Construction method of characteristic chromatogram of ginkgo leaf standard decoction and content determination method of multi-index components

Abstract

The invention relates to a construction method of a specific chromatogram of a ginkgo leaf standard decoction and a content determination method of multi-index components. The construction method of the characteristic chromatogram of the ginkgo leaf standard decoction comprises the following steps: mixing the ginkgo leaf standard decoction with a first solvent, and extracting to prepare a test solution; performing high performance liquid chromatography detection on the test solution, and constructing a characteristic chromatogram of the ginkgo leaf standard decoction; wherein the conditions of the high performance liquid chromatography detection are as follows: a chromatographic column is a T3 chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises methanol and acetonitrile according to a volume ratio of (3-5): 1, the mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.08-0.12%, and a gradient elution procedure is adopted. The specific chromatogram constructed by the method has many peaks, prototype components of flavonol glycoside effective components are detected, the detection is rapid and stable, and the specificity is strong.

Description

Technical Field

[0001] This application relates to the technical field of traditional Chinese medicine analysis, and particularly to a method for constructing a characteristic chromatogram of a standard decoction of Ginkgo biloba leaves and a method for determining the contents of multiple index components. Background Art

[0002] Ginkgo biloba leaves are the dried leaves of the Ginkgo biloba L. plant of the Ginkgoaceae family. They are sweet, bitter, astringent, and neutral in flavor, and belong to the heart and lung meridians. They have the effects of promoting blood circulation to remove blood stasis, dredging collaterals to relieve pain, relieving cough and asthma, and reducing turbidity and lipid. They are used for blood stasis blocking collaterals, chest pain due to chest impediment, hemiplegia due to stroke, cough and asthma due to lung deficiency, and hyperlipidemia. Modern pharmacological research believes that the main active components of Ginkgo biloba leaves are flavonoids and terpene lactones, which have effects such as antidiarrheal, antioxidant, hepatoprotective, lipid-lowering and blood sugar-lowering, enhancing immune activity, and anti-tumor. In the 2020 edition of the Chinese Pharmacopoeia and existing literature reports, acid hydrolysis methods are mostly used to indirectly calculate the amount of total flavonol glycosides in Ginkgo biloba leaves by measuring the contents of quercetin, kaempferol, and isorhamnetin, so as to achieve quantitative analysis of the prototype component total flavonol glycosides. There are few reports on the direct determination of flavonol glycosides, which are prototype components in Ginkgo biloba leaves. At the same time, the specificity of the acid hydrolysis method is also poor.

[0003] Traditional Chinese medicine has complex components, and there are synergistic effects among multiple components. Traditional Chinese medicine fingerprint / characteristic chromatograms can comprehensively consider the composition of traditional Chinese medicine components from an overall perspective, and are particularly suitable for the quality evaluation of traditional Chinese medicine. Currently, there are few research reports on the fingerprint / characteristic chromatograms of Ginkgo biloba leaves, and they are basically concentrated on the research of the fingerprint or characteristic chromatogram of Ginkgo biloba medicinal materials / Ginkgo biloba ethanol extracts, while there are few research reports on using fingerprint / characteristic chromatograms for traditional Chinese medicine decoctions and their related preparations with Ginkgo biloba leaves as raw materials.

[0004] Traditional Chinese medicine uses decoctions as the main form of medication. Decoctions are the material basis for traditional Chinese medicine to exert its efficacy. Traditional Chinese medicine standard decoctions are standardized preparation forms of traditional Chinese medicine decoctions. They are single-herb decoctions prepared by standardized processes with reference to modern extraction methods. As a standard substance and standard system, they are used to standardize the quality of modern traditional Chinese medicine preparations such as formula granules. Traditional Chinese medicine decoctions are the material basis for traditional Chinese medicine to exert its efficacy. Since standard decoctions use water as a solvent, water-soluble components should be the key points for the quality control of decoctions. In the aforementioned acid hydrolysis method, the solubility of aglycone components such as quercetin, kaempferol, and isorhamnetin obtained by acid hydrolysis in water is not high. Therefore, it is particularly important to determine the prototype components with good water solubility for the quality control of Ginkgo biloba leaf standard decoctions.

[0005] In addition, there are also methods for providing quality evaluation research on the standard decoction of Ginkgo biloba leaves and establishing a high-performance liquid chromatography fingerprint. It involves using high-performance liquid chromatography (HPLC) to establish the fingerprint of the standard decoction of Ginkgo biloba leaves and establishing a method for determining the contents of quercetin, kaempferol, and isorhamnetin. However, the number of common peaks in the fingerprint of this method is small, the resolution of chromatographic peaks is poor, and the main common peaks cannot be effectively identified. Therefore, the specificity and exclusivity of the method are not strong, and the significance of quality control is not great. In the content determination method, an acid hydrolysis method is still used to determine the contents of three aglycone components, namely quercetin, kaempferol, and isorhamnetin, and indirectly calculate the content of total flavonol glycosides. The exclusivity of the method is also not strong. Summary of the Invention

[0006] Based on this, the present application provides a method for constructing a characteristic fingerprint of the standard decoction of Ginkgo biloba leaves and a method for determining the contents of multiple index components. The characteristic fingerprint constructed by this method has many common peaks, and the detected prototype components are the active components of flavonol glycosides, with fast, stable, and strong specificity detection.

[0007] In the first aspect of the present application, there is provided a method for constructing a characteristic fingerprint of the standard decoction of Ginkgo biloba leaves, including the following steps:

[0008] Mix the standard decoction of Ginkgo biloba leaves with a first solvent for extraction to prepare a test solution.

[0009] Perform high-performance liquid chromatography detection on the test solution to construct the characteristic fingerprint of the standard decoction of Ginkgo biloba leaves.

[0010] Among them, the conditions for the high-performance liquid chromatography detection include:

[0011] The chromatographic column is a T3 chromatographic column.

[0012] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixture of methanol and acetonitrile with a volume ratio of (3 - 5):1, and mobile phase B is an aqueous phosphoric acid solution with a volume percentage of 0.08% - 0.12%. A gradient elution program is used.

[0013] In one embodiment, the gradient elution program includes:

[0014] From 0 min to 30 min, the volume percentage of mobile phase A is maintained at 28%, and the volume percentage of mobile phase B is maintained at 72%.

