A method for detecting fingerprint of ginkgo decoction and its fingerprint

Abstract

The present invention provides a method for detecting the fingerprint of a ginkgo decoction and its fingerprint, the method comprising the following steps: (1) preparing a test ginkgo decoction into a test solution; (2) subjecting the test solution to high performance liquid chromatography to obtain a fingerprint, and performing a quality evaluation on the ginkgo decoction based on the fingerprint. The present invention provides a method for detecting the fingerprint of a ginkgo decoction, which can comprehensively detect the types and quantities of chemical components contained in the ginkgo decoction, thereby establishing a fingerprint of the ginkgo decoction. The fingerprint method has strong specificity, good separation of chromatographic peaks, a stable baseline, and a good peak shape, and can comprehensively reflect the types and quantities of chemical components contained therein, thereby providing an overall description and evaluation of the ginkgo decoction.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting a fingerprint of a ginkgo decoction and the fingerprint thereof. Background Art

[0002] Currently, identification of Chinese medicinal materials under the Chinese Pharmacopoeia primarily involves identification of properties, microscopy, and physicochemical identification. Physicochemical identification includes physical identification, chemical identification, spectroscopy, and chromatography, with thin-layer chromatography still the primary method of chromatographic identification. However, identification of different varieties of Chinese medicinal materials is rarely addressed in the Chinese Pharmacopoeia. This is primarily due to insufficient research on the differences in the material composition of different varieties of Chinese medicinal materials, making it difficult to control differences in chemical information.

[0003] Traditional Chinese medicine fingerprints are a comprehensive, quantifiable identification method primarily used to evaluate the authenticity, quality, and stability of traditional Chinese medicines and their preparations. By providing rich identification information, the establishment of a traditional Chinese medicine fingerprint will comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, thereby providing a holistic description and evaluation of their quality. To better control the quality of traditional Chinese medicines and their preparations and ensure their clinical efficacy, component detection methods are being developed to comprehensively evaluate their quality.

[0004] Ginkgo, also known as gingko, contains a variety of nutrients, such as protein and carbohydrates. These nutrients not only provide energy for human life but also promote antibody production and enhance immunity. Ginkgo is neutral in nature, with a sweet, bitter, and astringent taste. It has the effects of astringing lung qi and calming wheezing, helping to relieve coughs, sputum, asthma, and other symptoms. Studies have found that the flavonoids and terpene lactones in ginkgo kernels can be used to prevent and treat conditions such as cerebral thrombosis and arteriosclerosis. Currently, the only quality control measures for ginkgo in pharmacopoeias include identification of properties, microscopy, thin-layer chromatography, and extracts. Due to the complex multi-component nature of traditional Chinese medicine, quality assessment should utilize appropriate methods. Ginkgo contains numerous chemical components, primarily organic acids, phenols, and alcohols. Testing these chemical components is necessary to better control and evaluate the quality of ginkgo nuts and their medicinal preparations. At present, most of the research on ginkgo is based on alcohol extraction. Studies have shown that the following compounds have been isolated from the seed coat and kernel so far, namely octadecanoic acid, palmitic acid, nonacosan-10-ol, β-sitosterol, n-triacontanol, n-docosanol, 6-(pentadecyl)-salicylic acid, 6-(8-pentadecenyl)-salicylic acid, n-docosanoic acid-1-glyceride, n-eicosanoic acid-1-glyceride, n-hexadecanoic acid-1-glyceride Oily esters, 1,3-hexadecanoic acid-glyceride, 1,3-dilinoleic acid-glyceride, tristearin, tripalmitin, ginkgolide B, ginkgolide C, daucin, hexacosanoic acid, palmitic acid, nonacosan-10-ol, n-hexadecanoic acid-1-glyceride, ursolic acid, cinnamoflavone, ginkgetin, isoginkgetin, daucin, ginkgolide A, uracil, coniferin, ginkgolide, liquiritin, adenosine, glucose, and sucrose. ("Chemical Research on Ginkgo Seed Resources," Zhou Guisheng, Nanjing University of Chinese Medicine, 2013). However, in clinical practice, Chinese medicinal materials are mostly extracted with water, so research on the components of ginkgo decoctions is relatively lacking.

[0005] CN114487204A discloses an ultra-high performance liquid chromatography-fluorimetry (UPLC-FLD) method for the simultaneous detection of pyridoxal and pyridoxine in ginkgo nuts. This method uses a ZIF-8 organometallic framework (ZIF-8) as a purification material, combined with UPLC-FLD analysis technology, to detect pyridoxal and pyridoxine in ginkgo nuts. The instrumental detection limits are 0.012 mg / L and 0.010 mg / L, respectively. While these low limits are significant, the ZIF-8 organometallic framework is required, leading to high testing costs.

