Method for separating dihydromyricetin, myricetin and quercetin from raspberry tea and application of dihydromyricetin, myricetin and quercetin

Abstract

The invention relates to the technical field of natural product extraction, in particular to a method for separating dihydromyricetin, myricetin and quercetin from raspberry tea and application, and the separation method comprises the following steps: pretreating raspberry tea powder, mixing with an extracting agent 1, an extracting agent 2 and an extracting agent 3 in sequence, and carrying out ultrasonic treatment, suction filtration, concentration and purification to obtain dihydromyricetin, myricetin and quercetin; the extracting agent 1 comprises an ethanol solution with the volume concentration of 50%-55% and a citrate buffer solution; the extracting agent 2 comprises an ethanol solution with the volume concentration of 40%-45% and a phosphate buffer solution; and the extracting agent 3 comprises an ethanol solution with the volume concentration of 75-80% and a sodium hydroxide solution. According to the physical and chemical properties of dihydromyricetin, myricetin and quercetin, an extracting agent with specific ethanol solution concentration and pH is set to adapt to the physical and chemical properties (polarity, heat sensitivity, pH sensitivity and the like) of the dihydromyricetin, myricetin and quercetin, and the defects of low separation efficiency and large activity loss of a traditional process are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and in particular to a method for separating dihydromyricetin, myricetin and quercetin from berry tea and application thereof. Background Art

[0002] Berry tea has attracted much attention because it is rich in flavonoids, among which dihydromyricetin (DHM), myricetin and quercetin are the core active ingredients, which have significant biological activities such as antioxidant, anti-inflammatory, hypoglycemic and liver protection. Therefore, berry tea has attracted much attention in the fields of food, health products, medicine and cosmetics in recent years. However, due to the differences in the physical and chemical properties of the flavonoid components in berry tea (such as polarity, heat sensitivity, pH sensitivity, light sensitivity, etc.), traditional extraction and drying processes are difficult to take into account their efficient separation, activity retention and stability. This results in significant technical defects in separation efficiency, activity retention and product stability, which restricts the high-quality development of the berry tea industry.

[0003] At present, the traditional extraction process has the following defects: (1) Single solvent extraction: Single solvent extraction cannot adapt to the polarity differences of DHM (hydrophilic) and quercetin (hydrophobic) at the same time, resulting in low total yield and high cross-contamination rate between active ingredients; (2) Insufficient adsorption selectivity: For example, MOFs materials are used to adsorb flavonoids, but the HPLC peak overlap rate of DHM, myricetin and quercetin is high and the separation degree is low; (3) High temperature process: High temperature will lead to the loss of activity of heat-sensitive components. For example, when the temperature is greater than 120°C, the thermal decomposition loss of DHM is as high as 15%, resulting in serious waste of resources. For example, when the extraction temperature reaches 85°C, the activity retention rate of quercetin is only 85%. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for separating dihydromyricetin, myricetin and quercetin from berry tea and its application to solve the defects of low separation efficiency and serious activity degradation caused by ignoring the differences in the physical and chemical properties of flavonoid components in traditional extraction processes.

[0005] like Figure 1 As shown, to achieve the above purpose, the present invention provides a method for separating dihydromyricetin, myricetin and quercetin from berry tea, comprising the following preparation steps:

[0006] S1, mixing berry tea powder with ethanol solution, ultrasonicating the mixture, filtering, concentrating the filtered liquid 1 to obtain an extract, redissolving the extract to obtain a reconstituted solution, adjusting the pH of the reconstituted solution to 4.2-4.8, and centrifuging to obtain centrifuge 1, adding ethanol to the centrifuge 1, allowing the mixture to stand, and centrifuging to obtain centrifuge 2, adding an adsorbent to the centrifuge 2, stirring, and filtering to obtain liquid 2, and concentrating liquid 2 to obtain pretreated berry tea;

[0007] S2. The pretreated berry tea and extractant 1 are mixed, ultrasonicated, and filtered to obtain a filter residue A and a filtrate A, and the filtrate A is concentrated to obtain a crude dihydromyricetin extract;

[0008] S3, mixing the crude dihydromyricetin extract with the ethanol solution to obtain a mixed solution, adjusting the pH of the mixed solution to 3.3-3.7, stirring, standing, and centrifuging to obtain a precipitate, washing the precipitate, freeze-drying, and purifying to obtain dihydromyricetin;

[0009] S4, mixing the filter residue A with the extractant 2, microwave-treated, and filtered to obtain filter residue B and filtrate B, and concentrating the filtrate B to obtain a crude myricetin extract;

[0010] S5, mixing the crude myricetin extract with the ethanol solution to obtain a mixed solution, adjusting the pH of the mixed solution to 6-7, stirring, standing, centrifuging to obtain a precipitate, washing the precipitate, freeze-drying, and purifying to obtain myricetin;

[0011] S6, after mixing the filter residue B with the extractant 3, reflux extraction is performed to obtain a dissolution, the dissolution is filtered to obtain a filter residue C and a filtrate C, and the filtrate C is concentrated to obtain a crude quercetin extract;

[0012] S7, mixing the crude quercetin extract with the ethanol solution to obtain a mixed solution, adjusting the pH of the mixed solution to 9.3-9.7, stirring, then adjusting the pH of the mixed solution to 2-3 using a gradient pH adjustment method, allowing to stand, centrifuging to obtain a precipitate, washing the precipitate, freeze-drying, and purifying to obtain quercetin;

[0013] Extractant 1 includes an ethanol solution and a citrate buffer solution with a volume concentration of 50%-55%;

[0014] Extractant 2 includes an ethanol solution and a phosphate buffer solution with a volume concentration of 40%-45%;

[0015] The extractant 3 includes an ethanol solution and a sodium hydroxide solution with a volume concentration of 75%-80%.

[0016] In the present invention, the preparation process of berry tea powder comprises: drying berry tea to a moisture content of ≤8% to obtain dried berry tea, crushing the dried berry tea, and passing the berry tea through a 40-mesh sieve to obtain berry tea powder.

[0017] In the present invention, the total flavonoid content of the berry tea powder in S1 is ≥20% (calculated as dihydromyricetin), the volume concentration of the ethanol solution is 65%-75%, the mass volume ratio of the berry tea powder to the ethanol solution is 1g:15mL, the ultrasonic temperature is 45-50°C, the ultrasonic time is 50-70min, and the adsorbent is a polyvinyl pyrrolidone solution with a mass concentration of 3%-5%.

[0018] In the present invention, the purpose of adjusting the pH of the reconstituted solution to 4.2-4.8 is to remove protein from berry tea; the purpose of adding ethanol to the centrifuge 1 is to remove polysaccharides from berry tea; the purpose of adding an adsorbent to the centrifuge 2 is to remove polyphenols from berry tea.

[0019] In the present invention, the S1 process can effectively remove proteins, polysaccharides and polyphenols in berry tea, that is, the present invention can also achieve the effect of extracting proteins, polysaccharides and polyphenols in berry tea.

[0020] In the present invention, the specific process of mixing the berry tea powder and the ethanol solution, ultrasonicating the mixture, filtering the mixture, and concentrating the filtered liquid 1 to obtain the extract is as follows: mixing the berry tea powder and the ethanol solution, ultrasonicating the mixture, filtering the mixture to obtain liquid a and filter residue a, ultrasonicating the filter residue again, filtering the mixture to obtain liquid b and filter residue b, mixing liquid a and liquid b to obtain liquid 1, and then concentrating the liquid 1 to obtain the extract.

[0021] In the present invention, the mass volume ratio of the pretreated berry tea to the extractant 1 in S2 is 1 g:18-22 mL; the pH of the extractant 1 is 4.9-5.1; the concentration of the citrate buffer is 0.08-0.12 mol / L, and the citric acid buffer includes citric acid and sodium citrate; the frequency of ultrasound in S2 is 35-45 kHz, the power of ultrasound is 180-220 W, the temperature of ultrasound is 49-51° C., and the time of ultrasound is 25-45 min.

