CN111153909A - A double-template molecular imprinting purification method for alpha-and beta-carotene in Stephania tetrandra - Google Patents

A double-template molecular imprinting purification method for alpha-and beta-carotene in Stephania tetrandra Download PDF

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CN111153909A
CN111153909A CN202010051769.1A CN202010051769A CN111153909A CN 111153909 A CN111153909 A CN 111153909A CN 202010051769 A CN202010051769 A CN 202010051769A CN 111153909 A CN111153909 A CN 111153909A
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tetrandrine
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ethanol
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CN111153909B (en
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陈金龙
黄文超
黄丽莉
揭建林
朱培林
杨燕欢
郜杰
康月凤
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Shiyao Group Jiangxi Jinfurong Pharmaceutical Co.,Ltd.
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Abstract

The invention relates to a medicine extraction and purification technology, in particular to a technical method for efficiently separating tetrandrine and tetrandrine simultaneously; the method comprises the steps of synthesizing methacrylic acid, ethylene glycol dimethacrylate, azodiisobutyronitrile and the like with target molecules of tetrandrine and tetrandrine, eluting the target molecules to obtain a double-template molecularly imprinted polymer, simultaneously extracting the tetrandrine and the tetrandrine from the polymer, and further separating the tetrandrine and the tetrandrine from the polymer by using a reversed phase chromatographic column and a preparative chromatograph, wherein the purity of the obtained tetrandrine and the tetrandrine is more than 99%.

Description

A double-template molecular imprinting purification method for alpha-and beta-carotene in Stephania tetrandra
Technical Field
The invention relates to a technology for extracting and purifying natural medicinal raw material medicines, in particular to a technology for simultaneously purifying tetrandrine and fangchinoline in tetrandrine by utilizing double-template molecular imprinting.
Background
Stephania tetrandra, called Stephania tetrandra and Aristolochia lappa, is the root of Shichang Bufo siccus of Menispermaceae. Tetrandrine, also known as tetrandrine, is also called tetrandrine and tetrandrine, is an antihypertensive drug, and is mainly used for treating mild and moderate hypertension and also can be used for treating hypertensive crisis clinically. Tetrandrine has anti-inflammatory, analgesic, blood pressure lowering, and anti-tumor effects.
With the deepened understanding of tetrandrine and the effective components in tetrandrine, tetrandrine and tetrandrine are applied more and more; at present, the tetrandrine and the tetrandrine are extracted mainly by a solvent reflux or percolation method, and then further purified by column chromatography to achieve medicinal purity; however, the method needs a large amount of organic solvent as a mobile phase, the recovery cost of the organic solvent is high, the adsorption quantity of the reversed phase adsorption filler to the crude extract is small, the loss of target components is high, the repeated utilization rate of the adsorption filler is low, the adsorption filler needs to be frequently replaced, and the method is not economical and environment-friendly; in addition, the chemical components of the tetrandrine medicinal materials are complex, and the tetrandrine medicinal materials are often accompanied by plant components with strong hydrophilicity, such as saccharides, tannins and the like, and other small molecular compounds with similar polarities in the extracts of the tetrandrine components, so that the difficulty is increased for separating and purifying the tetrandrine components, the purity of the separated tetrandrine and tetrandrine is low, the requirements of the pharmaceutical industry (the purity reaches more than 99 percent) cannot be met, and the application of the tetrandrine and the tetrandrine is limited.
The molecular imprinting technology is used for identifying specific target molecules, firstly, a host-guest complex is formed by template molecules and polymerizable functional monomers through non-covalent bond or covalent bond actions such as electrostatic interaction, van der waals force, hydrophobic interaction, hydrogen bond and the like, an initiator and a cross-linking agent are added for polymerization reaction to prepare a high molecular polymer, and then the template molecules are removed by a chemical or physical method; at present, single molecular imprinting is used as a solid phase extraction technology, corresponding technical reports exist, extraction is carried out by the method, the target molecule is single, and the purification degree is high.
