CN112043700B

CN112043700B - Application of demethylenetetrahydroberberine hydrochloride in preparation of medicines for preventing or treating neurodegenerative diseases

Info

Publication number: CN112043700B
Application number: CN202011007191.6A
Authority: CN
Inventors: 张玉彬; 闻婧; 刘东晴; 张元强; 聂伟芬; 请求不公布姓名; 王宇航
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2023-04-11
Anticipated expiration: 2040-09-23
Also published as: CN112043700A

Abstract

The invention relates to application of demethylenetetrahydroberberine hydrochloride in preparation of a medicament for preventing or treating neurodegenerative diseases. Experiments prove that the demethylenetetrahydroberberine hydrochloride can effectively prevent and treat neurodegenerative diseases, and the new clinical application of the demethylenetetrahydroberberine hydrochloride is increased.

Description

Application of demethylenetetrahydroberberine hydrochloride in preparation of medicines for preventing or treating neurodegenerative diseases

The technical field is as follows:

the invention relates to the field of biological medicines, in particular to application of demethylenetetrahydroberberine hydrochloride in preparation of medicines for preventing and/or treating neurodegenerative diseases.

Background art:

the pathogenic factors of neurodegenerative diseases are diverse and difficult to cure, and great troubles are brought to patients and families. As the disease progresses, the neuronal structure and function of neurodegenerative disease patients are gradually lost, involving neuronal death and glial cell balance, leading to cognitive impairment such as dementia. Among the disease-inducing factors, the incidence of alzheimer's disease and parkinson's disease will increase with age.

Alzheimer's Disease (AD) is a common neurodegenerative disease, affecting nearly 5000 million people worldwide, bringing a huge burden to the medical system, and analyzing the number of AD patients according to scientific data will continue to steadily increase. How to deal with the medical pressure brought by AD becomes a serious task for medical research and development personnel at present.

Two major theories are currently involved with the pathogenesis of AD: 1. a neuritic plaque; 2. and (4) neuro-entanglement. In addition, scientists have found that various degrees of oxidative stress and neuroinflammation occur in brain tissues of AD patients, and that improvement of neuronal cell oxidative stress and inflammatory effects are probably the key to the treatment of AD.

Among neurodegenerative diseases, in addition to alzheimer's disease, parkinson's disease PD is a major concern of current research, and is a common neurodegenerative disease with the number of onset second to alzheimer's disease, and the majority of patients with the disease are elderly. With the aging of the population gradually appearing in the human society, the patients are continuously enlarged in scale and show the trend of developing towards young people, and the treatment of the disease becomes a difficult point and a key point in the current medical field.

The disease process of the Parkinson's disease is long, the treatment cost is high, and the population base of patients is large, so that the research on the mechanism of the Parkinson's disease is very important. It is known from the existing reports that the pathogenesis of the Parkinson's disease is mostly related to the reduction and loss of dopaminergic (dopamine DA), and patients often have the phenomenon of abnormal performance such as myotonia, resting tremor, slow movement and the like in the pathogenesis process.

The existing research shows that the onset of the Parkinson's disease is often greatly related to the apoptosis of dopaminergic neurons in the substantia nigra of a patient. PD has genetic susceptibility, for example, PD patients have single nucleotide polymorphism of growth factor signal transduction kinase Akt gene, and the appearance phenomena of slow action, muscle tremor and the like caused by Parkinson disease and the phenomenon of remarkable reduction of Tyrosine hydroxylase (Tyrosine hydroxylase TH) in brain can be effectively improved in the modes of anti-inflammation, anti-oxidative stress, anti-apoptosis and the like. Interestingly, researchers find that the occurrence of oxidative stress phenomena further damages DA neurons, and currently, antioxidant therapy has become an important means for treating PD and has achieved certain results in experiments and clinics.

The hydrochloric acid demethylenetetrahydroberberine is shown as a formula (I).

Desmethylenetetrahydroberberine hydrochloride (formula I) is known under the English name Demethylene tetrahydrobererine. The invention patent refers to it as DMTHB for short. The structural formula in the formula (I) is demethylenetetrahydroberberine hydrochloride, and the structural formula is further modified on the basis of the demethyleneberberine hydrochloride, so that DMTHB has stronger antioxidant activity and biological activity.

So far, no record and report about the effect of the demethylenetetrahydroberberine hydrochloride on preventing or treating neurodegenerative diseases exists in the prior art.

The invention content is as follows:

in order to overcome the defects of the prior art, the invention aims to provide a compound for preventing or treating neurodegenerative diseases, and particularly provides application of demethylenetetrahydroberberine hydrochloride (DMTHB) shown as a formula (I) as a medicament for preventing or treating the neurodegenerative diseases.

According to the invention, whether the hydrochloric acid Demethylenetetrahydroberberine (DMTHB) has a treatment effect on the Alzheimer disease is observed by establishing an Alzheimer disease animal model. Research results show that the hydrochloric acid Demethylenetetrahydroberberine (DMTHB) has preventive, protective and therapeutic effects on Alzheimer disease.

The invention researches that the demethylenetetrahydroberberine hydrochloride has a treatment effect on a neurodegenerative disease model caused by induction of 5mg/kg AlCl3+100mg/kg D-Gla by taking Kunming mice as an animal model. The hydrochloric acid demethylenetetrahydroberberine administration groups are respectively administrated for three months of intragastric administration at 50mg/kg, 100mg/kg and 150mg/kg, and the model group and each administration group are simultaneously intraperitoneally injected with 5mg/kg AlCl3+100mg/kg D-Gla for three months. After dosing was completed, behavioral experiments were performed, and after completion of the experiments, animal mice were sacrificed. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride administration group can effectively improve the cognition problem of mice no matter the dosage is high or low, reduce the damage of hippocampal neuronal cells and show that the demethylenetetrahydroberberine hydrochloride has a certain effect on treating neurodegenerative diseases. The results of animal behavioral experiments show that the escape time of mice after intraperitoneal injection of 5mg/kg AlCl3+100mg/kg D-Gla is obviously prolonged for three months, and the escape time after treatment of the hydrochloric acid demethylenetetrahydroberberine is obviously shortened. The experimental results fully indicate that the demethylenetetrahydroberberine hydrochloride can effectively prevent and treat neurodegenerative diseases.

