US20230047648A1 - Method for Isolation of Cytisine - Google Patents
Method for Isolation of Cytisine Download PDFInfo
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- US20230047648A1 US20230047648A1 US17/969,859 US202217969859A US2023047648A1 US 20230047648 A1 US20230047648 A1 US 20230047648A1 US 202217969859 A US202217969859 A US 202217969859A US 2023047648 A1 US2023047648 A1 US 2023047648A1
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- cytisine
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- acid
- aqueous concentrate
- liquid mixture
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- ANJTVLIZGCUXLD-BDAKNGLRSA-N (-)-Cytisine Natural products C1NC[C@@H]2CN3C(=O)C=CC=C3[C@H]1C2 ANJTVLIZGCUXLD-BDAKNGLRSA-N 0.000 title claims abstract description 46
- 229930017327 cytisine Natural products 0.000 title claims abstract description 46
- 229940027564 cytisine Drugs 0.000 title claims abstract description 46
- ANJTVLIZGCUXLD-UHFFFAOYSA-N ent-cytisine Natural products C1NCC2CN3C(=O)C=CC=C3C1C2 ANJTVLIZGCUXLD-UHFFFAOYSA-N 0.000 title claims abstract description 46
- ANJTVLIZGCUXLD-DTWKUNHWSA-N cytisine Chemical compound C1NC[C@H]2CN3C(=O)C=CC=C3[C@@H]1C2 ANJTVLIZGCUXLD-DTWKUNHWSA-N 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000002955 isolation Methods 0.000 title description 5
- 239000012141 concentrate Substances 0.000 claims abstract description 30
- 239000002253 acid Substances 0.000 claims abstract description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000000203 mixture Substances 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract 8
- 239000007864 aqueous solution Substances 0.000 claims abstract 7
- 230000003113 alkalizing effect Effects 0.000 claims abstract 6
- 239000003795 chemical substances by application Substances 0.000 claims abstract 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 16
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 8
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 6
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 239000000908 ammonium hydroxide Substances 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims 3
- 230000020477 pH reduction Effects 0.000 claims 1
- 240000004752 Laburnum anagyroides Species 0.000 description 23
- 239000000284 extract Substances 0.000 description 13
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 238000000605 extraction Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000003808 methanol extraction Methods 0.000 description 7
- 239000000725 suspension Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 241000246091 Thermopsis Species 0.000 description 5
- 241000188851 Thermopsis lanceolata Species 0.000 description 5
- 238000004821 distillation Methods 0.000 description 5
- 239000011260 aqueous acid Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000012620 biological material Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 2
- 235000006693 Cassia laevigata Nutrition 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 229930013930 alkaloid Natural products 0.000 description 2
- 150000003797 alkaloid derivatives Chemical class 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000002021 butanolic extract Substances 0.000 description 2
- 239000002026 chloroform extract Substances 0.000 description 2
- 238000002481 ethanol extraction Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 2
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 2
- 229960002715 nicotine Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 229940122578 Acetylcholine receptor agonist Drugs 0.000 description 1
- 208000007848 Alcoholism Diseases 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000245573 Anagyris Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 241000220486 Caesalpinioideae Species 0.000 description 1
- 241000219758 Cytisus Species 0.000 description 1
- 241000246169 Genista Species 0.000 description 1
- 241000522163 Gymnocladus Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010057852 Nicotine dependence Diseases 0.000 description 1
- 102000019315 Nicotinic acetylcholine receptors Human genes 0.000 description 1
- 108050006807 Nicotinic acetylcholine receptors Proteins 0.000 description 1
- 241000522652 Ormosia <angiosperm> Species 0.000 description 1
- 241000220435 Papilionoideae Species 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- 241000219784 Sophora Species 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 208000025569 Tobacco Use disease Diseases 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- TVQGDYNRXLTQAP-UHFFFAOYSA-N ethyl heptanoate Chemical compound CCCCCCC(=O)OCC TVQGDYNRXLTQAP-UHFFFAOYSA-N 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 235000021388 linseed oil Nutrition 0.000 description 1
- 239000000944 linseed oil Substances 0.000 description 1
- 238000002803 maceration Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000004692 metal hydroxides Chemical class 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000010773 plant oil Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
- C07D471/18—Bridged systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/04—Solvent extraction of solutions which are liquid
- B01D11/0492—Applications, solvents used
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
Definitions
- the invention concerns a method for isolating cytisine from plant material.
