A method for extracting, separating, and purifying senna alkaloids from mountain tortoises

Abstract

This invention discloses a method for extracting, separating, and purifying senna-based alkaloids from *Trionyx sinensis*. The method employs an alcohol-water system for extraction from *Trionyx sinensis* raw materials. Based on the properties of alkaloids, a stepwise acid-base precipitation method is used to obtain crude senna-based alkaloids. Molecular imprinting technology is then used to further purify the crude senna-based alkaloids. A high-purity senna-based alkaloid product with a purity greater than 98% can be prepared by a single recrystallization. Compared with traditional extraction and refining processes, this method effectively avoids repeated material transfers, shortens production time, and reduces solvent consumption. Compared with traditional separation and purification methods, it effectively shortens the process route, increases the yield of active ingredients, and reduces the production cycle.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, and more specifically, to a method for extracting, separating and purifying cinnamic acid from mountain tortoises. Background Technology

[0002] Betaine is a benzyl isoquinoline alkaloid extracted and isolated from the tuberous root of *Lysimachia christinae*, a plant belonging to the Menispermaceae family. Studies have shown that it is a dopamine receptor blocker in the brain; it possesses antihypertensive, antiarrhythmic, myocardial infarction protective, analgesic, sedative, antispasmodic, and antipyretic effects. Clinically, it is mainly used to treat vascular headaches, such as migraines; ADHD; and ADHD in children. Research indicates a high incidence of ADHD in children. Surveys show that the incidence of behavioral problems among school-aged children reaches 12.9%, with ADHD ranking highest, reaching an incidence of 4.3-5.8%. These research results indicate that betaine has significant clinical application value and promising development prospects.

[0003] Currently reported techniques for preparing quinquefolin are mostly synthetic or semi-synthetic, with almost no literature or patent reports on the extraction and isolation of quinquefolin from *Trionyx sinensis*. Methods for extracting alkaloids from plants mainly include acid-water extraction and organic solvent extraction; methods for purifying alkaloids mainly include recrystallization, column chromatography, and alkaloid precipitation.

[0004] Acid extraction typically involves reflux extraction using a 1-2% inorganic acid aqueous solution at 80-90℃. The extract is then adjusted to alkaline, cooled, and allowed to stand to precipitate crude alkaloids. Further purification yields a high-purity alkaloid product. Alcohol extraction utilizes a 60%-100% (v / v) ethanol / methanol aqueous solution for reflux extraction. The extract is concentrated until alcohol-free, the pH is adjusted to alkaline, and the precipitate is allowed to stand, resulting in crude alkaloids. Further purification yields a high-purity alkaloid product. Recrystallization, a purification method, primarily utilizes the different solubilities of alkaloids in different solvents or at different temperatures of the same solution for purification and separation. Column chromatography currently uses cation exchange resins, silica gel, and alumina as packing materials. Alkaloid precipitants are mainly used to separate water-soluble quaternary ammonium alkaloids. However, recrystallization purification of alkaloids generally requires multiple crystallizations to obtain a high-purity product, resulting in low yields and numerous steps. Chromatographic separation of alkaloids has poor selectivity. Generally, crude extracts have low purity after one chromatographic separation and multiple crystallizations are required to prepare high-purity products. In addition, a large amount of organic solvent is used.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting, separating and purifying senna alkaloids from mountain tortoises, so as to improve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for extracting, separating, and purifying senna alkaloids from *Trionyx sinensis*, comprising:

[0009] The alcoholic solution of the crude extract of senna was passed through a molecularly imprinted polymer column of senna, and then methanol-glacial acetic acid was added for further column chromatography to collect the senna fraction.

[0010] Optionally, the preparation method of the quinquefolin-based molecularly imprinted polymer column includes: adding a functional monomer, a crosslinking agent and an initiator to an acetonitrile solution of quinquefolin-based base, purging with nitrogen gas, stirring evenly, and allowing to stand to obtain a white hard solid polymer. Then, the polymer is pulverized to obtain uniform particles which are packed into the column. The column is washed with a methanol-glacial acetic acid system until no template molecules are found in the effluent to obtain the quinquefolin-based molecularly imprinted polymer column.

