CN108409807B - Method for separating and preparing malvidin-3-O-glucoside - Google Patents
Method for separating and preparing malvidin-3-O-glucoside Download PDFInfo
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- PXUQTDZNOHRWLI-OXUVVOBNSA-O malvidin 3-O-beta-D-glucoside Chemical compound COC1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)=C1 PXUQTDZNOHRWLI-OXUVVOBNSA-O 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 139
- 229930002877 anthocyanin Natural products 0.000 claims abstract description 93
- 235000010208 anthocyanin Nutrition 0.000 claims abstract description 93
- 239000004410 anthocyanin Substances 0.000 claims abstract description 93
- 150000004636 anthocyanins Chemical class 0.000 claims abstract description 89
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- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 18
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 15
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- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 2
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- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims 1
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- KZMACGJDUUWFCH-UHFFFAOYSA-O malvidin Chemical compound COC1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O)=C1 KZMACGJDUUWFCH-UHFFFAOYSA-O 0.000 description 4
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- 235000006251 pelargonidin Nutrition 0.000 description 2
- XVFMGWDSJLBXDZ-UHFFFAOYSA-O pelargonidin Chemical compound C1=CC(O)=CC=C1C(C(=C1)O)=[O+]C2=C1C(O)=CC(O)=C2 XVFMGWDSJLBXDZ-UHFFFAOYSA-O 0.000 description 2
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- XFDQJKDGGOEYPI-UHFFFAOYSA-O peonidin Chemical compound C1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O)=C1 XFDQJKDGGOEYPI-UHFFFAOYSA-O 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- RKWHWFONKJEUEF-GQUPQBGVSA-O Cyanidin 3-O-glucoside Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 RKWHWFONKJEUEF-GQUPQBGVSA-O 0.000 description 1
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- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- 235000009715 Rubus hirsutus Nutrition 0.000 description 1
- 235000012511 Vaccinium Nutrition 0.000 description 1
- 241000736767 Vaccinium Species 0.000 description 1
- 244000077233 Vaccinium uliginosum Species 0.000 description 1
- 244000077923 Vaccinium vitis idaea Species 0.000 description 1
- 235000017606 Vaccinium vitis idaea Nutrition 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 235000019216 blueberry extract Nutrition 0.000 description 1
- YTMNONATNXDQJF-UBNZBFALSA-N chrysanthemin Chemical compound [Cl-].O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 YTMNONATNXDQJF-UBNZBFALSA-N 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
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- 235000007336 cyanidin Nutrition 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
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- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 235000019990 fruit wine Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
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- 230000004054 inflammatory process Effects 0.000 description 1
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- IFYOHQQBIKDHFT-ASZXTAQUSA-N pelargonidin 3-O-rutinoside betaine Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](OC=2C(=[O+]C3=CC(O)=CC([O-])=C3C=2)C=2C=CC(O)=CC=2)O1 IFYOHQQBIKDHFT-ASZXTAQUSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
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- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Saccharide Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a method for separating and preparing malvidin-3-O-glucoside, which comprises the steps of alcohol extraction and concentration, macroporous resin adsorption, preparation liquid chromatography purification and high-speed countercurrent chromatography separation, and malvidin-3-O-glucoside monomer is separated and prepared from complex blueberry raw materials consisting of anthocyanin. The preparation method combines the preparation liquid chromatography and the high-speed counter-current chromatography technology for the first time, and obtains the high-purity malvidin-3-O-glucoside purity monomer separated from the blueberries by optimizing the process parameters, wherein the purity of the monomer can reach 99%.
Description
Technical Field
The invention relates to the field of separation and purification of natural products, in particular to a method for separating and preparing malvidin-3-O-glucoside.
Background
Anthocyanin is a water-soluble pigment widely present in plants, and is a polyphenol compound formed by combining anthocyanin with one or more glycosyl groups, such as glucose, galactose, arabinose and the like, through glycosidic bonds. The anthocyanins commonly found in nature are mainly 6 kinds, namely Pelargonidin (Pelargonidin), cyanidin (cyanianidin), Delphinidin (Delphinidin), Peonidin (Peonidin), petuniadin (petuniadin) and Malvidin (Malvidin). In recent years, a large number of researches prove that natural anthocyanin has biological activities of resisting oxidation and tumors, preventing cardiovascular diseases, relieving diabetes, controlling obesity and the like. Along with the continuous improvement of living standard of people, people have higher and higher call for functional components which are natural in source and have no toxic or side effect, and anthocyanin is favored by researchers and food and medicine enterprises due to good coloring function and excellent biological activity as the most representative functional factor of natural source.
However, due to the similar structure and small polarity difference of anthocyanin compounds, the separation and purification of high-purity anthocyanin monomers are extremely difficult. However, at present, it has been reported that high-purity anthocyanin monomers are obtained by separation and purification.
