CN116120307A - A method for extracting, separating and purifying stepherine from mountain turtle - Google Patents

Abstract

The invention discloses a method for extracting, separating and purifying stephanine from tortoise, which adopts an alcohol-water system to extract the raw materials of the tortoise, utilizes a stepwise precipitation method of acid and alkali firstly to obtain a stephanine crude product according to the property of alkaloid substances, adopts a molecular imprinting technology to further purify the stephanine crude product, and can prepare a high-purity stephanine product with the purity of more than 98 percent through one-time recrystallization. Compared with the traditional extraction and crude refining process, the method effectively avoids repeated transfer of materials, shortens the production time and reduces the solvent consumption; compared with the traditional separation and purification mode, the method effectively shortens the process route, improves the yield of the effective components and reduces the production period.

Description

A method for extracting, separating and purifying stepherine from mountain turtle

technical field

The invention relates to the technical field of natural product extraction, in particular to a method for extracting, separating and purifying stepherine from mountain turtle.

Background technique

Stephine is a benzylisoquinoline alkaloid extracted and isolated from the root tubers of Stephaniaceae. Studies have shown that it is a dopamine receptor blocker in the brain; it has the effects of lowering blood pressure, antiarrhythmia, protecting myocardial infarction, analgesia, sedation, antispasmodic and antipyretic; it is mainly used clinically to treat vascular headache, Such as migraine; hyperkinetic movement disorder, hyperactive Tourette's syndrome in children. Studies have shown that the incidence of ADHD in children is higher. According to the survey, the incidence of behavioral problems in school-age children reaches 12.9%, among which ADHD ranks the top, with an incidence rate of 4.3-5.8%. The above research results show that stephenidine has great value in clinical application and has a good development prospect.

The currently reported preparation techniques of stephenine are mainly synthetic or semi-synthetic methods, and there are almost no related literature and patent reports on the extraction and isolation of stephenine from mountain turtle. The methods for extracting alkaloid natural components from plants mainly include acid water extraction and organic solvent extraction; the methods for purifying alkaloids mainly include recrystallization, column chromatography, and alkaloid precipitant precipitation.

The acid water extraction method generally uses 1-2% inorganic acid aqueous solution under the condition of heating at 80-90°C, reflux extraction, the extract is adjusted to alkaline, and the crude alkaloid is precipitated after cooling and standing, and the crude product is further purified to obtain a high-purity alkaloid Product: Alcohol extraction method uses ethanol/methanol aqueous solution with a volume fraction of 60%-100% for reflux extraction, the extract is concentrated to no alcohol, the pH is adjusted to alkaline, the crude alkaloid is precipitated after standing, and the crude product is further purified to obtain High-purity alkaloid products; the recrystallization method in the purification method mainly utilizes the different solubility of alkaloid products in different solvents or at different temperatures for the same solution to carry out purification and separation; the fillers used in column chromatography currently mainly include cation exchange resins, silica gel, oxidation There are three kinds of aluminum; alkaloid precipitant is mainly used to separate water-soluble quaternary ammonium alkaloids. However, the purification of alkaloids by recrystallization generally requires multiple repeated crystallizations to prepare high-purity alkaloid products, with low yield and many steps. Chromatographic separation of alkaloids has poor selectivity. Generally, the purity of the crude extract is low after one pass of chromatography, and multiple crystallizations are still required to prepare high-purity products, and the amount of organic solvent is large.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a method for extracting, separating and purifying stepherine from the mountain turtle, so as to improve the above-mentioned technical problems.

The present invention is achieved like this:

In the first aspect, the present invention provides a method for extracting, isolating and purifying stepherine from mountain tortoise, comprising:

The alcohol solution of the crude extract of stepheridine is passed through the molecularly imprinted polymer column of stepheridine, and then methanol-glacial acetic acid is added to pass through the column, and the fraction of stepheridine is collected.

