CN113845422B - Process for preparing L-chicoric acid from Echinacea purpurea in batches and application thereof - Google Patents
Process for preparing L-chicoric acid from Echinacea purpurea in batches and application thereof Download PDFInfo
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- YDDGKXBLOXEEMN-IABMMNSOSA-N chicoric acid Chemical compound O([C@@H](C(=O)O)[C@@H](OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C(O)=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 YDDGKXBLOXEEMN-IABMMNSOSA-N 0.000 title claims abstract description 113
- YDDGKXBLOXEEMN-WOJBJXKFSA-N dicaffeoyl-L-tartaric acid Natural products O([C@@H](C(=O)O)[C@@H](OC(=O)C=CC=1C=C(O)C(O)=CC=1)C(O)=O)C(=O)C=CC1=CC=C(O)C(O)=C1 YDDGKXBLOXEEMN-WOJBJXKFSA-N 0.000 title claims abstract description 113
- 235000014134 echinacea Nutrition 0.000 title claims abstract description 38
- 240000004530 Echinacea purpurea Species 0.000 title claims abstract description 37
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 230
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- YDDGKXBLOXEEMN-IABMMNSOSA-L Chicoric acid Natural products C1=C(O)C(O)=CC=C1\C=C\C(=O)O[C@@H](C([O-])=O)[C@H](C([O-])=O)OC(=O)\C=C\C1=CC=C(O)C(O)=C1 YDDGKXBLOXEEMN-IABMMNSOSA-L 0.000 claims description 83
- YDDGKXBLOXEEMN-UHFFFAOYSA-N Di-E-caffeoyl-meso-tartaric acid Natural products C=1C=C(O)C(O)=CC=1C=CC(=O)OC(C(O)=O)C(C(=O)O)OC(=O)C=CC1=CC=C(O)C(O)=C1 YDDGKXBLOXEEMN-UHFFFAOYSA-N 0.000 claims description 83
- 229930016920 cichoric acid Natural products 0.000 claims description 83
- YDDGKXBLOXEEMN-PMACEKPBSA-N dicaffeoyl-D-tartaric acid Natural products O([C@H](C(=O)O)[C@H](OC(=O)C=CC=1C=C(O)C(O)=CC=1)C(O)=O)C(=O)C=CC1=CC=C(O)C(O)=C1 YDDGKXBLOXEEMN-PMACEKPBSA-N 0.000 claims description 83
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/52—Separation; Purification; Stabilisation; Use of additives by change in the physical state, e.g. crystallisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
Abstract
The invention provides a process for preparing L-chicoric acid from Echinacea purpurea in batches and application thereof, and belongs to the technical field of separation, purification and refining of traditional Chinese medicines. According to the invention, through optimizing the process scheme, the alcohol extraction and water precipitation treatment, column chromatography and ethyl acetate extraction and impurity removal are carried out, the L-chicoric acid raw material with the purity of more than 98% can be obtained in a large scale through the preparation liquid phase separation and crystallization operation, and the method is very suitable for industrial large-scale production, so that the method has good practical application value.
Description
Technical Field
The invention belongs to the technical field of separation, purification and refining of traditional Chinese medicines, and particularly relates to a process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches and application thereof.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
In the process of global new coronavirus prevention and treatment, traditional Chinese medicine plays an increasingly important role in antiviral, and is favored by people at home and abroad. The novel coronavirus and the RSV belonging to respiratory viruses are common pathogens of respiratory infections, are the primes of diseases such as infant pneumonia, senile pneumonia, asthma and the like, are lack of effective therapeutic drugs at present, and the side effects of the representative drug ribavirin on the market are large, and the safety and the curative effect of the drug ribavirin are still to be further examined. The chicoric acid extracted from the traditional Chinese medicine has remarkable anti-RSV curative effect, small toxic and side effects, high safety, definite in-vivo and in-vitro anti-RSV action mechanism and wide prospect in the development of novel anti-RSV candidate medicines, however, the yield and purity of the chicoric acid extraction process in the currently marketed Echinacea extract are lower, and the development requirement of preparing the novel anti-RSV medicine by taking the chicoric acid as the raw material cannot be met.
At present, chicoric acid is mainly used in two major industries of medical raw materials and health-care foods, the content of chicoric acid in a commercial echinacea extract is low, and a production process for preparing high-purity chicoric acid on a large scale by taking echinacea as a raw material is not reported so far, so that further development and utilization of chicoric acid are limited to a certain extent. Therefore, optimizing a set of scheme of preparation process of chicoric acid monomer, which is economical, environment-friendly, low in cost and suitable for large-scale industrialized production, has important significance.
