Methods for constructing characteristic spectra of standard Ginkgo biloba decoction and determining the content of multiple index components.

Abstract

This application relates to a method for constructing a characteristic chromatogram of a standard Ginkgo biloba decoction and a method for determining the content of multiple index components. The method for constructing the characteristic chromatogram of the standard Ginkgo biloba decoction includes the following steps: extracting the standard Ginkgo biloba decoction with a first solvent to prepare a test solution; performing high-performance liquid chromatography (HPLC) on the test solution to construct the characteristic chromatogram of the standard Ginkgo biloba decoction; wherein the HPLC conditions include: a T3 column; mobile phases A and B, wherein mobile phase A is methanol and acetonitrile in a volume ratio of (3~5):1, and mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.08%~0.12%, using a gradient elution program. The characteristic chromatogram constructed by this method has many common peaks and detects the prototype components of flavonol glycosides, exhibiting rapid, stable, and highly specific detection.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for constructing a characteristic spectrum of a standard decoction of Ginkgo biloba leaves and a method for determining the content of multiple index components. Background Technology

[0002] Ginkgo leaves are the dried leaves of the Ginkgo biloba L. plant, belonging to the Ginkgoaceae family. They are sweet, bitter, astringent, and neutral in nature, and enter the heart and lung meridians. They possess the functions of promoting blood circulation and removing blood stasis, clearing the meridians and relieving pain, astringing the lungs and relieving asthma, and resolving turbidity and lowering lipids. They are used for blood stasis obstructing the meridians, chest pain, hemiplegia due to stroke, cough and asthma due to lung deficiency, and hyperlipidemia. Modern pharmacological studies suggest that the main active components of ginkgo leaves are flavonoids and terpenoid lactones, which have antidiarrheal, antioxidant, hepatoprotective, lipid-lowering, blood glucose-lowering, immune-enhancing, and anti-tumor effects. The 2020 edition of the Chinese Pharmacopoeia and existing literature reports mostly use acid hydrolysis to indirectly calculate the total flavonol glycosides in ginkgo leaves by determining the contents of quercetin, kaempferol, and isorhamnetin, thus achieving quantitative analysis of the original components. Direct determination of the original components of flavonol glycosides in ginkgo leaves is currently less reported. At the same time, the specificity of acid hydrolysis method is also poor.

[0003] Traditional Chinese medicine (TCM) has complex components, with synergistic effects among multiple components. TCM fingerprinting / characteristic spectroscopy can comprehensively consider the composition of TCM components from a holistic perspective, making it particularly suitable for TCM quality evaluation. Currently, there are few research reports on Ginkgo biloba leaf fingerprinting / characteristic spectroscopy, and they mainly focus on the fingerprinting or characteristic spectroscopy of Ginkgo biloba leaf raw materials / ethanol extracts. There are also few reports on the application of fingerprinting / characteristic spectroscopy to traditional TCM decoctions and related preparations made from Ginkgo biloba leaves.

[0004] Traditional Chinese medicine (TCM) primarily utilizes decoctions as its main form of administration. Decoctions form the material basis for the efficacy of TCM. Standardized TCM decoctions are standardized formulations of traditional TCM decoctions, prepared using modern extraction methods and standardized processes. These single-herb decoctions serve as a standard substance and system for standardizing the quality of modern TCM preparations such as granules. Since standard TCM decoctions use water as a solvent, water-soluble components are crucial for quality control. However, in the aforementioned acid hydrolysis method, aglycones such as quercetin, kaempferol, and isorhamnetin, obtained through acid hydrolysis, have low water solubility. Therefore, determining the water-soluble precursor components is particularly important for quality control of Ginkgo biloba leaf decoctions.

[0005] In addition, other methods have been developed for quality evaluation of Ginkgo biloba leaf standard decoctions and the establishment of high-performance liquid chromatography (HPLC) fingerprint spectra. These methods involve establishing fingerprint spectra of Ginkgo biloba leaf standard decoctions using HPLC and determining the content of quercetin, kaempferol, and isorhamnetin. However, the fingerprint spectra obtained by these methods have few common peaks, poor peak resolution, and the failure to effectively identify the main common peaks. Therefore, the methods lack specificity and are not very meaningful for quality control. Furthermore, the content determination methods still rely on acid hydrolysis to determine the content of the three aglycones (quercetin, kaempferol, and isorhamnetin), indirectly calculating the total flavonol glycoside content, which also indicates weak specificity. Summary of the Invention

[0006] Based on this, this application provides a method for constructing a characteristic spectrum of a standard Ginkgo biloba decoction and a method for determining the content of multiple index components. The characteristic spectrum constructed by this method has many common peaks and detects the prototype components of flavonol glycosides, which are rapid, stable, and highly specific.

[0007] The first aspect of this application provides a method for constructing a characteristic spectrum of a standard Ginkgo biloba decoction, comprising the following steps:

[0008] The standard decoction of ginkgo leaves was mixed with the first solvent for extraction to prepare the test solution;

[0009] The test solution was analyzed by high performance liquid chromatography to construct a characteristic chromatogram of the Ginkgo biloba leaf standard decoction;

[0010] The conditions for the high-performance liquid chromatography detection include:

[0011] The chromatographic column is a T3 column;

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

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

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

[0015] Between 30 and 40 minutes, the volume percentage of mobile phase A changed from 28% to 30%, and the volume percentage of mobile phase B changed from 72% to 70%.