[0011] Optionally, the functional monomer includes methacrylic acid.

[0012] Optionally, the crosslinking agent includes ethylene glycol dimethacrylate.

[0013] Alternatively, the initiator may include azobisisobutyronitrile (AIBN).

[0014] Optionally, the mass ratio of cypermethrin, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 1:3-4:15-20:0.1-0.3.

[0015] Optionally, the standing conditions are to allow the reaction to proceed at 55-65°C for 20-25 hours.

[0016] Optionally, the volume ratio of methanol to glacial acetic acid in the methanol-glacial acetic acid system is 100:0.5-2.

[0017] Optionally, the mass-to-volume ratio of the crude extract of senna to the alcohol solution in the alcohol solution is 1:5-6.

[0018] Optionally, the above-mentioned alcohol solution is ethanol.

[0019] Optionally, the above alcohol solution is anhydrous ethanol.

[0020] Optionally, the adsorption flow rate of the alcoholic solution of the crude extract of senna is 0.8-1.5 BV / h during adsorption elution.

[0021] Optionally, the purification steps also include: reducing the pressure of the cymbidium alkaloid component to recover methanol to 2-3 times the weight of the raw material, then adding activated carbon, heating under reflux, filtering while hot, then adding hot water to the filtrate, adjusting the pH to 8-10, letting it stand, and allowing it to cool to precipitate a large amount of crystals, which is cymbidium alkaloid.

[0022] Optionally, the amount of activated carbon added is 0.1%-0.3% of the mass of the quinine base component after methanol recovery under reduced pressure.

[0023] Optionally, the heating reflux time is 0.5-2 hours.

[0024] Optionally, the volume ratio of filtrate to hot water is 1:0.8-1.2.

[0025] Optionally, the extraction method of the crude extract of senna alkaloids includes extracting the raw material of mountain tortoise with an ethanol aqueous solution. After the first extraction, the extraction residue is added to the ethanol aqueous solution for a second extraction. The two extracts are combined and then allowed to stand under acidic conditions to precipitate. The first solid-liquid separation is performed to obtain a clear liquid. The clear liquid is then allowed to stand under alkaline conditions to precipitate. The second solid-liquid separation is performed, and the precipitate from the second solid-liquid separation is collected to obtain the crude extract of senna alkaloids.

[0026] Optionally, the volume content of ethanol in the aqueous ethanol solution is 60-95%.

[0027] Optionally, in the first extraction, the volume-to-mass ratio of the ethanol-water solution to the tortoise raw material is 5-10:1, and the extraction time is 3-5 hours.

[0028] Optionally, in the second extraction, the volume-to-mass ratio of the ethanol-water solution to the tortoise raw material is 3-6:1, and the extraction time is 2-3 hours.

[0029] Optionally, standing under acidic conditions includes: concentrating the combined extract under reduced pressure to an alcohol-free state, then adding an inorganic acid solution to bring the pH to 2-3, and standing for ≥4 hours.

[0030] The alkaline standing condition includes adding an inorganic alkaline solution to the clear liquid until the pH is 8-9, and standing for ≥4 hours.

[0031] Secondly, the present invention provides a purifying alkaloid obtained by the above-described method for extracting, separating and purifying ...

[0032] Thirdly, the present invention provides the application of the above-mentioned senna alkaloid in the preparation of related drugs, including drugs for treating and preventing vascular headaches, wherein vascular headaches include migraines, hyperactivity disorder and childhood hypercoagulable disorder.

[0033] The present invention has the following beneficial effects:

[0034] This invention provides a method for extracting, separating, and purifying quinquefolin from *Trionyx sinensis*. The method involves extracting the raw material from *Trionyx sinensis* using an alcohol-water system. Based on the properties of alkaloids, a stepwise acid-base precipitation method is employed to obtain crude quinquefolin. Compared to traditional extraction and refining processes, this method effectively avoids repeated material transfers, shortens production time, and reduces solvent consumption. Furthermore, this invention utilizes molecular imprinting technology to further purify the crude quinquefolin, allowing for the preparation of a high-purity quinquefolin product with a purity greater than 98% after a single recrystallization. Compared to traditional repeated recrystallization and column chromatography methods, this method effectively reduces material loss and minimizes production steps. Detailed Implementation

