Preparation method of anthocyanin reference substance

Abstract

The invention discloses a preparation method of an anthocyanin reference substance, and relates to the field of anthocyanin preparation, and the preparation method comprises the following steps: S1, dissolving an anthocyanin raw material with acid water, centrifuging, and filtering to obtain an anthocyanin clarified solution; s2, loading the anthocyanin clarified liquid into a medium-pressure preparative column for reversed-phase chromatography preparation, and adopting step isocratic elution by taking an acetonitrile-acid water system as an elution solvent to obtain a plurality of anthocyanin components; s3, respectively loading a sample into a medium-pressure preparation column for reversed-phase chromatography preparation, adopting isocratic elution, taking an acetonitrile-acid water system as an elution solvent, and carrying out vacuum concentration to remove acetonitrile so as to obtain a plurality of anthocyanin component concentrated solutions; and S4, respectively loading a sample into a medium-pressure preparation column for reversed-phase chromatography preparation, carrying out column concentration to remove acetonitrile and acid water, then carrying out step isocratic elution, carrying out halogenation by using an organic phase-hydrochloric acid water system, carrying out reduced-pressure concentration to remove acetonitrile until the solution is dry, and carrying out vacuum drying to prepare a plurality of anthocyanin reference substances. The method can be used for rapidly and efficiently separating and purifying the anthocyanin reference substance with the purity of 98% or above, and large-scale production is realized.

Description

Technical Field

[0001] The invention relates to the technical field of anthocyanin preparation, in particular to a method for preparing anthocyanin reference substance. Background Art

[0002] Anthocyanins are a class of compounds formed by anthocyanidins in a natural state and various monosaccharides with glycosidic bonds. They are widely present in the cell sap of flowers, fruits, stems, leaves and root organs of plants, making them appear in different colors from red, purple to blue. Anthocyanins are a class of substances based on flavonoid nuclei, which show different color changes under different pH conditions. There are more than 20 known types of anthocyanidins, and there are mainly 6 types existing in plants: cyanidin, malvidin, petunidin, delphinidin, peonydin, and pelargonidin. Under natural conditions, free anthocyanidins are extremely rare and mostly exist in the form of glycosides. Due to its unique functionality, it is used to remove free radicals in the body, anti-tumor, anti-cancer, anti-inflammatory, inhibit lipid peroxidation and platelet aggregation, prevent diabetes, lose weight, protect eyesight, etc. As a natural pigment, anthocyanins are safe, non-toxic, and have many health functions for the human body. They have been used in food, health products, cosmetics, medicine and other industries.

[0003] At present, domestic and foreign scholars have conducted a lot of research on the separation and purification of anthocyanins. Zhang Lixia et al.'s research showed that the macroporous resin AB-8 has good adsorption and resolution capabilities for blackberry anthocyanins, and under this condition, the purity of anthocyanins is 86.50%; Zheng Hongyan et al.'s research showed that the purity of blueberry anthocyanins after purification by XDA-7 macroporous resin increased from 2.20% to 24.54%; Liu Jingbo et al.'s research showed that after Amberlite XAD-7HP macroporous resin chromatography, Sep-Pak C18 solid phase extraction, Sephadex LH-20 gel chromatography, the purity of blueberry anthocyanins increased from 2.20% to 24.54%. Column separation, and finally semi-preparative high performance liquid chromatography technology was used to separate delphinidin-3-O-galactoside and malvidin-3-O-galactoside with purities of 96.98% and 95.63% respectively; Suo Yourui's authorized patent 201310658346.6 discloses the use of macroporous resin and high-efficiency semi-preparative chromatographic columns to separate anthocyanin monomers with a purity of ≥90% from Nitraria sibiricum fruit; Xin Xiulan's authorized patent 201010563334.1 discloses the use of blackberries as raw materials, pulping, extraction and concentration , impurity removal, and high-speed countercurrent chromatography purification to obtain a preparation method of cyanidin-3-glucoside with a purity of ≥95%; Mi Jia's patent application 202210434472.2 discloses the use of macroporous resin and gel column to separate and purify petunidin-3-O-rutinoyl (coumaryl)-5-O-diglucoside and petunidin-3-O-rutinoyl (coumaryl)-5-O-glucoside in black wolfberry, and the purity of both is between 80% and 85%; Xu Zhenzhen's patent application 2016106 71719.7 discloses the extraction of purple cabbage with acidified ethanol, the removal of alcohol from the extract, adsorption by macroporous resin, and purification of anthocyanins by high-speed countercurrent chromatography, and the purity of anthocyanins is only greater than 90%; Yue Huilan's patent application 201910081143.2 discloses the use of macroporous resin, SPE column enrichment, and reverse phase preparative column purification to obtain petunidin-3-O-rutinoside (trans-coumaric acid)-5-O-glucoside with a purity of ≥92.0% from black wolfberry. The above methods generally use macroporous resins to separate and purify anthocyanins, but the purity of the obtained anthocyanin monomers does not reach 98%.

