Separation preparation method and application of cyanidin-3-O-galactoside

Abstract

The invention relates to the technical field of natural product separation, in particular to a separation preparation method and application of cyanidin-3-O-galactoside. The high-purity cyanidin-3-O galactoside monomer is separated and prepared from a cranberry raw material with complex anthocyanin composition by combining preparative liquid chromatography and high-speed counter-current chromatography technologies and optimizing process parameters, and the purity of the cyanidin-3-O galactoside monomer can reach 99%. The separation and preparation method has the advantages of large sample treatment capacity, good repeatability and the like, and a large amount of high-purity cyanidin-3-O-galactoside monomers can be prepared, so that industrial production can be realized; animal experiments find that the cyanidin-3-O-galactose can reduce fasting blood-glucose of mice with type 2 diabetes and improve insulin resistance, and provides potential application for the field of preparation of medicaments for treating type 2 diabetes or hypoglycemic food.

Description

Technical Field

[0001] The invention relates to the technical field of natural product separation, and in particular to a separation and preparation method of cyanidin-3-O-galactoside and application thereof. Background Art

[0002] Diabetes is one of the most 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 the occurrence of a variety of acute and chronic complications. At present, different pharmacological methods such as stimulating insulin release, increasing glucose transport activity, inhibiting gluconeogenesis and reducing intestinal glucose absorption have been used to control diabetes. Oral hypoglycemic drugs such as biguanides, thiazolidinediones and glucosidase inhibitors are currently the main drugs for the treatment of type 2 diabetes. However, these drugs can only maintain blood sugar control, and long-term use may be accompanied by serious side effects. Therefore, finding and studying more effective and safer natural hypoglycemic drugs has become an important scientific issue at present. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a separation and preparation method of cyanidin-3-O-galactoside and its application. The cyanidin-3-O-galactoside obtained by the separation and preparation method can be used to prepare medicines or foods for treating type 2 diabetes.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for separating and preparing cyanidin-3-O-galactoside, comprising the following steps:

[0006] The cranberries are subjected to alcohol extraction to obtain a crude cranberry anthocyanin extract;

[0007] The crude cranberry anthocyanin extract is adsorbed by a macroporous resin and then freeze-dried to obtain a freeze-dried cranberry anthocyanin extract powder;

[0008] Sequentially subjecting the freeze-dried powder of cranberry anthocyanin extract to liquid chromatography purification and high-speed countercurrent chromatography separation to obtain cyanidin-3-O-galactoside;

[0009] The chromatographic column used for the liquid chromatography purification is a C18 chromatographic column, and the mobile phase includes phase A and phase B, wherein the phase A is pure methanol or a methanol solution of an acid with a volume percentage concentration of 0.1 to 1.5%; the phase B is an aqueous solution of formic acid with a volume percentage concentration of 1 to 5%; the elution procedure of the liquid chromatography purification is gradient elution, wherein the volume percentage concentration of phase A remains unchanged at 5% within 0 to 5 minutes, and increases from 5% to 60% within 5 to 24 minutes, and the eluate is collected for 17 to 18 minutes;

[0010] The two-phase solvent system for preparing the stationary phase and the mobile phase in the high-speed countercurrent chromatography separation comprises n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid.

[0011] Preferably, the volume ratio of n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid is 2:2:1:5:(0.01-0.1).

[0012] Preferably, before the liquid chromatography purification, the method further comprises mixing the cranberry anthocyanin extract with water to obtain a cranberry anthocyanin extract solution;

[0013] The concentration of the cranberry anthocyanin extract solution is 20 to 120 mg / mL, and the injection volume is 1 to 4 mL;

[0014] When the phase A is a methanol solution of an acid with a volume percentage concentration of 0.1 to 1.5%, the acid in the phase A is formic acid or trifluoroacetic acid.

[0015] Preferably, in the mobile phase of the liquid chromatography purification, phase A is pure methanol, and phase B is an aqueous solution of formic acid with a volume percentage concentration of 1.5 to 5%;

[0016] The flow rate of the mobile phase is 5-10 mL / min.

