CN108997366B - Preparation method of anti-breast cancer active ingredient in macleaya cordata - Google Patents
Preparation method of anti-breast cancer active ingredient in macleaya cordata Download PDFInfo
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- CN108997366B CN108997366B CN201810617108.3A CN201810617108A CN108997366B CN 108997366 B CN108997366 B CN 108997366B CN 201810617108 A CN201810617108 A CN 201810617108A CN 108997366 B CN108997366 B CN 108997366B
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Abstract
The invention discloses a method for separating alkaloid compounds from macleaya cordata by pH-zone refining countercurrent chromatography, wherein an environment-friendly n-hexane/ethyl acetate/isopropanol/water solvent system is adopted for separation, and the volume ratio of reagents in the solvent system is 1:3:1.5: 4.5. The method can efficiently separate sanguinarine and chelerythrine with the initial purity of more than 98 percent from macleaya cordata. And the evaluation test of the anti-breast cancer effect proves that the sanguinarine and the chelerythrine obtained by the extraction method have the anti-breast cancer activity and the synergistic effect, and a new thought is provided for the research and development of anti-breast cancer drugs.
Description
Technical Field
The invention relates to a method for separating and purifying effective components of traditional Chinese medicines, in particular to a method for separating alkaloid compounds (sanguinarine and chelerythrine) in macleaya cordata by using pH zone countercurrent chromatography.
Background
Macleaya cordata is the overground part of macleaya cordata (Willd.) r.br. of macleaya of papaveraceae, is mainly distributed in china and south-east asia, and is mainly distributed in China in Hunan, Anhui, Jiangsu, Zhejiang and other provinces. Macleaya cordata is rich in alkaloid, and modern pharmacology indicates that macleaya cordata has the effects of promoting animal growth, resisting bacteria, inflammation, tumors and the like. Products developed based on macleaya cordata mainly comprise the traditional Chinese medicines of macleaya cordata, macleaya cordata injection, macleaya cordata tincture for swelling and itching and the like. The traditional separation method of alkaloid monomer components comprises silica gel column chromatography, preparative liquid chromatography, macroporous resin and the like, has the limitations of time and labor waste, environmental pollution, low sample purity, irreversible adsorption on the sample by repeated column chromatography, low preparation efficiency and high cost of the alkaloid monomer obtained by separation.
High-speed Counter-current Chromatography (HSCCC) is a continuous High-efficiency and rapid liquid-liquid distribution Chromatography separation technology which is developed in 30 years, does not need any solid support, avoids various problems of easy dead adsorption, loss, denaturation and the like of a sample caused by a solid support or a carrier, and obviously reduces the distribution efficiency and the solvent consumption when other liquid Chromatography is used for preparative separation, ensures higher peak resolution of HSCCC, has large separation amount, no sample loss, High recovery rate, mild separation environment and saves the solvent. The high-speed counter-current chromatography can directly carry out a large amount of crude extraction samples or synthesis mixtures, the separation result can reach quite high purity, and the method is widely applied to the preparation, separation and purification of chemical substances in the fields of biology, medicine, environmental protection and the like. The pH Zone-reflecting Counter-current Chromatography (PZRCCC) is a novel preparative separation technology developed on the basis of Counter-current Chromatography, utilizes the difference of material dissociation constant and hydrophobicity for separation, has the advantages of large separation amount, good separation effect and the like, and is widely applied to separation of alkaloid and organic acid components. Liu et al (Liu Q, Sun C, Meng F, et. preliminary Separation of Chelerythrine and Sanguinarine from Macleayaccordidata by pH-Zone-Refining Counter-current Chromatography, journal of liquid Chromatography & Related Technologies,2015,38(20): 1789) used PZRCCC technology to separate the alkaloids in macleaya cordata with chloroform/ethyl acetate/methanol/water as the solvent system, which is limited in that chloroform is an environmentally unfriendly organic solvent and is sensitive to light and gradually decomposes with oxygen in the air to produce virulent phosgene (phosgene) and hydrogen chloride. It is mainly used for central nervous system, has anesthetic effect, and can be used for treating heart injury, liver injury, and kidney injury.
Although the prior art reports that pH-zone refining countercurrent chromatography is used for separating alkaloid from traditional Chinese medicines, the solvent system used in the pH-zone refining countercurrent chromatography is known in the art to be one of the key factors influencing the separation result. Different Chinese medicine extracts contain different components and different types and amounts of alkaloids, so different Chinese medicines, different extracts, the types of solvents of solvent systems and the dosage ratio of the solvents have no reference value.
