A composition for stroke

Abstract

The present invention relates to a composition for preventing cerebral stroke, and in particular to a composition containing effective amounts of B vitamins. The composition provided by the present invention has a significant synergistic therapeutic effect on cerebral stroke, and belongs to the field of pharmacy.

Description

Technical Field

[0001] The invention relates to a product for treating cerebral stroke and belongs to the field of pharmacy. Background Art

[0002] Stroke is an acute cerebrovascular disease. It is a group of diseases that cause brain tissue damage due to sudden rupture of brain blood vessels or blood vessel blockage that prevents blood from flowing into the brain, including ischemic and hemorrhagic strokes. The incidence of ischemic stroke is higher than that of hemorrhagic stroke, accounting for 60% to 70% of the total number of strokes. Occlusion and stenosis of the internal carotid artery and vertebral artery can cause ischemic stroke, which mostly occurs in people over 40 years old, more often in men than in women, and can cause death in severe cases. Stroke is characterized by high incidence, high mortality and high disability rate.

[0003] B vitamins are composed of compounds with similar biological activities but different chemical compositions. They can exist in free form and are widely involved in various physiological processes in the form of coenzymes. They cannot be synthesized in animals and some microorganisms, so they must be obtained from the outside. Generally, plants can synthesize various vitamins. The demand for vitamins in biological organisms is not high, but they are indispensable.

[0004] Borneol is a commonly used Chinese medicine. It is a colorless, transparent or white, translucent, flaky, crisp crystal. It has a light fragrance, a pungent and cool taste. It is volatile, easily sublimated, and produces thick smoke and a bright flame when ignited. Borneol has a light fragrance and has the effects of refreshing the mind, clearing away heat and toxins, improving eyesight and removing cataracts. It is mainly used to treat fever, high fever, coma, summer heat and dampness obstructing the clear mind, throat paralysis and deafness, mouth sores, tooth swelling, sores, carbuncle, malnutrition, hemorrhoids, red and swollen eyes, and cataracts covering the eyes.

[0005] 3-Methyl-1-phenyl-2-pyrazolin-5-one is a free radical scavenger and inhibitor, which can easily enter the brain tissue through the blood-brain barrier to work. 3-Methyl-1-phenyl-2-pyrazolin-5-one inhibits the activity of xanthine oxidase and hypoxanthine oxidase, reduces the concentration of hydroxyl free radicals, inhibits lipid peroxidation, reduces oxidative cell damage caused by lipid peroxides, the metabolic intermediate of arachidonic acid in the brain, prevents and treats vascular endothelial damage, and has the effect of resisting ischemia and reducing the area of ​​infarction. Summary of the invention

[0006] The purpose of the present invention is to provide a pharmaceutical composition for treating cerebral stroke, comprising B vitamins.

[0007] The present invention provides a composition for treating cerebral stroke, comprising an effective amount of B vitamins, wherein the B vitamins are selected from vitamin B6, vitamin B 12 Or folic acid substances, among which the effective content of vitamin B6 is 0.5-50㎎, the effective content of folic acid substances is 0.2-10㎎, and the effective content of vitamin B 12 The effective content is 0.1-2 mg.

[0008] In the present invention, the vitamin B6 is selected from pyridoxine, pyridoxal, pyridoxamine, pyridoxal phosphate, pyridoxamine phosphate and derivatives of the above substances and substances that can release / generate such compounds in vivo.

[0009] In the present invention, the vitamin B 12 Selected from methylcobalamin, 5'-deoxyadenosylcobalamin, hydroxocobalamin, cyanocobalamin or other substances that can release / generate cobalamin in vivo.

[0010] In the present invention, the folic acid substances include folic acid, calcium folate, 5-methyltetrahydrofolate, etc. Various salts or active metabolites of the above substances and substances that can release / generate folic acid in vivo are also included in the protection scope of the present invention.

[0011] In the present invention, the composition may further contain borneol or its extract. The borneol described in the present invention includes natural borneol and synthetic borneol, and natural borneol is preferably used. In the present invention, the effective content of borneol is 0.004-6g, preferably 4-15mg.

