Application of strychnine nitric oxide in preparation of medicine for preventing and treating cerebral arterial thrombosis and composition for preventing and treating cerebral arterial thrombosis

Abstract

The invention provides application of strychnine nitric oxide in preparation of a medicine for preventing and treating cerebral arterial thrombosis and a composition for preventing and treating cerebral arterial thrombosis, and belongs to the technical field of medicines. The invention provides application of strychnine nitric oxide in preparation of a medicine for preventing and treating cerebral arterial thrombosis. According to the present invention, the strychnine nitrogen oxide retains the biological activity and the effect of strychnine, can be used for preventing and treating cerebral arterial thrombosis, has less toxicity than the strychnine, and can significantly improve cerebral arterial thrombosis neurological function impairment and reduce the infarct volume when the strychnine nitrogen oxide is applied to treatment of cerebral arterial thrombosis, such that the strychnine nitrogen oxide can be used for preventing and treating cerebral arterial thrombosis. The traditional Chinese medicine composition can be used for preventing and treating cerebral arterial thrombosis, reducing inflammatory cell infiltration of brain tissues, protecting the integrity of blood-brain barriers and adjusting tight junction proteins, has a potential effect of preventing and treating cerebral arterial thrombosis, has obvious curative effect advantages, and can improve the defects of the current clinical treatment technology for cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of strychnine nitrogen oxide in the preparation of a drug for preventing and treating ischemic stroke, and a composition for preventing and treating ischemic stroke. Background Art

[0002] Ischemic stroke refers to a clinical syndrome caused by a variety of inducing factors that lead to cerebral blood circulation disorders, local brain tissue ischemic hypoxic necrosis, and destruction of cellular homeostasis, which ultimately leads to neuronal death and corresponding neurological deficits. It has the characteristics of high morbidity, high disability rate, high mortality rate, high recurrence rate and high economic burden, and seriously threatens human health. At present, clinical treatment mainly uses thrombolysis, fiber reduction, antiplatelet and other methods, but these methods are usually limited by the time window of infarction treatment (within 4.5 to 6 hours of the acute phase of onset) and serious bleeding complications. After active treatment, many patients are still disabled, which brings a huge burden to their families and society. Therefore, it is urgent to find truly effective and safe new drugs to provide new strategies for the clinical treatment of ischemic stroke.

[0003] Strychnos nux-vomica L., also known as Strychnos nux-vomica, is the dried mature seed of the plant Strychnos nux-vomica L. of the Logarithaceae family. It is bitter, warm in nature, enters the liver and spleen meridians, and is highly toxic. It has a great effect of clearing the meridians, and is the best at removing phlegm in the meridians and removing blood stasis in the meridians. Because Strychnos nux-vomica is extremely toxic, its processed products have been used clinically since ancient times, such as frying to reduce toxicity and making roasted Strychnos nux-vomica, but there is still a problem of high toxicity. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a use of strychnine nitrogen oxide in the preparation of a drug for preventing and treating ischemic stroke, and a composition for preventing and treating ischemic stroke. In the present invention, strychnine nitrogen oxide retains the biological activity and efficacy of strychnine, and has less toxicity than strychnine.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an application of strychnine nitrogen oxide (BNO) in preparing a medicine for preventing and treating ischemic stroke.

[0007] Preferably, the pharmaceutical dosage of strychnine N-oxide is 200-800 mg / kg / day.

[0008] Preferably, the pharmaceutical dosage of strychnine N-oxide is 400-600 mg / kg / day.

[0009] Preferably, the strychnine nitrogen oxide is prepared by strychnine oxidation with hydrogen peroxide.

[0010] The invention also provides a composition for preventing and treating ischemic stroke, comprising strychnine nitrogen oxide and pharmaceutically acceptable auxiliary materials.

[0011] Preferably, the composition is in the form of a suspension, capsule, tablet, pill, pellet, granule, powder, injection or sustained-release agent.

[0012] Preferably, the content of strychnine nitrogen oxide in the composition for preventing and treating ischemic stroke is 90-95 wt %.

[0013] The invention provides an application of strychnine nitrogen oxide in preparing a medicine for preventing and treating ischemic stroke.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] In the present invention, strychnine nitrogen oxide retains the biological activity and efficacy of strychnine, can be used for preventing and treating ischemic stroke, and has less toxicity than strychnine. The application of strychnine nitrogen oxide in the treatment of ischemic stroke can significantly improve the neurological damage caused by ischemic stroke, reduce the infarct volume, reduce the infiltration of inflammatory cells in brain tissue, protect the integrity of the blood-brain barrier and regulate tight junction proteins, has the potential to prevent and treat ischemic stroke, has obvious therapeutic advantages, and can improve the current clinical treatment technology deficiencies of ischemic stroke.