[0017] Preferably, the process of high speed countercurrent chromatography separation is:

[0018] The two-phase solvent system is prepared, with the upper phase being the stationary phase and the lower phase being the mobile phase. The stationary phase is pumped into a high-speed countercurrent chromatograph at a flow rate of 20 to 30 mL / min. Under the conditions of 25 to 35° C. and a main engine speed of 700 to 1000 r / min, the mobile phase is pumped in at a flow rate of 1 to 8 mL / min. After the two phases reach equilibrium, the crude lyophilized powder of cyanidin-3-O-galactoside is dissolved in the mobile phase and then injected. After liquid phase detection, the effluent containing only the target product is collected.

[0019] Preferably, the concentration of the cyanidin-3-O-galactoside crude freeze-dried powder solution obtained after the dissolution is 10 to 30 mg / mL;

[0020] The injection volume of the cyanidin-3-O-galactoside crude lyophilized powder solution is 1 to 15 mL;

[0021] The wavelength of the liquid phase detection is 280 nm.

[0022] Preferably, the extracting solution used in the alcohol extraction is an acidic ethanol solution;

[0023] The volume percentage concentration of the acid in the acidic ethanol solution is 0.1% to 1.5%;

[0024] The volume ratio of ethanol in the acidic ethanol solution to the acidic ethanol solution is (50-95):100.

[0025] Preferably, the ratio of the cranberry to the acidic ethanol solution is 1 g: (4-10) mL;

[0026] The alcohol extraction is carried out under light-proof and ultrasonic conditions.

[0027] Preferably, the macroporous resin adsorption process is: injecting the cranberry anthocyanin crude extract into the macroporous resin, first washing the macroporous resin with deionized water, then gradient eluting with an acidic alcohol solution, collecting the eluate of the acidic alcohol solution with a volume percentage concentration of 14-18% for rotary evaporation;

[0028] The volume ratio of the alcohol in the acidic alcohol solution to the acidic alcohol solution is (2-40):100.

[0029] The present invention also provides the use of cyanidin-3-O-galactoside prepared by the separation and preparation method described in the above technical scheme in the preparation of hypoglycemic drugs or hypoglycemic foods.

[0030] The present invention provides a separation and preparation method of cyanidin-3-O-galactoside, which is characterized by comprising the following steps: performing alcohol extraction on cranberries to obtain a crude cranberry anthocyanin extract; performing macroporous resin adsorption on the crude cranberry anthocyanin extract, and then freeze-drying to obtain a lyophilized cranberry anthocyanin extract powder; performing liquid chromatography purification and high-speed countercurrent chromatography separation on the lyophilized cranberry anthocyanin extract powder in sequence to obtain the cyanidin-3-O-galactoside; the chromatographic column used for the liquid chromatography purification is a C18 chromatographic column, and the mobile phase comprises an A phase and a B phase. The phase A is pure methanol or a methanol solution of an acid with a volume percentage concentration of 0.1 to 1.5%; the phase B is an aqueous solution of formic acid with a volume percentage concentration of 1 to 5%; the elution procedure of the liquid chromatography purification is gradient elution, and the volume percentage concentration of the phase A is kept constant at 5% within 0 to 5 minutes, and increases from 5% to 60% within 5 to 24 minutes, and the eluate is collected for 17 to 18 minutes; the two-phase solvent system for preparing the stationary phase and the mobile phase in the high-speed countercurrent chromatography separation includes n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid. The present invention combines preparative liquid chromatography and high-speed countercurrent chromatography technology, and then through the optimization of process parameters, separates and prepares high-purity cyanidin-3-O-galactoside monomers from cranberry raw materials with complex anthocyanin composition, and its purity can reach 99%. The separation and preparation method has the advantages of large sample processing capacity and good repeatability, and can prepare a large amount of high-purity cyanidin-3-O-galactoside monomers, so that it can be industrially produced. Animal experiments have found that cyanidin-3-O-galactose can reduce the fasting blood sugar and improve insulin resistance in type 2 diabetic mice, which provides potential applications in the preparation of drugs for treating type 2 diabetes or hypoglycemic foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the HPLC chromatogram of the freeze-dried powder of anthocyanin extract described in Example 1;