[0006] Therefore, developing an identification method for ginkgo decoction that is simple to operate, low in cost, highly characteristic, and highly accurate is a research focus in this field. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for detecting the fingerprint of ginkgo decoction and its fingerprint. The obtained fingerprint is highly characteristic and accurate, and can be used for comprehensive quality evaluation of ginkgo medicinal materials.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for detecting a fingerprint of a ginkgo decoction, the method comprising the following steps:

[0010] (1) Prepare the test medicinal material ginkgo into a test solution;

[0011] (2) The test solution is subjected to high performance liquid chromatography to obtain a fingerprint, and the quality of the ginkgo biloba medicinal material to be tested is evaluated based on the fingerprint.

[0012] The present invention provides a method for detecting the fingerprint of ginkgo biloba medicinal material. The method can be used to comprehensively detect the types and quantities of chemical components contained in the ginkgo biloba medicinal material, and a fingerprint of the ginkgo biloba medicinal material is established based on the method. The fingerprint method has strong specificity, good separation of chromatographic peaks, a stable baseline, and a good peak shape. It can comprehensively reflect the types and quantities of the chemical components contained therein, and further provide an overall description and evaluation of the ginkgo biloba medicinal material.

[0013] Preferably, the test solution is prepared by the following method, which includes: separating the ginkgo shell and ginkgo kernel of the ginkgo test medicinal material, crushing them, combining them to obtain a mixture, subjecting the mixture to a single water extraction and a secondary water extraction to obtain a water extract, and diluting the water extract to obtain the test solution.

[0014] Preferably, after the pulverization, the ginkgo medicinal material is selected to pass through the No. 1 sieve but not the No. 2 sieve.

[0015] Preferably, the mass ratio of ginkgo shell to ginkgo kernel in the mixture is 1:(1.5-2.5), for example, it can be 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, etc.

[0016] The present invention attempts to directly crush the ginkgo nuts before extraction, but when performing a repeatability experiment, the repeatability is poor, and the RSD values ​​of the main common peaks are all greater than 3.0%. It is speculated that this may be caused by uneven sampling. Because the ginkgo shells are lighter after crushing and are not easy to mix with the crushed ginkgo kernels, it is considered to sample the ginkgo shells and ginkgo kernels separately in proportion. The results show that the repeatability is improved, but during the extraction process, it is found that the ginkgo kernels have a fine particle size, are prone to violent boiling and gelatinization, and affect the extraction efficiency. The effect of ginkgo kernels of different particle sizes on the repeatability test is screened. The results show that when the ginkgo shells and ginkgo kernels are both between the No. 1 and No. 2 sieves, the repeatability is better. Therefore, when performing fingerprint spectrum detection in the present invention, the ginkgo nuts need to be crushed, and the ginkgo shells and ginkgo kernels are proportionally taken as medicinal materials with a particle size between the No. 1 and No. 2 sieves.

[0017] Preferably, the material-liquid ratio during the first water extraction is 1:(7-9), for example, it can be 1:7.5, 1:8, 1:8.5, etc., and the water content is 7-9 mL based on 1 g of the mixture of ginkgo shells and ginkgo kernels.

[0018] Preferably, the material-liquid ratio during the secondary water extraction is 1:(5-7), for example, it can be 1:5.5, 1:6, 1:6.5, etc., based on 1g of the mixture of ginkgo shells and ginkgo kernels, the amount of water is 5-7mL.

[0019] Preferably, the first water extraction further includes a soaking step.

[0020] Preferably, the soaking time is 20-40 min, for example, 25 min, 30 min, 35 min, etc.

[0021] Preferably, the first water extraction and the second water extraction are each independently kept at a slight boil for 25-50 minutes, for example, 30 minutes, 35 minutes, 40 minutes, 45 minutes, etc.

[0022] Preferably, the diluent used for the dilution is methanol.

[0023] Preferably, the volume ratio of the water extract to the diluent during the dilution is 1:(0.8-1.2), for example, it can be 1:0.9, 1:1, 1:1.1, etc.

