CN108362809B - Quality evaluation method of ginkgo leaf and extract and preparation thereof - Google Patents

Abstract

A quality evaluation method for Ginkgo biloba leaves and its extracts and preparations belongs to the technical field of quality detection of Chinese medicinal materials. The invention realizes the determination of the content of various flavonoid chemical components in Ginkgo biloba by constructing the high-performance liquid phase fingerprint of the chemical components of Ginkgo biloba, and calculating the correction factor by means of a method of one measurement and multiple evaluations. The method is easy to operate and has good stability. It can not only objectively, comprehensively and accurately evaluate the quality of Ginkgo biloba leaves and their extracts and preparations, and use them for quality control, but also solve the problem of inability to objectively and rationally control medicinal materials due to lack of reference substances. The question of the quality of its extract or preparation is of great significance to control the quality and ensure the curative effect.

Description

A kind of quality evaluation method of Ginkgo biloba and its extract and preparation

technical field

The invention relates to the technical field of quality detection of traditional Chinese medicinal materials, in particular to a quality evaluation method of Ginkgo biloba leaves and its extracts and preparations based on high-performance liquid phase fingerprint and one-measurement-multiple-evaluation method.

technical background

Ginkgo biloba is the dry leaf of Ginkgo biloba L., a plant of the family Ginkgoaceae. Ginkgo biloba is rich in chemical components, including flavonoids, lactones and phenolic acids, among which Ginkgo biloba has the most flavonoids and various compositions. Ginkgo biloba extract with a total flavonoid content of 24% can improve various diseases of the cardiovascular and cerebrovascular system. And no side effects.

Today, when Ginkgo extract and Ginkgo preparation-related products have been recognized by domestic and foreign pharmaceutical fields and received a large number of market demands, the quality evaluation method of Ginkgo is very important. At present, in the 2015 edition of "Chinese Pharmacopoeia" Ginkgo biloba quality standard: the total flavonoid content is evaluated and controlled by measuring the content of quercetin, kaempferol and isorhamnetin after acid hydrolysis. , time-consuming, and cannot fully reflect the true and accurate content of flavonoids. In addition, some studies reported that an analytical evaluation method for various flavonoids in Ginkgo biloba leaves and its extracts was established by HPLC-UV technology, but the measured index reference substance was difficult to separate and prepare, and the analysis cost was high. The control method is not easy to generalize. Therefore, in order to fully control the quality and clinical safety of Ginkgo biloba medicinal materials and their extracts and their preparations, a simple and easy-to-implement, low-cost detection method that can quantitatively analyze multiple active components at the same time was developed. The quality control of its extracts and preparations has practical significance.

To this end, the present invention provides a method for evaluating the quality of Ginkgo biloba leaves and its extracts and preparations based on high-performance liquid phase fingerprint and one-measurement-multiple-assessment method. A flavonoid component in Ginkgo biloba was used as an internal reference, and the content of various flavonoids in Ginkgo biloba was determined by calculating the relative correction factor. The method is easy to operate and has good stability, which can not only objectively, comprehensively and accurately evaluate the quality of Ginkgo biloba leaves and their extracts and preparations, but also solve the problem that the quality cannot be evaluated objectively and reasonably due to the lack of reference substances. Guaranteed efficacy is of great significance.

SUMMARY OF THE INVENTION

The object of the present invention is to aim at the deficiencies of the prior art, and to provide a method for measuring the content of various flavonoid components in Ginkgo biloba. Solve the problem that the quality of Chinese herbal medicines and their preparations cannot be controlled objectively and reasonably due to the lack of reference substances.

In order to achieve the above object, the present invention adopts the following technical solutions:

Provided is a method for evaluating the quality of Ginkgo biloba and its extracts and preparations. The method uses high performance liquid chromatography and a method of one measurement and multiple evaluations to measure the content of a plurality of flavonoid chemical components in Ginkgo biloba, so as to achieve Ginkgo biloba and Ginkgo biloba. The objective, comprehensive and accurate quality evaluation of its extracts and preparations, the content determination of flavonoid chemical components refers to the content determination of various flavonoid reference substances in Ginkgo biloba and its extracts and preparations, and the number of flavonoid chemical components is 8-16 species.

