CN112250724B - Preparation method of reference substance of cornflower-3-coumaroyl-diglucose-5-glucoside - Google Patents
Preparation method of reference substance of cornflower-3-coumaroyl-diglucose-5-glucoside Download PDFInfo
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Abstract
The invention provides a preparation method of a reference substance of cornflower-3-coumaroyl-diglucose-5-glucoside with purity of more than 98 percent, which comprises an enrichment concentration process, a chromatographic column purification process and a solidification process, wherein a solution of a target object is obtained in the chromatographic column preparation process and is concentrated until the concentration of the cornflower-3-coumaroyl-diglucose-5-glucoside is 5-30 percent, the concentrated solution is frozen and dried to obtain solid powder of the cornflower-3-coumaroyl-diglucose-5-glucoside, the stability of the cornflower-3-coumaroyl-diglucose-5-glucoside is verified, formate can exist stably, and the current anthocyanin standard product is found to obtain the reference substance with the property of the solid powder, the storage and distribution of the reference substance are very facilitated.
Description
Technical Field
The invention relates to a preparation method of cornflower-3-coumaroyl-diglucose-5-glucoside, in particular to a preparation method of a high-purity solid reference substance of cornflower-3-coumaroyl-diglucose-5-glucoside from purple cabbage.
Background
Cornflower-3-coumaroyl-diglucose-5-glucoside is an anthocyanin, the structure of which is shown in formula 1 below, which was originally reported by Ole-p. Johansen et al (Phytochemistry, 1991, 30, 4137-.
Researchers have also found its presence in purple cabbage. The purple cabbage is a common vegetable, also called purple cabbage, red cabbage, purple broccoli and red cabbage, is a variant of common head cabbage (cabbage), belongs to cruciferae, brassica and cabbage species, is a variant of common head cabbage, is originally originated from the Mediterranean sea and western and southern European regions, is planted and eaten all over the world at present, and is a research hotspot of medicinal and edible products. In recent years, many studies have been made on anthocyanins, and it is considered that anthocyanins have certain physiological activities, such as improvement of visual function, improvement of brain function, anti-obesity, anti-diabetes, anti-cardiovascular disease, anti-cancer, and the like.
The purple cabbage is greatly different from the common cabbage in anthocyanin content, the anthocyanin and the purple cabbage have close relationship in food therapy and medicinal value, and in order to deeply research the component content and the nutritional activity of the purple cabbage and in the exploration of formulating corresponding quality standards, the qualitative and quantitative detection of the purple cabbage extract by using the reference substance of the anthocyanin is the most basic means.
However, cornflower-3-coumaroyl-diglucose-5-glucoside, which is an important component of purple cabbage, is not available on the market at present, so that the related research on purple cabbage is difficult to advance. Research and development of a preparation method of cornflower-3-coumaroyl-diglucose-5-glucoside reference substance are urgently needed in both scientific research and industrial circles.
Anthocyanin extraction methods have been reported as follows: solvent extraction, microwave and ultrasonic assisted extraction, biological enzyme or biological fermentation, and ultra high pressure. However, many anthocyanins in purple cabbage have acylated residues, and the method has low resolution when extraction of acylated anthocyanins from purple cabbage is performed, so that satisfactory effects are difficult to obtain. The inventor of the invention researches a large amount of existing documents, and finds that no document provides a technical scheme for preparing high-purity samples of cornflower-3-coumaroyl-diglucose-5-glucoside.
Patent document CN106317145A discloses a scheme for separating and preparing anthocyanin of two acyl groups, namely Cyanidin-3- (silapoyl) diglucoside-5-glucoside and Cyanidin-3- (caffeoyl) (silapoyl) diglucoside-5-glucoside, in purple cabbage. However, cyanidin-3-coumaroyl-diglucose-5-glucoside is not mentioned, and the scheme provided by the method only can obtain anthocyanin with the purity of 92.4-94.3%, and cannot provide anthocyanin with the purity of more than 98%. The inventor of the invention utilizes the recorded scheme to separate and prepare cornflower-3-coumaroyl-diglucose-5-glucoside from purple cabbage, and finds that the separation effect of the countercurrent chromatography on the cornflower-3-coumaroyl-diglucose-5-glucoside is very poor, and a high-purity product cannot be obtained at all.
