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

Abstract

The application belongs to the field of Chinese herbal medicines, and particularly relates to a method for preparing acylated flavonoid glycoside B (kaempferol-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, and then uses activated white clay to decolorize the ginkgo leaf extract. After that, the decolorized flow fraction is separated by using flash medium-pressure preparative chromatography and silica gel column chromatography, and a large amount of acylated flavonoid glycoside B is successfully prepared. The purity of the separated acylated flavonoid glycoside B reaches UV >= 98%, ELSD >= 98%, and the recovery rate is high, wherein the acylated flavonoid glycoside B is 75%. The method has the advantages of large preparation amount, high stability, strong repeatability, simple process steps, high purity of the separated monomeric compound, and the like, and is an ideal separation process, which lays a foundation for subsequent industrial scale production.

Description

TECHNICAL FIELD

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

[0002] Ginkgo biloba L. is a gymnosperm, a single-species plant of Ginkgoaceae and Ginkgo, also known as duck foot, Gong Sun tree and white fruit. Ginkgo 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 was on the brink of extinction, and only some parts of 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 acids, etc. Among them, flavonoids and terpenoid lactones are the main active ingredients of ginkgo biloba extract.

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

[0005] Macroporous adsorbing resins (MAR) are a kind of high molecular adsorbent, which has the structural characteristics of macroporous structure without exchange groups. The 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 high molecular adsorbent began to develop in the 1960s, and is now 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 B monomers 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] A method for preparing acylated flavonoid glycoside B from ginkgo leaf extract, comprising the following steps:

[0009] (1) The macroporous adsorption resin is used for the preliminary purification of the ginkgo leaf extract, and the specific steps are as follows: the macroporous adsorption resin is pretreated and columned; the ginkgo leaf extract is taken and prepared into a test solution of a certain concentration, and is loaded and eluted with ethanol water;

[0010] (2) The acylated flavonoid glycoside B enriched stream obtained in step (1) is subjected to decolorization with activated clay, and the specific steps are as follows: the decolorizing agent is pretreated and standby; the macroporous adsorption resin enriched stream is taken and prepared into a test solution, the pretreated decolorizing agent is added, the temperature is adjusted, and the time is decolorized;

[0011] (3) The decolorized stream obtained in step (2) is subjected to enrichment and purification with Flash medium pressure preparative chromatography to obtain a crude product of glycoside B with higher purity; the specific steps are as follows: the decolorized stream is prepared into a test solution, and is separated and recovered by using Flash medium pressure preparative chromatography.

[0012] (4) The acylated flavonoid glycoside B crude product obtained in step (3) is subjected to separation and purification by silica gel column chromatography to obtain acylated flavonoid glycoside B; (5) The acylated flavonoid glycoside B obtained in step (4) is subjected to purity analysis by high-performance liquid chromatography.

[0013] The macroporous adsorption resin in step (1) is HPD-100 type macroporous adsorption resin.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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 for 2 minutes.

[0021] The flash medium pressure preparation 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(80 g, 40 μm), manufacturer: BUCHI, Switzerland.

[0022] Preparation of the test solution: 4.0 g of the decolorized sample was precisely weighed into a 10 mL volumetric flask, and diluted to the calibration mark with 50% ethanol water, the concentration of the solution was 400 mg / mL. The dissolution process could be assisted by ultrasonic. The solution was filtered by 0.45 μm microporous filter membrane.

[0023] Preparation of the medium pressure chromatography: column C 18 40 μm irregular 80 g, detection wavelength was 254, 315 nm, single injection was 5 mL, acetonitrile (A) - water was used as the mobile phase, the elution flow rate was 60 mL / min, and the elution gradient was: 0-2 BV, 5%-5% (A), 2-10 BV, 22%-22% (A), 10-14 BV, 25%-25% (A), 14-16 BV, 50%-50% (A), 16-18 BV, 50%-100% (A).

[0024] Collection of the fractions: the crude products of acylated flavonoid glycosides A and B were obtained by concentration and freeze-drying after peak collection.

[0025] Preparation of the sample for the silica gel column chromatography: dry mixing was used, the crude product of acylated flavonoid glycoside B was dissolved in methanol, and the sample was adsorbed on the mixed silica gel by grinding on a water bath in a fume hood. The mixed silica gel was selected to be 100-200 mesh, and the amount of silica gel was 1-1.5 times the amount of the sample.

