Method for extracting flavone from ginkgo leaves by water immersion

Abstract

The invention discloses a method for extracting flavonoids in ginkgo leaves by water immersion, which comprises the following steps of: adding crushed ginkgo leaf raw materials into a complexing agent solution, stirring and extracting in an extraction tank, and filtering after extraction; the complexing agent is a boron-containing electron-deficient compound, and specifically can be borate, and the boron anion group of the borate is [BO3] < 3-> or [BO4] < 5->; according to the extraction method, column chromatography adsorption and desorption are adopted, an ethanol solution is used as a desorption solution in the desorption process, a complexing agent can be desorbed, and then flavone is obtained. According to the method, the complexing agent aqueous solution is used for extraction, and the ethanol solution is used as a desorption solution in column chromatography desorption, so that the condition that the solubility of flavonoid components in water is relatively low can be improved, the extraction rate is increased, the extraction temperature and the water consumption can be greatly reduced, and compared with organic solvent extraction, the cost and environmental pollution are reduced; the impurity content of extracted species is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of plant flavonoid extraction, and specifically relates to a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. Background Art

[0002] Flavonoids are secondary metabolites in plants. Natural flavonoids often contain different substituents such as hydroxyl groups, methoxy groups, hydrocarbonoxy groups, and isopentenoxy groups on their parent nuclei, and their physicochemical properties also vary significantly. Flavonoids have significant curative effects in the treatment of diseases such as hypertension, diabetes, and atherosclerosis. Therefore, the market demand for flavonoids is huge.

[0003] Ginkgo biloba leaves are rich in flavonoids, but the solubility of its flavonoid compounds in water is not ideal. Currently, the processes for extracting flavonoids from Ginkgo biloba leaves mainly include water extraction and ethanol extraction. For example, in the water extraction method disclosed in the prior art, "Study on the Process of Extracting Total Flavonoids from Ginkgo biloba Leaves by Water Immersion Method", Yan Gaoying et al., "Northwest Pharmaceutical Journal", Vol. 31, No. 6, November 2016, pp. 560-561, it is disclosed that since the extraction process of Ginkgo biloba leaves mainly focuses on extraction using organic solvents, but the extraction cost is high, many manufacturers have stopped production one after another. In order to reduce costs, the optimal extraction process provided is 26 times the amount of water (1:10:8:8), extracted 3 times at 100 °C, each time for 1 h, and the process is stable and feasible and has been applied to large-scale production; another example is the invention patent with the authorization announcement number CN108703981B in the prior art, which discloses a method for extracting ginkgo flavonoids from Ginkgo biloba leaves. The Ginkgo biloba leaves are dried and crushed, then first extracted with ethanol, and then extracted with ethyl acetate to extract ginkgolide. After that, the remaining filter residue after extraction is extracted with methanol, and then loaded onto an adsorption resin column, and ginkgo flavonoids are obtained after elution; during the ethanol extraction process, the mass ratio of the crushed Ginkgo biloba leaves to the ethanol is 1:3-5, the extraction temperature is 50-75 °C, the number of extraction times is 3-6 times, and the extraction time for each extraction is 10-60 min; during the ethyl acetate extraction process, the mass ratio of the crushed Ginkgo biloba leaves to the ethyl acetate is 1:1-5, the extraction temperature is 45-75 °C, the number of extraction times is 2-6 times, and the extraction time for each extraction is 10-60 min; in the step of extracting with methanol, the mass ratio of the filter residue to the methanol is 1:2-5, the extraction temperature is 50-60 °C, the number of extraction times is 2-6 times, and the extraction time for each extraction is 10-60 min. Another example is the invention patent with the authorization announcement number CN105154252B in the prior art, which discloses a method for extracting Ginkgo biloba leaf extract, including the following steps: 1) drying and crushing Ginkgo biloba leaves; 2) taking the Ginkgo biloba leaf powder, adding a sodium acetate-acetic acid buffer solution with a pH of 4.0-5.5, adding cellulase, and enzymolyzing in a water bath at 50-60 °C for 1-2 h; 3) adding absolute ethanol to make the volume fraction of ethanol in the system 50-80%, performing microwave extraction in a microwave reactor, controlling the microwave power at 130 W, the microwave temperature at 78 °C, and the cumulative microwave time at 4 min; 4) filtering by suction, extracting the filtrate with petroleum ether to remove impurities, taking the water layer, rotary evaporating and concentrating, and drying to obtain the product.