In addition, the compound reference substance needs to be provided in a solid form as a reagent which needs to be distributed, sold and stored, otherwise, the stable state is difficult to maintain, however, how to provide a preparation method of the solid reference substance of cornflower-3-coumaroyl-diglucose-5-glucoside which is in a solid form and has the purity of more than 98 percent is a technical problem which needs to be solved urgently in the industry.
Disclosure of Invention
The purpose of the present invention is to provide a method for producing a control of cornflower-3-coumaroyl-diglucose-5-glucoside (hereinafter, sometimes referred to as a target) having a purity of 98% or more.
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 cornflower-3-coumaroyl-diglucose-5-glucoside with high purity through a simple and efficient operation process. The separation method of the invention comprises the following steps: the method sequentially comprises the following steps:
a high-pressure reversed-phase chromatographic column separation procedure, namely loading a crude product containing cornflower-3-coumaroyl-diglucose-5-glucoside to a high-pressure reversed-phase chromatographic column, eluting with water containing 0.05-1% of formic acid and 15% -20% of acetonitrile under the pressure of 5-15.0Mpa to obtain a solution of a target substance,
and a solidification step, namely concentrating the solution of the target object obtained in the reverse phase chromatographic column preparation step until the concentration of the cornflower-3-coumaroyl-diglucoside-5-glucoside is 5-50%, and freeze-drying the concentrated solution to obtain solid powder of formate of the cornflower-3-coumaroyl-diglucoside-5-glucoside.
As a preferred production method of the present invention, the above crude product containing cornflower-3-coumaroyl-diglucose-5-glucoside as a raw material in the high-pressure reverse phase chromatography column separation step is prepared by a method comprising the following steps in this order:
and (3) an enrichment and concentration process, wherein a cabbage red extract is used as a raw material, an acidified alcohol solvent is used for extraction, a first raw material concentrated solution is obtained through concentration, the first raw material concentrated solution is loaded onto styrene skeleton macroporous resin, water is used for elution to remove impurities, the acidified alcohol solvent is used for elution to obtain a solution containing a target substance, and a second raw material concentrated solution is obtained through concentration.
A chromatographic column purification step, namely loading the second raw material concentrated solution to a medium-pressure reversed-phase chromatographic column, performing gradient elution at the pressure of 0.05-0.5 Mpa, wherein the elution process comprises the steps of eluting with an acidic 4-5% acetonitrile water solution to remove impurities, eluting with acidic 7-9% acetonitrile water, collecting the eluent, and concentrating to obtain a first crude product concentrated solution; and loading the first crude product concentrated solution to a hydroxypropyl sephadex chromatographic column, eluting by using acidic water containing 18-22% of acetonitrile, detecting by using an HPLC ultraviolet detector, collecting a sample solution according to an absorption peak shape, concentrating and drying to obtain a crude product containing cornflower-3-coumaroyl-diglucoside-5-glucoside.
In a preferred production method of the present invention, in the column purification step, the acidic 4% to 5% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03% to 0.07% trifluoroacetic acid, the acidic 7% to 9% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03% to 0.07% trifluoroacetic acid, and the acidic 18% to 22% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03% to 0.07% trifluoroacetic acid.
As a preferred preparation method of the invention, the filler of the medium-pressure reverse-phase chromatographic column is octadecylsilane chemically bonded silica with the particle size of 40-60 μm, and the filler of the high-pressure reverse-phase chromatographic column is octadecylsilane chemically bonded silica with the particle size of 5-10 μm.
Preferably, the pressure of the high-pressure reversed-phase chromatographic column is 8.0-10.0 MPa.
As a preferred preparation method of the present invention, the pressure of the high pressure reverse phase chromatographic column is 0.1MPa to 0.2 MPa.