[0032] Figure 2 is the HPLC chromatogram of cyanidin-3-O-galactoside monomer described in Example 1;

[0033] Figure 3 The effect of cyanidin-3-O-galactoside on the body weight of type 2 diabetic mice described in the test example;

[0034] Figure 4 The effect of cyanidin-3-O-galactoside on fasting blood glucose in type 2 diabetic mice described in the test example;

[0035] Figure 5 The effect of cyanidin-3-O-galactoside on glucose sensitivity of type 2 diabetic mice described in the test example;

[0036] Figure 6 The effect of cyanidin-3-O-galactoside on insulin resistance in type 2 diabetic mice described in the test example;

[0037] Figure 7 The test example is the effect of cyanidin-3-O-galactoside on serum lipid levels (including triglycerides, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol) in type 2 diabetic mice. DETAILED DESCRIPTION

[0038] The present invention provides a method for separating and preparing cyanidin-3-O-galactoside, comprising the following steps:

[0039] The cranberries are subjected to alcohol extraction to obtain a crude cranberry anthocyanin extract;

[0040] The crude cranberry anthocyanin extract is adsorbed by a macroporous resin and then freeze-dried to obtain a freeze-dried cranberry anthocyanin extract powder;

[0041] Sequentially subjecting the freeze-dried powder of cranberry anthocyanin extract to liquid chromatography purification and high-speed countercurrent chromatography separation to obtain cyanidin-3-O-galactoside;

[0042] The chromatographic column used for the liquid chromatography purification is a C18 chromatographic column, and the mobile phase includes phase A and phase B, wherein the phase A is pure methanol or a methanol solution of an acid with a volume percentage concentration of 0.1 to 1.5%; the phase B is an aqueous solution of formic acid with a volume percentage concentration of 1 to 5%; the elution procedure of the liquid chromatography purification is gradient elution, wherein the volume percentage concentration of phase A remains unchanged at 5% within 0 to 5 minutes, and increases from 5% to 60% within 5 to 24 minutes, and the eluate is collected for 17 to 18 minutes;

[0043] The two-phase solvent system for preparing the stationary phase and the mobile phase in the high-speed countercurrent chromatography separation comprises n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid.

[0044] In the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0045] The invention carries out alcohol extraction on cranberries to obtain a crude cranberry anthocyanin extract.

[0046] Before performing the alcohol addition, the present invention preferably washes the cranberries; the present invention does not have any special limitation on the washing process, and the washing process may be performed using a process well known to those skilled in the art.

[0047] In the present invention, the extracting solution used in the alcohol extraction is preferably an acidic ethanol solution; the volume percentage concentration of the acid in the acidic ethanol solution is preferably 0.1-1.5%, more preferably 0.5-1.0%. In the present invention, the acid solution is preferably one or more of hydrochloric acid, formic acid, acetic acid and oxalic acid. When the acid solution is two or more of the above specific selections, the present invention has no special restrictions on the ratio of the above specific substances, and they can be mixed in any ratio. In the present invention, the volume ratio of ethanol in the acidic ethanol solution to the acidic ethanol solution is preferably (50-90):100. In the present invention, the volume ratio of ethanol to water in the acidic ethanol solution is preferably (50-95):(50-5), more preferably (60-70):(40-30).

[0048] In the present invention, during the alcohol extraction process, the usage ratio of cranberry to extract is preferably 1 g: (4-10) mL, more preferably 1 g: (4-6) mL.

[0049] In the present invention, the temperature of the alcohol extraction is preferably 25 to 49° C., more preferably 30 to 40° C.; the alcohol extraction is preferably carried out under light-proof and ultrasonic conditions, and the ultrasonic time is preferably 60 to 240 minutes, more preferably 100 to 200 minutes. The present invention does not have any special limitation on the frequency of the ultrasound, and any frequency known to those skilled in the art can be used.

