CN1565441A - Use of levobutylphthalide in prevention and cure of dementia - Google Patents

Abstract

The invention discloses the use of L-n-butylphthalide represented by the general formula (I) in the preparation of drugs for preventing and treating dementia, especially Alzheimer's disease and vascular dementia.

Description

Use of L-n-butylphthalide for prevention and treatment of dementia

technical field

The invention relates to the use of L-n-butylphthalide and the composition containing L-n-butylphthalide for treating dementia.

Background technique

Senile dementia or Alzheimer's disease (Alzheimer's disease, AD) is a progressive degenerative neurological disease characterized by clinical and pathological features. Its clinical manifestations are mainly memory (especially near memory) loss, low cognitive ability, slow thinking, spatial orientation disorder and so on. The pathological manifestations are β-amyloid peptide (Aβ) combined with other molecules, neurons and non-neural cells to form senile plaques and the formation of intracellular neurofibrillary tangles (neurofibrillary tangles, NFT). The prevalence of AD in my country is between 0.2% and 5.98%, and the disease mostly occurs in people over 60 years old. It increases with age, and it is estimated that there are more than 3.6 million AD patients in my country. In the survey of the incidence of dementia in Beijing, it was found that vascular dementia (VD) was more than AD. (Zhang Mingyuan et al.: Incidence of dementia and Alzheimer's disease. Chinese Journal of Psychiatry 1998; 31(4): 195-196) As my country has entered an aging period, dementia patients are increasing year by year. Moreover, the incidence of cerebrovascular disease is very high in the elderly population. It is estimated that the incidence of post-stroke dementia is about 9-30.8%. Long-term cerebral insufficiency is also an important cause of vascular dementia. In short, dementia patients are not only extremely painful to themselves, but also a great burden to their families and society. Therefore, it is extremely important to find effective drugs to delay and control the development of AD and VD.

Alzheimer's disease (AD) is the leading cause of progressive cognitive decline in the elderly. The main pathological changes are the formation of senile plaques and nerve fiber knots centered on the deposition of β-amyloid peptide (Aβ). Studies have shown that the cholinergic system in the brain is related to human learning and memory functions. The level of ACh in the brain of AD patients decreases, and the activity of choline acetyltransferase (ChAT), which catalyzes the synthesis of ACh, decreases, which is closely related to the decline of cognitive function. In addition, the involvement of oxidative stress and inflammatory response in the etiology of AD has also received increasing attention. Aβ consists of 39-43 amino acids and is a degradation product of β precursor protein (APP). The extent of Aβ deposition is closely related to neurological damage and cognitive impairment. Previous studies have confirmed that: continuous lateral ventricle perfusion (i.c.v.) Aβ(1-40) or Aβ(1-42) can cause learning and memory impairment in rats (Nitts.et al.β-Amyloid protein-induced Alzheimer's disease animalmodel.Neurosci . Lett.1994; 170:63-66), suggesting that the memory impairment caused by Aβ accumulation in the brain can simulate the symptoms of AD patients. According to research, L-n-butylphthalide (L-NBP) can significantly improve mitochondrial function, improve brain microcirculation and energy metabolism, inhibit nerve cell apoptosis, resist oxidative damage, inhibit inflammation, resist thrombosis, and reduce intracellular calcium , Inhibit the release of glutamate and other brain protection. For this purpose, continuous intraventricular perfusion (i.c.v.) Aβ(1-40) can be chosen as a model. Morris water maze and biochemical methods were used to detect the effects of the tested compounds on the animals' near memory and spatial position memory functions and on oxidative damage.

Vascular dementia (VD) is dementia caused by brain dysfunction caused by cerebrovascular diseases, most of which are accompanied by multiple large cerebral artery infarctions or lacunar infarctions or cerebral hypoperfusion. The degree of reduction in cerebral blood flow correlates with the severity of dementia (Roman et al. Vascular dementia: diagnostic criteria for research studies. Neurology 1993; 43: 250-260). Chronic progressive cerebral insufficiency reduces the utilization of oxygen, glucose and other essential metabolic substances, resulting in oxidative damage, impairment of mitochondrial function and neuronal biosynthesis, blockage of synaptic transmission, and finally neuropathological changes , that is, neurodegenerative changes occur (Beal et al. Do defects in mitochondrial metabolism underlie the pathology of neurodegenerative disease Trends Neurosci. 1993; 16: 125-131). Patients with VD mainly show progressive decline in near memory and spatial perception and cognitive impairment. The occurrence and development of vascular dementia are closely related to the information transmission of the cholinergic nervous system, and are also related to the oxidative damage of nerve cells. Pathologically, the subcortical white matter is sparse in VD patients. Numerous studies have shown that ACh is considered an important neurotransmitter for learning and memory. The function of the cholinergic pathway in AD patients is reduced, which is manifested as a decrease in the level of the neurotransmitter ACh, which is one of the important reasons for the impairment of memory function and cognitive impairment (Toghi et al. Cerebrospinal fluid acetylcholine and choline in vascular dementia of Binswanger and multiple small infarct types as compared with Alzheimer-type dementia. J. Neural Transm. 1996; 103: 1211-1220). The tested compounds can improve the activity of ChAT, indicating that they can increase the level of ACh in cholinergic nerves, which is beneficial to improve memory function.

In the past ten years, many laboratories have used the Morris water maze to test the near memory and spatial position memory of rats, which can sensitively reflect the damage and functional changes of the central nervous system of animals (Richard Morris.Developments of a water-maze procedure for studying spatial learning in the rat J.Neurosci Methods.1984;11:47-60) and observing the effect of drugs on this model. Because dementia patients mainly show cognitive impairment, especially progressive damage to near memory and spatial perception. Therefore, this model is an ideal model to observe whether the drug has a therapeutic effect on Alzheimer's disease (AD) and VD. However, continuous hypoperfusion in rat bilateral common carotid artery occlusion (2-VO) model can simulate clinical vascular dementia caused by blood supply insufficiency, so this method can reflect the effect of drug treatment on dementia (Ni.J.W.et al. Neuronal damage and decrease of central acetylcholine level following permanent occlusion bilateral common carotid arteries in rats. Brain Res. 1995; 673: 290-296).