[0016] Over 40-45 minutes, the volume percentage of mobile phase A changed from 30% to 35%, and the volume percentage of mobile phase B changed from 70% to 65%.

[0017] For 45-55 minutes, 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 embodiment, the conditions for the high-performance liquid chromatography detection further include at least one of the following:

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

[0020] (2) The column temperature is 33℃~37℃;

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

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

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

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

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

[0026] (3) Extraction time is 10 min to 50 min.

[0027] In one embodiment, the method for constructing the characteristic spectrum of the Ginkgo biloba leaf standard decoction further includes a characteristic peak identification step:

[0028] A reference solution is prepared by mixing the reference standard with a second solvent; the reference standard includes one or more of the following: typhain, kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucopyranoside reference standards;

[0029] The reference standard was subjected to the high-performance liquid chromatography detection described above;

[0030] Optionally, the second solvent is methanol.

[0031] A second aspect of this application provides a method for determining the content of multiple index components in a standard Ginkgo biloba leaf decoction, comprising the following steps:

[0032] The standard decoction of Ginkgo biloba leaves to be tested was mixed with the first solvent for extraction to prepare the test solution;

[0033] The reference standard is mixed with a second solvent to prepare reference standard solutions of different concentrations; the reference standard includes one or more of the following: typhaein, kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucoside (1-2)-α-L-rhamnoside reference standards;

[0034] The reference solutions of different concentrations were detected by high performance liquid chromatography, and a standard curve of the reference standards was constructed based on the detection results;

[0035] The test solution was subjected to high performance liquid chromatography (HPLC) for detection, and the detection results were substituted into the standard curve of the reference standard to calculate the content of the corresponding index components.

[0036] The conditions for the high-performance liquid chromatography detection include:

[0037] The chromatographic column is a T3 column;

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

[0039] In one embodiment, the gradient elution process includes:

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

[0041] Between 30 and 40 minutes, the volume percentage of mobile phase A changed from 28% to 30%, and the volume percentage of mobile phase B changed from 72% to 70%.

[0042] Over 40-45 minutes, the volume percentage of mobile phase A changed from 30% to 35%, and the volume percentage of mobile phase B changed from 70% to 65%.

[0043] For 45-55 minutes, 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 embodiment, the conditions for the high-performance liquid chromatography 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℃~37℃;

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

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

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

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

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

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

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

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

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

[0056] The standard curve for the kaempferol-3-O-rutin glycoside is: y = 4530.9x - 5766.7;

[0057] The standard curve for narcisin is: y = 5258.8x + 12960;

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

[0059] A third aspect of this application provides the application of the characteristic spectrum of the Ginkgo biloba leaf standard decoction obtained by the construction method described in the first aspect or the method for determining the content of multiple index components in the Ginkgo biloba leaf standard decoction described in the second aspect in the quality detection of the Ginkgo biloba leaf standard decoction.

[0060] The method for constructing the characteristic chromatogram of the above-mentioned Ginkgo biloba leaf standard decoction, by using appropriate chromatographic conditions, yields a characteristic chromatogram with numerous common peaks. These peaks detect the prototype components of flavonol glycosides, fully reflecting the characteristic peak information of the Ginkgo biloba leaf standard decoction and demonstrating its chemical composition characteristics. The method is rapid, stable, and highly specific, enabling the identification and quality control of Ginkgo biloba leaves. It provides a new technical method for quality control and offers important reference for the formulation of quality standards for Ginkgo biloba leaves and related preparations.

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

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

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

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

[0065] Figure 4 This is the chromatogram of the standard decoction of ginkgo leaves in Example 1 when the injection volume was 2 μl.

[0066] Figure 5 This is the chromatogram of the standard decoction of ginkgo leaves in Example 1 when the injection volume was 5 μl.

[0067] Figure 6 This is the chromatogram of the standard decoction of ginkgo leaves in Example 1 when the injection volume was 10 μl.

[0068] Figure 7 The chromatograms are of different extraction solvents used in the construction of the characteristic chromatogram of the standard Ginkgo leaf decoction in Example 1;

[0069] Figure 8 These are chromatograms of different extraction methods used in constructing the characteristic chromatogram of the standard Ginkgo leaf decoction in Example 1;

[0070] Figure 9 The chromatograms are those obtained at different extraction times during the construction of the characteristic chromatogram of the standard Ginkgo leaf decoction in Example 1.

[0071] Figure 10 This is the chromatogram used for specificity assessment during the construction of the characteristic chromatogram of the standard Ginkgo leaf decoction in Example 1;

[0072] Figure 11 This is a superimposed image of the characteristic spectra of 18 batches of Ginkgo biloba leaf standard decoction in the construction of the characteristic spectra of Ginkgo biloba leaf standard decoction in Example 1;

[0073] Figure 12The characteristic spectrum of Ginkgo biloba leaf reference material in the construction of the characteristic spectrum of Ginkgo biloba leaf standard decoction in Example 1 is shown in the following figures: Peak 2: kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside); Peak 3: typhain; Peak 4 (S1): rutin; Peak 7: kaempferol-3-O-rutin; Peak 8 (S2): narcissin; Peak 9: kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside.

[0074] Figure 13 The characteristic spectrum of Ginkgo biloba leaf decoction in Example 1 is a control characteristic spectrum of the standard decoction of Ginkgo biloba leaf. Peak 2: kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside); Peak 3: typhain; Peak 4 (S1): rutin; Peak 7: kaempferol-3-O-rutin; Peak 8 (S2): narcissin; Peak 9: kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside.

[0075] Figure 14 The chromatograms are comparisons of the test solution of Ginkgo biloba leaf standard decoction and the reference solution of typhaein in Example 1.

[0076] Figure 15 The chromatograms are shown below for comparison between the test solution of the standard decoction of Ginkgo biloba leaves in Example 1 and the reference solution of kaempferol-3-O-(2,6-α-L-dipyranorhamnetosyl-β-D-glucopyranoside);

[0077] Figure 16 The chromatograms are compared between the test solution of the standard decoction of Ginkgo biloba leaves in Example 1 and the reference solutions of rutin, kaempferol-3-O-rutin, and narcissin.

[0078] Figure 17 The chromatograms are comparisons between the test solution of the standard decoction of Ginkgo biloba leaves in Example 1 and the reference solution of kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnoside. Detailed Implementation

[0079] The following detailed description, in conjunction with specific embodiments, illustrates the method for constructing the characteristic chromatogram of the Ginkgo biloba leaf standard decoction and the method for determining the content of multiple index components. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

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

[0082] In this application, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only 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, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0083] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0084] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0085] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0086] Unless otherwise specified, all percentage concentrations mentioned in this application refer to final concentrations. The final concentration refers to the percentage of the added component in the system after its addition. Unless otherwise specified, water is used as the solvent in all solutions.

[0087] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0088] In this application, room temperature generally refers to 4℃~30℃, and preferably 20±5℃.

[0089] Some examples in this application provide a method for constructing the characteristic spectrum of a standard Ginkgo biloba decoction, including the following steps:

[0090] The standard decoction of ginkgo leaves was mixed with the first solvent for extraction to prepare the test solution;

[0091] The test solution was analyzed by high performance liquid chromatography to construct a characteristic chromatogram of the Ginkgo biloba leaf standard decoction;

[0092] The conditions for the high-performance liquid chromatography detection include:

[0093] The chromatographic column is a T3 column;

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

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

[0096] In some of these examples, the gradient elution procedure includes:

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

[0098] Between 30 and 40 minutes, the volume percentage of mobile phase A changed from 28% to 30%, and the volume percentage of mobile phase B changed from 72% to 70%.

[0099] Over 40-45 minutes, the volume percentage of mobile phase A changed from 30% to 35%, and the volume percentage of mobile phase B changed from 70% to 65%.

[0100] For 45-55 minutes, 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, the flow rate in the high-performance liquid chromatography detection is 0.2 mL / min to 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, and 0.4 mL / min.

[0102] In some of these examples, the column temperature in the high-performance liquid chromatography (HPLC) detection is 33°C to 37°C. Specifically, the column temperature includes, but is not limited to, 33°C, 34°C, 35°C, 36°C, and 37°C.

[0103] In some examples, the detection wavelength in the high-performance liquid chromatography (HPLC) detection is 363 nm to 367 nm. Specifically, the detection wavelengths include, but are not limited to, 363 nm, 364 nm, 365 nm, 366 nm, and 367 nm.

[0104] In some of these examples, the injection volume in the high-performance liquid chromatography (HPLC) detection 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, and 10 μL.

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

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

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

[0108] In some examples, the method for constructing the characteristic spectrum of the Ginkgo biloba leaf standard decoction also includes a characteristic peak identification step:

[0109] A reference solution is prepared by mixing the reference standard with a second solvent; the reference standard includes one or more of the following: typhain, kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucopyranoside reference standards;

[0110] The reference standard was subjected to the high-performance liquid chromatography detection.

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

[0112] In some examples, the method for constructing the characteristic spectrum of the Ginkgo biloba leaf standard decoction also includes a reference solution detection step:

[0113] The reference sample is mixed with a third solvent to prepare a reference sample solution;

[0114] The reference solution was subjected to high-performance liquid chromatography for detection; the reference solution included rutin and narcisin.

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

[0116] Other examples of this application provide a method for determining the content of multiple index components in a standard Ginkgo biloba decoction, comprising the following steps:

[0117] The standard decoction of Ginkgo biloba leaves to be tested was mixed with the first solvent for extraction to prepare the test solution;

[0118] The reference standard is mixed with a second solvent to prepare reference standard solutions of different concentrations; the reference standard includes one or more of the following: typhaein, kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucoside (1-2)-α-L-rhamnoside reference standards;

[0119] The reference solutions of different concentrations were detected by high performance liquid chromatography, and a standard curve of the reference standards was constructed based on the detection results;

[0120] The test solution was subjected to high performance liquid chromatography (HPLC) for detection, and the detection results were substituted into the standard curve of the reference standard to calculate the content of the corresponding index components.

[0121] The conditions for the high-performance liquid chromatography detection include:

[0122] The chromatographic column is a T3 column;

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

[0124] Understandably, the gradient elution procedure, the conditions for high-performance liquid chromatography detection, and the conditions for preparing the test solution are the same as those for constructing the aforementioned characteristic chromatograms, and will not be repeated here.

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

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

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

[0128] The standard curve for the kaempferol-3-O-rutin glycoside is: y = 4530.9x - 5766.7;

[0129] The standard curve for narcisin is: y = 5258.8x + 12960;

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

[0131] Other examples of this application also provide the application of the characteristic spectrum of the Ginkgo biloba leaf standard decoction obtained by the construction method described above, or the method for determining the content of multiple index components in the Ginkgo biloba leaf standard decoction as described above, in the quality detection of the Ginkgo biloba leaf standard decoction.

[0132] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0133] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0134] Example 1

[0135] This embodiment describes a method for constructing the characteristic spectrum of a standard Ginkgo biloba decoction.

[0136] 1. Instruments, reagents and reagents

[0137] Instruments: Waters high-performance liquid chromatograph (e2695, Waters Corporation); Waters Hss T3 column (2.1mm×150mm, 1.8μm); 1 / 1000 electronic analytical balance (JJ600, Changshu Shuangjie Test Instrument Factory), 0.001% electronic analytical balance (ME204E, Mettler Toledo), 0.1% electronic analytical balance (XP26, Mettler Toledo), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.); constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.); ultrapure water system (Milli-Q Direct, Merck KGaA).

[0138] Reagents: Methanol (Xilong Technology Co., Ltd.) was analytical grade; phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd.) and methanol (Merck Corporation) were chromatographic grade; water was ultrapure water (prepared in the laboratory). Ginkgo biloba leaf reference material (batch number: 121160-201304, China National Institutes for Food and Drug Control); batch number information of 18 batches of Ginkgo biloba leaf standard decoction is shown in Table 1.

[0139] Table 1 Information on 18 batches of Ginkgo Leaf Standard Decoction Freeze-dried Powder

[0140]

[0141] 2. Preparation of Ginkgo Leaf Slices

[0142] The Chinese Pharmacopoeia 2020 edition lists Ginkgo biloba leaves under the category of processed medicinal materials, therefore the processing method is "removing impurities." The specific processing method is as follows: Take the raw Ginkgo biloba leaves and remove impurities, deteriorated products, etc.

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

[0144] Take 100g of ginkgo leaf slices, add water and decoct twice. For the first decoction, add 12 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter while 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 (500W), then simmer over low heat (200W) for 25 minutes. Filter while hot through a 350-mesh sieve, and quickly cool the filtrate with cold water. Combine the two filtrates. Transfer the decoction to a 2000ml round-bottom flask and concentrate under reduced pressure at low temperature using a rotary evaporator (temperature: 65℃; vacuum: -0.10MPa) to 150ml. Dispense into 10ml vials, each with a volume of 2ml, half-stop, and then freeze-dry in a vacuum freeze dryer to obtain a standard lyophilized powder.

[0145] 4. Chromatographic conditions and preparation of test solution

[0146] 4.1 Chromatographic conditions

[0147] A Waters Hss T3 column (2.1 mm × 150 mm, 1.8 μm) was used with methanol-acetonitrile (4:1) as mobile phase A and 0.1% phosphoric acid as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.3 mL per minute, the column temperature was 35 °C, the detection wavelength was 365 nm, and the injection volume was 5 μl.

[0148] Table 2 Gradient Elution Table

[0149]

[0150] 4.2 Preparation of reference solution

[0151] Take appropriate amounts of rutin and narcisin reference standards, weigh them accurately, and add methanol to prepare a mixed solution containing 5 μg of each per 1 ml, which is used as the reference solution.

[0152] 4.3 Preparation of test solution

[0153] Take an appropriate amount of standard decoction of ginkgo leaves, grind it into a fine powder, take about 0.2g, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 250W, frequency 40kHz) for 15 minutes, cool, centrifuge, evaporate the supernatant to dryness, add 50% methanol to the residue and make up to 5ml in a volumetric flask, filter, and take the filtrate to obtain the final product.

[0154] 4.4 Determination Method

[0155] Accurately pipette 5 μl of the test solution and the reference solution into the liquid chromatograph for determination.

[0156] 5. Investigation of chromatographic conditions

[0157] 5.1 Determination of the optimal absorption wavelength

[0158] Take an appropriate amount of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it into a fine powder, and take about 0.2g. Prepare the test solution according to the method in section "4.3". Except for different absorption wavelengths (set to 254nm, 260nm, and 365nm), all other analyses are performed according to the specifications in section "4.1". The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown in the figure. The results show that when 365nm is selected as the detection wavelength, the response values ​​of each characteristic peak are larger, the baseline is stable, and the interference is smaller. Therefore, 365nm is selected as the detection wavelength.

[0159] 5.2 Investigation of injection volume

[0160] Take an appropriate amount of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it into a fine powder, and take about 0.2g. Prepare the test solution according to the method in section "4.3". Except for the injection volume (2μl, 5μl, 10μl), all other analyses are performed according to the provisions in section "4.1". The results are as follows: Figure 4 , Figure 5 and Figure 6 As shown in the figure. The results show that by comparing the chromatograms of three different injection volumes, it can be found that when 5 μl is selected as the injection volume, the peak response values ​​are better and the separation 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 Solvents

[0163] Take appropriate amounts of the same batch of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind them finely, and accurately weigh approximately 0.2g. Place the weighed amounts in stoppered conical flasks, and accurately add 25ml each of 70% methanol, 50% methanol, 30% methanol, and 10% methanol. Weigh the flasks, sonicate (250W, 40kHz) for 30 minutes, centrifuge, collect the supernatant, evaporate to dryness, and dilute to 5ml in volumetric flasks with 70% methanol, 50% methanol, 30% methanol, and 10% methanol, respectively. Filter the solution and collect the filtrate. Perform the determination according to the chromatographic conditions specified in section "4.1". The results are as follows: Figure 7 As shown in the figure. The results indicate that using 50% methanol as the extraction solvent can fully represent the characteristic spectrum of the standard Ginkgo biloba decoction. Therefore, 50% methanol was used as the extraction solvent for the characteristic spectrum of the standard Ginkgo biloba decoction.

[0164] 6.2 Examination of Extraction Methods

[0165] Take an appropriate amount of the same batch of Ginkgo biloba leaf standard decoction (number: YXY-1), grind it into a fine powder, take about 0.2g, divide into two parallel portions, weigh accurately, place in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh, sonicate (power 250W, frequency 40kHz) for 30 minutes, heat under reflux for 30 minutes, remove, centrifuge to collect the supernatant, evaporate to dryness, dilute to 5ml volumetric flask with 50% methanol, filter, and collect the filtrate to obtain the final product. The results are as follows. Figure 8 As shown in the figure. The results showed that different extraction methods had little effect on the response and peak shape of the characteristic spectrum of Ginkgo biloba leaf standard decoction. Considering ease of operation, ultrasonic treatment was selected as the extraction method for the characteristic spectrum of Ginkgo biloba leaf standard decoction.

[0166] 6.3 Examination of Extraction Time

[0167] Take an appropriate amount of the same batch of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it into a fine powder, take about 0.2g, divide into three parallel portions, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh them, and sonicate (power 250W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Remove them, centrifuge them, collect the supernatant, evaporate to dryness, and dilute to 5ml with 50% methanol in a volumetric flask. Filter, and collect the filtrate. Determine the chromatographic conditions specified in section "4.1". The results are as follows: Figure 9 As shown in the figure. The results showed that different extraction times had little effect on the characteristic maps of Ginkgo biloba leaves, so ultrasonic extraction for 15 minutes was chosen.

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

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

[0170] Take an appropriate amount of standard Ginkgo biloba leaf decoction, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, sonicate (power 250W, frequency 40kHz) for 15 minutes, centrifuge to collect the supernatant, evaporate to dryness, dilute to 5ml volume with 50% methanol, filter, and collect the filtrate to obtain the final product.

[0171] 7. Methodological Examination

[0172] 7.1 Specificity Examination

[0173] Take an appropriate amount of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it into a fine powder, and take about 0.2g. Prepare the test solution according to the method in section "4.3". Accurately pipette 5μl each of the test solution, the reference solution in section "4.2", and the blank solvent, and inject them for analysis according to the chromatographic conditions in section "4.1". The results are as follows: Figure 10 As shown in the figure. The results indicate that the chromatogram of the test sample shows the same chromatographic peak at the corresponding retention time as that of the reference sample, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0174] 7.2 Precision Test

[0175] Take an appropriate amount of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it into a fine powder, take about 0.2g, and prepare the test solution according to the method in section "4.3". Inject the sample 6 times repeatedly under the chromatographic conditions in section "4.1". Using rutin as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1-3 and 5 with peak S1. Using narcisin as the reference peak S2, calculate the relative retention time and relative peak area of ​​peaks 6, 7, 9, and 10 with peak S2, and calculate the RSD value. The RSD value of the relative retention time of the 10 common peaks is in the range of 0.05%~1.23%, and the RSD value of the relative peak area is in the range of 2.50%~2.91%, indicating that the instrument precision is good.

[0176] 7.3 Repeatability Test

[0177] Take an appropriate amount of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it into a fine powder, and take about 0.5g. Prepare 6 test solutions according to the method in section "4.3", and inject them for determination according to the chromatographic conditions in section "4.1". Using rutin as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1-3 and 5 with peak S1. Using narcisin as the reference peak S2, calculate the relative retention time and relative peak area of ​​peaks 6, 7, 9, and 10 with peak S2, and calculate the RSD value. The RSD values ​​of the relative retention time of the 10 common peaks are in the range of 0.03% to 0.17%, and the RSD values ​​of the relative peak area are in the range of 0.13% to 1.27%, all less than 3.0%, indicating good instrument precision and good repeatability of the method.

[0178] 7.4 Stability Test

[0179] Take an appropriate amount of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it finely, and take about 0.2g. Prepare the test solution according to the method in section "4.3". Inject the sample and determine it at 0, 4, 8, 12, and 24 hours according to the chromatographic conditions in section "4.1". Using rutin as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1-3 and 5 with peak S1. Using narcisin as the reference peak S2, calculate the relative retention time and relative peak area of ​​peaks 6, 7, 9, and 10 with peak S2. Calculate the RSD values. The RSD values ​​of the relative retention times of the 10 common peaks are in the range of 0.04% to 0.23%, all less than 3%, and the RSD values ​​of the relative peak areas are in the range of 0.25% to 2.2%, indicating that the test solution is relatively stable within 24 hours.

[0180] 8. Establishment of a characteristic chromatogram for a standard Ginkgo leaf decoction

[0181] 8.1 Results of Characteristic Spectrum Determination of Ginkgo Leaf Standard Decoction

[0182] Eighteen batches of Ginkgo biloba leaf standard decoction were prepared according to the chromatographic conditions in section "4.1" and the test solution preparation method determined in section "6.4". Using rutin as the reference peak S1, the relative retention times of characteristic peaks 1-5 with peak S1 were calculated as follows: 0.49 (peak 1), 0.75 (peak 2), 0.80 (peak 3), and 1.25 (peak 5). The peak corresponding to the narcissin reference peak was peak S2. The relative retention times of characteristic peaks 6, 7-10 with peak S2 were calculated as follows: 0.76 (peak 6), 0.85 (peak 7), 1.24 (peak 9), and 1.32 (peak 10). The characteristic chromatograms of the 18 batches of standard decoction are shown below. Figure 11 As shown.

[0183] 8.2 Establishment of a common pattern in the characteristic spectrum of Ginkgo biloba leaf standard decoction

[0184] Ginkgo biloba leaf reference material (batch number: 121606-201602, China National Institutes for Food and Drug Control) was used. A reference solution was prepared according to the method described in section "4.2". Eighteen batches of Ginkgo biloba leaf standard decoction were used. Test solutions were prepared according to the method described in section "4.3". The samples were then injected and analyzed under the chromatographic conditions described in section "4.1" to obtain the characteristic chromatograms of the Ginkgo biloba leaf reference material. Figure 12 The "Similarity Evaluation Software for Chromatographic Fingerprints of Traditional Chinese Medicine" was used to generate a reference chromatogram using the mean method, and a reference characteristic chromatogram of Ginkgo biloba leaf standard decoction was established. Figure 13 ).

[0185] The chromatogram of the test sample should show 10 characteristic peaks, which should correspond to the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 4 and 8 should correspond to the retention times of the corresponding reference material peaks, respectively. The peak corresponding to the rutin reference peak is peak S1. Calculate the relative retention times of characteristic peaks 1-3 and peak 5 with peak S1. The peak corresponding to the narcissin reference peak is peak S2. Calculate the relative retention times of characteristic peaks 6, 7, 9, and 10 with peak S2. The relative retention times should be within ±10% of the specified values, which 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), and 1.36 (peak 10).

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

[0187] 9.1 High Performance Liquid Chromatography Conditions

[0188] Take an appropriate amount of Ginkgo biloba leaf standard decoction (No.: YXY-1), grind it into a fine powder, take about 0.2g, prepare the test solution according to the method in section “4.3”, and analyze it according to the provisions in section “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 provisions in Table 2; the flow rate is 0.3ml per minute, the column temperature is 35 ℃, and the detection wavelength is 365nm.

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

[0190] The preparation of the test solution is the same as under section “4.3”; separately, take appropriate amounts of typhain, kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucopyranoside reference standards, accurately weigh them, and add methanol to prepare solutions containing 5 μg of each of the following per ml: typhain, kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucopyranoside, as reference solutions.

[0191] 9.3 Sample Determination

[0192] Accurately pipette 5 μl of the test solution and inject it into the liquid chromatograph. Analyze the test solution under the above-described liquid chromatographic conditions. The UV absorption comparison chromatograms of the test solution and the reference solution are shown below. Figure 14 , Figure 15 , Figure 16 and Figure 17 .

[0193] Example 2

[0194] This embodiment describes a method for determining the content of multiple components in a standard Ginkgo biloba leaf decoction.

[0195] 1. Instruments, reagents, and reagents

[0196] Same as Example 1.

[0197] 2. Preparation of Ginkgo Leaf Slices

[0198] Same as Example 1.

[0199] 3. Preparation of Standard Decoction of Ginkgo Leaf Slices

[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] Take appropriate amounts of kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), typhain, rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucoside (1-2)-α-L-rhamnoside reference standards, accurately weigh them, and add methanol to prepare standard solutions containing approximately 112 μg~19 μg, 75 μg~7 μg, 218~54 μg, 185~18 μg, 156~18 μg, and 106~26 μg per ml, respectively, as reference solutions.

[0206] 4.3 Preparation of test solution

[0207] Same as Example 1.

[0208] 4.5 Determination Method

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

[0210] 5. Examination of linear relationships

[0211] Accurately weigh an appropriate amount of kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside) reference standard and prepare a stock solution of reference standard with a mass content of 371.812 μg / ml using methanol. Then, dilute the stock solution with methanol to prepare reference standard solutions with mass contents 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 typhaoside reference standard and prepare a reference standard stock solution with a mass content of 378.859 μg / ml using methanol. Then, dilute the stock solution with methanol to prepare reference standard solutions with mass contents 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 standard and prepare a reference standard stock solution with a mass content of 362.9192 μg / ml using methanol. Then, dilute the stock solution with methanol to prepare reference standard solutions with masses 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-rutin reference standard and prepare a reference standard stock solution with a mass content of 368.000 μg / ml using methanol. Then, dilute the stock solution with methanol to prepare reference standard solutions with masses 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 standard and prepare a reference standard stock solution with a mass content of 388.786 μg / ml using methanol. Then, dilute the stock solution with methanol to prepare reference standard solutions with masses 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 standard and prepare a reference standard stock solution with a mass content of 352.000 μg / ml using methanol. Then, dilute the stock solution with methanol to prepare reference standard solutions with masses 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 inject 1 μl of each of the above-mentioned reference solutions of different concentrations and record the chromatographic peak areas. Plot the peak area as the ordinate (y) and the reference concentration as the abscissa (x), as shown in Table 3.

[0218] Table 3 Regression Equations and Linear Range

[0219]

[0220] 6. Precision test

[0221] The following reference solutions with concentrations of 74.362 μg / ml were injected and analyzed (using the same chromatographic conditions as described above). The solutions containing 74.362 μg / ml kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), 30.309 μg / ml typhain, 108.876 μg / ml rutin, 73.696 μg / ml kaempferol-3-O-rutin, 62.206 μg / ml narcissin, and 52.879 μg / ml kaempferol-3-O-β-D-glucose(1-2)-α-L-rhamnose were analyzed repeatedly six times to evaluate the instrument precision. The results showed that the RSD of the peak areas of kaempferol-3-O-(2,6-α-L-dipyranoramosyl-β-D-glucopyranoside), typhain, rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucopyranoside (1-2)-α-L-rhamnoside were all less than 5%, indicating that the instrument precision was good.

[0222] 7. Repeatability testing

[0223] Take approximately 0.2 g of Ginkgo biloba leaf standard decoction (batch number: YXY-1), accurately weigh it, and prepare 6 parallel portions. Prepare the test solution according to the determined test solution preparation method, inject the sample and determine the content of kaempferol-3-O-(2,6-α-L-dipyranoraminosyl-β-D-glucopyranoside), typhain, rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucose (1-2)-α-L-rhamnose in the test solution (test method is the same as the above chromatographic conditions). The experimental results showed that when the same batch of samples was measured repeatedly for 6 times, the RSD of the peak area of ​​kaempferol-3-O-(2,6-α-L-dipyranorhamnosyl-β-D-glucopyranoside), typhain, rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucopyranoside (1-2)-α-L-rhamnoside was less than 5%, indicating that the analytical method had good repeatability.

[0224] 8. Stability test

[0225] Approximately 0.2 g of Ginkgo biloba leaf standard decoction (batch number: YXY-1) was accurately weighed and prepared in six parallel portions according to the established test solution preparation method. The peak areas were measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and recorded (test method was the same as the chromatographic conditions described above). The results showed that the RSD values ​​of kaempferol-3-O-(2,6-α-L-dipyranoramosyl-β-D-glucopyranoside), typhain, rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucopyranoside were all less than 5%, indicating that the test solutions were stable at room temperature for 24 h.

[0226] 9. Content determination results

[0227] Ginkgo leaf standard decoction was used to prepare test solutions according to the above-mentioned test solution preparation method. The solutions were then injected and analyzed under the above-mentioned chromatographic conditions. The contents of kaempferol-3-O-(2,6-α-L-dipyranopyranosyl-β-D-glucopyranoside), typhain, rutin, kaempferol-3-O-rutin, narcissin, and kaempferol-3-O-β-D-glucose (1-2)-α-L-rhamnose were calculated using the external standard method. The results are shown in Table 4 below.

[0228] Table 4. Results of content determination in three batches of Ginkgo leaf standard decoction

[0229]

[0230] The technical features of the above embodiments can be combined in any way. For the sake of brevity, 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, they should be considered to be within the scope of this specification.

