阅读说明：本技术 一种海龙甾醇固体分散体及其在治疗脑卒中的应用 (Syngnathus sterol solid dispersion and application thereof in treating cerebral apoplexy ) 是由 许东晖 梅雪婷 于 2020-06-15 设计创作，主要内容包括：本发明属于生物医药技术领域,具体涉及一种海龙甾醇固体分散体及其在治疗脑卒中的应用。所述海龙甾醇固体分散体包括海龙甾醇提取物和聚乙烯吡咯烷酮,其中,所述海龙甾醇提取物为海龙经碱性甲醇水溶液提取后,加入可溶性钙盐溶液沉淀,再丙酮回流、重结晶制得,所得海龙甾醇固体分散体溶解性好,对脑卒中具有显著的治疗作用,可以显著提高脑卒中所引起的外周免疫抑制,同时具有毒性低,安全性高的优点；并且制备方法简单,适用于大规模产业化生产,应用范围广。(The invention belongs to the technical field of biological medicines, and particularly relates to a solid dispersion of Syngnathus sterol and application thereof in treating cerebral apoplexy. The solid dispersion of the syngnathus sterol comprises a syngnathus sterol extract and polyvinylpyrrolidone, wherein the syngnathus sterol extract is prepared by adding a soluble calcium salt solution for precipitation after the syngnathus is extracted by an alkaline methanol aqueous solution, and then carrying out acetone reflux and recrystallization; and the preparation method is simple, is suitable for large-scale industrial production and has wide application range.)

1. The solid dispersion of the syngnathus sterol is characterized by comprising a syngnathus sterol extract and polyvinylpyrrolidone, wherein the syngnathus sterol extract is prepared by adding a soluble calcium salt solution for precipitation after the syngnathus is extracted by an alkaline methanol aqueous solution, and then refluxing and recrystallizing by acetone;

the preparation method of the alkaline methanol aqueous solution comprises the following steps: methanol and water are mixed according to the volume ratio (0.125-10): 1, and adding 1-20 wt% of sodium hydroxide or potassium hydroxide to obtain the catalyst.

2. The solid dispersion of the Syngnathus sterol according to claim 1, wherein the mass ratio of the Syngnathus sterol extract to the polyvinylpyrrolidone is 1 (1-40).

3. The solid dispersion of the Syngnathus sterol according to claim 2, wherein the mass ratio of the Syngnathus sterol extract to the polyvinylpyrrolidone is 1 (2-16).

4. The Syngnathus sterol solid dispersion according to any one of claims 1 to 3, wherein the preparation method of the Syngnathus sterol solid dispersion comprises the steps of:

s1, crushing the dried sea dragon, adding an alkaline methanol aqueous solution, heating to reflux for 4-12 h, and filtering at normal temperature to obtain a filtrate;

s2, adding soluble calcium salt dropwise into the filtrate obtained in the step S2 while stirring until no precipitate is separated out, standing, and filtering to obtain a precipitate;

s3, adding acetone into the precipitate, heating to reflux, filtering, concentrating the filtrate to 10-30% of the original volume, cooling and crystallizing, washing the crystals with ethanol at 4 ℃, filtering, collecting the crystals, and drying to obtain a Syngnathus sterol extract;

s4, dissolving the Syngnathus sterol extract obtained in the step S3 in ethanol, adding polyvinylpyrrolidone for dissolving, uniformly mixing, and performing spray drying to obtain the Syngnathus sterol solid dispersion.

5. The Syngnathus sterol solid dispersion according to claim 4, wherein in step S1, the weight volume ratio of the dried Syngnathus to the alkaline aqueous methanol solution is (0.05-0.5): 1 g/ml.

6. The Syngnathus sterol solid dispersion according to claim 4, wherein in step S3, the weight to volume ratio of the precipitate to acetone is 1 (10-20) g/ml.