[0004] From the perspective of the prior art, the improvement of extracting flavonoids from Ginkgo biloba with organic solvents is based on the selection of organic solvents and the improvement of extraction processes on the one hand, and on the other hand, the introduction of other auxiliary extraction means, such as the addition of enzymes. However, in the extraction process with organic solvents, there are problems such as high costs and solvent residues that cannot be changed. In addition, the use of organic solvents for extraction will also produce a large amount of chlorophyll, proteins, and other lipophilic impurities, which will bring difficulties to the purification of subsequent flavonoid components. For the water immersion extraction process, although the use of organic solvents is avoided, the extraction temperature is 100 °C and the material-liquid ratio is 1:26, resulting in too high a temperature and a large amount of water used, and high costs.

[0005] It can also be seen from the prior art that the extraction of flavonoids from Ginkgo biloba leaves is a research hotspot, but how to reduce the extraction cost and reduce the use of organic solvents is the pursuit of the industry. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. This extraction method can greatly reduce the cost problem of extraction with organic solvents. While ensuring the extraction rate, it significantly reduces the extraction temperature, not only greatly reducing the production cost, but also being environmentally friendly and suitable for industrial production.

[0007] One concept of the present invention is to provide a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. The method solves the problem of poor water solubility of flavonoid components when extracted with water by introducing a specific complexing agent, thereby reducing the water consumption and extraction temperature in the water immersion extraction process and significantly reducing the extraction production cost.

[0008] Furthermore, another concept of the present invention also lies in providing a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. The complexing agent used in the method has good water solubility and is relatively stable, so that a lower extraction temperature and extraction solution dosage can be used during the extraction process to ensure the transfer rate of the extracted flavonoids.

[0009] Furthermore, another concept of the present invention is to provide a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. The complexing agent used in this method is an electron-deficient compound. The empty orbital on the electron-deficient compound forms a coordination bond with the carbonyl group at the 4th position of flavonoid, and then chelates with the hydroxyl group at the 5th or 3rd position to form a complex salt. The formed complex salt can, on the one hand, prevent the oxidation of flavonoids, and on the other hand, promote the dissolution of flavonoids and improve the transfer rate of flavonoids.

[0010] Furthermore, another concept of the present invention also lies in providing a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. This extraction method uses water as the extraction solvent. The total amount of the aqueous solution used is less than 20 times the material-liquid ratio of the raw material, and the extraction temperature is 40 - 50 °C. Using this extraction method can ensure the extraction rate while significantly reducing the extraction cost.

[0011] Furthermore, another concept of the present invention also lies in providing a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. After water immersion extraction, adsorption and desorption are carried out by macroporous resin column chromatography. Among them, the desorption is carried out with 70% ethanol. When the present invention uses macroporous resin for purification, flavonoid components will be adsorbed on the macroporous resin during the column chromatography adsorption process, while the complexing agent will flow out with the column effluent, resulting in a decomplexing effect. Finally, a high-purity flavonoid extract without complexing agent can be obtained after column chromatography desorption.

[0012] Specifically, the present invention provides a method for extracting flavonoids from Ginkgo biloba leaves by water immersion. The method comprises the following steps: (1) Extraction The pulverized Ginkgo biloba leaf raw material is added to the complexing agent solution, stirred and extracted in an extraction tank, and filtered after extraction to obtain a clear liquid; (2) Column chromatography adsorption The clear liquid obtained in step (1) is loaded onto a resin chromatograph for adsorption; (3) Column chromatography desorption Use an ethanol solution as the desorbing solution to desorb the column chromatography of the adsorbed clear liquid in step (2), and collect the desorbing solution; (4) Drying The desorbing solution obtained in step (4) is dried to obtain a Ginkgo biloba leaf flavonoid extract.

[0013] Among them, the concentration of the complexing agent solution in step (1) is 0.4% - 1.2%; Preferably, the concentration of the complexing agent solution is 0.5 - 1.0%.

[0014] More preferably, the concentration of the complexing agent solution is 0.6 - 0.8%.

[0015] The complexing agent is an electron-deficient compound.

[0016] Preferably, the complexing agent is an electron-deficient compound containing boron or aluminum element.

[0017] The electron-deficient compound containing boron element is borate, and the boron anion group of the borate is [BO3] 3- or [BO4] 5- .

[0018] Furthermore, the borate cation in the electron-deficient compound containing boron element is one or more of alkali metals, alkaline earth metals and transition metals.

[0019] Furthermore, the borate cation in the electron-deficient compound containing boron element is Li + , K + , Cs+ , Be 2+ , Mg 2 + , Ba 2+ , Ti 4+ , Ta 5+ , Mn 6+ , Fe 3+ , Ni 3+ , Cu 2+ or one or more of them.

[0020] Furthermore, the boron-containing electron-deficient compound is one or more of Mg[B2O(OH)6], Li(H2O)4B(OH)4, TaBO4, Ca(BO2)2, K3B3O6, Ba3(B3O6)2, Mg2B3O5, Fe2B2O5, Mg3(BO3)2, Rb2B4O5(OH)4, K[B5O6(OH)4], Ca[B3O3(OH)5].