[0012] In the present invention, the composition may further contain 3-methyl-1-phenyl-2-pyrazol-5-one. In the present invention, the effective content of 3-methyl-1-phenyl-2-pyrazol-5-one is 10-100 mg, preferably 30-60 mg.

[0013] As a preferred embodiment, the composition provided by the present invention is composed of B vitamins, borneol and 3-methyl-1-phenyl-2-pyrazol-5-one. The B vitamins are 5-methyltetrahydrofolate and vitamin B 12 One or two of the above, wherein the borneol is borneol.

[0014] As a preferred embodiment, the ratio of 3-methyl-1-phenyl-2-pyrazol-5-one to borneol in the present invention is 4:1-1:4.

[0015] As a preferred embodiment, the group B vitamins provided by the present invention are 5-methyltetrahydrofolate, wherein the effective content of 5-methyltetrahydrofolate is preferably 0.8 mg-1.6 mg.

[0016] As a preference, in the present invention, the ratio of 5-methyltetrahydrofolate, 3-methyl-1-phenyl-2-pyrazol-5-one and borneol is preferably 1:36:9-2:36:9.

[0017] As a preferred embodiment, the vitamin B group provided by the present invention is folic acid, wherein the effective content of folic acid is preferably 0.8mg-1.6mg. In the present invention, the ratio of folic acid, 3-methyl-1-phenyl-2-pyrazol-5-one and borneol is preferably 1:36:9-2:36:9.

[0018] The present invention also provides use of the above composition in preparing a product for treating cerebral stroke.

[0019] Preferably, the present invention uses the above composition in preparing a product for ischemic stroke or cerebral infarction.

[0020] In the use provided by the present invention, the above composition may also contain pharmaceutical excipients to prepare products that can be conveniently used by users. The products include oral products and injection products. Oral products include tablets, capsules, granules, oral liquids, films or patches, etc. Pharmaceutical excipients include but are not limited to excipients, fillers, binders, disintegrants, etc. Excipients include mannitol, lactose, dextran, cysteine, cyclodextrin, etc.; fillers include lactose, starch, sucrose, mannitol, sorbitol, microcrystalline cellulose, calcium dihydrogen phosphate, etc.; binders include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, starch, ethyl cellulose, methyl cellulose, etc.; disintegrants include sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked povidone, starch, etc. Injection products may contain a solvent, and the solvent may be a water-soluble organic solvent or a mixture of a water-soluble organic solvent and water. Commonly used aqueous solvents and organic solvents include alcohol solvents, ether solvents, ketone solvents, etc. Commonly used alcohol solvents include ethanol, isopropanol, ethylene glycol, propylene glycol, etc.; commonly used ether solvents include ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, etc.; commonly used ketone solvents include acetone and N-methyl-2-pyrrolidone. Propylene glycol is preferably used as the aqueous solvent organic solvent.

[0021] The pharmaceutical composition provided by the present invention has a significant therapeutic effect on cerebral stroke, especially after B vitamins are combined with borneol or 3-methyl-1-phenyl-2-pyrazol-5-one or a combination of borneol and 3-methyl-1-phenyl-2-pyrazol-5-one, the therapeutic effect can be significantly enhanced. Not only is the effective dose of the components low, the toxicity is small, and there is a significant synergistic effect, so it is a composition for treating cerebral stroke.

[0022] The present invention is further described below in conjunction with specific implementation modes, which is not intended to limit the present invention. Any equivalent replacements in the art made according to the contents of the present invention shall fall within the protection scope of the present invention. DETAILED DESCRIPTION

[0023] Example 1

[0024] Take 30 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one and 120 mg of synthetic borneol, add them to propylene glycol solution, stir to dissolve completely, slowly add water for injection containing 5 mg of folic acid to dissolve it, adjust the water for injection to 100 ml, and you have it.

[0025] Example 2

[0026] Take 30 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one and 7.5 mg of natural borneol, add them to propylene glycol solution, stir to dissolve completely, slowly add water for injection containing 0.8 mg of folic acid to dissolve it, adjust the water for injection to 100 ml, and you have it.

