Method for extracting cytisine

Abstract

The invention relates to the technical field of cytisine extraction, in particular to a cytisine extraction method which comprises the following steps: mixing hemerocallis lanceolata seeds with a first solvent, heating from room temperature to a reflux state, concentrating an extracting solution to obtain a cytisine-containing total alkaloid mixture, adding water to dilute, adding acid to adjust the pH value to 1-6, standing, and filtering to obtain a cytisine extract; then filtering the total alkaloid mixture containing the cytisine, collecting filtrate, adding alkali to adjust the pH value to 8-10, then concentrating to an extract state, adding a second solvent, standing for crystallization, and finally carrying out solid-liquid separation and drying to obtain the cytisine, the total alkaloid mixture containing the cytisine is obtained after the extracting solution is concentrated, impurities are separated out under the acidic condition, the mixture is filtered and then crystallized under the alkaline condition, and the phenomenon of product emulsification caused by extraction and concentration by using a large amount of solvent is avoided, so that the yield of the product is improved, and environmental protection is facilitated; and the final product cytisine is high in purity, and the yield is improved.

Description

Technical Field

[0001] This invention relates to the field of cytisine extraction technology, and more particularly to a method for extracting cytisine. Background Technology

[0002] *Thermopsis lanceolata* R.Br., also known as wild cassia seed or wild cassia, is a perennial herb belonging to the genus *Thermopsis* in the family Leguminosae. Its mature seeds are used medicinally. It is abundant in Northeast, North, and Northwest China. This plant contains various alkaloids, with cytisine being the most abundant, accounting for about half of the total alkaloid content. Cytisine acts on the respiratory system similarly to nicotine, reflexively stimulating respiration. Its stimulating effect is stronger than that of the same dose of lobeline. Clinically, a 0.15% aqueous solution is administered intramuscularly or intravenously to treat reflex respiratory arrest caused by surgical trauma, asphyxiation from toxic agents such as cyanide, and anesthetics, or respiratory and cardiovascular failure, shock, collapse, and neonatal asphyxia caused by infectious diseases. In recent years, cytisine has attracted international attention as a significant therapeutic agent for smoking cessation.

[0003] Current technical reports describe a method involving the crushing and solvent extraction of *Gynostemma pentaphyllum* seeds. The solvent volume is 2-20 times the weight of the seeds. The extraction temperature is room temperature to reflux. Extraction is performed 1-5 times, with each extraction lasting at least 30 minutes. The solvent is at least one of methanol, ethanol, propanol, isopropanol, and water. The extract is concentrated to obtain a total alkaloid mixture containing cytisine. This mixture is then diluted with water, and the pH is adjusted to neutral or acidic. Impurities are then extracted using an organic solvent, preferably at least one of chloroform, dichloromethane, ethyl acetate, diethyl ether, benzene, toluene, and xylene, with dichloromethane and chloroform being preferred. The pH of the aqueous phase of the extracted total alkaloid mixture is adjusted to neutral or alkaline, and then di-tert-butyl dicarbonate is added. The mixture is stirred to convert cytisine to... The N-tert-butyloxycarbonyl cytisine is converted into a less polar and less water-soluble form to facilitate the subsequent extraction process. The amount of di-tert-butyl dicarbonate is 0.8 to 2 times the cytisine content in the seeds of *Gynostemma pentaphyllum*. The reaction temperature is below 100°C, and the reaction time is at least 30 minutes. The aqueous phase is extracted with an organic solvent, and the organic phase is concentrated to obtain N-tert-butyloxycarbonyl cytisine. The organic solvent is at least one selected from chloroform, dichloromethane, ethyl acetate, diethyl ether, benzene, toluene, and xylene, with ethyl acetate being preferred. The N-tert-butyloxycarbonyl cytisine is deprotected with an acid, and then the acid is neutralized with a base to obtain cytisine. The acid used to deprotect the tert-butyloxycarbonyl group is at least one selected from hydrochloric acid, sulfuric acid, and trifluoroacetic acid. The solvent is at least one selected from methanol, ethyl acetate, and ethanol. The base is an inorganic or organic base. In the above steps, the inorganic base is sodium hydroxide, sodium carbonate, sodium bicarbonate, or ammonia, and the organic base is triethylamine. The reaction temperature in the above steps is 0℃~100℃.

[0004] The aforementioned existing technologies use a large number of different solvents, which can easily cause environmental and water pollution, and subsequent treatment is complicated. In addition, the yield of cytisine is low. Summary of the Invention

[0005] To address the aforementioned technical problems, embodiments of the present invention provide a method for extracting cytisine.

