CN112964804A - Method for preparing 5 anthocyanin standard products from blueberry anthocyanin extract - Google Patents
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Abstract
The invention relates to a method for preparing 5 anthocyanin standard products from a blueberry anthocyanin extract, which comprises the steps of treating a blueberry anthocyanin solution containing 5 anthocyanin components with an organic filter membrane, and performing semi-preparative high performance liquid chromatography to obtain the 5 anthocyanin standard products. The 5 kinds of anthocyanin are delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peoniflorin-3-O-glucoside and malvidin-3-O-glucoside. According to the method, different anthocyanin substances can be collected at different retention times by optimizing the treatment conditions of the semi-preparative high performance liquid chromatography, so that the method for simultaneously preparing 5 main anthocyanin standard substances from the blueberry anthocyanin extract is realized, the purity is high, and the yield is high; and the method is simple and convenient to operate, and can meet the large-scale preparation of anthocyanin in blueberries and the market demand on anthocyanin monomers.
Description
Technical Field
The invention belongs to the technical field of preparation of standard anthocyanin, and particularly relates to a method for preparing 5 anthocyanin standard products from a blueberry anthocyanin extract.
Background
Anthocyanidin or anthocyanidin is water soluble natural pigment, and the basic structure mother nucleus is 2-phenyl benzopyran, i.e. anthocyanidin (Flavonium). There are over 22 and over 500 classes of anthocyanins known in nature, the most common of which are 6, Cyanidin or hibiscus pigment (Cyanidin), Delphinidin or Delphinidin, Delphinidin (Delphinidin), Peonidin (Peonidin), Petunidin or petuniadin (petuniadin), Malvidin (Malvidin) and Pelargonidin (Pelargonidin). Anthocyanins are generally present in the form of glycosides, and are often glycosidically bonded to one or more of glucose, rhamnose, galactose, arabinose, etc. to form anthocyanins or anthocyanosides (Anthocyanin), the major anthocyanins being anthocyanidin-3-O-glucoside.
Anthocyanins are a family of compounds capable of presenting red color in flavonoid substances, and widely exist in cell sap of flowers, fruits, stems, leaves and root organs of plants, so that the anthocyanins present different colors from red, purple red to blue and the like. Due to its unique functionality, anthocyanins are used for scavenging free radicals in vivo, proliferating xanthophyll, resisting tumor, resisting cancer, resisting inflammation, inhibiting lipid peroxidation and platelet aggregation, preventing diabetes, reducing weight, protecting vision, etc. As a natural pigment, anthocyanin is safe and nontoxic, has a plurality of health care functions for human body, and is applied to industries of food, health care products, cosmetics, medicines and the like.
From the chemical structure, the anthocyanin has the characteristic of lacking one electron, contains a plurality of phenolic hydroxyl groups, and is easily attacked by active oxygen groups or free electrons, so the anthocyanin is considered as a powerful natural antioxidant, but due to the characteristic of the chemical structure, the anthocyanin has larger instability and is easy to degrade. Besides the structure of the anthocyanin, the stability of the anthocyanin can be affected by external pH, temperature, light, metal ions, auxiliary pigments, food additives and the like. This makes it very difficult to prepare and store pure anthocyanin standards, so that usually anthocyanin standards are expensive, and many anthocyanins are not yet available, thus bringing a great obstacle to the development and utilization of anthocyanins.
The blueberry fruit is rich in anthocyanin, and mainly comprises 5 kinds of delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, paeoniflorin-3-O-glucoside and malvidin-3-O-glucoside. Blueberry anthocyanin is called natural antioxidant, has the oxidation resistance 30 times of that of common pigment, and is a precious functional natural pigment. Therefore, the method for preparing anthocyanin monomers from blueberries by adopting a simple and easy method has important significance for basic research and industrial development of the blueberries.
The research on the preparation of anthocyanin monomers is reported at present, but the purity of the anthocyanin monomers can not reach the standard product, the types of the anthocyanin contained in different plants are different, and the preparation conditions are also different. At present, no report is found on a technology for simultaneously preparing 5 blueberry standard anthocyanins from blueberries.