[0022] In the present invention, dihydromyricetin is pH-sensitive and easily oxidized. Its structure is more stable under weakly acidic conditions and is less susceptible to oxidation or degradation. Therefore, the pH of extractant 1 is controlled at 4.9-5.1. At this pH, dihydromyricetin is more easily dissolved, ensuring the purity of the subsequent dihydromyricetin crude extract.

[0023] In the present invention, the funnel used for filtration in S2 is a Bush funnel, and the pore size of the filter paper is 5 μm.

[0024] In the present invention, the concentration in S2 is carried out under reduced pressure using a rotary evaporator, the vacuum degree of the reduced pressure concentration is -0.08 MPa, and the temperature of the reduced pressure concentration is 45-55°C.

[0025] In the present invention, the mass volume ratio of the crude dihydromyricetin extract to the ethanol solution in S3 is 1 g: 10-15 mL; the purification process in S3 includes: (1) soaking an AB-8 type macroporous resin in an ethanol solution, taking it out, washing it, and performing a balancing treatment to obtain a pretreated AB-8 type macroporous resin; (2) preparing the crude dihydromyricetin into a sample solution, and adjusting the pH of the sample solution to 4.5; (3) dynamically adsorbing the sample solution using the pretreated AB-8 type macroporous resin, and then performing a gradient elution to obtain an eluent, concentrating the eluent to obtain a paste, allowing the paste to stand for crystallization, centrifuging it to obtain crystals, washing the crystals, and vacuum drying it to obtain dihydromyricetin.

[0026] In the present invention, the pore size of the AB-8 macroporous resin used in the purification process of S3 is 10-15 nm, the volume concentration of the ethanol solution is 90%-95%, the immersion time is 20-28 h, the flow rate of dynamic adsorption is 2-2.2 BV / h, and the gradient elution reagent is an ethanol solution with a volume concentration of 10%-95%.

[0027] In the present invention, the gradient elution process includes: first eluting with a 10% volume concentration ethanol solution to remove water-soluble impurities, then eluting with a 30% volume concentration ethanol solution to remove weak polar impurities, then eluting with a 50% volume concentration ethanol solution to collect the dihydromyricetin enriched section, then eluting with a 70% volume concentration ethanol solution to remove strong polar impurities, and finally eluting with a 95% volume concentration ethanol solution, the purpose of which is to restore the adsorption capacity of the AB-8 macroporous resin.

[0028] In the present invention, the polarity of the AB-8 macroporous resin matches that of dihydromyricetin, and dihydromyricetin can be preferentially adsorbed, and the dynamic adsorption flow rate is limited to 2-2.2 BV / h. The dynamic adsorption capacity of the AB-8 macroporous resin for dihydromyricetin is ≥80 mg / g.

[0029] In the present invention, the structural formula of dihydromyricetin is as follows:

[0030]

[0031] The structural formula of dihydromyricetin contains six hydroxyl groups: 5,7-dihydroxy on Ring A, 3',4',5'-trihydroxy on Ring B, and 3-hydroxy on Ring C (with a C2-C3 single bond). The 3',4',5'-trihydroxy groups on Ring B provide strong hydrophilicity, while the conjugated double bonds in Ring C reduce electron density. A weakly acidic environment can inhibit the ionization (-OH→O-) of the 3',4',5'-trihydroxy groups on Ring B, maintaining moderate hydrophobicity. Therefore, the pH of Extractant 1 was controlled between 4.9 and 5.1, and the volume concentration of the ethanol solution was controlled between 50% and 55%.

[0032] In the present invention, the mass volume ratio of the filter residue A to the extractant 2 in S4 is 1 g:13-17 mL, the pH of the extractant 2 is 6.4-6.6, the phosphate buffer comprises a sodium phosphite solution and a potassium dihydrogen phosphate solution, the volume ratio of the sodium phosphite solution to the potassium dihydrogen phosphate solution is 0.5-1:1, the concentration of the sodium phosphite solution is 0.08-0.12 mol / L, and the concentration of the potassium dihydrogen phosphate solution is 0.08-0.12 mol / L; the power of the microwave in S4 is 280-320 W, the temperature of the microwave is 58-62° C., the microwave time is 4-6 min, the microwave mode is pulsed, and the pulse mode is pulsed for 10 s and rested for 5 s.

[0033] In the present invention, the molecular structure of myricetin contains multiple phenolic hydroxyl groups. Under pH conditions of 6-7, the degree of dissociation of the phenolic hydroxyl groups is low, the intramolecular hydrogen bond structure is stable, and it is not easily oxidized, degraded or changed in configuration, and its biological activity can be retained to the greatest extent.

[0034] In the present invention, the funnel used for filtration in S4 is a Bush funnel, and the pore size of the filter paper is 5 μm.

[0035] In the present invention, the mass volume ratio of the crude myricetin extract to the ethanol solution in S5 is 1 g: 10-15 mL; the purification process in S5 includes: (1) soaking the polyamide in an ethanol solution with a volume concentration of 95% to fully swell it, washing it with water after taking it out, and then balancing the polyamide with a phosphate buffer solution with a pH of 6.5 to obtain a pretreated polyamide; (2) preparing the crude myricetin into a sample solution, and adjusting the pH of the sample solution to 6.5; (3) using the pretreated polyamide as a chromatography column to perform chromatographic separation on the sample solution, and then performing linear gradient elution, and collecting the target segment in the linear gradient elution process; (4) adding activated carbon to the target segment, stirring, filtering, obtaining a filtrate, evaporating and concentrating the filtrate under reduced pressure to obtain a paste, freezing the paste, and vacuum drying to obtain myricetin.

[0036] In the present invention, during the purification of myricetin, the flow rate of the equilibrium treatment is 2 BV / L.

[0037] In the present invention, the linear gradient elution process includes: using a volume concentration of 40%-80% ethanol solution for linear gradient elution, first taking a 40% volume concentration ethanol solution for elution to remove water-soluble impurities, and then eluting with a 40%-80% volume concentration ethanol solution (excluding 40%), during which the myricetin in the 50%-60% volume concentration ethanol segment is collected to obtain the target segment, completing the linear gradient elution process.

[0038] In the present invention, the surface of the polyamide is rich in amide groups (-CONH2), which can form hydrogen bonds with the hydroxyl groups in myricetin and have similar polarity, so myricetin can be eluted preferentially.

[0039] In the present invention, the structural formula of myricetin is as follows:

[0040]

[0041] The structural formula of myricetin contains six hydroxyl groups: Ring A: 5,7-dihydroxy, Ring B: 3',4',5'-trihydroxy (key for high polarity), and Ring C: 3-hydroxy (C2=C3 double bond). The 3',4',5'-trihydroxy group provides strong hydrophilicity, while the conjugated double bond of Ring C reduces electron density. The pH of Extractant 2 is controlled at 6.4-6.6 to maintain a neutral environment, keeping the C2=C3 double bond of Ring C stable (avoiding ring-opening reactions caused by alkaline environments). Furthermore, a lower ethanol concentration (40-45%) can enhance the polarity of the aqueous phase and improve the solubility of myricetin.

[0042] In the present invention, the mass volume ratio of the filter residue B to the extractant 3 in S6 is 1 g:8-12 mL; the pH of the extractant 3 in S6 is 8.8-9.2, and the concentration of the sodium hydroxide solution is 0.8-1.2 mol / L.

[0043] In the present invention, alkaline conditions promote quercetin dissolution, but excessive alkalinity (pH > 10) can lead to excessive dissociation of the phenolic hydroxyl groups in quercetin, triggering side reactions such as intramolecular cyclization and oxidation, and destroying its structural integrity. Controlling the pH of extractant 3 between 8.8 and 9.2 ensures quercetin dissolution efficiency while avoiding structural degradation.