Optionally, the preparation method of the stepperidine molecularly imprinted polymer column includes: adding a functional monomer, a cross-linking agent and an initiator to the acetonitrile solution of stepharidine, introducing nitrogen gas, stirring evenly, and standing still to obtain White hard solid polymer, then pulverize the polymer to obtain uniform particles and pack it into the column, wash with methanol-glacial acetic acid system until the effluent has no template molecules, and then obtain the stepheridine molecularly imprinted polymer column.

Optionally, the functional monomer includes methacrylic acid.

Optionally, the crosslinking agent includes ethylene glycol dimethacrylate.

Optionally, the initiator includes azobisisobutyronitrile.

Optionally, the mass ratio of stepheridine, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 1:3-4:15-20:0.1-0.3.

Optionally, the standing condition is standing and reacting at 55-65° C. for 20-25 hours.

Optionally, the volume ratio of methanol to glacial acetic acid in the methanol-glacial acetic acid system is 100:0.5-2.

Optionally, the mass-to-volume ratio of the crude stepheridine extract to the alcohol solution in the alcohol solution of the stepheridine crude extract is 1:5-6.

Optionally, the above alcohol solution is ethanol.

Optionally, the above alcohol solution is absolute ethanol.

Optionally, the adsorption flow rate of the alcohol solution of the crude stephenine extract is 0.8-1.5 BV/h during adsorption and elution.

Optionally, the purification step also includes: decompressing the stephenidine component to recover methanol to 2-3 times the weight of the raw material, then adding activated carbon, heating to reflux, filtering while hot, and then adding hot water to the filtrate to adjust the pH To 8-10, let it stand, let cool and precipitate a large number of crystals, which is stephine.

Optionally, the amount of activated carbon added is 0.1%-0.3% of the mass of the stepheridine component after recovering the methanol under reduced pressure.

Optionally, the time for heating to reflux is 0.5-2h.

Optionally, the volume ratio of filtrate to hot water is 1:0.8-1.2.

Optionally, the method for extracting the crude extract of stephenidine includes extracting the mountain turtle raw material with ethanol aqueous solution. After the first extraction is completed, the extraction residue is added with ethanol aqueous solution for the second extraction, and the two extracts are combined, and then Stand still under the conditions to precipitate the precipitate, the first solid-liquid separation to obtain the clear liquid, and then stand the clear liquid under alkaline conditions, precipitate the precipitate, the second solid-liquid separation, collect the second solid-liquid separation precipitate, and get a daughter Crude extract of phenidine.

Optionally, the volume content of ethanol in the ethanol aqueous solution is 60-95%.

Optionally, in the first extraction, the volume-to-mass ratio of the ethanol aqueous solution to the mountain turtle raw material is 5-10:1, and the extraction time is 3-5 hours.

Optionally, in the second extraction, the volume-to-mass ratio of the aqueous ethanol solution to the raw material of mountain turtle is 3-6:1, and the extraction time is 2-3 hours.

Optionally, standing under acidic conditions includes: concentrating the combined extracts under reduced pressure to an alcohol-free state, and then adding an inorganic acid solution to pH = 2-3, and the standing time is ≥ 4 hours.

Standing under alkaline conditions includes: adding an inorganic alkali solution to the clear liquid until pH = 8-9, and the standing time is ≥ 4 hours.

In a second aspect, the present invention provides stepropine obtained by the method for extracting, isolating, and purifying stepropine from the mountain tortoise, and the purity of the stepropine is greater than 98%.

In a third aspect, the present invention provides the application of the above-mentioned stephenidine in the preparation of related drugs, the related drugs include drugs for the treatment and prevention of vascular headache, where vascular headache includes migraine, hyperkinetic movement disorder and hyperactivity in children Tourette Syndrome.

