CN116236491A

CN116236491A - Application of rhamnose in preparing medicine for treating or preventing neurodegenerative diseases, pharmaceutical composition and application thereof

Info

Publication number: CN116236491A
Application number: CN202310124122.0A
Authority: CN
Inventors: 裴钢; 黄世超; 周悦; 董晓旭; 夏鹏
Original assignee: Hainan Simcere Pharmaceutical Co ltd
Current assignee: Shanghai Dongdong Zhiyi Biomedical Co ltd
Priority date: 2022-07-29
Filing date: 2023-02-15
Publication date: 2023-06-09
Anticipated expiration: 2043-02-15
Also published as: WO2024022468A1; TW202404616A; CN116236491B

Abstract

The invention discloses application of rhamnose in preparing a medicament for treating or preventing neurodegenerative diseases, a pharmaceutical composition and application thereof, and belongs to the technical field of medicines. The invention provides application of rhamnose or hydrate thereof in preparing a medicament for treating or preventing neurodegenerative diseases. Based on the above uses, there is provided a composition for the treatment or prevention of neurodegenerative diseases, comprising rhamnose and/or its hydrate, cinnamic acid derivative or a pharmaceutically acceptable salt thereof; a flavonoid compound; the composition can obviously improve the cognitive behaviors of mice, and can be used for treating or preventing neurodegenerative diseases.

Description

Application of rhamnose in preparing medicine for treating or preventing neurodegenerative diseases, pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of pharmacy, and in particular relates to application of rhamnose in preparing a medicament for treating or preventing neurodegenerative diseases, a pharmaceutical composition and application thereof.

Background

Alzheimer's Disease (AD), also known as mental retardation, senile dementia, hypomnesis, language expression disorder, difficulty in identifying objects and figures, hypodirectional strength, loss of emotion and behavior control ability, etc., even if life is completely impossible to self-care. With the progressive prominence of the aging problem, the incidence of alzheimer's disease is increasing, which represents a very serious disease burden for patients and caregivers. The AD treatment market has grown from nearly 50 billion dollars in 2014 to about 104 billion dollars in 2021. Clearly, there is still a great unmet need in the field of AD treatment.

To date, the FDA has only approved 6 drugs for AD treatment, including acetylcholinesterase (AChE) inhibitors such as tacrine (which has been marketed off today), rivastigmine, galantamine, NMDA receptor antagonists memantine, and monoclonal antibodies available in 2021, aducanaumab (a Du Nashan antibody); among acetylcholinesterase inhibitors, donepezil is exemplified by administration starting at a dose of 5mg once a day, and after 4 to 6 weeks at an increased dose of 10mg, and is administered in the form of solid preparations for oral administration (such as film-coated tablets, capsules and granules). Memantine was developed as a therapeutic agent for moderate to severe alzheimer's disease and was administered in the form of a film coating or liquid formulation, and the drug was administered at a dose of 5mg daily and then at a maximum dose of 20 mg/day (i.e. 10mg each in the morning and evening), with 5mg increase per week, but due to the long half-life of 60 to 80 hours and relatively high side effects, many clinical researchers currently administer drugs once daily. Monoclonal antibody Aducanaumab (A Du Nashan antibody), a monoclonal antibody targeting amyloid beta, was the first drug approved by the FDA since 2003 to treat AD. Except for the al Du Nashan antibody, donepezil, rivastigmine, galantamine, memantine are used only to relieve the symptoms of the patient, and do not alleviate the pathological changes of AD nor reverse or slow down the disease process. The green cereal pharmaceutical mannite capsules (GV-971, trade name: nine-period one) recently obtained in China are prepared from marine brown algae extract as raw materials, and the obtained low molecular acidic oligosaccharide mixture plays a role through the brain intestinal axis. Because of the great market demand in the area of AD treatment, and the lack of effective and safe methods of treatment, there is an urgent need for safe and effective therapeutic agents for the treatment of AD.

Furthermore, given the very complex pathogenesis involved in AD, the trigger and detailed mechanisms of neurodegeneration remain unclear, although substantial progress has been made. Based on this:

there is a problem that the existing medicines hardly achieve satisfactory curative effects;

there is a problem in that undesired side effects are reduced to produce as little as possible of the desired therapeutic effect.

Disclosure of Invention

A first object of the present invention is to provide the use of rhamnose and/or its hydrates for the manufacture of a medicament for the treatment or prevention of neurodegenerative diseases;

based on the first object of the present invention, it is a further object to provide the use of rhamnose and/or its hydrates for the manufacture of a medicament for the treatment or prevention of alzheimer's disease;

based on the first object of the present invention, it is a further object to provide the use of rhamnose and/or its hydrates for the manufacture of a medicament for inhibiting phosphorylated tau proteins;

based on the first object of the present invention, it is a further object to provide the use of rhamnose and/or its hydrates for the preparation of a medicament for activating AMPK;

a second object of the present invention is to provide a medicament containing rhamnose or a hydrate thereof for treating or preventing a neurodegenerative disease; alternatively, the medicament is for inhibiting phosphorylated tau protein; alternatively, the medicament is for activating AMPK;

A third object of the present invention is to provide a pharmaceutical composition comprising rhamnose and/or its hydrates;

a fourth object of the present invention is to provide a pharmaceutical composition containing rhamnose and/or its hydrate for the treatment or prevention of neurodegenerative diseases.

Based on the above objects, the core problems to be solved by the present invention are: solves the problem of unsatisfactory treatment effect of the existing medicines;

further solves the problem of great side effect of the existing medicine.

[ pharmaceutical composition ]

[1] Based on the first object of the present invention, the use of rhamnose or its hydrate, the use of it in the manufacture of a medicament for the treatment or prevention of a neurodegenerative disease in order to treat or prevent or reduce the risk of occurrence of a disease or a symptom in a subject. According to the invention, the rhamnose can obviously improve the cognitive behaviors of mice on an APP/PS1 mouse model for the first time, and can be used for treating or preventing neurodegenerative diseases.

As used herein, "treating a neurodegenerative disease" refers to delaying the onset of the disease, slowing the progression of the disease, and/or ameliorating symptoms of the disease.

By "delaying" the onset of a neurodegenerative disease is meant that the drugs, compositions and methods of treatment provided herein are capable of delaying, impeding or slowing the onset of the disease such that the likelihood of manifestation of early disease symptoms in one patient or the likelihood of occurrence of the disease in multiple patients is reduced over a given timeframe as compared to the absence of the drugs, compositions or methods provided herein.

By "slowing" the progression of a neurodegenerative disease is meant that the medicaments, compositions and methods of treatment provided herein are effective to inhibit progressive decline in learning, memory, or language ability or other somatic functions.

By "ameliorating" a symptom of a neurodegenerative disease is meant that the pharmaceutical compositions and methods of treatment provided herein reduce the symptoms of the disease, and/or improve learning, memory, or linguistic ability or other somatic function.

As described herein, "rhamnose" as described herein, also known as mannose, is a methyl-pentasaccharide having the L-and D-2 isomers. The crystals of "L-form/D-form rhamnose" both take two forms: alpha and beta forms. The alpha type contains one molecule of crystal water, namely rhamnose hydrate; after heating, the alpha form loses crystal water and is converted into beta form. The beta type is extremely easy to absorb moisture, and is converted into the alpha type in the air. Furthermore, α -L-rhamnose is the most common type.

As used herein, "preventing a neurodegenerative disease" refers to delaying the onset of the disease, slowing the progression of the disease, and/or ameliorating symptoms of the disease.

As used herein, "preventing" or "prevention" as used herein refers to the administration of a drug, composition of the present invention to a subject to avoid the occurrence of a disease or condition before the occurrence of the disease or condition.

As used herein, "reducing the risk of developing a disease or symptom in a subject" means that the subject has a lower likelihood of developing the disease or symptom than an equivalent control individual, e.g., the subject has been administered a pharmaceutical composition of the invention and the control has not been treated or received a drug.

[1.1] use of rhamnose or a hydrate thereof according to any one of the embodiments of the first aspect of the present invention, for the manufacture of a medicament for the treatment or prevention of:

neurodegenerative diseases characterized by neuroinflammation occurring in the brain; or (b)

Neurodegenerative diseases characterized by a significant increase in aβ production; or (b)

Neurodegenerative diseases characterized by a significant decline in learning and memory; or (b)

Neurodegenerative diseases characterized by decreased function of neural stem cells; or (b)

Neurodegenerative diseases characterized by reduced motor coordination; or (b)

Neurodegenerative diseases characterized by a decrease in the number of nigral dopaminergic neurons; or (b)

Neurodegenerative diseases characterized by reduced striatal dopaminergic nerve fiber content.

According to the invention, rhamnose or the hydrate thereof can promote the proliferation of nerve cells, and the proliferation of the nerve cells is beneficial to the improvement of learning and memory capacity, the improvement of nerve stem cell functions, the improvement of movement coordination capacity, the improvement of the quantity of nigral dopaminergic neurons and the improvement of the content of striatal dopaminergic nerve fibers. Accordingly, rhamnose or a hydrate thereof may be used to treat or prevent: neurodegenerative diseases characterized by neuroinflammation occurring in the brain; or a neurodegenerative disease characterized by a significant increase in aβ production; or neurodegenerative diseases characterized by a significant decline in learning and memory; or a neurodegenerative disease characterized by reduced function of neural stem cells; or neurodegenerative diseases characterized by reduced motor coordination; or neurodegenerative diseases characterized by a decrease in the number of nigral dopaminergic neurons; or neurodegenerative diseases characterized by reduced striatal dopaminergic nerve fiber content.

[1.2] use of rhamnose or a hydrate thereof according to any one of the embodiments of the first aspect of the present invention, for the manufacture of a medicament for the treatment or prevention of Alzheimer's disease.

As described herein, alzheimer's Disease (AD) is the most common type of neurodegenerative disease, and its symptoms are clinically recognized. Early symptoms include the inability to acquire new memory (e.g., difficulty in recall of recent events) and the inability to acquire new information. As the disease progresses, disorders of learning and memory become more pronounced, and symptoms may include language disorders (including difficulty speaking and loss of read-write ability), long-term memory loss, loss of motor coordination, and behavioral and neuropsychiatric symptoms such as confusion, irritability, aggressiveness, mood swings, and general withdrawal symptoms (general withdrawal). Late stage is characterized by loss of language ability, decline in muscle mass and activity, and loss of other somatic functions.

As used herein, "treating alzheimer's disease" refers to delaying the onset of the disease, slowing the progression of the disease, and/or ameliorating symptoms of the disease.

By "delaying" the onset of Alzheimer's disease, it is meant that the medicaments, compositions and methods of treatment provided herein are capable of delaying, impeding or slowing the onset of the disease such that the likelihood of manifestation of early disease symptoms in one patient or the likelihood of occurrence of the disease in multiple patients is reduced over a given timeframe as compared to the absence of the compositions or methods provided herein.

By "slowing" the progression of Alzheimer's disease is meant that the medicaments, compositions and methods of treatment provided herein are effective in inhibiting progressive decline in learning, memory, or language ability or other somatic functions.

By "ameliorating" a symptom of Alzheimer's disease, it is meant that the medicaments, compositions and methods of treatment provided herein reduce the symptoms of the disease, and/or improve learning, memory, or linguistic ability or other somatic function.