For example, chinese patent publication No. CN 106366141 a discloses a method for separating and preparing geraniin-3-O-glucoside monomer, which is prepared from strawberry as a raw material by freeze drying, alcohol extraction and concentration, fractional extraction, and purification with AB-8 macroporous resin. Also, for example, chinese patent publication No. CN 106831911 a discloses a method for separating and purifying pelargonidin-3-O-glucoside monomer from Tephrodis hirsutus, which comprises alcohol extraction and concentration, ethyl acetate extraction, AB-8 macroporous resin and high-speed countercurrent chromatography.
In the technical scheme, high-purity anthocyanin monomers are prepared, but the main reason is that the anthocyanin in the strawberries and the rubus hirsutus is simple in composition, and only 3 anthocyanin compounds of cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside are contained, wherein the pelargonidin-3-O-glucoside accounts for more than 80% of the total anthocyanin content. However, for the raw materials with complicated anthocyanin composition, the technical scheme is difficult to realize the purification and preparation of the high-purity anthocyanin monomer.
Blueberry, also known as cowberry and blueberry, belongs to Ericaceae and is a plant of Vaccinium, not only contains essential nutrients required by human body, but also contains various kinds of anthocyanin, and has effects of activating retina, reducing blood sugar, resisting inflammation and resisting tumor. The blueberry cultivation in China starts late, the direct fresh food consumption is mainly used at present, or the blueberry cultivation is processed into primary products such as jam, fruit juice, fruit wine and the like, and related blueberry products with high additional values are few in the markets at home and abroad. Because the anthocyanin in the blueberry is complex in composition and contains at least 12 anthocyanin compounds with similar structures, the high-purity anthocyanin prepared by single column chromatography or chromatographic technology at present is an anthocyanin mixture, but not a high-purity anthocyanin monomer.
For example, in chinese patent publication No. CN 106905391a, a method for extracting, separating and purifying blueberry anthocyanin is disclosed, which comprises mixing blueberry juice with an extracting agent, performing homogeneous extraction for 1-4 times under the conditions of normal temperature and pressure of 100-160 MPa, filtering, combining filtrates to obtain a crude extract of blueberry anthocyanin, and then performing separation and purification by using HPD600 macroporous resin. According to the technical scheme, an 80% ethanol solution with the pH value of 1-2 is used as an extracting agent, the blueberry functional components are released from biological cells by using high pressure, the problem that the effective components of the blueberry are not damaged by high temperature on the premise of keeping high extraction rate is solved, the obtained extract is an anthocyanin mixture, and the content of anthocyanin is only 46.45%.
Also, as disclosed in chinese patent publication No. CN 104109403a, a new method for extracting and purifying wild blueberry anthocyanin is disclosed, and the preparation process includes: biological enzymolysis, microwave reflux extraction, collection, coarse filtration, microfiltration, ultrafiltration, vacuum freeze drying, phase separation and high-speed countercurrent chromatography purification. The technical scheme adopts a non-thermal efficient extraction and separation technology, improves the extraction speed, but the extraction and purification process is too complex to realize large-scale industrial production, the obtained extract is still an anthocyanin mixture, and the purity of the anthocyanin is only 42.7 percent at most.
Guodanni and the like (the glucosan gel chromatography is combined with high-speed counter-current chromatography to extract anthocyanin in blueberries, Guodanni is used for separating and purifying anthocyanin in wild blueberries by combining the glucosan gel chromatography and the high-speed counter-current chromatography in tests of Qinhui, Chenyang et al, food industry, 2016, 2 nd year), crude blueberry extracts are primarily separated by the glucosan gel chromatography to obtain components with high anthocyanin content, then the components are separated by the high-speed counter-current chromatography, MTBE-n-butanol-acetonitrile-water (volume ratio of 1: 3: 1: 5) is used as a two-phase solvent system, separation is carried out under the conditions of flow rate of 0.5m L/min, host rotation speed of 1860 r/min and detection wavelength of 280nm, and two kinds of anthocyanin are obtained by one-time separation from the glucosan anthocyanin gel chromatography column separation product of the blueberries, wherein the purities of the anthocyanin are 65.0% and 90.0% respectively. Although the technical scheme discloses that two kinds of anthocyanins are obtained by separation, from the UPLC analysis chromatogram of the technical scheme 2, only the fact that the sample 1 and the sample 2 are probably anthocyanins can be inferred, the conclusion that the two kinds of samples are the anthocyanins cannot be accurately confirmed, and the chemical components of the two kinds of samples cannot be further confirmed.
