WO2017128291A1 - 小檗碱用于预防和治疗阿尔茨海默病 - Google Patents

小檗碱用于预防和治疗阿尔茨海默病 Download PDF

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WO2017128291A1
WO2017128291A1 PCT/CN2016/072763 CN2016072763W WO2017128291A1 WO 2017128291 A1 WO2017128291 A1 WO 2017128291A1 CN 2016072763 W CN2016072763 W CN 2016072763W WO 2017128291 A1 WO2017128291 A1 WO 2017128291A1
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berberine
medicament
protein
disease
alzheimer
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French (fr)
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郭毅
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深圳市人民医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines

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  • the invention relates to the technical field of medicines, in particular to the technical field of medicine for preventing and treating Alzheimer's disease, in particular to a new application of berberine and a medicament for preventing and treating Alzheimer's disease.
  • AD Alzheimer's disease
  • the incidence rate is about 10% in the 65-year-old population, and can be as high as 50% in the 85-year-old population.
  • the situation in China is even more serious.
  • AD will become the first killer of the elderly. Due to the high disability rate of AD, it poses a serious hazard to human health.
  • the medical and care of AD also consumes a lot of manpower and financial resources. Therefore, AD has become the No. 4 killer, which seriously affects human health except cardiovascular and cerebrovascular diseases, diabetes and cancer, and has become a difficult social and medical problem.
  • the pathological features of AD mainly include two aspects: one is the formation of senile plaques (SP) with b-amyloid (A ⁇ ) deposition as the core, and SP is the abnormal degradation of A ⁇ precursor protein (APP).
  • SP senile plaques
  • a ⁇ b-amyloid
  • APP A ⁇ precursor protein
  • the insoluble A ⁇ 42 plaque is toxic to neurons; on the other hand, it is the neurofibrillary tangles (NFT) formed by hyperphosphorylation of Tau protein in nerve cells.
  • NFT neurofibrillary tangles
  • the main drugs commonly used in clinical practice are as follows: the drugs that affect the choline system (including cholinesterase inhibitors, Ach). Receptor agonist); cerebral blood circulation improver; brain metabolism activator; correct calcium homeostasis, anti-oxidation, anti-inflammatory drugs; neurotrophic factors.
  • These drugs have many disadvantages such as inaccurate curative effect or large toxic side effects, poor oral absorption, and they are all based on the corresponding pathogenesis hypothesis. They are some methods of "treating the symptoms" and cannot effectively prevent the rapid development of AD.
  • the lipid-lowering drugs and hormonal substances that are in the clinical research and have the effect of preventing and treating AD are not expected to have good application prospects because of the lack of specificity or difficulty in penetrating the blood-brain barrier.
  • Berberine also known as berberine, is a natural isoquinoline alkaloid extracted from the traditional Chinese medicine Coptidis Rhizoma. It has a wide range of pharmacological effects, including anti-inflammatory, antibacterial, anti-oxidative stress, and inhibition. Cholinesterase activity and neuroprotective effects on brain tissue ischemia-reperfusion injury. Berberine has an important protective effect on the central nervous system.
  • the present invention provides a novel application of berberine in the treatment and/or prevention of Alzheimer's disease and a medicament for controlling Alzheimer's disease.
  • the present invention provides a use of berberine for the preparation of a medicament for the prevention and treatment of Alzheimer's disease.
  • the drug in the above application includes at least berberine as an active ingredient of the drug.
  • the medicament in the above application further comprises a pharmaceutically acceptable carrier.
  • the medicament in the above application comprises berberine and a pharmaceutically acceptable carrier.
  • the drug in the above application is formulated into a tablet, an injection, a capsule, a granule, a pill, a pellet, a powder, a pill, a decoction, a syrup, a mixture, a decoction or an extract.
  • each of the drugs in the above application contains 0.2 to 1 gram of berberine.
  • the present invention provides a medicament for controlling Alzheimer's disease, which comprises at least berberine as an active ingredient of a medicament.
  • the above medicament further comprises a pharmaceutically acceptable carrier.
  • the above-mentioned medicament is formulated into a tablet, an injection, a capsule, a granule, a pill, a pellet, a powder, a pill, a decoction, a syrup, a mixture, an ointment or an extract.
  • each of the above-mentioned drugs contains 0.2 to 1 gram of berberine.
  • the present invention provides the use of berberine in the treatment and/or prevention of Alzheimer's disease and a medicament for the prevention and treatment of Alzheimer's disease.
  • berberine is used for the prevention and treatment of AD diseases, and the prophylactic and therapeutic effects of berberine on AD are verified at two levels of primary nerve cells and AD model mice. Accordingly, the present invention provides a novel use of berberine for the preparation of a medicament for the prevention and treatment of Alzheimer's disease.
  • Figure 1 is a graph showing the results of berberine promoting the growth of primary nerve cells in Example 1 of the present invention.
  • Figure 2 is a graph showing the results of berberine inhibiting the level of tau protein phosphorylation (PS404) in primary neurons in Example 1 of the present invention.
  • Fig. 3 is a result of immunoblotting experiment in which berberine inhibits the level of A ⁇ protein in primary nerve cells in Example 2 of the present invention.
  • Fig. 4 is a quantitative result of inhibition of primary neuronal A ⁇ protein levels by berberine in Example 2 of the present invention, and the ordinate indicates the expression level of A ⁇ protein.
  • Fig. 5 is a result of immunoblotting experiment in which berberine inhibits the level of APP protein in primary nerve cells in Example 2 of the present invention.
  • Fig. 6 is a quantitative result of inhibiting the level of APP protein in primary nerve cells by berberine in Example 2 of the present invention, and the ordinate indicates the expression level of APP protein.
  • Figure 7 is a result of immunoblotting experiments in which berberine inhibits the level of tau phosphorylation (PS422) in primary neural cells in Example 2 of the present invention.
  • Figure 8 is a quantitative result of berberine inhibiting the level of tau phosphorylation (PS422) in primary neurons in Example 2 of the present invention, and the ordinate indicates the expression level of P-tau422 protein.
  • Figure 9 is a result of immunoblot assay for inhibition of primary neuronal tau phosphorylation (PS396) levels by berberine in Example 2 of the present invention.
  • Figure 10 is a quantitative result of inhibition of primary neuron tau phosphorylation (PS396) levels by berberine in Example 2 of the present invention, and the ordinate indicates the expression level of P-tau396 protein.