[0035] To make the objectives, 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0036] Alkaloids are a class of nitrogen-containing basic organic compounds widely found in nature. They are effective components of plant-derived traditional Chinese medicine and possess significant physiological activities. There are dozens of types of alkaloids, broadly categorized as organic amines, pyrrolidines, pyridines, isoquinolines, indoles, and hyoscyamines. Among them, sennatin belongs to the isoquinoline alkaloid class and is often found in plant-derived traditional Chinese medicine. Due to its low content, the extraction and purification of this alkaloid are relatively difficult. Conventional separation and purification methods require combining multiple methods, which not only involves numerous steps and operational inconvenience but also makes it difficult to effectively separate sennatin from plants, and the purification effect is difficult to achieve a purity of over 95%. Furthermore, there are currently no reports on the extraction and separation of sennatin from *Trionyx sinensis*. Therefore, in order to obtain high-purity sennatin from *Trionyx sinensis*, the inventors of this invention provide a method for extracting, separating, and purifying sennatin from *Trionyx sinensis*, the method comprising:

[0037] The raw material of *Trionyx sinensis* was extracted using an ethanol-water solution. After the first extraction, the residue was subjected to a second extraction with an ethanol-water solution. The two extracts were combined, and then allowed to stand under acidic conditions to precipitate. A first solid-liquid separation was performed to obtain a clear liquid. The clear liquid was then allowed to stand under alkaline conditions to precipitate again, followed by a second solid-liquid separation. The precipitate from the second solid-liquid separation was collected to obtain the crude extract of *Trionyx sinensis* alkaloid. An alcoholic solution of the crude extract was then passed through a *Trionyx sinensis* alkaloid molecularly imprinted polymer column, followed by methanol-glacial acetic acid column chromatography, and the *Trionyx sinensis* alkaloid fraction was collected.

[0038] In some embodiments, the volume content of ethanol in the aqueous ethanol solution can be 60%, 75%, 80%, or 95%, or any value between 60% and 95%.

[0039] In some embodiments, the volume-to-mass ratio of the ethanol-water solution to the tortoise raw material in the first extraction can be 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, and the extraction time can be 3, 4, or 5 hours, etc.

[0040] In some embodiments, the volume-to-mass ratio of the ethanol-water solution to the tortoise 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, etc.

[0041] In some embodiments, standing under acidic conditions includes: concentrating the combined extract under reduced pressure to an alcohol-free state, and then adding an inorganic acid solution, wherein the inorganic acid can be sulfuric acid or hydrochloric acid, and the pH can be adjusted to pH=2, pH=2.2, pH=2.5 or pH=3, or any value between pH=2 and 3; the standing time can be 4h, 5h, 6h, 7h or 8h, etc.

[0042] In some embodiments, standing under alkaline conditions includes: adding an inorganic alkali solution to the clear liquid until the solution becomes alkaline, wherein the inorganic alkali can be sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, or ammonia water, and the pH adjusted can be pH=8, pH=8.2, pH=8.5, or pH=9, or any value between pH=8 and 9; the standing time can be 4h, 5h, 6h, 7h, or 8h, etc.

[0043] Most alkaloids can form salts with acids and are therefore soluble in acidic water. Acid precipitation can remove acid-insoluble impurities, such as fat-soluble impurities carried over from alcohol extraction. Acid precipitation removes some acid-insoluble impurities, and then the solution is adjusted to alkaline conditions, allowing the alkaloids containing sennae to precipitate out freely. Based on the properties of alkaloids, this invention utilizes a stepwise acid-to-alkali precipitation method to obtain a crude product with fewer impurities and a higher sennae content.

[0044] In some embodiments, the preparation method of the quinquefolin-based molecularly imprinted polymer column includes: adding a functional monomer, a crosslinking agent and an initiator to an acetonitrile solution of quinquefolin-based base, purging with nitrogen gas, stirring until homogeneous, and allowing to stand to obtain a white hard solid polymer. Then, the polymer is pulverized to obtain uniform particles which are packed into the column. The column is washed with a methanol-glacial acetic acid system until no template molecules are present in the effluent to obtain the quinquefolin-based molecularly imprinted polymer column.

