CN107929367B - Method for separating and preparing gelsemium elegans alkaloid from gelsemium elegans by ion exchange method - Google Patents
Method for separating and preparing gelsemium elegans alkaloid from gelsemium elegans by ion exchange method Download PDFInfo
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- 241001391115 Gelsemium elegans Species 0.000 title claims abstract description 52
- 229930013930 alkaloid Natural products 0.000 title claims abstract description 36
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000005342 ion exchange Methods 0.000 title claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 130
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000000706 filtrate Substances 0.000 claims abstract description 26
- 239000000287 crude extract Substances 0.000 claims abstract description 21
- 238000001035 drying Methods 0.000 claims abstract description 20
- 238000001914 filtration Methods 0.000 claims abstract description 19
- 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 claims abstract description 18
- 239000012488 sample solution Substances 0.000 claims abstract description 18
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 14
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 14
- 238000010992 reflux Methods 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 13
- 239000000284 extract Substances 0.000 claims abstract description 13
- 238000010438 heat treatment Methods 0.000 claims abstract description 13
- 229940079593 drug Drugs 0.000 claims abstract description 12
- 239000002253 acid Substances 0.000 claims abstract description 11
- 239000011347 resin Substances 0.000 claims abstract description 9
- 229920005989 resin Polymers 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 8
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- 230000007935 neutral effect Effects 0.000 claims abstract description 6
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 4
- 241001113926 Gelsemium Species 0.000 claims description 20
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- 238000011068 loading method Methods 0.000 claims description 10
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- 238000007873 sieving Methods 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 5
- 239000003729 cation exchange resin Substances 0.000 claims description 4
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- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000219321 Caryophyllaceae Species 0.000 description 1
- VJWIXTLPDOVKPN-UHFFFAOYSA-N Gelsemine Natural products CN1CC2(C=C)C3CC4OC3C1C2CC45C(=O)Nc6ccccc56 VJWIXTLPDOVKPN-UHFFFAOYSA-N 0.000 description 1
- 241000282376 Panthera tigris Species 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Chemical compound C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
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- NFYYATWFXNPTRM-QJICHLCESA-N gelsemine Chemical compound OC1=NC2=CC=CC=C2[C@@]21[C@H]1[C@H]3[C@H]4CO[C@@H]2C[C@H]4[C@]1(C=C)CN3C NFYYATWFXNPTRM-QJICHLCESA-N 0.000 description 1
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000008569 process Effects 0.000 description 1
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
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- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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Abstract
The invention discloses a method for separating and preparing gelsemium elegans alkaloid from gelsemium elegans by an ion exchange method, which comprises the following steps: 1) heating and refluxing herba Gelsemii Elegantis with ethanol; obtaining a crude extract; adding hydrochloric acid into the crude extract to adjust the pH to 2-5, and filtering; dissolving the precipitate in hydrochloric acid aqueous solution with the pH value of 2-5, filtering, combining the filtrates, and preparing a sample solution with the concentration of 0.75-1.5 g crude drug/mL; passing the sample solution through a pretreated strong acid type ion exchange resin column, wherein the flow rate of the sample solution is 1-5 BV/h, washing the sample solution to be neutral after the exchange is finished, removing impurities by using 80% ethanol in volume concentration, and eluting the resin column by using 5% ammonia water in volume concentration-90% ethanol in volume concentration; collecting eluate, recovering ethanol under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract. The invention improves the content of the gelsemium elegans from 4.6 percent of the crude extract to more than 20 percent, and lays a foundation for further separating and preparing the high-purity gelsemium elegans.
Description
Technical Field
The invention relates to the technical field of preparation of medicinal plant gelsemium elegans alkaloid, in particular to a method for preparing gelsemium elegans alkaloid extract from gelsemium elegans by taking preparation of gelsemium elegans as a guide.