In order to obtain the chicoric acid monomer with high purity, the technology is domesticA number of experimental studies have been developed by the external scholars. Nigel B.Perry et al [ J.Agric.food Chem,2001,49 (4): 1702-1706)]The chicoric acid reference substance with the purity of 95 percent is obtained by utilizing the preparation chromatography, but the method is not easy for large-scale production, has high cost and toxic reagents, and can only be used for producing standard substances, but can not be used for industrial production; the method comprises the steps of treating Echinacea purpurea with chloroform, extracting with methanol and ethyl acetate, and purifying with acetonitrile reversed phase chromatographic column to obtain high purity chicoric acid product. Chloroform and methanol used in the process are harmful to the environment and are easy to remain, and the production efficiency of the acetonitrile reversed phase chromatographic column is low and the cost is high; the extraction solvent and the recrystallization solvent adopted by the Siamice information technology Co., ltd (patent number: CN 109748796A) are respectively methanol and acetone, and have certain toxicity; the report of Hunan university (patent number: CN 102161620A) reports that chicoric acid with the content of 25-40% can be obtained by adopting an organic solvent for ultrasonic extraction, drying and purifying by chloroform and other treatments, the purity is low, and the ultrasonic treatment method is not suitable for large-scale industrial production; the Guilin Sanbao pharmaceutical industry Co Ltd (patent number: CN 105294440A) adopts a plurality of silica gel column chromatography, and is eluted by methanol and acetone, so that the operation steps are complex, the time and the labor are wasted, and the large-scale industrial production is difficult to realize. The purity of chicoric acid obtained by the process reported by China university of medical science (patent number: CN 104693034A), xinjiang Terui biotechnology Co Ltd (patent number: CN 103641716A) and Hunan Langline biological resource Co Ltd (patent number: CN 111233950A) is lower (less than 85 percent); the Shenyang Shuangding technology Co., ltd (patent No. CN102249917A, CN 102391118A) reports the extraction, separation and identification method of chicoric acid, which is used for the content measurement and quality control of herba Ixeritis Sonchifoliae medicinal materials and preparations. Nanjing Zealand pharmaceutical technologies Co., ltd (patent number: CN 102060706A) reported the use of supercritical CO 2 And the entrainer is extracted, ion exchanged and other separation and purification methods to prepare the chicoric acid with high content, so that the cost is high, and the method is not suitable for industrial production. Wu Qilin (patent number: CN 1587251A) is prepared from Echinacea purpurea root powder with high chicoric acid content, and has less liposoluble impurities such as chlorophyll, but is unfavorable for resource utilizationContinuous utilization; the medicinal materials are crushed and extracted, so that the extraction efficiency can be improved, but the production procedures are increased, dust is easy to fly, and the workshop environment and safety are influenced. When the pH value is=3, the chicoric acid is more stable, the pH value of the liquid medicine is regulated to be=1-3 in the acidification treatment process, the pH fluctuation is larger, the process stability is poorer, the degradation of the chicoric acid and the precipitation conditions of impurities and the like in the experiment process of different batches are different, and the quality control is inconvenient. Ethyl acetate is extracted for 3 times, each time for 2 hours, the extraction times are more, the extraction time is long, the solvent consumption is large, the extraction efficiency is affected, and the economic cost is increased. Only deionized water is adopted in the column chromatography process to remove water-soluble impurities, and the impurity removal is incomplete; and then, 10-50% ethanol is used as an eluent to enrich the chicoric acid, the eluting capacities of the eluent with different concentrations on the chicoric acid and impurities are different, the volume change of the eluent consumed for enriching the chicoric acid is large, the purity of the eluent chicoric acid is changed, and the repeatability and the quality stability of the product are not controlled. The chicory acid solution obtained by extraction and column chromatography is dried into solid and then is processed in the next step, which not only consumes time but also increases the production cost. Although chicoric acid with higher purity can be obtained by adopting the process, the variety and the content of impurities contained in the extract can be changed due to different raw materials of the echinacea, so that the separation and purification process procedures and effects can be different. The Hunan university of teachers (patent number: CN 109678706A) discloses a chemical synthesis method for preparing L-chicoric acid crystal forms, and the X-ray powder diffraction patterns of the crystal forms show diffraction at 6.36 degrees, 12.60 degrees, 19.24 degrees, 25.80 degrees, 32.46 degrees and 36.32 degrees, however, the crystal forms of chicoric acid extracted from plants are not reported at home and abroad at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a process for preparing high-purity L-chicoric acid from plant Echinacea purpurea in batches and application thereof, and the chicoric acid raw material with the purity of more than 98% can be obtained in batches by adopting the technical scheme of the invention, so that the process is very suitable for industrial mass production.
In a first aspect of the present invention, there is provided a process for the batch preparation of high purity L-chicoric acid from Echinacea purpurea, the process comprising: the preparation method comprises the steps of taking dry aerial parts of the echinacea purpurea medicinal material as raw materials, carrying out alcohol extraction and water precipitation treatment, column chromatography and ethyl acetate extraction and impurity removal, and preparing liquid phase separation and crystallization operation.
In the case of chicoric acid, since it is affected by light, heat, etc., it is likely to cause deterioration in stability, and therefore, it is preferable to select a light-shielding treatment in the whole of the above-mentioned operation steps, thereby reducing the deterioration of the components during the production thereof.
Specifically, the process method comprises the following steps:
extracting: reflux-extracting aerial parts of Echinacea purpurea with ethanol water solution for several times, mixing filtrates, and concentrating to obtain concentrated solution;
water sedimentation and column pretreatment: carrying out water sedimentation treatment on the concentrated solution to obtain a water sedimentation supernatant, centrifuging, concentrating the centrifugate, and carrying out suction filtration to obtain a sample liquid for later use;
purifying by macroporous resin: wet column packing; fixing the sample loading amount, loading the liquid medicine at the flow rate of 1-3 BV/h, circularly adsorbing for 2-3 h, respectively carrying out gradient impurity removal by using 3-6 BV of acidic purified water, 3-6 BV of acidic 10% ethanol solution and 1-2 BV of 20% ethanol solution, eluting enriched chicoric acid by using 6-12 BV of 30-35% ethanol solution, collecting eluent, and concentrating under reduced pressure;
ethyl acetate extraction: extracting 30% -35% concentrated solution with C=10-18 mg/mL and pH=1-3 with ethyl acetate; the organic phase is extracted reversely by acid water, the organic phase is concentrated to thick paste under reduced pressure, the ethyl acetate phase is replaced by high-purity ethanol, and the mixture is continuously concentrated to thick paste and dried;
high pressure preparation of liquid phase and crystallization: dissolving chicoric acid coarse powder with 10-20% methanol solution, centrifuging, vacuum filtering supernatant, separating by sample injection, collecting effluent corresponding to chromatographic peak of 30-50 min according to chicoric acid peak time, vacuum concentrating, crystallizing at low temperature, filtering, washing, and drying.
In a second aspect of the invention, there is provided the use of the above preparation method in the industrial preparation of L-chicoric acid.
The beneficial technical effects of one or more of the technical schemes are as follows:
the technical scheme provides a process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches, and the process scheme is optimized, so that alcohol extraction and water precipitation treatment, column chromatography and ethyl acetate extraction and impurity removal are carried out, and the L-chicoric acid raw material with the purity of more than 98% can be obtained in a large scale through preparation liquid phase separation and crystallization operation, so that the process is very suitable for industrial mass production, and has good practical application value.
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.