- cytisine is a potent nicotinic acetylcholine receptor agonist that shows pharmacological effects similar to nicotine. It is used in fighting nicotine and alcohol dependence. Its presence has been established in various plant species like Faboideae subfamily that includes Laburnum, Anagyris, Thermopsis, Cytisus, Genista and Sophora . Cytisine is also found in Gymnocladus , a member of Caesalpinioideae subfamily, etc. Extraction of cytisine from Laburnum seeds was reported more than a century ago (1). Cytisine was later extracted by processing milled seeds with alkali to the obtaining of a paste extracted for several hours using Soxhlet apparatus (2).
- the proposal includes the use of a mixture of dichloromethane, methanol and ammonium hydroxide (25%) for extraction of cytisine in two similar variants (3) and (4).
- a method for isolation of alkaloids from a mixture of biomaterial with 0.1 N HCl and subsequent elution of ion-exchange resin was described (5).
- a method for isolation of cytisine from biomaterials which includes extraction of aqueous alkaline solution (carbonate, hydrogen carbonate or hydroxide of alkaline metal or ammonium hydroxide) of the biomaterial with plant oil (rapeseed oil, sunflower oil, flax oil, grape oil, peanut oil, etc.), re-extraction in acid-aqueous phase (preferably with sulfuric acid), recovery of cytisine by alkaline extraction with dichloromethane (dichloromethane plus ammonium), removal of water and evaporation of the solvent.
- aqueous alkaline solution carbonate, hydrogen carbonate or hydroxide of alkaline metal or ammonium hydroxide
- plant oil rapeseed oil, sunflower oil, flax oil, grape oil, peanut oil, etc.
- re-extraction in acid-aqueous phase preferably with sulfuric acid
- recovery of cytisine by alkaline extraction with dichloromethane (dichloromethane plus ammonium) removal
- thermopsis lanceolata There is a semi-synthetic method for obtaining alkaloid derivatives of cytisine for treatment of CNS diseases, the initial stage consisting of maceration of powdered Ormosia , powdered roots and stems in particular, in methanol, followed by filtration and evaporation to dryness; distribution of the obtained extract between water solution of hydrochloric acid and ethyl acetate; separation of water and organic phases; alkalization of the aqueous phase to pH 10-12 with sodium carbonate; addition of dichloromethane; and repeated separation of phases and isolation of compounds which are later subjected to chemical reactions to the obtaining of the target products (7).
- a method for extraction of cytisine from thermopsis lanceolata has been protected as well (8).
- the method includes extraction of the plant raw material with a lower alcohol (preferably methanol or ethanol), acidified with a mineral acid to pH in the range 1.5-3.5 and temperature of 20-50° C.
- a lower alcohol preferably methanol or ethanol
- the combined alcohol-aqueous extracts which include 50-80% of lower alcohol are concentrated under vacuum to eliminate the alcohol, and after filtration they are extracted with chloroform, methylene chloride, butyl acetate or normal butanol in a ratio of 1:1.
- the acidified aqueous concentrate thus purified from ballast substances is alkalized to pH 9-12 with alkaline (sodium or potassium) hydroxide or ammonium hydroxide and the alkaline aqueous concentrate is extracted at least twice with chloroform, methylene chloride, butyl acetate or normal butanol in a ratio of concentrate:extractant from 1:5 to 1:10.
- the combined organic extracts are evaporated to dryness, then acetone or ethyl acetate are added to obtain a suspension that is allowed to crystallize completely at 5-10° C., then it is filtered and it is dried.
- Example 1 the milled seeds of Golden Chain tree ( Cytisus laburnum L.) are extracted with 60% methanol, acidified with hydrochloric acid.
- the acid aqueous concentrate is purified with chloroform, according to Example 1, alkalized with 25% of sodium hydroxide solution to pH 11, and then it is extracted seven times each with 20 L of methylene chloride.
- the combined organic extracts are subjected to distillation until the methylene chloride is completely eliminated and 2 L of acetone is added to the obtained residue to obtain acetone suspension which rested for 10 hours at 5-10 2C, then the obtained precipitate is filtered and it is dried. 121.5 g of Cytisine HPLC grade of 98.72% and yield of 81% are obtained.