  • Figure 11 is a graph showing the results of berberine in improving the spatial exploration and memory ability of AD model mice in Example 3 of the present invention.
  • the present invention provides a novel application of berberine and a medicament for controlling Alzheimer's disease.
  • the present invention will be further described in detail below. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • Berberine also known as berberine hydrochloride, is a natural isoquinoline alkaloid extracted from Rhizoma Coptidis. It has a variety of pharmacological effects, including anti-inflammatory, anti-cancer, anti-viral, antibacterial and hypoglycemic effects. , lipid regulation, etc. Berberine has been used as an anti-inflammatory drug for research and development in the past.
  • berberine is used for the prevention and development of AD diseases.
  • the prophylactic and therapeutic effects of berberine on AD were verified at both primary and AD model mice.
  • Berberine reduces A ⁇ protein levels; (2) berberine reduces APP protein levels; (3) berberine reduces phosphorylated tau422 protein levels; (5) berberine reduces phosphorylated tau396 protein levels; 5) Berberine improves the learning and memory ability of mice. Therefore, berberine can inhibit the occurrence and development of AD at multiple targets, and is a stable, safe and effective drug for the treatment of AD. Accordingly, one aspect of the present invention provides a novel use of berberine for the preparation of a medicament for the prevention and treatment of Alzheimer's disease; and a medicament for the prevention and treatment of Alzheimer's disease.
  • berberine has the effect of preventing Alzheimer's disease
  • control refers to treatment and/or prevention, and broadly includes any means for controlling a condition, such as prevention and cure. And relieve the condition, alleviate the condition and stop its progression.
  • the treatment can be performed after Alzheimer's disease has developed.
  • prevention can be performed asymptomatically. For example, prevention can be performed prior to the onset of symptoms in susceptible individuals (eg, considering the history of symptoms and/or considering genetic factors or other susceptibility factors). It is also possible to continue treatment after the symptoms have been overcome to prevent or delay their recurrence.
  • berberine is an active ingredient of a drug, that is, an active ingredient effective for Alzheimer's disease, but does not exclude the presence of other active ingredients, such as other pairs of Alzheim.
  • a pharmaceutically acceptable carrier is also included.
  • Such a carrier refers to a non-toxic carrier, adjuvant or vehicle which does not destroy the pharmacological activity of the compound (i.e., berberine) with which it is formulated.
  • Pharmaceutically acceptable carriers, adjuvants or vehicles which may be used in the medicament of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances Such as phosphates, glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated plant fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc Salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based materials, polyethylene glycol, cyclodextrin, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene - Polyoxypropylene-block copolymers, polyethylene glycol and lanolin.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum album
  • the "drug” in the present invention is understood to be a substance which contains at least berberine and has an effect (ie, treatment and/or prevention) against Alzheimer's disease.
  • the active ingredient berberine and the carrier may be mixed in any ratio, and are not particularly limited as long as the treatment of Alzheimer's disease is exerted. Or the preventive effect is sufficient, and can be appropriately determined according to the daily intake of the active ingredient, and the amount of the active ingredient is preferably 0.0005 to 100% by mass, more preferably, based on the total mass of the drug. It is selected from 0.005 to 90% by mass, and particularly preferably from 0.05 to 80% by mass.
  • a pharmaceutically active ingredient together with a pharmaceutically acceptable carrier into a suitable dosage form is well known to those skilled in the art of pharmaceutical technology, and thus the berberines mentioned in the present invention can be prepared according to a known method for preparing a drug.
  • the carrier is made into a finished drug that can be taken.
  • the medicament of the present invention can be prepared into various dosage forms, for example, into tablets, injections, capsules, granules, pills, pellets, powders, pills, decoctions, syrups, mixtures, ointments or extracts, etc. .
  • the content of berberine in each dosage form can be determined according to the therapeutic requirements, for example, 0.01 to 10 g of berberine per dosage form, preferably 0.1 to 2 g of berberine per dosage form, and most preferably 0.2 to 2 per dosage form. 1 gram of berberine.
  • the medicament of the present invention can be administered by various administration methods as long as the therapeutic or preventive effect of berberine on Alzheimer's disease can be ensured, for example, oral administration, injection, transdermal absorption and the like.
  • the dose to be administered and the administration time of the drug are not particularly limited, and may be appropriately selected depending on the age of the patient, the severity of the patient's symptoms, and other conditions.
  • Example 1 Immunofluorescence was used to detect the effect of berberine on the growth of primary neural cells and the effect on the phosphorylation of tau protein (PS404).
  • the primary neural cells used in the present invention were obtained from a 3 ⁇ Tg AD model mouse, which was transformed into three AD-related genes, APPswe, PS1M146V and TauP301L, and exhibited typical AD pathological features.
  • the epithelial tissue was obtained from the brain of 3 ⁇ Tg AD fetal rats born one day, and the primary nerve cells were obtained by papain digestion and boiled in 24-well plates with cell slides. After the seventh day of cell culture, the cells were treated with berberine at a final concentration of 1 ⁇ M for 24 hours, and then subjected to immunofluorescence. Primary nerve cells treated with berberine under the same culture conditions were used as a control group.
  • the blocking solution is blocked for more than 1 h, 300 ⁇ L per well, and placed on a refrigerator/ice at 4 ° C;
  • the nucleus was stained with DAPI (4',6-diamidino-2-phenylindole), and the neural cytoskeleton was stained with anti-MAP-2 antibody.
  • DAPI 4,',6-diamidino-2-phenylindole
  • the results are shown in Figure 1.
  • the left panel shows the blue channel and only shows DAPI.
  • the middle picture is the red channel, only MAP-2 is shown; the right picture is the top two pictures superimposed), as can be seen from Figure 1, the primary nerve cells treated with berberine, the cell filaments are longer, indicating small Scopolamine promotes the growth of nerve cells and the development of neurofilaments.
  • microtubule-associated protein tau Abnormal structural dysfunction of the microtubule-associated protein tau is an important factor in the induction of Alzheimer's disease.
  • the microtubule system is a major component of the neural cytoskeleton and plays an important role in maintaining cell function.
  • Microtubules are composed of ⁇ , ⁇ tubulin and microtubule-associated proteins.