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is a graph showing the characterization of the crystalline form of L-chicoric acid obtained in example 1, according to the invention, by means of X-ray powder diffraction;
FIG. 3 is a graph showing the characterization of the crystalline form of L-chicoric acid obtained in example 1 by differential scanning calorimetry;
FIG. 4 is a graph showing the characterization of the crystalline form of L-chicoric acid obtained in example 1, according to the invention, by means of thermogravimetric analysis.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. 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 invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
In an exemplary embodiment of the present invention, there is provided a process for preparing L-chicoric acid in high purity from Echinacea purpurea in batch, the process comprising: the preparation method comprises the steps of taking dry aerial parts of the echinacea purpurea medicinal material as raw materials, carrying out alcohol extraction and water precipitation treatment, column chromatography and ethyl acetate extraction and impurity removal, and preparing liquid phase separation and crystallization operation.
In the case of chicoric acid, since it is affected by light, heat, etc., it is likely to cause deterioration in stability, and therefore, it is preferable to select a light-shielding treatment in the whole of the above-mentioned operation steps, thereby reducing the deterioration of the components during the production thereof.
In yet another embodiment of the present invention, the process comprises:
extracting: reflux-extracting aerial parts of Echinacea purpurea with ethanol water solution for several times, mixing filtrates, and concentrating to obtain concentrated solution;
water sedimentation and column pretreatment: carrying out water sedimentation treatment on the concentrated solution to obtain a water sedimentation supernatant, centrifuging, concentrating the centrifugate, and carrying out suction filtration to obtain a sample liquid for later use;
purifying by macroporous resin: wet column packing; fixing the sample loading amount, loading the liquid medicine at the flow rate of 1-3 BV/h, circularly adsorbing for 2-3 h, respectively carrying out gradient impurity removal by using 3-6 BV of acidic purified water, 3-6 BV of acidic 10% ethanol solution and 1-2 BV of 20% ethanol solution, eluting enriched chicoric acid by using 6-12 BV of 30-35% ethanol solution, collecting eluent, and concentrating under reduced pressure;
ethyl acetate extraction: extracting 30% -35% concentrated solution with C=10-18 mg/mL and pH=1-3 with ethyl acetate; the organic phase is extracted reversely by acid water, the organic phase is concentrated to thick paste under reduced pressure, the ethyl acetate phase is replaced by high-purity ethanol, and the mixture is continuously concentrated to thick paste and dried;
high pressure preparation of liquid phase and crystallization: dissolving chicoric acid coarse powder with 10-20% methanol solution, centrifuging, vacuum filtering supernatant, separating by sample injection, collecting effluent corresponding to chromatographic peak of 30-50 min according to chicoric acid peak time, vacuum concentrating, crystallizing at low temperature, filtering, washing, and drying.
In a further specific embodiment of the present invention, in the extraction step, an aqueous solution of 20-40% (preferably 30%) ethanol is used for reflux extraction for 2-4 times, preferably 3 times, the ratio of feed to liquid is controlled to be 1:8-15, such as 1:10, 1:12, 1:14, and the extraction time is controlled to be 0.1-2 hours, further 0.5-1.5 hours; in one embodiment of the present invention, the extracting step includes: the aerial parts of the Echinacea purpurea medicinal materials are dried, reflux-extracted for 3 times with 30% ethanol water solution, the feed-liquid ratio is 1:14, 1:12 and 1:10 respectively, the extraction time is 1.5h, 0.5h and 0.5h respectively, the 3 times of filtrates are combined, and the filtrate is concentrated to 1/3 of the original volume under reduced pressure.
In still another embodiment of the present invention, in the water sedimentation and column pretreatment steps, the water sedimentation treatment method includes: water-sinking treatment is carried out for 5-8h, preferably 6h under the condition of low temperature (such as 4 ℃); concentrating the centrifugate to 1/2-1/4 of the original volume, preferably 1/3;
in another specific embodiment of the invention, water sedimentation is adopted to remove part of fat-soluble impurities, and a large amount of yellow-green sediment appears after the treatment process, so that the invention has obvious impurity removal effect and is environment-friendly and economical.
In the macroporous resin purification step in a further specific embodiment of the invention, the wet method column packing leads the diameter-to-height ratio of the resin bed to be 1:5-1: 10;
in a further embodiment of the invention, the ethyl acetate extraction step is carried out using the same volume of ethyl acetate for 2-3, preferably 2, extractions; the high-purity ethanol is ethanol with concentration of not less than 90%, such as 95% ethanol;
the drying is carried out by adopting reduced pressure vacuum drying, specifically, the temperature is controlled to be 50-60 ℃, and the pressure is controlled to be-0.07-0.10 MPa.
In the separation and purification process, experiments and comparison show that the purification effect of the method is obviously better than that of the method of extracting and purifying by column chromatography. In the column chromatography process, gradient impurity removal is carried out by adopting water, 10% ethanol and 20% ethanol, impurities with different polarities are removed in a grading way, and chicoric acid is enriched by adopting 30-35% ethanol with more specific ethanol concentration range, so that the purification effect is better. In the ethyl acetate extraction process, water back extraction is adopted to further remove water-soluble impurities in the ethyl acetate phase, so that the purity of chicoric acid is improved by 2% -5%, and the experimental process has continuity, so that the method is more suitable for industrial production.
In the high-pressure preparation liquid phase and crystallization step, the specific method for concentrating under reduced pressure, crystallizing at low temperature, filtering, washing and drying comprises the following steps: concentrating under reduced pressure to 1/30-1/40 of the original volume, crystallizing at low temperature (such as 4deg.C) for 4-10 hr, filtering white or quasi-white needle-like and needle-cluster crystals, and repeatedly washing filter cake with low temperature (such as 4deg.C) water (purified water); and drying the filter cake (in vacuum) to obtain the product.
In the process of preparing the liquid-phase purified chicoric acid, although the process cost is relatively high, the purity of the chicoric acid coarse powder can be more than 80% by further purifying through pretreatment, the sample is relatively pure, the pollution to the filler for preparing the liquid phase is less, the filler can be recycled, the process is stable, and a batch of L-chicoric acid crystal form monomers with the purity of 98% can be obtained to meet the market demand, so that higher social benefit and economic benefit can be obtained.