Preferably, the preparation method of the invention comprises eluting the high-pressure reversed-phase chromatographic column by using 0.1-0.2% formic acid and 15% -20% acetonitrile in water.
In the preferred production method of the present invention, in the solidification step, when the solution of the target product obtained in the column production step is concentrated, acetonitrile is removed under reduced pressure at a temperature of 45 ℃ or lower, and then the concentration step is controlled to 60 minutes or less, and then freeze-drying is directly performed.
The invention has the following characteristics:
the purity of the solid powder of the cornflower-3-coumaroyl-diglucose-5-glucoside obtained by the method can reach more than 98 percent, and the method is a preparation scheme for providing a feasible reference substance of the cornflower-3-coumaroyl-diglucose-5-glucoside for the first time, is simple and feasible, is suitable for process amplification in all steps, and can be used for industrial preparation and production.
Anthocyanin can generally form solid powder in salt-like form with acid anion in the form of pyrane cation, and in the process of the invention, the stability of cyanidin-3-coumaroyl-diglucose-5-glucoside is verified, and formate thereof can be stably existed, so that a control product of the solid powder property is obtained, and the storage and distribution of the control product are very facilitated. It is also found that the hydrochloride common in the existing anthocyanin reference substance is difficult to exist stably, and the hydrochloride of the cornflower-3-coumaroyl-diglucose-5-glucoside with the purity of solid powder of more than 98 percent is difficult to provide.
Drawings
FIG. 1 is an HPLC chromatogram before and after lyophilization of a target substance in the solid product recovery procedure in example 1;
FIG. 2 is an HPLC chromatogram of a target substance after lyophilization in the solid product recovery procedure in example 2;
FIG. 3 is an HPLC chromatogram of a lyophilized target substance in the solid product recovery process in example 3;
FIG. 4 is an HPLC chromatogram of the lyophilized target substance in the solid product recovery process in example 4;
FIG. 5 is an HPLC chromatogram before and after lyophilization of a target substance in the solid product recovery process in comparative example 1;
FIG. 6 is a mass spectrum of cornflower-3-coumaroyl-diglucose-5-glucoside;
FIG. 7 shows cyanidin-3-coumaroyl-diglucose-5-glucoside1H-NMR spectrum;
FIG. 8 shows cyanidin-3-coumaroyl-diglucose-5-glucoside13C-NMR spectrum.
Detailed Description
The following describes specific embodiments of the present invention.
The inventor of the invention researches extensively and deeply and develops a preparation method of a high-purity cornflower-3-coumaroyl-diglucose-5-glucoside reference substance for the first time. The separation method of the invention comprises the following steps: the method sequentially comprises the following steps:
and a high-pressure reverse phase chromatographic column separation step, wherein a crude product containing cornflower-3-coumaroyl-diglucose-5-glucoside is loaded onto a high-pressure reverse phase chromatographic column, and is eluted by water containing 0.05-1% of formic acid and 15% -20% of acetonitrile under the pressure of 5-15.0Mpa to obtain a solution of a target substance.
And a solidification step, namely concentrating the solution of the target object obtained in the reverse phase chromatographic column preparation step until the concentration of the cornflower-3-coumaroyl-diglucoside-5-glucoside is 5-50%, and freeze-drying the concentrated solution to obtain solid powder of formate of the cornflower-3-coumaroyl-diglucoside-5-glucoside.
The separation method of the present invention is characterized by the organic combination and synergy of the high-pressure reverse-phase chromatography column separation step and the solidification step, and is specifically described below.
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 method, a target crude product concentrated solution is loaded to a high-pressure reversed-phase chromatographic column, and is eluted by water containing 0.05-1% of formic acid and 15% -20% of acetonitrile under the pressure of 5-15.0Mpa to obtain a target solution. The targets can be combined according to the data of the ultraviolet detector of the high-pressure chromatographic column separation. The purity of the target product can reach more than 98% after the step of carrying out the high-pressure reverse phase chromatographic column separation process.
