A method for extracting flavonoids from ginkgo leaves by water immersion

Abstract

The present invention discloses a method for extracting flavonoids from ginkgo leaves by water immersion, wherein the extraction comprises adding crushed ginkgo leaves into a complexing agent solution, stirring and extracting in an extraction tank, and filtering after extraction; the complexing agent is an electron-deficient compound containing a boron element, specifically a borate, and the boron anion group of the borate is [BO3] 3‑ or [BO4] 5‑ The extraction method adopts column chromatography adsorption and decomposition, and an ethanol solution is used as a decomposition liquid during the decomposition process, which can decompose the complexing agent and thereby obtain flavonoids. The present invention uses a complexing agent aqueous solution for extraction and an ethanol solution as a decomposition liquid during column chromatography decomposition, which can improve the low solubility of flavonoid components in water, improve the extraction rate, and significantly reduce the extraction temperature and water usage. Compared with organic solvent extraction, it reduces costs and environmental pollution, and reduces the impurity content of the extract.

Description

Technical Field

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

[0002] Flavonoids are secondary metabolites in plants. The parent nucleus of natural flavonoids often contains different substituents such as hydroxyl, methoxy, alkoxy, and isopentenyloxy groups, and their physical and chemical properties also have significant differences. Flavonoids have significant therapeutic effects in the treatment of diseases such as hypertension, diabetes, and atherosclerosis. Therefore, there is a huge demand for flavonoids in the market.

[0003] Ginkgo leaves are rich in flavonoids, but their flavonoids have poor solubility in water. Currently, the processes for extracting flavonoids from ginkgo leaves mainly include water extraction and ethanol extraction. For example, the water extraction method disclosed in the prior art, "Study on the Extraction Technology of Total Flavonoids from Ginkgo Leaves by Water Immersion", Yan Gaoying et al., "Northwest Journal of Pharmacy", November 2016, Vol. 31, No. 6, pp. 560-561, discloses that since the extraction process of ginkgo leaves mainly focuses on the use of organic solvents for extraction, but the extraction cost is high, many manufacturers have stopped production. In order to reduce costs, the solution provided is that the optimal extraction process for ginkgo leaves is 26 times the amount of water (1:10:8:8), extracted 3 times at 100°C, each time for 1 hour. The process is stable and feasible and has been applied to large-scale production; for example, the invention patent with the prior art authorization announcement number CN108703981B discloses a method for extracting ginkgo flavonoids from ginkgo leaves, which comprises drying and crushing the ginkgo leaves, and then extracting them with ethanol, and then extracting the ginkgo lactones with ethyl acetate, and then using the remaining extraction residue to extract the ginkgo lactones. The method comprises extracting the ginkgo biloba leaves with methanol, applying the ginkgo biloba leaves to an adsorption resin column, and eluting the ginkgo flavonoids. During the ethanol extraction, 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 extractions is 3-6, and the time for each extraction is 10-60 minutes. During the ethyl acetate extraction, 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 extractions is 2-6, and the time for each extraction is 10-60 minutes. During the methanol extraction step, the mass ratio of the filter residue to the methanol is 1:2-5, the extraction temperature is 50-60°C, the number of extractions is 2-6, and the time for each extraction is 10-60 minutes. For example, the invention patent with prior art authorization announcement number CN105154252B discloses a method for extracting ginkgo leaf extract, comprising the following steps: 1) drying and crushing the ginkgo leaves; 2) taking the ginkgo leaf powder, adding a sodium acetate-acetic acid buffer solution with a pH of 4.0 to 5.5, adding cellulase, and enzymatically hydrolyzing in a water bath at 50 to 60°C for 1 to 2 hours; 3) adding anhydrous ethanol to adjust the volume fraction of ethanol in the system to 50 to 80%, and performing microwave extraction in a microwave reactor, controlling the microwave power to 130 W, the microwave temperature to 78°C, and the cumulative microwave time to 4 minutes; 4) filtering, extracting the filtrate with petroleum ether to remove impurities, concentrating the water layer by rotary evaporation, and drying to obtain the extract.

