A method for extracting and purifying dihydroquercetin from larch xylem

Abstract

The present invention belongs to the technical field of extraction and purification of fat-soluble chemical components, and particularly relates to a method for extracting and purifying dihydroquercetin from larch xylem. The present invention uses tea saponin aqueous micelles (tea saponin aqueous solution) to greenly extract dihydroquercetin from larch xylem, and then, according to the interfacial charge characteristics of the micelles formed by tea saponin and dihydroquercetin in the aqueous solution, utilizes the charge characteristics of chitosan to directionally enrich dihydroquercetin in the extract, and combines anti-solvent biphasic crystallization and water-solvent single-phase recrystallization to purify dihydroquercetin. The extraction and purification method provided by the present invention has a high extraction rate of dihydroquercetin and a high purity of the obtained pure product, and has the characteristics of simple operation, green safety, high efficiency and low cost. It not only improves the utilization rate of larch resources, but also has great significance for giving play to the high-value application of dihydroquercetin.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and purification of fat-soluble chemical components, and particularly relates to a method for extracting and purifying dihydroquercetin from larch xylem. Background Art

[0002] Larch is an arbor of the Pinaceae family, and its xylem is the main supply part of dihydroquercetin. Dihydroquercetin is a natural and potent antioxidant with broad-spectrum biological activities such as anti-inflammatory, antibacterial, anti-cancer, immune regulation, and improvement of microcirculation, and is an important raw material for the production of food, health products, and pharmaceuticals.

[0003] At present, Chinese patents CN116354918A and CN103360359A both disclose methods for extracting, separating, and purifying dihydroquercetin. However, in the process of purifying dihydroquercetin in Chinese patents CN103360359A and CN116354918A, resin adsorption is used for purification. Although dihydroquercetin with a high purity can be obtained, the purification efficiency is low, the cycle is long, and the process cost is high, which is not suitable for industrial production. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting and purifying dihydroquercetin from larch xylem. The extraction and purification method provided by the present invention has a high extraction rate of dihydroquercetin and a high purity of the obtained pure product, and has the characteristics of simple operation, green safety, high efficiency, and low cost. It not only improves the utilization rate of larch resources but also has great significance for giving play to the value application of dihydroquercetin.

[0005] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a method for extracting and purifying dihydroquercetin from larch xylem, comprising the following steps:

[0007] (1) Mixing larch xylem powder and tea saponin aqueous micelles for extraction to obtain a dihydroquercetin extraction solution;

[0008] (2) Mixing the dihydroquercetin extraction solution, organic acid, and chitosan for enriching dihydroquercetin to obtain a dihydroquercetin-enriched precipitate;

[0009] (3) Extracting the dihydroquercetin-enriched precipitate with an organic acid ester solvent, and drying the obtained organic acid ester layer to obtain a crude dihydroquercetin product;

[0010] (4) Dissolving the crude dihydroquercetin product in a lower-carbon alcohol solvent, mixing the obtained crude dihydroquercetin product alcohol solution with water, and performing anti-solvent biphasic recrystallization on the obtained mixed solution to obtain a refined dihydroquercetin product;

[0011] (5) Mix the dihydroquercetin product with water, heat and dissolve to obtain an aqueous solution of dihydroquercetin product; subject the aqueous solution of dihydroquercetin product to hot filtration, cool the obtained filtrate for single-phase recrystallization to obtain pure dihydroquercetin.

[0012] Preferably, the mass percentage of saponin in the saponin aqueous micelles is 0.1 - 10 wt%; the mass ratio of the larch xylem powder to the volume of the saponin aqueous micelles is 1 g: 5 - 30 mL.

[0013] Preferably, the organic acid includes one or more of glacial acetic acid, citric acid, tartaric acid and oxalic acid; the dosage of the organic acid accounts for 0.1 - 0.8% of the dihydroquercetin extraction solution.

[0014] Preferably, the mass of the chitosan accounts for 1 - 5 wt% of the mass of the dihydroquercetin extraction solution; the time for enriching dihydroquercetin is 4 - 6 h.

[0015] Preferably, the organic acid ester solvent is ethyl acetate;

[0016] The mass ratio of the dihydroquercetin enrichment precipitate to the volume of the organic acid ester solvent is 1 g: 3 - 8 mL; the extraction time is 15 - 30 min.

[0017] Preferably, the lower alcohol solvent is ethanol;

[0018] The mass ratio of the crude dihydroquercetin product to the volume of the lower alcohol solvent is 1 g: 1 - 5 mL.

[0019] Preferably, in step (4), the volume ratio of water to the lower alcohol solvent is 3 - 10: 1; the temperature of the anti-solvent biphasic recrystallization is 1 - 4 °C, and the time is 4 - 12 h.

[0020] Preferably, in step (5), the mass ratio of the dihydroquercetin product to the volume of water is 1 g: 3 - 10 mL; the temperature of the single-phase recrystallization is 1 - 4 °C, and the time is 4 - 12 h.