[0044] In the present invention, the temperature of reflux extraction in S6 is 65-75° C., the number of reflux extractions is ≥1, and the single reflux extraction time is 50-70 min.

[0045] In the present invention, the funnel used for filtration in S6 is a Bush funnel, and the pore size of the filter paper is 5 μm.

[0046] In the present invention, the mass volume ratio of the quercetin crude extract to the ethanol solution in S7 is 1 g:10-15 mL.

[0047] In the present invention, the purification process in S7 includes: (1) activating silica gel and packing the obtained silica gel column by wet method to obtain a silica gel chromatography column, balancing the silica gel chromatography column to obtain a balanced silica gel chromatography column; (2) preparing a sample solution from a crude quercetin product, performing chromatographic separation on a balanced silica gel column chromatography column, and collecting a target segment in the linear gradient elution process by linear gradient elution; (3) mixing the target segment with a methanol solution, cooling the solution for crystallization, allowing the solution to stand, and filtering the solution to obtain crystals, which are then vacuum dried to obtain quercetin.

[0048] In the present invention, during the purification of quercetin, the activation temperature is 110-120°C, the activation time is 1-2h, the solvent for the equilibrium treatment is the initial eluent, the initial eluent includes a mixed solution of chloroform and methanol in a volume ratio of 8:3, the target segment in the linear gradient elution process is the target segment with a retention time of 12-15min during elution, and the volume concentration of the methanol solution is 70%.

[0049] In the present invention, the structural formula of quercetin is shown below:

[0050]

[0051] The structural formula of quercetin contains five hydroxyl groups: Ring A: 5,7-dihydroxy, Ring B: 3',4'-dihydroxy (lacking a 5'-OH group), and Ring C: 3-hydroxy (C2=C3 double bond). The lack of one hydroxyl group in the 3',4'-dihydroxy group on Ring B significantly reduces polarity, while the planar conjugated system enhances hydrophobicity. Extractant 3 maintains a pH between 8.8 and 9.2. The alkaline environment ionizes the 3',4'-dihydroxy group on Ring B, disrupting intramolecular hydrogen bonds and increasing polarity. High ethanol concentrations (75-80%) compensate for hydrophobicity, achieving dissolution equilibrium and improving the dissolution rate of quercetin.

[0052] The present invention also provides application of the method for separating dihydromyricetin, myricetin and quercetin from berry tea in the fields of pharmaceuticals and cosmetics.

[0053] The present invention has the following beneficial effects:

[0054] The present invention provides a method for separating dihydromyricetin, myricetin and quercetin from berry tea, comprising the following preparation steps: S1, mixing berry tea powder with an ethanol solution, ultrasonicating the mixture, filtering the mixture, concentrating the filtered liquid 1 to obtain an extract, redissolving the extract to obtain a reconstituted solution, adjusting the pH of the reconstituted solution to 4.2-4.8, and centrifuging the solution to obtain a centrifuge 1, adding ethanol to the centrifuge 1, allowing the solution to stand, and centrifuging the solution to obtain a centrifuge 2, adding an adsorbent to the centrifuge 2, stirring the solution, and filtering the solution to obtain a liquid 2, and concentrating the liquid 2 to obtain a pre- Processing berry tea; S2, mixing the pretreated berry tea with extractant 1, ultrasonicating and filtering to obtain filter residue A and filtrate A, concentrating filtrate A to obtain a crude dihydromyricetin extract; S3, mixing the crude dihydromyricetin extract with ethanol solution to obtain a mixed solution, adjusting the pH of the mixed solution to 3.3-3.7, stirring, standing, centrifuging to obtain a precipitate, washing the precipitate, freeze-drying, and purifying to obtain dihydromyricetin; S4, mixing the filter residue A with extractant 2, microwave-treating, and filtering to obtain filter residue B and filtrate B, and concentrating the filtrate B is concentrated to obtain a crude myricetin extract; S5, the crude myricetin extract is mixed with an ethanol solution to obtain a mixed solution, the pH of the mixed solution is adjusted to 6-7, stirred, allowed to stand, centrifuged to obtain a precipitate, the precipitate is washed, freeze-dried, and purified to obtain myricetin; S6, the filter residue B is mixed with the extractant 3, refluxed to obtain an eluate, the eluate is filtered to obtain a filter residue C and a filtrate C, the filtrate C is concentrated to obtain a crude quercetin extract; S7, the crude quercetin extract is mixed with an ethanol solution to obtain a mixed solution, and the pH value of the mixed solution is adjusted. The mixed solution is adjusted to a pH of 9.3-9.7, stirred, and then adjusted to a pH of 2-3 by a gradient pH adjustment method, allowed to stand, centrifuged to obtain a precipitate, washed, freeze-dried, and purified to obtain quercetin; extractant 1 includes an ethanol solution with a volume concentration of 50%-55% and a citrate buffer; extractant 2 includes an ethanol solution with a volume concentration of 40%-45% and a phosphate buffer; and extractant 3 includes an ethanol solution with a volume concentration of 75%-80% and a sodium hydroxide solution.

[0055] In the present invention, protein is removed from the berry tea by adjusting the pH of the reconstituted solution during step S1. The presence of protein can interact with the target flavonoid components (e.g., hydrogen bonding, hydrophobic interactions, etc.), causing the target components to bind to the protein during subsequent separation, affecting separation efficiency and causing cross-contamination. After protein removal, the purity and separation efficiency of each isolate (dihydromyricetin, myricetin, and quercetin) are improved.

[0056] In the present invention, during step S1, ethanol is added to the centrifuge 1 to remove polysaccharides from the berry tea. Polysaccharides are highly viscous and can increase the viscosity of the extract, making separation operations such as filtration and centrifugation difficult. Removing the polysaccharides significantly improves the fluidity of the filtrate during subsequent separations and increases the purity of the resulting isolates.

[0057] In the present invention, during step S1, an adsorbent is added to the centrifuge 2 to remove polyphenols from the berry tea. The physicochemical properties of polyphenols are similar to those of flavonoids, making them easily extracted or adsorbed along with the target component during the separation and purification process. This removal effectively improves the purity and separation efficiency of each isolate.

[0058] The present invention utilizes the differences in polarity between dihydromyricetin, myricetin, and quercetin, creating extractants with varying ethanol solution concentrations to improve the dissolution rates of the three substances. The volume concentration of the ethanol solution in Extractant 1 is 50%-55%. Dihydromyricetin is a highly polar flavonoid compound (containing multiple hydroxyl groups in its molecule, with a high proportion of hydrophilic groups). A 50%-55% ethanol solution has moderate polarity, which can both dissolve dihydromyricetin and reduce the dissolution of more polar impurities, thereby improving dihydromyricetin dissolution. The volume concentration of the ethanol solution in Extractant 2 is 40%-45%. Myricetin is more polar than dihydromyricetin (due to its slightly higher hydrophilicity due to its hydroxyl distribution and molecular structure). A 40%-45% ethanol solution has a higher polarity than Extractant 1, facilitating the dissolution of myricetin. At this concentration, myricetin fully dissolves in Extractant 2, while other less polar components (such as quercetin) are less readily dissolved. The volume concentration of the ethanol solution in Extractant 3 is 75%-80%. Quercetin has the weakest polarity of the three components (fewer hydroxyl groups and a more hydrophobic molecular structure). The 75%-80% volume concentration of ethanol solution is less polar and effectively dissolves quercetin, while the more polar impurities remaining in Residue B are difficult to dissolve, thereby improving the purity of quercetin.