As used herein, "preventing" or "prevention" as used herein refers to the administration of a drug or composition of the invention to a subject prior to the occurrence of the disease or condition, to avoid the occurrence of the disease or condition.

[1.3] use of rhamnose or a hydrate thereof according to any one of the embodiments of the first aspect of the present invention for the manufacture of a medicament for activating AMPK.

The invention discovers that rhamnose or hydrate thereof can activate an AMPK signal pathway for the first time. AMPK acts as a regulator of cellular energy receptors and metabolic homeostasis, and its activation can regulate SIRT1, thereby inhibiting the accumulation of phosphorylated tau protein, slowing down further progression of AD.

[1.4] use of rhamnose or a hydrate thereof according to any of the embodiments of the first aspect of the present invention for the preparation of a medicament for the inhibition of phosphorylated tau protein.

Since the invention discovers that rhamnose or a hydrate thereof can activate an AMPK signal path, AMPK is used as a receptor of cell energy and a regulator of metabolic homeostasis, and the activation of AMPK can regulate SIRT1, thereby inhibiting accumulation of phosphorylated tau protein and slowing down further development of AD, and further the invention further relates to application of the rhamnose or the hydrate thereof in preparing medicines for inhibiting the phosphorylated tau protein.

It is noted here that neurofibrillary tangles are one of the main brain pathological features of neurodegenerative diseases such as AD. The main component of the prior studies is paired helical filaments (paired helical filament, PHF) formed by abnormal hyperphosphorylation of tau protein.

As used herein, "preventing" or "prevention" as used herein refers to the administration of a medicament or composition of the invention to a subject prior to the occurrence of a disease or condition associated with misfolding, accumulation, aggregation or deposition of beta amyloid, beta amyloid oligomers, tau proteins or other proteins, to avoid the occurrence of the disease or condition.

[2] Based on the second object of the present invention, the second aspect of the present invention provides a medicament containing rhamnose and/or its hydrate, wherein the amount of rhamnose or its hydrate in the medicament or the size of the dose to be administered of rhamnose or its hydrate contained in the medicament may be any one value chosen from any one of the following sets of values: 3-2000mg, 5-2000mg, 10-2000mg, 15-2000mg, 100-2000mg, 200-2000mg, 300-2000mg, 400-2000mg, 500-2000mg, 600-2000mg, 700-2000mg, 800-2000mg, 900-2000mg, 1000-2000mg, 1100-2000mg, 1200-2000mg, 1300-2000mg, 1400-2000mg, 1500-2000mg, 1600-2000mg, 1700-2000mg, 1800-2000mg, 1900-2000mg;

3-1900mg、3-1800mg、3-1700mg、3-1600mg、3-1500mg、3-1400mg、3-1300mg、3-1200mg、3-1100mg、3-1000mg、3-900mg、3-800mg、3-700mg、3-690mg、3-680mg、3-670mg、3-650mg、3-600mg、3-500mg、3-400mg、3-300mg、3-200mg、3-100mg、3-80mg、3-50mg、3-30mg、3-10mg；

15-1900mg、15-1800mg、15-1700mg、15-1600mg、15-1500mg、15-1400mg、15-1300mg、15-1200mg、15-1100mg、15-1000mg、15-900mg、15-800mg、15-700mg、15-690mg、15-680mg、15-670mg、15-650mg、15-600mg、15-500mg、15-400mg、15-300mg、15-200mg、15-170mg、15-150mg、15-130mg、15-110mg、15-100mg、15-80mg、15-50mg、15-30mg；

50-1900mg、50-1800mg、50-1700mg、50-1600mg、50-1500mg、50-1400mg、50-1300mg、50-1200mg、50-1100mg、50-1000mg、50-900mg、50-800mg、50-700mg、50-690mg、50-680mg、50-670mg、50-650mg、50-600mg、50-500mg、50-450mg、50-400mg、50-350mg、50-300mg、50-250mg、50-200mg、50-150mg、50-100mg、50-80mg；

Preferably, the amount of rhamnose or its hydrate in the medicament or the dose to be administered of rhamnose or its hydrate contained in the medicament is 200mg.

When administered in the above-mentioned doses, satisfactory results are obtained, preferably in 1, 2, 3 total or divided doses per day, or in a sustained release form. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

It should be noted that rhamnose is a substance widely existing in plant polysaccharide, glycoside, vegetable gum and bacterial polysaccharide, has very few toxic and side effects, is also used as a food additive, has been used for eating, and has not presented safety problems in canadian and other researchers using 25 g/day dose for serum propionate study in healthy people. And after single large-dose animal administration, the safety of rhamnose is proved without any toxic or side effect, and the safety of applying the rhamnose to life is extremely high. Therefore, the use of the composition for treating or preventing neurodegenerative diseases or the combination with other active ingredients capable of treating or preventing neurodegenerative diseases or known therapeutic drugs can be expected to improve the additive effect or synergistic effect to treat or prevent or reduce the risk of occurrence of diseases or symptoms in a subject without substantially causing increased side effects.

[3] Based on a third object of the present invention, a third aspect of the present invention provides a pharmaceutical composition comprising:

a first component: rhamnose and/or its hydrates;

and a second component: cinnamic acid derivatives, or pharmaceutically acceptable salts and hydrates thereof; and

and a third component: flavonoid compounds having a neuronal regeneration promoting effect.

It should be noted that cinnamic acid, also called beta-phenylacrylic acid, 3-phenyl-2-acrylic acid, cinnamic acid and analogues thereof have simple structures, and are mainly prepared by extraction and chemical synthesis of plants, namely organic acids which can be separated from cinnamomum cassia bark or benzoin, and are generated in plants through deamination and degradation of phenylalanine. Cinnamic acid and its derivatives are used as common aromatic compounds, have the characteristics of fragrance, corrosion resistance and the like, and are widely applied to the fields of foods, cosmetics and the like, and based on the cinnamic acid and its derivatives, the application to living bodies has extremely high safety. Therefore, the use of the composition for treating or preventing neurodegenerative diseases or the combination with other active ingredients capable of treating or preventing neurodegenerative diseases or known therapeutic drugs can be expected to improve the additive effect or synergistic effect to treat or prevent or reduce the risk of occurrence of diseases or symptoms in a subject without substantially causing increased side effects.

The cinnamic acid derivative may be a substance having the following formula 1:

in the formula ：

r is independently selected from hydrogen or contains one or more substituents on the benzene ring, wherein the substituents are independently selected from hydroxy, alkoxy, ester, aryl (hetero) group, halogen and glucoside group;

flavonoid compounds (flavonoids) are generic names of compounds derived from a skeleton of 2-phenylchromone, and are generic names of a series of compounds formed by connecting two benzene rings with each other through three carbon atoms, and are widely existing in the plant kingdom. It usually binds to sugars in plants to form aglycones in ligand form, and a small fraction exists in free aglycone form. At present, flavonoid compounds are mainly produced by plant extraction and chemical synthesis. In addition, flavonoids have low toxicity, and thus, they are expected to be used in medicaments for treating or preventing neurodegenerative diseases, or in combination with other active ingredients capable of treating or preventing neurodegenerative diseases or known therapeutic drugs, to have an enhanced additive effect or synergistic effect to treat or prevent or reduce the risk of occurrence of diseases or symptoms in a subject, without substantially causing increased side effects.

The flavonoid may be a substance having the following formula 2:

in the formula ：

2 and 3 are single bond or double bond;

R ₁ selected from hydrogen, hydroxy, alkoxy, ester, glycoside or halogen;

R ₂ selected from hydrogen, or from 5 to 8 containing one or more substituents, selected from hydroxy groupsAlkoxy, ester, aryl (hetero) groups, halogen, glucosidic groups;

R ₃ respectively selected from hydrogen or one or more substituents on the benzene ring, wherein the substituents are respectively selected from hydroxyl, alkoxy, ester, aryl (hetero) group, halogen and glucoside.

[3.1] a pharmaceutical composition according to any one of the embodiments of the third aspect of the invention, the rhamnose comprising rhamnose in form L and/or rhamnose in form D;

the cinnamic acid derivative comprises any one or more of ferulic acid, isoferulic acid, caffeic acid, p-hydroxy coumaric acid, o-hydroxy coumaric acid and m-hydroxy coumaric acid;

the flavonoid compound comprises one or more of chrysin, apigenin, farnesin and genkwanin.

As described herein, the ferulic acid (CAS: 537-98-4) has the structure (formula 3) shown below:

the chrysin (CAS: 480-40-0) has the structure shown below (formula 4)

[3.2] the pharmaceutical composition according to any one of the embodiments of the third aspect of the present invention, based on the sum of the weights of the first component, the second component and the third component, as the total weight; wherein,

the first component may be present in an amount selected from any one of the following ranges of values: 5-90wt%, 5-85wt%, 5-80wt%, 5-75wt%, 5-70wt%, 5-65wt%, 5-60wt%, 5-55wt%, 5-50wt%, 5-45wt%, 5-40wt%, 5-35wt%, 5-30wt%, 5-25wt%, 5-20wt%, 5-15wt%, 5-10wt% and one or more of the following components;

10-90wt％、10-85wt％、10-80wt％、10-75wt％、10-70wt％、10-65wt％、10-60wt％、10-55wt％、10-50wt％、10-45wt％、10-40wt％、10-35wt％、10-30wt％、10-25wt％、10-20wt％、10-15wt％；

15-90wt％、15-85wt％、15-80wt％、15-75wt％、15-70wt％、15-65wt％、15-60wt％、15-55wt％、15-50wt％、15-45wt％、15-40wt％、15-35wt％、15-30wt％、15-25wt％、15-20wt％；

20-90wt％、20-85wt％、20-80wt％、20-75wt％、20-70wt％、20-65wt％、20-60wt％、20-55wt％、20-50wt％、20-45wt％、20-40wt％、20-35wt％、20-30wt％、20-25wt％；

25-90wt％、25-85wt％、25-80wt％、25-75wt％、25-70wt％、25-65wt％、25-60wt％、25-55wt％、25-50wt％、25-45wt％、25-40wt％、25-35wt％、25-30wt％；

30-90wt％、30-85wt％、30-80wt％、30-75wt％、30-70wt％、30-65wt％、30-60wt％、30-55wt％、30-50wt％、30-45wt％、30-40wt％、30-35wt％；

35-90wt％、35-85wt％、35-80wt％、35-75wt％、35-70wt％、35-65wt％、35-60wt％、35-55wt％、35-50wt％、35-45wt％、35-40wt％；

40-90wt％、40-85wt％、40-80wt％、40-75wt％、40-70wt％、40-65wt％、40-60wt％、40-55wt％、40-50wt％、40-45wt％；

45-90wt％、45-85wt％、45-80wt％、45-75wt％、45-70wt％、45-65wt％、45-60wt％、45-55wt％、45-50wt％；

50-90wt％、50-85wt％、50-80wt％、50-75wt％、50-70wt％、50-65wt％、50-60wt％、50-55wt％；

55-90wt％、55-85wt％、55-80wt％、55-75wt％、55-70wt％、55-65wt％、55-60wt％；

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％；

the second component may be present in an amount selected from any one of the following ranges of values: 5-90wt%, 5-85wt%, 5-80wt%, 5-75wt%, 5-70wt%, 5-65wt%, 5-60wt%, 5-55wt%, 5-50wt%, 5-45wt%, 5-40wt%, 5-35wt%, 5-30wt%, 5-25wt%, 5-20wt%, 5-15wt%, 5-10wt% and one or more of the following components;

