CN112940056A - Preparation method of crocin reference substance - Google Patents
Preparation method of crocin reference substance Download PDFInfo
- Publication number
- CN112940056A CN112940056A CN202110152394.2A CN202110152394A CN112940056A CN 112940056 A CN112940056 A CN 112940056A CN 202110152394 A CN202110152394 A CN 202110152394A CN 112940056 A CN112940056 A CN 112940056A
- Authority
- CN
- China
- Prior art keywords
- crocin
- phase chromatography
- reference substance
- pressure
- methanol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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 crocin is easy to be oxidized into carboxyl due to the existence of aldehyde group, and the stability is poor, so that the method is a reason for limiting the obtaining of the high-purity crocin reference substance.
Therefore, it is very urgent to develop a method for preparing a reference substance of crocetin 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 crocin reference substance, which has high flux, can provide high-purity crocin and is suitable for large-scale 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 objective substance in the present invention is the content by mass ratio, if not specifically stated. 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 sequentially comprises the following steps:
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: 21-2: 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 at 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 bitter crocin semi-finished product powder;
a high-pressure reversed-phase chromatography purification procedure: dissolving the semi-finished powder of the crocin by using a methanol aqueous solution, loading the solution to a high-pressure reverse phase chromatographic column, eluting the solution by using 40-60% methanol aqueous solution under the pressure of 5-15.0 Mpa, removing pigment and a small amount of front and rear impurities, and collecting eluent containing a target substance;
a target enrichment and recovery process: concentrating the eluate containing the target substance at 45 deg.C in the dark until no alcohol smell exists, 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 18 to 22:0.8 to 1.5:0.01 to 0.15.
In a preferred embodiment of the present invention, the medium-pressure reversed-phase chromatography purification step is performed by eluting 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 performed by eluting with 45 to 55% methanol aqueous solution under 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 chemically bonded silica having a particle size of 40-60um, and the packing of the high pressure reverse phase chromatography column is octadecylsilane chemically 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.
According to the experimental scheme, the highly HPLC-pure crocin can be separated, and in a preferred embodiment, the crocin monomer with the purity of more than 98.5 percent can be obtained.
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 chromatography purification step, namely loading the crude raw material extract containing the crocin into a normal phase chromatography 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: 21-2: 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, wherein the first crude product concentrated solution is subjected to sample loading to medium-pressure reverse-phase chromatography, an acetonitrile water solution with the concentration of 12-16% is used for elution at the pressure of 0.05-0.5 Mpa, an eluent is collected according to HPLC detection, the eluent is concentrated to obtain a semi-finished product concentrated solution, and the concentrated solution is subjected to freeze drying to obtain a crocin semi-finished product powder;
a high-pressure reversed-phase chromatography purification process, dissolving the crocin semi-finished product powder with a methanol aqueous solution, loading the solution to a high-pressure reversed-phase chromatography column, eluting the column with a 40-60% methanol aqueous solution under the pressure of 5-15.0 Mpa, removing pigments and a small amount of impurities, and collecting the eluate 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 the preparative separation is further 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 chromatography purification step, the crude material extract containing the crocin is loaded on a normal phase chromatography column, and is eluted by an eluent containing ethyl acetate, methanol and water, wherein the ratio of ethyl acetate to methanol to water is 15-25: 21-2: 0.05-0.15. 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 Al2O3Polar 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 better obtainThe separation efficiency is preferably that in the normal phase chromatography purification step, the mixing ratio of ethyl acetate, methanol and water is 18-22: 0.8-1.5: 0.01-0.15.
In the invention, the medium-pressure reverse-phase chromatographic separation is used for separating other components with polarity difference with a target object, the elution pressure of 0.05-0.5 Mpa is adopted to realize the balance between the resolution and the speed, and the pressure of the medium-pressure reverse-phase chromatographic column in the medium-pressure column separation process is preferably 0.1 Mpa-0.2 Mpa, and is particularly preferred 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, elution is carried out at a pressure of 5-15.0 Mpa, so that the balance between resolution and 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 of the high pressure reversed phase chromatography of the present invention, it is preferable to elute the column 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 C18(ODS) silica gel column, i.e. octadecylsilane bonded silica, preferably with a filler of 40-60 μm, reasonable particle size is advantageous for maintaining proper column pressure and resolution, octadecylsilane bonded silica is easily available on the market. From the viewpoint of efficiency and easy availability of the filler, in the preferred preparation method of the invention, the filler of the reversed phase chromatographic column is octadecylsilane chemically bonded silica with the particle size of 40-60 μm.