The invention researches an Abeta pair by taking an ICR mouse as an animal model _25-35 The induced Alzheimer disease model has therapeutic effect. The administration groups of demethylenetetrahydroberberine hydrochloride are administered with 50mg/kg and 150mg/kg respectively for 7 days for intragastric administration for prevention, and after 7 days, 1mg/ml Abeta is injected intracranially in the model group and each administration group at the same time _25-35 2ul, and administration 14 days after 3 days of postoperative recovery, and after the administration, performing a behavioural experiment, and killing animal mice after the experiment is completed. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride administration group can effectively improve the cognition problem of mice, reduce the damage of hippocampal neuronal cells and show that the demethylenetetrahydroberberine hydrochloride has a certain effect on treating Alzheimer disease. The results of animal behavioral experiments show that mice are injected with 1mg/ml Abeta intracranially _25-35 After 2ul, the escape time is obviously prolonged, and the escape time is obviously shortened after the treatment of the demethylenetetrahydroberberine hydrochloride. The results of these experiments fully show that the hydrochloric acid demethylenetetrahydroberberine can effectively prevent and treat Alzheimer disease.

The invention is through H ₂ O ₂ Inducing PC12 cells to establish a cell-level oxidative damage model and carrying out DMTHB on 1mM H ₂ O ₂ Induced PC12 cell damage was treated and cellular ROS levels were detected by DCFH-DA probe and visualized by photography using a fluorescence microscope.

The invention is based on Abeta _25-35 Inducing PC12 cells to establish a cell level oxidative damage model and carrying out 1mM H-mediated oxidative damage by DMTHB ₂ O ₂ Induced PC12 cell damageWounds were treated and cellular ROS levels were detected by DCFH-DA probes and visualized by photography using a fluorescence microscope.

The invention is achieved by Abeta _25-35 + LPS-induced BV-2 cells mimic the neuroinflammation model and Abeta by DMTHB _25-35 Induced BV2 cell damage is treated. IL-1 beta and TNF-alpha expression levels were detected by qRT-PCR technology.

The invention researches that the demethylenetetrahydroberberine hydrochloride has prevention and treatment effects on a mouse subacute Parkinson disease model caused by MPTP by taking a C57BL/6 mouse as an animal model. The hydrochloric acid demethylenetetrahydroberberine administration group is respectively administered with 15mg/kg, 50mg/kg and 150mg/kg for intragastric administration for 1 week, the model group is administered with excipient with the same dose for intragastric administration for one week, then the mice are injected with MPTP (30 mg/kg) in the abdominal cavity for 5 days, the drug treatment is continued, the behavioural experiment is carried out after the administration is finished, and the animals and mice are killed after the experiment is finished. After MPTP injection, the mice have the conditions of tail straightening, feet standing and body shaking all over, and the mice in the administration group have fewer symptoms and have lighter symptoms. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride administration group can effectively reduce the phenomenon of reduction of the number of dopaminergic neurons in the substantia nigra of mice no matter the dosage is high or low, and shows that the demethylenetetrahydroberberine hydrochloride has certain effect on preventing and treating the Parkinson disease. The results of animal behavior experiments show that the movement balance and exploration capacity of the mice are reduced after MPTP injection, and the movement capacity of the mice is recovered after the treatment of the hydrochloric acid demethylenetetrahydroberberine. According to TH staining analysis, the number of TH neurons in the substantia nigra region of the brain of the mouse Parkinson model group is obviously reduced, and the substantia nigra region of the brain of the mouse Parkinson model group is recovered after the treatment of the demethylenetetrahydroberberine hydrochloride. The results of the experiments fully show that the hydrochloric acid demethylenetetrahydroberberine can effectively prevent and treat the Parkinson disease.

The invention researches that the demethylenetetrahydroberberine hydrochloride has prevention and treatment effects on the chronic mouse Parkinson disease model caused by MPTP by taking a C57BL/6 mouse as an animal model. The hydrochloric acid demethylenetetrahydroberberine administration group is respectively administered with 15mg/kg, 50mg/kg and 150mg/kg for intragastric administration and prevention for 3 days, the model group is administered with excipient with the same dose for intragastric administration for 3 days, the mice are injected with probenecid 200mg/kg intraperitoneally, MPTP (20 mg/kg) is injected into the intraperitoneally for two times a week for ten weeks, the drug treatment is continued, the ethological experiment is carried out after the administration is finished, and the animals and the mice are killed after the experiment is finished. After MPTP injection, the mice have the conditions of tail straightening, feet standing and body shaking all over, and the mice in the administration group have fewer symptoms and have lighter symptoms. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride administration group can effectively reduce the phenomenon of reduction of the number of dopaminergic neurons in the substantia nigra of mice no matter the dosage is high or low, and shows that the demethylenetetrahydroberberine hydrochloride has certain effect on preventing and treating the Parkinson disease. The results of animal behavior experiments show that the movement balance and exploration capacity of mice are reduced after MPTP injection, and the movement capacity is recovered after the treatment of the demethylenetetrahydroberberine hydrochloride. According to TH staining analysis, the number of TH neurons in the substantia nigra region of the brain of the mouse Parkinson model group is obviously reduced, and the substantia nigra region of the brain of the mouse Parkinson model group is recovered after the treatment of the demethylenetetrahydroberberine hydrochloride. The results of these experiments fully show that the demethylenetetrahydroberberine hydrochloride can effectively prevent and treat Parkinson's disease.