[0021] Preferably, in step (4), the crude dihydroquercetin alcohol solution is mixed with water to directly obtain an initial mixed solution, further including: subjecting the initial mixed solution to solid-liquid separation, and taking the liquid phase as the mixed solution; the solid-liquid separation is centrifugal separation, and the rotation speed of the centrifugal separation is 4000 - 8000 rpm, and the time is 6 - 10 min.

[0022] Preferably, the particle size of the larch xylem powder is 10 - 100 mesh; the extraction includes one or more of heating reflux extraction, ultrasonic extraction, microwave extraction and pressurized extraction.

[0023] The present invention provides a method for extracting and purifying dihydroquercetin from larch xylem, comprising the following steps: (1) Mixing larch xylem powder with tea saponin aqueous micelles for extraction to obtain a dihydroquercetin extract; (2) Mixing the dihydroquercetin extract, organic acid and chitosan for enriching dihydroquercetin to obtain a dihydroquercetin-enriched precipitate; (3) Extracting the dihydroquercetin-enriched precipitate with an organic acid ester solvent, drying the obtained organic acid ester layer to obtain crude dihydroquercetin; (4) Dissolving the crude dihydroquercetin in a lower-carbon alcohol solvent, mixing the obtained crude dihydroquercetin alcohol solution with water, and subjecting the obtained mixed solution to anti-solvent biphasic recrystallization to obtain high-quality dihydroquercetin; (5) Mixing high-quality dihydroquercetin with water, heating and dissolving to obtain an aqueous solution of high-quality dihydroquercetin; subjecting the aqueous solution of high-quality dihydroquercetin to hot filtration, and cooling the obtained filtrate for single-phase recrystallization to obtain pure dihydroquercetin. The present invention uses tea saponin aqueous micelles to extract dihydroquercetin from larch xylem powder. Tea saponin belongs to a non-ionic surfactant of glycoside compounds and has a relatively high solubility in dihydroquercetin. During the extraction process, tea saponin in the tea saponin aqueous micelles exists in the form of micelles. Due to the hydrogen bond interaction between tea saponin and dihydroquercetin, dihydroquercetin can be wrapped in the hydrophobic core to form a supramolecular micelle for extraction. Then, according to the interfacial charge characteristics of the micelle formed by tea saponin and dihydroquercetin, with the assistance of an organic acid, the electrostatic adsorption between chitosan and tea saponin is used to directionally enrich dihydroquercetin in the extract. Finally, the combination of anti-solvent biphasic recrystallization and single-phase recrystallization is used to purify dihydroquercetin, effectively improving the extraction rate and purity of dihydroquercetin. At the same time, tea saponin in the tea saponin aqueous micelles used in the present invention can be degraded by soil microorganisms and belongs to an environmentally friendly extraction solvent. Its recovery avoids environmental pollution and safety problems caused by solvent volatilization. In summary, the extraction and purification method provided by the present invention is simple in operation, high in extraction efficiency, low in energy consumption, safe and reliable in the extraction process, and has high economic and environmental benefits, and is suitable for industrial production. The data from the examples show that the maximum extraction rate of dihydroquercetin extracted by the present invention is 91.13%, and the maximum purity of the pure dihydroquercetin obtained by directional enrichment, anti-solvent biphasic crystallization and water solvent single-phase recrystallization is 98.31%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the flowchart of the operation of the method for extracting and purifying dihydroquercetin from larch xylem provided by the present invention;

[0025] Figure 2 is the high performance liquid chromatography (HPLC) chart of the extract obtained in Example 6 of the present invention;

[0026] Figure 3 is the high performance liquid chromatography (HPLC) chart of the crude dihydroquercetin obtained in Example 2 of the present invention;

[0027] Figure 4 This is the high performance liquid chromatography (HPLC) chromatogram of the dihydroquercetin fine product obtained in Example 5 of the present invention;

[0028] Figure 5 This is the high performance liquid chromatography (HPLC) chromatogram of the dihydroquercetin pure product obtained in Example 3 of the present invention. Detailed implementation manners

[0029] The present invention provides a method for extracting and purifying dihydroquercetin from larch xylem, comprising the following steps:

[0030] (1) Mix larch xylem powder and tea saponin aqueous micelles for extraction to obtain a dihydroquercetin extract;

[0031] (2) Mix the dihydroquercetin extract, organic acid and chitosan for enriching dihydroquercetin to obtain a dihydroquercetin enriched precipitate;

[0032] (3) Extract the dihydroquercetin enriched precipitate with an organic acid ester solvent, dry the obtained organic acid ester layer to obtain a crude dihydroquercetin product;

[0033] (4) Dissolve the crude dihydroquercetin product in a lower carbon alcohol solvent, mix the obtained crude dihydroquercetin product alcohol solution and water, and perform anti-solvent biphasic recrystallization on the obtained mixed solution to obtain a dihydroquercetin fine product;

[0034] (5) Mix the dihydroquercetin fine product and water, heat and dissolve to obtain a dihydroquercetin fine product aqueous solution; perform hot filtration on the dihydroquercetin fine product aqueous solution, and cool the obtained filtrate for single-phase recrystallization to obtain a dihydroquercetin pure product.