Patent application No. CN201610979230 discloses a method for extracting, separating and purifying four main anthocyanins from blackcurrant pomace, which adopts medium-pressure flash reverse-phase chromatography to separate anthocyanin monomers: dissolving a blackcurrant anthocyanin resin concentrate in water, methanol and formic acid solution according to a certain proportion, feeding the solution to an ODS-C18 reversed phase silica gel medium-pressure column, performing equal gradient elution by using a mobile phase A-water: formic acid (90: 10), a mobile phase B-acetonitrile: methanol (85: 15), and an A: B ratio of 93: 7 at an elution flow rate of 20-35 mL/min, and collecting corresponding eluent to obtain a corresponding anthocyanin monomer; and performing low-temperature crystallization and recrystallization to obtain anthocyanin monomer components with purity of 95%, which are delphinidin 3-O-glucoside, delphinidin 3-O-rutinoside, cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside respectively. The patented technology has the following disadvantages: (1) the anthocyanin monomer prepared by chromatographic separation can reach 95 percent of purity only by low-temperature crystallization and recrystallization; (2) the method is used for preparing blackcurrant anthocyanin monomers, and the prepared anthocyanin types are few (4, 2 of which are common); (3) the composition of different plant anthocyanins is different, the types of blackcurrant anthocyanins are few, the types of blueberry anthocyanins are many (at least 13), and the blueberry anthocyanins cannot be well separated by using the condition of separating the blackcurrant anthocyanins; (4) the extraction and separation process of the patent technology is complex, and the process is subjected to homogenate crushing enzymolysis, negative pressure enhanced extraction, medium pressure flash chromatography separation and the like, so that the production cost is high, and the process cannot be done in a common laboratory.
The patent with the application number of CN201710293701.2 discloses a preparation method of an anthocyanin standard substance, namely petunidin-3-trans-p-coumaroyl rutinoside-5-glucoside, which is prepared by removing insoluble solid impurities and flavonoid impurities from anthocyanin and then extracting and purifying the insoluble solid impurities and the flavonoid impurities through glucan gel resin and a preparation type liquid phase, and can obtain the petunidin-3-trans-p-coumaroyl rutinoside-5-grape with the purity of more than 98 percent. The technical scheme is limited to the petunidin-3-trans-p-coumaroyl rutinoside-5-glucoside, and cannot be applied to the preparation of other monomer anthocyanins. The literature, chromatographic separation and purification of anthocyanin monomers in blueberry fruits (food science, 2017), applies a semi-preparative high performance liquid chromatography technology to prepare two galactosylated anthocyanin monomers with lower content in blueberry fruits from 3 anthocyanin purification components, and the anthocyanin monomers are identified as delphinidin-3-O-galactoside and malvidin-3-O-galactoside through analytical high performance liquid chromatography, wherein the purities are 96.98% and 95.63% respectively; the conditions of the semi-preparative high performance liquid chromatography are as follows: column Shim-pack PREP-ODS (H) KIT (20 mm. times.25 cm, 5 μm), normal temperature, detection wavelength 520nm, mobile phase: the phase A is methanol, the phase B is 3% formic acid solution, the flow rate is 2mL/min, the sample to be tested is dissolved by 3% formic acid, the sample amount is 2000 muL, and gradient elution is carried out. The technology has the following defects: (1) the chromatographic separation technology has complicated anthocyanin monomer process, adopts multiple column chromatography technologies such as macroporous resin chromatography, solid phase extraction, gel chromatography and semi-preparative high performance liquid chromatography, causes the loss of over 50 percent of total anthocyanin, has lower recovery rate, and has less (2) kinds of prepared anthocyanin, low purity (less than 97 percent) and higher production cost. (2) The chromatographic separation technology has poor separation effect, and can not well separate 5 common main anthocyanins.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for preparing 5 anthocyanin standard products from a blueberry anthocyanin extract, which is realized by the following technical scheme.