The present invention has the following beneficial effects:

The method for extracting, separating and purifying stephine from the mountain turtle provided by the present invention is to extract the raw material of the mountain turtle by using an alcohol-water system, and according to the properties of the alkaloid substances, use the step-by-step precipitation method of acid first and then alkali to obtain stephine Crude statinine; compared with the traditional extraction and crude refining process, it effectively avoids the repeated transfer of materials, shortens the production time, and reduces the amount of solvent used. Moreover, the present invention also adopts molecular imprinting technology to further purify the crude product of stepheridine, and a high-purity stepheridine product with a purity of more than 98% can be prepared after one recrystallization. Compared with the traditional repeated recrystallization method and column chromatography method, it effectively reduces material loss and production steps.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Alkaloids are a class of nitrogen-containing basic organic compounds that widely exist in nature. They are active ingredients of plant-derived traditional Chinese medicines and have significant physiological activities. There are dozens of alkaloids, including organic amines, pyrrolidines, pyridines, isoquinolines, indoles, and tropanes. Among them, stephenidine belongs to the isoquinoline alkaloids and often exists in plant-derived traditional Chinese medicines. Due to its low content, it is relatively difficult to extract and purify the alkaloids. Conventional separation and purification requires a combination of multiple methods, which not only brings inconvenience to the operation due to the many steps, but also makes it difficult to effectively separate stepheridine from plants, and the purification effect is also difficult to achieve a purity of more than 95%. Related reports on the extraction and separation of stepropine from mountain tortoise, in view of this, in order to obtain high-purity stepceptine from mountain tortoise, the inventor of the present invention provides a kind of extracting, separating and purifying stepropine from mountain tortoise An alkaline method, the method comprising:

Use ethanol aqueous solution to extract the mountain turtle raw material. After the first extraction is completed, the extraction residue is added with ethanol aqueous solution for the second extraction. The two extracts are combined, and then left to stand under acidic conditions to precipitate precipitation. The first solid-liquid separation obtains clear liquid, put the clear liquid under alkaline conditions, precipitate out, separate solid-liquid for the second time, collect the precipitate from the second solid-liquid separation, and obtain the crude extract of stephenidine. Then, the alcohol solution of the crude steppenidine extract is passed through the stepropine molecularly imprinted polymer column, and then methanol-glacial acetic acid is added to pass through the column to collect the stepropine component.

In some embodiments, the volume content of ethanol in the aqueous ethanol solution may be 60%, 75%, 80% or 95%, or any value between 60-95%.

In some embodiments, the volume-to-mass ratio of the ethanol aqueous solution and the mountain turtle raw material in the first extraction can be 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, etc., and the extraction time It can be 3, 4 or 5 hours etc.

In some embodiments, the volume-to-mass ratio of the ethanol aqueous solution and the mountain turtle raw material in the second extraction can be 3:1, 4:1, 5:1 or 6:1, etc., and the extraction time can be 2, 2.5 or 3 hours wait.

In some embodiments, standing under acidic conditions includes: concentrating the combined extracts under reduced pressure to an alcohol-free state, and then adding an inorganic acid solution thereto, wherein the inorganic acid can be sulfuric acid or hydrochloric acid, and the adjusted pH can be pH =2, pH=2.2, pH=2.5 or pH=3, and any value in pH=2-3; the standing time can be 4h, 5h, 6h, 7h or 8h, etc.

In some embodiments, standing under alkaline conditions includes: adding an inorganic alkali solution to the clear liquid until the solution is alkaline, wherein the inorganic alkali can be sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate , potassium bicarbonate or ammonia water, the adjusted pH can be pH=8, pH=8.2, pH=8.5 or pH=9, or any value in pH=8-9; the standing time can be 4h, 5h, 6h, 7h or 8h etc.

Most of the alkaloids can form salts with acids and are soluble in acid water. Acid water precipitation can remove acid-water insoluble impurities such as fat-soluble impurities brought out by alcohol extraction. The alkaloids containing stepherine are separated out. According to the properties of the alkaloid substances, the present invention utilizes the step-by-step precipitation method of acid first and then alkali to obtain the crude product with less impurities and higher content of stepherine.

In some embodiments, the preparation method of the stepperine molecularly imprinted polymer column comprises: adding a functional monomer, a cross-linking agent and an initiator to the acetonitrile solution of stepperidine, feeding nitrogen gas, stirring evenly, and standing After obtaining a white hard solid polymer, the polymer was pulverized to obtain uniform particles and packed into a column, washed with methanol-glacial acetic acid system until the effluent had no template molecules, and then a stepherine molecularly imprinted polymer column was obtained.