According to the invention, by establishing a Parkinson disease animal model, under the condition of keeping the intragastric administration dosage of 50mg/kg, the prevention and treatment effects of demethylenetetrahydroberberine hydrochloride (DMTHB) and Tetrahydroberberine Hydrochloride (THB) on subacute Parkinson disease are observed. The demethylenetetrahydroberberine hydrochloride administration group and the tetrahydroberberine hydrochloride administration group are respectively administered with 50mg/kg for intragastric administration for 1 week, the model group is administered with excipient with the same dosage for intragastric administration for one week, then MPTP (30 mg/kg) is intraperitoneally injected for 5 days, the drug treatment is continuously carried out in the period, the ethological experiment is carried out after the administration is finished, and the animal mice are killed after the experiment is finished. After MPTP injection, the mice have the conditions of tail straightening, feet standing and body shaking all over, and the mice in the administration group have fewer symptoms and have lighter symptoms. The analysis of experimental results shows that the hydrochloric acid demethylenetetrahydroberberine has certain effect on preventing and treating the Parkinson disease. The results of animal behavioral experiments show that the movement balance and exploration capacity of a mouse are reduced after MPTP injection, the movement capacity is recovered after the treatment of the demethylenetetrahydroberberine hydrochloride, and the research results show that the demethylenetetrahydroberberine hydrochloride (DMTHB) has better effects on preventing, protecting and treating the behavioral disorders caused by the Parkinson's disease compared with the Tetrahydroberberine Hydrochloride (THB). According to TH staining analysis, the number of TH neurons in the substantia nigra region of the brain of the mouse Parkinson model group is obviously reduced, and the substantia nigra region of the brain of the mouse Parkinson model group is recovered after being treated by the demethylenetetrahydroberberine hydrochloride. The results of these experiments fully show that the demethylenetetrahydroberberine hydrochloride can effectively prevent and treat Parkinson's disease.

The invention analyzes the prevention effect of the hydrochloric acid demethylenetetrahydroberberine on MPP + caused by cell injury at a cell level by an MTT method, and research results show that the improvement effect of the cell injury is obvious when the hydrochloric acid demethylenetetrahydroberberine concentration is 25 and 50 mu M.

The hydrochloric acid demethylenetetrahydroberberine product is prepared by conventional chemistry and separation and purification. The purity of the product reaches over 99 percent by adopting High Performance Liquid Chromatography (HPLC) analysis and detection in the laboratory, and the chemical method, the mass spectrometry and the nuclear magnetic resonance method are used for analysis and identification, which shows that the chemical structure of the product of the hydrochloric acid demethylenetetrahydroberberine is correct. The research shows that the purity and the chemical structure of the hydrochloric acid demethylenetetrahydroberberine meet the research requirements of developing in vivo and in vitro biological activities and pharmacological actions.

The invention also relates to a pharmaceutical composition containing the demethylenetetrahydroberberine hydrochloride as an active ingredient and conventional pharmaceutical excipients or auxiliary agents. Generally, the pharmaceutical composition of the present invention comprises 0.1 to 95% by weight of demethylenetetrahydroberberine hydrochloride. The compounds of the invention are generally present in an amount of 0.1 to 100mg in a unit dosage form.

Pharmaceutical compositions of the compounds of the present invention may be prepared according to methods well known in the art. For this purpose, the compounds of the invention can, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants and brought into a suitable administration form or dosage form for use as human or veterinary medicine.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form by enteral or parenteral routes, such as oral, intramuscular, subcutaneous, nasal, oromucosal, dermal, peritoneal or rectal administration.

The route of administration of the compounds of the invention or the pharmaceutical compositions containing them may be by injection. The injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, etc.

The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.

The compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.

For example, in order to form a unit dosage form into a tablet, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

For example, to form the dosage unit into a pill, a wide variety of carriers known in the art are used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, glycerin monostearate, kaolin, talc or the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agent such as agar powder, dried powder, alginate, sodium dodecylsulfate, methylcellulose, and ethyl cellulose.

For example, in order to encapsulate the administration units, the active ingredient, demethyleneberberine hydrochloride, a compound of the invention, is mixed with the various carriers described above and the mixture thus obtained is placed in hard gelatin capsules or soft capsules. The effective component of the compound can also be prepared into microcapsules, suspended in an aqueous medium to form a suspension, and also can be filled into hard capsules or prepared into injections for application.

For example, the compound of the invention, demethyleneberberine hydrochloride, can be prepared into injection preparations, such as solution, suspension solution, emulsion, lyophilized powder injection, which can be aqueous or non-aqueous, and can contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, dispersing agents, osmotic pressure regulators, solubilizers and pH regulators. For example, water, ethanol, polyethylene glycol, 1, 3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, fatty acid ester, etc. can be used for dilution. The osmotic pressure regulator can be sodium chloride, mannitol, glycerol, glucose, phosphate, acetate, etc.; the solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl beta-cyclodextrin, etc.; the pH regulator may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc. For example, mannitol and glucose can be added as propping agent for preparing lyophilized powder for injection.

In addition, a coloring agent, a preservative, a flavor, a corrigent, a sweetener, a flavor, or the like may be added to the pharmaceutical preparation, if necessary. These adjuvants are commonly used in the art.

