CN112940056B - Preparation method of crocin reference substance - Google Patents
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Abstract
The invention provides a preparation method of a crocin reference substance, which comprises a normal phase chromatography purification process, a medium pressure reverse phase chromatography purification process, a high pressure reverse phase chromatography purification process and a target substance enrichment and recovery process, wherein in the target substance enrichment and recovery process, eluent containing a target substance obtained in the high pressure reverse phase chromatography purification process is concentrated at 45 ℃ in the dark until no alcohol smell exists, all the eluent is loaded onto a medium pressure reverse phase chromatography column and eluted with acetonitrile or methanol at the pressure of 0.05-0.5 Mpa to obtain a target fraction, and the target fraction is concentrated at 50 ℃ in the dark and is freeze-dried to obtain the crocin reference substance.
Description
Technical Field
The invention relates to a preparation method of crocin, in particular to a preparation method of a solid reference substance capable of preparing high-purity crocin for TCL.
Background
Crocus sativus L (also called crocus sativus L) is a plant of crocus genus of Iridaceae family, is native to southern Europe to Iran, is imported to Tibet via India, and is also called saffron, is commonly used up to now, is mainly produced in Iran, greece, italy, spain, india, china, japan and other countries, and can be used for food dyes and spices. At present, zhejiang, jiangxi, jiangsu, beijing and Shanghai in China have a small amount of cultivation. The dry stigma is used as a medicine in the traditional Chinese medicine, and has the effects of promoting blood circulation, removing blood stasis, cooling blood, removing toxic substances, resolving stagnation and tranquilizing. Is used for amenorrhea, obstruction in the abdomen, postpartum stasis, toxic heat and speckle, melancholy and stuffiness, palpitation and mania, and the modern pharmacological research shows that the saffron has the efficacy of strengthening the body. At present, more than 100 compounds are separated from saffron at home and abroad, mainly comprise mushrooms, flavonoids, anthrone and the like, and have various activities of treating mental diseases, neurodegenerative diseases, learning and memory disorders, cardiovascular diseases, atherosclerosis, hyperlipidemia, diabetes, hypertension, gastric ulcer, fatty liver, epilepsy resistance, convulsion resistance and the like.
As a famous and precious medicinal material and a famous and precious spice, the price of saffron is high, but the products in the market are unsmooth, so that the quality control of the products by strict quality control means is imperative. The method for detecting the content of the saffron in the Chinese and western medicine of 2015 edition of Chinese pharmacopoeia only stipulates the method for detecting the content of the two components of the crocin-I and the crocin-II. According to the published literature, the content measurement of the saffron component can not meet the actual requirement of related products. Recently, it is being studied to include the active ingredient crocin of another chinese medicine in saffron also in the scope of ingredient detection.
The structural formula of the crocin is as follows:
as one of index components of the crocus sativus, the crocin reference substance is very necessary to obtain. However, the reference substance of the highly purified crocetin is not sold in the market, and the reference substance of the crocetin with the purity of about 98 percent can be purchased only in general. Through structural analysis, the bitter crocin is easy to be oxidized into carboxyl due to the existence of aldehyde group, and the stability is poor, so that the reference substance of the bitter crocin with high purity is limited.
Therefore, it is very urgent to develop a method for preparing the reference substance of the crocin, which provides high-throughput, high-purity, especially high-TLC, crocin and is suitable for mass production.
Disclosure of Invention
The invention aims to provide a preparation method of a bitter crocin reference substance, which has high flux, can provide high-purity bitter crocin and is suitable for mass preparation. In the present invention, crocin is sometimes referred to as a target substance or an isolated target substance.
The percentage of the purity and content of the object in the present invention is the content by mass ratio, unless otherwise specified. Unless otherwise specified, the percentage of the liquid to liquid ratio in the present invention, for example, the ratio of formic acid to acetonitrile in water, is measured in volume fraction.
The method can separate and obtain the crocin with high purity by a simple and efficient operation process. The preparation and separation method of the invention sequentially comprises the following steps:
the preparation method of the crocin reference substance is characterized by sequentially comprising the following steps of:
a normal phase chromatography purification procedure: loading the crude raw material extract containing the crocin into a normal phase chromatographic column, eluting by using an eluent containing ethyl acetate, methanol and water, wherein the ratio of ethyl acetate to methanol to water is 15-25;
a medium-pressure reversed-phase chromatography purification procedure: loading the first crude product concentrated solution to a medium-pressure reverse phase chromatography, eluting with 12-16% acetonitrile aqueous solution under the pressure of 0.05-0.5 Mpa, collecting eluent according to HPLC detection, concentrating the eluent to obtain a semi-finished product concentrated solution, and freeze-drying the concentrated solution to obtain the semi-finished product powder of the crocetin;
a high-pressure reversed-phase chromatography purification procedure: dissolving the semi-finished powder of the crocin with a methanol aqueous solution, loading the solution to a high-pressure reversed-phase chromatographic column, eluting the solution with 40 to 60 percent methanol aqueous solution under the pressure of 5 to 15.0Mpa, removing pigment and a small amount of impurities in front and back, and collecting the eluent containing the target substance;
a target enrichment and recovery process: concentrating the eluate containing the target substance at 45 deg.C in the dark to remove alcohol smell, loading onto medium pressure reverse phase chromatographic column, eluting with acetonitrile or methanol under 0.05-0.5 Mpa to obtain target fraction, concentrating at 50 deg.C in the dark, and freeze drying to obtain crocin reference substance.
In a preferred embodiment of the present invention, the crude extract of raw material containing crocin is prepared as follows: pulverizing stigma croci Sativi, soaking in alcohol water solution, filtering, concentrating the filtrate at below 50 deg.C in dark, and freeze drying.
In a preferred embodiment of the present invention, in the normal phase chromatography purification/purification step, the mixing ratio of ethyl acetate, methanol and water is from 18 to 22.
In a preferred embodiment of the present invention, in the purification step by medium-pressure reverse phase chromatography, elution is carried out with an aqueous acetonitrile solution having a concentration of 13 to 15% under a pressure of 0.1 to 0.3 MPa.
In a preferred embodiment of the present invention, the purification step by high pressure reverse phase chromatography is carried out by eluting with 45 to 55% methanol aqueous solution at a pressure of 8 to 10 MPa.
In a preferred embodiment of the present invention, in the normal phase chromatography purification step, the filler of the normal phase chromatography column is silica gel having a particle size of 100 to 300 mesh.
In a preferred embodiment of the invention, the packing of the medium pressure reverse phase chromatography column is octadecylsilane bonded silica having a particle size of 40-60um, and the packing of the high pressure reverse phase chromatography column is octadecylsilane bonded silica having a particle size of 5-10 um.
In a preferred embodiment of the present invention, the alcohol-based aqueous solution is a 60% to 85% methanol aqueous solution or a 60% to 85% ethanol aqueous solution
In a preferred embodiment of the present invention, the target enrichment recovery step comprises: eluting with 90-98% acetonitrile or 90-98% methanol.
The invention also provides a solid reference substance of the crocetin with the purity of more than 98.5 percent, which is prepared by the preparation method, and the reference substance prepared by the method has high HPLC purity.
The invention has the following characteristics:
the method is simple and efficient to operate, and can amplify and separate the process to obtain a large amount of the crocetin monomer.
The product obtained by separation is subjected to nuclear magnetism and mass spectrum to determine the structure information of the product, and the structure is not identified by mass spectrum conjecture in other literatures.
The bitter crocin with high HPLC purity can be separated and obtained through the experimental scheme of the invention, and the bitter crocin monomer with the purity of more than 98.5 percent can be obtained in the preferred embodiment.
Drawings
FIG. 1 is an HPLC chromatogram of pure crocin obtained in example 1 of the present invention;
FIG. 2 is an HPLC chromatogram of the pure crocetin obtained in comparative example 1 of the present invention;
FIG. 3 is an H-NMR spectrum of pure crocin;
FIG. 4 is a C-NMR spectrum of pure crocin;
FIG. 5 is a mass spectrum of pure crocin.
Detailed Description
The following describes specific embodiments of the present invention.