Therefore, the research and development of a process for purifying a complex anthocyanin raw material such as blueberries has important significance for promoting the anthocyanin standard market and developing deep-processed products of the blueberries.
Malvidin-3-O-glucoside (Malvidin-3-O-glucoside) has the following structural formula, is one of main anthocyanins in blueberries and is also an important component for exerting bioactivity of the anthocyanins in the blueberries.
However, no research and report on the separation and preparation of the malvidin-3-O-glucoside monomer from the blueberries is found.
Disclosure of Invention
The invention aims to solve the technical problems and provides a method for separating and preparing malvidin-3-O-glucoside, which combines the preparation liquid chromatography and high-speed counter-current chromatography, and then separates and prepares a high-purity malvidin-3-O-glucoside monomer from a blueberry raw material with complex anthocyanin composition by optimizing process parameters.
The specific technical scheme is as follows:
a method for separating and preparing malvidin-3-O-glucoside comprises the following steps:
(1) alcohol extraction and concentration: blueberry is taken as a raw material, and is subjected to alcohol extraction and concentration to obtain a blueberry anthocyanin crude extract;
(2) macroporous resin adsorption: injecting the blueberry anthocyanin crude extract into macroporous resin, eluting and post-treating to obtain blueberry anthocyanin extract freeze-dried powder;
(3) and (3) purifying by preparative liquid chromatography: gradient elution is carried out by adopting a C18 chromatographic column through a mobile phase, and then the malvidin-3-O-glucoside crude product freeze-dried powder is obtained through post-treatment;
the mobile phase is as follows: the phase A is pure methanol or an acid-methanol system with the acid volume percentage concentration of 0.1-1.5%, and the phase B is a formic acid-water system with the formic acid volume percentage concentration of 1.5-5%;
the procedure for the gradient elution was: keeping the volume percentage concentration of the phase A unchanged within 0-5 min, increasing the volume percentage concentration of the phase A from 5% to 60% within 5-30 min, and collecting the eluate for 22-23 min;
(4) high-speed countercurrent chromatographic separation: separating n-butyl alcohol-methyl tert-butyl ether-methanol-water-trifluoroacetic acid as a two-phase solvent system to obtain a malvidin-3-O-glucoside monomer;
the volume ratio of the n-butyl alcohol to the methyl tert-butyl ether to the methanol to the water to the trifluoroacetic acid is 2: 2: 1: 5: 0.01 to 0.1.
All percentages of starting materials present in the present invention are by volume unless otherwise specified.
The various solutions present in the present invention, unless otherwise specified, all use water as the solvent.
In the step (1), the alcohol extraction and concentration specifically comprises the following steps:
mixing the cleaned blueberries with an acidic ethanol solution, filtering after complete ultrasonic extraction, collecting filtrate, and performing vacuum rotary evaporation on the filtrate at 40-50 ℃ to remove ethanol and concentrating to obtain a blueberry anthocyanin crude extract;
the acidic ethanol solution is an ethanol solution with the acid volume percentage concentration of 0.1-1.5%;
the acid is at least one of hydrochloric acid, formic acid, acetic acid and oxalic acid;
the volume percentage concentration of the ethanol solution is 50-95%;
the mass volume ratio (namely the material-liquid ratio) of the blueberries to the acidic ethanol solution is 1: 5-12 g/mL.
Preferably, the ultrasonic extraction time is 60-240 min, and the process is carried out at 25-49 ℃ under the condition of keeping out of the sun.
In order to ensure complete extraction of anthocyanin, the filter residue obtained after the first extraction is repeatedly extracted for a plurality of times according to the same conditions.
Preferably, the volume percentage concentration of the ethanol solution is 60-70%, and the mass volume ratio of the blueberries to the acidic ethanol is 1: 5-8 g/mL.
In the step (2), the macroporous resin adsorption specifically comprises the following steps:
injecting the blueberry anthocyanin extracting solution into macroporous resin, firstly washing the macroporous resin with deionized water, then carrying out gradient elution with an acidic alcohol solution with the volume percentage concentration of 2-22%, collecting an eluent of the acidic alcohol solution with the volume percentage concentration of 14-18%, carrying out vacuum rotary evaporation at 40-50 ℃ to remove alcohol, and carrying out vacuum freeze drying to obtain blueberry anthocyanin extract freeze-dried powder;
preferably, the macroporous resin is selected from AB-8, HPD-100, D101 or DM-130;
preferably, the acidic alcohol solution is selected from alcohol solutions with the acid concentration of 0.1-1.5% by volume;
the alcohol solution is selected from a methanol solution or an ethanol solution, and the volume percentage concentration is 2-22%;
the acid is at least one selected from hydrochloric acid, formic acid and acetic acid.
