 8 minutes. The maze was cleaned with 70% ethanol between the tests of two mice to remove the influence of odor. Each test was recorded by video and analyzed with SMART software. The mouse entered three different arms in turn as a correct alternation, and the spontaneous alternation rate (%) = number of correct alternations / (total number of arms entered-2) × 100%. The higher the spontaneous alternation rate, the better the short-term working memory of the mouse.

[0102] Data analysis: All data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used to detect differences between three or more groups, and Bonferroni correction was used for comparisons between two groups. The differences were considered significant when P < 0.05. GraphPad Prism 8.0.1 software was used to analyze the data and plot the graphs.

[0103] The results are as follows Figure 1 As shown, Figure 1The results showed that the spontaneous alternation rate of the solvent control group was significantly lower than that of the wild group, the spontaneous alternation rate of each drug group was significantly higher than that of the solvent control group, and the spontaneous alternation rate of the combination group was slightly higher than that of the single drug group, which was equivalent to the positive control group. This suggests that the combination of (-)-donkey herbivorous phenol and caryophyllene can significantly improve the short-term working memory of AD mice, and the effect is better than that of the single drug group, which is equivalent to the positive control drug donepezil hydrochloride.

[0104] Example 2: (-)-Carnosyl phenol combined with carnosyl phenol significantly improves cognitive function (specifically, spatial learning and memory ability) of AD mice

[0105] After the Y-maze test, the mice in each drug administration group in Example 1 were subjected to the Morris water maze test to detect the spatial learning and memory abilities of the mice.

[0106] The Morris water maze detection system consists of a cylindrical pool with a diameter of 120 cm and a water depth of 50 cm and an image acquisition and analysis system. The SMART software divides the water surface into four quadrants: northeast (NE), southeast (SE), southwest (SW), and northwest (NW). A platform with a diameter of 8-10 cm is placed in the center of the NE quadrant (target quadrant), and the water temperature is maintained at around 22°C. The test was conducted over a 6-day period, including a 5-day training phase and a 1-day probe test. The platform was located about 1 cm above the water on the first day, about 1 cm below the water on the 2nd to 5th day, and the platform was removed on the 6th day. During the training phase, the mouse was placed in the water facing the pool wall according to the entry point in Table 2. If the mouse climbed onto the platform within 60 seconds, it was allowed to stay on the platform for 5 seconds. If the mouse failed to find the platform in the target quadrant within 60 seconds, it was manually guided to the platform and allowed to stay on the platform for 15 seconds. The training was conducted 4 times a day from different entry points, with at least 30 minutes between two training sessions, and the training was conducted for 5 consecutive days. The time it takes for mice to find the platform is the escape latency, and the average of the escape latency of mice for 4 training sessions (the escape latency of mice that did not reach the platform within 60 seconds was recorded as 60 seconds) was used as the learning performance of the mice for that day. The 6th day was the probe test. At this time, the platform was removed, and the mice were put into the water from the farthest entry point SW from the platform position. The exploration time and distance in the target quadrant were recorded, and the average swimming speed was calculated. Each test was recorded by video and analyzed by SMART software. The shorter the escape latency and the longer the exploration time and distance of the target quadrant, the stronger the spatial learning and memory ability of the mice. The no difference in average swimming speed means that the differences in the escape latency, exploration time and distance of the target quadrant of the mice are not caused by different motor abilities.

[0107] Table 2. Sequence of water entry points in the water maze

[0108] Trial 1 Trial 2 Trial 3 Trial 4 Day 1 S W NW SE Day 2 NW S SE W Day 3 SE NW W S Day 4 W SE S NW Day 5 S NW W SE Day 6 SW

[0109] Data analysis: All data are expressed as mean ± SEM. For escape latency, two-way ANOVA was used to detect differences between three or more groups, and Bonferroni correction was used for comparison between two groups. For other indicators, one-way ANOVA was used to detect differences between three or more groups, and Bonferroni correction was used for comparison between two groups. The difference was significant when P < 0.05. GraphPad Prism 8.0.1 software was used to analyze data and plot.