The sterile media used in the present invention may be prepared by standard techniques well known to those skilled in the art. They may be sterilized, for example, by filtration through a bacteria filter, by adding a sterilizing agent to the composition, by irradiating the composition, or by sterilizing the composition by heating. They may also be prepared as sterile injectable media just prior to use.

For the purpose of administration, to increase the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method. The route of administration used to administer the compounds of the present invention will of course depend on the disease and the site in need of treatment. Because the pharmacokinetic and pharmacodynamic profiles of the compounds of the invention may vary somewhat, the most preferred method of achieving therapeutic concentrations in tissues is to gradually increase the dosage and monitor the clinical effect. For such escalating therapeutic doses, the initial dose will depend on the route of administration.

The dosage of the pharmaceutical composition of the compound of the present invention to be administered to any particular patient depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, character and individual response of the patient or animal, the route of administration, the frequency of administration, the purpose of treatment, and thus the therapeutic dosage of the present invention may vary widely. Depending on the condition of the patient to be treated, some variation in dosage may be necessary and in any case, the appropriate dosage for the individual patient is determined by the physician.

The dose administered refers to the weight of the compound excluding the weight of the carrier when used. Generally, the dosage of the pharmaceutical ingredients of the present invention used is well known to those skilled in the art. The prophylactic or therapeutic objectives of the present invention can be accomplished by appropriate adjustment of the actual amount of drug contained in the final formulation of the compound composition of the present invention to achieve the desired therapeutically effective amount. Can be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms; this is limited by the clinical experience of the administering physician and by the dosage regimen that includes the use of other therapeutic means. The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents and adjusted in dosage.

Term(s)

DMTHB: hydrochloric acid demethylenetetrahydroberberine

BBR: berberine hydrochloride

THB: hydrochloric acid tetrahydroberberine

MPTP: 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine

MPP +: 1-methyl-4-phenylpyridinium

TH: tyrosine hydroxylase

IP: abdominal injection

IG: gavage stomach

Drawings

FIG. 1 inhibition of MPP + -induced ROS in PC12 cells by demethylenetetrahydroberberine hydrochloride

FIG. 2 prevention of MPP + induced cell damage by demethylenetetrahydroberberine hydrochloride

FIG. 3 MPTP-induced pathological morphological changes of substantia nigra part of brain tissue of mouse model of chronic Parkinson's disease

Wherein A represents the Niger staining pattern (x 400) of the substantia nigra part of the brain tissue of a normal mouse, B represents the Niger staining pattern (x 400) of the substantia nigra part of the brain tissue of a model mouse with chronic Parkinson disease induced by MPTP, C represents the administration group of 5mg/kg of demethylenetetrahydroberberine hydrochloride, and D represents the administration group of 50mg/kg of demethylenetetrahydroberberine hydrochloride

FIG. 4 MPTP-induced pathological morphological changes of substantia nigra part of chronic Parkinson disease model mouse brain tissue

Wherein A represents the TH staining pattern (x 400) of the substantia nigra part of the brain tissue of a normal mouse, B represents the TH staining pattern (x 400) of the substantia nigra part of the brain tissue of a mouse model of chronic Parkinson disease induced by MPTP, C represents the administration group of 5mg/kg of demethylenetetrahydroberberine hydrochloride, and D represents the administration group of 50mg/kg of demethylenetetrahydroberberine hydrochloride

FIG. 5 shows the results of protein immunoblotting experiment of MPTP-induced chronic Parkinson disease by using demethylenetetrahydroberberine hydrochloride

FIG. 6 MPTP-induced pathological morphological changes of substantia nigra part of brain tissue of subacute Parkinson disease model mouse

Wherein A represents the Niger staining pattern (x 400) of the substantia nigra part of normal mouse brain tissue, B represents the Niger staining pattern (x 400) of the substantia nigra part of the brain tissue of a subacute Parkinson disease model mouse induced by MPTP, C represents the 50mg/kg administration group of the demethyleneberberine hydrochloride, D represents the 5mg/kg administration group of the demethylenetetrahydroberberine hydrochloride, E represents the 50mg/kg administration group of the demethylenetetrahydroberberine hydrochloride, and F represents the 15mg/kg administration group of the demethylenetetrahydroberberine hydrochloride

FIG. 7 MPTP-induced pathological morphological changes of substantia nigra part of brain tissue of subacute Parkinson disease model mouse

Wherein A represents a TH staining pattern (x 400) of the substantia nigra part of a brain tissue of a normal mouse, B represents a TH staining pattern (x 400) of the substantia nigra part of the brain tissue of a mouse model of subacute Parkinson disease induced by MPTP, C represents a 50mg/kg administration group of demethyleneberberine hydrochloride, D represents a 5mg/kg administration group of demethylenetetrahydroberberine hydrochloride, E represents a 50mg/kg administration group of demethylenetetrahydroberberine hydrochloride, and F represents a 15mg/kg administration group of demethylenetetrahydroberberine hydrochloride

FIG. 8 shows the result of experiments on protein immunoblotting of subacute Parkinson disease induced by MPTP treatment with demethylenetetrahydroberberine hydrochloride

FIG. 9 trace diagram of water maze behavior of mice induced by improvement of AlCl3+ D-Gla by demethylenetetrahydroberberine hydrochloride

Wherein A represents the behavior trace map of the normal control group on the sixth day, B represents the behavior trace map of the model group on the sixth day, C represents the behavior trace map of the low dose 50mg/kg group on the sixth day, D represents the behavior trace map of the medium dose 100mg/kg group on the sixth day, and E represents the behavior trace map of the medium dose 150mg/kg group on the sixth day

FIG. 10 quantitative graph of water maze behavior of mice induced by AlCl3+ D-Gla improved by demethylenetetrahydroberberine hydrochloride