[0027] Example 3

[0028] Take 30 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one and 7.5 mg of natural borneol, add them to propylene glycol solution, stir to dissolve completely, slowly add water for injection containing 0.8 mg of 5-methyltetrahydrofolate to dissolve it, adjust the water for injection to 100 ml, and you have it.

[0029] Example 4

[0030] Take 30 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one and 7.5 mg of natural borneol, add them to propylene glycol solution, stir to dissolve completely, slowly add water for injection containing 1.6 mg of 5-methyltetrahydrofolate to dissolve it, adjust the water for injection to 100 ml, and you have it.

[0031] Example 5

[0032] Take 30 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one and 7.5 mg of natural borneol, add them to propylene glycol solution, stir to dissolve completely, slowly add water for injection containing 5 mg of 5-methyltetrahydrofolate and 0.25 mg of vitamin B12 to dissolve it, adjust the water for injection to 100 ml, and you have it.

[0033] Example 6

[0034] Take 30 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one and 15 mg of natural borneol, add them to propylene glycol solution, stir to dissolve completely, slowly add water for injection containing 5 mg of 5-methyltetrahydrofolate and 0.25 mg of vitamin B12 to dissolve it, adjust the water for injection to 100 ml, and you have it.

[0035] Example 7

[0036] Take 15 mg of 3-methyl-1-phenyl-2-pyrazolin-5-one and 15 mg of natural borneol, add them to propylene glycol solution, stir to dissolve completely, slowly add water for injection containing 0.4 mg of 5-methyltetrahydrofolate and 0.25 mg of vitamin B12 to dissolve, adjust the water for injection to 100 ml, and you have it.

[0037] Example 8

[0038] Take 30 g of 3-methyl-1-phenyl-2-pyrazolin-5-one, 7.5 g of natural borneol, 800 mg of 5-methyltetrahydrofolate, 35 g of mannitol, 4 g of hydroxypropyl methylcellulose, 4 g of cross-linked carboxymethyl cellulose and 0.8 g of magnesium stearate, mix them evenly, and tablet them to obtain 1000 sublingual tablets containing 3-methyl-1-phenyl-2-pyrazolin-5-one, natural borneol and 5-methyltetrahydrofolate.

[0039] Example 9

[0040] Take 40g of 3-methyl-1-phenyl-2-pyrazol-5-one, 10g of natural borneol, 5g of 5-methyltetrahydrofolate, and 250mg of vitamin B 12 , lactose 25g, hydroxypropyl methylcellulose 4g, cross-linked carboxymethyl cellulose 4g and magnesium stearate 0.8g are mixed evenly and tableted to obtain 1000 tablets containing 3-methyl-1-phenyl-2-pyrazol-5-one, natural borneol, 5-methyltetrahydrofolate and vitamin B 12 Sublingual tablet.

[0041] Example 10

[0042] Take 60g of 3-methyl-1-phenyl-2-pyrazol-5-one, 15g of natural borneol, 1g of 5-methyltetrahydrofolate, and 500mg of vitamin B 12 , mannitol 50g, hydroxypropyl methylcellulose 4g, cross-linked carboxymethyl cellulose 4g and magnesium stearate 0.8g were mixed evenly and tableted to obtain 1000 tablets containing 3-methyl-1-phenyl-2-pyrazol-5-one, natural borneol, 5-methyltetrahydrofolate and vitamin B 12 tablets.

[0043] Example 11 Effect of the composition on rats with cerebral ischemia-reperfusion injury

[0044] 1. Methods

[0045] Male SD rats about 7-8 weeks old were randomly divided into sham operation group, model group, 3-methyl-1-phenyl-2-pyrazolin-5-one group (referred to as ketone group) (3 mg / kg), natural borneol group (0.75 mg / kg), 5-methyltetrahydrofolate group (0.08 mg / kg), 3-methyl-1-phenyl-2-pyrazolin-5-one + 5-methyltetrahydrofolate group (referred to as ketone leaf group) (3 mg / kg + 0.08 mg / kg), 3-methyl-1-phenyl-2-pyrazolin-5-one + natural borneol group (referred to as ketone ice group) (3 mg / kg + 0.75 mg / kg), 3-methyl-1-phenyl-2-pyrazolin-5-one + natural borneol + 5-methyltetrahydrofolate group (referred to as ketone ice leaf group) (3 mg / kg + 0.75 mg / kg), 12 rats in each group. The above doses were administered by oral gavage 3 days before and 2 hours after the establishment of the cerebral ischemia-reperfusion model.

[0046] Model building

[0047] Rats were anesthetized, the neck was opened, the external carotid artery and the internal carotid artery were separated, and the thread plug was inserted into the common carotid artery, advanced to about 16-18mm from the internal artery, fixed, and the wound was sutured. After the thread plug was inserted for 2 hours, the thread plug was removed and the common carotid artery was ligated. In the sham operation group, only the common carotid artery was freed after anesthesia.

[0048] Neurological assessment

[0049] 24 hours after the suture plug was removed, the neurological function of rats in each group was evaluated.

[0050] According to the Bederson neurological function grading standard, the patients were divided into the following 5 levels and observed once a day.

[0051] Grade 0 (normal): No symptoms of neurological deficit.

[0052] Grade 1 (mild): Forelimb curling or flexion, mainly persistent wrist flexion or shoulder adduction with elbow extension.

[0053] Grade 2 (moderate): The forelimbs are continuously curled up or flexed, with complete flexion of the wrist and elbow, and adduction or internal rotation of the shoulder.

[0054] Grade 3 (severe): Continuous limb curling or flexion, continuous weakening of the limb's thrust resistance on one side, and no circling behavior during autonomous movement.

[0055] Grade 4 (very severe): The bilateral limbs are persistently curled up or flexed, the thrust resistance of one limb is persistently weakened, and the limbs make circles during autonomous activities.

[0056] Cerebral infarction assessment

[0057] The whole brain tissue of rats was isolated and frozen, cut into 5 slices of about 2 mm thick, immersed in 2% triphenyltetrazolium chloride solvent, incubated in the dark for 30 minutes, and fixed with 4% paraformaldehyde at 37°C for analysis.

[0058] Normal brain tissue appears red after staining, and cerebral infarction appears white. The white tissue is weighed, and the percentage of infarct tissue weight to total brain weight is used as the percentage of infarct volume for comparison.

[0059] Statistical analysis

[0060] The experimental data were analyzed using SPSS22.0 software.

[0061] In order to verify the scientific nature of the pharmaceutical composition provided by the present invention, and to illustrate that the three components of the pharmaceutical composition are reasonably matched and can play a synergistic role in combination, rather than simply superimposing pharmacological effects, the Jin Zhengjun Q value method is introduced for analysis. The Jin Zhengjun Q value method is also known as the probability addition method. According to the pharmacological effects of the combination of two drugs and the pharmacological effects of the two drugs alone in the dose-effect curve area, the following calculation formula is used to calculate: Q = E A+B / (E A +E B -E A *E B ), where the numerator represents the "measured combined effect" and the denominator represents the "expected combined effect". (In order to analyze the pharmacological effects of components and combinations, their pharmacological effects are converted into effects that can intuitively reflect the strength of the pharmacological effects. The calculation formula is: E i =1-P i / P 模型组 , P i is the pharmacological index of each component, P 模型组 is the pharmacological index of the model group), Q is the ratio of the two: when Q is less than 0.85, the combination of the two drugs is considered to be antagonistic; when it is less than 1.15 and greater than 0.85, it is considered to be additive; when it is greater than 1.15, it is considered to be synergistic.