7. The Syngnathus sterol solid dispersion according to any one of claims 1 to 6, wherein said Syngnathus sterol extract comprises the following sterol components: 24-dehydrocholesterol, cholesterol, 7-cholesta (sterol) alkanol, 26-nor-cholest-5-cholesten 3 beta-OL-25 ketone, cholest-5-en-3-one, cholest-4-en-3-one, cholest-3, 5-diene, cholest-4, 6-diene-3-one, beta-sitosterol, (3 beta, 5 alpha) cholest-7-en-3-hydroxy.

8. The Syngnathus sterol solid dispersion of claim 7, wherein said sterol component and parts by weight thereof are: 1-16 parts of 24-dehydrocholesterol, 3-14 parts of cholesterol, 1-16 parts of cholesterol, 2-10 parts of 7-cholesta (sterol) alkanol, 1-15 parts of 26-cholesta-5-cholestene 3 beta-OL-25 ketone, 2-60 parts of cholest-5-ene-3-ketone, 5-150 parts of cholest-4-ene-3-ketone, 5-40 parts of cholest-3, 5-diene, 5-40 parts of cholest-4, 6-diene-3-ketone, 4-60 parts of beta-sitosterol and 5-60 parts of (3 beta, 5 alpha) cholest-7-ene-3-hydroxyl.

9. The Syngnathus sterol solid dispersion of claim 8, wherein the sterol component further comprises a pharmaceutically acceptable salt, ester, glycoside, or hydrate.

10. The use of the solid dispersion of Syngnathus sterol according to any one of claims 1-9 in the preparation of a medicament for the prevention or treatment of stroke or stroke-induced peripheral immunosuppression.

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, relates to a solid dispersion of Syngnathus sterol and its application in treating cerebral apoplexy.

Background

Syngnathus is a marine traditional Chinese medicinal material in China, has a habit of taking Syngnathus in folk, is used for stewing soup, has the effects of relieving swelling and resolving masses, and can be used for treating diseases such as abdominal mass accumulation, scrofula and gall, traumatic injury, carbuncle, swelling, furuncle and the like. The existing research finds that the active substances contained in the sea dragon have better curative effect on diseases such as tumor, hyperplasia and the like, for example, Chinese patent application CN1793174A discloses an anti-tumor active protein of the sea dragon, and experiments prove that the active protein extracted from the sea dragon has anti-tumor activity alone or in combination with other medicines, and has the characteristics of low toxicity and immunity enhancement; chinese patent application CN101019892A discloses a syngnathus solid dispersion, its preparation method and application, the solid dispersion is prepared from syngnathus zymolyte obtained through enzymolysis, deodorization and other steps and polyvinylpyrrolidone, and has good curative effect on prostatic hyperplasia and hyperplasia of mammary glands, and low toxicity. However, the application of the Syngnathus, the Syngnathus extract or the preparation in the aspect of cerebral apoplexy diseases has not been researched, and the effect is not clear.

Stroke (Stroke), also known as cerebrovascular accident or Stroke, is an acute cerebrovascular disease manifested by circulatory disturbance of cerebral blood flow and functional or structural damage of brain tissue due to cerebrovascular occlusion and rupture, and has become the second most common cause of death worldwide in recent years, and precedes cancer. It is reported that the risk of stroke in people over 65 years old is as high as two thirds, ranked after heart disease and before cancer, with south asian people having a particularly high risk of stroke accounting for 40% of global stroke deaths. In addition, the stroke not only harms the health of people with high morbidity, high mortality and high disability rate, but only about 10 percent of the surviving stroke patients accounting for 80 percent of the patients can completely recover the normal functions, and most of the patients have sequelae of hemiplegia, aphasia and the like, thereby causing serious burden to the society and families.

At present, the method for clinically treating the cerebral apoplexy mainly comprises the steps of taking aspirin, clopidogrel, a brain active tablet, Mikebao and the like, and the drug effect of the method is mainly to eliminate local embolism of the cerebral apoplexy. However, most of the above drugs are chemical drugs, and after ingestion, the body has various side effects, which is poor in safety, and also brings ischemia-reperfusion injury while relieving embolism, resulting in reduction of peripheral immunosuppression. Therefore, it is necessary to develop a novel medicament for treating stroke, which can improve peripheral immunosuppression while treating stroke, and achieve better therapeutic effect.

