CN115536542B - Preparation method of colchicine - Google Patents
Preparation method of colchicine Download PDFInfo
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- CN115536542B CN115536542B CN202110727203.0A CN202110727203A CN115536542B CN 115536542 B CN115536542 B CN 115536542B CN 202110727203 A CN202110727203 A CN 202110727203A CN 115536542 B CN115536542 B CN 115536542B
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- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 title claims abstract description 378
- 229960001338 colchicine Drugs 0.000 title claims abstract description 189
- 238000002360 preparation method Methods 0.000 title abstract description 16
- 229920005989 resin Polymers 0.000 claims abstract description 58
- 239000011347 resin Substances 0.000 claims abstract description 58
- 238000000605 extraction Methods 0.000 claims abstract description 51
- 229920000642 polymer Polymers 0.000 claims abstract description 45
- 238000001914 filtration Methods 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 35
- 238000001035 drying Methods 0.000 claims abstract description 31
- 239000006228 supernatant Substances 0.000 claims abstract description 31
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 28
- 238000001556 precipitation Methods 0.000 claims abstract description 23
- 239000012043 crude product Substances 0.000 claims abstract description 21
- 239000000047 product Substances 0.000 claims abstract description 21
- 239000003463 adsorbent Substances 0.000 claims abstract description 20
- 239000000706 filtrate Substances 0.000 claims abstract description 19
- 239000002994 raw material Substances 0.000 claims abstract description 15
- 239000002904 solvent Substances 0.000 claims abstract description 15
- 238000010298 pulverizing process Methods 0.000 claims abstract description 14
- 238000011068 loading method Methods 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 102
- 239000000243 solution Substances 0.000 claims description 72
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 66
- 241000131505 Gloriosa Species 0.000 claims description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 239000003480 eluent Substances 0.000 claims description 27
- 239000012535 impurity Substances 0.000 claims description 21
- 239000003960 organic solvent Substances 0.000 claims description 21
- 238000001179 sorption measurement Methods 0.000 claims description 20
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 17
- 239000007864 aqueous solution Substances 0.000 claims description 16
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 239000000945 filler Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 5
- 241000234280 Liliaceae Species 0.000 claims description 2
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical group O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 claims 1
- 238000012546 transfer Methods 0.000 abstract description 23
- 238000000746 purification Methods 0.000 abstract description 12
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 26
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 24
- 238000012360 testing method Methods 0.000 description 22
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 239000002244 precipitate Substances 0.000 description 9
- 238000001953 recrystallisation Methods 0.000 description 9
- 238000001291 vacuum drying Methods 0.000 description 9
- 239000013078 crystal Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- 238000011097 chromatography purification Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 201000005569 Gout Diseases 0.000 description 3
- 206010018634 Gouty Arthritis Diseases 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 2
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000000194 supercritical-fluid extraction Methods 0.000 description 2
- DURPTKYDGMDSBL-UHFFFAOYSA-N 1-butoxybutane Chemical compound CCCCOCCCC DURPTKYDGMDSBL-UHFFFAOYSA-N 0.000 description 1
- 241000189665 Colchicum autumnale Species 0.000 description 1
- 240000008609 Gloriosa superba Species 0.000 description 1
- 244000056623 Iphigenia indica Species 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003113 alkalizing effect Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 229920000193 polymethacrylate Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/30—Ortho- or ortho- and peri-condensed systems containing three rings containing seven-membered rings
- C07C2603/34—Benzoheptalenes; Hydrogenated benzoheptalenes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a preparation method of colchicine, which comprises the following steps: (1) pretreatment: drying and pulverizing the raw materials; (2) extraction: extracting the pretreated raw materials by adopting an extraction solvent, filtering, and concentrating the filtrate to obtain a concentrated solution; (3) precipitation: standing the concentrated solution, and filtering to obtain supernatant; (4) macroporous resin purification: loading the supernatant onto macroporous adsorbent resin, eluting in segments, and concentrating the eluate to obtain colchicine extract; (5) polymer chromatography: dissolving colchicine extract, filtering, performing polymer chromatography, eluting, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product; (6) Recrystallizing colchicine crude product, drying, and pulverizing to obtain colchicine pure product. The method has the advantages of high colchicine extraction rate, high transfer rate, high extraction purity, low production cost, safe process, environmental friendliness and extremely strong practicability.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a preparation method of colchicine.
Background
Colchicine is an alkaloid, and the chemical structure of colchicine is shown in the following formula I. Colchicine was originally extracted from colchicine (colchicum autumnale) which is a lily family plant. Pure colchicine is in yellow needle-like crystal with a melting point of 157 ℃; is easy to dissolve in water, ethanol and chloroform; bitter and toxic. Colchicine is widely used in cytology, genetics research and plant breeding work. Colchicine is clinically used for treating acute attacks of gouty arthritis and preventing acute attacks of recurrent gouty arthritis.