[0004] From the existing perspective, improvements to the organic solvent extraction of flavonoids from Ginkgo biloba have been based on, on the one hand, the selection of organic solvents and improvements to the extraction process, and on the other hand, the introduction of other auxiliary extraction methods, such as the use of enzymes. However, the organic solvent extraction process is subject to high costs and unresolved solvent residue issues. In addition, the use of organic solvents also produces large amounts of chlorophyll, protein, and other fat-soluble impurities, which makes the subsequent purification of flavonoid components difficult. Although the water immersion extraction process avoids the use of organic solvents, the extraction temperature is 100°C and the solid-liquid ratio is 1:26. The high temperature and large amount of water used are also high, resulting in high costs.

[0005] It can be seen from the existing technology that the extraction of flavonoids from ginkgo 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 leaves by water immersion. The extraction method can significantly reduce the cost of organic solvent extraction, significantly reduce the extraction temperature while ensuring the extraction rate, and not only greatly reduce the production cost, but also is more environmentally friendly and suitable for industrial production.

[0007] One of the concepts of the present invention is to provide a method for water-immersion extraction of flavonoids from ginkgo leaves. The method solves the problem of poor water solubility of flavonoid components when extracted with water by introducing a specific chelating agent, thereby reducing the water usage and extraction temperature during the water-immersion extraction process, and significantly reducing the cost of extraction production.

[0008] Furthermore, another concept of the present invention is to provide a method for extracting flavonoids from ginkgo leaves by water immersion. The complexing agent used in the method has a complex salt that has good water solubility and is relatively stable, thereby allowing the use of lower extraction temperatures and extraction solution amounts during the extraction process, thereby ensuring 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 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 coordinate bond with the carbonyl group at the 4-position of the flavonoid, and then chelates with the hydroxyl group at the 5-position or 3-position to form a complex salt. The generated complex salt can, on the one hand, prevent the flavonoids from being oxidized, and on the other hand, promote the dissolution of the flavonoids and improve the transfer rate of the flavonoids.

[0010] Furthermore, another concept of the present invention is to provide a method for extracting flavonoids from ginkgo leaves by water immersion. The extraction method uses water as the extraction solvent, the total amount of the aqueous solution is less than 20 times the material-liquid ratio of the raw material, and the extraction temperature is 40-50°C. The use of this extraction method can not only ensure the extraction rate but also significantly reduce the extraction cost.

[0011] Furthermore, another concept of the present invention is to provide a method for extracting flavonoids from ginkgo leaves by water leaching. After water extraction, the method uses macroporous resin column chromatography for adsorption and analysis, wherein the analysis is 70% ethanol analysis. When the present invention uses macroporous resin for purification, the 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 liquid, resulting in decomplexation. Finally, after column chromatography analysis, a high-purity flavonoid extract free of complexing agent can be obtained.

[0012] Specifically, the present invention provides a method for extracting flavonoids from ginkgo leaves by water immersion, the method comprising the following steps:

[0013] (1) Extraction

[0014] The crushed 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;

[0015] (2) Column chromatography adsorption

[0016] The clear solution obtained in step (1) is loaded onto a resin chromatograph for adsorption;

[0017] (3) Column chromatography analysis

[0018] Using ethanol solution as an analytical solution to analyze the adsorption clear solution in step (2) by column chromatography, and collecting the analytical solution;

[0019] (4) Drying

[0020] The analytical solution obtained in step (4) is dried to obtain the ginkgo leaf flavonoid extract.

[0021] Wherein, the concentration of the complexing agent solution in step (1) is 0.4% to 1.2%;

[0022] Preferably, the concentration of the complexing agent solution is 0.5-1.0%.

[0023] More preferably, the concentration of the complexing agent solution is 0.6-0.8%.

[0024] The complexing agent is an electron-deficient compound.

[0025] Preferably, the complexing agent is an electron-deficient compound containing boron or aluminum.

[0026] The electron-deficient compound containing boron is a borate, and the boron anion group of the borate is [BO3] 3- or [BO4] 5- .

[0027] Furthermore, the borate cation in the electron-deficient compound containing the boron element is one or more of an alkali metal, an alkaline earth metal and a transition metal.