A method for preparing 5 anthocyanin standard products from blueberry anthocyanin extract comprises treating blueberry anthocyanin solution containing 5 anthocyanin components with organic filter membrane, and subjecting to semi-preparative high performance liquid chromatography to obtain 5 anthocyanin standard products; the chromatographic column conditions were: preparing a column: c18; flow rate: 4 mL/min; detection wavelength: 520 nm; detecting the temperature: 25 ℃; detection time: 80 min; mobile phase: the water phase B is 0.1 percent trifluoroacetic acid water solution, and the organic phase A is acetonitrile; gradient elution.
Preferably, the C18 column is Venusil MP C18 with the specification of 5 μm
10×250mm。
Preferably, the conditions of the gradient elution are: 0-19 min 91% B9% A; 20-29 min, 90% B, 10% A; 89% B11% A in 30-39 min; 87% B13% A at 40-49 min; 50-59 min 86% B14% A; 60-69 min 85% B15% A; 70-77 min 80% B20% A; 78min 91% B9% A.
Preferably, the 5 kinds of anthocyanin refers to delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peoniflorin-3-O-glucoside and malvidin-3-O-glucoside.
Preferably, the blueberry anthocyanin to-be-prepared solution is prepared by dissolving a blueberry anthocyanin extract in a methanol water solution, wherein the content of methanol in the methanol water solution is 0-50 w%.
Preferably, the concentration of the blueberry anthocyanin extract in the blueberry anthocyanin ready-to-prepare solution is 1mg/ml-50mg/ml
Preferably, the sample loading amount of the blueberry anthocyanin solution to be prepared is 1mg-10000 mg.
Preferably, the blueberry anthocyanin extract is prepared by extracting and enriching according to the prior patent or non-patent literature technology and contains 5 anthocyanin of delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunianin-3-O-glucoside, paeoniflorin-3-O-glucoside and malvidin-3-O-glucoside. Such as the document "Qi Xu, et al, enzyme-associated solvent extraction for extraction of blue and separation using solvent incorporated with extraction technologies" (int.J. food Sci.Tech.2016).
More preferably, the blueberry anthocyanin extract is prepared by the following technology: adding 0.3% of complex enzyme into fresh blueberry fruits, wherein the complex enzyme consists of cellulase and pectinase according to the mass ratio of 2: 1, and performing enzymolysis for 4 hours at 37 ℃ in a dark place; then adding 75% ethanol containing 1% citric acid, extracting for 6 hr, and filtering to obtain filtrate and residue; adding 75% ethanol containing 1% citric acid into the residue, extracting for 6 hr, and repeating for 2 times; combining the three filtrates, concentrating under reduced pressure to half volume, adding 2 times volume of ethyl acetate, and extracting for 3 times; combining the lower layers, concentrating under reduced pressure, performing anthocyanin enrichment through D101 macroporous adsorption resin, eluting with 40% ethanol at a rate of 2BV/h for 3BV, and freeze-drying (freezing end temperature-40 ℃, vacuum degree of 1Pa, material liquid thickness of 11mm) to obtain blueberry anthocyanin extract.
Preferably, the pore size of the organic filter membrane is 0.22 μm.
The invention has the beneficial effects that:
the invention carries out optimization research through the conditions of semi-preparative high performance liquid chromatography, provides a semi-preparative high performance liquid chromatography for preparing blueberry anthocyanin monomers, collects different anthocyanin substances at different retention times, and realizes the method for simultaneously preparing 5 main anthocyanin (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunianin-3-O-glucoside, paeoniflorin-3-O-glucoside, malvidin-3-O-glucoside) standards from blueberry anthocyanin extracts, wherein the purity is high and can reach 99%; the yield is high and can reach 22.5%, 6.8%, 9.9%, 12.8% and 20.5% respectively. And the method is simple and convenient to operate, and can meet the large-scale preparation of anthocyanin in blueberries and the market demand on anthocyanin monomers.
Drawings
FIG. 1 is a high performance liquid chromatography chromatogram of blueberry anthocyanin extract.
FIG. 2 is a spectrum diagram of a semi-preparative high performance liquid chromatography for separating blueberry anthocyanin extract.
FIG. 3 is a high performance liquid chromatography spectrum diagram for determining the purity of isolated anthocyanin 1 (delphinidin-3-O-glucoside).
FIG. 4 is a high performance liquid chromatography spectrum diagram for determining the purity of isolated anthocyanin 3 (petunidin-3-O-glucoside).