[0059] The present invention is by regulating the pH of mixed solution in step S3, step S5 and step S7, improves separation efficiency and the purity of dihydromyricetin, myricetin, quercetin. Under the condition of pH to 3.3-3.7, the hydroxyl of dihydromyricetin (DHM) is protonated, and molecular polarity is reduced, and hydrophobicity is enhanced, and preferential precipitation is achieved. Quercetin and myricetin still maintain higher solubility because of retaining the partially dissociated hydroxyl, and it is impossible to precipitate. Under the condition of pH being 6-7, myricetin is close to the isoelectric point, and hydrogen bond reorganization forms microcrystal precipitation, and quercetin still maintains higher solubility under neutral conditions because of its weaker polarity (needing higher pH to significantly dissociate), thus realizing selective precipitation and separation of myricetin. Under the condition of pH being 9.3-9.7, quercetin is deprotonated, forms water-soluble phenol oxide anion, is protonated again under the condition of pH being 2-3 subsequently, has increased its hydrophobicity, and precipitation is separated.

[0060] The present invention adopts a gradient pH adjustment method in the S7 process to prevent the rapid nucleation of microcrystals caused by excessive pH changes, which increases the specific surface area, adsorbs more impurities, and reduces the purity of subsequent quercetin. In addition, direct acidification can also cause intramolecular cyclization of the o-diphenolic hydroxyl groups in quercetin to form dihydroflavonoid heterocyclic derivatives, increasing the difficulty of subsequent purification.

[0061] The present invention solves the defects of low separation efficiency and large activity loss of traditional processes by adapting the physical and chemical properties (polarity, heat sensitivity, pH sensitivity, etc.) of dihydromyricetin, myricetin and quercetin based on the differences in their physical and chemical properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The present invention provides a flow chart of a method for separating dihydromyricetin, myricetin and quercetin from berry tea. DETAILED DESCRIPTION

[0063] The present invention will be further described below with reference to the following embodiments. Unless otherwise defined, technical or scientific terms used herein shall have the same meanings as those commonly understood by persons of ordinary skill in the art to which the present invention pertains. The above-mentioned features or features described in the specific examples of the present invention may be combined in any manner. These specific embodiments are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0064] Example 1

[0065] A method for separating dihydromyricetin, myricetin, and quercetin from berry tea comprises the following preparation steps:

[0066] Extractant 1 was prepared by mixing 550 mL of ethanol with 450 mL of water to obtain an ethanol solution having a volume concentration of 55%. 0.1 mol / L of citrate buffer (comprising sodium citrate and citric acid) was added to the ethanol solution until the pH of the ethanol solution reached 5.0 to obtain extractant 1.

[0067] Extractant 2 was prepared by mixing 450 mL of ethanol with 550 mL of water to obtain a 45% ethanol solution. A 1:1 volume ratio of 0.1 mol / L sodium phosphite solution and 0.1 mol / L potassium dihydrogen phosphate solution were added to the ethanol solution until the pH of the ethanol solution reached 6.5 to obtain extractant 2.

[0068] Extractant 3 was prepared by mixing 800 mL of ethanol with 200 mL of water to obtain an ethanol solution with a volume concentration of 80%. 1 mol / L sodium hydroxide solution was added to the ethanol solution until the pH of the ethanol solution reached 9.0 to obtain extractant 3.

[0069] Preparation of berry tea powder: Select berry tea with a total flavonoid content of 20% based on dihydromyricetin, dry the berry tea to a moisture content of ≤8% to obtain dried berry tea, crush the dried berry tea, and pass it through a 40-mesh sieve to obtain berry tea powder.

[0070] An AB-8 macroporous resin with a pore size of 15 nm was immersed in an ethanol solution with a volume concentration of 95%, taken out after soaking for 24 hours, washed with clean water, and then the AB-8 macroporous resin was equilibrated with a buffer solution with a pH of 4.5 to obtain a pretreated AB-8 macroporous resin.

[0071] The polyamide was placed in a 95% volume concentration ethanol solution, soaked for 24 hours, taken out and washed with water, and then the polyamide was balanced with a phosphate buffer solution with a pH of 6.5 at a flow rate of 2 BV / L to obtain a pretreated polyamide.

[0072] Silica gel was activated at 110° C. for 2 h, wet packed (column diameter to height ratio 1:15) to obtain a silica gel chromatography column, and equilibrated with an initial eluent (chloroform to methanol volume ratio of 8:3) to obtain an equilibrated silica gel chromatography column.

[0073] S1. After mixing 100 g of berry tea powder with 1500 mL of 70% ethanol solution by volume, ultrasonically treat at a temperature of 50° C. for 30 min, and filter to obtain liquid a and filter residue b. Keeping the temperature at 50° C. unchanged, ultrasonically treat the filter residue b again for 30 min, and filter to obtain liquid b and filter residue b. Mix liquid a and liquid b to obtain liquid 1, and then concentrate liquid 1 to obtain an extract with a solid content of 25%. The extract is redissolved in an ethanol solution with a volume concentration of 70% to obtain a resolution. After adjusting the pH of the resolution to 4.2, centrifuge at a speed of 4000 rpm for 15 min to obtain centrifuge 1. Ethanol is added to the centrifuge 1 until the volume concentration of ethanol is 70%, let it stand for 2 h, and centrifuge at a speed of 4000 rpm for 15 min to obtain centrifuge 2. Polyvinyl pyrrolidone with a mass concentration of 5% is added to the centrifuge 2, stirred for 30 min, and filtered to obtain liquid 2. Liquid 2 is concentrated to obtain pretreated berry tea;

[0074] S2. 1 kg of pretreated berry tea was mixed with 20 L of extractant 1, and the mixture was ultrasonically filtered at a frequency of 40 kHz, a power of 200 W, and a temperature of 50° C. for 30 min. The mixture was filtered using a Buchner funnel with a pore size of 5 μm to obtain a filter residue A and a filtrate A. The filtrate A was transferred to a rotary evaporator and concentrated under reduced pressure at a temperature of 50° C. and a vacuum degree of -0.08 MPa to obtain a crude dihydromyricetin extract with a solid content of 20% (dissolution rate of 94%).

[0075] S3. The crude dihydromyricetin extract was mixed with a 55% ethanol solution at a mass volume ratio of 1 g: 10 mL to obtain a mixed solution, and the pH of the mixed solution was adjusted to 3.5 with 0.1 mol / L hydrochloric acid. The mixture was stirred at a speed of 200 rpm for 10 min, allowed to stand at 25° C. for 1 h, and centrifuged at a speed of 4000 rpm for 15 min at a temperature of 4° C. to obtain a precipitate. The obtained precipitate was washed twice with a 60% ethanol solution by volume, and freeze-dried to obtain a crude dihydromyricetin product (purity 75%).

[0076] The crude dihydromyricetin was prepared into a loading solution, the pH of the loading solution was adjusted to 4.5, and the loading solution was dynamically adsorbed using a pretreated AB-8 macroporous resin. The flow rate of the dynamic adsorption was 2 mL / min, and then a gradient elution was performed using an ethanol solution with a volume concentration of 10%-95%. Specifically, the elution was first performed with a 10% volume concentration ethanol solution (elution conditions were 3BV, 2.0 mL / min), and then with a 30% volume concentration ethanol solution (elution conditions were 10%-95%). 3BV, 1.5mL / min), then eluted with a 50% volume concentration of ethanol solution (elution conditions are 4BV, 1.0mL / min, pH is 4.5), collected the dihydromyricetin enriched section, then eluted with a 70% volume concentration of ethanol solution (elution conditions are 2BV, 1.5mL / min), and finally eluted with a 95% ethanol solution (elution conditions are 2BV, 2.0mL / min), completed the gradient elution, and obtained the eluent. The eluent was concentrated to obtain a paste with a solid content of 30%, and the paste was allowed to stand at 4°C for crystallization for 12h to precipitate dihydromyricetin crystals, which were then centrifuged at 4000rpm for 15min to obtain crystals. After the crystals were washed with ethanol, they were vacuum dried at 50°C to obtain dihydromyricetin (purity of 92%).