The Chinese application publication No. CN101602686A discloses a method for preparing colchicine, which comprises the steps of taking fine powder of raw materials, adding the fine powder into an alkali aqueous solution for soaking overnight, drying and placing the mixture in CO 2 Supercritical extraction in a supercritical extraction device; dissolving the extract in 2% hydrochloric acid aqueous solution, filtering, extracting the filtrate with chloroform, collecting the lower layer solution, adjusting pH to 13, extracting with chloroform, separating the chloroform layer with macroporous adsorbent resin column, eluting with acid aqueous solution as eluent, collecting eluate, concentrating, recrystallizing with acetone, washing, and drying. The method has the advantages of high preparation cost, low extraction rate, unsafe and environment-friendly chloroform.
Chinese patent publication No. CN102276493a discloses a colchicine extraction method comprising 1) pretreating lijiang iphigenia indica; 2) Drying; 3) Reflux extraction; 4) Recovering ethanol; 5) Precipitating with water to obtain supernatant; 6) Regulating the pH value to 9-13 after the supernatant is eluted, alkalizing, and then carrying out secondary elution; 7) Drying to remove methanol; 8) Adding sulfuric acid for dissolving and filtering; 9) Extracting with ethyl acetate, filtering, and vacuum drying the precipitate; 10 Recrystallizing, filtering, vacuum drying the precipitate, and pulverizing the crystal to obtain colchicine. The method has the defects of incomplete extraction, low extraction rate, low purity, safety and environmental protection.
Therefore, a preparation method of colchicine with complete extraction, high extraction rate, high purity, safety and environmental protection is needed.
The present invention has been made in view of this.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the preparation method of colchicine, which has the advantages of high extraction rate, high transfer rate, high extraction purity, low production cost, safe process, environmental friendliness and extremely strong practicability.
In order to solve the technical problems, the invention adopts the basic conception of the technical scheme that:
the invention provides a preparation method of colchicine, which comprises the following steps:
(1) Pretreatment: drying and crushing gloriosa seed;
(2) Extracting: extracting the pretreated raw materials by adopting an extraction solvent, filtering, and concentrating the filtrate to obtain a concentrated solution;
(3) Precipitation: standing the concentrated solution, and filtering to obtain supernatant;
(4) Purifying by macroporous resin: loading the supernatant onto macroporous adsorbent resin, eluting in segments, and concentrating the eluate to obtain colchicine extract;
(5) Polymer chromatography: dissolving colchicine extract, filtering, performing polymer chromatography, eluting, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product;
(6) Recrystallizing colchicine crude product, drying, and pulverizing to obtain colchicine pure product.
According to the preparation method disclosed by the invention, the pretreated gloriosa seeds are sequentially subjected to extraction, precipitation, macroporous resin purification, polymer chromatography, recrystallization, drying and crushing to obtain the colchicine pure product, the method is simple, water/alcohol systems can be used in the process, the cost is low, the environment is protected, and the extraction rate, the transfer rate and the extraction purity of colchicine can be improved by the whole method.
In a further scheme, in the step (1), the pretreatment step specifically includes: collecting raw materials, cleaning, removing impurities, drying, and pulverizing.
Colchicine can be obtained by extracting and purifying different materials, such as rhizoma Sagittariae Sagittifoliae and colchicine. The extraction method is completely different due to different impurities in different raw materials. The preparation method is particularly suitable for extracting and preparing colchicine from seeds of gloriosa (Gloriosa superba L) which is a lily plant.
In the present invention, the seed of gloriosa can be wild or cultivated gloriosa seed.
The collected gloriosa seeds generally need to be subjected to pretreatment such as cleaning, cutting and/or crushing. The purification is to remove impurities such as dust, sediment and the like carried in the process of collecting the gloriosa seeds. The collected gloriosa seeds are prepared into dry products by adopting a method of air drying, sun drying or machine drying, and the obtained dry products are crushed to facilitate extraction.
In a further aspect, in step (2), the extraction solvent comprises water, or an organic solvent, or an aqueous solution of an organic solvent; the organic solvent includes alcohols, ketones, ethers containing 1 to 4 carbon atoms, and mixtures thereof;
preferably, the organic solvent comprises at least one of methanol, ethanol, acetone, n-butyl ether or diethyl ether;
preferably, the extraction solvent is an aqueous solution of ethanol or methanol, more preferably an aqueous ethanol solution, and even more preferably an aqueous ethanol solution having a concentration of 45% to 97% by volume.