[0045] Specifically, the functional monomer can be methacrylic acid, the crosslinking agent can be ethylene glycol dimethacrylate, and the initiator can be azobisisobutyronitrile.

[0046] In some embodiments, the molar ratio of cypermethrin, 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, or any value in the range of 1:3-4:15-20:0.1-0.3.

[0047] In some embodiments, the settling temperature during the preparation of the quinine-based 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 settling time can be 20h, 21h, 22h, 23h, 23h, 24h, or 25h, or any value between 20h and 25h.

[0048] In some implementations, the volume ratio of methanol to glacial acetic acid in the methanol-glacial acetic acid system can be 99:1.

[0049] In some embodiments, the mass-to-volume ratio of the crude extract of senna to the alcohol solution in the alcohol solution of senna can be 1:5 or 1:6, or any value between 1:5 and 6.

[0050] In some embodiments, the alcohol solution is ethanol. More preferably, the alcohol solution is anhydrous ethanol.

[0051] In some embodiments, the adsorption flow rate of the alcoholic solution of the crude extract of sennae can be 0.5-1.5 BV / h during adsorption elution.

[0052] In some embodiments, the purification steps further include: recovering methanol from the cylindrical component under reduced pressure, then adding activated carbon, heating under reflux, filtering while hot, then adding hot water to the filtrate, adjusting the pH to 8-10, letting it stand, and allowing it to cool to precipitate a large amount of crystals, which is cylindrical.

[0053] In some embodiments, the amount of activated carbon added can be 0.1%, 0.2%, or 0.3% of the mass of the cynomorine base component after reduced pressure recovery of methanol, or any value between 0.1% and 0.3%.

[0054] In some embodiments, the reflux heating time can be 0.5h, 1.0h, 1.5h, or 2h, or any value within the range of 0.5-2h. More preferably, the reflux heating time is 1.0h.

[0055] In some implementations, the volume ratio of filtrate to hot water can be 1:0.8-1.2.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1

[0058] This embodiment provides a method for extracting, separating, and purifying cinnamine from *Trionyx sinensis*, the steps of which include:

[0059] 1) Take dried mountain tortoise slices, coarsely crush them, and pass them through a 10-mesh sieve.

[0060] 2) Soak and extract with 5 times the amount of 95% ethanol aqueous solution for 5 hours, separate the extract, and continue to soak and extract the residue with 6 times the amount of 95% ethanol aqueous solution for 3 hours. Combine the two extracts.

[0061] 3) Concentrate the extract under reduced pressure until alcohol-free, release it, add concentrated sulfuric acid solution to adjust the pH to 2, let it cool, let it stand for 5 hours, filter it, add liquid alkali solution to adjust the pH to 9, let it stand for 5 hours, and a large amount of precipitate will precipitate.

[0062] 4) Filter the liquid from 3), precipitate and dry to obtain crude cypermethrin.

[0063] 5) Preparation of molecularly imprinted polymer of quinacrine: Weigh 1 part of commercially available quinacrine with a purity greater than 98% and dissolve it completely in an appropriate amount of acetonitrile. Add 4 parts of methacrylic acid and stir for 8 hours. Add 20 parts of ethylene glycol dimethacrylate and 0.3 parts of azobisisobutyronitrile. Purge with nitrogen and stir until homogeneous. Let stand at 60°C for 25 hours to obtain a white, hard solid polymer. Crush the polymer and pass it through a 60-mesh sieve to obtain uniform particles. Pack the particles into a column and wash with a methanol-glacial acetic acid (99:1) system until the effluent is free of template quinacrine molecules. The molecularly imprinted polymer column is ready for use.

[0064] 6) Dissolve the crude *Euphorbia lathyris* in 5 times the amount of anhydrous ethanol, filter, and pass the filtrate through a molecularly imprinted polymer column at 1 BV / h. Add methanol-glacial acetic acid (99:1) to the column and collect the pyridine base fraction of *Euphorbia lathyris*.

[0065] 7) Collect the cynomorine component from 6) and recover methanol under reduced pressure to an appropriate amount. Add 0.3% powdered activated carbon, heat under reflux for 1 hour, filter while hot, add hot water to the filtrate at a volume ratio of 1:1, adjust the pH to 8.5, let stand, and let cool to precipitate a large amount of crystals, which is cynomorine.