However, because tetrandrine and tetrandrine have similar structural characteristics to each other, mutual component insertion is inevitable during the purification process of single molecular imprinting, which leads to further purification after single molecular imprinting purification, thus making the purification steps more complicated and limiting the application of molecular imprinting in purifying tetrandrine and tetrandrine.
Therefore, the inventor proposes to establish the tetrandrine and tetrandrine double-template molecularly imprinted polymer, simultaneously enrich the tetrandrine and the tetrandrine, extract a high-purity mixture of the tetrandrine and the tetrandrine, effectively reduce the interference of other analogues, further purify the tetrandrine and the tetrandrine by using reverse phase column chromatography, effectively avoid the interpenetration of two components in the single extraction process, and improve the yield.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of a double-template molecularly imprinted polymer of tetrandrine and tetrandrine.
The second purpose of the invention is to provide a method for purifying tetrandrine and tetrandrine by combining the double-template molecularly imprinted polymer and reversed-phase column chromatography.
The invention specifically comprises the following contents:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and B, mixing with methacrylic acid, placing into a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile at a volume ratio of 1:1-5, sealing, and shaking in a shaker for 1-4h for sufficient reaction; adding ethylene glycol dimethacrylate and azodiisobutyronitrile, shaking thoroughly, transferring the solution into a glass ampoule, introducing nitrogen for 5-20min to remove oxygen; reacting in a constant-temperature water bath at 40-60 ℃ for 30-48 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 50-100 mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 50-80% ethanol aqueous solution at an elution flow rate of 1-4ml/min, and detecting by using a high performance liquid chromatography until the tetrandrine and the tetrandrine are not obtained; drying in a vacuum drying oven at 40-60 ℃ for 18-24h to obtain the double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 6-10 times volume of 70-90% ethanol for 3-5 times, each for 2-5 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 7-9, mixing the filtrate with double-template molecularly imprinted polymer, and standing for 2-18 hr; loading the column, eluting with a mixed solvent of dichloromethane and methanol at a volume ratio of 1:1-5, and leaching the solid phase for extraction at a flow rate of 1-4 ml/min; until the tetrandrine and the tetrandrine B are not detected in the leacheate;
3) collecting tetrandrine and tetrandrine: detecting by ultraviolet-visible spectrophotometry, collecting eluate containing tetrandrine and tetrandrine B, and recovering solvent; concentrating to obtain crude extract of tetrandrine and fangchinoline;
4) further purifying the tetrandrine and the tetrandrine B: dissolving the crude extract with ethanol, and purifying with reversed phase chromatographic column to obtain tetrandrine and B.
Wherein, in the step 1), the tetrandrine, the methacrylic acid, the ethylene glycol dimethacrylate and the azodiisobutyronitrile are mixed according to the molar ratio of 1: 3-5:15-25:10-15.
Wherein, the filtrate in the step 2) and the double-template molecularly imprinted polymer are mixed according to the proportion of 10 ml: mixing at a ratio of 2-5 mg.
Wherein, in the step 4) of the reverse phase chromatographic column for further purification, the quantity of the reverse phase C18 filler used is 200-300 times of the mass of the crude extract in the step 3).
Wherein, during the further purification of the reversed phase chromatographic column in the step 4), the crude extract of the tetrandrine and the ethyl is dissolved by 20 to 30 percent ethanol.
Wherein, in the step 4) of further purification by the reversed phase chromatographic column, the sample loading volume is 1-5% of the column volume.
Wherein, in the step 4) of further purification of the reversed phase chromatographic column, the elution procedure is that 20 to 30 percent of ethanol elutes 4 to 8BV, 50 to 60 percent of ethanol elutes 4 to 8BV, and 70 to 90 percent of ethanol elutes 4 to 8 BV.
Wherein, in the step 4) of further purifying the reversed phase chromatographic column, the eluent is collected according to an ultraviolet spectrum.