Apigenin A (3-n-butylphthalide, Ag-1), reported by Yu Shuren in 1988, is a synthetic racemic 3-n-butylphthalide, which caused learning and memory impairment in rats to masangolactone It has an improvement effect and a protective effect on rat hippocampal cells (Yu Shuren et al.: The effect of apigenin A on enhancing learning and memory, Chinese Journal of Pharmacology 1988, 9(5): 385-388). Since then, it has been reported that celery extract can improve the learning and memory of middle-aged and elderly mice (Li Jing et al. The Effect of Celery Extract on Learning and Memory of Middle-aged and Aged Mice. Chinese Herbal Medicine 1996, 27(2): 104-105; Liu Luosheng et al.: An Research on the quality standard of Qingyizhi Capsules (Journal of Shandong Medical University 2001, 39(6): 562-564), but so far no optical stereoisomer - L-n-butylphthalide (L-NBP) has been seen in the treatment of senile dementia reports.

Contents of the invention

In order to overcome the deficiencies in the prior art, the present invention provides as shown in general formula (I)

The application of L-n-butylphthalide (hereinafter referred to as L-NBP) as an anti-dementia drug.

The L-n-butylphthalide used in the present invention is chemically synthesized, first to obtain racemic n-butylphthalide, and then chemically resolved to become L-n-butylphthalide, an optical isomer. Analysis, especially with Hp5890 gas chromatograph, chiral gas chromatographic column (Chiraldex G-TA) is analyzed, proves that the optical purity and chemical purity of this product are single optical stereoisomer---Levorotatory n-butylphthalide (specific optical rotation degree>-66.49°, optical purity>98%, chemical purity>98%), the resolution method refers to the Chinese patent "Method for preparing optically active 3-n-butylphthalide", application number 99109673.8, publication number CN1283621. The chemical structural formula of this product is the same as that of apigenin A contained in edible celery and its seeds.

The present invention uses an internationally recognized method, that is, the Morris water maze test to detect the animal's near memory and spatial orientation.

The present invention causes persistent hypoperfusion by permanently blocking bilateral common carotid arteries (2-VO) in rats, and uses the Morris water maze method to detect the effect of L-NBP on the animal's recent memory and spatial position function, and detects it with biochemical methods The effects of L-NBP on some indicators of oxidative damage and on the cholinergic nervous system were investigated. Whereas cerebral hypoperfusion induces behavioral changes accompanied by activation of glial cells and thinning of white matter. Therefore, this study used pathological and immunohistochemical methods, using glial cell fibrillary acidic protein (GFAP) and K-B staining (reflecting the pathological changes of neuron myelin sheath) as indicators, and observed the effect of drugs on it.

The experiment of the present invention shows that the L-n-butylphthalide of the present invention can obviously improve the near memory and spatial position dysfunction of rats with cerebral insufficiency. The learning and retention assay of the water maze test is often used to evaluate spatial memory in hypoperfused rats. The experimental results showed that in the water maze experiment, in the first day of training, there was no significant difference in the latency between the groups, indicating that all animals were not familiar with the experimental operation on the first day. After 5 days of training, the search strategy of the sham operation group changed from marginal and random to trend and linear, and the latency (12.6±3.34 seconds) was significantly shortened, indicating that the animals had a certain memory and spatial orientation after training. The search strategy of the solvent control group did not change significantly, it was still marginal and random, and the latency (47.6±5.88 seconds) was not significantly shortened, and the difference between the two groups was significant (P<0.01). The search strategy of the L-NBP10mg/kg group changed from marginal and random to trend and linear, and the latency (26.85±5.98 seconds) was significantly shortened, which was significantly different from that of the solvent control group (P<0.001 two-factor analysis of variance). Compared with the sham operation group, there was no significant difference, indicating that the dose group had a significant improvement effect on memory and spatial orientation. Other drugs such as DL-NBP10mg/kg, DL-NBP30mg/kg and D-NBP30mg/kg had no significant improvement.

After the 5 days of learning and training, the platform exploration experiment was carried out, and the safety island was removed to test whether the rats had formed the spatial memory of the safety island. Except for the solvent control group, all rats stayed in the target quadrant for more than 25% of the time, indicating that they had formed memory of the relative position of the safety island. The residence time of the sham operation group was 17.73±1.19 seconds, while that of the solvent group and the control group (14.40±0.73 seconds) was significantly shortened. Through one-way analysis of variance statistics, there was a significant difference between the two groups (P<0.05). The residence time of the L-NBP10mg/kg group in the quadrant of the platform was significantly longer than that of the solvent control group (17.62±1.27 seconds), and there was a significant difference between the two groups (P<0.05). DL-NBP group (10mg/kg and 30mg/kg) had no effect. In order to exclude the influence of the animal's athletic ability on it, it was determined that there was no difference in swimming speed among the groups. The above shows that only L-n-butylphthalide has a significant effect on the short-term memory and spatial position functional impairment of rats with cerebral insufficiency, while racemization and D-n-butylphthalide have no effect.

SOD is an important antioxidant enzyme. The activity of SOD in the cortex tissue of the normal control group was 100.07±3.64 (NU/mg protein) and that in the hippocampal tissue was 57.90±7.41 (U/mg protein). After permanent ligation of bilateral common carotid arteries in rats, the activity of SOD in the hippocampus was significantly higher than that in the control group (P<0.05), which may be a compensatory reaction. After treatment with L-NBP (10 mg/kg), the enzyme activity was significantly close to the normal level (P<0.05). MDA is a sign of lipid peroxidation, which can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage. In this experiment, the cortical MDA content of the model group increased by 19.9%, which was significantly different from that of the normal control group (P<0.001). After treatment with L-NBP (10mg/kg), the cortical MDA content was significantly reduced by 20.7% (P<0.001). After permanent ligation of bilateral common carotid arteries in rats, the activity of ChAT in the cortex decreased significantly, compared with the normal control group, it decreased by 34.4% (P<0.05), indicating that hypoperfusion can cause cholinergic nerve function impairment. However, after 16 days of continuous administration of L-NBP (10mg/kg), the activity of ChAT in the cortex tissue increased by 37.1% compared with the model group, with a significant difference (P<0.05). The following conclusions can be drawn from the results: L-NBP10mg/kg group can significantly improve the memory impairment of the near memory and spatial position of 2-VO animals, while racemic butylphthalide and d-butylphthalide have both effects on improving memory dysfunction. invalid. The present invention starts administration on the 10th day after 2-VO (administration continues until the 35th day), and the purpose is to observe the therapeutic effect of the drug on the neuron degeneration caused by cerebral hypoperfusion, so as to rule out the effect on acute hypoperfusion ischemia. Impact.