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％；

the third component may be present in an amount selected from any one of the following ranges of values: 5-90wt%, 5-85wt%, 5-80wt%, 5-75wt%, 5-70wt%, 5-65wt%, 5-60wt%, 5-55wt%, 5-50wt%, 5-45wt%, 5-40wt%, 5-35wt%, 5-30wt%, 5-25wt%, 5-20wt%, 5-15wt%, 5-10wt% and one or more of the following components;

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％。

[3.3] the pharmaceutical composition according to any one of the embodiments of the third aspect of the present invention, the content ratio of the first component, the content of the second component and the content of the third component is A: b: c, wherein, the method comprises the steps of,

the A may take the form of any of the group of values of (0.5-18), (1-18), (3-18), (4-18), (5-18) (7-18), (9-18), (10-18), (12-18), (14-18), (16-18), (0.05-15), (1-15), (3-15), (4-15), (5-15), (7-15), (9-15), (10-15), (12-15), (14-15), (0.05-10), (1-10), (3-10), (4-10), (5-10), (7-10), (9-10), (0.05-8), (1-8), (3-8), (4-8), (5-8), (0.05-3), (0.5-3), (1-3), (0.05-2), (0.5-2), (1-2) and (1) in the range of values;

the B may be any of the group of values of (0.5-18), (1-18), (3-18), (4-18), (5-18) (7-18), (9-18), (10-18), (12-18), (14-18), (16-18), (0.05-15), (1-15), (3-15), (4-15), (5-15), (7-15), (9-15), (10-15), (12-15), (14-15), (0.05-10), (1-10), (3-10), (4-10), (5-10), (7-10), (9-10), (0.05-8), (1-8), (3-8), (4-8), (5-8), (0.05-3), (0.5-3), (1-3), (0.05-2), (0.5-2), (1-2) and (1) in the range of values;

The C may take the form of any of the group of numerical ranges of (0.5-18), (1-18), (3-18), (4-18), (5-18) (7-18), (9-18), (10-18), (12-18), (14-18), (16-18), (0.05-15), (1-15), (3-15), (4-15), (5-15), (7-15), (9-15), (10-15), (12-15), (14-15), (0.05-10), (1-10), (3-10), (4-10), (5-10), (7-10), (9-10), (0.05-8), (1-8), (3-8), (4-8), (5-8), (0.05-3), (0.5-3), (1-3), (0.05-2), (0.5-2), (1-2) and (1).

[4] In a fourth aspect, the present invention provides a pharmaceutical composition comprising:

a first component: rhamnose and/or its hydrates;

and a second component: ferulic acid and/or isomers thereof, and/or pharmaceutically acceptable salts thereof, and hydrates thereof;

and a third component: chrysin and/or derivatives thereof.

[4.1] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention comprises

A first component: rhamnose or rhamnose hydrate;

and a second component: ferulic acid or sodium ferulate hydrate;

and a third component: chrysin; and

pharmaceutically acceptable carriers or adjuvants.

[4.2] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention, based on the sum of the weights of the first component, the second component and the third component as the total weight; wherein,

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％；

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％；

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％。

[4.3] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention, the content ratio of the first component, the content of the second component and the content of the third component is A: b: c, wherein, the method comprises the steps of,

[4.4] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention, further comprises a pharmaceutically acceptable carrier or adjuvant.

[4.5] a pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention, comprising:

the amount of the first component or the dose to be administered may be any value taken from any of the following sets of value ranges: 3-2000mg, 5-2000mg, 10-2000mg, 15-2000mg, 100-2000mg, 200-2000mg, 300-2000mg, 400-2000mg, 500-2000mg, 600-2000mg, 700-2000mg, 800-2000mg, 900-2000mg, 1000-2000mg, 1100-2000mg, 1200-2000mg, 1300-2000mg, 1400-2000mg, 1500-2000mg, 1600-2000mg, 1700-2000mg, 1800-2000mg, 1900-2000mg;

the amount of the second component or the dose to be administered may be any value taken from any of the following sets of value ranges: 3-2000mg, 5-2000mg, 10-2000mg, 15-2000mg, 100-2000mg, 200-2000mg, 300-2000mg, 400-2000mg, 500-2000mg, 600-2000mg, 700-2000mg, 800-2000mg, 900-2000mg, 1000-2000mg, 1100-2000mg, 1200-2000mg, 1300-2000mg, 1400-2000mg, 1500-2000mg, 1600-2000mg, 1700-2000mg, 1800-2000mg, 1900-2000mg;

The amount of the third component or the dose to be administered may be any value taken from any of the following sets of value ranges: 3-2000mg, 5-2000mg, 10-2000mg, 15-2000mg, 100-2000mg, 200-2000mg, 300-2000mg, 400-2000mg, 500-2000mg, 600-2000mg, 700-2000mg, 800-2000mg, 900-2000mg, 1000-2000mg, 1100-2000mg, 1200-2000mg, 1300-2000mg, 1400-2000mg, 1500-2000mg, 1600-2000mg, 1700-2000mg, 1800-2000mg, 1900-2000mg;

50-1900mg, 50-1800mg, 50-1700mg, 50-1600mg, 50-1500mg, 50-1400mg, 50-1300mg, 50-1200mg, 50-1100mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-690mg, 50-680mg, 50-670mg, 50-650mg, 50-600mg, 50-500mg, 50-450mg, 50-400mg, 50-350mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-100mg, 50-80 mg. When administered in the above-mentioned doses, satisfactory results are obtained, preferably in 1-3 times daily, in whole or divided doses, or in sustained release form. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

[4.6] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention, the dose to be administered is:

dosage of single administration; or, a daily dosage.

[4.7] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention is administered once, twice or three times daily.

[4.8] the pharmaceutical composition according to any of the fourth embodiments of the present invention is not particularly limited as long as it can be formulated directly or as a raw material for medicines, external medicines for medical department, foods and drinks, additives for foods and drinks, cosmetics, etc., as a form (dosage form) of the pharmaceutical composition for preventing action and/or the pharmaceutical composition for treating action of the present invention, as long as the extract of the present invention can act on nerve cells in brain. Preferred dosage forms of the pharmaceutical composition include powders, tablets, pills, capsules, sustained release agents, immediate release agents, injections, infusion solutions or suspensions.

[4.9] the pharmaceutical composition according to any one of the embodiments of the present invention, which is a medicament for administration via the gastrointestinal tract, or a medicament for administration via injection, or a medicament for administration via subcutaneous embedding, or preferably a medicament for administration via subcutaneous embedding.

[4.10] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention is administered clinically by gastrointestinal tract, or by injection, or by subcutaneous embedding, or preferably by subcutaneous embedding. The administration by injection includes intravenous administration, intramuscular administration and subcutaneous administration.

[4.11] use of a pharmaceutical composition according to any one of the embodiments of the fourth aspect of the invention in the manufacture of a medicament for the treatment or prevention of a neurodegenerative disease.

[4.12] the pharmaceutical composition according to any one of the embodiments of the fourth aspect of the present invention, the neurodegenerative disease comprises:

Neurodegenerative diseases characterized by reduced motor coordination; or (b)

[4.13] pharmaceutical compositions according to any of the embodiments of the fourth aspect of the invention, said neurodegenerative diseases comprise Alzheimer's disease, parkinson's disease, lewy body dementia, huntington's disease, amyotrophic lateral sclerosis; preferably Alzheimer's disease.

[5] In a fifth aspect, the present invention provides a pharmaceutical composition comprising:

a first active ingredient: rhamnose;

a second active ingredient: ferulic acid;

third active ingredient: chrysin; and

pharmaceutically acceptable carriers or adjuvants.

[5.1] the pharmaceutical composition according to any one of the embodiments of the fifth aspect of the present invention, based on the sum of the weights of the first active ingredient, the second active ingredient and the third active ingredient, as the total weight; wherein,

the first active ingredient may be present in an amount ranging between any of the values recited in any of the following groups: 5-90wt%, 5-85wt%, 5-80wt%, 5-75wt%, 5-70wt%, 5-65wt%, 5-60wt%, 5-55wt%, 5-50wt%, 5-45wt%, 5-40wt%, 5-35wt%, 5-30wt%, 5-25wt%, 5-20wt%, 5-15wt%, 5-10wt% and one or more of the following components;

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％；

the amount of the second active ingredient may be any value selected from any of the following groups of values: 5-90wt%, 5-85wt%, 5-80wt%, 5-75wt%, 5-70wt%, 5-65wt%, 5-60wt%, 5-55wt%, 5-50wt%, 5-45wt%, 5-40wt%, 5-35wt%, 5-30wt%, 5-25wt%, 5-20wt%, 5-15wt%, 5-10wt% and one or more of the following components;

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％；

The value of the content of the third active ingredient may be any value selected from any of the following groups of values: 5-90wt%, 5-85wt%, 5-80wt%, 5-75wt%, 5-70wt%, 5-65wt%, 5-60wt%, 5-55wt%, 5-50wt%, 5-45wt%, 5-40wt%, 5-35wt%, 5-30wt%, 5-25wt%, 5-20wt%, 5-15wt%, 5-10wt% and one or more of the following components;

60-90wt％、60-85wt％、60-80wt％、60-75wt％、60-70wt％、60-65wt％；

65-90wt％、65-85wt％、65-80wt％、65-75wt％、65-70wt％；

70-90wt％、70-85wt％、70-80wt％、70-75wt％；

75-90wt％、75-85wt％、75-80wt％；

80-90wt％、80-85wt％；

85-90wt％。

[5.2] the pharmaceutical composition according to any one of the embodiments of the fifth aspect of the present invention, the content ratio of the first active ingredient, the content of the second active ingredient, and the content of the third active ingredient is A: b: c, wherein, the method comprises the steps of,

[5.3] a pharmaceutical composition according to any one of the embodiments of the fifth aspect of the present invention, comprising:

the amount of the first active ingredient or the numerical size of the dose to be administered may be any number taken from any one of the following sets of numerical ranges: 3-2000mg, 5-2000mg, 10-2000mg, 15-2000mg, 100-2000mg, 200-2000mg, 300-2000mg, 400-2000mg, 500-2000mg, 600-2000mg, 700-2000mg, 800-2000mg, 900-2000mg, 1000-2000mg, 1100-2000mg, 1200-2000mg, 1300-2000mg, 1400-2000mg, 1500-2000mg, 1600-2000mg, 1700-2000mg, 1800-2000mg, 1900-2000mg, 3-1900mg, 3-1800mg, 3-1700mg, 3-1600mg, 3-1500mg, 3-1400mg, 3-1300mg, 3-1200mg, 3-1100mg, 3-1000mg, 3-900mg, 3-800mg, 3-700mg, 3-690mg, 3-680mg, 3-670mg, 3-650mg, 3-600mg, 3-500mg, 3-1900mg 3-400mg, 3-300mg, 3-200mg, 3-100mg, 3-80mg, 3-50mg, 3-30mg, 3-10mg, 15-1900mg, 15-1800mg, 15-1700mg, 15-1600mg, 15-1500mg, 15-1400mg, 15-1300mg, 15-1200mg, 15-1100mg, 15-1000mg, 15-900mg, 15-800mg, 15-700mg, 15-690mg, 15-680mg, 15-670mg, 15-650mg, 15-600mg, 15-500mg, 15-400mg, 15-300mg, 15-200mg, 15-170mg, 15-150mg, 15-130mg, 15-110mg, 15-100mg, 15-80mg, 15-50mg, 15-30mg, 50-1900mg, 50-1800mg, 50-1700mg, 50-1600mg, 50-1500mg, 50-1400mg, 50-1300mg, 50-1200mg, 50-1100mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-690mg, 50-680mg, 50-670mg, 50-650mg, 50-600mg, 50-500mg, 50-450mg, 50-400mg, 50-350mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-100mg, 50-80mg, 100-1900mg, 100-1800mg, 100-1700mg, 100-1600mg, 100-1500mg, 100-1400mg, 100-1300mg, 100-1200mg, 100-1100mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-690mg, 100-680mg, 100-670mg, 100-650mg, 100-600mg, 100-500mg, 100-450mg, 100-400mg, 100-350mg, 100-300mg, 100-250mg, 100-200mg, 100-120mg, 100-100 mg.

