CN113995776A

CN113995776A - Ginkgo leaf flavone extract, preparation method thereof and characteristic map construction method

Info

Publication number: CN113995776A
Application number: CN202010732010.XA
Authority: CN
Inventors: 何厚洪; 姚建标; 丁楠; 平夏婷; 王建方; 郑亚纯; 朱小翠
Original assignee: Zhejiang Conba Pharmaceutical Co ltd
Current assignee: Zhejiang Conba Pharmaceutical Co ltd
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2022-02-01
Anticipated expiration: 2040-07-27
Also published as: CN113995776B

Abstract

The invention provides a ginkgo biloba leaf flavone extract, a preparation method and a characteristic map construction method thereof, wherein the pharmaceutical effect components of the ginkgo biloba leaf flavone extract comprise the following components: 3-6% (w/w) of myricetin-3-O-rutinose, 3-6% (w/w) of myricetin-3-O-glucose, 8-11% (w/w) of rutin, 5-7% (w/w) of tagetenin-3-O-rutinose, 5-7% (w/w) of quercetin-3-O-rutinose, 6-9% (w/w) of kaempferol-3-O-rutinose, 6-10% (w/w) of isorhamnetin-3-O-rutinose, 13-15% (w/w) of quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), 11-15% (w/w) of kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside) w). The flavone extract is obtained by separating folium Ginkgo extract with polyamide column twice, and can be used as antioxidant in food, medicine, and health product. The invention solves the problem that the effective components of the ginkgo leaf flavone extract are unknown, reduces the quality control difficulty, and establishes the ultra-high performance liquid characteristic spectrum which has specificity on quality detection.

Description

Ginkgo leaf flavone extract, preparation method thereof and characteristic map construction method

Technical Field

The invention relates to the technical field of biotechnology and medicine, in particular to a ginkgo leaf flavone extract, a preparation method thereof and a characteristic map construction method.

Background

The ginkgo leaf extract is a natural plant medicine widely used for cardiovascular and cerebrovascular diseases, and comprises main components of flavone, lactone, organic acid and the like, wherein the flavone is one of main active substances and index substances. The flavone in semen Ginkgo has aglycone including quercetin, kaempferol, isorhamnetin, myricetin, luteolin, apigenin, eugenin, dihydrokaempferol, etc.; most of the aglycone is 3-position or 7-position glucoside, and the glycosyl mainly has monosaccharide, disaccharide and trisaccharide structures such as D-glucose, L-rhamnose, rutinose and the like. In addition, cinnamoyl-containing flavonoid glycosides, such as quercitrin-2 "- (6" -p-cinnamoyl glucosyl) (2 "- (6" -p-cinnamoyl glucosyl) quercitrin), Kaempferol-3- (6 '"-p-cinnamoyl glucosyl-rhamnoside) (Kaempferol 3- (6'" -p-cinnamoyl-rhamnoside)), which are specific components of ginkgo biloba and are considered as pharmacologically active components in ginkgo biloba. U.S. Pat. No. 5,430,220,6, 6 "Compositions and methods for the prevention and treatment of circulatory conditions" reports quercetin-2 "- (6" -p-cinnamoyl glucosyl) and kaempferol-3- (6' "-p-cinnamoyl glucosyl-rhamnoside) as platelet aggregation inhibitors for the prevention and treatment of certain cardiovascular diseases. Wherein the chemical structural formulas of quercetin-2 ' - (6 ' -p-cinnamoyl glucosyl) and kaempferol-3- (6 ' -p-cinnamoyl glucosyl-rhamnoside) are as follows:

the flavonoids show heavy metal complexing and antioxidant effects due to the phenolic hydroxyl structure of the flavonoids; reports have shown that quercetin glycoside in ginkgo can lower blood pressure, and the mechanism of the quercetin glycoside can accelerate the elimination of superoxide or hydrogen peroxide in the vasoconstriction process; other clinical research reports show that the quercetin related glycoside can enhance the functions of blood vessel function, immunity resistance, infection resistance and the like; the kaempferol-related flavonoid glycoside in the ginkgo has an anticoagulant effect through the regulation and control of a PI3K/Akt/PKB signal path. Therefore, the ginkgo leaf flavonoids have more and obvious pharmacological and medicinal effects, and the application value of improving the content of the ginkgo leaf flavonoids is very high.

The extraction method for preparing ginkgo biloba flavone by using ginkgo biloba extract (the total flavonol glycoside content is not less than 24% (w/w) and the terpene lactone content is not less than 6% (w/w)) which meets the standard of national formulary as a raw material has been reported. Zhengzhwei and the like prepare high-purity ginkgetin glycoside by using a modified macroporous resin (LSA-12S) column combined with polarity and acidity in the 'modified macroporous resin column chromatography combined with acidity and polarity' (journal of Chinese medical industry, 2018,49(9): 1283-1288), packing the column by a wet method (the diameter-height ratio is 1: 5), loading the sample by a wet method (the loading ratio is 1: 30), sequentially eluting by water and 0.1% (v/v) sodium carbonate solution (5 times of the column volume respectively), collecting 0.1% (v/v) sodium carbonate solution elution products, concentrating under reduced pressure to a proper volume, and adjusting the pH to 1-3 by using dilute hydrochloric acid. Loading the acidic product on a polyamide column (the diameter-height ratio is 1: 10, the loading ratio is 1: 30), sequentially using water and 20% (v/v) and 80% (v/v) ethanol solutions (5 times of the column volume respectively), collecting 80% (v/v) ethanol elution parts, concentrating under reduced pressure, and drying to obtain a ginkgo flavonol glycoside product with the purity of more than or equal to 80% (w/w); the content of total flavonol glycosides is 83.1 + -7.5% (w/w), and the transfer rate of total flavonol glycosides is 20.9 + -7.7%. The types and corresponding contents of the prepared ginkgo flavonoid glycosides are not disclosed.

Zhang in the research on ginkgo flavone purification and biological activity (Jiangnan university, 2010), a polyamide resin column is adopted for purifying ginkgo flavone, the loading concentration of the flavone is 1.41mg/mL, the loading speed is 2mL/min, 30% (v/v) ethanol is used as a constant composition elution solvent, and the total flavone content in the product is increased from 24% (w/w) to more than 50% (w/w); meanwhile, preparing flavonoid aglycone by an acid hydrolysis process, wherein hydrolysis temperature is 80 ℃, hydrochloric acid is 25% (v/v), and the flavonoid aglycone and methanol are prepared into a solution with the volume fraction of 1:4, and the flavonoid yield is 90% (w/w) after hydrolysis for 2h under the condition that the raw material concentration is 1 mg/mL; the liquid chromatography and LC-MS are adopted to detect the hydrolyzed sample, the result shows that the flavone mainly exists in the form of glucoside before hydrolysis, and the product after hydrolysis is identified to mainly contain quercetin, kaempferol and isorhamnetin. The method adopts acid hydrolysis to prepare flavone aglycone, the prepared components are different from alcohol extraction, and the used acid water has great pollution to the environment.

Zhang et al in "preparation of high purity ginkgetin" (Anhui agricultural science, 2011,39(2): 764-765,769) "a chromatographic column with ODS as solid phase filler to purify ginkgetin, the fractions of methanol, 0.5% (V/V) phosphoric acid, 5: 5 and 7: 3 (V: V) are target fractions, the concentration and drying temperature is 45 ℃, the ginkgetin mass percentage content is more than or equal to 90%, and the yield is 78.3%. The process adopts ODS chromatographic column to prepare ginkgetin, has high production cost, and is not suitable for industrial production. In addition, the prepared ginkgetin and the hydrolyzed flavone aglycone component thereof are not disclosed.

It can be seen that, the preparation of high purity ginkgetin is a hot point of research at present, but the research on the identification method of high content ginkgetin component is still rare, and more, the identification method of Ginkgo leaf extract component is more, such as Yi-Bing Ji et al in "Development, optimization and validation of a finger print of Ginkgo leaf extract high performance liquid chromatography fingerprint, optimization and verification" (j.chromatography.a 1066(2005): 97-104) which adopts C18 column, acetonitrile (a) -0.1% (v/v) phosphoric acid (B) as mobile phase, 0-40 min, mobile phase a of 14% → 30%, column temperature of 30 ℃, detection wavelength of 350 nm. The flavone types in the fingerprint are determined by standard substance contrast as follows: rutin, quercetin, kaempferol-3-O-beta-D-glucoside, kaempferol-3-O- (6-O- (alpha-L-rhamnosyl) -beta-D-glucosyl), and quercetin-3-O- (alpha-L-rhamnosyl).

CN 107884483A' method for measuring content of flavonoid components in folium Ginkgo and its preparation, and its application (application 2017.09.27, published 2018.04.06) comprises selecting chromatographic column with octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, 0.08% -0.12% (v/v) acetic acid-acetonitrile solution as mobile phase B, flow rate of 0.9-1.1 mL/min, detection wavelength of 355-365 nm, and column temperature of 38-42 deg.C. Elution gradient: 0-25 min, and 10% → 25% of mobile phase A; 25-40 min, and 25% → 35% of mobile phase A; 40 min-45 min, 35% → 80% of mobile phase A; 45-60 min, and 80% of mobile phase A. The flavone types in the fingerprint are as follows: quercetin-3-O- (2 ', 6 ' -alpha-L-dirhamnosyl) -beta-D-glucose, quercetin-3-O-alpha-L-rhamnose-2 ' - (6-p-coumaroyl) -beta-D-glucose-7-O-beta-D-glucose, kaempferide-3-O-alpha-L-rhamnose-2 ' - (6 ' -p-coumaroyl) -beta-D-glucoside, isorhamnetin-3-O- [2,6-di-O- (alpha-L-dirhamnosyl) ] -beta-D-glucose, beta-D-glucose and their pharmaceutically acceptable salts, Rutin, quercetin-3-O-glucoside, quercetin-3-O- (2' -beta-D-glucose) -alpha-L-rhamnoside, nicotiflorin, narcissus, isorhamnetin-3-O-beta-D-glucoside, and kaempferide-3-O-glucose rhamnoside.