[0035] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well-known to those skilled in the art.

[0036] The present invention mixes larch xylem powder and tea saponin aqueous micelles for extraction to obtain a dihydroquercetin extract. In the present invention, the preparation method of the larch xylem powder includes the following steps: drying, pulverizing, and screening the larch xylem in sequence to obtain the larch xylem powder. The larch xylem is preferably one or more of the roots, trunks, and sawdust of larch. The drying is preferably drying by baking. The present invention has no special requirements for the specific implementation manner of the pulverizing. The diameter of the sieve mesh used for the screening is preferably 10 to 100 mesh, more preferably 20 to 80 mesh. The particle size of the obtained larch xylem powder is preferably 10 to 100 mesh, more preferably 20 to 80 mesh. In the present invention, the tea saponin aqueous micelles are an aqueous solution of tea saponin micelles self-assembled when tea saponin is dispersed in water. The mass percentage content of tea saponin in the tea saponin aqueous micelles is preferably 0.1 to 10 wt%, more preferably 0.5 to 8 wt%. The mass ratio of the larch xylem powder to the volume of the tea saponin aqueous micelles is preferably 1 g:5 to 30 mL, more preferably 1 g:6 to 15 mL. In the present invention, the extraction preferably includes one or more of heating reflux extraction, ultrasonic extraction, microwave extraction, and pressurized extraction, more preferably heating reflux extraction, ultrasonic extraction, or pressurized extraction. When the extraction is preferably heating reflux extraction, the number of times of heating reflux extraction is preferably 1 to 3 times, and the time of each heating reflux extraction is preferably 45 to 60 min. When the extraction is preferably ultrasonic extraction, the number of times of ultrasonic extraction is preferably 1 to 3 times, and the time of each ultrasonic extraction is preferably 45 to 50 min. When the extraction is preferably pressurized extraction, the pressure of the pressurized extraction is preferably 50 to 250 MPa, specifically preferably 50 MPa, 150 MPa, or 250 MPa; the number of times of pressurized extraction is preferably 1 to 3 times, and the time of each pressurized extraction is preferably 3 to 8 min, specifically preferably 3 min, 4 min, or 8 min. The extraction is carried out in an extraction device, and the extraction device includes one or more of a heating reflux extraction device, an ultrasonic extraction device, a microwave extraction device, and a pressurized extraction device, more preferably a heating reflux extraction device, an ultrasonic extraction device, or a pressurized extraction device.

[0037] In the present invention, the extraction directly obtains an extraction slurry. The present invention further includes solid-liquid separation of the extraction slurry, and the obtained liquid phase is the dihydroquercetin extract. The solid-liquid separation is preferably centrifugal separation. The rotation speed of the centrifugal separation is preferably 4000 to 8000 rpm, and the time is preferably 6 to 10 min.

[0038] After obtaining the dihydroquercetin extract, the present invention mixes the dihydroquercetin extract, an organic acid, and chitosan for dihydroquercetin enrichment to obtain a dihydroquercetin-enriched precipitate. In the present invention, the organic acid preferably includes one or more of glacial acetic acid, citric acid, tartaric acid, and oxalic acid, and more preferably glacial acetic acid. The dosage of the organic acid accounts for 0.1-0.8% of the dihydroquercetin extract. In a specific embodiment of the present invention, when the organic acid is glacial acetic acid, the volume of the glacial acetic acid accounts for 0.1-0.8% of the volume of the dihydroquercetin extract, more preferably 0.2%, 0.3%, 0.4%, or 0.8%. When the organic acid is one or more of citric acid, tartaric acid, and oxalic acid, the mass of the organic acid accounts for 0.1-0.8% of the mass of the dihydroquercetin extract, more preferably 0.2%, 0.3%, 0.4%, or 0.8%. The mass of the chitosan preferably accounts for 1-5 wt% of the mass of the dihydroquercetin extract, more preferably 1 wt%, 2 wt%, 3 wt%, 4 wt%, or 5 wt%. The time for dihydroquercetin enrichment is preferably 4-6 h.

[0039] In the present invention, the dihydroquercetin enrichment directly yields an enriched slurry. The present invention further includes solid-liquid separation of the enriched slurry, and the obtained solid phase is the dihydroquercetin-enriched precipitate. The solid-liquid separation is preferably centrifugal separation, and the rotation speed of the centrifugal separation is preferably 4000-8000 rpm, and the time is preferably 6-10 min.

[0040] After obtaining the dihydroquercetin-enriched precipitate, the present invention extracts the dihydroquercetin-enriched precipitate with an organic acid ester solvent, and dries the obtained organic acid ester layer to obtain a crude dihydroquercetin product. In the present invention, the organic acid ester solvent is preferably ethyl acetate. The mass ratio of the dihydroquercetin-enriched precipitate to the volume of the organic acid ester solvent is preferably 1 g: 3-8 mL, more preferably 1 g: 3 mL, 1 g: 4 mL, 1 g: 5 mL, 1 g: 6 mL, 1 g: 7 mL, or 1 g: 8 mL. The temperature of the extraction is preferably room temperature, and the time of the extraction is preferably 15-30 min.