Background
Kiss (Gelsemium elegansBenth.) is evergreen woody vine of genus Caryophyllaceae, and is used as medicineHas the functions of relieving swelling and pain, dispelling wind and removing blood stasis, counteracting toxic materials and killing parasites. The gelsemium elegans contains indole alkaloid as main effective component, and has antiinflammatory, analgesic, antitumor, psoriasis resisting, and growth promoting effects. The gelsemium elegans alkaloid comprises gelsemium elegans, formazan, tiger, Mao and other nearly hundred monomers. In recent years, a series of researches show that the gelsemium has remarkable effects of relieving pain, resisting anxiety and the like, and has good industrial medicine application prospect. The preparation of gelsemium elegans relies on the separation and purification of the natural gelsemium elegans alkaloid extract, so the preparation technology of the gelsemium elegans alkaloid extract is an important basic step.
Reference 1 (Zhang lan. extraction and separation of gelsemin from total alkaloids of gelsemium elegans [ J ] journal of first university of military medical science, 2004,24(9): 1006-. The method adopts hot alcohol reflux extraction and then chloroform extraction to obtain the gelsemium elegans total alkaloids. The method uses a large amount of chloroform in the extraction process, has high reagent cost, generates great toxic action on the liver of operators and causes environmental pollution, and the extraction operation is not suitable for large-scale industrialized production.
Comparison document 2 (research on the separation and purification of gelsemium elegans total alkaloids by using macroporous adsorption resin [ J ]. pharmacy of China, 2006,17(13):969 and 971.) discloses that 1kg of water decoction of gelsemium elegans is separated and purified by using macroporous adsorption resin to obtain the gelsemium total alkaloids. The extraction efficiency of the water decoction is low, the amount of the water decoction is large, and the inconvenience and difficulty are brought to the later treatment.
Comparison document 3 (comparison of the method for extracting total alkaloids from gelsemium elegans (xujian, Liang Chong., Gelsemiun) (J. pharmaceutical bulletin, 1988,23(6):341 + 342.) discloses a method for separating total alkaloids from gelsemium elegans by ion exchange resin method after medicinal materials are soaked in acid water. The acid water immersion extraction method takes a long time and the volume of the extracting solution is large.
The invention discloses a method for separating and preparing gelsemium elegans alkaloid extract with the final purpose of preparing gelsemium elegans.
Disclosure of Invention
In order to overcome the defects of the prior art for preparing the gelsemium alkaloid by separation, the invention aims to provide a method for preparing the gelsemium alkaloid by separation by adopting ion exchange resin from plant gelsemium and taking preparation of gelsemium as guidance.
The invention aims to realize the method for separating and preparing gelsemium alkaloid from gelsemium by an ion exchange method, which comprises the following steps:
1) drying a gelsemium elegans medicinal material at the temperature of 50-70 ℃, crushing, sieving with a 20-60-mesh sieve, adding 65-95% ethanol by volume concentration, and heating, refluxing and extracting; extracting for 1-3 h, and collecting filtrate; repeatedly extracting for 1-3 times, combining the filtrates, and concentrating under reduced pressure to recover ethanol until no ethanol smell exists to obtain a crude extract;
2) adding hydrochloric acid into the crude extract obtained in the step 1) to adjust the pH to 2-5, and filtering; dissolving the precipitate with hydrochloric acid aqueous solution with the pH value of 2-5, filtering, operating for 1-2 times, combining the filtrates, and preparing a sample loading solution with the concentration of 0.75-1.5 g crude drug/mL;
3) passing the sample solution through a pretreated strong acid type ion exchange resin column, wherein the flow rate of the sample solution is 1-5 BV/h, washing the sample solution to be neutral after the exchange is finished, removing impurities by using 80% ethanol in volume concentration, and eluting the resin column by using 5% ammonia water in volume concentration-90% ethanol in volume concentration; collecting eluate, recovering ethanol under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract.
The above-mentioned addition volume concentration is preferably 95% ethanol, and the extraction is performed by heating and refluxing.
The flow rate of the above-mentioned sample is preferably 3 BV/h.