Wherein A represents the times of each experimental group mouse crossing the platform, B represents the escape time of each experimental group mouse, C represents the swimming speed of each experimental group mouse, and D represents the staying time of each experimental group mouse in the target area

FIG. 11 AlCl3+D-Gla induced mice maze results on the first five days

Wherein A represents the escape time change of each experimental group of mice within five days, and B represents the swimming speed change of each experimental group of mice within five days

FIG. 12 AlCl3 Sc+ D-Gla induced mouse brain histopathological section map

Wherein A represents a normal control group, B represents a model group, C represents a low dose 50mg/kg group, D represents a medium dose 100mg/kg group, and E represents a high dose 150mg/kg group

FIG. 13 Abeta _25-35 Water maze quantification map of induced mice on day six

Wherein A represents the swimming speed of each experimental group of mice, B represents the times of each experimental group of mice crossing the platform, C represents the escape time of each experimental group of mice, and D represents the staying time of each experimental group of mice in the target area

FIG. 14 Abeta _25-35 Induced mice water maze results in the first five days

FIG. 15 Abeta _25-35 Induced mouse water maze behavior trace diagram

Wherein A represents the behavior trace map of the normal control group on the sixth day, B represents the behavior trace map of the model group on the sixth day, C represents the behavior trace map of the low dose 50mg/kg group on the sixth day, and D represents the behavior trace map of the medium dose 150mg/kg group on the sixth day

FIG. 16 improvement of Abeta by demethylenetetrahydroberberine hydrochloride _25-35 Induction of oxidative stress in PC12 cells

FIG. 17 improvement of H by demethylenetetrahydroberberine hydrochloride ₂ O ₂ Induction of oxidative stress profiles in PC12 cells

FIG. 18 improvement of Abeta by demethylenetetrahydroberberine hydrochloride _25-35 + LPS-induced IL-1. Beta. And TNF-. Alpha.expression levels in BV2 cells

Detailed Description

The following examples may assist those skilled in the art in a more complete understanding of the present invention, but are not intended to limit the invention in any way.

Example 1 protective Effect of cell level of Desmethylenetetrahydroberberine hydrochloride (DMTHB) on oxidative damage caused by MPP +

The method comprises the following steps: the ROS probe method is used for detecting the ROS elimination effect of the demethylenetetrahydroberberine hydrochloride on the cell level. 1-methyl-4-phenylpyridine ion MPP + (1 mM) stimulates a murine pheochromocytoma cell strain (PC 12) for 24h and then generates ROS to damage cells. In cell culture, the demethylenetetrahydroberberine hydrochloride is given for pretreatment for 6h at 12.5 mu M and 25 mu M, and the ROS scavenging action is observed under a fluorescence microscope by utilizing the property that a fluorescence probe DCFH-DA (50 mu M) is combined with ROS to generate fluorescence.

As a result: fluorescence microscopy showed that 12.5, 25 μ M DMTHB significantly inhibited the production of ROS by MPP + in PC12 cells, as shown in figure 1.

Example 2 prevention of cellular injury by MPP + at the cell level of Demethylenetetrahydroberberine hydrochloride

The method comprises the following steps: and (3) detecting the cell damage caused by MPP + by using an MTT method and a cell level detection method. Inoculating a normal mouse pheochromocytoma cell strain (PC 12) into a 96-well plate according to the cell number of 4 multiplied by 104/cm < 2 > respectively, taking out after preventing for 6h, treating the cells with MPP + (1 mM) for 24h, adding 20 mu l of MTT (5 mg/ml) into each hole, continuously incubating for 4h, taking out, completely absorbing the culture medium, adding 150 mu l of DMSO into each hole, shaking on a shaking table for 10min, and measuring the absorbance at 490 nm.

As a result: as shown in figure 2, the demethylenetetrahydroberberine hydrochloride has better prevention and protection effects on cell damage caused by MPP + at the concentrations of 25 μ M and 50 μ M.

Example 3 protective Effect of Desmethylenetetrahydroberberine hydrochloride on behavioural changes in models of Chronic Parkinson's disease

The method comprises the following steps: male C57BL/6 mice, weighing 20-25g, were randomly assigned to 5 groups of 6 mice each. The 5 groups are normal control group, model group, low dose group (5 mg/kg) of demethylenetetrahydroberberine hydrochloride, medium dose group (15 mg/kg) of demethylenetetrahydroberberine hydrochloride, and high dose group (50 mg/kg) of demethylenetetrahydroberberine hydrochloride. The administration group was administered prophylactically for 3 consecutive days, once a day, and the same volume of vehicle was administered to each of the remaining groups. After the prevention administration is finished, except for a normal control group, animals of other groups are subjected to intraperitoneal injection of probenecid at an amount of 200mg/kg, and are subjected to intraperitoneal injection of MPTP at an interval of 20mg/kg after 1h, twice a week and ten times in total, and the administration is continued during the molding period. The experimental experiment of behaviours is carried out after the last injection to test the active behavioural ability of the mice, and the behavioural experimental items comprise a rotating rod experiment, a pole climbing experiment and an open field experiment.

As a result: as shown in tables 1,2 and 3, compared with the control group, the balance ability and the exploration ability of the model group mice are obviously reduced, the exercise ability of the mice in the administration groups with various doses is improved to a certain extent, and the experimental results show that the improvement of the exercise ability of the medium-dose group (15 mg/kg) of demethylenetetrahydroberberine hydrochloride and the high-dose group (50 mg/kg) of demethylenetetrahydroberberine hydrochloride on the mice is obvious and has statistical difference.