[0024] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0025] Preferably, the HPLC detection conditions are as follows: the detection wavelength is 200-250 nm, for example, 210 nm, 220 nm, 230 nm, 240 nm, etc., the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, gradient elution is performed, the flow rate is 0.8-1.2 mL / min, for example, 0.9 mL / min, 1 mL / min, 1.1 mL / min, etc., and the column temperature is 25-35°C, for example, 26°C, 28°C, 30°C, 32°C, 34°C, etc.

[0026] Preferably, the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%, for example, it can be 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, etc.

[0027] Preferably, the gradient elution process is: 0-10 min, for example, 2 min, 4 min, 6 min, 8 min, etc., mobile phase A: 5%, mobile phase B: 95%; 10-45 min, for example, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, 42 min, 44 min, etc., mobile phase A: 5-15%, for example, 6%, 8%, 10%, 12 %, 14%, etc., mobile phase B: 95-85%, for example, it can be 94%, 92%, 90%, 88%, 86%, etc.; 45-50min, for example, it can be 46min, 48min, 49min, etc., mobile phase A: 15-25%, for example, it can be 16%, 18%, 20%, 22%, 24%, etc., mobile phase B: 85-75%, for example, it can be 84%, 82%, 80%, 78%, 76%, etc.; 50-60min, for example, it can be 52min, 54min, 56min, 58min, etc., mobile phase A: 25%, mobile phase B: 75%.

[0028] Preferably, the detection method further comprises the steps of preparing a reference solution, and detecting the reference solution using the same detection method as that for the test solution to obtain a characteristic spectrum.

[0029] Preferably, the reference solution is prepared by the following method, which comprises: extracting the ginkgo reference medicinal material with water, concentrating the obtained filtrate, diluting it, and centrifuging it to obtain the reference solution.

[0030] Preferably, the material-liquid ratio during water extraction is 1:(45-55), for example, it can be 1:46, 1:48, 1:50, 1:52, 1:54, etc.

[0031] Preferably, the water extraction is heated under reflux for 0.8-1.2 h, for example, 0.9 h, 1 h, 1.1 h, etc.

[0032] Preferably, the concentration is to 1 / 4 volume.

[0033] Preferably, methanol is used for dilution, and the material-liquid ratio during dilution is 1:(0.8-1.2), for example, 1:0.9, 1:1, 1:1.1, etc. Based on 1 mL of the concentrated solution, the volume of methanol is 0.8-1.2 mL.

[0034] As a preferred technical solution, the method includes the following steps:

[0035] (1) Separate the ginkgo nut shell and ginkgo kernel of the ginkgo nut to be tested, grind them until they pass through a No. 1 sieve but not a No. 2 sieve, combine them to obtain a mixture, perform a first water extraction and a second water extraction on the mixture, and keep the first water extraction and the second water extraction independently at a slight boil for 25-50 minutes to obtain a water extract, and dilute the water extract to obtain a test solution;

[0036] The mass ratio of ginkgo shell to ginkgo kernel in the mixture is 1:(1.5-2.5); the material-liquid ratio during the first water extraction is 1:(7-9), and the material-liquid ratio during the second water extraction is 1:(5-7); the diluent used in the dilution is methanol, and the volume ratio of the water extract to the diluent is 1:(0.8-1.2);

[0037] (2) subjecting the test solution to high performance liquid chromatography to obtain a fingerprint, and performing quality evaluation on the ginkgo biloba medicinal material to be tested based on the fingerprint;

[0038] The conditions for the high performance liquid chromatography detection are as follows: the detection wavelength is 200-250 nm, the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, the gradient elution is performed, the flow rate is 0.8-1.2 mL / min, the column temperature is 25-35° C., and the gradient elution process is: 0-10 min, mobile phase A: 5%, mobile phase B: 95%; 10-45 min, mobile phase A: 5-15%, mobile phase B: 95-85%; 45-50 min, mobile phase A: 15-25%, mobile phase B: 85-75%; 50-60 min, mobile phase A: 25%, mobile phase B: 75%;

[0039] The mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%.

[0040] In a second aspect, the present invention provides a fingerprint spectrum detected by the method described in the first aspect, wherein the fingerprint spectrum includes three characteristic peaks.

[0041] Preferably, in the order of peak appearance, with peak 1 as the reference peak, the relative retention times of the three characteristic peaks are 1.00, 1.09-1.22 (for example, 1.10, 1.11, 1.12, 1.14, 1.16, 1.18, 1.20, etc.), and 1.20-1.34 (for example, 1.22, 1.24, 1.26, 1.28, 1.30, 1.32, etc.).

[0042] Preferably, according to the order of peak appearance, with Peak 1 as the reference peak, the relative retention times of the three characteristic peaks are 1.00, 1.11, and 1.21.