In a further embodiment of the present invention, there is provided the use of the above preparation method in the industrial preparation of L-chicoric acid.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are noted, and are generally conducted under conventional conditions.
Example 1
A process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches comprises the following steps:
(1) Extracting 500g of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and extracting under reflux for 1.5h; the filtered residues are subjected to secondary reflux extraction and tertiary reflux extraction (both 0.5 h) by using 30% ethanol with the amount of l2 times and the amount of l0 times, and the filtrates of 3 times are combined and concentrated to 1/3 of the original volume under reduced pressure.
(2) Water sedimentation and column pretreatment: transferring the concentrated solution to water at 4 ℃ for 6h. Centrifuging the supernatant fluid after water precipitation (v=5000 r/min, t=10 min), concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration c=2.90 mg/mL, regulating the pH value to be 3, and storing at 4 ℃, and centrifuging and filtering to obtain a filtrate as a sample loading liquid.
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in absolute ethanol solution for 24h, and activating the resin. Removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, rinsing the resin with purified water until no alcoholic smell.
(4) Macroporous resin purification, namely loading at a loading flow rate of 2BV/h, circularly adsorbing for 3h, respectively carrying out gradient impurity removal by using 10% ethanol solution 3BV with pH=3 and 3 and 20% ethanol solution 2BV with pH=3, eluting enriched chicoric acid by using 10BV with 35% ethanol solution, and concentrating the eluent under reduced pressure until no alcohol smell exists.
(5) Ethyl acetate extraction: taking the concentrated solution with the concentration of C=13.56 mg/mL and the pH=2, and extracting with ethyl acetate with the same volume for 2 times, each time for 40min; the organic phases were combined and back-extracted 1 time with water of ph=2 for 40min each. The organic phase was concentrated to a thick paste under reduced pressure and then transferred to a tray for vacuum drying under reduced pressure (t=50 ℃, p= -0.09 MPa).
(6) Preparing and crystallizing: dissolving the chicoric acid coarse powder with methanol, centrifuging, performing vacuum suction filtration on supernatant, performing sample injection separation, collecting effluent corresponding to a chromatographic peak of 36-44 min according to the time of the chicoric acid peak, concentrating under reduced pressure to 4 ℃ after the time, performing refrigerator overnight crystallization, filtering, washing a filter cake with purified water at 4 ℃, and performing vacuum drying on the filter cake (T=60 ℃ and P= -0.10 MPa).
Example 2
A process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches comprises the following steps:
(1) Extracting 500g of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and extracting under reflux for 1.5h; filtering, and extracting residues with 1-2 times of 30% ethanol under reflux for 0.5 hr; reflux-extracting the residue with l0 times of 30% ethanol for 0.5 hr, mixing the filtrates, and concentrating under reduced pressure to 1/3 of the original volume.
(2) Water sedimentation and column pretreatment: transferring the concentrated solution to water at 4 ℃ for 6h. Centrifuging the supernatant fluid after water precipitation (v=5000 r/min, t=10 min), concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration c=2.57 mg/mL, regulating the pH value to be 3, and storing at 4 ℃, and centrifuging and filtering to obtain a filtrate as a sample loading liquid.
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in absolute ethanol solution for 24h, and activating the resin. Removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, rinsing the resin with purified water until no alcoholic smell.
(4) Macroporous resin purification, namely loading at a loading flow rate of 2BV/h, circularly adsorbing for 3h, respectively carrying out gradient impurity removal by using 10% ethanol solution 3BV with pH=3 and 3 and 20% ethanol solution 2BV with pH=3, eluting enriched chicoric acid by using 10BV with 35% ethanol solution, and concentrating the eluent under reduced pressure until no alcohol smell exists.
(5) Ethyl acetate extraction: taking the concentrated solution with the concentration of C=15.15 mg/mL and the pH value of 1.99, and extracting with ethyl acetate with the same volume for 2 times, each time for 40min; the organic phases were combined and back-extracted 1 time with water of ph=2 for 40min each. The organic phase was concentrated to a thick paste under reduced pressure and then transferred to a tray for vacuum drying under reduced pressure (t=55 ℃, p= -0.08 MPa).
(6) Preparing and crystallizing: dissolving the chicoric acid coarse powder in methanol, centrifuging, performing vacuum suction filtration on the supernatant, performing sample introduction separation, collecting effluent corresponding to 32-42 min chromatographic peaks, concentrating under reduced pressure to a refrigerator at the temperature of 4 ℃ for overnight, filtering crystals, washing a filter cake with purified water at the temperature of 4 ℃, and performing vacuum drying on the filter cake (T=60 ℃, P= -0.09 MPa).
Example 3
A process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches comprises the following steps:
(1) Extracting 500g of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and extracting under reflux for 1.5h; filtering, and extracting residues with 1-2 times of 30% ethanol under reflux for 0.5 hr; reflux-extracting the residue with l0 times of 30% ethanol for 0.5 hr, mixing the filtrates, and concentrating under reduced pressure to 1/3 of the original volume.
(2) Water sedimentation and column pretreatment: transferring the concentrated solution to water at 4 ℃ for 6h. Centrifuging the supernatant fluid after water precipitation (v=5000 r/min, t=10 min), concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration c=2.71 mg/mL, regulating the pH value to be 2.99, and storing at 4 ℃, centrifuging and filtering, wherein the filtrate is used as a loading liquid.
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in absolute ethanol solution for 24h, and activating the resin. Removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, rinsing the resin with purified water until no alcoholic smell.
(4) Macroporous resin purification, namely loading at a loading flow rate of 2BV/h, circularly adsorbing for 3h, respectively carrying out gradient impurity removal by using 10% ethanol solution 6BV with pH=3 and 3 and 20% ethanol solution 2BV with pH=3, eluting enriched chicoric acid by using 35% ethanol solution 9BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.
(5) Ethyl acetate extraction: taking the concentrated solution C=13.34 mg/mL and pH=2.00, extracting with ethyl acetate with the same volume for 2 times, and 40min each time; the organic phases were combined and back-extracted 1 time with water of ph=2 for 40min each. The organic phase was concentrated to a thick paste under reduced pressure and then transferred to a tray for vacuum drying under reduced pressure (t=50 ℃, p= -0.09 MPa).