[0050] After the alcohol extraction is completed, the present invention also preferably includes filtration, repeated alcohol extraction, combined filtrate and rotary evaporation performed in sequence; in the present invention, the filtering method is preferably suction filtration, and the present invention has no special restrictions on the process of suction filtration, and the process of suction filtration well known to those skilled in the art can be used. In the present invention, the process of repeated alcohol extraction is preferably referred to the above-mentioned alcohol extraction process and will not be repeated here. In the present invention, the rotary evaporation is preferably vacuum rotary evaporation at 45°C to remove ethanol.

[0051] After obtaining the crude cranberry anthocyanin extract, the present invention adsorbs the crude cranberry anthocyanin extract with a macroporous resin and then freeze-dries it to obtain a freeze-dried cranberry anthocyanin extract powder.

[0052] In the present invention, the process of macroporous resin adsorption is preferably: injecting the crude extract of cranberry anthocyanins into the macroporous resin, first rinsing the macroporous resin with deionized water, then gradient eluting with an acidic alcohol solution, collecting the eluate of the acidic alcohol solution with a volume percentage concentration of 14-18% for rotary evaporation; the volume ratio of alcohol in the acidic alcohol solution to the acidic alcohol solution is (2-40):100.

[0053] In the present invention, the brand of the macroporous resin is preferably AB-8, HPD-100, D101 or DM-130.

[0054] In the present invention, the volume percentage concentration of the acid in the acidic alcohol solution is preferably 0.1-1.5%, more preferably 0.5-1.0%, and most preferably 0.5%. In the present invention, the type of the acid solution is preferably one or more of hydrochloric acid, formic acid and acetic acid. When the acid solution is two or more of the above specific selections, the present invention has no special restrictions on the ratio of the above specific substances, and they can be mixed in any ratio. In the present invention, the volume ratio of the alcohol in the acidic alcohol solution to the acidic alcohol solution is preferably (2-40):100. In the present invention, the alcohol in the acidic alcohol solution is preferably methanol and / or ethanol; the volume ratio of the alcohol and water in the acidic alcohol solution is preferably (2-22):(98-78), more preferably (5-15):(95-85), and most preferably 2:98, 6:94, 10:90, 14:86, 18:82 or 22:78.

[0055] In the present invention, during the gradient elution, the volume of the acidic alcohol solution is preferably 1 to 4 times, more preferably 2 times, the volume of the macroporous resin column.

[0056] After the adsorption of the macroporous resin is completed, the present invention also preferably includes rotary evaporation, dissolution and freeze-drying performed in sequence. In the present invention, the rotary evaporation is preferably vacuum rotary evaporation, and the temperature of the vacuum rotary evaporation is preferably 40 to 50° C., more preferably 45° C. In the present invention, the dissolution is preferably carried out using deionized water. The present invention does not have any special restrictions on the freeze-drying process, and the process well known to those skilled in the art can be used.

[0057] After obtaining the freeze-dried powder of cranberry anthocyanin extract, the present invention sequentially performs liquid chromatography purification and high-speed countercurrent chromatography separation on the freeze-dried powder of cranberry anthocyanin extract to obtain the cyanidin-3-O-galactoside; the chromatographic column used in the liquid chromatography purification is a C18 chromatographic column, and the mobile phase includes phase A and phase B, the phase A is pure methanol or a methanol solution of an acid with a volume percentage concentration of 0.1-1.5%; the phase B is an aqueous solution of formic acid with a volume percentage concentration of 1-5%; the elution procedure of the liquid chromatography purification is gradient elution, the volume percentage concentration of the phase A is kept constant at 5% within 0-5 minutes, and increases from 5% to 60% within 5-24 minutes, and the eluate is collected for 17-18 minutes; the two-phase solvent system for preparing the stationary phase and the mobile phase in the high-speed countercurrent chromatography separation includes n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid.

[0058] In the present invention, before the liquid chromatography purification, the cranberry anthocyanin extract and water are mixed to obtain a cranberry anthocyanin extract solution; in the present invention, the concentration of the cranberry anthocyanin extract solution is preferably 20 to 120 mg / mL, more preferably 50 to 120 mg / mL, and most preferably 50 mg / mL; the injection volume is preferably 1 to 4 mL, more preferably 3 to 4 mL.