FIG. 5 is a high performance liquid chromatography chromatogram for determining the purity of isolated anthocyanin 4 (phytochrome-3-O-glucoside).
FIG. 6 is a high performance liquid chromatography spectrum diagram for determination of purity of isolated anthocyanin 5 (malvidin-3-O-glucoside).
Detailed Description
The technical solution of the present invention is further limited by the following specific embodiments, but the scope of the claims is not limited to the description.
Example 1
First, materials and reagents: the tested blueberries were collected from the blueberry planting base in Majiang county, Kaili, Guizhou, and the blueberry varieties were rabbit eye series. The reagent comprises chromatographically pure acetonitrile, methanol, trifluoroacetic acid, analytically pure ethanol, citric acid, ethyl acetate and the like
II, instrument equipment: analytical and semi-preparative high performance liquid chromatography system (Thermo Scientific UltiMate 3000); LGJ-50F vacuum freeze dryer (Beijing Songyuan Huaxing science and technology development Co., Ltd.); BEATL-10 ultrapure water preparation instrument (Shandong Tinglan environmental protection science and technology Co., Ltd.); in addition, the device also relates to instruments such as a rotary evaporator, an ultrasonic cleaning instrument, a digital display constant temperature water bath, an ultrafiltration device and the like.
Thirdly, preparing the blueberry anthocyanin extract:
adding 0.3% of complex enzyme into fresh blueberry fruits, wherein the complex enzyme consists of cellulase and pectinase according to the mass ratio of 2: 1, and performing enzymolysis for 4 hours at 37 ℃ in a dark place; then adding 5-10 times volume of 75% ethanol containing 1% citric acid, extracting for 6 hr, and filtering to obtain filtrate and residue; adding 5-10 times volume of 75% ethanol containing 1% citric acid into the residue, extracting for 6 hr, and repeating for 2 times; combining the three filtrates, concentrating under reduced pressure to half volume, adding 2 times volume of ethyl acetate, and extracting for 3 times; and combining the lower layers, concentrating under reduced pressure, enriching anthocyanin through D101 macroporous adsorption resin, eluting with 30-50% ethanol, and carrying out freeze drying treatment to obtain the blueberry anthocyanin extract.
High performance liquid chromatography analysis of blueberry anthocyanin extract
High performance liquid chromatography parameters: venusil MP C18 column (5 μm,
4.6X 250 mm); flow rate: 0.8 mL/min; detection wavelength: 520nm and 254 nm; detecting the temperature: 25 ℃; sample introduction volume: 10 mu L of the solution; detection time: and (5) 60 min.
Mobile phase: the water phase B is 0.3% phosphoric acid water solution, and the organic phase A is acetonitrile.
And (3) an elution mode: gradient elution is shown in Table 1
TABLE 1 high Performance liquid chromatography gradient elution mode
Anthocyanin has unique absorption characteristics, and can be simply determined by spectral characteristics. Through chromatographic analysis, the blueberry anthocyanin extract is mainly provided with 13 anthocyanin types, as shown in figure 1.
Research on preparation of blueberry anthocyanin monomer by using semi-preparative high performance liquid chromatography
1. Preparing a blueberry anthocyanin sample solution to be detected: dissolving blueberry anthocyanin extract 10mg with solvent, diluting to a constant volume of 10mL to obtain a sample solution, and filtering the sample solution with a 0.22-micron organic filter membrane to obtain a preparation solution for later use. The solvent is methanol water solution, wherein the content of methanol is 30%.
2. Screening of semi-preparative HPLC separation conditions
2.1 chromatographic column screening Studies
The experiment used Shim-pack PREP-ODS (H) KIT (20 mm. times.25 cm, 5 μm,
)、Agilent ZORBAX SB-C18(10×250mm，5μm，
) And Venusi MP C18(10 x 250mm, 5 μm,
) The blueberry anthocyanin extract was separated by three different chromatographic columns, and the results are shown in table 2.