In a further scheme, in the step (3), adding water into the concentrated solution, mixing, stirring and standing; wherein, the volume ratio of the concentrated solution to the water is 1:0.5-2.
In a further scheme, after adding water into the concentrated solution and mixing, stirring for half an hour, standing at 40 ℃ for 8 hours, and filtering to obtain supernatant. Thus, the precipitation effect is good, and part of fat-soluble components and impurities are separated out in the standing precipitation.
And (3) carrying out proper post-treatment on the extracting solution of the raw material obtained in the step (2) to obtain the colchicine extract. The invention discovers that the organic solvent in the extracting solution is recovered in the step (2), or the extracting solution is properly concentrated, and then the water precipitation in the step (3) can separate out partial fat-soluble components and impurities from the extracting solution, and colchicine is remained in the water solution due to better water solubility, so that the transfer rate can reach more than 85 percent.
In the invention, after the concentrated solution is mixed with water, stirring is carried out for half an hour, and the mixture is stood at 40 ℃, wherein the temperature is the optimal process after conditional screening, and the dissolution of colchicine can be increased at the temperature, so that the transfer rate of colchicine is improved.
In a further scheme, in the step (4), the macroporous adsorption resin comprises weak-polarity or medium-polarity macroporous adsorption resin;
preferably, the macroporous adsorbent resin comprises D101, ADS-8, ADS-5, LK2MGL type adsorbent resin.
Further, the step (4) of eluting in segments comprises: washing with a first eluent to remove water-soluble impurities, and eluting colchicine adsorbed on macroporous adsorption resin with a second eluent;
preferably, the first eluent comprises water, or an ethanol water solution with the volume concentration of 10-30% or a methanol water solution with the volume concentration of 10-50%; more preferably, the first eluent comprises water, or an ethanol water solution with the volume concentration of 10-20% or a methanol water solution with the volume concentration of 20-35%;
preferably, the second eluent comprises 30% -50% ethanol water solution or 40% -60% methanol water solution; more preferably, the second eluent comprises 30% -35% ethanol water solution or 40% -60% methanol water solution.
The colchicine extract obtained in the step (4) contains 50-80% of colchicine by weight percent.
In the invention, colchicine in the supernatant is purified by macroporous adsorption resin chromatography. In the step (4), along with the recovery of the organic solvent, when the supernatant is loaded on the macroporous adsorption resin column, colchicine contained in the supernatant is easy to be adsorbed by the resin column bed; after loading and adsorption, the resin bed can be washed by water and a low-concentration organic solvent aqueous solution to further remove impurities with larger polarity; then the organic solvent water solution with higher concentration is adopted to step and elute the needed chemical components, and the obtained eluent is concentrated to obtain the colchicine extract.
When colchicine contained in the supernatant is adsorbed by using macroporous resin, the volume of the column bed is generally set to adsorb 1-4g of the supernatant per 100mL of the adsorption resin (the volume after column loading), and then good adsorption transfer rate can be obtained.
After the supernatant is loaded and adsorbed, an aqueous solution, or an ethanol aqueous solution with the volume concentration of 10-30 percent, or a methanol aqueous solution with the volume concentration of 10-50 percent can be used for washing to remove most water-soluble impurities; then eluting colchicine adsorbed on macroporous resin by adopting 30-50% ethanol water solution or 40-60% methanol water solution. Preferred macroporous resins of the present invention are weak polarity, or medium polarity resins such as commercial type D101, ADS-8, ADS-5, LK2MGL type adsorbent resins. Under the condition of the invention, the colchicine content in the extract obtained by the macroporous resin adsorption method can reach about 50-80 percent by weight percent, and the transfer rate can reach 85-95 percent.
In a further scheme, in the step (5), the filler used for polymer chromatography takes polystyrene, divinylbenzene or polymethacrylate as a matrix;
preferably, the filler used for polymer chromatography is selected from one of PS40, PMN40, PSN30, and PSA 30.
In a further embodiment, in step (5), the polymer chromatography is performed, and the polymer is eluted with an eluent selected from one of an aqueous ethanol solution, an aqueous methanol solution, and an aqueous acetonitrile solution.
In a further scheme, in the step (5), the eluent is an ethanol water solution with the volume concentration of 10-60% or a methanol water solution with the volume concentration of 20-60% or an acetonitrile solution with the volume concentration of 10-50%.
Preferably, in the step (5), the eluent is 30% ethanol water solution, 20% -40% methanol water solution, or 20% acetonitrile solution. In a further scheme, in the step (5), adding water, an organic solvent or an aqueous solution of the organic solvent into the colchicine extract for dissolution;
preferably, the colchicine extract is dissolved in water.