Disclosure of Invention

The invention aims to solve the technical problems that the existing clinical treatment medicine for cerebral apoplexy has poor safety and is easy to cause the problems of reduction of peripheral immune suppression and the like, and provides a novel solid dispersion of Syngnathus sterol for treating cerebral apoplexy, which can improve peripheral immune suppression while treating cerebral apoplexy, achieves better treatment effect and has higher safety.

The invention aims to provide a solid dispersion of Syngnathus sterol.

The invention also aims to provide the application of the solid dispersion of the Syngnathus sterol in preparing the drugs for preventing and treating cerebral apoplexy or peripheral immunosuppression caused by cerebral apoplexy.

The above purpose of the invention is realized by the following technical scheme:

a Syngnathus sterol solid dispersion comprises Syngnathus sterol extract and polyvinylpyrrolidone, wherein the Syngnathus sterol extract is prepared by extracting Syngnathus with alkaline methanol water solution, adding soluble calcium salt solution for precipitation, refluxing with acetone, and recrystallizing;

the preparation method of the alkaline methanol aqueous solution comprises the following steps: methanol and water are mixed according to the volume ratio (0.125-10): 1, and adding 1-20 wt% of sodium hydroxide or potassium hydroxide to obtain the catalyst.

Further, the mass ratio of the Syngnathus sterol extract to the polyvinylpyrrolidone is 1 (1-40). In practice, the solid dispersion prepared from the Syngnathus sterol extract and polyvinylpyrrolidone can remarkably improve the solubility of Syngnathus sterol in an aqueous solution and remarkably improve the absorption and pharmacological effects of Syngnathus sterol.

Preferably, the mass ratio of the Syngnathus sterol extract to the polyvinylpyrrolidone is 1 (2-25).

Preferably, the mass ratio of the Syngnathus sterol extract to the polyvinylpyrrolidone is 1 (2-16).

More preferably, the mass ratio of the Syngnathus sterol extract to the polyvinylpyrrolidone is 1 (4-15).

Further, the preparation method of the solid dispersion of the Syngnathus sterol specifically comprises the following steps:

s1, crushing the dried sea dragon, adding an alkaline methanol aqueous solution, heating to reflux for 4-12 h, and filtering at normal temperature to obtain a filtrate;

s2, adding soluble calcium salt into the filtrate obtained in the step S2 dropwise while stirring until no precipitate is separated out, standing, and filtering to obtain precipitate Syngnathus sterol calcium salt;

s3, adding acetone into the precipitate, heating to reflux, filtering, concentrating the filtrate to 10-30% of the original volume, cooling and crystallizing, washing the crystals with ethanol at 4 ℃, filtering, collecting the crystals, and drying to obtain a Syngnathus sterol extract;

s4, dissolving the Syngnathus sterol extract obtained in the step S3 in ethanol, adding polyvinylpyrrolidone for dissolving, uniformly mixing, and performing spray drying to obtain the Syngnathus sterol solid dispersion.

Further, in step S1, the weight-to-volume ratio of the dried sea dragon product to the alkaline methanol aqueous solution is (0.05-0.5): 1 g/ml.

Furthermore, in step S3, the weight/volume ratio of the precipitate to acetone is 1 (10-20) g/ml.

Further, the Syngnathus sterol extract comprises the following sterol components: 24-dehydrocholesterol, cholesterol, 7-cholesta (sterol) alkanol, 26-nor-cholest-5-cholesten 3 beta-OL-25 ketone, cholest-5-en-3-one, cholest-4-en-3-one, cholest-3, 5-diene, cholest-4, 6-diene-3-one, beta-sitosterol, (3 beta, 5 alpha) cholest-7-en-3-hydroxy.