More preferably, the elution is performed by gradient elution with 2%, 6%, 10%, 14%, 18%, 22% ethanol solution containing 0.5% (v/v, the same applies hereinafter) hydrochloric acid at 2 column volumes (2BV), and the elution of 0.5% hydrochloric acid-ethanol solution with volume percentage concentration of 14-18% is collected.
The concentration of the acidic alcohol solution is 2% by volume of 2% ethanol solution containing 0.5% hydrochloric acid, and the volume ratio of hydrochloric acid to ethanol solution is 0.5: 99.5. in the step (3), the blueberry anthocyanin extract freeze-dried powder is redissolved by deionized water and then injected into a preparative liquid chromatograph for purification; after purification, the post-treatment includes concentration under reduced pressure and vacuum freeze-drying.
Preferably:
the concentration after re-dissolution is 20-120 mg/mL, and the sample injection amount is 1-4 mL;
the specification of the C18 chromatographic column is 20mm multiplied by 250mm, and the temperature is 30 ℃;
the acid in phase a is selected from formic acid or trifluoroacetic acid.
Further preferably:
the concentration after re-dissolution is 50-120 mg/mL, and the sample injection amount is 3-4 mL;
the mobile phase is as follows: the phase A is pure methanol, the phase B is a formic acid-water system with the volume percentage concentration of formic acid of 1.5-5%, and the flow rate of the mobile phase is 5-10 mL/min.
The phase B is a formic acid-water system, taking the volume percentage concentration of formic acid as 1.5% as an example, and specifically means that the volume ratio of formic acid to water is 1.5: 98.5.
in the step (4), the high-speed counter-current chromatography separation specifically comprises:
preparing the two-phase solvent system, wherein an upper phase is a stationary phase, a lower phase is a mobile phase, pumping the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min, pumping the mobile phase at a flow rate of 1-8 mL/min under the conditions of 25-35 ℃ and a main engine rotation speed of 700-1000 r/min, dissolving the malvidin-3-O-glucoside crude product freeze-dried powder with the mobile phase after the two phases are balanced, injecting a sample, collecting an effluent liquid only containing a target product after liquid phase detection, and obtaining the malvidin-3-O-glucoside monomer after reduced pressure concentration and freeze drying.
Preferably, the concentration of the malvidin-3-O-glucoside crude product freeze-dried powder after being dissolved by using a mobile phase is 10-30 mg/mL;
the wavelength of the detection is 280 nm.
Further preferably, in the two-phase solvent system, the volume ratio of n-butanol-methyl tert-butyl ether-methanol-water-trifluoroacetic acid is 2: 2: 1: 5: 0.01;
pumping the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min, and pumping the mobile phase at a flow rate of 3-4 mL/min under the conditions of 25-30 ℃ and a main engine rotation speed of 850-910 r/min;
the concentration of the malvidin-3-O-glucoside crude product freeze-dried powder after being dissolved by using the mobile phase is 13-27 mg/mL.
Compared with the prior art, the invention has the following advantages:
the preparation method combines the preparation liquid chromatography and the high-speed counter-current chromatography technology for the first time, and separates the high-purity malvidin-3-O-glucoside monomer from the blueberries with complex anthocyanin composition by optimizing process parameters, wherein the purity of the malvidin-3-O-glucoside monomer can reach 99%. The separation method has the advantages of large sample processing capacity, good repeatability and the like, can prepare a large amount of high-purity malvidin-3-O-glucoside monomers, can realize industrial production, and provides a new idea for developing and utilizing blueberry resources in China.
Drawings
FIG. 1 is a high performance liquid chromatogram of the lyophilized powder of blueberry anthocyanin in example 1;
FIG. 2 is a high performance liquid chromatogram of the purified product of the blueberry anthocyanin freeze-dried powder by preparative liquid chromatography in example 1;
FIG. 3 is a high performance liquid chromatogram of the final product of example 1;
FIG. 4 is a high performance liquid chromatogram of the final product in comparative example 2;
FIG. 5 is a high performance liquid chromatogram of the final product in comparative example 5;
FIG. 6 is a high performance liquid chromatogram of the final product in comparative example 6.
Detailed Description
The invention will be further described with reference to specific examples, which are given below only by way of illustration, without limiting the scope of the invention:
example 1
Cleaning 1kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: adding 70% ethanol water solution containing 0.1% (v/v) hydrochloric acid (volume ratio of ethanol to water is 70: 30) at a ratio of 8(w/v, g/mL), mixing, ultrasonic extracting for 90min, (controlling temperature below 45 deg.C, keeping out of the sun), vacuum filtering after ultrasonic processing, extracting the obtained residue once again according to the above conditions, mixing filtrates, and vacuum rotary evaporating at 45 deg.C to remove ethanol to obtain crude anthocyanin extract.