[0004] Anthocyanins have very strong antioxidant activity. Qi Xiangyang et al. studied the extraction process of bayberry anthocyanins and their antioxidant activity. The results showed that the crude extract of bayberry had strong antioxidant activity, among which anthocyanins and other phenolic substances may be important antioxidant components in bayberry. Xue Hongwei et al. compared the in vitro antioxidant activity of different varieties of purple sweet potatoes and found that purple sweet potato anthocyanins had higher free radical scavenging ability and iron ion reduction ability than grape skin and purple rice anthocyanins. Li Yang et al. studied the in vitro free radical scavenging activity of several natural plant anthocyanins and found that litchi skin anthocyanins had the strongest activity in scavenging DPPH free radicals, paper mulberry anthocyanins had the strongest activity in scavenging hydroxyl free radicals, and mulberry anthocyanins had the strongest activity in scavenging superoxide anion free radicals, and each plant anthocyanin showed different antioxidant activities in different free radical systems. Bowen-Forbes et al. found that anthocyanin concentration of 50umol / mL can effectively control lipid peroxidation, and the inhibition rates of intestinal cancer, breast cancer, gastric cancer and lung cancer can reach 50%, 24%, 37% and 54% respectively.

[0005] Although scholars from various countries have made certain achievements in the study of anthocyanins, the purification methods that are currently being studied have mixed advantages and disadvantages, low efficiency and unclear purity, and cannot meet the needs of industrial production.

[0006] Moreover, anthocyanins exist in aqueous solution in the form of 2-phenyl-benzopyran cation, quinone base, pseudobase, and chalcone. These four forms undergo reversible or irreversible changes with the pH of the aqueous solution, and the color of the solution also changes with the change of structure (e.g. Figure 1 ), which puts higher requirements on the purification and separation technology of anthocyanins. Under the natural pH of fresh and processed vegetables and fruits, various forms of anthocyanins will exist in the form of equilibrium mixtures. Anthocyanins are also easily affected by temperature, light, oxygen, etc., which leads to a decrease in purity. Therefore, even after anthocyanins are separated and purified, their preservation is still a difficult point.

[0007] Therefore, it is particularly important to research and develop a fast and efficient anthocyanin separation and purification technology, and to make it stable and preserve it, so as to finally achieve industrial production. The purpose of the present invention is to develop a method for preparing a stable anthocyanin natural product standard product that can be quickly and efficiently separated and purified with a purity of more than 98%, so as to achieve scaled-up production. Summary of the invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing anthocyanin reference substances, which can quickly and efficiently separate and purify a stable anthocyanin natural product standard substance with a purity of more than 98%, so as to achieve scaled-up production.

[0009] To achieve the above object, the present invention provides the following technical solutions: A method for preparing anthocyanin reference substance comprises the following steps: S1. Raw material pretreatment: dissolving the anthocyanin raw material in acid water, centrifuging, filtering, and obtaining anthocyanin clarified liquid; S2 Anthocyanin segmented preparation: The anthocyanin clarified solution was loaded onto a medium pressure preparative column for reverse phase chromatography preparation, using a step isocratic elution method, the elution solvent was an acetonitrile-acid water system, and the target segment components were segmented and collected according to the chromatographic peak elution time to obtain multiple anthocyanin components; Preparation of S3 anthocyanin reference substance: the samples were loaded onto medium pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, the elution solvent was acetonitrile-acid water system, and the target segments were collected according to the chromatographic peak elution time, and the acetonitrile was removed by vacuum concentration to obtain multiple anthocyanin component concentrates; S4 anthocyanin column concentration and halogenation: load the samples onto a medium pressure preparative column for reverse phase chromatography preparation, perform column concentration to remove acetonitrile and acid water, then use a step isocratic elution method and use an organic phase-hydrochloric acid water system for halogenation, detect via HPLC, collect the target segment according to the elution time of the chromatographic peak, concentrate under reduced pressure to remove acetonitrile to dryness, and vacuum dry to prepare multiple anthocyanin reference substances.

[0010] Preferably, according to the determination of the types of anthocyanins in the anthocyanin raw material, when the number of anthocyanins is ≤4, step S2 can be omitted.

[0011] Preferably, the anthocyanin raw material is an anthocyanin extract, which is an extract of bilberry, blackcurrant or bilberry.

[0012] Preferably, in the pretreatment of S1 raw material, filtration is first performed by suction filtration using filter paper and then by suction filtration using a 0.45 um microporous filter membrane; the acid water for dissolving the anthocyanin raw material is 1.0% formic acid water or trifluoroacetic acid water.