The specific steps for obtaining the crude extract in the step 1) are as follows: drying and crushing a gelsemium elegans medicinal material, adding an extracting agent ethanol, wherein the volume-to-mass ratio of the extracting agent to the gelsemium elegans medicinal material is 4-10 mL/g, the volume concentration of an extraction solvent ethanol is 60-95%, and heating and refluxing for 1-3 hours; collecting the filtrate, adding ethanol into the filter residue, repeatedly extracting for 1-3 times, filtering, combining the filtrates, and recovering ethanol until no alcohol smell exists;
the ion exchange resin in the step 3) is a strong acid type cation exchange resin, including but not limited to 732 type;
in the step 3), the sample loading flow rate is 1-5 BV/h, the effluent is detected by silicotungstic acid and potassium bismuth iodide, and the sample loading is stopped when a positive reaction occurs; washing the resin column with distilled water to neutral, removing impurities with 80% ethanol, eluting with ammonia water-ethanol, recovering ethanol from the eluate under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract.
The method realizes the enrichment effect on the koumine, lays a foundation for further separating and preparing the high-purity koumine, and lays a foundation for the production raw material source for the industrial preparation of the koumine and the acceptable salt thereof when the koumine is applied to the aspects of preparing medicines, health care products, feeds and the like.
Specifically, the technical scheme adopted by the invention is as follows: a method for separating and preparing gelsemium elegans alkaloid from gelsemium elegans by adopting ion exchange resin comprises the following steps:
1) adding herba Gelsemii Elegantis into ethanol, heating, reflux extracting, and recovering ethanol until no ethanol smell is detected to obtain crude extractive solution.
2) Adding 5% hydrochloric acid into the crude extract to adjust the pH to 2-5, and filtering; and dissolving the precipitate in hydrochloric acid aqueous solution with the pH value of 2-5, filtering, carrying out operation for 1-2 times, mixing to obtain acid aqueous filtrate, and preparing a sample loading solution with a certain concentration.
3) And (3) carrying out exchange adsorption on the sample loading solution through pretreated strong acid type ion exchange resin, and eluting with 3-5% ammonia water-ethanol after adsorption balance is achieved, wherein the ethanol concentration is 70-95%. Recovering ethanol from the eluate under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract.
The specific steps for obtaining the crude extract in the step 1) are as follows: drying and crushing a gelsemium elegans medicinal material, adding an extracting agent ethanol, wherein the volume-to-mass ratio of the extracting agent to the gelsemium elegans medicinal material is 4-10 mL/g, the volume concentration of an extraction solvent ethanol is 60-95%, and heating and refluxing for 1-3 hours; and collecting the filtrate, adding ethanol into the filter residue, repeatedly extracting for 2-3 times, mixing the filtrates after filtering, and recovering the ethanol until no alcohol smell exists.
The ion exchange resin in the step 3) is a strong acid type cation exchange resin, including but not limited to 732 type.
In the step 3), the sample loading flow rate is 1-5 BV/h, the effluent is detected by silicotungstic acid and potassium bismuth iodide, and the sample loading is stopped when a positive reaction occurs. Washing the resin column with distilled water to neutrality, removing impurities with 80% ethanol, and eluting with ammonia water-ethanol. Recovering ethanol from the eluate under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract.
The invention has the advantages that: the method has the good characteristics of simple and convenient process, economy, environmental protection and the like, and is more suitable for industrial large-scale production. The gelsemium elegans alkaloid prepared by the invention realizes the enrichment effect on gelsemium elegans, improves the content of the gelsemium elegans from 4.6 percent of the crude extract to more than 20 percent, and lays a foundation for further separating and preparing high-purity gelsemium elegans. The invention lays a foundation for the industrial preparation of the koumine and the acceptable salt thereof in the preparation of medicines, health products, feeds and the like.
Drawings
FIG. 1 is a high performance liquid chromatogram of the content of koumine in crude extract.
FIG. 2 is a high performance liquid chromatogram of the content of koumine in gelsemium alkaloid prepared by the invention.