[0026] Column packing of the silica gel column chromatography: dry loading was used, and the mixed sample was evenly added to the top of the blank silica gel without disturbing the surface of the column bed. After the sample layer was settled, a small amount of methanol was added to wash the residual sample in the inner wall of the chromatography column. After the inner wall of the chromatography column was washed clean, a certain liquid level was reserved on the column surface, and an appropriate amount of protective silica gel was added. The blank silica gel was selected to be 200-300 mesh 10% inactivated silica gel, and the amount of the blank silica gel was 20 times the mass of the sample layer. The 10% inactivated silica gel was prepared by adding water in an amount of one-tenth of the mass of the silica gel, shaking well, and standing for 48 h. The diameter-height ratio of the glass column should be less than 0.15.

[0027] Elution of the silica gel column chromatography: after the sample was loaded, chloroform (A), methanol (B), and water (C) were selected to form a mixed solvent as the mobile phase, and the elution was performed at a flow rate of 2 BV / h. The elution gradient was: 0-18 BV, V A :V B :V C = 9:2:0.2; 18-22 BV, V A :V B :VC = 5:2:0.2; 22-26 BV, pure methanol.

[0028] The flow fraction collection in step (4) is as follows: A The flow fraction collection in step (4) is as follows: B The flow fraction collection in step (4) is as follows: C The flow fraction eluted under the gradient of = 9:2:0.2 is acylated flavonoid glycoside B, and the refined acylated flavonoid glycoside B is obtained by purifying the sample by liquid chromatography.

[0029] The purity analysis of acylated flavonoid glycoside B by high performance liquid chromatography in step (5) is as follows: the chromatographic column is Agilent Eclipse XDB C 18 (250mm x 4.6mm, 5um), the detection wavelength is 315nm, the column temperature is 25℃, the sample injection is 10uL, the mobile phase is acetonitrile-0.1% formic acid aqueous solution (22:78, V / V) for 30min, and the flow rate is 1.0mL / min.

[0030] The present application has the following advantages in preparing a large amount of acylated flavonoid glycoside B from ginkgo biloba extract:

[0031] The present application develops a process for preparing acylated flavonoid glycoside B from ginkgo biloba extract. The process first purifies ginkgo biloba extract by using macroporous adsorption resin, decolorizes the purified extract by activated white clay, and then successfully prepares a large amount of acylated flavonoid glycoside B by using Flash medium pressure preparative chromatography and silica gel column chromatography. The purity of the separated acylated flavonoid glycoside B reaches UV≥98% and ELSD≥98%, and the recovery rate is high, among which the acylated flavonoid glycoside B is 75%. The process has the advantages of large preparation amount, high stability, strong reproducibility, simple process steps, high purity of separated monomer compounds, etc., and is an ideal separation process for preparing acylated flavonoid glycoside B from ginkgo biloba extract, which lays a foundation for subsequent industrial scale production.

[0032] The present application uses macroporous adsorption resin, Flash medium pressure preparative chromatography, silica gel column chromatography separation means, and activated white clay decolorization to prepare monomer acylated flavonoid glycoside, which provides a train of thought for preparing flavonoid compounds in laboratory and industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a macroporous adsorption resin purification process flow chart of acylated flavonoid glycoside B.

[0034] Figure 2 Figure 2 is a comparison chart of sample color before and after decolorization of acylated flavonoid flow fraction.

[0035] Figure 3 Figure 3 is a decolorization process flow chart of acylated flavonoid flow fraction.

[0036] Figure 4Flash middle pressure preparative chromatogram.

[0037] Figure 5 is the Flash middle pressure preparative chromatography process flow chart of acylated flavonoid glycoside B.

[0038] Figure 6 is the high performance liquid chromatogram of acylated flavonoid glycoside B purity analysis.

[0039] Figure 7 is the H NMR chart of acylated flavonoid glycoside B. 1 H NMR chart.

[0040] Figure 8 is the C NMR chart of acylated flavonoid glycoside B. 13 C NMR chart. Embodiment

[0041] The following is a specific embodiment of the present application, which is further described in the present application, but the scope of the present application is not limited to the following embodiments.