- Example 1 the milled seeds of Golden Chain tree ( Cytisus laburnum L.) are extracted with 70% ethanol acidified with sulfuric acid, instead of acid methanol.
- the acid aqueous concentrate, purified with chloroform is alkalized according to Example 1, with 25% of ammonium hydroxide solution to pH 11, then it is extracted six times each with 25 L of chloroform.
- the obtained chloroform extracts are further treated as in Example 1. 125 g of Cytisine HPLC grade of 98.7% and yield of 83% are obtained.
- Example 1 the milled seeds of Golden Chain tree ( Cytisus laburnum L.) are extracted with acidified with sulfuric acid 70% ethanol instead of acid methanol.
- the acid aqueous concentrate is purified with chloroform using 40% of sodium hydroxide solution to pH 11, then it is extracted five times each with 25 L of chloroform.
- the obtained chloroform extracts further are treated as in Example 1. 120 g of Cytisine HPLC grade of 98.3% and yield of 80% are obtained.
- Thermopsis lanceolata R. Br 15 kg of milled seeds of Thermopsis ( Thermopsis lanceolata R. Br) are extracted three times each with 45 L of acidified with 1.09 L of hydrochloric acid 80% ethanol for 5 hours at pH 2.5-3 and temperature of 28° C.
- the combined alcohol-aqueous acidified extracts are concentrated under vacuum to a volume of 20 L, the acid concentrate is filtered, then it is extracted twice each with 10 L of methylene chloride at pH range of 2.5-3.0.
- the acid purified aqueous concentrate is alkalized with sodium hydroxide to pH 12, then it is extracted six times each with 20 L of methylene chloride.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Alternative & Traditional Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
A method for isolating cytisine from plant material includes dissolving the plant material in an alcohol to form a liquid mixture and acidifying the liquid mixture by addition of an acid. The method also includes concentrating the liquid mixture to form a concentrated aqueous solution and extracting the concentrated aqueous solution with a first extractant to form a purified aqueous concentrate. The method includes alkalizing the purified aqueous concentrate with an alkalizing agent to form an alkaline aqueous concentrate. The method further includes extracting the alkaline aqueous concentrate with a second extractant and removing the second extractant to obtain cytisine.
Description
- This application is a continuation of U.S. patent application Ser. No. 16/965,790, filed on Jul. 29, 2020, which is a 35 USC § 371 National Stage application of International Application No. PCT/BG2018/000044, filed on Nov. 19, 2018, which claims the benefit of Bulgarian Patent Application No. 112671, filed on Jan. 29, 2018, the disclosure of which are incorporated herein by reference in their entireties.
- The invention concerns a method for isolating cytisine from plant material.
- Cytisine-(R,55)-1,2,3,4,5,6-hexahydro1.5-methano-8H-pyrido[1,2-a] (1,5) diazocin-8-one, with the following configuration:
- is a potent nicotinic acetylcholine receptor agonist that shows pharmacological effects similar to nicotine. It is used in fighting nicotine and alcohol dependence. Its presence has been established in various plant species like Faboideae subfamily that includes Laburnum, Anagyris, Thermopsis, Cytisus, Genista and Sophora. Cytisine is also found in Gymnocladus, a member of Caesalpinioideae subfamily, etc. Extraction of cytisine from Laburnum seeds was reported more than a century ago (1). Cytisine was later extracted by processing milled seeds with alkali to the obtaining of a paste extracted for several hours using Soxhlet apparatus (2). The proposal includes the use of a mixture of dichloromethane, methanol and ammonium hydroxide (25%) for extraction of cytisine in two similar variants (3) and (4). A method for isolation of alkaloids from a mixture of biomaterial with 0.1 N HCl and subsequent elution of ion-exchange resin was described (5). A method is known for isolation of cytisine from biomaterials, which includes extraction of aqueous alkaline solution (carbonate, hydrogen carbonate or hydroxide of alkaline metal or ammonium hydroxide) of the biomaterial with plant oil (rapeseed oil, sunflower oil, flax oil, grape oil, peanut oil, etc.), re-extraction in acid-aqueous phase (preferably with sulfuric acid), recovery of cytisine by alkaline extraction with dichloromethane (dichloromethane plus ammonium), removal of water and evaporation of the solvent. However, the purity of cytisine obtained in the above examples is very low (28%) (6). There is a semi-synthetic method for obtaining alkaloid derivatives of cytisine for treatment of CNS diseases, the initial stage consisting of maceration of powdered Ormosia, powdered roots and stems in particular, in methanol, followed by filtration and evaporation to dryness; distribution of the obtained extract between water solution of hydrochloric acid and ethyl acetate; separation of water and organic phases; alkalization of the aqueous phase to pH 10-12 with sodium carbonate; addition of dichloromethane; and repeated separation of phases and isolation of compounds which are later subjected to chemical reactions to the obtaining of the target products (7). A method for extraction of cytisine from thermopsis lanceolata has been protected as well (8).
