阅读说明：本技术 一种从紫锥菊中批量制备高纯度l-菊苣酸的工艺及应用 (Process for preparing high-purity L-chicoric acid from echinacea purpurea in batches and application ) 是由 李峰 冯帅 李健 张天锡 杨敏 于 2021-11-04 设计创作，主要内容包括：本发明提供一种从紫锥菊中批量制备高纯度L-菊苣酸的工艺及其应用,属于中药分离纯化精制技术领域。本发明通过优化工艺方案,从而进行醇提水沉处理,柱层析及乙酸乙酯萃取除杂,制备液相分离及结晶操作即可大批量获得纯度98％以上的L-菊苣酸原料,非常适合工业化大规模生产,因此具有良好的实际应用之价值。(The invention provides a process for preparing high-purity L-chicoric acid from echinacea purpurea in batches and application thereof, belonging to the technical field of separation, purification and refining of traditional Chinese medicines. According to the invention, by optimizing the process scheme, the L-chicoric acid raw material with the purity of more than 98% can be obtained in a large batch by carrying out alcohol extraction and water precipitation treatment, column chromatography and ethyl acetate extraction impurity removal, 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.)

1. A process for preparing high-purity L-chicoric acid from echinacea purpurea in batches, which is characterized by comprising the following steps: taking dry aerial parts of echinacea purpurea as raw materials, carrying out alcohol extraction and water precipitation treatment, carrying out column chromatography and ethyl acetate extraction for impurity removal, and carrying out liquid phase separation and crystallization operation.

2. The process of claim 1 wherein each of the steps of the process is protected from light.

3. The process of claim 1, wherein the process comprises:

extraction: drying aerial parts of Echinacea purpurea, extracting with ethanol water solution under reflux for several times, mixing filtrates, and concentrating to obtain concentrated solution;

water precipitation and column pretreatment: carrying out water precipitation treatment on the concentrated solution to obtain a supernatant of the water precipitation, centrifuging, and concentrating and filtering the centrifugate to obtain a sample solution for later use;

and (3) macroporous resin purification: packing columns by a wet method; fixing the sample loading quantity to be unchanged, loading the liquid medicine at the flow rate of 1-3 BV/h, circularly adsorbing for 2-3 h, performing 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 and enriching chicoric acid by using 30-35% ethanol solution for 6-12 BV, and collecting eluent for decompression and concentration;

and (3) ethyl acetate extraction: extracting 30-35% of a concentrated solution with the concentration of 10-18 mg/mL of C and the pH of 1-3 with ethyl acetate; extracting organic phase with acidic water, concentrating organic phase under reduced pressure to obtain soft extract, replacing ethyl acetate phase with high purity ethanol, concentrating to obtain soft extract, and drying;

high pressure preparation of liquid phase and crystallization: dissolving coarse chicoric acid powder in 10-20% methanol solution, centrifuging, performing vacuum filtration on the supernatant, performing sample injection separation, collecting the effluent corresponding to a chromatographic peak for 30-50 min according to the chicoric acid peak time, performing vacuum concentration and low-temperature crystallization, filtering, washing and drying to obtain the cichoric acid.

4. The process method as claimed in claim 3, wherein in the extraction step, 20-40% (preferably 30%) ethanol aqueous solution is used for reflux extraction for 2-4 times, preferably 3 times, the material-liquid ratio is controlled to be 1:8-15, and the extraction time is controlled to be 0.1-2h, preferably 0.5-1.5 h.

5. The process method as claimed in claim 3, wherein in the step of pre-treatment of the water precipitation and column, the specific method of water precipitation treatment comprises: performing water sedimentation treatment for 5-8h, preferably 6h, at low temperature; the centrate is concentrated to an original volume of 1/2-1/4, preferably 1/3.

6. The process method according to claim 3, wherein in the macroporous resin purification step, the column is filled by a wet method so that the ratio of the diameter to the height of a resin bed is 1: 5-1: 10.

7. the process according to claim 3, wherein in the ethyl acetate extraction step, the same volume of ethyl acetate is used for 2-3 times, preferably 2 times; the high-purity ethanol is ethanol with the concentration of not less than 90%.

8. The process of claim 7, wherein the drying is performed by vacuum drying under reduced pressure, preferably at 50-60 ℃ and-0.07-0.10 MPa.

9. The process of claim 3, wherein in the steps of preparing the liquid phase under high pressure and crystallizing, the specific steps of concentrating under reduced pressure and crystallizing at low temperature, filtering, washing and drying comprise: concentrating under reduced pressure to 1/30-1/40 of the original volume, crystallizing for 4-10 h in a low-temperature environment, filtering white or off-white needle-like and needle cluster crystals, and repeatedly washing a filter cake with low-temperature water; and drying the filter cake to obtain the filter cake.