(6) Preparing and crystallizing: dissolving the organic phase powder with 15% methanol to prepare the concentration of about 28mg/mL, centrifuging, carrying out vacuum suction filtration on the supernatant, carrying out sample injection separation, collecting effluent corresponding to a chromatographic peak of 26-36 min according to the time for which chicoric acid appears, concentrating the effluent to the temperature of 4 ℃ after the concentration under reduced pressure, standing overnight in a refrigerator, carrying out vacuum filtration on crystals, washing a filter cake with purified water at the temperature of 4 ℃, and carrying out vacuum drying on the filter cake (T=60 ℃ and P= -0.09 MPa).
Comparative example 1
A process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches comprises the following steps:
(1) Extracting by taking 500g of dry aerial parts of Echinacea purpurea, soaking in 10 times of 60% ethanol water solution for 0.5h, and extracting under reflux for 1.5h for the first time; filtering, and reflux extracting the residue with 8 times of 60% ethanol for 1 hr; reflux-extracting the residue with 6 times of 60% ethanol for 1 hr, mixing the filtrates, and concentrating under reduced pressure to 1/3 of the original volume.
(2) Water sedimentation and column pretreatment: transferring the concentrated solution to water at 4 ℃ for 6h. Centrifuging the supernatant fluid after water precipitation (v=5000 r/min, t=10 min), concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration C=2.98 mg/mL, regulating the pH value to be 3.00, and storing at 4 ℃, centrifuging and filtering, wherein the filtrate is used as a loading liquid.
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in absolute ethanol solution for 24h, and activating the resin. Removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, rinsing the resin with purified water until no alcoholic smell.
(4) And (3) purifying by macroporous resin, namely loading at a loading flow rate of 2BV/h, circularly adsorbing for 3h, removing impurities by using purified water with pH=3 and 3BV, eluting and enriching chicoric acid by using a 10% ethanol solution and 6BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.
(5) Ethyl acetate extraction: taking the concentrated solution with the concentration of C=17.19 mg/mL and the pH value of 3.00, and extracting with ethyl acetate with the same volume for 2 times, each time for 40min; the combined organic phases were concentrated to a thick paste under reduced pressure and then transferred to a tray for vacuum drying under reduced pressure (t=50 ℃, p= -0.09 MPa).
(6) Preparing and crystallizing: dissolving the organic phase powder with 100% methanol, preparing the concentration to be about 80mg/mL, centrifuging, carrying out vacuum suction filtration on the supernatant, carrying out sample injection separation, collecting effluent corresponding to a chromatographic peak of 26-39 min according to the time for which chicoric acid appears, concentrating under reduced pressure to the temperature of 4 ℃ after the time for which the effluent is subjected to cooling, carrying out vacuum suction filtration on the crystals, washing the filter cake with purified water at the temperature of 4 ℃, and carrying out vacuum drying on the filter cake (T=60 ℃ and P= -0.10 MPa).
Comparative example 2
A process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches comprises the following steps:
(1) Extracting 500g of dried aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution with pH=2 for 0.5h, and extracting under reflux for 1.5h; filtering, and extracting residues with l2 times of 30% ethanol with pH=2 under reflux for 0.5 hr; the residue after filtration was subjected to a third reflux extraction with l0 times of 30% ethanol at ph=2 for 0.5h, and the 3 filtrates were combined and concentrated under reduced pressure to 1/3 of the original volume.
(2) Water sedimentation and column pretreatment: transferring the concentrated solution to water at 4 ℃ for 12h. Centrifuging the supernatant fluid after water precipitation (v=5000 r/min, t=10 min), concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration C=2.71 mg/mL, regulating the pH value to be 2.99, and storing at 4 ℃, centrifuging and filtering, wherein the filtrate is used as a loading liquid.
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in absolute ethanol solution for 24h, and activating the resin. Removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, rinsing the resin with purified water until no alcoholic smell.
(4) And (3) purifying by macroporous resin, namely loading at a loading flow rate of 2BV/h, circularly adsorbing for 2h, removing impurities by using purified water with pH=3 and 3BV, eluting and enriching chicoric acid by using a 20% ethanol solution and 6BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.
(5) Ethyl acetate extraction: taking the concentrated solution with the concentration of C=17.19 mg/mL and the pH value of 4.00, and extracting with ethyl acetate with the same volume for 2 times, each time for 40min; the combined organic phases were concentrated to a thick paste under reduced pressure and then transferred to a tray for vacuum drying under reduced pressure (t=50 ℃, p= -0.09 MPa).
(6) Preparing and crystallizing: dissolving the chicoric acid coarse powder with 100% methanol to prepare the chicoric acid coarse powder with the concentration of about 80mg/mL, carrying out vacuum suction filtration on supernatant after centrifugation, carrying out sample introduction separation, collecting effluent liquid corresponding to chromatographic peaks at 28-37 min, concentrating the effluent liquid to the temperature of 4 ℃ after decompression, carrying out refrigerator overnight crystallization, carrying out vacuum suction filtration on the effluent liquid, washing a filter cake with purified water at the temperature of 4 ℃, and carrying out vacuum drying on the filter cake (T=60 ℃ and P= -0.10 MPa).
Comparative example 3
A process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches comprises the following steps:
(1) Extracting by taking 500g of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution with pH=3 for 0.5h, and extracting under reflux for 1.5h; filtering, and extracting residues with l2 times of 30% ethanol with pH=3 under reflux for 0.5 hr; the residue after filtration was subjected to a third reflux extraction with l0 times of 30% ethanol at ph=3 for 0.5h, and the 3 filtrates were combined and concentrated under reduced pressure to 1/3 of the original volume.
(2) Water sedimentation and column pretreatment: transferring the concentrated solution to water at 25 ℃ for 6h. Centrifuging the supernatant fluid after water precipitation (v=5000 r/min, t=10 min), concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration C=2.71 mg/mL, regulating the pH value to be 2.99, and storing at 4 ℃, centrifuging and filtering, wherein the filtrate is used as a loading liquid.
