CN108586308B - Method for refining amphoteric alkaloid by membrane combination - Google Patents
Method for refining amphoteric alkaloid by membrane combination Download PDFInfo
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- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Abstract
The invention provides a method for refining amphoteric alkaloid by membrane combination, which provides a novel method for separating amphoteric alkaloid, aims at the difference between the physicochemical property of amphoteric alkaloid and the components such as alkaloid, phenolic acid, saponin and the like in traditional Chinese medicines, adjusts the existing state of the components, introduces an organic solvent to strengthen or weaken the acting force between the amphoteric alkaloid and a nanofiltration membrane, and amplifies the difference between the amphoteric alkaloid and other components in the nanofiltration acting force, thereby realizing the purpose of refining amphoteric alkaloid. The invention can quickly realize the enrichment of amphoteric alkaloid in complex component environment, has no influence on thermosensitive components in the separation process, avoids the environmental pollution caused by organic reagents generated by chromatographic separation, reduces the energy consumption and saves the production cost of pharmaceutical enterprises.
Description
Technical Field
The invention relates to an amphoteric alkaloid refining method, in particular to a method for refining amphoteric alkaloid components by adopting a membrane combination technology based on physicochemical characteristics of the alkaloid components, and belongs to the field of medicines.
Background
Alkaloids are basic organic compounds containing nitrogen, which are present in nature (mainly plants, but also in animals). Compared with conventional alkaloids, the amphoteric alkaloids mostly have carboxyl or phenolic hydroxyl in the structure, and have the physicochemical properties of acidic compounds while having alkalinity.
The traditional Chinese medicine components are complex and various, and comprise flavone, saponin, alkaloid, phenolic acid, tannin, protein, polysaccharide, inorganic ions and the like, and the alkaloid components are enriched by adopting preparative chromatography, ion exchange resin, silica gel column chromatography and the like, the enrichment technologies all have certain technical defects, the preparative chromatography can obtain target components with higher relative purity, but the enrichment efficiency and the cost are higher, and the subsequent solution recovery treatment of the enrichment part can also cause component conversion; ion exchange resin and chromatographic chromatography, low separation efficiency, and organic solvents such as trichloromethane and toluene used for eluting alkaloid components, which not only bring harm to operators, but also cause pollution to the environment. How to enrich acid-base amphoteric alkaloids under the condition of normal temperature or low temperature, reduce organic solvent pollution and improve refining efficiency is a scientific and technical problem which needs to be solved urgently in the pharmaceutical industry.
The membrane separation technology realizes separation by means of molecular cutting of membrane pore size, has the advantages of simplicity, convenience, high efficiency, no heat effect and the like, can pertinently remove macromolecular substances and reserve micromolecular components when filtering a sample, but has poor selectivity. The invention carries out the separation behavior research of alkaloid components in different solution environments and dissociation states at the beginning of the research, and accidentally finds that the interception efficiency of the alkaloid in the dissociation state can be enhanced in the environment of the organic solvent with lower concentration, and the correlation between the dissociation behavior of the amphoteric alkaloid and the solution is obviously different from other components of the traditional Chinese medicine, thereby achieving the targeted refining and enrichment of the acid-base amphoteric alkaloid.
Based on the background, the method for refining the amphoteric alkaloid has the advantages of normal temperature, low pollution, low energy consumption and low cycle, retains the original component structure, and is beneficial to development of related work of component purification and new drug research and development.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a membrane combination refining method of amphoteric alkaloid, which can be used for quickly separating and enriching amphoteric alkaloid in a complex component system.
The technical scheme is as follows:
a membrane combination refining method of amphoteric alkaloid is characterized in that: comprises the following steps:
a. detecting the isoelectric point of amphoteric alkaloid to be separated, and calculating the pH value of the amphoteric alkaloid compound when an acid group is in a dissociated state and the pH value of the amphoteric alkaloid compound when a basic group is in a dissociated state; b. dissolving the extract to be separated in purified water, performing pretreatment by adopting an ultrafiltration membrane with the cut-off molecular weight of 10 KDa-100 KDa to remove macromolecular protein and polysaccharide, and collecting ultrafiltrate; c. gradually diluting the ultrafiltrate in the step b by using a water-soluble organic solvent until the amount of the water-soluble organic solvent in the solution reaches 5-40%, adjusting the pH to the pH of the alkaline group dissociation in the step a by using a sodium hydroxide aqueous solution with the mass concentration of 1-10%, separating by using a nanofiltration membrane with the molecular weight cutoff of 100-800 Da, diluting the trapped liquid by using purified water when the trapped liquid cannot be continuously separated, repeating for 1-3 times, and collecting the terminal trapped liquid when the trapped liquid cannot be continuously separated; d. and (c) gradually diluting the terminal trapped fluid in the step (c) by using a water-soluble organic solvent until the amount of the water-soluble organic solvent in the solution reaches 5-40%, adjusting the pH to the pH of the acid group dissociation in the step (a) by using a 1-10% hydrochloric acid aqueous solution, separating by using a nanofiltration membrane with the molecular weight cutoff of 100-800 Da, diluting the trapped fluid by using purified water when the trapped fluid volume cannot be continuously separated, repeating for 1-3 times, and collecting the trapped fluid when the trapped fluid volume cannot be continuously separated, thereby obtaining the amphoteric alkaloid part.