10. Use of a process according to any one of claims 1 to 9 for the industrial preparation of L-chicoric acid.

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 of the process.

Background

The information disclosed in this 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 acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

In the process of global prevention and treatment of new coronavirus, traditional Chinese medicine plays an increasingly important role in antivirus and is favored by people at home and abroad. The RSV, which belongs to respiratory tract virus together with the novel coronavirus, is a common pathogen of respiratory tract infection, is a fierce of diseases such as infant pneumonia, senile pneumonia, asthma and the like, and an effective treatment medicament is lacked at present, while a representative medicament ribavirin on the market at present has large side effect, and the safety and the curative effect of the ribavirin are still required to be further examined. The traditional Chinese medicine extract, namely the chicoric acid, has the advantages of remarkable anti-RSV curative effect, small toxic and side effect, high safety, clear in-vivo and in-vitro anti-RSV action mechanism and wide prospect in the development of anti-RSV novel candidate drugs, but the yield and purity of the chicoric acid extraction process in the existing commercially available echinacea extract are lower, and the development requirement of preparing anti-RSV new drugs by taking the chicoric acid as a raw material cannot be met.

At present, chicoric acid is mainly used in two industries of medicine raw materials and health-care food, while the content of the chicoric acid in the commercially available echinacea extract is low, and a production process for preparing high-purity chicoric acid in a large scale by taking echinacea as a raw material is not reported so far, so that the further development and utilization of the chicoric acid are limited to a certain extent. Therefore, the scheme for optimizing the preparation process of the cichoric acid monomer, which is economic, environment-friendly, low in cost and suitable for large-scale industrial production, has important significance.

In order to obtain high-purity cichoric acid monomers, a large number of experimental studies are carried out by scholars at home and abroad. 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 preparative chromatography, but the method is not easy to enlarge production, has high cost and toxic and harmful reagents, and can only be used for producing standard products but not be used for industrial production; the high purity chicoric acid product was obtained by treating Echinacea purpurea with chloroform, extracting with methanol and ethyl acetate, and purifying with acetonitrile reverse phase chromatography, as reported by Beijing university of chemistry (patent No: CN 1587251). The processChloroform and methanol used in the method are harmful to the environment and easy to remain, and the acetonitrile reverse phase chromatographic column has low production efficiency and high cost; the extraction solvent and the recrystallization solvent adopted by the Xiange information technology company Limited (patent number: CN109748796A) are methanol and acetone respectively, and have certain toxicity; the reports of the university of Hunan (patent number: CN102161620A) report that chicoric acid with the content of 25-40 percent can be obtained by adopting an organic solvent for ultrasonic extraction and drying and purifying by using chloroform, and the like, the purity is lower, and the ultrasonic treatment method is not suitable for large-scale industrial production; guilin Sanbao pharmaceutical industry Co., Ltd (patent No. CN105294440A) adopts silica gel column chromatography for many times, and uses methanol and acetone for elution, so that the operation steps are complicated, 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 processes reported by the university of Chinese pharmacy (patent number: CN104693034A), Sinkiang Terry biotechnology limited (patent number: CN103641716A) and Hunan Lanolin biological resource GmbH (patent number: CN111233950A) is lower (less than 85%); shenyang Shuangding science and technology Limited (patent numbers: CN102249917A, CN 102391118A) reports a method for extracting, separating and identifying chicoric acid, which is used for content determination and quality control of the picrorhiza scrophulariiflora Pennell medicinal material and preparation. Nanjing Zeron pharmaceutical science and technology Co., Ltd (patent No.: CN102060706A) reported the use of supercritical CO₂And the entrainer is used for extraction, ion exchange and other separation and purification methods to prepare the high-content chicoric acid, so the cost is higher, and the method is not suitable for industrial production. Wuqi Lin (patent No. CN1587251A) process uses Echinacea purpurea root powder with high content of chicoric acid as raw material, although fat-soluble impurities such as chlorophyll and the like in the extract are less, the process is not beneficial to the sustainable utilization of resources; although the extraction efficiency can be improved by crushing and extracting the medicinal materials, the production process is increased, and dust is easy to fly, so that the workshop environment and safety are affected. When the pH is 3, the chicoric acid is stable, the pH of the liquid medicine is adjusted to 1-3 in the acidification treatment process, the pH fluctuation is large, the stability of the process is poor, the degradation of the chicoric acid and the precipitation conditions of impurities and the like are different in the experiment processes of different batches, and the quality control is not convenient. The ethyl acetate is extracted for 3 times, each time is 2 hours, the extraction times are more, the extraction time is long, the solvent dosage is large, the extraction efficiency is influenced, and the solvent dosage is increasedAdding economic cost. In the column chromatography process, only deionized water is used for removing water-soluble impurities, and the impurities are not completely removed; then, 10-50% ethanol is used as eluent to enrich the chicoric acid, the eluents with different concentrations have different elution capacities on the chicoric acid and impurities, the volume change of the eluent consumed for enriching the chicoric acid is large, and the purity of the chicoric acid in the eluent is changed, so that the reproducibility and the quality stability of the product are not favorably controlled. The chicoric 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 production cost. Although the chicoric acid with high purity can be obtained by adopting the process, the types and the contents of impurities contained in the extract can be changed due to different echinacea raw materials, so that the processes and the effects of the separation and purification process can be different. The Hunan university (patent No. CN109678706A) discloses a chemical synthesis method for preparing L-chicoric acid crystal form, wherein the X-ray powder diffraction pattern of the crystal form shows diffraction at 6.36 degrees, 12.60 degrees, 19.24 degrees, 25.80 degrees, 32.46 degrees and 36.32 degrees, however, the crystal form of chicoric acid extracted from plants is 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 in batches from plant echinacea purpurea and application thereof.