[0110] Figure 2-6 The escape latency during the training phase, the percentage of exploration time in the target quadrant in the probe test, the percentage of exploration distance in the target quadrant, the average swimming speed, and the representative route map in the probe test of mice in different drug administration groups are shown respectively.

[0111] Figure 2-6 The results showed that the escape latency, target quadrant exploration time and distance of the solvent control group were significantly lower than those of the wild group, while the escape latency, target quadrant exploration time and distance of the combination group and donepezil hydrochloride group were significantly higher than those of the solvent control group. The effect of the combination group was better than that of the single drug group and was equivalent to that of the positive control group, indicating that the combination of (-)-donkey herbivorous phenol and caryophyllene can significantly improve the spatial learning and memory ability of AD mice, and the effect is better than that of the single drug group and is equivalent to that of the positive control drug donepezil hydrochloride.

[0112] Example 3: Synergistic effect of (-)-donkey herbivorous phenol and carnosol in improving Aβ pathology in the brain of AD mice (specifically, reducing Aβ plaque deposition in the cerebral cortex and hippocampus of AD mice)

[0113] The mice in each drug administration group in Example 2 were killed after the Morris water maze test, and cardiac perfusion was performed after anesthesia. After the perfusion was clean, the brain tissue was taken, and the left brain was made into paraffin sections for Aβ immunohistochemical staining, and the right brain homogenate was used for Aβ ELISA detection. A brain tissue paraffin section was randomly selected from each mouse for immunohistochemical staining with 6E10 (Abcam, 803001) antibody, photographed with an optical microscope, and the percentage of Aβ plaque area in the cortex and hippocampus was calculated using Fiji software.

[0114] Data analysis: All data are expressed as mean ± SEM. One-way ANOVA was used to detect differences between three or more groups, and Bonferroni correction was used for comparisons between two groups. The differences were considered significant when P < 0.05. GraphPad Prism 8.0.1 software was used to analyze data and plot.

[0115] Figure 7-9The percentage of Aβ plaque area in the cerebral cortex and hippocampus of mice in different drug administration groups, as well as representative tissue sections of Aβ plaques in the cerebral cortex and hippocampus are shown respectively; these results show that there is no Aβ plaque deposition in the cortex and hippocampus of wild-type (WT) mice, a large amount of Aβ plaque deposition in the solvent control group, the Aβ plaque deposition in each drug group is significantly reduced compared with the solvent control group, and the Aβ plaque deposition in the combination group is significantly reduced compared with the single drug group, which is equivalent to the positive control group, indicating that the combination of (-)-donkey herbivorous phenol and carnosol ketone can significantly improve the deposition of Aβ plaques in the brain of AD mice, and its improvement effect is significantly better than that of the single drug group at the same dose (equivalent to donepezil hydrochloride), which shows that (-)-donkey herbivorous phenol and carnosol ketone have a synergistic effect in improving the Aβ pathology of Aβ plaque deposition in the brain of AD mice.

[0116] Example 4: Synergistic effect of (-)-donkey herbivorous phenol and carnosol in improving Aβ pathology in the brain of AD mice (specifically, reducing three types of toxic Aβ in the brain)

[0117] In order to further verify the therapeutic effect of (-)-donkey herbivorous phenol and carnosol ketone on AD, this example further detected the three toxic Aβ (including soluble Aβ) in the brain of mice in each drug administration group in the above examples. 1-40 , soluble Aβ 1-42 The three types of toxic Aβ were detected using ELISA kits according to the instructions.

[0118] Data analysis: All data are expressed as mean ± SEM. One-way ANOVA was used to detect differences between three or more groups, and Bonferroni correction was used for comparisons between two groups. The differences were considered significant when P < 0.05. GraphPad Prism 8.0.1 software was used to analyze data and plot.