In the step (5), the colchicine crude product obtained by polymer chromatography purification contains 85-98% of colchicine by weight percent.
The colchicine extract obtained in the step (4) is purified by reverse high performance liquid chromatography (polymer chromatography), and the selected filler is a polymer filler such as PS40, PMN40, PSN30, PSA30 polymer and the like. Adding water, an organic solvent or an aqueous solution of the organic solvent, preferably water, into the colchicine extract obtained in the step (4), dissolving, filtering, performing polymer chromatography on the filtrate, eluting the required chemical components by adopting low-concentration ethanol, methanol or acetonitrile aqueous solution, and concentrating the obtained eluent to obtain a colchicine crude product.
When colchicine is purified by reverse-phase high performance liquid chromatography, the bed volume is generally set to adsorb 3-15g of colchicine extract per 1000mL, and good adsorption transfer rate can be obtained.
After loading, the sample can be eluted by adopting 10-60% ethanol water solution, or 20-60% methanol water solution with volume concentration, or 10-50% acetonitrile solution with volume concentration. The preferred filler of the present invention is a reverse polymer filler. Under the condition of polymer chromatography, the colchicine content in the product obtained by polymer chromatography purification can reach 85-98% by weight percent, and the transfer rate can reach 70-95%.
In a further scheme, in the step (6),
and (5) recrystallizing: recrystallizing the dried colchicine crude product with ethyl acetate, recovering the organic solvent after recrystallization, filtering, and vacuum drying the precipitate to obtain colchicine containing 50-100% of colchicine by weight percent;
crushing: pulverizing dried colchicine to obtain colchicine pure product.
After the purification in the five steps, colchicine is enriched, and then the colchicine pure product is obtained through recrystallization refining purification. In the present invention, the purity of colchicine pure product is not less than 90%, preferably not less than 95%, more preferably 98%.
The colchicine extract and colchicine pure product prepared by the invention have wide application in preparing the therapeutic drugs for treating the acute episode of gouty arthritis.
By adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects.
1. The colchicine prepared by the invention has clear structure and definite composition, is beneficial to quality detection and control of products at all levels, and accords with the development direction and requirements of natural product composite preparations. In addition, the preparation method of colchicine is simple and easy to implement, low in production cost and extremely high in practicability.
2. The preparation method comprises the steps of sequentially extracting pretreated gloriosa seed, precipitating, purifying by macroporous resin, carrying out polymer chromatography, recrystallizing, drying and crushing to obtain colchicine pure product; the whole method is simple, water/alcohol systems can be used in the process, the cost is low, the method is environment-friendly, and the precipitation is matched with macroporous resin purification and polymer chromatography purification which are sequentially carried out, so that the extraction rate, the transfer rate and the extraction purity of colchicine can be effectively improved.
3. In the invention, the extraction purity of colchicine pure product is not lower than 90%, preferably not lower than 95%, more preferably 98% and above; the transfer rate is 70-95%.
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. It is evident that the drawings in the following description are only examples, from which other drawings can be obtained by a person skilled in the art without the inventive effort. In the drawings:
FIG. 1 shows the result of thin layer discrimination (iodine color development) in test example 1 of the present invention,
among them, lane 1 is water washing, 2 is 10% ethanol, 3 is 20% ethanol, 4 is 30% ethanol, 5 is 40% ethanol, 6 is 50% ethanol, 7 and 8 are STD.
FIG. 2 is a HPLC typical spectrum (colchicine, RT: 5.860) of a sample of a gloriosa seed material in test example 1.
FIG. 3 is a typical HPLC profile of a sample solution of colchicine extract (colchicine, RT: 5.835).
FIG. 4 shows the HPLC profile of colchicine pure prepared in example 1 (colchicine, RT: 7.463).
It should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
Example 1: colchicine extraction
Pretreatment: cleaning gloriosa seed, removing silt and foreign matter, drying, and pulverizing.
Extracting: reflux-extracting crushed gloriosa seed with water for 3 times and 2 hr each time, filtering to obtain filtrate, and concentrating the filtrate.
Water precipitation: mixing the concentrated solution with water (the volume ratio of the material to the water is 1:0.5), stirring for half an hour, standing at 40 ℃ for 8 hours, and filtering to obtain supernatant.
Macroporous adsorption resin: the supernatant was loaded onto a D101 macroporous adsorbent resin column (phi 5.0cm,105cm, column bed volume 2000 mL) and the effluent was tested by HPLC without colchicine leakage. The resin column was eluted with 10% aqueous ethanol and the eluate was checked by HPLC and contained substantially no or only a small amount of colchicine. Eluting with 30% ethanol solution, collecting eluate, and concentrating under reduced pressure to obtain colchicine extract.