[0059] In the present invention, the specification of the C18 chromatographic column is preferably 20 mm×250 mm, and the column temperature of the C18 chromatographic column is preferably 30° C.

[0060] In the present invention, when the phase A is a methanol solution of an acid with a volume percent concentration of 0.1 to 1.5%, the acid in the phase A is preferably formic acid or trifluoroacetic acid.

[0061] In the present invention, when phase A in the mobile phase of the liquid chromatography purification is pure methanol, phase B is preferably an aqueous solution of formic acid with a volume percentage concentration of 1.5-5%; the flow rate of the mobile phase is preferably 5-10 mL / min.

[0062] In the present invention, the process of high-speed countercurrent chromatography separation is preferably: prepare the two-phase solvent system, the upper phase is the stationary phase, and the lower phase is the mobile phase, the stationary phase is pumped into the high-speed countercurrent chromatograph at a flow rate of 20 to 30 mL / min, and the mobile phase is pumped at a flow rate of 1 to 8 mL / min under the conditions of 25 to 35°C and a main engine speed of 700 to 1000 r / min. After the two phases reach equilibrium, the crude lyophilized powder of cyanidin-3-O-galactoside is dissolved in the mobile phase and then injected, and after liquid phase detection, the effluent containing only the target product is collected. More preferably, the two-phase solvent system is prepared, the upper phase is the stationary phase, and the lower phase is the mobile phase, the stationary phase is pumped into the high-speed countercurrent chromatograph at a flow rate of 20-30 mL / min, and the mobile phase is pumped in at a flow rate of 3-4 mL / min under the conditions of 25-35°C and a main engine speed of 850-910 r / min. After the two phases reach equilibrium, the crude lyophilized powder of cyanidin-3-O-galactoside is dissolved in the mobile phase and then injected, and after liquid phase detection, the effluent containing only the target product is collected.

[0063] In the present invention, the concentration of the crude lyophilized powder solution of cyanidin-3-O-galactoside obtained after the dissolution is preferably 10 to 30 mg / mL, more preferably 13 to 27 mg / mL; the injection volume of the crude lyophilized powder solution of cyanidin-3-O-galactoside is preferably 1 to 15 mL; and the wavelength of the liquid phase detection is preferably 280 nm.

[0064] In the present invention, the volume ratio of n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid is preferably 2:2:1:5:(0.01-0.1), more preferably 2:2:1:5:0.01.

[0065] After the high-speed countercurrent chromatography separation is completed, the present invention also preferably includes collecting the effluent containing the target product, and then performing reduced pressure concentration and freeze drying in sequence; the present invention does not have any special limitations on the reduced pressure concentration and freeze drying process, and the process familiar to those skilled in the art can be used.

[0066] The present invention also provides the use of cyanidin-3-O-galactoside prepared by the separation and preparation method described in the above technical solution in the preparation of hypoglycemic drugs or hypoglycemic foods. The present invention does not have any special limitation on the process of the application, and the process well known to those skilled in the art can be used.

[0067] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] Example 1

[0069] 1 kg of fresh cranberries were washed, and then an ethanol aqueous solution containing 0.1% hydrochloric acid (the volume ratio of ethanol to water was 70:30) was added at a ratio of 1 g:4 mL, and the mixture was fully mixed. The mixture was ultrasonically extracted for 60 min (below 45° C., protected from light). After the ultrasonic extraction, vacuum filtration was performed, and the obtained residue was extracted again under the above conditions. The filtrate was combined, and the ethanol was removed by vacuum rotary evaporation at 45° C. to obtain a crude anthocyanin extract.

[0070] Soak the AB-8 macroporous resin in ethanol for 24 hours and then load it into the chromatography column. Wash it with pure water until there is no alcohol smell, then rinse it with 0.5 mol / L sodium hydroxide solution for 1 hour, then wash it with deionized water until the effluent is centered, then wash it with 0.5 mol / L hydrochloric acid solution for 1 hour, and finally rinse it with deionized water until it is neutral.