Soaking AB-8 macroporous resin in ethanol for 24h, loading into a chromatographic column, washing with pure water until no alcohol smell exists, washing with 0.5M sodium hydroxide solution at the flow rate of 2BV/h for 1h, and washing with deionized water until the effluent is neutral; then washed with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1h, and then washed with deionized water to neutrality. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.4BV/h until the adsorption volume reaches 1/3 of the total volume of the resin. The resin was washed with deionized water at a flow rate of 2BV/h, then 2BV/h with 2%, 6%, 10%, 14%, 18%, 22% aqueous ethanol containing 0.5% (v/v) hydrochloric acid, respectively, and 14% -18% of the eluted fractions were collected. Removing ethanol by vacuum rotary evaporation at 45 ℃ to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and freeze-drying to obtain blueberry anthocyanin freeze-dried powder.
The preparation liquid phase column, uniformity C1820 mm X250 mm, was used, and the mobile phase was: phase A: pure methanol, phase B: formic acid (5%): water (95%); the column temperature was 30 ℃, the flow rate was 10mL/min, and the gradient was: 5% of phase A for 0-5 min, and 5% -60% of phase A for 5-30 min. Dissolving the anthocyanin freeze-dried powder with deionized water to make the concentration of the anthocyanin freeze-dried powder reach 50mg/mL, injecting the anthocyanin freeze-dried powder into a prepared liquid phase for separation, wherein the single sample injection amount is 4 mL. Detecting under an ultraviolet detector, collecting peaks for 22-23 min, concentrating under reduced pressure, and freeze-drying to obtain malvidin-3-O-glucoside crude extract freeze-dried powder.
And (2) adding n-butyl alcohol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as 2: 2: 1: 5: placing into a separating funnel at a volume ratio of 0.01, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 30 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 30 ℃, pumping the stationary phase into the instrument at a flow rate of 30mL/min, positively rotating, and starting the instrument to enable the rotating speed of a host to reach 850 r/min. Pumping the mobile phase at a flow rate of 3mL/min after the rotation speed is stable, dissolving 200mg of malvidin-3-O-glucoside crude extract freeze-dried powder in 15mL of the mobile phase after the two phases are balanced in a pipeline, injecting a sample and detecting under an ultraviolet detector, collecting target peak components, carrying out reduced pressure concentration, and freeze-drying to obtain 12.8mg of malvidin-3-O-glucoside monomer with the purity of 99.30%.
As can be seen by comparing the high performance liquid chromatograms in fig. 1 to 3, after blueberry is subjected to extraction and concentration and gradient elution by macroporous resin, the obtained blueberry anthocyanin freeze-dried powder is mainly a mixture containing 9 anthocyanin monomers, the mixture containing malvidin-3-O-glucoside and other 2 anthocyanins can be obtained by further purifying the mixture by preparative liquid chromatography and collecting the eluate for 22 to 23min, and finally the monomer anthocyanin only containing malvidin-3-O-glucoside can be obtained by high-speed countercurrent chromatography separation, and the purity is 99.30%.
Example 2
Cleaning 5kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: adding 70% ethanol solution containing 0.5% (v/v) hydrochloric acid at a ratio of 7(w/v), mixing, ultrasonic extracting for 150min, (controlling temperature below 45 deg.C, keeping out of the sun), vacuum filtering after ultrasonic processing, extracting the obtained residue once again according to the above conditions, mixing filtrates, and vacuum rotary evaporating at 45 deg.C to remove ethanol to obtain crude anthocyanin extract.
Soaking AB-8 macroporous resin in ethanol for 24h, loading into a chromatographic column, washing with pure water until no alcohol smell exists, washing with 0.5M sodium hydroxide solution at the flow rate of 2BV/h for 1h, and washing with deionized water until the effluent is neutral; then washed with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1h, and then washed with deionized water to neutrality. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.4BV/h until the adsorption volume reaches 1/3 of the total volume of the resin. The resin was washed with deionized water at a flow rate of 2BV/h, then washed with 2%, 6%, 10%, 14%, 18%, 22% acidic ethanol containing 0.5% (v/v) hydrochloric acid at a flow rate of 2BV/h for 2h, and 14% -18% of the eluted fractions were collected. Removing ethanol by vacuum rotary evaporation at 50 ℃ to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and freeze-drying to obtain blueberry anthocyanin freeze-dried powder.