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, said process comprising: taking dry aerial parts of echinacea purpurea as raw materials, carrying out alcohol extraction and water precipitation treatment, carrying out column chromatography and ethyl acetate extraction for impurity removal, and carrying out liquid phase separation and crystallization operation.

In the case of chicoric acid, the stability of chicoric acid is damaged due to the influence of light, heat and the like, and therefore, the whole operation steps are preferably protected from light, so that the damage of the ingredients in the preparation process is reduced.

Specifically, the process method comprises the following steps:

extraction: drying aerial parts of Echinacea purpurea, extracting with ethanol water solution under reflux for several times, mixing filtrates, and concentrating to obtain concentrated solution;

water precipitation and column pretreatment: carrying out water precipitation treatment on the concentrated solution to obtain a supernatant of the water precipitation, centrifuging, and concentrating and filtering the centrifugate to obtain a sample solution for later use;

and (3) macroporous resin purification: packing columns by a wet method; fixing the sample loading quantity to be unchanged, loading the liquid medicine at the flow rate of 1-3 BV/h, circularly adsorbing for 2-3 h, performing 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 and enriching chicoric acid by using 30-35% ethanol solution for 6-12 BV, and collecting eluent for decompression and concentration;

and (3) ethyl acetate extraction: extracting 30-35% of a concentrated solution with the concentration of 10-18 mg/mL of C and the pH of 1-3 with ethyl acetate; extracting organic phase with acidic water, concentrating organic phase under reduced pressure to obtain soft extract, replacing ethyl acetate phase with high purity ethanol, concentrating to obtain soft extract, and drying;

high pressure preparation of liquid phase and crystallization: dissolving coarse chicoric acid powder in 10-20% methanol solution, centrifuging, performing vacuum filtration on the supernatant, performing sample injection separation, collecting the effluent corresponding to a chromatographic peak for 30-50 min according to the chicoric acid peak time, performing vacuum concentration and low-temperature crystallization, filtering, washing and drying to obtain the cichoric acid.

In a second aspect of the present invention, there is provided the use of the above process for the industrial preparation of L-chicoric acid.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme provides a process for preparing high-purity L-chicoric acid in batches from echinacea purpurea, and the process scheme is optimized, so that the L-chicoric acid raw material with the purity of more than 98 percent can be obtained in batches by carrying out alcohol extraction and water precipitation treatment, column chromatography and ethyl acetate extraction impurity removal, and preparation liquid phase separation and crystallization operations, and the process is very suitable for industrial large-scale production, and has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a process flow diagram of the present invention;

FIG. 2 is a graph of the characterization of the crystal form of L-chicoric acid obtained in example 1 by X-ray powder diffraction according to the present invention;

FIG. 3 is a representation of the crystal form of L-chicoric acid obtained in example 1 according to the present invention by differential scanning calorimetry;

FIG. 4 is a graph representing the crystal form of L-chicoric acid obtained in example 1 by thermogravimetric analysis according to the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

In an exemplary embodiment 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: taking dry aerial parts of echinacea purpurea as raw materials, carrying out alcohol extraction and water precipitation treatment, carrying out column chromatography and ethyl acetate extraction for impurity removal, and carrying out liquid phase separation and crystallization operation.

In the case of chicoric acid, the stability of chicoric acid is damaged due to the influence of light, heat and the like, and therefore, the whole operation steps are preferably protected from light, so that the damage of the ingredients in the preparation process is reduced.