  • Tau protein is a microtubule-associated protein that binds to microtubules and maintains microtubule stability. In the case of AD, tau is hyperphosphorylated and binds to other tau protein fibers. This excessive binding eventually leads to the formation of neurofibrillary tangles in the cell bodies of the nerve cells, which in turn leads to the collapse of the microtubule system of the nerve cells.
  • Example 2 Western blot analysis of the effect of berberine on the expression levels of various AD-related proteins in primary neurons
  • Western blotting is Western Blot. It is an experimental method commonly used in molecular biology, biochemistry and immunogenetics. The basic principle is to color a gel-electrophoresis-treated cell or biological tissue sample by a specific antibody. Information on the expression of a particular protein in the cells or tissues analyzed is obtained by analyzing the location of the staining and the depth of staining. It is generally composed of three parts: gel electrophoresis, sample imprinting and immunological detection. The first step is to perform SDS polyacrylamide gel electrophoresis, so that the protein in the sample to be tested is divided into bands in the gel according to the molecular weight. In the second step, the protein that has been separated into bands in the gel is transferred to a solid support.
  • the most used materials are nitrocellulose membrane (NC membrane) and PVDF membrane.
  • the method of protein transfer is mostly electrophoretic transfer. There are also semi-dry and wet methods, and most of them are now wet.
  • the third step is to detect the corresponding antigen to be studied that has been blotted on the membrane with a specific antibody.
  • Methods of immunoassay can be direct and indirect. Now using the indirect immunolabeling method, after hybridization with a specific first antibody, the second antibody (alkaline phosphatase (AP) or horseradish peroxidase (HRP)-labeled anti-first is used.
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • the anti-antibody hybridizes to the substrate, and the substrate of the added enzyme shows either the presence of the antigen by the color on the film or the strip exposed on the X-ray film. This technique is widely used in the detection of protein expression levels. Specific steps are as follows:
  • the corresponding protein content ( ⁇ g) of the sample to be tested can be found, and then divided by the total volume of the sample diluent (20 ⁇ L), multiplied by the dilution factor of the sample to obtain the protein sample. Actual concentration ( ⁇ g/ ⁇ L).
  • Preparation before gel preparation Calculate the concentration of the gel according to the molecular weight of the original antigen corresponding to the antibody to be detected, and calculate the amount of each component of the gel (Table 2).
  • the general electrophoresis time is about 1.5 hours.
  • the inner tank must use the newly configured 1 ⁇ SDS electrophoresis fluid, and the outer tank can be recycled.
  • the gel plate is immersed in the running buffer for a few minutes.
  • the buffer tank was placed in an ice box, and the electrophoresis transfer buffer precooled at 4 ° C was filled in the electrophoresis tank.
  • the transfer electrode was connected to a constant current of 100 mA for 1.5 h.
  • the post-transfer gel was stained with Coomassie brilliant blue for 30 min-1 h, and after decolorization to decolorization without background color, it was observed whether there was any residual protein band on the gel for detecting the effect of the transfer film. Or stain the PVDF membrane with Li Chunhong, observe the film strip and wash off the Ponceau before blocking.
  • 5% skim milk (1 x TBST dissolved) was prepared before the end of the transfer. After the transfer, the membrane was placed in 5% skim milk for 2 h, and placed at 37 ° C on a horizontal shaker at appropriate speed for closure. Note: The skimmed milk powder solution should be sufficient to cover the membrane and try to avoid contamination.
  • the membrane was taken out and placed in a clean dish, washed with 1 ⁇ TBST at a suitable speed of the shaker for about 15 minutes, and changed for 3-5 times.
  • the washing is to wash away the non-specific binding of the primary antibody to the antigen, and the effect of the washing directly affects whether the result has a band.
  • the dilution ratio of the secondary antibody should not be too high, otherwise it will easily lead to non-specific binding. According to the experimental needs and design, select the appropriate enzyme standard secondary antibody and dilution concentration (5% BSA dilution), after the completion of washing, add the secondary antibody 3ml at room temperature for 2h on the shaker, taking care to ensure that all parts of the membrane are in contact with the solution.
  • Discard the secondary antibody place the strip in a clean Petri dish, add 1 ⁇ TBST, wash on a shaker, wash for about 15 minutes, change the solution, repeat 3 times.
  • the washing is to remove the non-specific binding of the secondary antibody, and the washing effect directly affects the depth of the background of the result, so the washing must be clean.
  • the X-ray film into a suitable size in the dark room, cover the PVDF film, clip the clip, and expose the film.
  • the exposure time is determined according to the fluorescence brightness of the protein, or it can be pressed several times at different times.
  • the gray value analysis of the images was performed using the quantity one optical density analysis software, and the t-test between the groups was performed.
  • the data analysis was performed by Graphpad software, and p ⁇ 0.05 was statistically significant.
  • AD Alzheimer's disease
  • SP amyloid deposits in the brain
  • senil plaque The core component of senile plaques is a small neuropeptide consisting of 39-43 amino acid residues - ⁇ -amyloid peptide, which is passed from ⁇ -secretase and ⁇ -amyloid precursor protein (APP).
  • APP ⁇ -amyloid precursor protein
  • the secretase is hydrolyzed and secreted outside the cell.
  • Western blot results showed that (Fig. 3-6), after treatment with berberine, the levels of A ⁇ and APP protein in primary neurons were significantly decreased, indicating that berberine can inhibit the expression of A ⁇ and APP or have a certain ability to scavenge it. .
  • tau hyperphosphorylation is an important factor in the occurrence and development of AD.
  • Western blotting showed (Fig. 7-10) that berberine significantly inhibited the phosphorylation levels of the tau protein PS422 and PS396 sites in primary neurons.
  • berberine can promote the growth of primary nerve cells, reduce the expression of A ⁇ and hyperphosphorylation of tau protein, thus inhibiting the formation of senile plaques and neurofibrillary tangles, thereby inhibiting the development of AD. .
  • Example 3 Morris water maze test for the effect of berberine on spatial exploration and memory in AD model mice
  • One of the subjects of the experiment in this example is a three-transgenic mouse (3 ⁇ TG Deer Mice containing the Psen1 mutant gene, and the APPSwe and tauP301L susceptibility genes (Tg(APPSwe, tauP301L)1Lfa)), which have fertility and no A physiological feature of congenital diseases and behavioral abnormalities that are capable of overexpressing the transferred genes, particularly in the AD-related areas, the hippocampus, and the cerebral cortex, resulting in behavioral abnormalities confined to the central nervous system.