[0066] HPLC analysis showed that the purity of the obtained cypermethrin was 98.6%.

[0067] Example 2

[0068] This embodiment provides a method for extracting, separating, and purifying cinnamine from *Trionyx sinensis*, the steps of which include:

[0069] 1) Take dried mountain tortoise slices, coarsely crush them, and pass them through a 10-mesh sieve.

[0070] 2) Soak and extract with 10 times the amount of 60% ethanol aqueous solution for 3 hours, separate the extract, and continue to soak and extract the residue with 6 times the amount of 60% ethanol aqueous solution for 2 hours. Combine the two extracts.

[0071] 3) Concentrate the extract under reduced pressure until alcohol-free, release it, add concentrated sulfuric acid solution to adjust the pH to 2, let it cool, let it stand for 5 hours, filter it, add liquid alkali solution to adjust the pH to 9, let it stand for 5 hours, and a large amount of precipitate will precipitate.

[0072] 4) Filter the liquid from 3), precipitate and dry to obtain crude cypermethrin.

[0073] 5) Preparation of molecularly imprinted polymer of quinacrine: Weigh 1 part of commercially available quinacrine with a purity greater than 98% and dissolve it completely in an appropriate amount of acetonitrile. Add 4 parts of methacrylic acid and stir for 8 hours. Add 20 parts of ethylene glycol dimethacrylate and 0.3 parts of azobisisobutyronitrile. Purge with nitrogen gas and stir until homogeneous. Let stand at 60°C for 25 hours to obtain a white, hard solid polymer. Crush the polymer and pass it through a 60-mesh sieve to obtain uniform particles. Pack the particles into a column and wash with a methanol-glacial acetic acid (99:1) system until the effluent is free of template quinacrine molecules. The molecularly imprinted polymer column is ready for use.

[0074] 6) Dissolve the crude *Euphorbia lathyris* in 5 times the amount of anhydrous ethanol, filter, and pass the filtrate through a molecularly imprinted polymer column at 1 BV / h. Add methanol-glacial acetic acid (99:1) to the column and collect the pyridine base fraction of *Euphorbia lathyris*.

[0075] 7) Collect the cynomorine component from 6) and recover methanol under reduced pressure to an appropriate amount. Add 0.3% powdered activated carbon, heat under reflux for 1 hour, filter while hot, add hot water to the filtrate at a volume ratio of 1:1, adjust the pH to 9.0, let stand, and let cool to precipitate a large amount of crystals, which is cynomorine.

[0076] HPLC analysis showed that the purity of the obtained cypermethrin was 99.1%.

[0077] Example 3

[0078] This embodiment provides a method for extracting, separating, and purifying cinnamine from *Trionyx sinensis*, the steps of which include:

[0079] 1) Take dried mountain tortoise slices, coarsely crush them, and pass them through a 10-mesh sieve.

[0080] 2) Soak and extract with 5 times the amount of 95% ethanol aqueous solution for 5 hours, separate the extract, and continue to soak and extract the residue with 3 times the amount of 95% ethanol aqueous solution for 3 hours. Combine the two extracts.

[0081] 3) Concentrate the extract under reduced pressure until alcohol-free, release it, add concentrated sulfuric acid solution to adjust the pH to 2, let it cool, let it stand for 5 hours, filter it, add liquid alkali solution to adjust the pH to 9, let it stand for 5 hours, and a large amount of precipitate will precipitate.

[0082] 4) Filter the liquid from 3), precipitate and dry to obtain crude cypermethrin.

[0083] 5) Preparation of molecularly imprinted polymer of quinacrine: Weigh 1 part of commercially available quinacrine with a purity greater than 98% and dissolve it completely in an appropriate amount of acetonitrile. Add 3 parts of methacrylic acid and stir for 8 hours. Add 15 parts of ethylene glycol dimethacrylate and 0.1 parts of azobisisobutyronitrile. Purge with nitrogen gas and stir until homogeneous. Let stand at 60°C for 20 hours to obtain a white, hard solid polymer. Crush the polymer and pass it through a 60-mesh sieve to obtain uniform particles. Pack the particles into a column and wash with a methanol-glacial acetic acid (99:1) system until the effluent is free of template quinacrine molecules. The molecularly imprinted polymer column is ready for use.