The amount of the second active ingredient or the numerical size of the dose to be administered may be any number taken from any of the following sets of numerical ranges: 3-2000mg, 5-2000mg, 10-2000mg, 15-2000mg, 100-2000mg, 200-2000mg, 300-2000mg, 400-2000mg, 500-2000mg, 600-2000mg, 700-2000mg, 800-2000mg, 900-2000mg, 1000-2000mg, 1100-2000mg, 1200-2000mg, 1300-2000mg, 1400-2000mg, 1500-2000mg, 1600-2000mg, 1700-2000mg, 1800-2000mg, 1900-2000mg, 3-1900mg, 3-1800mg, 3-1700mg, 3-1600mg, 3-1500mg, 3-1400mg, 3-1300mg, 3-1200mg, 3-1100mg, 3-1000mg, 3-900mg, 3-800mg, 3-700mg, 3-690mg, 3-680mg, 3-670mg, 3-650mg, 3-600mg, 3-500mg, 3-1900mg 3-400mg, 3-300mg, 3-200mg, 3-100mg, 3-80mg, 3-50mg, 3-30mg, 3-10mg, 15-1900mg, 15-1800mg, 15-1700mg, 15-1600mg, 15-1500mg, 15-1400mg, 15-1300mg, 15-1200mg, 15-1100mg, 15-1000mg, 15-900mg, 15-800mg, 15-700mg, 15-690mg, 15-680mg, 15-670mg, 15-650mg, 15-600mg, 15-500mg, 15-400mg, 15-300mg, 15-200mg, 15-170mg, 15-150mg, 15-130mg, 15-110mg, 15-100mg, 15-80mg, 15-50mg, 15-30mg, 50-1900mg, 50-1800mg, 50-1700mg, 50-1600mg, 50-1500mg, 50-1400mg, 50-1300mg, 50-1200mg, 50-1100mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-690mg, 50-680mg, 50-670mg, 50-650mg, 50-600mg, 50-500mg, 50-450mg, 50-400mg, 50-350mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-100mg, 50-80mg, 100-1900mg, 100-1800mg, 100-1700mg, 100-1600mg, 100-1500mg, 100-1400mg, 100-1300mg, 100-1200mg, 100-1100mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-690mg, 100-680mg, 100-670mg, 100-650mg, 100-600mg, 100-500mg, 100-450mg, 100-400mg, 100-350mg, 100-300mg, 100-250mg, 100-200mg, 100-120mg, 100-100 mg.

The amount of the third active ingredient or the numerical size of the dose to be administered may be any number taken from any of the following sets of numerical ranges: 3-2000mg, 5-2000mg, 10-2000mg, 15-2000mg, 100-2000mg, 200-2000mg, 300-2000mg, 400-2000mg, 500-2000mg, 600-2000mg, 700-2000mg, 800-2000mg, 900-2000mg, 1000-2000mg, 1100-2000mg, 1200-2000mg, 1300-2000mg, 1400-2000mg, 1500-2000mg, 1600-2000mg, 1700-2000mg, 1800-2000mg, 1900-2000mg, 3-1900mg, 3-1800mg, 3-1700mg, 3-1600mg, 3-1500mg, 3-1400mg, 3-1300mg, 3-1200mg, 3-1100mg, 3-1000mg, 3-900mg, 3-800mg, 3-700mg, 3-690mg, 3-680mg, 3-670mg, 3-650mg, 3-600mg, 3-500mg, 3-1900mg 3-400mg, 3-300mg, 3-200mg, 3-100mg, 3-80mg, 3-50mg, 3-30mg, 3-10mg, 15-1900mg, 15-1800mg, 15-1700mg, 15-1600mg, 15-1500mg, 15-1400mg, 15-1300mg, 15-1200mg, 15-1100mg, 15-1000mg, 15-900mg, 15-800mg, 15-700mg, 15-690mg, 15-680mg, 15-670mg, 15-650mg, 15-600mg, 15-500mg, 15-400mg, 15-300mg, 15-200mg, 15-170mg, 15-150mg, 15-130mg, 15-110mg, 15-100mg, 15-80mg, 15-50mg, 15-30mg, 50-1900mg, 50-1800mg, 50-1700mg, 50-1600mg, 50-1500mg, 50-1400mg, 50-1300mg, 50-1200mg, 50-1100mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-690mg, 50-680mg, 50-670mg, 50-650mg, 50-600mg, 50-500mg, 50-450mg, 50-400mg, 50-350mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-100mg, 50-80mg, 100-1900mg, 100-1800mg, 100-1700mg, 100-1600mg, 100-1500mg, 100-1400mg, 100-1300mg, 100-1200mg, 100-1100mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-690mg, 100-680mg, 100-670mg, 100-650mg, 100-600mg, 100-500mg, 100-450mg, 100-400mg, 100-350mg, 100-300mg, 100-250mg, 100-200mg, 100-120mg, 100-100 mg.

[5.3] the pharmaceutical composition according to any one of the embodiments of the fifth aspect of the present invention, the dose to be administered is:

dosage of single administration; or, a daily dosage.

[5.4] the pharmaceutical composition according to any one of the embodiments of the fifth aspect of the present invention is administered once, twice or three times daily.

[5.5] pharmaceutical compositions according to any of the embodiments of the fifth aspect of the present invention, in the form of powders, tablets, pills, capsules, sustained release agents, immediate release agents, injections, infusion solutions or suspensions.

[5.6] the pharmaceutical composition according to any one of the embodiments of the present invention is a medicament for administration via the gastrointestinal tract, or a medicament for administration via injection, or a medicament for administration via subcutaneous embedding, or preferably a medicament for administration via subcutaneous embedding.

[5.7] the pharmaceutical composition according to any one of the embodiments of the fifth aspect of the present invention is administered clinically by gastrointestinal tract, or by injection, or by subcutaneous embedding, or preferably by subcutaneous embedding.

[5.8] use of a pharmaceutical composition according to any one of the embodiments of the fifth aspect of the invention in the manufacture of a medicament for the treatment or prevention of a neurodegenerative disease.

[5.9] the pharmaceutical composition according to any one of the embodiments of the fifth aspect of the present invention, the neurodegenerative disease comprises:

Neurodegenerative diseases characterized by reduced motor coordination; or (b)

[5.10] pharmaceutical compositions according to any of the embodiments of the fifth aspect of the invention, said neurodegenerative diseases comprise Alzheimer's disease, parkinson's disease, lewy body dementia, huntington's disease, amyotrophic lateral sclerosis; preferably Alzheimer's disease.

The dosage form of the pharmaceutical composition of the invention may be varied and is any form that allows the active ingredient to reach the mammalian body effectively. For example, it may be selected from: powder, tablet, pill, capsule, sustained release agent, quick release agent, injection, infusion solution, and suspension. The type of disease to be treated with the composition of the present invention can be selected by those skilled in the art to be conveniently used. Preferred pharmaceutical compositions are solid compositions, especially tablets and solid filled or liquid filled capsules, from the point of view of ease of preparation and storage. From the viewpoint of ease of administration, the preferred pharmaceutical composition is an oral preparation. The pharmaceutical compositions of the present invention may also be stored in a disinfection device suitable for injection or instillation.

Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they may be prepared with a coating.

Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs.

The formulation and preparation of these pharmaceutical compositions of the invention in the form of pharmaceutical formulations can be accomplished by methods and experience well known to those skilled in the art.

[6] In fact, whether "rhamnose and its hydrate", or "containing rhamnose and its hydrate, cinnamic acid derivatives, or a pharmaceutically acceptable salt thereof" as described above; and flavonoid compounds, or a composition containing rhamnose and/or a hydrate thereof, ferulic acid and/or an isomer thereof, and/or a pharmaceutically acceptable salt thereof, chrysin and/or a derivative thereof, and/or a pharmaceutically acceptable salt thereof, or a composition containing rhamnose, ferulic acid and chrysin, is not particularly limited in form as long as it is capable of passing through the blood-brain barrier, and for example, the composition can be suitably used as a pharmaceutical composition, a food or beverage composition.

The pharmaceutical composition can be widely used as a pharmaceutical product, and the like. Specifically, examples thereof include tablets (including plain tablets, sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, and the like), pills, powders, granules, capsules (including soft capsules and microcapsules), buccal tablets, syrups, liquids, emulsions, suspensions, controlled release preparations (e.g., immediate release preparations, sustained release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa films), percutaneous absorption preparations, ointments, lotions, patches, pellets, nasal preparations, pulmonary preparations (inhalants), and the like. The pharmaceutical composition of the present invention can be prepared by appropriately blending, according to a conventional method, a carrier, a base, and/or an additive, etc., which are generally used in the field of preparations, within a range where the object of the present invention can be achieved, as long as the pharmaceutical composition contains the preventive and/or therapeutic agent of the present invention.

The food and beverage composition is widely used as an additive for food and beverage. Specifically, examples of the food include various foods such as bread, cake, noodle, snack, jelly, frozen food, ice cream, dairy product, beverage, soup, and edible oil, and juice beverage, carbonated beverage, tea beverage, sports beverage, milk beverage, alcoholic beverage, and refreshing beverage. The food and drink further includes functional labeling food, nutritional functional food, food for specific health, food for specific use, food for aged people, food for patients, and health auxiliary food (supplement). These forms are not particularly limited, and may be those of oral administration type of the above-mentioned drugs and external products of the medical department. These may be compositions obtained by adding the prophylactic and/or therapeutic agent of the present invention to conventional foods and beverages at the time of or after production, and the addition timing and addition method are not particularly limited.

[ use for treating or preventing neurodegenerative diseases ]

In a sixth aspect the present invention provides a method of treating or preventing a neurodegenerative disease comprising the step of administering to a mammal in need thereof an effective amount of a medicament, composition according to any one or more of the second to fifth aspects.