The development of the extraction and separation method for efficiently extracting high-purity sanguinarine and chelerythrine from macleaya cordata is also of great significance for the development of antitumor drugs.
Disclosure of Invention
The invention aims to provide a method for separating and preparing alkaloid monomeric compounds with purity of more than 98% from macleaya cordata in large batches, which has the advantages of low cost, simple and convenient operation and high efficiency.
In order to achieve the purpose, the invention provides the following technical scheme:
one of the purposes of the invention is to provide a method for separating sanguinarine and chelerythrine from macleaya cordata, which comprises the following steps:
(1) carrying out secondary separation treatment on the macleaya cordata alkaloid crude sample by adopting an acid dissolution and alkali precipitation method;
(2) separating and enriching the macleaya cordata alkaloid total sample subjected to secondary treatment by adopting pH zone countercurrent chromatography,
obtaining corresponding components, wherein the separation adopts a normal hexane/ethyl acetate/isopropanol/water solvent system.
The application of pH zone countercurrent chromatography to the extraction of alkaloid is a common means for extracting active ingredients of natural medicines, and the solvent system used by the pH zone countercurrent chromatography is known in the art to be an important factor influencing the separation result, and different medicines and different alkaloid general samples have no reference significance. The n-hexane/ethyl acetate/isopropanol/water solvent system adopted by the invention has the characteristics of environmental friendliness, high extraction efficiency and high purity of the separated product.
Preferably, the solvent system employed in the present invention is n-hexane/ethyl acetate/isopropanol/water (1:3:1.5:4.5, v/v).
In a preferred embodiment of the present invention, the step (1) may specifically be: dissolving the macleaya cordata alkaloid crude sample in an aqueous solution with the pH value of 2, filtering through medium-speed filter paper with the speed of 30-50 mu m, adjusting the pH value of filtrate to 9, fully precipitating alkaloid, centrifuging by adopting a centrifuge at 3000 rpm, removing the upper-layer solution, and obtaining the macleaya cordata alkaloid total sample from the lower-layer precipitate.
Further, the aqueous solution was adjusted to pH 2 with HCl.
Further, the pH of the filtrate was adjusted to 9 with 10% diluted ammonia water.
In another preferred embodiment of the present invention, the specific operation of step (2) may be: placing the solvent system in a separating funnel, shaking uniformly, standing for layering, and separating an upper phase from a lower phase; adding triethylamine as a retention alkali into the upper phase, adding trifluoroacetic acid into the lower phase as washing deacidification, and dissolving the macleaya cordata alkaloid total sample into 10mL of alkalized upper phase and 10mL of non-acidified lower phase for later use; adopting a semi-preparative high-speed counter-current chromatograph, firstly enabling a sample injection valve to be in a sample injection state, filling a chromatographic separation column with a stationary phase at a certain flow velocity by using a pump, and stopping the pump; starting a speed controller to enable a chromatographic separation column of a high-speed countercurrent chromatograph to rotate forwards, injecting a dissolved sample into a sample injection valve of the countercurrent chromatograph by using an injector when the rotating speed reaches 800 revolutions per minute, and enabling the sample to enter the chromatographic separation column by rotating the sample injection valve to be in a column connection state; setting the flow rate of the mobile phase to be 2.0mL/min, starting pumping the mobile phase, receiving each component according to a detector ultraviolet spectrogram, and separating to obtain sanguinarine and chelerythrine.
Further, 10mmol of triethylamine was added to the upper phase as a retained base.
Further, 10mmol of trifluoroacetic acid was added to the lower phase as a wash deacidification.
Furthermore, the chromatographic separation column is a spiral column formed by winding polytetrafluoroethylene tubes in multiple layers, and the capacity of the chromatographic separation column is 300 mL.
Further, the liquid phase analysis conditions of the high performance countercurrent chromatography are as follows: waters SymmetryC18Chromatographic column (4.6mm × 250mm,5 μmi.d.) with mobile phase acetonitrile/0.1% triethylamine water solution, pH adjusted to 2.5(35:75, v/v) with phosphoric acid, flow rate, 1.0mL/min, detection wavelength 254 nm.
The macleaya cordata alkaloid total sample, sanguinarine, chelerythrine and total sample knockout obtained by separation through the method are proved to have anti-breast cancer activity evaluation, the macleaya cordata total sample, sanguinarine and chelerythrine obtained by the method have anti-breast cancer activity, the anti-breast cancer activity is sequentially the macleaya cordata total sample > sanguinarine > chelerythrine > knockout, and the macleaya cordata alkaloid anti-breast cancer activity is realized through the synergistic effect of the sanguinarine and the chelerythrine.