[0043] After characterization, the compound to which peak 1 belongs is indoleacetic acid-glutamine glucose (IAA-Asp-N-Glc), and its structural formula is shown below:

[0044]

[0045] After characterization, the compound to which peak 3 belongs is indoleacetic acid-aspartic acid glucose (IAA-Glu-N-Glc), and its structural formula is shown below:

[0046]

[0047] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention provides a method for detecting the fingerprint of ginkgo biloba medicinal material. The method can be used to comprehensively detect the types and quantities of chemical components contained in the ginkgo biloba medicinal material, and a fingerprint of the ginkgo biloba medicinal material is established based on the method. The fingerprint method has strong specificity, good separation of chromatographic peaks, a stable baseline, and a good peak shape. It can comprehensively reflect the types and quantities of the chemical components contained therein, and further provide an overall description and evaluation of the ginkgo biloba medicinal material.

[0050] When processing a ginkgo sample to be tested, the present invention samples the ginkgo shell and the ginkgo kernel separately in proportion and controls the particle size of the samples, thereby effectively improving the repeatability of the test results. Simultaneously, the present invention separates two components that have not been reported to be contained in ginkgo during the test process, which is of great significance for the quality control of clinical ginkgo medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the full wavelength 3D chromatogram in Test Example 1;

[0052] Figure 2 This is the characteristic spectrum obtained from test case 3;

[0053] Figure 3This is the characteristic spectrum obtained from test example 4;

[0054] Figure 4 This is the characteristic spectrum obtained from test case 5;

[0055] Figure 5 This is the characteristic spectrum obtained by detection in Example 1;

[0056] Figure 6 This is the characteristic spectrum obtained from the precision verification test in Test Example 6;

[0057] Figure 7 This is the characteristic spectrum obtained from the repeatability verification test in Test Example 6;

[0058] Figure 8 This is the characteristic spectrum obtained from the stability verification test in Test Example 6;

[0059] Figure 9 This is the characteristic spectrum obtained from the verification test of multiple batches of ginkgo medicinal materials in Test Example 6. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0061] As used herein, the terms "comprises," "including," "having," "containing" or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0062] "Optional" or "either" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event occurs and instances where it does not.

[0063] The indefinite articles "a" and "an" before the elements or components of the present invention do not limit the quantity requirement (i.e. the number of times they appear). Therefore, "a" or "an" should be interpreted as including one or at least one, and elements or components in the singular also include plural forms, unless the quantity clearly refers to only the singular form.

[0064] The terms "one embodiment," "some embodiments," "exemplarily," "specific examples," or "some examples" used in the present invention mean that a specific feature, structure, material, or characteristic described in conjunction with the embodiment or example is included in at least one embodiment or example of the present invention. In this document, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example.

[0065] The sources of reagents or instruments in the following examples are as follows:

[0066] High performance liquid chromatograph: LC-2030C 3D Plus, Shimadzu;

[0067] Electronic analytical balance: XRP2.METTLER.TOLEDO, Mettler-Toledo, Switzerland;

[0068] Electronic analytical balance: ML204T / 02.METTLER.TOLEDO, Mettler-Toledo, Switzerland;

[0069] Ginkgo biloba reference medicinal material: provided by China Food and Drug Inspection Institute.

[0070] Test Example 1

[0071] Selection of detection wavelength

[0072] Take an appropriate amount of ginkgo nuts, separate the ginkgo shells from the ginkgo kernels, crush them separately, take 5g of the ginkgo shells that pass through the No. 1 sieve but not the No. 2 sieve, and 10g of the ginkgo kernels, put them in the same round-bottom flask, add 8 times the amount of water (120mL), soak for 30min, heat to boiling and keep it slightly boiling for 45min, filter while hot, collect the filtrate, add 6 times the amount of water (90mL) to the residue, heat to boiling and keep it slightly boiling for 30min, filter while hot, combine the two filtrates, and obtain a water extract. Take 2mL of the water extract, add 2mL of methanol, shake well, and centrifuge. Take the supernatant for full-wavelength scanning. After comparative analysis of multiple wavelengths, the full-wavelength 3D chromatogram is as shown below. Figure 1 As shown, according to the detection results, the final wavelength selected is 225nm.

