CN108516999B - method for separating and preparing petunidin-3-O-galactoside - Google Patents
method for separating and preparing petunidin-3-O-galactoside Download PDFInfo
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- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
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Abstract
The method for separating and preparing the petunidin-3-O-galactoside comprises the steps of alcohol extraction and concentration, macroporous resin adsorption, preparation of liquid chromatography and purification and high-speed countercurrent chromatography separation, and the high-purity petunidin-3-O-galactoside monomer is separated and prepared from the blueberry raw material which is composed of anthocyanin and is complex.
Description
Technical Field
The invention relates to the field of separation and purification of natural products, in particular to a method for preparing petunidin-3-O-galactoside by separation.
Background
Anthocyanin is kinds of water-soluble pigments which are kinds existing in plants, and is a polyphenol compound formed by combining anthocyanin and or more glycosyl groups, such as glucose, galactose, arabinose and the like, through glycosidic bonds, 6 kinds of common anthocyanin in nature are Pelargonidin (Pelargonidin), cyanidin (cyanianin), Delphinidin (Delphinidin), Peonidin (Peonidin), petuniadin (petuniadin) and Malvidin (Malvidin), and in recent years, a great deal of research proves that natural anthocyanin has biological activities of antioxidation, antitumor, cardiovascular disease prevention, diabetes alleviation, obesity control and the like, along with the improvement of living standard of people, the sound of functional components of natural origin and without toxic and side effects is higher and higher, and the anthocyanin is taken as the most representative functional factor of natural origin, and is favored by researchers and food due to good coloring function and biological activity.
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, the Chinese patent publication No. CN 106366141A discloses a method for separating and preparing pelargonidin-3-O-glucoside monomers, which uses strawberries as raw materials and is prepared by freeze drying, alcohol extraction concentration, fractional extraction and AB-8 macroporous resin purification, and for example, the Chinese patent publication No. CN 106831911A discloses methods for separating and purifying pelargonidin-3-O-glucoside monomers from Tephrodia hirsuta, which comprises alcohol extraction 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 cowberry, not only is rich in basic nutrient components required by human bodies, but also is rich in various kinds of anthocyanin, and has the effects of activating retina, reducing blood sugar, resisting inflammation and resisting tumors.
For example, chinese patent publication No. CN 106905391 a discloses a method for extracting, separating and purifying blueberry anthocyanins, which comprises mixing blueberry juice with an extractant, performing homogeneous extraction for 1-4 times under the conditions of normal temperature and 100-160 MPa, filtering, combining filtrates to obtain a crude blueberry anthocyanin extract, and performing separation and purification by using HPD600 macroporous resin.
Also, for example, Chinese patent publication No. CN 104109403A discloses a novel method for extracting and purifying wild blueberry anthocyanins, the preparation process comprises 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, the extraction speed is improved, 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% at most.
Guodanni and the like ("Sephadex chromatography combines with high-speed countercurrent chromatography to extract anthocyanin in blueberry", Guodanni, splenghes, Chenyang et al, food industry, 2016, stage 2) tests combine Sephadex chromatography and high-speed countercurrent chromatography to separate and purify anthocyanin in wild blueberry, crude blueberry extract is firstly subjected to preliminary separation by Sephadex chromatography to obtain components with high anthocyanin content, and then is subjected to high-speed countercurrent chromatography, MTBE-n-butanol-acetonitrile-water (volume ratio of 1: 3: 1: 5) is taken 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, times of two anthocyanidins are separated from a separation product of a Sephadex chromatographic column of blueberry, the purities of 65.0% and 90.0% respectively, the technical scheme discloses that the two anthocyanidins are separated and obtained, but only 352 times of a chromatogram of a sample can be inferred from UPLC analysis of FIG. 2, and the two anthocyanidins can not be chemically confirmed, and even more accurate anthocyanin sample can be obtained.
Therefore, the research and development of processes for purifying complex anthocyanin raw materials such as blueberries are of great significance for promoting the anthocyanin standard market and developing deep-processed products of blueberries.
petunianin-3-O-galactoside (petuniadin-3-O-galactoside) has the following structural formula, is of main anthocyanin in blueberry, and is also an important component of blueberry anthocyanin for exerting bioactivity.
However, no research and report on the isolation and preparation of the petunidin-3-O-galactoside monomer from the blueberry is found at present.