Therefore, the invention also provides application of the sanguinarine, the chelerythrine or the mixture thereof obtained by the preparation method in anti-breast cancer drugs.
The invention has the advantages of
The invention adopts pH zone countercurrent chromatography to separate alkaloid in macleaya cordata, provides an environment-friendly n-hexane/ethyl acetate/isopropanol/water solvent system, and is superior to the prior art in single preparation amount. The separation method of the invention has simple operation and high efficiency, and the purity of the alkaloid monomeric compound obtained by separation is more than 98 percent.
Meanwhile, MCF-7 human breast cancer cells are used as a model, the anti-breast cancer activity of the separated alkaloid monomers is evaluated, and the evaluation result proves that the sanguinarine and the chelerythrine which are separated by the method have the anti-breast cancer activity and the synergistic effect, so that a new idea is provided for the development of anti-breast cancer medicines.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is a zonal countercurrent chromatogram of the separation of macleaya cordata total alkaloids;
FIG. 3 is a high performance liquid chromatogram of the total alkaloids from Macleaya cordata and the countercurrent fractions.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The present invention will be described with reference to the following embodiments and drawings.
Example 1:
1. sample extraction
Dissolving the macleaya cordata alkaloid crude sample in a water solution with the pH value of 2 (adjusted by HCl), filtering the solution through medium-speed filter paper with the speed of 30-50 mu m, adjusting the pH value of filtrate to 9 by using 10% dilute ammonia water, centrifuging the solution by adopting a centrifuge at 3000 rpm after the alkaloid is fully precipitated, removing the upper-layer solution, and obtaining the macleaya cordata alkaloid total sample by using the lower-layer precipitate. 2. Separation of macleaya cordata alkaloid total sample by pH zone countercurrent chromatography
Preparing a solvent system from a two-phase solvent system of n-hexane/ethyl acetate/isopropanol/water (1:3:1.5:4.5, v/v) according to the solvent ratio, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, separating an upper phase and a lower phase after balancing for a period of time, adding 10mmol of triethylamine into an upper phase as reserved alkali, adding 10mmol of trifluoroacetic acid into a lower phase as washing deacidification, and dissolving 2.5g of macleaya cordata alkaloid total sample into 10mL of alkalized upper phase and 10mL of non-acidified lower phase for later use. A semi-preparative high-speed counter-current chromatograph is used, which is composed of plunger pump, sampling valve, ultraviolet detector, recorder and chromatographic separation column (spiral pipe column formed by winding polytetrafluoroethylene tubes in multiple layers with capacity of 300 mL). And starting a speed controller to enable a chromatographic separation column of the high-speed countercurrent chromatograph to rotate forwards, and when the rotating speed reaches 800 revolutions per minute, injecting the dissolved sample into a sample injection valve of the countercurrent chromatograph by using an injector, wherein the sample injection valve is rotated to be in a column connection state, so that the sample enters the chromatographic separation column. Setting the flow rate of the mobile phase to be 2.0mL/min, starting pumping the mobile phase, receiving each component according to a detector ultraviolet spectrogram, and analyzing the pH value and the purity. Finally, 0.45 g and 0.59g of sanguinarine and chelerythrine are obtained by separation, the yield is 18.10 and 23.64 percent, and the purity is higher than 98 percent.
3. Evaluation of anti-Breast cancer Effect
MCF-7 human breast cancer cells were activated and cultured at 2 × 104Coating the position/position on 96-position plate, culturing for 12h, treating cells with macleaya cordata alkaloid total sample, sanguinarine, chelerythrine, knockout and positive control (vincristine) at different gradient concentrations, culturing for 24h, adding 5mg/mL MTT to each well, discarding the culture medium,adding 150 mu L of DMSO into each hole, incubating for 4h at 37 ℃, detecting at 570nm by using a microplate reader, and calculating the breast cancer inhibition rate by adopting the following formula: inhibition ratio (%) (a 570)control-A570sample)/A570control× 100% the result shows that the anti-breast cancer effect is the macleaya cordata total sample in turn>Sanguinarine>Positive control>Chelerythrine>>A knockout. The anti-breast cancer activity of macleaya cordata alkaloid is realized by the synergistic effect of sanguinarine and chelerythrine, and specific results are shown in table 1.