Specifically, the functional monomer may be methacrylic acid, the crosslinking agent may be ethylene glycol dimethacrylate, and the initiator may be azobisisobutyronitrile.

In some embodiments, the molar ratio of stepheridine, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile can be 1:3:15:0.1, 1:4:20:0.3 Or 1:3.5:18:0.2, etc., or any value in 1:3-4:15-20:0.1-0.3.

In some embodiments, the standing temperature during the preparation of the stepheridine molecularly imprinted polymer column can be 55°C, 58°C, 60°C, 62°C or 65°C, or any value between 55°C and 65°C . The standing time can be 20h, 21h, 22h, 23h, 23h, 24h or 25h, or any value in 20-25h.

In some embodiments, the volume ratio of methanol to glacial acetic acid in the methanol-glacial acetic acid system may be 99:1.

In some embodiments, the mass-to-volume ratio of the crude steppenidine extract to the alcohol solution in the alcoholic solution of the stephenine crude extract can be 1:5 or 1:6, or 1:5-6. any value of .

In some embodiments, the aforementioned alcoholic solution is ethanol. More preferably, the alcohol solution is absolute ethanol.

In some embodiments, the adsorption flow rate of the alcohol solution of the crude stephenine extract during adsorption and elution can be 0.5-1.5 BV/h.

In some embodiments, the purification step further includes: recovering methanol from the stephenidine component under reduced pressure, then adding activated carbon, heating to reflux, and filtering while hot, then adding hot water to the filtrate, adjusting the pH to 8-10, and statically Set it aside, let it cool and precipitate a large amount of crystals, which is Stephine.

In some embodiments, the amount of activated carbon added can be 0.1%, 0.2% or 0.3% of the mass of the stephanine component after recovering methanol under reduced pressure, and can also be any value in the range of 0.1-0.3%.

In some embodiments, the time for heating to reflux may be 0.5h, 1.0h, 1.5h or 2h, or any value in 0.5-2h. More preferably, the time of heating to reflux is 1.0h.

In some embodiments, the volume ratio of filtrate to hot water may be 1:0.8-1.2.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

Example 1

This embodiment provides a method for extracting, separating and purifying stepheridine from mountain turtle, the steps of which include:

1) Take the dry slices of tortoise, coarsely crush them, and pass through a 10-mesh sieve.

2) Soak and extract with 5 times the amount of 95% ethanol aqueous solution for 5 hours, separate the extract, add 6 times the amount of 95% ethanol aqueous solution to soak and extract for 3 hours, and combine the two extracts.

3) Concentrate the extract under reduced pressure until there is no alcohol, release and add concentrated sulfuric acid solution to adjust the pH to 2, let cool, let stand for 5 hours, filter, add liquid alkali solution to the clear liquid to adjust the pH to 9, stand for 5 hours, and precipitate a large amount of precipitate.

4) The liquid in 3) is filtered, and the precipitate is dried to obtain the crude product of stepheridine.

5) Preparation of steproprine molecularly imprinted polymer: Weigh 1 part of steproprine with a commercial purity greater than 98% and fully dissolve it in an appropriate amount of acetonitrile, add 4 parts of methacrylic acid, stir for 8 hours, add 20 Parts of ethylene glycol dimethacrylate, 0.3 parts of azobisisobutyronitrile, nitrogen gas, stirring evenly, standing at 60 ° C for 25 hours to obtain a white hard solid polymer, the polymer was crushed through a 60-mesh sieve , the uniform particles were packed into the column, washed with methanol-glacial acetic acid (99:1) system until the effluent was free of the template molecule stepheridine, and the molecularly imprinted polymer column was set aside.

6) Add 5 times the amount of absolute ethanol to dissolve the crude Stephania vine, filter it, and pass the filtrate through 5) a molecularly imprinted polymer column at 1 BV/h, add methanol-glacial acetic acid (99:1) to pass through the column, and collect the stephenidine group point.