TABLE 1 Effect of demethylenetetrahydroberberine hydrochloride on the rotating rod experiment of Parkinson model mice

(n＝8,mean±variance，#p<0.05、##p<0.01、###p<0.001、####p<0.0001VS control，*p<0.05、**p<0.01、 ***p<0.001、****p<0.0001VS model)

TABLE 2 Effect of demethylenetetrahydroberberine hydrochloride on the Pole-climbing experiment of Parkinson's model mice

(n＝10,mean±variance，#p<0.05、##p<0.01、###p<0.001、####p<0.0001VS control，*p<0.05、**p<0.01、 ***p<0.001、****p<0.0001VS model)

TABLE 3 Effect of demethylenetetrahydroberberine hydrochloride on open field experiments in Parkinson's model mice

(n＝10,mean±variance，#p<0.05、##p<0.01、###p<0.001、####p<0.0001VS control，*p<0.05、**p<0.01、***p<0.001、****p<0.0001VS model)

Example 4 protective Effect of Desmethylenetetrahydroberberine hydrochloride on brain pathological changes in model of Chronic Parkinson's disease

The method comprises the following steps: male C57BL/6 mice, weighing 20-25g, were randomly assigned to 5 groups of 6 mice each. The 5 groups are normal control group, model group, low dose group (5 mg/kg) of demethylenetetrahydroberberine hydrochloride, medium dose group (15 mg/kg) of demethylenetetrahydroberberine hydrochloride, and high dose group (50 mg/kg) of demethylenetetrahydroberberine hydrochloride. The administration group was administered prophylactically for 3 consecutive days, once a day, and the same volume of vehicle was administered to each of the remaining groups. After the prevention administration is finished, except for a normal control group, animals of other groups are subjected to intraperitoneal injection of probenecid at an amount of 200mg/kg, and are subjected to intraperitoneal injection of MPTP at an interval of 20mg/kg after 1h, twice a week and ten times in total, and the administration is continued during the molding period. The animal was sacrificed after completion of the experiment, brain tissue was taken from different mice, fixed with 10% formaldehyde, and subjected to Nissel and TH staining for pathological examination of the substantia nigra part of the brain.

As a result: pathological section research on the substantia nigra region of the mouse brain shows that the morphology of the substantia nigra region of the brain of a normal control group is not abnormal, the numbers of nissl bodies and dopaminergic neurons of an MPTP (20 mg/kg) model group are obviously reduced, and the reduction of the numbers of the nissl bodies and the dopaminergic neurons of a demethylenetetrahydroberberine hydrochloride low-dose group (5 mg/kg), a demethylenetetrahydroberberine hydrochloride medium-dose group (15 mg/kg) and a demethylenetetrahydroberberine hydrochloride high-dose group (50 mg/kg) are all alleviated, as shown in fig. 3 and 4.

Example 5 therapeutic Effect of Desmethylenetetrahydroberberine hydrochloride on models of Chronic Parkinson's disease

The method comprises the following steps: male C57BL/6 mice, weight 20-25g, randomly divided into 5 groups, each group of 6. The 5 groups are normal control group, model group, low dose group (5 mg/kg) of demethylenetetrahydroberberine hydrochloride, medium dose group (15 mg/kg) of demethylenetetrahydroberberine hydrochloride, and high dose group (50 mg/kg) of demethylenetetrahydroberberine hydrochloride. The administration group was administered prophylactically for 3 consecutive days, once a day, and the same volume of vehicle was administered to each of the remaining groups. After the prevention administration is finished, 200mg/kg probenecid is injected into abdominal cavities of other animals except a normal control group, 20mg/kg MPTP is injected into abdominal cavities after 1h interval, the administration is continued twice a week for ten times, and the administration is continued during the molding period. And (3) performing a behavioral experiment after the last injection, killing animals after the experiment is completed, taking brain tissues of different mice, extracting proteins at the substantia nigra part in the brain tissues, performing a protein immunoblotting experiment, and detecting the protein expression levels of alpha synuclein and TH in the substantia nigra brain region of the mice.

As a result: the result of a protein immunoblotting experiment on the substantia nigra protein of a mouse brain shows that the MPTP (20 mg/kg) model group has obvious alpha synuclein accumulation compared with a control group, and the abnormal accumulation phenomenon of the protein of an administration group is obviously improved. The expression level of TH protein at substantia nigra part of the model group is also obviously reduced, which indicates that dopaminergic neurons at substantia nigra part of brain are seriously injured, and the injury condition of each administration group is obviously improved, as shown in figure 5

Example 6 protective Effect of Desmethylenetetrahydroberberine hydrochloride on behavioral changes of subacute Parkinson's disease

The method comprises the following steps: male C57BL/6 mice, weighing 20-25g, were randomly assigned to 6 groups of 8 mice each. The 6 groups are normal control group, model group, tetrahydroberberine hydrochloride group (50 mg/kg), demethylenetetrahydroberberine hydrochloride low dose group (15 mg/kg), demethylenetetrahydroberberine hydrochloride medium dose group (50 mg/kg) and demethylenetetrahydroberberine hydrochloride high dose group (150 mg/kg). The administration group was continuously administered for 7 days for prophylaxis once a day, and the same volume of vehicle was administered to the remaining groups. After the preventive administration, 30mg/kg MPTP was intraperitoneally injected into the animals of each group except the normal control group once a day for five times. The experimental experiment of behaviours is carried out after the last injection to test the active behavioural ability of the mice, and the behavioural experimental items comprise a rotating rod experiment, a pole climbing experiment and an open field experiment.