The ultrafiltration membrane in the method for refining the amphoteric alkaloid by the membrane combination is made of one of cellulose, polyether sulfone and cellulose acetate.
The water-soluble organic solvent in the membrane combination refining method of the amphoteric alkaloid is one of methanol, ethanol, acetonitrile and tetrahydrofuran.
The nanofiltration membrane material in the method for refining the amphoteric alkaloid by membrane combination is one of organic solvent resistant materials such as polyimide, polyamide and polyacrylonitrile.
Has the advantages that: (1) provides a novel amphoteric alkaloid separation method, quickly realizes the enrichment of amphoteric alkaloid in complex component environment, and has no influence on thermosensitive components in the separation process. (2) The environmental pollution caused by organic reagents generated by chromatographic separation is avoided, the energy consumption is reduced, and the production cost of pharmaceutical enterprises is saved.
Drawings
FIG. 1 is a chromatogram of a stachydrine control.
FIG. 2 is a chromatogram of stachydrine prepared by membrane separation.
FIG. 3 is a chromatogram of stachydrine prepared by resin separation.
Detailed Description
The foregoing aspects of the present invention are described in further detail below by way of examples, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and that all the technologies implemented based on the above-described aspects of the present invention are within the scope of the present invention.
Example 1:
a membrane combination refining method of amphoteric alkaloid comprises the following specific steps:
the step a is as follows: detecting the isoelectric point of amphoteric alkaloid to be separated, and calculating the pH value of the amphoteric alkaloid compound when an acid group is in a dissociated state and the pH value of the amphoteric alkaloid compound when a basic group is in a dissociated state;
the step b is as follows: dissolving the extract to be separated in purified water, performing pretreatment by adopting an ultrafiltration membrane with the cut-off molecular weight of 10 KDa-100 KDa to remove macromolecular protein and polysaccharide, and collecting ultrafiltrate;
the step c is as follows: gradually diluting the ultrafiltrate in the step b by using a water-soluble organic solvent until the amount of the water-soluble organic solvent in the solution reaches 5-40%, adjusting the pH to the pH of the alkaline group dissociation in the step a by using a sodium hydroxide aqueous solution with the mass concentration of 1-10%, separating by using a nanofiltration membrane with the molecular weight cutoff of 100-800 Da, diluting the trapped liquid by using purified water when the trapped liquid cannot be continuously separated, repeating for 1-3 times, and collecting the terminal trapped liquid when the trapped liquid cannot be continuously separated;
step d is: and (c) gradually diluting the terminal trapped fluid in the step (c) by using a water-soluble organic solvent until the amount of the water-soluble organic solvent in the solution reaches 5-40%, adjusting the pH to the pH of the acid group dissociation in the step (a) by using a 1-10% hydrochloric acid aqueous solution, separating by using a nanofiltration membrane with the molecular weight cutoff of 100-800 Da, diluting the trapped fluid by using purified water when the trapped fluid volume cannot be continuously separated, repeating for 1-3 times, and collecting the trapped fluid when the trapped fluid volume cannot be continuously separated, thereby obtaining the amphoteric alkaloid part.
EXAMPLE 2 purification of the amphoteric alkaloid stachydrine
Collecting herba Leonuri water extractive solution, adopting example 1 and cation exchange resin to enrich stachydrine, and performing comparative analysis with stachydrine purity and separation period as indexes.