[0013] Preferably, the medium-pressure preparation column filler in the segmented preparation of anthocyanins in S2 is a reverse phase pure water-resistant C18AQ filler, which is an octadecylsilane bonded silica filler with a particle size of 10um, a pore size of 100A, and a sample loading of 5.0%-10.0%, wherein the sample loading is calculated by the mass of the clarified liquid to the mass of the filler; The acid water in the acetonitrile-acid water system is 1.0% trifluoroacetic acid, the flow rate is 300 ml / min, and the wavelength is 535 nm.

[0014] Preferably, when the anthocyanin raw material is bilberry extract, the elution conditions in S2 are 0-47 min, 15% acetonitrile; 47-67 min, 20% acetonitrile; 67-77 min, 50% acetonitrile for elution; when the anthocyanin raw material is blackcurrant extract, the elution conditions in S2 are 0-50 min, 25% acetonitrile for elution.

[0015] Preferably, the filler used in the preparation of the anthocyanin reference substance in S3 is a reverse phase strong acid resistant C18SAT filler, which is an octadecylsilane bonded silica filler with a particle size of 5 or 10 um and a pore size of 100A; The acid water in the acetonitrile-acid water system is 1.0% trifluoroacetic acid water, the acetonitrile elution ratio is 15%-30%, the elution time is 30 minutes, the flow rate is 300 ml / min, and the wavelength is 535 nm.

[0016] Preferably, when the number of anthocyanin types in the anthocyanin raw material is ≤4, step S2 is omitted and the preparation of S3 is directly carried out, the sample loading amount in S3 is 1%, and the sample loading amount is calculated by the mass of the clarified liquid to the mass of the filler.

[0017] Preferably, the filler in the anthocyanin column concentration process and the halogenation process in S4 is a reverse phase water-resistant C18AQ filler with a particle size of 10um and a pore size of 100A. The halogenation is eluted with an acetonitrile-hydrochloric acid water system with a flow rate of 300ml / min, 0-8min, 0.01% hydrochloric acid water, 8-30min, 50%-80% acetonitrile-0.01% hydrochloric acid water, and a wavelength of 535nm.

[0018] Preferably, the temperature of the reduced pressure concentration is controlled at 30°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention can quickly obtain a series of high-purity anthocyanin reference substances, providing high-quality samples for further research on the pharmacological activity of anthocyanins.

[0020] 2. The present invention greatly simplifies the process flow of anthocyanin separation and purification: anthocyanin segmentation process - anthocyanin reference substance purification process - column concentration and halogenation process, which has the advantages of high efficiency and high yield, and can quickly obtain gram-level anthocyanin series reference substances.

[0021] 3. The whole process of the present invention uses only acetonitrile and acid water system as solvent, which is more environmentally friendly and conducive to production. The acetonitrile organic solvent is reused by concentration and recovery, saving solvent costs; the acid water removed by column concentration and the acid water flowing out of the column after halogenation are concentrated to remove acetonitrile, and the remaining acid water is recovered and then used to mix the mobile phase in the segmentation process of the next batch of products for reuse.

[0022] 4. The significance of halogenation is to allow the compound to exist in the form of hydrochloric acid to ensure stability.

[0023] 5. The present invention has strong versatility and is suitable for the separation of anthocyanin reference substances from a variety of anthocyanin extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the changes in the structure and color of anthocyanins in different pH ranges; Figure 2 Process flow chart of the preparation method of bilberry anthocyanin reference substance; Figure 3 HPLC analysis of bilberry anthocyanin series reference substances Figure a; Figure 4 Figure b: HPLC analysis of bilberry anthocyanin series reference substances; Figure 5 HPLC analysis chart of black currant anthocyanin series reference substances; Figure 6 HPLC analysis chart of the series of reference substances of anthocyanins from malvaceae. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0026] The present invention provides the following technical solutions: A method for preparing anthocyanin reference substance comprises the following steps: S1. Raw material pretreatment: dissolving the anthocyanin raw material in acid water, centrifuging, filtering, and obtaining anthocyanin clarified liquid; S2 Anthocyanin segmented preparation: The anthocyanin clarified solution was loaded onto a medium pressure preparative column for reverse phase chromatography preparation, using a step isocratic elution method, the elution solvent was an acetonitrile-acid water system, and the target segment components were segmented and collected according to the chromatographic peak elution time to obtain multiple anthocyanin components; Preparation of S3 anthocyanin reference substance: the samples were loaded onto medium pressure preparative columns for reverse phase chromatography preparation, isocratic elution was used, the elution solvent was acetonitrile-acid water system, and high performance liquid chromatography was used for detection. The target segments were collected according to the chromatographic peak elution time, and the acetonitrile was removed by vacuum concentration to obtain multiple anthocyanin component concentrates; too much acetonitrile organic solvent in S3 would cause the concentrated sample on the S4 column to flow through, so vacuum concentration was performed; S4 anthocyanin column concentration and halogenation: load the samples onto a medium pressure preparative column for reverse phase chromatography preparation, perform column concentration to remove acetonitrile and acid water, then use a step isocratic elution method and an organic phase-hydrochloric acid water system for halogenation, detect by HPLC, collect the target segment according to the elution time of the chromatographic peak, concentrate under reduced pressure to dryness to remove acetonitrile, and vacuum dry to prepare multiple anthocyanin reference substances; since the sample volume after preparation in S3 is large, it will take a long time to concentrate only through S3 reduced pressure concentration, and the sample is unstable and easily degraded, so it is necessary to perform column concentration through S4 step; in addition, hydrochloric acid water is used for halogenation to remove the formic acid or trifluoroacetic acid used previously.