Polymer chromatography: dissolving colchicine extract in water, filtering, loading onto PS40 polymer chromatographic column, eluting with 30% ethanol solution, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product.
And (5) recrystallizing: recrystallizing the colchicine crude product with ethyl acetate, recovering the organic solvent after the recrystallization is finished, filtering, and carrying out vacuum drying on the precipitate.
And (3) crushing the dried colchicine crystals to obtain a colchicine pure product with the purity of 98.8 percent.
Example 2: colchicine extraction
Pretreatment: cleaning gloriosa seed, removing silt and foreign matter, drying, and pulverizing.
Extracting: reflux-extracting crushed gloriosa seed with 50% ethanol for 3 times each for 2 hr, filtering to obtain filtrate, concentrating the filtrate, and recovering ethanol.
Water precipitation: mixing the concentrated solution with water (the volume ratio of the material to the water is 1:1), stirring for half an hour, standing at 40 ℃ for 8 hours, and filtering to obtain supernatant.
Macroporous adsorption resin: the supernatant was loaded onto an ADS-8 macroporous adsorbent resin column (phi 5.0cm,105cm, column bed volume 2000 mL) and the effluent was tested by HPLC without colchicine leakage. The resin column was eluted with 20% aqueous ethanol and the eluate was checked by HPLC and contained substantially no or only a small amount of colchicine. Eluting with 35% ethanol solution, collecting eluate, and concentrating under reduced pressure to obtain colchicine extract.
Polymer chromatography: dissolving colchicine extract in water, filtering, loading onto PSN30 polymer chromatographic column, eluting with 30% ethanol solution, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product.
And (5) recrystallizing: recrystallizing the colchicine crude product with ethyl acetate, recovering the organic solvent after the recrystallization is finished, filtering, and carrying out vacuum drying on the precipitate.
And (3) crushing the dried colchicine crystals to obtain a colchicine pure product with the purity of 98.5 percent.
Example 3: extraction of colchicine.
Pretreatment: cleaning gloriosa seed, removing silt and foreign matter, drying, and pulverizing.
Extracting: reflux-extracting crushed gloriosa seed with 70% ethanol for 3 times each for 2 hr, filtering to obtain filtrate, concentrating the filtrate, and recovering ethanol.
Water precipitation: mixing the concentrated solution with water (the volume ratio of the material to the water is 1:2), stirring for half an hour, standing at 40 ℃ for 8 hours, and filtering to obtain supernatant.
Macroporous adsorption resin: the supernatant was loaded onto an ADS-5 macroporous adsorbent resin column (phi 5.0cm,105cm, column bed volume 2000 mL) and the effluent was tested by HPLC without colchicine leakage. The resin column was eluted with 20% aqueous methanol and the eluent was checked by HPLC and contained substantially no or only a small amount of colchicine. Eluting with 40% methanol water solution, collecting eluate, and concentrating under reduced pressure to obtain colchicine extract.
Polymer chromatography: dissolving colchicine extract in water, filtering, loading onto PMN40 polymer chromatographic column, eluting with 20% methanol water solution, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product.
And (5) recrystallizing: recrystallizing the colchicine crude product with ethyl acetate, recovering the organic solvent after the recrystallization is finished, filtering, and carrying out vacuum drying on the precipitate.
And (3) crushing the dried colchicine crystals to obtain a colchicine pure product with the purity of 98.5 percent.
Example 4: extraction of colchicine.
Pretreatment: cleaning gloriosa seed, removing silt and foreign matter, drying, and pulverizing.
Extracting: reflux-extracting crushed gloriosa seed with 90% ethanol for 3 times each for 2 hr, filtering to obtain filtrate, concentrating the filtrate, and recovering ethanol.
Water precipitation: mixing the concentrated solution with water (the volume ratio of the material to the water is 1:0.5), stirring for half an hour, standing at 40 ℃ for 8 hours, and filtering to obtain supernatant.
Macroporous adsorption resin: the supernatant was applied to LK2MGL type macroporous adsorbent resin column (phi 5.0cm,105cm, column bed volume 2000 mL) and the effluent was checked by HPLC, and no leakage of colchicine was seen. The resin column was eluted with 35% aqueous methanol and the eluent was checked by HPLC and contained substantially no or only a small amount of colchicine. Eluting with 60% methanol water solution, collecting eluate, and concentrating under reduced pressure to obtain colchicine extract.