Disclosure of Invention
The invention provides a method for separation and preparation of petunidin-3-O-galactoside for solving the technical problems, which combines the preparation liquid chromatography and high-speed countercurrent chromatography technology, and then separates and prepares the high-purity petunidin-3-O-galactoside monomer from the blueberry raw material with complex anthocyanin composition by optimizing process parameters.
The specific technical scheme is as follows:
A method for preparing petunidin-3-O-galactoside by separation, which 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 lyophilized powder of the crude product of the petunidin-3-O-galactoside 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%;
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 18-19 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, and separating to obtain petunidin-3-O-galactoside;
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 selected from 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 is 1: 6-12 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 70-90%, and the mass volume ratio of the blueberries to the acidic ethanol is 1: 7-9 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 grade of the macroporous resin is selected from AB-8, HPD-100, D101 or DM-130, and the grade of the macroporous resin is preferably AB-8 macroporous resin in step .
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 selected from hydrochloric acid, formic acid, and acetic acid.
preferably, the elution solution is gradient eluted by 2 times column volume (2BV) with 2%, 6%, 10%, 14%, 18% and 22% ethanol solution containing 0.5% (v/v, the same below) hydrochloric acid, and the elution solution of hydrochloric acid-ethanol solution with 14-18% concentration by volume 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.
, preferably:
the concentration after re-dissolution is 30-50 mg/mL, and the sample injection amount is 2 mL;
the mobile phase is as follows: the phase A is pure methanol or a formic acid-methanol system with the formic acid volume percentage concentration of 0.1%, the phase B is a formic acid-water system with the formic acid volume percentage concentration of 3-5%, and the flow rate of the mobile phase is 5-10 mL/min.
The phase A is a formic acid-methanol system with the volume percentage concentration of formic acid of 0.1 percent, and specifically means that the volume ratio of formic acid to methanol is 0.1: 99.9.
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 petunidin-3-O-galactoside crude product freeze-dried powder with the mobile phase after the two phases are balanced, injecting a sample, detecting the liquid phase, collecting an effluent liquid only containing a target product, and carrying out reduced pressure concentration and freeze drying to obtain the petunidin-3-O-galactoside monomer.
Preferably, the concentration of the petunidin-3-O-galactoside crude product freeze-dried powder after being dissolved by using a mobile phase is 15-35 mg/mL;
the wavelength of the detection is 280 nm.
preferably, the volume ratio of n-butanol-methyl tert-butyl ether-methanol-water-trifluoroacetic acid in the two-phase solvent system 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 3mL/min under the conditions of 25-30 ℃ and a main engine rotation speed of 850-1000 r/min.
Compared with the prior art, the invention has the following advantages:
the method combines the preparation liquid chromatography and the high-speed counter-current chromatography technology for the first time, and separates the high-purity petunidin-3-O-galactoside monomer from the blueberry with complex anthocyanin composition by optimizing the process parameters, wherein the purity of the monomer can reach 99 percent at most. The separation method has the advantages of large sample processing capacity, good repeatability and the like, can be used for preparing a large amount of high-purity petunidin-3-O-galactoside monomer, 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 1;
FIG. 5 is a high performance liquid chromatogram of the final product in comparative example 2;
FIG. 6 is a high performance liquid chromatogram of the final product in comparative example 5;
FIG. 7 is a high performance liquid chromatogram of the final product in comparative example 6.