[0084] 6) Dissolve the crude *Euphorbia lathyris* in 5 times the amount of anhydrous ethanol, filter, and pass the filtrate through a molecularly imprinted polymer column at 1 BV / h. Add methanol-glacial acetic acid (99:1) to the column and collect the pyridine base fraction of *Euphorbia lathyris*.

[0085] 7) Collect the cynomorine component from 6) and recover methanol under reduced pressure to an appropriate amount. Add 0.3% powdered activated carbon, heat under reflux for 1 hour, filter while hot, add hot water to the filtrate at a volume ratio of 1:1, adjust the pH to 9.5, let stand, and let cool to precipitate a large amount of crystals, which is cynomorine.

[0086] HPLC analysis showed that the purity of the obtained cypermethrin was 99.3%.

[0087] Example 4

[0088] This embodiment provides a method for extracting, separating, and purifying cinnamine from *Trionyx sinensis*, the steps of which include:

[0089] 1) Take dried mountain tortoise slices, coarsely crush them, and pass them through a 10-mesh sieve.

[0090] 2) Soak and extract with 5 times the amount of 80% ethanol aqueous solution for 5 hours, separate the extract, and continue to soak and extract the residue with 6 times the amount of 80% ethanol aqueous solution for 3 hours. Combine the two extracts.

[0091] 3) Concentrate the extract under reduced pressure until alcohol-free, release it, add concentrated sulfuric acid solution to adjust the pH to 2, let it cool, let it stand for 8 hours, filter it, add liquid alkali solution to the clear liquid to adjust the pH to 9.5, let it stand for 5 hours, and a large amount of precipitate will precipitate.

[0092] 4) Filter the liquid from 3), precipitate and dry to obtain crude cypermethrin.

[0093] 5) Preparation of molecularly imprinted polymer of quinacrine: Weigh 1 part of commercially available quinacrine with a purity greater than 98% and dissolve it completely in an appropriate amount of acetonitrile. Add 4 parts of methacrylic acid and stir for 8 hours. Add 20 parts of ethylene glycol dimethacrylate and 0.3 parts of azobisisobutyronitrile. Purge with nitrogen gas and stir until homogeneous. Let stand at 60°C for 25 hours to obtain a white, hard solid polymer. Crush the polymer and pass it through a 60-mesh sieve to obtain uniform particles. Pack the particles into a column and wash with a methanol-glacial acetic acid (99:1) system until the effluent is free of template quinacrine molecules. The molecularly imprinted polymer column is ready for use.

[0094] 6) Dissolve the crude *Euphorbia lathyris* in 10 times the amount of anhydrous ethanol, filter, and pass the filtrate through a molecularly imprinted polymer column at 1 BV / h. Add methanol-glacial acetic acid (99:1) to the column and collect the pyridine base fraction of *Euphorbia lathyris*.

[0095] 7) Collect the cynomorine component from 6) and recover methanol under reduced pressure to an appropriate amount. Add 0.1% powdered activated carbon, heat under reflux for 1 hour, filter while hot, add hot water to the filtrate at a volume ratio of 1:1, adjust the pH to 8.5, let stand, and let cool to precipitate a large amount of crystals, which is cynomorine.

[0096] The purity of the obtained cypermethrin was determined to be 98.9% by HPLC.

[0097] Example 5

[0098] This embodiment provides a method for extracting, separating, and purifying cinnamine from *Trionyx sinensis*, the steps of which include:

[0099] 1) Take dried mountain tortoise slices, coarsely crush them, and pass them through a 10-mesh sieve.

[0100] 2) Soak and extract with 8 times the amount of 75% ethanol aqueous solution for 5 hours, separate the extract, and continue to soak and extract the residue with 5 times the amount of 65% ethanol aqueous solution for 3 hours. Combine the two extracts.