The present invention proves that L-NBP has obvious therapeutic effect on vascular dementia through pathological and immunohistochemical studies. After permanent ligation of bilateral common carotid arteries, neurons in the cortex and hippocampus CA1 area were significantly reduced in the model group, cells shrunk and neurons were deeply stained, and L-NBP (10mg/kg) treatment could significantly improve the induction of hypoperfusion of neuronal damage. It has been reported that bilateral common carotid artery ligation can induce glial cell activation in the brain, accompanied by white matter thinning. White matter thinning is generally divided into 4 grades according to its severity: grade 0, normal; grade 1, nerve fiber misalignment; grade 2, obvious vacuolation; grade 3, myelinated fibers disappeared. In our experiment, compared with the normal control group, the optic tract of the model group showed obvious thinning of the white matter and a large number of vacuoles. After long-term administration of L-NBP (10mg/kg), this situation could be significantly improved. Cavitation is significantly reduced. Immunohistochemical experiments found that GFAP-positive astrocytes were rarely detected in the hippocampus, caudate nucleus, and corpus callosum of the normal control group, but after 4 weeks of bilateral common carotid artery ligation, many GFAP-positive astrocytes Glial cells and microglia appear. After treatment with L-NBP (10 mg/kg), GFAP-positive glial cells were significantly reduced (see Figures 3 and 4). In conclusion, L-NBP can increase the activity of ChAT, indicating that it can increase the level of ACh in cholinergic nerves, which is beneficial to improve memory function. In addition, L-NBP can significantly inhibit oxidative damage, indicating that L-NBP can reduce the damage of nerve cells. The pathological features of cerebral hypoperfusion caused by 2-VO are sparse white matter, vacuoles and increased glial cells (Narri.et al. Chronic cerebral hypoperfusion-induced neuropathological changes in rats.Jpn.J.Psychopharmacol.1998;18:181- 188), and L-NBP can improve these pathological changes. The above mechanisms provide a basis for L-NBP to improve the memory impairment caused by 2-VO rats. According to the above results, L-NBP has obvious therapeutic effect on vascular dementia.

The experimental results of the present invention show that the L-n-butylphthalide of the present invention has an obvious improvement effect on the memory and spatial orientation disturbance of rats caused by the β-amyloid peptide (1-40). In the water maze experiment after continuous perfusion of Aβ in the ventricles of rats, there was no significant difference in latency among the groups on the first day of training. After 5 days of training, the search strategy of the sham operation group changed from marginal and random to trend and linear, and the latency (13.02±2.77 seconds) was significantly shortened. However, the search strategy of the model group did not change significantly, it was still marginal and random, and the latency (30.18±4.81 seconds) was not significantly shortened, and the difference between the two groups was significant (P<0.01). After L-NBP treatment, the latency period of the rats in the water maze test was significantly shortened, and the search strategy of the L-NBP10mg/kg group and the 30mg/kg group changed from the edge type and the random type to the trend type and the linear type, and the latency was respectively 27.28±6.42 seconds and 25.88±5.51 seconds, which are significantly different from the model group (P<0.05 two-factor analysis of variance), but not significantly different from the sham operation group, indicating that the rats treated with 1-NBP are close to the normal level , see Figure 4. The latencies for the first trial and trials 2-5 in the working memory test are shown in Figure 5A,B. Although in the first experiment, there was no significant difference in the latency of each group, but in the next four experiments, the latency of the sham operation group was 9.15±0.91 seconds, and the latency of the model group was 14.05±1.88 seconds. There was a significant difference between the two (P<0.01). L-NBP group can significantly improve working memory ability in a dose-dependent manner (P<0.01).

L-NBP can also increase the activity of GSH-Px and reduce the content of MDA. GSH-Px is an important antioxidant enzyme. In our experiment, the activity of GSH-PX in the cortical tissue of the sham operation group was 15.86±0.91 (U/mg protein); the activity in the hippocampal tissue was 16.19±1.19 (U/mg protein). mg protein). After continuous perfusion of Aβ(1-40) in rats, the activity of GSH-Px decreased by 29.5% and 42.4% in the cortex and hippocampus, respectively, compared with the sham operation group, with significant differences (P<0.01 and P<0.001). After treatment with L-NBP, the activity of the enzyme was significantly increased in the 30mg/kg group (P<0.01), and L-NBP (10mg/kg) also increased the activity of the enzyme, but there was no statistical significance. MDA is a sign of lipid peroxidation, which reflects the degree of lipid peroxidation in the body and indirectly reflects the degree of cell damage. In this experiment, after continuous perfusion of Aβ(1-40) in rats, the contents of MDA in cortex and hippocampus increased by 25.7% and 23.6%, respectively, which was significantly different from that in sham operation group (P<0.05 and P<0.01 ). After L-NBP treatment, the content of MDA in the cortex and hippocampus decreased significantly, and the 10mg/kg group decreased by 28.4% and 24.3% respectively (P<0.05 and P<0.01). The effect of 30mg/kg was weaker than that of the 10mg/kg group. However, compared with the Aβ(1-40) model group, there was still a significant difference (P<0.05).

The experiments of the present invention can draw the following conclusions: the memory impairment caused by continuous lateral ventricle perfusion of Aβ(1-40) in rats is a recognized model for observing the therapeutic effect of drugs on AD. From the above results, it can be seen that L-NBP not only has a significant effect on the model of vascular dementia caused by insufficient blood supply caused by 2-VO, but also has a significant effect on the memory impairment of near memory and spatial position caused by continuous lateral ventricle perfusion Aβ(1-40) in rats There is also a significant improvement. This shows that L-NBP can significantly improve the impairment of near memory and spatial position memory caused by different reasons. In addition, L-NBP has the effect of blocking oxidative damage (increasing the activity of GSH-Px and reducing the content of MDA), combined with its obvious brain protection effect, it is suggested that L-NBP has the effect of treating senile dementia.

