CN112684087A - Method for measuring content of total flavonol glycosides and total ginkgolic acid in ginkgo leaf extracting solution - Google Patents
Method for measuring content of total flavonol glycosides and total ginkgolic acid in ginkgo leaf extracting solution Download PDFInfo
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Abstract
The invention relates to a method for measuring the content of total flavonol glycosides and total ginkgolic acid in a ginkgo leaf extract, which comprises the following steps: preparing a sample solution by using a methanol hydrochloric acid solution, and measuring the content of the total flavonol glycosides in the sample solution by using HPLC (high performance liquid chromatography), wherein the chromatographic conditions when the content of the total flavonol glycosides is detected are as follows: a C18 chromatographic column, taking methanol-0.4% phosphoric acid solution as a mobile phase, and detecting the wavelength of 360 nm; the method for measuring the content of the total ginkgoic acid in the ginkgo leaf extract comprises the following steps: preparing a test solution by using methanol, measuring the content of total ginkgoic acid in the test solution by using HPLC, wherein the chromatographic conditions for detecting the content of the total ginkgoic acid are as follows: a C18 chromatography column; performing gradient elution by using acetonitrile containing 0.1% of trifluoroacetic acid as a mobile phase A and water containing 0.1% of trifluoroacetic acid as a mobile phase B; the detection wavelength is 310 nm. The invention establishes a method for measuring the content of total flavonol glycosides and total ginkgolic acid in the ginkgo biloba extract, and can be used for the quality control research of the ginkgo biloba extract.
Description
Technical Field
The invention discloses a method for measuring the content of total flavonol glycoside and total ginkgolic acid in a ginkgo leaf extracting solution, belonging to the technical field of plant extraction and detection.
Background
Folium Ginkgo is dry leaf of Ginkgo biloba L. of Ginkgo of Ginkgoaceae, has effects of promoting blood circulation for removing blood stasis, dredging collaterals, relieving pain, astringing lung, relieving asthma, eliminating turbid pathogen, and reducing blood lipid, and can be widely used for treating blood stasis, obstruction of collaterals, thoracic obstruction, cardiodynia, apoplexy, hemiplegia, cough and asthma due to lung deficiency, and hyperlipidemia. The folium Ginkgo contains flavonoids, terpene lactones, carboxylic acids, alkylphenols, polypentenol, etc., and has effects of dilating blood vessel, improving brain circulation, and inhibiting platelet activating factor. The ginkgo biloba extract is an extract prepared by processing dry leaves of ginkgo biloba, is registered and sold in more than 70 countries in the world as one of the most mature plant medicines, and most of the ginkgo biloba medicines and preparations are prepared by adopting the ginkgo biloba extract at present and play an important role in treating cardiovascular and cerebrovascular diseases. Meanwhile, ginkgo biloba extract is also used for the development of various ginkgo drugs, beverages, health products, foods, cosmetics and other products. The ginkgetin is one of the main active components of ginkgo leaf extract, and has effects of dilating coronary artery blood vessel, increasing blood flow, improving brain nutrition and resisting bacteria. The ginkgolides compounds include: the bilobalide A, the bilobalide B, the bilobalide C, the bilobalide M, the bilobalide J and the bilobalide, and literature researches show that the bilobalide is a platelet activating factor receptor antagonist, wherein the bilobalide B has the strongest activity. The total ginkgolic acid in the ginkgo biloba extract is considered as a toxic substance, and can cause gastrointestinal adverse reactions and even increase transaminase.
At present, the extraction process of ginkgo leaves is reported in a lot of but not the same, the extraction of active ingredients of the ginkgo leaves is usually carried out by using solvents such as ethanol, water and the like, and the extraction rate of the ethanol is higher. In recent years, the extraction method for ginkgetin comprises organic solvent extraction, steam distillation, supercritical fluid extraction, ultrasonic extraction, microwave-assisted extraction and the like, wherein an ethanol reflux method is the most widely used and mature method at present. In the process of the ethanol reflux method, the main factors influencing the extraction rate of the total flavonol glycosides of the effective components of the extract comprise the concentration of an extraction solvent, the dosage of the extraction solvent, the extraction times and the extraction time. But different processes lead to more obvious differences in the effect of the final extraction.
In addition, the method for measuring the content of total flavonol glycosides and total ginkgoic acid in different ginkgo leaf extracting solutions has an important influence on the research of the ginkgo leaf extracting solutions, so that the establishment of a proper measuring standard for the content of total flavonol glycosides and total ginkgoic acid in the ginkgo leaf extracting solutions is very important for accurately researching the ginkgo leaf extracting solutions.