As a result: as a result: as shown in tables 4, 5 and 6, compared with the control group, the balance ability and the exploration ability of the model group mice are both significantly reduced, the exercise ability of the mice of the administration groups with various doses is improved to a certain extent, and the experimental results show that the low dose group (15 mg/kg) of the demethylenetetrahydroberberine hydrochloride, the medium dose group (50 mg/kg) of the demethylenetetrahydroberberine hydrochloride and the high dose group (150 mg/kg) of the demethylenetetrahydroberberine hydrochloride can improve the exercise ability of the mice, have statistical differences, and the effects are better than those of the tetrahydroberberine hydrochloride (50 mg/kg), which indicates that the demethylenetetrahydroberberine hydrochloride has better effects on improving behavior disorders of Parkinson's disease.

TABLE 4 Effect of demethylenetetrahydroberberine hydrochloride on the rotating rod experiment of Parkinson model mice

TABLE 5 Effect of demethylenetetrahydroberberine hydrochloride on the Pole-climbing experiment of Parkinson's model mice

TABLE 6 Effect of demethylenetetrahydroberberine hydrochloride on open field experiments in Parkinson's model mice

Example 7 protective Effect of Desmethylenetetrahydroberberine hydrochloride on brain pathological changes of subacute Parkinson's disease

The method comprises the following steps: male C57BL/6 mice, weight 20-25g, randomly divided into 6 groups, each group of 8. The 6 groups are normal control group, model group, tetrahydroberberine hydrochloride group (50 mg/kg), demethylenetetrahydroberberine hydrochloride low dose group (15 mg/kg), demethylenetetrahydroberberine hydrochloride medium dose group (50 mg/kg), and demethylenetetrahydroberberine hydrochloride high dose group (150 mg/kg). The administration group was continuously administered for 7 days for prophylaxis once a day, and the same volume of vehicle was administered to the remaining groups. After the preventive administration, 30mg/kg MPTP was intraperitoneally injected into the animals of each group except the normal control group once a day for five times. The animal was sacrificed after completion of the experiment, brain tissue was taken from different mice, fixed with 10% formaldehyde, and subjected to Nissel and TH staining for pathological examination of the substantia nigra part of the brain.

As a result: pathological section research on the substantia nigra region of the mouse brain shows that the morphology of the substantia nigra region of the brain of a normal control group is not abnormal, the numbers of nissl bodies and dopaminergic neurons of an MPTP (20 mg/kg) model group are obviously reduced, and the reduction of the numbers of the nissl bodies and the dopaminergic neurons of a demethylenetetrahydroberberine hydrochloride low-dose group (5 mg/kg), a demethylenetetrahydroberberine hydrochloride medium-dose group (15 mg/kg) and a demethylenetetrahydroberberine hydrochloride high-dose group (50 mg/kg) are all alleviated, as shown in fig. 6 and 7.

Example 8 therapeutic Effect of Desmethylenetetrahydroberberine hydrochloride on subacute Parkinson disease model

Male C57BL/6 mice, weighing 20-25g, were randomly assigned to 6 groups of 8 mice each. The 6 groups are normal control group, model group, tetrahydroberberine hydrochloride group (50 mg/kg), demethylenetetrahydroberberine hydrochloride low dose group (15 mg/kg), demethylenetetrahydroberberine hydrochloride medium dose group (50 mg/kg) and demethylenetetrahydroberberine hydrochloride high dose group (150 mg/kg). The administration group was continuously administered for 7 days for prophylaxis once a day, and the same volume of vehicle was administered to the remaining groups. After the preventive administration, 30mg/kg MPTP was intraperitoneally injected into the animals of each group except the normal control group once a day for five times. And (3) performing a behavioral experiment after the last injection, killing animals after the experiment is finished, taking brain tissues of different mice, extracting proteins at the substantia nigra part in the brain tissues to perform a protein immunoblotting experiment, and detecting the expression levels of alpha synuclein and TH proteins in the substantia nigra brain region of the mice.

As a result: the result of a protein immunoblotting experiment on the substantia nigra protein of a mouse brain shows that the MPTP (20 mg/kg) model group has obvious alpha synuclein accumulation compared with a control group, and the abnormal accumulation phenomenon of the protein of an administration group is obviously improved. The expression level of TH protein at substantia nigra part of the model group is also obviously reduced, which indicates that dopaminergic neurons at substantia nigra part of brain are seriously damaged, and the damage condition of each administration group is obviously improved, as shown in figure 8.

Example 9 Demethylenetetrahydroberberine hydrochloride improves AlCl3+ D-Gla-induced behavioral changes in mice

The method comprises the following steps: male Kunming mice, weighing 20-22g, were randomly divided into 5 groups of 8 mice each. The 5 groups are normal control group, model group, low dose group (50 mg/kg) of demethylenetetrahydroberberine hydrochloride, medium dose group (100 mg/kg) of demethylenetetrahydroberberine hydrochloride, and high dose group (150 mg/kg) of demethylenetetrahydroberberine hydrochloride. Except for normal control group, animals of other groups are injected with 5mg/kg AlCl3+100mg/kg D-Gla in the abdominal cavity once a day, and each administration group is administered by gastric gavage once a day for three months. After the experiment, a water maze behavioural experiment is carried out to test the cognitive behavioural ability of the mice.

As a result: the water maze escaping time of the model group mouse is not improved after 5 days of learning, but the escaping time of the model group mouse is obviously improved after DMTHB administration treatment, the result is shown in figure 11, after the test on the 6 th day, the model group mouse cannot identify the platform position, the normal control group and each administration group memorize the platform position, the platform position is searched in the experimental process, and the ethological result is shown in figure 9 and figure 10. The combination of the above results indicates that DMTHB can improve the cognitive impairment induced by AlCl3+ D-Gla.