[0015] From 30 min to 40 min, the volume percentage of mobile phase A changes from 28% to 30%, and the volume percentage of mobile phase B changes from 72% to 70%.

[0016] From 40 min to 45 min, the volume percentage of mobile phase A changes from 30% to 35%, and the volume percentage of mobile phase B changes from 70% to 65%.

[0017] From 45 min to 55 min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%.

[0018] In one of the embodiments, the conditions for the high performance liquid chromatography detection further include at least one of the following:

[0019] (1) The flow rate is from 0.2 mL / min to 0.4 mL / min;

[0020] (2) The column temperature is from 33 °C to 37 °C;

[0021] (3) The detection wavelength is from 363 nm to 367 nm;

[0022] (4) The injection volume is from 3 μL to 10 μL.

[0023] In one of the embodiments, the preparation of the test solution has at least one of the following characteristics:

[0024] (1) The first solvent is an aqueous methanol solution with a volume percentage of 10% to 60%;

[0025] (2) The extraction method is ultrasonic extraction or reflux extraction;

[0026] (3) The extraction time is from 10 min to 50 min.

[0027] In one of the embodiments, the method for constructing the characteristic chromatogram of the standard ginkgo leaf decoction further includes a characteristic peak identification step:

[0028] Mix the reference substance with the second solvent to prepare a reference substance solution; the reference substance includes one or more of the reference substances of typhaneoside, kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutinoside, narcissin and kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside;

[0029] Perform the high performance liquid chromatography detection on the reference substance;

[0030] Optionally, the second solvent is methanol.

[0031] In the second aspect of the present application, a method for determining the content of multiple target components in the standard ginkgo leaf decoction is provided, including the following steps:

[0032] Mix the test standard ginkgo leaf decoction with the first solvent for extraction to prepare a test solution;

[0033] Mix the reference substance with the second solvent to prepare reference substance solutions with different concentrations; the reference substance includes one or more of typhaneoside, kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucopyranosyl-(1→2)-α-L-rhamnoside reference substances;

[0034] Perform high performance liquid chromatography (HPLC) detection on the reference substance solutions with different concentrations, and construct the standard curve of the reference substance according to the detection results;

[0035] Perform HPLC detection on the test sample solution, substitute the detection results into the standard curve of the reference substance, and calculate the content of the corresponding index components;

[0036] Among them, the conditions for the HPLC detection include:

[0037] The chromatographic column is a T3 chromatographic column;

[0038] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixture of methanol and acetonitrile with a volume ratio of (3-5):1, and mobile phase B is an aqueous phosphoric acid solution with a volume percentage of 0.08%-0.12%. A gradient elution program is adopted.

[0039] In one of the embodiments, the gradient elution program includes:

[0040] From 0 min to 30 min, the volume percentage of mobile phase A is maintained at 28%, and the volume percentage of mobile phase B is maintained at 72%;

[0041] From 30 min to 40 min, the volume percentage of mobile phase A changes from 28% to 30%, and the volume percentage of mobile phase B changes from 72% to 70%;

[0042] From 40 min to 45 min, the volume percentage of mobile phase A changes from 30% to 35%, and the volume percentage of mobile phase B changes from 70% to 65%;

[0043] From 45 min to 55 min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%.

[0044] In one of the embodiments, the conditions for the HPLC detection further include at least one of the following:

[0045] (1) The flow rate is 0.2 mL / min to 0.4 mL / min;

[0046] (2) The column temperature is 33°C to 37°C;

[0047] (3) The detection wavelength is 363 nm to 367 nm;

[0048] (4) The sample injection volume is 3 μL to 10 μL.

[0049] In one of the embodiments, the test solution has at least one of the following characteristics:

[0050] (1) The first solvent is an aqueous methanol solution with a volume percentage of 10% to 60%;

[0051] (2) The extraction method is ultrasonic extraction or reflux extraction;

[0052] (3) The extraction time is 10 min to 50 min.

[0053] In one of the embodiments, the standard curve of typhaneoside is: y = 4369.4x + 2509.2;

[0054] The standard curve of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside) is: y = 3505.4x - 4244.3;

[0055] The standard curve of rutin is: y = 6044x - 28958;

[0056] The standard curve of kaempferol-3-O-rutinoside is: y = 4530.9x - 5766.7;

[0057] The standard curve of narcissin is: y = 5258.8x + 12960;

[0058] The standard curve of kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside is: y = 3535.2x + 1324.3.

[0059] In the third aspect of the present application, there is provided the use of the characteristic fingerprint of the Ginkgo biloba standard decoction constructed by the construction method described in the first aspect or the content determination method of multiple index components in the Ginkgo biloba standard decoction described in the second aspect in the quality detection of the Ginkgo biloba standard decoction.

[0060] The above-mentioned construction method of the characteristic fingerprint of the Ginkgo biloba standard decoction, by adopting appropriate chromatographic conditions, has many common peaks in the constructed characteristic fingerprint of the Ginkgo biloba standard decoction, and the detected prototype components are flavonol glycoside active components, fully reflecting the characteristic peak information of the Ginkgo biloba standard decoction, showing the chemical composition characteristics of the Ginkgo biloba standard decoction, with fast, stable and specific detection, capable of realizing the identification and quality control of Ginkgo biloba, providing a new technical method for its quality control, and also providing an important reference for the formulation of the quality standards of Ginkgo biloba and its related preparations.

[0061] The content determination method for multiple index components of the above-mentioned Ginkgo biloba standard decoction can detect the content of the prototype components of multiple index components in the Ginkgo biloba standard decoction by adopting appropriate chromatographic conditions, comprehensively reflecting the quality attributes of the Ginkgo biloba standard decoction, and providing a new scientific method for the quality control of the Ginkgo biloba standard decoction. Description of the Drawings

[0062] Figure 1 It is the chromatogram with a wavelength of 254 nm in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0063] Figure 2 It is the chromatogram with a wavelength of 260 nm in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0064] Figure 3 It is the chromatogram with a wavelength of 365 nm in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0065] Figure 4 It is the chromatogram with an injection volume of 2 μl in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0066] Figure 5 It is the chromatogram with an injection volume of 5 μl in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0067] Figure 6 It is the chromatogram with an injection volume of 10 μl in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0068] Figure 7 It is the chromatogram of different extraction solvents in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0069] Figure 8 It is the chromatogram of different extraction methods in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0070] Figure 9 It is the chromatogram of different extraction times in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0071] Figure 10 It is the chromatogram for specificity investigation in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0072] Figure 11 It is the superimposed chromatogram of the characteristic chromatograms of 18 batches of Ginkgo biloba standard decoctions in the construction of the characteristic chromatogram of the Ginkgo biloba standard decoction in Example 1;