Chinese patent CN109381496A discloses ginkgo leaf extract and its quality control method and use. The ginkgo leaf extract contains 24.0-37.0% of total flavonol glycosides and 6.0-12.0% of terpene lactones according to the dry product, preferably, the rutin content is not more than 4.0%, the free quercetin content is not more than 0.4%, more preferably, the total ginkgoic acid content is not more than 2mg/kg, and further preferably, the heavy metal content is as follows: lead is not more than 5mg/kg, cadmium is not more than 0.3mg/kg, arsenic is not more than 2mg/kg, mercury is not more than 0.2mg/kg, and copper is not more than 20 mg/kg. However, the above patent does not provide a systematic method for measuring the content of total flavonol glycosides and total ginkgolic acids in the ginkgo biloba extract.
The detection method of total ginkgolic acid in ginkgo biloba extract in the Chinese pharmacopoeia comprises the following steps: weighing 2g of the powder, precisely weighing, placing into a conical flask with a plug, precisely adding 10ml of methanol, weighing, ultrasonically dissolving, cooling, shaking uniformly with methanol of less than the initial weight, filtering, and removing the filtrate. Taking 50uL sample of the sample filtered by the filter membrane for detection (the mobile phase is 0.1% trifluoroacetic acid water solution and 0.1% trifluoroacetic acid acetonitrile solution). In order to ensure that the total ginkgoic acid in the produced ginkgo biloba extract is qualified, production process samples (mostly solutions in different production procedures) need to be monitored, if a pharmacopeia method is adopted for control, a certain amount of uniform solution needs to be taken for concentration and drying, then the dry powder samples are pretreated and detected for a long time, and in the actual production process, the condition of production discontinuity often occurs in order to wait for a detection result, so that the production period is prolonged, and the production cost is increased.
Chinese patent CN111487345A discloses a method for rapidly detecting total ginkgoic acid in samples during the production process of ginkgo biloba extract, which comprises steps of rapid pretreatment and detection, and can rapidly detect total ginkgoic acid in liquid samples and solid samples during the production process of ginkgo biloba extract, thereby greatly shortening the detection time of the index, and effectively ensuring the product quality and realizing high continuity of production by monitoring the samples in the process in real time. However, no measurement of total flavonol glycosides is given in the above patent.
Disclosure of Invention
The invention aims to provide a method for measuring the content of total flavonol glycosides and total ginkgolic acid in a ginkgo leaf extracting solution, which is used for deeply researching the ginkgo leaf extracting solution.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a method for measuring the content of total flavonol glycosides and total ginkgolic acid in a ginkgo leaf extracting solution, which comprises the steps of measuring the content of the total flavonol glycosides in a ginkgo leaf extract and the content of the total ginkgolic acid in the ginkgo leaf extract,
the method for measuring the content of the total flavonol glycosides in the ginkgo leaf extract comprises the following steps:
taking folium Ginkgo extract, preparing into sample solution with methanol hydrochloric acid solution, measuring total flavonol glycoside content in the sample solution by HPLC,
the chromatographic conditions for detecting the content of the total flavonol glycosides are as follows: a chromatographic column: dikma Diamonsil Plus C18 column with methanol-0.4% phosphoric acid solution (50:50) as mobile phase, detection wavelength: 360 nm;
the method for measuring the content of the total ginkgoic acid in the ginkgo leaf extract comprises the following steps:
taking ginkgo leaf extract, preparing a test solution by using methanol, measuring the content of total ginkgoic acid in the test solution by using HPLC,
the chromatographic conditions for detecting the total ginkgoic acid content are as follows: a chromatographic column: a Dikma Diamonsil Plus C18 chromatography column; performing gradient elution by using acetonitrile containing 0.1% of trifluoroacetic acid as a mobile phase A and water containing 0.1% of trifluoroacetic acid as a mobile phase B; detection wavelength: 310 nm.
In one embodiment of the present invention, the test solution is prepared by the following steps:
precisely weighing folium Ginkgo extract, adding mixed solution of methanol-25% hydrochloric acid solution (4:1), heating and refluxing in water bath, rapidly cooling to room temperature, transferring to a measuring flask, adding methanol to desired volume, shaking, filtering, and collecting filtrate to obtain test solution for detecting total flavonol glycoside content.