Detailed Description
The present invention is further described in conjunction with the following specific examples, which are set forth merely for the purpose of describing particular embodiments of the present invention, but the scope of the present invention is not limited thereto:
example 1
(1) Cleaning 1000g of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: 7(w/v, g/mL) is added with 70% ethanol water solution containing 0.1% (v/v) hydrochloric acid (the volume ratio of ethanol to water is 70: 30), fully mixed, ultrasonically extracted for 90min, (45 ℃, and protected from light), vacuum filtered, the filter residue is repeatedly extracted for 2 times according to the above conditions, the filtrate is combined, and vacuum rotary evaporation is carried out at 48 ℃ to remove ethanol, thus obtaining crude anthocyanin extract;
(2) loading the AB-8 macroporous resin soaked in ethanol for 24 hours into a chromatographic column, washing with deionized water until no alcohol smell exists, washing with 4% hydrochloric acid solution at the flow rate of 2BV/h for 1 hour, and washing with deionized water until the effluent is neutral; the solution is washed by 4 percent sodium hydroxide solution at the flow rate of 2BV/h for 1h and then is washed by deionized water to be neutral. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.5 BV/h. The resin is washed by deionized water at the flow rate of 2BV/h, then by an ethanol aqueous solution containing 2%, 6%, 10%, 14%, 18% and 22% of 0.5% (v/v) hydrochloric acid at the flow rate of 2BV/h for 1h, and 14-18% of elution parts are collected. Performing vacuum rotary evaporation at 48 ℃ to remove ethanol to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and performing freeze drying to obtain blueberry anthocyanin freeze-dried powder;
(3) and (3) purifying by preparative liquid chromatography: dissolving anthocyanin freeze-dried powder with deionized water to a concentration of 50mg/mL by using a Unitry C1820 mm multiplied by 250mm chromatographic column, injecting into a preparative liquid chromatography with a sample volume of 2mL, wherein the used mobile phase is phase A: 99.9% methanol + 0.1% formic acid; phase B: 97% water + 3% formic acid; 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. And detecting at 280nm, collecting the eluate for 18-19 min, combining, concentrating under reduced pressure, and freeze-drying under vacuum to obtain the crude petunidin-3-O-galactoside freeze-dried powder.
(4) Preparing petunidin-3-O-galactoside by high-speed countercurrent chromatography:
placing n-butyl alcohol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid in a separating funnel according to the volume ratio of 2: 2: 1: 5: 0.01, fully shaking up, standing for 30min, separating an upper phase from a lower phase, ultrasonically degassing for 35min, taking the upper phase as a stationary phase and the lower phase as a mobile phase, starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 25 ℃, pumping the stationary phase into an instrument at the flow rate of 30mL/min, rotating the instrument forward, starting the instrument, stabilizing the rotating speed of the host to 850r/min, pumping the mobile phase at the flow rate of 2mL/min, dissolving 200mg of freeze-dried powder into 10mL of mobile phase after the two phases are balanced in a pipeline, sampling, collecting pipes every 9mL of the freeze-dried substances, collecting and merging galactosides in a pipe liquid phase solution only containing a target product-petunidin-3-O-galactosides after liquid phase detection (the detection wavelength is 280nm), and then carrying out reduced pressure concentration to obtain a 15mg of a final product, wherein the high-efficient liquid phase can be clear through a graph 3, the final product is petunidin purity of 98.25.
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, steps are further carried out, the prepared liquid chromatography is used for purification, 18 to 19min of eluate is collected, a mixture containing petunidin-3-O-galactoside and other 2 anthocyanins can be obtained, and finally, the monomer anthocyanin only containing the petunidin-3-O-galactoside can be obtained through high-speed countercurrent chromatography separation, wherein the purity is 98.25%.
Example 2
(1) Cleaning 3kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: 9(w/v), adding 80% ethanol aqueous solution containing 1.0% (v/v) hydrochloric acid, mixing, ultrasonic extracting for 120min, (45 deg.C below, in dark place), filtering under reduced pressure, extracting the residue for 3 times, mixing filtrates, and vacuum rotary evaporating at 46 deg.C to remove ethanol to obtain crude anthocyanin extractive solution;
(2) loading the AB-8 macroporous resin soaked in ethanol for 24 hours into a chromatographic column, washing with deionized water until no alcohol smell exists, washing with 4% hydrochloric acid solution at the flow rate of 2BV/h for 1 hour, and washing with deionized water until the effluent is neutral; the solution is washed by 4 percent sodium hydroxide solution at the flow rate of 2BV/h for 1h and then is washed by deionized water to be neutral. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.5 BV/h. The resin is washed by deionized water at the flow rate of 2BV/h, then by an ethanol aqueous solution containing 2%, 6%, 10%, 14%, 18% and 22% of 0.5% (v/v) hydrochloric acid at the flow rate of 2BV/h for 1h, and 14-18% of elution parts are collected. Performing vacuum rotary evaporation at 48 ℃ to remove ethanol to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and performing freeze drying to obtain blueberry anthocyanin freeze-dried powder;
(3) and (3) purifying by preparative liquid chromatography: dissolving anthocyanin freeze-dried powder with deionized water to a concentration of 30mg/mL by using a Unitry C1820 mm multiplied by 250mm chromatographic column, injecting into a preparative liquid chromatography with a sample volume of 3mL, wherein the used mobile phase is phase A: 99.9% methanol + 0.1% formic acid; phase B: 96% water + 4% formic acid; the solvent gradient was: and (3) carrying out gradient elution at the flow rate of 10mL/min for 0-5 min on the 5% A phase and 5-30 min on the 5% -60% A phase, detecting at 280nm, collecting the eluate for 18-19 min, combining, concentrating under reduced pressure, and carrying out vacuum freeze drying to obtain the petunidin-3-O-galactoside crude product freeze-dried powder.