[0028] Furthermore, the borate cation in the electron-deficient compound containing boron is Li + , K + 、Cs + 、Be 2+ Mg 2 + 、Ba 2+ 、Ti 4+ 、Ta 5+ 、Mn 6+ 、Fe 3+ 、Ni 3+ 、Cu 2+ One or more of .

[0029] Furthermore, the electron-deficient boron-containing 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], and Ca[B3O3(OH)5].

[0030] The extraction temperature in step (1) is 40-50°C, preferably 45-50°C.

[0031] The amount of the complexing agent solution used in the extraction of step (1) is 15 to 22 times the weight of the ginkgo leaf raw material, preferably 18 to 20 times; the extraction time in step (1) is 6 to 8 hours, preferably 7 to 8 hours.

[0032] Furthermore, in the step (1), the complexing agent solution is extracted multiple times, preferably three times;

[0033] 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.

[0034] Furthermore, the extraction in step (1) is performed by mechanical stirring, preferably by heating and stirring.

[0035] In some embodiments, the extraction and filtration in step (1) are performed using filter cloth, wherein the filter cloth is a 200-400 mesh filter cloth;

[0036] Furthermore, the filtrate is centrifuged in a centrifuge at a rate of 3000-5000 rpm for 5-20 min.

[0037] In some embodiments, the column chromatography adsorption operation in step (2) is to adsorb the centrifugal supernatant using a D101 macroporous resin column chromatography at a flow rate of 1.5 to 2 column volumes / h, i.e., 1.5 to 2 column volumes per hour, followed by washing with water for 2 to 4 column volumes.

[0038] In some embodiments, the concentration of ethanol in the column chromatography analysis in step (3) is 65-75% ethanol aqueous solution, preferably 70-75% ethanol aqueous solution.

[0039] Furthermore, the amount of ethanol used for analysis is 1.5 to 2.5 times the column volume.

[0040] In some embodiments, the drying in step (4) is performed by vacuum drying, the drying temperature is 60-70° C., and the vacuum pressure is -0.08 MPa--0.09 MPa.

[0041] Compared with the prior art, the present invention uses a complexing agent aqueous solution for extraction, which greatly reduces the amount of organic solvent used compared to ethanol solution extraction, saves costs and protects the environment. However, when using ethanol solution for extraction, a large amount of chlorophyll, protein and other fat-soluble components will be extracted, which brings difficulties to the subsequent purification of ginkgo leaf flavonoids.

[0042] In addition, the present invention uses a complexing agent aqueous solution for extraction, which can improve the low solubility of flavonoid components in water compared to water extraction. While improving the extraction rate, it can significantly reduce the extraction temperature and water usage. For industrial production, it can significantly reduce energy consumption and make the production process safer and more stable.

[0043] Thirdly, the complexing agent forms a relatively stable complex with the flavonoids during the extraction process. Unlike other alkaline substances, which can act as solubilizers but may also cause structural changes in the flavonoids, the complexing agent will be absorbed by the macroporous resin during subsequent chromatographic adsorption. The flavonoids will be adsorbed on the resin due to the adsorption effect of the macroporous resin, while the complexing agent will flow out with the column liquid. This will achieve the purpose of decomposition during purification and achieve a good flavonoid extraction rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 High performance liquid chromatography test spectrum of ginkgo leaf raw material.

[0045] Figure 2 HPLC test spectrum of Ginkgo biloba flavonoids extract in Examples 1-3.

[0046] Figure 3 HPLC test spectrum of Ginkgo biloba extract residue in Examples 1-3. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0048] This embodiment provides a method for extracting flavonoids from ginkgo leaves by water immersion, the method comprising the following steps:

[0049] (1) Extraction

[0050] The crushed 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;

[0051] Wherein, the concentration of the complexing agent solution in step (1) 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%; and can be 0.4%, 0.5%, 0.6%, 0.9%, 1.2%.

[0052] The specific concentration of the complexing agent should be considered and adjusted according to the region of acquisition, storage time, humidity and other specifications of the ginkgo leaves to be extracted. The flavonoid content of ginkgo leaves of different specifications varies to a certain extent, and the flavonoid extraction effect can be ensured by adjusting the dosage of the complexing agent.

[0053] The complexing agent is an electron-deficient compound;

[0054] The complexing agent is an electron-deficient compound containing boron or aluminum.