Further, the sterol component and the weight portion thereof are as follows: 1-16 parts of 24-dehydrocholesterol, 3-14 parts of cholesterol, 1-16 parts of cholesterol, 2-10 parts of 7-cholesta (sterol) alkanol, 1-15 parts of 26-cholesta-5-cholestene 3 beta-OL-25 ketone, 2-60 parts of cholest-5-ene-3-ketone, 5-150 parts of cholest-4-ene-3-ketone, 5-40 parts of cholest-3, 5-diene, 5-40 parts of cholest-4, 6-diene-3-ketone, 4-60 parts of beta-sitosterol and 5-60 parts of (3 beta, 5 alpha) cholest-7-ene-3-hydroxyl.

Further, the sterol component also includes pharmaceutically acceptable salts, esters, glycosides or hydrates.

Further, the soluble calcium salt is a saturated soluble calcium salt, including a saturated calcium chloride solution, a saturated calcium acetate solution, and a saturated calcium gluconate solution.

In addition, the invention also provides application of the solid dispersion of the Syngnathus sterol in preparation of drugs for preventing and treating stroke or peripheral immunosuppression caused by stroke.

Similarly, the solid dispersion of the Syngnathus sterol can also be applied to the fields of health food, food and the like.

Further, the solid dispersion of the Syngnathus sterol can be made into appropriate dosage forms such as tablets, powders, granules, capsules, syrups, suppositories, oral liquids and the like according to requirements.

Furthermore, the dose of the Syngnathus sterol is generally 1-30000 mg per day, and the dose is usually 5-10000 mg per day, preferably 30-3000 mg per adult.

The invention has the following beneficial effects:

the solid dispersion of the syngnathus sterol comprises a syngnathus sterol extract and polyvinylpyrrolidone, wherein the syngnathus sterol extract is prepared by extracting syngnathus with an alkaline methanol aqueous solution, adding a soluble calcium salt solution for precipitation, then refluxing with acetone and recrystallizing, and the obtained solid dispersion of the syngnathus sterol has good solubility, has a remarkable treatment effect on stroke, can remarkably improve peripheral immunosuppression caused by the stroke, and has the advantages of low toxicity and high safety; and the preparation method is simple, is suitable for large-scale industrial production and has wide application range.

Drawings

FIG. 1 is a standard curve diagram of cholesterol obtained by determining the purity of Syngnathus sterol according to the present invention.

FIG. 2 is a gas mass spectrum of the Syngnathus sterol extract of Experimental example 1 of the present invention.

FIG. 3 is a schematic view of the Morris water maze platform used in example 2 of the present invention.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Wherein the dried sea dragon product is prepared by mixing 1kg of acutus sea dragon, 1kg of quasi-sea dragon and 1kg of cunninghamia paniculata;

the preparation method of the alkaline methanol aqueous solution comprises the following steps: mixing methanol and water according to the volume ratio of 1:0.25, and adding 10 wt% of sodium hydroxide to obtain the catalyst.

Determination method of the purity of the Syngnathus sterol (based on the content of cholesterol):

(1) preparation of a standard curve: accurately weighing cholesterol, dissolving in n-hexane, and preparing into standard substances with different concentrations; accurately measuring 2ml of acetic anhydride in a 10ml test tube with a plug, dripping 1 drop of concentrated sulfuric acid, immediately adding 1ml of standard solution with each concentration, shaking up by vortex oscillation, standing for 10min, and measuring the absorbance of the standard solution with each concentration at the wavelength of 650 nm. The concentration of the cholesterol solution was plotted on the abscissa and the corresponding OD value of the cholesterol solution was plotted on the ordinate to calculate a regression equation, the result of which is shown in FIG. 1.

(2) Sample detection: accurately weighing 65.2mg of the Syngnathus sterol extract in a 50ml volumetric flask, adding n-hexane for dissolving, and fixing the volume to obtain a sample solution to be detected; accurately sucking 2ml of acetic anhydride, placing the acetic anhydride in a 10ml test tube with a plug, dripping 1 drop of concentrated sulfuric acid, immediately adding 1ml of diluted sample solution, shaking and uniformly mixing in a vortex, standing for 10 minutes, measuring the light absorption value at the wavelength of 650nm, and calculating the content of total sterol according to a cholesterol standard curve.

The remaining reagents and materials used in the following examples are all commercially available unless otherwise specified.