Method for constructing lignan fingerprint of ginkgo leaf medicinal materials, extracts and their single-herb preparations

Abstract

The present invention relates to a method for constructing a lignan fingerprint spectrum in a ginkgo leaf medicinal material, an extract and a single-flavor preparation thereof, and can provide a technical method for quality research and quality control of ginkgo leaf medicinal materials, extracts and single-flavor preparations thereof. In the present invention, 14 common peaks of lignan components are identified, and 16 lignan compounds are identified in the common peaks of the 14 lignan components, which more comprehensively clarifies the material basis in the ginkgo leaf medicinal materials, extracts and preparations thereof, and provides a basis for their quality control. The UPLC lignan fingerprint spectrum detection method established by the present invention can be used for component detection of ginkgo leaf medicinal materials, extracts and preparations thereof, which is conducive to more comprehensive quality control thereof, and provides a guarantee for its clinical efficacy and safety.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis, and particularly relates to a method for constructing a lignan fingerprint of Ginkgo biloba medicinal materials, extracts and their single-ingredient preparations. Background Art

[0002] Ginkgo biloba extract and its preparations are one of the main traditional Chinese medicines clinically used for treating cardiovascular and cerebrovascular diseases, and have definite curative effects. The chemical components contained in Ginkgo biloba and its preparations are complex. Research shows that they are mainly flavonoids and ginkgolides. The latest research finds that Ginkgo biloba and its preparations contain lignan components, and studies show that lignan components have good therapeutic effects on cerebral ischemic injury, hyperlipidemia, myocardial ischemia-reperfusion injury, etc. However, in the existing quality research and quality standards of Ginkgo biloba medicinal materials, extracts and their preparations, the quality control indicators are mainly flavonol glycosides and terpene lactones, lacking a quality control method for lignan components, and unable to comprehensively and scientifically control the quality of Ginkgo biloba extract and its preparations, as well as ensure the clinical effectiveness and safety of Ginkgo biloba extract and its preparations. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: The present invention provides a method for constructing a lignan fingerprint of Ginkgo biloba medicinal materials, extracts and their preparations, which can effectively control the quality of Ginkgo biloba medicinal material extracts and their preparations, thereby ensuring their clinical efficacy.

[0004] The technical solution adopted by the present invention to solve its technical problems is: A method for constructing a lignan fingerprint of Ginkgo biloba medicinal materials, extracts and their single-ingredient preparations, comprising the following steps:

[0005] Step 1: Prepare a control sample solution

[0006] Respectively prepare solutions with a concentration of 0.01 mg / mL - 2 mg / mL from each control sample with 20% - 100% methanol-aqueous solution, and set aside;

[0007] Step 2: Prepare a test sample solution

[0008] Step 2.1, Select different batches of Shuxuening injection and dilute it with pure water;

[0009] Step 2.2, Activate a neutral alumina extraction column, and respectively rinse the neutral alumina extraction column with 4 - 8 mL of methanol with a concentration of 50% - 100% and 4 - 8 mL of pure water, with a flow rate of 0.5 - 5 mL / min;

[0010] Step 2.3, Then place the diluted Shuxuening injection into the activated neutral alumina extraction column respectively, and load the sample at a flow rate of 0.1 - 3 mL / min, and collect the eluate;

[0011] Step 2.4: Then elute with 1 - 5 mL of methanol at a flow rate of 0.1 - 3 mL / min. Combine the eluates in Step 2.3 and transfer them to a volumetric flask. Add water to make up the volume to a fixed scale to obtain the test solution.

[0012] Step 3: Inject the prepared test solution into the chromatograph to obtain a fingerprint. Then inject each reference solution into the chromatograph to obtain the chromatogram of the reference sample under the same chromatographic conditions. The chromatographic conditions are as follows: Use a C18 chromatographic column; mobile phase A is acetonitrile containing 0% - 1% formic acid or acetic acid, and mobile phase B is ultrapure water containing 0% - 1% formic acid or acetic acid. Perform gradient elution using mobile phase A and mobile phase B. The elution conditions are: 0 - 5 min, 5 - 15% A; 5 - 25 min, 5% - 15A → 15 - 25% A; 25 - 34 min, 15 - 25% A → 30 - 40% A; the flow rate is 0.1 mL / min - 1 mL / min. Use a PDA detector, the detection wavelength is 200 nm - 300 nm, the column temperature is 20°C - 45°C, and the injection volume is 1 μL - 20 μL.

[0013] Step 4: Calibrate the common peaks in the fingerprint of the test solution.