[0042] Example 1 Macroporous adsorption resin acylated flavonoid glycoside B purification process

[0043] Pretreatment of macroporous adsorption resin: wash the macroporous adsorption resin 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; take 95% industrial ethanol to soak for 24h, then pass through the macroporous resin column with 95% industrial ethanol at a elution flow rate of 2BV / h (1BV is the macroporous resin loading volume), until the effluent does not appear white turbidity after adding water; elute with water until the effluent has no alcohol taste, add 2-5% hydrochloric acid solution to soak the macroporous resin for 2-4h, add water and pass through the macroporous resin chromatography column at a elution flow rate of 2-3BV / h, wash until the effluent pH is neutral; add 2-5% sodium hydroxide solution to soak the macroporous resin for 2-4h, add water and pass through the macroporous resin chromatography column at a elution flow rate of 2-3BV / h, wash until the effluent pH is neutral, which is the available resin.

[0044] Preparation of ginkgo leaf extract test solution: take ginkgo leaf extract, add 50% ethanol solution to dissolve, prepare a concentration of 12.5mg / ml original solution, which can be accelerated by ultrasonic dissolution. After dissolution, add distilled water to dilute the sample solution to a concentration of 1.25mg / ml. Add dilute hydrochloric acid to adjust the sample solution to pH=5.

[0045] Column loading: take the pretreated macroporous adsorption resin, wash with pure water before use until the effluent has no alcohol taste. The loaded macroporous resin column must be uniform, and the generation of "knots" and air bubbles must be prevented. The diameter-height ratio is controlled within 0.2.

[0046] Loading: The prepared solution of Ginkgo biloba extract was loaded onto the macroporous adsorption resin column at a flow rate of 6 BV / h. The flow rate was controlled by a peristaltic pump.

[0047] Elution: After loading, the column was first eluted with pure water at a flow rate of 4 BV / h until the effluent was colorless. Then, the column was eluted with a mixture of ethanol and water as the mobile phase at a flow rate of 2 BV / h. The fractions were collected. In Table 1, A represents ethanol and B represents water. The 50% ethanol aqueous fraction is the acylated flavonoid glycoside fraction.

[0048] Table 1: Gradient elution conditions

[0049]

[0050] Fraction collection: The 50% ethanol aqueous elution fraction was concentrated by rotary evaporation to obtain the acylated flavonoid glycoside B fraction.

[0051] The purification process flow chart of the macroporous adsorption resin for acylated flavonoid glycoside B is shown in Figure 1 .

[0052] Example 2: Decolorization process of acylated flavonoid B fraction

[0053] Preparation of test solution: The 50% ethanol aqueous elution fraction of the macroporous adsorption resin was dissolved in 50% ethanol water to prepare a test solution with a concentration of 4 mg / ml.

[0054] 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.

[0055] Sample decolorization: A certain amount of decolorizing agent was mixed with the sample solution. The decolorization conditions were as follows: decolorizing agent dosage was 0.32 g / ml, decolorization time was 5.8 h, and decolorization temperature was 42°C. The test results showed that the decolorization rate was 43%.

[0056] Sample recovery: First, the solution part was taken, and the precipitate 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 at 10,000 rpm for 2 minutes, and the upper solution was taken. The precipitate 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 use.

[0057] Color comparison of acylated flavonoid glycoside B fraction before and after decolorization is shown in Figure 2 .

[0058] The decolorization process flow chart of acylated flavonoid glycoside B fraction is shown in Figure 3 .

[0059] Flash prep HPLC purification of Example 3

[0060] Preparation of sample solution: 4.0 g of the decolorized sample was accurately weighed into a 10 mL volumetric flask, and diluted to the mark with 50% ethanol water, the concentration of the solution was 400 mg / mL. The sample was dissolved with the aid of ultrasonic, and filtered through a 0.45 μm microporous filter.

[0061] Preparation of sample solution: 4.0 g of the decolorized sample was accurately weighed into a 10 mL volumetric flask, and diluted to the mark with 50% ethanol water, the concentration of the solution was 400 mg / mL. The sample was dissolved with the aid of ultrasonic, and filtered through a 0.45 μm microporous filter. 18 40 μm irregular 80 g, acetonitrile-water was used as the mobile phase, the elution flow rate was 60 mL / min, the detection wavelength was 254, 315 nm, 5 mL of sample was injected at a time, and the elution gradient was as shown in Table 2, A was acetonitrile, and B was water.