[0101] 3) Concentrate the extract under reduced pressure until alcohol-free, release it, add concentrated sulfuric acid solution to adjust the pH to 2, let it cool, let it stand for 5 hours, filter it, add liquid alkali solution to adjust the pH to 9, let it stand for 5 hours, and a large amount of precipitate will precipitate.

[0102] 4) Filter the liquid from 3), precipitate and dry to obtain crude cypermethrin.

[0103] 5) Preparation of molecularly imprinted polymer of quinacrine: Weigh 1 part of commercially available quinacrine with a purity greater than 98% and dissolve it completely in an appropriate amount of acetonitrile. Add 4 parts of methacrylic acid and stir for 8 hours. Add 20 parts of ethylene glycol dimethacrylate and 0.3 parts of azobisisobutyronitrile. Purge with nitrogen and stir until homogeneous. Let stand at 60°C for 25 hours to obtain a white, hard solid polymer. Crush the polymer and pass it through a 60-mesh sieve to obtain uniform particles. Pack the particles into a column and wash with a methanol-glacial acetic acid (99:1) system until the effluent is free of template quinacrine molecules. The molecularly imprinted polymer column is ready for use.

[0104] 6) Dissolve the crude *Euphorbia lathyris* in 5 times the amount of anhydrous ethanol, filter, and pass the filtrate through a molecularly imprinted polymer column at 1 BV / h. Add methanol-glacial acetic acid (99:1) to the column and collect the pyridine base fraction of *Euphorbia lathyris*.

[0105] 7) Collect the cynomorine component from 6) and recover methanol under reduced pressure to an appropriate amount. Add 0.3% powdered activated carbon, heat under reflux for 1 hour, filter while hot, add hot water to the filtrate at a 1:1 (volume ratio), adjust the pH to 10, let stand, and let cool to precipitate a large amount of crystals, which is cynomorine.

[0106] HPLC analysis showed that the purity of the obtained cypermethrin was 98.5%.

[0107] Comparative Example 1

[0108] The difference from Example 1 is that the extraction method in this comparative example uses acid extraction and alkali precipitation to obtain a crude extract, which is then further purified by multiple recrystallization processes. The steps include:

[0109] 1) Take dried mountain tortoise slices, coarsely crush them, and pass them through a 10-mesh sieve.

[0110] 2) Soak the residue in 8 times the amount of 2% sulfuric acid aqueous solution at room temperature for 24 hours, separate the extract, add 7 times the amount of 2% sulfuric acid aqueous solution to the residue and soak in it at room temperature for 24 hours, separate the extract, add 5 times the amount of 2% sulfuric acid aqueous solution to the residue and soak in it at room temperature for 24 hours, and combine the three extracts.

[0111] 3) Adjust the pH of the extract to 9 with an alkaline solution, let it stand for 5 hours, and a large amount of precipitate will be formed.

[0112] 4) Filter the liquid from 3), precipitate and dry to obtain crude cypermethrin.

[0113] 5) Add 5 times the amount of methanol to the crude cypermethrin product from 4) and heat under reflux for 1-2 hours. Filter while hot, let the filtrate cool to allow crystals to precipitate, and collect the crystals by filtration. Repeat this crystallization process 4 times to obtain the cypermethrin product.

[0114] HPLC analysis showed that the purity of the obtained cynomorium alkaloid was 90%. Each methanol crystallization step in this method results in the loss of approximately 30% of the active ingredient, and the final product purity can only reach about 90%. Subsequent fifth methanol crystallization steps show almost no change in the content, making it difficult to further improve the product purity.

[0115] Comparative Example 2

[0116] The difference from Example 1 is that the purification method in this comparative example uses existing silica gel column chromatography. The extraction method is the same as in Example 1, and the purification method is performed using silica gel column chromatography. The specific steps are as follows:

[0117] 1) Take the crude product of acid extraction and alkali precipitation of cypermethrin, dissolve it fully in methanol, add 2-3 times the amount of silica gel, stir evenly, and pulverize the solvent for later use.

[0118] 2) Pack the silica gel column using the chloroform wet packing method. Spread the powder from step 1) evenly on the top of the silica gel column.

[0119] 3) Analysis: Elution was performed using a chloroform:methanol 50:1 column chromatography. The fraction containing sennae was collected, and the solvent was recovered to obtain sennae. HPLC analysis showed that the purity of sennae was 60-70%.