[0027] Preferably, according to the determination of the types of anthocyanins in the anthocyanin raw material, when the number of anthocyanin types is ≤4, step S2 can be omitted; for raw materials with fewer types of anthocyanins, the steps can be simplified; Preferably, the anthocyanin raw material is an anthocyanin extract, which is an extract of bilberry, blackcurrant or bilberry.

[0028] Preferably, in the pretreatment of S1 raw material, filtration is first performed by suction filtration using filter paper and then by suction filtration using a 0.45 um microporous filter membrane; the acid water for dissolving the anthocyanin raw material is 1.0% formic acid water or trifluoroacetic acid water.

[0029] Preferably, the medium-pressure preparation column filler in the segmented preparation of anthocyanins in S2 is a reverse phase pure water-resistant C18AQ filler, which is an octadecylsilane bonded silica filler with a particle size of 10um, a pore size of 100A, and a sample loading of 5.0%-10.0%, wherein the sample loading is calculated by the mass of the clarified liquid to the mass of the filler; The acid water in the acetonitrile-acid water system is 1.0% trifluoroacetic acid, the flow rate is 300 ml / min, and the wavelength is 535 nm.

[0030] Preferably, when the anthocyanin raw material is bilberry extract, the elution conditions in S2 are 0-47 min, 15% acetonitrile; 47-67 min, 20% acetonitrile; 67-77 min, 50% acetonitrile for elution; when the anthocyanin raw material is blackcurrant extract, the elution conditions in S2 are 0-50 min, 25% acetonitrile for elution.

[0031] Preferably, the filler used in the preparation of the anthocyanin reference substance in S3 is a reverse phase strong acid resistant C18SAT filler, which is an octadecylsilane bonded silica filler with a particle size of 5 or 10 um and a pore size of 100A; The acid water in the acetonitrile-acid water system is 1.0% trifluoroacetic acid water, the acetonitrile elution ratio is 15%-30%, the elution time is 30 minutes, the flow rate is 300 ml / min, and the wavelength is 535 nm.

[0032] Preferably, when the number of anthocyanin types in the anthocyanin raw material is ≤4, step S2 is omitted and the preparation of S3 is directly carried out, the sample loading amount in S3 is 1%, and the sample loading amount is calculated by the mass of the clarified liquid to the mass of the filler.

[0033] Preferably, the filler in the anthocyanin column concentration process and the halogenation process in S4 is a reverse phase water-resistant C18AQ filler with a particle size of 10um and a pore size of 100A. The halogenation is eluted with an acetonitrile-hydrochloric acid water system with a flow rate of 300ml / min, 0-8min, 0.01% hydrochloric acid water, 8-30min, 50%-80% acetonitrile-0.01% hydrochloric acid water, and a wavelength of 535nm.

[0034] Preferably, the temperature of the reduced pressure concentration is controlled at 30°C.

[0035] Example 1 like Figures 2 to 4 S1 raw material pretreatment: the anthocyanin raw material is bilberry extract, named European bilberry, specification HPLC36%, manufacturer Green Health; 1KG bilberry extract is dissolved in 20L of 1.0% formic acid water, centrifuged, filtered, and anthocyanin clarified liquid is obtained; the filtration is first filtered with filter paper, and then filtered with a 0.45um microporous filter membrane.

