Process for the preparation of acylated flavonoid glycoside a from ginkgo biloba leaf extract

Abstract

The application belongs to the field of Chinese herbal medicines, and relates to a method for preparing acylated flavonoid glycoside A (quercetin-3-O-[6'''-O-E-p-coumaroyl-beta-D-glucosyl-(1->2)-alpha-L-rhamnose glycoside]) from ginkgo leaf extract. The method first uses macroporous adsorption resin to preliminarily purify the ginkgo leaf extract, carries out decolorization through activated white clay, and then uses flash medium-pressure preparative chromatography and recrystallization means to separate acylated flavonoid glycoside A, so that the purity of the separated acylated flavonoid glycoside A reaches UV >= 98%, ELSD >= 98%, and the recovery rate is high, wherein the acylated flavonoid glycoside A is 70%. The method has the advantages of large preparation amount, high stability, strong repeatability, simple process steps, high purity of separated monomeric compounds, etc., and is an ideal separation process for preparing acylated flavonoid glycoside A from ginkgo leaf extract, and lays a foundation for subsequent industrial scale production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of Chinese herbal medicine, and relates to a preparation process of acylated flavonoid glycoside A (quercetin-3-O-[6'''-O-E-p-coumaroyl-β-D-glucosyl-(1→2)-α-L-rhamnopyranoside]) in ginkgo biloba extract, in particular to the separation and purification of macroporous adsorption resins, the decolorization process of activated white clay, the use of flash medium pressure preparation chromatography, recrystallization and other technical means. BACKGROUND

[0002] Ginkgo biloba L. is a gymnosperm, Ginkgoaceae, Ginkgo biloba L. single species plant, also known as duck foot, Gong Sun tree, white fruit. Ginkgo biloba first appeared in the Carboniferous period more than 300 million years ago, mainly distributed in the northern hemisphere. Due to the influence of the ice age, ginkgo biloba was on the brink of extinction, and only some areas in China were preserved, becoming one of the oldest surviving plants in the world.

[0003] Ginkgo biloba leaves and extracts contain a variety of chemical components, including flavonoids, terpenoid lactones, ginkgo acid, etc. Among them, flavonoids and terpenoid lactones are the main active ingredients of ginkgo biloba extract.

[0004] Literature research shows that quercetin-3-O-[6'''-O-E-p-coumaroyl-β-D-glucosyl-(1→2)-α-L-rhamnopyranoside] (acylated flavonoid glycoside A) is an acylated flavonoid glycoside containing coumaroyl group, and is one of the main components in ginkgo biloba extract, with a high content and rarely reported in other plants. It is a relatively "unique component" in ginkgo biloba extract, and acylated flavonoid glycoside A is contained in ginkgo biloba leaf clinical drugs prepared from ginkgo biloba extract. Modern pharmacological activity research shows that acylated flavonoid glycoside A can stimulate growth hormone secretion, and the compound rich in acylated flavonoid glycoside A has good activity, such as relieving atherosclerosis, neuroprotection, anti-tumor, anti-aging, etc. It can be seen that acylated flavonoid glycoside A has potential excellent pharmacological activity, but its preparation process has not been reported.

[0005] Macroporous adsorption resins (MAR) are a kind of high molecular adsorbent, which has no exchange group macroporous structure. This adsorbent has good macroporous network structure and large specific surface area, and can screen organic matter in aqueous solution by physical adsorption. This new type of organic polymer adsorbent began to develop in the 1960s and is widely used in the separation of effective components of Chinese herbal medicine and the development and research of new drugs.