The eluent used in the high-pressure reverse-phase chromatographic column separation process is the key technical characteristic of the invention, and the eluent used is very important. The inventors have tried various acids as additives to 15% to 20% acetonitrile and found that although the respective acid components have a small influence on the separation result at the time of separation, the influence on the purity of the finally obtained solid object is very large.
Specifically, the present inventors have found that a solid pure product having a purity of 98% or more can be obtained in the solidification step by performing high-pressure column separation using an aqueous acetonitrile solution of 0.05 to 1% formic acid. If the other acid is, for example, hydrochloric acid corresponding to the hydrochloride salt most commonly found in anthocyanin control. A high purity solid product cannot be obtained. If hydrochloric acid is used as an acidifying agent as an eluent in the high-pressure column separation, it is difficult to obtain a high-purity solid pure product by the solidification step even if the hydrochloric acid concentration is set to be very low (0.0075% (volume fraction) of hydrochloric acid is confirmed in the example described later). This may be due to the poor stability of cyanidin-3-coumaroyl-diglucose-5-glucoside hydrochloride itself.
In summary, although many anthocyanin standards are commercially available as hydrochloride, the present inventors have verified the stability of cyanidin-3-coumaroyl-diglucose-5-glucoside, and found that the hydrochloride thereof does not exist stably. The freeze-drying purity of the hydrochloride is obviously reduced, wherein the purity is 99 percent before freeze-drying and the maximum purity is 97 percent after freeze-drying. A solid control of cyanidin-3-coumaroyl-diglucose-5-glucoside, preferably provided in the form of a formate salt.
In the high-pressure column separation step, the pressure of the high-pressure reverse phase column is preferably 8.0 to 10.0MPa, and this pressure is particularly preferable because the separation effect and the separation speed can be balanced. The elution solution used in the elution of the high-pressure reversed-phase chromatographic column is preferably used because the elution is performed by using 0.1-0.2% of formic acid and 15% -20% of acetonitrile in water, which is favorable for the separation effect and speed and the service life of the chromatographic column.
In the solidification step, when the solution of the target product obtained in the column preparation step is concentrated, it is preferable to remove acetonitrile at a temperature of 45 ℃ or lower under reduced pressure, control the concentration process within 60 minutes, and then directly freeze-dry the solution, whereby stability can be improved, degradation of the pure target product can be suppressed, and a solid reference product with higher purity can be obtained. Concentration to too high a concentration is not preferable because there is a fear that the target substance is degraded by increasing the acid concentration in the eluent, and the preferred concentration should be 5% to 30%, but is not limited thereto.
In order to better exert the effect of the above separation method and provide a more efficient separation scheme as a whole, it is preferable to prepare the above crude product containing procyanidin-3-coumaroyl-diglucose-5-glucoside as a raw material in the high-pressure reverse phase chromatography column separation process by a method comprising the following steps in order:
and (3) an enrichment and concentration process, namely taking the cabbage red extract as a raw material, extracting by using an acidified alcohol solvent, concentrating to obtain a first raw material concentrated solution, loading the first raw material concentrated solution to styrene skeleton macroporous resin, eluting by using water to remove impurities, eluting by using the acidified alcohol solvent to obtain a solution containing a target substance, and concentrating to obtain a second raw material concentrated solution.
A chromatographic column purification step, namely loading the second raw material concentrated solution to a medium-pressure reversed-phase chromatographic column, performing gradient elution at the pressure of 0.05-0.5 Mpa, wherein the elution process comprises the steps of eluting with an acidic 4-5% acetonitrile water solution to remove impurities, eluting with acidic 7-9% acetonitrile water, collecting the eluent, and concentrating to obtain a first crude product concentrated solution; and loading the first crude product concentrated solution to a hydroxypropyl sephadex chromatographic column, eluting by using acidic water containing 18-22% of acetonitrile, detecting by using an HPLC ultraviolet detector, collecting a sample solution according to an absorption peak shape, concentrating and drying to obtain a crude product containing cornflower-3-coumaroyl-diglucoside-5-glucoside.
The enrichment and concentration process and the chromatographic column purification process are organically combined, so that the crude target product suitable for the method can be quickly provided.