In one embodiment of the present invention, the test solution is prepared by the following steps:
taking 0.1g of ginkgo leaf extract, precisely weighing, adding 25ml of a mixed solution of methanol-25% hydrochloric acid solution (4:1), placing in a water bath, heating and refluxing for 30 minutes, rapidly cooling to room temperature, transferring to a 50ml measuring flask, fixing the volume to a scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution for detecting the content of the total flavonol glycosides.
In one embodiment of the present invention, the specification of the Dikma diamond Plus C18 chromatographic column when determining the total flavonol glycoside content is: 5 μm, 4.6 mm. times.250 mm.
In one embodiment of the present invention, the theoretical plate number is not less than 8000 as calculated from the peak of quercetin when the total flavonol glycoside content is measured.
In one embodiment of the present invention, the total flavonol glycoside extraction rate is calculated as follows:
the total flavonol glycoside extraction rate (%) - (%) of the total flavonol glycoside in the extracting solution/the total flavonol glycoside content (mg) in the ginkgo leaf.
In one embodiment of the present invention, the test solution is prepared by the following steps:
precisely weighing folium Ginkgo extract, adding methanol, weighing, dissolving with ultrasound, cooling, adding methanol to supplement the weight loss, shaking, filtering, and collecting the filtrate to obtain the sample solution.
In one embodiment of the invention, the theoretical plate number is not less than 6000 calculated according to ginkgolic acid when the total ginkgolic acid content is detected.
In one embodiment of the present invention, when detecting the total ginkgoic acid content, the HPLC mobile phase gradient for determining the total ginkgoic acid content in the ginkgo biloba extract is as follows: within 0-30 minutes, the content of the mobile phase A is 75-90%, the content of the mobile phase B is 25-10%, within 30-35 minutes, the content of the mobile phase A is 90%, within 10% of the mobile phase B, within 35-36 minutes, the content of the mobile phase A is 90-75%, within 10-25% of the mobile phase B, within 36-45 minutes, the content of the mobile phase A is 75%, and the content of the mobile phase B is 25%.
In one embodiment of the present invention, when the total ginkgolic acid content is measured,
absorbing a test solution, a ginkgo neoacid reference solution and a total ginkgoic acid reference solution, injecting the test solution, the ginkgo neoacid reference solution and the total ginkgoic acid reference solution into a high performance liquid chromatograph, respectively obtaining total peak areas of chromatographic peaks corresponding to the total ginkgoic acid reference in the test solution, determining 5 chromatographic peaks of the total ginkgoic acid in a chromatogram of a ginkgo leaf extract by using the total ginkgoic acid positioning reference solution, and calculating the content of the total ginkgoic acid by using the ginkgo neoacid reference by using an external standard method;
wherein the concentration of the neoacid reference solution is 322.0 μ g/mL-1(ii) a The total ginkgoic acid reference solution refers to: a solution containing 20.21 mug of total ginkgolic acid per l ml made of methanol.
In the process of establishing the method for measuring the content of the total ginkgoic acid in the ginkgo biloba extract, the selection of an extraction solvent in the preparation of a test sample is key, ether or methanol extraction is reported in documents, and the fact that the content of the ginkgoic acid in an ethanol-extracted sample is higher than that of the ether extraction in the experimental process is found, so that the establishment of an accurate content measuring method is more facilitated, therefore, the method refers to the pharmacopoeia of 2015 edition, and selects methanol as the extraction solvent of the test sample. Meanwhile, a preliminary experiment also finds that the sampling amount of a sample is also a key, the content of the total ginkgoic acid is slightly reduced along with the increase of the sampling amount, but the total ginkgoic acid concentration is possibly lower than the detection limit due to the over-small sampling amount, and the accurate quantification cannot be realized. Therefore, on the premise of ensuring certain extraction efficiency, the detection limit of the total ginkgoic acid is considered, and the optimized sampling amount is 2 g. The content of the total ginkgoic acid in the 3 batches of ginkgo biloba extracts measured by the method is not higher than 10 mug/g, and the content of the total ginkgoic acid meets the requirement according to the limit range of the total ginkgoic acid content in the 2015 edition of pharmacopoeia.
The invention adopts high performance liquid chromatography to establish an external standard determination method taking quercetin, kaempferol and isorhamnetin as standard substances, and the result shows that the established content determination method is stable and reliable through experimental methodology researches such as linear relation, precision, stability, repeatability, sample adding recovery rate and the like. Literature studies show that the method has no significant difference from the one-test-multiple-evaluation method in the pharmacopoeia of 2015 edition. The total flavonoid glycoside content in the three batches of ginkgo extracts is not less than 100 mg/g.