[0016] The data in the examples show that feeding strychnine nitrogen oxide to ischemic stroke model mice can significantly reduce the infarct volume of mice, improve the neurological damage of mice, reduce the infiltration of inflammatory cells in brain tissue, and effectively alleviate the clinical syndrome complicated by ischemic stroke damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a reaction schematic diagram of preparing strychnine nitrogen oxide from strychnine oxidation in Example 3;

[0018] Figure 2 is a high-resolution mass spectrum of the strychnine nitrogen oxide sample obtained in Example 4;

[0019] Figure 3 The figure shows the therapeutic effect of strychnine nitrogen oxide on brain tissue damage induced by middle cerebral artery occlusion in mice with ischemic stroke in Example 5, wherein A is the longa score, B is the water content of mouse brain tissue, C is a representative picture of TTC staining of cerebral infarction, D is the volume of cerebral infarction, and E is the levels of MPO, TNF-α, IL-1β and IL-6 in mouse serum. DETAILED DESCRIPTION

[0020] The present invention provides an application of strychnine nitrogen oxide in the preparation of a drug for preventing and treating ischemic stroke. The application of strychnine nitrogen oxide in the treatment of ischemic stroke can significantly improve ischemic stroke neurological damage, reduce infarct volume, reduce brain tissue inflammatory cell infiltration, protect the integrity of the blood-brain barrier and regulate tight junction proteins. It has a potential role in preventing and treating ischemic stroke, has obvious therapeutic advantages, and can improve the current clinical treatment technology deficiencies of ischemic stroke.

[0021] In order to explore the therapeutic effect of strychnine N-oxide on stroke, a series of studies were conducted on strychnine N-oxide using the most commonly used mammalian model mice. The results showed that feeding strychnine N-oxide to ischemic stroke model mice significantly reduced the infarct volume of mice, improved the neurological damage of mice, reduced the infiltration of inflammatory cells in brain tissue, and effectively alleviated the clinical syndrome complicated by ischemic stroke damage.

[0022] Based on the above research, the mechanism of action of strychnine N-oxide in improving ischemic stroke was studied in depth. By feeding strychnine N-oxide to model mice, it was found that strychnine N-oxide can effectively regulate tight junction proteins and protect the integrity of the blood-brain barrier. It can be seen that strychnine N-oxide plays a preventive and therapeutic role in ischemic stroke by acting on tight junction proteins and protecting the integrity of the blood-brain barrier.

[0023] The present invention conducts a series of studies on strychnine nitrogen oxide using model mice, indicating that strychnine nitrogen oxide can be effectively applied in ischemic stroke diseases, thus providing a new use of strychnine nitrogen oxide.

[0024] In the present invention, the pharmaceutical dosage of strychnine nitrogen oxide is preferably 200-800 mg / kg / day, more preferably 400-600 mg / kg / day, specifically 200, 400, 600 or 800 mg / kg / day. In the experiment of the effect of strychnine nitrogen oxide on ischemic stroke model mice induced by middle cerebral artery occlusion reperfusion, it was observed that the dosage of strychnine nitrogen oxide was 200-800 mg / kg / day to achieve the pharmaceutical effect.

[0025] In the present invention, the strychnine nitrogen oxide is prepared by strychnine oxidation with hydrogen peroxide.

[0026] The present invention preferably uses an alcohol extraction method to extract strychnine total alkaloids from Strychnine; then uses a column chromatography method to separate and purify strychnine from the strychnine total alkaloids, and finally oxidizes the strychnine with hydrogen peroxide to obtain strychnine nitrogen oxide. The present invention does not specifically limit the specific parameters of the alcohol extraction method, column chromatography method and hydrogen peroxide oxidation method, and methods familiar to those skilled in the art can be used. The preparation method of strychnine nitrogen oxide proposed in the present invention can efficiently convert strychnine, a toxic component of Strychnine, into low-toxic strychnine nitrogen oxide.

[0027] The invention also provides a composition for preventing and treating ischemic stroke, comprising strychnine nitrogen oxide and pharmaceutically acceptable auxiliary materials.

[0028] In the present invention, the dosage form of the composition is preferably a suspension, capsule, tablet, pill, drop pill, granule, powder, injection or sustained-release agent.

[0029] In the present invention, the pharmaceutically acceptable excipients preferably include one or more of carriers, excipients and diluents. The present invention has no particular limitation on the specific form of the pharmaceutically acceptable excipients, and any type familiar to those skilled in the art may be used.

[0030] In the present invention, the content of strychnine nitrogen oxide in the composition for preventing and treating ischemic stroke is preferably 90-95 wt %.