[0006] Medium Pressure Liquid Chromatography (MPLC) is widely used in the fields of natural product chemistry, biochemistry, medicinal chemistry and organic synthesis for separation and purification. MPLC has the advantages of rapid separation and high efficiency, and can quickly prepare kilogram samples. Compared with traditional column chromatography, MPLC has a real-time detector and a flow phase driven metering pump, so the separation is more accurate and faster. Compared with high-performance liquid preparation, MPLC can handle larger sample quantities and take less time. In addition, the use of different types of fillers can further improve the selectivity of separation, so MPLC is often used for the separation and purification of natural products. It is particularly suitable for large-scale enrichment of compounds in industrial production. SUMMARY

[0007] In order to overcome the shortcomings of the prior art, the technical scheme of the present application provides a purification process for preparing acylated flavonoid glycoside A monomer from ginkgo leaf extract, which has the characteristics of simple and convenient process and strong repeatability. In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] The preparation process of acylated flavonoid glycoside A in ginkgo leaf extract includes the following steps:

[0009] (1) The macroporous adsorption resin is used for preliminary purification of the ginkgo leaf extract, and the specific steps are as follows: pretreatment of the macroporous adsorption resin, column loading; taking the ginkgo leaf extract to prepare a test solution of a certain concentration, sample loading, and elution with ethanol water;

[0010] (2) The enriched fraction containing acylated flavonoid glycoside A obtained in step (1) is subjected to decolorization with activated clay, and the specific steps are as follows: pretreatment of the decolorizing agent, standby; taking the macroporous adsorption resin enriched fraction to prepare a test solution, adding the pretreated decolorizing agent, adjusting the temperature, and decolorizing for a certain time;

[0011] (3) The decolorized fraction obtained in step (2) is subjected to enrichment and purification by Flash medium pressure preparative chromatography to obtain a crude product of acylated flavonoid glycoside A with high purity; the specific steps are as follows: preparing the decolorized fraction into a test solution, separating and recovering by using Flash medium pressure preparative chromatography.

[0012] (4) The crude acylated flavonoid glycoside A obtained in step (3) is refined by recrystallization to obtain acylated flavonoid glycoside A;

[0013] (5) The acylated flavonoid glycoside A obtained in step (4) is subjected to purity analysis by high-performance liquid chromatography.

[0014] The macroporous adsorption resin in step (1) is HPD-100 resin.

[0015] The macroporous adsorption resin pretreatment in step (1) is performed as follows: the macroporous adsorption resin is washed with warm water for 3-5 times to remove part of the pore-forming agent and crosslinking agent on the surface of the macroporous adsorption resin; 95% industrial ethanol is used to soak the macroporous adsorption resin for 24 hours, and then 95% industrial ethanol is used to elute the macroporous adsorption resin column at a flow rate of 2 BV / h (1 BV is the volume of the macroporous resin filled), until the effluent does not appear white turbidity after adding water; water is used to elute until the effluent has no alcohol taste, 2-5% hydrochloric acid solution is added to soak the macroporous adsorption resin for 2-4 hours, water is used to elute the macroporous adsorption resin column at a flow rate of 2-3 BV / h, and the effluent is washed until the pH is neutral; 2-5% sodium hydroxide solution is added to soak the macroporous adsorption resin for 2-4 hours, water is used to elute the macroporous adsorption resin column at a flow rate of 2-3 BV / h, and the effluent is washed until the pH is neutral, which is the available resin.

[0016] The test sample solution preparation method in step (1) is as follows: 25.0 mg of ginkgo leaf extract is accurately weighed and placed in a 10 mL brown volumetric flask, and then methanol is added to the calibration line, and then shaken and filtered with a 0.45 μm microporous filter, and then sealed and stored at 4°C.

[0017] The pH value of the test sample solution in step (1) is 5, which is adjusted by dilute hydrochloric acid, the sample volume is 100 BV, the sample flow rate is 6 BV / h, the sample concentration is 1.25 mg / mL, the impurity removal elution solvent is 8% ethanol water, the elution volume is 20 BV, the elution solvent is 50% ethanol water, the flow rate is 2 BV / h, and the elution volume is 6 BV.

[0018] The test sample solution preparation method in step (2) is as follows: 40 mg of macroporous adsorption resin 50% ethanol water elution fraction is accurately weighed and placed in a 10 mL volumetric flask, and then 50% ethanol water is added to the calibration line, and then shaken and used.