CN108362809A "quality evaluation method of ginkgo leaf and its extract and preparation" (application date 2018.02.01, published date 2018.08.03) using octa-or octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase a, 0.1% (v/v) formic acid aqueous solution as mobile phase B, gradient elution: 0-20 min, and 15% → 20% of mobile phase A; 20 min-25 min, and 20% → 30% of mobile phase A; 25 min-30 min, and the mobile phase A is 30% → 35%; 30-45 min, and 35% → 40% of mobile phase A; detecting the wavelength of 210 nm-360 nm by using an ultraviolet detector; the flow rate is 0.5mL/min to 1.5mL/min, and the column temperature is kept between 20 ℃ and 45 ℃. Simultaneously quantifying quercetin-3-O- (2 ', 6 ' -di-O-alpha-L-rhamnosyl) -beta-D-glucoside, kaempferol-3-O- (2 ', 6 ' -di-O-alpha-L-rhamnosyl) -beta-D-glucoside, quercetin-3-O-rutinoside-7-O-glucoside, 3' -methyl-myricetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-beta-D-rutinoside, kaempferol-3-O-beta-D-rutinoside, kaempferol-3-O-L-glucoside, kaempferol-3-O-beta-D-rutinoside, and kaempferol-3-O-L-D-glucoside in ginkgo by high performance liquid phase method, Kaempferol 3- (2 ' -beta-D-glucoside) -alpha-L-rhamnoside, quercetin 3-O-alpha- (6 ' -p-coumaroyl-glucoside-beta-1, 4-rhamnoside), and kaempferol 3-O-alpha- (6 ' -p-coumaroyl-glucoside-beta-1, 2-rhamnoside).

The flavone component belonging to the technical scheme is not the specific flavone of ginkgo, so that the problems of identification of flavone doped with other sources in the ginkgo preparation and full reflection of the quality of the ginkgo flavone cannot be solved. Moreover, the liquid phase peak-off time is long, and the rapid detection within 16min cannot be realized. In addition, the characteristic spectrum and the construction method of the flavone extract in the ginkgo leaves are not reported, and compared with the ginkgo leaf extract, the flavone component and the content of the flavone extract in the ginkgo leaves are changed, so that the characteristic spectrum construction method of the ginkgo leaf extract can not be directly used for realizing the purpose.

Therefore, it is necessary to provide a high-purity flavone extract with definite components and content from ginkgo biloba extract, and establish a characteristic map analysis method capable of fully reflecting the quality of the high-purity flavone extract on the basis of the high-purity flavone extract, so as to identify the source of the flavone component and solve the problems of unclear effective components and difficult quality control.

Disclosure of Invention

The invention provides a high-purity flavone extract obtained from a ginkgo biloba leaf extract (meeting the national pharmacopoeia standard), which is named as ginkgo biloba leaf flavone extract, and provides a preparation method and a characteristic map construction method of the ginkgo biloba leaf flavone extract, which solve the problems of unclear effect components and difficult quality control of the ginkgo biloba leaf flavone extract.

The invention provides a ginkgo leaf flavone extract, the content of total flavone of the extract is not less than 85% (w/w), and the effective components of the extract consist of the following parts: 3-6% (w/w) of myricetin-3-O-rutinose, 3-6% (w/w) of myricetin-3-O-glucose, 8-11% (w/w) of rutin, 5-7% (w/w) of tagetenin-3-O-rutinose, 5-7% (w/w) of quercetin-3-O-rutinose, 6-9% (w/w) of kaempferol-3-O-rutinose, 6-10% (w/w) of isorhamnetin-3-O-rutinose, 13-15% (w/w) of quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), 11-15% (w/w) of kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside) w), the content ratio of quercetin-2 ' - (6 ' -p-cinnamoyl glucosyl) to rutin is 1.2-1.8, the content ratio of kaempferol-3- (6 ' -p-cinnamoyl glucosyl-rhamnoside) to rutin is 1.0-1.8, and the content ratio of quercetin-3-O-rutinose to kaempferol-3-O-rutinose is 0.6-1.15.

Preferably, the content ratio of the quercetin-2 ' - (6 ' -p-cinnamoyl glucosyl) to the rutin is 1.33-1.64, the content ratio of the kaempferol-3- (6 ' -p-cinnamoyl glucosyl-rhamnoside) to the rutin is 1.16-1.57, and the content ratio of the quercetin-3-O-rutinose to the kaempferol-3-O-rutinose is 0.75-0.91.

Further, the preparation method of the ginkgo biloba leaf flavone extract comprises the following steps:

1) dissolving folium Ginkgo extract in boiling water or ethanol solution to obtain a sample solution; further, the boiling water is boiling purified water, and the concentration of the ethanol solution is not higher than 10% (v/v), preferably, 8% (v/v) ethanol solution is used for dissolving to prepare a sample solution.

2) Passing the sample solution through a polyamide column, eluting with purified water with the volume of 3-5 times of the column volume, discarding the eluent, eluting with 0.1-0.5% (v/v) sodium carbonate solution with the volume of 5-8 times of the column volume, and collecting the sodium carbonate solution eluent; preferably, the column is eluted with 4 column volumes of purified water, the eluate is discarded and then eluted with 7 column volumes of 0.15% (v/v) sodium carbonate solution.

3) Adjusting the pH of the sodium carbonate solution eluent to 1-5 to prepare a secondary sample loading solution; preferably, the pH is adjusted to 3.

4) Passing the secondary sample solution through a polyamide column, eluting with purified water with the volume of 3-5 times of the column volume, discarding the eluent, eluting with 40% -80% (v/v) ethanol solution with the volume of 5-8 times of the column volume, and collecting the ethanol solution eluent; preferably, the column is eluted with 4 column volumes of purified water, the eluate is discarded and the column is eluted with 7 column volumes of 60% (v/v) ethanol solution.

5) Collecting ethanol solution eluate, concentrating, and drying to obtain folium Ginkgo flavone extract.

Preferably, the elution flow rate in the preparation method is 1-4 times of column volume (BV)/h;

preferably, the polyamide column in the preparation method is 10-30 meshes, 30-60 meshes and 80-100 meshes.

Further, the method for constructing the characteristic map of the ginkgo biloba leaf flavonoid extract comprises the following steps:

1) preparing a test sample: taking 5-10 mg of ginkgo leaf flavone extract, precisely weighing, placing in a10 mL measuring flask, adding 50% (v/v) methanol for dissolving, diluting to scale, filtering with a filter membrane, and taking subsequent filtrate to obtain the ginkgo leaf flavone extract;

2) preparation of control: taking a proper amount of rutin reference substance, and adding methanol to prepare a reference substance solution;

3) performing Ultra Performance Liquid Chromatography (UPLC) analysis on the test solution to obtain an UPLC characteristic spectrum, determining a common peak according to the relative retention time of the UPLC-MS method, indicating the chemical components of the characteristic peak by using UPLC-MS method, and establishing the UPLC characteristic spectrum of the ginkgo leaf flavone extract by using standard substance contrast; the conditions for the UPLC analysis were:

the chromatographic column is a Phenomenx Luna Omega (2.1X 100mm, 1.6 μm) chromatographic column with octadecylsilane chemically bonded silica as a filler.

Mass spectrum conditions: adopting a Q-TOF method, wherein the parameters of a primary mass spectrum are that the temperature of drying gas is 300-400 ℃, the flow rate of the drying gas is 10-15L/min, the pressure of an atomizer is 42-46 psig, and the pressure of a capillary tube is 4500-5500V; the secondary mass spectrum parameters are that the temperature of the drying gas is 300-400 ℃, the flow rate of the drying gas is 10-15L/min, the pressure of an atomizer is 40-50 psig, the pressure of a capillary tube is 4500-5500V, and the collision energy is 90-120V;

preferably, the first-stage mass spectrum parameters are 350 ℃ of drying gas temperature, 12L/min of drying gas flow rate, 45psig of atomizer pressure and 5000V of capillary pressure; the secondary mass spectrum parameters are 350 ℃ of drying gas temperature, 12L/min of drying gas flow rate, 45psig of atomizer pressure, 5000V of capillary pressure and 100V of collision energy.

The mobile phase is acetonitrile (A) and 0.08-0.12% (v/v) formic acid aqueous solution (B), the detection wavelength is 300-370 nm, and the column temperature is 27-33 ℃;

preferably, the mobile phase is acetonitrile (A) and 0.10% (v/v) formic acid aqueous solution (B), the detection wavelength is 360nm, and the column temperature is 30 ℃.