[0073] Figure 12It is the characteristic chromatogram of the Ginkgo biloba reference medicinal material in the construction of the characteristic chromatogram of the standard decoction of Ginkgo biloba in Example 1. Among them, Peak 2: Kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside); Peak 3: Typhaneoside; Peak 4 (S1): Rutin; Peak 7: Kaempferol-3-O-rutinoside; Peak 8 (S2): Narcissoside; Peak 9: Kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside;

[0074] Figure 13 It is the control characteristic chromatogram of the standard decoction of Ginkgo biloba in the construction of the characteristic chromatogram of the standard decoction of Ginkgo biloba in Example 1. Among them, Peak 2: Kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside); Peak 3: Typhaneoside; Peak 4 (S1): Rutin; Peak 7: Kaempferol-3-O-rutinoside; Peak 8 (S2): Narcissoside; Peak 9: Kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside;

[0075] Figure 14 It is the comparative chromatogram of the test solution of the standard decoction of Ginkgo biloba and the reference substance solution of typhaneoside in Example 1;

[0076] Figure 15 It is the comparative chromatogram of the test solution of the standard decoction of Ginkgo biloba and the reference substance solution of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside) in Example 1;

[0077] Figure 16 It is the comparative chromatogram of the test solution of the standard decoction of Ginkgo biloba and the reference substance solutions of rutin, kaempferol-3-O-rutinoside, and narcissoside in Example 1;

[0078] Figure 17 It is the comparative chromatogram of the test solution of the standard decoction of Ginkgo biloba and the reference substance solution of kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside in Example 1. Specific Embodiments

[0079] The following further elaborates in detail on the construction method of the characteristic chromatogram and the content determination method of multiple index components of the standard decoction of Ginkgo biloba in this application in combination with specific examples. This application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of this application more thorough and comprehensive.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0081] In this text, "one or more" refers to any one, any two, or any two or more of the listed items.

[0082] In this application, "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are only for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "the first", "the second", "the third", "the fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0083] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features and also includes an open-ended technical solution containing the listed features.

[0084] In this application, regarding numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0085] In this application, for the percentage content involved, unless otherwise specified, it refers to the mass percentage for solid-liquid mixing and solid-solid mixing, and the volume percentage for liquid-liquid mixing.

[0086] In this application, for the percentage concentration involved, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component. Unless otherwise specified, the solvent used for the solution is water.

[0087] In this application, for the temperature parameter, unless otherwise specified, it allows for isothermal treatment and also allows treatment within a certain temperature range. The isothermal treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0088] In this application, room temperature generally refers to 4°C to 30°C, preferably 20 ± 5°C.

[0089] In some examples of this application, a method for constructing a characteristic fingerprint of a standard ginkgo leaf decoction is provided, including the following steps:

[0090] Mix the standard ginkgo leaf decoction with a first solvent for extraction to prepare a test solution.

[0091] Perform high performance liquid chromatography (HPLC) detection on the test sample solution to construct the characteristic chromatogram of the standard ginkgo leaf decoction;

[0092] Among them, the conditions for the HPLC detection include:

[0093] The chromatographic column is a T3 chromatographic column;

[0094] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixture of methanol and acetonitrile with a volume ratio of (3 - 5):1, and mobile phase B is an aqueous phosphoric acid solution with a volume percentage of 0.08% - 0.12%. A gradient elution program is used.

[0095] In some examples, the chromatographic column is Waters HSS T3. Without limitation, the diameter of the chromatographic column is 1.8 mm - 2.5 mm, the length is 140 mm - 160 mm, and the particle size of the packing is 1.5 μm - 2 μm.

[0096] In some examples, the gradient elution program includes:

[0097] From 0 min to 30 min, the volume percentage of mobile phase A is maintained at 28%, and the volume percentage of mobile phase B is maintained at 72%;

[0098] From 30 min to 40 min, the volume percentage of mobile phase A changes from 28% to 30%, and the volume percentage of mobile phase B changes from 72% to 70%;

[0099] From 40 min to 45 min, the volume percentage of mobile phase A changes from 30% to 35%, and the volume percentage of mobile phase B changes from 70% to 65%;

[0100] From 45 min to 55 min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%.

[0101] In some examples, in the HPLC detection, the flow rate is 0.2 mL / min - 0.4 mL / min. Specifically, the flow rate includes but is not limited to: 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min, 0.4 mL / min.

[0102] In some examples, in the HPLC detection, the column temperature is 33°C - 37°C. Specifically, the column temperature includes but is not limited to: 33°C, 34°C, 35°C, 36°C, 37°C.

[0103] In some of these examples, in the high performance liquid chromatography (HPLC) detection, the detection wavelength is 363 nm to 367 nm. Specifically, the detection wavelength includes, but is not limited to: 363 nm, 364 nm, 365 nm, 366 nm, 367 nm.

[0104] In some of these examples, in the high performance liquid chromatography (HPLC) detection, the injection volume is 3 μL to 10 μL. Specifically, the injection volume includes, but is not limited to: 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL.

[0105] In some of these examples, the first solvent is an aqueous methanol solution with a volume percentage of 10% to 60%. Specifically, the volume percentage of the aqueous methanol solution includes, but is not limited to: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%.

[0106] In some of these examples, the extraction method is ultrasonic extraction or reflux extraction. Without limitation, the power of ultrasonic extraction is 200 W to 300 W, and the frequency is 35 kHz to 45 kHz.

[0107] In some of these examples, the extraction time is 10 min to 50 min. Specifically, the extraction time includes, but is not limited to: 10 min, 20 min, 30 min, 40 min, 50 min.

[0108] In some of these examples, the method for constructing the characteristic fingerprint of the standard Ginkgo biloba decoction further includes a characteristic peak identification step:

[0109] Mix a reference substance with a second solvent to prepare a reference substance solution; the reference substance includes one or more of typhaneoside, kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucopyranosyl-(1→2)-α-L-rhamnoside reference substances;

[0110] Perform the high performance liquid chromatography (HPLC) detection on the reference substance.

[0111] In some of these examples, the second solvent is methanol.