[0019] The decolorizing agent pretreatment in step (2) is as follows: 100 g of activated clay is accurately weighed, soaked with 95% ethanol, and then naturally settled until the supernatant is clear, and then the reagent is dried, and then placed in a 50°C oven for drying and storage.

[0020] The decolorizing agent dosage in step (2) is 0.32 g / mL, the decolorizing time is 5.8 hours, and the decolorizing temperature is 42°C.

[0021] The sample recovery in the decolorizing process in step (2) is performed by centrifugation and then filtration and rotary drying, and the centrifugation conditions are 10,000 revolutions per minute and 2 minutes.

[0022] The flash medium pressure preparative chromatography in step (3) is of the type BUCHI X2 produced by the Swiss BUCHI company, and the chromatography column type is Flash pure ID C 18(80g, 40μm), manufacturer: BUCHI, Switzerland.

[0023] Step (3) Preparation of the test sample solution: 4.0g of the decolorized sample was precisely weighed and placed in a 10mL volumetric flask, which was then diluted to the calibration line with 50% ethanol water, and the solution concentration was 400mg / mL. The dissolution process could be assisted by ultrasonic dissolution, and the solution was filtered with a 0.45μm microporous filter.

[0024] Step (3) Medium pressure preparative chromatography conditions: chromatographic column C 18 40μm irregular 80g, detection wavelength: 254nm, 315nm, single injection: 5mL, acetonitrile (A)-water was used as the mobile phase, the elution flow rate was 60mL / min, and the elution gradient was: 0-2BV, 5%-5%(A), 2-10BV, 22%-22%(A), 10-14BV, 25%-25%(A), 14-16BV, 50%-50%(A), 16-18BV, 50%-100%(A).

[0025] Step (3) Collection of the fractions: after concentration and freeze-drying, the acylated flavonoid glycoside A crude product was obtained.

[0026] Step (4) Recrystallization of the acylated flavonoid glycoside A: the acylated flavonoid glycoside A crude product was taken, and chromatographic grade methanol was added in a solution volume to sample mass ratio of 5:1, and then it was placed at room temperature for volatilization. When the solvent volume was 1 times the sample amount or a large amount of precipitate was precipitated, the upper solution was sucked out, and the remaining precipitate was repeatedly subjected to the above steps for one to two times, to obtain the refined acylated flavonoid glycoside A.

[0027] Step (5) High performance liquid chromatography purity analysis chromatography conditions of the acylated flavonoid glycoside A: chromatographic column Agilent Eclipse XDB C 18 (250mm×4.6mm, 5μm), detection wavelength: 315nm, column temperature: 25℃, injection: 10μL, mobile phase: acetonitrile-0.1% formic acid aqueous solution (20:80, V / V) for 30min, flow rate: 1.0mL / min.

[0028] The present application has the following advantages in preparing a large amount and high purity acylated flavonoid glycoside A from ginkgo leaf extract:

[0029] The application develops a process for preparing acylated flavonoid glycoside A from ginkgo biloba extract, which firstly uses macroporous adsorption resin to preliminarily purify the ginkgo biloba extract, carries out decolorization through activated white clay, and then uses Flash medium-pressure preparative chromatography and recrystallization to successfully prepare a large amount of quercetin-3-O-[6'''-O-E-p-coumaroyl-beta-D-glucosyl-(1→2)-alpha-L-rhamnose glycoside], the purity of the separated acylated flavonoid glycoside A reaches UV≥98%, ELSD≥98%, and the recovery rate is high, wherein the acylated flavonoid glycoside A is 70%; the process has the advantages of large preparation amount, high stability, strong repeatability, simple process steps, high purity of the separated monomer compound and the like, is an ideal separation process for preparing acylated flavonoid glycoside A from ginkgo biloba extract, and lays a foundation for subsequent industrial scale production.

[0030] The application uses the separation means of macroporous adsorption resin, Flash medium-pressure preparative chromatography, recrystallization and activated white clay decolorization to prepare monomer acylated flavonoid glycoside, and provides a train of thought for preparing flavonoid compounds in laboratory and industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a macroporous adsorption resin purification process flow chart of acylated flavonoid glycoside A.