A method of treating or preventing a neurodegenerative disease according to any one of the embodiments of the sixth aspect of the invention, wherein the pharmaceutical composition is applied to a mammal, in particular a human, wherein rhamnose and/or its hydrates are administered to the patient at a dose of 0.1 to 50 mg/kg/day/human, ferulic acid and/or its pharmaceutically acceptable salts and its hydrates at a dose of 0.1 to 50 mg/kg/day/human, chrysin at a dose of 0.1 to 50 mg/kg/day/human. Preferably, wherein rhamnose and/or its hydrate in the pharmaceutical composition is administered to the patient at a dose of 0.16 to 33.3 mg/kg/day/person, ferulic acid and/or its pharmaceutically acceptable salt and its hydrate at a dose of 0.16 to 33.3 mg/kg/day/person, chrysin at a dose of 0.16 to 33.3 mg/kg/day/person. Preferably, wherein rhamnose and/or its hydrate in the pharmaceutical composition is administered to the patient at a dose of 1 to 20 mg/kg/day/person, ferulic acid and/or its pharmaceutically acceptable salts and its hydrate at a dose of 1 to 20 mg/kg/day/person, chrysin at a dose of 1 to 20 mg/kg/day/person. Preferably, wherein rhamnose and/or its hydrate in the pharmaceutical composition is administered to the patient at a dose of 1 to 10 mg/kg/day/person, ferulic acid and/or its pharmaceutically acceptable salts and its hydrate at a dose of 1 to 10 mg/kg/day/person, chrysin at a dose of 1 to 10 mg/kg/day/person. The pharmaceutical composition does not show toxic or side effects when administered within the above dosage range.

In the context of the present invention, the content ratio refers to the mass ratio between the different components, such as the pharmaceutical composition of the embodiment, the content ratio of the first component, the content ratio of the second component and the content ratio of the third component being a: b: c, the mass ratio of the first component, the second component and the third component is A: b: C.

in the context of the present invention, "ferulic acid" includes ferulic acid as a free acid and as a pharmaceutically acceptable salt. Particularly mentioned are sodium salts and hydrates thereof, as well as isomers of the various forms thereof or combinations thereof and pharmaceutically acceptable salts thereof.

In the context of the present invention, "rhamnose" includes various forms of isomers or combinations thereof; l-rhamnose is particularly mentioned. Also included are solvates thereof, particularly the hydrates.

In the context of the present invention, "chrysin" includes various forms of isomers or combinations thereof.

Any of the embodiments of any of the aspects of the invention may be combined with other embodiments without contradiction. Furthermore, in any of the embodiments of any of the aspects of the present invention, any feature may be applied to the feature in other embodiments without contradiction.

Any technical feature provided in any aspect of the present invention or any embodiment of any aspect thereof is equally applicable to any other embodiment or any other embodiment of any other aspect, unless contradictory, although appropriate modifications of the corresponding features may be made as appropriate as applicable to each other. Various aspects and features of the invention are described further below.

All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.

The term "isomer" as used herein includes: geometric isomers, enantiomers, diastereomers (e.g., cis-trans isomers, conformational isomers).

"analog" as described herein. Such as the ferulic acid has analogs including, but not limited to, isoferulic acid, caffeic acid, p-hydroxy coumaric acid, o-hydroxy coumaric acid, m-hydroxy coumaric acid, and the like;

The chrysin has analogues including, but not limited to, apigenin, farnesin, genkwanin and the like.

The term "solvate" as used herein refers to a compound carrying a solvent molecule, for example, the solvate may be a hydrate.

In the present invention, the term "comprising" or "containing" means that the various ingredients can be used together in the compositions of the present invention. Thus, the terms "consisting essentially of and" consisting of "are encompassed by the terms" comprising "or" containing.

The compositions of the present invention also include isomers, solvates, precursors, or pharmaceutically acceptable salts thereof of the particular compounds in the compositions described above, provided that they also have the same or substantially the same function as the particular compounds in the compositions.

In the present invention, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response)), commensurate with a reasonable benefit/risk ratio.

In the present invention, the term "pharmaceutically acceptable salt" refers to an acid addition salt of a compound with a nontoxic acid, and includes salts formed by the reaction of inorganic acids, organic acids, alkali metals or alkaline earth metals, etc. These salts include, but are not limited to:

(1) Salts with the following mineral acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid;

(2) Salts with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium) in the form of esters, carbamates, or other conventional "prodrugs".

The compounds in the compositions of the invention have one or more asymmetric centers. These compounds can therefore exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis-or trans-isomers.

In the present invention, a "pharmaceutically acceptable carrier" is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering the composition, isomer, solvate, or pharmaceutically acceptable salt thereof of the present invention to an animal or human. The carrier may be a liquid or a solid.

The "pharmaceutically acceptable carrier or adjuvant" in the present invention includes, but is not limited to, preservatives, wetting agents, emulsifying agents, dispersing agents and the like.

The pharmaceutical compositions of the present invention may be used as the sole drug or may be used in combination with one or more other drugs having synergistic and/or synergistic effects with the substances of the present invention. Combination therapy may be achieved by simultaneous, sequential or separate administration of the individual therapeutic components.

The actual dosage levels and mode of administration of the individual active ingredients (ferulic acid, rhamnose, and chrysin) in the pharmaceutical compositions of the present invention may be varied so that the resulting active amounts are effective to achieve the desired therapeutic response for a particular patient. The dosage level will be selected based on the activity of the particular active agent, the route of administration, the severity of the condition being treated, and the condition and past history of the patient being treated. However, it is practiced in the art that the dosage of the active substance is gradually increased from a level below that required to achieve the desired therapeutic effect until the desired effect is achieved.

When used in the above-described treatment and/or prophylaxis of the present invention or other treatments and/or prophylaxis, a therapeutically and/or prophylactically effective amount of one of the pharmaceutical compositions of the present invention may be employed in isolation. Alternatively, the pharmaceutical compositions of the present invention may be administered in combination with one or more pharmaceutically acceptable carriers or excipients.

The term "treatment" in the present invention means managing and caring for a patient for the purpose of combating a condition (e.g., a disease or disorder). The term is intended to include the full scope of treatment for a given condition to which a patient is exposed, such as administration of a composition to alleviate symptoms or complications, to delay the progression of the condition, and/or to cure or eliminate the condition. The patient to be treated is preferably a mammal, in particular a human.

The term "therapeutically and/or prophylactically effective amount" of a pharmaceutical composition of the invention in the present invention refers to an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications in a therapeutic intervention involving administration of the composition. An amount sufficient to achieve the above is defined as a "therapeutically and/or prophylactically effective amount". The effective amount for each purpose will depend on the severity of the disease or injury and the weight and general state of the subject. It will be appreciated that the total daily dose of the pharmaceutical composition of the invention must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the particular therapeutically effective dose level will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical composition employed; the specific pharmaceutical composition employed; age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular pharmaceutical composition employed; duration of treatment; other drugs used in combination with or simultaneously with the pharmaceutical composition employed; and similar factors well known in the medical arts. For example, it is common in the art to start doses of pharmaceutical compositions below the level required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is obtained.

The compounds (active ingredients) in the compositions used in the present invention may be administered as pure compounds alone in single or multiple doses, or in combination with pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions according to the invention may be formulated according to conventional techniques using pharmaceutically acceptable carriers or excipients and any other known adjuvants and excipients.

In the present invention, the combined administration of ferulic acid, rhamnose and chrysin in the pharmaceutical composition may take the form of simultaneous administration. In some embodiments, ferulic acid, rhamnose and chrysin are administered to the patient at substantially the same time in a form comprising ferulic acid, rhamnose and chrysin, or in separate forms, i.e. a first form comprising rhamnose, a second form comprising ferulic acid and a third form comprising chrysin. When administered in a separate administration form, it is desirable that pharmacologically relevant amounts of ferulic acid, rhamnose and chrysin are present in the body simultaneously, i.e. that the time gap between administration of ferulic acid, rhamnose and chrysin is as short as possible, in order to ensure a synergistic effect, which is likely to be reduced if the time gap between administration of the three is large.

To ensure a synergistic effect, the synergistic effect between the three compounds (active ingredients) in the composition can be exploited in at least two ways. The dosages normally applied in monotherapy with ferulic acid and chrysin can be maintained in combination therapy and larger effects than the intended clinical effects can be achieved. Alternatively, either or both of ferulic acid and chrysin in the composition are at lower doses than those typically employed in monotherapy with ferulic acid and chrysin, in order to maintain the clinical effect, but with lower drug exposure. Lower drug exposure is advantageous because adverse events can be expected to decrease with decreasing drug loading.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) According to the invention, rhamnose can obviously improve the cognitive behaviors of mice on an APP/PS1 mouse model, and can be used for treating or preventing neurodegenerative diseases, and further treating or preventing Alzheimer's disease.

(2) The experiment of the invention shows that the rhamnose can enhance the phosphorylation of the AMPK, the AMPK is used as a receptor of cell energy and a regulator of metabolic homeostasis, and the activation of the AMPK can regulate SIRT1, so that the accumulation of phosphorylated tau protein is inhibited, and the cognitive behaviors of mice are further improved.

(3) When the rhamnose or the hydrate thereof is used for preparing the medicine for treating or preventing the neurodegenerative diseases, the amount of the rhamnose or the hydrate thereof in the medicine or the dosage of the rhamnose or the hydrate thereof contained in the medicine to be applied is 3-2000mg; or 10-2000mg; or 3-670mg; or 15-500mg; or 50-500mg; or 15-170mg; which exhibits the safety of high dose administration.

(4) Rhamnose, cinnamic acid derivatives and flavonoids are administered in a composition with an enhanced effect of slowing down the further progression of neurodegenerative diseases such as AD compared to the single active ingredient or the multi-target compounds of the prior art;

(5) The present invention further found that when the combination of rhamnose, ferulic acid and chrysin (hereinafter abbreviated as "Wei Libai") is applied as an active ingredient, the symptom of neurodegenerative diseases can be significantly improved and proliferation of neural stem cells can be effectively promoted compared to a single active ingredient or a multi-target compound in the prior art; it can be used for preventing and treating aging and neurodegenerative diseases;

(6) When the combination of rhamnose, ferulic acid and chrysin (hereinafter referred to as Wei Libai) is taken as an active ingredient and applied in a certain proportion, the invention surprisingly finds that the combination of rhamnose, ferulic acid and chrysin has favorable synergistic effect on learning, memorizing and cognitive behaviors of mice, can obviously improve the symptom of neurodegenerative diseases and can effectively enhance the functions of neural stem cells.