  • intracellular A ⁇ deposition began to appear in 3-4 months, and obvious senile plaques were seen in 6 months, and synaptic transmission impaired at 6 months, and after 6 months.
  • the hippocampus and subsequent cerebral cortical tangles are visible.
  • mice divided into three groups, one group was 6-month-old wild-type mice, one group was 6-month-old three-transgenic AD mice not bred with berberine, and one group was small at 50 mg/Kg/day. 6-month-old three-transgenic AD mice fed by purine.
  • mice were selected from each group for testing and placed in different breeding cages.
  • the second day to the fifth day of the navigation is the detection period: water is entered into the water in a certain quadrant order every day, and four rounds are recorded every day, and relevant data is recorded.
  • Space exploration is divided into 24-hour memory and 72-hour memory: it is timed from the end of the positioning navigation, the platform is removed, and the number of times the mouse stays in the quadrant of the original platform and the number of times across the original platform are recorded.
  • the escape latency of the AD model mice was significantly increased in the four quadrants compared to the wild type mice, whereas the AD model mice fed berberine were in the escape latency of the four quadrants.
  • the mice were significantly lower than the untreated AD model mice, indicating that berberine can significantly improve the learning and memory ability of AD model mice.
  • the present invention verifies the prophylactic and therapeutic effects of berberine on AD at two levels of primary nerve cells and AD model mice: (1) berberine reduces the protein level of A ⁇ ; (2) Berberine reduces the protein level of APP; (3) berberine reduces the protein level of phosphorylated tau422; (4) berberine reduces the protein level of phosphorylated tau396; (5) berberine improves learning and memory ability in mice . Therefore, berberine can inhibit the occurrence and development of AD at multiple targets, and can be used as a stable, safe and effective treatment for AD.

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Abstract

小檗碱在制备预防和/或治疗阿尔茨海默病药物中的应用。小檗碱可降低Aβ、APP、磷酸化tau422和磷酸化tau396的蛋白水平，并且可提高小鼠的学习记忆能力，可多靶点起效用于预防和/或治疗阿尔茨海默病。

Description

小檗碱的新应用及用于防治阿尔茨海默病的药物 技术领域
本发明涉及药物技术领域，尤其涉及防治阿尔茨海默病的药物技术领域，特别涉及小檗碱的新应用及用于防治阿尔茨海默病的药物。
背景技术
阿尔茨海默症(Alzheimer's disease,AD)是一组病因尚未明确的原发性退行性脑变性疾病。目前全球已经有超过3500万的AD患者。随着社会人口老龄化逐步加剧，AD发病率呈逐渐上升趋势，65岁人群中发病率大约为10％，而85岁人群中这一比例可高达50％。中国这方面情况更为严重，目前60岁以上的人口已达1.78亿，预测到2050年将达到4.37亿，届时AD将成为老年人的第一杀手。由于AD的致残率较高，对人类健康造成严重危害。同时，对AD的医疗和照料还耗费了大量人力和财力，因此AD已成为除心脑血管病、糖尿病、癌症外严重影响人类健康的第4号杀手，成为一个棘手的社会和医疗卫生问题。