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in absolute ethanol solution for 24h, and activating the resin. Removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, rinsing the resin with purified water until no alcoholic smell.
(4) And (3) purifying by macroporous resin, namely loading at a loading flow rate of 2BV/h, circularly adsorbing for 3h, removing impurities by using purified water with pH=3 and 3BV, eluting and enriching chicoric acid by using a 50% ethanol solution and 6BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.
(5) Ethyl acetate extraction: taking the concentrated solution with the concentration of C=17.19 mg/mL and the pH value of 2.00, and extracting with ethyl acetate with the same volume for 3 times, each time for 10min; the combined organic phases were concentrated to a thick paste under reduced pressure and then transferred to a tray for vacuum drying under reduced pressure (t=50 ℃, p= -0.10 MPa).
(6) Preparing and crystallizing: dissolving the chicoric acid coarse powder with 15% methanol to prepare the solution with the concentration of about 17.55mg/mL, carrying out vacuum suction filtration on supernatant after centrifugation, carrying out sample injection separation, collecting effluent liquid corresponding to chromatographic peaks of 40-50 min according to the peak-out time of the chicoric acid, concentrating the effluent liquid to a refrigerator at the temperature of 4 ℃ for overnight after the effluent liquid is decompressed and filtered, washing a filter cake with purified water at the temperature of 4 ℃, and carrying out vacuum drying on the filter cake (T=60 ℃ and P= -0.10 MPa).
Analysis of results
The results (international standard) of examples and comparative examples were measured using the extraction yield and purity of chicoric acid as indicators, respectively, and the measurement results were as follows.
From the results, the chicoric acid content in the obtained echinacea extract product is high, and compared with a comparative example, the whole process has good extraction effect, and compared with comparative example, the invention has better effects in the aspects of chicoric acid extraction, water precipitation process optimization, gradient impurity removal of resin, ethyl acetate extraction process improvement and the like.
Pilot test
As shown in FIG. 1, a process for preparing high-purity L-chicoric acid from Echinacea purpurea in batches comprises the following steps:
(1) Extracting 125kg of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and reflux-extracting for 1.5h for the first time; filtering, and extracting with l2 times of 30% ethanol under reflux for the second time; extracting the filtered medicinal materials with L0 times of 30% ethanol under reflux for 0.5 hr for the third time, mixing the filtrates, and concentrating under reduced pressure to 1604L (first-effect temperature T) 1 =70 ℃, p= -0.048MPa; two-effect temperature T 2 ＝70℃、P＝-0.066MPa)。
(2) Water sedimentation and column pretreatment: transferring the concentrated solution into a precipitation tank, and water-precipitating for 6h at 4 ℃. Centrifuging the water precipitation supernatant at a feed rate of 30-50 mL/s, concentrating under reduced pressure (T=74 ℃, P= -0.062 MPa) to 482L, adjusting pH=3 before loading the concentrated solution, transferring to a cold storage at 4 ℃ for storage, centrifuging and filtering, and taking filtrate as loading solution.
(3) Pretreatment of macroporous adsorption resin: and (3) taking 75Kg of AB-8 type macroporous adsorption resin, soaking the resin in 1.5 times of 95% ethanol solution for 24 hours, and activating the resin. Removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; the activated resin was transferred to a chromatography column and the resin was rinsed with purified water until no alcoholic taste was observed.
(4) Macroporous resin purification, namely converting the diameter height of a stainless steel macroporous adsorption column (h=160 cm, d=25 cm) according to actual conditions of a production workshop and laboratory process parameters, and determining the diameter-to-height ratio of a resin bed to be 1:6; the actual concentration range of the chicoric acid liquid is 1.2-1.7 mg/mL, and the loading volume is adjusted according to the loading concentration under the condition of keeping the loading amount unchanged in consideration of the concentration loss. Loading at the flow rate of 2BV/h, circularly adsorbing for 3h, respectively carrying out gradient impurity removal by using 3BV of purified water with pH=3, 3BV of 10% ethanol solution with pH=3 and 2BV of 20% ethanol solution, eluting enriched chicoric acid by using 6 BV-12 BV of 35% ethanol solution, and concentrating the eluent under reduced pressure until no alcohol smell exists.
(5) Ethyl acetate extraction: taking the concentrated solution (C=11-16 mg/mL, pH=1.91), extracting with ethyl acetate with the same volume for 2 times, and 40min each time; the organic phases were combined and back-extracted 1 time with water of ph=2 for 40min each. The organic phase is concentrated to thick paste under reduced pressure, the ethyl acetate phase is replaced by 95% ethanol, the thick paste is continuously concentrated, and the thick paste is transferred into a tray for vacuum drying under reduced pressure (T=50-60 ℃, P= -0.10 MPa).