In another embodiment of the present invention, the process comprises:

extraction: drying aerial parts of Echinacea purpurea, extracting with ethanol water solution under reflux for several times, mixing filtrates, and concentrating to obtain concentrated solution;

water precipitation and column pretreatment: carrying out water precipitation treatment on the concentrated solution to obtain a supernatant of the water precipitation, centrifuging, and concentrating and filtering the centrifugate to obtain a sample solution for later use;

and (3) macroporous resin purification: packing columns by a wet method; fixing the sample loading quantity to be unchanged, loading the liquid medicine at the flow rate of 1-3 BV/h, circularly adsorbing for 2-3 h, performing 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 and enriching chicoric acid by using 30-35% ethanol solution for 6-12 BV, and collecting eluent for decompression and concentration;

and (3) ethyl acetate extraction: extracting 30-35% of a concentrated solution with the concentration of 10-18 mg/mL of C and the pH of 1-3 with ethyl acetate; extracting organic phase with acidic water, concentrating organic phase under reduced pressure to obtain soft extract, replacing ethyl acetate phase with high purity ethanol, concentrating to obtain soft extract, and drying;

high pressure preparation of liquid phase and crystallization: dissolving coarse chicoric acid powder in 10-20% methanol solution, centrifuging, performing vacuum filtration on the supernatant, performing sample injection separation, collecting the effluent corresponding to a chromatographic peak for 30-50 min according to the chicoric acid peak time, performing vacuum concentration and low-temperature crystallization, filtering, washing and drying to obtain the cichoric acid.

In another embodiment of the present invention, in the extracting step, 20-40% (preferably 30%) ethanol aqueous solution is used for reflux extraction for 2-4 times, preferably 3 times, with a feed-liquid ratio of 1:8-15, such as 1:10, 1:12, 1:14, and an extraction time of 0.1-2h, further 0.5-1.5 h; in one embodiment of the present invention, the extracting step comprises: drying aerial parts of Echinacea purpurea, extracting with 30% ethanol water solution under reflux for 3 times at material-to-liquid ratio of 1:14, 1:12, and 1:10 for 1.5 hr, 0.5 hr, and 0.5 hr, mixing the 3 filtrates, and concentrating under reduced pressure to 1/3.

In another embodiment of the present invention, in the step of pre-treating the water-settling and column, the water-settling treatment method includes: performing water precipitation treatment at low temperature (such as 4 deg.C) for 5-8 hr, preferably 6 hr; the centrate is concentrated to an original volume of 1/2-1/4, preferably 1/3;

in another embodiment of the invention, water precipitation is adopted to remove part of fat-soluble impurities, and after the treatment process, a large amount of yellow-green precipitates appear, so that the impurity removal effect is obvious, and the environment is protected and economical.

In another embodiment of the present invention, in the step of purifying the macroporous resin, the column is packed by a wet method so that the ratio of the diameter to the height of the resin bed is 1: 5-1: 10;

in another embodiment of the present invention, in the ethyl acetate extraction step, the same volume of ethyl acetate is used for 2-3 times, preferably 2 times; the high-purity ethanol is ethanol with the concentration of not less than 90 percent, such as 95 percent ethanol;

the drying is carried out by vacuum drying under reduced pressure, 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 show that water-soluble impurities are further removed by column chromatography, and then the purification effect of ethyl acetate extraction is obviously superior to that of extraction and column chromatography purification. In the column chromatography process, water, 10% ethanol and 20% ethanol are adopted for gradient impurity removal, impurities with different polarities are removed in a grading mode, the chicoric acid is enriched by 30-35% ethanol with a more specific ethanol concentration range, and the purification effect is good. And water back extraction is adopted in the ethyl acetate extraction process to further remove water-soluble impurities in the ethyl acetate phase, so that the purity of the chicoric acid is improved by 2-5%, and the experimental process has continuity, and is more suitable for industrial production.

In the steps of preparing the liquid phase at high pressure and crystallizing, the specific method of decompression concentration and low-temperature crystallization, filtration, washing and drying comprises the following steps: concentrating under reduced pressure to 1/30-1/40 of the original volume, crystallizing for 4-10 h at low temperature (such as 4 ℃), filtering white or off-white needle-like and needle cluster-like crystals, and repeatedly washing a filter cake with low temperature (such as 4 ℃) water (purified water); and drying the filter cake (in vacuum) to obtain the filter cake.

It is noted that, in the process of preparing liquid phase purified chicoric acid, although the process cost is relatively high, the purity of the chicoric acid coarse powder can be above 80% through further purification by 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 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 still another embodiment of the present invention, there is provided the use of the above process for 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 thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out 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 for 0.5h by using 14 times of 30% ethanol aqueous solution, and carrying out first heating reflux extraction for 1.5 h; after filtration, the dregs are subjected to reflux extraction for the second time and the third time (0.5 h respectively) by 30% ethanol with the amount which is equal to l2 times and l0 times, and 3 times of filtrates are combined and concentrated under reduced pressure to 1/3 with the original volume.