[0071] The anthocyanin crude extract is slowly injected into the AB-8 macroporous resin until the adsorption volume reaches 1 / 3 of the total volume of the resin, and the resin is first rinsed with deionized water, and then rinsed with an ethanol aqueous solution containing 0.5% hydrochloric acid by volume (the volume ratio of ethanol to water is 2:98, 6:94, 10:90, 14:86, 18:82 and 22:78) for 2 hours, and 14% to 18% (ethanol concentration of the ethanol aqueous solution during the elution process) elution portion is collected, and ethanol is removed by vacuum rotary evaporation at 45° C., and the obtained anthocyanin extract is mixed with deionized water, and then freeze-dried to obtain anthocyanin extract freeze-dried powder;

[0072] A preparative liquid phase column Unitary C18 20mm×250mm was used, and the mobile phase was: phase A: alcohol methanol, phase B was formic acid and water in a volume ratio of 5:95; the column temperature was 30°C, and the flow rate was 10mL / min; the gradient was 5% phase A for 0-5min, and 5%-60% phase A for 5-24min; the anthocyanin extract lyophilized powder was dissolved in deionized water to obtain an anthocyanin extract solution with a concentration of 50mg / mL, and then separated using the above-mentioned preparative liquid phase column Unitary C18 20mm×250mm, with a single injection volume of 4mL; the peak of 17-18min was collected and concentrated under reduced pressure to remove methanol, and freeze-dried to obtain a cyanidin-3-O-galactoside crude extract lyophilized powder;

[0073] n-Butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid were mixed in a volume ratio of 2:2:1:5:0.01 and placed in a separatory funnel. After being fully shaken and allowed to stand for 30 minutes, the upper and lower phases were separated and ultrasonically degassed for 30 minutes. 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 instrument was started in a forward and reverse direction, and the main engine speed was increased to 850 rpm. After the speed was stable, the mobile phase was pumped into the instrument at a flow rate of 3 mL / min. After the two phases reached equilibrium in the pipeline, 200 mg of the cyanidin-3-O-galactoside crude extract freeze-dried powder was dissolved in 15 mL of the mobile phase, the sample was injected and detected under a UV detector, the target peak components were collected and the organic phase was removed by reduced pressure concentration, and freeze-dried to obtain cyanidin-3-O-galactoside monomer with a purity of 99.13%;

[0074] Figure 1 is a high performance liquid chromatogram of the anthocyanin extract freeze-dried powder, Figure 1 It can be seen that after cranberry is extracted and concentrated and gradient eluted with macroporous resin, the obtained cranberry anthocyanin freeze-dried powder is mainly a mixture containing 11 anthocyanin monomers;

[0075] Figure 2is the HPLC chromatogram of the cyanidin-3-O-galactoside monomer, Figure 2 It can be seen that after liquid chromatography purification and collection of the eluate for 17 to 18 minutes, cyanidin-3-O-galactoside can be obtained, and finally, by high-speed countercurrent chromatography separation, monomer anthocyanins containing only cyanidin-3-O-galactoside can be obtained, with a purity of up to 98.13%. 。

[0076] Test Case

[0077] Cyanidin-3-O-galactoside in the treatment of type 2 obese diabetic animal model experiment:

[0078] The animal model of type 2 obese diabetes was established by injection of low-dose STZ and feeding of high-fat diet; 32 mice were randomly divided into model group and normal group, and fed with high-fat experimental diet 40g / kg and basic diet 40g / kg respectively; 24 mice in model group were intraperitoneally injected with 4% STZ solution at a low dose of 25mg / kg body weight, and 8 mice in normal control group were intraperitoneally injected with the same dose of distilled water; after the model group was fed for 4 weeks, fasting blood glucose and glucose tolerance were measured, and those with abnormal blood glucose were divided into model group, low-dose test group and high-dose test group, each with 8 mice;

[0079] The mice in the low-dose test group were gavaged with 50 mg / kg body weight of cyanidin-3-O-galactoside daily, and the mice in the high-dose test group were gavaged with 150 mg / kg body weight of cyanidin-3-O-galactoside daily. The rats in the model group and the normal group were gavaged with normal saline at the same dose daily. The rats in each group were intervened every day for 8 weeks.