[0032] Figure 2 It is a sample color comparison chart before and after decolorization of acylated flavonoid A flow.

[0033] Figure 3 It is a decolorization process flow chart of acylated flavonoid A flow.

[0034] Figure 4 Flash medium-pressure preparative chromatogram.

[0035] Figure 5 It is a Flash medium-pressure preparative chromatography purification process flow chart.

[0036] Figure 6 It is a sample color comparison chart before and after recrystallization.

[0037] Figure 7 It is a purity analysis high performance liquid chromatogram of acylated flavonoid glycoside A.

[0038] Figure 8 It is a H NMR chart of acylated flavonoid glycoside A. 1

[0039] Figure 9 It is a C NMR chart of acylated flavonoid glycoside A. 13 DETAILED EMBODIMENT

[0040] ​​The following is a specific embodiment of the present application, which is further described to illustrate the present application, but the scope of protection claimed by the present application is not limited to the following embodiments.

[0041] Example 1 Purification process of acylated flavonoid glycoside A by macroporous adsorption resin

[0042] Pretreatment of macroporous adsorption resin: The macroporous adsorption resin was washed with warm water for 3-5 times to remove part of the pore-forming agent and crosslinking agent on the surface of the macroporous adsorption resin; 95% industrial ethanol was taken to soak for 24 h, and then 95% industrial ethanol was used to elute through the macroporous resin column at a flow rate of 2 BV / h (1 BV is the volume of macroporous resin loading), until no white turbidity appeared in the effluent after adding water; water was used to elute until no alcohol taste was present in the effluent, 2-5% hydrochloric acid solution was added to soak the macroporous resin for 2-4 h, water was added to pass through the macroporous resin chromatography column at a flow rate of 2-3 BV / h, and the effluent was washed until the pH was neutral; 2-5% sodium hydroxide solution was added to soak the macroporous resin for 2-4 h, water was added to pass through the macroporous resin chromatography column at a flow rate of 2-3 BV / h, and the effluent was washed until the pH was neutral, which was the available resin.

[0043] Preparation of ginkgo leaf extract test solution: The ginkgo leaf extract was dissolved in 50% ethanol solution to prepare a raw solution with a concentration of 12.5 mg / ml, which could be accelerated by ultrasonic dissolution. After dissolution, distilled water was added to dilute the sample solution to a concentration of 1.25 mg / ml. Dilute hydrochloric acid was added to adjust the pH of the sample solution to 5.

[0044] Column loading: The pretreated macroporous adsorption resin was washed with pure water until no alcohol taste was present in the effluent before use. The loaded macroporous resin column must be evenly loaded to prevent the generation of "knots" and air bubbles. The diameter-height ratio was controlled to be less than 0.2.

[0045] Sample loading: The prepared ginkgo leaf extract solution was loaded, and the sample volume was 100 times that of the macroporous adsorption resin. The sample was loaded at a flow rate of 6 BV / h. A peristaltic pump was used to control the flow rate.

[0046] Elution: After sample loading, pure water was used for elution, and 2-4 column volumes were eluted at a flow rate of 4 BV / h until no obvious color was present in the effluent. Then, a mixed solvent of ethanol and water was used as the mobile phase to elute at a flow rate of 2 BV / h. The gradient fractions were collected. In Table 1, A is ethanol and B is water. The 50% ethanol water fraction is the acylated flavonoid glycoside A fraction.

[0047] Table 1 Gradient elution conditions:

[0048]

[0049] Fraction collection: The 50% ethanol water elution fraction was concentrated by rotary evaporation to obtain the acylated flavonoid glycoside A fraction.

[0050] The process flow chart of macroporous adsorption resin for purifying acylated flavonoid glycoside A is shown in Figure 1

[0051] Example 2: Decolorization process of acylated flavonoid glycoside A stream

[0052] Preparation of test sample solution: The stream eluted by 50% ethanol water from macroporous adsorption resin was dissolved in 50% ethanol water. The test sample solution with a concentration of 4 mg / ml was prepared.