[0073] Test Example 2

[0074] Choice of mobile phase

[0075] Methanol-0.4% phosphoric acid, methanol-0.5% formic acid, acetonitrile-0.1% trifluoroacetic acid, acetonitrile-0.5% formic acid, and acetonitrile-0.1% phosphoric acid were selected for investigation. Based on the number of chromatographic peaks and baseline conditions presented in the test results, the acetonitrile-0.1% phosphoric acid system was finally determined, and the elution process was determined as shown in the following table:

[0076]

[0077]

[0078] Test Example 3

[0079] Selection of test solution preparation method - alcohol ratio

[0080] Option 1:

[0081] (1) Take an appropriate amount of ginkgo nuts, separate the ginkgo nuts shell and ginkgo kernel, crush them separately, take 5g of ginkgo nuts shell that pass through a No. 1 sieve but not through a No. 2 sieve, and 10g of ginkgo kernel, put them into the same round-bottom flask, add 8 times the amount of water (120mL), soak for 30min, heat to boiling and keep slightly boiling for 45min, filter while hot, collect the filtrate, add 6 times the amount of water (90mL) to the residue, heat to boiling and keep slightly boiling for 30min, filter while hot, combine the two filtrates to obtain an aqueous extract;

[0082] (2) The aqueous extract was concentrated to dryness, re-dissolved in 70% methanol, filtered, and sampled for analysis.

[0083] Option 2:

[0084] The only difference between this method and Scheme 1 is step (2). Step (2) of this method is: concentrate the water extract to 1 / 4 volume, add 1 times volume of methanol, mix well, filter, and sample for detection and analysis.

[0085] Option 3:

[0086] The only difference between this method and Scheme 1 is step (2). Step (2) of this method is: add 3 times the volume of methanol to the water extract, mix well, filter, and sample for detection and analysis.

[0087] Option 4:

[0088] The only difference between this method and Scheme 1 is step (2). Step (2) of this method is: add 2 times the volume of methanol to the water extract, mix well, filter, and sample for detection and analysis.

[0089] Option 5:

[0090] The only difference between this method and Scheme 1 is step (2). Step (2) of this method is: add 1 times the volume of methanol to the water extract, mix well, filter, and sample for detection and analysis.

[0091] The characteristic spectra obtained by schemes 1 to 5 are as follows Figure 2 As shown in the figure, it can be seen that the separation of the characteristic peaks, the baseline situation, and the peak shape in the spectrum obtained by Scheme 5 are slightly better than those of other spectra. Therefore, the present invention selects the test sample preparation method of Scheme 5.

[0092] Test Example 4

[0093] Selection of test solution preparation method - crushing method

[0094] After directly crushing the ginkgo nuts, take 15g and place it in the same round-bottom flask. Add 8 times the amount of water (120mL) and soak for 30 minutes. Heat to boiling and maintain a slight boil for 45 minutes. Filter while hot and collect the filtrate. Add 6 times the amount of water (90mL) to the residue and heat to boiling and maintain a slight boil for 30 minutes. Filter while hot and combine the two filtrates. Take 2mL of the aqueous extract, add 2mL of methanol, shake well, and centrifuge. The supernatant is used as the test solution. Prepare 5 replicates for testing.

[0095] The characteristic spectrum obtained by detection is as follows Figure 3 As shown in the figure, the repeatability of directly crushing the ginkgo nuts and then extracting them is poor, and the RSD values ​​of the main common peaks are all greater than 3.0%. It is speculated that this may be caused by uneven sampling. Because the ginkgo shell is lighter after crushing, it is not easy to mix with the crushed ginkgo kernels. Therefore, it is considered to sample the ginkgo shells and ginkgo kernels separately in proportion.

[0096] Test Example 5

[0097] Selection of test solution preparation method - medicinal material particle size

[0098] Take an appropriate amount of ginkgo nuts, separate the shells from the kernels, and pass each through a No. 1 sieve. Weigh 5g of the shells and 10g of the kernels, place them in a round-bottom flask, add 8 times the volume of water (120mL), soak for 30 minutes, heat to boiling and maintain a slight boil for 45 minutes, filter while hot, collect the filtrate, add 6 times the volume of water (90mL) to the residue, heat to boiling and maintain a slight boil for 30 minutes, filter while hot, and combine the two filtrates. Take 2mL of the aqueous extract, add 2mL of methanol, shake well, and centrifuge. Take the supernatant as the test solution, and prepare 5 replicates for testing.