[0014] Step 5: Identify the common peaks in the fingerprint of the test solution using the chromatogram of the reference sample.

[0015] Step 6: Import the fingerprints of 10 batches of test solutions into the similarity evaluation system software for traditional Chinese medicine chromatographic fingerprints and calculate the similarity.

[0016] Further, the C18 chromatographic column is Waters BEH C18 (2.1×100 mm, 1.7 μm).

[0017] Further, the neutral alumina solid phase extraction column is Cleanert Alumina N neutral alumina solid phase extraction column (1 g / 6 mL).

[0018] Further, the detection wavelength of the PDA detector is 225 nm.

[0019] Further, the column temperature of the chromatographic column is 40°C.

[0020] Further, the flow rate is 0.2 mL / min.

[0021] Further, the injection volume is 2 μL.

[0022] Furthermore, a total of 29 common peaks are calibrated in the fingerprint of the test sample solution. The relative retention times of the 29 common peaks are as follows: 0.145 ± 0.001 (peak 1), 0.171 ± 0.001 (peak 2), 0.219 ± 0.001 (peak 3), 0.287 ± 0.001 (peak 4), 0.330 ± 0.005 (peak 5), 0.347 ± 0.001 (peak 6), 0.388 ± 0.002 (peak 7), 0.438 ± 0.007 (peak 8), 0.511 ± 0.002 (peak 9), 0.530 ± 0.002 (peak 10), 0.675 ± 0.002 (peak 11), 0.779 ± 0.002 (peak 12), 0.852 ± 0.001 (peak 13), 0.892 ± 0.001 (peak 14), 0.915 ± 0.001 (peak 15), 0.940 ± 0.001 (peak 16), 0.963 ± 0.002 (peak 17), 1.000 (S, peak 18), 1.130 ± 0.002 (peak 19), 1.196 ± 0.002 (peak 20), 1.270 ± 0.002 (peak 21), 1.320 ± 0.013 (peak 22), 1.370 ± 0.002 (peak 23), 1.389 ± 0.002 (peak 24), 1.418 ± 0.002 (peak 25), 1.576 ± 0.003 (peak 26), 1.599 ± 0.003 (peak 27), 1.751 ± 0.005 (peak 28), 1.826 ± 0.006 (peak 29).

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention firstly establishes a method for constructing the lignan fingerprint in ginkgo leaf medicinal materials, extracts and their single - ingredient preparations, which can provide a technical method for the quality research and quality control of ginkgo leaf medicinal materials, extracts and their single - ingredient preparations. A total of 14 common peaks of lignan components are co - identified in the present invention, and 16 lignan compounds are co - identified among the 14 common peaks of lignan components, which can more comprehensively clarify the material basis in ginkgo leaf medicinal materials, extracts and their preparations, and provide a basis for the quality control of ginkgo leaf medicinal materials, extracts and their preparations. The UPLC lignan fingerprint detection method established by the present invention can be used for the component detection of ginkgo leaf medicinal materials, extracts and their preparations, which is conducive to more comprehensively controlling the quality of ginkgo leaf medicinal materials, extracts and their preparations, and providing guarantee for the clinical efficacy and safety of ginkgo leaf medicinal materials, extracts and their preparations. Description of the Drawings

[0025] The present invention will be further described below in conjunction with the drawings and embodiments.

[0026] Figure 1 It is the superposition diagram of the fingerprints of 10 batches of Shuxuening test samples;

[0027] Figure 2 It is the relative fingerprint of lignans in Ginkgo biloba medicinal extract and its single preparation. DETAILED DESCRIPTION

[0028] The instruments involved in the present invention are: Waters H-Class ultra-high performance liquid chromatography system (Waters Corporation, USA); Mettler Toledo MS105 electronic analytical balance (Mettler Toledo, Switzerland); Milli-Q Advantange A10 water purifier (Millipore, USA); KQ-250DE ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd.). The reagents used in the present invention are: methanol (chromatographically pure, Admas, China); acetonitrile (chromatographically pure, Admas, China); phosphoric acid (analytical pure, Shanghai Test, China). Cleanert Alumina N neutral alumina solid phase extraction column (1g / 6mL) was purchased from Tianjin Bona Aiger Technology Co., Ltd.