TABLE 2
As can be seen from Table 2, the difference of the chromatographic columns has a certain influence on the separation effect of blueberry anthocyanins, and the three chromatographic columns can simultaneously separate 5 kinds of blueberry anthocyanins, so that compared with Agilent ZORBAX SB-C18 and Venusi MP C18 chromatographic columns, the separation effect is better, and the Venusi MP C18 chromatographic column is preferred.
2.2 Mobile phase screening Studies
And respectively carrying out mobile phase screening on the A-phase methanol, the acetonitrile, the B-phase formic acid water, the trifluoroacetic acid water, the acetic acid water and the phosphoric acid water, carrying out gradient elution, and inspecting the separation effect of chromatographic peaks. Table 3 shows the separation of the blueberry anthocyanidin extract in different mobile phases.
TABLE 3
As can be seen from Table 3, the different proportions of the mobile phase have a great influence on the separation effect of blueberry anthocyanin. The methanol-water system cannot well separate 5 types of blueberry anthocyanins simultaneously, the acetonitrile-water system has a good separation effect on the blueberry anthocyanins, and in view of the fact that a large amount of phosphoric acid water system blocks a chromatographic column, experiments preferably adopt 0.1% trifluoroacetic acid water-acetonitrile as a mobile phase to prepare the anthocyanins.
2.3 screening study of elution conditions
The study was performed with acetonitrile (solution A) and 0.1% trifluoroacetic acid (solution B) from 90% to 80% in 0-100 min. Other elution conditions were: the sample amount was 10mg, the flow rate was 4mL/min, the column temperature was 25 ℃ and the monitoring wavelength was 520 nm. The initial gradient composition was then adjusted to 5% of solvent a and 95% of solvent B. Finally, the initial gradient composition was further adjusted to 9% a and 91% solvent B. By comparison, the elution gradients obtained by the optimization screen are shown in table 4.
TABLE 4 semi-preparative HPLC gradient elution mode
2.4 investigation of flow Rate
Through comparison of three flow rates of 2mL/min, 4mL/min and 6mL/min, the flow rate has no great influence on the peak separation effect of anthocyanin, but the large or small flow rate can cause incomplete separation of certain components, and when the flow rate is small, the elution time is prolonged, and the elution capacity is poor; at higher flow rates, the elution capacity increases and component stacking occurs. By comparison, 4mL/min was selected as the flow rate for blueberry anthocyanin production.
2.5 investigation of preparation temperature
The temperature affects the physical properties of the solvent, such as density, viscosity, etc. In general, as the column temperature increases, the time to peak decreases, but component stacking occurs. In view of the similarity of structure and polarity of 5 anthocyanins, and the sensitivity to temperature. Higher temperatures can affect the separation effect and degrade anthocyanin. The separation effect of 20 ℃, 25 ℃ and 30 ℃ is examined in the experiment, and the separation temperature is determined to be 25 ℃ according to the experimental result.
Determination of detection wavelength
The maximum absorption wavelength of 5 anthocyanins at about 520nm is experimentally observed for 515nm, 520nm and 530 nm. The results show that the peak shape and absorption at 520nm are good, and 520nm is selected as the detection wavelength.
3. Preparation of blueberry anthocyanin
Preparing a semi-preparative high performance liquid chromatography according to the chromatographic strip configuration optimized and screened in the step 2, wherein the chromatographic conditions are as follows: venusil MP C18 with specification of 5 μm
10X 250 mm. Flow rate: 4 mL/min; detection wavelength: 520 nm; detecting the temperature: 25 ℃; detection time: 80 min; mobile phase: the water phase B is 0.1 percent trifluoroacetic acid water solution, and the organic phase A is acetonitrile prepared mobile phase; the gradient elution pattern is shown in Table 4.
And (3) loading the blueberry anthocyanin to-be-detected sample solution processed in the step (1) into a semi-preparative high performance liquid chromatography to obtain a chromatogram shown in a figure 2, which is very similar to an analysis spectrum shown in a figure 1. Collecting eluate flowing out in 35min, 46min, 52min, 63min and 68min periods according to chromatogram, concentrating, and drying to obtain 5 main anthocyanins with high purity and high yield, which correspond to semi-preparative high performance liquid chromatography chromatogram shown in FIG. 2.