And an enrichment and concentration process, wherein a cabbage red extract is used as a raw material, an acidified alcohol solvent is used for extraction and concentration to obtain a first raw material concentrated solution, the first raw material concentrated solution is loaded onto styrene skeleton macroporous resin, water is used for elution to remove impurities, the acidified alcohol solvent is used for elution to obtain a solution containing a target substance, and concentration is performed to obtain a second raw material concentrated solution, wherein the acidified alcohol solvent can be methanol or ethanol containing 0.03-0.06% of hydrochloric acid in terms of volume ratio.
A chromatographic column purification step, namely loading the second raw material concentrated solution to a medium-pressure reversed-phase chromatographic column, performing gradient elution at the pressure of 0.05-0.5 Mpa, wherein the elution process comprises the steps of eluting with an acidic 4-5% acetonitrile water solution to remove impurities, eluting with acidic 7-9% acetonitrile water, collecting the eluent, and concentrating to obtain a first crude product concentrated solution; and loading the first crude product concentrated solution to a hydroxypropyl sephadex chromatographic column, eluting by using acidic water containing 18-22% of acetonitrile, detecting by using an HPLC ultraviolet detector, collecting a sample solution according to an absorption peak shape, concentrating and drying to obtain a crude product.
In the present invention, the combination and order of the above steps are also very important.
Enrichment and concentration process through continuous screening and test in conventional anthocyanin enrichment, find that the utilization of the styrene-divinylbenzene skeleton macroporous resin can improve the extraction yield and is also beneficial to improving the corresponding purity, the concrete reason is not clear, probably because the specific structure of the styrene-divinylbenzene skeleton macroporous resin has stronger adsorption and elution specificity to cornflower-3-coumaroyl-diglucoside-5-glucoside, the inventor also tries the styrene-acrylate macroporous resin, and finds that the yield is worse than that of the styrene-divinylbenzene skeleton macroporous resin. Thus, in the enrichment and concentration steps, a styrene-divinylbenzene backbone is preferably used. As a commonly used styrene-divinylbenzene skeleton macroporous resin, there can be used macroporous resins of AB-8 type, HPD-100 type, D-101 type, and the like.
In the chromatographic column purification process, the two steps of medium-pressure column separation and gel separation are divided, and the combination and the sequence of the two steps are particularly important. In this step, the term "medium-pressure column separation" generally refers to a column chromatography separation at an elution pressure of less than 1 MPa. In the invention, the medium-pressure column separation is used for separating other components with polarity difference with a target object, and the balance between resolution and speed can be realized by adopting the elution pressure of 0.05-0.5 Mpa. And for the eluent of the medium-pressure column, eluting with acidic 4-5% acetonitrile water solution to remove impurities, eluting with acidic 7-9% acetonitrile water, collecting the eluent, concentrating to obtain a first crude product concentrated solution, preferably eluting with 5% acetonitrile water solution to remove impurities, eluting with acidic 8% acetonitrile water, collecting the eluent, and concentrating to obtain the first crude product concentrated solution. The middle reversed phase packing of the medium pressure column may be prepared from known nonpolar silica gel (e.g., C18(ODS), C8, C4, etc.) in which the bonded functional group is an alkane. Preferably C18(ODS) silica gel column, i.e., octadecylsilane bonded silica, and more preferably octadecylsilane bonded silica having a particle size of 40-60 μm, is used to further balance resolution and separation speed. In an aqueous solution of acidic acetonitrile, a weak acid is generally added to improve the resolution, and trifluoroacetic acid is preferred, and formic acid may be used. The inventors have found that the resolution of separation is best when the acidic 4% to 5% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03% to 0.07% trifluoroacetic acid and the acidic 7% to 9% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03% to 0.07% trifluoroacetic acid. The purity of the target substance obtained after the working procedure can reach about 35-40%. In the medium-pressure column separation step, the pressure of the medium-pressure reverse-phase column is preferably 0.1 to 0.2MPa, and this pressure is particularly preferable because the separation effect and the separation speed can be balanced.