[0062] result

[0063] Table 1 Comparison of neurological function grades in stroke patients (n=1)

[0064]

[0065] Compared with the sham operation group, the survival number of rats in the model group was lower than that in the sham operation group, and there was 1 rat of grade 2, 3 rats of grade 3, and 3 rats of grade 4, indicating that the model of cerebral ischemia-reperfusion injury was successfully established. Compared with the model group, the survival number of rats in the ketone group increased, not only grade 1 rats, but also the percentage of grade 2 rats was higher than that of the model group, and the percentage of grade 3 and grade 4 was lower than that of the model group, indicating that 3-methyl-1-phenyl-2-pyrazolin-5-one can improve the neurological function grade of rats with cerebral ischemia-reperfusion injury. There was no grade 1 rat in the natural borneol group, the percentage of grade 2 and grade 4 rats was lower than that of the model group, and the percentage of grade 3 rats was higher than that of the model group, indicating that natural borneol had a slight effect on improving the neurological function grade of rats with cerebral ischemia-reperfusion injury. The percentage of grade 2 rats in the 5-methyltetrahydrofolate group was the same as that of the model group, the percentage of grade 3 rats was higher than that of the model group, and the percentage of grade 4 rats was lower than that of the model group, indicating that 5-methyltetrahydrofolate had a slight effect on improving the neurological function grade of rats. The number of rats surviving in the keto ice group was higher than that in the control group, and there were 2 grade 1 rats. The percentage of grade 2 rats was higher than that in the model group, and the percentages of grade 3 and grade 4 were lower than those in the model group, indicating that the keto ice composition can significantly improve the neurological function grade of rats; compared with the keto group, the number of rats surviving in the keto ice group increased, the percentage of grade 1 rats increased, and the percentage of grade 4 rats decreased, indicating that the keto ice group has a stronger effect on improving the neurological function grade of rats than the keto group. There was 1 grade 1 rat in the keto leaf group, the percentage of grade 2 rats was higher than that in the model group, and the percentages of grade 3 and grade 4 were lower than those in the model group, indicating that the keto leaf composition can significantly improve the neurological function grade of rats; compared with the keto group, the percentage of grade 3 rats in the keto leaf group increased, and the percentage of grade 4 rats decreased, indicating that the effect of the keto leaf composition on improving the neurological function grade of rats is slightly stronger than that in the keto group. The keto ice leaf group not only has a higher number of rats surviving, but also has the highest percentage of grade 1 and grade 2 rats among all groups, and the lowest percentage of grade 3 and grade 4 rats among all groups, indicating that the keto ice leaf composition is the most effective product among all products that improve the neurological function grade of rats.

[0066] Table 2 Comparison of cerebral infarction volume in rats of each group

[0067]

[0068] Compared with the sham operation group, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0069] After staining, the brain tissue sections of the rats in the sham operation group appeared red, indicating that there was no infarction area in the rats in the sham operation group. The ischemic side of the brain tissue of the rats in the model group was almost all stained white, and the volume of cerebral infarction accounted for 43.8%. Compared with the model group, there was no significant change in the natural borneol group and the 5-methyltetrahydrofolate group, but the volume of cerebral infarction in the rats in the ketone group and the ketone leaf group decreased significantly, with significant statistical differences. The volume of cerebral infarction in the rats in the ketone ice group not only decreased significantly compared with the model group, but also had a very significant statistical difference; compared with the ketone group, the volume of cerebral infarction in the rats in the ketone ice group was further reduced. The Q value of the volume of cerebral infarction in the ketone ice group, the ketone group, and the natural borneol group was 1.11, indicating that the two are additive. The volume of cerebral infarction in the rats in the ketone ice leaf group not only had a very significant statistical difference with the model group, but also was further reduced compared with the ketone group, the ketone ice group, or the ketone leaf group. The Q value of the proportion of cerebral infarction volume among the keto ice leaf group, the keto leaf group and the natural borneol group was 1.45, and the Q value of the proportion of cerebral infarction volume among the keto ice leaf group, the keto ice group and the 5-methyltetrahydrofolate group was 1.16, indicating that whether 5-methyltetrahydrofolate is added to the combination of 3-methyl-1-phenyl-2-pyrazol-5-one and natural borneol, or natural borneol is added to the combination of 3-methyl-1-phenyl-2-pyrazol-5-one and 5-methyltetrahydrofolate, the combination of 3-methyl-1-phenyl-2-pyrazol-5-one + natural borneol + 5-methyltetrahydrofolate produces a stronger synergistic effect in treating cerebral infarction than the original dual or single drug, indicating that the combination of 3-methyl-1-phenyl-2-pyrazol-5-one + natural borneol + 5-methyltetrahydrofolate is a more effective drug for treating stroke.