AD的病理特征主要包括两方面：一是神经细胞外以b-淀粉样蛋白(Aβ)沉积为核心形成老年斑(senile plaques，SP)，SP是Aβ前体(amyloid precursor protein,APP)异常降解形成的不溶解性Aβ42斑块，对神经元有毒性作用；另一方面则是神经细胞内Tau蛋白过度磷酸化形成的神经纤维缠结(NFT)。AD的发病机理十分复杂，可能是多种因素相互作用的结果，迄今为止其确切的发病机制尚不清楚。
近年来对AD的治疗研究已取得一定的进展，研发出不少新的药物，归纳目前临床上常用的药物主要有以下几类：影响胆碱系统功能药物(包括胆碱酯酶抑制剂、Ach受体激动剂)；脑血循环改善剂；脑代谢激活剂；纠正钙稳态失调、抗氧化、抗炎药物；神经营养因子等。这些药物多存在疗效不确切或毒副作用大、口服吸收差的缺点，并且它们均是依据相应发病机理假说而进行的尝试，都是一些“治标”的方法，不能有效防止AD的迅速发展。正处于临床研究中的具有防治AD作用的降脂药、激素类物质则由于特异性不强或不易透过血脑屏障而预测不到良好的应用前景。
小檗碱(Berberine，BBR)又称黄连素，是从中药黄连中提取的一种天然的异喹啉类生物碱，具有广泛的药理作用，包括抗炎、抑菌、抗氧化应激、抑 制胆碱酯酶活性、及对脑组织缺血再灌注损伤的神经保护作用等。小檗碱对中枢神经系统具有重要的保护作用。
发明内容
本发明提供一种小檗碱在治疗和/或预防阿尔茨海默病中的新应用及用于防治阿尔茨海默病的药物。
根据本发明的第一方面，本发明提供一种小檗碱在制备防治阿尔茨海默病的药物中的应用。
作为本发明的优选方案，上述应用中的药物至少包括小檗碱作为药物的有效成分。
作为本发明的优选方案，上述应用中的药物还包括药学上可接受的载体。
作为本发明的优选方案，上述应用中的药物包括小檗碱和药学上可接受的载体。
作为本发明的优选方案，上述应用中的药物制成片剂、注射剂、胶囊剂、颗粒剂、丸剂、微丸、散剂、滴丸剂、汤剂、糖浆剂、合剂、煎膏剂或浸膏剂剂型。
作为本发明的优选方案，上述应用中的药物的每种剂型含0.2～1克小檗碱。
根据本发明的第二方面，本发明提供一种用于防治阿尔茨海默病的药物，该药物至少包括小檗碱作为药物的有效成分。
作为本发明的优选方案，上述药物还包括药学上可接受的载体。
作为本发明的优选方案，上述药物制成片剂、注射剂、胶囊剂、颗粒剂、丸剂、微丸、散剂、滴丸剂、汤剂、糖浆剂、合剂、煎膏剂或浸膏剂剂型。
作为本发明的优选方案，上述药物的每种剂型含0.2～1克小檗碱。
本发明提供小檗碱在治疗和/或预防阿尔茨海默病中的应用及用于防治阿尔茨海默病的药物。在本发明中，将小檗碱用于防治AD疾病的开发与研究，并在原代神经细胞和AD模型鼠两个水平上验证了小檗碱对AD的预防和治疗效果。因此，本发明提供小檗碱的新应用，将小檗碱用于制备防治阿尔茨海默病的药物。
附图说明
图1是本发明实施例1中小檗碱促进原代神经细胞生长的结果。
图2是本发明实施例1中小檗碱抑制原代神经细胞tau蛋白磷酸化(PS404)水平的结果。
图3是本发明实施例2中小檗碱抑制原代神经细胞Aβ蛋白水平的免疫印迹实验结果。
图4是本发明实施例2中小檗碱抑制原代神经细胞Aβ蛋白水平的定量结果，纵坐标表示Aβ蛋白表达水平。
图5是本发明实施例2中小檗碱抑制原代神经细胞APP蛋白水平的免疫印迹实验结果。
图6是本发明实施例2中小檗碱抑制原代神经细胞APP蛋白水平的定量结果，纵坐标表示APP蛋白表达水平。
图7是本发明实施例2中小檗碱抑制原代神经细胞tau蛋白磷酸化(PS422)水平的免疫印迹实验结果。
图8是本发明实施例2中小檗碱抑制原代神经细胞tau蛋白磷酸化(PS422)水平的定量结果，纵坐标表示P-tau422蛋白表达水平。
图9是本发明实施例2中小檗碱抑制原代神经细胞tau蛋白磷酸化(PS396)水平的免疫印迹实验结果。
图10是本发明实施例2中小檗碱抑制原代神经细胞tau蛋白磷酸化(PS396)水平的定量结果，纵坐标表示P-tau396蛋白表达水平。
图11是本发明实施例3中小檗碱提高AD模型小鼠的空间探索和记忆能力的结果。
具体实施方式
本发明提供小檗碱的新应用及用于防治阿尔茨海默病的药物，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
AD作为公认的发病率及危害性最高的老年性疾病之一，目前尚无有效的治疗药物。黄连素(berberine)又称盐酸小檗碱，是从黄连中提取的一种天然的异喹啉类生物碱，具有多种药理作用，包括抗炎、抗癌、抗病毒、抑菌及降糖、调脂等。小檗碱在过去一直作为抗炎药物作为研究以及开发。
而本发明中，则将小檗碱用于防治AD疾病的开发与研究。在原代神经细胞和AD模型鼠两个水平上验证了小檗碱对AD的预防和治疗效果。发现：(1) 小檗碱降低Aβ的蛋白水平；(2)小檗碱降低APP的蛋白水平；(3)小檗碱降低磷酸化tau422的蛋白水平；(5)小檗碱降低磷酸化tau396的蛋白水平；(5)小檗碱提高小鼠的学习记忆能力。因此，小檗碱可在多靶点抑制AD的发生和发展，是一种稳定、安全、有效的AD治疗药物。因此，本发明一方面提供一种小檗碱的新应用，将小檗碱用于制备防治阿尔茨海默病的药物；另一方面还提供一种用于防治阿尔茨海默病的药物。
在本发明的应用和/或药物中，小檗碱具有防治阿尔茨海默病的作用，所谓“防治”是指治疗和/或预防，广义上包括了控制病症的任何手段，例如预防、治愈和缓解该病症，减轻病症和阻停其进展。在某些实施方案中，可以在阿尔茨海默病已经形成之后实施治疗。在其它实施方案中，可以在无症状时实施预防。例如，可以在易发个体发生症状之前实施预防(例如，考虑症状的历史和/或考虑遗传因素或其它易感因素)。还可以在已经克服症状之后继续治疗，以预防或延迟其复发。