4. Anthocyanin structure identification
And comparing the prepared chromatogram of the sample with a preset standard chromatogram, and performing mass spectrum identification by HPLC-ESI-MS to obtain the variety of anthocyanin in the sample to be detected. Standard delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, paeoniflorin-3-O-glucoside and malvidin-3-O-glucoside are all HPLC chromatographically pure. The obtained 5 major anthocyanins corresponding to the eluents at each peak time of the semi-preparative HPLC chromatogram are shown in Table 5.
TABLE 5 semi-preparative 5 major anthocyanins corresponding to the peak time of HPLC chromatogram
Note: indicates the markers in semi-preparative HPLC chromatogram 2
5. Determination of anthocyanin purity and yield
And respectively analyzing the purity of the high performance liquid chromatography according to the prepared anthocyanin monomers.
High performance liquid chromatography parameters: venusil MP C18 column (5m, 100, 4.6X 250 mm); flow rate: 0.8 mL/min; detection wavelength: 520 nm; detecting the temperature: 25 ℃; sample introduction volume: 10 mu L of the solution; detection time: 60min mobile phase: the water phase B is 0.3 percent phosphoric acid water solution, and the organic phase A is acetonitrile; and (3) an elution mode: the gradient elution pattern is shown in Table 1.
Dissolving a certain amount of anthocyanin dry powder prepared in the step 3 with 30% methanol, detecting by using HPLC, obtaining the purity of 5 anthocyanins according to the detection result as shown in figure 3, and calculating to obtain the yield of the 5 anthocyanins prepared from the blueberry anthocyanin extract as shown in table 6.
TABLE 6 anthocyanin purity and yield
Note: indicates the markers in semi-preparative HPLC chromatogram 2
# shows that the preparation is performed by two times of semi-preparative high performance liquid chromatography
And (3) comparative analysis:
comparative analysis 1:
the preparation method is characterized by comprising the following steps of preparing a semi-preparation high-performance chromatography method for preparing blueberry anthocyanin monomers, which is disclosed in the literature 'chromatographic separation and purification of anthocyanin monomers in blueberry fruits' (food science, 2017):
preparing a sample to be tested: taking blueberry anthocyanin extract 10mg, adding 3% formic acid solution, ultrasonically dissolving for 5min, fixing the volume to 10mL, filtering by a 0.45 mu m microporous filter membrane, injecting a sample, and performing semi-preparative high performance liquid chromatography separation.
The conditions of the semi-preparative high performance liquid chromatography are as follows: column Shim-pack PREP-ODS (H) KIT (20 mm. times.25 cm, 5 μm); normal temperature; the detection wavelength is 520 nm; mobile phase: the phase A is methanol, and the phase B is 3% formic acid solution; the flow rate is 2 mL/min; the sample size was 2000. mu.L. Gradient elution: 0-5 min, 85% -80% B; for 6-10 min, 80-75% of B; 11-15 min, 75% B; 16-55 min, 75% -30% of B; 56-60 min, 30% B; 61-65 min, 85% B; 66-78 min, 85% B.
TABLE 7 anthocyanin purity and yield
Comparing table 6, it is clear that the process is inferior in both purity of isolation and yield to the process.
Comparative analysis 2:
according to the patent CN201610979230, a method for extracting, separating and purifying four main anthocyanins from blackcurrant pomace is disclosed:
separating anthocyanin monomers by medium-pressure flash reversed-phase chromatography: taking blueberry anthocyanin extract. Adding the initial mobile phase for dissolution, and filtering and sampling by a 0.45-micron filter membrane; the medium-pressure chromatographic column is filled with ODS-C18 reversed-phase silica gel, the loading volume is 1/8 of the column volume, mobile phase A-water: formic acid (90: 10), mobile phase B-acetonitrile: methanol (85: 15), A: B is 93: 7, and the like, and the elution flow rate is 20 ml/min. The effluent components were checked by HPLC at a detection wavelength of 520nm, and the same fractions were combined and then concentrated to dryness under reduced pressure, respectively.
TABLE 8 anthocyanin purity and yield
Comparing table 6, it is clear that the purity and yield of the isolation of this method are far inferior to that of the present method.