In a word, the present invention uses the Morris water maze to detect the memory of rats' near memory and spatial position, and uses the rat 2-VO model for continuous hypoperfusion to simulate clinically the vascular dementia caused by insufficient blood supply, so this method can fully reflect the drug treatment of dementia. role. The experimental results of the present invention show that the L-NBP10mg/kg group can significantly improve the memory impairment of the near memory and spatial position of 2-VO animals, while racemic butylphthalide and d-butylphthalide have no effect on improving memory dysfunction. ACh is considered an important neurotransmitter for learning and memory. The function of cholinergic pathways in AD patients is reduced, which is manifested as a decrease in the level of neurotransmitter ACh, which is one of the important reasons for the impairment of memory function and cognitive impairment. However, L-NBP can increase the activity of ChAT, indicating that it can increase the level of ACh in cholinergic nerves, which is beneficial to improve memory function. L-NBP can significantly inhibit oxidative damage, indicating that L-NBP can reduce the damage of nerve cells. The pathological features of cerebral hypoperfusion caused by 2-VO are sparse white matter, vacuoles and increased glial cells, and L-NBP can improve these pathological changes. Therefore, the compound L-n-butylphthalide represented by the general formula (I) of the present invention has preventive and therapeutic effects on vascular dementia.

Continuous intracerebroventricular perfusion of Aβ(1-40) induced learning and memory impairment in rats, which could simulate the symptoms of Alzheimer's patients. The near memory and spatial location memory function of rats were tested by Morris water maze. L-NBP can significantly improve the impairment of near memory and spatial position memory, while D-NBP and DL-NBP have no effect. GSH-Px is an important antioxidant enzyme; MDA reflects the degree of lipid peroxidation in the body and indirectly reflects the degree of brain cell damage. L-NBP can increase the activity of GSH-Px and reduce the content of MDA, indicating that L-NBP can block oxidative damage and protect brain damage, while D-NBP and DL-NBP have no effect. Therefore, the compound L-n-butylphthalide shown in the general formula (I) of the present invention has preventive and therapeutic effects on Alzheimer's disease. In short, L-n-butylphthalide has the effect of preventing and treating senile dementia, while rac-butylphthalide and D-butylphthalide have no effect.

The present invention therefore also relates to pharmaceutical compositions comprising the compound according to the invention as active ingredient together with customary pharmaceutical excipients or adjuvants. Typically the pharmaceutical compositions of the invention contain 0.1-95% by weight of the compound of the invention.

Pharmaceutical compositions of compounds of the present invention can be prepared according to methods well known in the art. When used for this purpose, the compound of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if necessary, to prepare an appropriate administration form or dosage that can be used as human or veterinary medicine form.

The compound of the present invention or the pharmaceutical composition containing it can be administered in the form of unit dosage, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal or rectal, etc.

The route of administration of the compound of the present invention or the pharmaceutical composition containing it may be injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection.

The dosage forms for administration may be liquid dosage forms or solid dosage forms. For example, the liquid dosage forms can be true solutions, colloids, particulate dosage forms, emulsion dosage forms, and suspension dosage forms. Other dosage forms such as tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, etc.

The compound of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

Various carriers known in the art can be widely used for tableting unit dosage forms. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid Aluminum, etc.; wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin paste, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbate Sugar alcohol fatty acid esters, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers , such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

For example, in order to form a dosage unit into a pellet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, Ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecylsulfonate, methylcellulose, ethylcellulose, etc.

For example, to form a dosage unit into a capsule, the compound of the present invention as an active ingredient is mixed with the above-mentioned various carriers, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule. The active ingredient compound of the present invention can also be made into microcapsules, suspended in an aqueous medium to form a suspension, and can also be packed into hard capsules or made into injections for application.

For example, the compound of the present invention is made into injection preparations, such as solutions, suspension solutions, emulsions, and freeze-dried powder injections. This preparation can be aqueous or non-aqueous, and can contain one and/or more drugs A pharmaceutically acceptable carrier, diluent, binder, lubricant, preservative, surfactant or dispersant. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like. In addition, in order to prepare isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the preparation for injection, and in addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. These excipients are commonly used in the field

In addition, colorants, preservatives, fragrances, correctives, sweeteners or other materials can also be added to the pharmaceutical preparations, if necessary.

In order to achieve the purpose of medication and enhance the therapeutic effect, the medicine or pharmaceutical composition of the present invention can be administered by any known administration method.

The dosage of the compound pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, personality and individual reaction of the patient or animal, the route of administration, the frequency of administration, Therapeutic purposes, and thus the therapeutic doses of the present invention, can vary widely. Generally speaking, the dosages of the pharmaceutical ingredients in the present invention are well known to those skilled in the art. According to the actual amount of drug contained in the final preparation of the compound composition of the present invention, appropriate adjustments can be made to achieve the requirement of its therapeutically effective dose, so as to achieve the preventive or therapeutic purpose of the present invention. The suitable daily dosage range of the compound of the present invention is preferably 0.1-100 mg/kg body weight, more preferably 0.1-100 mg/day/person. The above-mentioned doses may be administered in single-dose form or divided into several, eg, two, three or four dose forms, which are limited by the clinical experience of the administering physician and the dosing regimen including the use of other therapeutic means.

The total dosage required for each treatment may be divided into multiple doses or administered in a single dose. The compound or composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs and the dosage can be adjusted.

the term:

L-NBP: L-n-butylphthalide

2-VO model: permanent bilateral common carotid artery ligation

Description of drawings

Figure 1. Water maze experiment, 1A the search route of rats on the fifth day; 1B the search route of rats in the platform exploration experiment on the fifth day.

Figure 2. Effect of oral administration of L-NBP after permanent bilateral common carotid artery ligation on spatial memory impairment in rats in the water maze test. Figure 2A shows the change in latency during the training phase; Figure 2B shows the platform exploration experiment after the platform was removed, the rats swam freely for 60 seconds, and the time they stayed in the quadrant (Q4) where the platform was located. All values are expressed as mean ± standard error. 12-14 rats per group; #P<0.05 vs. sham group. P<0.05 compared with the vehicle control group (vehicle).

