CN112494491B

CN112494491B - Use of Spanish in preparing medicine for preventing or treating neurodegenerative disease

Info

Publication number: CN112494491B
Application number: CN202011471876.6A
Authority: CN
Inventors: 侯悦; 李宁; 何大阔; 杨艳秋
Original assignee: Northeastern University China
Current assignee: Northeastern University China
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-04-01
Anticipated expiration: 2040-12-14
Also published as: CN112494491A

Abstract

The invention relates to a medical application of Spanish in preventing or treating neurodegenerative diseases, belongs to the field of medicines, and particularly relates to a method for improving ischemic stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis or Huntington's disease by Spanish. Pharmacological experiments show that the Spanish has a good treatment effect on ischemic cerebral apoplexy caused by arterial ischemia reperfusion and has a remarkable treatment effect on Alzheimer disease model mice caused by LPS, so that the Spanish can be used for preparing medicines for preventing or treating neurodegenerative diseases.

Description

Use of Spanish in preparing medicine for preventing or treating neurodegenerative disease

Technical Field

The invention belongs to the field of medicines, and particularly relates to a new medical application of Spanish in preparation of medicines for preventing or treating neurodegenerative diseases.

Background

Neurodegenerative diseases are a class of progressively-developing disabling, severely lethal complex diseases characterized primarily by massive loss of specific neurons. It can be divided into acute neurodegenerative disease and chronic neurodegenerative disease, the former mainly includes apoplexy and brain injury; the latter mainly includes Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease (HD), and the like. Although the disease is different in the pathological part and the etiology, irreversible degeneration of neuronal cells is one of the common characteristics of the diseases. The patients with the diseases can have dysfunction of movement or memory along with the prolongation of the disease duration, and the living standard and quality of the patients and family members are seriously affected. The diseases mainly occur in middle-aged and elderly people, the incidence of neurodegenerative diseases is increasing along with the aging of the population and the improvement of living standard, and the incidence of the neurodegenerative diseases in the young and middle-aged people tends to increase year by year. Therefore, the search for safe and effective medicaments is of great significance.

Apoplexy, also known as stroke, is a common cerebrovascular disease in which the blood supply to brain tissue is insufficient due to rupture or infarction of cerebral vessels, and the brain tissue is damaged. The diseases have the characteristics of high morbidity, high mortality and high disability rate, are the first cause of death of residents in China at present, and have obvious younger morbidity. Alzheimer's Disease (AD) is also a common disease, with four million people in the united states suffering from AD, the more hundreds of thousands of people dying from AD each year, and medical costs as high as $ 600 billion per year. The epidemiological research on AD in China is incomplete, and generally, the prevalence rate of dementia in people over 65 years old is about 4%, and the annual incidence rate is 0.6-1.2%. The prevalence of PD is second only to AD, occurring mainly in people over the middle age, with prevalence of 2% in people over 65 years old. In addition, ALS, HD and other diseases occur in severe neurodegenerative diseases of different age groups.

For many years, the treatment of such diseases has been a problem due to the complexity of brain function. In recent decades, with the rapid development of knowledge and research means of molecular biology, neurobiology, behavioral science and other subjects, the research of the pathological mechanism of neurodegenerative diseases has been found. Based on the research on the mechanism of neurodegenerative diseases, researchers find that the neurodegenerative diseases are prevented by protecting nerve cells, so that the purpose of treating diseases is achieved, and the life quality of patients can be effectively improved.

The medicines for treating neurodegenerative diseases on the market mainly aim at improving the symptoms of the diseases in an auxiliary way, but cannot radically prevent the disease from deteriorating. In essence, over the past decades, new drug development in this area has been almost completely covered by the army. Many of the drugs in the pharmaceutical profunds of fevered and asikang failed in the 3 rd clinical stage. In 2019, the drug administration of our country conditionally approves that the mannitol sodium is marketed for treating the alzheimer disease, and the success of the mannitol sodium brings encouragement to Chinese scholars to search for research and development ideas of drugs for preventing or treating neurodegenerative diseases from natural products.

The genus Spanish is an aporphine alkaloid of isoquinoline alkaloids, and is widely present in plants of multiple genera in multiple families, such as Menispermaceae and Magnoliaceae. Its molecular weight is 339 and molecular formula is C₂₀H₂₁NO₄The molecular structural formula is shown as formula (I).