[0029] Step 1: Prepare control sample solution

[0030] The control samples required for the present invention are: oleuropein-4,4'-diglucoside, pinoresinol D, neoglycinoside B, lariciresinol-4,4'-diglucoside, Glehlinoside C, pinoresinol diglucoside, β-D-Glucopyranoside,3-[2-[4-(β-D-glucopyranosyloxy)-3-methoxyphenyl]-2,3-dihydro-3-(hydroxymethyl)-7-methoxy-5-benzofuranyl]propyl,(2R-trans)-(9CI), acanthoside E, neoglycinoside A, Urolignoside, β-D-Glucopyranoside,4-[3-hydroxy-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]propyl]-2-methoxyphenyl, (+)-pinoresinol-β-D-glucopyranoside, dihydrodehydrodiconiferyl alcohol 9-O-β-D-glucoside, (+)-syringaresinol-4-O-β-D-glucoside, (-)-syringaresinol-4-O-β-D-glucoside, Dihydrodehydrodiconiferyl alcohol 9'-O-α-L-rhamnoside. Among them, pinoresinol diglucoside (purity 91.7%, batch number 111537-201706) and acanthoside E (purity 97.9%, batch number 111713-201804) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products, China (for content determination), (+)-pinoresinol-β-D-glucopyranoside was purchased from Chengdu Efar Biotechnology Co., Ltd., and the rest of the reference substances were self-made, and their purities were determined by HPLC area normalization method to be greater than 97%. The above reference substances are all chemical components in the extracts of Ginkgo biloba medicinal materials and their single-herb preparations, and their structures were identified by spectral analysis in the research.

[0031] Accurately measure appropriate amounts of the above control samples respectively, and place them in 5 mL volumetric flasks. Add methanol-aqueous solution with a concentration of 20%-100% to the volumetric flasks to prepare a solution with a concentration of 0.01 mg / mL-2 mg / mL.

[0032] Step 2: Preparation of the test solution

[0033] Step 2.1, Select different batches of Shuxuening injection and dilute it with pure water. Specifically, select 10 batches of Shuxuening injection, and then accurately measure 2.0 mL of the 10 batches of Shuxuening injection respectively, and dilute the Shuxuening injection with pure water by 0-10 times, specifically 5 times;

[0034] Step 2.2: Activate the neutral alumina extraction column. Rinse the neutral alumina extraction column with 4 - 8 mL of methanol with a concentration of 50% - 100% and 4 - 8 mL of pure water successively, with a flow rate of 0.5 - 5 mL / min. Specifically, rinse the neutral alumina extraction column twice with 6 mL of methanol with a concentration of 60% and 6 mL of water successively, with a flow rate of 2 mL / min.

[0035] Step 2.3: Then place the diluted 10 batches of Shuxuening injection into the activated neutral alumina extraction column respectively, and load the sample at a flow rate of 0.1 - 3 mL / min. Specifically, the flow rate is 2 mL / min, and collect the eluate.

[0036] Step 2.4: Then elute with 1 - 5 mL of methanol at a flow rate of 0.1 - 3 mL / min. Specifically, elute with 3 mL of methanol at a flow rate of 1 mL / min, combine the eluate in Step 2.3 and transfer it to a volumetric flask, add water to make the volume up to 5 mL to obtain the test solution.

[0037] Among the selection of the dilution solvent and the elution flow rate, when Shuxuening injection is diluted 5 - fold with pure water and eluted at a flow rate of 2 mL / min, no obvious flavonoid components are detected in the eluate. When eluted at a flow rate of 4 mL / min, a small amount of flavonoid components can be detected in the eluate. When Shuxuening injection is diluted 5.0 - fold with methanol and eluted at a flow rate of 1 mL / min, flavonoid components can still be detected in the eluate. Therefore, in order to adsorb the flavonoid components onto the neutral alumina solid-phase extraction column as much as possible, it is diluted 5 - fold with pure water and eluted at a flow rate of 2 mL / min.

[0038] Step 3: Inject the prepared test solution into the chromatograph to obtain a fingerprint chromatogram. Then, inject each reference substance solution into the chromatograph to obtain the chromatogram of the reference sample under the same chromatographic conditions. The chromatographic conditions are as follows: Use Waters BEHC18 (2.1×100 mm, 1.7 μm) as the chromatographic column; mobile phase A is acetonitrile containing 0%-1% formic acid or acetic acid, and mobile phase B is ultrapure water containing 0%-1% formic acid or acetic acid. Gradient elution is performed using mobile phase A and mobile phase B. The elution conditions are: 0-5 min, 10% A; 5-25 min, 10% A → 20% A; 25-34 min, 20% A → 35% A; the flow rate is 0.1 mL / min - 1 mL / min, preferably 0.2 mL / min, the detection wavelength is 200 nm - 300 nm, preferably 225 nm; the column temperature is 20 °C - 45 °C, preferably 40 °C; the injection volume is 1 μL - 3 μL, preferably 2 μL. With this optimal mobile phase system and elution ratio, the peak information in the chromatogram is comprehensive, the peak shape is good, and the baseline is stable. Using 225 nm as the detection wavelength has a strong absorption for lignan compounds, and the best number of peaks, peak height, peak area, and resolution in each chromatogram can be obtained. When the flow rate is 0.2 mL / min, the resolution of the chromatogram is better, and the elution time is shorter without affecting the resolution and the number of peaks.