Specifically include the following steps:

1) Preparation of the reference substance solution: Precisely weigh 1-5 mg of each flavonoid reference substance and place it in a volumetric flask, dissolve it with 80-100% methanol to volume, and then filter to prepare each single reference substance stock solution, and then measure the Take a single standard solution of different volumes, mix and dilute to prepare a mixed sample containing a variety of flavonoids, which is used as a mixed reference solution for future use;

2) Preparation of the test solution: Precisely weigh Ginkgo biloba leaves, Ginkgo biloba extract or Ginkgo biloba preparation, add methanol with a concentration of 50-100%, extract and filter by ultrasonic or heating, take the continuous filtrate and pass it through a 0.45um or 0.22um filter membrane, get the sample solution;

3) Calculation of correction factor RCF: Take 2-20 μL of the mixed reference solution prepared in step 1), measure the chromatogram with high performance liquid chromatography and calculate the peak area, select one of the compounds as the internal reference, and calculate the internal reference respectively. and the relative correction factor f _k/s between other compounds.

The formula for calculating the relative correction factor is: f _k/s =f _k /f _s =A _k C _s /(A _s C _k ). In the formula: A _s is the peak area of the internal reference substance; C _s is the concentration of the internal reference substance; Ak is the peak area of a certain component _{k to be measured; C k is} the concentration of a certain component k to be measured.

4) Determination of active ingredients in the sample: take 2-20 μL of the test solution prepared in step 2), measure the chromatogram with high performance liquid chromatography, and calculate the quality of other flavonoids according to the following formula;

The formula is: W _k =(W _s ╳A _k )/(f _k/s ╳A _s ), W _k is the mass of the tested component, W _s is the mass of the internal reference, and A _k is the tested component The peak area of the fraction, f _k/s is the correction factor, and A _s is the peak area of the internal reference.

The invention accurately evaluates the total flavonoid content of Ginkgo biloba by measuring the total amount of 8-16 kinds of flavonoid glycosides with high content and good activity in Ginkgo biloba leaves and its extracts and preparations and various flavonoid reference substances of aglycone components.

Flavonoid controls included quercetin-3-O-(2'',6"-di-O-α-L-rhamnosyl)-β-D-glucoside, kaempferol-3-0-(2 '',6"-di-O-α-L-rhamnosyl)-β-D-glucoside, kaempferol-3-O-β sophoroside, quercetin-3-O-rutose-7 -O-glucoside, 3'-methyl-myricetin-3-O-rutinoside, quercetin 3-O-glucoside, quercetin 3-O-(2-β-D-glucosyl-α -L-rhamnoside), kaempferol-3-O-rutinoside, isorhamnetin-3-O-β-D-rutinoside, kaempferol 3-(2''-β-D-glucoside) -α-L-rhamnoside, quercetin 3-O-α-(6'''-p-coumaroyl-glucoside-β-1,4-rhamnoside), kaempferol 3-O- α-(6'''-p-coumaroyl-glucoside-β-1,2-rhamnoside).

The present invention constructs a characteristic liquid phase fingerprint through high-performance liquid chromatography, and ensures that the flavonoid reference components have a good degree of separation (degree of separation ≥ 1.2).

The detection conditions of high performance liquid chromatography are as follows: the detector of the high performance liquid chromatograph uses a diode array detector or a general-purpose detector, with octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, and 0.1% formic acid aqueous solution is mobile phase B, gradient elution, during gradient elution, the ratio of mobile phase A and B changes as follows: 0-20min, phase A 15%-20%, phase B 85%-80%; 20 -25min, A phase 20%-30%, B phase 80%-70%; 25-30min, A phase 30%-35%, B phase 70%-65%; 30-45min, A phase 35%-40% , B phase 65%-60%. The UV detector is used to detect wavelengths of 210nm-360nm. The flow rate was 0.5ml/min-1.5ml/min, and the column temperature was maintained at 20-45°C.

Applying the above conditions to obtain high performance liquid phase fingerprints, the similarity of each spectrum is not less than 0.90, and the RSD of each peak area is not higher than 2%.