TABLE 1 anti-Breast cancer IC of Total alkaloid sample and countercurrent chromatography samples50Value (MCF-7 human breast cancer cells)
The liquid phase analysis employed the following conditions: waters SymmetryC18Chromatographic column (4.6mm × 250mm,5 μmi.d.) with mobile phase acetonitrile/0.1% triethylamine water solution, pH adjusted to 2.5(35:75, v/v) with phosphoric acid, flow rate, 1.0mL/min, detection wavelength 254 nm.
And (3) structural identification: mass spectrometry analysis is carried out on the separated alkaloid component monomers, and the results show that the molecular weights of the compounds 1 and 2 are respectively M/z 322.0946[ M + H ]]+And 348.1257[ M + H ]]+The control literature identifies sanguinarine and chelerythrine.
Claims (8)
1. A method for separating sanguinarine and chelerythrine from Macleaya cordata comprises the following steps:
(1) carrying out secondary separation treatment on the macleaya cordata alkaloid crude sample by adopting an acid dissolution and alkali precipitation method;
(2) separating and enriching the macleaya cordata alkaloid total sample subjected to secondary treatment by adopting pH zone countercurrent chromatography to obtain corresponding components, wherein a solvent system is n-hexane: ethyl acetate: isopropyl alcohol: water in a volume ratio of 1:3:1.5: 4.5; placing the solvent system in a separating funnel, shaking uniformly, standing for layering, and separating an upper phase from a lower phase; adding triethylamine into the upper phase to retain alkali, adding trifluoroacetic acid into the lower phase to wash and deacidify, and dissolving the macleaya cordata alkaloid total sample into 10mL of alkalized upper phase and 10mL of non-acidified lower phase for later use; the upper phase is a stationary phase and the lower phase is a mobile phase.
2. The method for separating sanguinarine and chelerythrine from macleaya cordata according to claim 1, wherein said step (1) is practiced to: dissolving the macleaya cordata alkaloid crude sample in an aqueous solution with the pH value of 2, filtering by using medium-speed filter paper with the speed of 30-50 mu m, adjusting the pH value of filtrate to 9, fully precipitating alkaloid, centrifuging by adopting a centrifuge at 3000 rpm, removing the upper-layer solution, and obtaining the macleaya cordata alkaloid total sample by using the lower-layer precipitate.
3. The process for the separation of sanguinarine and chelerythrine from macleaya cordata according to claim 2, wherein the aqueous solution is adjusted to a pH of 2 with HCl.
4. The process for the separation of sanguinarine and chelerythrine from macleaya cordata as claimed in claim 2, wherein the filtrate is adjusted to pH 9 with 10% dilute ammonia.
5. The method for separating sanguinarine and chelerythrine from macleaya cordata according to claim 1, wherein the step (2) is specifically performed by: adopting a semi-preparative high-speed counter-current chromatograph, firstly enabling a sample injection valve to be in a sample injection state, filling a chromatographic separation column with a stationary phase at a certain flow velocity by using a pump, and stopping the pump; starting a speed controller to enable a chromatographic separation column of a high-speed countercurrent chromatograph to rotate forwards, injecting a dissolved sample into a sample injection valve of the countercurrent chromatograph by using an injector when the rotating speed reaches 800 revolutions per minute, and enabling the sample to enter the chromatographic separation column by rotating the sample injection valve to be in a column connection state; setting the flow rate of the mobile phase to be 2.0mL/min, starting pumping the mobile phase, receiving each component according to a detector ultraviolet spectrogram, and separating to obtain sanguinarine and chelerythrine.
6. The process of claim 1, wherein 10mmol triethylamine is added to the upper phase.
7. The process for the separation of sanguinarine and chelerythrine from macleaya cordata according to claim 1, wherein 10mmol of trifluoroacetic acid are added to the lower phase.
8. The method of separating sanguinarine and chelerythrine from macleaya cordata according to claim 5, wherein the countercurrent chromatography comprises the following liquid phase analysis conditions: a Waters symmetry C18 chromatographic column with the specification of 4.6mm multiplied by 250mm and 5 mu mi.d., wherein the mobile phase is acetonitrile/0.1 percent triethylamine water solution, the volume ratio is 35:75, the pH value is adjusted to 2.5 by phosphoric acid, the flow rate is 1.0mL/min, and the detection wavelength is 254 nm.
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| CN105131005A (en) * | 2015-08-25 | 2015-12-09 | 长沙博拓生物科技有限公司 | Preparation method for high content macleaya cordata alkaloid |
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