And a gel separation step, namely loading the first crude product concentrated solution to a hydroxypropyl glucan gel chromatographic column, eluting by using acidic water containing 18-22% of acetonitrile, detecting by using an HPLC (high performance liquid chromatography) ultraviolet detector, collecting a sample solution according to an absorption peak shape, and concentrating to obtain a second crude product concentrated solution. This step is a conventional step, but the inventors have found that the order of separation with the medium-pressure column is very important, and the ultraviolet absorption state of each component in the second raw material concentrated solution obtained as described above is similar, and if the gel step is performed before the medium-pressure separation, it is difficult to collect a sample solution based on the absorption peak shape with an efficient ultraviolet detector, and the effect of separation with the gel separation column is hardly exhibited. In this step, the acidic 18 to 22% acetonitrile aqueous solution is preferably an acetonitrile aqueous solution containing 0.03 to 0.07% trifluoroacetic acid, since the resolution of the separation is the best. The purity of the target substance obtained after the working procedure can reach about 80-90%.
In conclusion, the cornflower-3-coumaroyl-diglucose-5-glucoside compound with the solid purity of more than 98% 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.
Example 1
A. Raw material extraction: adding 0.5kg of cabbage red powder into 5L of 0.05% hydrochloric acid methanol solution, performing ultrasonic extraction for 3 times, each time for 1 hour, cooling to room temperature, performing suction filtration, mixing filtrates, and concentrating at 45 deg.C until no alcohol smell exists, wherein the total volume is 500 mL;
B. and (3) macroporous resin enrichment: weighing 8Kg of AB-8 macroporous adsorption resin, loading the concentrated solution obtained in the step A into a column, dynamically adsorbing and sampling the concentrated solution by using the macroporous adsorption resin, eluting impurities by using 20L of water, eluting the target substance by using 30L of 0.05% hydrochloric acid methanol solution, collecting the eluent, and concentrating to 1L;
C. medium pressure preparation: one medium pressure column was loaded with about 100g solids crude concentrate, target content about 10g, and 4 in parallel.
Medium pressure column preparation: daiso RPS C18, 460X 100 mm, 1.6-1.7kg of filler. Flow rate: 70 mL/min. The anthocyanin sample has good solubility, and the pressure is basically 0.1-0.2 MPa.
Eluting with 5% acetonitrile (containing 0.05% trifluoroacetic acid) for about 4h, a volume of about 20L to remove the precursor impurity, eluting with 8% acetonitrile (containing 0.05% trifluoroacetic acid) for a target 3-4h, a volume of about 15L, and concentrating to obtain a mixture powder of about 36% cornflower-3-coumaroyl-diglucoside-5-glucoside, about 60g, and a target content of about 25 g;
D. gel purification: sephadex LH-20 chromatographic column, 1100 × 55 mm, 500g-600g packing is adopted. Controlling the flow rate to be 1 drop in 2-3 seconds;
dissolving 60g of the target crude product obtained in the step C with 20% acetonitrile (containing 0.05% trifluoroacetic acid) and purifying the target crude product by a gel column, eluting with 20% acetonitrile (containing 0.05% trifluoroacetic acid) to obtain about 500mL of the target crude product, observing an obvious color band and starting HPLC tracking detection, collecting target fractions, and concentrating the target fractions to obtain cornflower-3-coumaroyl-diglucose-5-glucoside (about 150mL of the target crude product, 6g of powder after concentration and about 4g of the target crude product) with the purity of about 89%;
E. high-pressure preparation: HPLC 89% cornflower-3-coumaroyl-diglucose-5-glucoside from step D was dissolved in 10mL of 15% acetonitrile and loaded once with about 1.2g of powder (about 800mg of the title compound). The column used YMC-Triart C18, 100X 250 mm, 5 μm. The flow rate is controlled to be 70mL/min, the detection wavelength is 280/540nm, the column pressure is controlled to be 8.0-10.0Mpa, and the linear gradient elution is carried out for 0-20min by using 15-20% acetonitrile (containing 0.05% formic acid). The target fraction was collected at about 16-17 min. Obtaining cornflower-3-coumaroyl-diglucose-5-glucoside solution with the HPLC purity of 98.4 percent;
F. and (3) solid product recovery: and E, concentrating the high-pressure fluid obtained in the step E for 20min at 45 degrees, and freeze-drying to obtain 0.50g of cornflower-3-coumaroyl-diglucoside-5-glucoside solid with the purity of 98.4 percent. The structure can be confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Please refer to fig. 6-8 for mass spectra and nuclear magnetic spectra.