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 lifetime 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, it is preferable that the concentration and lyophilization are performed under a dark condition in consideration of the possibility that the target substance may be unstable, and it is preferable that the concentration is performed for 60 minutes or less after removing acetonitrile under reduced pressure at a temperature of 45 ℃ or less, and then the concentration is directly lyophilized, so that the stability is improved, the degradation of the pure target substance is 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) into 910g stigma croci Sativi, ultrasonic extracting for 2 times, each time for 1 hr, cooling to room temperature, vacuum filtering, mixing filtrates, concentrating at 45 deg.C in dark place until there is no alcohol smell, and concentrating to 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 Yangttai jiangyou, adding 25L of ethyl acetate, and packing with a wet method (the ethyl acetate is purchased from Shanghai Tantake technology Co., Ltd., 30L/barrel); 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, the target substance is eluted with ethyl acetate, methanol and water at the ratio of 20:1:0.1 in an isocratic manner, the eluent is collected according to HPLC detection to obtain 90% target fraction of HPLC, and the target fraction is concentrated to 400ml at 45 ℃ in a dark place;
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 of packing, column size 460X 100mm, purchased from Congo chromatography, isolation and purification, Suzhou) at a 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 crocin powder obtained in step D with about 98.5% HPLC with 40% methanol (methanol is purchased from Shanghai Kangshi Kogyo Co., Ltd., 30L/barrel); prepared by LC-20AP preparative Lixiang (purchased from Shimadzu corporation), chromatographic column using 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 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, loading the high-pressure target fraction onto 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 Virging chromatography separation and purification Co., Ltd.), adsorbing, eluting the target fraction 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) into 910g stigma croci Sativi, ultrasonic extracting for 2 times, each time for 1 hr, cooling to room temperature, vacuum filtering, mixing filtrates, concentrating at 45 deg.C in dark place until there is no alcohol smell, and concentrating to 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 Yangttai jiangyou, adding 25L of ethyl acetate, and packing with a wet method (the ethyl acetate is purchased from Shanghai Tantake technology Co., Ltd., 30L/barrel); 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, the target substance is eluted with ethyl acetate, methanol and water at the ratio of 20:1:0.1 in an isocratic manner, the eluent is collected according to HPLC detection to obtain 90% target fraction of HPLC, and the target fraction is concentrated to 400ml at 45 ℃ in a dark place;
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 of packing, column size 460X 100mm, purchased from Congo chromatography, isolation and purification, Suzhou) at a 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, 7um, flow rate of 120ml/min, 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:
1H-NMR
13C-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 (10)
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: 21-2: 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 at 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 bitter crocin semi-finished product powder;
a high-pressure reversed-phase chromatography purification process, dissolving the crocin semi-finished product powder with a methanol aqueous solution, loading the solution to a high-pressure reversed-phase chromatography column, eluting the column with a 40-60% methanol aqueous solution under the pressure of 5-15.0 Mpa, removing pigments and a small amount of impurities, and collecting the eluate 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.
2. The method of preparing a crocin reference substance of claim 1, wherein the crocin reference substance is obtained by subjecting crocin to a reaction,
the preparation method of the crude material extract containing the crocin comprises the following steps,
pulverizing stigma croci Sativi, soaking in alcohol water solution, filtering, concentrating the filtrate at below 50 deg.C in dark, and freeze drying.
3. The method for preparing a crocin reference substance according to claim 1, wherein the mixing ratio of ethyl acetate, methanol and water in the normal phase chromatography purification step is 18-22: 0.8-1.5: 0.01-0.15.
4. The method of preparing a crocin reference substance of claim 1, wherein the crocin reference substance is obtained by subjecting crocin to a reaction,
in the medium-pressure reversed-phase chromatography purification step, 13-15% acetonitrile aqueous solution is used for elution at a pressure of 0.1-0.3 MPa.
5. The method for preparing crocin reference substance of claim 1, wherein the high pressure reverse phase chromatography is performed under 8-10 MPa and 45-55% methanol aqueous solution.