(4) High-speed countercurrent chromatography for preparing petunidin-3-O-galactoside
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 45 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 25mL/min, rotating the main machine forward, and starting the instrument to enable the rotating speed of the main machine to reach 950 r/min. And pumping the mobile phase at the flow rate of 3mL/min after the rotation speed is stable, dissolving 350mg of freeze-dried powder in the 12mL 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 40mg of petunidin-3-O-galactoside with the purity of 98.79%.
Example 3
(1) Cleaning 5kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: adding 90% ethanol water solution containing 1.5% (v/v) hydrochloric acid at a ratio of 8(w/v), mixing, ultrasonic extracting for 200min, (below 45 deg.C, in dark place), filtering under reduced pressure, extracting the residue for 4 times, mixing filtrates, and vacuum rotary evaporating at 47 deg.C to remove ethanol to obtain crude anthocyanin extract;
(2) loading the AB-8 macroporous resin soaked in ethanol for 24 hours into a chromatographic column, washing with deionized water until no alcohol smell exists, washing with 4% hydrochloric acid solution at the flow rate of 2BV/h for 1 hour, and washing with deionized water until the effluent is neutral; the solution is washed by 4 percent sodium hydroxide solution at the flow rate of 2BV/h for 1h and then is washed by deionized water to be neutral. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.5 BV/h. The resin is washed by deionized water at the flow rate of 2BV/h, then by an ethanol aqueous solution containing 2%, 6%, 10%, 14%, 18% and 22% of 0.5% (v/v) hydrochloric acid at the flow rate of 2BV/h for 1h, and 14-18% of elution parts are collected. Performing vacuum rotary evaporation at 48 ℃ to remove ethanol to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and performing freeze drying to obtain blueberry anthocyanin freeze-dried powder;
(3) and (3) purifying by preparative liquid chromatography: dissolving anthocyanin freeze-dried powder with deionized water to a concentration of 30mg/mL by using a Unitry C1820 mm multiplied by 250mm chromatographic column, injecting into a preparative liquid chromatography with a sample volume of 3mL, wherein the used mobile phase is phase A: pure methanol; phase B: 95% water + 5% formic acid; the solvent gradient was: and (3) carrying out gradient elution on 5% of phase A for 0-5 min and 5% -60% of phase A for 5-30 min, detecting at 280nm, collecting eluate for 18-19 min at the flow rate of 10mL/min, and carrying out vacuum freeze drying to obtain the crude petunidin-3-O-galactoside freeze-dried powder.
(4) High-speed countercurrent chromatography for preparing petunidin-3-O-galactoside
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 45 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting the high-speed counter-current chromatograph, preheating for 30min, setting the circulating water bath at 28 ℃, pumping the stationary phase into the instrument at the flow rate of 20mL/min, rotating the main machine forward, and starting the instrument to enable the rotating speed of the main machine to be 1000 r/min. And pumping the mobile phase at the flow rate of 3mL/min after the rotation speed is stable, dissolving 500mg of 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 60mg of petunidin-3-O-galactoside with the purity of 99.12%.