[0036] S2 anthocyanin segmented preparation: The anthocyanin clarified liquid was loaded onto a medium-pressure preparative column at a loading volume of 10.0%, model 100×650mm, and the filler was a reversed-phase pure water-resistant C18AQ filler, which is an octadecylsilane bonded silica filler with a particle size of 10um and a pore size of 100A. The loading volume was calculated based on the mass of the clarified liquid to the mass of the filler; a step isocratic elution method was used, the elution solvent was an acetonitrile-acid water system, the acid water was 1.0% trifluoroacetic acid water, and the elution conditions were 0-47min , 15% acetonitrile; 47-67min, 20% acetonitrile; 67-77min, 50% acetonitrile; flow rate 300ml / min, wavelength 535nm; by high performance liquid chromatography detection, the target segment components were collected in segments according to the elution time of the chromatographic peaks, and 11 anthocyanin components were obtained, which were named YJ-1, YJ-2, YJ-3, YJ-4, YJ-5, YJ-6, YJ-7, YJ-8, YJ-9, YJ-10, and YJ-11 respectively.

[0037] Preparation of S3 anthocyanin reference substance: The above 11 anthocyanin components were loaded onto medium pressure preparative columns for reverse phase chromatography preparation. The filler was reverse phase strong acid resistant C18SAT filler, which is an octadecylsilane bonded silica filler with a particle size of 5 or 10um and a pore size of 100A. The isocratic elution method was adopted, and the elution solvent was an acetonitrile-acid water system. The acid water was 1.0% trifluoroacetic acid water, the acetonitrile elution ratio was 15%-30%, the elution time was 30min, the flow rate was 300ml / min, and the wavelength was 535nm. The target segment components were collected according to the chromatographic peak elution time, and the acetonitrile was removed by vacuum concentration to obtain a total of 20 anthocyanin component concentrates, as follows: YJ-1, YJ-2, YJ-3, and YJ-4: 10um reverse phase strong acid resistant C18SAT filler was used for preparation, and the mobile phase was 15% acetonitrile and 1.0% trifluoroacetic acid water. A total of 99.1% HPLC purity of delphinidin-3-O-galactoside was prepared, with a mass of 50.1g; 98.6% purity of delphinidin-3-O-glucoside, with a mass of 49.6g; 99.8% purity of cyanidin-3-O-galactoside, with a mass of 35.1g; 99.7% purity of delphinidin-3-O-arabinoside, with a mass of 40.5g; 99.2% purity of cyanidin-3-O-glucoside, with a mass of 35.3g; 99.1% purity of petunidin-3-O-galactoside, with a mass of 12.3g; YJ-5, YJ-6, YJ-7, and YJ-8 were prepared using 5um reverse-phase strong acid-resistant C18SAT filler, and the mobile phase was 20% acetonitrile and 1.0% trifluoroacetic acid water. A total of 98.8% HPLC purity of petunidin-3-O-glucoside was prepared, with a mass of 32.3 g; 99.3% purity of cyanidin-3-O-arabinoside was prepared, with a mass of 25.9 g; and 99.5% purity of delphinidin-3-O-glucoside was prepared, with a mass of 1.5 g; 99.2% purity of peonidin-3-O-galactoside, mass 4.2g; 98.8% purity of petunidin-3-O-arabinoside, mass 10.8g; 99.8% purity of peonidin-3-O-glucoside, mass 14.7g; 99.2% purity of malvidin-3-O-galactoside, mass 12.9g; 98.8% purity of malvidin-3-O-glucoside, mass 30.1g; YJ-9 and YJ-10: 10um reverse phase strong acid resistant C18SAT filler was used for preparation, and the mobile phase was 25% acetonitrile and 1.0% trifluoroacetic acid water. A total of 98.5% HPLC purity of peony-3-O-galactoside was prepared, with a mass of 3.1 g; 99.2% purity of peony-3-O-arabinoside was prepared, with a mass of 2.2 g; 99.7% purity of malvidin-3-O-glucoside was prepared, with a mass of 1.3 g; and 98.6% purity of cyanidin was prepared, with a mass of 5.1 g; YJ-11: 5um reverse phase strong acid resistant C18SAT filler was used for preparation, and the mobile phase was 30% acetonitrile and 1.0% trifluoroacetic acid water. A total of 99.3% cyanidin with a purity of HPLC was prepared, with a mass of 2.5g; 99.9% malvidin-3-O-arabinoside with a purity of HPLC was prepared, with a mass of 2.1g; 99.6% HPLC purity of petunidin with a mass of 1.5g; 99.5% HPLC purity of paeoniflorin with a mass of 1.3g; 98.3% HPLC purity of malvidin with a mass of 1.0g; S4 Anthocyanin column concentration and halogenation: The above 20 anthocyanin samples were loaded onto a medium-pressure preparative column for reverse phase chromatography preparation. The model was 100×650mm, and the filler was a reverse phase water-resistant C18AQ filler with a particle size of 10um and a pore size of 100A. The column was concentrated to remove acetonitrile and acid water. Then, a step isocratic elution method was used, and elution was performed using an acetonitrile-hydrochloric acid water system. The flow rate was 300ml / min, 0-8min, 0.01% hydrochloric acid water, 8-30min, 80% acetonitrile-0.01% hydrochloric acid water, and the wavelength was 535nm. HPLC detection was performed, and the target segment samples were collected according to the elution time of the chromatographic peak. Finally, the obtained samples were concentrated under reduced pressure at 30°C to remove acetonitrile to dryness, and vacuum dried to prepare 20 anthocyanin reference substances.