Example 10 protective Effect of demethylenetetrahydroberberine hydrochloride on AlCl3+ D-Gla induced pathological changes in the brain of mice

The method comprises the following steps: male Kunming mice, weighing 20-22g, were randomly divided into 5 groups of 8 mice each. The 5 groups are normal control group, model group, low dose group (50 mg/kg) of demethylenetetrahydroberberine hydrochloride, medium dose group (100 mg/kg) of demethylenetetrahydroberberine hydrochloride, and high dose group (150 mg/kg) of demethylenetetrahydroberberine hydrochloride. Except for the normal control group, animals in each group were injected with 5mg/kg of AlCl3+100mg/kg of D-Gla intraperitoneally once a day, and each administration group was administered by intragastric gavage once a day for three months. After the experiment, the animal was sacrificed, and brain tissues of different mice were fixed with 10% formaldehyde, and HE-stained to observe pathological changes.

As a result: brain tissue sections of all groups of mice are analyzed, and AlCl3+ D-Gla is found to induce the hippocampus tissues of the mice to have obvious damage and the hippocampus nerve cell morphology is irregular, as shown in figure 12, the hippocampus tissue damage of all administration groups is obviously improved, and the hippocampus nerve cell morphology is relatively regular. The result shows that the hydrochloric acid demethylenetetrahydroberberine has obvious improvement effect on AlCl3+ D-Gla induced mouse brain lesion.

Example 11 Demethylenetetrahydroberberine hydrochloride amelioration of Abeta _25-35 Induced behavioral changes in mice

The method comprises the following steps: male ICR mice, weighing 20-22g, were randomized into 4 groups of 6 mice each. The 4 groups are normal control group, model group, low dose (50 mg/kg) of demethylenetetrahydroberberine hydrochloride group, and high dose (150 mg/kg) of demethylenetetrahydroberberine hydrochloride group. Each administration group was administered for 7 days for prophylactic administration, and after 7 days, surgery was performed, wherein 2ul of physiological saline was injected intracranially in the normal group, and 2ul of 1mg/ml Abeta was injected intracranially in the model group and the administration group _25-35 . Postoperative recovery was 3 days, administration was continued for 14 days, and water maze experiments were performed after the administration was completed. To test the cognitive performance ability of the mice.

As a result: after 5 days of learning of each experimental mouse, the water maze escape time of the model group is not improved, but after DMTHB administration treatment,the escape time is obviously improved, the result is shown in fig. 14, after the test on the 6 th day, the model group mice can not identify the platform position, the normal control group and each administration group memorize the platform position, the platform position is searched in the experimental process, and the behavioral result is shown in fig. 13 and fig. 15. The combination of the above results indicates that DMTHB can improve the efficacy of the drug by Abeta _25-35 Induced cognitive impairment.

Example 12 improvement of H with demethylenetetrahydroberberine hydrochloride ₂ O ₂ Induced oxidative stress of PC12 cells

The method comprises the following steps: the PC12 cells were cultured by recovery, passaged into 6-well plates until the start of the experiment, and totally divided into four groups, each of which was: normal control group, 200uM H ₂ O ₂ Induction model group, 200uM H2O2+20uM DMTHB administration group, 200uM H ₂ O ₂ +40uM DMTHB administration group. After 24 hours, the DCFH-DA probe was used for ROS detection and visualized by photography using a fluorescence microscope.

As a result: as shown in FIG. 17, it was found that the ROS levels of the model group were significantly increased and the ROS levels of the remaining administered groups were significantly improved upon examination by fluorescence microscopy. The DMTHB can obviously improve the oxidative stress of the PC12 cells induced by H2O 2.

Example 13 Demethylenetetrahydroberberine hydrochloride amelioration of Abeta _25-35 Induced oxidative stress of PC12 cells

The method comprises the following steps: the PC12 cells were cultured by recovery, passaged into 6-well plates until the start of the experiment, and divided into four groups in total, each of which was: normal control group, 0.06uM Abeta _25-35 Induction model group, 0.06uM Abeta _25-35 +5uM DMTHB administration group, 0.06uM Abeta _25-35 +10uM DMTHB administration group. After 24 hours, the DCFH-DA probe was used for ROS detection and visualized by photography using a fluorescence microscope.

As a result: as shown in FIG. 16, it was found that the ROS levels in the model group were significantly increased and the ROS levels in the remaining groups were significantly improved upon examination by fluorescence microscopy. The result shows that DMTHB can obviously improve the oxidative stress of the PC12 cells induced by H2O 2.

Example 14 Demethylenetetrahydroberberine hydrochloride ameliorationAbeta _25-35 + LPS-induced neuroinflammation mimicked by BV2 cells

The method comprises the following steps: recovering and culturing BV2 cells, subculturing the BV2 cells into a 6-well plate until the beginning of an experiment, totally dividing the BV2 cells into 3 groups, namely: normal control group, 0.06uM Abeta _25-35 +200ng/ml LPS Induction model group, 0.06uM Abeta _25-35 +200ng/ml LPS +10uM DMTHB administration group. After 24 hours of administration, the expression levels of IL-1 beta and TNF-alpha were measured by qRT-PCR to determine the effect of improving inflammation.

As a result: as shown in FIG. 18, the IL-1. Beta. And TNF-. Alpha. Expression levels in the model group were improved after administration, indicating that DMTHB was significantly improved by Abeta _25-35 + LPS-induced neuroinflammation mimicked by BV2 cells.

Claims

1. Application of demethylenetetrahydroberberine hydrochloride in preparing medicine for preventing or treating neurodegenerative diseases, wherein demethylenetetrahydroberberine hydrochloride is

；

The neurodegenerative disease is Alzheimer disease and Parkinson disease induced by Abeta protein.

2. Use according to claim 1, characterized in that: the demethylenetetrahydroberberine also comprises phosphate or sulfate of demethylenetetrahydroberberine.