In the present invention, the model of the high performance liquid chromatography column is preferably Gemini C18 (250 mm×4.6 mm, 5.0 μm), the flow rate is preferably 0.8 mL/min, the detection wavelength is preferably 360 nm, and the column temperature is preferably 35ºC.

The present invention adopts the method of one measurement and multiple evaluations, one of the components is used as an internal reference, and the content of each compound is calculated respectively through the relative correction factor. The formula for calculating the relative correction factor is: f _k/s =f _k /f _s =A _k C _s /(A _s C _k ). In the formula: A _s is the peak area of the internal reference substance; C _s is the concentration of the internal reference substance; Ak is the peak area of a certain component _{k to be measured; C k is} the concentration of a certain component k to be measured.

The present invention has the following advantages:

1) Compared with the traditional method, the present invention solves the problems of complicated operation and lack of reference substances in the multi-component quantification and multi-index quality control of traditional Chinese medicine. Quality of Chinese medicine products.

2) The present invention combines high performance liquid chromatography technology with a multi-evaluation method, and the double-standard products that meet both the fingerprint similarity requirements and the content requirements are confirmed as qualified products, and the quality of products in different batches is controlled to be consistent. Sex is important.

3) The method of the present invention uses a flavonoid component in Ginkgo biloba as a reference substance, and simultaneously quantifies quercetin and its glycosides, kaempferol and its glycosides, and isorhamnetin and its glycosides in Ginkgo biloba through a multi-evaluation method. As well as the content of various flavonoids such as Ginkgo biflavonoids, the content of total flavonoids in Ginkgo biloba leaves and their extracts and preparations can be accurately evaluated.

4) The one-test-multi-evaluation method provided by the present invention is easy to operate, and the multi-index quality evaluation method has good stability. The evaluation method for determining the content of hydrolyzate of flavonoids can effectively eliminate the problems of product counterfeiting and process modification.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of Ginkgo biloba extract.

Fig. 2 is the high performance liquid chromatogram of the mixed standard product of Ginkgo biloba composed of 8 kinds of flavonoids.

Figure 3 is a high performance liquid chromatogram of a mixed standard of Ginkgo biloba consisting of 10 flavonoids.

Fig. 4 is the high performance liquid chromatogram of the mixed standard product of Ginkgo biloba composed of 12 kinds of flavonoids.

Detailed ways

The present invention is further clarified below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to go straight to the scope of the invention. Those skilled in the art can modify colonies of various equivalent forms of the present invention and the present application. Scope limited by ownership requirements.

Chromatographic conditions used in the following examples:

1) Instrument: high performance liquid chromatograph (Huapu S6000, Dalian Huapu Technology Development Co., Ltd.);

2) Analytical chromatographic column: Gemini C ₁₈ (250mm×4.6mm, 5.0μm, Philomon Corporation, USA);

3) Mobile phase: use acetonitrile as mobile phase A, and use 0.1% formic acid aqueous solution as mobile phase B,

4) Gradient conditions: The ratio of mobile phase A and B changes as follows: 0—23min, 16—17%A, 23—25min, 17—20%A, 25—40min, 20%A, 40—45min, 20—28 %A, 45—48min, 28—35%A, 48—60min, 35%A

5) Detector condition: UV detector, detection wavelength is 360nm

6) The flow rate is 0.8ml/min;

7) The column temperature is 35°C.

Example 1

2 and the embodiment are described in detail below:

1) Reference substance

Quercetin-3-O-(2'',6"-di-O-α-L-rhamnosyl)-β-D-glucoside (1), Kaempferol-3-0-(2' ',6"-di-O-α-L-rhamnosyl)-β-D-glucoside (2), quercetin-3-O-rutino-7-O-glucoside (3), Quercetin 3-O-glucoside (4), Isorhamnetin-3-O-β-D-rutinoside (5), Kaempferol 3-(2''-β-D-glucoside)-α -L-rhamnoside (6), quercetin 3-O-α-(6'''-p-coumaroyl-glucoside-β-1,4-rhamnoside) (7), kaempferol 3-O-α-(6'''-p-coumaroyl-glucoside-β-1,2-rhamnoside) (8).