Detailed Description
The invention relates to a method for separating and preparing gelsemium elegans alkaloid extract from gelsemium elegans by taking obtained gelsemium elegans as a target product, which mainly comprises the following steps:
1) drying a gelsemium elegans medicinal material at 50-70 ℃, crushing, sieving with a 20-60-mesh sieve, adding 65-95% ethanol by volume concentration, heating and refluxing for extraction, and preferably selecting the concentration as 95%; extracting for 1-3 h, and collecting filtrate; and (3) repeatedly extracting for 1-3 times, combining the filtrates, and carrying out reduced pressure concentration to recover ethanol until no alcohol smell exists, thereby obtaining a crude extract.
2) Adding 5% hydrochloric acid into the crude extract obtained in the step 1) to adjust the pH to 2-5, and filtering; dissolving the precipitate in hydrochloric acid aqueous solution with pH of 2-5, filtering, operating for 1-2 times, and combining the filtrates. Preparing a sample solution with the concentration of 0.75-1.5 g crude drug/mL.
3) The sample solution was passed through a pretreated strong acid type ion exchange resin (Shanghai leaf Biotech, 732 cation exchange resin). The flow rate of the sample liquid is 1-5 BV/h, preferably 3 BV/h. After the exchange is finished, 6BV water is used for washing to be neutral, after the 6BV ethanol with the volume concentration of 80 percent is used for removing impurities, the resin column is eluted by ammonia water with the volume concentration of 5 percent and ethanol with the volume concentration of 90 percent. Collecting eluate, recovering ethanol under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract.
Example 1
Drying the gelsemium elegans medicinal material at 60 ℃, crushing and sieving the gelsemium elegans medicinal material with a 60-mesh sieve, adding ethanol with the volume concentration of 95%, heating and refluxing for extraction for 1-3 h, and collecting filtrate; and (3) repeatedly extracting for 1-3 times, combining the filtrates, and carrying out reduced pressure concentration to recover ethanol until no alcohol smell exists, thereby obtaining a crude extract. Adding 5% hydrochloric acid into the obtained crude extract to adjust pH to 3, and filtering; dissolving the precipitate with hydrochloric acid solution (pH = 2), filtering, performing 1-2 times, mixing filtrates, and preparing into a sample solution with a concentration of 1.0 g crude drug/mL.
Passing the sample solution with concentration of 1.0 g crude drug/mL at room temperature at flow rate of 2 BV/h through chromatography column filled with pretreated 732 ion exchange resin (Shanghai-derived leaf Biotechnology), eluting with 6BV distilled water, 6BV 80% ethanol and 8 BV 5% ammonia-90% ethanol. Collecting eluate, recovering ethanol under reduced pressure, and drying to obtain gelsemium alkaloid (containing gelsemium 20.8% by high performance liquid chromatography).
Example 2
Drying the gelsemium elegans medicinal material at 60 ℃, crushing and sieving the gelsemium elegans medicinal material with a 60-mesh sieve, adding ethanol with the volume concentration of 95%, heating and refluxing for extraction for 1-3 h, and collecting filtrate; and (3) repeatedly extracting for 1-3 times, combining the filtrates, and carrying out reduced pressure concentration to recover ethanol until no alcohol smell exists, thereby obtaining a crude extract. Adding 5% hydrochloric acid into the obtained crude extract to adjust pH to 3, and filtering; dissolving the precipitate with hydrochloric acid solution (pH = 2), filtering, performing 1-2 times, mixing filtrates, and preparing into a sample solution with a concentration of 1.0 g crude drug/mL.
Passing the sample solution with concentration of 1.0 g crude drug/mL at room temperature at flow rate of 3 BV/h through chromatographic column filled with pretreated 732 ion exchange resin (Shanghai-derived leaf Biotechnology), eluting with 6BV distilled water, 6BV 80% ethanol and 8 BV 5% ammonia-90% ethanol. Collecting eluate, recovering ethanol under reduced pressure, and drying to obtain gelsemium alkaloid (containing gelsemium 20.5% by high performance liquid chromatography).