[0053] Pre-treatment of decolorizing agent: A certain amount of activated clay was soaked in 95% ethanol and then naturally settled. The supernatant was clear, and the reagent was dried and placed in a 50°C oven for drying.

[0054] Decolorization of sample: A certain amount of decolorizing agent was mixed with the sample solution, and the decolorization conditions were as follows: the amount of decolorizing agent was 0.32 g / ml, the decolorization time was 5.8 h, and the decolorization temperature was 42°C. The test results showed that the decolorization rate was 43%.

[0055] Sample recovery: First, the solution part was taken, and the precipitated part was immersed in 50% ethanol water with a volume of one time. Slow stirring was performed, and the upper solution was taken after standing. The process was repeated once. All the solvent parts were centrifuged under the conditions of 10,000 revolutions per minute for 2 minutes, and the upper solution was taken. The precipitated part after centrifugation was immersed in 50% ethanol water with a volume of one time, slowly stirred, and the upper solution was taken after centrifugation. The solutions were combined, hot suction filtered, and rotary dried for standby.

[0056] Comparison of color before and after decolorization of acylated flavonoid glycoside A stream Figure 2

[0057] Process flow of decolorization of acylated flavonoid glycoside A stream Figure 3

[0058] Example 3: Purification by flash medium pressure chromatography

[0059] Preparation of test sample solution: 4.0 g of the sample after decolorization was accurately weighed and placed in a 10 mL volumetric flask. The solution was diluted to the calibration line with 50% ethanol water, and the concentration of the solution was 400 mg / mL. The dissolution process could be assisted by ultrasonic dissolution, and a 0.45 μm microporous filter was used for filtration.

[0060] Medium pressure chromatography conditions: A chromatographic column C 18 40 μm irregular 80 g was selected, acetonitrile-water was used as the mobile phase, the elution flow rate was 60 mL / min, the detection wavelength was 254, 315 nm, the single injection was 5 mL, and the elution gradient was shown in Table 2, A was acetonitrile, and B was water.

[0061] Table 2: Gradient elution conditions

[0062] ​​​

[0063] Fractions were collected: Figure 4 The acylated flavonoid glycoside A was obtained after concentration and lyophilization.

[0064] The flow chart of the flash chromatography purification process is shown in Figure 5 .

[0065] Example 4 Recrystallization of acylated flavonoid glycoside A

[0066] The acylated flavonoid glycoside A was obtained after concentration and lyophilization.

[0067] The color comparison of the sample before and after recrystallization is shown in Figure 6 .

[0068] The acylated flavonoid glycoside A was obtained after concentration and lyophilization.

[0069] The high performance liquid chromatogram of the purity analysis of acylated flavonoid glycoside A is shown in Figure 7 .

[0070] Example 6 Nuclear magnetic identification of acylated flavonoid glycoside A