[0112] In some of these examples, the method for constructing the characteristic fingerprint of the standard Ginkgo biloba decoction further includes a reference substance solution detection step:

[0113] Mix a reference sample with a third solvent to prepare a reference substance solution;

[0114] Perform the high performance liquid chromatography (HPLC) detection on the reference substance solution; the reference sample includes rutin and narcissin;

[0115] In some of these examples, the third solvent is methanol.

[0116] Some other examples of this application provide a method for determining the contents of multiple index components in a standard Ginkgo biloba decoction, including the following steps:

[0117] Mix the standard Ginkgo biloba decoction to be tested with a first solvent for extraction to prepare a test solution;

[0118] Mix a reference substance with a second solvent to prepare reference substance solutions with different concentrations; the reference substance includes one or more of the reference substances of typhaneoside, kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucose(1→2)-α-L-rhamnoside;

[0119] Perform high performance liquid chromatography (HPLC) detection on the reference substance solutions with different concentrations, and construct a standard curve of the reference substance according to the detection results;

[0120] Perform HPLC detection on the test solution, and substitute the detection results into the standard curve of the reference substance to calculate the contents of the corresponding index components;

[0121] Among them, the conditions for the HPLC detection include:

[0122] The chromatographic column is a T3 chromatographic column;

[0123] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixture of methanol and acetonitrile with a volume ratio of (3-5):1, and mobile phase B is an aqueous phosphoric acid solution with a volume percentage of 0.08%-0.12%. A gradient elution program is adopted.

[0124] It can be understood that the gradient elution program, the conditions for the HPLC detection, and the conditions for preparing the test solution are the same as those in the aforementioned method for constructing the characteristic chromatogram, and will not be elaborated here.

[0125] In some of these examples, the standard curve of typhaneoside is: y = 4369.4x + 2509.2;

[0126] The standard curve of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside) is: y = 3505.4x - 4244.3;

[0127] The standard curve of rutin is: y = 6044x - 28958;

[0128] The standard curve of kaempferol-3-O-rutinoside is: y = 4530.9x - 5766.7;

[0129] The standard curve of narcissin is: y = 5258.8x + 12960;

[0130] The standard curve of kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside is: y = 3535.2x + 1324.3.

[0131] Some other examples of this application also provide the characteristic chromatogram of the standard ginkgo leaf decoction constructed by the construction method as described above, or the application of the content determination method of multiple index components in the standard ginkgo leaf decoction as described above in the quality inspection of the standard ginkgo leaf decoction.

[0132] For the experimental parameters not specified in the following specific examples, preferably refer to the guidance given in this application document, and also refer to the experimental manuals in this field or other experimental methods known in this field, or refer to the experimental conditions recommended by the manufacturers.

[0133] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known means.

[0134] Example 1

[0135] This example is about the construction method of the characteristic chromatogram of the standard ginkgo leaf decoction.

[0136] 1. Instruments, reagents and test drugs

[0137] Instruments: Waters high performance liquid chromatograph (e2695, Waters Corporation); Waters Hss T3 chromatographic column (2.1mm×150mm, 1.8μm); One percent electronic analytical balance (JJ600, Changshu Shuangjie Testing Instrument Factory), One ten-thousandth electronic analytical balance (ME204E, Mettler-Toledo), One millionth electronic analytical balance (XP26, Mettler-Toledo); Numerical control ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.); Constant temperature water bath (Model HWS28, Shanghai Yiheng Technology Co., Ltd.); Ultra-pure water system (Milli-Q Direct, Merck KGaA).

[0138] Reagents: Methanol (Xilong Science Co., Ltd.) is of analytical grade; Phosphoric acid (Tianjin Kemiou Chemical Reagent Co., Ltd.), methanol (Merck KGaA) are of chromatographic grade, and water is ultra-pure water (self-made in the laboratory). Ginkgo leaf reference crude drug (Batch number: 121160 - 201304, National Institutes for Food and Drug Control); The batch number information of 18 batches of standard ginkgo leaf decoctions is shown in Table 1.

[0139] Table 1 Information Table of Freeze-dried Powders of 18 Batches of Standard Decoctions of Ginkgo Biloba Leaves

[0140]

[0141] 2. Preparation of Ginkgo Biloba Leaf Slices

[0142] For the slices of Ginkgo biloba leaves in the Chinese Pharmacopoeia (2020 Edition), the same method as that for the medicinal materials is adopted, so "removing impurities" is carried out. The specific processing method is as follows: Take the original medicinal materials of Ginkgo biloba leaves, and pick out impurities, deteriorated products, etc.

[0143] 3. Preparation of Standard Decoction of Ginkgo Biloba Leaf Slices

[0144] Take 100 g of Ginkgo biloba leaf slices, decoct twice with water. For the first decoction, add 12 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500 W), then keep it simmering gently over low heat (200 W) for 30 minutes, filter while it is hot through a 350-mesh sieve, and quickly cool the filtrate with cold water. For the second decoction, add 10 times the amount of water, bring to a boil over high heat (500 W), then keep it simmering gently over low heat (200 W) for 25 minutes, filter while it is hot through a 350-mesh sieve, and quickly cool the filtrate with cold water. Combine the two filtrates. Transfer the decoction to a 2000-ml round-bottom flask, and concentrate it under reduced pressure and low temperature (temperature: 65 °C; vacuum degree: -0.10 MPa) using a rotary evaporator to 150 ml. Dispense it into 10-ml vials, with 2 ml dispensed into each vial. Half-cork it, and after dispensing, transfer it to a vacuum freeze-dryer for freeze-drying to obtain the freeze-dried powder of the standard decoction.

[0145] 4. Chromatographic Conditions and Preparation of Test Solution

[0146] 4.1 Chromatographic Conditions

[0147] Use a Waters Hss T3 chromatographic column (2.1 mm × 150 mm, 1.8 μm). Methanol-acetonitrile (4:1) is used as mobile phase A, and 0.1% phosphoric acid is used as mobile phase B. Gradient elution is carried out according to the regulations in Table 2; the flow rate is 0.3 ml per minute, the column temperature is 35 °C; the detection wavelength is 365 nm; the injection volume is 5 μl.

[0148] Table 2 Gradient Elution Table

[0149]

[0150] 4.2 Preparation of Reference Substance Solution

[0151] Take appropriate amounts of rutin reference substance and narcissin reference substance, weigh accurately, and dissolve them in methanol to prepare a mixed solution containing 5 μg of each per 1 ml as the reference substance solution.