Drawings

FIG. 1 shows the effect of L-rhamnose and metformin on the expression level of SK-N-SH cell AMPK phosphorylated protein, total AMPK in example 1; (xp <0.05,/p <0.01, vs control group)

FIG. 2 is the effect of L-rhamnose in example 2 on the Morris water maze index of AD mice; (< 0.05, <0.01, <0.001, <0.0001AD solvent group vs L-rhamnose administration group)

FIG. 3 is the effect of L-rhamnose on the number of neurons in the hippocampal DG region of AD mice in example 2; (/ p <0.001, vs AD solvent group)

FIG. 4 shows the effect of ferulic acid on TNFa, IL-6 expression in example 3;

FIG. 5 shows the effect of chrysin, apigenin, farnesoid, and genkwanin on cell viability of 3L NSC in example 4;

FIG. 6 is a graph showing the effect of chrysin on Morris water maze index in AD mice in example 5; (< p <0.05, < p <0.01, < p <0.001, vs AD solvent group)

FIG. 7 is a graph showing the effect of Wei Libai on SK-N-SH and HEK293-APPswe cell viability in example 6;

FIG. 8 is a graph showing the change in body weight of mice after a single administration Wei Libai in example 7;

FIG. 9 is the effect of combination of rhamnose, ferulic acid and chrysin on the effect of anti-inflammatory (inflammatory factor TNFa) in example 8 (where no LPS is a non-stimulated negative control, veh is a positive control, FA is ferulic acid, L-Rha is rhamnose, chr is chrysin, FRC 10. Mu.M represents a composition consisting of rhamnose, ferulic acid and chrysin each 10. Mu.M);

FIG. 10 is the effect of the amount of chrysin on the anti-inflammatory (inflammatory factor IL-6) effect of the combination of rhamnose, ferulic acid and chrysin of example 9 (where no LPS is a non-stimulated negative control, veh is a positive control, FA is ferulic acid, L-Rha is rhamnose, and Chr is chrysin);

FIG. 11 shows the effect of the rhamnose monohydrate, ferulic acid and chrysin composition in a mass ratio of 1:1:1 in example 10 on the Morris water maze index of AD mice (FIG. 11 (a) shows the plateau time in training; FIG. 11 (b) shows the plateau time in the seventh day of the bench withdrawal experiment); fig. 11 (c) shows the time for the mice to search in each quadrant region (TR is the quadrant in which the platform is located, AR is the quadrant on the right side of the quadrant in which the platform is located, AL is the quadrant on the left side of the quadrant in which the platform is located, and OP is the quadrant on the opposite side of the quadrant in which the platform is located) in the Morris water maze test.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, metric or value. The degree of flexibility of a particular variable can be readily determined by one skilled in the art.

Concentrations, amounts, percentages, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

Through intensive researches, the inventor of the present invention has conducted a study on pharmaceutical compositions of AMPK activator, ferulic acid or pharmaceutically acceptable salts thereof, hydrates thereof and neuron regeneration promoter, especially a combination of rhamnose, ferulic acid and chrysin (Wei Libai), capable of significantly improving the symptoms of neurodegenerative diseases. In vitro and in vivo experiments, the pharmaceutical composition can effectively promote the regeneration of neurons; it can be used for preventing and treating nerve regeneration, and can be used for treating cognitive decline associated with neurodegenerative diseases.

According to our latest experimental data, it is shown that L-rhamnose can activate the AMPK signaling pathway in vitro experiments. AMPK acts as a regulator of cellular energy receptors and metabolic homeostasis, and its activation can regulate SIRT1, thereby inhibiting the accumulation of phosphorylated tau protein, slowing down further progression of AD. In addition, the L-rhamnose can improve the performance of an APP/PS1 mouse in water maze behaviours, enhance the learning and memory functions of the AD mouse, and show better AD treatment potential. A large number of documents show that the ferulic acid has remarkable anti-inflammatory effect, and the characteristic is also confirmed in the latest experimental data of us. Meanwhile, the medicine has excellent curative effect in an AD animal model. Chrysin also has obvious improvement on the behavior cognition ability of APP/PS1 mice. All three compounds have better potential in the aspect of AD treatment, so we combine the three compounds, namely L-rhamnose, ferulic acid and chrysin, according to mass 1:1:1 forms a new composition Wei Libai which shows no significant toxicity on SK-N-SH cells, HEK 293-appwe cells and in the preliminary safety assessment of mice.

Meanwhile, the influence of the strain on the learning and memory capacity of APP/PS1 mice is observed. The APP/PS1 mice composition Wei Libai, i.e. 5mg/kg each of ferulic acid, rhamnose monohydrate and chrysin, 10mg/kg each of ferulic acid, rhamnose monohydrate and chrysin, and 20mg/kg each of ferulic acid, rhamnose monohydrate and chrysin, were given daily intragastrically for 3 months, administration of Wei Libai resulted in AD mice exhibiting improved learning and memory capacity in the water maze behavioural test.

The invention is further described below in connection with specific embodiments. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

Statistical analysis of data

All experimental data are expressed as mean ± standard error. Comparisons were made between different treatment groups using t-test. Between sets of results, one-way ANOVA was used for analysis and Fisher's protected least significant difference test or Bonferroni t test for post hoc testing, or two-way ANOVA was used for analysis and Tukey post hoc test for post hoc testing. Significant differences between groups were considered at P < 0.05.

Statistical analysis

Data were analyzed by Prism 6.0 (GraphPad Software inc., san Diego, CA) and two or more data sets were compared using single-factor analysis of variance (ANOVA) for post-test of Bonferroni, receiving statistical significance at p < 0.05.

Reagent(s)

L-rhamnose (L-rhamnose monohydrate) was purchased from the national drug group (BR) Walker, cat: 63010284; metformin is purchased from beyotime, cat: s1741; ferulic acid was purchased from the national drug group, cat No.: 30089423; chrysin was purchased from MCE, cat No.: HY-W013372. Other reagents are commercially available.

Dosage of

For the administration or dosage or effective amount of mice, the corresponding administration or dosage or effective amount of the human obtained by cross-species conversion criteria of a pharmaceutically equivalent dosage can be theoretically employed. For example, the daily dosage of L-rhamnose in the pharmacodynamic test of mice is 10mg/kg and 30mg/kg, the mice and the human are assumed to have the same pharmacodynamic effect on the same surface area, the dosages of the mice are converted into human equivalent dosages according to the body surface area, and the equivalent dosages are respectively 10/30mg/kg, 0.08 x 60 kg=48/144 mg, namely, the corresponding administration dosage of the human is 48 mg/day or 144 mg/day theoretically. The dose conversion formula for humans and animals is: dose of mice (mg/kg) =12.3 dose of human (mg/kg), reference: ( Nair AB, jacob s.a simple practice guide for dose conversion between animals and human j Basic Clin pharm.2016mar;7 (2):27-31. )

However, in practical cases, great differences in absorption, metabolism, distribution, etc. of humans and animals need to be considered, and therefore, the results of cross-species conversion (abbreviated as M hereinafter) cannot be strictly followed by pharmaceutically equivalent doses _{Conversion of} ) Is used for human consumption. Based on this, the actual amount of people (hereinafter abbreviated as M _{Actual practice is that of} )，M _{Actual practice is that of} M _{Conversion of} It is reasonable to have a 1-3 times relationship with the other.

Example 1

L-rhamnose activates AMPK of SK-N-SH (human neuroblastoma) cells

Experimental cells: SK-N-SH cells

Experimental instrument: bio-Rad electrophoresis apparatus, SEQ ID NO: 042BR10743

The experimental method comprises the following steps: after SK-N-SH cells are plated and cultured, L-rhamnose (30 mu M,100 mu M,300 mu M) is added for incubation, simultaneously metformin (metformin-1 mM) is used as a positive control, after incubation for 24 hours, supernatant is discarded, and the expression quantity of the AMPK phosphorylated protein and the total AMPK of the SK-N-SH cells is detected through western blotting;

conclusion of experiment: as shown in fig. 1, the results showed that 30 μm,100 μm,300 μm of L-rhamnose increased the amount of phosphorylated AMPK to total AMPK in a dose-dependent manner.

Example 2

L-rhamnose improves learning and memory ability of APP/PS1 mice

Experimental animals: 45 APP/PS1 mice of 5-6 months of age, about half of each male and female, and about half of each female and male, 12 wild type (wild type) in total.

Experimental instrument: morris water maze; model: the method is universal; matching software: ji measuring software

Experimental protocol: the mice of the experimental animals were randomly divided into 3 groups (wild type (WT) solvent group, AD solvent group and L-rhamnose group), APP/PS1 mice of the L-rhamnose group were administered 30mg/kg of L-rhamnose (sterile water preparation) by stomach infusion, and wild type The form (WT) solvent set and the AD solvent set were given equal doses of sterile water. After 4 months of continuous dosing, the effect of L-rhamnose on learning and memory capacity of APP/PS1 mice was examined using Morris water maze behavioural experiments. All animals were continuously intraperitoneally injected with Brdu (5-Bromo-2' -deoxyundene, sigma, HMBG 3845V) for 7 days before performing the water maze behavioral test, for labeling cells newly added to the hippocampal region, at a dose of 50mg/kg per day. After the study memory capacity of the experimental mice is observed through Morris water maze, the animals are anesthetized, brain tissues are obtained through heart perfusion, and Brdu is sliced and marked ⁺ /NeuN ⁺ Double positive cells, calculating the total Brdu of double positive cells ⁺ Percentage of cells.

Conclusion of experiment: as shown in fig. 2, mice had a shorter platform finding time with increasing training days, whereas WT solvent group mice had a greater degree of latency shortening than AD solvent group mice, as evidenced by faster platform finding by WT solvent group mice. In ten days of training, the time to first find the platform was significantly reduced from the second day for the 30mg/kg L-rhamnose dosed AD mice group compared to the AD solvent group, and the significant differences were maintained until the tenth day of training. The intervention of L-rhamnose is shown, so that an APP/PS1 mouse can be positioned to a platform more quickly, and the learning and memory capacity of an AD mouse is obviously improved.

Cells newly added to the DG region of the hippocampus were labeled with BrdU by immunofluorescence staining, and the ratio of the numbers of BrdU and NeuN biscationic cells (proliferating neurons) to the numbers of newly added cells was counted. As shown in FIG. 3, the number ratio of newly added neurons in the hippocampal DG region of the AD mice after 30mg/kg of L-rhamnose administration is extremely remarkably higher than that of the AD solvent control group, which shows that 30mg/kg of L-mice Li Tanggan has prognosis and promotes the proliferation of the neurons in the hippocampal DG region.

Example 3

Ferulic acid has antiinflammatory effect on mice microglial BV2 cells

Experimental cells: mouse microglial cell BV2

The experimental method comprises the following steps: compound treatment of microglial cells

1) 12X 10 inoculation of 12 well plate ⁴ Microglial cells BV2 (DMEM)HG+10％FBS)；

2) Attaching for 48 hours, and replacing serum-free culture medium to starve for 12 hours, wherein the concentration is 700 mu L/hole;

3) After 24 hours of treatment with 100. Mu.M and 300. Mu.M ferulic acid, respectively, the supernatants were collected, centrifuged at 12000rpm for 5min to remove dead cells and cell debris, and the TNFα and IL-6 were detected by ELISA, and the remaining samples were stored at 4 ℃.

4) Detection was performed according to the instructions of the ELISA kit.

Tnfα ELISA kit: cargo number: 88-7024; brand: invitrogen

IL-6ELISA kit: cargo number: 88-7064, brand: invitrogen

Another 0.05% Tween-20/PBS wash and 2N H were prepared ₂ SO ₄ The reaction solution was terminated.

Conclusion of experiment: as shown in FIG. 4, after the cells are treated for 24 hours by 100 mu M ferulic acid and 300 mu M ferulic acid respectively, 300 mu M ferulic acid can significantly down-regulate the expression of TNF alpha and IL-6 on BV2 background level, which indicates that ferulic acid has anti-inflammatory effect.

Example 4

Chrysin, apigenin, farnesin and genkwanin promote proliferation of 3L NSC of human neural stem cells

Experimental cells: human neural stem cell 3L NSC

The experimental method comprises the following steps: cell Counting-Lite 2.0reagent (cat# DD1101-01; brand: vazyme) was used for cellular ATP level detection to indicate Cell number.