[0021] In the extraction of step (1), the extraction temperature is 40 - 50 °C, preferably 45 - 50 °C.

[0022] In the extraction of step (1), the dosage of the complexing agent solution is 15 - 22 times the weight of the ginkgo leaf raw material, preferably 18 - 20 times; the extraction time in the extraction of step (1) is 6 - 8 h, preferably 7 - 8 h.

[0023] Furthermore, in the extraction of step (1), the complexing agent solution is divided into multiple extractions, preferably three extractions; Furthermore, when the complexing agent solution is extracted three times, the dosage ratio of the complexing agent is (1.6 - 2) : (1.6 - 2) : 1, and the extraction time ratio is (1.3 - 1.8) : (1 - 1.2) : 1.

[0024] Furthermore, in the extraction of step (1), mechanical stirring extraction is adopted, preferably heating and stirring extraction.

[0025] In some embodiments, after the extraction in step (1), filtration is carried out using a filter cloth, and the filter cloth is a 200 - 400 mesh filter cloth for filtration; Furthermore, the filtrate is centrifuged using a centrifuge, the centrifugation rate is 3000 - 5000 rpm, and the centrifugation time is 5 - 20 min.

[0026] In some embodiments, the operation of column chromatography adsorption in step (2) is to adsorb the centrifuged supernatant with a D101 macroporous resin column chromatography, the flow rate of loading onto the column is 1.5 - 2 times the column volume / h, that is, 1.5 - 2 times the column volume per hour, and then wash with water for 2 - 4 times the column volume.

[0027] In some embodiments, the concentration of ethanol in the column chromatography elution in step (3) is an aqueous ethanol solution of 65-75%, preferably an aqueous ethanol solution of 70-75%.

[0028] Furthermore, the amount of ethanol for elution is 1.5-2.5 times the column volume.

[0029] In some embodiments, in step (4), drying is performed by vacuum drying, the drying temperature is 60-70 °C, and the vacuum pressure is -0.08 MPa to -0.09 MPa.

[0030] Compared with the prior art, the present invention uses an aqueous solution of a complexing agent for extraction. Compared with extraction using an ethanol solution, the amount of organic solvent used is significantly reduced, which saves costs and protects the environment. Moreover, when using an ethanol solution for extraction, a large amount of chlorophyll, proteins, and other liposoluble components will be extracted, which brings difficulties to the subsequent purification of ginkgo leaf flavonoids.

[0031] In addition, the present invention uses an aqueous solution of a complexing agent for extraction. Compared with extraction using water, it can improve the low solubility of flavonoid components in water. While increasing the extraction rate, it can significantly reduce the extraction temperature and the amount of water used. For industrial production, it can greatly reduce energy consumption and make the production process safer and more stable.

[0032] Furthermore, the complexing agent forms a relatively stable complex with flavonoid components during the extraction process. Unlike other alkaline substances, although they can play a solubilizing role, they may also cause changes in the flavonoid structure. When using macroporous resin column chromatography for adsorption subsequently, the flavonoid components are adsorbed on the resin due to the adsorption of the macroporous resin, while the complexing agent will flow out with the eluate. During purification, the complex is also dissociated, and a good extraction rate of flavonoids can be achieved. Description of the Drawings

[0033] Figure 1 HPLC test spectrum of ginkgo leaf raw material.

[0034] Figure 2 HPLC test spectra of ginkgo leaf flavonoid extracts in Examples 1-3.

[0035] Figure 3 HPLC test spectra of ginkgo leaf extraction residues in Examples 1-3. Detailed Embodiments

[0036] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the protection scope of the present invention.

[0037] A method for extracting flavonoids from ginkgo leaves by water immersion provided by the solution of this embodiment includes the following steps: (1) Extraction The pulverized ginkgo leaf raw material is added to the complexing agent solution, stirred and extracted in an extraction tank, and filtered after extraction to obtain a clear liquid; Among them, in step (1), the concentration of the complexing agent solution is 0.4% - 1.2%; the concentration of the complexing agent solution is 0.5 - 1.0%; the concentration of the complexing agent solution is 0.6 - 0.8%; it can be 0.4%, 0.5%, 0.6%, 0.9%, 1.2%.

[0038] Specifically, when selecting the concentration of the complexing agent, it is necessary to consider the acquisition region, storage time, humidity and other specifications of the ginkgo leaf raw material to be extracted, and adjustments can be made. There are certain differences in the flavonoid content of different specifications of ginkgo leaves, and the extraction effect of flavonoids can be ensured by adjusting the dosage of the complexing agent.