And (3) purifying by preparative liquid chromatography: the preparation liquid phase column, uniformity C1820 mm X250 mm, was used, and the mobile phase was: phase A: pure methanol, phase B: formic acid (4%): water; the column temperature was 30 ℃, the flow rate was 10mL/min, and the gradient was: 5% of phase A for 0-5 min, and 5% -60% of phase A for 5-30 min. Dissolving the anthocyanin freeze-dried powder with deionized water to make the concentration of the anthocyanin freeze-dried powder reach 80mg/mL, injecting the anthocyanin freeze-dried powder into a prepared liquid phase for separation, wherein the single sample injection amount is 4 mL. Detecting under an ultraviolet detector, collecting peaks for 22-23 min, concentrating under reduced pressure, and freeze-drying to obtain malvidin-3-O-glucoside crude extract freeze-dried powder.
And (2) adding n-butyl alcohol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as 2: 2: 1: 5: placing into a separating funnel at a volume ratio of 0.01, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 30 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 25 ℃, pumping the stationary phase into the instrument at a flow rate of 30mL/min, positively rotating, and starting the instrument to enable the rotating speed of a host to reach 900 r/min. Pumping the mobile phase at a flow rate of 3mL/min after the rotation speed is stable, dissolving 300mg of malvidin-3-O-glucoside crude extract freeze-dried powder in the 15mL of mobile phase after the two phases are balanced in a pipeline, injecting and detecting under an ultraviolet detector, collecting target peak components, concentrating under reduced pressure, and freeze-drying to obtain 62.0mg of malvidin-3-O-glucoside with the purity of 98.74%.
Example 3
Cleaning 10kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: adding 60% ethanol solution containing 0.1% (v/v) hydrochloric acid at a ratio of 5(w/v), mixing, ultrasonic extracting for 200min, (controlling temperature below 45 deg.C, keeping out of the sun), vacuum filtering after ultrasonic processing, extracting the obtained residue once again according to the above conditions, mixing filtrates, and vacuum rotary evaporating at 45 deg.C to remove ethanol to obtain crude anthocyanin extract.
Soaking AB-8 macroporous resin in ethanol for 24h, loading into a chromatographic column, washing with pure water until no alcohol smell exists, washing with 0.5M sodium hydroxide solution at the flow rate of 2BV/h for 1h, and washing with deionized water until the effluent is neutral; then washed with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1h, and then washed with deionized water to neutrality. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.4BV/h until the adsorption volume reaches 1/3 of the total volume of the resin. The resin was washed with deionized water at a flow rate of 2BV/h, then washed with 2%, 6%, 10%, 14%, 18%, 22% acidic ethanol containing 0.5% (v/v) hydrochloric acid at a flow rate of 2BV/h for 2h, and 14% -18% of the eluted fractions were collected. Removing ethanol by vacuum rotary evaporation at 45 ℃ to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and freeze-drying to obtain blueberry anthocyanin freeze-dried powder.
And (3) purifying by preparative liquid chromatography: the preparation liquid phase column, uniformity C1820 mm X250 mm, was used, and the mobile phase was: phase A: pure methanol, phase B: formic acid (1.5%): water; the column temperature was 30 ℃, the flow rate was 10mL/min, and the gradient was: 5% of phase A for 0-5 min, and 5% -60% of phase A for 5-30 min. Dissolving the anthocyanin freeze-dried powder with deionized water to make the concentration of the anthocyanin freeze-dried powder reach 120mg/mL, injecting the anthocyanin freeze-dried powder into a prepared liquid phase for separation, wherein the single sample injection amount is 4 mL. Detecting under an ultraviolet detector, collecting peaks for 22-23 min, concentrating under reduced pressure, and freeze-drying to obtain malvidin-3-O-glucoside crude extract freeze-dried powder.
And (2) adding n-butyl alcohol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as 2: 2: 1: 5: placing into a separating funnel at a volume ratio of 0.01, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 30 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 35 ℃, pumping the stationary phase into the instrument at a flow rate of 30mL/min, positively rotating, and starting the instrument to enable the rotating speed of a host to reach 910 r/min. Pumping the mobile phase at the flow rate of 4mL/min after the rotation speed is stable, dissolving 400mg of malvidin-3-O-glucoside crude extract freeze-dried powder in the 15mL of mobile phase after the two phases are balanced in a pipeline, injecting and detecting under an ultraviolet detector, collecting target peak components, concentrating under reduced pressure, and freeze-drying to obtain 122.2mg of malvidin-3-O-glucoside with the purity of 98.26%.
Comparative example 1
Cleaning 10kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: adding 60% ethanol solution containing 0.1% (v/v) hydrochloric acid at a ratio of 7(w/v), mixing, ultrasonic extracting for 200min, (controlling temperature below 45 deg.C, keeping out of the sun), vacuum filtering after ultrasonic processing, extracting the obtained residue once again according to the above conditions, mixing filtrates, and vacuum rotary evaporating at 45 deg.C to remove ethanol to obtain crude anthocyanin extract.