[0062] Table 2 Gradient elution conditions:

[0063]

[0064] Collection of fractions: Figure 4 The components were collected according to the peaks, concentrated and lyophilized to obtain acylated flavonoid glycoside B.

[0065] The process flow chart of the Flash prep HPLC purification is shown in Figure 1. Figure 5

[0066] Example 4 Silica gel column chromatography of acylated flavonoid glycoside B

[0067] Sample mixing: dry mixing was used, the crude acylated flavonoid glycoside B was dissolved in methanol, and the sample was ground on a water bath in a fume hood in a clockwise direction, and then adsorbed on the mixing silica gel; the mixing silica gel was 100-200 mesh, and the amount of silica gel was 1-1.5 times the amount of the sample.

[0068] Column loading: dry loading was used, the mixed sample was evenly added to the top of the blank silica gel, the surface of the column bed was not disturbed, after the sample layer was settled, a small amount of methanol was added to wash the residual sample in the inner wall of the column, after the inner wall of the column was washed clean, a certain liquid level was reserved on the column surface, and an appropriate amount of protective silica gel was added; the blank silica gel was 200-300 mesh 10% inactivated silica gel, and the amount of blank silica gel was 20 times the mass of the sample layer. The 10% inactivated silica gel was prepared by adding water in an amount of 1 / 10 of the mass of the silica gel, shaking well, and standing for 48 h. The ratio of the diameter to the height of the glass column should be less than 0.15.

[0069] Elution: after the sample was loaded, a mixture of chloroform, methanol and water was used as the mobile phase, and elution was performed at a flow rate of 2 BV / h. The elution gradient is shown in Table 3, wherein A represents chloroform, B represents methanol, and C represents water.

[0070] Table 3 Gradient elution conditions​

[0071]

[0072] Fractions collected: V A :V B :V C The fraction eluted under the gradient of 9:2:0.2 was acylated flavonoid B, which was purified by HPLC, concentrated and dried to obtain purified acylated flavonoid B.

[0073] The acylated flavonoid B obtained by separation was analyzed by HPLC, and the purity UV was ≥98%, and the purity ELSD was ≥99%.

[0074] The HPLC purity analysis of acylated flavonoid B is shown in Figure 6

[0075] Example 5 Identification of acylated flavonoid B by nuclear magnetic resonance

[0076] The compound is a yellow amorphous powder (methanol), mp 177-179°C. 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, indicating that it is a flavonoid compound. The molecular formula is C 36 H 36 O 17 , and the molecular weight is 740. 1 In the H NMR spectrum, a group of aromatic ring proton signals δ7.74 (2H, d, J = 9.0 Hz, H-2', H-6') and 6.92 (2H, d, J = 9.0 Hz, H-3', H-5') indicate that the B ring of flavone is 1,4-disubstituted. δ6.32 (1H, d, J = 2.0 Hz, H-8) and 6.18 (1H, d, J = 2.0 Hz, H-6) indicate that the A ring is 5,7-disubstituted. 13 In the C NMR spectrum, there are 36 carbon signals, among which δ177.8 is the carbonyl carbon signal, δ130.7, 130.7, 115.5, 115.5, 98.8, 93.8 are six methine signals of the flavonoid aglycone, and δ164.3, 161.5, 160.2, 156.7, 156.5, 134.5, 120.5, 104.2 are the carbon signals of the substituted groups of the flavonoid aglycone. The above signals indicate that the compound is a 5,7,4'-trisubstituted flavone compound. In addition, 1 HNMR shows δ7.38 (2H, d, J = 8.5 Hz, H-2"", H-6"") and 6.70 (2H, d, J = 9.0 Hz, H-3"", H-5"") indicate that the benzene ring is 1,4-disubstituted. δ7.44 (1H, d, J = 15.5 Hz, H-7"") and 6.19 (1H, d, J = 15.5 Hz, H-8"") indicate that there is a trans double bond. 13 ​The C NMR spectrum revealed a carbonyl carbon signal at δ166.5, a group of olefinic carbon signals at δ144.8 and 114.0, and a group of 1,4-disubstituted aromatic carbon signals at δ159.9, 130.2×2, 125.1, and 115.7×2. These characteristics suggest that the compound contains an EP-coumaroyl group. The NMR spectrum also revealed two sugar signals: a terminal hydrogen signal at δ5.63 (1H, br s, Rha-1”), a terminal carbon signal at δ100.7, and methyl hydrogen carbon signals at δ0.90 (3H, d, J = 6.0 Hz) and 17.5, suggesting the presence of rhamnose. A terminal hydrogen signal at δ4.33 (1H, d, J = 8.0 Hz, Glc-1”’) and a terminal carbon signal at δ106.2 suggest the presence of glucose. Based on the above data, the isolated acylated flavonoid glycoside B is kaempferol-3-O-[6″′-OEp-coumaroyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside].