[0039] The complexing agent is an electron-deficient compound;

[0040] The complexing agent is an electron-deficient compound containing boron or aluminum elements.

[0041] The boron-containing electron-deficient compound is borate, and the boron anion group of the borate is [BO3] 3- or [BO4] 5- ; The creativity of the present invention selects an electron-deficient compound in terms of the complexing agent, especially an electron-deficient compound containing boron elements. Among them, the electron configuration of the boron atom is 1s 2 2s 2 2p 1 , and the number of valence electrons is less than the number of valence orbitals. When coordinating with a cationic atom, it can adopt sp3 hybridization to coordinate with three oxygen atoms, or it can also adopt sp4 to coordinate with oxygen atoms.

[0042] When the present invention extracts flavonoids from ginkgo leaves, the structure of the flavonoids is mostly a diketone structure, with low polarity and poor solubility in water. Generally, weakly polar solvents are used for dissolution, including ethanol, methanol, propanol, ethyl acetate, etc. The structure of ginkgetin is generally as follows: ; In this structure, the phenolic hydroxyl group on the benzene ring of flavonoids is easily oxidized, making flavonoids unstable and thus affecting the extraction rate of flavonoids during the extraction process. In addition, the carbonyl group in flavonoids makes the solubility of the entire flavonoid very low. The electron-deficient compound complexing agent used in the present invention is based on the above two groups. The empty orbital of the complexing agent forms a coordination bond with the carbonyl group at the 4th position of flavonoids, and then chelates with the phenolic hydroxyl group at the 5th or 3rd position to form a complex salt. The formed complex salt can, on the one hand, prevent the oxidation of flavonoid substances, and on the other hand, promote solubility and help improve the extraction efficiency.

[0043] Further, in the present invention, preferably, the boron anion group of the borate adopts a tetrahedral structure [BO4] coordinated with four oxygen atoms in a sp3 hybridization mode. 5- Anionic structure.

[0044] The present invention finds that the [BO4] 5- anionic structure coordinated with four oxygen atoms in a sp3 hybridization mode shows more excellent extraction effects during the extraction process and can further reduce the dosage of the complexing agent during the extraction process.

[0045] In the boron-containing electron-deficient compound, the borate cation is one or more of alkali metals, alkaline earth metals, and transition metals; Further, the borate cation in the boron-containing electron-deficient compound is Mg 2+ 、Li + 、K + 、Cs + 、Be 2 + 、Ba 2+ 、Ti 4+ 、Ta 5+ 、Mn 6+ 、Fe 3+ 、Ni 3+ 、Cu 2+ one or more of;

[0046] When used as a complexing agent, the solubility of the complexing agent compound needs to be ensured. Generally speaking, the key complexing group is determined by the anionic group. Therefore, for cations that can ensure the solubility of the complexing agent, they are generally feasible and can ensure the extraction of flavonoid substances from Ginkgo biloba leaves.

[0047] Further, the preferred complexing agents of the present invention are one or more of Mg[B2O(OH)6], Li(H2O)4B(OH)4, TaBO4, Ca(BO2)2, K3B3O6, Ba3(B3O6)2, Mg2B3O5, Fe2B2O5, Mg3(BO3)2, Rb2B4O5(OH)4, K[B5O6(OH)4], Ca[B3O3(OH)5].

[0048] More preferably, it is one or more of Mg[B2O(OH)6], Li(H2O)4B(OH)4, K[B5O6(OH)4].

[0049] In some embodiments, the extraction temperature in the extraction of step (1) is 40 - 50 °C, preferably 45 - 50 °C. The extraction temperature can be 40 °C, 45 °C, 48 °C, 50 °C.

[0050] After using the complexing agent in the present invention, the complexing agent and flavonoids undergo a coordination reaction, which can improve the solubility of flavonoids. Therefore, the transfer and dissolution of flavonoids can be ensured at normal or low temperatures. According to the change in the solubility of flavonoids, the present invention can select a lower dissolution temperature, which can greatly reduce the extraction cost in terms of energy.

[0051] In some embodiments, the dosage of the complexing agent solution in step (1) of the present invention is 17 - 22 times the weight of the ginkgo leaf raw material. Here, the solution dosage is based on weight, preferably 18 - 20 times. The dosage of the complexing agent solution can be 18 times, 19 times, 20 times, 22 times of the ginkgo leaf raw material.

[0052] The extraction time in the extraction of step (1) is 6 - 9 h, preferably 7 - 8 h. Specifically, the extraction time can be 6 h, 7 h, 8 h, 9 h.