在本发明的应用和/或药物中，小檗碱是药物的有效成分，即对阿尔茨海默病具有疗效的活性成分，但并不排除其它有效成分的存在，例如其它的对阿尔茨海默病具有疗效的活性成分，只要不发生对小檗碱药效的抑制作用即可。此外，还包括药学上可接受的载体。这样的载体是指无毒的载体、佐剂或媒介物，其不会破坏与其一起配制的化合物(即小檗碱)的药理学活性。可用于本发明的药物中的药学上可接受的载体、佐剂或媒介物包括但不限于：离子交换剂，氧化铝，硬脂酸铝，卵磷脂，血清蛋白例如人血清白蛋白，缓冲物质如磷酸盐类，甘氨酸，山梨酸，山梨酸钾，饱和植物脂肪酸的偏甘油酯混合物，水，盐类或电解质类例如硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐，胶体二氧化硅，三硅酸镁，聚乙烯吡咯烷酮，基于纤维素的物质，聚乙二醇，环糊精类，羧甲基纤维素钠，聚丙烯酸酯类，蜡类，聚乙烯-聚氧丙烯-嵌段共聚物，聚乙二醇和羊毛脂等。
基于以上说明，本发明中的“药物”应当理解为至少包含小檗碱，同时对阿尔茨海默病具有疗效(即治疗和/或预防)的物质。
在本发明的应用和/或药物中，在包括药学上可接受的载体的情况下，有效成分小檗碱与载体可以按照任意比例混合，没有特别限制，只要发挥对阿尔茨海默病的治疗或预防效果即可，并且可以根据每天优选的有效成分摄入量适当确定，以药物的总质量计，该有效成分的量优选为0.0005至100质量％，更优 选为0.005至90质量％，并且特别优选为0.05至80质量％。将药物有效成分与药学上可接受的载体一起制成适当的剂型的技术，是药物技术领域的技术人员公知的，因此可以按照公知的制备药物的方法将本发明中提到的小檗碱与载体制成可服用的成品药物。
本发明的药物可以制成多种剂型，例如制成片剂、注射剂、胶囊剂、颗粒剂、丸剂、微丸、散剂、滴丸剂、汤剂、糖浆剂、合剂、煎膏剂或浸膏剂剂型等。每种剂型中小檗碱的含量可以根据疗效需求确定，例如每种剂型中含0.01～10克小檗碱，优选每种剂型中含0.1～2克小檗碱，最优选每种剂型含0.2～1克小檗碱。相应地，本发明的药物可以通过各种不同的服用方式服用，只要能够保证小檗碱对阿尔茨海默病的治疗或预防效果即可，例如口服、注射、经皮吸收等。
本发明中，对于药物的给药剂量和给药时间没有特别限制，可以是根据患者年龄、患者症状的严重性以及其他条件等适当选择。
以下通过实施例对本发明做进一步说明。
实施例1以免疫荧光检测小檗碱对原代神经细胞生长的影响以及对tau蛋白磷酸化(PS404)水平的影响
a.原代细胞培养
本发明中所用的原代神经细胞取自3×Tg AD模型小鼠，该小鼠转入APPswe、PS1M146V和TauP301L三种AD相关基因，表现出典型的AD病理特征。
从出生一天的3×Tg AD胎鼠大脑中分别获取上皮层组织，经过木瓜酶消化吹打后得到原代神经细胞，培养在放有细胞爬片的24孔板中。细胞培养到第七天后以终浓度为1μM的小檗碱处理24小时后进行免疫荧光检测。以相同培养条件下无小檗碱处理的原代神经细胞作为对照组。
实验前准备工作：制备4％PFA、0.2％Triton、10％山羊血清封闭液、DAPI、封片剂(甘油/PBS)、指甲油、计时器(Timer)，PBS预冷，准备冰盒与冰袋。具体操作步骤如下：
(1)提前半小时将4％PFA解冻，60℃水浴溶解；
(2)加4％PFA固定15min，每孔1ml，冰上进行；
(3)预冷PBS洗3次，每孔1ml，分别洗5min、10min、15min，冰上进行；
(4)0.2％Triton透膜10min，每孔500μL，冰上进行；
(5)封闭液封闭1h以上，每孔300μL，置于4℃冰箱/冰上；
(6)加一抗4℃过夜，母液300μL封闭液；
(7)一抗回收，PBS洗3次，每孔1ml，每次5min，冰上进行；
(8)加二抗1：200，母液300μL封闭液，常温下避光放置1h以上；
(9)回收二抗，PBS洗3次，分别洗5min、10min、15min，在冰上避光进行；
(10)5μg/mlDAPI染色2min(1:500)，每孔500μL，在冰上避光进行；
(11)PBS洗3次，分别洗5min、10min、15min，在冰上避光进行；
(12)甘油/PBS封片，指甲油固定；
(13)避光4℃保存，待拍片；
(14)最后在激光共聚焦显微镜下观察。
b.小檗碱对原代神经细胞生长状况的影响
以DAPI(4',6-二脒基-2-苯基吲哚)染细胞核，以抗MAP-2抗体染神经细胞骨架，结果如图1所示(左图为蓝色通道，只显示DAPI；中图为红色通道，只显示MAP-2；右图为前面两张图叠加)，从图1可以看出，原代神经细胞经小檗碱处理后，细胞丝更多更长，表明小檗碱促进了神经细胞的生长和神经丝的发育。
c.小檗碱抑制原代神经细胞tau蛋白磷酸化(PS404)水平
微管结合蛋白tau的结构功能异常是诱发阿尔茨海默症的一个重要因素。微管系统是神经细胞骨架的主要成分，在维持细胞功能上起了重要作用，微管由α、β微管蛋白和微管相关蛋白组成。tau蛋白是一种微管相关蛋白，可与微管相结合并保持微管的稳定性。AD病例状态下，tau过度磷酸化，并与其它的tau蛋白纤维配对结合。这种过度结合最终会在神经细胞胞体内形成神经纤维缠结，进而导致神经细胞微管骨架系统瓦解。这不但会影响神经细胞间正常的信号传导，最终必然还会引起神经细胞的死亡。关于AD病理状态下，微管结合蛋白tau的细胞毒性的机理，目前主要有两种观点。一种认为是由于过度磷酸化的tau与微管骨架分离从而失去其生理功能；另一种认为过度磷酸化tau组成的神经纤维缠结会破坏正常tau蛋白的生理功能，并直接成为胞浆运输的障碍。因此抑制tau蛋白的过度磷酸化，是抑制AD发展的一个重要策略。
从图2(左图为红色通道，只显示MAP-2；中图为绿色通道，只显示PS404；右图为前面两张图叠加)中可以看出，小檗碱处理后，原代神经细胞tau蛋白在 其Ser404位点的磷酸化水平显著降低，表明小檗碱可抑制tau蛋白在该位点的过度磷酸化，抑制神经纤维缠结的形成，维护神经细胞微管骨架的稳定性，保护神经细胞。
实施例2：以免疫印迹(western blot)检测小檗碱对原代神经细胞各AD相关蛋白表达水平的影响