[0152] 4.3 Preparation of Test Solution

[0153] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves, grind it finely, take about 0.2 g, place it in a stoppered conical flask, add 25 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 15 minutes, let it cool, centrifuge, evaporate the supernatant to dryness, dissolve the residue in 50% methanol and dilute to a volume of 5 ml in a volumetric flask, filter, and take the subsequent filtrate to obtain the solution.

[0154] 4.4 Assay method

[0155] Precisely pipette 5 μl of the test solution and the reference solution respectively, inject them into the liquid chromatograph, and determine.

[0156] 5. Investigation of chromatographic conditions

[0157] 5.1 Determination of the optimal absorption wavelength

[0158] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves (No.: YXY - 1), grind it finely, take about 0.2 g, prepare the test solution according to the method under item "4.3", and perform analysis according to the provisions under item "4.1" except for different absorption wavelengths (set at 254 nm, 260 nm, 365 nm). The results are as Figure 1 、 Figure 2 and Figure 3 shown. The results show that when 365 nm is selected as the detection wavelength, the response values of each characteristic peak are relatively large, the baseline is stable, and the interference is small. Therefore, 365 nm is selected as the detection wavelength.

[0159] 5.2 Investigation of injection volume

[0160] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves (No.: YXY - 1), grind it finely, take about 0.2 g, prepare the test solution according to the method under item "4.3", and perform analysis according to the provisions under item "4.1" except for the injection volume (2 μl, 5 μl, 10 μl). The results are as Figure 4 、 Figure 5 and Figure 6 shown. The results show that by comparing the chromatograms of 3 different injection volumes, it can be found that when 5 μl is selected as the injection volume, the response value of the peak is better and the separation effect of each peak is better. Therefore, the injection volume of 5 μl is selected.

[0161] 6. Investigation of the preparation method of the test solution

[0162] 6.1 Investigation of extraction solvent

[0163] Appropriately take an equal amount of the same batch of standard Ginkgo biloba leaf decoction (No.: YXY-1), grind it finely, take about 0.2 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, 50% methanol, 30% methanol, and 10% methanol respectively, weigh it, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, centrifuge to obtain the supernatant, evaporate to dryness, and accurately dilute to 5 ml with 70% methanol, 50% methanol, 30% methanol, and 10% methanol respectively in a volumetric flask, filter, and take the subsequent filtrate to obtain the solution. Determine according to the chromatographic conditions specified under item "4.1", and the results are as Figure 7 shown. The results show that using 50% methanol as the extraction solvent can fully exhibit the characteristic chromatogram of the standard Ginkgo biloba leaf decoction. Therefore, 50% methanol is used as the extraction solvent for the characteristic chromatogram of the standard Ginkgo biloba leaf decoction.

[0164] 6.2 Investigation of extraction methods

[0165] Appropriately take an equal amount of the same batch of standard Ginkgo biloba leaf decoction (No.: YXY-1), grind it finely, take about 0.2 g, in two parallel portions, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes and heat under reflux for 30 minutes, take out, centrifuge to obtain the supernatant, evaporate to dryness, accurately dilute to 5 ml with 50% methanol in a volumetric flask, filter, and take the subsequent filtrate to obtain the solution, and the results are as Figure 8 shown. The results show that different extraction methods have little effect on the response and peak shape of the characteristic chromatogram of the standard Ginkgo biloba leaf decoction. Considering the convenience of operation, ultrasonic treatment is selected as the extraction method for the characteristic chromatogram of the standard Ginkgo biloba leaf decoction.

[0166] 6.3 Investigation of extraction time

[0167] Appropriately take an equal amount of the same batch of standard Ginkgo biloba leaf decoction (No.: YXY-1), grind it finely, take about 0.2 g, in three parallel portions, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, ultrasonically treat (power 250 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 45 minutes, take out, centrifuge to obtain the supernatant, evaporate to dryness, accurately dilute to 5 ml with 50% methanol in a volumetric flask, filter, and take the subsequent filtrate to obtain the solution. Determine according to the chromatographic conditions specified under item "4.1", and the results are as Figure 9 shown. The results show that different extraction times have little effect on the characteristic chromatogram of Ginkgo biloba. Therefore, ultrasonic extraction for 15 minutes is selected.

[0168] 6.4 Determination of the preparation method of the test solution

[0169] According to the above experimental results, the sample pretreatment method for the characteristic chromatogram of the standard Ginkgo biloba leaf decoction can be determined as:

[0170] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves, grind it finely, take about 0.2 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 15 minutes, centrifuge to obtain the supernatant, evaporate to dryness, dissolve it in 50% methanol and dilute to a volume of 5 ml in a volumetric flask, filter, and take the subsequent filtrate, that is obtained.

[0171] 7. Methodological investigation

[0172] 7.1 Specificity investigation

[0173] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves (No.: YXY-1), grind it finely, take about 0.2 g, prepare the test solution according to the method under item "4.3", accurately pipette 5 μl of the test solution, the reference substance solution of the reference substance under item "4.2" and the blank solvent respectively, inject the sample for analysis according to the chromatographic conditions under item "4.1", and the results are as Figure 10 shown. The results show that the test solution chromatogram has the same chromatographic peaks at the retention times corresponding to the reference substance chromatogram, and the blank solvent has no interference, indicating that the method has good specificity.

[0174] 7.2 Precision investigation

[0175] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves (No.: YXY-1), grind it finely, take about 0.2 g, prepare the test solution according to the method under item "4.3", and inject the sample repeatedly 6 times according to the chromatographic conditions under item "4.1". Using rutin as the reference peak S1, calculate the relative retention times and relative peak areas of peaks 1-3, peak 5 and the S1 peak; using narcissin as the reference peak S2, calculate the relative retention times and relative peak areas of peaks 6, 7, 9, 10 and the S2 peak, and calculate the RSD values. The RSD values of the relative retention times of 10 common peaks are in the range of 0.05% - 1.23%, and the RSD values of the relative peak areas are in the range of 2.50% - 2.91%, indicating that the instrument precision is good.