[0041] In the present invention, the extraction directly yields an extraction slurry. The present invention further includes solid-liquid separation of the extraction slurry, and the obtained liquid phase is the organic acid ester layer. The specific embodiment of the drying is preferably rotary evaporation drying. The solid-liquid separation is preferably centrifugal separation, and the rotation speed of the centrifugal separation is preferably 4000-8000 rpm, and the time is preferably 6-10 min.

[0042] After obtaining the crude dihydroquercetin, the present invention dissolves the crude dihydroquercetin in a lower-carbon alcohol solvent, mixes the obtained crude dihydroquercetin alcohol solution with water, and performs anti-solvent biphasic recrystallization on the obtained mixed solution to obtain high-quality dihydroquercetin. In the present invention, the lower-carbon alcohol solvent is preferably ethanol. The water is preferably ultrapure water. The mass ratio of the crude dihydroquercetin to the volume of the lower-carbon alcohol solvent is preferably 1 g: 1-5 mL, more preferably 1 g: 1 mL, 1 g: 2 mL, 1 g: 3 mL, 1 g: 4 mL or 1 g: 5 mL. The volume ratio of the water to the lower-carbon alcohol solvent is 3-10: 1. The mixing is preferably stirring mixing, and the stirring mixing time is preferably 5-15 min. The crude dihydroquercetin alcohol solution and water are mixed to directly obtain an initial mixed solution. The present invention preferably further includes: separating the solid and liquid of the initial mixed solution, and taking the liquid phase as the mixed solution; the solid-liquid separation is centrifugal separation, the rotation speed of the centrifugal separation is 4000-8000 rpm, and the time is 6-10 min. The temperature of the anti-solvent biphasic recrystallization is 1-4 °C, and the time is preferably 4-12 h.

[0043] In the present invention, the anti-solvent biphasic recrystallization directly obtains an anti-solvent biphasic recrystallization slurry. The present invention preferably includes separating the solid and liquid of the anti-solvent biphasic recrystallization slurry, and drying the obtained solid phase to obtain high-quality dihydroquercetin. The solid-liquid separation is preferably centrifugal separation, the rotation speed of the centrifugal separation is preferably 4000-8000 rpm, and the time is preferably 6-10 min. The present invention has no special requirements for the specific implementation manner of the drying.

[0044] After obtaining the high-quality dihydroquercetin, the present invention mixes the high-quality dihydroquercetin with water, heats and dissolves it to obtain an aqueous solution of high-quality dihydroquercetin; filters the aqueous solution of high-quality dihydroquercetin while it is hot, and cools the obtained filtrate for single-phase recrystallization to obtain pure dihydroquercetin. The water is preferably ultrapure water. The mass ratio of the high-quality dihydroquercetin to the volume of water is preferably 1 g: 3-10 mL, more preferably 1 g: 3 mL, 1 g: 4 mL, 1 g: 5 mL, 1 g: 6 mL, 1 g: 7 mL, 1 g: 8 mL, 1 g: 9 mL or 1 g: 10 mL. The temperature of the single-phase recrystallization is preferably 1-4 °C, more preferably 4 °C; the time is preferably 4-12 h.

[0045] In the present invention, the single-phase recrystallization directly obtains a single-phase recrystallization slurry. The present invention preferably includes separating the solid and liquid of the single-phase recrystallization slurry, and drying the obtained solid phase to obtain pure dihydroquercetin. The solid-liquid separation is preferably centrifugal separation, the rotation speed of the centrifugal separation is preferably 4000-8000 rpm, and the time is preferably 6-10 min. The present invention has no special requirements for the specific implementation manner of the drying.

[0046] A method for extracting and purifying dihydroquercetin from larch xylem provided by the present invention uses tea saponin aqueous micelles to greenly extract dihydroquercetin from larch xylem. Then, according to the interfacial charge characteristics of the micelles formed by tea saponin and dihydroquercetin in the aqueous solution, the charge characteristics of chitosan are used to directionally enrich dihydroquercetin in the extract, and dihydroquercetin is purified by combining anti-solvent biphasic crystallization and water-solvent single-phase recrystallization. The extraction and purification method provided by the present invention has a high extraction rate of dihydroquercetin and a high purity of the obtained pure product, and has the characteristics of simple operation, green safety, high efficiency and low cost. It not only improves the utilization rate of larch resources, but also has great significance for giving play to the value application of dihydroquercetin.

[0047] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0048] In the following examples, all are carried out according to Figure 1 the operation process of the method for extracting and purifying dihydroquercetin from larch xylem to carry out the extraction.