[0031] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1 Extraction of total alkaloids from Strychnos nux vomica

[0033] S1. By weight, 130 g of Strychnos nux vomica was added to 1.5 L of 85 wt% ethanol with a pH value of 2, and refluxed for extraction for 2 h. After filtering, the filter cake was taken for later use;

[0034] S2, by weight, add 85wt% ethanol (8 times the amount of pH value = 2) to the residue obtained in S1, reflux and extract for 1h, filter, combine the filtrate, and concentrate to 100mL;

[0035] S3, adjust the pH value of 100 mL of the concentrated solution of S2 to 10, extract with chloroform for 3 times, combine the chloroform layers, recover the chloroform under reduced pressure, and obtain a total alkaloid extract of Strychnos nux vomica. Dissolve it in water and adjust the volume to 50 mL to obtain a total alkaloid solution of Strychnos nux vomica with a content of 2.60 g / mL.

[0036] Example 2 Isolation and purification of strychnine

[0037] S1. Take 50 g of column chromatography silica gel (200-300 mesh) and wet-pack it into a chromatography column; prepare the eluent with petroleum ether and ethyl acetate.

[0038] S2. Take 500 mg of strychnine total alkaloid extract, mix the sample on silica gel, add eluent and start gradient elution: start with a ratio of petroleum ether to ethyl acetate of 4:1 (v / v), gradually increase the ratio of ethyl acetate to 1:1 (v / v), and collect the eluate in units of 15 mL. Monitor by thin layer chromatography, and combine and collect the eluate of strychnine components.

[0039] S3. Concentrate the combined eluate, recover the solvent under reduced pressure, and obtain strychnine.

[0040] Example 3 Preparation of strychnine nitrogen oxide

[0041] S1. Disperse strychnine (2.19 g, 2.16 mmol) in deionized water, and slowly add 0.2 mol / L NaOH solution under ice bath condition to adjust the pH value to 12-13.

[0042] S2. After the white solid no longer precipitates, continue stirring for 10 min in an ice bath and filter. The filter cake is dried and set aside; the filtrate is extracted with dichloromethane three times, the organic layer is washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a white solid, which is combined with the filter cake and dried.

[0043] S3. Dissolve the dried white solid in acetonitrile (15 mL) and deionized water (15 mL), add 30 wt% H 2 O 2 (0.9 mL, 8.81 mmol), stirred for 15 min, then heated to 25 °C and continued to react for 48 h. The solvent was removed under reduced pressure and recrystallized from methanol / dichloromethane to obtain 1.67 g of a white solid with a yield of 93.8%. ESI-HRMS (m / z) [M+H] + calculated for C 23 H 27 N 2 O 5 411.1914, found 411.1922.

[0044] Figure 1 This is a reaction schematic diagram for preparing strychnine nitrogen oxide from strychnine oxidation in Example 3.

[0045] Example 4 Identification of strychnine nitrogen oxide components

[0046] Take the strychnine nitrogen oxide solution prepared in Example 3, dilute it with pure water to 24 g / mL, and perform LC-MS / MS detection on the liquid phase conditions: BEH C18 chromatographic column (2.1×100 mm, 1.7 μm), water (A)-acetonitrile (B) containing 0.1 vol% formic acid gradient elution (0-0.5 min, 5% B; 0.5-12 min, 5-20% B; 12-15 min, 20-30% B; 15-18 min, 30-60% B; 18-18.5 min, 60-90% B; 18.5-22 min, 95% B). Mass spectrometry conditions: positive ion mode, SCAN mode scanning to obtain total ion chromatogram, MRM mode to extract compound ion pairs. Ion source temperature, 150°C; desolvation gas temperature, 400°C; desolvation gas flow rate, 800 L / Hr.

[0047] Figure 2 This is the high-resolution mass spectrum of the strychnine nitrogen oxide sample obtained in Example 4.

[0048] Example 5 Effect of strychnine nitric oxide on the improvement of ischemic stroke in mice induced by middle cerebral artery occlusion (MCAO)