Gradient elution: in terms of volume fraction, as shown in table 1:

TABLE 1 mobile phase elution gradient

Further, in the step (3), the characteristic peaks are 12, wherein the peak 1 is myricetin-3-O-rutinose, and the relative retention time is 0.578; peak 2 is myricetin-3-O-glucose, the relative retention time is 0.644; peak 3 is disharmonidin with a relative retention time of 0.725; peak 4 is rutin, relative retention time 1.000; peak 5 is Tagetin-3-O-rutinose with a relative retention time of 1.083; peak 6 is quercetin-3-O-rutinose, with a relative retention time of 1.166; peak 7 is Kaempferol-3-O-rutinose, relative retention time is 1.441; peak 8 is isorhamnetin-3-O-rutinose, relative retention time is 1.490; peak 9 is quercetin, relative retention time is 1.632; peak 10 is kaempferol-3-O-glucose- (1-2) rhamnoside with a relative retention time of 2.099; peak 11 is quercetin-2 "- (6" -p-cinnamoyl glucosyl) with a relative retention time of 2.551; peak 12 was kaempferol-3- (6' "-p-cinnamoyl glucosyl-rhamnoside) with a relative retention time of 2.835.

Further, the method for identifying the ginkgo biloba leaf flavonoid extract by utilizing the constructed ginkgo biloba leaf flavonoid extract characteristic spectrum is characterized in that a ginkgo biloba leaf flavonoid extract sample is taken and operated according to the method of the invention to obtain the ginkgo biloba leaf flavonoid extract sample characteristic spectrum, the ginkgo biloba leaf flavonoid extract characteristic spectrum of the invention is analyzed by adopting traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, and the qualified product is obtained when the similarity is more than 0.9.

The invention has the following specific beneficial effects:

1. the invention provides a ginkgo flavone extract with high flavone purity, definite effect components and strong oxidation resistance. The invention discloses medicinal components in ginkgo leaf flavone extract, which comprise 3-6% (w/w) of myricetin-3-O-rutinose, 3-6% (w/w) of myricetin-3-O-glucose, 8-11% (w/w) of rutin, 5-7% (w/w) of tagetetin-3-O-rutinose, 5-7% (w/w) of quercetin-3-O-rutinose, 6-9% (w/w) of kaempferol-3-O-rutinose, 6-10% (w/w) of isorhamnetin-3-O-rutinose, 13-15% (w/w) of quercetin-2 '- (6' -p-cinnamoyl glucose), the content of kaempferol-3- (6' -p-cinnamoyl glucosyl-rhamnoside) is 11-15% (w/w). The content ratio of quercetin-2 ' - (6 ' -p-cinnamoyl glucosyl) to rutin is 1.2-1.8, the content ratio of kaempferol-3- (6 ' -p-cinnamoyl glucosyl-rhamnoside) to rutin is 1.0-1.8, the content ratio of quercetin-3-O-rutinose to kaempferol-3-O-rutinose is 0.6-1.15, and the total flavone content (determined by taking CGQ as a reference substance) is more than 85% (w/w). The invention solves the problems of low purity of flavone and unknown effective components in the ginkgo leaf flavone extract, and reduces the difficulty of quality control. Through UPLC comparative analysis, the folium Ginkgo flavone extract basically retains flavone species in its raw material folium Ginkgo extract, provides a method for refining total flavone from folium Ginkgo extract, and improves the use efficiency of folium Ginkgo flavone substances. Meanwhile, under the variety and content of the flavonol glycosides of the extract, the oxidation resistance of the extract is 1.86 times of that of a standard ginkgo leaf extract.

The flavonoid compound has antioxidant activity, and the type and content of the flavone directly influence the antioxidant activity. In the research of the ginkgo flavone components, besides 12 main characteristic peaks shown by a characteristic spectrum, a plurality of other flavones exist, so that the total flavone content is different from the total value of the 9 flavonol glycosides. Response values in the characteristic map also show that the proportion of the 9 flavonol glycosides is higher, so that the characteristic map has a better reference effect in antioxidant activity research. By combining the comparison of the total content of 9 kinds of flavonol glycosides under different processes, as shown in Table 2, the total content of ginkgo flavonol glycosides of lots 3-6 indicates that the total content of 9 kinds of flavonol glycosides of lots 4-6 are 68.37% (w/w), 68.97% (w/w), 67.31% (w/w), respectively, slightly higher than the total content of ginkgo flavonol glycosides of lot 3 of 64.14% (w/w), but the clearance rate IC of lots 4-6 is₅₀IC with value of 0.010-0.011 mg/mL, higher than batch No. 3₅₀The value was 0.008 mg/mL. It can be seen that the flavone components of run No. 3 have a synergistic effect on antioxidant function. Comparison of bound flavonol glycosides contentIt can be seen that 9 flavone components represented by ginkgetin glycosides in the ginkgo biloba leaf flavone extracts obtained in examples 1-3 have a synergistic effect on antioxidant activity in the corresponding content range, resulting in a synergistic effect.

TABLE 2 comparison of flavone extracts from Ginkgo biloba leaves under different preparation methods

Note: IC (integrated circuit)₅₀The smaller the value, the better the antioxidant activity.

2. The invention also provides a preparation method of the high-purity ginkgo flavone extract, which has the advantages of high ginkgo flavone transfer rate of more than 65.8 percent, low production cost, low raw material consumption and suitability for industrial mass production. The ginkgo biloba extract is loaded on a polyamide column and eluted by pure water and 0.1-0.50% (v/v) sodium carbonate solution, and the eluent is loaded on the polyamide column again and eluted again by pure water and 40-80% (v/v) ethanol, so that the ginkgo biloba flavone extract is obtained. Wherein the content of the total flavonol glycosides (determined by a pharmacopoeia method) is 56-63% (w/w), which is greatly improved compared with the content of the total flavonol glycosides 24% (w/w) of the ginkgo leaf extract (raw material), the transfer rate of the total flavonol glycosides in the ginkgo leaf flavone extract is 65-72%, the consumption of the raw material is reduced, and the waste of resources is reduced. The invention adopts ginkgo leaf extract, and obtains the high-purity ginkgo flavone extract by two-step polyamide column chromatography: firstly, polyamide chromatography is eluted by using a sodium carbonate solution, the flavonol glycoside molecules are dissociated and then eluted, and undissociated impurities are primarily separated; secondly, acidifying the flavonol glycoside dissociation molecule, reforming the molecular morphology, eluting after hydroxyl displacement during ethanol elution, and separating impurities with weak hydroxyl displacement capacity again; and finally concentrating the eluent to obtain the high-purity ginkgo flavone extract. The polyamide column can be subjected to alkali washing for impurity removal and acid washing for activation, and can be repeatedly used, so that the production cost is reduced.

The extract is refined by adopting LSA-12S macroporous resin column in the prior art, and the particle size is small (the particle size is 0.3 mm-0.6 mm) and has blockage phenomenon, so the only particle size is slightly usedRefining the flavone extract from ginkgo leaves by using an enlarged LSA-12 macroporous resin column (particle size 0.8 mm-1.3 mm) (example 4). The content of total flavone (determined by CGQ as reference substance) of the flavone extract obtained by LSA-12 macroporous resin column is 75.94% (w/w), which is lower than the content of total flavone of the ginkgo leaf flavone extract obtained by the invention; the flavone extract obtained by LSA-12 has a big difference from the ginkgo flavone extract obtained by the invention in myricetin-3-O-rutinoside, myricetin-3-O-glucose, rutin, quercetin-3-O-rutinoside and isorhamnetin-3-O-rutinoside through the content measurement of flavonol glycosides; in addition, on antioxidant function, the clearance rate IC of flavone extract obtained by LSA-12 macroporous resin column₅₀The value is 0.011mg/mL, which is higher than the clearance rate IC of the ginkgo biloba leaf flavone extract obtained by the invention₅₀The value is 0.007-0.008 mg/mL, and the ginkgo leaf flavone extract obtained by the method has a better antioxidant effect. The inventor finds that in the process of elution by adopting the LSA-12 macroporous resin column, the polarities of all flavones are different, and partial flavones cannot be eluted due to small polarities, so that the content of flavonol glycosides in the final extract is different, and the polyamide column can be used for separating antioxidant active ingredients better under the elution condition of the invention.

The invention optimizes the alkaline solution eluted for the first time, and discovers that the total flavone content (determined by taking CGQ as a reference substance) of the flavone extract obtained by eluting with 0.15 percent (v/v) sodium acetate solution (example 5) under the same condition is 89.82 percent (w/w), the total content of 9 flavonol glycosides is 68.97 percent (w/w) and the clearance rate IC is₅₀The value of 0.010mg/mL, compared to 0.15% (v/v) sodium carbonate (example 1) used under the same conditions in the present invention, was lower in total flavone content, total content of 9 flavonol glycosides, and antioxidant activity, wherein the content of tagetenin-3-O-rutinose, quercetin-2 "- (6" -p-cinnamoyl glucose group) was significantly reduced. The sodium acetate is adopted to replace sodium carbonate, so that the content and the transfer rate of the total flavone are influenced, and the content of the total flavone is reduced and the transfer rate is reduced because the alkalinity of the sodium acetate is lower than that of the sodium carbonate under the same concentration, and the elution capacity is weaker as the alkalinity is smaller.