蛋白质印迹法即Western Blot。它是分子生物学、生物化学和免疫遗传学中常用的一种实验方法。其基本原理是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行着色。通过分析着色的位置和着色深度获得特定蛋白质在所分析的细胞或组织中的表达情况的信息。一般由凝胶电泳、样品的印迹和免疫学检测三个部分组成。第一步是做SDS聚丙烯酰胺凝胶电泳，使待测样品中的蛋白质按分子量大小在凝胶中分成带。第二步把凝胶中已分成条带的蛋白质转移到一种固相支持物上，用得最多的材料是硝酸纤维素膜(NC膜)和PVDF膜，蛋白转移的方法多用电泳转移，它又有半干法和湿法之分，现在大多用湿法。第三步是用特异性的抗体检测出已经印迹在膜上的所要研究的相应抗原。免疫检测的方法可以是直接的和间接的。现在多用间接免疫酶标的方法，在用特异性的第一抗体杂交结合后，再用酶标的第二抗体(碱性磷酸酶(AP)或辣根过氧化物酶(HRP)标记的抗第一抗的抗体)杂交结合，再加酶的底物显示或者通过膜上的颜色或X光底片上曝光的条带来显示抗原的存在。该技术被广泛应用于蛋白表达水平的检测中。具体步骤如：
1、组织蛋白样品的制备：
1)以浓度为1μM的小檗碱处理24小时后的细胞为实验组，以无小檗碱处理的细胞为对照组。
2)提蛋白，按一定比例加入RIPA裂解液，并加入蛋白酶及磷酸酶抑制剂。
3)超声破碎细胞，按照超声1S停1S，超声2min。
4)4℃、12000rpm离心1h，取上清蛋白，分装其余样品冻存置-80℃冰箱。
5)在蛋白中加入适量蛋白4×上样缓冲液，沸水煮5min使蛋白变性。样品制好后，保存于-20℃备用。
2、蛋白定量：
1)标准曲线的制作：取一块酶标板，按表1加入试剂。
表1：蛋白的浓度梯度
Figure PCTCN2016072763-appb-000001
2)根据样品数量，按50体积BCA试剂A加1体积BCA试剂B(50:1)配制适量BCA工作液，充分混匀。
3)各孔加入200μL BCA工作液。
4)将酶标板放在振荡器上轻微振荡30s，37℃放置30分钟，酶标仪562nm下进行比色测定，记录数据后，以蛋白含量(μg)为横坐标，吸光值为纵坐标，做标准曲线。
5)取待测样品稀释至20μL加入酶标板孔中，再加入200μL配制好的BCA工作液，轻微振荡混匀，37℃放置30分钟，酶标仪562nm波长下进行比色测定，记录吸光值。
6)根据吸光值与标准曲线进行比对即可查得待测样品相应的蛋白含量(μg)，然后再除以样品稀释液的总体积(20μL)，乘以样品稀释倍数即得蛋白样品的实际浓度(μg/μL)。
3、SDS-聚丙烯酸胺凝胶电泳(SDS－PAGE)
制胶前准备：按将要检测的抗体对应的原始抗原的分子量大小，计算出胶的浓度，并计算出分离胶各组分的用量(表2)。
表2：分离胶及浓缩胶的配制
Figure PCTCN2016072763-appb-000002
Figure PCTCN2016072763-appb-000003
1)先配制分离胶，按照配方将各试剂一次加入烧杯中混匀(注：APS与TEMED混合后会快速凝固)，迅速加入先放好的胶板缝隙中，加至2/3处停止，在胶上面加入一层去离子水，压齐分离胶并隔绝空气促进胶的凝集。一般凝胶需30min。待分离胶凝集后，吸去水，配制浓缩胶(1/3)加在上面，倒好后插入预先准备好的梳子。
2)待胶凝集好后，上样，每孔上样蛋白5-10μg左右，蛋白标准分子量(Marker)上样5μL，上样时要避免产生气泡。电泳，上层浓缩胶用80V电压，当样品至下层分离胶时，用120V电压。
3)一般电泳时间在1.5小时左右。电泳时，内槽一定要用新配置的1×SDS电泳液，外槽可用回收的。
4、切胶转膜
实验条件的选择：电流1mA-2mA/cm2，我们通常100mA/膜，按照目的蛋白分子大小、胶浓度选择转移时间，具体可以根据实际适当调整(表3)。
表3：转膜条件
Figure PCTCN2016072763-appb-000004
(1)待电泳完成后将凝胶板放在电泳缓冲液中浸泡几分钟。
(2)打开胶板，将胶放置于平板上，切除浓缩胶，根据Marker大小分离出所要的分离胶并泡在电转液中。
(3)根据所取分离胶的大小剪取滤纸(三层)，并根据滤纸大小剪取PVDF膜，转膜前先将膜浸泡一下甲醇，尽量避免污染滤纸和膜，将裁好的滤纸和膜完全浸泡于电泳转移缓冲液中以驱除留于滤纸的气泡。
(4)打开转移盒，将浸透后的海绵垫放于转移盒壁上，再放一张电泳转移缓冲液浸湿的滤纸于海绵上。
(5)按“黑色胶板—海绵—3层滤纸(大)—凝胶—膜—3层滤纸(小)—海绵”的顺序装置好(注意每放一层都需要赶走气泡)，再倒一些电转液到膜上，保持海绵，滤纸，膜的湿润。
(6)将缓冲液槽装入冰盒，将4℃预冷的电泳转移缓冲液注满电泳槽。连接好转移电极恒流100mA转移1.5h。
5、转移后效果的鉴定
将转膜后凝胶用考马斯亮兰染色30min-1h，经脱色液脱色至无背景色后，观察胶上是否还有残余的蛋白条带，用于检测转膜的效果。或用丽春红对PVDF膜进行染色，观察转膜条带并于封闭前洗掉丽春红。
6、封闭
在转移结束前配好5％脱脂牛奶(1×TBST溶解)。转移结束后将膜放入5％脱脂牛奶中封闭2h，封闭时放置在37℃水平摇床上适当转速进行封闭。注意：脱脂奶粉溶液要足以覆盖膜，操作尽量避免污染。
7、一抗孵育
将所需检测抗体稀释至适当倍数加入封闭液中达到工作浓度，注意保证膜的所有部分同溶液接触。一般采用37℃摇床孵育2h或4℃过夜。可根据抗体量和膜上抗原量适当延长或缩短时间。
8、洗涤
一抗孵育完成后，将膜取出来放置于干净平皿中，用1×TBST在摇床适当转速洗涤15min左右，换液，反复3-5次。洗涤是为了洗去一抗与抗原的非特异性结合，洗涤的效果直接影响结果是否有杂带。
9、二抗孵育
二抗的稀释比例不能太高，否则容易导致非特异性的结合。根据实验需要和设计选择合适的酶标二抗和稀释浓度(5％BSA稀释)，洗涤完成后加入二抗3ml左右室温下于摇床孵育2h，注意保证膜的所有部分同溶液接触。
10、洗涤
弃去二抗，膜条置于干净的培养皿中，加入1×TBST，在摇床上洗涤，洗涤15min左右，换液，重复3次。洗涤是为了去除二抗的非特异性结合，洗涤的效果直接影响结果背景的深浅，所以洗涤一定要干净。
11、显色——增强化学发光法(ECL)
1)将两种显色底物A液1ml，B液2μL混合。
2)将混合物均匀滴加覆盖在膜表面1-2分钟，可见发光的条带(避光)。
3)取出PVDF膜并用保鲜膜把PVDF膜包起来，擦去多余的显色液，放入夹板中。
4)在暗室中将X光片剪至合适大小，覆盖在PVDF膜的上面，夹好夹子，进行压片曝光，曝光时间根据蛋白显现的荧光亮度决定，也可不同时间多次压片。