[0176] 7.3 Repeatability investigation

[0177] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves (No.: YXY-1), grind it finely, take about 0.5 g, prepare 6 test solutions according to the method under item "4.3", inject the sample for determination according to the chromatographic conditions under item "4.1". Using rutin as the reference peak S1, calculate the relative retention times and relative peak areas of peaks 1-3, peak 5 and the S1 peak; using narcissin as the reference peak S2, calculate the relative retention times and relative peak areas of peaks 6, 7, 9, 10 and the S2 peak, and calculate the RSD values. The RSD values of the relative retention times of 10 common peaks are in the range of 0.03% - 0.17%, and the RSD values of the relative peak areas are in the range of 0.13% - 1.27%, both less than 3.0%, indicating that the instrument precision is good and the method has good repeatability.

[0178] 7.4 Stability Study

[0179] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves (batch number: YXY-1), grind it finely, take about 0.2 g, prepare the test solution according to the method under "4.3", and inject samples for determination at 0, 4, 8, 12, and 24 hours respectively according to the chromatographic conditions under "4.1". Using rutin as the reference peak S1, calculate the relative retention time and relative peak area of peaks 1-3, peak 5 and peak S1; using naringin as the reference peak S2, calculate the relative retention time and relative peak area of peaks 6, 7, 9, 10 and peak S2, and calculate the RSD value. The RSD values of the relative retention times of the 10 common peaks are in the range of 0.04% - 0.23%, all less than 3%, and the RSD values of the relative peak areas are in the range of 0.25% - 2.2%, indicating that the test solution is relatively stable within 24 hours.

[0180] 8. Establishment of the Characteristic Chromatogram of the Standard Decoction of Ginkgo biloba Leaves

[0181] 8.1 Determination Results of the Characteristic Chromatogram of the Standard Decoction of Ginkgo biloba Leaves

[0182] Take 18 batches of the standard decoction of Ginkgo biloba leaves, according to the chromatographic conditions under "4.1" and the preparation method of the test solution determined under "6.4", using rutin as the reference peak S1, the relative retention times of characteristic peaks 1-5 and peak S are calculated as: 0.49 (peak 1), 0.75 (peak 2), 0.80 (peak 3), 1.25 (peak 5); the peak corresponding to the naringin reference substance peak is peak S2, and the relative retention times of characteristic peaks 6, 7-10 and peak S2 are calculated as: 0.76 (peak 6), 0.85 (peak 7), 1.24 (peak 9), 1.32 (peak 10). The characteristic chromatograms of 18 batches of the standard decoction are shown in Figure 11 as follows.

[0183] 8.2 Establishment of the Common Pattern of the Characteristic Chromatogram of the Standard Decoction of Ginkgo biloba Leaves

[0184] Take the reference medicinal material of Ginkgo biloba leaves (batch number: 121606-201602, National Institutes for Food and Drug Control), prepare the reference substance solution of the reference medicinal material according to the method under "4.2", for 18 batches of the standard decoction of Ginkgo biloba leaves, prepare the test solution according to the method under "4.3", inject samples for determination respectively according to the chromatographic conditions under "4.1", and obtain the characteristic chromatogram of the reference medicinal material of Ginkgo biloba leaves ( Figure 12 ); use the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" to generate the reference chromatogram by the average method, and establish the reference characteristic chromatogram of the standard decoction of Ginkgo biloba leaves ( Figure 13 ).

[0185] In the chromatogram of the test sample, 10 characteristic peaks should be presented, and their retention times should correspond to those of the 10 characteristic peaks in the chromatogram of the reference medicinal material. Among them, the retention times of peak 4 and peak 8 should correspond to those of the corresponding reference peaks of the reference substances respectively; the peak corresponding to the rutin reference peak is peak S1, and the relative retention times of characteristic peaks 1 - 3, peak 5 and peak S1 are calculated; the peak corresponding to the narcissin reference peak is peak S2, and the relative retention times of characteristic peaks 6, 7, 9, 10 and peak S2 are calculated. The relative retention times should be within the range of ±10% of the specified values, and the specified values are: 0.50 (peak 1), 0.77 (peak 2), 0.83 (peak 3), 1.25 (peak 5), 0.76 (peak 6), 0.84 (peak 7), 1.27 (peak 9), 1.36 (peak 10).

[0186] 9. Identification of reference substances for characteristic peaks

[0187] 9.1 High performance liquid chromatography conditions

[0188] Take an appropriate amount of the standard decoction of Ginkgo biloba leaves (No.: YXY - 1), grind it finely, take about 0.2 g, prepare the test solution according to the method under item "4.3", and conduct analysis according to the regulations under item "4.1". Use methanol - acetonitrile (4:1) as mobile phase A and 0.1% formic acid as mobile phase B, and perform gradient elution according to the regulations in Table 2; the flow rate is 0.3 ml per minute, the column temperature is 35 °C; the detection wavelength is 365 nm.

[0189] 9.2 Preparation of test solution and preparation of reference solution

[0190] The preparation of the test solution is the same as that under item "4.3"; separately take appropriate amounts of reference substances of typhaneoside, kaempferol - 3 - O - (2,6 - α - L - dirhamnosyl - β - D - glucopyranoside), rutin, kaempferol - 3 - O - rutinoside, narcissin, kaempferol - 3 - O - β - D - glucopyranosyl(1→2)-α - L - rhamnoside, accurately weigh them, and separately dissolve them in methanol to prepare solutions containing 5 μg of typhaneoside, kaempferol - 3 - O - (2,6 - α - L - dirhamnosyl - β - D - glucopyranoside), rutin, kaempferol - 3 - O - rutinoside, narcissin, kaempferol - 3 - O - β - D - glucopyranosyl(1→2)-α - L - rhamnoside per 1 ml as reference substance solution.

[0191] 9.3 Sample determination

[0192] Precisely pipette 5 μl of the test solution, inject it into the liquid chromatograph, and detect the test solution under the above liquid chromatography conditions. The ultraviolet absorption comparison chromatograms of the test solution and the reference solution are shown in Figure 14 、 Figure 15 、 Figure 16 and Figure 17 .

[0193] Example 2

[0194] This example is a method for determining the contents of multiple index components in the standard decoction of Ginkgo biloba leaves.

[0195] 1. Instruments, reagents, and test drugs

[0196] Same as Example 1.

[0197] 2. Preparation of Ginkgo biloba leaf pieces

[0198] Same as Example 1.

[0199] 3. Preparation of the standard decoction of Ginkgo biloba leaf pieces

[0200] Same as Example 1.