A simultaneous examination of the percentage concentration of sodium carbonate revealed that the elution was carried out with a 2% (v/v) sodium carbonate solution under the same conditions (example)6) The obtained total flavone (determined by CGQ as reference substance) content is 73.64% (w/w), the total content of 9 kinds of flavonol glycosides is 67.31% (w/w), and clearance rate IC₅₀The value was 0.010mg/mL, and the total flavone content, the total content of 9 flavonol glycosides, and the antioxidant activity were lower than those of 0.15% (v/v) sodium carbonate (example 1) used under the same conditions in the present invention. Too high a sodium carbonate content results in elution of part of the impurities, resulting in a reduction of the total flavone content. According to the judgment of the embodiment 1-3, the sodium carbonate with the content of 0.1-0.5% (v/v) has specificity in the extraction process of the ginkgo biloba leaf flavone.

In addition, pilot test results show that (examples 6-7), the content and transfer rate of the total flavonoids produced in a large scale by the preparation method are similar to those of small batches; the comparison of the contents of the flavonol glycosides shows that in the batch precision investigation of the contents of the flavonol glycosides of batch numbers 8-10, the RAD is less than 3%, and the process stability is good.

3. The invention discloses a method for establishing a characteristic spectrum of a ginkgo leaf flavone extract, which realizes the rapid identification of main 12 flavonol glycosides in the ginkgo leaf flavone extract and can realize component detection within 16 min. The sample characteristic spectrum is compared with the characteristic spectrum of the invention by the traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system software, and the sample quality can be judged to be qualified if the similarity is more than 0.9.

General flavone components such as quercetin, isorhamnetin, kaempferol and the like belonging to a characteristic map of the existing ginkgo leaf extract widely exist in various plants, whether other plant components are mixed in the extract or not cannot be identified, and effective quality detection cannot be realized. The characteristic spectrum constructed by the invention separates and belongs to characteristic peaks of quercetin-2 ' - (6 ' -p-cinnamoyl glucosyl) and kaempferol-3- (6 ' -p-cinnamoyl glucosyl-rhamnoside). The cinnamoyl flavonoid glycoside is used as a platelet aggregation inhibitor for preventing and treating certain cardiovascular diseases, and belongs to pharmacodynamic active ingredients; meanwhile, the compound is a specific component of ginkgo leaves. The characteristic map constructed by the method can synchronously detect the pharmacodynamic active ingredients, and has specificity in quality detection.

4. The ginkgo biloba flavone extract has no ginkgolic acid detected, meets the requirement that the ginkgolic acid in the ginkgo biloba extract is less than 1ppm required by Chinese pharmacopoeia, is safe to apply, and can be used as an antioxidant in food and health care products.

Drawings

FIG. 1 is a characteristic map of a ginkgo biloba leaf flavonoid extract, wherein 1 is myricetin-3-O-rutinoside, 2 is myricetin-3-O-glucose, 3 is trabeculetin, 4 is rutin, 5 is tagetedin-3-O-rutinoside, 6 is quercetin-3-O-rutinoside, 7 is kaempferol-3-O-rutinoside, 8 is isorhamnetin-3-O-rutinoside, 9 is quercetin, 10 is kaempferol-3-O-glucose- (1-2) rhamnoside, 11 is quercetin-2 "- (6" -p-cinnamoyl glucose), and 12 is kaempferol-3- (6' "-p-cinnamoyl glucose-rhamnoside).

FIG. 2 shows the result of optimizing the conditions of the characteristic spectrum mobile phase of ginkgo biloba leaf flavone extract, wherein the mobile phase is acetonitrile (A) -0.12% (v/v) formic acid aqueous solution (B), the mobile phase is acetonitrile (A) -0.10% (v/v) methanol aqueous solution (B), and the mobile phase is acetonitrile (A) -0.08% (v/v) methanol aqueous solution (B). Wherein 1 is myricetin-3-O-rutinose, 2 is myricetin-3-O-glucose, 3 is carvacrol, 4 is rutin, 5 is tagetetin-3-O-rutinose, 6 is quercetin-3-O-rutinose, 7 is kaempferol-3-O-rutinose, 8 is isorhamnetin-3-O-rutinose, 9 is quercetin, 10 is kaempferol-3-O-glucose- (1-2) rhamnoside, 11 is quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), and 12 is kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside).

FIG. 3 shows the optimized results of the column temperature conditions of the characteristic spectrum of ginkgo leaf flavone extract, wherein the column temperature is 27 ℃, 30 ℃ and 33 ℃. Wherein 1 is myricetin-3-O-rutinose, 2 is myricetin-3-O-glucose, 3 is carvacrol, 4 is rutin, 5 is tagetetin-3-O-rutinose, 6 is quercetin-3-O-rutinose, 7 is kaempferol-3-O-rutinose, 8 is isorhamnetin-3-O-rutinose, 9 is quercetin, 10 is kaempferol-3-O-glucose- (1-2) rhamnoside, 11 is quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), and 12 is kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside).

FIG. 4 shows the optimized results of the chromatographic columns of the characteristic spectrum of ginkgo biloba leaf flavonoid extract, wherein the chromatographic columns include a Waters BEH Shield RP18 chromatographic column, a Waters ACQUITY UPLC HSS T3 chromatographic column, and a Phenomenx Luna Omega chromatographic column. Wherein 1 is myricetin-3-O-rutinose, 2 is myricetin-3-O-glucose, 3 is carvacrol, 4 is rutin, 5 is tagetetin-3-O-rutinose, 6 is quercetin-3-O-rutinose, 7 is kaempferol-3-O-rutinose, 8 is isorhamnetin-3-O-rutinose, 9 is quercetin, 10 is kaempferol-3-O-glucose- (1-2) rhamnoside, 11 is quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), and 12 is kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside).

FIG. 5 is a UPLC graph comparing folium Ginkgo extract and folium Ginkgo flavone extract, wherein the first is folium Ginkgo extract UPLC graph, and the second is folium Ginkgo flavone extract UPLC graph.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

Example 1: preparation method of ginkgo leaf flavone-1

1) Taking 10g of ginkgo biloba extract meeting the national pharmacopoeia standard, and adding 8% (v/v) ethanol solution to prepare 2% (v/v) loading solution;

2) passing the sample solution through a polyamide column (30-60 meshes), purifying and washing with 4 times of column volume (BV) at a flow rate of 2BV/h, discarding the eluent, eluting with 7 times of column volume 0.15% (v/v) of sodium carbonate solution at a flow rate of 2BV/h, and collecting the sodium carbonate solution eluent;

3) adjusting pH of the sodium carbonate solution eluent to 3 with hydrochloric acid to obtain a secondary sample solution;

4) passing the secondary sample solution through a polyamide column (parameters are the same as above) at a flow rate of 2BV/h, eluting with 4 times of column volume of purified water at a flow rate of 4BV/h, discarding the eluent, eluting with 7 times of column volume of 60% (v/v) ethanol at a flow rate of 4BV/h, and collecting the ethanol solution eluent;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. Set to batch number 1.

Example 2: preparation method of ginkgo biloba leaf flavone-2

1) Taking 10g of ginkgo biloba extract meeting the national pharmacopoeia standard, and adding 10% (v/v) ethanol solution to prepare 2% (v/v) loading solution;

2) passing the sample solution through a polyamide column (10-30 meshes), purifying and washing with 3 times of column volume (BV) at a flow rate of 1BV/h, discarding the eluent, eluting with 5 times of column volume 0.5% (v/v) of sodium carbonate solution at a flow rate of 1BV/h, and collecting the sodium carbonate solution eluent;

3) adjusting pH of the sodium carbonate solution eluent to 5 with hydrochloric acid to obtain a secondary sample solution;

4) passing the secondary sample solution through a polyamide column (parameters are the same as above) at a flow rate of 1BV/h, eluting with 5 times of column volume of purified water at a flow rate of 3BV/h, discarding the eluent, eluting with 5 times of column volume of 80% (v/v) ethanol at a flow rate of 1BV/h, and collecting the ethanol solution eluent;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. Set to batch number 2.

Example 3: preparation method of ginkgo biloba leaf flavone-3

1) Dissolving folium Ginkgo extract 10g in boiling water (boiling purified water), and adding water to obtain 2% (v/v) sample solution;

2) passing the sample solution through a polyamide column (80-100 meshes), purifying and washing with 5 times of column volume (BV) at a flow rate of 3BV/h, discarding the eluent at a flow rate of 4BV/h, eluting with 8 times of column volume of 0.1% (v/v) of sodium carbonate solution at a flow rate of 4BV/h, and collecting the sodium carbonate solution eluent;

3) adjusting pH of the sodium carbonate solution eluent to 1 with hydrochloric acid to prepare a secondary sample solution;

4) passing the secondary sample solution through a polyamide column (parameters are the same as above) at a flow rate of 3BV/h, eluting with 3 times of column volume of purified water at a flow rate of 1BV/h, discarding the eluent, eluting with 8 times of column volume of 40% (v/v) ethanol at a flow rate of 3BV/h, and collecting the ethanol solution eluent;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. Set to run No. 3.

Example 4: preparation method of ginkgo biloba leaf flavone-4

The influence of column chromatography packing on flavone content and transfer rate was compared on the basis of the process of example 1 by using the prior art. In the technical scheme, the LSA-12S macroporous resin column adopted in the prior art is used for replacing a polyamide column, and the LSA-12 macroporous resin column (with the particle size of 0.8-1.3 mm) with only slightly adjusted particle size is adopted because the particle size is smaller (0.3-0.6 mm) and has a blockage phenomenon.