2) Preparation of sample solution

Precisely weigh 5.0 mg of Ginkgo biloba extract powder into a volumetric flask, add methanol to dissolve, ultrasonicate for 5 min, dilute to 20 mL, shake well, filter with 0.22 μm microporous membrane, and use high performance liquid chromatography as the test solution. Perform analytical testing.

3) Preparation of reference solution

Precisely weigh 1.0 mg of each of the 8 Ginkgo flavonoid monomer compounds in step 1), add methanol to dissolve and dilute to 2 ml in a volumetric flask, shake well to prepare a stock solution of each reference substance with a concentration of 0.5 mg/mL . Precisely measure 2.0mL of the stock solution of each reference substance, put it in the same 25mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well to obtain the reference substance mixed solution, and filter the membrane into the high performance liquid chromatograph.

4) Calculation of correction factor

Take 10 μL of the mixed reference substance solution prepared in step 3), measure the chromatogram with high performance liquid chromatography and calculate the peak area, select No. 4 reference substance as the internal reference substance, and calculate the relative correction factor between the internal reference substance and other compounds respectively f _k/s . The calculation formula of the relative correction factor is: f _k/s =f _k /f _s =A _k C _s /(A _s C _k ). In the formula: A _s is the peak area of the internal standard; C _s is the concentration of the internal standard; A _k is the peak area of a certain component k to be measured; C _k is the concentration of a certain component k to be measured.

4) Determination of other flavonoids

Take 10 μL of the test solution prepared in step 2), measure the chromatogram by high performance liquid chromatography, and calculate the mass of other flavonoids according to the following formula;

The formula is: W _k =(W _s ╳A _k )/(f _k/s ╳A _s ), W _k is the mass of the tested component, W _s is the mass of the internal reference, and A _k is the tested component The peak area of the fraction, f _k/s is the correction factor, and A _s is the peak area of the internal reference.

Its gradient conditions: The ratio of mobile phase A and B changes as follows: 0—23min, 16—17%A, 23—25min, 17—20%A, 25—40min, 20%A, 40—45min, 20—28% A, 45—48min, 28—35%A, 48—60min, 35%A;

Example 2

3 and the embodiment are described in detail below:

1) Reference substance

Quercetin-3-O-(2'',6"-di-O-α-L-rhamnosyl)-β-D-glucoside (1), Kaempferol-3-0-(2' ',6"-di-O-α-L-rhamnosyl)-β-D-glucoside (2), quercetin-3-O-rutino-7-O-glucoside (3), 3'-Methyl-myricetin-3-O-rutinoside (4), Quercetin 3-O-glucoside (5), Kaempferol-3-O-rutinoside (6), Isorhamnetin- 3-O-β-D-rutinoside (7), kaempferol 3-(2''-β-D-glucoside)-α-L-rhamnoside (8), quercetin 3-O-α -(6'''-p-coumaroyl-glucoside-β-1,4-rhamnoside) (9), kaempferol 3-O-α-(6'''-p-coumaroyl- glucoside-beta-1,2-rhamnoside) (10).

2) Preparation of sample solution

Precisely weigh 5.0 mg of Ginkgo biloba extract powder into a volumetric flask, add methanol to dissolve, ultrasonicate for 5 min, dilute to 20 mL, shake well, filter with 0.22 μm microporous membrane, and use high performance liquid chromatography as the test solution. Perform analytical testing.

3) Preparation of reference solution

Precisely weigh 1.0 mg of each of the 10 Ginkgo flavonoid monomer compounds in step 1) into a volumetric flask, add methanol to dissolve and dilute to 2.0 ml, shake well, and prepare a stock solution of each reference substance with a concentration of 0.5 mg/ mL. Precisely measure 2.0mL of the stock solution of each reference substance, put it in the same 25mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well to obtain the reference substance mixed solution, and filter the membrane into the high performance liquid chromatograph.

4) Calculation of correction factor

Take 10 μL of the mixed reference solution prepared in step 3), measure the chromatogram with high performance liquid chromatography and calculate the peak area, select No. 5 reference substance as the internal reference, and calculate the relative correction factor between the internal reference and other compounds respectively f _k/s . The calculation formula of the relative correction factor is: f _k/s =f _k /f _s =A _k C _s /(A _s C _k ). In the formula: A _s is the peak area of the internal standard; C _s is the concentration of the internal standard; A _k is the peak area of a certain component k to be measured; C _k is the concentration of a certain component k to be measured.