The invention has the advantages that:
1. the invention adopts double-template molecular imprinting to simultaneously purify the tetrandrine and the tetrandrine, compared with single-template imprinting, the cost of synthesizing the polymer template is not increased, but the purification of double compounds can be realized, and the prepared double-template molecular imprinting polymer can be repeatedly utilized after leaching, compared with the characteristics of difficult washing, small adsorption quantity and high loss of a chromatographic column, the method is economic and environment-friendly;
2. the high-selectivity molecularly imprinted polymer process related in the disclosure is subjected to system research, and comprises system optimization of a functional monomer, a cross-linking agent, an initiator, a pore-forming agent (a toluene and acetonitrile mixed solvent) and the like;
3. the invention combines the technologies of double-template molecular imprinting, reversed-phase chromatographic column and preparative chromatograph, so that the content of the tetrandrine and the content of the tetrandrine B after separation and purification are both more than 99 percent, the purity requirement of the current medical industry is met, the safety of the derivative product is improved, and the development and the application of the tetrandrine are promoted.
Detailed Description
Example 1:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 160mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:1, sealing, and shaking in a shaker for 4h to allow full reaction; adding 800mmol of ethylene glycol dimethacrylate and 480mmol of azodiisobutyronitrile, shaking up fully, transferring the solution into a glass ampoule, introducing nitrogen for 20min, and removing oxygen; reacting in a constant-temperature water bath at 60 ℃ for 40 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 100-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 70% ethanol aqueous solution at an elution flow rate of 3ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying the mixture for 20 hours in a vacuum drying oven at 50 ℃ to obtain a double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 8 times volume of 80% ethanol for 3 times, each time for 2 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 8 with ammonia water, mixing the filtrate with double-template molecularly imprinted polymer 10 ml: mixing at a ratio of 2mg, and standing for 8 h; loading the column, eluting with a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and leaching the solid phase for extraction at a flow rate of 3 ml/min; until the tetrandrine and the tetrandrine B are not detected in the leacheate; detecting by ultraviolet-visible spectrophotometry, collecting eluate, and concentrating to obtain crude extract of tetrandrine and tetrandrine;
3) further purifying the tetrandrine and the tetrandrine B: weighing the collected crude extract, dissolving with 30% ethanol, loading the crude extract into a column with 250 times of C18 reversed phase filler, balancing the column with 30% ethanol water solution, loading the column with 5% column volume balance solution, eluting with 5BV of 30% ethanol, 50% ethanol and 70% ethanol in sequence, collecting and combining eluates according to ultraviolet spectrum to obtain tetrandrine and ethyl; the purity of tetrandrine and tetrandrine measured by high performance liquid chromatography is 99.1% and 99.3%, respectively.
Example 2:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 120mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:5, sealing, and shaking in a shaker for 1h to allow full reaction; adding 1000mmol of ethylene glycol dimethacrylate and 600mmol of azodiisobutyronitrile, shaking up, transferring the solution into a glass ampoule, introducing nitrogen for 5min to remove oxygen; reacting in a constant-temperature water bath at 40 ℃ for 48 hours; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 50-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 50% ethanol aqueous solution at an elution flow rate of 1ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying for 24h in a vacuum drying oven at 60 ℃ to obtain a double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 6 times volume of 70% ethanol for 5 times, each time for 5 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 7 with ammonia water, mixing the filtrate with double-template molecularly imprinted polymer 10 ml: 5mg, mixing and standing for 2 hours; loading the column, eluting with a mixed solvent of dichloromethane and methanol in a volume ratio of 1:5, and leaching the solid phase for extraction at a flow rate of 1 ml/min; until the tetrandrine and the tetrandrine B are not detected in the leacheate; detecting by ultraviolet-visible spectrophotometry, collecting eluate, and concentrating to obtain crude extract of tetrandrine and tetrandrine;
3) further purifying the tetrandrine and the tetrandrine B: weighing the collected crude extract, dissolving with 20% ethanol, loading the crude extract into a column with 300 times of C18 reversed phase filler, balancing the column with 30% ethanol water solution, loading the column with 1% column volume balance solution, eluting with 4BV of 20% ethanol, 60% ethanol and 90% ethanol in sequence, collecting and combining eluates according to ultraviolet spectrum to obtain tetrandrine and B; the purity of tetrandrine and tetrandrine measured by high performance liquid chromatography is 99.2% and 99.3%, respectively.