[0080] During the experiment, body weight and fasting blood glucose levels were recorded weekly. After weekly administration, mice were fasted but not watered for more than 12 hours, and blood samples were collected from the tails of mice. Fasting blood glucose levels were measured using a blood glucose monitor and blood glucose test strips.

[0081] Figure 3 The effect of cyanidin-3-O-galactoside on the body weight of type 2 diabetic mice is as follows: Figure 4 It can be seen that after 8 weeks of intervention, compared with the model group, the weight of mice in the high-dose experimental group was significantly reduced (P < 0.05), and the differences were statistically significant;

[0082] Figure 4 The effect of cyanidin-3-O-galactoside on fasting blood glucose in type 2 diabetic mice is as follows: Figure 4It can be seen that after 8 weeks of intervention, compared with the model group, the fasting blood glucose level of mice in the high-dose experimental group was significantly decreased (P < 0.05), and the differences were statistically significant, indicating that cyanidin-3-O-galactose can improve the body weight and fasting blood glucose level of type 2 diabetes;

[0083] Oral glucose tolerance test: Prepare 10mL of 20% glucose solution in advance, then fast the mice for 12h, measure the fasting blood glucose of the mice, record it as the blood glucose value at 0min, and after the mice adapt for 30min, gavage the mice at 2g glucose / kg body weight. Then, measure the blood glucose values ​​at 30min, 60min and 120min after gavage;

[0084] in, Figure 5 The effect of cyanidin-3-O-galactoside on glucose sensitivity of type 2 diabetic mice; Figure 5 It can be seen that the blood glucose level of the model group at each time point was significantly higher than that of the normal control group (P<0.01). Compared with the model group, the blood glucose level of the high-dose experimental group at 30min, 60min and 120min was significantly lower than that of the model group (P<0.01), which indicates that cyanidin-3-O-galactoside can improve glucose tolerance in type 2 diabetes.

[0085] Insulin tolerance test:

[0086] The mice were fasted for 6 hours before insulin injection, and the fasting basal blood glucose of the mice was measured, recorded as the blood glucose value at 0 min. Insulin was diluted in a sodium chloride solution with a mass concentration of 0.9% at a mass ratio of 1:1000, and insulin was injected intraperitoneally (0.75 U insulin / kg body weight). The blood glucose values ​​at 30 min, 60 min and 120 min after intraperitoneal injection were measured;

[0087] Figure 6 The effect of cyanidin-3-O-galactoside on insulin resistance in type 2 diabetic mice; Figure 6 It can be seen that the blood glucose level of the model group at each time point was significantly higher than that of the normal control group (P<0.01). Compared with the model group, the blood glucose level of the high-dose experimental group at 30min, 60min and 120min was significantly lower than that of the model group (P<0.01), which indicates that cyanidin-3-O-galactoside can improve the insulin sensitivity of type 2 diabetes;

[0088] After the insulin tolerance experiment, blood was collected from the eyeball, and then centrifuged at 3000 rpm for 30 minutes to collect the supernatant. The levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in the serum were then detected by an automatic biochemical analyzer. Figure 7The effect of cyanidin-3-O-galactoside on serum lipid levels (including triglycerides, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol) in type 2 diabetic mice is described. Figure 7 It can be seen that compared with the normal control group mice, the blood triglyceride, total cholesterol and low-density lipoprotein cholesterol of the model group mice were at high levels, while the high-density lipoprotein cholesterol was at low levels (p<0.01), indicating that they may suffer from hyperlipidemia. Compared with the model group mice, the high-density lipoprotein cholesterol content in the high-dose experimental group was significantly increased (p<0.01), and the levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol were significantly reduced (p<0.01). This shows that cyanidin-3-O-galactoside can improve the hyperlipidemia of type 2 diabetes.