Fig. 3. Changes in H-E staining microscopic images of rat hippocampal CA1 region (A, B, C) and cortex (D, E, F) after 5 weeks of bilateral common carotid artery ligation (40 times). Sham operation group (A, D); solvent control group (B, E); L-NBP (10mg/kg) treatment group (C, F)

Figure 4. After 5 weeks of bilateral common carotid artery ligation, the rat optic tract K-B staining (A, B, C magnified 40 times) and caudate nucleus GFAP immunohistochemical staining (D, E, F magnified 20 times) under the microscope Image changes. Sham operation group (A, D); solvent control group (B, E); L-NBP (10mg/kg) treatment group (C, F)

Figure 5. Effect of L-NBP on memory impairment in water maze test after continuous lateral ventricle perfusion of Aβ(1-40) in rats. Shown is the change in escape latency during the training phase; all values are expressed as mean ± standard error. 10 rats in each group.

Figure 6. Effect of L-NBP on memory impairment caused by continuous lateral ventricle perfusion of Aβ(1-40) in rats. The working memory test (5 times a day) was performed on the 14th-16th day after Aβ(1-40) perfusion. Figure 5A shows the change in latency in the first experiment; Figure 5B shows the change in latency in the next four experiments. All values are expressed as mean ± standard error. 10 rats in each group. ##P<0.01 compared with the sham operation group; ^* P<0.05 compared with the Aβ(1-40) model group.

Fig. 7. The mice of Example 1 were infused with drugs or solvents from the 10th day after the operation. The water maze test was carried out on the 29th-33th day after operation, and the dark avoidance test was carried out on the 34th-35th day. Animals were sacrificed on day 36 for biochemical assays or pathological examinations. In the behavioral experiment, all the drugs were administered 40 minutes before the experiment.

Fig. 8. The mice of Example 2 were infused with drugs and solvents from day 2 after operation. The water maze training test was performed on postoperative days 9-13, the platform exploration test was performed on day 13, and the working memory test was performed on day 14-16. The animals were sacrificed on the 17th day, and the brains were decapitated for biochemical determination. In the behavioral experiment, all the drugs were administered 40 minutes before the experiment.

Detailed ways

1. Example 1 The therapeutic effect of L-n-butylphthalide on one of senile dementia, vascular dementia

Materials and methods

Reagents and Medicines

L-, D-, DL-NBP were provided by the synthesis laboratory of our institute, the optical and chemical purity were >98%, and the optical rotations were -66.49, +66.88 and 0 degrees in turn. Formulated with vegetable oil.

instrument

Morris water maze automatic monitor, avoiding the obscura was developed by the Instrument and Electricity Department of the Institute of Materia Medica, Chinese Academy of Medical Sciences

2-VO model establishment

Male Wistar rats, 10 weeks old, with a weight of about 280 grams, were placed in a cage for every 5 rats, and the room temperature was kept at 23°C, with free access to food and water. Rats were anesthetized with sodium pentobarbital (40 mg/kg), the bilateral common carotid arteries were exposed, and the capsule of the common carotid arteries and the vagus nerve were carefully separated. In the hypoperfusion model group, the bilateral common carotid arteries were ligated with 5-0 silk thread. The sham operation group underwent the same operation except that the bilateral common carotid arteries were not ligated. Sprinkle a little sterilized crystalline sulfa powder on the wound after the operation, and suture the skin. One month after the operation, the water maze test and dark avoidance test were carried out.

Experiment grouping and design

Rats were randomly divided into 8 groups, 10 in each group. 1) Sham operation group: except that the bilateral common carotid arteries were not ligated, the other operations were the same as the hypoperfusion group; 2) Solvent control group: only oral vegetable oil; 3) DL-NBP10mg/kg group; 4) DL-NBP30mg/kg 5) L-NBP10mg/kg group; 6) L-NBP30mg/kg group; 7) D-NBP30mg/kg group; drugs or solvents were fed from the 10th day after operation. The water maze test was carried out on the 29th-33th day after operation, and the dark avoidance test was carried out on the 34th-35th day. Animals were sacrificed on day 36 for biochemical assays or pathological examinations. In the behavioral experiment, all the drugs were administered 40 minutes before the experiment.

water maze experiment

The Morris water maze is mainly composed of a metal cylindrical pool (pool height 60cm, diameter 120cm), automatic display, monitoring, recording devices and a safety island (platform with a diameter of 10cm). Inject clear water in the pool in advance, then add the aqueous solution that dissolves 1000g of fresh milk powder, make the pool water become opaque milky white, and the water surface is 15mm higher than the platform. This way the animal cannot reach the platform by hearing, seeing and smelling, in order to test the animal's sensitivity to spatial position. The water temperature is maintained at 23±1°C, the pool is divided into 4 quadrants (east, south, west, north), and the platform is placed in the center of the southwest quadrant. The swimming activity of each rat is monitored by a TV and directly connected to a computer for processing and analysis. The water maze experiment was carried out for 5 consecutive days. Each rat was trained twice a day to find the platform, and entered the water from the midpoint of the northeast quadrant and the northwest quadrant respectively, with its head facing the pool wall, with an interval of 10 minutes between the two trainings. The time to find the platform (latency period) was recorded, and the results of the two experiments were averaged. If the rat did not find the platform within 60 seconds, the latency was counted as 60 seconds. Whether the platform was found within 60 seconds or not, the rat stayed on the platform for 10 seconds. Rats were placed on the platform for 10 seconds to acclimate before the start of the first experiment. With the increase of training times, the latency period of rats in each group to find the safety island was shortened. Exploration experiments are performed after the last training run. The platform was removed and the rat swam freely for 60 seconds to find the platform, and the time the rat spent in each quadrant was recorded. The rats stayed in the quadrant where the original platform was located for a long time, suggesting that the rats had memory for this spatial object.

We divided the search strategies into four categories: (1) edge type, the rats moved along the edge of the pool without motivation to search; (2) random type, the rats searched without a clear direction; (3) trend type, the rats had remembered The approximate location of the safety island, turning less than 4 times before finding the safety island; (4) straight line, the rat has clearly remembered the location of the safety island, and swam directly to the safety island. The experimental results are represented by the time for rats to find the safety island, that is, the latency and search strategy

Detection of choline acetyltransferase, antioxidant enzymes and MDA

The brains of the rats were decapitated, and the cortex and hippocampus were peeled off in an ice bath. After weighing, they were quickly frozen in liquid nitrogen for later use. Add 0.05 mol L ^-1 of pre-cooled pH 7.0 potassium phosphate buffer solution (containing 0.5 mol L ^-1 EDTA and 7% glycerin) to the sample, make 10% tissue homogenate in ice bath, and use Lowry colorimetric method for protein quantification Determination.