[0077] S4. After the filter residue A was mixed with the extractant 2 at a mass volume ratio of 1 g: 15 mL, the mixture was pulsed at a power of 300 W for 5 min (10 s on / 5 s off) at 60° C. using a pulse mode, and filtered using a Bush funnel with a pore size of 5 μm filter paper to obtain filter residue B and filtrate B. The filtrate B was transferred to a rotary evaporator and concentrated to obtain a crude myricetin extract with a solid content of 25% (dissolution rate of 80%).

[0078] S5. The crude myricetin extract was mixed with a 45% ethanol solution at a mass volume ratio of 1 g: 10 mL to obtain a mixed solution. A 0.1 mol / L sodium hydroxide solution was added to the mixed solution, and the pH of the mixed solution was adjusted to 6.5. The mixture was stirred for 30 min, allowed to stand at 25° C. for 1 h, and then centrifuged at 4000 rpm for 15 min to obtain a precipitate. The precipitate was washed with deionized water at a pH of 6.5 to obtain a crude myricetin product (purity 75%).

[0079] The crude myricetin product is prepared into a loading solution, the pH of the loading solution is adjusted to 6.5, and the loading solution is chromatographically separated using a pretreated polyamide as a chromatography column at a loading flow rate of 1.5 BV / h. A linear gradient elution is performed using an ethanol solution with a volume concentration of 40%-80%, wherein the elution conditions of the 40% elution segment are 3 BV, 1.5 BV / h, and then elution is performed with a 40%-80% ethanol solution concentration (excluding 40%) at an elution condition of 8 BV, 1.5 BV / h. During this period, the myricetin in the 50%-60% volume concentration ethanol segment is collected to obtain the target segment, thereby completing the linear gradient elution process. Activated carbon was added to the target segment, stirred at 25°C for 30 min, filtered using a 0.22 μm filter membrane to obtain a filtrate, and the filtrate was evaporated under reduced pressure to a paste with a solid content of 25%. The paste was frozen at -40°C for 24 h, and then vacuum dried at -50°C and a vacuum degree of 10 Pa to obtain myricetin (purity 92%);

[0080] S6. After mixing the filter residue B with the extractant 3 at a mass volume ratio of 1 g:10 mL, reflux extraction was performed at a temperature of 70° C. for 60 min. The above reflux extraction process was repeated, i.e., reflux extraction was performed twice to obtain an extract. The extract was filtered using a Bush funnel with a pore size of 5 μm to obtain a filter residue C and a filtrate C. The filtrate C was transferred to a rotary evaporator and concentrated to obtain a crude quercetin extract with a solid content of 25% (dissolution rate of 70%).

[0081] S7. The crude quercetin extract was mixed with an 80% ethanol solution at a mass volume ratio of 1 g:10 mL to obtain a mixed solution. A 1 mol / L sodium hydroxide solution was added to adjust the pH of the mixed solution to 9.5, and the mixture was stirred for 30 min. Subsequently, 0.1 mol / L hydrochloric acid was added to adjust the pH of the mixed solution to 2.5 using a gradient pH adjustment method (gradient pH adjustment method: pH changes: 9.5-7-5-2.5, with an interval of 5 min for each pH adjustment). The mixture was allowed to stand at 25° C. for 2 h, and centrifuged at 4000 rpm for 20 min to obtain a precipitate. The precipitate was washed with hydrochloric acid at a pH of 2.5, and freeze-dried to obtain a crude quercetin product (purity 75%).

[0082] The crude quercetin product was dissolved in the initial eluent (chloroform to methanol volume ratio of 8:3), then filtered through a 0.45μm filter membrane to obtain the sample solution, which was then chromatographed on a balanced silica gel column. Elution was then performed using a linear gradient elution method with a mixed solution of chloroform and methanol as the eluent. The linear gradient elution process was as follows: first, a mixed solution of chloroform to methanol volume ratio of 8:3 was used as the eluent, and then the volume ratio of the two was linearly reduced to 7:2, that is, the volume ratio of chloroform to methanol was 7:2, completing the linear gradient elution. The conditions during this period were: 10BV, 1.0mL / min (excluding the elution stage with a volume ratio of chloroform to methanol of 7:2). When the volume ratio of chloroform to methanol in the eluent was 7:2, the conditions for linear gradient elution were 2BV, 1.0mL / min. During the period, the target segment of 12-15 minutes during the linear gradient elution was retained, and the target segment was mixed with a methanol solution with a volume concentration of 70% at a volume ratio of 1:1 at a temperature of 60°C, and then the temperature was lowered for crystallization. The solution was allowed to stand at 4°C for 24 hours, and filtered with a 0.22 μm filter membrane to obtain crystals. The crystals were washed three times and then vacuum dried at 40°C and 50 Pa to obtain quercetin (purity 98%).

[0083] test

[0084] The protein removal rate, polysaccharide precipitation rate and polyphenol adsorption rate of the S1 process in Example 1 were tested respectively, and the calculation formulas are as follows:

[0085]

[0086] Calculations showed that the protein removal rate in step S1 was 90%, the polysaccharide precipitation rate was 88%, and the polyphenol adsorption rate was 85%.

[0087] Comparative Example 1

[0088] A method for separating dihydromyricetin, myricetin, and quercetin from berry tea comprises the following preparation steps:

[0089] Extractant 1 was prepared by mixing 550 mL of ethanol with 450 mL of water to obtain an ethanol solution having a volume concentration of 55%. 0.1 mol / L of citrate buffer (comprising sodium citrate and citric acid) was added to the ethanol solution until the pH of the ethanol solution reached 5.0 to obtain extractant 1.

[0090] Extractant 2 was prepared by mixing 450 mL of ethanol with 550 mL of water to obtain a 45% ethanol solution. A 1:1 volume ratio of 0.1 mol / L sodium phosphite solution and 0.1 mol / L potassium dihydrogen phosphate solution were added to the ethanol solution until the pH of the ethanol solution reached 6.5 to obtain extractant 2.

[0091] Extractant 3 was prepared by mixing 800 mL of ethanol with 200 mL of water to obtain an ethanol solution with a volume concentration of 80%. 1 mol / L sodium hydroxide solution was added to the ethanol solution until the pH of the ethanol solution reached 9.0 to obtain extractant 3.

[0092] Preparation of berry tea powder: Select berry tea with a total flavonoid content of 20% based on dihydromyricetin, dry the berry tea to a moisture content of ≤8% to obtain dried berry tea, crush the dried berry tea, and pass it through a 40-mesh sieve to obtain berry tea powder.

[0093] An AB-8 macroporous resin with a pore size of 15 nm was immersed in an ethanol solution with a volume concentration of 95%, taken out after soaking for 24 hours, washed with clean water, and then the AB-8 macroporous resin was equilibrated with a buffer solution with a pH of 4.5 to obtain a pretreated AB-8 macroporous resin.

[0094] The polyamide was placed in a 95% volume concentration ethanol solution, soaked for 24 hours, taken out and washed with water, and then the polyamide was balanced with a phosphate buffer solution with a pH of 6.5 at a flow rate of 2 BV / L to obtain a pretreated polyamide.

[0095] Silica gel was activated at 110° C. for 2 h, wet packed (column diameter to height ratio 1:15) to obtain a silica gel chromatography column, and equilibrated with an initial eluent (chloroform to methanol volume ratio of 8:3) to obtain an equilibrated silica gel chromatography column.