Example 3
Drying the gelsemium elegans medicinal material at 60 ℃, crushing and sieving the gelsemium elegans medicinal material with a 60-mesh sieve, adding ethanol with the volume concentration of 95%, heating and refluxing for extraction for 1-3 h, and collecting filtrate; and (3) repeatedly extracting for 1-3 times, combining the filtrates, and carrying out reduced pressure concentration to recover ethanol until no alcohol smell exists, thereby obtaining a crude extract. Adding 5% hydrochloric acid into the obtained crude extract to adjust pH to 3, and filtering; dissolving the precipitate with hydrochloric acid solution (pH = 2), filtering, performing 1-2 times, mixing filtrates, and preparing into a sample solution with a concentration of 1.0 g crude drug/mL.
Passing the sample solution with concentration of 1.0 g crude drug/mL at the flow rate of 5BV/h through a chromatographic column filled with pretreated 732 ion exchange resin (Shanghai-derived leaf Biotechnology) at room temperature for exchange adsorption, and eluting with 6BV distilled water, 6BV 80% ethanol by volume concentration, and 8 BV 5% ammonia water-90% ethanol by volume concentration. Collecting eluate, recovering ethanol under reduced pressure, and drying to obtain gelsemium alkaloid (containing gelsemium 20.1% by high performance liquid chromatography).
The embodiments 1 to 3 are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention without departing from the technical spirit of the present invention.
Claims (3)
1. A method for separating and preparing gelsemium elegans alkaloid from gelsemium elegans by ion exchange method comprises the following steps:
1) drying a gelsemium elegans medicinal material at the temperature of 50-70 ℃, crushing, sieving with a 20-60-mesh sieve, adding 65-95% ethanol by volume concentration, and heating, refluxing and extracting; extracting for 1-3 h, and collecting filtrate; repeatedly extracting for 1-3 times, combining the filtrates, and concentrating under reduced pressure to recover ethanol until no ethanol smell exists to obtain a crude extract;
2) adding hydrochloric acid into the crude extract obtained in the step 1) to adjust the pH value to 2-5, and filtering; dissolving the precipitate with hydrochloric acid aqueous solution with pH of 2-5, filtering, operating for 1-2 times, combining the filtrates, and preparing a sample loading solution with a concentration of 0.75-1.5 g crude drug/mL;
3) passing the sample solution through a pretreated 732 strong acid type ion exchange resin column, wherein the flow rate of the sample solution is 1-5 BV/h, washing the sample solution to be neutral after the exchange is finished, removing impurities by using 80% ethanol in volume concentration, and eluting the resin column by using 5% ammonia water in volume concentration-90% ethanol in volume concentration; collecting eluate, recovering ethanol under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract.
2. The method for separating and preparing gelsemium alkaloid from gelsemium according to the ion exchange method of claim 1, characterized in that: adding 95% ethanol by volume, heating and refluxing for extraction.
3. The method for separating and preparing gelsemium alkaloid from gelsemium according to the ion exchange method of claim 1 or 2, characterized in that: the specific steps for obtaining the crude extract in the step 1) are as follows: drying and crushing a gelsemium elegans medicinal material, adding an extracting agent ethanol, wherein the volume-to-mass ratio of the extracting agent to the gelsemium elegans medicinal material is 4-10 mL/g, the volume concentration of an extraction solvent ethanol is 65-95%, and heating and refluxing for 1-3 hours; collecting the filtrate, adding ethanol into the filter residue, repeatedly extracting for 1-3 times, filtering, combining the filtrates, and recovering ethanol until no alcohol smell exists;
the ion exchange resin in the step 3) is 732 type strong acid type cation exchange resin;
in the step 3), the sample loading flow rate is 1-5 BV/h, the effluent is detected by silicotungstic acid and potassium bismuth iodide, and the sample loading is stopped when a positive reaction occurs; washing the resin column with distilled water to neutral, removing impurities with 80% ethanol, eluting with ammonia water-ethanol, recovering ethanol from the eluate under reduced pressure, and drying to obtain herba Gelsemii Elegantis alkaloid extract.
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