[0070] Example 12 Effect of the composition on rats with cerebral ischemia-reperfusion injury

[0071] 1. Methods

[0072] Male SD rats aged about 7-8 weeks were randomly divided into sham operation group, model group, keto ice group (3-methyl-1-phenyl-2-pyrazolin-5-one group + natural borneol, 3 + 0.75 mg / kg), combination group 1 (3-methyl-1-phenyl-2-pyrazolin-5-one group + natural borneol + 5-methyltetrahydrofolate, 3 + 0.75 + 0.02 mg / kg), combination group 2 (3-methyl-1-phenyl-2-pyrazolin-5-one group + natural borneol + 5-methyltetrahydrofolate, 3 + 0.75 + 0.08 mg / kg), combination group 3 (3-methyl-1-phenyl-2-pyrazol-5-one group + natural borneol + 5-methyltetrahydrofolate, 3 + 0.75 + 0.16 mg / kg), combination group 4 (3-methyl-1-phenyl-2-pyrazol-5-one group + natural borneol + 5-methyltetrahydrofolate, 3 + 0.75 + 1 mg / kg), 12 mice in each group. Three days before and 2 hours after the establishment of the cerebral ischemia-reperfusion model, the above doses were administered by gavage.

[0073] Model building

[0074] See Example 11.

[0075] Biochemical testing

[0076] The ischemic tissue of the rat was taken and mixed with PBS for homogenization. After centrifugation, the supernatant was taken and MDA, SOD and GSH were detected according to the instructions of the kit (the kit was purchased from Shanghai ELISA Biotechnology Co., Ltd.).

[0077] Compared with the sham operation group, the MDA in the model group increased significantly, and the SOD and GSH decreased significantly, with extremely significant statistical differences, indicating that the model group rats were damaged by oxygen free radicals. The ketone ice group can significantly reduce the MDA value, increase the SOD activity and GSH level, and its effect is similar to that of the composition group 1. Compared with the ketone ice group, the composition groups 2 and 3 further significantly reduced the MDA value, increased the SOD activity and GSH level on the basis of the ketone ice group, and compared with the ketone ice group, there were extremely significant statistical differences, indicating that the 3-methyl-1-phenyl-2-pyrazol-5-one + natural borneol + 5-methyltetrahydrofolate composition produced a stronger synergistic effect than the original two-combination to improve oxygen free radical damage. The effect of the composition group 4 was comparable to that of the composition group 3, but the GSH level did not increase but decreased, indicating that the addition of 5-methyltetrahydrofolate to the 3-methyl-1-phenyl-2-pyrazol-5-one and natural borneol composition was dose-dependent.

[0078] Table 3 Effect of the combination on biochemical factors in rats with cerebral ischemia-reperfusion injury

[0079]

[0080] Compared with the sham operation group, **P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the keto ice group, & P<0.05, && P<0.01.

Claims

1. A composition for treating stroke, comprising B vitamins, borneol and 3-methyl-1-phenyl-2-pyrazol-5-one, wherein the B vitamins are folic acid substances, the effective content of the folic acid substances is 0.2-10 mg, and the folic acid substance is 5-methyltetrahydrofolate or its salt, wherein the ratio of 5-methyltetrahydrofolate, 3-methyl-1-phenyl-2-pyrazol-5-one and borneol is 0.08:3:0.

75.

2. The composition according to claim 1, characterized in that The effective content of 5-methyltetrahydrofolate is 0.8-1.6 mg.

3. The composition according to claim 2, characterized in that The borneol is borneol.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for treating stroke.

5. The use according to claim 4, characterized in that The dosage form of the drug is an oral preparation or an injection preparation.

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