[0099] The characteristic spectrum obtained by detection is as follows Figure 4 As shown in the figure, it can be seen that when ginkgo shells and ginkgo kernels are only passed through No. 1 sieve, the particle size differences of the obtained medicinal materials are still large, and the repeatability is poor. The RSD values ​​of the main common peaks are all greater than 3.0%. It is speculated that this may be caused by uneven particle size. Therefore, it is considered to further control the particle size of the medicinal materials by passing the ginkgo shells and ginkgo kernels through the No. 1 sieve but not the No. 2 sieve, that is, the No. 1-2 sieve.

[0100] Example 1

[0101] This embodiment provides a method for detecting the fingerprint of ginkgo decoction, the method comprising the following steps:

[0102] (1) Take about 1 g of ginkgo biloba reference medicinal material, add 50 mL of water, heat and reflux for 1 hour, filter, concentrate the filtrate to about 12 mL, take 1 mL of the concentrate, add 1 mL of methanol, mix, centrifuge (4000 rpm) for 5 minutes, take the supernatant, filter, and obtain the reference solution, which is sampled for detection and analysis;

[0103] (2) Take an appropriate amount of ginkgo nuts, separate the ginkgo shells from the ginkgo kernels, crush them separately, take 5g of the ginkgo shells that pass through the No. 1 sieve but not the No. 2 sieve, and 10g of the ginkgo kernels, put them in the same round-bottom flask, add 8 times the amount of water (120mL), soak for 30min, heat to boiling and keep it slightly boiling for 45min, filter while hot, collect the filtrate, add 6 times the amount of water (90mL) to the residue, heat to boiling and keep it slightly boiling for 30min, filter while hot, combine the two filtrates to obtain a water extract; take 2mL of the water extract, add 2mL of methanol, mix well and filter to obtain the test solution, and inject the sample for detection and analysis;

[0104] (3) An Agilent Poroshell 120EC column was used, the column temperature was 30°C, the detection wavelength was 225 nm, the mobile phase A was acetonitrile, the mobile phase B was 0.1% phosphoric acid aqueous solution, and gradient elution was performed at a flow rate of 1.0 mL / min. The elution process is shown in the following table:

[0105] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-10 5 95 10-45 5→15 95→85 45-50 15→25 85→75 50-60 25 75

[0106] The characteristic spectrum obtained by detection is as follows Figure 5 shown.

[0107] Test Example 6

[0108] Methodological validation

[0109] 1. Precision

[0110] The test solution obtained in Example 1 was injected 6 times continuously for measurement, and the characteristic spectrum obtained by detection was as follows Figure 6 The relative retention time and relative peak area of ​​each characteristic peak in the detection results are shown in Table 1.

[0111] It can be seen from the chromatogram and table data that the relative standard deviation of the relative retention time and relative peak area of ​​each common peak in the test solution is less than 0.23%, and the similarity is not less than 0.999, indicating that the method has good precision.

[0112] Table 1

[0113]

[0114]

[0115] 2. Repeatability

[0116] The test solution obtained in Example 1 was prepared in parallel for 5 times and the characteristic spectrum obtained was as follows. Figure 7 The relative retention time and relative peak area of ​​each characteristic peak in the detection results are shown in Table 2.

[0117] It can be seen from the spectral and tabular data that the relative standard deviations of the relative retention times and relative peak areas of the common peaks in the test solutions are less than 0.94%, and the similarities of the test solutions are not less than 0.999, indicating that the method has good stability.

[0118] Table 2

[0119]

[0120] 3. Stability

[0121] The test solution obtained in Example 1 was sampled and tested at 0h, 3h, 6h, 9h, 15h, and 24h, and the characteristic spectra obtained were as follows: Figure 8 The relative retention time and relative peak area of ​​each characteristic peak in the detection results are shown in Table 3.

[0122] It can be seen from the spectra and table data that the similarity is calculated with 0h as the reference, and the similarity of the test solutions is not less than 0.999, indicating that the method has good stability.

[0123] Table 3

[0124]

[0125] 4. Multi-batch preparation

[0126] The preparation method of Example 1 was used to test 21 batches of ginkgo medicinal materials that met the quality requirements of the quality analysis. The test results were as follows: Figure 9 As shown in the figure, the obtained fingerprints were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 A Edition" issued by the State Pharmacopoeia Committee, and the similarity (Mark peak matching) was calculated. The similarity results were all greater than 0.9. The chromatographic peaks with good stability and appropriate response values ​​in the fingerprints of 21 batches of samples were selected as common peaks and used as the characteristic peaks of ginkgo. A total of 3 characteristic peaks were calibrated.