[0120] 4) Repeating step 3) twice with methanol recrystallization yields a product of approximately 90% purity, namely, cypermethrin. The purity is difficult to further improve through recrystallization.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting, separating, and purifying cinnamine from *Trionyx sinensis*, characterized in that, It involves passing an alcoholic solution of crude extract of gentianine from mountain tortoise through a gentianine molecularly imprinted polymer column, then adding methanol-glacial acetic acid for further column chromatography, and collecting the gentianine fraction. The preparation method of the quinquercetin molecularly imprinted polymer column includes: adding functional monomers, crosslinking agents and initiators to an acetonitrile solution of quinquercetin, introducing nitrogen gas, stirring evenly, and allowing it to stand to obtain a white hard solid polymer. Then, the polymer is crushed to obtain uniform particles which are packed into the column. The column is washed with a methanol-glacial acetic acid system until no template molecules are present in the effluent to obtain the quinquercetin molecularly imprinted polymer column. Wherein, the functional monomer is selected from methacrylic acid; the crosslinking agent is selected from ethylene glycol dimethacrylate; and the initiator is selected from azobisisobutyronitrile. The molar ratio of the cypermethrin base, methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 1:3-4:15-20:0.1-0.3; The conditions for standing are to allow the reaction to proceed at 55-65°C for 20-25 hours. The volume ratio of methanol to glacial acetic acid in the methanol-glacial acetic acid system is 100:0.5-2; The extraction method of the crude extract of cynomorium alkaloids includes: extracting the raw material of mountain tortoise with ethanol aqueous solution; after the first extraction, adding ethanol aqueous solution to the extraction residue for a second extraction; combining the two extracts; then allowing the mixture to stand under acidic conditions to precipitate; performing a first solid-liquid separation to obtain a clear liquid; then allowing the clear liquid to stand under alkaline conditions to precipitate; performing a second solid-liquid separation; and collecting the precipitate from the second solid-liquid separation to obtain the crude extract of cynomorium alkaloids. The volume content of ethanol in the aqueous ethanol solution is 60-95%. In the first extraction, the volume-to-mass ratio of ethanol aqueous solution to mountain tortoise raw material is 5-10:1, and the extraction time is 3-5 hours. In the second extraction, the volume-to-mass ratio of ethanol aqueous solution to mountain tortoise raw material is 3-6:1, and the extraction time is 2-3 hours. The acidic standing condition includes: concentrating the combined extract under reduced pressure to an alcohol-free state, then adding an inorganic acid solution to adjust the pH to 2-3, and standing for ≥4 hours; the inorganic acid is selected from hydrochloric acid and sulfuric acid; The alkaline standing condition includes: adding an inorganic alkali solution to the clear liquid until the pH is 8-9, and standing for ≥4 hours; the inorganic alkali is selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate and ammonia.

2. The method according to claim 1, characterized in that, The mass-to-volume ratio of crude extract of gentianine to alcohol solution in the alcohol solution is 1:5-6.

3. The method according to claim 2, characterized in that, The alcohol solution is ethanol.

4. The method according to claim 2, characterized in that, The alcohol solution is anhydrous ethanol.

5. The method according to claim 2, characterized in that, The adsorption flow rate of the alcoholic solution of the crude extract of cypermethrin during adsorption elution is 0.8-1.5 BV / h.

6. The method according to claim 5, characterized in that, The method further includes: reducing the pressure of the cynomorium alkaloid component to recover methanol to 2-3 times the weight of the raw material, then adding activated carbon, heating under reflux, filtering while hot, then adding hot water to the filtrate, adjusting the pH to 8-10, letting it stand, and allowing it to cool to precipitate a large amount of crystals, which is cynomorium alkaloid.

7. The method according to claim 6, characterized in that, The amount of activated carbon added is 0.1%-0.3% of the mass of the cinnamic acid base component after methanol is recovered under reduced pressure.

8. The method according to claim 6, characterized in that, The heating and reflux time is 0.5-2 hours.

9. The method according to claim 6, characterized in that, The volume ratio of the filtrate to the hot water is 1:0.8-1.2.