(6) High-pressure preparation and crystallization: will be 1010-C 18 Filling stuffing into DAC-150 dynamic axial compression column, dissolving the chicoric acid powder with 15% methanol solution to obtain 15mg/mL medicinal liquid, centrifuging (v=10000 r/min, t=7min), vacuum filtering supernatant, sampling with chicoric acid content of 10g, separating, collecting effluent corresponding to 40-50 min chromatographic peak according to chicoric acid peak time of 6L, vacuum concentrating the effluent to 1/30 of original volume (T=60 ℃, P= -0.10 MPa), crystallizing the concentrated solution in 4 deg.C environment, filtering white or white needle-like and needle-cluster crystals, washing filter cake with 4 deg.C purified water, vacuum drying (T=60 ℃, P= -0.10 MPa) the filter cake to obtain the final product with purity of 98% or aboveThe L-chicoric acid raw material of (2) and the crystallization mother liquor are recrystallized for recovery treatment.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (4)
1. A process method for preparing L-chicoric acid from Echinacea purpurea in batches is characterized by comprising the following steps:
(1) Extracting 500g of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and extracting under reflux for 1.5h; reflux extracting the filtered residue with 12 times and 10 times of 30% ethanol for the second and third times for 0.5 hr, mixing the 3 filtrates, and concentrating under reduced pressure to 1/3 of the original volume to obtain concentrated solution;
(2) Water sedimentation and column pretreatment: transferring the concentrated solution in the step (1) to water sedimentation 6h under the condition of 4 ℃; centrifuging the supernatant fluid of water precipitation, concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration c=2.90 mg/mL under reduced pressure, regulating the pH value to be 3, and transferring to 4 ℃ for storage, and centrifuging and suction filtering to obtain a filtrate as a sample loading liquid; the centrifugation conditions were: v=5000 r/min, t=10 min;
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorption resin in absolute ethanol solution for 24-h to activate the resin; removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, washing the resin with purified water until no alcohol smell exists;
(4) Purifying by macroporous resin: loading at a loading flow rate of 2BV/h, circularly adsorbing 3h, respectively carrying out gradient impurity removal by using 10% ethanol solution 3BV with pH=3 and 3 purified water 3BV with pH=3 and 20% ethanol solution 2BV, eluting enriched chicoric acid by using 35% ethanol solution 10BV, and concentrating the eluent under reduced pressure until no alcohol smell exists;
(5) Ethyl acetate extraction: taking C=13.56 mg/mL and pH=2 concentrated solution in the step (4), and extracting with ethyl acetate with the same volume for 2 times, each time for 40min; the organic phases were combined and back-extracted 1 time with the same volume of water at ph=2 for 40min each; concentrating the organic phase under reduced pressure to obtain thick paste, transferring into a tray, and vacuum drying under reduced pressure to obtain coarse powder of chicoric acid, wherein the specific conditions of vacuum drying under reduced pressure are as follows: t=50 ℃, p= -0.09MPa;
(6) Preparing and crystallizing: dissolving the chicoric acid coarse powder with methanol, centrifuging, carrying out vacuum suction filtration on supernatant, carrying out sample introduction and separation, collecting effluent corresponding to a chromatographic peak of 36-44 min according to the time of the chicoric acid peak, concentrating under reduced pressure to 4 ℃ after the time of standing overnight in a refrigerator for crystallization, filtering, washing a filter cake with purified water at 4 ℃, and carrying out vacuum drying on the filter cake, wherein the specific conditions of vacuum drying are as follows: t=60 ℃, p= -0.10MPa.
2. A process method for preparing L-chicoric acid from Echinacea purpurea in batches is characterized by comprising the following steps:
(1) Extracting 500g of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and extracting under reflux for 1.5h; the filtered residues are subjected to secondary reflux extraction for 0.5h by using 12 times of 30% ethanol; reflux extracting the residue with 10 times of 30% ethanol for 0.5 hr for the third time, mixing the filtrates, and concentrating under reduced pressure to 1/3 of the original volume to obtain concentrated solution;
(2) Water sedimentation and column pretreatment: transferring the concentrated solution in the step (1) to water sedimentation 6h under the condition of 4 ℃; centrifuging the supernatant fluid of water precipitation, concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration c=2.57 mg/mL under the condition that the liquid medicine chicoric acid concentration c=2.57 mg/mL is then adjusted to pH=3, and then transferring the liquid medicine to a condition of 4 ℃ for storage, and centrifuging the filtrate after suction filtration to obtain a sample loading liquid, wherein the centrifuging condition is as follows: v=5000 r/min, t=10 min;
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorption resin in absolute ethanol solution for 24-h to activate the resin; removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, washing the resin with purified water until no alcohol smell exists;
(4) Macroporous resin purification, namely loading at a loading flow rate of 2BV/h, circularly adsorbing 3h, respectively carrying out gradient impurity removal by using 10% ethanol solution 3BV with pH=3 and 3 and 20% ethanol solution 2BV with pH=3, eluting and enriching chicoric acid by using 10BV with 35% ethanol solution, and concentrating the eluent under reduced pressure until no alcohol smell exists;
(5) Ethyl acetate extraction: taking the concentrated solution with the concentration of C=15.15 mg/mL and the pH=1.99 in the step (4), and extracting for 2 times with ethyl acetate with the same volume for 40min each time; the organic phases were combined and back-extracted 1 time with the same volume of water at ph=2 for 40min each; concentrating the organic phase under reduced pressure to obtain thick paste, transferring into a tray, and vacuum drying under reduced pressure to obtain coarse powder of chicoric acid, wherein the specific conditions of vacuum drying under reduced pressure are as follows: t=55 ℃, p= -0.08MPa;
(6) Preparing and crystallizing: dissolving the chicoric acid coarse powder in methanol, centrifuging, carrying out vacuum suction filtration on supernatant, carrying out sample introduction separation, collecting effluent corresponding to 32-42 min chromatographic peaks, concentrating under reduced pressure to a refrigerator at the temperature of 4 ℃ for overnight, filtering crystals, washing filter cakes with purified water at the temperature of 4 ℃, and carrying out vacuum drying on the filter cakes, wherein the specific conditions of the vacuum drying are as follows: t=60 ℃, p= -0.09MPa.