The preparation and separation method sequentially comprises the following steps:
a normal phase chromatographic purification step, namely loading the crude raw material extract containing the crocin into a normal phase chromatographic column, eluting by using an eluent containing ethyl acetate, methanol and water, wherein the ratio of ethyl acetate to methanol to water is 15-25;
a medium-pressure reversed-phase chromatography purification process, namely loading the first crude product concentrated solution into a medium-pressure reversed-phase chromatography, eluting the first crude product concentrated solution by using an acetonitrile aqueous solution with the concentration of 12 to 16 percent at the pressure of 0.05 to 0.5Mpa, collecting eluent according to HPLC detection, concentrating the eluent to obtain a semi-finished product concentrated solution, and freeze-drying the concentrated solution to obtain semi-finished product powder of the crocin;
a high-pressure reversed phase chromatography purification process, dissolving the semi-finished powder of the crocin with a methanol water solution, loading the solution to a high-pressure reversed phase chromatography column, eluting with 40-60% methanol water solution under the pressure of 5-15.0 Mpa, removing pigment and a small amount of impurities, and collecting the eluent containing the target substance;
and a target substance enrichment and recovery process, namely concentrating the eluent containing the target substance obtained in the high-pressure reverse phase chromatography purification process at 45 ℃ in the dark until no alcohol smell exists, loading all the eluent into a medium-pressure reverse phase chromatography column, eluting with acetonitrile or methanol at the pressure of 0.05-0.5 Mpa to obtain a target fraction, concentrating at the temperature of below 50 ℃ in the dark, and freeze-drying to obtain the crocin reference substance.
The inventor of the invention finds that the general standard preparation idea is to continuously increase the resolution of chromatographic separation and continuously improve the purity by using a chromatographic column with higher pressure and better separation degree. However, the inventors of the present invention found that this technical idea is basically applicable and practical for obtaining crocin having a purity of 98% or less. However, when the purity of the crude product obtained by the column purification process reaches 98.5% or more, it is very difficult to increase the purity even if the resolution of preparative separation is continued to be increased. The reason is not clear, probably because the stability of the target compound is not good, so that the method can only obtain about 98 percent of pure products at most due to self decomposition in the extraction and separation process.
In the conventional recovery process, after the eluent purified by high pressure reverse phase chromatography is obtained, since the eluent contains a large amount of water, a long time of concentration and drying process is required, and even though the inventor carelessly uses a low temperature, light shielding and freeze drying process, a pure product with more than 98.5 percent can not be obtained. The inventor improves the final target recovery process, obtains the eluent purified by high-pressure reverse phase chromatography, does not carry out concentration and drying on the eluent, only concentrates the eluent at 45 ℃ in the dark till no alcohol smell, and then quickly and completely loads the eluent on a medium-pressure reverse phase chromatography column which is completely filled with a new column and washed clean by a syringe pump. The concentration method is not particularly limited, and heating evaporation concentration, rotary evaporator concentration, and the like can be used. By concentrating to no alcohol smell, the methanol in the substantially mixed solution is substantially evaporated off (content less than 5%), and only water which is difficult to evaporate is left. In this case, an aqueous solution of the target substance is applied directly to the reversed-phase column chromatography, and almost all of the target substance is adsorbed on the column due to the polarity. At this time, the target compound is eluted with an eluent of an organic solvent, such as methanol or acetonitrile, whereby an eluate in which the target compound is dissolved in methanol or acetonitrile can be obtained, and the eluate can be rapidly concentrated. Probably because the recovery time of the purified sample is greatly shortened, the decomposition and the deterioration of the compound are more obviously inhibited, the obstacle that the purity cannot exceed 98 percent is broken through, the crocin with the purity higher than 99 percent is obtained, and the crocin is particularly suitable for being used as a high-quality reference substance. Meanwhile, the whole process is suitable for amplification, and the preparation flux of the reference substance can be greatly improved.
In the normal phase chromatographic purification process, the crude material extract containing the crocin is loaded on a normal phase chromatographic column and eluted by an eluent containing ethyl acetate, methanol and water, wherein the ratio of ethyl acetate to methanol to water is 15-25. The filler of the normal phase chromatographic column can be known normal phase filler, i.e. filler with stationary phase having polarity greater than that of mobile phase, and if the mobile phase is organic solvent, commonly used filler is silica gel or Al 2 O 3 Polar bonded phase fillers, etc., silica gel is preferably used in the present invention. The particle size of the silica gel is not particularly limited, and in a preferred production method of the present invention, from the viewpoint of efficiency and availability of the filler, the filler of the normal phase chromatography column in the first normal phase chromatography column purification step is silica gel having a particle size of 100 to 300 mesh. In order to obtain a better separation efficiency, in the normal phase chromatography purification step, the mixing ratio of ethyl acetate to methanol to water is preferably from 18 to 22.