4) Determination of other flavonoids

Take 10 μL of the test solution prepared in step 2), measure the chromatogram by high performance liquid chromatography, and calculate the mass of other flavonoids according to the following formula;

The formula is: W _k =(W _s ╳A _k )/(f _k/s ╳A _s ), W _k is the mass of the tested component, W _s is the mass of the internal reference, and A _k is the tested component The peak area of the fraction, f _k/s is the correction factor, and A _s is the peak area of the internal reference.

Its gradient conditions: The ratio of mobile phase A and B changes as follows: 0—23min, 16—17%A, 23—25min, 17—20%A, 25—40min, 20%A, 40—45min, 20—28% A, 45—48min, 28—35%A, 48—60min, 35%A;

Example 3

4 and the embodiment are described in detail below:

1) Reference substance

Quercetin-3-O-(2'',6"-di-O-α-L-rhamnosyl)-β-D-glucoside (1), Kaempferol-3-0-(2' ',6"-di-O-α-L-rhamnosyl)-β-D-glucoside (2), kaempferol-3-O-β sophoroside (3), quercetin-3-O - Rutino-7-O-glucoside (4), 3'-methyl-myricetin-3-O-rutinoside (5), Quercetin 3-O-glucoside (6), Quercetin 3 -O-(2-β-D-glucosyl-α-L-rhamnoside) (7), kaempferol-3-O-rutinoside (8), isorhamnetin-3-O-β-D - Rutinoside (9), Kaempferol 3-(2''-β-D-glucoside)-α-L-rhamnoside (10), Quercetin 3-O-α-(6'''- p-coumaroyl-glucoside-β-1,4-rhamnoside) (11), kaempferol 3-O-α-(6'''-p-coumaroyl-glucoside-β-1, 2-rhamnoside) (12).

2) Preparation of sample solution

Precisely weigh 5.0 mg of Ginkgo biloba extract powder into a volumetric flask, add methanol to dissolve, ultrasonicate for 5 min, dilute to 20 mL, shake well, filter with 0.22 μm microporous membrane, and use high performance liquid chromatography as the test solution. Perform analytical testing.

3) Preparation of reference solution

Precisely weigh 1.0 mg of each of the 12 Ginkgo flavonoid monomer compounds in step 1) into a volumetric flask, add methanol to dissolve and dilute to 2 ml, shake well, and prepare a stock solution of each reference substance with a concentration of 0.5 mg/mL . Precisely measure 1.5mL of the stock solution of each reference substance, put it in the same 25mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well to obtain the reference substance mixed solution, and filter the membrane into the high performance liquid chromatograph.

4) Calculation of correction factor

Take 10 μL of the mixed reference solution prepared in step 3), measure the chromatogram with high performance liquid chromatography and calculate the peak area, select No. 6 reference substance as the internal reference, and calculate the relative correction factor between the internal reference and other compounds respectively f _k/s . The calculation formula of the relative correction factor is: f _k/s =f _k /f _s =A _k C _s /(A _s C _k ). In the formula: A _s is the peak area of the internal standard; C _s is the concentration of the internal standard; A _k is the peak area of a certain component k to be measured; C _k is the concentration of a certain component k to be measured.

4) Determination of other flavonoids

Take 10 μL of the test solution prepared in step 2), measure the chromatogram by high performance liquid chromatography, and calculate the mass of other flavonoids according to the following formula;

The formula is: W _k =(W _s ╳A _k )/(f _k/s ╳A _s ), W _k is the mass of the tested component, W _s is the mass of the internal reference, and A _k is the tested component The peak area of the fraction, f _k/s is the correction factor, and A _s is the peak area of the internal reference.