[0055] The electron-deficient compound containing boron is a borate, and the boron anion group of the borate is [BO3] 3- or [BO4] 5- ;

[0056] The present invention creatively selects electron-deficient compounds as complexing agents, especially electron-deficient compounds containing boron elements, wherein the electronic configuration of the boron atom is 1s 2 2s 2 2p 1 The number of valence electrons is less than the number of valence orbitals. When coordinating with a positive atom, it can adopt sp3 hybridization to coordinate with three oxygen atoms, or it can adopt sp4 to coordinate with oxygen atoms.

[0057] When extracting flavonoids from ginkgo leaves, the structure of the flavonoids is mostly diketones, which have low polarity and poor solubility in water. Generally, weakly polar solvents are used to dissolve them, including ethanol, methanol, propanol, ethyl acetate, etc. The structure of ginkgo biflavonoids is generally as follows:

[0058] ;

[0059] The phenolic hydroxyl group on the benzene ring in this structure is easily oxidized, making the flavonoid unstable, thereby affecting the extraction rate of the flavonoid during the extraction process; in addition, the carbonyl group on the flavonoid 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 is used to form a coordinate bond with the carbonyl group on the 4 position of the flavonoid, and then chelates with the phenolic hydroxyl group on the 5 position or 3 position to form a complex salt. The generated complex salt can not only prevent the oxidation of flavonoid substances, but also promote solubility and help improve extraction efficiency.

[0060] Furthermore, the present invention preferably adopts the boron anion group of the borate to coordinate with four oxygen atoms in an sp3 hybrid manner to form a tetrahedral structure [BO4] 5- Anionic structure.

[0061] The present invention discovered that [BO4] 5- The anionic structure shows a more excellent extraction effect during the extraction process, and can further reduce the amount of complexing agent used during the extraction process.

[0062] The borate cation in the electron-deficient boron-containing compound is one or more of an alkali metal, an alkaline earth metal and a transition metal;

[0063] Furthermore, the borate cation in the electron-deficient compound containing boron 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;

[0064] When used as a chelating agent, it is necessary to ensure the solubility of the chelating agent compound. Generally speaking, the key chelating group is determined by the anionic group. Therefore, as long as the cation can ensure the solubility of the chelating agent, it is generally feasible to ensure the extraction of flavonoids in ginkgo leaves.

[0065] Furthermore, the preferred complexing agent of the present invention 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], and Ca[B3O3(OH)5].

[0066] More preferred are one or more of Mg[B2O(OH)6], Li(H2O)4B(OH)4, and K[B5O6(OH)4].

[0067] In some embodiments, the extraction temperature in step (1) is 40-50°C, preferably 45-50°C, and the extraction temperature can be 40°C, 45°C, 48°C, or 50°C.

[0068] After the complexing agent is used in the present invention, a coordination reaction occurs between the complexing agent and flavonoids, which can improve the solubility of the flavonoids. Therefore, the transfer and dissolution of the flavonoids can be guaranteed under normal temperature or low temperature conditions. According to the change of the solubility of the flavonoids, a lower dissolution temperature can be selected in the present invention, which can greatly reduce the extraction cost in terms of energy.

[0069] In some embodiments, the amount of the complexing agent solution in step (1) of the present invention is 17-22 times the weight of the ginkgo leaf raw material. The amount of the solution used here is based on weight, preferably 18-20 times. The amount of the complexing agent solution can be 18 times, 19 times, 20 times, or 22 times the weight of the ginkgo leaf raw material.

[0070] The extraction time in step (1) is 6-9 hours, preferably 7-8 hours. Specifically, the extraction time can be 6 hours, 7 hours, 8 hours, or 9 hours.

[0071] Furthermore, in some embodiments, the complexing agent solution in the step (1) is extracted multiple times, preferably three times;

[0072] The present invention further adopts a multiple extraction process, which can further affect the solubility balance of flavonoids after complexation with the complexing agent, facilitate 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 flavonoids to a certain extent and improve the extraction rate.