1. Determination of choline acetyltransferase (ChAT): add sodium phosphate buffer 0.5mol L ^-1 , acetyl-CoA 0.0062mol L ^-1 , choline chloride 1.0mol L ^-1 , methylsulfate to the reaction system Neostigmine 76μmol L ^-1 , NaCL 3mol L ^-1 and EDTA 0.011mol L ^-1 , creatinine hydrochloride 0.5mol·L ^-1 40μl each, and finally add distilled water to each tube to 0.8ml. After warming at 37°C for 5 minutes, add 200 μl tissue homogenate to each tube, then add boiling water to each tube for 2 minutes, then add 0.8 ml of 2.5 mmol L ^-1 sodium arsenate, centrifuge at 15,000 × g for 3 min at room temperature, take 2.0 ml of supernatant and add 40 μl of 3 mmol L ^-1 4-PDS was incubated at 25°C for 15 min, and the OD value (λ=324 nm) was measured to calculate the ChAT activity, expressed as nmol CoA SH/mgprotein/hr.

2. The determination of superoxide dismutase (SOD) and malondialdehyde (MDA) was carried out according to the kit instructions of Nanjing Jiancheng Reagent Company.

Pathology and Immunohistochemical Testing

Randomly select 4-6 animals in each group, after the behavioral experiment, anesthetize with pentobarbital (100mg/kgip), cut the skin and chest cavity in turn, fully expose the heart, cut the left apex, and point the perfusion needle towards the aorta Insert in the direction, clamp the hemostat, infuse normal saline, cut the right atrium at the lower part of the right atrial appendage, perfuse with normal saline for 15-20min (200-300ml), the effluent becomes clear, and continue perfusion with 4% paraformaldehyde in PBS 15-20min (150-200ml), the whole body of the animal is stiff until the liver turns white, then decapitate and take the brain, cut off the front part of the telencephalon and the back part of the cerebellum with a razor blade, put in 4% paraformaldehyde and continue to fix for 48h (paraffin section ) or 20% sucrose paraformaldehyde solution for 48h (frozen sections).

The paraffin sections were fixed and embedded, and stained with Hematoxylin-Eosin (HE) and K-B (Klüver-Barrera Luxol fast blue) for pathological examination after sectioning. The changes of GFAP content in brain tissue were detected by immunohistochemistry.

Statistical Analysis

All results are expressed as mean ± standard error. In the water maze experiment, the differences in latency between the groups were compared using two-factor analysis of variance with repeated measures. Differences between groups were analyzed by post hoc LSD or Turkey test. The water maze platform exploration experiment was performed by one-way analysis of variance. The dark avoidance test was performed by Kruskal-Wallis test and Mann-Whitney U test. Biochemical assays were performed using one-way analysis of variance. P<0.05 means significant difference.

Experimental results

Effects of L-NBP on the learning and memory of rats in the water maze In the water maze experiment, learning and retention tests are often used to evaluate the spatial memory ability of hypoperfusion rats. On the first day of training, there were no significant differences between the groups. After 5 days of training, the search strategy of the sham operation group changed from marginal and random to trend and linear, and the latency (12.6±3.34 seconds) was significantly shortened. The search strategy of the solvent control group did not change significantly, it was still marginal and random, and the latency (47.6±5.88 seconds) was not significantly shortened, and the difference between the two groups was significant (P<0.01). The search strategy of the L-NBP10mg/kg group changed from marginal and random to trend and linear, and the latency (26.85±5.98 seconds) was significantly shortened, which was significantly different from that of the solvent control group (P<0.001 two-factor analysis of variance). There was no significant difference compared with the sham operation group. The improvement effects of other drugs such as DL-NBP10mg/kg, DL-NBP30mg/kg and D-NBP30mg/kg were not obvious, see Figure 1A, B and Figure 2. After the 5 days of learning and training, the platform exploration experiment was carried out, and the safety island was removed to test whether the rats had formed the spatial memory of the safety island. Except for the solvent control group, all rats stayed in the target quadrant for more than 25% of the time, indicating that they had formed memory of the relative position of the safety island. The residence time of the sham operation group (17.73±1.19 seconds) was significantly longer than that of the solvent control group (14.40±0.73 seconds), and there was a significant difference (P<0.05) as shown in Figure 3 through one-way analysis of variance. The residence time of the L-NBP10mg/kg group in the quadrant of the platform was significantly longer than that of the solvent control group (17.62±1.27 seconds, P<0.05). DL-NBP group (10mg/kg and 30mg/kg) had no effect. In order to exclude the influence of the animal's athletic ability on it, it was determined that there was no difference in swimming speed among the groups. The above shows that only L-n-butylphthalide has a significant effect on the short-term memory and spatial position functional impairment of rats with cerebral insufficiency, while racemization and D-n-butylphthalide have no effect.

Effects on SOD, ChAT activity and MDA content SOD is an important antioxidant enzyme. The activity of SOD in the cortex tissue of the normal control group was 100.07±3.64 (NU/mg protein); the activity in the hippocampal tissue was 57.90±7.41 (U/mg protein). mg protein). After permanent ligation of bilateral common carotid arteries in rats, the activity of SOD in the hippocampus was significantly higher than that in the control group (P<0.05), which may be a compensatory reaction. After treatment with L-NBP (10mg/kg), the enzyme activity is close to the normal level (P<0.05). MDA is a sign of lipid peroxidation, which can reflect the degree of lipid peroxidation in the body and indirectly reflect the the extent of the damage. In this experiment, the cortical MDA content of the model group increased by 19.9%, which was significantly different from that of the normal control group (P<0.001). After treatment with L-NBP (10mg/kg), the cortical MDA content was significantly reduced by 20.7% (P<0.001). After permanent ligation of bilateral common carotid arteries in rats, the activity of ChAT in the cortex decreased significantly, compared with the normal control group, it decreased by 34.4% (P<0.05), indicating that hypoperfusion can cause cholinergic nerve function impairment. After 16 days of continuous administration of L-NBP (10mg/kg), the activity of ChAT in the cortex tissue was increased by 37.1% compared with the model group, with a significant difference (P<0.05). (See Table 1