The Klaining has stronger biological activity and is reported in the prior art. For example, Spanisin is used in the treatment of acute and subacute inflammation, including xylene-induced ear swelling and carrageenan-induced toe swelling in mice; subacute inflammation includes cotton ball granuloma (CN 108524512A). As another example, as an active ingredient in a transdermal patch, it is used for the treatment of cardiac arrhythmias, including rat arrhythmias induced by barium chloride (CN 107019683A). However, to date, no report has been made on the preventive or therapeutic effects of Spanish on neurodegenerative diseases.

In view of the above, there is still a continuous urgent need for a novel drug for preventing or treating neurodegenerative diseases, which has good efficacy, low side effects and good tolerance worldwide. In view of the above, the present inventors have developed a drug for preventing or treating neurodegenerative diseases.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, realize the development and utilization of natural products by adopting a modern drug research method, and provide a new medical application of the Spanish by combining a large amount of pharmacodynamic experiment screening, namely the application of the Spanish in preventing or treating neurodegenerative diseases.

In particular, the invention relates to the use of Spanish for the preparation of a medicament for the prevention or treatment of neurodegenerative diseases.

Preferably, the neurodegenerative disease is ischemic stroke, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis or huntington's disease.

Preferably, the use of Spanisin as the sole active ingredient in the manufacture of a medicament as described above.

Alternatively, the use of Spanish together with other active ingredients in the manufacture of a medicament; wherein the other active ingredient is selected from one or more of donepezil, galantamine, rivastigmine, memantine, prednisone, rofecoxib, nimesulide, diclofenac, rhodanine, ginkgo biloba extract, ginsenoside, huperzine A, stilbene glycoside, levodopa, carbidopa, benserazide, phenmedic, phentolin, prohexadine, prophenamine, butyphthalide, dipyridamole, low molecular weight dextran, heparin, urokininogenase, citicoline, butyphthalide, edaravone, nimodipine or aspirin.

Preferably, the further active ingredient is selected from butylphthalide or donepezil.

Preferably, the said flibanserin is a flibanserin modification or a pharmaceutically acceptable salt thereof. More preferably, the spanish modification is bromospanish.

Preferably, the medicament is selected from one or more of oral preparation or non-oral preparation.

Preferably, the oral preparation is one or more of capsules, tablets, oral liquid, granules, pills, powder, pellets or paste.

Preferably, the non-oral formulation is one or more of an injection, a cream or a patch.

Preferably, the injection is administered by one or more of subcutaneous administration, intramuscular administration or intravenous administration.

Preferably, the pharmaceutically acceptable excipients of the present invention include, but are not limited to: (1) diluents, for example, starch, sugar powder, dextrin, lactose, pregelatinized starch, microcrystalline fiber, inorganic calcium salts (e.g., calcium sulfate, calcium hydrogen phosphate, calcium carbonate for pharmaceutical use, etc.), mannitol, etc., vegetable oils, polyethylene glycol, cacao butter, semisynthetic or fully synthetic fatty acid glycerides, glycerogelatin, etc.; (2) binders, for example, distilled water, ethanol, starch slurry, povidone, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hypromellose, and the like; (3) disintegrants, for example, dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crospovidone, croscarmellose sodium, crospovidone, and the like; (4) lubricants, for example, magnesium stearate, aerosil, talc, hydrogenated vegetable oils, polyethylene glycols, magnesium lauryl sulfate, etc.; (5) solvents such as water for injection, ethanol, etc.; (6) examples of the preservative include benzoic acid and salts thereof, sorbic acid and salts thereof, and parabens.

Preferably, the subject of use is a mammal.

Compared with the prior art, the invention has the following beneficial effects: the inventor firstly discovers that the Spanish can be used for preventing or treating neurodegenerative diseases. Pharmacological experiments show that the Spanish has a good treatment effect on ischemic cerebral apoplexy caused by arterial ischemia reperfusion and has a remarkable treatment effect on AD model mice caused by LPS, so that the Spanish can be used for preparing medicines for preventing or treating neurodegenerative diseases.

Detailed Description

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example 1: function of Klaining in treating cerebral arterial thrombosis of rat caused by ischemia-reperfusion of middle artery

1. Purpose of experiment

Establishing an ischemic stroke model caused by middle artery ischemia reperfusion, and evaluating the treatment effect of the Spanish on the ischemic stroke by observing the indexes of the nerve dysfunction and the brain injury degree of a rat, the survival quantity of neuron cells, the high and low oxidative stress level and the like.