It should be noted that the above examples and test examples are only for further illustration and understanding of the technical solutions of the present invention, and are not to be construed as further limitations of the technical solutions of the present invention, and the invention which does not highlight essential features and significant advances made by those skilled in the art still belongs to the protection scope of the present invention.
Claims (10)
1. A method for preparing 5 anthocyanin standard products from a blueberry anthocyanin extract is characterized in that a blueberry anthocyanin solution containing 5 anthocyanin components is treated by an organic filter membrane and then subjected to semi-preparative high performance liquid chromatography to prepare 5 anthocyanin standard products; the chromatographic column conditions were: preparing a column: c18; flow rate: 4 mL/min; detection wavelength: 520 nm; detecting the temperature: 25 ℃; detection time: 80 min; mobile phase: the water phase B is 0.1 percent trifluoroacetic acid water solution, and the organic phase A is acetonitrile; gradient elution.
2. The method for preparing 5 anthocyanin standards from blueberry anthocyanin extract as claimed in claim 1, wherein the C18 column is Venusil MP C18 with specification of 5 μm
10×250mm。
3. The method for preparing 5 anthocyanin standards from blueberry anthocyanin extracts as claimed in claim 1, wherein the gradient elution conditions are as follows: 0-19 min 91% B9% A; 20-29 min, 90% B, 10% A; 89% B11% A in 30-39 min; 40-49 min 87% B13% A; 50-59 min 86% B14% A; 60-69 min 85% B15% A; 70-77 min 80% B20% A; 78min 91% B9% A.
4. The method for preparing 5 anthocyanin standards from a blueberry anthocyanin extract as set forth in claim 1, wherein the 5 anthocyanins are delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunia-3-O-glucoside, peoniflorin-3-O-glucoside and malvidin-3-O-glucoside.
5. The method for preparing 5 anthocyanin standards from a blueberry anthocyanin extract as claimed in claim 1, wherein the blueberry anthocyanin ready-to-prepare solution is prepared by dissolving a blueberry anthocyanin extract in a methanol aqueous solution, wherein the content of methanol in the methanol aqueous solution is 0-50 w%.
6. The method for preparing 5 anthocyanin standards from a blueberry anthocyanin extract as claimed in claim 5, wherein the concentration of the blueberry anthocyanin extract in the blueberry anthocyanin ready-to-prepare solution is 1mg/ml to 50 mg/ml.
7. The method for preparing 5 anthocyanin standards from blueberry anthocyanin extracts as claimed in claim 1, wherein the loading amount of the blueberry anthocyanin solution to be prepared is lmg-10000 mg.
8. The method for preparing 5 anthocyanin standards from blueberry anthocyanin extract as claimed in claim 5, wherein the blueberry anthocyanin extract is extracted and enriched according to the existing patent or non-patent literature technology.
9. The method for preparing 5 anthocyanin standards from a blueberry anthocyanin extract as set forth in claim 8, wherein the blueberry anthocyanin extract is prepared by the following technology: adding 0.1-0.5% of complex enzyme into fresh blueberry fruit, wherein the complex enzyme consists of cellulase and pectinase according to the mass ratio of 1: 1-4: 1, and performing enzymolysis for 4 hours at 37 ℃ in a dark place; then adding 75% ethanol containing 0.5-1.5% citric acid, extracting for 6 hr, and filtering to obtain filtrate and residue; adding 75% ethanol containing 1% citric acid into the residue, extracting for 6 hr, and repeating for 2 times; combining the three filtrates, concentrating under reduced pressure to half volume, adding 2 times volume of ethyl acetate, and extracting for 3 times; combining the lower layers, concentrating under reduced pressure, enriching anthocyanin by using D101 macroporous adsorption resin, eluting with 30-50% ethanol at the flow rate of 1-3BV/h and the using amount of 2-4BV ethanol solution, and freeze-drying at the freezing end temperature of-30 ℃ -50 ℃, the vacuum degree of 1-5Pa and the material liquid thickness of 10-15mm to obtain the blueberry anthocyanin extract.
10. The method for preparing 5 anthocyanin standards from blueberry anthocyanin extracts as claimed in claim 1, wherein the pore size of the organic filter membrane is 0.22 μm.
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