Table 1. Effect of L-NBP on SOD and ChAT activity and MDA content in hypoperfusion rat cortex and hippocampus (n=7-9)

Group SOD activity MDA content ChAT activity

(NU/mg protein) (nmol/mg protein) (% of sham)

Brain Regions Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus

Sham operation group 100.07±3.64 57.90±7.41 3.13±0.10 3.03±0.12 100.0±13.3 100.0±7.2

Solvent control group 114.42±7.82 81.16±6.84 ^# 3.91±0.22 ^### 3.63±0.45 65.6±15.1 ^# 102.6±15.1

L-NBP 98.84±5.53 57.60±3.86 ^* 3.10±0.09 ^*** 3.42±0.12 104.3±8.2 ^* 110.0±13.5

(10mg/kg)

^# P<0.05, ^### P<0.001 vs. sham group; ^* P<0.05, ^*** P<0.001 vs. vehicle control group.

Effects on pathology and histochemistry After permanent ligation of the bilateral common carotid arteries, neurons in the cortex and hippocampus CA1 area in the model group were significantly reduced, with shrunken cells and deep staining of neurons, while L-NBP (10mg/kg) After treatment, hypoperfusion-induced neuronal damage can be significantly improved. It has been reported in the literature that bilateral common carotid artery ligation can induce glial cell activation, accompanied by white matter thinning. White matter thinning is generally divided into 4 grades according to its severity: grade 0, normal; grade 1, nerve fiber misalignment; grade 2, obvious vacuolation; grade 3, myelinated fibers disappeared. In our experiment, compared with the normal control group, the optic tract of the model group showed obvious thinning of the white matter and a large number of vacuoles. After long-term administration of L-NBP (10mg/kg), this situation could be significantly improved. Cavitation is significantly reduced. Immunohistochemical experiments found that GFAP-positive astrocytes were rarely detected in the hippocampus, caudate nucleus, and corpus callosum of the normal control group, but after 4 weeks of bilateral common carotid artery ligation, many GFAP-positive astrocytes Glial cells and microglia appear. After treatment with L-NBP (10 mg/kg), GFAP-positive glial cells were significantly reduced (see Figures 3 and 4).

Experimental results

The following conclusions can be drawn from the results: L-NBP10mg/kg group can significantly improve the memory impairment of the near memory and spatial position of 2-VO animals, while racemic butylphthalide and d-butylphthalide have both effects on improving memory dysfunction. invalid. In this study, the drug was administered on the 10th day after 2-VO (administration continued until the 35th day), the purpose of which was to observe the therapeutic effect of the drug on neuronal degeneration caused by cerebral hypoperfusion, so as to rule out the effect of the drug on the acute hypoperfusion-ischemic period. Impact.

L-NBP can increase the activity of ChAT, indicating that it can increase the level of ACh in cholinergic nerves, which is beneficial to improve memory function. In addition, L-NBP can significantly inhibit oxidative damage, indicating that L-NBP can reduce the damage of nerve cells. The pathological features of cerebral hypoperfusion caused by 2-VO are sparse white matter, vacuoles and increased glial cells (Narri.et al. Chronic cerebral hypoperfusion-induced neuropathological changes in rats.Jpn.J.Psychopharmacol.1998;18:181- 188), and L-NBP can improve these pathological changes. The above mechanisms provide a basis for L-NBP to improve the memory impairment caused by 2-VO rats. According to the above results, it is suggested that L-NBP may have obvious therapeutic and preventive effects on vascular dementia.

Example 2 L-n-butylphthalide can significantly improve the beta-amyloid peptide (1-40) caused

Alzheimer's Symptoms

Materials and methods

Reagents and medicines: L-NBP is provided by the synthesis laboratory of our institute and prepared with vegetable oil. Aβ(1-40) was purchased from Sigma. The Alzet brain micro-osmotic pump perfusion device was purchased from Durect Company of the United States. Instrument: Morris water maze automatic monitoring instrument and experimental methods, see the above experiments on learning and memory impairment in rats with cerebral hypoperfusion

Model establishment: Male Wistar rats, 10 weeks old, weighing about 280 grams, placed one animal in each cage, kept the room temperature at 23°C, and had free access to food and water. Rats were anesthetized with pentobarbital sodium (40 mg/kg), fixed on a stereotaxic apparatus in the abdominal position, cut the skin of the head, and implanted a cannula for perfusing Aβ (1-40) into the right ventricle. According to the stereotaxic atlas of the rat brain by Paxions and Watson, the implantation site was 0.3mm behind bregma, 1.1mm to the right, and 4.0mm deep. The cannula is connected to a micro-osmotic pump. An osmotic micropump was placed on the back of the rat. Aβ(1-40) was dissolved in 35% acetonitrile/0.1 trifluoroacetic acid, and was continuously perfused (i.c.v) at 300 pmol/day. The control group was only perfused with solvent 35% acetonitrile/0.1% trifluoroacetic acid. Previous experiments confirmed that solvents did not cause behavioral and neurochemical changes in rats at this flow rate.

Experimental grouping and design: Rats were randomly divided into 4 groups, 10 in each group. 1) Sham operation group: rats' ventricles were only perfused with 35% acetonitrile/0.1 trifluoroacetic acid + solvent; 2) Model group: Aβ(1-40)+solvent; 3) Aβ(1-40)+L-NBP10mg/kg group; 4) Aβ(1-40)+L-NBP30mg/kg group. Drugs and solvents were administered from day 2 after operation. The water maze training test was performed on postoperative days 9-13, the platform exploration test was performed on day 13, and the working memory test was performed on day 14-16. The animals were sacrificed on the 17th day, and the brains were decapitated for biochemical determination. In the behavioral experiment, all the drugs were administered 40 minutes before the experiment. (See Figure 8)

Water maze test: after icv Aβ(1-40), the water maze training test was performed on the 9th to 13th day, and the platform exploration test was performed on the 13th day. On the 14th-16th day after icv Aβ(1-40), the working memory test (see the figure above) was carried out. The test was based on the fact that the animal had acquired a certain memory after the training test and the platform exploration test, and observed after changing the platform and quadrant Rapid learning of spatial memory in animals. The experimental procedure was similar to the standard water maze training trial, except that the platform changed position daily. For 5 experiments every day, the rats started to swim from 5 water entry points. In the first trial of each day, called the demonstration trial, rats were allowed to swim to the platform in a new location and stay there for 10 seconds. For the next four trials, the platform position remained the same, only the quadrant of the entry point was different. The latency period of working memory ability is the average value of the second to fifth trials, and the working memory ability of each rat is calculated by the average value of the 3-day experiment.