2. Experimental Material

2.1. Experimental animals: healthy 6-8 week old SPF-grade male Sprague-Dawley (SD) rats weighing 280-300g were purchased from Shenyang pharmaceutical university laboratory animal center.

2.2. The tested drugs are:

spray formulation of banning: the self-made dosage is 250 mu g/kg and 500 mu g/kg respectively based on the content of the Spanish. Blank spray formulation: the self-made spray is prepared in parallel with the spray preparation of the Spanish spray, and the main components are diluted ethanol and medical high-purity water. Butyl phthalide: shiyao group Enbipu pharmaceutical Co., Ltd, the national Standard H20050299.

3. Experimental methods

3.1. Establishment and experimental grouping of middle artery ischemic infarction (MCAO) model

After anesthesia of SD rats, the right common carotid artery, external carotid artery and internal carotid artery of the rats were exposed. The smooth thread plug is inserted into the bifurcation of the external carotid artery 3mm away from the bifurcation, and is pushed gently to lead the plug thread to enter the starting end of the middle cerebral artery along the internal carotid artery, and the plug thread is fixed by a standby silk thread. The suture is fixed by a suture thread through a thread plug and ligation of external carotid artery, and then the suture is sutured. After 1h, the plug was pulled out by about 1cm to restore blood supply and form reperfusion injury. Sham rats were not inserted with a wire plug and the rest of the procedure was as above. All successfully molded SD rats were randomly assigned to 11 model groups, 11 butylphthalide (80mg/kg) groups, 11 Spanisin low dose (250. mu.g/kg) groups, 11 Spanisin high dose (500. mu.g/kg) groups, and 11 Spanisin and butylphthalide combination (250. mu.g/kg +40mg/kg) groups; the other 11 false operation groups are provided.

3.2. Administration of drugs

After molding for 1h, the nasal feed is given to the Spanish at 20 mu L/time, the single action group is 250 mu g/kg/time or 500 mu g/kg/time, and the combined action group is given at 250 mu g/kg/time; the butylphthalide is administrated by intragastric administration, the single action group is 80 mg/kg/time, and the combined action group is 40 mg/kg/time; the model group and sham-operated group rats were given the same volume of placebo blank.

3.3. Observation index

After 24h of administration, the neurological function of each group of rats was assessed using a grade 5 scoring system reported by Zea Longa; detecting the water content of the brain by a dry-wet weight method, and evaluating the cerebral edema of the rat; TTC staining method for detecting cerebral infarction volume for evaluating cerebral infarction of rat; detecting changes in neurons using immunofluorescence; DHE staining was used to detect oxidative stress in the cortical region.

4. Results of the experiment

4.1. Effects of Spanish on neurological dysfunction and brain injury in MCAO model rats

The results are shown in Table 1.

TABLE 1 effects of Spanish on neurological dysfunction and brain injury in MCAO model rats (mean + -SEM)

Note:^###p<0.001 represents comparison to sham group;^***p<0.001 represents comparison to model set; n.s. no significant difference.

As can be seen from table 1, after MCAO surgery for 24h, the neurological score of the model rats was significantly increased, and the brain water content and the cerebral infarction volume were significantly increased, which indicates that the MCAO model rats had severe neurological impairment. Both the combination of Spanish (500. mu.g/kg) and Spanish and butylphthalide were effective in reducing neurological impairment in MCAO model rats.

4.2. Effects of Spanish on ischemic lateral cortical neurons in MCAO model rats

The results are shown in Table 2.

TABLE 2 effects of Spanisin on ischemic lateral cortical neurons in MCAO rats (mean. + -. SEM)

Note:^###p<0.001 represents comparison to sham group;^**p<0.05，^**p<0.01 represents comparison to the model set.

As can be seen from table 2, the NeuN fluorescence intensity in the ischemic side cortex region was significantly reduced in the model group rats compared to the sham operation group, indicating that neurons in the MCAO model rats had been severely damaged. Both Spanism (500. mu.g/kg) and the combined effect of Spanism and butylphthalide were effective in reversing NeuN neuronal damage in MCAO model rats.