[0127] Test Example 7

[0128] Characteristic peak attribution

[0129] The three characteristic peaks identified were compared with control medicinal materials and existing reference substances and were temporarily considered to be unknown components. Therefore, the control medicinal materials were temporarily selected as reference substances, and Peak 1, which had a strong response, a moderate retention time, and achieved baseline separation, was selected as the reference peak and labeled as Peak S. The three unknown components were separated, purified, and identified by high-resolution mass spectrometry (high resolution mass spectrometry) and nuclear magnetic resonance spectroscopy (NMR). Peak 1 represented the component IAA-Asp-N-Glc (Compound 1), and Peak 3 represented the component IAA-Glu-N-Glc (Compound 3). Their structures are as follows:

[0130]

[0131] The positive source high resolution electrospray ionization mass spectrometry (PHRESIMS) of compound 1 showed that its quasi-molecular ion peak was m / z 453.1504 [M+H] +, and the corresponding molecular formula was C 20 H 24 N2O 10 (Theoretical calculated value is 453.1486; unsaturation degree is 10). By comparison with the literature, the compound was identified as the known compound IAA-Asp-N-Glc, and its NMR data are shown in Table 4:

[0132] Table 4

[0133] position. <![CDATA[δ H a (J in Hz)]]> <![CDATA[δ C b ,a lot.]]> <![CDATA[HMBC c (H→C)]]> 2 7.331,s 124.47,CH 3,3a,7a,8,1′ 3 109.34,qC 3a 128.11,qC 4 7.549,d(7.8) 119.04,CH 3,6,7a 5 7.015,t(7.2) 119.16,CH 3a,7 6 7.121,t(7.8) 121.29,CH 4,7a, 7 7.511,d(8.4) 110.52,CH 3a,5 7a 136.50,qC 8 3.563,d(6.0) <![CDATA[32.13,CH2]]> 2,3,3a,9 9 170.18,qC 10 4.489,d(6.6) 48.76,CH 12,13 11 172.69,qC 12a 2.686,dd(16.2,6.6) <![CDATA[36.85,CH2]]> 10,13 12b 10,13 13 2.574,dd(16.2,6.0) 171.92,qC 1′ 84.43,CH 2,7a,2′,3′ 2′ 5.392,d(9.0) 72.51,CH 1′,3′ 3′ 3.662-3.772,m 77.62,CH 4′ 3.401-3.458,m 3.255, 69.91,CH 3′,5′,6′ 5′ t(9.0) 79.38,CH 6′a 3.401-3.458,m <![CDATA[60.93,CH2]]> 6′b 3.662-3.772,m OH 3.401-3.458,m OH 4.593,br s NH 5.083,br s 9 OH 8.292,d(6.0) 12.994,br s

[0134] The positive source high resolution electrospray ionization mass spectrometry (PHRESIMS) of compound 3 showed that its quasi-molecular ion peak was m / z 467.1653 [M+H] +, and the corresponding molecular formula was C 21 H 26 N2O 10 (Theoretical calculated value is 467.1660; unsaturation degree is 10). By comparison with the literature, the compound was identified as the known compound IAA-Glu-N-Glc, and its NMR data are shown in Table 5:

[0135] Table 5

[0136] position. <![CDATA[δ H a (J in Hz)]]> <![CDATA[δ C b ,a lot.]]> <![CDATA[HMBC c (H→C)]]> 2 7.339,s 124.48,CH 3,3a,7a, 3 109.45,qC 3a 128.09,qC 4 7.555,d(7.8) 118.99,CH 3,6,7a 5 7.024,t(7.2) 119.13,CH 3a,7 6 7.131,t(7.8) 121.27,CH 4,7a, 7 7.510,d(8.4) 110.53,CH 3a,5 7a 136.49,qC 8 3.557,s <![CDATA[32.20,CH2]]> 2,3,3a,9 9 170.17,qC 10 4.179,d(5.4) 51.73,CH 11 174.09,qC 12 1.854,dd(16.2,7.8) <![CDATA[27.11,CH2]]> 13 2.268,s <![CDATA[30.89,CH2]]> 14 173.62,qC 1′ 5.392,d(9.0) 84.43,CH 2,7a,2′ 2′ 3.660-3.720,m 71.86,CH 1′,3′ 3′ 3.374-3.454,m 3.252,t 77.60,CH 4′ (9.0) 69.90,CH 5′,6′ 5′ 3.374-3.454,m 79.37,CH 6′a 3.660-3.720,m <![CDATA[60.93,CH2]]> 6′b 3.374-3.454,m OH 5.092,br s NH 8.110,s