[0201] 4. Chromatographic conditions and preparation of test solution

[0202] 4.1 Chromatographic conditions

[0203] Same as Example 1.

[0204] 4.2 Preparation of reference solution

[0205] Weigh an appropriate amount of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), typhaneoside, rutin, kaempferol-3-O-rutinoside, narcissin, kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside reference substances accurately, and dissolve them separately in methanol to prepare standard solutions containing approximately 112 μg - 19 μg, 75 μg - 7 μg, 218 - 54 μg, 185 - 18 μg, 156 - 18 μg, 106 - 26 μg per 1 ml respectively as reference solutions.

[0206] 4.3 Preparation of test solution

[0207] Same as Example 1.

[0208] 4.5 Determination method

[0209] Precisely pipette 5 μl of the test solution and inject it into the liquid chromatograph for determination.

[0210] 5. Investigation of linear relationship

[0211] Accurately weigh an appropriate amount of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside) reference substance, dissolve it in methanol to prepare a reference substance stock solution with a mass concentration of 371.812 μg / ml, and then dilute it with methanol to prepare reference substance solutions with concentrations of 111.544 μg / ml, 92.953 μg / ml, 74.362 μg / ml, 46.476 μg / ml, 37.316 μg / ml, and 18.658 μg / ml respectively.

[0212] Accurately weigh an appropriate amount of typhaneoside reference substance, dissolve it in methanol to prepare a reference substance stock solution with a mass concentration of 378.859 μg / ml, and then dilute it with methanol to prepare reference substance solutions with concentrations of 75.771 μg / ml, 53.040 μg / ml, 37.886 μg / ml, 30.309 μg / ml, 15.154 μg / ml, and 7.578 μg / ml respectively.

[0213] Accurately weigh an appropriate amount of rutin reference substance, dissolve it in methanol to prepare a reference substance stock solution with a mass concentration of 362.9192 μg / ml, and then dilute it with methanol to prepare reference substance solutions with concentrations of 217.752 μg / ml, 181.460 μg / ml, 163.314 μg / ml, 108.876 μg / ml, 81.660 μg / ml, and 54.438 μg / ml respectively.

[0214] Accurately weigh an appropriate amount of kaempferol-3-O-rutinoside reference substance, dissolve it in methanol to prepare a reference substance stock solution with a mass concentration of 368.000 μg / ml, and then dilute it with methanol to prepare reference substance solutions with concentrations of 184.240 μg / ml, 138.180 μg / ml, 92.120 μg / ml, 73.696 μg / ml, 36.848 μg / ml, and 18.424 μg / ml respectively.

[0215] Accurately weigh an appropriate amount of narcissin reference substance, dissolve it in methanol to prepare a reference substance stock solution with a mass concentration of 388.786 μg / ml, and then dilute it with methanol to prepare reference substance solutions with concentrations of 155.514 μg / ml, 116.636 μg / ml, 87.477 μg / ml, 62.206 μg / ml, 37.323 μg / ml, and 18.662 μg / ml respectively.

[0216] Accurately weigh an appropriate amount of kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnose reference substance, dissolve it in methanol to prepare a reference substance stock solution with a mass concentration of 352.000 μg / ml, and then dilute it with methanol to prepare reference substance solutions with concentrations of 105.759 μg / ml, 88.132 μg / ml, 63.455 μg / ml, 52.879 μg / ml, 42.303 μg / ml, and 26.440 μg / ml respectively.

[0217] Precisely pipette 1 μl of the reference substance solutions with different concentrations prepared above for injection and determination, and record the chromatographic peak areas. Taking the peak area as the ordinate (y) and the injection concentration of the reference substance as the abscissa (x), as shown in Table 3.

[0218] Table 3 Regression Equation and Linear Range

[0219]

[0220] 6. Precision Investigation

[0221] Precisely pipette 1 μl of the kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside) reference substance solution with a content of 74.362 μg / ml, typhaneoside reference substance solution with a content of 30.309 μg / ml, rutin reference substance solution with a content of 108.876 μg / ml, kaempferol-3-O-rutinoside reference substance solution with a content of 73.696 μg / ml, narcissin reference substance solution with a content of 62.206 μg / ml, and kaempferol-3-O-β-D-glucose(1→2)-α-L-rhamnose reference substance solution with a content of 52.879 μg / ml prepared above for injection and determination (the test method is the same as the above chromatographic conditions). The same reference substance solution was continuously and repeatedly determined 6 times to evaluate the precision of the instrument. The results showed that the RSDs of the peak areas of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), typhaneoside, rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucose(1→2)-α-L-rhamnoside were all less than 5%, indicating good precision of the instrument.

[0222] 7. Repeatability Investigation

[0223] Take about 0.2 g of the standard decoction of Ginkgo biloba leaves (batch number: YXY-1), precisely weigh it, make 6 parallel portions, prepare the test solution according to the determined preparation method of the test solution, inject it for determination, and determine the contents of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), typhaneoside, rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucose(1→2)-α-L-rhamnose in the test solution (the test method is the same as the above chromatographic conditions). The experimental results showed that when the same batch of samples was repeatedly determined 6 times, the RSDs of the peak areas of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), typhaneoside, rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucose(1→2)-α-L-rhamnoside were all less than 5%, indicating good repeatability of this analytical method.

[0224] 8. Stability Investigation

[0225] Take about 0.2 g of the standard decoction of Ginkgo biloba leaves (batch number: YXY-1), accurately weigh it, make 6 parallel portions, prepare the test solution according to the determined preparation method of the test solution, and measure it at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively, and record the peak areas (the test method is the same as the above chromatographic conditions). The results show that the RSD values of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), typhaneoside, rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside are all less than 5%, indicating that the test solution is stable within 24 h when placed at room temperature.