The HPLC chromatogram of the target solution before the solid product recovery process and the HPLC chromatogram of the lyophilized solid can be referred to FIG. 1, and the high-purity formate of the target product can be stably provided.
Example 2
The same procedures as in example 1 were repeated except that in the E.high pressure preparation step, gradient elution was performed using an eluent of 15% to 20% acetonitrile (containing 0.1% formic acid), to obtain approximately 0.55g of cornflower-3-coumaroyl-diglucoside having a purity of 98.4%. The HPLC chromatogram of the liquid before the solid product recovery process and the HPLC chromatogram of the solid after freeze-drying can be referred to FIG. 2, and the high-purity formate of the target product can be stably provided.
Example 3
The same procedures as in example 1 were repeated except that in the E.high pressure preparation step, gradient elution was performed using an eluent of 15% to 20% acetonitrile (containing 0.5% formic acid), to obtain approximately 0.55g of cornflower-3-coumaroyl-diglucose-5-glucoside, which had a purity of 98.4%. The HPLC chromatogram of the liquid before the solid product recovery process and the HPLC chromatogram of the solid after freeze-drying can be referred to FIG. 3, and the high-purity formate of the target product can be stably provided.
Example 4
The same procedures as in example 1 were repeated except that in the E.high pressure preparation step, gradient elution was carried out using an eluent of 15% to 20% acetonitrile (containing 1% formic acid), to obtain approximately 0.55g of cornflower-3-coumaroyl-diglucose-5-glucoside with a purity of 98.4%. The HPLC chromatogram of the liquid before the solid product recovery process and the HPLC chromatogram of the solid after freeze-drying can be referred to FIG. 4, and the high-purity formate of the target product can be stably provided.
Comparative example 1
The same procedures as in example 1 were repeated except that in the E.high pressure preparation step, gradient elution was performed using an eluent of 15% to 20% acetonitrile (containing 0.075% hydrochloric acid), to obtain about 0.45g of cornflower-3-coumaroyl-diglucoside having a purity of 96.5%. The HPLC chromatogram of the liquid before the solid product recovery process and the HPLC chromatogram of the solid after lyophilization can be referred to FIG. 5. The hydrochloride of the target product cannot be stably provided in high purity, and degradation thereof is difficult to avoid even by freeze-drying.
The acidity of the 1% formic acid in acetonitrile used in example 4 was much higher than that of the 0.075% hydrochloric acid in acetonitrile used in the comparative example, suggesting that the stability of cyanidin-3-coumaroyl-diglucoside is not solely related to the acidity, and it should be assumed that the different anionic moieties have a greater effect on the stability of cyanidin-3-coumaroyl-diglucoside-5-glucoside.
At present, no nuclear magnetic spectrum of cornflower-3-coumaroyl-diglucose-5-glucoside is disclosed, and the nuclear magnetic spectrum of the target with very high definition is obtained by the preparation method (refer to fig. 7 and 8).