Polymer chromatography: dissolving colchicine extract in water, filtering, loading onto PSA30 polymer chromatographic column, eluting with 40% methanol water solution, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product.
And (5) recrystallizing: recrystallizing the colchicine crude product with ethyl acetate, recovering the organic solvent after the recrystallization is finished, filtering, and carrying out vacuum drying on the precipitate.
And (3) crushing the dried colchicine crystals to obtain a colchicine pure product with the purity of 98.4 percent.
Example 5: colchicine extraction
Pretreatment: cleaning gloriosa seed, removing silt and foreign matter, drying, and pulverizing.
Extracting: reflux-extracting crushed gloriosa seed with 50% methanol for 3 times each for 2 hr, filtering to obtain filtrate, concentrating the filtrate, and recovering methanol.
Water precipitation: mixing the concentrated solution with water (the volume ratio of the material to the water is 1:1), stirring for half an hour, standing at 40 ℃ for 8 hours, and filtering to obtain supernatant.
Macroporous adsorption resin: the supernatant was loaded onto a D101 macroporous adsorbent resin column (phi 5.0cm,105cm, column bed volume 2000 mL) and the effluent was tested by HPLC without colchicine leakage. The resin column was eluted with 20% aqueous methanol and the eluent was checked by HPLC and contained substantially no or only a small amount of colchicine. Eluting with 40% methanol water solution, collecting eluate, and concentrating under reduced pressure to obtain colchicine extract.
Polymer chromatography: dissolving colchicine extract in water, filtering, loading onto PS40 polymer chromatographic column, eluting with 20% acetonitrile solution, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product.
And (5) recrystallizing: recrystallizing the colchicine crude product with ethyl acetate, recovering the organic solvent after the recrystallization is finished, filtering, and carrying out vacuum drying on the precipitate.
And (3) crushing the dried colchicine crystals to obtain a colchicine pure product with the purity of 98.7 percent.
Example 6: extraction of colchicine.
Pretreatment: cleaning gloriosa seed, removing silt and foreign matter, drying, and pulverizing.
Extracting: reflux-extracting crushed gloriosa seed with 90% methanol for 3 times each for 2 hr, filtering to obtain filtrate, concentrating the filtrate, and recovering methanol.
Water precipitation: mixing the concentrated solution with water (the volume ratio of the material to the water is 1:2), stirring for half an hour, standing at 40 ℃ for 8 hours, and filtering to obtain supernatant.
Macroporous adsorption resin: the supernatant was loaded onto an ADS-5 macroporous adsorbent resin column (phi 5.0cm,105cm, column bed volume 2000 mL) and the effluent was tested by HPLC without colchicine leakage. The resin column was eluted with 30% aqueous methanol and the eluent was checked by HPLC and contained substantially no or only a small amount of colchicine. Eluting with 50% methanol water solution, collecting eluate, and concentrating under reduced pressure to obtain colchicine extract.
Polymer chromatography: dissolving colchicine extract in water, filtering, loading onto PSN30 polymer chromatographic column, eluting with 20% acetonitrile solution, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product.
And (5) recrystallizing: recrystallizing the colchicine crude product with ethyl acetate, recovering the organic solvent after the recrystallization is finished, filtering, and carrying out vacuum drying on the precipitate.
And (3) crushing the dried colchicine crystals to obtain a colchicine pure product with the purity of 98.6 percent.
Test example 1 qualitative identification and quantitative detection method of colchicine in a colchicine extract of a gloriosa seed raw material.
(1) Preparation of reference substance and test sample
Control: colchicine, purchased from: middle check hospital, purity: 93.6%, lot number: 101176-202003
Preparing a reference substance solution: colchicine 8mg was weighed and dissolved in 50% methanol to 100ml.
Preparing a sample solution of a gloriosa seed raw material: weighing about 0.4g of seeds, placing into cone, adding 50ml of 50% methanol, weighing, ultrasound for 30min, cooling, weighing again, supplementing weight loss with 50% methanol, shaking, filtering, and collecting subsequent filtrate.
Preparing colchicine extract test sample solution: about 8mg of colchicine extract was weighed, diluted to 100mL (about 80. Mu.g/mL) with a solvent, filtered using an organic filter, and the filtrate was removed.
(2) Qualitative identification: thin layer chromatography inspection
The method adopts silica gel thin layer chromatography, the reference substance liquid and the sample liquid are respectively spotted on the same silica gel thin layer chromatography plate (silica gel G) plate, and chloroform is used for preparing the reference substance liquid and the sample liquid: methanol (volume ratio, 9:0.6) is used as developing agent, taken out, dried and placed in an iodine jar for developing. The results show that the test samples all have the same spots at the same positions of the reference sample.