[0073] Furthermore, when the complexing agent solution is extracted three times, the ratio of the complexing agent dosage for the three extractions is (1.6-2):(1.6-2):1, and the ratio of the extraction time is (1.3-1.8):(1-1.2):1. Specifically, for the three extractions, the ratio of the complexing agent dosage is 8:8:4, or 8:7:5, or 8:8:5; and the ratio of the extraction time can be 3:2:2.

[0074] In the present invention, the number of extractions affects the extraction efficiency. Generally speaking, the complexing agent undergoes a coordination reaction with the flavonoid compound, which not only improves the stability of the flavonoid compound but also improves the solubility of the flavonoid substance. During the extraction process, the flavonoid content in the initial raw material is relatively the highest. As time goes by, the flavonoid content in the raw material decreases, and the dissolution difficulty becomes more difficult. When the flavonoid content in the complexing agent solution increases, the dissolution becomes even more difficult. Therefore, the present invention adopts multiple extractions to dissolve the flavonoids. After process adjustments, it is found that the extraction rate is most excellent when the complexing agent solution is extracted three times, the amount of the complexing agent is (1.6-2): (1.6-2): 1 of the ginkgo leaf raw material, and the extraction time is (1.3~1.8): (1~1.2): 1.

[0075] The extraction in step (1) is performed by mechanical stirring extraction, specifically heating stirring extraction.

[0076] In some embodiments, the extraction and filtration in step (1) are performed using filter cloth, and the filter cloth is 200-400 mesh; specifically, the filter cloth is 200 mesh, 300 mesh, or 400 mesh.

[0077] Generally speaking, after the extraction in step (1), large impurities need to be removed. The filtration of the present invention is to remove large particles of impurities, facilitate the subsequent column chromatography adsorption and analysis of the extract, and extract, separate and purify the flavonoids.

[0078] Furthermore, the filtrate is centrifuged in a centrifuge at a rate of 3000-5000 rpm for 5-20 min.

[0079] In some embodiments, a method for extracting flavonoids from Ginkgo biloba leaves by water immersion further comprises (2) a column chromatography adsorption step,

[0080] The specific operation is to adsorb the clear solution obtained in step (1) using D101 macroporous resin column chromatography at a flow rate of 1.5 to 2 column volumes / h, and then wash with water for 2 to 4 column volumes.

[0081] In some embodiments, a method for extracting flavonoids from Ginkgo biloba leaves by water immersion further comprises (3) column chromatography analysis,

[0082] The specific operation is to use ethanol solution as an analytical solution to analyze the adsorption clear solution of step (2) by column chromatography, and collect the analytical solution;

[0083] Wherein, the concentration of ethanol in the column chromatography analysis in step (3) is 65-75% ethanol aqueous solution, preferably 70-75% ethanol aqueous solution; specifically, it can be 65%, 70%, or 75% ethanol aqueous solution;

[0084] The ethanol analysis dosage is 1.5 to 2.5 times the column volume.

[0085] In some embodiments, a method for extracting flavonoids from Ginkgo biloba leaves through water immersion further comprises (4) drying;

[0086] The specific operation is to vacuum dry the analytical solution obtained in step (3) at a drying temperature of 60 to 70° C. and a vacuum pressure of -0.08 MPa to -0.09 MPa to obtain the ginkgo leaf flavonoid extract.

[0087] For the test of total flavonoid content, refer to the test method for total flavonol glycosides in Ginkgo biloba extract in the 2020 edition of the Chinese Pharmacopoeia. The specific implementation method is as follows:

[0088] (1) Experimental instruments: high performance liquid chromatograph (Thermo Fisher Ultimate 3000), analytical balance (Shimadzu AUW200D), microporous filter membrane (0.45), pinhole filter membrane;

[0089] (2) Reagents: methanol (chromatographic grade, analytical grade), phosphoric acid (analytical grade), hydrochloric acid (analytical grade), purified water;

[0090] (3) Test conditions:

[0091] Chromatographic column: C18, 4.6×150 mm, 5 μm (Agilent);

[0092] Detection wavelength: 370 nm, flow rate: 1.0 ml / min, column temperature: 25 °C;

[0093] Mobile phase: methanol: 0.4% phosphoric acid aqueous solution = 50:50.