[0038] Anthocyanin sample purity analysis Figure 3 Figure 4 .

[0039] Example 2 like Figure 5 S1 raw material pretreatment: the anthocyanin raw material is black currant extract, named black currant, manufacturer: Daxinganling Lingbei, specification UV25%; 200g black currant extract is dissolved in 4L of 1.0% trifluoroacetic acid water, centrifuged, filtered, and anthocyanin clarified liquid is obtained; the filtration is first filtered with filter paper, and then filtered with a 0.45um microporous filter membrane.

[0040] S2 anthocyanin segmented preparation: The anthocyanin clarified liquid was loaded with a 5.0% sample loading onto a medium pressure preparation column, model 100×650mm, and the filler was a reverse phase pure water resistant C18AQ filler, which is an octadecylsilane bonded silica filler, with a particle size of 10um and a pore size of 100A. The sample loading amount was calculated by the mass of the clarified liquid to the mass of the filler; a step isocratic elution method was used, the elution solvent was an acetonitrile-acid water system, the acid water was 1.0% trifluoroacetic acid water, the elution conditions were 0-50min, 25% acetonitrile was used for elution, the flow rate was 300ml / min, the wavelength was 535nm, and the target segment components were collected segmentally according to the chromatographic peak elution time to obtain an anthocyanin component, named HJL-A segment; Preparation of S3 anthocyanin reference substance: The HJL-A section sample was loaded onto a medium-pressure preparative column for reverse phase chromatography preparation. The model was 100×650mm, and the filler was a reverse phase strong acid resistant C18SAT filler, which is an octadecylsilane bonded silica gel filler with a particle size of 10um and a pore size of 100A. The isocratic elution method was adopted, and the elution solvent was an acetonitrile-acid water system. The acid water was 1.0% trifluoroacetic acid water, the acetonitrile elution ratio was 15%, the elution time was 30min, the flow rate was 300ml / min, and the wavelength was 535nm. The target section anthocyanin components were collected according to the chromatographic peak elution time, and the acetonitrile was removed by vacuum concentration to obtain 4 anthocyanin component concentrates, as follows; Delphinidin-3-O-glucoside with an HPLC purity of 98.9% was prepared, with a mass of 10.6 g; delphinidin-3-O-rutinoside with an HPLC purity of 99.9% was prepared, with a mass of 15.1 g; cyanidin-3-O-glucoside with an HPLC purity of 99.4% was prepared, with a mass of 5.6 g; cyanidin-3-O-rutinoside with an HPLC purity of 98.7% was prepared, with a mass of 6.2 g.

[0041] S4 anthocyanin column concentration and halogenation: The four anthocyanin component concentrates were separately loaded onto a medium-pressure preparative column, model 100×650mm, for reverse phase chromatography preparation. The filler was a reverse phase water-resistant C18AQ filler with a particle size of 10um and a pore size of 100A. The column was concentrated to remove acetonitrile and acid water, and then the step isocratic elution method was used to use an acetonitrile-hydrochloric acid water system for halogenation. The flow rate was 300ml / min, 0-8min, 0.01% hydrochloric acid water, 8-30min, 50% acetonitrile-0.01% hydrochloric acid water, wavelength 535nm, and high performance liquid chromatography was used for detection. The target segment samples were collected according to the elution time of the chromatographic peak. Finally, the obtained samples were concentrated under reduced pressure at 30°C to remove acetonitrile to dryness, and vacuum dried to prepare 4 anthocyanin reference substances.

[0042] Preferred solution: Determine the number of anthocyanin types in the anthocyanin raw material by liquid phase analysis or by consulting data. If there are 4 types of anthocyanins in the black currant extract, step S2 can be omitted and preparation of S3 can be directly performed. The sample loading amount in S3 is 1%, and the sample loading amount is calculated by the mass of the clarified liquid to the mass of the filler.