7) Recover the stephenidine component collected in 6) to an appropriate amount of methanol under reduced pressure, add 0.3% powdered activated carbon, heat and reflux for 1 hour, filter while hot, add hot water to the filtrate at a ratio of 1:1 (volume ratio), and adjust the pH To 8.5, stand still, let cool and precipitate a large number of crystals, which is stephenidine.

As detected by HPLC, the purity of the obtained stepheridine was 98.6%.

Example 2

This embodiment provides a method for extracting, separating and purifying stepheridine from mountain turtle, the steps of which include:

1) Take the dry slices of tortoise, coarsely crush them, and pass through a 10-mesh sieve.

2) Soak and extract with 10 times the amount of 60% ethanol aqueous solution for 3 hours, separate the extract, add 6 times the amount of 60% ethanol aqueous solution to soak and extract for 2 hours, and combine the two extracts.

3) Concentrate the extract under reduced pressure until there is no alcohol, release and add concentrated sulfuric acid solution to adjust the pH to 2, let cool, let stand for 5 hours, filter, add liquid alkali solution to the clear liquid to adjust the pH to 9, stand for 5 hours, and precipitate a large amount of precipitate.

4) The liquid in 3) is filtered, and the precipitate is dried to obtain the crude product of stepheridine.

5) Preparation of steproprine molecularly imprinted polymer: Weigh 1 part of steproprine with a commercial purity greater than 98% and fully dissolve it in an appropriate amount of acetonitrile, add 4 parts of methacrylic acid, stir for 8 hours, add 20 Parts of ethylene glycol dimethacrylate, 0.3 parts of azobisisobutyronitrile, nitrogen gas, stirring evenly, standing at 60 ° C for 25 hours to obtain a white hard solid polymer, the polymer was crushed through a 60-mesh sieve , the uniform particles were packed into the column, washed with methanol-glacial acetic acid (99:1) system until the effluent was free of the template molecule stepheridine, and the molecularly imprinted polymer column was set aside.

6) Add 5 times the amount of absolute ethanol to dissolve the crude Stephania vine, filter it, and pass the filtrate through 5) a molecularly imprinted polymer column at 1 BV/h, add methanol-glacial acetic acid (99:1) to pass through the column, and collect the stephenidine group point.

7) Recover the stephenidine component collected in 6) to an appropriate amount of methanol under reduced pressure, add 0.3% powdered activated carbon, heat and reflux for 1 hour, filter while hot, add hot water to the filtrate at a ratio of 1:1 (volume ratio), and adjust the pH to 9.0, stand still, let cool and precipitate a large number of crystals, which is stephenidine.

As detected by HPLC, the purity of the obtained stepheridine was 99.1%.

Example 3

This embodiment provides a method for extracting, separating and purifying stepheridine from mountain turtle, the steps of which include:

1) Take the dry slices of tortoise, coarsely crush them, and pass through a 10-mesh sieve.

2) Soak and extract with 5 times the amount of 95% ethanol aqueous solution for 5 hours, separate the extract, add 3 times the amount of 95% ethanol aqueous solution to the extraction residue for 3 hours, and combine the two extracts.

3) Concentrate the extract under reduced pressure until there is no alcohol, release and add concentrated sulfuric acid solution to adjust the pH to 2, let cool, let stand for 5 hours, filter, add liquid alkali solution to the clear liquid to adjust the pH to 9, stand for 5 hours, and precipitate a large amount of precipitate.

4) The liquid in 3) is filtered, and the precipitate is dried to obtain the crude product of stepheridine.

5) Preparation of steproprine molecularly imprinted polymer: Weigh 1 portion of commercially available steproprine with a purity greater than 98% and fully dissolve it in an appropriate amount of acetonitrile, add 3 portions of methacrylic acid, stir for 8 hours, add 15 Parts of ethylene glycol dimethacrylate, 0.1 part of azobisisobutyronitrile, nitrogen gas, stirring evenly, standing at 60 ° C for 20 hours to obtain a white hard solid polymer, the polymer was crushed through a 60-mesh sieve , the uniform particles were packed into the column, washed with methanol-glacial acetic acid (99:1) system until the effluent was free of the template molecule stepheridine, and the molecularly imprinted polymer column was set aside.