[0094] (4) Preparation of reference solution: Accurately weigh appropriate amounts of quercetin, kaempferol, and isorhamnetin reference substances and dissolve them in methanol to make up the volume to obtain reference solution.

[0095] (5) Preparation of test solution: Accurately weigh an appropriate amount of the sample to be tested, add 25 ml of a mixture of methanol and 25% hydrochloric acid solution (4:1), heat and reflux in a water bath for 30 minutes, cool to room temperature, transfer to a volumetric flask and make up to volume to obtain the test solution.

[0096] (6) Determination method: Accurately pipette 10 μl of each reference and test sample into a liquid chromatograph. Record the chromatogram and calculate the peak area using the external standard method.

[0097] (7) Calculation of total flavonoid content: Calculate by peak area according to external standard method

[0098]

[0099] ;

[0100] Where: A 样 ——Peak area of the test sample; A 对 ——Peak area of reference substance;

[0101] W 对 ——Weighing amount of reference substance; W 样 ——Weighing amount of test sample;

[0102] V 样 ——Dilution multiple of the test sample; V 对 ——Dilution multiple of reference substance;

[0103] P——reference substance content (quercetin 99.1%, kaempferol 97.4%, isorhamnetin 99.1%);

[0104] The present invention will be described in detail below through specific examples.

[0105] Example 1

[0106] A method for extracting flavonoids from ginkgo leaves by water immersion comprises the following steps: first, preparing a complexing agent solution: weighing an appropriate amount of complexing agent, and preparing the complexing agent solution with water; then performing extraction, wherein the extraction comprises the following operations:

[0107] (1) Extraction: Add the chelating agent solution to the crushed ginkgo leaves, heat and stir in an extraction tank, extract at a temperature of 40-50°C, filter the extract through a 300-mesh filter cloth, and centrifuge at 4000 rpm for 10 min to obtain the supernatant.

[0108] (2) Column chromatography adsorption: The centrifugal supernatant was adsorbed on a D101 macroporous resin column chromatography at a flow rate of 1.5 to 2 column volumes / h, and then washed with water for 2 column volumes.

[0109] (3) Column chromatography analysis: Use 70% ethanol to analyze until the analytical solution is almost colorless, and analyze a total of 1.5 to 2.5 times the column volume, and collect the analytical solution.

[0110] (4) Drying: The analytical solution was evaporated to dryness in a 60°C water bath, and then transferred to a 60°C vacuum drying oven and dried under a vacuum of -0.08 MPa to -0.09 MPa to obtain the total flavonoids extract of Ginkgo biloba leaves.

[0111] Content detection: The EP-SOP-QC-05-046 (Rev1.00) detection method was used to detect the total flavonoid content of the centrifugal supernatant, centrifugal precipitate, medicinal residue and total flavonoid extract.

[0112] Specific process parameters are shown in the table below.

[0113]

[0114] From Examples 1-1 to 1-14, it can be seen that after using the complexing agent, 1. the extraction of flavonoids from ginkgo leaves can be achieved at a relatively low temperature, and the extraction effect is relatively excellent; 2. a good extraction rate can be achieved at a relatively low amount of extract (complexing agent solution), about 20 times the amount.

[0115] Further analysis shows that the extraction effects obtained in Examples 1-1, 1-2, and 1-3 show that the extraction rate increases significantly with increasing temperature. For example, when comparing Examples 1-2 and 1-3, the extraction rate increases by nearly 5%, which is a significant improvement. This indicates that the complexing agent can promote the dissolution of flavonoids on the one hand, and that increasing the temperature under certain conditions is also beneficial to the extraction. From the results of Example 1-2, the flavonoid extraction rate will also increase relatively with a slight increase in the amount of complexing agent and the temperature. However, from Example 1-14, increasing the temperature does not have a positive effect on the flavonoid extraction rate. Instead, the flavonoid extraction rate decreases. The analysis suggests that, on the one hand, the flavonoids may be unstable under high temperature conditions, resulting in a decrease in the extraction rate. On the other hand, high temperature conditions may affect the binding stability of the complexing agent and flavonoids, reduce the solubility of flavonoids, and comprehensively lead to an adverse effect on the extraction of flavonoids under high temperature conditions.