[0096] S1. After mixing 1 kg of berry tea powder with 20 L of extractant 1, ultrasonically filtration was performed at a frequency of 40 kHz, a power of 200 W, and a temperature of 50° C. for 30 min, and filtration was performed using a Buchner funnel with a pore size of filter paper of 5 μm to obtain a filter residue A and a filtrate A. The filtrate A was transferred to a rotary evaporator and concentrated under reduced pressure at a temperature of 50° C. and a vacuum degree of -0.08 MPa to obtain a crude dihydromyricetin extract with a solid content of 20% (dissolution rate of 67%).

[0097] S2. The crude dihydromyricetin extract was mixed with a 55% ethanol solution by volume at a mass volume ratio of 1 g: 10 mL to obtain a mixed solution, and the pH of the mixed solution was adjusted to 3.5 with 0.1 mol / L hydrochloric acid, stirred at a speed of 200 rpm for 10 min, allowed to stand at 25° C. for 1 h, and centrifuged at a rate of 4000 rpm for 15 min at a temperature of 4° C. to obtain a precipitate, which was washed twice with a 60% ethanol solution by volume, and freeze-dried to obtain a crude dihydromyricetin product (purity 65%).

[0098] The crude dihydromyricetin was prepared into a loading solution, the pH of the loading solution was adjusted to 4.5, and the loading solution was dynamically adsorbed using a pretreated AB-8 macroporous resin. The flow rate of the dynamic adsorption was 2 mL / min, and then a gradient elution was performed using an ethanol solution with a volume concentration of 10%-95%. Specifically, the elution was first performed with a 10% volume concentration ethanol solution (elution conditions were 3BV, 2.0 mL / min), and then with a 30% volume concentration ethanol solution (elution conditions were 10%-95%). 3BV, 1.5mL / min), then eluted with a 50% volume concentration of ethanol solution (elution conditions are 4BV, 1.0mL / min, pH is 4.5), collected the dihydromyricetin enriched section, then eluted with a 70% volume concentration of ethanol solution (elution conditions are 2BV, 1.5mL / min), and finally eluted with a 95% ethanol solution (elution conditions are 2BV, 2.0mL / min), completed the gradient elution, and obtained the eluent. The eluent was concentrated to obtain a paste with a solid content of 30%, and the paste was allowed to stand at 4°C for crystallization for 12h to precipitate dihydromyricetin crystals, which were then centrifuged at 4000rpm for 15min to obtain crystals. After the crystals were washed with ethanol, they were vacuum dried at 50°C to obtain dihydromyricetin (purity of 75%).

[0099] S3. After the filter residue A was mixed with the extractant 2 at a mass volume ratio of 1 g: 15 mL, the mixture was filtered using a Busch funnel with a pore size of 5 μm at a power of 300 W for 5 min (10 s on / 5 s off) at a temperature of 60°C in pulse mode to obtain a filter residue B and a filtrate B. The filtrate B was transferred to a rotary evaporator and concentrated to obtain a crude myricetin extract with a solid content of 25% (dissolution rate of 58%).

[0100] S4, the crude myricetin extract was mixed with a 45% ethanol solution at a mass volume ratio of 1 g: 10 mL to obtain a mixed solution, a 0.1 mol / L sodium hydroxide solution was added to the mixed solution, the pH of the mixed solution was adjusted to 6.5, the mixture was stirred for 30 min, and the mixture was allowed to stand at a temperature of 25° C. for 1 h, followed by centrifugation at a rate of 4000 rpm for 15 min to obtain a precipitate, and the precipitate was washed with deionized water having a pH of 6.5 to obtain a crude myricetin product (purity 68%);

[0101] The crude myricetin product is prepared into a loading solution, the pH of the loading solution is adjusted to 6.5, and the loading solution is chromatographically separated using a pretreated polyamide as a chromatography column at a loading flow rate of 1.5 BV / h. A linear gradient elution is performed using an ethanol solution with a volume concentration of 40%-80%, wherein the elution conditions of the 40% elution segment are 3 BV, 1.5 BV / h, and then elution is performed with a 40%-80% ethanol solution concentration (excluding 40%) at an elution condition of 8 BV, 1.5 BV / h. During this period, the myricetin in the 50%-60% volume concentration ethanol segment is collected to obtain the target segment, thereby completing the linear gradient elution process. Activated carbon was added to the target segment, stirred at 25°C for 30 min, filtered using a 0.22 μm filter membrane to obtain a filtrate, and the filtrate was evaporated under reduced pressure to a paste with a solid content of 25%. The paste was frozen at -40°C for 24 h, and then vacuum dried at -50°C and a vacuum degree of 10 Pa to obtain myricetin (purity 70%);

[0102] S5. The residue B was mixed with the extractant 3 at a mass volume ratio of 1 g: 10 mL, and the mixture was refluxed at 70° C. for 60 min. The reflux extraction process was repeated twice to obtain an extract. The extract was filtered using a Bush funnel with a pore size of 5 μm to obtain a residue C and a filtrate C. The filtrate C was transferred to a rotary evaporator and concentrated to obtain a crude quercetin extract with a solid content of 25% (dissolution rate of 60%).

[0103] S6. The crude quercetin extract was mixed with an 80% ethanol solution at a mass volume ratio of 1 g:10 mL to obtain a mixed solution. A 1 mol / L sodium hydroxide solution was added to adjust the pH of the mixed solution to 9.5, and the mixture was stirred for 30 min. Subsequently, 0.1 mol / L hydrochloric acid was added to adjust the pH of the mixed solution to 2.5 using a gradient pH adjustment method (gradient pH adjustment method: pH changes: 9.5-7-5-2.5, with an interval of 5 min for each pH adjustment). The mixture was allowed to stand at 25° C. for 2 h, and centrifuged at 4000 rpm for 20 min to obtain a precipitate. The precipitate was washed with hydrochloric acid at a pH of 2.5, and freeze-dried to obtain a crude quercetin product (purity 70%).

[0104] The crude quercetin product was dissolved in the initial eluent (the volume ratio of chloroform to methanol was 8:3), and then filtered through a 0.45μm filter membrane to obtain the sample solution, which was then chromatographed on a balanced silica gel column. Subsequently, during the linear gradient elution process, the eluent was a mixed solution of chloroform and methanol. The linear gradient elution process was as follows: first, a mixed solution of chloroform and methanol with a volume ratio of 8:3 was used as the eluent, and then the volume ratio of the two was linearly reduced to 7:2 to complete the linear gradient elution. The conditions during this period were: 10BV, 1.0mL / min (excluding the elution stage with a volume ratio of chloroform to methanol of 7:2). When the volume ratio of chloroform to methanol in the eluent was 7:2, the conditions for the linear gradient elution were 2BV, 1.0mL / min. During the period, the target segment of 12-15 minutes during the linear gradient elution was retained, and the target segment was mixed with a methanol solution with a volume concentration of 70% at a volume ratio of 1:1 at a temperature of 60°C, and then the temperature was lowered for crystallization. The mixture was allowed to stand at 4°C for 24 hours, and filtered with a 0.22 μm filter membrane to obtain crystals. The crystals were washed three times and then vacuum dried at 40°C and 50 Pa to obtain quercetin (purity 80%).

[0105] Comparative Example 2

[0106] A method for separating dihydromyricetin, myricetin, and quercetin from berry tea comprises the following preparation steps:

[0107] Extractant 1 was prepared by mixing 550 mL of ethanol with 450 mL of water to obtain an ethanol solution having a volume concentration of 55%. 0.1 mol / L of citrate buffer (comprising sodium citrate and citric acid) was added to the ethanol solution until the pH of the ethanol solution reached 2.0 to obtain extractant 1.

[0108] Extractant 2 was prepared by mixing 450 mL of ethanol with 550 mL of water to obtain a 45% ethanol solution. A 1:1 volume ratio of 0.1 mol / L sodium phosphite solution and 0.1 mol / L potassium dihydrogen phosphate solution were added to the ethanol solution until the pH of the ethanol solution reached 6.5 to obtain extractant 2.