[0089] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating and preparing cyanidin-3-O-galactoside, characterized in that: The following steps are involved: The cranberry is subjected to alcohol extraction to obtain a crude cranberry anthocyanin extract; The crude cranberry anthocyanin extract is adsorbed by a macroporous resin and then freeze-dried to obtain a freeze-dried cranberry anthocyanin extract powder; Sequentially subjecting the freeze-dried powder of cranberry anthocyanin extract to liquid chromatography purification and high-speed countercurrent chromatography separation to obtain cyanidin-3-O-galactoside; The chromatographic column used for the liquid chromatography purification is a C18 chromatographic column, and the mobile phase includes phase A and phase B, wherein the phase A is pure methanol or a methanol solution of an acid with a volume percentage concentration of 0.1 to 1.5%; the phase B is an aqueous solution of formic acid with a volume percentage concentration of 1 to 5%; the elution procedure of the liquid chromatography purification is gradient elution, wherein the volume percentage concentration of phase A remains unchanged at 5% within 0 to 5 minutes, and increases from 5% to 60% within 5 to 24 minutes, and the eluate is collected for 17 to 18 minutes; The two-phase solvent system for preparing the stationary phase and the mobile phase in the high-speed countercurrent chromatography separation comprises n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid.

2. The separation and preparation method according to claim 1, characterized in that: The volume ratio of n-butanol, methyl tert-butyl ether, methanol, water and trifluoroacetic acid is 2:2:1:5:(0.01-0.1).

3. The separation and preparation method according to claim 1, characterized in that: Before the liquid chromatography purification, the method further comprises mixing the cranberry anthocyanin extract with water to obtain a cranberry anthocyanin extract solution; The concentration of the cranberry anthocyanin extract solution is 20 to 120 mg / mL, and the injection volume is 1 to 4 mL; When the phase A is a methanol solution of an acid with a volume percentage concentration of 0.1 to 1.5%, the acid in the phase A is formic acid or trifluoroacetic acid.

4. The separation and preparation method according to claim 3, characterized in that: In the mobile phase of the liquid chromatography purification, phase A is pure methanol, and phase B is an aqueous solution of formic acid with a volume percentage concentration of 1.5 to 5%; The flow rate of the mobile phase is 5-10 mL / min.

5. The separation and preparation method according to claim 1, characterized in that: The process of high speed countercurrent chromatography separation is: The two-phase solvent system is prepared, with the upper phase being the stationary phase and the lower phase being the mobile phase. The stationary phase is pumped into a high-speed countercurrent chromatograph at a flow rate of 20 to 30 mL / min. Under the conditions of 25 to 35° C. and a main engine speed of 700 to 1000 r / min, the mobile phase is pumped in at a flow rate of 1 to 8 mL / min. After the two phases reach equilibrium, the crude lyophilized powder of cyanidin-3-O-galactoside is dissolved in the mobile phase and then injected. After liquid phase detection, the effluent containing only the target product is collected.

6. The separation and preparation method according to claim 5, characterized in that: The concentration of the cyanidin-3-O-galactoside crude freeze-dried powder solution obtained after the dissolution is 10 to 30 mg / mL; The injection volume of the cyanidin-3-O-galactoside crude lyophilized powder solution is 1 to 15 mL; The wavelength of the liquid phase detection is 280 nm.

7. The separation and preparation method according to claim 1, characterized in that: The extracting solution used in the alcohol extraction is an acidic ethanol solution; The volume percentage concentration of the acid in the acidic ethanol solution is 0.1% to 1.5%; The volume ratio of ethanol in the acidic ethanol solution to the acidic ethanol solution is (50-95):

100.

8. The separation and preparation method according to claim 7, characterized in that: The ratio of cranberry to acidic ethanol solution is 1 g: (4-10) mL; The alcohol extraction is carried out under light-proof and ultrasonic conditions.

9. The separation and preparation method according to claim 1, characterized in that: The macroporous resin adsorption process is as follows: injecting the cranberry anthocyanin crude extract into the macroporous resin, first washing the macroporous resin with deionized water, then gradient eluting with an acidic alcohol solution, collecting the eluate of the acidic alcohol solution with a volume percentage concentration of 14-18% for rotary evaporation; The volume ratio of the alcohol in the acidic alcohol solution to the acidic alcohol solution is (2-40):

100.

10. Use of cyanidin-3-O-galactoside prepared by the separation and preparation method according to any one of claims 1 to 9 in the preparation of hypoglycemic drugs or hypoglycemic foods.

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