(2) Water precipitation and column pretreatment: the concentrated solution is transferred to 4 ℃ for precipitation in water for 6 h. Centrifuging the supernatant after water precipitation (v is 5000r/min, t is 10min), concentrating the filtrate under reduced pressure until the concentration c of chicoric acid in the liquid medicine is 2.90mg/mL, adjusting the pH value to 3, transferring to 4 ℃, storing, centrifuging, filtering, and taking the filtrate as the supernatant.

(3) Pretreating macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in anhydrous ethanol solution for 24 hr to activate the resin. Removing the turbid matter floating on the ethanol in time during the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatographic column to ensure that the height ratio of the resin bed diameter to the height is 1:10, washing the resin with purified water until no alcohol smell exists.

(4) And (3) macroporous resin purification, namely sampling at the sampling flow rate of 2BV/h, circularly adsorbing for 3h, performing gradient impurity removal by using 10% ethanol solution 3BV with the pH value of 3 and 10% ethanol solution 2BV with the pH value of 3 respectively, eluting and enriching chicoric acid by using 35% ethanol solution 10BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.

(5) And (3) ethyl acetate extraction: extracting the concentrated solution with C of 13.56mg/mL and pH of 2 with ethyl acetate of the same volume for 40min for 2 times; the organic phases were combined and back-extracted 1 time 40min with the same volume of water at pH 2. Concentrating the organic phase under reduced pressure to obtain soft extract, transferring into tray, and vacuum drying under reduced pressure (T50 deg.C, P-0.09 MPa).

(6) Preparation and crystallization: dissolving the coarse powder of the chicoric acid in methanol, centrifuging, performing vacuum filtration on supernate, performing sample injection and separation, collecting an effluent corresponding to a chromatographic peak for 36-44 min according to the peak emergence time of the chicoric acid, concentrating the effluent under reduced pressure until the effluent is crystallized in a refrigerator at 4 ℃ overnight, filtering, washing a filter cake with purified water at 4 ℃, and performing vacuum drying on the filter cake (T is 60 ℃, P is-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 for 0.5h by using 14 times of 30% ethanol aqueous solution, and carrying out first heating reflux extraction for 1.5 h; carrying out secondary reflux extraction on the filtered medicine residues by using 30 percent ethanol which is 2 times of the amount of the medicine residues for 0.5 hour; the filtered dregs are subjected to reflux extraction for the third time for 0.5h by 30 percent ethanol which is the same as the amount of the filtered dregs in l0 times, and 3 times of filtrates are combined and concentrated to 1/3 of the original volume under reduced pressure.

(2) Water precipitation and column pretreatment: the concentrated solution is transferred to 4 ℃ for precipitation in water for 6 h. Centrifuging the supernatant after water precipitation (v is 5000r/min, t is 10min), concentrating the filtrate under reduced pressure until the concentration c of chicoric acid in the liquid medicine is 2.57mg/mL, adjusting the pH value to 3, transferring to 4 ℃, storing, centrifuging, filtering, and taking the filtrate as the supernatant.

(3) Pretreating macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in anhydrous ethanol solution for 24 hr to activate the resin. Removing the turbid matter floating on the ethanol in time during the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatographic column to ensure that the height ratio of the resin bed diameter to the height is 1:10, washing the resin with purified water until no alcohol smell exists.

(4) And (3) macroporous resin purification, namely sampling at the sampling flow rate of 2BV/h, circularly adsorbing for 3h, performing gradient impurity removal by using 10% ethanol solution 3BV with the pH value of 3 and 10% ethanol solution 2BV with the pH value of 3 respectively, eluting and enriching chicoric acid by using 35% ethanol solution 10BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.

(5) And (3) ethyl acetate extraction: extracting the concentrated solution with C15.15 mg/mL and pH 1.99 with ethyl acetate of the same volume for 40min for 2 times; the organic phases were combined and back-extracted 1 time 40min with the same volume of water at pH 2. Concentrating the organic phase under reduced pressure to obtain soft extract, transferring into tray, and vacuum drying under reduced pressure (T ═ 55 deg.C, P ═ 0.08 MPa).

(6) Preparation and crystallization: dissolving the coarse chicoric acid powder in methanol, centrifuging, performing vacuum filtration on a supernatant, performing sample separation, collecting an effluent corresponding to a chromatographic peak for 32-42 min, concentrating under reduced pressure to a temperature of 4 ℃ for overnight in a refrigerator, filtering crystals, washing a filter cake with purified water at 4 ℃, and performing vacuum drying on the filter cake (the temperature is 60 ℃ and the pressure is-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 for 0.5h by using 14 times of 30% ethanol aqueous solution, and carrying out first heating reflux extraction for 1.5 h; carrying out secondary reflux extraction on the filtered medicine residues by using 30 percent ethanol which is 2 times of the amount of the medicine residues for 0.5 hour; the filtered dregs are subjected to reflux extraction for the third time for 0.5h by 30 percent ethanol which is the same as the amount of the filtered dregs in l0 times, and 3 times of filtrates are combined and concentrated to 1/3 of the original volume under reduced pressure.