[0049] Example 1

[0050] Using dried larch xylem as raw material, it is pulverized and sieved through a 20-mesh sieve. Weigh 10 kg of larch xylem powder, add 6 times the amount of 0.5 wt% tea saponin aqueous micelle extraction solvent, place it in a heat reflux extraction device, heat and reflux for 45 min, and extract 3 times. After each extraction, centrifuge at 5000 rpm for 8 min to obtain the dihydroquercetin extract. Through high performance liquid chromatography determination and analysis, according to the content of dihydroquercetin in larch xylem and the content of dihydroquercetin in the extract, the extraction rate of dihydroquercetin is calculated to be 84.67%.

[0051] Add 0.2% glacial acetic acid according to the volume of the dihydroquercetin extract, stir well and then add 1 wt% chitosan of the extract, and carry out directional enrichment at 4°C for 4 hours. After the enrichment is completed, centrifuge to obtain the dihydroquercetin enriched precipitate.

[0052] Add the dihydroquercetin enriched precipitate to 3 times the amount of ethyl acetate and extract for 15 min. After the extraction is completed, centrifuge at 4000 rpm for 7 min, and rotary evaporate the ethyl acetate extract to obtain the crude dihydroquercetin. Through high performance liquid chromatography determination and analysis, the purity of dihydroquercetin is measured to be 21.37%.

[0053] The obtained crude dihydroquercetin was added with 1 volume of ethanol for dissolution, and then 3 volumes of ultrapure water based on the volume of ethanol was added for thorough stirring for 5 min and centrifugation. The supernatant was taken for recrystallization at 4 °C for 4 h, and finally centrifuged at 8000 rpm for 10 min. The crystalline precipitate was dried to obtain high-purity dihydroquercetin. By determination and analysis using high-performance liquid chromatography, the purity of dihydroquercetin was measured to be 46.54%.

[0054] The high-purity dihydroquercetin was added with 3 volumes of ultrapure water and heated. After complete dissolution, it was immediately filtered while hot. The filtrate was recrystallized at 4 °C for 4 h, and finally centrifuged at 8000 rpm for 10 min. The crystalline precipitate was dried to obtain the finished product of dihydroquercetin. By determination and analysis using high-performance liquid chromatography, the purity of dihydroquercetin was measured to be 93.41%, and the yield calculated based on larch powder was 1.75%.

[0055] Example 2

[0056] Using the dried larch xylem as raw material, it was pulverized and passed through a 20-mesh sieve. 10 kg of larch xylem powder was weighed, added with 6 volumes of 4 wt% tea saponin aqueous micelle extraction solvent, placed in an ultrasonic extraction device, and ultrasonically extracted for 45 min, with extraction carried out 3 times. After each extraction, it was centrifuged at 5000 rpm for 8 min to obtain the dihydroquercetin extract. By determination and analysis using high-performance liquid chromatography, based on the content of dihydroquercetin in larch xylem and the content of dihydroquercetin in the extract, the extraction rate of dihydroquercetin was calculated to be 88.67%.

[0057] 0.2% glacial acetic acid was added according to the volume of the dihydroquercetin extract. After thorough stirring, 2 wt% of chitosan based on the mass of the extract was added, and directional enrichment was carried out at 4 °C for 6 hours. After the enrichment was completed, it was centrifuged at 4000 rpm for 8 min to obtain the dihydroquercetin enriched precipitate.

[0058] The dihydroquercetin enriched precipitate was added with 4 volumes of ethyl acetate and extracted for 20 min. After the extraction was completed, it was centrifuged at 4000 rpm for 7 min, and the ethyl acetate extract was rotary evaporated to obtain the crude dihydroquercetin. By determination and analysis using high-performance liquid chromatography, the purity of dihydroquercetin was measured to be 22.71%. The high-performance liquid chromatography chart of the crude dihydroquercetin obtained in Example 2 is as Figure 3 shown.

[0059] The obtained crude dihydroquercetin was added with 2 volumes of ethanol for dissolution, and then 3 volumes of ultrapure water based on the volume of ethanol was added for thorough stirring for 10 min and centrifugation. The supernatant was taken for recrystallization at 4 °C for 6 h, and finally centrifuged at 8000 rpm for 10 min. The crystalline precipitate was dried to obtain high-purity dihydroquercetin. By determination and analysis using high-performance liquid chromatography, the purity of dihydroquercetin was measured to be 48.71%.

[0060] High-purity dihydroquercetin was added to 5 times the amount of ultrapure water and heated. After being fully dissolved, it was immediately filtered while hot, and the filtrate was recrystallized at 4°C for 6 hours. Finally, it was centrifuged at 8000rpm for 10 minutes, and the crystal precipitate was dried to obtain the finished dihydroquercetin. The purity of dihydroquercetin was 95.73% by high performance liquid chromatography, and the yield was 1.63% based on larch powder.

[0061] Example 3

[0062] The dried larch wood is used as raw material and crushed through a 40-mesh sieve. 10 kg of larch wood powder is weighed, 8 times the amount of 2 wt% tea saponin water-based micellar extraction solvent is added, placed in a hot reflux extraction device, heated and refluxed for 1 hour, and extracted 3 times. After each extraction, centrifuge at 5000 rpm for 8 minutes to obtain a dihydroquercetin extract. The dihydroquercetin extraction rate is calculated to be 87.57% based on the dihydroquercetin content in the larch wood and the dihydroquercetin content in the extract by high performance liquid chromatography.