[0116] From the extraction ratio, an excellent extraction effect can be achieved when the amount of complexing agent solution is 20-21 times that of the ginkgo leaf raw material. Referring to Example 1-9, when the amount of complexing agent solution in the extract is reduced to 15 times, the extraction rate drops sharply. However, when the amount of complexing agent solution is further increased, as shown in Example 1-13, it has no obvious effect on the extraction rate of flavonoids. It may be that the soluble flavonoids in the ginkgo leaf have already dissolved, and the dissolution of flavonoids cannot be further improved based on this formula. Compared with the results of Example 1-10, it can be seen that increasing the extraction temperature under low extraction ratio conditions can increase the flavonoid extraction rate in ginkgo leaves, and the increase is relatively significant. Further combined with 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 influence is the effect of high temperature on the stability of flavonoids; and for the results of Example 1-10, it can be seen that under the conditions of lower extraction solution (complexing agent solution), the key influencing the extraction rate may be the dissolution rate of flavonoids, that is, even if the dissolution temperature is greatly increased under the conditions of lower extraction solution dosage in Example 1-10, it is still impossible to ensure that the flavonoids have a good extraction effect. At the same time, high temperature will increase the dissolution of water-soluble impurities, which will affect the final extraction purity of flavonoids.

[0117] In addition, from the analysis of the concentration of the complexing agent, as shown in Examples 1-11 and 1-12, when the concentration of the complexing agent changes, it will have an impact on the flavonoids in the ginkgo leaves. This is mainly because when the amount of the complexing agent is too small, insufficient complexation with the flavonoids may occur, thereby reducing the extraction rate. However, when the amount of the complexing agent is increased to exceed the complete complexation with the flavonoids, there is no obvious adverse effect on the extraction effect.

[0118] Furthermore, based on the extraction effects of Examples 1-3, the present invention also conducted other experiments, using electron-deficient compounds as complexing agents to extract flavonoids from ginkgo leaves. It can be seen that the electron-deficient compounds of Examples 1-4 to 1-8 can all achieve the extraction of flavonoid components from ginkgo leaves using water as a medium under low temperature conditions. Furthermore, we can also find that the [BO4] 5- Under this specific condition, the extraction effect is more excellent, which may be due to the influence of the atomic structure, resulting in a difference in the coordination effect between the complexing agent of the electron-deficient compound and the flavonoids.

[0119] From Examples 1-1 to 1-14, it can be seen that in the method for extracting flavonoids from ginkgo leaves by water immersion provided by the present invention, a better degree of complexation between the flavonoids and the complexing agent can be achieved when the concentration of the complexing agent is greater than 0.4; and a better extraction effect can be achieved when the amount of the complexing agent is greater than 16 times the mass of the ginkgo leaf raw material.

[0120] In the above experiment, the high performance liquid chromatography test spectrum of the ginkgo leaf raw material was detected by the test method provided above. Figure 1 As shown; and the extracts and the residues after extraction of Examples 1-3 were tested, wherein, Figure 2 This is the HPLC test spectrum of the Ginkgo biloba flavonoids extract in Example 1-3; Figure 3 This is the HPLC test spectrum of the Ginkgo biloba extract residue in Examples 1-3.

[0121] Example 2

[0122] Refer to the operating steps and operating parameters of Examples 1-3, the difference is that in step (1) of Example 2, heating ultrasonic extraction is performed in an extraction tank for 3 times, and the total flavonoid extraction rate obtained by testing is 74.93%.

[0123] From Example 2, it can be seen that ultrasonic extraction is not favorable after using the complexing agent solution as the extraction solution. This may be because the ultrasonic effect will prevent the complexation of the complexing agent with flavonoids, or promote the decomplexation of the already complexed flavonoids.

[0124] Example 3

[0125] Refer to the operating steps and operating parameters of Examples 1-3, the difference being that no complexing agent is used for extraction, and water is directly used as the extraction medium. The total flavonoid extraction rate obtained by testing is 51.63%.

[0126] From Example 3, it can be seen that it is almost difficult to extract flavonoids under low temperature conditions using water as a medium, the cost of industrial extraction is high, and the industrial value is low.