[0071] The compound is a yellow amorphous powder (methanol), mp 177-179°C. The molecular formula is C 36 H 36 O 18 , and the molecular weight is 756. It is easily soluble in dimethyl sulfoxide, has a dark spot under UV 254 nm, shows yellow color in concentrated sulfuric acid-vanillin reaction, and is positive in hydrochloric acid-magnesium powder reaction and Molish reaction, suggesting that it is a flavonoid glycoside compound. 1 The ABX coupled hydrogen signals of the B ring of flavone are δ7.25 (1H, dd, J = 8.5 Hz, 2.0 Hz, H-6'), 7.36 (1H, d, J = 2.0 Hz, H-2') and 6.88 (1H, d, J = 8.5 Hz, H-5'). The signals of δ6.16 (1H, d, J = 2.0 Hz, H-6) and 6.31 (1H, d, J = 2.0 Hz, H-8) suggest that the A ring is 5,7-disubstituted. 13The C NMR spectrum has 36 carbon signals, among which δ178.1 is the carbonyl carbon signal, δ121.0, 116.0, 116.0, 98.6, 93.5 are five methine signals of the flavonoid aglycone, δ164.1, 161.7, 157.0, 156.8, 149.0, 145.6, 134.8, 121.3, 106.6 are nine quaternary carbon signals of the flavonoid aglycone. The above signals suggest that the compound is a 5,7,3',4'-tetra-oxygenated flavonoid. In addition, 1 The H NMR shows δ7.40 (2H, d, J = 8.5 Hz, H-2"", H-6"") and 6.70 (2H, d, J = 8.5 Hz, H-3"", H-5"") suggesting that the benzene ring is 1,4-disubstituted. δ7.44 (1H, d, J = 16.0 Hz, H-7"") and 6.22 (1H, d, J = 16.0 Hz, H-8"") suggest that there is a trans double bond. 13 The C NMR spectrum shows δ166.8 is the carbonyl carbon signal δ145.6, 114.3 is a group of olefinic carbon signals, δ160.1, 130.5 x 2, 125.4, 115.9 x 2 is a group of 1,4-disubstituted aromatic carbon signals, from the above characteristics can be concluded that the compound contains E-P-coumaroyl. The NMR spectrum also shows two sugar signals, including δ5.53 (1H, br s, Rha-1") of the end group hydrogen signal, δ101.1 of the end group carbon signal, δ0.92 (3H, d, J = 6.0 Hz) and 17.8 methyl hydrogen carbon signal, suggesting that it contains rhamnose. δ4.30 (1H, d, J = 8.0 Hz, Glc-1"') is the end group hydrogen signal and δ104.3 is the end group carbon signal, it is speculated that it contains glucose. In summary, the acylated flavonoid A isolated is determined to be quercetin-3-O-[6"-O-trans-p-coumaroyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside], the English name (quercetin-3-O-[6"-O-trans-p-coumaroyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside]).

[0072] The acylated flavonoid A of 1 H NMR as Figure 8 indicated.

[0073] The acylated flavonoid A of 13 C NMR as Figure 9 indicated.

Claims

1. A method for preparing acylated flavonoid glycoside A from ginkgo biloba leaf extract, the acylated flavonoid glycoside A being quercetin-3-O-[6'''-O-E-p-coumaroyl-β-D-glucosyl-(1→2)-α-L-rhamnopyranoside], comprising the following steps: (1) applying macroporous adsorption resin to preliminarily purify the ginkgo biloba leaf extract, the specific steps being: pretreating the macroporous adsorption resin, loading the column; taking the ginkgo biloba leaf extract to prepare a test solution a, loading, and eluting with ethanol water; (2) applying activated white clay to decolorize the enriched fraction containing the acylated flavonoid glycoside A obtained in step (1), the specific steps being: pretreating the decolorizing agent, reserving; taking the macroporous adsorption resin enriched fraction to prepare a test solution b, adding the pretreated decolorizing agent, adjusting the temperature, and decolorizing for a time; (3) applying Flash medium-pressure preparative chromatography to the decolorized fraction obtained in step (2) to enrich and purify, to obtain acylated flavonoid glycoside A crude product; the specific steps being: taking the decolorized fraction to prepare a test solution c, applying Flash medium-pressure preparative chromatography to separate, collecting components according to peaks, to obtain acylated flavonoid glycoside A crude product; (4) recrystallizing the acylated flavonoid glycoside A crude product obtained in step (3) to refine, to obtain acylated flavonoid glycoside A; The preliminary purification process parameters are that the pH value of the sample solution loaded is 5, the pH value is adjusted by dilute hydrochloric acid, the loading solution volume is 100 BV, the loading flow rate is 6 BV / h, the loading concentration is 1.25 mg / mL, the impurity-removing elution solvent is 8% ethanol water, the elution volume is 20 BV, the elution solvent is 50% ethanol water, the flow rate is 2 BV / h, and the elution volume is 6 BV; The medium-pressure preparative chromatography conditions are that the chromatographic column is C18 40 μm irregular 80 g, the detection wavelength is 254 nm and 315 nm, the single sample injection is 5 mL, acetonitrile-water is used as the mobile phase, the elution flow rate is 60 mL / min, and the elution gradient is: 0-2 BV, 5%-5% acetonitrile, 2-10 BV, 22%-22% acetonitrile, 10-14 BV, 25%-25% acetonitrile, 14-16 BV, 50%-50% acetonitrile, 16-18 BV, 50%-100% acetonitrile.