[0039] Step 4: Calibration of common peaks

[0040] Inject 10 batches of Shuxuening test solutions respectively by the above method. All chromatographic peaks elute completely within 40 minutes. A total of 29 common peaks are calibrated by comparing the fingerprint chromatograms of 10 batches of Shuxuening. Taking the 18th peak as the reference peak with a retention time of 1.000, the relative retention times of other common fingerprint peaks are shown in Table 1. The superposition diagram of the fingerprint chromatograms of 10 batches of Shuxuening is shown in Figure 1 .

[0041] Table 1 Fingerprint chromatogram of Shuxuening test solution (relative retention time)

[0042]

[0043]

[0044] Step 5: Identification of common peaks

[0045] The prepared control sample solution was injected and the liquid phase spectrum of the control sample was obtained. The fingerprints of 10 batches of Shuxuening injection test samples were compared with the liquid phase spectra of the control samples. A total of 14 common peaks of lignan components were identified in the fingerprints of the 10 batches of Shuxuening test samples, and a total of 16 lignan compounds were identified in the 14 common peaks of lignan components. Because there are many lignan compounds in Shuxuening injection with similar polarity, peak 7 identified three compounds, namely, larch resinol-4,4'-diglucoside, Glehlinoside C, and neoginkgolignan B. The identification of the common peaks of the remaining lignan compounds is shown in Table 2.

[0046] Table 2 Identification of common peaks of lignan compounds

[0047]

[0048]

[0049] Step 6: Import the fingerprints of 10 batches of Shuxuening test products into the Chinese medicine chromatographic fingerprint similarity evaluation system software, and generate the fingerprint superposition maps of 10 batches of Shuxuening test products in the software, see Figure 1 , and then generate a relative fingerprint in the software, see Figure 2 Then, the similarity between the fingerprints of 10 batches of Shuxuening test samples and the relative fingerprints was calculated. The results are shown in Table 3. The similarity of the fingerprints of the 10 batches of Shuxuening test samples is greater than 0.97. The fingerprint of the Shuxuening test sample of the present invention can be used as a standard fingerprint for identifying the quality of Ginkgo biloba medicinal materials, extracts and preparations thereof.

[0050] When using the above-mentioned standard fingerprint to identify the quality of Ginkgo biloba medicinal materials, extracts and preparations thereof, it is only necessary to prepare the sample solution according to the above-mentioned method for preparing the test sample, and prepare the sample for injection according to the above-mentioned chromatographic conditions to obtain the fingerprint of the sample to be tested, and then compare the fingerprint of the sample to be tested with the above-mentioned standard fingerprint, and calculate the similarity, so as to complete the quality identification of the sample to be tested.

[0051] The lignan fingerprint of ginkgo biloba medicinal materials, extracts and single preparations established by the present invention can provide a technical method for quality research and quality control of ginkgo biloba medicinal materials, extracts and single preparations. The UPLC lignan fingerprint detection method established by the present invention can be used for component detection of ginkgo biloba medicinal materials, extracts and preparations, which is conducive to more comprehensive quality control of ginkgo biloba medicinal materials, extracts and preparations, and provides guarantee for the clinical efficacy and safety of ginkgo biloba medicinal materials, extracts and preparations.

[0052] Table 3 Fingerprint numbers and batches of 10 batches of Shuxuening test products

[0053]

[0054] Methodology Verification

[0055] 1. Repeatability Experiment

[0056] Take 6 portions of Shuxuening Injection (batch number 1906192), and prepare the test solution according to the above-mentioned preparation method of the test solution. Inject the samples respectively, and the RSD of the relative retention time of each common peak measured is less than 1%, indicating that the repeatability of this method is good. The test results are shown in Table 4.

[0057] Table 4 Repeatability Investigation (Relative Retention Time)

[0058]

[0059]

[0060] 2. Stability Experiment

[0061] For the same test solution (batch number 1906192), measure it at certain intervals. The RSD of the relative retention time of each common peak measured is less than 2%, and the results show that the sample is stable for at least 24 hours. The test results are shown in Table 5.