[0137] The applicant states that while the above-described embodiments illustrate the process of the present invention, the present invention is not limited to the above-described process steps, nor does it imply that the present invention must rely on the above-described process steps for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for the raw materials used, additions of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for detecting the fingerprint of ginkgo decoction, characterized in that: The method comprises the following steps: (1) Prepare the test solution of ginkgo biloba medicinal material; (2) subjecting the test solution to high performance liquid chromatography to obtain a fingerprint, and evaluating the quality of the ginkgo biloba medicinal material according to the fingerprint; The test solution is prepared by the following method, which includes: separating the ginkgo nut shell and ginkgo kernel of the ginkgo nut to be tested medicinal material, crushing them, combining them to obtain a mixture, subjecting the mixture to a single water extraction and a secondary water extraction to obtain a water extract, and diluting the water extract to obtain the test solution; the conditions for the high performance liquid chromatography detection are as follows: the detection wavelength is 200-250 nm, the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, gradient elution is performed, the flow rate is 0.8-1.2 mL / min, an Agilent Poroshell 120EC column is used, and the column temperature is 25-35°C; The gradient elution process of the HPLC method is as follows: 0-10 min, mobile phase A: 5%, mobile phase B: 95%; 10-45 min, mobile phase A: 5-15%, mobile phase B: 95-85%; 45-50 min, mobile phase A: 15-25%, mobile phase B: 85-75%; 50-60 min, mobile phase A: 25%, mobile phase B: 75%; The fingerprint spectrum detected by the method includes three characteristic peaks. In the order of peak appearance, with peak 1 as the reference peak, the relative retention times of the three characteristic peaks are 1.00, 1.09-1.22, and 1.20-1.34; the compound to which characteristic peak 1 belongs is indoleacetic acid-glutamine glucose, and the compound to which characteristic peak 3 belongs is indoleacetic acid-aspartic acid glucose.

2. The method according to claim 1, characterized in that The grinding is performed until it passes through a No. 1 sieve but does not pass through a No. 2 sieve.

3. The method according to claim 1, characterized in that The mass ratio of ginkgo nut shells to ginkgo nut kernels in the mixture is 1:(1.5-2.5).

4. The method according to claim 1, wherein The material-liquid ratio during the first water extraction is 1:(7-9).

5. The method according to claim 1, wherein The material-liquid ratio during the secondary water extraction is 1:(5-7).

6. The method according to claim 1, characterized in that The first water extraction and the second water extraction are each independently kept at a slight boiling temperature for 25-50 minutes.

7. The method according to claim 1, characterized in that The diluent used in the dilution is methanol.

8. The method according to claim 1, characterized in that During the dilution, the volume ratio of the water extract to the diluent is 1:(0.8-1.2).

9. The method according to claim 1, characterized in that The mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%.

10. The method according to claim 1, characterized in that The detection method further includes the steps of preparing a reference solution and detecting the reference solution using the same detection method as that for the test solution to obtain a characteristic spectrum.

11. The method according to claim 1, wherein The method comprises the following steps: (1) Separate the ginkgo nut shell and ginkgo kernel of the ginkgo nut to be tested, grind them until they pass through a No. 1 sieve but not a No. 2 sieve, combine them to obtain a mixture, perform a first water extraction and a second water extraction on the mixture, and keep the first water extraction and the second water extraction independently at a slight boil for 25-50 minutes to obtain a water extract, and dilute the water extract to obtain a test solution; The mass ratio of ginkgo shell to ginkgo kernel in the mixture is 1:(1.5-2.5); the material-liquid ratio during the first water extraction is 1:(7-9), and the material-liquid ratio during the second water extraction is 1:(5-7); the diluent used in the dilution is methanol, and the volume ratio of the water extract to the diluent is 1:(0.8-1.2); (2) subjecting the test solution to high performance liquid chromatography to obtain a fingerprint, and evaluating the quality of the ginkgo biloba medicinal material according to the fingerprint; The conditions for the high performance liquid chromatography detection are as follows: the detection wavelength is 200-250 nm, the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, the gradient elution is performed, the flow rate is 0.8-1.2 mL / min, an Agilent Poroshell 120EC column is used, the column temperature is 25-35°C, and the gradient elution process is: 0-10 min, mobile phase A: 5%, mobile phase B: 95%; 10-45 min, mobile phase A: 5-15%, mobile phase B: 95-85%; 45-50 min, mobile phase A: 15-25%, mobile phase B: 85-75%; 50-60 min, mobile phase A: 25%, mobile phase B: 75%; The mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%.

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