The invention comprises the following steps: detecting that the isoelectric point of the stachydrine is about 7.60, and calculating that the pH value of acid-base group dissociation in the stachydrine structure is 10.50 and the pH value of the alkaline group dissociation state is 5.50 by adopting a titration method; taking the leonurus water extract, adopting an ultrafiltration membrane made of cellulose with the molecular weight cutoff of 50KDa for pretreatment to remove macromolecular protein and polysaccharide, and collecting ultrafiltrate; diluting the ultrafiltrate with ethanol until the ethanol concentration is 25%, adjusting pH to 10.50 with 5% sodium hydroxide aqueous solution, separating with nanofiltration membrane of 500Da molecular weight cut-off polyacrylonitrile material, diluting the cut-off solution with purified water when the cut-off solution can not be continuously separated, repeating for 2 times, and collecting the terminal cut-off solution when the cut-off solution can not be continuously separated; gradually diluting the terminal trapped fluid with ethanol until the ethanol content in the solution reaches 40%, adjusting pH to 5.50 with 5% hydrochloric acid aqueous solution, separating with a nanofiltration membrane of trapped molecule 300Da, diluting the trapped fluid with purified water when the trapped fluid cannot be continuously separated, repeating for 2 times, and collecting the trapped fluid when the trapped fluid cannot be continuously separated to obtain stachydrine. A Waters2695 high performance liquid chromatograph and an Alltech 6000 evaporative light scattering detector which are respectively used for detecting a stachydrine reference substance and refined and separated stachydrine, wherein the chromatogram of the stachydrine reference substance is shown in a figure 1, the chromatogram of the enriched amphiprotic alkaloid stachydrine is shown in a figure 2, and the result of calculating the purity of the stachydrine is shown in a table 1;
the cation exchange resin method comprises collecting herba Leonuri extract 1000mL, purifying with 1000mL 732 type cation exchange resin, detecting liquid phase of the refined part, and calculating stachydrine purity as shown in Table 1, wherein the chromatogram is shown in FIG. 3.
TABLE 1
| Refining method | Stachydrine purity (%) | Separation period |
| Example 1 | 77.6 | 3 hours |
| Adsorption with cationic resin | 42.5 | 20 hours |
By changing the existence state of the stachydrine, adjusting the acting force between the stachydrine and the nanofiltration membrane and amplifying the action difference between the stachydrine and other alkaloid, sterol and phenolic acid components in the motherwort and the nanofiltration membrane, the rapid separation of the stachydrine and other components in the motherwort is realized in a targeted manner, and the purity is 77.6 percent. In the refining process, because normal temperature operation is adopted, the stachydrine hardly has conversion loss. The method adopts cation resin adsorption, wherein sample loading adsorption and elution are continuously operated, the separation period is longer than 20 hours, the separation solution needs thermal concentration, the amphoteric alkaloid cannot be subjected to characteristic treatment, the stachydrine purity in the prepared total alkaloid part is 42.5 percent, and the method in the embodiment 1 has obvious technical advancement.
Example 3 refining of the amphoteric alkaloid trigonelline
Taking the water extract of the fenugreek medicinal material, adopting the embodiment 1 and cation exchange resin to enrich the trigonelline, and taking the purity and the production period of the trigonelline as indexes to carry out comparative analysis.
The invention comprises the following steps: detecting that the isoelectric point of trigonelline is about 6.10, and calculating the pH value of acid-base group dissociation in a stachydrine structure to be 8.50 and the pH value of a basic group dissociation state to be 4.00 by adopting a titration method; taking the fenugreek water extract, pretreating by adopting an ultrafiltration membrane made of cellulose with the cut-off molecular weight of 100KDa to remove macromolecular protein and polysaccharide, and collecting ultrafiltrate; diluting the ultrafiltrate with methanol until the concentration of methanol is 30%, adjusting pH to 8.50 with 5% sodium hydroxide aqueous solution, separating with nanofiltration membrane of 800Da molecular weight cutoff, diluting the cutoff liquid with purified water when the cutoff liquid cannot be continuously separated, repeating for 2 times, and collecting the terminal cutoff liquid when the cutoff liquid cannot be continuously separated; gradually diluting the terminal trapped fluid by adopting methanol until the methanol content in the solution reaches 35%, adjusting the pH to 4.00 by adopting a hydrochloric acid aqueous solution with the mass concentration of 5%, separating by adopting a trapped molecule 300Da nanofiltration membrane, diluting the trapped fluid by using purified water when the trapped fluid cannot be continuously separated, repeating for 3 times, and collecting the trapped fluid when the trapped fluid cannot be continuously separated, thus obtaining the trigonelline part. Waters2695 high performance liquid chromatograph, PAD ultraviolet detector, detect and calculate trigonelline purity result and see Table 2;
the cation exchange resin method comprises collecting semen Trigonellae extract 1000mL, adsorbing and refining with 1000mL 732 type cation exchange resin, and calculating trigonelline purity at the refined part, and the result is shown in Table 2.