6. The method of preparing crocin reference substance of claim 1,
in the normal phase chromatographic purification procedure, the filler of the normal phase chromatographic column is silica gel with the particle size of 100-300 meshes.
7. The method of claim 1, wherein the medium pressure reverse phase column is packed with octadecylsilane chemically bonded silica having a particle size of 40-60um, and the high pressure reverse phase column is packed with octadecylsilane chemically bonded silica having a particle size of 5-10 um.
8. The method for preparing crocin reference substance of claim 2, wherein the alcohol-based aqueous solution is 60-85% methanol aqueous solution or 60-85% ethanol aqueous solution
9. The method of preparing a crocin reference substance of claim 2, wherein,
a target enrichment and recovery process: eluting with 90-98% acetonitrile or 90-98% methanol.
10. A solid control of crocin having a purity of 98.5% or more, which is prepared by the preparation method of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110152394.2A CN112940056B (en) | 2021-02-03 | 2021-02-03 | Preparation method of crocin reference substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110152394.2A CN112940056B (en) | 2021-02-03 | 2021-02-03 | Preparation method of crocin reference substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112940056A true CN112940056A (en) | 2021-06-11 |
| CN112940056B CN112940056B (en) | 2023-02-03 |
Family
ID=76243595
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110152394.2A Active CN112940056B (en) | 2021-02-03 | 2021-02-03 | Preparation method of crocin reference substance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112940056B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114870428A (en) * | 2022-04-08 | 2022-08-09 | 张齐杰 | Production device and working method of preparation for promoting synthesis of gardenia crocetin |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2130066B1 (en) * | 1997-04-15 | 2000-04-01 | Univ Castilla La Mancha | METHOD OF ISOLATION OF COLORING AND FLAVORING SUBSTANCES OF SAFFRON SPECIES: CROCINAS AND PICROCROCINA. |
| CN104083544A (en) * | 2014-05-09 | 2014-10-08 | 杭州九如堂生物科技有限公司 | Extraction preparation method of Crocus sativus active component |
| CN104610214A (en) * | 2013-11-04 | 2015-05-13 | 中国科学院大连化学物理研究所 | Method for rapidly preparing six compounds in Stellera chamaejasme L. |
| CN108948108A (en) * | 2018-09-04 | 2018-12-07 | 山东中医药大学附属医院 | A kind of new compound being isolated from oldenlandia diffusa |
| US20190099464A1 (en) * | 2016-04-18 | 2019-04-04 | Pharmactive Biotech Products, S.L. | Saffron extract and its use for the prevention of mood disorders related to depression |
| CN110526952A (en) * | 2019-09-20 | 2019-12-03 | 大连民族大学 | The preparation method of flavonoid glycoside is extracted in a kind of coarse brake fern |
| CN110746474A (en) * | 2019-11-14 | 2020-02-04 | 广西师范大学 | Dammarane type triterpenoid saponin compound, preparation method thereof and application thereof in preparation of anti-inflammatory drugs |
| CN111689935A (en) * | 2020-06-20 | 2020-09-22 | 中国科学院新疆理化技术研究所 | Preparation method of lactuca sativa bitter element |
-
2021
- 2021-02-03 CN CN202110152394.2A patent/CN112940056B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2130066B1 (en) * | 1997-04-15 | 2000-04-01 | Univ Castilla La Mancha | METHOD OF ISOLATION OF COLORING AND FLAVORING SUBSTANCES OF SAFFRON SPECIES: CROCINAS AND PICROCROCINA. |
| CN104610214A (en) * | 2013-11-04 | 2015-05-13 | 中国科学院大连化学物理研究所 | Method for rapidly preparing six compounds in Stellera chamaejasme L. |
| CN104083544A (en) * | 2014-05-09 | 2014-10-08 | 杭州九如堂生物科技有限公司 | Extraction preparation method of Crocus sativus active component |
| US20190099464A1 (en) * | 2016-04-18 | 2019-04-04 | Pharmactive Biotech Products, S.L. | Saffron extract and its use for the prevention of mood disorders related to depression |
| CN108948108A (en) * | 2018-09-04 | 2018-12-07 | 山东中医药大学附属医院 | A kind of new compound being isolated from oldenlandia diffusa |
| CN110526952A (en) * | 2019-09-20 | 2019-12-03 | 大连民族大学 | The preparation method of flavonoid glycoside is extracted in a kind of coarse brake fern |