Example 3:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 200mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:3, sealing, and shaking in a shaker for 2h to allow full reaction; adding 600mmol of ethylene glycol dimethacrylate and 400mmol of azodiisobutyronitrile, shaking up, transferring the solution into a glass ampoule, introducing nitrogen for 15min to remove oxygen; reacting in a constant-temperature water bath at 50 ℃ for 30 hours; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 100-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 80% ethanol water solution at an elution flow rate of 4ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying for 18h in a vacuum drying oven at 40 ℃ to obtain a double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 10 times volume of 90% ethanol for 4 times, each for 3 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 9 with ammonia water, mixing the filtrate with double-template molecularly imprinted polymer 10 ml: 3mg, and standing for 18 hours; loading the column, eluting with a mixed solvent of dichloromethane and methanol in a volume ratio of 1:3, and leaching the solid phase for extraction at a flow rate of 4 ml/min; until the tetrandrine and the tetrandrine B are not detected in the leacheate; detecting by ultraviolet-visible spectrophotometry, collecting eluate, and concentrating to obtain crude extract of tetrandrine and tetrandrine;
3) further purifying the tetrandrine and the tetrandrine B: weighing the collected crude extract, dissolving with 30% ethanol, loading into a column with 200 times of C18 reversed phase filler, balancing the column with 30% ethanol water solution, loading with 3% column volume balance solution, eluting with 8BV of 30% ethanol, 55% ethanol and 80% ethanol, collecting combined eluates according to ultraviolet spectrum to obtain tetrandrine and B; the purity of tetrandrine and tetrandrine measured by high performance liquid chromatography is 99.3% and 99.1%.
Comparative example 1:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 160mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:1, sealing, and shaking in a shaker for 4h to allow full reaction; adding 800mmol of ethylene glycol dimethacrylate and 480mmol of azodiisobutyronitrile, shaking up fully, transferring the solution into a glass ampoule, introducing nitrogen for 20min, and removing oxygen; reacting in a constant-temperature water bath at 60 ℃ for 40 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 100-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 70% ethanol aqueous solution at an elution flow rate of 3ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying the mixture for 20 hours in a vacuum drying oven at 50 ℃ to obtain a double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 8 times volume of 80% ethanol for 3 times, each time for 2 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 8 with ammonia water, mixing the filtrate with double-template molecularly imprinted polymer 10 ml: mixing at a ratio of 2mg, and standing for 8 h; loading the column, eluting with a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and leaching the solid phase for extraction at a flow rate of 3 ml/min; until the tetrandrine and the tetrandrine B are not detected in the leacheate; detecting by ultraviolet-visible spectrophotometry, collecting eluate, and concentrating to obtain crude extract of tetrandrine and tetrandrine;
3) the purity of tetrandrine and tetrandrine in the concentrate was measured by high performance liquid chromatography to be 37% and 29%.
Comparative example 2:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 110mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:1, sealing, and shaking in a shaker for 4h to allow full reaction; adding 590mmol of ethylene glycol dimethacrylate and 610mmol of azodiisobutyronitrile, shaking up sufficiently, transferring the solution into a glass ampoule, introducing nitrogen for 20min, and removing oxygen; reacting in a constant-temperature water bath at 60 ℃ for 40 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 100-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 70% ethanol aqueous solution at an elution flow rate of 3ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying the mixture for 20 hours in a vacuum drying oven at 50 ℃ to obtain a double-template molecularly imprinted polymer; otherwise, the same as example 1; the contents of tetrandrine and tetrandrine in the concentrate were measured by high performance liquid chromatography and were 17% and 12%, respectively.
Comparative example 3:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 210mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:1, sealing, and shaking in a shaker for 4h to allow full reaction; adding 1100mmol of ethylene glycol dimethacrylate and 390mmol of azodiisobutyronitrile, shaking up, transferring the solution into a glass ampoule, introducing nitrogen for 20min to remove oxygen; reacting in a constant-temperature water bath at 60 ℃ for 40 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 100-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 70% ethanol aqueous solution at an elution flow rate of 3ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying the mixture for 20 hours in a vacuum drying oven at 50 ℃ to obtain a double-template molecularly imprinted polymer; otherwise, the same as example 1; the contents of tetrandrine and tetrandrine in the concentrate were measured by high performance liquid chromatography to be 7% and 9%, respectively.
Comparative example 4:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 160mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:1, sealing, and shaking in a shaker for 4h to allow full reaction; adding 800mmol of ethylene glycol dimethacrylate and 480mmol of azodiisobutyronitrile, shaking up fully, transferring the solution into a glass ampoule, introducing nitrogen for 20min, and removing oxygen; reacting in a constant-temperature water bath at 60 ℃ for 40 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 100-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 70% ethanol aqueous solution at an elution flow rate of 3ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying the mixture for 20 hours in a vacuum drying oven at 50 ℃ to obtain a double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 8 times volume of 80% ethanol for 3 times, each time for 2 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 4 with hydrochloric acid, mixing the filtrate with double-template molecularly imprinted polymer 10 ml: mixing at a ratio of 2mg, and standing for 8 h; loading the column, eluting with a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and leaching the solid phase for extraction at a flow rate of 3 ml/min; until the tetrandrine and the tetrandrine B are not detected in the leacheate; detecting by ultraviolet-visible spectrophotometry, collecting eluate, and concentrating to obtain crude extract of tetrandrine and tetrandrine;
3) further purifying the tetrandrine and the tetrandrine B: weighing the collected crude extract, dissolving with 30% ethanol, loading the crude extract into a column with 250 times of C18 reversed phase filler, balancing the column with 30% ethanol water solution, loading the column with 5% column volume balance solution, eluting with 5BV of 30% ethanol, 50% ethanol and 70% ethanol in sequence, collecting and combining eluates according to ultraviolet spectrum to obtain tetrandrine and ethyl; the purity of tetrandrine and tetrandrine measured by high performance liquid chromatography was 69.1% and 69.3%, respectively.
Comparative example 5:
a method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and 40mmol of ethyl, mixing with 160mmol of methacrylic acid, placing in a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile in a volume ratio of 1:1, sealing, and shaking in a shaker for 4h to allow full reaction; adding 800mmol of ethylene glycol dimethacrylate and 480mmol of azodiisobutyronitrile, shaking up fully, transferring the solution into a glass ampoule, introducing nitrogen for 20min, and removing oxygen; reacting in a constant-temperature water bath at 60 ℃ for 40 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 100-mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 70% ethanol aqueous solution at an elution flow rate of 3ml/min, and detecting by using a high performance liquid chromatography until no tetrandrine and no ethyl; drying the mixture for 20 hours in a vacuum drying oven at 50 ℃ to obtain a double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 8 times volume of 80% ethanol for 3 times, each time for 2 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 8 with ammonia water, mixing the filtrate with double-template molecularly imprinted polymer 10 ml: mixing at a ratio of 2mg, and standing for 8 h; loading the column, leaching the solid phase with 95% ethanol for extraction, and leaching the solid phase for extraction; until the tetrandrine and the tetrandrine B are not detected in the leacheate; detecting by ultraviolet-visible spectrophotometry, collecting eluate, and concentrating to obtain crude extract of tetrandrine and tetrandrine;
3) further purifying the tetrandrine and the tetrandrine B: weighing the collected crude extract, dissolving with 30% ethanol, loading the crude extract into a column with 250 times of C18 reversed phase filler, balancing the column with 30% ethanol water solution, loading the column with 5% column volume balance solution, eluting with 5BV of 30% ethanol, 50% ethanol and 70% ethanol in sequence, collecting and combining eluates according to ultraviolet spectrum to obtain tetrandrine and ethyl; the purity of tetrandrine and tetrandrine measured by high performance liquid chromatography is 79.1% and 79.1%.

Claims (7)

1. A method for purifying alpha and beta-carotene in tetrandra root by double-template molecular imprinting is characterized by comprising the following steps of: comprises the following steps:
1) preparing a double-template molecularly imprinted polymer: weighing tetrandrine and B, mixing with methacrylic acid, placing into a conical flask with a stopper, dissolving with a mixed solvent of toluene and acetonitrile at a volume ratio of 1:1-5, sealing, and shaking in a shaker for 1-4h for sufficient reaction; adding ethylene glycol dimethacrylate and azodiisobutyronitrile, shaking thoroughly, transferring the solution into a glass ampoule, introducing nitrogen for 5-20min to remove oxygen; reacting in a constant-temperature water bath at 40-60 ℃ for 30-48 h; filtering the generated polymer to remove the solvent, cooling to room temperature, grinding, and sieving with a 50-100 mesh sieve; loading the sieved polymer into a column, washing the template molecules and unreacted or excessive functional monomers by using 50-80% ethanol aqueous solution at an elution flow rate of 1-4ml/min, and detecting by using a high performance liquid chromatography until the tetrandrine and the tetrandrine are not contained; drying for 18-24h in a vacuum drying oven at 40-60 ℃ to obtain a double-template molecularly imprinted polymer;
2) extracting tetrandrine and tetrandrine: pulverizing radix Stephaniae Japonicae, reflux-extracting with 6-10 times volume of 70-90% ethanol for 3-5 times, each for 2-5 hr, mixing extractive solutions, filtering to obtain extract, dispersing in 4 times volume of water, filtering, collecting filtrate, adjusting pH to 7-9, mixing the filtrate with double-template molecularly imprinted polymer, and standing for 2-18 hr; loading the column, eluting with a mixed solvent of dichloromethane and methanol at a volume ratio of 1:1-5, and leaching the solid phase for extraction at a flow rate of 1-4 ml/min; until the tetrandrine and the tetrandrine B are not detected in the leacheate;
3) collecting tetrandrine and tetrandrine: detecting by ultraviolet-visible spectrophotometry, collecting eluate containing tetrandrine and tetrandrine B, and recovering solvent; concentrating to obtain crude extract of tetrandrine and fangchinoline;
4) further purifying the tetrandrine and the tetrandrine B: dissolving the crude extract with 20-30% ethanol, and purifying with reversed phase chromatographic column to obtain tetrandrine and tetrandrine.
2. The method for purifying stephania tetrandra medium alpha and B by double-template molecular imprinting as claimed in claim 1, wherein the method comprises the following steps: in the step 1), tetrandrine, methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile are mixed according to a molar ratio of 1: 3-5:15-25:10-15.
3. The method for purifying stephania tetrandra medium alpha and B by double-template molecular imprinting as claimed in claim 1, wherein the method comprises the following steps: the filtrate in the step 2) and the double-template molecularly imprinted polymer are mixed according to the proportion of 10 ml: mixing at a ratio of 2-5 mg.
4. The method for purifying stephania tetrandra medium alpha and B by double-template molecular imprinting as claimed in claim 1, wherein the method comprises the following steps: in the step 4) of the reverse phase chromatographic column for further purification, the amount of the reverse phase C18 filler used is 200-fold and 300-fold of the crude extraction mass in the step 3).
5. The method for purifying stephania tetrandra medium alpha and B by double-template molecular imprinting as claimed in claim 1, wherein the method comprises the following steps: in the step 4) of further purification by the reversed phase chromatographic column, the sample loading volume is 1-5% of the column volume.
6. The method for purifying stephania tetrandra medium alpha and B by double-template molecular imprinting as claimed in claim 1, wherein the method comprises the following steps: in the step 4) of further purification of the reversed phase chromatographic column, the elution procedure is that 20-30% ethanol is used for eluting 4-8BV, 50-60% ethanol is used for eluting 4-8BV, and 70-90% ethanol is used for eluting 4-8 BV.
7. The method for purifying stephania tetrandra medium alpha and B by double-template molecular imprinting as claimed in claim 1, wherein the method comprises the following steps: and in the step 4) of further purifying the reversed phase chromatographic column, collecting eluent according to an ultraviolet spectrum.
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