[0043] Preparation of S3 anthocyanin reference substance: The anthocyanin clarified solution in S1 was directly loaded with a medium-pressure preparative column at a sample loading of 1% for reverse phase chromatography preparation. The model was 100×650mm, and the filler was a reverse phase strong acid resistant C18SAT filler, which is an octadecylsilane bonded silica gel filler with a particle size of 10um and a pore size of 100A. The isocratic elution method was adopted, and the elution solvent was an acetonitrile-acid water system. The acid water was 1.0% trifluoroacetic acid water, the acetonitrile elution ratio was 15%, the elution time was 30min, the flow rate was 300ml / min, and the wavelength was 535nm. The target anthocyanin components were collected according to the chromatographic peak elution time, and the acetonitrile was removed by vacuum concentration to obtain 4 anthocyanin component concentrates, as follows; Delphinidin-3-O-glucoside with an HPLC purity of 98.9% was prepared, with a mass of 10.6 g; delphinidin-3-O-rutinoside with an HPLC purity of 99.9% was prepared, with a mass of 15.1 g; cyanidin-3-O-glucoside with an HPLC purity of 99.4% was prepared, with a mass of 5.6 g; cyanidin-3-O-rutinoside with an HPLC purity of 98.7% was prepared, with a mass of 6.2 g.

[0044] S4 anthocyanin column concentration and halogenation: The four anthocyanin component concentrates were separately loaded onto a medium-pressure preparative column, model 100×650mm, for reverse phase chromatography preparation. The filler was a reverse phase water-resistant C18AQ filler with a particle size of 10um and a pore size of 100A. The column was concentrated to remove acetonitrile and acid water, and then the step isocratic elution method was used to use an acetonitrile-hydrochloric acid water system for halogenation. The flow rate was 300ml / min, 0-8min, 0.01% hydrochloric acid water, 8-30min, 50% acetonitrile-0.01% hydrochloric acid water, wavelength 535nm, and high performance liquid chromatography was used for detection. The target segment samples were collected according to the elution time of the chromatographic peak. Finally, the obtained samples were concentrated under reduced pressure at 30°C to remove acetonitrile to dryness, and vacuum dried to prepare 4 anthocyanin reference substances.

[0045] Anthocyanin sample purity analysis Figure 5 .

[0046] Example 3 like Figure 6 S1 raw material pretreatment: the anthocyanin raw material is berry extract, named berry, manufacturer Heilongjiang Lvshang, specification: UV15%; 100g berry extract is dissolved in 2L of 1.0% formic acid water, centrifuged, filtered, and anthocyanin clarified liquid is obtained; the filtration is first filtered with filter paper, and then filtered with a 0.45um microporous filter membrane.

[0047] The number of anthocyanin types in the anthocyanin raw material was determined by liquid phase analysis or reference to data. There were two types of anthocyanins in the berry extract, so step S2 of segmented preparation of anthocyanins was omitted and the preparation of S3 anthocyanin reference substance was directly performed.

[0048] Preparation of S3 anthocyanin reference substance: directly load the anthocyanin clarified solution with 1% sample loading onto a medium pressure preparative column, model 100×650mm, for reverse phase chromatography preparation, the filler is reverse phase strong acid resistant C18SAT filler, which is octadecylsilane bonded silica filler, 10um, pore size 100A; adopt isocratic elution mode, the elution solvent is acetonitrile-acid water system, the acid water is 1.0% trifluoroacetic acid water, the acetonitrile elution ratio is 18%, elution 30min, flow rate 300ml / min, wavelength 535nm, through high performance liquid chromatography detection, collect the target segment according to the chromatographic peak elution time, reduce pressure and concentrate to remove acetonitrile, and obtain 2 anthocyanin component concentrates, as follows: Cyanidin-3-O-galactoside was prepared with a HPLC purity of 99.4% and a mass of 5.2 g; The cyanidin-3-O-arabinoside with a HPLC purity of 99.1% was prepared and the mass was 3.5 g.

[0049] S4 anthocyanin column concentration and halogenation: The above two anthocyanin component concentrates were separately loaded onto a medium-pressure preparative column for reverse phase chromatography preparation. The filler was a reverse phase water-resistant C18AQ filler with a particle size of 10um and a pore size of 100A. The column was concentrated to remove acetonitrile and acid water. Then, a step isocratic elution method was used, and elution was performed using an acetonitrile-hydrochloric acid water system. The flow rate was 300ml / min, 0-8min, 0.01% hydrochloric acid water, 8-30min, 50% acetonitrile-0.01% hydrochloric acid water, and the wavelength was 535nm. HPLC detection was performed, and the target segment samples were collected according to the elution time of the chromatographic peak. Finally, the obtained samples were concentrated under reduced pressure at 30°C to remove acetonitrile to dryness, and vacuum dried to prepare two anthocyanin reference substances.

[0050] Anthocyanin sample purity analysis Figure 6 .

[0051] in conclusion: The present invention can realize rapid separation of anthocyanin compounds at high sample loadings, and obtain gram-level anthocyanin compounds, wherein the maximum sample loading of the segmentation process can reach 10%, thereby realizing the preparation of compounds at high sample loadings, and obtaining chlorinated anthocyanin monomer compounds, providing reference substances for their pharmacological activity or quality research.