(2) Water precipitation and column pretreatment: the concentrated solution is transferred to 4 ℃ for precipitation in water for 6 h. Centrifuging the supernatant after water precipitation (v is 5000r/min, t is 10min), filtering, concentrating the filtrate under reduced pressure until the concentration c of chicoric acid in the liquid medicine is 2.71mg/mL, adjusting the pH value to 2.99, transferring to 4 ℃, storing, centrifuging, filtering, and taking the filtrate as a supernatant.

(3) Pretreating macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in anhydrous ethanol solution for 24 hr to activate the resin. Removing the turbid matter floating on the ethanol in time during the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatographic column to ensure that the height ratio of the resin bed diameter to the height is 1:10, washing the resin with purified water until no alcohol smell exists.

(4) And (3) macroporous resin purification, namely sampling at the sampling flow rate of 2BV/h, circularly adsorbing for 3h, performing gradient impurity removal by using 10 percent ethanol solution with the pH value of 3 and 6BV and 20 percent ethanol solution with the pH value of 3 and 2BV respectively, eluting and enriching chicoric acid by using 35 percent ethanol solution with the pH value of 9BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.

(5) And (3) ethyl acetate extraction: collecting the above concentrated solution C13.34 mg/mL and pH 2.00), and extracting with the same volume of ethyl acetate for 2 times each for 40 min; the organic phases were combined and back-extracted 1 time 40min with the same volume of water at pH 2. Concentrating the organic phase under reduced pressure to obtain soft extract, transferring into tray, and vacuum drying under reduced pressure (T50 deg.C, P-0.09 MPa).

(6) Preparation and crystallization: dissolving the organic phase powder with 15% methanol, preparing the concentration of the solution to be about 28mg/mL, centrifuging, performing vacuum filtration on the supernate, injecting and separating, collecting the effluent corresponding to a chromatographic peak for 26-36 min according to the peak emergence time of chicoric acid, performing vacuum concentration on the effluent until the effluent is kept in a refrigerator at 4 ℃ for standing overnight, performing vacuum filtration on crystals, washing a filter cake with purified water at 4 ℃, and performing vacuum drying on the filter cake (T is 60 ℃ and P is-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 soaking 500g of dry aerial parts of Echinacea purpurea with 10 times of 60% ethanol aqueous solution for 0.5 hr, and extracting under reflux for the first time for 1.5 hr; carrying out secondary reflux extraction on the filtered dregs of a decoction for 1 hour by using 8 times of 60% ethanol; and carrying out reflux extraction on the filtered residue for the third time for 1h by using 6 times of 60% ethanol, combining 3 times of filtrates, and concentrating under reduced pressure to 1/3 of the original volume.

(2) Water precipitation and column pretreatment: the concentrated solution is transferred to 4 ℃ for precipitation in water for 6 h. Centrifuging the supernatant after water precipitation (v is 5000r/min, t is 10min), filtering, concentrating the filtrate under reduced pressure until the concentration of chicoric acid in the liquid medicine C is 2.98mg/mL, adjusting the pH value to 3.00, transferring to 4 ℃, storing, centrifuging, filtering, and collecting the filtrate as the supernatant.

(3) Pretreating macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in anhydrous ethanol solution for 24 hr to activate the resin. Removing the turbid matter floating on the ethanol in time during the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatographic column to ensure that the height ratio of the resin bed diameter to the height is 1:10, washing the resin with purified water until no alcohol smell exists.

(4) And (3) macroporous resin purification, namely sampling at the sampling flow rate of 2BV/h, circularly adsorbing for 3h, removing impurities by using purified water with the pH value of 3 for 3BV, eluting and enriching chicoric acid by using 10 percent ethanol solution with the pH value of 6BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.

(5) And (3) ethyl acetate extraction: extracting the concentrated solution with C17.19 mg/mL and pH 3.00 with ethyl acetate of the same volume for 40min for 2 times; the organic phases are combined and concentrated under reduced pressure to be thick paste, and then transferred to a tray for vacuum drying under reduced pressure (T is 50 ℃, P is-0.09 MPa).

(6) Preparation and crystallization: dissolving the organic phase powder with 100% methanol, preparing the concentration of the solution to be about 80mg/mL, centrifuging, performing vacuum filtration on the supernate, injecting and separating, collecting the effluent corresponding to a chromatographic peak of 26-39 min according to the peak emergence time of chicoric acid, concentrating the effluent under reduced pressure until the effluent is crystallized in a refrigerator at 4 ℃ overnight, performing vacuum filtration on the crystals, washing the filter cake with purified water at 4 ℃, and performing vacuum drying on the filter cake (T is 60 ℃ and P is-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 dry aerial parts of echinacea purpurea, soaking in 14 times of 30% ethanol aqueous solution with pH of 2 for 0.5h, and extracting under reflux for 1.5 h; filtering, and extracting the residue with l2 times of 30% ethanol with pH of 2 under reflux for 0.5 hr; the filtered residue was subjected to a third reflux extraction with l0 times of 2-pH 30% ethanol for 0.5h, and the 3 filtrates were combined and concentrated under reduced pressure to 1/3 of the original volume.