| CN110746474A (en) * | 2019-11-14 | 2020-02-04 | 广西师范大学 | Dammarane type triterpenoid saponin compound, preparation method thereof and application thereof in preparation of anti-inflammatory drugs |
| CN111689935A (en) * | 2020-06-20 | 2020-09-22 | 中国科学院新疆理化技术研究所 | Preparation method of lactuca sativa bitter element |
Non-Patent Citations (2)
| Title |
|---|
| 童应鹏: "中药西红花烘干工艺及有效成分的提取工艺和网络药理学研究", 《中国优秀博硕士学位论文全文数据库(博士) 医药卫生科技辑》 * |
| 陈天翱等: "西红花中苦藏花素的分离及抑菌研究", 《生物化工》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114870428A (en) * | 2022-04-08 | 2022-08-09 | 张齐杰 | Production device and working method of preparation for promoting synthesis of gardenia crocetin |
| CN114870428B (en) * | 2022-04-08 | 2023-12-05 | 张齐杰 | Production device and working method of preparation for promoting synthesis of gardenia crocin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112940056B (en) | 2023-02-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | Extraction of sesamin from sesame oil using macroporous resin | |
| CN108358991B (en) | Triterpenoid in chengshuanghua, and preparation method and application thereof | |
| CN107964031B (en) | Triterpenoid extracted and separated from rhizoma alismatis, and method and application thereof | |
| WO2020063894A1 (en) | Industrial utilization method for stevia rebaudiana and stevioside and chlorogenic acid of stevia rebaudiana | |
| Dawa et al. | Targeted isolation of 1, 1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata using medium-and high-pressure liquid chromatography combined with online high performance liquid chromatography–1, 1-diphenyl-2-picrylhydrazyl detection | |
| Denderz et al. | Using of molecularly imprinted polymers for determination of gallic and protocatechuic acids in red wines by high performance liquid chromatography | |
| CN112940056B (en) | Preparation method of crocin reference substance | |
| JP7305870B2 (en) | Method for producing tetragalloyl glucose | |
| CN105566414B (en) | The method that four kinds of flavone glycosides are isolated and purified from waxberry flesh | |
| Wang et al. | An efficient method for the preparative separation and isolation of ginkgolic acids from the sarcotesta of Ginkgo biloba L by pH-zone-refining counter-current chromatography coupled with inner-recycling mode | |
| Dong et al. | A general separation method of phenolic acids using pH-zone-refining counter-current chromatography and its application to oat bran | |
| Wei et al. | An efficient preparative procedure for main flavone aglycones from Equisetum palustre L. using macroporous resin followed by gel resin flash chromatography | |
| CN109320571B (en) | Method for extracting luteolin compound and cynaropicrin | |
| CN108129544B (en) | Compound with antimicrobial activity and preparation method and application thereof | |
| Liao et al. | Recovery of value-added anthocyanins from mulberry by a cation exchange chromatography | |
| CN110590882B (en) | Method for simultaneously separating and purifying 6 flavone compounds from sunflower seeds | |
| Zou et al. | An efficient protocol for preparation of gallic acid from Terminalia bellirica (Gaertn.) Roxb by combination of macroporous resin and preparative high-performance liquid chromatography | |
| Hou et al. | Preparative purification of corilagin from Phyllanthus by combining ionic liquid extraction, prep-HPLC, and precipitation | |
| CN109180622B (en) | Method for extracting guaiane type sesquiterpene compound from artichoke | |
| Xie et al. | Recovery of chlorogenic acid from haskap leaves (Lonicera caerulea) using aqueous two-phase extraction | |
| CN111440184B (en) | Method for preparing high-purity carnosol | |
| CN112321664B (en) | Method for extracting, separating and purifying inonotus obliquus alcohol | |
| CN114988983A (en) | Method for separating sclareol from sclareol extract | |
| CN110240585B (en) | Preparation method of agilawood tetraol | |
| Lv et al. | Preparative separation of steviol glycosides from Stevia rebaudiana bertoni by macroporous resin and preparative HPLC |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |