CN108409805B - Separation and purification method of delphinidin-3-O-galactoside and application thereof - Google Patents

Abstract

The invention discloses a separation and purification method of delphinidin-3-O-galactoside and application thereof in preparation of an alpha-glucosidase inhibitor, wherein the separation and purification method takes complex blueberries formed by anthocyanin as raw materials, and comprises alcohol extraction concentration, macroporous resin adsorption, preparative liquid chromatography purification and high-speed countercurrent chromatography separation, the preparative liquid chromatography and the high-speed countercurrent chromatography technology are combined, and then the high-purity delphinidin-3-O-galactoside monomer is obtained by separation and preparation through optimization of technological parameters, wherein the purity is as high as 99%; activity tests show that the delphinidin-3-O-galactoside monomer can obviously inhibit the activity of alpha-glucosidase and can be used for preparing alpha-glucosidase inhibitors.

Description

A kind of separation and purification method of delphinidin-3-O-galactoside and its application

technical field

The invention relates to the field of separation and purification of natural products, in particular to a method for separation and purification of delphinidin-3-O-galactoside and application thereof.

Background technique

Diabetes is one of the common chronic diseases in the world. Diabetic patients are usually accompanied by symptoms of hyperglycemia. Long-term hyperglycemia can cause damage to tissues and organs such as nerves, heart, blood vessels and kidneys, and cause a variety of acute and chronic complications. . According to statistics from the World Health Organization (WHO), there were 422 million people with diabetes worldwide in 2014, of which type 2 diabetes is the most common. In recent years, with the changes in people's eating habits and lifestyles and the acceleration of the aging process, the prevalence of diabetes in my country has risen rapidly. By the end of 2016, the number of diabetic patients in my country had reached 110 million. Therefore, research on the prevention and treatment of diabetes is of great significance.

Alpha-D-glucoside glucohydrolase (EC 3.2.1.20), also known as α-D-glucosidase hydrolase, is a membrane-bound enzyme in the GH31 family of glycoside hydrolase, including sucrase, maltase, Isomaltase, etc., mainly exists in the brush border cells of the small intestinal villi mucosa. After eating, α-glucosidase can hydrolyze carbohydrates in food into glucose, and the glucose is absorbed into the blood circulation and leads to an increase in blood sugar. Therefore, α-glucosidase is one of the main target enzymes for controlling postprandial blood sugar. At present, the domestic marketed α-glucosidase inhibitors mainly include acarbose, voglibose and miglitol. The use of these inhibitors usually causes adverse reactions in the gastrointestinal tract, such as abdominal distension, gas, etc. . Therefore, mining safe and effective food functional factors and targeting to inhibit α-glucosidase activity will be an effective strategy for reducing the incidence of diabetes and improving the health status of diabetic patients. At present, a large number of researchers at home and abroad have focused on food-derived functional factors, trying to screen out α-glucosidase inhibitors with excellent activity and no side effects on the human body from food. At present, it has been reported that food-derived flavonoids, alkaloids, phenolic compounds, curcuminoids, and terpenoids have strong α-glucosidase inhibitory activity in vitro, and the activity is better than that of positive drugs. Acarbose, but there are not many reports on the α-glucosidase inhibitory activity of anthocyanins. It has been found that cyanidin-3-O glucoside has α-glucosidase activity.

Blueberries, also known as lingonberries and blueberries, belong to the Rhododendron family and the bilberry genus. They are not only rich in basic nutrients needed by the human body, but also rich in a variety of different types of anthocyanins, which have the functions of activating the retina, lowering blood sugar, Anti-inflammatory and antitumor effects. The cultivation of blueberries in my country started relatively late. At present, it is mainly used for direct consumption of fresh food, or processed into primary products such as jam, fruit juice, and fruit wine. However, there are very few blueberry products with high added value in the domestic and foreign markets.

Therefore, it is of great significance to explore a method to separate and prepare high-purity anthocyanin monomers from blueberries for the in-depth research and application of blueberries. However, the separation and purification of high-purity anthocyanin monomers is extremely difficult due to the similar structures of anthocyanins and small differences in polarity.

But at present, it has been reported that high-purity anthocyanin monomers can be obtained by separation and purification.

For example, the Chinese patent document with publication number CN 106366141A discloses a method for separating and preparing pelargonidin-3-O-glucoside monomer, using strawberry as raw material, through freeze-drying, alcohol extraction and concentration, fractional extraction, AB-8 large The pore resin was purified and prepared. Another example is the Chinese patent document with the publication number of CN 106831911A, which discloses a method for separating and purifying pelargonidin-3-O-glucoside monomer from P. Resin and high-speed countercurrent chromatography. Although high-purity anthocyanin monomers are all prepared in the above technical solutions, the main reason is that the anthocyanins in strawberry and P. Three anthocyanin compounds of O-glucoside and pelargonidin-3-O-rutinoside, among which pelargonidin-3-O-glucoside accounts for more than 80% of the total anthocyanin content.

However, for raw materials with complex anthocyanin composition such as blueberries (containing at least 12 structurally similar anthocyanin compounds), the above technical solution is difficult to achieve purification and preparation of high-purity anthocyanin monomers. At present, high-purity anthocyanins prepared by a single column chromatography or chromatography technique are all anthocyanin mixtures, rather than high-purity anthocyanin monomers.

For example, a Chinese patent document with publication number CN 106905391A discloses a blueberry anthocyanin extraction, separation and purification method. The blueberry juice is mixed with an extractant, and 1-4 1-4 Second, filter and combine the filtrates to obtain a crude extract of blueberry anthocyanins, which is then separated and purified by HPD600 macroporous resin. The technical scheme uses 80% ethanol solution with pH=1-2 as the extracting agent, and uses high pressure to release the functional components of blueberry from biological cells, which solves the problem that the effective components of blueberry will not be damaged by high temperature under the premise of maintaining a high extraction rate. However, the obtained extract was a mixture of anthocyanins, and the content of anthocyanins was only 46.45%.

Another example is the Chinese patent document with publication number CN 104109403 A, which discloses a new method for extracting and purifying wild blueberry anthocyanins. Filtration, vacuum freeze-drying, phase separation, and purification by high-speed countercurrent chromatography. The technical scheme adopts non-thermal high-efficiency extraction and separation technology, which improves the extraction speed, but the extraction and purification processes are too complicated to realize large-scale industrial production, and the obtained extract is still a mixture of anthocyanins, and the highest purity of anthocyanins is only 42.7%.

Guo Danni et al. (“Extraction of anthocyanins from blueberries by dextran gel chromatography combined with high-speed countercurrent chromatography”, Guo Danni, Xiang Canhui, Chen Yang et.al, Food Industry, No. 2, 2016) test combined with dextran gel Chromatography and high-speed countercurrent chromatography were used to separate and purify anthocyanins in wild blueberries. Blueberry crude extracts were initially separated by glucan gel chromatography to obtain components with high anthocyanin content, and then separated by high-speed countercurrent chromatography. Using MTBE-n-butanol-acetonitrile-water (volume ratio 1:3:1:5) as a two-phase solvent system, separation was carried out under the conditions of flow rate 0.5mL/min, main engine speed 1860r/min and detection wavelength 280nm, Two kinds of anthocyanins were separated from blueberries separated by Sephadex column at one time, with purities of 65.0% and 90.0%, respectively. Although the technical solution discloses that two kinds of anthocyanins are obtained by separation, but from the UPLC analysis chromatogram of Fig. 2, it can only be inferred that sample 1 and sample 2 may be anthocyanins, and it is impossible to confirm that the two samples are anthocyanins. For the conclusion of anthocyanin, the chemical composition of the two samples could not be further confirmed.

Delphinidin-3-O-galactoside, with the following structural formula, is one of the main anthocyanins in blueberry, and is also an important component of blueberry anthocyanin to exert biological activity.

However, there is no research or report on the preparation of delphinidin-3-O-galactoside monomers from blueberries, nor has the delphinidin-3-O-galactoside monomers been used in the preparation of α-galactoside monomers. - Use of glucosidase inhibitors.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a method for separation and purification of delphinidin-3-O-galactoside, which combines liquid chromatography purification and high-speed countercurrent chromatography separation technology, and then optimizes process parameters. , and isolated and prepared high-purity delphinidin-3-O-galactoside monomer from blueberry raw materials with complex anthocyanin composition; It can significantly inhibit the activity of α-glucosidase and can be used to prepare α-glucosidase inhibitors.

The specific technical solutions are as follows:

A method for separating and purifying delphinidin-3-O-galactoside, comprising:

(1) alcohol extraction and concentration: take blueberry as raw material, obtain blueberry anthocyanin crude extract through alcohol extraction and concentration;

(2) macroporous resin adsorption: the blueberry anthocyanin crude extract is injected into the macroporous resin, and the blueberry anthocyanin extract freeze-dried powder is obtained through elution and post-processing;

(3) purification by preparative liquid chromatography: adopt C18 chromatographic column, carry out gradient elution through mobile phase, and then obtain delphinidin-3-O-galactoside crude lyophilized powder through post-processing;

The mobile phase: Phase A is pure methanol or an acid-methanol system with a volume percent concentration of acid of 0.1-1.5%, and Phase B is a formic acid-water system with a volume percent concentration of formic acid of 1.5-5%;

The procedure of the gradient elution is as follows: the volume percentage concentration of phase A remains unchanged at 5% within 0-5 minutes, increases from 5% to 60% within 5-30 minutes, and collects the eluate for 15-16.5 minutes;

(4) High-speed countercurrent chromatographic separation: using n-butanol-methyl tert-butyl ether-methanol-water-trifluoroacetic acid as a two-phase solvent system, the monomer of delphinidin-3-O-galactoside is obtained by separation ;

The volume ratio of n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid is 2:2:1:5:0.01-0.1.

Unless otherwise specified, the percentages of all raw materials in the present invention are volume percentage concentrations.

Various solutions appearing in the present invention, unless otherwise specified, use water as a solvent.

In step (1), described alcohol extraction is concentrated, specifically:

Mix the washed blueberries with an acidic ethanol solution, filter and collect the filtrate after ultrasonic extraction is complete, remove the ethanol by vacuum rotary evaporation of the filtrate at 40-50° C. and concentrate to obtain a blueberry anthocyanin crude extract;

The acidic ethanol solution is an ethanol solution with an acid volume percentage concentration of 0.1-1.5%;

The acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid;

The volume percent concentration of the ethanol solution is 50-95%;

The mass-volume ratio (that is, the material-to-liquid ratio) of the blueberry and 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° C. in a dark condition.

In order to ensure the complete extraction of anthocyanins, the filter residue obtained after the first extraction was repeated several times under the same conditions.

Further preferably, the volume percentage concentration of the ethanol solution is 60-70%, and the mass-volume ratio of blueberries to acidic ethanol is 1:5-8 g/mL.

In step (2), the adsorption of the macroporous resin is specifically:

The blueberry anthocyanin extract is injected into the macroporous resin, the macroporous resin is first rinsed with deionized water, and then gradient elution is performed with an acidic alcohol solution with a volume percent concentration of 2-22%, and the collected volume percent concentration is 14-18% of the eluate of the acidic alcohol solution, after removing alcohol by vacuum rotary evaporation at 40-50° C., 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;

Preferably, the acidic alcohol solution is selected from alcohol solutions with an acid concentration of 0.1-1.5% by volume;

The alcohol solution is selected from methanol solution or ethanol solution, and the volume percentage concentration is 2-22%;

The acid is selected from at least one of hydrochloric acid, formic acid, and acetic acid.

Further preferably, 2%, 6%, 10%, 14%, 18%, and 22% ethanol solutions containing 0.5% (v/v, the same below) hydrochloric acid were used for gradient elution at 2 column volumes (2BV). , and collect the eluate of 0.5% hydrochloric acid-ethanol solution with a volume percent concentration of 14-18%.

In the present invention, the acidic alcohol solution is 2% ethanol solution containing 0.5% hydrochloric acid as an example, the volume percent concentration of the ethanol solution is 2%, and the volume ratio of hydrochloric acid to ethanol solution is 0.5:99.5.

In step (3), first reconstitute the blueberry anthocyanin extract freeze-dried powder in deionized water, and then inject it into a preparative liquid chromatograph for purification; after purification, the post-processing includes concentration under reduced pressure and Vacuum freeze-dried.

Preferably:

The reconstituted concentration is 20-120 mg/mL, and the injection volume is 1-4 mL;

The specification of the C18 chromatographic column is 20mm×250mm, and the temperature is 30°C;

The acid in the phase A is selected from formic acid or trifluoroacetic acid.

Further preferred:

The reconstituted concentration is 50-120 mg/mL, and the injection volume is 3-4 mL;

The mobile phase: phase A is pure methanol, phase B is a formic acid-water system with a volume percent concentration of formic acid of 1.5-5%, and the flow rate of the mobile phase is 5-10 mL/min.

In step (4), described high-speed countercurrent chromatographic separation is specifically:

To prepare the two-phase solvent system, the upper phase is the stationary phase, the lower phase is the mobile phase, and the stationary phase is pumped into the high-speed countercurrent chromatograph at a flow rate of 20-30 mL/min, at 25-35 ° C, the main engine speed is Under the condition of 700～1000r/min, the mobile phase is pumped at a flow rate of 1～8mL/min, and after the two phases reach equilibrium, the crude lyophilized powder of the delphinidin-3-O-galactoside is prepared. After dissolving with the mobile phase and injecting the sample, after liquid phase detection, the effluent containing only the target product is collected, and then concentrated under reduced pressure and freeze-dried to obtain delphinidin-3-O-galactoside monomer.

Preferably, the concentration of the crude delphinidin-3-O-galactoside lyophilized powder dissolved in the mobile phase is 10-30 mg/mL;

The detection wavelength was 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;

The stationary phase was pumped into the high-speed countercurrent chromatograph at a flow rate of 20-30mL/min, and the stationary phase was pumped into the high-speed countercurrent chromatograph at a flow rate of 3-4mL/min under the conditions of 25-35°C and a main engine speed of 850-910r/min. the mobile phase;

The concentration of the crude delphinidin-3-O-galactoside lyophilized powder dissolved in the mobile phase is 13-27 mg/mL.

After the above preparation process is optimized, the purity of delphinidin-3-O-galactoside monomer obtained by separation and purification is as high as 99%.

After further activity test, it was found that the isolated and purified delphinidin-3-O-galactoside monomer has a significant inhibitory effect on α-glucosidase, and the IC ₅₀ value is 0.11mM, which is significantly better than the positive drug A. Carbose (IC ₅₀ =0.52mM), can be used as a novel natural source of α-glucosidase inhibitor for controlling postprandial blood sugar.

Compared with the prior art, the present invention has the following advantages:

The present invention combines preparative liquid chromatography and high-speed countercurrent chromatography technology for the first time, and through optimization of process parameters, high-purity delphinidin-3-O-galactoside monomer is separated from blueberries, and its purity can be as high as 99%. The separation method has the advantages of large sample processing capacity and good repeatability, and can prepare a large number of high-purity delphinidin-3-O-galactoside monomers, enabling industrial production. The delphinidin-3-O-galactoside monomer can significantly inhibit the activity of α-glucosidase, and can be used for preparing the α-glucosidase inhibitor.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of blueberry anthocyanin extract freeze-dried powder in embodiment 1;

Fig. 2 is the high performance liquid chromatogram of blueberry anthocyanin extract freeze-dried powder after purification by preparative liquid chromatography in Example 1;

Fig. 3 is the high performance liquid chromatogram of final product in embodiment 1;

Figure 4 is the α-glucosidase inhibitory activity curve (a) of delphinidin-3-O-galactoside isolated and purified in Example 1, and the α-glucosidase inhibitory activity curve of acarbose is given (b) as a comparison;

Fig. 5 is the high performance liquid chromatogram of final product in Comparative Example 2;

Fig. 6 is the high performance liquid chromatogram of final product in Comparative Example 5;

FIG. 7 is a high-performance liquid chromatogram of the final product in Comparative Example 6. FIG.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, the following enumerates are only specific embodiments of the present invention, but the protection scope of the present invention is not limited to this:

Example 1

Wash 1kg of fresh blueberries, add a 70% ethanol aqueous solution containing 0.1% (v/v) hydrochloric acid (the volume ratio of ethanol to water is 70%) according to the ratio of material to liquid ratio of 1:8 (w/v, g/mL). : 30) fully mixed, ultrasonically extracted for 90min, (control temperature below 45 ℃, avoid light), vacuum filtration after ultrasonication, the obtained filter residue was extracted once under the above conditions, and the filtrate was combined and removed by vacuum rotary evaporation at 45 ℃ Ethanol to obtain the crude extract of anthocyanins.

The AB-8 macroporous resin was soaked in ethanol for 24 hours and then loaded into a chromatography column. After washing with pure water until there was no alcohol smell, it was washed with 0.5M sodium hydroxide solution at a flow rate of 2BV/h for 1 hour, and then washed with deionized water. until the effluent is neutral; then rinse with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1 h, and then rinse with deionized water until neutral. The crude anthocyanin extract was injected into the AB-8 macroporous resin at a flow rate of 0.4 BV/h until the adsorption volume reached 1/3 of the total resin volume. Rinse the resin with deionized water at a flow rate of 2BV/h, and then use 2%, 6%, 10%, 14%, 18%, and 22% ethanol aqueous solution containing 0.5% (v/v) hydrochloric acid at 2BV/h, respectively. The flow rate of h was washed for 2 hours, and the eluted fraction of 14% to 18% was collected. The ethanol was removed by vacuum rotary evaporation at 45° C. to obtain an anthocyanin extract, which was dissolved in a small amount of deionized water, and then freeze-dried to obtain a blueberry anthocyanin extract freeze-dried powder.

Use a preparative liquid column Unitary C18 20mm×250mm, the mobile phase is: A phase: pure methanol, B phase: formic acid (5%): water (95%); the column temperature is 30 °C, the flow rate is 10 mL/min, and the gradient is : 5% A phase 0～5min, 5%～60% A phase 5～30min. The lyophilized powder of anthocyanin extract was dissolved in deionized water to make its concentration reach 50 mg/mL, and injected into the preparation liquid phase for separation, and the single injection volume was 4 mL. Detected under an ultraviolet detector, collected the peaks of 15-16.5 min, concentrated under reduced pressure to remove methanol, and freeze-dried to obtain the crude lyophilized powder of delphinidin-3-O-galactoside.

Place n-butanol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid in a separatory funnel in a volume ratio of 2:2:1:5:0.01, shake it well, and let it stand for 30 minutes. The phases were separated and degassed by ultrasonic for 30 min. The upper phase was used as the stationary phase and the lower phase as the mobile phase. After the high-speed countercurrent chromatograph was started and preheated for 30 minutes, the circulating water bath was set to 30 °C, the stationary phase was pumped into the instrument at a flow rate of 30 mL/min, and then the forward rotation was connected, and the instrument was started to make the main engine rotate to 850 r/min. After the rotation speed was stabilized, the mobile phase was pumped at a flow rate of 3 mL/min. After the two phases reached equilibrium in the pipeline, 200 mg of delphinidin-3-O-galactoside crude lyophilized powder was dissolved in 15 mL of mobile phase. The sample was injected and detected under a UV detector, the target peak components were collected and concentrated under reduced pressure to remove the organic phase, and lyophilized to obtain 30.1 mg of delphinidin-3-O-galactoside monomer with a purity of 99.47%.

By comparing the high performance liquid chromatograms in Figures 1 to 3, it can be seen that the blueberry anthocyanin freeze-dried powder is mainly a mixture containing 9 anthocyanin monomers after extraction and concentration of blueberries and gradient elution of macroporous resin. Purify by liquid chromatography, collect the eluate for 15 to 16.5 minutes, and obtain a mixture containing delphinidin-3-O-galactoside and a small amount of impurities. Monomeric anthocyanin of oxalin-3-O-galactoside with a purity of up to 99.47%.

Example 2

Wash 5kg of fresh blueberries, add a 70% ethanol aqueous solution containing 0.5% (v/v) hydrochloric acid according to the ratio of material to liquid ratio of 1:7 (w/v), and mix thoroughly, ultrasonically extract for 150 min, (control the temperature at 45° C. Hereinafter, protect from light), vacuum filtration after ultrasonication, the obtained filter residue is repeatedly extracted once according to the above conditions, the filtrates are combined, and the ethanol is removed by vacuum rotary evaporation at 45° C. to obtain a crude extract of anthocyanins.

The AB-8 macroporous resin was soaked in ethanol for 24 hours and then loaded into a chromatography column. After washing with pure water until there was no alcohol smell, it was washed with 0.5M sodium hydroxide solution at a flow rate of 2BV/h for 1 hour, and then washed with deionized water. until the effluent is neutral; then rinse with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1 h, and then rinse with deionized water until neutral. The crude anthocyanin extract was injected into the AB-8 macroporous resin at a flow rate of 0.4 BV/h until the adsorption volume reached 1/3 of the total resin volume. Rinse the resin with deionized water at a flow rate of 2BV/h, and then use 2%, 6%, 10%, 14%, 18%, and 22% ethanol aqueous solution containing 0.5% (v/v) hydrochloric acid at 2BV/h, respectively. The flow rate of h was washed for 2h, and the 14%-18%% eluted fraction was collected. The ethanol was removed by vacuum rotary evaporation at 50° C. to obtain an anthocyanin extract, which was dissolved in a small amount of deionized water, and then freeze-dried to obtain blueberry anthocyanin freeze-dried powder.

Use a preparative liquid column Unitary C18 20mm×250mm, the mobile phase is: A phase: pure methanol, B phase: formic acid (4%): water; the column temperature is 30 ° C, the flow rate is 10 mL/min, the gradient is: 5% A phase 0～5min, 5%～60% A phase 5～30min. The lyophilized powder of anthocyanin extract was dissolved in deionized water to make its concentration reach 80 mg/mL, and injected into the preparation liquid phase for separation, and the single injection volume was 4 mL. Detected under an ultraviolet detector, collected the peaks of 15-16.5 min, concentrated under reduced pressure, and freeze-dried to obtain the crude lyophilized powder of delphinidin-3-O-galactoside.

Place n-butanol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid in a separatory funnel in a volume ratio of 2:2:1:5:0.01, shake it well, and let it stand for 30 minutes. The phases were separated and degassed by ultrasonic for 30 min. The upper phase was used as the stationary phase and the lower phase as the mobile phase. After the high-speed countercurrent chromatograph was started and preheated for 30 minutes, the circulating water bath was set to 25 °C, the stationary phase was pumped into the instrument at a flow rate of 30 mL/min, and then the positive rotation was connected, and the instrument was started to make the main engine rotate to 900 r/min. After the rotation speed is stable, pump into the mobile phase at a flow rate of 3mL/min. After the two phases reach equilibrium in the pipeline, 300mg of delphinidin-3-O-galactoside crude lyophilized powder is dissolved in 15mL of mobile phase. The sample was injected and detected under an ultraviolet detector, the target peak components were collected, concentrated under reduced pressure, and lyophilized to obtain 147 mg delphinidin-3-O-galactoside with a purity of 98.85%.

Example 3

Wash 10kg of fresh blueberries, add a 60% ethanol aqueous solution containing 0.1% (v/v) hydrochloric acid according to the ratio of material to liquid ratio of 1:5 (w/v) and mix thoroughly, extract by ultrasonic for 200min, (control the temperature at 45°C) Hereinafter, protect from light), vacuum filtration after ultrasonication, the obtained filter residue is repeatedly extracted once according to the above conditions, the filtrates are combined, and the ethanol is removed by vacuum rotary evaporation at 45° C. to obtain a crude extract of anthocyanins.

The AB-8 macroporous resin was soaked in ethanol for 24 hours and then loaded into a chromatography column. After washing with pure water until there was no alcohol smell, it was washed with 0.5M sodium hydroxide solution at a flow rate of 2BV/h for 1 hour, and then washed with deionized water. until the effluent is neutral; then rinse with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1 h, and then rinse with deionized water until neutral. The crude anthocyanin extract was injected into the AB-8 macroporous resin at a flow rate of 0.4 BV/h until the adsorption volume reached 1/3 of the total resin volume. Rinse the resin with deionized water at a flow rate of 2BV/h, and then use 2%, 6%, 10%, 14%, 18%, and 22% ethanol aqueous solution containing 0.5% (v/v) hydrochloric acid at 2BV/h, respectively. The flow rate of h was washed for 2 hours, and the eluted fraction of 14% to 18% was collected. The ethanol was removed by vacuum rotary evaporation at 45° C. to obtain an anthocyanin extract, which was dissolved in a small amount of deionized water, and then freeze-dried to obtain a blueberry anthocyanin freeze-dried powder.

Use a preparative liquid column Unitary C18 20mm×250mm, the mobile phase is: A phase: pure methanol, B phase: formic acid (1.5%): water; the column temperature is 30 °C, the flow rate is 10 mL/min, the gradient is: 5% A phase 0～5min, 5%～60% A phase 5～30min. The lyophilized powder of the anthocyanin extract was dissolved in deionized water to make the concentration reach 120 mg/mL, and injected into the preparation liquid phase for separation, and the single injection volume was 4 mL. Detected under an ultraviolet detector, collected the peaks of 15-16.5 min, concentrated under reduced pressure, and freeze-dried to obtain the crude lyophilized powder of delphinidin-3-O-galactoside.

Place n-butanol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid in a separatory funnel in a volume ratio of 2:2:1:5:0.01, shake it well, and let it stand for 30 minutes. The phases were separated and degassed by ultrasonic for 30 min. The upper phase was used as the stationary phase and the lower phase as the mobile phase. After the high-speed countercurrent chromatograph was started and preheated for 30 minutes, the circulating water bath was set to 35 °C, the stationary phase was pumped into the instrument at a flow rate of 30 mL/min, and then the positive rotation was connected, and the instrument was started to make the main engine rotate to 910 r/min. After the rotation speed is stable, the mobile phase is pumped at a flow rate of 4 mL/min. After the two phases reach equilibrium in the pipeline, 400 mg of delphinidin-3-O-galactoside crude lyophilized powder is dissolved in 15 mL of mobile phase. The sample was injected and detected under an ultraviolet detector, the target peak components were collected, concentrated under reduced pressure, and lyophilized to obtain 291.8 mg delphinidin-3-O-galactoside with a purity of 98.64%.

Comparative Example 1

Wash 10kg of fresh blueberries, add 60% ethanol aqueous solution containing 0.1% (v/v) hydrochloric acid according to the ratio of material to liquid ratio of 1:7 (w/v), and mix thoroughly, ultrasonically extract for 200min, (control the temperature at 45°C) Hereinafter, protect from light), vacuum filtration after ultrasonication, the obtained filter residue is repeatedly extracted once according to the above conditions, the filtrates are combined, and the ethanol is removed by vacuum rotary evaporation at 45° C. to obtain a crude extract of anthocyanins.

The AB-8 macroporous resin was soaked in ethanol for 24 hours and then loaded into a chromatography column. After washing with pure water until there was no alcohol smell, it was washed with 0.5M sodium hydroxide solution at a flow rate of 2BV/h for 1 hour, and then washed with deionized water. until the effluent is neutral; then rinse with 0.5M hydrochloric acid solution at a flow rate of 2BV/h for 1 h, and then rinse with deionized water until neutral. The crude anthocyanin extract was injected into the AB-8 macroporous resin at a flow rate of 0.4 BV/h until the adsorption volume reached 1/3 of the total resin volume. Rinse the resin with deionized water at a flow rate of 2BV/h, and then use 2%, 6%, 10%, 14%, 18%, and 22% ethanol aqueous solution containing 0.5% (v/v) hydrochloric acid at 2BV/h, respectively. The flow rate of h was washed for 2 hours, and the eluted fraction of 14% to 18% was collected. The ethanol was removed by vacuum rotary evaporation at 45° C. to obtain an anthocyanin extract, which was dissolved in a small amount of deionized water, and then freeze-dried to obtain a blueberry anthocyanin freeze-dried powder.

Use a preparative liquid column Unitary C18 20mm×250mm, the mobile phase is: A phase: pure methanol, B phase: formic acid (0.1%): water; the column temperature is 30 ° C, the flow rate is 10 mL/min, the gradient is: 5% A phase 0～5min, 5%～60% A phase 5～30min. The lyophilized powder of anthocyanin extract was dissolved in deionized water to make its concentration reach 120 mg/mL, and injected into the preparation liquid phase for separation, and the single injection volume was 3 mL. Detected under an ultraviolet detector, collected the peaks of 15-16.5 min, concentrated under reduced pressure, and freeze-dried to obtain the crude lyophilized powder of delphinidin-3-O-galactoside.

Place n-butanol:methyl tert-butyl ether:methanol:water:trifluoroacetic acid in a separatory funnel in a volume ratio of 2:2:1:5:0.1, shake well, and let stand for 30 minutes, then put the upper and lower The phases were separated and degassed by ultrasonic for 30 min. The upper phase was used as the stationary phase and the lower phase as the mobile phase. After the high-speed countercurrent chromatograph was started and preheated for 30 minutes, the circulating water bath was set to 35 °C, the stationary phase was pumped into the instrument at a flow rate of 30 mL/min, and then the positive rotation was connected, and the instrument was started to make the main engine rotate to 910 r/min. After the rotation speed is stable, the mobile phase is pumped at a flow rate of 4 mL/min. After the two phases reach equilibrium in the pipeline, 400 mg of delphinidin-3-O-galactoside crude lyophilized powder is dissolved in 15 mL of mobile phase. The sample was injected and detected under an ultraviolet detector, the target peak components were collected, concentrated under reduced pressure, and lyophilized to obtain 315.6 mg delphinidin-3-O-galactoside with a purity of 89.61%.

Comparative Example 2

Compared with Example 1, the step of purification by preparative liquid chromatography is removed, and other steps remain unchanged. After testing, only a mixture containing delphinidin-3-O-galactoside can be obtained, but delphinidin-3 cannot be obtained. -O-galactoside monomer, the high performance liquid chromatogram of the final product is shown in Figure 5.

Comparative Example 3

The preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with: n-butanol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid at 1:3:1:5: Mix at a volume ratio of 0.01. After testing, delphinidin-3-O-galactoside monomer could not be obtained.

Comparative Example 4

The preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with: n-butanol: methyl tert-butyl ether: acetonitrile: water: trifluoroacetic acid at 2:2:1:5: Mix at a volume ratio of 0.01. After testing, it is possible to separate and obtain delphinidin-3-O-galactoside monomer, but its purity is only 92.48%, which is lower than the delphinidin-3-O-galactoside monomer prepared in Example 1. The purity of the body (99.47%).

Comparative Example 5

The preparation process is the same as that of Example 1, except that the mobile phase B in the liquid chromatography purification process is prepared, and the formic acid-aqueous solution system is replaced with an aqueous solution, that is, no formic acid is added, and other steps remain unchanged. The monomer purity of arvenin-3-O-galactoside is only 71.69%, and the high performance liquid chromatogram of the final product is shown in Figure 6.

Comparative Example 6

The preparation process is the same as that of Example 1, and the difference is only in that the component collection time in the purification process of the preparative liquid chromatography is changed. O-galactoside monomer, if the component collection time is included and wider than the range of 15-16.5min, the purity of the obtained delphinidin-3-O-galactoside monomer is less than 98%. High performance liquid chromatography The diagram is shown in Figure 7.

Application example 1

The delphinidin-3-O-galactoside prepared in Example 1 was dissolved in water, and made into a series of concentrations (0.1mmol/L, 0.2mmol/L, 0.5mmol/L, 0.7mmol/L, 1mmol/L). L) as an inhibitor. The α-glucosidase was diluted with 0.1 mol/L phosphate buffer solution (PBS, pH=6.9) until the enzyme activity was 0.5 U/mL. The substrate p-nitrophenyl-α-D-glucopyranoside (PNPG) was prepared with 0.1 mol/L phosphate buffer (PBS, pH=6.9) to a concentration of 1 mmol/L. The enzymatic reaction system was 20 μL of The enzyme was mixed with 10 μL of inhibitor, 130 μL of buffer and 40 μL of substrate were added, and the reaction was carried out at 37 °C for 30 min. After that, 200 μL of 1 mol/L sodium carbonate solution was added, and the absorbance was detected at 405 nm. In the blank group, the inhibitor was replaced with buffer. The enzyme activity inhibition rate was calculated according to the following formula: Inhibition rate %=(A _blank -A _experiment )/A _blank *100%. The IC ₅₀ value of delphinidin-3-O-galactoside measured in this reaction system was 0.11 mM, and the positive control acarbose was 0.52 mM.

Application example 2

The cyanidin-3-O-glucoside (commercially available) that has been reported to have α-glucosidase inhibitory activity was prepared into a series of concentrations (0.1mmol/L, 1mmol/L, 2mmol/L, 5mmol/L, 10 mmol/L) as an inhibitor. The α-glucosidase was diluted with 0.1 mol/L phosphate buffer solution (PBS, pH=6.9) until the enzyme activity was 0.5 U/mL. The substrate p-nitrophenyl-α-D-glucopyranoside (PNPG) was prepared with 0.1 mol/L phosphate buffer (PBS, pH=6.9) to a concentration of 1 mmol/L. The enzymatic reaction system was 20 μL of The enzyme was mixed with 10 μL of inhibitor, 130 μL of buffer and 40 μL of substrate were added, and the reaction was carried out at 37 °C for 30 min. After that, 200 μL of 1 mol/L sodium carbonate solution was added, and the absorbance was detected at 405 nm. In the blank group, the inhibitor was replaced with buffer. The enzyme activity inhibition rate was calculated according to the following formula: Inhibition rate %=(A blank-A experiment)/A blank*100%. The IC ₅₀ value of cyanidin-3-O-glucoside measured in this reaction system was 1.03 mM, and the positive control acarbose was 0.52 mM.

By comparison, it can be seen that the inhibitory effect of delphinidin-3-O-galactoside prepared by the present invention on α-glucosidase is significantly better than that of positive control acarbose and cyanidin-3-O- glucoside. Therefore, delphinidin-3-O-galactoside isolated and prepared from blueberry can be used as a novel natural source of α-glucosidase inhibitor.

Claims (9)

1. a separation and purification method of delphinidin-3-O-galactoside, is characterized in that, comprises: (1) alcohol extraction and concentration: take blueberry as raw material, obtain blueberry anthocyanin crude extract through alcohol extraction and concentration; (2) macroporous resin adsorption: the blueberry anthocyanin crude extract is injected into the macroporous resin, and the blueberry anthocyanin extract freeze-dried powder is obtained through elution and post-processing; (3) purification by preparative liquid chromatography: adopt C18 chromatographic column, carry out gradient elution through mobile phase, and then obtain delphinidin-3-O-galactoside crude lyophilized powder through post-processing; The mobile phase: Phase A is pure methanol or an acid-methanol system with a volume percent concentration of acid of 0.1-1.5%, and Phase B is a formic acid-water system with a volume percent concentration of formic acid of 1.5-5%; The procedure of the gradient elution is as follows: the volume percentage concentration of phase A remains unchanged at 5% within 0-5 minutes, increases from 5% to 60% within 5-30 minutes, and collects the eluate for 15-16.5 minutes; (4) High-speed countercurrent chromatographic separation: using n-butanol-methyl tert-butyl ether-methanol-water-trifluoroacetic acid as a two-phase solvent system, the monomer of delphinidin-3-O-galactoside is obtained by separation ; The volume ratio of n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid is 2:2:1:5:0.01. 2. the separation and purification method of delphinidin-3-O-galactoside according to claim 1, is characterized in that, in step (1), described alcohol extraction concentrates, is specially: Mix the washed blueberries with an acidic ethanol solution, filter and collect the filtrate after ultrasonic extraction is complete, remove the ethanol by vacuum rotary evaporation of the filtrate at 40-50° C. and concentrate to obtain a blueberry anthocyanin crude extract; The acidic ethanol solution is an ethanol solution with an acid volume percentage concentration of 0.1-1.5%; The acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid; The volume percent concentration of the ethanol solution is 50-95%; The mass-volume ratio of the blueberry to the acidic ethanol solution is 1:5-12 g/mL. 3. the separation and purification method of delphinidin-3-O-galactoside according to claim 1, is characterized in that, in step (2), described macroporous resin adsorption, is specially: The blueberry anthocyanin extract is injected into the macroporous resin, the macroporous resin is first rinsed with deionized water, and then gradient elution is performed with an acidic alcohol solution with a volume percent concentration of 2-22%, and the collected volume percent concentration is 14-18% of the eluate of the acidic alcohol solution, after removing alcohol by vacuum rotary evaporation at 40-50° C., 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 the alcohol solution with an acid volume percentage concentration of 0.1-1.5%, the alcohol solution is selected from methanol solution or ethanol solution, and the acid is selected from at least one of hydrochloric acid, formic acid and acetic acid. 4. the method for separation and purification of delphinidin-3-O-galactoside according to claim 1, is characterized in that, in step (3), first described blueberry anthocyanin extract freeze-dried powder is removed After redissolving in ionized water, it is injected into a preparative liquid chromatograph for purification; The reconstituted concentration is 20-120 mg/mL, and the injection volume is 1-4 mL; The specification of the C18 chromatographic column is 20mm×250mm, and the temperature is 25～30℃; The acid in the phase A is selected from formic acid or trifluoroacetic acid. 5. the separation and purification method of delphinidin-3-O-galactoside according to claim 4, is characterized in that: The reconstituted concentration is 50-120 mg/mL; The mobile phase: phase A is pure methanol, phase B is a formic acid-water system with a volume percent concentration of formic acid of 1.5-5%, and the flow rate of the mobile phase is 5-10 mL/min. 6. the separation and purification method of delphinidin-3-O-galactoside according to claim 1, is characterized in that, in step (3), described post-processing comprises decompression concentration and vacuum freeze-drying. 7. the separation and purification method of delphinidin-3-O-galactoside according to claim 1, is characterized in that, in step (4), described high-speed countercurrent chromatographic separation, is specially: To prepare the two-phase solvent system, the upper phase is the stationary phase, the lower phase is the mobile phase, and the stationary phase is pumped into the high-speed countercurrent chromatograph at a flow rate of 20-30 mL/min, at 25-35 ° C, the main engine speed is Under the condition of 700～1000r/min, the mobile phase is pumped at a flow rate of 1～8mL/min, and after the two phases reach equilibrium, the crude lyophilized powder of the delphinidin-3-O-galactoside is prepared. After dissolving with the mobile phase and injecting the sample, after liquid phase detection, the effluent containing only the target product is collected, and then concentrated under reduced pressure and freeze-dried to obtain delphinidin-3-O-galactoside monomer. 8. the separation and purification method of delphinidin-3-O-galactoside according to claim 7, is characterized in that, described stationary phase is pumped in high-speed countercurrent chromatograph with the flow velocity of 20～30mL/min , under the conditions of 25-35° C. and the rotational speed of the main engine being 850-910 r/min, the mobile phase is pumped at a flow rate of 3-4 mL/min. 9. the separation and purification method of delphinidin-3-O-galactoside according to claim 8, is characterized in that, described delphinidin-3-O-galactoside crude product freeze-dried powder flows with flow The concentration after phase dissolution is 10-30 mg/mL, and the injection volume is 1-15 mL; The wavelength of the liquid phase detection was 280 nm.

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