6) Add 5 times the amount of absolute ethanol to dissolve the crude Stephania vine, filter it, and pass the filtrate through 5) a molecularly imprinted polymer column at 1 BV/h, add methanol-glacial acetic acid (99:1) to pass through the column, and collect the stephenidine group point.

7) Recover the stephenidine component collected in 6) to an appropriate amount of methanol under reduced pressure, add 0.3% powdered activated carbon, heat and reflux for 1 hour, filter while hot, add hot water to the filtrate at a ratio of 1:1 (volume ratio), and adjust the pH To 9.5, stand still, let cool and precipitate a large number of crystals, which is stephenidine.

As detected by HPLC, the purity of the obtained stepheridine was 99.3%.

Example 4

This embodiment provides a method for extracting, separating and purifying stepheridine from mountain turtle, the steps of which include:

1) Take the dry slices of tortoise, coarsely crush them, and pass through a 10-mesh sieve.

2) Soak and extract with 5 times the amount of 80% ethanol aqueous solution for 5 hours, separate the extract, add 6 times the amount of 80% ethanol aqueous solution to soak and extract for 3 hours, and combine the two extracts.

3) Concentrate the extract under reduced pressure until there is no alcohol, release and add concentrated sulfuric acid solution to adjust the pH to 2, let cool, let stand for 8 hours, filter, add liquid alkali solution to the clear liquid to adjust the pH to 9.5, stand for 5 hours, and precipitate a large amount of precipitate.

4) The liquid in 3) is filtered, and the precipitate is dried to obtain the crude product of stepheridine.

5) Preparation of steproprine molecularly imprinted polymer: Weigh 1 part of steproprine with a commercial purity greater than 98% and fully dissolve it in an appropriate amount of acetonitrile, add 4 parts of methacrylic acid, stir for 8 hours, add 20 Parts of ethylene glycol dimethacrylate, 0.3 parts of azobisisobutyronitrile, nitrogen gas, stirring evenly, standing at 60 ° C for 25 hours to obtain a white hard solid polymer, the polymer was crushed through a 60-mesh sieve , the uniform particles were packed into the column, washed with methanol-glacial acetic acid (99:1) system until the effluent was free of the template molecule stepheridine, and the molecularly imprinted polymer column was set aside.

6) Add 10 times the amount of absolute ethanol to dissolve the crude Stephania vine, filter it, and pass the filtrate at 1BV/h through the 5) molecularly imprinted polymer column, add methanol-glacial acetic acid (99:1) to pass through the column, and collect the stephenidine group point.

7) Recover the stephenidine component collected in 6) to an appropriate amount of methanol under reduced pressure, add 0.1% powdered activated carbon, heat and reflux for 1 hour, filter while hot, and add hot water to the filtrate at 1:1 (volume ratio) to adjust the pH To 8.5, stand still, let cool and precipitate a large number of crystals, which is stephenidine.

As detected by HPLC, the purity of the obtained stepheridine is 98.9%.

Example 5

This embodiment provides a method for extracting, separating and purifying stepheridine from mountain turtle, the steps of which include:

1) Take the dry slices of tortoise, coarsely crush them, and pass through a 10-mesh sieve.

2) Soak and extract with 8 times the amount of 75% ethanol aqueous solution for 5 hours, separate the extract, add 5 times the amount of 65% ethanol aqueous solution to soak and extract for 3 hours, and combine the two extracts.

3) Concentrate the extract under reduced pressure until there is no alcohol, release and add concentrated sulfuric acid solution to adjust the pH to 2, let cool, let stand for 5 hours, filter, add liquid alkali solution to the clear liquid to adjust the pH to 9, stand for 5 hours, and precipitate a large amount of precipitate.

4) The liquid in 3) is filtered, and the precipitate is dried to obtain the crude product of stepheridine.