[0063] 0.3% glacial acetic acid was added according to the volume of the dihydroquercetin extract, and after sufficient stirring, chitosan (2 wt% of the extract) was added, and directional enrichment was carried out at 4° C. for 6 hours. After the enrichment was completed, centrifugation was performed to obtain a dihydroquercetin enriched precipitate.

[0064] The dihydroquercetin enriched precipitate was added with 5 times the amount of ethyl acetate and extracted for 20 minutes. After the extraction, the mixture was centrifuged at 4000 rpm for 7 minutes, and the ethyl acetate extract was spin-dried to obtain a crude dihydroquercetin product. The purity of the dihydroquercetin was 25.47% as determined by high performance liquid chromatography.

[0065] The obtained crude dihydroquercetin was dissolved by adding 2 times the volume of ethanol, and then ultrapure water with a volume of 5 times the volume of ethanol was added for full stirring for 10 minutes and centrifugation. The clear liquid was recrystallized at 4°C for 12 hours, and finally centrifuged at 8000 rpm for 10 minutes. The crystal precipitate was dried to obtain high-purity dihydroquercetin. The purity of dihydroquercetin was determined to be 51.54% by high performance liquid chromatography.

[0066] Add 5 times the amount of ultrapure water to high-purity dihydroquercetin and heat it. After it is fully dissolved, filter it while hot immediately. Recrystallize the filtrate at 4°C for 12 hours. Finally, centrifuge it at 8000rpm for 10 minutes. Dry the crystalline precipitate to obtain the finished product of dihydroquercetin. The purity of dihydroquercetin was determined to be 97.41% by high-performance liquid chromatography, and the yield was 1.69% based on larch powder. The high-performance liquid chromatogram of the pure dihydroquercetin (finished product) obtained in Example 3 is as follows: Figure 5 shown.

[0067] Example 4

[0068] The dried larch wood is used as raw material and crushed through a 60-mesh sieve. 100g of larch wood powder is weighed, 10 times the amount of 4wt% tea saponin water-based micellar extraction solvent is added, and the mixture is placed in an ultra-high pressure extraction device with an extraction pressure of 50MPa, an extraction time of 3min, and extraction for 3 times. After each extraction, the mixture is centrifuged at 5000rpm for 8min to obtain a dihydroquercetin extract. The dihydroquercetin extraction rate is calculated to be 88.67% based on the dihydroquercetin content in the larch wood and the dihydroquercetin content in the extract by high performance liquid chromatography.

[0069] 0.4% glacial acetic acid was added according to the volume of the dihydroquercetin extract, and after sufficient stirring, 4wt% chitosan was added to the extract, and directional enrichment was carried out at 4°C for 6 hours. After the enrichment, the dihydroquercetin enriched precipitate was obtained by centrifugation at 4000rpm for 8min.

[0070] The dihydroquercetin enriched precipitate was added with 4 times the amount of ethyl acetate and extracted for 30 minutes. After the extraction, the mixture was centrifuged at 4000 rpm for 7 minutes, and the ethyl acetate extract was spin-dried to obtain a crude dihydroquercetin product. The purity of the dihydroquercetin was determined to be 25.43% by high performance liquid chromatography.

[0071] The obtained crude dihydroquercetin was dissolved by adding 2 times the volume of ethanol, and then ultrapure water with a volume of 7 times the volume of ethanol was added for full stirring for 10 minutes and centrifugation. The clear liquid was recrystallized at 4°C for 6 hours, and finally centrifuged at 8000 rpm for 10 minutes. The crystal precipitate was dried to obtain high-purity dihydroquercetin. The purity of dihydroquercetin was 57.74% as determined by high performance liquid chromatography.

[0072] High-purity dihydroquercetin was added to 8 times the amount of ultrapure water and heated. After being fully dissolved, it was immediately filtered while hot, and the filtrate was recrystallized at 4°C for 8 hours. Finally, it was centrifuged at 8000 rpm for 10 minutes, and the crystal precipitate was dried to obtain the finished dihydroquercetin. The purity of dihydroquercetin was 96.31% by high performance liquid chromatography, and the yield was 1.63% based on larch powder.

[0073] Example 5

[0074] The dried larch wood is used as raw material and crushed through a 60-mesh sieve. 100g of larch wood powder is weighed, 10 times the amount of 4wt% tea saponin water-based micellar extraction solvent is added, and the mixture is placed in an ultra-high pressure extraction device with an extraction pressure of 150MPa, an extraction time of 4min, and 3 extractions. After each extraction, the mixture is centrifuged at 5000rpm for 8min to obtain a dihydroquercetin extract. The dihydroquercetin extraction rate is calculated to be 89.67% based on the dihydroquercetin content in the larch wood and the dihydroquercetin content in the extract by high performance liquid chromatography.