[0062] Table 5 Stability Investigation (Relative Retention Time)

[0063]

[0064]

[0065] 3. Precision Test

[0066] For the same test solution (batch number 1906192), inject it continuously for 6 times. The RSD of the relative retention time of each common peak measured is less than 1%. The test results are shown in Table 6.

[0067] Table 6 Precision Investigation (Relative Retention Time)

[0068]

[0069]

[0070] Taking the ideal embodiments of the present invention as the inspiration, through the above description, relevant staff can make various changes and modifications without departing from the scope of the present invention. The technical scope of this invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A method for constructing the lignan fingerprint of Ginkgo biloba medicinal materials, extracts and single-herb preparations, comprising the following steps: Step 1: Preparation of control sample solutions Each control sample was made into a solution with a concentration of 0.01 mg / mL - 2 mg / mL using a 20% - 100% methanol - aqueous solution, and set aside; Step 2: Preparation of test sample solution Step 2.1, Select different batches of Shuxuening injection and dilute it with pure water; Step 2.2, Activate the neutral alumina extraction column, and rinse the neutral alumina extraction column with 4 - 8 mL of methanol with a concentration of 50% - 100% and 4 - 8 mL of pure water respectively, with a flow rate of 0.5 - 5 mL / min; Step 2.3, Then place the diluted Shuxuening injection into the activated neutral alumina extraction column respectively, and load the sample at a flow rate of 0.1 - 3 mL / min, and collect the eluate; Step 2.4, Then elute with 1 - 5 mL of methanol at a flow rate of 0.1 - 3 mL / min, combine the eluates in Step 2.3 and transfer them to a volumetric flask, add water to a fixed scale, and use it as the test sample solution; Step 3: Inject the prepared test sample solution into the chromatograph to obtain a fingerprint, and then inject each reference solution into the chromatograph to obtain the chromatogram of the control sample under the same chromatographic conditions. The chromatographic conditions are: Use a C18 chromatographic column; Mobile phase A is acetonitrile containing 0% - 1% formic acid or acetic acid, Mobile phase B is ultrapure water containing 0% - 1% formic acid or acetic acid, and gradient elution is carried out using the Mobile phase A and the Mobile phase B. The elution conditions are: 0 - 5 min, 5 - 15% A; 5 - 25 min, 5% - 15A → 15 - 25% A; 25 - 34 min, 15 - 25% A → 30 - 40% A; The flow rate is 0.1 mL / min - 1 mL / min, use a PDA detector, the detection wavelength is 200 nm - 300 nm, the column temperature is 20°C - 45°C, and the injection volume is 1 μL - 20 μL; Step 4: Calibrate the common peaks in the fingerprint of the test sample solution; Step 5: Identify the common peaks in the fingerprint of the test sample using the chromatogram of the control sample; Step 6: Import the fingerprint of the test sample solution into the software for evaluating the similarity of traditional Chinese medicine chromatographic fingerprints and calculate the similarity; The C18 chromatographic column is Waters BEH C18(2.1×100mm, 1.7μm); The neutral alumina solid phase extraction column is Cleanert Alumina N neutral alumina solid phase extraction column (1g / 6mL); The detection wavelength of the PDA detector is 225 mm; The column temperature of the chromatographic column is 40°C; The flow rate is 0.2 mL / min; The injection volume is 2 μL.

2. A method for constructing the lignan fingerprint of ginkgo leaf medicinal materials, extracts and single - herb preparations according to claim 1. The fingerprint of the test solution was calibrated with 29 common peaks, and the relative retention times of the 29 common peaks are as follows: 0.145±0.001 (peak 1), 0.171±0.001 (peak 2), 0.219±0.001 (peak 3), 0.287±0.001 (peak 4), 0.330±0.005 (peak 5), 0.347±0.001 (peak 6), 0.388±0.002 (peak 7), 0.438±0.007 (peak 8), 0.511±0.002 (peak 9), 0.530±0.002 (peak 10), 0.675±0.002 (peak 11), 0.779±0.002 (peak 12), 0.852±0.001 (peak 13), 0.892±0.001 (peak 14), 0.915±0.001 (peak 15), 0.940±0.001 (peak 16), 0.963±0.002 (peak 17), 1.000 (S, peak 18), 1.130±0.002 (peak 19), 1.196±0.002 (peak 20), 1.270±0.002 (peak 21), 1.320±0.013 (peak 22), 1.370±0.002 (peak 23), 1.389±0.002 (peak 24), 1.418±0.002 (peak 25), 1.576±0.003 (peak 26), 1.599±0.003 (peak 27), 1.751±0.005 (peak 28), 1.826±0.006 (peak 29).

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