[0006] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions: This invention provides a method for extracting cytisine, comprising the following steps: The seeds of *Gynostemma pentaphyllum* were mixed with the first solvent and heated from room temperature to reflux. The extract was concentrated to obtain a total alkaloid mixture containing cytisine. Water was added to dilute the mixture, and acid was added to adjust the pH to 1-6. The mixture was then allowed to stand. The total alkaloid mixture containing cytisine was then filtered, and the filtrate was collected. Alkali was added to adjust the pH to 8-10. The mixture was then concentrated to a paste state and a second solvent was added. The mixture was allowed to stand and crystallize. Finally, the solid and liquid were separated and dried to obtain cytisine.

[0007] In some embodiments, the lanceolate yellow-flowered seeds have a mesh size of 10.

[0008] In some embodiments, the first solvent includes at least one of methanol, ethanol, propanol, isopropanol, and water.

[0009] In some embodiments, the amount of the first solvent is 2 to 20 times the weight of the lanceolate yellow flower seeds, the number of extractions is 1 to 5, and each extraction lasts 2 to 6 hours.

[0010] In some embodiments, the amount of water added is equal to the weight of the lanceolate yellow flower seeds.

[0011] In some embodiments, the acid includes at least one of hydrochloric acid, sulfuric acid, and trifluoroacetic acid.

[0012] In some embodiments, the alkali includes at least one of sodium hydroxide, sodium carbonate, and sodium bicarbonate.

[0013] In some embodiments, the amount of the second solvent added is 0.05-0.3 times the weight of the lanceolate yellow flower seeds.

[0014] In some embodiments, the second solvent is ethyl acetate.

[0015] In some embodiments, the solid-liquid separation is performed after static crystallization for 24-40 hours, and the drying temperature is 45-65°C with a vacuum degree of less than or equal to -0.05 MPa. Compared with the prior art, the beneficial effects of the present invention are as follows: the extract of the present invention is concentrated to obtain a total alkaloid mixture containing cytisine. Impurities are precipitated under acidic conditions and filtered, and then crystallized under alkaline conditions. This avoids the use of a large amount of solvent for extraction and concentration, which leads to emulsification of the product, thereby improving the product yield, which is beneficial to the environment. Moreover, the final product has high purity of cytisine and the yield is improved. Attached Figure Description

[0016] Figure 1 The HPLC chromatogram of cytisine was obtained in Example 1; Figure 2 The HPLC chromatogram of cytisine was obtained in Example 2. Detailed Implementation

[0017] The technical solutions in some embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments provided in this disclosure are within the scope of protection of this disclosure.

[0018] Example 1 20 kg of lanceolate yellow flower seeds, pulverized and sieved through a 10-mesh sieve, were extracted with 100 kg of methanol (80% by volume) under reflux for 3 hours. The residue was filtered, and another 100 kg of methanol (80% by volume) was added, followed by reflux extraction for 2 hours. The mixture was filtered again, and all methanol extracts were combined and concentrated under reduced pressure to a paste-like consistency to obtain a total alkaloid mixture containing cytisine. This mixture was diluted with 20 kg of water, and the pH was adjusted to 5 using trifluoroacetic acid. After standing for 3 hours, the mixture was filtered. The pH of the aqueous phase was adjusted to 10 using sodium bicarbonate, and the mixture was concentrated to a paste-like consistency. 1 kg of ethyl acetate was added, and the mixture was allowed to crystallize for 24 hours. After solid-liquid separation, the crystals were dried under negative pressure (-0.05 MPa) at 45°C, yielding 1010 g of cytisine, with a yield of 5.05%.

[0019] Organic solvent recovery: The liquid was concentrated by heating to 78°C to recover 0.9 kg of ethyl acetate with a purity of 98%.

[0020] from Figure 1 The area percentage report shows that the final product, cytisine, has a high purity of 99.9%, with single impurities such as other alkaloids below 0.1%.

[0021] Example 2 20 kg of lanceolate yellow flower seeds, pulverized and sieved through a 10-mesh sieve, were extracted with 200 kg of methanol (50% by volume) under reflux for 4 hours. The residue was filtered, and another 100 kg of methanol (50% by volume) was added, followed by reflux extraction for 3 hours. All methanol was combined and concentrated under reduced pressure to a paste-like mixture containing cytisine and total alkaloids. The mixture was diluted with 20 kg of water, and the pH was adjusted to 5 using sulfuric acid. After standing for 5 hours, the mixture was filtered. The pH of the aqueous phase was adjusted to 8 using sodium bicarbonate, and the mixture was concentrated to a paste. 4 kg of ethyl acetate was added, and the mixture was allowed to crystallize for 40 hours. After solid-liquid separation, the crystals were dried under negative pressure (-0.05 MPa) at 65°C, yielding 1037 g of cytisine, with a yield of 5.18%.

[0022] Organic solvent recovery: The liquid was concentrated at 78°C to recover 3.8 kg of ethyl acetate with a purity of 99%.

[0023] from Figure 2 The area percentage report shows that the final product, cytisine, has a high purity of 99.9%, with single impurities such as other alkaloids below 0.1%.

