Method for rapidly detecting content of terpene lactones in ginkgo biloba extract

Abstract

The invention discloses a method for rapidly detecting the content of terpene lactones in a ginkgo biloba extract, which comprises the following steps: directly carrying out reflux extraction on an extract sample by using a 90% methanol solution, and analyzing and detecting by using an ultra-high performance liquid chromatography-evaporative light detector combined technology. The method can remarkably shorten the sample pretreatment time and the instrument analysis time, improve the detection efficiency and improve the detection sensitivity, is suitable for industrial production, and plays an important role in guaranteeing the product quality of the ginkgo biloba extract.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for rapidly detecting the content of terpene lactones in Ginkgo biloba leaf extract. Background Technology

[0002] Ginkgo leaf extract is derived from the plant Ginkgo biloba (Ginkgoaceae family). Ginkgo biloba Extracts made from the dried leaves of L. are a widely used traditional Chinese medicine and dietary supplement. They have the effects of promoting blood circulation, removing blood stasis, clearing the meridians and relaxing muscles. In modern medicine, they are mainly used to treat cardiovascular and cerebrovascular diseases, improve cognitive function and circulatory disorders.

[0003] Terpene lactones are among the main pharmacologically recognized active ingredients in Ginkgo biloba leaf extract. They primarily include ginkgolide A, B, and C, and bilobalide. These components have attracted significant attention due to their unique pharmacological activities; for example, ginkgolide B is the most potent known platelet-activating factor (PAF) antagonist. Therefore, the content of terpene lactones is a key indicator for evaluating the quality, authenticity, and efficacy of Ginkgo biloba leaf extract and its related preparations (such as tablets and injections). Pharmacopoes of various countries (such as the Chinese Pharmacopoeia, the United States Pharmacopeia (USP), and the European Pharmacopoeia (EP)) all use its content determination as a mandatory quality control standard. Currently, high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) is widely used in pharmacopoeias both domestically and internationally. This method has been validated over many years and possesses high accuracy and specificity, making it the industry-recognized "gold standard."

[0004] The existing terpene lactone detection has the following main shortcomings: (1) long analysis time and low efficiency. In order to fully separate ginkgo biloba lactone A, B, C and ginkgo biloba lactone, which are similar in structure, the traditional chromatographic method usually adopts a longer chromatographic column (such as 250 mm) and a slower gradient elution program. A complete analysis cycle usually takes more than 45 minutes, even 1 hour. For modern quality control laboratories (such as pharmaceutical manufacturers and testing institutions) that require high-throughput detection, the efficiency is too low, which seriously limits the analysis throughput. (2) Large consumption of organic solvents, high cost and not environmentally friendly. The mobile phase used in Chinese Pharmacopoeia is n-propyl alcohol, tetrahydrofuran and water. N-propyl alcohol can explode when mixed with air, and is flammable when exposed to fire, high temperature and oxidizing agents. Tetrahydrofuran is relatively active in chemical properties and is easily oxidized to form explosive peroxide in air, which needs to be carefully stored and used. Its toxicity is higher than that of acetonitrile and methanol. (3) The pretreatment process is too complex. Chinese Pharmacopoeia requires 9 liquid-liquid extraction pretreatment steps, which further increases the analysis time and operation difficulty. The existing International Business Standard for Plant Extracts Ginkgo Biloba Leaf Extract uses an acidic alumina chromatographic column for component purification, which requires a complex pretreatment process such as sample weighing, column loading, sample loading, elution, evaporation, and transfer. The column loading process is prone to cause sample processing unevenness, and the elution process causes component loss. The existing patent (such as CN201410321649.3) uses ethyl acetate solution for magnetic stirring extraction three times, and then combines, rotary evaporation, transfer and constant volume. Ethyl acetate has a lower boiling point of 76.5-77.5℃, and is prone to loss of components during rotary evaporation.

[0005] Therefore, it is of great significance to establish a more rapid, scientific and accurate detection method. SUMMARY

[0006] The purpose of the present application is to provide a method for rapidly detecting the content of terpene lactones in ginkgo biloba leaf extract, which can significantly shorten the sample pretreatment time and sample analysis time, improve the detection efficiency, and improve the detection sensitivity by using methanol-water cooperative extraction and gradient elution conditions of ultra-high performance liquid chromatography.