(2) Water precipitation and column pretreatment: the concentrated solution is transferred to 4 ℃ for water precipitation for 12 h. Centrifuging the supernatant after water precipitation (v is 5000r/min, t is 10min), filtering, concentrating the filtrate under reduced pressure until the concentration of chicoric acid in the liquid medicine C is 2.71mg/mL, adjusting the pH value to 2.99, transferring to 4 ℃, storing, centrifuging, filtering, and collecting the filtrate as the supernatant.

(3) Pretreating macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in anhydrous ethanol solution for 24 hr to activate the resin. Removing the turbid matter floating on the ethanol in time during the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatographic column to ensure that the height ratio of the resin bed diameter to the height is 1:10, washing the resin with purified water until no alcohol smell exists.

(4) And (3) macroporous resin purification, namely, sampling at the sampling flow rate of 2BV/h, circularly adsorbing for 2h, removing impurities by using purified water with the pH value of 3 for 3BV, eluting and enriching chicoric acid by using 20 percent ethanol solution with the pH value of 6BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.

(5) And (3) ethyl acetate extraction: extracting the concentrated solution with C17.19 mg/mL and pH 4.00 with ethyl acetate of the same volume for 40min for 2 times; the organic phases are combined and concentrated under reduced pressure to be thick paste, and then transferred to a tray for vacuum drying under reduced pressure (T is 50 ℃, P is-0.09 MPa).

(6) Preparation and crystallization: dissolving the coarse powder of the chicoric acid in 100% methanol, preparing the solution with the concentration of about 80mg/mL, centrifuging, performing vacuum filtration on a supernatant, performing sample separation, collecting an effluent corresponding to a chromatographic peak for 28-37 min, concentrating the effluent under reduced pressure until the effluent is crystallized in a refrigerator at 4 ℃ overnight, performing vacuum filtration, washing a filter cake with purified water at 4 ℃, and performing vacuum drying on the filter cake (the temperature is 60 ℃ and the pressure is-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 soaking 500g dried aerial parts of Echinacea purpurea with 14 times of 30% ethanol aqueous solution with pH of 3 for 0.5 hr, and extracting under reflux for 1.5 hr; filtering, and extracting the residue with l2 times of 30% ethanol with pH of 3 under reflux for 0.5 hr; the filtered residue was subjected to a third reflux extraction with l0 times of 3-pH 30% ethanol for 0.5h, and the 3 filtrates were combined and concentrated under reduced pressure to 1/3 of the original volume.

(2) Water precipitation and column pretreatment: the concentrated solution is transferred to 25 ℃ for precipitation in water for 6 h. Centrifuging the supernatant after water precipitation (v is 5000r/min, t is 10min), filtering, concentrating the filtrate under reduced pressure until the concentration of chicoric acid in the liquid medicine C is 2.71mg/mL, adjusting the pH value to 2.99, transferring to 4 ℃, storing, centrifuging, filtering, and collecting the filtrate as the supernatant.

(3) Pretreating macroporous adsorption resin: soaking AB-8 type macroporous adsorbent resin in anhydrous ethanol solution for 24 hr to activate the resin. Removing the turbid matter floating on the ethanol in time during the activation process, replacing the ethanol and keeping the ethanol higher than the resin surface; transferring the activated resin into a chromatographic column to ensure that the height ratio of the resin bed diameter to the height is 1:10, washing the resin with purified water until no alcohol smell exists.

(4) And (3) macroporous resin purification, namely sampling at the sampling flow rate of 2BV/h, circularly adsorbing for 3h, removing impurities by using purified water with the pH value of 3 for 3BV, eluting and enriching chicoric acid by using 50 percent ethanol solution with the pH value of 6BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.

(5) And (3) ethyl acetate extraction: extracting the concentrated solution with C17.19 mg/mL and pH 2.00 with ethyl acetate of the same volume for 10min for 3 times; the organic phases are combined and concentrated under reduced pressure to be thick paste, and then transferred to a tray for vacuum drying under reduced pressure (T is 50 ℃, P is-0.10 MPa).