(3) Quantitative detection of colchicine content in a sample: high Performance Liquid Chromatography (HPLC).
Chromatographic conditions: a Thermo Acclaim 120C8 (250 x 4.6 mm) chromatographic column is adopted, 53% methanol-6.8 g/L potassium dihydrogen phosphate is taken as a mobile phase, the flow rate is 1mL/min, and the detection wavelength is 254nm.
Sample measurement: and respectively injecting 10uL of the sample solution of the gloriosa seed raw material and the sample solution of the colchicine extract into a liquid chromatograph, and calculating the content of colchicine in the gloriosa seed raw material extract and the colchicine extract.
The HPLC typical spectrum of the sample of the gloriosa seed raw material (colchicine, RT: 5.860) is shown in figure 2; a typical HPLC profile of the sample solution of colchicine extract (colchicine, RT: 5.835) is shown in FIG. 3.
The pure colchicine product prepared in example 1 was tested by the method of test example 1 and the HPLC chart obtained is shown in FIG. 4 (colchicine, RT: 7.463).
Test examples 2 to 6 below refer to the procedure of example 1, and the procedure of example 1 was followed except for the variables to examine the effect of different conditions on the results.
Experimental example 2 investigation of the influence of different extraction solvents on the extraction results
In this test example 2, the method of example 1 was followed by extraction with different extraction solvents, and the influence of the solvents on the extraction results was examined. The content of colchicine in the residue was measured by high performance liquid chromatography in test example 1, and the results are shown in Table 1.
Table 1: extraction experimental results of different extraction solvents.
Detecting the content of colchicine in the medicine residues by an HPLC method; and B, the transfer rate of colchicine.
In the extraction experiment, when water, 50% ethanol, 70% ethanol, 95% ethanol, 50% methanol and 90% methanol are used as extraction solvents, the transfer rate of colchicine in the extraction step is between 90% and 99%. It can be seen that they can be used as effective extraction solvents for the compounds, wherein ethanol water is preferred as the extraction solvent in view of the safety, low cost and high extraction rate of ethanol.
Test example 3 Water sedimentation test
This test example 3 was prepared by the method of example 1, and the steps of pretreatment, extraction and water precipitation were performed. In the water precipitation step, water with different volumes is added into each experimental component for standing, and the influence of the different volumes is examined. Other conditions in the steps of pretreatment, extraction and water precipitation were the same as in example 1.
The content of colchicine in the supernatant was measured by high performance liquid chromatography in test example 1, and the results are shown in Table 2.
Table 2: and (5) water sedimentation test results.
C, the content of colchicine in the supernatant; d, colchicine transfer rate.
The data in Table 2 shows that the above steps can generate precipitation after adding water with different volumes and standing, and the transfer rate of colchicine is between 80% and 95%, so that the treatment in the step can effectively remove fat-soluble impurities, and the loss of colchicine is smaller. The present invention therefore takes this step as a key step in the preparation of colchicine.
Test example 4 Water deposition rest temperature experiment
In this test example 4, the influence of the water settlement temperature on the result was examined by referring to the method of example 1, and the other conditions were the same as in example 1.
The content of colchicine in the supernatant was measured by high performance liquid chromatography in test example 1, and the results are shown in Table 3.
Table 3: water sinking temperature experimental result
C, the content of colchicine in the supernatant; d, colchicine transfer rate.
The data in Table 3 shows that the above steps can all produce precipitation after standing at different temperatures, and the colchicine transfer rate is between 40% and 60%, wherein the colchicine transfer rate is the highest after standing at 40 ℃. The present invention therefore takes this step as a key step in the preparation of colchicine.
Experimental example 5 macroporous resin purification experiments
This test example 5 was prepared by the method of example 1, and the pretreatment, extraction, water precipitation and macroporous adsorbent resin purification steps were performed. In the macroporous adsorption resin purification step, different eluents are adopted for each experimental component, and the influence of the different eluents is examined. Other conditions in the steps of pretreatment, extraction, water precipitation and macroporous adsorption resin were the same as in example 1.
The content of colchicine in the macroporous resin purification eluent was determined by high performance liquid chromatography in test example 1, and the results are shown in table 4.
Table 4: and purifying the experimental result by macroporous resin.
E, the content of colchicine in the eluent; f, colchicine transfer rate.
The data in Table 4 shows that colchicine content eluted from the adsorption resin column is between 50% and 80% and colchicine transfer rate is between 85% and 96%. Among them, in view of safety and low cost of ethanol, the ethanol aqueous solution is preferable as an eluting solvent, and the eluting concentration is between 10% and 50%.