[0053] Further, in some embodiments, the complexing agent solution in the extraction of step (1) is divided into multiple extractions, preferably three extractions;

[0054] The present invention further adopts a multiple - extraction process. The multiple - extraction process can further affect the solubility balance of flavonoids after complexation with the complexing agent, contribute to the dissolution of flavonoids, and improve the flavonoid extraction rate. The present invention preferably divides the complexing agent solution into three extractions, which can improve the dissolution of flavonoid substances to a certain extent and increase the extraction rate.

[0055] Further, when the complexing agent solution is extracted three times, the dosage ratio of the complexing agent for the three extractions is (1.6 - 2):(1.6 - 2):1, and the extraction time ratio is (1.3 - 1.8):(1 - 1.2):1. Specifically, for the three extractions, the dosage ratio of the complexing agent can be 8:8:4, or 8:7:5, or 8:8:5; the extraction time ratio can be 3:2:2.

[0056] In the present invention, the number of extractions has an impact on the extraction efficiency. Generally, when the complexing agent undergoes a coordination reaction with flavonoid compounds, on the one hand, it improves the stability of flavonoid compounds, and on the other hand, it improves the solubility of flavonoid substances. During the extraction process, the content of flavonoids in the initial raw material is relatively the highest, and as time goes by, the content of flavonoids in the raw material decreases, making the dissolution more difficult. When the content of flavonoids in the complexing agent solution increases, this dissolution becomes even more difficult. Therefore, the present invention uses multiple extractions to dissolve flavonoids. Through process adjustment, it is found that under the conditions that the complexing agent solution is extracted three times, the dosages of the complexing agent are (1.6 - 2):(1.6 - 2):1 of the ginkgo leaf raw material respectively, and the extraction time is (1.3 - 1.8):(1 - 1.2):1, the extraction rate is the most excellent.

[0057] In the extraction of step (1), mechanical stirring extraction is adopted, specifically heating and stirring extraction.

[0058] In some embodiments, after the extraction in step (1), filtration is carried out using a filter cloth, and the filter cloth is a 200 - 400 mesh filter cloth; specifically, the filter cloth can be 200 mesh, 300 mesh, or 400 mesh.

[0059] Generally speaking, for step (1), after extraction, substances with large impurities need to be removed. The filtration in the present invention is to remove large - particle impurities, which is convenient for the subsequent column chromatography adsorption and analysis of the extract, and for the extraction, separation, and purification of flavonoid substances.

[0060] Further, the filtrate is centrifuged using a centrifuge, the centrifugation rate is 3000 - 5000 rpm, and the centrifugation time is 5 - 20 min.

[0061] In some embodiments, a method for water - immersion extraction of flavonoids from ginkgo leaves further includes a step (2) of column chromatography adsorption. The specific operation is to adsorb the clear liquid obtained in step (1) using a D101 macroporous resin column chromatography, with an upper - column flow rate of 1.5 - 2 times the column volume / h, and then wash with water for 2 - 4 times the column volume.

[0062] In some embodiments, a method for water - immersion extraction of flavonoids from ginkgo leaves further includes a step (3) of column chromatography elution. Specifically, ethanol solution is used as the eluent to elute the column chromatography of the adsorbed clear liquid in step (2), and the eluent is collected. Among them, in the column chromatography elution in step (3), the concentration of ethanol in the ethanol solution is 65-75% aqueous ethanol solution, preferably 70-75% aqueous ethanol solution; specifically, it can be 65%, 70%, or 75% aqueous ethanol solution. The dosage of ethanol elution is 1.5-2.5 times the column volume.

[0063] In some embodiments, a method for water extraction of flavonoids from Ginkgo biloba leaves further includes (4) drying. Specifically, the eluent obtained in step (3) is vacuum dried at a drying temperature of 60-70°C and a vacuum pressure of -0.08 MPa to -0.09 MPa to obtain the Ginkgo biloba leaf flavonoid extract.

[0064] For the test of the total content in flavonoids, referring to the test method for total flavonol glycosides in Ginkgo biloba leaf extract in the Chinese Pharmacopoeia 2020 Edition, the specific implementation method is as follows: (1) Experimental instruments: high performance liquid chromatograph (Thermo Fisher Ultimate 3000), analytical balance (Shimadzu AUW200D, Japan), microfiltration membrane (0.45), syringe filter. (2) Reagents: methanol (chromatographic grade, analytical grade), phosphoric acid (analytical grade), hydrochloric acid (analytical grade), purified water. (3) Detection conditions: Chromatographic column: C18, 4.6×150 mm, 5 μm (Agilent); Detection wavelength: 370 nm, flow rate: 1.0 ml / min, column temperature: 25°C; Mobile phase: methanol: 0.4% phosphoric acid aqueous solution = 50:50.