[0007] In order to achieve the above-mentioned purpose of the application, the following technical solutions are adopted: A method for rapidly detecting the content of terpene lactones in ginkgo biloba leaf extract, comprising the following steps: (1) Preparation of test solution: take 150 mg of the product, accurately weigh and place in a glass bottle with a cover, add 90% methanol 20 mL, dissolve, tightly seal, heat at 90℃ for 30 minutes, mix, heat at 90℃ for another 30 minutes, cool at room temperature, filter through a filter membrane, take the filtrate as the test solution; (2) Preparation of the control solution: Take ginkgo leaf total lactone control extract, accurately weigh, add methanol to prepare a solution containing 2.5 mg per 1 ml, and then accurately take 2.0, 4.0, 6.0, 8.0, and 10.0 mL of the solution into 10 mL volumetric flasks, respectively, add methanol to the mark, and shake well to obtain the solution. (3) Content determination: accurately take 1.0 μL and 2.0 μl of the control extract solution and 2.0 μl of the test sample solution, respectively, inject into the ultra-high performance liquid chromatograph, determine, and calculate the contents of ginkgolide, ginkgolide A, ginkgolide B, and ginkgolide C by the external standard two-point method logarithmic equation.

[0008] Preferably, the chromatographic column of the ultra-high performance liquid chromatograph in step (3) is filled with octadecylsilane-bonded silica gel.

[0009] Preferably, the particle size of the filler in step (3) is 1.7 µm, and the chromatographic column specification is 2.1*50 mm.

[0010] Preferably, in step (3), methanol-water is used for gradient elution, the flow rate is 0.3 mL / min, the column temperature is 30°C, the drift tube temperature is 100°C, the nitrogen flow rate is 2 L / min, and the theoretical plate number calculated according to the terpene lactone peak should not be less than 2500.

[0011] Preferably, the gradient elution is as follows:

[0012] Preferably, in step (1), a 0.22 µm filter membrane is used for filtration.

[0013] Compared with the prior art, the technical scheme of the present application has at least the following beneficial effects: (1) Simple sample detection pretreatment: 90% methanol is used as the sample extraction solvent, which has better polarity than water solution and can fully disperse and dissolve the sample, thus simplifying the pretreatment.

[0014] (2) Simple and efficient chromatographic conditions: the use of ultra-high performance liquid chromatography technology and methanol and water gradient elution can realize the separation and determination of target components in 12 min, effectively avoid the use of n-propyl alcohol as a normal solvent, and avoid the use of toxic reagent tetrahydrofuran, greatly improve the analysis efficiency, and improve the detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The sample liquid chromatogram of the present application is shown in the following figure: Figure 2 The sample liquid chromatogram of the prior art "Chinese Pharmacopoeia" is shown in the following figure: Figure 3 Sample liquid chromatogram of prior art "Business Standard"; Figure 4 Sample liquid chromatogram prepared by the method of prior art CN201410321649.3. DETAILED DESCRIPTION

[0016] The application will be described in detail below with reference to the accompanying drawings and specific examples.

[0017] The equipment and materials used in the following examples are as follows: (1) Instruments and equipment Waters ACQUITY ultra-high performance liquid chromatograph, AllChrom ELSD6000 evaporative light detector; XS105 electronic balance (Mettler); SK8200HP ultrasonic extractor (Shanghai Keduo Ultrasonic Instrument Co., Ltd.); electric heating constant temperature water bath (Beijing Yongguangming Medical Instrument Co., Ltd.).

[0018] (2) Reagents and materials Ginkgo biloba extract (batch number C0253180202); standard product: total ginkgo biloba lactone control extract, China Institute for Drug Control, batch number 610017-201501; methanol: chromatographically pure; purified water.