5)曝光完成，打开夹子，迅速将胶片放入显影液中，待出现条带后，即可终止，取出放入定影液中，以胶片变透明为止，再用自来水冲洗，晾干，扫描拍照。
12、数据图片整理及分析
利用quantity one光密度分析软件对图片进行灰度值分析，并进行组间t检验；利用Graphpad软件进行数据整理作图分析，p<0.05具有显著性的统计学意义。
a.小檗碱抑制原代神经细胞Aβ和APP水平
AD的另一个主要病理学标志是脑部出现淀粉样沉积，即老年斑(senil plaque,SP)。老年斑的核心成分是一种由39-43个氨基酸残基构成的小神经肽—Aβ(β-amyloid peptide)，Aβ由淀粉样前体蛋白(amyloidprecursor protein,APP)经过β-分泌酶及γ-分泌酶水解并分泌至细胞外。免疫印迹结果表明(图3-图6)，小檗碱处理后，原代神经细胞中Aβ和APP蛋白水平显著降低，表明小檗碱可抑制Aβ和APP的表达或对其有一定的清除能力。
b.小檗碱抑制原代神经细胞tau蛋白磷酸化(PS422和PS396)水平
上文已阐述tau蛋白过度磷酸化是AD发生、发展的重要因素。Tau蛋白上至少存在21个异常磷酸化位点。免疫印迹实验表明(图7-图10)，小檗碱可显著抑制原代神经细胞tau蛋白PS422和PS396位点的磷酸化水平。
以上本实施例结果表明，小檗碱可促进原代神经细胞的生长，降低Aβ的表达和tau蛋白的过度磷酸化，因此具有抑制老年斑和神经纤维缠结形成的功能，从而抑制AD的发生发展。
实施例3：Morris水迷宫检测小檗碱对AD模型小鼠的空间探索和记忆能力的影响
Morris水迷宫：直径为16cm，高5cm的圆形水池，池内水深26cm，水温保持在(22±2)℃，整个圆形水池用黑布笼罩，里面配备探照灯，光线恒定。水池 分为4个象限，在其中一个象限内，距离池壁3cm处放置一个直径为12cm，高24cm的圆形(白色表面)站台，水位淹没站台顶1-2cm。连接显示系统的摄像头安置于水迷宫正上方，用于同步记录小鼠的运动轨迹。采用Morris水迷宫视频分析系统进行相关信息处理。
本实施例实验的对象之一是一种三转基因小鼠(3×TG Deer Mice，含有Psen1突变基因，以及APPSwe和tauP301L易感基因(Tg(APPSwe,tauP301L)1Lfa))，具有可生育、无先天性疾病和行为异常的生理特征，其能够过量表达转入的基因，尤其是在AD相关区、海马区和大脑皮层，导致局限于中枢神经系统的行为学异常。在前期的饲喂过程中，3-4月时开始可见细胞内Aβ沉积，6个月胞外可见明显的老年斑块，6个月时突触传导受损现象开始出现，而且6个月后海马区、随后脑皮层神经缠结可见。
实验小鼠：分为三组，一组为6月龄野生型小鼠，一组为未以小檗碱饲养的6月龄三转基因AD小鼠，一组为以50mg/Kg/天的小檗碱饲养的6月龄三转基因AD小鼠。
每组分别挑选10只小鼠进行试验，并做好标记放置在不同的饲养笼中。
Morris水迷宫实验：分为定位航行及空间探索两部分。其中定位航行历时5天，空间探索两天。定位航行第一天为训练期：实验前将小鼠置于平台上适应10s，随后将小鼠从不同象限的对应位置面壁放入池内，小鼠登上平台2s后终止记录，最长记录时间为60s，若小鼠在60s内不能上台，引导其登上平台适应10s，最后将小鼠擦干放入鼠笼。如此将小鼠置入水池，从四个象限入水，每次间隔1h，进行定位航行训练。定位航行第二天到第五天为检测期：每天按照一定的象限顺序入水，每天四轮，并记录相关数据。空间探索分为24小时记忆和72小时记忆：是从定位航行结束开始计时，将平台撤去，记录小鼠在原平台所在象限停留时间及跨越原平台的次数。
Morris水迷宫实验数据指标参数及整理：定位航行逃避潜伏期、运动轨迹、平均速度；空间探索24小时、72小时记忆在原平台所在象限停留时间及跨越原平台的次数。通过水迷宫跟踪系统得出数据，利用Graphpad软件进行数据整理作图分析，单因素重复性方差分析逃避潜伏期，t检验分析组间差异，p<0.05具有显著性的统计学意义。
从图11可以看出，相对于野生型小鼠，AD模型小鼠在四个象限中的逃避潜伏期均显著提高，而饲喂小檗碱的AD模型小鼠在四个象限的逃避潜伏期均 明显低于未喂药AD模型小鼠小鼠，表明小檗碱可明显提高AD模型小鼠的学习记忆能力。
从上述实施例可以看出，本发明在原代神经细胞和AD模型鼠两个水平上验证了小檗碱对AD的预防和治疗效果：(1)小檗碱降低Aβ的蛋白水平；(2)小檗碱降低APP的蛋白水平；(3)小檗碱降低磷酸化tau422的蛋白水平；(4)小檗碱降低磷酸化tau396的蛋白水平；(5)小檗碱提高小鼠的学习记忆能力。因此，小檗碱可在多靶点抑制AD的发生、发展，可以作为一种稳定、安全、有效的AD治疗药物。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (10)

  1. 小檗碱在制备防治阿尔茨海默病的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述药物至少包括所述小檗碱作为药物的有效成分。
  3. 根据权利要求2所述的应用，其特征在于，所述药物还包括药学上可接受的载体。
  4. 根据权利要求1所述的应用，其特征在于，所述药物包括所述小檗碱和药学上可接受的载体。
  5. 根据权利要求1-4任一项所述的应用，其特征在于，所述药物制成片剂、注射剂、胶囊剂、颗粒剂、丸剂、微丸、散剂、滴丸剂、汤剂、糖浆剂、合剂、煎膏剂或浸膏剂剂型。
  6. 根据权利要求5所述的应用，其特征在于，所述药物的每种剂型含0.2～1克小檗碱。
  7. 一种用于防治阿尔茨海默病的药物，其特征在于，所述药物至少包括小檗碱作为药物的有效成分。
  8. 根据权利要求7所述的药物，其特征在于，所述药物还包括药学上可接受的载体。
  9. 根据权利要求7或8所述的药物，其特征在于，所述药物制成片剂、注射剂、胶囊剂、颗粒剂、丸剂、微丸、散剂、滴丸剂、汤剂、糖浆剂、合剂、煎膏剂或浸膏剂剂型。
  10. 根据权利要求9所述的药物，其特征在于，所述药物的每种剂型含0.2～1克小檗碱。
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