[0065] (4) Preparation of reference solution: Weigh appropriate amounts of quercetin, kaempferol, and isorhamnetin reference substances accurately, dissolve and make up the volume with methanol to obtain the reference solution.

[0066] (5) Preparation of test solution: Weigh appropriate amounts of the test sample accurately, add 25 ml of a mixed solution of methanol - 25% hydrochloric acid solution (4:1), heat under reflux in a water bath for 30 minutes, cool to room temperature, transfer to a volumetric flask and make up the volume to obtain the test solution.

[0067] (6) Determination method: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph. Record the chromatogram and calculate by the external standard method based on the peak area.

[0068] (7) Calculate the total flavonoid content: Calculate by the external standard method based on the peak area ; In the formula: A 样 —— Peak area of the test sample; A 对 —— Peak area of the reference substance; W 对 —— Weighing amount of the reference substance; W 样 —— Weighing amount of the test sample; V 样 —— Dilution factor of the test sample; V 对 —— Dilution factor of the reference substance; P—— Content of the reference substance (quercetin 99.1%, kaempferol 97.4%, isorhamnetin 99.1%); The present invention will be described in detail below through specific factual examples.

[0069] Example 1

[0070] A method for water extraction of flavonoids from Ginkgo biloba leaves, comprising the following steps. First, prepare a complexing agent solution: weigh an appropriate amount of the complexing agent and dissolve it in water to form a complexing agent solution. Subsequently, carry out the extraction work, and the extraction includes the following operations: (1) Extraction: Add the pulverized Ginkgo biloba leaf raw material to the complexing agent solution, heat and stir for extraction in an extraction tank, the extraction temperature is 40 - 50 °C, the extract is filtered through a 300 - mesh filter cloth, and centrifuged at 4000 rpm for 10 min using a centrifuge to obtain the centrifuged supernatant.

[0071] (2) Column chromatography adsorption: Adsorb the centrifuged supernatant with a D101 macroporous resin column chromatography, the loading flow rate is 1.5 - 2 times the column volume / h, and then wash with water for 2 times the column volume.

[0072] (3) Column chromatography elution: Elute with 70% ethanol until the eluate is nearly colorless, a total of 1.5 - 2.5 times the column volume is eluted, and collect the eluate.

[0073] (4) Drying: Evaporate the eluate to dryness on a 60 °C water bath, and then transfer it to a 60 °C vacuum drying oven for vacuum drying at - 0.08 MPa to - 0.09 MPa to obtain the total flavonoid extract of Ginkgo biloba leaves.

[0074] Content detection: Use the EP - SOP - QC - 05 - 046 (Rev1.00) detection method to detect the total flavonoid content of the centrifuged supernatant, centrifuged precipitate, medicinal residues, and total flavonoid extract.

[0075] Specific process parameters are shown in the following table

[0076] As can be seen from Examples 1-1 to 1-14, after using the complexing agent, 1. The extraction of flavonoids from Ginkgo biloba leaves can be achieved at a relatively low temperature, and the extraction effect is relatively excellent; 2. Under the condition of a relatively low dosage of the extraction solution (complexing agent solution), a good extraction rate can be achieved when the dosage is about 20 times.

[0077] For further analysis, in terms of the extraction effects obtained from Examples 1-1, 1-2, and 1-3, when the temperature increases, the increase in the extraction rate is relatively obvious. For example, when comparing Example 1-2 and Example 1-3, the increase in the extraction rate is close to 5%, which is significant. This shows that on the one hand, the complexing agent can promote the dissolution of flavonoids, and on the other hand, an increase in temperature under certain conditions is also beneficial to extraction; from the results of Example 1-2, when the dosage of the complexing agent and the temperature increase slightly, the extraction rate of flavonoids will also increase relatively; however, from Example 1-14, an increase in temperature does not have a positive impact on the extraction rate of flavonoids, but instead, the extraction rate of flavonoids will decrease. It is analyzed that on the one hand, flavonoids may be unstable under high-temperature conditions, resulting in a decrease in the extraction rate; on the other hand, high-temperature conditions will affect the binding stability between the complexing agent and flavonoids, reducing the solubility of flavonoids, which comprehensively leads to an adverse effect on the extraction of flavonoids under high-temperature conditions.