[0077] Acylated flavonoid glycoside B 1 H NMR Figure 7 shown.

[0078] Acylated flavonoid glycoside B 13 C NMR Figure 8 shown.

Claims

1. A method for preparing acylated flavonoid glycoside B from ginkgo biloba leaf extract, the acylated flavonoid glycoside B being kaempferol-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 macroporous adsorption resin-enriched fraction obtained in step (1), the specific steps being: pretreating the decolorizing agent, and 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 period of 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 B 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, and obtaining acylated flavonoid glycoside B crude product; (4) applying silica gel column chromatography to separate and purify the acylated flavonoid glycoside B crude product obtained in step (3), to obtain acylated flavonoid glycoside B; The process parameters of step (1) are that the pH value of the sample solution loaded is 5, the pH value is adjusted by dilute hydrochloric acid, the volume of the loading solution 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, and 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 cross-linking agent on the surface of the macroporous adsorption resin; 95% industrial ethanol is soaked for 24 hours, and then 95% industrial ethanol is eluted through the macroporous adsorption resin column at a flow rate of 2 BV / h until no white turbidity appears in the effluent after adding water; water is eluted until no alcohol taste appears in the effluent, 2-5% hydrochloric acid solution is added to soak the macroporous adsorption resin for 2-4 hours, water is added and eluted through the macroporous adsorption resin column 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, water is added and eluted through the macroporous adsorption resin column 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 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°C.

5. The method of claim 1, wherein: The preparation method of the test 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; 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 placed in a 50°C oven for drying and storage.

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

7. The method of claim 1, wherein: The preparation method of the test 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 solution concentration is 400 mg / mL; the dissolution process is assisted by ultrasonic dissolution, and the solution is filtered with a 0.45 μm microporous filter.

8. The method of claim 1, wherein: The sample mixing method for the silica gel column chromatography is as follows: dry mixing is adopted, the acylated flavonoid glycoside B crude product is dissolved in methanol, and the sample is adsorbed on the mixed silica gel by clockwise grinding on the water bath of a fume hood; the mixed silica gel is selected from 100-200 mesh, and the silica gel dosage is 1-1.5 times the sample amount.

9. The method of claim 1, wherein: The silica gel column chromatography loading method: dry loading, uniformly adding the mixed sample to the blank silica gel, not disturbing the column bed surface, after the sample layer is settled, adding a small amount of methanol to wash the residual sample in the column wall for several times, after the column wall is washed clean, reserving a certain liquid surface on the column surface, adding appropriate amount of protective silica gel; the blank silica gel is 200-300 mesh 10% inactivated silica gel, the blank silica gel amount is 20 times of the sample layer mass; 10% inactivated silica gel preparation: adding water with a volume of one tenth of the silica gel mass, shaking, standing for 48 h; the diameter-height ratio of the glass column should be less than 0.15; the silica gel column chromatography elution: after the sample is loaded, using chloroform A, methanol B, water C three solvents to form a mixed solvent as the mobile phase, and eluting at a flow rate of 2 BV / h; the elution gradient is: 0-18 BV, VA:VB:VC=9:2:0.2; 18-22 BV, VA:VB:VC=5:2:0.2; 22-26 BV, pure methanol; the silica gel column chromatography fraction collection: the fraction eluted under the gradient of VA:VB:VC=9:2:0.2 is acylated flavonoid glycoside B.

10. The method of claim 1, wherein: The purity of the acylated flavonoid glycoside B separated is analyzed by high performance liquid chromatography, and the 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 22:78, V / V, 30 min, and the flow rate is 1.0 mL / min.

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