[0231] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic map of a standard decoction of Ginkgo biloba leaves, characterized in that, The method comprises the following steps: mixing ginkgo leaf standard decoction with a first solvent to extract and prepare a test sample solution; the first solvent is a methanol aqueous solution with a volume percentage of 10-60%; performing ultra-high performance liquid chromatography detection on the test sample solution to construct a characteristic map of the ginkgo leaf standard decoction; wherein the conditions of the ultra-high performance liquid chromatography detection comprise: the chromatographic column is a Waters Hss T3 chromatographic column with a specification of 2.1mm×150mm, 1.8μm; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is methanol and acetonitrile with a volume ratio of 4:1, and the mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.08-0.12%, and a gradient elution program is adopted; the gradient elution program comprises: 0min-30min, the volume percentage of the mobile phase A is kept at 28%, and the volume percentage of the mobile phase B is kept at 72%; 30min-40min, the volume percentage of the mobile phase A changes from 28% to 30%, and the volume percentage of the mobile phase B changes from 72% to 70%; 40min-45min, the volume percentage of the mobile phase A changes from 30% to 35%, and the volume percentage of the mobile phase B changes from 70% to 65%; 45min-55min, the volume percentage of the mobile phase A is kept at 35%, and the volume percentage of the mobile phase B is kept at 65%; the detection wavelength is 363nm-367nm; the construction method further comprises a characteristic peak identification step: mixing reference substances with a second solvent to prepare reference substance solutions; the reference substances comprise calceolusoside, kaempferol-3-O-(2,6-α-L-dipyrano-rhamnose-β-D-glucopyranoside), rutin, kaempferol-3-O-rutinose, zephyranthine and kaempferol-3-O-β-D-glucose (1-2)-α-L-rhamnoside reference substances; performing the ultra-high performance liquid chromatography detection on the reference substance solutions.

2. The method for constructing the characteristic pattern of the standard decoction of Ginkgo biloba leaves according to claim 1, characterized in that, The conditions of the ultra-high performance liquid chromatography detection further comprise 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℃-37℃; (3) the injection volume is 3μL-10μL.

3. The method for constructing the characteristic pattern of the Ginkgo biloba leaf standard decoction according to any one of claims 1-2, characterized in that, The preparation of the test sample solution has at least one of the following characteristics: (1) the extraction method is ultrasonic extraction or reflux extraction; (2) the extraction time is 10min-50min.

4. The method for constructing the characteristic pattern of the Ginkgo biloba leaf standard decoction according to any one of claims 1-2, characterized in that, The second solvent is methanol.

5. A method for determining the contents of multiple index components in a standard decoction of Ginkgo biloba leaves, characterized in that, The method comprises the following steps: mixing a ginkgo leaf standard decoction to be tested with a first solvent to extract and prepare a test sample solution; the first solvent is a methanol aqueous solution with a volume percentage of 10-60%; mixing reference substances with a second solvent to prepare reference substance solutions with different concentrations; the reference substances comprise calceolusoside, kaempferol-3-O-(2,6-α-L-dipyrano-rhamnose-β-D-glucopyranoside), rutin, kaempferol-3-O-rutinose, zephyranthine and kaempferol-3-O-β-D-glucose (1-2)-α-L-rhamnoside reference substances; The different concentrations of the control solution are detected by ultra-high performance liquid chromatography, and a standard curve of the control is constructed according to the detection results; The test sample solution is detected by ultra-high performance liquid chromatography, and the content of the corresponding index component is calculated by substituting the detection results into the standard curve of the control; The conditions of the ultra-high performance liquid chromatography detection include: The chromatographic column is a Waters Hss T3 chromatographic column with a specification of 2.1mmx150mm, 1.8μm; The mobile phase includes mobile phase A and mobile phase B, the mobile phase A is methanol and acetonitrile with a volume ratio of 4:1, and the mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.08%-0.12%, and a gradient elution program is adopted; The gradient elution program includes: 0min-30min, the volume percentage of mobile phase A is kept at 28%, and the volume percentage of mobile phase B is kept at 72%; 30min-40min, 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%; 40min-45min, 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%; 45min-55min, the volume percentage of mobile phase A is kept at 35%, and the volume percentage of mobile phase B is kept at 65%; The detection wavelength is 363nm-367nm.

6. The method according to claim 5, wherein the method is characterized in that, The conditions of the ultra-high performance liquid chromatography detection further include at least one of the following: (1) the flow rate is 0.2mL / min-0.4mL / min; (2) the column temperature is 33℃-37℃; (3) the injection volume is 3μL-10μL.

7. The method according to any one of claims 5-6, wherein the method is for determining the contents of the multi-index components in the ginkgo leaf standard decoction. The preparation of the test sample solution has at least one of the following characteristics: (1) the extraction method is ultrasonic extraction or reflux extraction; (2) the extraction time is 10min-50min.

8. The method according to any one of claims 5 to 6, wherein the method is for determining the contents of the multi-index components in a standard decoction of Ginkgo biloba leaves. The standard curve of the typhaneoside is y=4369.4x+2509.2; The standard curve of kaempferol-3-O-(2,6-α-L-rhamnopyranosyl-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-rhamnopyranoside is y=3535.2x+1324.

3.

9. The application of the characteristic spectrum of the ginkgo leaf standard decoction constructed by the construction method of any one of claims 1-4 or the method for determining the content of multiple index components in the ginkgo leaf standard decoction of any one of claims 5-8 in the quality detection of the ginkgo leaf standard decoction.

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