2) passing the sample solution through LSA-12 macroporous resin (with the particle size of 0.8 mm-1.3 mm) at the flow rate of 2BV/h, purifying and washing with 4 times of column volume (BV), removing the eluent at the flow rate of 2BV/h, eluting with 7 times of column volume of 0.15% (v/v) sodium carbonate solution at the flow rate of 2BV/h, and collecting the sodium carbonate solution eluent;

4) passing the secondary sample loading solution through a polyamide column (30-60 meshes), eluting with 4 times of column volume of purified water at a flow rate of 2BV/h, discarding the eluent, eluting with 7 times of column volume of 60% (v/v) ethanol at a flow rate of 4BV/h, and collecting the ethanol solution eluent;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. Set to batch number 4.

Example 5: preparation method of ginkgo biloba leaf flavone-5

On the basis of the process of example 1, sodium acetate was used instead of sodium carbonate for elution, and the influence of the eluent on the flavone content and conversion rate was compared.

2) passing the sample solution through a polyamide column (30-60 meshes), purifying and washing with 4 times of column volume (BV) at a flow rate of 2BV/h, discarding the eluent, eluting with 7 times of column volume 0.15% (v/v) of sodium acetate solution at a flow rate of 2BV/h, and collecting the sodium acetate solution eluent;

3) adjusting pH of the sodium acetate solution eluent to 3 with hydrochloric acid to obtain a secondary sample liquid;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. Set to batch number 5.

Example 6: preparation method of ginkgo leaf flavone-6

On the basis of the process of example 1, the concentration of sodium carbonate was adjusted and the influence of the concentration of the eluent on the flavone content and the conversion rate was compared.

2) passing the sample solution through a polyamide column (30-60 meshes), purifying and washing with 4 times of column volume (BV) at a flow rate of 2BV/h, discarding the eluent, eluting with 7 times of column volume 2% (v/v) of sodium carbonate solution at a flow rate of 2BV/h, and collecting the sodium carbonate solution eluent;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. Set to batch number 6.

Example 7: preparation method of ginkgo leaf flavone-7

The mass of the ginkgo biloba extract is increased to 100g on the basis of the embodiment 1, and the process is enlarged.

1) Taking 100g of ginkgo biloba extract meeting the national pharmacopoeia standard, and adding 8% (v/v) ethanol solution to prepare 2% (v/v) loading solution;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. Set to run No. 7.

Example 8: preparation method of ginkgo biloba leaf flavone-8

Adding the mass of the ginkgo biloba extract to 6000g on the basis of the embodiment 1, and carrying out process amplification; meanwhile, 3 batches of ginkgo leaf extracts are set, and the stability of the preparation process is investigated.

1) 6000g of ginkgo biloba extract meeting the national pharmacopoeia standard is taken and added with 8 percent (v/v) ethanol solution to prepare 2 percent (v/v) sample loading solution;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain extract. The 3 batches of ginkgo biloba extract were used to obtain

lots

8, 9, 10, respectively.

Example 9: content determination of total flavone and total flavonol glycoside of ginkgo leaf flavone

1. Total Flavonoids determination (CGQ control method)

1.1 reference preparation

Taking about 20mg of quercetin-2 '- (6' -p-Cinnamoyl Glucosyl) (CGQ) reference substance, precisely weighing, placing into a 100mL measuring flask, adding 70% (v/v) ethanol solution to dissolve and dilute to scale, and shaking uniformly to obtain the final product (each 1mL contains 0.2mg of CGQ).

1.2 Standard Curve preparation

Preparation of a standard curve reference substance solutions of 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL and 1.2mL are precisely measured, and respectively placed in 10mL measuring bottles, 70% (v/v) ethanol is respectively added to 3mL, acetic acid-sodium acetate buffer solution (pH 4.5) and 0.1mol/L aluminum trichloride solution are respectively added to 2mL, shaking is carried out, 70% (v/v) ethanol is added to the scale, and shaking is carried out; the absorbance is measured at a wavelength of 270nm according to an ultraviolet-visible spectrophotometry (national pharmacopoeia 2015 edition four-part general rules 0401) test with a corresponding reagent as a blank, and a standard curve is drawn by taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate. And respectively drawing the CGQ as a standard curve of the reference substance according to the difference of the reference substance.

1.3 preparation of sample solutions

The determination method comprises weighing about 25mg, accurately weighing, placing in 50mL measuring flask, dissolving with 70% (v/v) ethanol solution, diluting to scale, and shaking.

1.4 measurement of content

Precisely measuring 0.5mL, placing in a10 mL measuring flask, measuring absorbance according to the method, reading out the concentration of the sample solution equivalent to the reference substance from the standard curve 1 and the standard curve 2 respectively, and calculating to obtain the total flavone transfer rate 1 according to the total flavone content in the raw materials.

2. Total flavonol glycoside determination (method 0512 of the four-part general rules of 2015 Chinese pharmacopoeia method)

Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.4% (v/v) phosphoric acid solution (50:50) is used as a mobile phase; the detection wavelength was 360 nm. The number of theoretical plates should not be lower than 2500 calculated from the peak of quercetin.

2.1 preparation of control 2 solution

Taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solutions containing 60 μ g of quercetin per 1 mL.

2.2 preparation of test solutions

Taking about 25mg of ginkgo biloba leaf flavone extract, precisely weighing, adding 25mL of a mixed solution of methanol-25% hydrochloric acid solution (4:1), placing in a water bath, heating and refluxing for 30min, rapidly cooling to room temperature, transferring to a 50mL measuring flask, diluting to scale with methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the ginkgo biloba leaf flavone extract.

2.3 assay

Respectively and precisely absorbing 10 mu L of reference substance solution and test substance solution, injecting into a liquid chromatograph, measuring, taking the peak area of the quercetin reference substance as a reference, respectively calculating the contents of quercetin, kaempferide and isorhamnetin according to the corresponding correction factors in the following table, and determining the peak positions of quercetin, kaempferide and isorhamnetin according to the relative retention time of the chromatographic peak of the component to be measured and the chromatographic peak of quercetin, wherein the relative retention time is within +/-5% of a specified value (if the relative retention time deviates by more than 5%, the corresponding substituted reference substance is confirmed as the reference), thus obtaining the target product. The relative retention times and correction factors (F) are given in the following table:

the total flavonol glycoside content (quercetin content + kaempferol content + isorhamnetin content) × 2.51. The total flavone transfer rate 2 is calculated according to the total flavone content in the raw materials.

2. And (3) test results: the test results of the total flavone content and transfer rate in ginkgo biloba leaf flavone are shown in table 3.

TABLE 3 Total Flavonoids content and transfer Rate

Test results show that the CGQ is used as a reference substance, the total flavone content in the ginkgo biloba leaf flavone of batch number 1 is 92.98% (w/w), the transfer rate is 72.31%, and the best result is obtained by calculation; the total flavone content and transfer rate in

run

2 and 3 were slightly lower than in run 1. The total flavone content obtained in the embodiments 1 to 3 is 85 to 93 percent (w/w), and the transfer rate is 65 to 73 percent. The content of total flavonol glycosides obtained by the pharmacopoeia method is smaller than that obtained by the CGQ method, but the trend is the same as that obtained by the CGQ method. The transfer rate is a ratio, so that the final result is similar to that of the standard product and the corresponding standard curve.

CGQ (molecular weight 756.7) was set by the United States Pharmacopeia (USP) as the average molecular weight of the ginkgo biloba extract flavonoids, and the chinese pharmacopeia, the united states pharmacopeia and the european pharmacopeia all use total flavonol glycosides as mass standards, which are expressed by the formula of total flavonol glycosides (quercetin + isorhamnetin + kaempferol) × average correction factor (2.51) or respective correction factors (quercetin 2.504, isorhamnetin 2.437, kaempferol 2.588), i.e., flavonol aglycones are converted into CGQ content. In terms of detection method, the quality of flavonol aglycone in ginkgo can be evaluated by converting the three aglycones according to the above multi-national pharmacopoeia, but other flavonol aglycones (such as myricetin, dihydrokaempferol and the like) cannot be detected, and the representativeness of total flavone is limited. It has also been reported that ultraviolet visible spectrophotometer is used for detection, total flavone content in related ginkgo preparation is determined by aluminium trichloride color development and rutin as contrast, but rutin has poor representativeness and can not reflect flavone content really. And CGQ is used as a representative substance with the strongest activity and higher content as a unique component of ginkgo. Therefore, CGQ can be selected as a reference, and the total flavone content can be better evaluated by detecting the total flavone of ginkgo biloba.

The total flavone content of batch 4 is 75.94% (w/w), the transfer rate is 67.67%, and is obviously lower than that of batch 1 ginkgo leaf flavone, which shows that the total flavone content and the transfer rate of the polyamide column under the process condition are obviously higher than those of LSA-12 macroporous resin column, and the polyamide adsorption belongs to hydrogen bond adsorption and is mainly related to the hydrogen bond association capacity; the macroporous resin adsorption is polar adsorption which is mainly related to the polarity of substances. The flavonoid compound contains a phenolic hydroxyl structure, can selectively destroy the phenolic hydroxyl molecular state to influence the adsorption state in the desorption process of sodium carbonate in the polyamide column, and the macroporous resin has low selectivity on the flavonoid, so that impurities can be eluted. And purifying the flavone in the ginkgo leaves by adopting a polyamide column to obtain an optimized result.