3. A process method for preparing L-chicoric acid from Echinacea purpurea in batches is characterized by comprising the following steps:
(1) Extracting 500g of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and extracting under reflux for 1.5h; the filtered residues are subjected to secondary reflux extraction for 0.5h by using 12 times of 30% ethanol; reflux extracting the residue with 10 times of 30% ethanol for 0.5 hr for the third time, mixing the filtrates, and concentrating under reduced pressure to 1/3 of the original volume to obtain concentrated solution;
(2) Water sedimentation and column pretreatment: transferring the concentrated solution in the step (1) to water sedimentation 6h under the condition of 4 ℃; centrifuging the supernatant fluid of water precipitation, concentrating the filtrate obtained by suction filtration to obtain a liquid medicine chicoric acid concentration c=2.71 mg/mL under reduced pressure, regulating the pH value to be 2.99, and storing at the temperature of 4 ℃, wherein the filtrate is used as a sample loading liquid after centrifugal suction filtration; the centrifugation conditions were: v=5000 r/min, t=10 min;
(3) Pretreatment of macroporous adsorption resin: soaking AB-8 type macroporous adsorption resin in absolute ethanol solution for 24-h to activate the resin; removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatography column allows the resin bed diameter to height ratio 1:10, washing the resin with purified water until no alcohol smell exists;
(4) Macroporous resin purification, namely loading at a loading flow rate of 2BV/h, circularly adsorbing 3h, respectively carrying out gradient impurity removal by using 10% ethanol solution 6BV with pH=3 and 3 and 20% ethanol solution 2BV with pH=3, eluting and enriching chicoric acid by using 35% ethanol solution 9BV, and concentrating the eluent under reduced pressure until no alcohol smell exists;
(5) Ethyl acetate extraction: taking the concentrated solution with the concentration of C=13.34 mg/mL and the pH value of 2.00 in the step (4), and extracting for 2 times with ethyl acetate with the same volume for 40min each time; the organic phases were combined and back-extracted 1 time with the same volume of water at ph=2 for 40min each; concentrating the organic phase under reduced pressure to obtain thick paste, transferring into a tray, and vacuum drying under reduced pressure, wherein the specific conditions of the vacuum drying under reduced pressure are as follows: t=50 ℃, p= -0.09MPa;
(6) Preparing and crystallizing: dissolving the organic phase powder with 15% methanol to prepare a supernatant with the concentration of about 28mg/mL, carrying out vacuum suction filtration and sample injection separation on the supernatant after centrifugation, collecting effluent corresponding to a chromatographic peak of 26-36 min according to the time for which chicoric acid appears, concentrating the effluent under reduced pressure to the temperature of 4 ℃ after the effluent is cooled to a refrigerator for standing overnight, carrying out vacuum filtration on crystals, washing a filter cake with purified water at the temperature of 4 ℃, and carrying out vacuum drying on the filter cake, wherein the specific conditions of vacuum drying are as follows: t=60 ℃, p= -0.09MPa.
4. A process method for preparing L-chicoric acid from Echinacea purpurea in batches is characterized by comprising the following steps:
(1) Extracting 125kg of dry aerial parts of Echinacea purpurea, soaking in 14 times of 30% ethanol water solution for 0.5h, and reflux-extracting for 1.5h for the first time; filtering, and extracting with 12 times of 30% ethanol under reflux for the second time; carrying out third reflux extraction on the filtered medicinal materials by using 10 times of 30% ethanol, wherein the second reflux extraction time and the third reflux extraction time are both 0.5h, combining 3 filtrates to obtain filtrate with total volume of 4296L, and concentrating under reduced pressure by a double-effect energy-saving concentrating evaporator to 1604L; the specific conditions of the decompression concentration of the double-effect energy-saving concentration evaporator are as follows: first-effect temperature T 1 =70 ℃, p= -0.048MPa; two-effect temperature T 2 =70℃、P=-0.066MPa；
(2) Water sedimentation and column pretreatment: transferring the concentrated solution prepared in the step (1) into a precipitation tank to sink 6h in water at 4 ℃; centrifuging the water precipitation supernatant at a feeding speed of 30-50 mL/s, concentrating to 482L under reduced pressure, adjusting pH=3 before loading the concentrated solution, transferring to a cold storage at 4 ℃ for storage, centrifuging, and filtering to obtain filtrate as loading solution; the specific conditions of the reduced pressure concentration are as follows: t=74 ℃, p= -0.062MPa;
(3) Pretreatment of macroporous adsorption resin: taking 75Kg of AB-8 type macroporous adsorption resin, soaking the resin 24 and h in 1.5 times of 95% ethanol solution, and activating the resin; removing the turbid matters floating on the ethanol in time in the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatographic column, and washing the resin with purified water until no alcohol smell exists;
(4) Macroporous resin purification, namely converting the diameter height of the stainless steel macroporous adsorption column according to the actual conditions of a production workshop and the technological parameters of a laboratory, and determining the diameter-to-height ratio of a resin bed to be 1:6; the actual concentration range of the chicoric acid in the liquid medicine is 1.2-1.7 mg/mL, the sample loading is carried out at the flow rate of 2BV/h, the 3h is circularly adsorbed, the 3BV of purified water with the pH value of=3, the 3BV of 10 percent ethanol solution with the pH value of=3 and the 2BV of 20 percent ethanol solution are respectively used for gradient impurity removal, the 6 BV-12 BV of 35 percent ethanol solution is used for eluting and enriching the chicoric acid, and the eluent is decompressed and concentrated until no alcohol smell exists; the stainless steel macroporous adsorption column h=160 cm, d=25 cm;
(5) Ethyl acetate extraction: taking the concentrated solution with C=11-16 mg/mL and pH=1.91 in the step (4), and extracting for 2 times with ethyl acetate with the same volume for 40min each time; the organic phases were combined and back-extracted 1 time with the same volume of water at ph=2 for 40min each; concentrating the organic phase under reduced pressure to obtain a thick paste, replacing the ethyl acetate phase with 95% ethanol, continuously concentrating the thick paste, transferring the thick paste into a tray, and drying under reduced pressure and vacuum under the specific conditions that: t=50 to 60 ℃, p= -0.10MPa;
(6) High-pressure preparation and crystallization: will be 1010-C 18 Filling into DAC-150 dynamic axial compression column, dissolving the chicoric acid powder with 15% methanol solution to obtain medicinal liquid with concentration of 15mg/mL, centrifuging under v=10000 r/min and t=7min, vacuum filtering supernatant, sampling with chicoric acid content of 10g, and separating according to the peak time of chicoric acidCollecting effluent liquid 6L corresponding to chromatographic peaks of 40-50 min, and concentrating the effluent liquid to 1/30 of the original volume under reduced pressure; the conditions of the reduced pressure concentration are as follows: t=60 ℃, p= -0.10MPa, the concentrated solution is transferred to 4 ℃ environment for crystallization, white or quasi-white needle-like and needle cluster-like body crystals are filtered, and the filter cake is washed by 4 ℃ purified water and dried in vacuum, the specific conditions of the vacuum drying are as follows: and (3) carrying out recrystallization on the crystallization mother liquor to obtain the L-chicoric acid raw material with the purity of more than 98 percent, wherein T=60 ℃ and P= -0.10MPa.
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