2. The method of claim 1, wherein: The macroporous adsorption resin is HPD-100 type macroporous adsorption resin.

3. The method of claim 1, wherein: The pre-treatment is performed by the following operations: the macroporous adsorption resin is washed with warm water for 3-5 times to remove part of the pore-forming agent and crosslinking agent on the surface of the macroporous adsorption resin; 95% industrial ethanol is used to soak the macroporous adsorption resin for 24 hours, and then the macroporous adsorption resin column is eluted with 95% industrial ethanol at a flow rate of 2 BV / h until no white turbidity appears in the effluent after adding water; the effluent is eluted with water until no alcohol smell is generated, 2-5% hydrochloric acid solution is added to soak the macroporous adsorption resin for 2-4 hours, and then the macroporous adsorption resin column is eluted with water at a flow rate of 2-3 BV / h until the effluent is neutral; 2-5% sodium hydroxide solution is added to soak the macroporous adsorption resin for 2-4 hours, and then the macroporous adsorption resin column is eluted with water at a flow rate of 2-3 BV / h until the effluent is neutral, which is the available resin.

4. The method of claim 1, wherein: The preparation method of the test sample solution a is as follows: 25.0 mg of ginkgo leaf extract is accurately weighed and placed in a 10 mL brown volumetric flask, which is then diluted to the calibration line with methanol, shaken uniformly, filtered with a 0.45 μm microporous filter, and sealed and stored at 4 ℃.

5. The method as claimed in claim 1, wherein: The preparation method of the test sample solution b is as follows: 40 mg of the macroporous adsorption resin 50% ethanol water elution fraction is accurately weighed and placed in a 10 mL volumetric flask, which is then diluted to the calibration line with 50% ethanol water, shaken uniformly, and used as needed; the pre-treatment method of the decolorizing agent is as follows: 100 g of activated clay is accurately weighed, soaked with 95% ethanol, and naturally settled until the supernatant is clear, the reagent is dried, and then dried in a 50 ℃ oven, and stored for use.

6. The method of claim 1, wherein: The decolorization process parameters are as follows: the dosage of the decolorizing agent is 0.32 g / mL, the decolorization time is 5.8 hours, and the decolorization temperature is 42 ℃.

7. The method of claim 1, wherein: The preparation method of the test sample solution c is as follows: 4.0 g of the decolorized sample is accurately weighed and placed in a 10 mL volumetric flask, which is then diluted to the calibration line with 50% ethanol water, and the concentration of the solution is 400 mg / mL; the dissolution process is assisted by ultrasonic, and the solution is filtered with a 0.45 μm microporous filter.

8. The method of claim 1, wherein: The recrystallization method is as follows: the crude acylated flavonoid glycoside A is taken, chromatographic grade methanol is added at a solution volume to sample mass ratio of 5:1, and the solution is left to stand at room temperature; when the solvent volume is 1 times the sample amount, the upper layer of the solution is sucked out, and the remaining precipitate is repeatedly subjected to the above steps for one to two times to obtain refined acylated flavonoid glycoside A.

9. The method as claimed in claim 1, wherein: The acylated flavonoid glycoside A separated in step (4) is subjected to purity analysis by high performance liquid chromatography, and the detection chromatographic conditions are as follows: the chromatographic column is Agilent Eclipse XDB C18, 250 mm×4.6 mm, 5 μm, the detection wavelength is 315 nm, the column temperature is 25 ℃, the sample injection is 10 μL, the mobile phase is acetonitrile-0.1% formic acid aqueous solution 20:80, V / V, 30 min, and the flow rate is 1.0 mL / min.

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