[0109] Extractant 3 was prepared by mixing 800 mL of ethanol with 200 mL of water to obtain an ethanol solution with a volume concentration of 80%. 1 mol / L sodium hydroxide solution was added to the ethanol solution until the pH of the ethanol solution reached 9.0 to obtain extractant 3.

[0110] Preparation of berry tea powder: Select berry tea with a total flavonoid content of 20% based on dihydromyricetin, dry the berry tea to a moisture content of ≤8% to obtain dried berry tea, crush the dried berry tea, and pass it through a 40-mesh sieve to obtain berry tea powder.

[0111] An AB-8 macroporous resin with a pore size of 15 nm was immersed in an ethanol solution with a volume concentration of 95%, taken out after soaking for 24 hours, washed with clean water, and then the AB-8 macroporous resin was equilibrated with a buffer solution with a pH of 4.5 to obtain a pretreated AB-8 macroporous resin.

[0112] The polyamide was placed in a 95% volume concentration ethanol solution, soaked for 24 hours, taken out and washed with water, and then the polyamide was balanced with a phosphate buffer solution with a pH of 6.5 at a flow rate of 2 BV / L to obtain a pretreated polyamide.

[0113] The silica gel column chromatography column was activated at a temperature of 120° C. for 2 h, and then equilibrated with a mixed solution of chloroform and methanol (the volume ratio of chloroform to methanol was 8:2) to obtain an equilibrated silica gel column chromatography column.

[0114] S1. After mixing 100 g of berry tea powder with 1500 mL of 70% ethanol solution by volume, ultrasonically treat at a temperature of 50° C. for 30 min, and filter to obtain liquid a and filter residue b. Keeping the temperature at 50° C. unchanged, ultrasonically treat the filter residue b again for 30 min, and filter to obtain liquid b and filter residue b. Mix liquid a and liquid b to obtain liquid 1, and then concentrate liquid 1 to obtain an extract with a solid content of 25%. The extract is redissolved in an ethanol solution with a volume concentration of 70% to obtain a resolution. After adjusting the pH of the resolution to 4.2, centrifuge at a speed of 4000 rpm for 15 min to obtain centrifuge 1. Ethanol is added to the centrifuge 1 until the volume concentration of ethanol is 70%, let it stand for 2 h, and centrifuge at a speed of 4000 rpm for 15 min to obtain centrifuge 2. Polyvinyl pyrrolidone with a mass concentration of 5% is added to the centrifuge 2, stirred for 30 min, and filtered to obtain liquid 2. Liquid 2 is concentrated to obtain pretreated berry tea;

[0115] S2. After mixing 1 kg of pretreated berry tea with 20 L of extractant 1, ultrasonically irradiate the mixture at a frequency of 40 kHz, a power of 200 W, and a temperature of 50° C. for 30 min, and filter the mixture using a Buchner funnel with a pore size of 5 μm to obtain a filter residue A and a filtrate A. The filtrate A is transferred to a rotary evaporator and concentrated under reduced pressure at a temperature of 50° C. and a vacuum degree of -0.08 MPa to obtain a crude dihydromyricetin extract with a solid content of 20% (dissolution rate of 38%).

[0116] S3. The crude dihydromyricetin extract was mixed with a 55% ethanol solution at a mass volume ratio of 1 g: 10 mL to obtain a mixed solution, and the pH of the mixed solution was adjusted to 3.5 with 0.1 mol / L hydrochloric acid, stirred at a speed of 200 rpm for 10 min, allowed to stand at 25° C. for 1 h, and centrifuged at a rate of 4000 rpm for 15 min at a temperature of 4° C. to obtain a precipitate, which was washed twice with a 60% ethanol solution by volume, and freeze-dried to obtain a crude dihydromyricetin product (purity 50%).

[0117] The crude dihydromyricetin was prepared into a loading solution, the pH of the loading solution was adjusted to 4.5, and the loading solution was dynamically adsorbed using a pretreated AB-8 macroporous resin. The flow rate of the dynamic adsorption was 2 mL / min, and then a gradient elution was performed using an ethanol solution with a volume concentration of 10%-95%. Specifically, the elution was first performed with a 10% volume concentration ethanol solution (elution conditions were 3BV, 2.0 mL / min), and then with a 30% volume concentration ethanol solution (elution conditions were 10%-95%). 3BV, 1.5mL / min), then eluted with a 50% volume concentration of ethanol solution (elution conditions are 4BV, 1.0mL / min, pH is 4.5), collected the dihydromyricetin enriched section, then eluted with a 70% volume concentration of ethanol solution (elution conditions are 2BV, 1.5mL / min), and finally eluted with a 95% ethanol solution (elution conditions are 2BV, 2.0mL / min), completed the gradient elution, and obtained the eluent. The eluent was concentrated to obtain a paste with a solid content of 30%, and the paste was allowed to stand at 4°C for crystallization for 12h to precipitate dihydromyricetin crystals, which were then centrifuged at 4000rpm for 15min to obtain crystals. After the crystals were washed with ethanol, they were vacuum dried at 50°C to obtain dihydromyricetin (purity of 70%).

[0118] S4. After the filter residue A was mixed with the extractant 2 at a mass volume ratio of 1 g: 15 mL, the mixture was pulsed at a power of 300 W for 5 min (10 s on / 5 s off) at 60° C. using a pulse mode, and filtered using a Bush funnel with a pore size of 5 μm filter paper to obtain filter residue B and filtrate B. The filtrate B was transferred to a rotary evaporator and concentrated to obtain a crude myricetin extract with a solid content of 25% (dissolution rate of 52%).

[0119] S5. The crude myricetin extract was mixed with a 45% ethanol solution at a mass volume ratio of 1 g: 10 mL to obtain a mixed solution. A 0.1 mol / L sodium hydroxide solution was added to the mixed solution, and the pH of the mixed solution was adjusted to 6.5. The mixture was stirred for 30 min, allowed to stand at 25° C. for 1 h, and then centrifuged at 4000 rpm for 15 min to obtain a precipitate. The precipitate was washed with deionized water at a pH of 6.5 to obtain a crude myricetin product (purity 45%).

[0120] The crude myricetin product is prepared into a loading solution, the pH of the loading solution is adjusted to 6.5, and the loading solution is chromatographically separated using a pretreated polyamide as a chromatography column at a loading flow rate of 1.5 BV / h. A linear gradient elution is performed using an ethanol solution with a volume concentration of 40%-80%, wherein the elution conditions of the 40% elution segment are 3 BV, 1.5 BV / h, and then elution is performed with a 40%-80% ethanol solution concentration (excluding 40%) at an elution condition of 8 BV, 1.5 BV / h. During this period, the myricetin in the 50%-60% volume concentration ethanol segment is collected to obtain the target segment, thereby completing the linear gradient elution process. Activated carbon was added to the target segment, stirred at 25°C for 30 min, filtered using a 0.22 μm filter membrane to obtain a filtrate, and the filtrate was evaporated under reduced pressure to a paste with a solid content of 25%. The paste was frozen at -40°C for 24 h, and then vacuum dried at -50°C and a vacuum degree of 10 Pa to obtain myricetin (purity 68%);

[0121] S6. The residue B was mixed with the extractant 3 at a mass volume ratio of 1 g: 10 mL, and the mixture was refluxed at 70° C. for 60 min. The reflux extraction process was repeated twice to obtain an extract. The extract was filtered using a Bush funnel with a pore size of 5 μm to obtain a residue C and a filtrate C. The filtrate C was transferred to a rotary evaporator and concentrated to obtain a crude quercetin extract with a solid content of 25% (dissolution rate of 72%).