Biochemical experiment: the determination of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) was the same as the above experiment.

Statistical analysis: All results are expressed as mean ± standard error. In the water maze experiment, the differences in latency between the groups were compared using two-factor analysis of variance with repeated measures. Differences between groups were analyzed by post hoc LSD or Turkey test. The water maze platform exploration test, working memory test, and biochemical determination were performed by one-way analysis of variance. P<0.05 means significant difference.

Experimental results

Effects of L-NBP on learning and memory in water maze in rats

On the first day of training, there were no significant differences between the groups. After 5 days of training, the search strategy of the sham operation group changed from marginal and random to trend and linear, and the latency (13.02±2.77 seconds) was significantly shortened. However, the search strategy of the model group did not change significantly, it was still marginal and random, and the latency (30.18±4.81 seconds) was not significantly shortened, and the difference between the two groups was significant (P<0.01). After L-NBP treatment, the latency period of the rats in the water maze test was significantly shortened, and the search strategy of the L-NBP10mg/kg group and the 30mg/kg group changed from the edge type and the random type to the trend type and the linear type, and the latency was respectively 27.28±6.42 seconds and 25.88±5.51 seconds, which are significantly different from the model group (P<0.05 two-factor analysis of variance), but not significantly different from the sham operation group, indicating that the rats treated with L-NBP are close to the normal level , see Figure 4. The latencies for the first trial and trials 2-5 in the working memory test are shown in Figure 5A,B. Although in the first demonstration experiment, there was no significant difference in the latency of each group, but in the following four experiments, the latency of the sham operation group was 9.15±0.91 seconds, the latency of the model group was 14.05±1.88 seconds, and the latency of the latter group was 9.15±0.91 seconds. Significantly prolonged, there was a significant difference between the two (P <0.01). L-NBP group can significantly improve working memory ability in a dose-dependent manner (P<0.01). L-NBP increases the activity of GSH-Px and decreases the content of MDA

GSH-Px is an important antioxidant enzyme. In our experiment, the activity of GSH-PX in the cortical tissue of the sham operation group was 15.86±0.91 (U/mg protein); the activity in the hippocampal tissue was 16.19±1.19 (U/mg protein). mg protein). After continuous perfusion of Aβ(1-40) in rats, the activity of GSH-Px decreased by 29.5% and 42.4% in the cortex and hippocampus, respectively, compared with the sham operation group, with significant differences (P<0.01 and P<0.001). After treatment with L-NBP, the activity of the enzyme was significantly increased in the 30mg/kg group (P<0.01), and L-NBP (10mg/kg) also increased the activity of the enzyme, but there was no statistical significance. MDA is a sign of lipid peroxidation, which reflects the degree of lipid peroxidation in the body and indirectly reflects the degree of cell damage. In this experiment, after continuous perfusion of Aβ(1-40) in rats, the contents of MDA in cortex and hippocampus increased by 25.7% and 23.6%, respectively, which was significantly different from that in sham operation group (P<0.05 and P<0.01 ). After L-NBP treatment, the content of MDA in the cortex and hippocampus decreased significantly, and the 10mg/kg group decreased by 28.4% and 24.3% respectively (P<0.05 and P<0.01). The effect of 30mg/kg was weaker than that of the 10mg/kg group. However, compared with the Aβ(1-40) model group, there is still a significant difference (P<0.05), see Table 2.

Table 2. Effect of L-NBP on SOD activity and MDA content in cortex and hippocampus after continuous perfusion of Aβ (1-40) in rats (n=7)

Group GSH-PX MDA

(U/mg protein) (U/mg protein) (nmol/mg protein)

Cortex Hippocampus Hippocampus Cortex Hippocampus

Sham operation group 15.86±0.91 16.19±1.19 1.10±0.09 1.13±0.06

Model group 11.18±1.32 ^## 9.32±1.01 ^### 1.48±0.14 ^# 1.48±0.12 ^##

Aβ(1-40)

L-NBP 11.73±0.53 12.73±1.34 1.06±0.05 ^* 1.12±0.07 ^**

(10mg/kg)

L-NBP 15.40±1.03 ^** 18.91±1.16 ^** 1.06±0.10 ^* 1.15±0.07 ^*

(30mg/kg)

^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the sham group; ^* P<0.05, ^** P<0.01 compared with the model group.

Experimental Discussion:

The memory impairment caused by Aβ(1-40) induced by continuous lateral ventricle perfusion in rats is a recognized model for observing the therapeutic effect of drugs on AD. From the above results, it can be seen that L-NBP not only has a significant effect on the model of vascular dementia caused by insufficient blood supply caused by 2-VO, but also has a significant effect on the memory impairment of near memory and spatial position caused by continuous lateral ventricle perfusion Aβ(1-40) in rats There is also a significant improvement. This shows that L-NBP can significantly improve the impairment of near memory and spatial position memory caused by different reasons. In addition, L-NBP has the effect of blocking oxidative damage (increasing GSH-Px activity and reducing MDA content), combined with its obvious brain protection effect, it is suggested that L-NBP has the effect of treating and preventing senile dementia.

Claims (6)

1, the application in the medicine of preparation prevention or treatment dementia of L-NBP shown in general formula (I).

According to the application of claim 1, it is characterized in that 2, described dementia is an Alzheimer.

According to the application of claim 1, it is characterized in that 3, described dementia is a vascular dementia.

4,, it is characterized in that the dose therapeutically effective of this chemical compound is: 0.1～100mg/kg/ days according to the application of claim 1.

5, a kind of pharmaceutical composition is characterized in that, contains the L-NBP and the pharmaceutical carrier shown in general formula (I) for the treatment of effective dose.

According to the pharmaceutical composition of claim 5, it is characterized in that 6, described pharmaceutical composition comprises tablet, capsule, pill, injection, slow releasing preparation, controlled release preparation and various particulate delivery system.

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