Its gradient conditions: The ratio of mobile phase A and B changes as follows: 0—23min, 16—17%A, 23—25min, 17—20%A, 25—40min, 20%A, 40—45min, 20—28% A, 45—48min, 28—35%A, 48—60min, 35%A;

Example 4

The present invention will be further described below in conjunction with FIG. 3

Implementation cases of methodological inspections

1) Linearity, lower limit of detection (LOD) and lower limit of quantification (LOQ) assays

Accurately draw the standard solutions of 10 compounds, 8, 20, 40, 80, 160, 320, 640ug/ml, and inject 10 uL into the liquid chromatograph, record the peak area, and take the concentration of each reference substance as The abscissa, the peak area as the ordinate, draw a standard curve, and determine the detection limit of the method with S/N =3, and determine the quantification limit of the method with S/N =10, the detection limit, the quantification limit and the linear regression equation and other parameters , the results showed a good linear relationship (r ² greater than or equal to 0.9993), the detection limit was 0.13-1.11, the quantification limit was 0.43-3.69, the RSD of the relative correction factor was 0.23-1.34, and the relative retention time was 0.05-0.72.

Regression equation, linear range, and LOD and LQ

2) Precision, repeatability, stability and accuracy test:

Precision: Take the reference solution, repeat the injection 6 times within one day and 6 times within 3 consecutive days under the determined chromatographic conditions, and calculate the relative standard deviation (RSD) and the peak area of each component analyzed. Retention time (RT) was used to evaluate intra-day and inter-day precision. The precision results showed that the RSDs of intra-day A (peak area) and RT (retention time) were lower than 0.89 and 0.75, respectively, and inter-day A (peak area) and RT (retention time) time), the RSDs were lower than 1.86 and 0.6, respectively, indicating good precision.

Stability test: Take the same mixed standard solution and inject it into the liquid chromatograph at 0, 2, 4, 6, 8, 12, 24, and 48, respectively, and investigate the peak area RSD and retention time (RT) of each component. Its stability, the RSD of stability A (peak area) and RT (retention time) were 0.66-1.92 and 0.13-1.18, respectively, indicating that the sample solution was stable within 48h.

Repeatability test: take the mixed standard solution (6 copies in parallel), inject and analyze under the above chromatographic conditions, and evaluate its stability with the RSD retention time (RT) value of each component content in the sample. Repeatability A (peak area) ) and RT (retention time) RSDs were 0.67-1.96 and 0.05-0.51, respectively, indicating that the sample solution had good repeatability.

Precision, Repeatability and Stability Testing

Accuracy experiment: To evaluate the accuracy of the method through the sample addition recovery experiment, accurately weigh 1 g of Ginkgo biloba powder with known content, add a certain amount of reference solution, prepare samples according to the test solution, and make low and medium , three high concentrations (50%, 80%, 100%) were measured, and the recovery rate of sample addition was calculated. Recovery rate = (measured value - original value) / added standard value, the recovery rate was 98.8-102.1, and the RSD was 0.32-1.76 , the results show that this method has good accuracy.

Applicability of the method to check:

In order to test the new method, the experiment examines the existing different liquid chromatographs, different chromatographic columns and other influencing factors (wavelength, flow rate, column temperature, gradient of mobile phase, gradient time program) in the laboratory. factor (RPF) and its relative retention time (RSD).

Accurate positioning of chromatographic peaks is the premise to ensure the application of the "one-test, multiple-evaluation" method. The relative retention time (RRT) parameter is used to locate the chromatogram in combination with the overall characteristics of the chromatogram. RRT=RT _x /RT _s , RT _x : retention time of other chromatographic peaks RT _s : retention time of chromatographic peaks of internal standard, the RSD range of RCF of different liquid chromatographs is 0.12-1.18, and the range of RSD of RRT is 0.12-1.18 0.2-1.36, so different liquid chromatographs have little effect on the method. The RSD range of RCF with different wavelengths is 0.26-2.03, and the RSD range of RRT is 0.53-1.83. The RSD of RCF with different flow rates is in the range of 0.53-1.72, the RSD of RRT is in the range of 0.33-1.65. The RSD of RCF with different column temperatures is in the range of 0.65-2.02, the RSD of RRT is in the range of 0.50-1.67, and the RCF of mobile phase acid is in the range of 0.65-2.02. Between 0.65-1.77, RRT is between 0.46-2.30, which has little effect on the method.