[0226] 9. Results of content determination

[0227] Take the standard decoction of Ginkgo biloba leaves, prepare the test solution according to the above preparation method of the test sample, inject the sample for determination according to the above chromatographic conditions, and calculate the contents of kaempferol-3-O-(2,6-α-L-dirhamnopyranosyl-β-D-glucopyranoside), typhaneoside, rutin, kaempferol-3-O-rutinoside, narcissin, and kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnose by the external standard method. The results are shown in Table 4 below:

[0228] Table 4 Results of content determination of 3 batches of standard decoctions of Ginkgo biloba leaves

[0229]

[0230] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0231] The above-described embodiments only represent several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided in the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the content of the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. A method for constructing a characteristic fingerprint of a standard decoction of Ginkgo biloba leaves, characterized in that, It includes the following steps: Mix the standard ginkgo leaf decoction with a first solvent for extraction to prepare a test solution; Perform high performance liquid chromatography (HPLC) detection on the test solution to construct the characteristic chromatogram of the standard ginkgo leaf decoction; Among them, the conditions for the HPLC detection include: The chromatographic column is a T3 chromatographic column; The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixture of methanol and acetonitrile with a volume ratio of (3 - 5):1, and mobile phase B is an aqueous phosphoric acid solution with a volume percentage of 0.08% - 0.12%. A gradient elution program is adopted.

2. The method for constructing the characteristic spectrum of the standard ginkgo leaf decoction according to claim 1, wherein, The gradient elution program includes: From 0 min to 30 min, the volume percentage of mobile phase A is maintained at 28%, and the volume percentage of mobile phase B is maintained at 72%; From 30 min to 40 min, the volume percentage of mobile phase A changes from 28% to 30%, and the volume percentage of mobile phase B changes from 72% to 70%; From 40 min to 45 min, the volume percentage of mobile phase A changes from 30% to 35%, and the volume percentage of mobile phase B changes from 70% to 65%; From 45 min to 55 min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%.

3. The method for constructing the characteristic fingerprint of the standard decoction of Ginkgo biloba leaves according to claim 1, wherein The conditions for the HPLC detection further include at least one of the following: (1) The flow rate is 0.2 mL / min - 0.4 mL / min; (2) The column temperature is 33°C - 37°C; (3) The detection wavelength is 363 nm - 367 nm; (4) The injection volume is 3 μL - 10 μL.

4. The method for constructing the characteristic spectrum of the standard ginkgo leaf decoction according to any one of claims 1 to 3, characterized in that, The preparation of the test solution has at least one of the following characteristics: (1) The first solvent is an aqueous methanol solution with a volume percentage of 10% - 60%; (2) The extraction method is ultrasonic extraction or reflux extraction; (3) The extraction time is 10 min - 50 min.

5. The method for constructing the characteristic chromatogram of the standard ginkgo leaf decoction according to any one of claims 1 to 3, characterized in that, It further includes the step of characteristic peak identification: Mix the reference substance with a second solvent to prepare a reference solution; the reference substance includes one or more of the reference substances of typhaneoside, kaempferol - 3 - O - (2,6 - α - L - dirhamnopyranosyl - β - D - glucopyranoside), rutin, kaempferol - 3 - O - rutinoside, narcissin, and kaempferol - 3 - O - β - D - glucose(1→2)-α - L - rhamnoside; Perform the HPLC detection on the reference substance; Optionally, the second solvent is methanol.

6. A method for determining the contents of multiple index components in a standard decoction of Ginkgo biloba leaves, characterized in that, It includes the following steps: Mix the to - be - tested standard ginkgo leaf decoction with a first solvent for extraction to prepare a test solution; Mix the reference substance with a second solvent to prepare reference solutions with different concentrations; the reference substance includes one or more of the reference substances of typhaneoside, kaempferol - 3 - O - (2,6 - α - L - dirhamnopyranosyl - β - D - glucopyranoside), rutin, kaempferol - 3 - O - rutinoside, narcissin, and kaempferol - 3 - O - β - D - glucose(1→2)-α - L - rhamnoside; Perform HPLC detection on the reference solutions with different concentrations and construct the standard curve of the reference substance according to the detection results; Perform high performance liquid chromatography (HPLC) detection on the test sample solution, substitute the detection results into the standard curve of the reference substance, and calculate the content of the corresponding index components; Among them, the conditions for the HPLC detection include: The chromatographic column is a T3 chromatographic column; The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixture of methanol and acetonitrile with a volume ratio of (3 - 5):1, and mobile phase B is an aqueous phosphoric acid solution with a volume percentage of 0.08% - 0.12%. A gradient elution program is adopted.

7. The content determination method of multiple index components in the standard ginkgo leaf decoction according to claim 6, characterized in that, The gradient elution program includes: From 0 min to 30 min, the volume percentage of mobile phase A is maintained at 28%, and the volume percentage of mobile phase B is maintained at 72%; From 30 min to 40 min, the volume percentage of mobile phase A changes from 28% to 30%, and the volume percentage of mobile phase B changes from 72% to 70%; From 40 min to 45 min, the volume percentage of mobile phase A changes from 30% to 35%, and the volume percentage of mobile phase B changes from 70% to 65%; From 45 min to 55 min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%.

8. The content determination method of multiple index components in the standard ginkgo leaf decoction according to claim 6, characterized in that, The conditions for the HPLC detection further include at least one of the following: (1) The flow rate is 0.2 mL / min - 0.4 mL / min; (2) The column temperature is 33°C - 37°C; (3) The detection wavelength is 363 nm - 367 nm; (4) The injection volume is 3 μL - 10 μL.

9. The content determination method of multi-index components in the standard ginkgo leaf decoction according to any one of claims 6 to 8, characterized in that, The preparation of the test sample solution has at least one of the following characteristics: (1) The first solvent is an aqueous methanol solution with a volume percentage of 10% - 60%; (2) The extraction method is ultrasonic extraction or reflux extraction; (3) The extraction time is 10 min - 50 min.

10. The method for determining the contents of multi-index components in the standard ginkgo leaf decoction according to any one of claims 6 to 8, characterized in that, The standard curve of typhaneoside is: y = 4369.4x + 2509.2; The standard curve of kaempferol - 3 - O - (2,6 - α - L - dirhamnopyranosyl - β - D - glucopyranoside) is: y = 3505.4x - 4244.3; The standard curve of rutin is: y = 6044x - 28958; The standard curve of kaempferol - 3 - O - rutinoside is: y = 4530.9x - 5766.7; The standard curve of narcissin is: y = 5258.8x + 12960; The standard curve of kaempferol - 3 - O - β - D - glucopyranosyl(1→2) - α - L - rhamnoside is: y = 3535.2x + 1324.

3.

11. Application of the characteristic fingerprint of the Ginkgo biloba standard decoction constructed by the construction method according to any one of claims 1 - 5 or the method for determining the content of multiple index components in the Ginkgo biloba standard decoction according to any one of claims 6 - 10 in the quality inspection of the Ginkgo biloba standard decoction.

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