[0024] Example 3 20 kg of lanceolate yellow flower seeds, pulverized and sieved through a 10-mesh sieve, were extracted with 300 kg of ethanol (70% by volume) under reflux for 5 hours. The residue was filtered, and another 200 kg of ethanol (70% by volume) was added, followed by reflux extraction for 6 hours. All ethanol was combined and concentrated under reduced pressure to a paste-like mixture containing cytisine and total alkaloids. This paste was diluted with 20 kg of water, and the pH was adjusted to 3 using hydrochloric acid. The mixture was allowed to stand for 4 hours, and then filtered. After filtration, the pH of the aqueous phase was adjusted to 10 using sodium hydroxide, and then concentrated to a paste. 6 kg of ethyl acetate was added, and the mixture was allowed to stand for crystallization for 38 hours. After solid-liquid separation, the crystals were dried under negative pressure (-0.1 MPa) at 60°C, yielding 1024 g of cytisine, with a yield of 5.12%.

[0025] Example 4 20 kg of lanceolate yellow flower seeds, pulverized and sieved through a 10-mesh sieve, were added to 40 kg of water and refluxed for 5 hours. The residue was filtered, and another 100 kg of water was added, followed by reflux extraction for 3 hours. All water was combined and concentrated under reduced pressure to a paste-like mixture containing cytisine and total alkaloids. This paste was diluted with 20 kg of water, and the pH was adjusted to 1 using trifluoroacetic acid. The mixture was allowed to stand for 5 hours, and then filtered. After filtration, the pH of the aqueous phase was adjusted to 9 using sodium carbonate, and then concentrated to a paste. 2 kg of ethyl acetate was added, and the mixture was allowed to stand for 30 hours to crystallize. After solid-liquid separation, the crystals were dried under negative pressure (-0.2 MPa) at 60°C, yielding 1041 g of cytisine, with a yield of 5.21%.

[0026] Example 5 20 kg of lanceolate yellow flower seeds, pulverized and sieved through a 10-mesh sieve, were extracted with 400 kg of isopropanol (80% by volume) under reflux for 6 hours. The residue was filtered, and another 400 kg of isopropanol (80% by volume) was added, followed by reflux extraction for 5 hours. All isopropanol was combined and concentrated under reduced pressure to a paste-like mixture containing cytisine and total alkaloids. 20 kg of water was added for dilution, and the pH was adjusted to 2 using sulfuric acid. The mixture was allowed to stand for 5 hours, and then filtered. After filtration, the pH of the aqueous phase was adjusted to 10 using sodium bicarbonate, and then concentrated to a paste. 6 kg of ethyl acetate was added, and the mixture was allowed to stand for 24 hours to crystallize. After solid-liquid separation, the crystals were dried under negative pressure (-0.2 MPa) at 65°C, yielding 1006 g of cytisine, with a yield of 5.03%.

[0027] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0028] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for extracting cytisine, characterized in that, Includes the following steps: The seeds of *Gynostemma pentaphyllum* were mixed with the first solvent and heated from room temperature to reflux. The extract was concentrated to obtain a total alkaloid mixture containing cytisine. Water was added to dilute the mixture, and acid was added to adjust the pH to 1-6. The mixture was then allowed to stand. The total alkaloid mixture containing cytisine was then filtered, and the filtrate was collected. Alkali was added to adjust the pH to 8-10. The mixture was then concentrated to a paste state and a second solvent was added. The mixture was allowed to stand and crystallize. Finally, the solid and liquid were separated and dried to obtain cytisine.

2. The method according to claim 1, characterized in that, The lanceolate yellow-flowered seeds have a mesh size of 10.

3. The method according to claim 1, characterized in that, The first solvent includes at least one of methanol, ethanol, propanol, isopropanol, and water.

4. The method according to claim 1, characterized in that, The amount of the first solvent is 2 to 20 times the weight of the lanceolate yellow flower seeds, and the extraction is performed 1 to 5 times, with each extraction lasting 2 to 6 hours.

5. The method according to claim 1, characterized in that, The amount of water added is equal to the weight of the lanceolate yellow flower seeds.

6. The method according to claim 1, characterized in that, The acid includes at least one of hydrochloric acid, sulfuric acid, and trifluoroacetic acid.

7. The method according to claim 1, characterized in that, The alkali includes at least one of sodium hydroxide, sodium carbonate, and sodium bicarbonate.

8. The method according to claim 1, characterized in that, The amount of the second solvent added is 0.05-0.3 times the weight of the lanceolate yellow flower seeds.

9. The method according to claim 1, characterized in that, The second solvent is ethyl acetate.

10. The method according to claim 1, characterized in that, The solid-liquid separation is carried out after static crystallization for 24-40 hours, and the drying temperature is 45-65℃ with a vacuum degree of less than or equal to -0.05MPa.

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