Example 4
(1) Cleaning 5kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: adding 90% ethanol solution containing 1.5% (v/v) hydrochloric acid at a ratio of 8(w/v), mixing, ultrasonic extracting for 200min, (below 45 deg.C, in dark place), filtering under reduced pressure, extracting the residue for 4 times, mixing filtrates, and vacuum rotary evaporating at 47 deg.C to remove ethanol to obtain crude anthocyanin extract;
(2) loading the AB-8 macroporous resin soaked in ethanol for 24 hours into a chromatographic column, washing with deionized water until no alcohol smell exists, washing with 4% hydrochloric acid solution at the flow rate of 2BV/h for 1 hour, and washing with deionized water until the effluent is neutral; the solution is washed by 4 percent sodium hydroxide solution at the flow rate of 2BV/h for 1h and then is washed by deionized water to be neutral. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.5 BV/h. The resin was washed with deionized water at a flow rate of 2BV/h, then washed with 2%, 6%, 10%, 14%, 18%, 22% ethanol containing 0.5% (v/v) hydrochloric acid at a flow rate of 2BV/h for 1h, and 14-18% of the eluted fractions were collected. Performing vacuum rotary evaporation at 48 ℃ to remove ethanol to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and performing freeze drying to obtain blueberry anthocyanin freeze-dried powder;
(3) and (3) purifying by preparative liquid chromatography: dissolving anthocyanin freeze-dried powder with deionized water to a concentration of 30mg/mL by using a Unitry C1820 mm multiplied by 250mm chromatographic column, injecting into a preparative liquid chromatography with a sample volume of 3mL, wherein the used mobile phase is phase A: pure methanol; phase B: 99% water + 1% formic acid; the solvent gradient was: and (3) carrying out gradient elution at the flow rate of 10mL/min for 0-5 min on the 5% A phase and 5-30 min on the 5% -60% A phase, detecting at 280nm, collecting the eluate for 18-19 min, and carrying out vacuum freeze-drying to obtain the crude petunidin-3-O-galactoside freeze-dried powder.
(4) High-speed countercurrent chromatography for preparing petunidin-3-O-galactoside
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 45 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting the high-speed counter-current chromatograph, preheating for 30min, setting the circulating water bath at 28 ℃, pumping the stationary phase into the instrument at the flow rate of 20mL/min, rotating the main machine forward, and starting the instrument to enable the rotating speed of the main machine to be 1000 r/min. Pumping the mobile phase at a flow rate of 3mL/min after the rotation speed is stable, dissolving 500mg of 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 the petunidin-3-O-galactoside with the purity of 94.08%.
Comparative example 1
(1) Cleaning 5kg of fresh blueberries, and mixing the raw materials according to a material-liquid ratio of 1: adding 90% ethanol solution containing 1.5% (v/v) hydrochloric acid at a ratio of 8(w/v), mixing, ultrasonic extracting for 200min, (below 45 deg.C, in dark place), filtering under reduced pressure, extracting the residue for 4 times, mixing filtrates, and vacuum rotary evaporating at 47 deg.C to remove ethanol to obtain crude anthocyanin extract;
(2) loading the AB-8 macroporous resin soaked in ethanol for 24 hours into a chromatographic column, washing with deionized water until no alcohol smell exists, washing with 4% hydrochloric acid solution at the flow rate of 2BV/h for 1 hour, and washing with deionized water until the effluent is neutral; the solution is washed by 4 percent sodium hydroxide solution at the flow rate of 2BV/h for 1h and then is washed by deionized water to be neutral. Injecting the crude anthocyanin extract into AB-8 macroporous resin at the flow rate of 0.5 BV/h. Washing the resin with deionized water at a flow rate of 2BV/h, washing with 2%, 6%, 10%, 14%, 18% and 22% ethanol containing 0.5% (v/v) hydrochloric acid at a flow rate of 2BV/h for 1h, and collecting 14-18% of the eluate. Performing vacuum rotary evaporation at 48 ℃ to remove ethanol to obtain anthocyanin extract, dissolving the extract with a small amount of deionized water, and performing freeze drying to obtain blueberry anthocyanin freeze-dried powder;
(3) and (3) purifying by preparative liquid chromatography: dissolving anthocyanin freeze-dried powder with deionized water to a concentration of 30mg/mL by using a Unitry C1820 mm multiplied by 250mm chromatographic column, injecting into a preparative liquid chromatography with a sample volume of 3mL, wherein the used mobile phase is phase A: pure methanol; phase B: 99.9% water + 0.1% formic acid; the solvent gradient was: and (3) carrying out gradient elution at the flow rate of 10mL/min for 0-5 min on the 5% A phase and 5-30 min on the 5% -60% A phase, detecting at 280nm, collecting the eluate for 18-19 min, and carrying out vacuum freeze-drying to obtain the crude petunidin-3-O-galactoside freeze-dried powder.