- It is evident from the overview that the matter of cytisine extraction from plants, although with a long history, has not received a satisfactory solution. The known approaches for isolation of cytisine are multistage, labor-intensive, low-effective and result in a product with very low purity. This determines the great need of a method that ensures good yield of cytisine with higher purity a need imposed by the increasing search and the expanding usage application of this alkaloid.
- It was established that from cytisine-containing plant material, using an accessible and efficient method, cytisine with high yield and purity grade of an active pharmaceutical ingredient (API) could be obtained. The method, according to the invention, includes extraction of the plant raw material with a lower alcohol (preferably methanol or ethanol), acidified with a mineral acid to pH in the range 1.5-3.5 and temperature of 20-50° C. The combined alcohol-aqueous extracts which include 50-80% of lower alcohol are concentrated under vacuum to eliminate the alcohol, and after filtration they are extracted with chloroform, methylene chloride, butyl acetate or normal butanol in a ratio of 1:1. The acidified aqueous concentrate thus purified from ballast substances is alkalized to pH 9-12 with alkaline (sodium or potassium) hydroxide or ammonium hydroxide and the alkaline aqueous concentrate is extracted at least twice with chloroform, methylene chloride, butyl acetate or normal butanol in a ratio of concentrate:extractant from 1:5 to 1:10. The combined organic extracts are evaporated to dryness, then acetone or ethyl acetate are added to obtain a suspension that is allowed to crystallize completely at 5-10° C., then it is filtered and it is dried. The use of the simple and elegant method, according to the invention, surprisingly obtains Cytisine HPLC grade of 98-99.9%, and yield of 80-85%. The Use of the simple and elegant method according to the invention surprisingly obtained a Cytisine HPLC grade of 98-99.9% and a yield of 80-85%. The following examples illustrate the essence of the method according to the invention, without limiting it.
- 15 kg of milled seeds of Golden Chain tree (Cytisus laburnum L.) are extracted twice, each with 45 L of acidified with 0.350 L of sulfuric acid 70% methanol for 5 hours at pH 2.5-3 and temperature of 30° C. The combined alcohol-aqueous acid extracts are concentrated under vacuum to a volume of 25 L (⅕) of the initial volume, the solid components are removed by filtration and the obtained concentrate is twice extracted with 10 L of chloroform each at pH range of 2.5-3.0. The obtained purified acid aqueous concentrate is alkalized with 30% sodium hydroxide solution to pH 11, then it is extracted five times each extraction with 25 L of chloroform. The combined organic extracts are subjected to distillation until the chloroform is completely eliminated and 2 L of acetone is added to the obtained residue to obtain an acetone suspension which rested for 11 hours at 5-10 2c, then is filtered and it is dried. 127 g of Cytisine with HPLC grade of 99.15% and yield of 85% are obtained.
- As in Example 1, the milled seeds of Golden Chain tree (Cytisus laburnum L.) are extracted with 60% methanol, acidified with hydrochloric acid. The acid aqueous concentrate is purified with chloroform, according to Example 1, alkalized with 25% of sodium hydroxide solution to pH 11, and then it is extracted seven times each with 20 L of methylene chloride. The combined organic extracts are subjected to distillation until the methylene chloride is completely eliminated and 2 L of acetone is added to the obtained residue to obtain acetone suspension which rested for 10 hours at 5-10 2C, then the obtained precipitate is filtered and it is dried. 121.5 g of Cytisine HPLC grade of 98.72% and yield of 81% are obtained.
- As in Example 1, the milled seeds of Golden Chain tree (Cytisus laburnum L.) are extracted with 70% ethanol acidified with sulfuric acid, instead of acid methanol. The acid aqueous concentrate, purified with chloroform is alkalized according to Example 1, with 25% of ammonium hydroxide solution to pH 11, then it is extracted six times each with 25 L of chloroform. The obtained chloroform extracts are further treated as in Example 1. 125 g of Cytisine HPLC grade of 98.7% and yield of 83% are obtained.
- As in Example 1, the milled seeds of Golden Chain tree (Cytisus laburnum L.) are extracted with acidified with sulfuric acid 70% ethanol instead of acid methanol. The acid aqueous concentrate is purified with chloroform using 40% of sodium hydroxide solution to pH 11, then it is extracted five times each with 25 L of chloroform. The obtained chloroform extracts further are treated as in Example 1. 120 g of Cytisine HPLC grade of 98.3% and yield of 80% are obtained.
- 15 kg of milled seeds of Golden Chain tree (Cytisus laburnum L.) are extracted three times each with 45 L of acidified with 1.09 L of hydrochloric acid 80% ethanol, for 5 hours at pH 2.5-3.0 and temperature of 28° C. The combined alcohol-aqueous acid extracts are concentrated under vacuum to a volume of 20 L, the acid concentrate is filtered, then it is extracted twice each with 10 L of methylene chloride at pH range of 2.5-3.0. The acid purified aqueous concentrate is alkalized with sodium hydroxide to pH 11, then it is extracted six times each with 20 L of methylene chloride. The combined organic extracts are subjected to distillation until the methylene chloride is completely eliminated and 2 L of ethyl acetate is added to the obtained residue to obtain ethyl acetate suspension, stirred at 5-10° C. and allowing to stay at rest for 10 hours, then the obtained precipitate is filtered and also it is dried. 119 g of Cytisine HPLC grade of 98.8% and yield of 79% are obtained.
- 15 kg of milled seeds of Golden Chain tree (Cytisus laburnum L.) are extracted three times each with 45 L of acidified with 0.350 L of sulfuric acid 70% methanol for 5 hours at pH 2.5-3 and temperature of 30° C. The combined alcohol-aqueous acid extracts are concentrated under vacuum to a volume of 25 L (⅕) of the initial volume, the solid components are removed by filtration and the obtained concentrate is extracted twice each with 10 L of butyl acetate at pH range of 2.5-3.0. The acid purified aqueous concentrate is alkalized with 30% of sodium hydroxide solution to pH 11, then it is extracted six times each with 25 L of butyl acetate. The combined organic extracts are subjected to distillation until the butyl acetate is completely eliminated and 2 L of acetone is added to the obtained residue, which is left at rest for 11 hours at 5-10° C. to obtain aimed acetone suspension, then it is filtered and it is dried. 125 g of Cytisine HPLC grade of 99.85% and yield of 81% are obtained.
- 15 kg of milled seeds of Golden Chain tree (Cytisus laburnum L.) are extracted three times each with 45 L of acidified with 0.350 L of sulfuric acid 70% methanol for 5 hours at pH 2.5-3 and temperature of 30° C. The combined alcohol-aqueous acid extracts are concentrated under vacuum to a volume of 25 L (⅕) of the initial volume, the solid components are removed by filtration and the obtained concentrate is extracted twice each with 10 L of n-butanol at pH range of 2.5-3.0. The acid purified aqueous concentrate is alkalized with 30% sodium hydroxide solution to pH 11, then it is extracted six times each with 25 L of n-butanol. The combined n-butanol extracts are further treated as in Example 6. 120 g of Cytisine HPLC grade of 99.66% and yield of 82% are obtained.
- 15 kg of powdered seeds of Thermopsis (Thermopsis lanceolata R. Br) are extracted three times each with 45 L of acidified with 0.350 L of sulfuric acid 70% methanol for 5 hours at pH 2.5-3.0 and temperature of 30° C. The combined alcohol-aqueous acidified extracts are concentrated under vacuum to a volume of 25 L (⅕) of the initial volume, the solid components are removed by filtration and the obtained concentrate is extracted twice each with 10 L of n-butanol at pH range of 2.5-3.0. The acid purified aqueous concentrate is alkalized with 30% sodium hydroxide solution to pH 11, then it is extracted six times each with 25 L of n-butanol. The combined n-butanol extracts are further treated as in Example 6. 121 g of Cytisine HPLC grade of 99.32% and yield of 81% are obtained.
- 15 kg of milled seeds of Thermopsis (Thermopsis lanceolata R. Br) are extracted three times each with 45 L of acidified with 1.09 L of hydrochloric acid 80% ethanol for 5 hours at pH 2.5-3 and temperature of 28° C. The combined alcohol-aqueous acidified extracts are concentrated under vacuum to a volume of 20 L, the acid concentrate is filtered, then it is extracted twice each with 10 L of methylene chloride at pH range of 2.5-3.0. The acid purified aqueous concentrate is alkalized with sodium hydroxide to pH 12, then it is extracted six times each with 20 L of methylene chloride. The combined organic extracts are subjected to distillation until the methylene chloride is completely eliminated and 2 L of ethyl acetate is added to the obtained residue to obtain ethyl acetate suspension, stirred it at 5-10° C., allowed to stay at rest for 12 hours, then it is filtered and the obtained precipitate is dried. 120 g of Cytisine HPLC grade of 98.9% and yield of 80% are obtained.
Claims (18)
1. A method for isolating cytisine from plant material, the method comprising:
(a) dissolving the plant material in an alcohol to form a liquid mixture;
(b) acidifying the liquid mixture by addition of an acid;
(c) concentrating the liquid mixture to form a concentrated aqueous solution;
(d) extracting the concentrated aqueous solution with a first extractant to form a purified aqueous concentrate;
(e) alkalizing the purified aqueous concentrate with an alkalizing agent to form an alkaline aqueous concentrate;
(f) extracting the alkaline aqueous concentrate with a second extractant; and
(g) removing the second extractant to obtain cytisine.
2. The method of claim 1 , further comprising crystallizing cytisine in an organic solvent.
3. The method of claim 2 , wherein crystallizing further comprises filtering cytisine from the organic solvent.
4. The method of claim 3 , wherein the organic solvent is acetone or ethyl acetate.
5. The method of claim 1 , further comprising filtering the liquid mixture after concentrating.
6. The method of claim 1 , wherein extracting the concentrated aqueous solution with the first extractant comprises adding the first extractant, wherein a ratio of the concentrated aqueous solution to the first extractant is 1:1.
7. The method of claim 1 , wherein extracting the alkaline aqueous concentrate with the second extractant comprises adding the second extractant, wherein a ratio of the alkaline aqueous concentrate to the second extractant is about 1:5 to about 1:10.
8. The method of claim 1 , wherein the alcohol is methanol or ethanol.
9. The method of claim 1 , wherein a pH of the liquid mixture after acidification is between about 1.5 and about 3.5.
10. The method of claim 1 , wherein the acid includes sulfuric acid or hydrochloric acid.
11. The method of claim 1 , wherein the first extractant and the second extractant are independently selected from chloroform, methylene chloride, butyl acetate, and n-butanol.
12. The method of claim 1 , wherein the alkalizing agent is sodium hydroxide or ammonium hydroxide.
13. The method of claim 1 , wherein a pH of the purified aqueous concentrate is about 9 to about 12 after addition of the alkalizing agent.
14. The method of claim 1 , wherein cytisine is obtained at a purity of at least about 98%.
15. The method of claim 14 , wherein cytisine is obtained at a purity of at least about 99%.
16. The method of claim 1 , wherein a yield of cytisine is at least about 80%.
17. The method of claim 16 , wherein the yield of cytisine is at least about 85%.
18. The method of claim 1 , wherein (d) extracting the concentrated aqueous solution with the second extractant to form the purified aqueous concentrate is performed at a temperature ranging from 20° C. to 50° C.
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US17/969,859 US20230047648A1 (en) | 2018-01-29 | 2022-10-20 | Method for Isolation of Cytisine |
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PCT/BG2018/000044 WO2019144204A1 (en) | 2018-01-29 | 2018-11-19 | Method for isolation of cytisine |
US202016965790A | 2020-07-29 | 2020-07-29 | |
US17/969,859 US20230047648A1 (en) | 2018-01-29 | 2022-10-20 | Method for Isolation of Cytisine |
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