5) Preparation of steproprine molecularly imprinted polymer: Weigh 1 part of steproprine with a commercial purity greater than 98% and fully dissolve it in an appropriate amount of acetonitrile, add 4 parts of methacrylic acid, stir for 8 hours, add 20 Parts of ethylene glycol dimethacrylate, 0.3 parts of azobisisobutyronitrile, nitrogen gas, stirring evenly, standing at 60 ° C for 25 hours to obtain a white hard solid polymer, the polymer was crushed through a 60-mesh sieve , the uniform particles were packed into the column, washed with methanol-glacial acetic acid (99:1) system until the effluent was free of the template molecule stepheridine, and the molecularly imprinted polymer column was set aside.

6) Add 5 times the amount of absolute ethanol to dissolve the crude Stephania vine, filter it, and pass the filtrate through 5) a molecularly imprinted polymer column at 1 BV/h, add methanol-glacial acetic acid (99:1) to pass through the column, and collect the stephenidine group point.

7) Recover the stephenidine component collected in 6) to an appropriate amount of methanol under reduced pressure, add 0.3% powdered activated carbon, heat and reflux for 1 hour, filter while hot, add hot water to the filtrate at a ratio of 1:1 (volume ratio), and adjust the pH To 10, let it stand, let it cool and precipitate a large amount of crystals, which is stephenidine.

As detected by HPLC, the purity of the obtained stepheridine was 98.5%.

Comparative example 1

The difference from Example 1 is that the extraction method of this comparative example is to obtain the crude extract by acid extraction and alkali precipitation, and the crude extract is further purified by multiple recrystallization methods. Its steps include:

1) Take the dry slices of tortoise, coarsely crush them, and pass through a 10-mesh sieve.

2) Soak in 8 times the amount of 2% sulfuric acid aqueous solution at room temperature for 24 hours, separate the extract, continue to add 7 times the amount of 2% sulfuric acid aqueous solution to the extraction residue and soak for 24 hours at room temperature, separate the extract, and continue to add 5 times the amount of 2% sulfuric acid aqueous solution to the extraction residue Soak at room temperature for 24 hours, and combine the three extracts.

3) Adjust the pH of the extract to 9 with the alkaline solution, and let it stand for 5 hours to precipitate a large amount of precipitate.

4) The liquid in 3) is filtered, and the precipitate is dried to obtain the crude product of stepheridine.

5) Add 5 times the amount of methanol to the crude product of 4) paternidine, heat it to reflux for 1-2 hours, filter it while it is hot, let the filtrate cool to precipitate crystals, and collect the crystals by filtration; repeat crystallization four times in this way to obtain the paternidine product.

As detected by HPLC, the purity of the obtained stepheridine is 90%. Each step of methanol crystallization operation in this method will lose about 30% of the active ingredients, and the final product purity can only reach about 90%. The subsequent further implementation of the fifth methanol crystallization content will hardly change, and the product purity will be difficult to further improve.

Comparative example 2

The difference with Example 1 is that the purification method of this comparative example adopts the prior art silica gel column chromatography, wherein the extraction method is the same as in Example 1, and the purification method is carried out by silica gel column chromatography, and the specific steps are:

1) Take the crude product of acid-extracted base and steppehnidine base, add methanol to fully dissolve, add 2-3 times the amount of silica gel, stir evenly, and shake the solvent to powder it for later use.

2) Pack silica gel column, chloroform wet packing column, and spread the powder in step 1) on the top of the silica gel column;

3) analysis, chloroform:methanol 50:1 column elution, collect the fraction containing stephine, and recover the solvent to get stephine. The purity of stephanidine is 60-70% as detected by HPLC.