In the present invention, the medium-pressure reverse-phase chromatography is used for separating other components having a polarity different from that of the target, and the elution pressure of 0.05 to 0.5Mpa is used to balance the resolution and the speed, and the pressure of the medium-pressure reverse-phase chromatography column in the medium-pressure column separation step is preferably 0.1 to 0.2Mpa, which is particularly preferable because the separation effect and the separation speed can be balanced. The high-pressure reverse phase chromatographic column separation generally refers to a chromatographic column separation technology with an elution pressure of more than 1 MPa. In the invention, the elution is carried out under the pressure of 5-15.0 Mpa, so that the balance between the resolution and the speed is realized. In the purification step by medium-pressure reverse-phase chromatography of the present invention, it is further preferable to elute with an acetonitrile aqueous solution having a concentration of 13 to 15% under a pressure of 0.1 to 0.3 MPa. In the purification step by high pressure reverse phase chromatography of the present invention, it is further preferable to elute the product with 45 to 55% methanol aqueous solution under a pressure of 8 to 10 MPa.
As the reversed-phase packing of the reversed-phase column used in the column preparation step of the present invention, known nonpolar silica gel having an alkane as a bonded functional group (e.g., C18 (ODS), C8, C4, etc.) can be used. Preferably a C18 (ODS) silica gel column, i.e. octadecylsilane bonded silica, preferably using 40-60 μm filler, reasonable particle size is advantageous for maintaining proper column pressure and resolution, octadecylsilane bonded silica is readily available on the market. In a preferred preparation method of the present invention, the packing of the reverse phase chromatography column is octadecylsilane chemically bonded silica having a particle size of 40 to 60 μm from the viewpoint of efficiency and easy availability of the packing.
According to the principle of the present invention, the medium-pressure reverse-phase chromatography column that can be used in the target enrichment recovery process may be any medium-pressure reverse-phase chromatography column as long as the packing loading capacity can satisfy the capacity of the target.
In the preparation method of the present invention, commercially available crocus extract can be used as it is as a crude material extract containing crocin, or can be extracted by a conventionally known method, and preferably by the following method:
pulverizing stigma croci Sativi, soaking in alcohol water solution, filtering, concentrating the filtrate at below 50 deg.C in dark, and freeze drying. The alcohol-based aqueous solution used herein is preferably an aqueous solution of methanol or ethanol, and more preferably an aqueous solution of 60% to 85% methanol or an aqueous solution of 60% to 85% ethanol.
In the target enrichment recovery step of the present invention, the target adsorbed on the reverse phase column may be directly eluted with acetonitrile using a reverse phase eluent such as methanol or acetonitrile, but in view of the life of the reverse phase column, it is preferable to use 90 to 98% acetonitrile for elution or 90 to 98% methanol for elution.
In the present invention, considering that the target substance may be unstable, it is preferable to perform all the concentration and lyophilization steps in the dark, and it is preferable to perform the concentration step for 60 minutes or less after removing acetonitrile under reduced pressure at a temperature of 45 ℃ or lower, and then to perform lyophilization directly, whereby stability can be improved, degradation of the pure target substance can be suppressed, and a solid reference substance with higher purity can be obtained.
In conclusion, the bitter crocin compound with the solid purity of more than 98.5 percent is obtained for the first time through the preparation method.
Examples
Hereinafter, a typical extraction method of the present invention will be described in further detail with reference to examples. The following experimental protocols are merely examples and are not intended to limit the present invention. Any modification can be made by those skilled in the art without departing from the principles and spirit of the invention.