[0019] Example 1 Precisely weigh 12.29 mg of total ginkgo biloba lactone control extract, add methanol to prepare a solution containing 2.458 mg per 1 ml, and obtain. Then precisely take 0.4, 0.8, 1.2, 1.6, and 2.0 mL of the solution into 10 mL volumetric flasks, respectively, add methanol to the mark, shake well, and obtain. Chromatographic condition analysis obtains the peak areas of ginkgo biloba lactone, ginkgolide C, ginkgolide A, and ginkgolide B at various concentrations, and establishes a standard curve with the logarithm of the concentration of each control solution as the abscissa and the logarithm of the peak area of each control solution as the ordinate. The concentrations and peak areas of the ginkgo biloba lactone, ginkgolide C, ginkgolide A, and ginkgolide B standard curve series solutions are as follows: Table 1 Linear equation, correlation coefficient, and linear range of ginkgo biloba lactone

[0020] Table 2 Linear equation, correlation coefficient, and linear range of ginkgolide C

[0021] Table 3 Linear equation, correlation coefficient, and linear range of ginkgolide A

[0022] Table 4 Linear equation, correlation coefficient and linear range of ginkgolide B

[0023] From the results, ginkgolide B has a good linear relationship in the concentration range of 165.8167-829.0834 mg / L, the linear equation is Y=1.5825*X+1.8943, and the correlation coefficient R is 0.9968.

[0024] Ginkgolide C has a good linear relationship in the concentration range of 67.4968-337.4834 mg / L, the linear equation is Y=1.7832*X+1.3846, and the correlation coefficient R is 0.9990.

[0025] Ginkgolide A has a good linear relationship in the concentration range of 139.8110-669.0552 mg / L, the linear equation is Y=1.6847*X+1.5993, and the correlation coefficient R is 0.9980.

[0026] Ginkgolide B has a good linear relationship in the concentration range of 79.7375-398.6876 mg / L, the linear equation is Y=1.7581*X+1.3884, and the correlation coefficient R is 0.9995.

[0027] Example 2 Prepare one test sample solution according to the preparation method of the test sample solution, continuously sample and analyze 6 times under the above chromatographic conditions, obtain the chromatographic peak area of each analysis, and the average content of terpene lactones is 6.77%, and the RSD value is 0.82%. The resolution should be greater than 1.5. The related data of the precision experiment are shown in Table 2.

[0028] Table 5 Precision experiment data of terpene lactones

[0029] Example 3 Prepare one test sample solution according to the preparation method of the test sample solution, sample and analyze at 0, 30, 60, 90, 120 and 240 minutes respectively under the above chromatographic conditions, obtain the chromatographic peak area of each analysis, and calculate the RSD value of terpene lactones respectively. The RSD of the stability experiment is 0.55% calculated. The related data of the stability experiment are shown in Table 3.

[0030] Table 6 Stability experiment data of terpene lactones

[0031] From the experimental data in the table, the test sample solution has good stability at room temperature for 6 hours.

[0032] Example 4 Prepare 6 test sample solutions according to the preparation method of the test sample solution, perform chromatographic analysis under the above chromatographic conditions, calculate the content of bilobalide, ginkgolide C, ginkgolide A and ginkgolide B in the test sample solution by using the established standard curve, obtain the content of terpene lactones in the ginkgo leaf extract sample, and calculate the RSD value of the repeatability experiment. The average content of terpene lactones in the ginkgo leaf extract sample is calculated to be 6.75%, and the RSD value of the repeatability experiment is 1.33%. The relevant data of the repeatability experiment are shown in Table 7.

[0033] Table 7 Terpene lactone repeatability experiment data

[0034] Example 5 Take 75 mg of ginkgo leaf extract (batch number C0253180202) in 9 conical flasks, and take 26.94 mg, 8.11 mg, 11.93 mg and 7.62 mg of bilobalide (purity 99.8%), ginkgolide C (purity 99.8%), ginkgolide A (purity 97.4%) and ginkgolide B (purity 95.6%) reference substances in a 50 mL volumetric flask, add methanol to dissolve and dilute to volume (the reference substance solution contains 537.7224 mg / L of bilobalide, 161.8756 mg / L of ginkgolide C, 232.3964 mg / L of ginkgolide A and 145.6944 mg / L of ginkgolide B). 2.3 mL, 4.6 mL and 6.9 mL of the reference substance solution are respectively taken and added to the 9 conical flasks containing the sample, 20 mL of 90% methanol is added to dissolve, tightly sealed, heated at 90°C for 30 minutes, mixed, and then heated at 90°C for another 30 minutes. Cool to room temperature, filter through a 0.22 µm filter membrane, take the filtrate as the test sample solution. Perform chromatographic analysis under the above chromatographic conditions to obtain the content of bilobalide, ginkgolide C, ginkgolide A and ginkgolide B in the test sample solution, then calculate the recovery rate of bilobalide, ginkgolide C, ginkgolide A and ginkgolide B in the sample according to the amount of the reference substance solution added to the sample and the content of bilobalide, ginkgolide C, ginkgolide A and ginkgolide B in the sample measured in the repeatability experiment, and calculate the RSD value of the experiment. The relevant data of the standard addition recovery experiment are shown in Table 8.