(6) Preparation and crystallization: dissolving the coarse powder of the chicoric acid in 15% methanol, preparing the concentration of the solution to be about 17.55mg/mL, centrifuging, performing vacuum filtration on the supernate, performing sample injection and separation, collecting the effluent corresponding to a chromatographic peak of the chicoric acid for 40-50 min, performing vacuum concentration on the effluent until the effluent is concentrated at the temperature of 4 ℃ for a night, performing vacuum filtration on crystals, washing a filter cake with purified water at the temperature of 4 ℃, and performing vacuum drying on the filter cake (the temperature is 60 ℃ and the pressure is-0.10 MPa).

Analysis of results

The results (international standard) of the examples and comparative examples were measured, respectively, in the following manner using the extraction rate and purity of chicoric acid as indices.

Therefore, the obtained echinacea purpurea extract product has high content of chicoric acid, compared with a comparative example, the whole process obtains good extraction effect, and the comparative analysis shows that the invention obtains better effects in the aspects of chicoric acid extraction, water precipitation process optimization, resin gradient impurity removal, ethyl acetate extraction process improvement and the like.

Pilot scale

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 0.5 hr, and extracting under reflux for the first time for 1.5 hr; after filtration, the medicinal materials are subjected to secondary reflux extraction by 30 percent ethanol which is the same as the amount of the medicinal materials in l2 times; extracting the filtered materials with 30% ethanol L0 times for 0.5 hr, mixing the filtrates to obtain a total volume of 4296L, and concentrating under reduced pressure to 1604L (first effect temperature T) in a double-effect energy-saving concentration evaporator₁70 ℃ and P-0.048 MPa; double effect temperature T₂＝70℃、P＝-0.066MPa)。

(2) Water precipitation and column pretreatment: transferring the concentrated solution into a precipitation tank, and precipitating with water at 4 deg.C for 6 hr. Centrifuging the supernatant obtained by water precipitation at a feeding speed of 30-50 mL/s, concentrating under reduced pressure (T is 74 ℃, P is-0.062 MPa) to 482L, adjusting the pH value to 3 before loading the concentrated solution, transferring the concentrated solution to a refrigerator at 4 ℃, and performing centrifugal filtration to obtain filtrate as a loading solution.

(3) Pretreating macroporous adsorption resin: soaking 75Kg of AB-8 type macroporous adsorbent resin in 1.5 times of 95% ethanol solution for 24h to activate the resin. Removing the turbid matter floating on the ethanol in time during 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 washed with purified water until free of alcohol smell.

(4) Purifying with macroporous resin, i.e. converting the diameter and height of a stainless steel macroporous adsorption column (h is 160cm, and d is 25cm) according to the actual conditions of a production workshop and the laboratory process parameters, and determining the ratio of the diameter to the height of a resin bed to be 1: 6; the actual concentration range of the medicinal liquid chicoric acid is 1.2-1.7 mg/mL, and the sample loading volume is adjusted according to the sample loading concentration under the condition of keeping the sample loading amount unchanged in consideration of concentration loss. Sampling at the flow rate of 2BV/h, circularly adsorbing for 3h, performing gradient impurity removal by using 10 percent ethanol solution 3BV with the pH value of 3 and 2BV with 20 percent ethanol solution to remove impurities, eluting and enriching chicoric acid by using 35 percent ethanol solution 6 BV-12 BV, and concentrating the eluent under reduced pressure until no alcohol smell exists.

(5) And (3) ethyl acetate extraction: extracting the concentrated solution (C is 11-16 mg/mL, pH is 1.91) with ethyl acetate of the same volume for 2 times, each time for 40 min; the organic phases were combined and back-extracted 1 time 40min with the same volume of water at pH 2. Concentrating the organic phase under reduced pressure to obtain a thick paste, replacing the ethyl acetate phase with 95% ethanol, continuously concentrating to obtain a thick paste, transferring the thick paste into a tray, and drying under reduced pressure and vacuum (T is 50-60 ℃, and P is-0.10 MPa).

(6) High-pressure preparation and crystallization: will 1010-C₁₈Filling the filling material into a DAC-150 dynamic axial compression column, dissolving the chicoric acid powder by using a 15% methanol solution to prepare a liquid medicine with the concentration of 15mg/mL, centrifuging (v is 10000r/min, T is 7min), performing vacuum filtration on a supernatant, injecting and separating by using the chicoric acid amount of 10g, collecting an effluent liquid 6L corresponding to a chromatographic peak for 40-50 min according to the chicoric acid peak time, performing vacuum concentration on the effluent liquid to 1/30(T is 60 ℃, P is-0.10 MPa) of the original volume, transferring the concentrated liquid to an environment of 4 ℃ for crystallization, filtering white or white needle-like and needle-cluster crystals, washing filter cakes by using purified water at 4 ℃, performing vacuum drying on the filter cakes (T is 60 ℃, P is-0.10 MPa) to obtain an L-chicoric acid raw material with the purity of more than 98%, and performing recrystallization on a crystallization mother liquid for recovery treatment.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.