TABLE 2
| Refining method | Trigonelline purity (%) | Separation period |
| Example 1 | 70.4 | 3 hours |
| Adsorption with cationic resin | 24.3 | 25 hours |
Compared with ion exchange resin, the separation technology for adjusting the acting force between the amphoteric alkaloid and the nanofiltration membrane by using the organic solvent in the embodiment 1 has obvious advantages, the purity of the trigonelline in the prepared alkaloid part is 70.4 percent, the trigonelline is higher than that of trigonelline prepared by adsorbing with cationic resin, the implementation period of the ultrafiltration-nanofiltration combined separation is short, the use amount of the organic reagent is far lower than that of resin separation, the method can carry out targeted separation on the amphoteric alkaloid, and the component loss is not generated in the normal-temperature refining process.
Claims (2)
1. A membrane combination refining method of amphoteric alkaloid is characterized in that: comprises the following steps:
a. detecting the isoelectric point of amphoteric alkaloid to be separated, and calculating the pH value of an acidic group in the structure of the amphoteric alkaloid compound when the acidic group is in a dissociated state and the pH value of a basic group when the basic group is in a dissociated state;
b. dissolving the extract to be separated in purified water, performing pretreatment by adopting an ultrafiltration membrane with the cut-off molecular weight of 10 KDa-100 KDa to remove macromolecular protein and polysaccharide, and collecting ultrafiltrate;
c. gradually diluting the ultrafiltrate in the step b by using a water-soluble organic solvent until the amount of the water-soluble organic solvent in the solution reaches 5-40%, adjusting the pH to the pH of the alkaline group dissociation in the step a by using a sodium hydroxide aqueous solution with the mass concentration of 1-10%, separating by using a nanofiltration membrane with the molecular weight cutoff of 100-800 Da, diluting the trapped liquid by using purified water when the trapped liquid cannot be continuously separated, repeating for 1-3 times, and collecting the terminal trapped liquid when the trapped liquid cannot be continuously separated;
d. gradually diluting the terminal trapped fluid in the step c by using a water-soluble organic solvent until the amount of the water-soluble organic solvent in the solution reaches 5-40%, adjusting the pH to the pH of acid group dissociation in the step a by using a 1-10% hydrochloric acid aqueous solution, separating by using a nanofiltration membrane with the molecular weight cutoff of 100-800 Da, diluting the trapped fluid by using purified water when the trapped fluid volume cannot be continuously separated, repeating for 1-3 times, and collecting the trapped fluid when the trapped fluid volume cannot be continuously separated, thereby obtaining an amphoteric alkaloid part;
the ultrafiltration membrane in the step b is made of one of cellulose, polyether sulfone and cellulose acetate;
the nanofiltration membrane in the step c and the step d is made of one of organic solvent resistant materials such as polyimide, polyamide and polyacrylonitrile;
the amphoteric alkaloid is stachydrine or trigonelline.
2. The membrane-combined refining method of amphoteric alkaloids according to claim 1, characterized in that: the water-soluble organic solvent in the step c and the step d is one of methanol, ethanol, acetonitrile and tetrahydrofuran.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103830298A (en) * | 2014-03-26 | 2014-06-04 | 南京中医药大学 | Method for preparing panax notoginseng saponins by adopting membrane coupling technology |
| CN106045890A (en) * | 2016-05-11 | 2016-10-26 | 南京泽朗医药科技有限公司 | A stachydrine preparing method |
| CN106631767A (en) * | 2017-01-04 | 2017-05-10 | 南京中医药大学 | Method for high-efficiency enrichment of phenolic acid components in traditional Chinese medicine through nanofiltration |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103830298A (en) * | 2014-03-26 | 2014-06-04 | 南京中医药大学 | Method for preparing panax notoginseng saponins by adopting membrane coupling technology |
| CN106045890A (en) * | 2016-05-11 | 2016-10-26 | 南京泽朗医药科技有限公司 | A stachydrine preparing method |
| CN106631767A (en) * | 2017-01-04 | 2017-05-10 | 南京中医药大学 | Method for high-efficiency enrichment of phenolic acid components in traditional Chinese medicine through nanofiltration |
Non-Patent Citations (1)
| Title |
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| 超滤-纳滤联用优化益母草生物碱的浓缩工艺;李存玉等;《中国中药杂志》;20170131;第42卷(第1期);100-106 * |
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