The experimental steps are as follows:

1) Human neural stem cell 3L NSC 2×10 inoculated by 96 well plate ³ Well (low growth factor conditioned medium);

2) After adhering for 24 hours, adding compounds (chrysin, apigenin, farnesin and genkwanin) with different concentrations for treatment;

3) After 48h of compound treatment, the supernatant was discarded, 100. Mu.L of Cell Counting-Lite 2.0Reagent was added to each well, and the mixture was shaken for 10min with an oscillator, and then transferred to a white opaque 96-well plate, and the luminescence intensity was detected by using an enzyme-labeled instrument (Bio tek Synergy NEO);

conclusion of experiment: as shown in fig. 5, after the cells were treated with 1 μm,3 μm and 10 μm compounds, respectively, it was found that chrysin, apigenin, farnesin and genkwanin significantly improved the cell viability of 3L NSC, indicating that chrysin, apigenin, farnesin and genkwanin had activity in promoting proliferation of human neural stem cells.

Example 5

Chrysin improves learning and memory capacity of APP/PS1 mice

Experimental animals: 28 APP/PS1 mice at 6 months of age were half-male and half-female, 15 wild type (wild type) and about half-male.

Experimental protocol: mice from experimental animals were randomized into 3 groups, with chrysin-interfered groups of APP/PS1 mice, and 25mg/kg chrysin (5% DMSO+0.5% CMC) was administered by gavage, while the wild-type (WT) solvent group and AD solvent group were given equal doses of solvent. After 3 months of continuous administration, the study memory ability of the experimental mice was observed through Morris water maze.

Conclusion of experiment: as shown in fig. 6, mice showed a trend of shortening the latency with increasing training days, whereas WT mice showed a greater degree of latency shortening than APP/PS1 solvent group mice, indicating that WT mice were able to find the platform faster. In six days of training, the time to find the platform for the first time was extremely significantly reduced from the second, third, fourth, and fifth days of APP/PS1 chrysin dosing group compared to APP/PS1 solvent group. Experimental results show that the study and memory capacity of APP/PS1 mice can be obviously improved after 25mg/kg chrysin is continuously administrated for 3 months.

Example 6

Wei Libai the activity of SK-N-SH cells and HEK293-APPswe cells is not affected

Experimental cells: SK-N-SH cells and HEK293-APPswe cells

Experimental instrument: perforated plate microwell detector Bio Tek Synergy NEO, luminescence

Composition Wei Libai formulation: mixing ferulic acid, rhamnose monohydrate and chrysin at a mass ratio of 1:1:1, preparing into 80mg/ml storage solutions with DMSO, and storing in a refrigerator at-80deg.C. When in use, a small tube is taken out, the culture solution is firstly diluted 160 times to 500 mu g/ml, then diluted 100 times until the final concentration of each component is 5 mu g/ml (high concentration), and finally diluted 3 times in gradient until the final concentration of each component is 1.7 mu g/ml (medium concentration) and 0.6 mu g/ml (low concentration).

The experimental method comprises the following steps:

1) Wei Libai treatment of SK-N-SH cells and HEK293-APPswe cells

Day 1: and (5) paving cells. mu.L of SK-N-SH or HEK293-APPswe cell suspension (5X 103cells/well;10% FBS MEM medium) was inoculated into 96-well plates at 37℃with 5% CO ₂ The cells were cultured overnight (18-24 h) in an incubator. (only the middle 60 wells are spread and the edge wells are used with ddH) ₂ O-tonifying flat

Day 2: the cells are treated with the composition. Firstly observing the cell state and density under a microscope, wherein the cell state is good and the density is 30% -50%. Culture supernatants were removed, low, medium, and high 3 doses of Wei Libai (10% FBS-containing MEM) were added to the well plates and at least 3 wells were made per group using medium with the corresponding concentration of DMSO as a control. Placing at 37deg.C and 5% CO ₂ Culturing in an incubator (24 h,48h,72 h).

2) Cell viability assay

Day 3: cell viability was checked (24 h,48h,72 h). The cell Titer Glo assay (Noruzan, 4 ℃) was equilibrated to room temperature half an hour in advance, then the 96-well plate was taken out of the incubator, the cell status was observed under a microscope, then left to equilibrate at room temperature for 3-5 minutes, then the supernatant of the culture was removed, 100. Mu.L of the cell Titer Glo assay was added to each well, and the mixture was placed on an shaker for 10 minutes, then all of the wells were transferred to the 96-well plate, left to stand at room temperature for 2-3 minutes, and detected on-machine (multi-well plate microwell detector Bio Tek Synergy NEO, lumineancence).

Conclusion of experiment: as shown in FIG. 7, with reference to control group 1, on SK-N-SH cells, the viability of cells was not affected after 24h,48h and 72h of treatment of low, medium and high concentrations Wei Libai, and was not different from that of the control group. This was also true on HEK 293-appwe cells, where low, medium, and high concentrations of Wei Libai incubated cells for 24h,48h, or 72h, all had no effect on cell viability, indicating that the low, medium, and high concentrations of Wei Libai were not toxic to SK-N-SH cells and HEK 293-appwe cells.

Example 7

The single administration mice Wei Libai have no obvious toxic and side effects

Experimental animals: APP/PS1 littermate wild mice, 4-5 months old, 4 total, 2 males, 2 females;

the experimental method comprises the following steps:

preparation and administration of composition Wei Libai comprises dissolving Wei Libai (33 mg of ferulic acid, rhamnose monohydrate and chrysin) in 60 μl DMSO, adding 1140 μl of 0.5% CMC-Na, shaking thoroughly, mixing, centrifuging, settling the powder on the tube wall, and shaking again to make the suspension uniformly distributed in the solvent. The stomach was irrigated orally and administered at 100. Mu.L/10 g body weight.

Experimental observation: the animals were continuously observed for 4 hours after the first administration, and the mental behavior, voluntary activity, hair, glandular secretion, feces, death, etc. were recorded with a focus on observation, and the animals were continuously observed for 13 days while observing the change in body weight. Dissecting at the end of the observation period or when the animal dies or is dying, and fixedly preserving abnormal tissues for pathological observation.

Experimental results: as shown in fig. 8, single administration Wei Libai (ferulic acid, rhamnose monohydrate, chrysin each 275 mg/kg) was not toxic to wild-type mice.

Example 8

The combined use of rhamnose, ferulic acid and chrysin increases (produces) anti-inflammatory (inflammatory factor TNFa) effect

Experimental cells: mouse microglial cell BV2;

the experimental method comprises the following steps: compound treatment of microglial cells;

1) 24 well plate inoculation 5X 10 ⁴ Microglial cells BV2 (DMEM hg+10% fbs);

2) Attaching the wall for 24 hours;

3) After pretreatment with 10. Mu.M rhamnose, 10. Mu.M ferulic acid, 10. Mu.M chrysin and 10. Mu.M each of rhamnose, ferulic acid and chrysin for 4 hours, 300ng/ml LPS was added to the mixture to be treated with each group of compounds for 24 hours, the supernatant was collected, centrifuged at 12000rpm for 5 minutes to remove dead cells and cell debris, TNF. Alpha. Was detected by ELISA, and the remaining samples were stored at 4 ℃.

4) Detection was performed according to the instructions of the ELISA kit.

Tnfα ELISA kit: cargo number: 88-7024; brand: invitrogen

Conclusion of experiment: as shown in fig. 9, after the cells were treated with 10 μm of rhamnose, 10 μm of ferulic acid, 10 μm of chrysin and 10 μm of each of rhamnose, ferulic acid and chrysin, respectively, the combination of rhamnose, ferulic acid and chrysin was more pronounced than the down-regulation of tnfα by rhamnose, ferulic acid or chrysin alone, indicating that the combination of rhamnose, ferulic acid and chrysin had better anti-inflammatory effect than the combination of rhamnose, ferulic acid or chrysin alone.

Example 9

When rhamnose, ferulic acid and chrysin are used in combination, the increase of chrysin can improve anti-inflammatory (inflammatory factor IL-6) effect experimental cells: mouse microglial cell BV2

1) 24 well plate inoculation 5X 10 ⁴ Microglial cells BV2 (DMEM hg+10% fbs);

2) Attaching the wall for 24 hours;

3) The method comprises the steps of respectively adopting 10 mu M-rhamnose 10 mu M of ferulic acid, 10 mu M-rhamnose 10 mu M-chrysin 3 mu M of ferulic acid, 10 mu M-rhamnose 10 mu M-chrysin 10 mu M of ferulic acid, 10 mu M-rhamnose 10 mu M-chrysin 30 mu M of chrysin, 10 mu M of chrysin and 30 mu M of chrysin for pretreatment for 24 hours, adding 300ng/ml LPS to jointly treat with each group of compounds for 24 hours, collecting supernatant, centrifuging at 12000rpm for 5min to remove dead cells and cell fragments, detecting IL-6 by ELISA, and preserving the rest samples at 4 ℃.

4) Detection was performed according to the instructions of the ELISA kit.

IL-6ELISA kit: cargo number: 88-7064, brand: invitrogen;

Conclusion of experiment: as shown in FIG. 10, increasing the concentration of chrysin in compositions of rhamnose, ferulic acid and chrysin significantly down-regulates IL-6 expression on BV2 after treatment with ferulic acid 10. Mu.M-rhamnose 10. Mu.M, ferulic acid 10. Mu.M-rhamnose 10. Mu.M-chrysin 3. Mu.M, ferulic acid 10. Mu.M-rhamnose 10. Mu.M-chrysin 10. Mu.M, ferulic acid 10. Mu.M-rhamnose 10. Mu.M, chrysin 10. Mu.M-rhamnose 10. Mu.M, chrysin 10. Mu.M and chrysin 30. Mu.M.

Example 10

Ferulic acid, chrysin and rhamnose combined administration for improving learning and memory capacity of APP/PS1 mice

Experimental animals: 61 APP/PS1 mice of 5-6 months of age were about half of each male and female, and 11 wild type (wild type) mice were about half of each male and female. AD solvent group (APP/PS1+veh) 17, 5mg/kg group 14, 10mg/kg group 17, 20mg/kg group 13.

Experimental protocol: mice from experimental animals were divided into 5 groups (wild type (WT) solvent group, AD solvent group, 5mg/kg group (ferulic acid, rhamnose monohydrate and chrysin each 5 mg/kg), 10mg/kg group (ferulic acid, rhamnose monohydrate and chrysin each 10 mg/kg), 20mg/kg group (ferulic acid, rhamnose monohydrate and chrysin each 20 mg/kg.) the influence of different doses of ferulic acid, rhamnose and chrysin compositions on learning memory of APP 1 mice was examined by gavage APP/PS1 mice three doses of ferulic acid, rhamnose monohydrate and chrysin compositions (sterile water formulation), whereas Wild Type (WT) solvent group and AD solvent group were dosed with equal doses of sterile water after 4 months of continuous administration using Morris water maze behavioural experiments.