Calculated with CGQ as a control, the total flavone content of run No. 5 was 89.82% (w/w), the transfer rate was 47.63%, the total flavone content was slightly lower than 92.98% (w/w) of run No. 1, and the transfer rate was significantly lower than 72.31% of run No. 1. The sodium acetate is adopted to replace sodium carbonate, so that the content and the transfer rate of the total flavone are influenced, and the content of the total flavone is reduced and the transfer rate is reduced because the alkalinity of the sodium acetate is lower than that of the sodium carbonate under the same concentration, and the elution capacity is weaker as the alkalinity is smaller.

Calculated with CGQ as a reference, the total flavone content of run No. 6 was 73.64% (w/w), the transfer rate was 69.70%, and the total flavone content and transfer rate were significantly lower than those of run No. 1. Too high a sodium carbonate concentration causes some of the impurities to be eluted, resulting in a reduction in the total flavone content. Therefore, the sodium carbonate with the concentration of 0.1-0.5% (v/v) has specificity in the extraction process of the ginkgetin.

Calculated with CGQ as a reference, the total flavone content of run No. 7 was 89.43% (w/w), the transfer rate was 66.19%, and the total flavone content and transfer rate were slightly lower than those of run No. 1, which were similar to runs Nos. 2 and 3. Therefore, the content of the total flavone and the transfer rate are not greatly different under the pilot test condition of the corresponding process of example 1.

The total flavone content of the batches 8-10 is respectively 90.33% (w/w), 88.43% (w/w) and 89.58% (w/w), and the transfer rates are respectively 67.46%, 66.91% and 66.34%, calculated by taking CGQ as a reference substance. Under the same process conditions, RAD of the total flavone content and the transfer rate obtained after process amplification are respectively 1.34% and 2.64%, and the RAD has no obvious difference compared with batch No. 1 and batch No. 7 of pilot amplification, which shows that the total flavone content and the transfer rate of the large-scale production of the preparation method are similar to those of a small batch, and the preparation method can be used for industrial production. The details are shown in Table 4.

Table 4 comparison of Total Flavonoids content and transfer Rate of batches No. 1, 7 to 10

Example 10: determination of flavone content in ginkgo leaf flavone extract

1. UPLC test method

1) Preparation of control solutions: uses myricetin-3-O-rutinose, myricetin-3-O-glucose, rutin, marigold-3-O-rutinose, quercetin-3-O-rutinose, kaempferol-3-O-rutinose, isorhamnetin-3-O-rutinose, quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside) as reference.

2) Preparation of a test solution: taking 10mg of each extract of batch numbers 1-10, accurately weighing, placing in a10 mL measuring flask, adding 50% (v/v) methanol for dissolving and diluting to scale, filtering, and taking the subsequent filtrate to obtain the final product.

3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filler (Phenomenx Luna Omega column is 10cm long, the inner diameter is 2.1mm, and the particle size is 1.6 mu m); acetonitrile is taken as a mobile phase A, 0.1 percent (v/v) formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the table 1; the flow rate is 0.4 mL/min; the column temperature is 30 ℃; the detection wavelength was 300 nm. Calculating the content of each flavone.

TABLE 1 mobile phase elution gradient

2. Test results

The results of measuring the flavone content of the extracts of lots 1 to 10 are shown in Table 5.

TABLE 5 Flavoneglycoside content of extracts of lots 1-10

Test results show that the extracts of the batches 1-3 and 7-10 contain 3-6% (w/w) of myricetin-3-O-rutinose, 3-6% (w/w) of myricetin-3-O-glucose, 8-11% (w/w) of rutin, 5-7% (w/w) of tagetein-3-O-rutinose, 5-7% (w/w) of quercetin-3-O-rutinose, 6-9% (w/w) of kaempferol-3-O-rutinose, 6-10% (w/w) of isorhamnetin-3-O-rutinose, 13-15% (w/w) of quercetin-2 '- (6' -p-cinnamoyl glucose), 11-15% (w/w) of kaempferol-3- (6' -p-cinnamoyl glucosyl-rhamnoside). The contents of 9 flavonol glycosides of runs No. 4-6 do not all fall within the above ranges.

The total of the 9 flavonol glycoside contents for run No. 1 was 85.14% (w/w) maximum and the total of the flavonol glycoside contents for run No. 3 was 64.14% (w/w) minimum compared to run No. 2 and 3. Compared with the types of the flavonol glycosides, the process variation corresponds to the fluctuation of the content of each flavonol glycoside, but the fluctuation range is +/-2 percent, and the preparation process of the example 1 is the optimal process.

The total content of the 9 flavonol glycosides of batch No. 4 was 68.37% (w/w), wherein myricetin-3-O-rutinose 0.62%, (w/w) myricetin-3-O-glucose 0.95% (w/w), rutin 6.23% (w/w) was significantly reduced compared to myricetin-3-O-rutinose 5.56% (w/w), myricetin-3-O-glucose 6.21% (w/w), and rutin 11.39% (w/w) of batch No. 1; Quercetin-3-O-rutinose 9.76% (w/w), higher than Quercetin-3-O-rutinose 6.60% (w/w) of batch No. 1. The reason is that the LSA-12 macroporous resin column adopted in example 4 is different from the polyamide column used in example 1 in terms of filler, so that the adsorption effect on different flavonol glycosides in the elution process is different, resulting in the difference of the content of the flavonol glycosides in the final extract.

The total of the 9 flavonol glycoside contents of run No. 5 was 68.97% (w/w), with 3.38% (w/w) of tagetetin-3-O-rutinose, 8.99% (w/w) of quercitrin-2 "- (6" -p-cinnamoyl glucosyl) being significantly reduced compared to the corresponding flavonol glycoside contents of run No. 1. Example 5 sodium acetate was used instead of sodium carbonate for elution, and sodium acetate at the same concentration was less basic than sodium carbonate, and the less basic, the weaker the elution ability, and the relatively reduced total flavonol glycoside content. Furthermore, sodium acetate differs from sodium carbonate in the type of base, resulting in a difference in the content of flavonols of the different types.

The total of the 9 flavonol glycosides content of run No. 6 was 67.31% (w/w), and the flavonol glycoside content was reduced compared to run No. 1. The reason is that batch No. 6 was eluted with 2% (v/v) sodium carbonate, and the content of sodium carbonate was so high that a part of impurities was eluted, resulting in a decrease in the content of each flavonol glycoside. Therefore, the concentration range of the sodium carbonate directly influences the content of each ginkgetin glycoside, the fluctuation range of the content of the ginkgetin glycoside is +/-2 percent when the concentration of the sodium carbonate is 0.1-0.5 percent (v/v), and the quality is stable.

Runs 7-10 of the extract are pilot runs of the process of example 1, run 7 is a process scale-up of 100g of ginkgo biloba extract, and runs 8-10 are a process scale-up of 6000g of ginkgo biloba extract. Batches 7 and 8-10 had similar levels of 9 flavonol glycosides, but were slightly less than batch 1, probably due to some loss of content in process scale-up. According to comparison of the contents of the flavonol glycosides, the content of RAD is less than 3% and the process stability is good in precision investigation among 9 flavonol glycoside contents of batches 8-10. The corresponding process in example 1 is stable, but a certain content loss exists in the process amplification, and if the content of each flavonol glycoside needs to be further improved, the process parameters need to be further optimized. Table 6 shows the inter-batch precision examination of flavonol glycosides between batches 8 to 10.

TABLE 6 precision investigation between lots of 8-10 folium Ginkgo flavone extract

Example 11: research on Oxidation resistance

1. Test method

In order to evaluate the antioxidation effect of the flavone extract of batch No. 1-10, the antioxidation effect of the flavone extract is compared with the antioxidation effect of the ginkgo leaf extract meeting the national pharmacopoeia standard, and the efficacy of the flavone extract in the antioxidation effect is determined by taking rutin as a positive control.

1) Preparing a1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution: taking about 25mg of DPPH, accurately weighing, placing in a 250mL measuring flask, adding ethanol to dissolve and dilute to a scale, and shaking up to obtain DPPH solution (0.1 mg in each 1 mL).

2) Preparing a test sample mother solution: taking rutin, ginkgo leaf extract and batch number 1-10 extract about 10mg, precisely weighing, placing into a 25mL measuring flask, adding ethanol to dissolve and dilute to scale, and shaking up to obtain rutin, ginkgo leaf extract and ginkgo leaf flavone extract mother liquor to be tested.

3) And (3) measuring absorbance: precisely measuring a test sample mother solution, diluting the test sample mother solution by using ethanol to prepare solutions with the concentrations of about 0, 0.018mg/mL, 0.035mg/mL, 0.070mg/mL, 0.139mg/mL, 0.278mg/mL and 0.557mg/mL, adding 2mL of solution and 4mL of DPPH solution in each portion in parallel by 3 portions, and shaking up. And (3) determining the absorbance at 519nm wavelength by using ethanol as a negative control through an ultraviolet-visible spectrophotometry.

A₀: blank control solution absorbance;

a: absorbance of the sample.

Calculate clearance and calculate clearance IC by SPSS (17.0) software₅₀，IC₅₀The smaller the value, the better the oxidation resistance.

2. Test results

The test results are shown in Table 7. The results show that the ICs of lots 1-3, 7-8₅₀The value was 0.007-0.008 mg/mL, where IC of run No. 1₅₀The value is the smallest and preferred. IC of lot number 4-6₅₀The value is 0.010-0.011 mg/mL.