4) The product of step 3) is further repeated twice by methanol recrystallization method, and the stepherine product with a purity of about 90% can be obtained. The purity is difficult to further improve by recrystallization.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A method for extracting, separating and purifying stepherine from the mountain tortoise, characterized in that it comprises passing the alcohol solution of the stepherine crude extract extracted from the mountain tortoise over the stepherine molecularly imprinted polymer Column, then add methanol-glacial acetic acid to pass through the column, and collect the stepheridine fraction. 2. The method according to claim 1, characterized in that, the preparation method of the stepheridine molecularly imprinted polymer column comprises: adding a functional monomer, a cross-linking agent, and an initiator to an acetonitrile solution of stepheridine. Nitrogen gas was passed through, stirred evenly, and after standing still, a white hard solid polymer was obtained, and then the polymer was pulverized to obtain uniform particles and packed into a column, and washed with methanol-glacial acetic acid system until the effluent had no template molecules to obtain the Stephania vine Pyridine base molecularly imprinted polymer column; Preferably, the functional monomer comprises methacrylic acid; Preferably, the crosslinking agent comprises ethylene glycol dimethacrylate; Preferably, the initiator includes azobisisobutyronitrile; Preferably, the molar ratio of stepheridine, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 1:3-4:15-20:0.1-0.3; Preferably, the standing condition is standing and reacting at 55-65°C for 20-25 hours; Preferably, the volume ratio of methanol to glacial acetic acid in the methanol-glacial acetic acid system is 100:0.5-2. 3. The method according to claim 2, characterized in that, the mass volume ratio of the stephardine crude extract to the alcoholic solution in the alcoholic solution of the stephardine crude extract is 1:5-6; Preferably, the alcoholic solution is ethanol; Preferably, the alcoholic solution is absolute ethanol; Preferably, the adsorption flow rate of the alcohol solution of the crude stephenine extract during adsorption and elution is 0.5-1.5 BV/h. 4. The method according to claim 3, characterized in that, the method also includes: recovering methanol to 2-3 times the weight of the raw material by the stephalidine component under reduced pressure, then adding activated carbon, heating to reflux, Filtrate while it is hot, then add hot water to the filtrate, adjust the pH to 8-10, let it stand, and let it cool to precipitate a large amount of crystals, which is stephenidine; Preferably, the amount of activated carbon added is 0.1%-0.3% of the mass of the stepherine component after recovering methanol under reduced pressure; Preferably, the time of heating to reflux is 0.5-2h; Preferably, the volume ratio of the filtrate to hot water is 1:0.8-1.2. 5. according to the method described in any one of claim 1-4, it is characterized in that, the extraction method of described Stephalidine crude extract comprises: Use ethanol aqueous solution to extract the mountain turtle raw material. After the first extraction is completed, the extraction residue is added with ethanol aqueous solution for the second extraction. The two extracts are combined, and then left to stand under acidic conditions to precipitate precipitation. The first solid-liquid separation obtains clear liquid, and then leave the clear liquid under alkaline conditions to precipitate a precipitate, separate solid-liquid for the second time, and collect the precipitate of the second solid-liquid separation to obtain a crude extract of stephine. 6. The method according to claim 5, characterized in that, the volume content of ethanol in the aqueous ethanol solution is 60-95%; Preferably, the volume-to-mass ratio of the aqueous ethanol solution to the mountain turtle raw material in the first extraction is 5-10:1, and the extraction time is 3-5 hours; Preferably, in the second extraction, the volume-to-mass ratio of the ethanol aqueous solution to the mountain turtle raw material is 3-6:1, and the extraction time is 2-3 hours. 7. The method according to claim 5, wherein standing under the acidic condition comprises: concentrating the combined extract under reduced pressure to an alcohol-free state, and then adding a mineral acid solution to it to pH=2- 3. The standing time is ≥4 hours; The inorganic acids include hydrochloric acid and sulfuric acid. 8. The method according to claim 5, wherein the standing under alkaline conditions comprises: adding an inorganic alkali solution to the clear liquid until pH=8-9, and the standing time is ≥4 hours; The inorganic base includes sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate and ammonia water. 9. The stephine obtained by the method of extracting, separating and purifying stephine from the mountain turtle according to any one of claims 1-8, characterized in that, the stephine has a purity >98%. 10. the application of Stephalidine as claimed in claim 9 in the preparation of related medicines, is characterized in that, said related medicines comprise the medicine of treatment prevention vascular headache, and said vascular headache comprises migraine, hyperactivity Movement disorders and hyperactive Tourette syndrome in children.