Example 1
A. Raw material extraction: adding 10L 80% methanol water (methanol is purchased from Shanghai Kangshi high-purity solvent Co., ltd., 30L/barrel), ultrasonically extracting for 2 times (1 hr each time), cooling to room temperature, vacuum filtering, mixing filtrates, and concentrating at 45 deg.C in dark place until no alcohol smell exists, and total 3L;
B. and (3) freeze drying: performing freeze drying treatment on the concentrated solution obtained in the step A (a freeze dryer is purchased from Ningbo Xinzhi Biotechnology GmbH) to obtain stigma croci Sativi extract powder;
C. normal phase purification: weighing 8.5kg of silica gel of Yangtze river friend of tobacco, adding 25L of ethyl acetate, and packing by wet method (the ethyl acetate is purchased from 30L/barrel of Shanghai Tantake technology Co., ltd.); dissolving the extract powder in step B with 1L methanol (30L/barrel of Shanghai Kanghao high purity solvent Co., ltd.), adding 1.5kg silica gel, mixing, naturally air drying in shade, and loading; then isocratic elution of the target substance is carried out with ethyl acetate methanol water =20 = 1.1, the eluent is collected according to HPLC detection to obtain 90% of target fraction by HPLC, and the target fraction is concentrated to 400ml at 45 ℃ in the dark;
D. medium-pressure preparation and purification: the concentrate obtained in step C was applied to a medium pressure preparative column (Daiso RPS-C18, 40-60um,1.8-2.0kg packing, column size 460X 100mm, purchased from Suzhou Congo chromatography, isolation and purification Co., ltd.), flow rate: performing isocratic elution with 14% acetonitrile water (30L/barrel acetonitrile is purchased from Shanghai Xingshi, high purity solvent Co., ltd.) at 50mL/min, and collecting eluate according to HPLC detection to obtain target fraction of HPLC about 98.5%; concentrating at 45 deg.C in dark to no acetonitrile smell, and freeze drying to obtain powder;
E. high-pressure preparation and purification: dissolving the powder of crocin with HPLC about 98.5% obtained in step D with 40% methanol (methanol is purchased from Shanghai Kangchi high purity solvent Co., ltd., 30L/barrel); prepared by LC-20AP preparative Lixiang (purchased from Shimadzu corporation), subjecting to chromatography with YMC-Triart C18, 250 x 50mm, 7um, flow rate of 120ml/min, detection wavelength of 254nm, isocratic elution with 50% methanol water, removing pigment and a small amount of impurities, and collecting the target fraction;
F. and (3) product recovery: concentrating the high-pressure target fraction obtained in the step E at 45 ℃ in the dark until no alcohol smell exists, using a medium-pressure C18 column (Daiso RPS-C18, 40-60um,1.8-2.0kg of filler, the column size is 460 multiplied by 100mm, purchased from Suzhou Vietui chromatography separation and purification Co., ltd.) to perform sample adsorption, eluting the target with 95% acetonitrile to obtain a target fraction, concentrating at 45 ℃ in the dark, and freeze-drying to obtain 35g of a target substance.
Comparative example 1
A. Raw material extraction: adding 10L 80% methanol water (methanol is purchased from Shanghai Kangshi high-purity solvent Co., ltd., 30L/barrel), ultrasonically extracting for 2 times (1 hr each time), cooling to room temperature, vacuum filtering, mixing filtrates, and concentrating at 45 deg.C in dark place until no alcohol smell exists, and total 3L;
B. and (3) freeze drying: performing freeze drying treatment on the concentrated solution obtained in the step A (a freeze dryer is purchased from Ningbo Xinzhi Biotechnology GmbH) to obtain stigma croci Sativi extract powder;
C. normal phase purification: weighing 8.5kg of silica gel of Yangtze river friend of tobacco, adding 25L of ethyl acetate, and packing by wet method (the ethyl acetate is purchased from 30L/barrel of Shanghai Tantake technology Co., ltd.); dissolving the extract powder in step B with 1L methanol (methanol is purchased from Shanghai Kagaku Kogyo Co., ltd., 30L/barrel), adding 1.5kg silica gel, mixing, naturally air drying in shade, and loading; then isocratic elution of the target substance is carried out with ethyl acetate methanol water =20 = 1.1, the eluent is collected according to HPLC detection to obtain 90% of target fraction by HPLC, and the target fraction is concentrated to 400ml at 45 ℃ in the dark;
D. medium-pressure preparation and purification: the concentrate obtained in step C was applied to a medium pressure preparative column (Daiso RPS-C18, 40-60um,1.8-2.0kg packing, column size 460X 100mm, purchased from Suzhou Congo chromatography, isolation and purification Co., ltd.), flow rate: 50mL/min, using 14% acetonitrile to elute isocratically (acetonitrile purchased from Shanghai Kagaku Kogyo, 30L/barrel), and collecting eluent according to HPLC detection to obtain target fraction of about 98.5% HPLC; concentrating at 45 deg.C in dark to no acetonitrile smell, and freeze drying to obtain powder;
E. high-pressure preparation and purification: dissolving the obtained HPLC 98.5% crocin powder in 40% methanol water, preparing by LC-20AP preparative Lixiang (purchased from Shimadzu corporation), subjecting to chromatographic column with YMC-Triart C18, 250 x 50mm and 7um at flow rate of 120ml/min and detection wavelength of 254nm, isocratically eluting with 50% methanol water, removing pigment and small amount of impurities, collecting the target fraction, concentrating at 45 deg.C in dark place, and freeze drying to obtain 40g of target.