A comparison of the ginkgo flavonol glycosides contents of batches 3 to 6 in Table 4 shows that the total amount of the 9 ginkgo flavonol glycosides of batches 4 to 6 is 68.37% (w/w), 68.97% (w/w) and 67.31% (w/w), respectively, which are slightly higher than the total amount of the 9 ginkgo flavonol glycosides of batch 3 by 64.14% (w/w), but the IC of batches 4 to 6 is₅₀IC with value of 0.010-0.011 mg/mL, higher than batch No. 3₅₀The value was 0.008mg/mL, and there was a synergistic effect of the 9 flavone components of batch No. 3 on antioxidant function.

The comparison of the total flavone content and the flavonol glycoside content is combined, and 9 flavone components represented by ginkgo flavonol glycosides in the ginkgo leaf flavone extracts obtained in the examples 1 to 3 have the coordination function of the antioxidant function under the corresponding content range, so that the synergistic result is generated.

In addition, the folium Ginkgo flavone extract has higher antioxidant effect than folium Ginkgo extract, and can be used as antioxidant, and can reduce usage amount.

TABLE 7 clearance IC₅₀

Note: delta indicates different batch number samples and rutin IC₅₀Value comparison p<0.05; delta indicates the samples of different batches and rutin IC₅₀Value comparison p<0.01; # denotes samples of different lot numbers and Ginkgo biloba extract IC₅₀Value comparison p<0.05; # indicates different lot number samples and Ginkgo biloba extract IC₅₀Value comparison p<0.01。

Example 12: feature map construction

1. Test method

1.1 instruments and reagents

1.1.1 instruments

Waters I-Class UPLC ultra-high performance liquid chromatograph; phenomenx Luna Omega (2.1 × 100mm, 1.6 μm) chromatographic column, and the number of theoretical plates is not less than 2500 in terms of rutin peak; MSA 225S-1CE-Du electronic analytical balance; Milli-QA10 water purifier.

1.1.2 test article

15 batches of ginkgo biloba extract meeting the national pharmacopoeia standard are taken, 5 batches of ginkgo biloba flavone extract batch numbers 1 are obtained by adopting the preparation process of the embodiment 1, 5 batches of ginkgo biloba extract batch numbers 2 are obtained by adopting the preparation of the embodiment 2, and 5 batches of ginkgo biloba flavone extract batch numbers 3 are obtained by adopting the preparation of the embodiment 3.

1.2 assay method

1.2.1 chromatographic conditions

Phenomenx Luna Omega (2.1 × 100mm, 1.6 μm) chromatographic column using octadecylsilane chemically bonded silica as filler; acetonitrile was used as mobile phase A, 0.1% (v/v) formic acid solution was used as mobile phase B, and gradient elution was carried out as specified in Table 1 at a flow rate of 0.4mL/min, a column temperature of 30 ℃ and a detection wavelength of 360 nm.

TABLE 1 elution gradient

1.2.2 Mass Spectrometry conditions

Adopting a Q-TOF method, wherein the parameters of a primary mass spectrum are that the temperature of a drying gas is 350 ℃, the flow rate of the drying gas is 12L/min, the pressure of an atomizer is 45psig, and the pressure of a capillary tube is 5000V; the secondary mass spectrum parameters are 350 ℃ of drying gas temperature, 12L/min of drying gas flow rate, 45psig of atomizer pressure, 5000V of capillary pressure and 100V of collision energy.

1.2.3 preparation of test solutions

Taking 10mg of each of the 1.1.2 reagent ginkgo leaf flavone extracts, precisely weighing, placing into a10 mL measuring flask, adding 50% (v/v) methanol for dissolving, diluting to scale, filtering with a filter membrane, and taking the subsequent filtrate to obtain the final product.

1.2.4 preparation of control solutions

Taking a proper amount of rutin reference substance, precisely weighing, and adding 50% (v/v) methanol to obtain a solution containing 0.5mg per 1 mL.

1.2.5 feature map construction

Respectively and precisely absorbing the test solution and the reference solution, injecting into a high performance liquid chromatograph for determination, and recording the chromatogram to obtain the UPLC spectrum of the ginkgo biloba leaf flavonoid extract.

2. Test results

15 batches of ginkgo leaf flavone extracts have 12 common chromatographic peaks, rutin of a peak 4 is determined as a reference peak, and the relative retention time of each characteristic peak and the reference peak (rutin, peak 4) of each batch is further calculated. Identifying the most main chromatographic peak by adopting a standard substance contrast, and adopting the relative retention time of 15 batches of ginkgo leaf flavone extracts, wherein the relative retention time is within +/-5% of a specified value. The compound assignments from peak 1 to peak 12 are shown in Table 8, and the characteristic map is shown in FIG. 1.

TABLE 8 mean values of the relative retention times of Ginkgo biloba leaf flavonoid extracts

Example 13: study on characteristic spectrum mass spectrum conditions

The mass spectrometry conditions were modified to determine the suitability of the mass spectrometry conditions in UPLC-MS according to the experimental procedure of example 12.

Mass spectrometry conditions 1: adopting a Q-TOF method, wherein the parameters of a primary mass spectrum are that the temperature of the drying gas is 300 ℃, the flow rate of the drying gas is 10L/min, the pressure of an atomizer is 42psig, and the pressure of a capillary is 4500V; the secondary mass spectrum parameters are that the temperature of the dry gas is 300 ℃, the flow rate of the dry gas is 10L/min, the pressure of the atomizer is 40psig, the pressure of the capillary tube is 4500V, and the collision energy is 90V.

Mass spectrometry conditions 2: adopting a Q-TOF method, wherein the parameters of a primary mass spectrum are that the temperature of the drying gas is 400 ℃, the flow rate of the drying gas is 15L/min, the pressure of an atomizer is 46psig, and the pressure of a capillary tube is 5500V; the secondary mass spectrum parameters are 400 ℃ of the drying gas temperature, 15L/min of the drying gas flow rate, 50psig of the atomizer, 5500V of the capillary pressure and 120V of collision energy.

The test results show that the

mass spectrometry conditions

1 and 2 can realize the construction of the characteristic spectrum by combining the technical schemes of 1.2.1 chromatographic conditions, 1.2.3 preparation of the test solution, 1.2.4 reference solution and 1.2.5 characteristic spectrum construction, and the construction results are the same as those of example 12 and are shown in figure 1.

Example 14: study of feature mapping methodology

1. Mobile phase optimization

Taking ginkgo biloba extract meeting the national pharmacopoeia standard, preparing 1 part of ginkgo biloba flavone extract according to the method of the embodiment 12, setting acetonitrile as a mobile phase A, and taking 0.8% (v/v), 0.10% (v/v) and 0.12% (v/v) formic acid solution as a mobile phase B, and carrying out sample injection detection under the test method of the embodiment 12, wherein the detection result is shown in figure 2.

Test results show that when acetonitrile is used as the mobile phase A and 0.08% (v/v), 0.10% (v/v) and 0.12% (v/v) formic acid solution is used as the mobile phase B, the characteristic peak type in the liquid phase map of the ginkgo leaf flavone extract is sharp and the separation degree is good. Preferably, an intermediate 0.10% (v/v) methanol solution is mobile phase B, further reducing the risk of errors.

2. Column temperature optimization

Taking ginkgo biloba extract meeting the national pharmacopoeia standard, preparing 1 part of ginkgo biloba flavone extract according to the method of the embodiment 12, setting the column temperature at 27 ℃, 30 ℃ and 33 ℃, and carrying out sample injection detection under the test method of the embodiment 12, wherein the detection result is shown in figure 3.

The test result shows that when the column temperature is 27 ℃, 30 ℃ and 33 ℃, the characteristic peak type in the liquid phase atlas of the ginkgo biloba leaf flavone extract is sharp and the separation degree is good. Preferably an intermediate value of 30 c column temperature, further reducing the risk of errors.

3. Column durability test

Taking ginkgo biloba extract meeting the national pharmacopoeia standard, preparing 1 part of test solution according to the method of the embodiment 12, and detecting in 3 chromatographic columns respectively, wherein the types of the chromatographic columns are shown in table 9. The results are shown in FIG. 4.

TABLE 9 chromatographic column type number

The retention time of the liquid chromatogram of the chromatographic columns No. 1 to No. 3 is different from the peak type, wherein the retention time difference of the liquid chromatogram corresponding to 3 chromatographic columns is larger. Compared with No. 3, the UPLC patterns obtained by the

chromatographic columns

1 and 2 are beyond the relative retention time range +/-0.050 specified by the characteristic spectrum. In addition, the separation degree of each characteristic peak of the column No. 1 is not high under the chromatographic conditions of example 12, and the pharmacopoeia specification that the separation degree R is greater than 1.5 cannot be satisfied, and the characteristic peak corresponding to the peak No. 3 of the column No. 2 cannot be separated effectively under the chromatographic conditions of example 12. Therefore, it is recommended to use a column # 3 (Phenomenx Luna Omega, 2.1X 100mm, 1.6 μm).

4. Repeatability test

Taking folium Ginkgo extract meeting national pharmacopoeia standard, preparing 6 parts of test solution according to the method of example 12, detecting by sample injection, matching with 12 common peaks by using traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723 version), calculating similarity, and finding the result in Table 10. The average value of the similarity is 1.000, and the RSD is 0.050%, which indicates that the sample has good repeatability.