(4) Preparing petunidin-3-O-galactoside by high-speed countercurrent chromatography: 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 0.5, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 45 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting the high-speed counter-current chromatograph, preheating for 30min, setting the circulating water bath at 28 ℃, pumping the stationary phase into the instrument at the flow rate of 20mL/min, rotating the main machine forward, and starting the instrument to enable the rotating speed of the main machine to be 1000 r/min. Pumping the mobile phase at a flow rate of 3mL/min after the rotation speed is stable, dissolving 500mg of 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 the petunidin-3-O-galactoside with the purity of 87.56%.
Comparing the high performance liquid chromatograms in fig. 3 and 4, it can be seen that after the components of the two-phase solvent system for high-speed countercurrent chromatography separation are finely adjusted, the purity of the finally separated petunidin-3-O-galactoside monomer can be significantly changed.
Comparative example 2
In comparison with example 3, the procedure of preparative liquid chromatography purification was omitted, and the other procedures were not changed, and the high performance liquid chromatogram of the final product was shown in fig. 5, which indicates that the separation process only yielded a mixture containing petunidin-3-O-galactoside, and that petunidin-3-O-galactoside monomer could not be obtained.
Comparative example 3
The preparation process is the same as in example 3, 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. Tests show that the petunidin-3-O-galactoside monomer cannot be obtained due to the low retention rate of a countercurrent system.
Comparative example 4
The preparation process is the same as in example 3, 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. Through tests, although the petunidin-3-O-galactoside monomer can be obtained, the purity of the petunidin-3-O-galactoside monomer is only 90.15 percent and is far lower than that of the petunidin-3-O-galactoside monomer prepared in example 3 (99.12 percent).
Comparative example 5
The preparation process is the same as in example 3, 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 the high performance liquid chromatogram of the final product is shown in figure 6, so that the petunidin-3-O-galactoside monomer is obtained, but the purity is lower than 90%.
Comparative example 6
The preparation process is the same as that of example 3, except that the component collection time in the purification process of preparative liquid chromatography is changed, if the component collection time is not in the range of 18-19 min, the petunidin-3-O-galactoside monomer cannot be obtained, and if the component collection time is included and is wider than the range of 18-19 min, the purity is lower than 98%, and the high performance liquid chromatogram of the final product is shown in FIG. 7.
Claims (6)
- The method for separating and preparing petunidin-3-O-galactoside by 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 30-50 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 lyophilized powder of the crude product of the petunidin-3-O-galactoside is obtained through post-treatment; the specification of the C18 chromatographic column is 20mm multiplied by 250mm, and the temperature is 30 ℃;the mobile phase is as follows: the phase A is pure methanol or a formic acid-methanol system with the formic acid volume percentage concentration of 0.1%, the phase B is a formic acid-water system with the formic acid volume percentage concentration of 3-5%, and the flow rate of the mobile phase is 5-10 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 18-19 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 1-3 mL/min under the conditions of 25-30 ℃ and the main engine rotation speed of 850-1000 r/min, dissolving the crude petunidin-3-O-galactoside 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 petunidin-3-O-galactoside monomer;the concentration of the lyophilized powder of the petunidin-3-O-galactoside crude product after being dissolved by the mobile phase is 15-35 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 petunidin-3-O-galactoside according to claim 1, wherein in the step (1), the alcohol extraction and concentration are as follows: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: 6-12 g/mL.
- 3. The method for separating and preparing petunidin-3-O-galactoside according to claim 2, wherein the acid is at least selected from hydrochloric acid, formic acid, acetic acid and oxalic acid;the volume percentage concentration of the ethanol solution is 50-95%.
- 4. The method for separating and preparing petunidin-3-O-galactoside according to claim 1, wherein in the step (2), the macroporous resin is used for adsorbing, 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 of hydrochloric acid, formic acid and acetic acid.
- 5. The method for separating and preparing petunidin-3-O-galactoside according to claim 1, wherein the post-treatment in the step (3) comprises concentration under reduced pressure and vacuum freeze drying.
- 6. The method for separating and preparing petunidin-3-O-galactoside according to claim 1, wherein the volume ratio of n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid in the two-phase solvent system is 2: 2: 1: 5: 0.01.
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