[0052] The acetonitrile-acid water system is used in the purification process and can be reused, instead of the normal phase organic solvents such as ethyl acetate and petroleum ether used in traditional plant chemical separation, which is more environmentally friendly and safe.

[0053] It should be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing anthocyanin reference substance, It is characterized in that The following steps are involved: S1. Raw material pretreatment: dissolving the anthocyanin raw material in acid water, centrifuging, filtering, and obtaining anthocyanin clarified liquid; S2 Anthocyanin segmented preparation: The anthocyanin clarified solution was loaded onto a medium pressure preparative column for reverse phase chromatography preparation, using a step isocratic elution method, the elution solvent was an acetonitrile-acid water system, and the target segment components were segmented and collected according to the chromatographic peak elution time to obtain multiple anthocyanin components; Preparation of S3 anthocyanin reference substance: the samples were loaded onto medium pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, the elution solvent was acetonitrile-acid water system, and the target segments were collected according to the chromatographic peak elution time, and the acetonitrile was removed by vacuum concentration to obtain multiple anthocyanin component concentrates; S4 anthocyanin column concentration and halogenation: load the samples onto a medium pressure preparative column for reverse phase chromatography preparation, perform column concentration to remove acetonitrile and acid water, then use a step isocratic elution method and use an organic phase-hydrochloric acid water system for halogenation, detect via HPLC, collect the target segment according to the elution time of the chromatographic peak, concentrate under reduced pressure to remove acetonitrile to dryness, and vacuum dry to prepare multiple anthocyanin reference substances.

2. The method for preparing anthocyanin reference substance according to claim 1, It is characterized in that According to the determination of the types of anthocyanins in the anthocyanin raw material, when the number of anthocyanins is ≤4, step S2 can be omitted.

3. The method for preparing anthocyanin reference substance according to claim 1 or 2, It is characterized in that The anthocyanin raw material is anthocyanin extract, which is an extract of bilberry, blackcurrant or bilberry.

4. The method for preparing anthocyanin reference substance according to claim 1 or 2, It is characterized in that In the pretreatment of S1 raw materials, filtration is first performed with filter paper and then with a 0.45 um microporous filter membrane; the acid water for dissolving the anthocyanin raw materials is 1.0% formic acid water or trifluoroacetic acid water.

5. The method for preparing anthocyanin reference substance according to claim 1 or 2, It is characterized in that The medium-pressure preparation column filler for the segmented preparation of anthocyanins in S2 is a reverse phase pure water-resistant C18AQ filler, which is an octadecylsilane bonded silica filler with a particle size of 10um, a pore size of 100A, and a sample loading of 5.0%-10.0%, where the sample loading is calculated by the mass of the clarified liquid to the mass of the filler; The acid water in the acetonitrile-acid water system is 1.0% trifluoroacetic acid, the flow rate is 300 ml / min, and the wavelength is 535 nm.

6. The method for preparing anthocyanin reference substance according to claim 5, It is characterized in that When the anthocyanin raw material is bilberry extract, the elution conditions in S2 are 0-47 min, 15% acetonitrile; 47-67 min, 20% acetonitrile; 67-77 min, 50% acetonitrile for elution; when the anthocyanin raw material is black currant extract, the elution conditions in S2 are 0-50 min, 25% acetonitrile for elution.

7. The method for preparing anthocyanin reference substance according to claim 1 or 2, It is characterized in that The filler used in the preparation of the anthocyanin reference substance in S3 was a reverse phase strong acid resistant C18SAT filler, which is an octadecylsilane bonded silica filler with a particle size of 5 or 10 μm and a pore size of 100A; The acid water in the acetonitrile-acid water system is 1.0% trifluoroacetic acid water, the acetonitrile elution ratio is 15%-30%, the elution time is 30 minutes, the flow rate is 300 ml / min, and the wavelength is 535 nm.

8. The method for preparing anthocyanin reference substance according to claim 7, It is characterized in that When the number of anthocyanin types in the anthocyanin raw material is ≤4, step S2 is omitted and S3 is directly prepared. The sample loading amount in S3 is 1%, and the sample loading amount is calculated by the mass of the clarified liquid to the mass of the filler.

9. The method for preparing anthocyanin reference substance according to claim 1 or 2, It is characterized in that The filler in the anthocyanin column concentration process and halogenation process in S4 is a reverse phase water-resistant C18AQ filler with a particle size of 10um and a pore size of 100A. The halogenation is eluted with an acetonitrile-hydrochloric acid water system with a flow rate of 300ml / min, 0-8min, 0.01% hydrochloric acid water, 8-30min, 50%-80% acetonitrile-0.01% hydrochloric acid water, and a wavelength of 535nm.

10. The method for preparing anthocyanin reference substance according to claim 1 or 2, It is characterized in that The temperature of the reduced pressure concentration was controlled at 30°C.

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