[0078] In terms of the extraction multiple, when the dosage of the complexing agent solution is 20-21 times that of the Ginkgo biloba leaf raw material, an excellent extraction effect can be achieved. Referring to Example 1-9, when the dosage of the extraction solution (complexing agent solution) is reduced to 15 times, the extraction rate drops sharply. However, further, when the complexing agent solution is increased further, as shown in Example 1-13, there is no obvious effect on the extraction rate of flavonoids. It may be that the soluble flavonoids in Ginkgo biloba leaves have already been dissolved, and it is impossible to further improve the dissolution of flavonoids based on this formula; comparing with the results of Example 1-10, it can be seen that under the condition of a low multiple, increasing the extraction temperature can increase the extraction rate of flavonoids from Ginkgo biloba leaves, and the increase amplitude is relatively obvious. Further combining the conclusions on the extraction temperature in Examples 1-1 to 1-3, on the one hand, the dissolution rate of flavonoids in Examples 1-1 to 1-3 is relatively high, and the relatively greater impact is the influence of high temperature on the stability of flavonoids; for the results of Example 1-10, it can be seen that under the condition of a relatively low extraction solution (complexing agent solution), the key factor affecting the extraction rate may be the dissolution rate of flavonoids. That is, in Example 1-10, even if the dissolution temperature is increased significantly under the condition of a relatively low dosage of the extraction solution, it is still impossible to ensure a good extraction effect of flavonoids. At the same time, on the other hand, high temperature will increase the dissolution of water-soluble impurities, which will affect the final extraction purity of flavonoids.

[0079] In addition, from the analysis of the concentration of the complexing agent, as can be seen from Examples 1-11 and 1-12, when the concentration of the complexing agent changes, it will have an impact on the flavonoids in Ginkgo biloba leaves. Specifically, when the amount of the complexing agent is too small, it may not complex with flavonoids sufficiently, thereby reducing the extraction rate. However, when the amount of the complexing agent increases beyond the amount required for complete complexation with flavonoids, there is no obvious adverse effect on the extraction effect.

[0080] Furthermore, based on the extraction effects of Examples 1-3, the present invention also used other electron-deficient compounds as complexing agents to extract flavonoids from Ginkgo biloba leaves. It can be seen that the electron-deficient compounds in Examples 1-4 to 1-8 can all extract the flavonoid components from Ginkgo biloba leaves using water as a medium under low-temperature conditions. Further, we can also find that [BO4] coordinated with oxygen atoms in the sp3 hybridization mode 5- has a more excellent extraction effect under these specific conditions. This may be due to the influence of the atomic structure, which causes differences in the coordination effects between the electron-deficient compound complexing agent and flavonoid substances.

[0081] As can be seen from Examples 1-1 to 1-14, in the method for water extraction of flavonoids from Ginkgo biloba leaves provided by the present invention, when the concentration of the complexing agent is greater than 0.4, a good complexation degree between the complexing agent and flavonoids can be obtained; and when the amount of the complexing agent is greater than 16 times the mass of the Ginkgo biloba leaf raw material, a good extraction effect can also be achieved.

[0082] In the above experiment, through the previously provided test method, the high-performance liquid chromatography test spectrum of the Ginkgo biloba leaf raw material was detected, as shown in Figure 1 shown; and the extracts and extraction residues after extraction in Examples 1-3 were tested. Among them, Figure 2 is the high-performance liquid chromatography test spectrum of the Ginkgo biloba leaf flavonoid extract in Examples 1-3; Figure 3 is the high-performance liquid chromatography test spectrum of the Ginkgo biloba leaf extraction residue in Examples 1-3.

[0083] Example 2

[0084] Referring to the operation steps and operation parameters of Examples 1-3, the difference is that in step (1) of Example 2, heating and ultrasonic extraction were performed 3 times in the extraction tank, and the total flavonoid extraction rate obtained through testing was 74.93%.

[0085] From Example 2, it can be seen that after using the complexing agent solution as the extraction solution, ultrasonic extraction is disadvantageous. This may be because the ultrasonic action will prevent the complexation of the complexing agent with flavonoids, or promote the dissociation of the already complexed flavonoid substances.

[0086] Example 3

[0087] Refer to the operation steps and operation parameters of Examples 1 - 3. The difference is that no complexing agent is used for extraction, and water is directly used as the extraction medium. The total flavonoid extraction rate obtained through testing is 51.63%.

[0088] From Example 3, when water is used as the medium, it is almost impossible to extract flavonoids under low - temperature conditions. The extraction cost in industry is high, and the industrial value is relatively low.

[0089] Example 4

[0090] Refer to the operation steps and operation parameters of Examples 1 - 3. The difference is that the extraction times are 2 times. The extraction dosage is 11 times for the first extraction for 4 hours, and 10 times for the second extraction for 3 hours. Finally, the total flavonoid extraction rate obtained through testing is 82.23%. It can be seen that the flavonoid extraction rate by the two - extraction method is less than that of the three - extraction method. This may be determined by the dissolution situation of flavonoids. Since the dissolution of flavonoids is an equilibrium dissolution, when the content of flavonoids in the solution is small, it is more conducive to the dissolution of flavonoids. When extracting in two times compared with three times, although the solution is relatively larger during the two - time dissolution, it is easier to reach the dissolution equilibrium, resulting in a poorer extraction effect.