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 (5)
1. A preparation method of a reference substance of cornflower-3-coumaroyl-diglucose-5-glucoside is characterized by sequentially comprising the following steps:
an enrichment and concentration process, which is a process that a cabbage red extract is used as a raw material, methanol or ethanol solvent containing 0.03-0.06% hydrochloric acid by volume ratio is used for extraction, first raw material concentrated solution is obtained by concentration, the first raw material concentrated solution is loaded onto styrene-divinylbenzene framework macroporous resin, water is used for elution to remove impurities, then methanol or ethanol containing 0.03-0.06% hydrochloric acid by volume ratio is used for elution to obtain solution containing a target substance, and a second raw material concentrated solution is obtained by concentration,
a chromatographic column purification step, namely loading the second raw material concentrated solution to a medium-pressure reversed-phase chromatographic column, performing gradient elution at the pressure of 0.05-0.5 Mpa, wherein the elution process comprises the steps of eluting with an acidic 4-5% acetonitrile water solution to remove impurities, eluting with acidic 7-9% acetonitrile water, collecting the eluent, and concentrating to obtain a first crude product concentrated solution; loading the first crude product concentrated solution to a hydroxypropyl sephadex chromatographic column, eluting by using acidic water containing 18-22% of acetonitrile, detecting by using an HPLC ultraviolet detector, collecting a sample solution according to an absorption peak shape, concentrating and drying to obtain a crude product containing cornflower-3-coumaroyl-diglucoside-5-glucoside,
a high-pressure reversed-phase chromatographic column separation step, wherein a crude product containing cornflower-3-coumaroyl-diglucose-5-glucoside obtained in the chromatographic column purification step is loaded onto a high-pressure reversed-phase chromatographic column, and is eluted by water containing 0.05-1% of formic acid and 15-20% of acetonitrile in volume ratio under the pressure of 8.0-10.0Mpa to obtain a solution of a target substance,
and a solidification step, namely, performing reduced pressure removal on the solution of the target object obtained in the reverse phase chromatographic column preparation step at the temperature below 45 ℃ to remove acetonitrile, concentrating until the concentration of the cornflower-3-coumaroyl-diglucoside-5-glucoside is 5-50%, controlling the concentration process within 60 minutes, performing freeze drying on the concentrated solution, and then directly performing freeze drying to obtain solid powder of formate of the cornflower-3-coumaroyl-diglucoside-5-glucoside.
2. The method of claim 1, wherein the acidic 4-5% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03-0.07% trifluoroacetic acid, the acidic 7-9% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03-0.07% trifluoroacetic acid, and the acidic 18-22% acetonitrile aqueous solution is an acetonitrile aqueous solution containing 0.03-0.07% trifluoroacetic acid, in terms of volume ratio.
3. The method of claim 1, wherein the medium pressure reverse phase chromatography column is packed with octadecylsilane bonded silica having a particle size of 40 to 60 μm, and the high pressure reverse phase chromatography column is packed with octadecylsilane bonded silica having a particle size of 5 to 10 μm.
4. The method of claim 1, wherein the medium pressure reverse phase chromatography column is at a pressure of 0.1Mpa to 0.2 Mpa.
5. The method for preparing a control of cornflower-3-coumaroyl-diglucose-5-glucoside according to claim 1, wherein the elution of the high pressure reverse phase chromatography column is carried out using water containing 0.1-0.2% by volume of formic acid and 15-20% by volume of acetonitrile, the amounts being given by volume.
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| CN102229632A (en) * | 2011-05-25 | 2011-11-02 | 北京市农林科学院 | Preparation method of cyaniding-3-O-glucoside chloride |
| CN104403358A (en) * | 2014-11-10 | 2015-03-11 | 云南瑞宝生物科技有限公司 | Preparation method of heatproof anthocyanin pigment |
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| JPS63243167A (en) * | 1987-03-30 | 1988-10-11 | Hidekazu Idaka | Acylated anthocyanin |
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| CN102229632A (en) * | 2011-05-25 | 2011-11-02 | 北京市农林科学院 | Preparation method of cyaniding-3-O-glucoside chloride |
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Denomination of invention: Preparation method of reference substance of Centaurea cyanus 3-coumaroyl-diglucoside-5-glucoside Effective date of registration: 20230531 Granted publication date: 20210305 Pledgee: Shanghai Rural Commercial Bank Co.,Ltd. Shanghai pilot Free Trade Zone Lingang xinpian District sub branch Pledgor: SHANGHAI STANDARD TECHNOLOGY CO.,LTD. Registration number: Y2023310000223 |
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