Soaking AB-8 macroporous resin in ethanol for 24h, loading into a chromatographic column, washing with pure water until no alcohol smell exists, washing with 0.5M sodium hydroxide solution at the flow rate of 2BV/h for 1h, and washing with deionized water until the effluent is neutral; then washed with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1h, and then washed with deionized water to neutrality. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.4BV/h until the adsorption volume reaches 1/3 of the total volume of the resin. The resin was washed with deionized water at a flow rate of 2BV/h, then washed with 2%, 6%, 10%, 14%, 18%, 22% acidic ethanol containing 0.5% (v/v) hydrochloric acid at a flow rate of 2BV/h for 2h, and 14% -18% of the eluted fractions were collected. Removing ethanol by vacuum rotary evaporation at 45 ℃ to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and freeze-drying to obtain blueberry anthocyanin freeze-dried powder.
Preparation and liquid phase purification: the preparation liquid phase column, uniformity C1820 mm X250 mm, was used, and the mobile phase was: phase A: pure methanol, phase B: formic acid (0.1%): water; the column temperature was 30 ℃, the flow rate was 10mL/min, and the gradient was: 5% of phase A for 0-5 min, and 5% -60% of phase A for 5-30 min. Dissolving the anthocyanin freeze-dried powder with deionized water to make the concentration of the anthocyanin freeze-dried powder reach 120mg/mL, injecting the anthocyanin freeze-dried powder into a prepared liquid phase for separation, wherein the single sample injection amount is 3 mL. Detecting under an ultraviolet detector, collecting peaks for 22-23 min, concentrating under reduced pressure, and freeze-drying to obtain malvidin-3-O-glucoside crude extract freeze-dried powder.
And (2) adding n-butyl alcohol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as 2: 2: 1: 5: placing into a separating funnel at a volume ratio of 0.1, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 30 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 35 ℃, pumping the stationary phase into the instrument at a flow rate of 30mL/min, positively rotating, and starting the instrument to enable the rotating speed of a host to reach 910 r/min. Pumping the mobile phase at the flow rate of 4mL/min after the rotation speed is stable, dissolving 400mg of malvidin-3-O-glucoside crude extract freeze-dried powder in the 15mL of mobile phase after the two phases are balanced in a pipeline, injecting a sample and detecting under an ultraviolet detector, collecting target peak components, concentrating under reduced pressure, and freeze-drying to obtain 145.8mg of malvidin-3-O-glucoside with the purity of 89.29%.
Comparative example 2
In comparison with example 1, the high performance liquid chromatogram of the final product obtained without the step of preparative liquid chromatography purification was as shown in fig. 4, and it was found that this comparative example can only obtain a mixture containing malvidin-3-O-glucoside, and cannot obtain a malvidin-3-O-glucoside monomer.
Comparative example 3
The preparation process is the same as in example 1, except that the solvent system of the high-speed countercurrent chromatography separation is replaced by: n-butanol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as follows: 3: 1: 5: mixing at a volume ratio of 0.01. The test shows that the malvidin-3-O-glucoside monomer cannot be obtained.
Comparative example 4
The preparation process is the same as in example 1, except that the solvent system of the high-speed countercurrent chromatography separation is replaced by: n-butanol: methyl tert-butyl ether: acetonitrile: water: trifluoroacetic acid was prepared as 2: 2: 1: 5: mixing at a volume ratio of 0.01. Tests show that although the malvidin-3-O-glucoside monomer can be obtained, the purity of the malvidin-3-O-glucoside monomer is 90.57 percent and is far lower than that of the malvidin-3-O-glucoside monomer prepared in example 1 (99.30 percent).
Comparative example 5
The preparation process is the same as example 1, except that: in the mobile phase B in the purification process of the preparative liquid chromatography, a formic acid-aqueous solution system is replaced by an aqueous solution, namely, formic acid is not added, other steps are not changed, and a high performance liquid chromatogram of a final product is shown in figure 5, so that the obtained malvidin-3-O-glucoside monomer has the purity of only 71.83 percent.
Comparative example 6
The preparation process is the same as that of example 1, except that the component collection time in the purification process of preparative liquid chromatography is changed, a malvidin-3-O-glucoside monomer cannot be obtained if the component collection time is not in the range of 22-23 min, and the purity of the separated malvidin-3-O-glucoside monomer is lower than 98% if the component collection time is wider than the range of 22-23 min, and the high performance liquid chromatogram of the product is shown in FIG. 6.