[0119] Figure 10-15 Figure 3 shows the Aβ in the cerebral cortex of mice in different drug administration groups. 1-40 Aβ in hippocampus 1-40 Aβ content in the cerebral cortex 1-42 Aβ in hippocampus 1-42 The results showed that the soluble Aβ in the cortex and hippocampus of mice in each treatment group was significantly higher than that in the control group. 1-40 Content, soluble Aβ 1-42 The content of Aβ oligomers and the content of soluble Aβ in the combined group were significantly lower than those in the solvent control group. 1-40 Content, soluble Aβ 1-42The content and Aβ oligomer content of (-)-donkey herbivorous phenol and carnosine ketone were significantly reduced compared with those of the single-drug group and were equivalent to those of the positive control group, indicating that the combined use of (-)-donkey herbivorous phenol and carnosine ketone can significantly reduce the toxic Aβ in the brain of AD mice, and its effect is significantly better than that of the single-drug group at the same dose (equivalent to donepezil hydrochloride). This shows that (-)-donkey herbivorous phenol and carnosine ketone have a synergistic effect in reducing the Aβ content in the brain of AD mice, a kind of Aβ pathology.

[0120] As can be seen from the above, the combination of (-)-donkey herbivorous phenol and carnosol ketone shows significant effects in improving the cognitive function of AD mice (including short-term working memory, spatial learning and memory ability) and improving the Aβ pathology of AD mice (including reducing Aβ plaque formation and reducing the toxic Aβ content in the brain), and its efficacy is comparable to that of donepezil hydrochloride, the current mainstream AD drug; in particular, in improving the Aβ pathology of AD mice, the combination of (-)-donkey herbivorous phenol and carnosol ketone shows obvious synergistic effects (as demonstrated in Examples 3 and 4). Therefore, the combination of (-)-donkey herbivorous phenol and carnosol ketone is expected to become one of the mainstream therapeutic drugs for AD diseases as a multi-target, multi-pathway AD therapeutic drug.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a combination of (-)-carnosin or a pharmaceutically acceptable salt thereof and carnosin ketone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating Alzheimer's disease.

2. The use according to claim 1, characterized in that The salt of (-)-herbivorous phenol is a salt type of (-)-herbivorous phenol selected from the following: hydrochloride, nitrate, sulfate, phosphate, bromate, hydrobromide, citrate, formate, acetate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzoate, phthalate, malonate, maleate, perchlorate, fumarate, succinate, tartrate, lactate, gluconate, pamoate, aspartate or glutamate; And / or, the salt of carnosol ketone is a salt type of carnosol ketone selected from the following salts: hydrochloride, nitrate, sulfate, phosphate, bromate, hydrobromide, citrate, formate, acetate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzoate, phthalate, malonate, maleate, perchlorate, fumarate, succinate, tartrate, lactate, gluconate, pamoate, aspartate or glutamate.

3. The use according to claim 1 or 2, characterized in that The mass ratio of the (-)-caryophyllene phenol or its pharmaceutically acceptable salt to the caryophyllene phenol or its pharmaceutically acceptable salt is 1: (0.8-1.2).

4. The use according to claim 3, characterized in that The mass ratio of the (-)-caryophyllene phenol or its pharmaceutically acceptable salt to the caryophyllene phenol or its pharmaceutically acceptable salt is 1:

1.

5. The use according to claim 1 or 2, characterized in that: The medicine comprises a preventive and / or therapeutically effective amount of a combination of the (-)-carnosin or a pharmaceutically acceptable salt thereof and the carnosin ketone or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

6. The use according to claim 1 or 2, characterized in that: The administration method of the drug is one or more selected from the following: oral administration, injection, implantation, spraying and / or inhalation.

7. The use according to claim 1 or 2, characterized in that: The drug is in the form of a preparation selected from the following: injection, oral solution, powder, tablet, granule, capsule, decoction, sustained-release preparation or aerosol.

8. The use according to claim 7, characterized in that The oral liquid is a syrup or a suspension; Alternatively, the sustained-release preparation is an enteric solvent.

9. The use according to claim 1 or 2, characterized in that: The treatment of Alzheimer's disease includes one or more of the following: (1) Slow down the progression of Alzheimer's disease; (2) Improve symptoms caused by Alzheimer's disease.

10. The use according to claim 9, characterized in that The improvement of symptoms caused by Alzheimer's disease is to improve cognitive dysfunction, neuropsychological symptoms, psychiatric symptoms, abnormal behavior and / or sleep disorders caused by Alzheimer's disease.

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