4.3. Effect of Spanish on oxidative stress of ischemic lateral cortex of MCAO model rats

The results are shown in Table 3.

TABLE 3 effects of Spanish on oxidative stress of ischemic lateral cortex of MCAO rats (mean. + -. SEM)

Note:^###p<0.001 represents comparison to sham group;^**p<0.01，^***p<0.001 represents comparison to the model set.

As can be seen from table 3, DHE fluorescence intensity of the ischemic lateral cortex cells was significantly increased in the model group rats compared to the sham-operated group, which indicates that the oxidative stress level of the cortex region was significantly increased in the MCAO model rats. Butylphthalide, cobinin (500 μ g/kg) and the combination of cobinin and butylphthalide were all effective in reducing oxidative stress in the cortical areas of MCAO model rats.

5. Conclusion

The research results show that the Spanish has good treatment effect on ischemic cerebral apoplexy caused by the ischemia-reperfusion of the middle artery, can effectively improve the neurological dysfunction, inhibit cerebral edema and cerebral infarction, reduce the loss of cortical neuron cells caused by MCAO and weaken the oxidative stress reaction. Low doses of the combination of clonine and clonine also provide significant improvement.

Example 2: therapeutic effect of Klaining on AD model mice injected with lipopolysaccharide from bilateral hippocampus

1. Purpose of experiment

Alzheimer's disease is one of the common neurodegenerative diseases in the middle-aged and the elderly. In the experiment, a mouse dementia model caused by Lipopolysaccharide (LPS) injected into ventricles of the hippocampus on two sides is used, and the treatment effect of the Spanisin on AD mice is evaluated by observing the learning and memory capacity and the change of the number of neurons of the mice.

2. Experimental Material

2.1. Experimental animals: healthy SPF grade male C57BL/6 mice, 6-8 weeks old, weigh 20-25 g. Purchased from the animal testing center of Shenyang pharmaceutical university.

2.2. The tested drugs are: a spanish suspension: the self-made coating is dissolved in CMC-Na solution according to the content of the Spanish, and has three specifications of 5mg/mL, 10mg/mL and 20mg/mL respectively. Donepezil hydrochloride: shanxi Fangzhou pharmacy Co., Ltd, the Standard character of the national medicine H20030583. Blank suspension: homemade, prepared in parallel with the above-mentioned flibaning suspension, only as a CMC-Na solution.

3. Experimental methods

3.1. Establishment of AD model by injecting LPS into bilateral hippocampal ventricles and grouping experiments

C57BL/6 mice were anesthetized and bregma was exposed and marked as the origin. Mice were fixed in a mouse stereotaxic apparatus, hippocampus was positioned according to brain pattern, LPS (40. mu.g/mL) was injected at a rate of 1. mu.L/min using a microsyringe, and the needle was left for 5 min. Mice in the sham operation group were injected with physiological saline. Mice were randomized into model groups of 8 mice each, 8 sham surgery groups, donepezil (50mg/kg), low, medium, and high doses of clonidine (5, 10, or 20mg/kg), and a combination of donepezil (25mg/kg) and clonidine (5 mg/kg).

3.2. Administration of drugs

Before 7d of modeling, the ketamine group, donepezil group, and combination of ketamine and donepezil are administered by gastric gavage, and the medicine is administered in an amount of 0.1mL/10g once a day; the model group was given placebo; until the end of the experiment.

3.3. Observation index

The influence of the drugs on the motor capacity of each group of mice is investigated by using an open field experiment; the influence of the drugs on the learning and memory abilities of all groups of mice is investigated by utilizing a Morris water maze experiment; detecting changes in neurons using immunofluorescence; DHE staining was used to detect oxidative stress in the cortical region.

4. Results of the experiment

4.1. Effect of Spanish on motor function of mice model injected with LPS from bilateral hippocampus

The results are shown in Table 4.

TABLE 4 effects of Spanism on locomotor function of mice from bilateral hippocampal LPS-injected model (mean. + -. SEM)

Note: n.s. no significant difference.

As can be seen from table 4, the mean movement velocity and total movement distance of each group of mice did not significantly change after 7d of surgery. This suggests that bilateral hippocampal LPS injection and administration of the spanin treatment had no significant effect on the locomotor ability of the mice.