The crocin reference substances of example 1 and comparative example 1 were subjected to the following HPLC chromatographic conditions,
taking a sample of 2.5mg of the test solution, adding 1mL of aqueous solution for dissolving, preparing a sample of 2.5mg/mL, and performing on-site detection.
Mobile phase composition: a-water, B-acetonitrile
| Time (min) | Flow rate (mL/min) | ％B |
| 0.0 | 1.000 | 13.0 |
| 22.0 | 1.000 | 13.0 |
| 28.0 | 1.000 | 100％ |
From the results of the HPLC chromatogram readings, it is understood that the purity of the target compound obtained in example 1 of the present invention is 100%, and the purity of comparative example 1 is 98.25%. The absolute value of the concentration here is limited by the resolution of the instrument and is not completely accurate, but the relative difference between the examples and the comparative examples illustrates the excellent technical effect of the extraction method of the present invention.
Referring to FIGS. 3 and 4, the obtained NMR assignment data of crocin are as follows:
1 H-NMR
13 C-NMR
mass spectrum information of the crocetin obtained by the invention is shown in figure 5.
The instrument comprises the following steps: sciex TripleTOF 4600 LC/MS
Detection mode: positive ion mode ESI source parameters were as follows:
the technical features disclosed above are not limited to the combinations with other features disclosed, and other combinations between the technical features can be performed by those skilled in the art according to the purpose of the invention to achieve the aim of the invention, and various modifications made to the technical scheme of the invention by those skilled in the art without departing from the design spirit of the invention shall fall within the protection scope defined by the claims of the invention.
Claims (4)
1. The preparation method of the crocin reference substance is characterized by sequentially comprising the following steps of:
normal phase chromatography purification procedure: loading the crude raw material extract containing the crocin into a normal phase chromatographic column, eluting by using an eluent containing ethyl acetate, methanol and water, wherein the ratio of ethyl acetate to methanol to water is 15-25: 1-2: 0.05-0.15, collecting the eluent according to HPLC detection, and concentrating the eluent to obtain a first crude concentrated solution;
a medium-pressure reverse-phase chromatography purification process: loading the first crude product concentrated solution to medium-pressure reverse phase chromatography, eluting with 12-16% acetonitrile water solution under the pressure of 0.05-0.5 Mpa, collecting eluent according to HPLC detection, concentrating the eluent to obtain semi-finished product concentrated solution, and freeze-drying the concentrated solution to obtain the semi-finished product powder of the crocin;
a high-pressure reversed-phase chromatography purification process, dissolving the crude powder of the crocin with methanol water solution, loading the solution to a high-pressure reversed-phase chromatography column, eluting with 40-60% methanol water solution under the pressure of 5-15.0 Mpa, removing pigment and a small amount of impurities, and collecting the eluent containing the target substance;
concentrating the eluate containing the target substance at 45 deg.C in the dark to remove alcohol smell, loading onto medium pressure reverse phase chromatographic column, eluting with acetonitrile or methanol under 0.05-0.5 Mpa to obtain target fraction, concentrating at 50 deg.C in the dark, and freeze drying to obtain crude extract containing crocin,
pulverizing saffron serving as a raw material, soaking the saffron in an alcohol aqueous solution, filtering, concentrating filtrate at the temperature of below 50 ℃ in a dark place, and freeze-drying, wherein in the normal-phase chromatographic purification process, a filler of a normal-phase chromatographic column is silica gel with the particle size of 100-300 meshes, a filler of a medium-pressure reverse-phase chromatographic column is octadecylsilane chemically bonded silica with the particle size of 40-60um, a filler of a high-pressure reverse-phase chromatographic column is octadecylsilane chemically bonded silica with the particle size of 5-10um, and an alcohol aqueous solution is a 60-85% methanol aqueous solution or a 60-85% ethanol aqueous solution for soaking, and the target is enriched and recovered: eluting with 90-98% acetonitrile or 90-98% methanol.
2. The method for preparing a reference substance of crocin according to claim 1, wherein in the normal phase chromatography purification step, the mixing ratio of ethyl acetate, methanol and water is 15-22.
3. The method for preparing crocin reference substance of claim 1, wherein the elution is performed with 13-15% acetonitrile water solution under a pressure of 0.1-0.3 Mpa in the middle pressure reversed phase chromatography purification process.
4. The method of claim 1, wherein the purification step of the crocin comprises eluting with 45-55% methanol aqueous solution under 8-10 MPa.
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