TABLE 10 evaluation results of the repeatability similarity

5. Stability test

Taking ginkgo biloba extract meeting the national pharmacopoeia standard, preparing 1 part of test solution according to the method of the embodiment 12, respectively detecting at 0 hour, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours, carrying out sample injection detection, calculating the similarity, and the result is shown in a table 11. The mean value of the similarity is 0.999, and the RSD is 0.052%, which indicates that the sample has good repeatability.

TABLE 11 calculation of stability similarity

6. Intermediate precision test

The ginkgo biloba extract meeting the national pharmacopoeia standard is taken, 6 parts of test solution is prepared by different persons at different time according to the method of the embodiment 12, the sample injection detection is carried out, the similarity is calculated, and the result is shown in the table 12. The mean value of the similarity is 0.999, the RSD is 0.075 percent, and the intermediate precision of the method is good.

TABLE 12 results of sample detection similarity

Example 15: fingerprint comparison of folium Ginkgo flavone extract and raw materials

In order to evaluate the total flavone characteristics of the ginkgo biloba leaf flavone extract, the fingerprint spectrum of the ginkgo biloba leaf flavone extract is compared with the fingerprint spectrum of the raw material ginkgo biloba leaf extract, and the change of different flavones in the extract is determined.

1. Test method

The ginkgo biloba leaf flavone extract (batch No. 1, prepared by the method of example 1) and the ginkgo biloba leaf extract (batch No. 181107, Zhejiang Connbei pharmaceutical Co., Ltd.) which meet the standard of national pharmacopoeia are respectively prepared into test solution, and UPLC spectrum comparison is carried out according to the characteristic spectrum method of example 12.

2. Test results

The result is shown in the figure, which shows that the ginkgo biloba leaf flavone extract and the raw material have obvious peak difference in 0-2 min and no obvious peak shape difference in 2-14 min. Further material confirmation proves that the extract is mainly organic acid materials such as quinic acid, shikimic acid, protocatechuic acid, gallic acid, 6-hydroxykynureninic acid, p-hydroxybenzoic acid and the like in 0-2 min; 2-14 min is mainly flavone substances, such as myricetin-3-O-rutinose, myricetin-3-O-glucose, rutin, tagetetin-3-O-rutinose, quercetin-3-O-rutinose, kaempferol-3-O-rutinose, isorhamnetin-3-O-rutinose, quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside), etc. The test result shows that the ginkgo biloba leaf flavonoid extract basically keeps the flavonoid variety in the raw materials, so that the ginkgo biloba leaf flavonoid extract is used for refining and purifying the total flavonoid from the ginkgo biloba leaf extract, and the use efficiency of the ginkgo biloba leaf flavonoid substance is improved.

Claims

1. The ginkgo leaf flavone extract is characterized in that the content of total flavone in the extract is not less than 85% (w/w), and the effective components of the extract consist of the following parts: 3-6% (w/w) of myricetin-3-O-rutinose, 3-6% (w/w) of myricetin-3-O-glucose, 8-11% (w/w) of rutin, 5-7% (w/w) of tagetenin-3-O-rutinose, 5-7% (w/w) of quercetin-3-O-rutinose, 6-9% (w/w) of kaempferol-3-O-rutinose, 6-10% (w/w) of isorhamnetin-3-O-rutinose, 13-15% (w/w) of quercetin-2 ' - (6 ' -p-cinnamoyl glucose group), 11-15% (w/w) of kaempferol-3- (6 ' -p-cinnamoyl glucose group-rhamnoside) w).

2. The ginkgo biloba leaf flavonoid extract of claim 1, wherein the content ratio of quercetin-2 "- (6" -p-cinnamoyl glucosyl) to rutin is 1.2 to 1.8, the content ratio of kaempferol-3- (6' "-p-cinnamoyl glucosyl-rhamnoside) to rutin is 1.0 to 1.8, and the content ratio of quercetin-3-O-rutinose to kaempferol-3-O-rutinose is 0.6 to 1.15.

3. The ginkgo biloba leaf flavonoid extract according to claim 1 or 2, wherein the content ratio of quercetin-2 "- (6" -p-cinnamoyl glucosyl) to rutin is 1.33 to 1.64, the content ratio of kaempferol-3- (6' "-p-cinnamoyl glucosyl-rhamnoside) to rutin is 1.16 to 1.57, and the content ratio of quercetin-3-O-rutinose to kaempferol-3-O-rutinose is 0.75 to 0.91.

4. A method for preparing the ginkgo biloba leaf flavonoid extract as claimed in any one of claims 1 to 3, which comprises the steps of:

1) dissolving folium Ginkgo extract in boiling water or ethanol solution to obtain a sample solution;

2) passing the sample solution through a polyamide column, eluting with purified water with the volume of 3-5 times of the column volume, discarding the eluent, eluting with sodium carbonate solution with the volume of 5-8 times of the column volume of 0.1-0.5% (v/v), and collecting the sodium carbonate solution eluent;

3) adjusting the pH of the sodium carbonate solution eluent to 1-5 to prepare a secondary sample loading solution;

4) passing the secondary sample solution through a polyamide column, eluting with purified water with the volume of 3-5 times of the column volume, discarding the eluent, eluting with 40% -80% (v/v) ethanol solution with the volume of 5-8 times of the column volume, and collecting the ethanol solution eluent;

5) concentrating the ethanol solution eluate, drying, and pulverizing to obtain folium Ginkgo flavone extract.

5. The method according to claim 4, wherein the boiling water in step 1) is boiling purified water, and the concentration of the ethanol solution is not higher than 10% (v/v); eluting with 4 times of column volume of purified water in step 2), discarding the eluate, and eluting with 7 times of column volume of 0.15% (v/v) sodium carbonate solution; adjusting the pH value of the sodium carbonate solution eluent to 3 in the step 3); eluting with 4 times of column volume of purified water in the step 4), discarding the eluent, and eluting with 7 times of column volume of 60% (v/v) ethanol solution.

6. The method according to claim 4 or 5, wherein the step 1) is performed by dissolving with an 8% (v/v) ethanol solution; the elution flow rate in the steps 1) to 4) is 1-4 times of the column volume/h, and the polyamide column is 10-30 meshes, 30-60 meshes or 80-100 meshes.

7. A method for constructing a characteristic spectrum of the ginkgo biloba leaf flavonoid extract according to any one of claims 1 to 3, which comprises the following steps:

3) performing ultra-high performance liquid chromatography analysis on a test solution to obtain an ultra-high performance liquid chromatography characteristic spectrum, determining a common peak according to the relative retention time of the test solution and a reference substance, indicating the chemical components of the characteristic peak by adopting a liquid chromatography-mass spectrometry method, and establishing the ultra-high performance liquid chromatography characteristic spectrum of the ginkgo leaf flavonoid extract by using a standard substance reference; the conditions of the ultra-high performance liquid chromatography analysis are as follows:

the chromatographic column is a Phenomenx Luna Omega (2.1 × 100mm, 1.6 μm) chromatographic column with octadecylsilane chemically bonded silica as filler, and the number of theoretical plates is not less than 2500 in terms of rutin peak;

gradient elution: in terms of volume fraction, as shown in table 1:

TABLE 1 mobile phase elution gradient

8. The method for constructing a feature map according to claim 7, wherein the primary mass spectrum parameters are a drying gas temperature of 350 ℃, a drying gas flow rate of 12L/min, an atomizer pressure of 45psig, and a capillary pressure of 5000V; the secondary mass spectrum parameters are 350 ℃ of the drying gas, 12L/min of the drying gas flow rate, 45psig of the atomizer, 5000V of the capillary pressure and 100V of collision energy; the mobile phase is acetonitrile (A) and 0.10% (v/v) formic acid aqueous solution (B), the detection wavelength is 360nm, and the column temperature is 30 ℃.

9. The feature map construction method according to claim 7 or 8, characterized in that: the characteristic peaks in the step 3) are 12, wherein the peak 1 is myricetin-3-O-rutinose, and the relative retention time is 0.578; peak 2 is myricetin-3-O-glucose, the relative retention time is 0.644; peak 3 is disharmonidin with a relative retention time of 0.725; peak 4 is rutin, relative retention time 1.000; peak 5 is Tagetin-3-O-rutinose with a relative retention time of 1.083; peak 6 is quercetin-3-O-rutinose, with a relative retention time of 1.166; peak 7 is Kaempferol-3-O-rutinose, relative retention time is 1.441; peak 8 is isorhamnetin-3-O-rutinose, relative retention time is 1.490; peak 9 is quercetin, relative retention time is 1.632; peak 10 is kaempferol-3-O-glucose- (1-2) rhamnoside with a relative retention time of 2.099; peak 11 is quercetin-2 "- (6" -p-cinnamoyl glucosyl) with a relative retention time of 2.551; peak 12 was kaempferol-3- (6' "-p-cinnamoyl glucosyl-rhamnoside) with a relative retention time of 2.835.

10. The method for identifying the ginkgo biloba leaf flavonoid extract by utilizing the ginkgo biloba leaf flavonoid extract characteristic spectrum constructed by the method of any one of claims 7 to 9 is characterized in that a ginkgo biloba leaf flavonoid extract sample is taken and operated according to the same method of any one of claims 7 to 8 to obtain the ginkgo biloba leaf flavonoid extract sample characteristic spectrum, the sample characteristic spectrum and the ginkgo biloba leaf flavonoid extract characteristic spectrum of claim 9 are analyzed by adopting traditional Chinese medicine chromatographic fingerprint similarity evaluation system software, and the qualified product is obtained when the similarity is more than 0.9.