Claims (6)
1. A method for separating and preparing malvidin-3-O-glucoside, which is characterized by comprising the following steps:
(1) alcohol extraction and concentration: blueberry is taken as a raw material, and is subjected to alcohol extraction and concentration to obtain a blueberry anthocyanin crude extract;
(2) macroporous resin adsorption: injecting the blueberry anthocyanin crude extract into macroporous resin, eluting and post-treating to obtain blueberry anthocyanin extract freeze-dried powder;
(3) and (3) purifying by preparative liquid chromatography:
redissolving the blueberry anthocyanin extract freeze-dried powder by using deionized water, and injecting the redissolved blueberry anthocyanin extract freeze-dried powder into a preparative liquid chromatograph for purification, wherein the concentration after redissolution is 50-120 mg/mL, and the sample injection amount is 1-4 mL;
gradient elution is carried out by adopting a C18 chromatographic column through a mobile phase, and then the malvidin-3-O-glucoside crude product freeze-dried powder is obtained through post-treatment; the specification of the C18 chromatographic column is 20mm multiplied by 250mm, and the temperature is 25-30 ℃;
the mobile phase is as follows: the phase A is pure methanol, the phase B is a formic acid-water system with the volume percentage concentration of formic acid of 1.5-5%, and the flow rate of the mobile phase is 10 mL/min;
the procedure for the gradient elution was: keeping the volume percentage concentration of the phase A unchanged within 0-5 min, increasing the volume percentage concentration of the phase A from 5% to 60% within 5-30 min, and collecting the eluate for 22-23 min;
(4) high-speed countercurrent chromatographic separation:
taking n-butyl alcohol-methyl tert-butyl ether-methanol-water-trifluoroacetic acid as a two-phase solvent system, preparing the two-phase solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase, pumping the stationary phase into a high-speed counter-current chromatograph at the flow rate of 20-30 mL/min, pumping the mobile phase at the flow rate of 3-4 mL/min under the conditions of 30-35 ℃ and the main engine rotation speed of 850-910 r/min, dissolving the malvidin-3-O-glucoside crude product freeze-dried powder by using the mobile phase after the two phases are balanced, injecting a sample, collecting an effluent liquid only containing a target product after liquid phase detection, and performing reduced pressure concentration and freeze drying on the effluent liquid to obtain a malvidin-3-O-glucoside monomer;
the concentration of the malvidin-3-O-glucoside crude product freeze-dried powder after being dissolved by using a mobile phase is 10-30 mg/mL, and the sample injection volume is 1-15 mL;
the wavelength of the liquid phase detection is 280 nm;
the volume ratio of the n-butyl alcohol to the methyl tert-butyl ether to the methanol to the water to the trifluoroacetic acid is 2: 2: 1: 5: 0.01 to 0.1.
2. The method for separating and preparing malvidin-3-O-glucoside according to claim 1, wherein in the step (1), the alcohol extraction is concentrated, and specifically comprises:
mixing the cleaned blueberries with an acidic ethanol solution, filtering after complete ultrasonic extraction, collecting filtrate, and performing vacuum rotary evaporation on the filtrate at 40-50 ℃ to remove ethanol and concentrating to obtain a blueberry anthocyanin crude extract;
the acidic ethanol solution is an ethanol solution with the acid volume percentage concentration of 0.1-1.5%;
the mass-volume ratio of the blueberries to the acidic ethanol solution is 1: 5-12 g/mL.
3. The method for separating and preparing malvidin-3-O-glucoside according to claim 2, wherein said acid is at least one selected from the group consisting of hydrochloric acid, formic acid, acetic acid, oxalic acid;
the volume percentage concentration of the ethanol solution is 50-95%.
4. The method for separating and preparing malvidin-3-O-glucoside according to claim 1, wherein in the step (2), the macroporous resin is used for adsorption, and specifically comprises the following steps:
injecting the blueberry anthocyanin crude extract into macroporous resin, washing the macroporous resin with deionized water, performing gradient elution with an acidic alcohol solution with the volume percentage concentration of 2-22%, collecting an eluent of the acidic alcohol solution with the volume percentage concentration of 14-18%, performing vacuum rotary evaporation at 40-50 ℃ to remove alcohol, and performing vacuum freeze drying to obtain blueberry anthocyanin extract freeze-dried powder;
the grade of the macroporous resin is selected from AB-8, HPD-100, D101 or DM-130;
the acidic alcohol solution is selected from an alcohol solution with the acid volume percentage concentration of 0.1-1.5%, the alcohol solution is selected from a methanol solution or an ethanol solution, and the acid is selected from at least one of hydrochloric acid, formic acid and acetic acid.
5. The process for separating and preparing malvidin-3-O-glucoside according to claim 1, wherein in the step (3), the post-treatment comprises concentration under reduced pressure and vacuum freeze-drying.
6. The process for the isolated preparation of malvidin-3-O-glucoside according to claim 1, wherein the volume ratio of n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid in said biphasic solvent system is 2: 2: 1: 5: 0.01.
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