[0049] 1. Experimental Methods

[0050] Sixty (24±2g) male C57BL / 6SPF mice were randomly divided into 6 groups (10 mice in each group): sham group (sham group), model group (Control group), edaravone and dextroborneol positive control group (ED group 10mg / kg), strychnine N-oxide groups: strychnine N-oxide low-dose group (BNO-200mg / kg), strychnine N-oxide medium-dose group (BNO-400mg / kg) and strychnine N-oxide high-dose group (BNO-800mg / kg). All mice underwent MCAO modeling, referring to the Zealonga thread plug method. Neurological deficits in mice in each group were evaluated according to the longa 5-point method 2 hours after MAAO. Mice with death and subarachnoid hemorrhage were excluded, and mice with scores of 1 to 3 points were selected as successful models for subsequent testing. Except for the insertion of thread plugs, the sham group was operated in the same way. 2 hours after surgery, mice in the strychnine N-oxide group were intraperitoneally injected with the corresponding dose of strychnine N-oxide, mice in the sham operation group and the model group were intraperitoneally injected with an equal volume of normal saline, and mice in the edaravone-dexamethasone group were intraperitoneally injected with edaravone-dexamethasone, with a dosage volume of 0.1 mL / 10 g. 24 hours after surgery, longa scores were performed. After scoring, mice were killed by cervical dislocation and anatomical sampling was performed. Whole blood was allowed to stand at room temperature for 2 hours, and serum was taken for biochemical index detection; the brain was quickly taken, and the brain tissue was cut into 5 slices (1 mm thick) for TTC staining to calculate the infarct volume. After staining, normal tissue was red and the infarct area was white. After anesthesia, the mice were decapitated and the brain was taken out. The two cerebral hemispheres and cerebellum were quickly weighed with an electronic balance (accuracy of 0.1 mg), placed in a 100°C oven, and weighed again after 24 hours to obtain the dry mass, and the water content of the brain tissue was calculated.

[0051] 2. Experimental results

[0052] The experimental results are as follows Figure 3 As shown, Figure 3 The figure is a graph showing the therapeutic effect of strychnine nitrogen oxide on brain tissue damage in mice with ischemic stroke induced by middle cerebral artery occlusion in the examples. Figure 3 A is the longa score, B is the water content of mouse brain tissue, C is a representative picture of TTC staining of cerebral infarction, D is the volume of cerebral infarction, and E is the levels of MPO, TNF-α, IL-1β and IL-6 in mouse serum. All data are expressed as mean ± standard deviation (n = 6). Compared with the sham operation group, #### p<0.0001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0053] Depend on Figure 3 As shown in Figure A, BNO can significantly improve the damage of neurological function in mice, especially in the high-dose BNO (800 mg / kg) group. Figure 3 As shown in Figure B, BNO significantly reduced the degree of brain edema after ischemic stroke in mice. Figure 3 C and D show that BNO can significantly reduce the cerebral infarct volume in mice with ischemic stroke. Figure 3 Figure E shows that BNO can significantly reduce the levels of MPO, TNF-α, IL-1β and IL-6 in mouse serum. In summary, BNO can significantly improve neurological function damage, reduce brain edema, reduce infarct volume, and reduce inflammatory damage in ischemic stroke mice. The therapeutic effect of the high-dose BNO (800 mg / kg) group is similar to that of the positive control group of edaravone and dexamethasone.

[0054] In summary, the strychnine nitrogen oxide prepared by the present invention can effectively achieve the "reduced toxicity and enhanced efficacy" of strychnine, not only can it significantly reduce the cerebral infarction volume of mice with ischemic stroke and improve neurological damage, but also can reduce the infiltration of inflammatory cells in brain tissue at the same time, and has a potential role in preventing and treating ischemic stroke. Further studies have also found that its mechanism of action is related to protecting the integrity of the blood-brain barrier and regulating tight junction proteins. The present invention can be applied to the clinical treatment and mechanism of action research of ischemic stroke, and provide a basis for exploring the development of new drugs for the treatment of ischemic stroke.

[0055] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A use of strychnine nitrogen oxide in the preparation of drugs for preventing and treating ischemic stroke.

2. The use according to claim 1, characterized in that: The medicinal dosage of strychnine nitrogen oxide is 200-800 mg / kg / day.

3. The use according to claim 2, characterized in that: The medicinal dosage of strychnine nitrogen oxide is 400-600 mg / kg / day.

4. The use according to claim 1, characterized in that: The strychnine nitrogen oxide is prepared by oxidizing strychnine with hydrogen peroxide.

5. A composition for preventing and treating ischemic stroke, characterized in that: The invention comprises strychnine nitrogen oxide and pharmaceutically acceptable excipients.

6. The composition for preventing and treating ischemic stroke according to claim 5, characterized in that: The dosage form of the composition is suspension, capsule, tablet, pill, drop pill, granule, powder, injection or sustained release agent.

7. The composition for preventing and treating ischemic stroke according to claim 5, characterized in that: The pharmaceutically acceptable excipients include one or more of carriers, excipients and diluents.

8. The composition for preventing and treating ischemic stroke according to claim 5, characterized in that: The content of strychnine nitrogen oxide in the composition for preventing and treating ischemic stroke is 90-95wt%.

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