CN112321655B - Method for separating and preparing petunidin-3-O- (6-O-p-coumaroyl) glucoside - Google Patents
Method for separating and preparing petunidin-3-O- (6-O-p-coumaroyl) glucoside Download PDFInfo
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Abstract
The invention discloses a method for separating and preparing petunidin-3-O- (6-O-p-coumaroyl) glucoside, which separates and purifies high-purity petunidin-3-O- (6-O-p-coumaroyl) glucoside monomers from grapes through the steps of extraction, macroporous resin purification, extraction, high-speed counter-current chromatography, solid phase extraction and purification and the like. By the method, at least 80mg of petunidin-3-O- (6-O-p-coumaroyl) glucoside can be obtained from 10kg of grape skin, and the purity can reach not less than 98%. The method has the advantages of simple operation, large treatment capacity, good repeatability and the like, and provides a new idea for developing and utilizing the grape resources in China.
Description
Technical Field
The invention relates to the technical field of separation and purification of natural products, in particular to a method for separating and preparing petunidin-3-O- (6-O-p-coumaroyl) glucoside.
Background
Grapes are the fruits of plants of the genus vitis of the family vitidae and are cultivated all over the world. The grape has high nutritive value. The traditional Chinese medicine considers that grapes are neutral in nature and sweet in taste, and can tonify qi and blood, strengthen heart and promote urination. Modern researches show that the grapes not only contain vitamins such as vitamin B1, vitamin B2, vitamin C, vitamin E and the like, but also contain rich polyphenol compounds such as resveratrol, procyanidine, anthocyanin and the like, and have biological activities of resisting oxidation, protecting heart vessels, resisting tumors, resisting bacteria and the like.
The anthocyanin is one of the important polyphenols in the grape, and researches show that the grape contains anthocyanin formed by combining anthocyanin aglycone such as delphinidin, malvidin, paeoniflorin, petuniain and the like with glucose. Recent researches prove that anthocyanin derived from natural fruits and vegetables has biological activities of resisting oxidation, resisting tumors, controlling obesity, preventing cardiovascular diseases and the like. However, because anthocyanins are sensitive to light, temperature and pH, their chemical properties are relatively unstable and their bioavailability is low, which greatly limits the practical application of anthocyanins. It has been shown that acylated anthocyanins are also biologically active, and have more stable chemical properties and higher bioavailability than non-acylated anthocyanins. The grapes contain a large amount of p-coumaroyl acylated anthocyanin, which shows that the grape anthocyanin has better market prospect.
In recent years, novel purification techniques such as Solid Phase Extraction (SPE), preparative high performance liquid chromatography (preparative-HPLC), and high-speed countercurrent chromatography (HSCCC) have been developed and used. Among them, the countercurrent chromatography is a liquid-liquid chromatography technique in which the stationary phase and the mobile phase are both in liquid state and the principle is that the separation is based on the difference of the partition coefficients of different compound molecules between the stationary phase and the mobile phase. The separation performance of countercurrent chromatography depends mainly on whether a two-phase solvent system is suitable. The countercurrent chromatography technology has the advantages of simple sample pretreatment, wide application range, less sample loss, large treatment capacity and the like. The solid phase extraction technology is to use a solid adsorbent to adsorb a target compound in a sample, separate the target compound from a matrix and other interfering compounds of the sample, and then elute the target compound with an eluent, so that the target compound can be enriched and separated. The solid phase extraction method has the advantages of time saving, small solvent consumption, easy scale-up production and the like. At present, in the preparation of the anthocyanin of the grape, extraction, macroporous resin and single column chromatography or chromatographic technology are mainly adopted for separation and purification.
For example, chinese patent publication No. CN104177460A discloses a method for preparing 3, 5-disaccharide anthocyanin, which uses ultrasonic-assisted extraction, purification, etc., wherein the use of an optimized ultrasonic-assisted extraction method reduces the use of an extractant, and the operation is simplified by a method of combining a gel column and macroporous resin. However, the obtained product is an anthocyanin mixture, containing three different disaccharide anthocyanins, and the product does not involve acylated anthocyanins.
Also, for example, chinese patent publication No. CN102229633A discloses a method for separating and preparing five high-purity anthocyanin monomers from grape skin. The method uses extraction, macroporous resin purification and liquid phase preparation method to obtain five kinds of anthocyanin, however, two steps of liquid phase preparation purification are used, so that sample loss is caused, and the purification yield is reduced, and the purities of two kinds of acylated anthocyanin (malvidin acetylated glucoside and malvidin trans-coumaroylated glucoside) are relatively low, and are only 91.7% and 95.5% respectively.
Chinese patent publication No. CN108976268A discloses a method for preparing two main anthocyanin standard products of Vitis davidii, which comprises adsorbing and enriching vitis davidii turbid juice with macroporous resin, eluting, freeze-drying to obtain crude anthocyanin, and separating with high-speed countercurrent chromatography using water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid (volume ratio 5: 4: 1: 2: 0.001 or 5: 3: 1: 1: 0.001) as two-phase solvent system to obtain two kinds of anthocyanin, the purity of which is 95.8% and 92.2%, respectively. Although the technical scheme discloses that two kinds of anthocyanins are obtained by separation, the anthocyanin composition is simple from the HPLC chart of the vitis amurensis turbid juice in FIG. 1, and the limitation of high-speed countercurrent chromatography can be conjectured, and when the separated anthocyanin component of a sample is complex, the target anthocyanins can be difficultly obtained by using the method.
Because of the difficulty in separating and purifying acylated anthocyanin, no commercial acylated anthocyanin standard product is available on the market at present. Therefore, research and development of a process for purifying acylated anthocyanin monomers from complex anthocyanin raw materials such as grapes are of great significance for promoting the anthocyanin standard market and developing grape deep-processed products. Therefore, the invention discloses a method based on the combination of high-speed countercurrent chromatography and solid phase extraction, which can realize the mass preparation of high-purity petunidin-3-O- (6-O-p-coumaroyl) glucoside monomers from grapes with complex anthocyanin components.
Disclosure of Invention
Aiming at the defects in the field, the invention provides a method for separating and preparing petunidin-3-O- (6-O-p-coumaroyl) glucoside, and provides a new idea for developing and utilizing grape resources in China.
A method for separating and preparing petunidin-3-O- (6-O-p-coumaroyl) glucoside comprises the following steps:
(1) alcohol extraction and concentration: taking grapes as raw materials, and performing alcohol extraction and concentration to obtain a crude anthocyanin extract of grape skin;
(2) and (3) macroporous resin purification: injecting the crude anthocyanin extract of the grape skin into macroporous resin, eluting and concentrating to obtain anthocyanin eluent;
(3) and (3) extraction: extracting the anthocyanin eluent by using an organic solvent, then carrying out reduced pressure concentration and freeze-drying to obtain anthocyanin freeze-dried powder;
(4) high-speed countercurrent chromatographic separation: mixing methyl tert-butyl ether, methanol, water and trifluoroacetic acid to form a two-phase solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase, pumping the stationary phase and the mobile phase into a high-speed countercurrent chromatographic instrument in sequence, dissolving anthocyanin freeze-dried powder by using the mobile phase after the two phases reach balance in a pipeline, injecting a sample and detecting under an ultraviolet detector, wherein the detection wavelength is 280nm, collecting components with the retention time of 100 plus of material for 120min, and concentrating under reduced pressure to obtain a crude anthocyanin monomer solution;
(5) and (3) solid phase extraction column purification: injecting the crude anthocyanin monomer solution into a solid phase extraction column, performing gradient elution by using an acidic acetonitrile solution with the acetonitrile volume percentage concentration of 0-40%, collecting an acidic acetonitrile eluent with the acetonitrile volume percentage concentration of 28-32%, and performing reduced pressure concentration and freeze drying to obtain a target compound, namely petunidin-3-O- (6-O-p-coumaroyl) glucoside;
the concentration of the crude anthocyanin monomer solution is 5-20mg/mL, and the sample amount is 2-10 mL;
the acid in the acidic acetonitrile solution is at least one selected from hydrochloric acid, formic acid, acetic acid and oxalic acid.
The percentages of the raw materials in the present invention refer to volume percentages unless otherwise specified, and the various solutions in the present invention refer to water as the solvent unless otherwise specified.
In the step (1), the alcohol extraction and concentration specifically comprises the following steps: cleaning fructus Vitis Viniferae, collecting peel, mixing with acidic ethanol solution, pulping, ultrasonically extracting at below 50 deg.C (preferably room temperature), filtering, and concentrating the filtrate at 40-50 deg.C under reduced pressure to remove ethanol to obtain crude extract of anthocyanin from pericarpium Vitis Viniferae;
the feed-liquid ratio of the grape skin to the acidic ethanol solution is 1g:4-8 mL;
in the acidic ethanol solution, the volume concentration of ethanol is 50-80%, preferably 60-70%, and the volume concentration of acid is 0.1-1%;
the ultrasonic extraction time is 40-120 min.
In the step (1), in the acidic ethanol solution, an acid is selected from at least one of hydrochloric acid, formic acid, acetic acid and oxalic acid.
In the step (2), the macroporous resin purification method specifically comprises the following steps:
injecting the crude anthocyanin extract of the grape skin into macroporous resin, then respectively eluting with 4 times of column volume (4BV) of acidic ethanol solutions with the ethanol volume concentration of 0, 5 percent, 20 percent and 40 percent, collecting the acidic ethanol eluent with the ethanol volume concentration of 40 percent, and removing the ethanol by reduced pressure evaporation at 40-50 ℃ to obtain anthocyanin eluent;
the macroporous resin is selected from AB-8, D101, XAD-7, HPD-100 or DM-130, and the specific surface area is 450-550m2Per g, the average pore diameter is 10-50nm, and the particle size range is 0.3-1.25 mm;
the acidic ethanol solution is selected from ethanol solution with the volume percentage concentration of acid of 0.1-1.5%, wherein the acid is selected from at least one of hydrochloric acid, formic acid and acetic acid.
In the step (3), the organic solvent is ethyl acetate.
In the step (3), the extraction is preferably carried out for more than 2 times at a volume ratio of the organic solvent to the anthocyanin eluent of 1: 1.
In the step (4), the volume ratio of the methyl tert-butyl ether to the methanol to the water to the trifluoroacetic acid in the two-phase solvent system is 2:2:3: 0.001.
In the step (4), the temperature of the high-speed countercurrent chromatography instrument is stabilized at 20-30 ℃, the stationary phase is pumped in by forward rotation, then the rotating speed is adjusted to 800-950r/min, the mobile phase is introduced at the flow rate of 3mL/min and balanced, and the sample amount of each time is 10-50mg counted by the anthocyanin freeze-dried powder.
In the step (5), a C18 solid phase extraction column is adopted, and gradient elution is carried out by respectively using acidic acetonitrile solutions with acetonitrile volume percentage concentration of 0, 4%, 8%, 12%, 16%, 20%, 24%, 28%, 32%, 36% and 40% by respectively using 2 times of columns;
in the acidic acetonitrile solution, the acid volume percentage concentration is 0.1-2%.
Compared with the prior art, the invention has the main advantages that:
1. the method for separating the petunidin-3-O- (6-O-p-coumaroyl) glucoside (the molecular structure is shown in figure 1) from the grape skin is established for the first time, the yield can reach not less than 8mg/kg of grape skin, and the purity can reach not less than 98%.
2. By combining high-speed counter-current chromatography and a solid-phase extraction column, the petunidin-3-O- (6-O-p-coumaroyl) glucoside can be prepared in large batch from grape raw materials with complex polyphenol components, has the advantages of large treatment capacity, good repeatability and the like, and is convenient for realizing industrial production.
Drawings
FIG. 1 is a molecular structure diagram of petunidin-3-O- (6-O-p-coumaroyl) glucoside;
FIG. 2 is a high performance liquid chromatogram of a crude anthocyanin extract from grape skin in example 1;
FIG. 3 is a high performance liquid chromatogram of a fraction containing petunidin-3-O- (6-O-p-coumaroyl) glucoside obtained by separation and purification with macroporous resin in example 1;
FIG. 4 is a high-speed countercurrent chromatogram of example 1;
FIG. 5 is a high performance liquid chromatogram of a fraction containing petunidin-3-O- (6-O-p-coumaroyl) glucoside obtained by countercurrent chromatography in example 1;
FIG. 6 is a high performance liquid chromatogram of the final product petunidin-3-O- (6-O-p-coumaroyl) glucoside in example 1;
FIG. 7 is a primary mass spectrum and a secondary mass spectrum of petunidin-3-O- (6-O-p-coumaroyl) glucoside in example 1;
FIG. 8 is a liquid chromatogram of the final product in comparative example 1;
FIG. 9 is a liquid chromatogram of the final product in comparative example 3.
Detailed Description
The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.
Example 1
Cleaning and peeling grapes to obtain 1kg of grape skin, wherein the material-liquid ratio is 1g: adding 70% ethanol solution containing 0.5% (v/v) hydrochloric acid at a ratio of 6mL, mixing thoroughly, ultrasonic extracting for 60min, (controlling temperature below 50 deg.C, keeping out of the sun), filtering with gauze, centrifuging the filtrate at 4000rpm for 10min, and collecting supernatant. Extracting the residue with the same method for 1 time. The filtrates were combined and filtered once more using a buchner funnel. Evaporating the filtrate at 45 deg.C under reduced pressure to remove ethanol, and concentrating to obtain crude extract of anthocyanin in grape skin. The high performance liquid chromatogram of the crude extract of anthocyanin from grape skin is shown in FIG. 2.
Loading AB-8 macroporous resin into a chromatographic column, sequentially using ethanol, 0.5mol/L hydrochloric acid solution and 0.5mol/L sodium hydroxide solution, washing with water, and injecting the crude anthocyanin extract into the chromatographic column at the flow rate of 0.2 BV/h. After the sample was applied, the column was eluted with 4 column volumes of acid water (containing 0.5% hydrochloric acid), 5%, 20%, and 40% acidic ethanol (containing 0.5% hydrochloric acid), respectively, and 40% acidic ethanol eluate was collected and evaporated under reduced pressure to remove ethanol. Then, the ratio of 1:1, extracting for 2 times by using ethyl acetate, taking a water phase, appropriately decompressing and concentrating, and freeze-drying to obtain the anthocyanin crude extract freeze-dried powder. The high performance liquid chromatogram of the fraction containing petunidin-3-O- (6-O-p-coumaroyl) glucoside after separation and purification by macroporous resin is shown in FIG. 3.
Mixing methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as 2:2:3: placing into a separating funnel at a volume ratio of 0.001, shaking thoroughly, standing for 30min, separating upper and lower phases, and ultrasonic degassing for 30min respectively. Stabilizing the temperature of an instrument of a high-speed counter-current chromatography system at 20 ℃, pumping a stationary phase, then adjusting the rotating speed to 850r/min, positively rotating, introducing a mobile phase at the flow rate of 3mL/min until balance is reached, dissolving anthocyanin freeze-dried powder in the proportion that every 3mg of freeze-dried powder is dissolved in 1mL of the mobile phase, filtering by using a microporous filter membrane, then injecting a sample, injecting 10mL once, and detecting under an ultraviolet detector, wherein the detection wavelength is 280 nm. Collecting the components (shown in figure 4) for 100-120min and concentrating under reduced pressure to obtain crude petunidin-3-O- (6-O-p-coumaroyl) glucoside monomer solution. After separation by countercurrent chromatography, the high performance liquid chromatogram of the fraction containing petunidin-3-O- (6-O-p-coumaroyl) glucoside is shown in FIG. 5.
Activating a solid phase extraction column by using methanol, then injecting pure water to replace the methanol in the column, then slowly injecting 4mL of crude anthocyanin monomer solution into the solid phase extraction column each time, then performing gradient elution by respectively using acetonitrile solutions containing 0.5% (v/v) hydrochloric acid and having the volume of 0, 4%, 8%, 12%, 16%, 20%, 24%, 28%, 32%, 36% and 40% by 2 times of the column volume, and collecting eluent with the volume percentage concentration of the acetonitrile of 28-32%. Then, the mixture is subjected to reduced pressure concentration and freeze-drying to obtain 9.2mg of the target compound petunidin-3-O- (6-O-p-coumaroyl) glucoside, and the high performance liquid chromatogram is shown in figure 6, wherein the HPLC purity is 99.1%.
And (3) injecting the prepared anthocyanin sample into a mass spectrometer, analyzing the sample according to a mass spectrogram (figure 7), and confirming that the mass number of the anthocyanin obtained by separation is normal.
Example 2
Cleaning and peeling grapes to obtain 4kg of grape skin, wherein the material-liquid ratio is 1g: adding 70% ethanol solution containing 0.5% (v/v) hydrochloric acid at a ratio of 6mL, mixing thoroughly, ultrasonic extracting for 90min, (controlling temperature below 50 deg.C, keeping out of the sun), filtering with gauze, centrifuging the filtrate at 4000rpm for 10min, and collecting supernatant. Extracting the residue with the same method for 1 time. The filtrates were combined and filtered once more using a buchner funnel. Evaporating the filtrate at 45 deg.C under reduced pressure to remove ethanol, and concentrating to obtain crude extract of anthocyanin in grape skin.
Loading AB-8 macroporous resin into a chromatographic column, sequentially using ethanol, 0.5mol/L hydrochloric acid solution and 0.5mol/L sodium hydroxide solution, washing with water, and injecting the crude anthocyanin extract into the chromatographic column at the flow rate of 0.2 BV/h. After the sample was applied, the column was eluted with 4 column volumes of acid water (containing 0.5% hydrochloric acid), 5%, 20%, and 40% acidic ethanol (containing 0.5% hydrochloric acid), respectively, and 40% acidic ethanol eluate was collected and evaporated under reduced pressure to remove ethanol. Then, the ratio of 1:1, extracting for 1 time by using ethyl acetate, taking a water phase, appropriately decompressing and concentrating, and freeze-drying to obtain the anthocyanin crude extract freeze-dried powder.
Mixing methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as 2:2:3: placing into a separating funnel at a volume ratio of 0.001, shaking thoroughly, standing for 30min, separating upper and lower phases, and ultrasonic degassing for 30min respectively. Stabilizing the temperature of an instrument of a high-speed counter-current chromatography system at 20 ℃, pumping a stationary phase, then adjusting the rotating speed to 850r/min, positively rotating, introducing a mobile phase at the flow rate of 3mL/min until balance is reached, dissolving anthocyanin freeze-dried powder in the proportion that every 5mg of freeze-dried powder is dissolved in 1mL of the mobile phase, filtering by using a microporous filter membrane, then injecting a sample, injecting 10mL once, and detecting under an ultraviolet detector, wherein the detection wavelength is 280 nm. Collecting the components of 100-120min and concentrating under reduced pressure to obtain crude petunidin-3-O- (6-O-p-coumaroyl) glucoside monomer solution.
Activating a solid phase extraction column by using methanol, then injecting pure water to replace the methanol in the column, then slowly injecting 4mL of crude anthocyanin monomer solution into the solid phase extraction column each time, then performing gradient elution by respectively using acetonitrile solutions containing 0.5% (v/v) hydrochloric acid and having the volume of 0, 4%, 8%, 12%, 16%, 20%, 24%, 28%, 32%, 36% and 40% by 2 times of the column volume, and collecting eluent with the volume percentage concentration of the acetonitrile of 28-32%. Then, the mixture is decompressed, concentrated and freeze-dried to obtain 40mg of target compound petunidin-3-O- (6-O-p-coumaroyl) glucoside, and the HPLC purity is 98.1%.
Example 3
Cleaning and peeling grapes to obtain 10kg of grape skin, wherein the material-liquid ratio is 1g: adding 70% ethanol solution containing 0.5% (v/v) hydrochloric acid at a ratio of 6mL, mixing thoroughly, ultrasonic extracting for 60min, (controlling temperature below 50 deg.C, keeping out of the sun), filtering with gauze, centrifuging the filtrate at 4000rpm for 10min, and collecting supernatant. Extracting the residue with the same method for 1 time. The filtrates were combined and filtered once more using a buchner funnel. Evaporating the filtrate at 45 deg.C under reduced pressure to remove ethanol, and concentrating to obtain crude extract of anthocyanin in grape skin.
Loading AB-8 macroporous resin into a chromatographic column, sequentially using ethanol, 0.5mol/L hydrochloric acid solution and 0.5mol/L sodium hydroxide solution, washing with water, and injecting the crude anthocyanin extract into the chromatographic column at the flow rate of 0.2 BV/h. After the sample was applied, the column was eluted with 4 column volumes of acid water (containing 0.5% hydrochloric acid), 5%, 20%, and 40% acidic ethanol (containing 0.5% hydrochloric acid), respectively, and 40% acidic ethanol eluate was collected and evaporated under reduced pressure to remove ethanol. Then, the ratio of 1:1, extracting for 2 times by using ethyl acetate, taking a water phase, appropriately decompressing and concentrating, and freeze-drying to obtain the anthocyanin crude extract freeze-dried powder.
Mixing methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as 2:2:3: placing into a separating funnel at a volume ratio of 0.001, shaking thoroughly, standing for 30min, separating upper and lower phases, and ultrasonic degassing for 30min respectively. Stabilizing the temperature of an instrument of a high-speed counter-current chromatography system at 20 ℃, pumping a stationary phase, then adjusting the rotating speed to 850r/min, positively rotating, introducing a mobile phase at the flow rate of 3mL/min until balance is reached, dissolving anthocyanin freeze-dried powder in the proportion that every 5mg of freeze-dried powder is dissolved in 1mL of the mobile phase, filtering by using a microporous filter membrane, then injecting a sample, injecting 10mL once, and detecting under an ultraviolet detector, wherein the detection wavelength is 280 nm. Collecting the components for 100-120min and concentrating under reduced pressure to obtain crude petunidin-3-O- (6-O-p-coumaroyl) glucoside monomer solution.
Activating a solid phase extraction column by using methanol, then injecting pure water to replace the methanol in the column, then slowly injecting 6mL of crude anthocyanin monomer solution into the solid phase extraction column each time, then performing gradient elution by respectively using acetonitrile solutions containing 0.5% (v/v) hydrochloric acid and having the volume of 0, 4%, 8%, 12%, 16%, 20%, 24%, 28%, 32%, 36% and 40% by 2 times of the column volume, and collecting eluent with the volume percentage concentration of the acetonitrile of 28-32%. Then, the mixture is decompressed, concentrated and freeze-dried to obtain 95mg of target compound petunidin-3-O- (6-O-p-coumaroyl) glucoside, and the HPLC purity is 98.6%.
Comparative example 1
The difference from example 1 is only that 10kg of grape skin is used, 15mL of crude anthocyanin monomer solution is used in each solid phase extraction and purification process, other steps are not changed, 87mg of target compound petunidin-3-O- (6-O-p-coumaroyl) glucoside is finally obtained, and the HPLC purity is only 92.2% due to the overlarge sample size in the solid phase extraction and purification process (FIG. 8).
Comparative example 2
In comparison with example 1, the procedure of the solid phase extraction column was omitted, and the other steps were not changed, and the HPLC chromatogram of the final product is shown in FIG. 5. It is understood that this comparative example can only obtain a mixture containing petunidin-3-O- (6-O-p-coumaroyl) glucoside, and cannot obtain a petunidin-3-O- (6-O-p-coumaroyl) glucoside monomer.
Comparative example 3
The preparation process differs from that of example 1 only in 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 2:2: 1: 5: a system of 0.001. Through tests, the target compound petunidin-3-O- (6-O-p-coumaroyl) glucoside cannot be collected within the period of 100-120min, and the part containing the target compound contains a large amount of impurities (as shown in FIG. 9).
Comparative example 4
Compared with the preparation process of the embodiment 1, the preparation process only has the difference of the collection range of the re-solid phase extraction column, and if the eluent with the acetonitrile volume percentage concentration of 28-32% is not collected, the petunidin-3-O- (6-O-p-coumaroyl) glucoside monomer cannot be obtained; if the collection range is more than 28-32%, the purity of the separated petunidin-3-O- (6-O-p-coumaroyl) glucoside monomer is lower than 90%.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.
Claims (6)
1. A method for separating and preparing petunidin-3-O- (6-O-p-coumaroyl) glucoside is characterized by comprising the following steps:
(1) alcohol extraction and concentration: taking grapes as raw materials, and carrying out alcohol extraction and concentration by using an acidic ethanol solution to obtain a crude anthocyanin extract of grape skin;
in the acidic ethanol solution in the step (1), the volume concentration of ethanol is 50-80%, and the volume concentration of acid is 0.1-1%;
(2) and (3) macroporous resin purification: injecting the crude anthocyanin extract of the grape skin into macroporous resin, eluting and concentrating to obtain anthocyanin eluent; the macroporous resin is selected from AB-8, D101, XAD-7, HPD-100 or DM-130, and the specific surface area is 450-550m2Per g, the average pore diameter is 10-50nm, and the particle size range is 0.3-1.25 mm;
(3) and (3) extraction: extracting the anthocyanin eluent by using ethyl acetate, then carrying out reduced pressure concentration and freeze-drying to obtain anthocyanin freeze-dried powder;
(4) high-speed countercurrent chromatographic separation: mixing methyl tert-butyl ether, methanol, water and trifluoroacetic acid in a volume ratio of 2:2:3:0.001 to form a two-phase solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase, sequentially pumping the stationary phase and the mobile phase into a high-speed countercurrent chromatography instrument, dissolving anthocyanin freeze-dried powder by using the mobile phase after the two phases reach balance in a pipeline, injecting a sample, detecting under an ultraviolet detector, wherein the detection wavelength is 280nm, collecting components with retention time of 100 plus of material for 120min, and concentrating under reduced pressure to obtain a crude anthocyanin monomer solution;
(5) and (3) solid phase extraction column purification: injecting the crude anthocyanin monomer solution into a C18 solid-phase extraction column, performing gradient elution by using an acid solution with the acetonitrile volume percentage concentration of 0-40%, collecting an acid acetonitrile eluent with the acetonitrile volume percentage concentration of 28-32%, and performing reduced pressure concentration and freeze drying to obtain a target compound petunidin-3-O- (6-O-p-coumaroyl) glucoside;
the concentration of the crude anthocyanin monomer solution is 5-20mg/mL, and the sample amount is 2-10 mL;
and (3) the acid in the acidic solution in the step (5) is at least one selected from hydrochloric acid, formic acid, acetic acid and oxalic acid.
2. The method according to claim 1, wherein in step (1), the alcohol extraction is concentrated, specifically: cleaning fructus Vitis Viniferae, collecting peel, mixing with acidic ethanol solution, pulping, ultrasonic extracting at below 50 deg.C, filtering, and concentrating the filtrate at 40-50 deg.C under reduced pressure to remove ethanol to obtain crude anthocyanin extract of pericarpium Vitis Viniferae;
the feed-liquid ratio of the grape skin to the acidic ethanol solution is 1g:4-8 mL;
the ultrasonic extraction time is 40-120 min.
3. The method according to claim 2, wherein in the acidic ethanol solution, the acid is at least one selected from hydrochloric acid, formic acid, acetic acid and oxalic acid.
4. The method according to claim 1, wherein in step (2), the macroporous resin purification method is specifically:
injecting the crude anthocyanin extract of the grape skin into macroporous resin, eluting by respectively using 4 times of column volumes of acidic solutions with the volume concentration of ethanol being 0, 5 percent, 20 percent and 40 percent, collecting acidic ethanol eluent with the volume concentration of ethanol being 40 percent, and removing the ethanol by reduced pressure evaporation at 40-50 ℃ to obtain anthocyanin eluent;
the acid solution in the step (2) is selected from a solution with the acid volume percentage concentration of 0.1-1.5%, wherein the acid is selected from at least one of hydrochloric acid, formic acid and acetic acid.
5. The method as claimed in claim 1, wherein in step (4), the temperature of the high-speed countercurrent chromatography apparatus is stabilized at 20-30 ℃, the stationary phase is pumped in forward rotation, then the rotation speed is adjusted to 800-950r/min, the mobile phase is pumped in at a flow rate of 3mL/min and balanced, and the sampling amount in each time is 10-50mg based on the anthocyanin freeze-dried powder.
6. The method of claim 1, wherein in step (5), the gradient elution is carried out by using 2 times of the acidic solution with acetonitrile of 0, 4%, 8%, 12%, 16%, 20%, 24%, 28%, 32%, 36% and 40% by volume respectively;
in the acidic solution in the step (5), the volume percentage concentration of the acid is 0.1-2%.
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| HPLC-ESI-MS/MS 鉴定几种提取物的主要花色苷成分;赵宇等;《中国食品添加剂》;20160731(第7期);第181-186页 * |
| 山葡萄花青素超高压提取工艺优化及其组分分析;杜月娇等;《食品科学》;20171031;第38卷(第10期);第258-263页 * |
| 红肉葡萄果实发育过程中花青苷组分变化与基因表达规律分析;王倩兰等;《西北植物学报》;20190430;第39卷(第4期);第648-659页 * |
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