[0127] Example 4

[0128] Referring to the operating steps and operating parameters of Examples 1-3, the difference is that the extraction number is 2 times, the extraction amount is 11 times the amount used in the first extraction, and the extraction time is 4 hours; the second extraction amount is 10 times the amount used in the second extraction, and the extraction time is 3 hours; and the total flavonoid extraction rate obtained by the final test is 82.23%. It can be seen that the flavonoid extraction rate of the two-extraction method is lower than that of the three-extraction method. This may be due to the dissolution of flavonoids. Since flavonoid dissolution is an equilibrium dissolution, when the content of flavonoids in the solution is low, it is more conducive to the dissolution of flavonoids. When the two-extraction method is compared with the three-extraction method, although the solution is relatively larger, it is easier to reach the dissolution equilibrium, resulting in a poorer extraction effect.

[0129] It should be noted that for more extractions, such as four extractions or even more, the total extraction rate of flavonoids is not greatly affected in the subsequent extraction process. At this time, the flavonoids are in a basic dissolution state, and increasing the number of dissolutions has little effect on the extraction effect.

[0130] Although the present invention has been described in detail in the embodiments and drawings, the scope of protection of the present invention is not limited to the specific embodiments and drawings. Those skilled in the art can implement various changes and modifications herein without departing from the concept of the present invention.

Claims

1. A method for extracting flavonoids from Ginkgo biloba leaves by water immersion, characterized in that: The method comprises the following steps: (1) Extraction The crushed 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; (2) Column chromatography adsorption The clear solution obtained in step (1) is loaded onto a resin chromatograph for adsorption; (3) Column chromatography analysis Using ethanol solution as an analytical solution to analyze the adsorption clear solution in step (2) by column chromatography, and collecting the analytical solution; (4) Drying Drying the analytical solution obtained in step (3) to obtain the ginkgo biloba flavonoid extract; Wherein, the concentration of the complexing agent solution in step (1) is 0.4% to 1.2%; The concentration of ethanol in the step (3) column chromatography analysis is 65% to 75% ethanol aqueous solution; Said step (1) wherein the complexing agent is one or more of Mg[B2O(OH)6], Li(H2O)4B(OH)4, Fe2B2O5, K[B5O6(OH)4]; The extraction temperature in step (1) is 40-50°C; The extraction time in the step (1) is 6 to 8 hours; The amount of the complexing agent solution in step (1) is 17 to 22 times the weight of the ginkgo leaf raw material; In the step (1), the complexing agent solution is extracted three times.

2. The method for extracting flavonoids from Ginkgo biloba leaves by water immersion according to claim 1, wherein: In the step (1), the complexing agent solution is divided into three extractions in a dosage ratio of (1.6~2):(1.6~2):1, and the extraction time ratio is (1.3~1.8):(1~1.2):

1.

3. The method for extracting flavonoids from Ginkgo biloba leaves by water immersion as claimed in claim 2, wherein: The extraction in step (1) is performed by heating and stirring.

4. The method for extracting flavonoids from Ginkgo biloba leaves by water immersion as claimed in claim 3, wherein: After the extraction in step (1), the extraction is performed by filtering with a filter cloth to obtain a filtrate, wherein the filter cloth is a 200-400 mesh filter cloth; The filtrate is centrifuged in a centrifuge at a rate of 3000-5000 rpm for 5-20 min.

5. The method for extracting flavonoids from Ginkgo biloba leaves by water immersion according to any one of claims 1 to 4, characterized in that: The column chromatography adsorption in step (2) is to adsorb the clear liquid obtained in step (1) using D101 macroporous resin column chromatography at a flow rate of 1.5 to 2 times the column volume / h, followed by washing with water for 2 to 4 times the column volume.

6. The method for extracting flavonoids from Ginkgo biloba leaves by water immersion according to any one of claims 1 to 4, characterized in that: The amount of ethanol-water solution used in the column chromatography analysis in step (3) is 1.5 to 2.5 times the column volume.

7. The method for extracting flavonoids from Ginkgo biloba leaves by water immersion according to any one of claims 1 to 4, characterized in that: The drying step (4) is to vacuum dry the analytical solution obtained in step (3) at a drying temperature of 60 to 70° C. and a vacuum pressure of -0.09 MPa to -0.08 MPa.

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