[0035] Table 8 Standard addition recovery experiment data

[0036] The experimental results show that the detection method of the content of terpene lactones in the ginkgo leaf extract is accurate and reliable, the average recovery rate of bilobalide, ginkgolide C, ginkgolide A and ginkgolide B is close to 100%, and the deviation is small (RSD% is less than 1%).

[0037] Example 6 The content of terpene lactones in the sample (production batch number: C0253180202) was detected by the method for determining the content of terpene lactones under the item of ginkgo leaf extract according to the preparation method and chromatographic conditions of the test product solution of the present application (new method), the method for determining the content of terpene lactones under the item of ginkgo leaf extract in Chinese Pharmacopoeia 2020 edition part one (current method), International Business Standard Plant Extract Ginkgo Leaf Extract, and CN201410321649.3, Example 2, respectively. The results are shown in Table 9 and Figures 1-4

[0038] Table 9 Comparison of terpene lactone content detection results of three methods

[0039] Compared with the prior art (Chinese Pharmacopoeia, Business Standard, CN201410321649.3, Example 2 method), the pretreatment of the present application is simpler, the detection sensitivity is higher, the detection limit is lower, and the recovery rate of the present application is closer to 100%. From the comparison of the chromatograms, it can be seen that the chromatographic condition analysis of Chinese Pharmacopoeia 2020 edition needs 60 min, the chromatographic condition analysis of Business Standard needs 45 min, the method of CN201410321649.3 needs 20 min in total for chromatographic gradient elution (12 min) and instrument post-equilibrium time, while the present application only needs 12 min. The present application greatly shortens the sample pretreatment time and instrument analysis time, and can improve the sample analysis efficiency.​

Claims

1. A method for rapid detection of terpene lactone content in Ginkgo biloba leaf extract, characterized in that: Includes the following steps: (1) Preparation of test solution: Weigh 150 mg of this product accurately, place it in a glass bottle with a cap, add 20 mL of 90% methanol to dissolve it, seal tightly, heat at 90°C and reflux for 30 minutes, mix well, heat at 90°C and reflux for 30 minutes, cool to room temperature, filter through a filter membrane, and take the filtrate as the test solution. (2) Preparation of reference solution: Take an appropriate amount of total lactone reference extract of Ginkgo biloba leaves, accurately weigh it, add methanol to prepare a solution containing 2.5 mg per 1 ml, then accurately pipette 2.0, 4.0, 6.0, 8.0 and 10.0 mL of the solution into 10 mL volumetric flasks, add methanol to the mark, shake well, and the solution is obtained. (3) Content determination: Accurately pipette 1.0 μL and 2.0 μL of the reference extract solution and 2.0 μL of the test sample solution into the ultra-high performance liquid chromatograph and determine the content of ginkgolide, ginkgolide A, ginkgolide B and ginkgolide C respectively using the external standard two-point logarithmic equation.

2. The method according to claim 1, characterized in that, The ultra-high performance liquid chromatograph in step (3) uses octadecylsilane-bonded silica gel as the packing material.

3. The method according to claim 2, characterized in that, The particle size of the packing agent in step (3) is 1.7µm, and the column size is 2.1*50mm.

4. The method according to claim 1, characterized in that, Gradient elution with methanol-water was performed at a flow rate of 0.3 mL / min and a column temperature of 30℃; the drift tube temperature was 100℃; the nitrogen flow rate was 2 L / min; and the theoretical plate number, calculated based on the terpene lactone peak, should be no less than 2500.

5. The method according to claim 1, characterized in that, The gradient elution specifically refers to: 。 6. The method according to claim 1, characterized in that, In step (1), a 0.22µm filter membrane is used for filtration.

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