[0122] S7. The crude quercetin extract was mixed with an 80% ethanol solution at a mass volume ratio of 1 g:10 mL to obtain a mixed solution. A 1 mol / L sodium hydroxide solution was added to adjust the pH of the mixed solution to 9.5, and the mixture was stirred for 30 min. Subsequently, 0.1 mol / L hydrochloric acid was added to adjust the pH of the mixed solution to 2.5 using a gradient pH adjustment method (gradient pH adjustment method: pH changes: 9.5-7-5-2.5, with an interval of 5 min for each pH adjustment). The mixture was allowed to stand at 25° C. for 2 h, and centrifuged at 4000 rpm for 20 min to obtain a precipitate. The precipitate was washed with hydrochloric acid at a pH of 2.5, and freeze-dried to obtain a crude quercetin product (purity 45%).

[0123] The crude quercetin product was dissolved in the initial eluent (chloroform to methanol volume ratio of 8:3), then filtered through a 0.45μm filter membrane to obtain the sample solution, which was then chromatographed on a balanced silica gel column. Elution was then performed using a linear gradient elution method with a mixed solution of chloroform and methanol as the eluent. The linear gradient elution process was as follows: first, a mixed solution of chloroform to methanol volume ratio of 8:3 was used as the eluent, and then the volume ratio of the two was linearly reduced to 7:2, that is, the volume ratio of chloroform to methanol was 7:2, completing the linear gradient elution. The conditions during this period were: 10BV, 1.0mL / min (excluding the elution stage with a volume ratio of chloroform to methanol of 7:2). When the volume ratio of chloroform to methanol in the eluent was 7:2, the conditions for linear gradient elution were 2BV, 1.0mL / min. During the period, the target segment of 12-15 minutes during the linear gradient elution was retained, and the target segment was mixed with a methanol solution with a volume concentration of 70% at a volume ratio of 1:1 at a temperature of 60°C, and then the temperature was lowered for crystallization. The mixture was allowed to stand at 4°C for 24 hours, and filtered with a 0.22 μm filter membrane to obtain crystals. The crystals were washed three times and then vacuum dried at 40°C and 50 Pa to obtain quercetin (purity 82%).

[0124] Comparative Example 1 does not include only the S1 process compared to Example 1, that is, the berry tea is not pre-treated, and the content of protein, polysaccharide and polyphenols in the berry tea is higher. From the results, the flavonoid dissolution rates of the three substances (dihydromyricetin crude extract, myricetin crude extract, quercetin crude extract) are all reduced, and the purity of the three flavonoids is all reduced (dihydromyricetin, myricetin, quercetin). This is because protein can be combined with dihydromyricetin and cannot be effectively separated, resulting in a decrease in the dissolution rate and purity of dihydromyricetin, polysaccharides can wrap around myricetin, resulting in a decrease in the dissolution rate and purity of myricetin, and polyphenols can be combined with quercetin and cannot be effectively dissociated, resulting in a decrease in dissolution rate and purity.

[0125] In Comparative Example 2, the pH of the extractant 1 was changed to 2 compared to Example 1. From the results, the purity of the three substances decreased. This is because dihydromyricetin itself degrades in an overly acidic environment, and more polar impurities increase in solubility as the acidity increases, destroying its purity and affecting the subsequent separation and purification steps.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for separating dihydromyricetin, myricetin and quercetin from berry tea, characterized in that: The method comprises the following preparation steps: S1, mixing berry tea powder with ethanol solution, ultrasonicating the mixture, filtering, concentrating the filtered liquid 1 to obtain an extract, redissolving the extract to obtain a reconstituted solution, adjusting the pH of the reconstituted solution to 4.2-4.8, and centrifuging to obtain centrifuge 1, adding ethanol to the centrifuge 1, allowing the mixture to stand, and centrifuging to obtain centrifuge 2, adding an adsorbent to the centrifuge 2, stirring, and filtering to obtain liquid 2, and concentrating liquid 2 to obtain pretreated berry tea; S2. The pretreated berry tea and extractant 1 are mixed, ultrasonicated, and filtered to obtain a filter residue A and a filtrate A, and the filtrate A is concentrated to obtain a crude dihydromyricetin extract; S3, mixing the crude dihydromyricetin extract with the ethanol solution to obtain a mixed solution, adjusting the pH of the mixed solution to 3.3-3.7, stirring, standing, centrifuging to obtain a precipitate, washing the precipitate, freeze-drying, and purifying to obtain dihydromyricetin; S4, mixing the filter residue A with the extractant 2, microwave-treated, and filtered to obtain filter residue B and filtrate B, and concentrating the filtrate B to obtain a crude myricetin extract; S5, mixing the crude myricetin extract with the ethanol solution to obtain a mixed solution, adjusting the pH of the mixed solution to 6-7, stirring, standing, centrifuging to obtain a precipitate, washing the precipitate, freeze-drying, and purifying to obtain myricetin; S6, after mixing the filter residue B with the extractant 3, reflux extraction is performed to obtain a dissolution, the dissolution is filtered to obtain a filter residue C and a filtrate C, and the filtrate C is concentrated to obtain a crude quercetin extract; S7, mixing the crude quercetin extract with the ethanol solution to obtain a mixed solution, adjusting the pH of the mixed solution to 9.3-9.7, stirring, then adjusting the pH of the mixed solution to 2-3 using a gradient pH adjustment method, allowing to stand, centrifuging to obtain a precipitate, washing the precipitate, freeze-drying, and purifying to obtain quercetin; Extractant 1 includes an ethanol solution and a citrate buffer solution with a volume concentration of 50%-55%; Extractant 2 includes an ethanol solution and a phosphate buffer solution with a volume concentration of 40%-45%; The extractant 3 includes an ethanol solution and a sodium hydroxide solution with a volume concentration of 75%-80%.

2. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The total flavonoid content of the berry tea powder in S1 is ≥20%, the volume concentration of the ethanol solution is 65%-75%, and the adsorbent is a polyvinyl pyrrolidone solution with a mass concentration of 3%-5%.

3. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The mass volume ratio of the pretreated berry tea to the extractant 1 in S2 is 1 g: 18-22 mL; the pH of the extractant 1 is 4.9-5.1; the concentration of the citrate buffer is 0.08-0.12 mol / L, and the citrate buffer includes citric acid and sodium citrate; The frequency of ultrasound in S2 is 35-45 kHz, the power of ultrasound is 180-220 W, the temperature of ultrasound is 49-51° C., and the time of ultrasound is 25-45 min.

4. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The mass volume ratio of the crude dihydromyricetin extract to the ethanol solution in S3 is 1 g:10-15 mL.

5. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The mass volume ratio of the filter residue A to the extractant 2 in S4 is 1 g:13-17 mL, the pH of the extractant 2 is 6.4-6.6, and the phosphate buffer comprises a sodium phosphite solution and a potassium dihydrogen phosphate solution; In S4, the power of the microwave is 280-320 W, the temperature of the microwave is 58-62° C., and the microwave time is 4-6 min.

6. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The mass volume ratio of the crude myricetin extract to the ethanol solution in S5 is 1 g:10-15 mL.

7. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The mass volume ratio of filter residue B to extractant 3 in S6 is 1 g:8-12 mL; The pH of the extractant 3 in S6 is 8.8-9.2, and the concentration of the sodium hydroxide solution is 0.8-1.2 mol / L.

8. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The temperature of the reflux extraction in S6 is 65-75° C., the number of reflux extractions is ≥1, and the single reflux extraction time is 50-70 min.

9. The method for separating dihydromyricetin, myricetin and quercetin from berry tea according to claim 1, wherein: The mass volume ratio of the crude quercetin extract to the ethanol solution in S7 is 1 g:10-15 mL.

10. Use of the method for separating dihydromyricetin, myricetin and quercetin from berry tea according to any one of claims 1 to 9 in the fields of pharmaceuticals and cosmetics.

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