4.2. Effect of Spanish on learning ability of bilateral hippocampal LPS-injected model mice

The results are shown in Table 5.

TABLE 5 impact of Spanish on learning ability of bilateral hippocampal LPS-injected model mice (mean. + -. SEM)

Note:^###p<0.001 represents comparison to sham group;^*p<0.05，^**p<0.01，^***p<0.001 represents comparison to model set; n.s. no significant difference.

As can be seen from table 5, the mean movement speed of each group of mice did not significantly change after the operation 12d, and the escape latency distance were significantly increased. This suggests that LPS causes a decrease in learning ability of mice. The escape latency and latency distance of the mice in the group of Spanish (10, 20mg/kg) were significantly reduced, with no significant change in locomotor speed. The combined effect group of low dose of the clobanine and the donepezil also has obvious improvement effect. 4.3. Effect of Spanish on memory of mice in bilateral hippocampal LPS-injected model

The results are shown in Table 6.

TABLE 6 effects of Spanish on memory of mice from bilateral hippocampal LPS-injected model (mean. + -. SEM)

Note:^###p<0.001 represents comparison to sham group;^*p<0.05，^**p<0.01，^***p<0.001 represents comparison to the model set.

As can be seen from table 6, after 20d of the operation, the number of times of platform crossing of the model group mice is significantly reduced in the target quadrant, compared with the number of times of platform crossing of the sham operation group mice; the number of landings, residence time and distance of movement in the target quadrant, was significantly increased in the mice of the Spanish (5, 10 or 20mg/kg) group. The combined effect group of low dose of the clobanine and the donepezil also has obvious improvement effect.

4.4. Effect of Spanish on neuronal expression in mice model with bilateral hippocampal LPS injection

The results are shown in Table 7.

TABLE 7 Effect of Spanish on neuronal expression in mice model by bilateral hippocampal LPS injection (mean. + -. SEM)

Note:^##p<0.01 represents comparison with sham group;^*p<0.05，^**p<0.01 represents comparison to the model set.

As can be seen from table 7, the number of neurons was significantly reduced in the model group mice compared to the sham-operated group mice after 20d of the operation; spanish (10 or 20mg/kg) was able to effectively inhibit LPS-induced neuronal loss. The combined effect group of low dose of the clobanine and the donepezil also has obvious improvement effect.

4.5. Effect of Spanish on oxidative stress response of mice model injected with LPS from bilateral hippocampus

The results are shown in Table 8.

TABLE 8 impact of Spanish on oxidative stress in mice from bilateral hippocampal LPS-injected model (mean. + -. SEM)

Note:^###p<0.001 represents comparison to sham group;^**p<0.01，^***p<0.001 represents comparison to model set; n.s. no significant difference.

As can be seen from table 8, the expression level of DHE was significantly increased in the model mice after 20d of surgery, compared to the sham-operated mice; the expression level of DHE was significantly reduced in mice from the group of Spanish (5, 10 or 20 mg/kg). The combined effect group of low dose of the clobanine and the donepezil also has obvious improvement effect.

5. Conclusion

The research results show that the Spanish has a remarkable treatment effect on the AD model mouse caused by the LPS, can effectively improve learning and memory damage caused by the LPS, and weakens neuron damage and oxidative stress reaction caused by the LPS. The low dose of the Spanish and the combined action of the Spanish and other medicines can also obviously improve the learning and memory disorder of a model mouse and inhibit the damage and the oxidative stress of neurons. The research suggests that the Spanish can be used for treating neurodegenerative diseases.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. Use of Spanish as the sole active ingredient in the manufacture of a medicament for the prevention or treatment of ischemic stroke.

2. Application of a composition of Spanish and butylphthalide in preparing a medicament for preventing or treating ischemic stroke.

3. The use according to claim 1 or claim 2, wherein the medicament is selected from one or more of an oral formulation or a non-oral formulation.

4. The use of claim 3, wherein the oral formulation is one or more of a capsule, a tablet, an oral liquid, a granule, a pill, a powder, a pellet, or an ointment.

5. The use according to claim 3, wherein the non-oral formulation is one or more of an injection, a cream, a patch, an ointment or a spray.

6. The use of claim 5, wherein the injectable formulation is administered by one or more of subcutaneous, intramuscular, or intravenous administration.

7. The use according to claim 1 or claim 2, wherein the subject of said medicament is a mammal.