[0075] 0.8% glacial acetic acid was added according to the volume of the dihydroquercetin extract, and after sufficient stirring, 5wt% chitosan of the extract was added, and directional enrichment was carried out at 4°C for 4 hours. After the enrichment, the dihydroquercetin enriched precipitate was obtained by centrifugation at 4000 rpm for 8 minutes.

[0076] The dihydroquercetin enriched precipitate was added with 8 times the amount of ethyl acetate and extracted for 20 minutes. After the extraction, the mixture was centrifuged at 4000 rpm for 7 minutes, and the ethyl acetate extract was spin-dried to obtain a crude dihydroquercetin product. The purity of the dihydroquercetin was determined to be 19.63% by high performance liquid chromatography.

[0077] The obtained crude dihydroquercetin was dissolved by adding 1 times the volume of ethanol, and then ultrapure water with a volume of 5 times the volume of ethanol was added for full stirring for 10 minutes and centrifugation. The clear liquid was recrystallized at 4°C for 6 hours, and finally centrifuged at 8000 rpm for 10 minutes. The crystal precipitate was dried to obtain high-purity dihydroquercetin. The purity of dihydroquercetin was measured and analyzed by high-performance liquid chromatography to be 57.74%. Figure 4 This is the HPLC chromatogram of the high-purity dihydroquercetin (dihydroquercetin premium product) obtained in Example 5.

[0078] High-purity dihydroquercetin was added to 6 times the amount of ultrapure water and heated. After being fully dissolved, it was immediately filtered while hot, and the filtrate was recrystallized at 4°C for 12 hours. Finally, it was centrifuged at 8000 rpm for 10 minutes, and the crystal precipitate was dried to obtain the finished dihydroquercetin. The purity of dihydroquercetin was 98.31% by high performance liquid chromatography, and the yield was 1.81% based on larch powder.

[0079] Example 6

[0080] The dried larch xylem was used as raw material and crushed through a 100-mesh sieve. Weigh 100g of larch xylem powder, add 15 times the amount of 8wt% tea saponin water-based micellar extraction solvent, place in an ultra-high pressure extraction device, extraction pressure 250MPa, extraction time 8min, extraction 3 times. After each extraction, centrifuge at 5000rpm for 8min to obtain a dihydroquercetin extract. HPLC determination and analysis, based on the dihydroquercetin content in the larch xylem and the dihydroquercetin content in the extract, calculated dihydroquercetin extraction rate is 91.13%. The high performance liquid chromatogram of the extract obtained in Example 6 is shown in Figure 2. Figure 2 shown.

[0081] 0.2% glacial acetic acid was added according to the volume of the dihydroquercetin extract, and after sufficient stirring, chitosan (3 wt% of the extract) was added, and directional enrichment was carried out at 4° C. for 4 hours. After the enrichment, the dihydroquercetin enriched precipitate was obtained by centrifugation at 4000 rpm for 8 minutes.

[0082] The dihydroquercetin enriched precipitate was added with 6 times the amount of ethyl acetate and extracted for 30 minutes. After the extraction, the mixture was centrifuged at 4000 rpm for 7 minutes, and the ethyl acetate extract was spin-dried to obtain a crude dihydroquercetin product. The purity of the dihydroquercetin was determined to be 25.57% by high performance liquid chromatography.

[0083] The obtained crude dihydroquercetin was dissolved by adding 2 times the volume of ethanol, and then adding ultrapure water with a volume of 7 times the volume of ethanol, stirring for 15 minutes, centrifuging, taking the clear liquid for recrystallization at 4°C for 12 hours, and finally centrifuging at 8000rpm for 10 minutes, and drying the crystal precipitate to obtain high-purity dihydroquercetin. The purity of dihydroquercetin was measured and analyzed by high-performance liquid chromatography to be 57.93%.

[0084] High-purity dihydroquercetin was added to 5 times the amount of ultrapure water and heated. After being fully dissolved, it was immediately filtered while hot, and the filtrate was recrystallized at 4°C for 12 hours. Finally, it was centrifuged at 8000rpm for 10 minutes, and the crystal precipitate was dried to obtain the finished dihydroquercetin. The purity of dihydroquercetin was 96.81% by high performance liquid chromatography, and the yield was 1.78% based on larch powder.

[0085] Example 7

[0086] The dried larch wood is used as raw material and crushed through a 20-mesh sieve. 10 kg of larch wood powder is weighed, 10 times the amount of 8 wt% tea saponin water-based micellar extraction solvent is added, placed in an ultrasonic extraction device, ultrasonic extraction for 60 minutes, and extracted 3 times. After each extraction, centrifuge at 5000 rpm for 8 minutes to obtain a dihydroquercetin extract. The dihydroquercetin extraction rate is calculated to be 90.67% based on the dihydroquercetin content in the larch wood and the dihydroquercetin content in the extract by high performance liquid chromatography.

[0087] 0.8% glacial acetic acid was added to the volume of the dihydroquercetin extract, and after sufficient stirring, 5wt% chitosan was added, and directional enrichment was carried out at 4°C for 5 hours. After the enrichment, the dihydroquercetin enriched precipitate was obtained by centrifugation at 4000 rpm for 8 minutes.