Conclusion of experiment: as shown in FIG. 11 (a), mice found the platform less frequently with increasing days of training, and the WT solvent group mice had a greater degree of latency reduction than the AD solvent group mice, indicating that the WT solvent group mice found the platform more rapidly. In 6 days of training, the time to first find the platform was significantly reduced from the second day for the 5mg/kg, 10mg/kg, 20mg/kg dosing group mice group compared to the AD solvent group, with significant differences maintained until day 6 of training. As shown in fig. 11 (b), in the seventh day of bench withdrawal experiments, AD mice administered with 5mg/kg, 10mg/kg, 20mg/kg of ferulic acid, rhamnose monohydrate and chrysin composition all showed a significant reduction in the time to find the bench, indicating that intervention with ferulic acid, rhamnose and chrysin composition allows APP/PS1 mice to be more rapidly located to the bench, significantly improving the learning and memory capacity of AD mice.

As shown in fig. 11 (c), the group of AD mice dosed with 5mg/kg, 10mg/kg, 20mg/kg ferulic acid, rhamnose monohydrate and chrysin composition all showed significantly more quadrant residence time of the platform relative to the AD solvent control group in the seventh day of withdrawal experiment.

The experiment shows that the three concentrations of the composition can reduce the time for reaching the platform for the first time in the water maze training process of the APP/PS1 mouse, and in the bench withdrawal experiment, compared with the AD solvent control group, the three concentrations of the composition administration group can shorten the latency time for reaching the platform, and the searching time of the quadrant where the platform is located is increased. It follows that ferulic acid, rhamnose and chrysin compositions can alleviate cognitive dysfunction in APP/PS1 model mice.

The above description of the invention and its embodiments has been given by way of illustration and not limitation, and the examples shown are merely examples of embodiments of the invention, without limitation to the actual embodiments. Therefore, if one of ordinary skill in the art is informed by this disclosure, embodiments and examples similar to the technical solution are not creatively devised without departing from the gist of the present invention, and all the embodiments and examples are considered to be within the protection scope of the present invention.

Claims

1. Use of rhamnose or its hydrates, characterised by the use in the manufacture of a medicament for the treatment or prophylaxis of neurodegenerative diseases.

2. Use of rhamnose or its hydrate according to claim 1, characterized in that it is used in the preparation of a medicament for the treatment or prevention of:

Neurodegenerative diseases characterized by reduced motor coordination; or (b)

3. Use of rhamnose or its hydrate according to claim 1, characterized in that it is used for the preparation of a medicament for the treatment or prevention of alzheimer's disease.

4. Use of rhamnose or its hydrate according to claim 1, characterized in that it is used for the preparation of a medicament for activating AMPK.

5. Use of rhamnose or its hydrate according to claim 1, characterized in that it is used for the preparation of a medicament for inhibiting phosphorylated tau protein.

6. A pharmaceutical composition for use according to any one of claim 1 to 5,

The amount of rhamnose or the hydrate thereof in the medicament or the dose to be administered of the rhamnose or the hydrate thereof contained in the medicament is as follows: 3-2000mg; or 10-2000mg; or 3-670mg; or 15-500mg; or 50-500mg;

preferably 100 to 300mg, further preferably 150 to 250mg; most preferably 200mg.

7. The medicament according to claim 6, characterized in that it comprises rhamnose and/or its hydrates; and pharmaceutically acceptable carriers or adjuvants.

8. A pharmaceutical composition according to claim 6 or 7, wherein,

the doses to be administered are:

dosage of single administration; or,

daily dosage of medication; or,

dosage of the medicine used every week.

9. A pharmaceutical composition according to any one of claims 6 to 8,

the medicament is administered once, twice or three times daily.

10. A pharmaceutical composition according to any one of claims 1 to 9,

the dosage forms of the medicine comprise powder, tablets, pills, capsules, sustained release agents, quick release agents, injections, infusion solutions or suspension agents.

11. A pharmaceutical composition according to any one of claims 1 to 10,

The medicine is a medicine which is administrated through gastrointestinal tract, or administrated through injection, or administrated through subcutaneous embedding, or preferably administrated through subcutaneous embedding.

12. A pharmaceutical composition according to any one of claims 1 to 11,

the medicine is clinically administered by gastrointestinal tract, or by injection, or by subcutaneous embedding, or preferably by subcutaneous embedding.

13. A pharmaceutical composition comprising:

a first component: rhamnose and/or its hydrates;

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition comprises,

the rhamnose comprises L-rhamnose and/or D-rhamnose:

15. The pharmaceutical composition according to claim 13 or 14, wherein,

taking the sum of the weights of the first component, the second component and the third component as the total weight; wherein,

the content of the first component is 5-90wt%;

the content of the second component is 5-90wt%;

the content of the third component is 5-90wt%.

16. The pharmaceutical composition according to any one of claims 13 to 15, wherein,

the ratio of the content of the first component, the content of the second component and the content of the third component is (1-18): (1-18): (1-18); preferably (1-8): (1-8): (1-8); further preferably (1-3): (1-3): (1-3); most preferably 1:1:1.

17. a pharmaceutical composition comprising:

a first component: rhamnose and/or its hydrates;

and a third component: chrysin and/or derivatives thereof.

18. The pharmaceutical composition of claim 17, wherein,

The content of the first component is 5-90wt%;

the content of the second component is 5-90wt%;

the content of the third component is 5-90wt%.

19. The pharmaceutical composition of claim 18, wherein,

the content of the first component is 10-80wt%;

the content of the second component is 10-80wt%;

the content of the third component is 10-80wt%.

20. The pharmaceutical composition according to any one of claims 13 to 19, wherein,

the content ratio of the first component, the content of the second component and the content of the third component is (1-18): (1-18): (1-18);

preferably, the content ratio of the first component, the content of the second component and the content of the third component is (1-8): (1-8): (1-8);

it is further preferred that the content ratio of the first component, the content of the second component and the content of the third component is (1-3): (1-3): (1-3);

more preferably, the content ratio of the first component, the content of the second component and the content of the third component is (1-2): (1-2): (1-2);

most preferably, the ratio of the content of the first component, the content of the second component and the content of the third component is 1:1:1.

21. The pharmaceutical composition according to any one of claim 13 to 20,

also comprises pharmaceutically acceptable carriers or auxiliary materials.

22. A pharmaceutical composition according to any one of claims 13 to 21, comprising

A first component: rhamnose or rhamnose hydrate;

and a second component: ferulic acid or sodium ferulate hydrate;

and a third component: chrysin; and

pharmaceutically acceptable carriers or adjuvants.

23. A pharmaceutical composition comprising

A first active ingredient: rhamnose;

a second active ingredient: ferulic acid;

third active ingredient: chrysin; and

pharmaceutically acceptable carriers or adjuvants.

24. The pharmaceutical composition according to claim 22 or 23, wherein,

taking the sum of the weights of the first active ingredient, the second active ingredient and the third active ingredient as the total weight; wherein,

the content of the first active ingredient is 5-90wt%;

the content of the second active ingredient is 5-90wt%;

the content of the third active ingredient is 5-90wt%.

25. The pharmaceutical composition according to claim 22 or 23, wherein,

The content of the first active ingredient is 10-80wt%;

the content of the second active ingredient is 10-80wt%;

the content of the third active ingredient is 10-80wt%.

26. The pharmaceutical composition according to any one of claim 13 to 25,

the content ratio of the first active ingredient, the content of the second active ingredient and the content of the third active ingredient is (1-18): (1-18): (1-18);

preferably, the ratio of the content of the first active ingredient, the content of the second active ingredient to the content of the third active ingredient is (1-8): (1-8): (1-8);

it is further preferred that the content ratio of the first active ingredient, the content of the second active ingredient to the content of the third active ingredient is (1-3): (1-3): (1-3);

more preferably, the ratio of the content of the first active ingredient, the content of the second active ingredient to the content of the third active ingredient is (1-2): (1-2): (1-2);

it is most preferable that the ratio of the content of the first active ingredient, the content of the second active ingredient to the content of the third active ingredient is 1:1:1.

27. the pharmaceutical composition according to any one of claim 13 to 26,

the pharmaceutical composition comprises:

The amount of the first active ingredient or first component or the dose to be administered is 3-2000mg;

the amount of the second active ingredient or second component or dose to be administered is 3-2000mg;

the amount of the third active ingredient or third component or dose to be administered is 3-2000mg.

28. The pharmaceutical composition according to any one of claims 13 to 27,

the pharmaceutical composition comprises:

the amount of the first active ingredient or first component or the dose to be administered is 10-2000mg;

the amount of the second active ingredient or second component or dose to be administered is 10-2000mg;

the amount of the third active ingredient or third component or dose to be administered is 10-2000mg;

preferably, the pharmaceutical composition comprises:

the amount of the first active ingredient or first component or the dose to be administered is 50-500mg;

the amount of the second active ingredient or second component or dose to be administered is 50-500mg;

the amount of the third active ingredient or third component or dose to be administered is 50-500mg.

29. The pharmaceutical composition according to any one of claims 13 to 28,

the pharmaceutical composition comprises:

the amount of the first active ingredient or first component or dose to be administered is 3-670mg;

The amount of the second active ingredient or second component or dose to be administered is 3-670mg;

the amount of the third active ingredient or third component or dose to be administered is 3-670mg;

preferably, the pharmaceutical composition comprises:

the amount of the first active ingredient or first component or the dose to be administered is 15-500mg;

the amount of the second active ingredient or second component or dose to be administered is 15-500mg;

the amount of the third active ingredient or third component or dose to be administered is 15-500mg;

further preferably, the pharmaceutical composition comprises:

the amount of the first active ingredient or first component or the dose to be administered is 100-300mg;

the amount of the second active ingredient or second component or dose to be administered is 100-300mg;

the amount of the third active ingredient or third component or dose to be administered is 100-300mg.

30. The pharmaceutical composition according to any one of claim 13 to 29,

the doses to be administered are:

dosage of single administration; or,

daily dosage of the medicine.

31. The pharmaceutical composition according to any one of claim 13 to 30,

the pharmaceutical composition is administered once, twice or three times daily.

32. The pharmaceutical composition according to any one of claims 13 to 31,

the dosage forms of the pharmaceutical composition comprise powder, tablets, pills, capsules, sustained release agents, quick release agents, injections, infusion solutions or suspension agents.

33. The pharmaceutical composition according to any one of claims 13 to 32,

the pharmaceutical composition is a medicament which is administrated through gastrointestinal alimentary canal, or an injection, or a subcutaneous embedding administration mode, or preferably a subcutaneous embedding administration mode.

34. The pharmaceutical composition according to any one of claims 13 to 33,

the pharmaceutical composition is clinically administered by gastrointestinal tract, or by injection, or by subcutaneous embedding, or preferably by subcutaneous embedding.

35. Use of a pharmaceutical composition according to any one of claims 13 to 34 for the manufacture of a medicament for the treatment or prophylaxis of neurodegenerative diseases.

36. The use of a pharmaceutical composition according to any one of claims 13 to 35, wherein the neurodegenerative disease comprises:

Neurodegenerative diseases characterized by reduced motor coordination; or (b)

37. The use of a pharmaceutical composition according to any one of claims 13 to 36, wherein the neurodegenerative disease comprises alzheimer's disease, parkinson's disease, lewy body dementia, huntington's disease, amyotrophic lateral sclerosis.

38. The use of a pharmaceutical composition according to any one of claims 13 to 37, wherein the neurodegenerative disease is alzheimer's disease.