Experimental example 6 polymer chromatography purification experiments
This test example 6 was prepared by the method of example 1, and subjected to pretreatment, extraction, water precipitation, macroporous adsorbent resin purification and polymer chromatography. In the step of polymer chromatography, different polymer chromatography is adopted for each experimental component, and the influence of chromatography by different polymers is examined. Other conditions in the pretreatment, extraction, water precipitation, macroporous adsorbent resin and polymer chromatography steps were the same as in example 1.
The content of colchicine in the polymer chromatography purification eluate was determined by high performance liquid chromatography in test example 1, and the results are shown in tables 5 (1) and (2).
Table 5: (1) Content and transfer rate of colchicine after chromatography of different types of polymers
G, the mass percentage of colchicine in the eluent; transfer rate of colchicine.
Table 5: (2) Levels of related substances after purification of the Polymer
Impurity limitation requirements:
EP9.4 limit: impurity A:3.0%; 0.25% of impurity G; 0.2% of impurity E; other unknown list: 0.1%; total impurities: 4.0%, and the impurity B is ignored. Impurity G correction factor: 1.6.
ALK new standard impurity A:1.5%; 1.0% of impurity B; other single impurities are 0.5% and total impurities are 5.0%.
The data in tables 5 (1) and (2) show that colchicine content eluted from different types of polymer chromatographic columns is between 85.0% and 98.5%, colchicine transfer rate is between 85% and 93%, and impurity limits are all in accordance with relevant requirements. Therefore, the preferred polymeric fillers of the present invention meet the process requirements.
The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.
Claims (9)
1. A method for preparing colchicine, which is characterized by comprising the following steps:
(1) Pretreatment: drying and crushing gloriosa seed;
(2) Extracting: extracting the pretreated raw materials by adopting an extraction solvent, filtering, and concentrating the filtrate to obtain a concentrated solution;
(3) Precipitation: standing the concentrated solution, and filtering to obtain supernatant;
(4) Purifying by macroporous resin: loading the supernatant onto macroporous adsorbent resin, eluting in segments, and concentrating the eluate to obtain colchicine extract;
(5) Polymer chromatography: dissolving colchicine extract, filtering, performing polymer chromatography, eluting, collecting eluate, concentrating under reduced pressure, and drying to obtain colchicine crude product;
(6) Recrystallizing colchicine crude product, drying, and pulverizing to obtain colchicine pure product;
in the step (2), the extraction solvent is water, or ethanol water solution with the volume concentration of 50% -95%, or methanol water solution with the volume concentration of 50% or 90%;
in the step (3), adding water into the concentrated solution, mixing, stirring and standing; wherein, the volume ratio of the concentrated solution to the water is 1:0.5-2, and standing at 40deg.C, 50deg.C or 60deg.C;
in step (4), the step of eluting includes: washing with a first eluent to remove water-soluble impurities, and eluting colchicine adsorbed on macroporous adsorption resin with a second eluent; the first eluent is ethanol water solution with volume concentration of 10% -20% or methanol water solution with volume concentration of 20%; the second eluent is ethanol water solution with volume concentration of 10% or 30% -35% or methanol water solution with volume concentration of 40% -50%;
in the step (5), the filler used for polymer chromatography is selected from one of PS40, PMN40, PSN30 and PSA 30.
2. The method for preparing colchicine according to claim 1, wherein in step (5), the polymer chromatography is performed, eluting with an eluent selected from one of an aqueous ethanol solution, an aqueous methanol solution or an aqueous acetonitrile solution.
3. The method for preparing colchicine according to claim 2, wherein in the step (5), the eluent is 10-60% ethanol aqueous solution, 20% -60% methanol aqueous solution, or 10% -50% acetonitrile solution.
4. The method for producing colchicine according to claim 1, wherein in the step (5), the colchicine extract is dissolved by adding water, an organic solvent or an aqueous solution of an organic solvent.
5. The method for preparing colchicine according to claim 4, wherein the colchicine extract is dissolved in water.
6. The method for preparing colchicine according to any one of claims 1 to 5, wherein in the step (4), the macroporous adsorbent resin is a weak polarity or medium polarity macroporous adsorbent resin.
7. The method for preparing colchicine according to claim 6, wherein the macroporous adsorbent resin is selected from one of D101, ADS-8, ADS-5 or LK2MGL type adsorbent resins.
8. The method for preparing colchicine according to any one of claims 1 to 5, wherein in step (1), the pretreatment step specifically comprises: collecting raw materials, cleaning, removing impurities, drying, and pulverizing.
9. The method for preparing colchicine according to claim 8, wherein the raw material is seeds of gloriosa of the family liliaceae.
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