[0091] It should be noted that for more extraction times, such as four - time extraction or even more, the influence on the total flavonoid extraction rate in the subsequent extraction process is not significant. At this time, flavonoids are in a basically dissolved state, and increasing the dissolution times has little effect on the extraction effect.

[0092] Although the present invention has made a detailed description of the invention scheme in the examples and drawings, the protection scope of the present invention is not limited to the specific examples and the display in the drawings. Based on this, those skilled in the art can make various changes and modifications in this article without departing from the inventive concept of the present invention.

Claims

1. A method for extracting flavonoids from Ginkgo biloba leaves by water immersion, characterized in that: The method described above includes the following steps: (1) Extraction The pulverized ginkgo leaf raw material is added to a complexing agent solution, stirred and extracted in an extraction tank, and filtered after extraction to obtain a clear liquid; (2) Column chromatography adsorption The clear liquid obtained in step (1) is loaded onto a resin chromatograph for adsorption; (3) Column chromatography elution An ethanol solution is used as the eluent to elute the column chromatography of the clear liquid adsorbed in step (2), and the eluate is collected; (4) Drying The eluate obtained in step (4) is dried to obtain a ginkgo leaf flavonoid extract; Among them, the concentration of the complexing agent solution in step (1) is 0.4% - 1.2%; The complexing agent is an electron-deficient compound containing boron element; The electron-deficient compound containing a boron element is a borate, and the boron anion group of the borate is [BO3] 3- or [BO4] 5- ; In the column chromatography elution in step (3), the ethanol concentration is an ethanol aqueous solution of 65% - 75%.

2. The method for water extraction of flavonoids from ginkgo leaves according to claim 1, characterized in that: In the boron element-containing electron-deficient compound, the borate cation is Mg 2+ , Li + , K + , Cs + , Be 2+ , Ba 2+ , Ti 4+ , Ta 5 + , Mn 6+ , Fe 3+ , Ni 3+ , Cu 2+ or more of them.

3. The method for water extraction of flavonoids from ginkgo leaves according to claim 1, characterized in that: In step (1), the complexing agent is one or more of Mg[B2O(OH)6], Li(H2O)4B(OH)4, TaBO4, Ca(BO2)2, K3B3O6, Ba3(B3O6)2, Mg2B3O5, Fe2B2O5, Mg3(BO3)2, Rb2B4O5(OH)4, K[B5O6(OH)4], Ca[B3O3(OH)5]; 4. The method for water extraction of flavonoids from ginkgo leaves according to claim 1, characterized in that: The dosage of the complexing agent solution in step (1) is 17 - 22 times the weight of the ginkgo leaf raw material; The extraction temperature in step (1) is 40 - 50 °C.

5. The method for water extraction of flavonoids from ginkgo leaves according to claim 4, characterized in that: In step (1), the complexing agent solution is extracted three times; The dosage ratio of the complexing agent is (1.6 - 2):(1.6 - 2):1, and the extraction time ratio is (1.3 - 1.8):(1 - 1.2):

1.

6. The method for water extraction of flavonoids from ginkgo leaves according to claim 4, characterized in that: In step (1), heating and stirring extraction is adopted.

7. The method for water extraction of flavonoids from ginkgo leaves according to claim 6, characterized in that: After extraction in step (1), filtration is carried out using a filter cloth, and the filtrate is obtained. The filter cloth is a 200 - 400 mesh filter cloth; The filtrate is centrifuged using a centrifuge, the centrifugation rate is 3000 - 5000 rpm, and the centrifugation time is 5 - 20 min.

8. The method for water extraction of flavonoids from ginkgo leaves according to any one of claims 1 - 7, characterized in that: In step (2), column chromatography adsorption is to adsorb the clear liquid obtained in step (1) using a D101 macroporous resin column chromatography, the loading flow rate is 1.5 - 2 times the column volume / h, and then washed with water for 2 - 4 times the column volume.

9. The method for water extraction of flavonoids from ginkgo leaves according to any one of claims 1 - 7, characterized in that: In the column chromatography elution in step (3), the ethanol concentration is an ethanol aqueous solution of 65% to 75%, and the ethanol elution amount is 1.5 to 2.5 times the column volume.

10. The method for water extraction of flavonoids from Ginkgo biloba leaves according to any one of claims 1-7, characterized in that: The drying in step (4) is to subject the eluate obtained in step (3) to vacuum drying, the drying temperature is 60 to 70 °C, and the vacuum pressure is -0.09 MPa to -0.08 MPa.

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