Comparison of measurement results between one test and multiple evaluation method and external standard method (mg/g)

Note: Peak 5 is quercetin 3-O-glucoside

The Pearson correlation coefficient is used to characterize the correlation between the results obtained by the method of the present invention and the external standard method, and it can be seen that there is no significant difference between the results obtained by the method of the present invention and the external standard method.

Claims (1)

1. A quality evaluation method of a ginkgo leaf extract is characterized in that the method is used for measuring the content of a plurality of flavonoid chemical components in the ginkgo leaf extract by utilizing a high performance liquid chromatography technology and a one-measurement-multiple-evaluation method, and comprises the following steps:

1) reference substance

quercetin-3-O- (2' ', 6' ' -di-O- α -L-rhamnosyl) -L-D-glucoside (1), kaempferol-3-O- (2' ', 6' ' -di-O-L2-L-rhamnosyl) -L-D-glucoside (2), kaempferol-3-O-L-sophoroside (3), quercetin-3-O-rutinoside-7-O-glucoside (4), 3' -methyl-myricetin-3-O-rutinoside (5), quercetin-3-O-glucoside (6), quercetin-3-O- (2-L-D-glucosyl-L-L-rhamnoside) (7), kaempferol-3-O-rutinoside (8), isorhamnetin-3-O- β -D-glucoside (9), kaempferol-3- (2' ' -34-D-rhamnoside) -736-rhamnosyl) -L-D-glucoside (1), kaempferol-3-O-glucoside (3-O-636), kaempferol-3-O-glucoside (3-O- α), kaempferol-3-O-glucoside (3- α), kaempferol-3-4-rhamnoside (3-p-366- α, 3-rhamnoside) (3611-rhamnoside-p-rhamnoside) (3-3611- α);

2) preparation of test solution

Precisely weighing folium Ginkgo extract powder 5.0mg in volumetric flask, adding methanol for dissolving, performing ultrasonic treatment for 5min, diluting to constant volume of 20m L, shaking, and filtering with 0.22 μm microporous membrane to obtain sample solution;

3) preparation of Mixed control solutions

Precisely weighing 1.0mg of each of the 12 reference substances in the step 1), respectively placing the reference substances in volumetric flasks, adding methanol for dissolving, fixing the volume to 2ml, shaking up to prepare stock solutions of the reference substances, wherein the concentration of the stock solutions is 0.5mg/m L, precisely weighing 1.5m L of the stock solutions of the reference substances, placing the stock solutions in the same volumetric flask with the volume of 25m L, adding methanol for dissolving, diluting to the scale, and shaking up to obtain mixed reference substance solutions;

4) calculation of correction factors

Taking 10 mu L of the mixed reference substance solution prepared in the step 3), measuring chromatogram by using high performance liquid chromatography, calculating peak area, selecting the No. 6 reference substance quercetin-3-O-glucoside as an internal reference substance, and calculating relative correction factor f between the internal reference substance and other compounds respectively_k/s；

The relative correction factor calculation formula is: f. of_k/s＝f_k/f_s＝A_kC_s/(A_sC_k) (ii) a In the formula: a. the_sPeak area of the internal standard substance; c_sIs the internal standard concentration; a. the_kThe peak area of a certain component k to be measured; c_kThe concentration of a certain component k to be measured;

5) determination of other flavone Components

Taking 10 mu L of the test solution prepared in the step 2), measuring a chromatogram by using a high performance liquid chromatography, and respectively calculating the mass of other flavone components according to the following formula;

the formula is: w_k=（W_s╳A_k)/(f_k/s╳A_s），W_kAs mass of the component to be measured, W_sIs the mass of the internal reference substance, A_kPeak area of the measured component, f_k/sTo correct the factor, A_sIs the peak area of the internal reference substance;

the high performance liquid chromatography adopts an analytical chromatographic column: gemini C₁₈；

Mobile phase: acetonitrile is taken as a mobile phase A, and 0.1% formic acid aqueous solution is taken as a mobile phase B;

mobile phase gradient conditions: the ratio of mobile phase A, B varies as: 0-23min, 16-17% A, 23-25min, 17-20% A, 25-40min, 20% A, 40-45min, 20-28% A, 45-48min, 28-35% A, 48-60min, 35% A.

Images