[0088] The dihydroquercetin enriched precipitate was added with 7 times the amount of ethyl acetate and extracted for 25 minutes. After the extraction, the mixture was centrifuged at 4000 rpm for 7 minutes, and the ethyl acetate extract was spin-dried to obtain a crude dihydroquercetin product. The purity of the dihydroquercetin was determined to be 23.43% by high performance liquid chromatography.

[0089] The obtained crude dihydroquercetin was dissolved by adding 5 times the volume of ethanol, and then ultrapure water with a volume of 10 times the volume of ethanol was added for full stirring for 10 minutes and centrifugation. The clear liquid was recrystallized at 4°C for 12 hours, and finally centrifuged at 8000 rpm for 10 minutes. The crystal precipitate was dried to obtain high-purity dihydroquercetin. The purity of dihydroquercetin was 56.71% as determined by high performance liquid chromatography.

[0090] High-purity dihydroquercetin was added to 8 times the amount of ultrapure water and heated. After being fully dissolved, it was immediately filtered while hot, and the filtrate was recrystallized at 4°C for 8 hours. Finally, it was centrifuged at 8000 rpm for 10 minutes, and the crystal precipitate was dried to obtain the finished dihydroquercetin. The purity of dihydroquercetin was 94.73% by high performance liquid chromatography, and the yield was 1.71% based on larch powder.

[0091] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting and purifying dihydroquercetin from larch xylem, characterized in that, It includes the following steps: (1) Mix larch xylem powder and tea saponin aqueous micelles for extraction to obtain a dihydroquercetin extract; (2) Mix the dihydroquercetin extract, organic acid, and chitosan for dihydroquercetin enrichment to obtain a dihydroquercetin-enriched precipitate, where the organic acid is one or more of glacial acetic acid, citric acid, tartaric acid, and oxalic acid; (3) Extract the dihydroquercetin-enriched precipitate with an organic acid ester solvent, where the organic acid ester solvent is ethyl acetate, and dry the obtained organic acid ester layer to obtain crude dihydroquercetin; (4) Dissolve the crude dihydroquercetin in a lower-carbon alcohol solvent, mix the obtained crude dihydroquercetin alcohol solution and water, and perform anti-solvent biphasic recrystallization on the obtained mixed solution to obtain high-quality dihydroquercetin; (5) Mix high-quality dihydroquercetin and water, heat and dissolve to obtain an aqueous solution of high-quality dihydroquercetin; perform hot filtration on the aqueous solution of high-quality dihydroquercetin, and cool the obtained filtrate for single-phase recrystallization to obtain pure dihydroquercetin.

2. The extraction and purification method according to claim 1, wherein The mass percentage of tea saponin in the tea saponin aqueous micelles is 0.1 - 10 wt%; the mass ratio of the larch xylem powder to the volume of the tea saponin aqueous micelles is 1 g:5 - 30 mL.

3. The extraction and purification method according to claim 1, characterized in that The dosage of the organic acid accounts for 0.1 - 0.8% of the dihydroquercetin extract.

4. The extraction and purification method according to claim 1 or 3, characterized in that, The mass of the chitosan accounts for 1 - 5 wt% of the mass of the dihydroquercetin extract; the time for dihydroquercetin enrichment is 4 - 6 h.

5. The extraction and purification method according to claim 1, characterized in that, The mass ratio of the dihydroquercetin-enriched precipitate to the volume of the organic acid ester solvent is 1 g:3 - 8 mL; the extraction time is 15 - 30 min.

6. The extraction and purification method according to claim 1, wherein The lower-carbon alcohol solvent is ethanol; The mass ratio of the crude dihydroquercetin to the volume of the lower-carbon alcohol solvent is 1 g:1 - 5 mL.

7. The extraction and purification method according to claim 1 or 6, characterized in that, In step (4), the volume ratio of water to the lower-carbon alcohol solvent is 3 - 10:1; the temperature of the anti-solvent biphasic recrystallization is 1 - 4 °C, and the time is 4 - 12 h.

8. The extraction and purification method according to claim 1, wherein In step (5), the mass ratio of high-quality dihydroquercetin to the volume of water is 1 g:3 - 10 mL; the temperature of the single-phase recrystallization is 1 - 4 °C, and the time is 4 - 12 h.

9. The extraction and purification method according to claim 1, wherein In step (4), when the crude dihydroquercetin alcohol solution and water are mixed to directly obtain an initial mixed solution, it further includes: separating the solid and liquid of the initial mixed solution, and taking the liquid phase as the mixed solution; the solid-liquid separation is centrifugal separation, the rotation speed of the centrifugal separation is 4000 - 8000 rpm, and the time is 6 - 10 min.

10. The extraction and purification method according to claim 1 or 2, characterized in that, The particle size of the larch xylem powder is 10 - 100 mesh; the extraction includes one or more of heating reflux extraction, ultrasonic extraction, microwave extraction, and pressurized extraction.

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