Based on the research of chemical components and related pharmacological actions of the ginkgo biloba extract, the invention adopts thin-layer chromatography to establish a quality control identification method of the ginkgo biloba extract, adopts high performance liquid chromatography to establish a content determination method of total flavonol glycosides and total ginkgoic acid in the ginkgo biloba extract, and can be used for the quality control research of the ginkgo biloba extract.
Drawings
FIG. 1 is an HPLC chromatogram of a mixed standard solution of quercetin, kaempferol, isorhamnetin and ginkgo biloba extract;
in fig. 1, a. mixed standard solution: 1, quercetin; 2, kaempferol; 3 isorhamnetin B, ginkgo biloba extract;
FIG. 2: HPLC chromatograms of neoginkgolic acid, total ginkgolic acid and folium Ginkgo extract;
in FIG. 2, A is ginkgolic acid; b, total ginkgoic acid; c, ginkgo leaf extract;
FIG. 3 the effect of ethanol concentration on the extraction rate of total flavonol from ginkgo biloba leaf extract
FIG. 4 shows the effect of different extraction times on the extraction rate of total flavonol from ginkgo leaf extract
FIG. 5 TLC chromatogram of ginkgo biloba extract
In fig. 5, 1 is ginkgo biloba extract reference 2, ginkgo biloba extract (2017032201)3, ginkgo biloba extract (2017032202)4, ginkgo biloba extract (2017032203).
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
The instruments used in the following examples are illustrated below:
an Agilent 1200series high performance liquid chromatograph (VWD detector, quaternary constant flow pump, online degasser, autosampler, column oven); an Agilent 1100series high performance liquid chromatograph (Alltech ELSD detector, quaternary constant flow pump, online degasser, autosampler, column oven); SB5200 type ultrasonic cleaner (Ningbo Xinzhi Biotech Co., Ltd.); XS105DU electronic balance (mertler-toledo international trade (shanghai) ltd); BS124S electronic balance (sydow sedolis, germany); DZF-6050 vacuum drying cabinet (Shanghai precision instruments & instruments).
The reagents used in the following examples are illustrated below:
ginkgo biloba extract (self-made in the laboratory, lot numbers: 2017032201, 2017032202, 2017032203); the reference substances such as neoginkgolic acid, quercetin, total ginkgolic acid and the like are purchased from China pharmaceutical and biological product institute; folium Ginkgo (lot number: 20170301, available from Shanghai Kangqiao Chinese medicinal decoction pieces Co., Ltd.); acetonitrile and methanol are used as chromatographic pure water (purified water, Hangzhou child Hahaha Baili food Co., Ltd.); the rest of the reagents are analytically pure (purchased from Shanghai chemical reagent company of China medicine (group)).
Wherein, the method for obtaining the ginkgo biloba extract batch number 2017032201 comprises the following steps:
accurately weighing 10g of folium ginkgo fine powder in a round bottom flask, adding 6 times volume amount of 60% ethanol, heating and refluxing for 1.5h, combining the extracting solutions, filtering, recovering ethanol, concentrating to a proper amount, accurately weighing 1ml, placing in a 100ml volumetric flask, and adding methanol to a constant volume.
The method for obtaining folium Ginkgo extract lot 2017032202 comprises:
accurately weighing 10g of folium ginkgo fine powder in a round bottom flask, adding 8 times volume of 70% ethanol, heating and refluxing for 2h, combining the extracting solutions, filtering, recovering ethanol, concentrating to a proper amount, accurately weighing 1ml, placing in a 100ml volumetric flask, and adding methanol to a constant volume.
The method for obtaining folium Ginkgo extract lot 2017032203 comprises:
accurately weighing 10g of folium ginkgo fine powder in a round bottom flask, adding 10 times volume of 80% ethanol, heating and refluxing for 2.5h, combining the extracting solutions, filtering, recovering ethanol, concentrating to a proper amount, accurately weighing 1ml, placing in a 100ml volumetric flask, and adding methanol to a constant volume.
Example 1
Establishment of method for measuring content of total flavonol glycosides in folium Ginkgo extract
(1) Chromatographic conditions
A chromatographic column: dikma Diamond Plus C18 column (5 μm, 4.6 mm. times.250 mm) with methanol-0.4% phosphoric acid solution (50:50) as mobile phase, detection wavelength: 360 nm. The theoretical plate number is not less than 8000 according to the peak calculation of quercetin.
(2) Preparation of control solutions
Precisely weighing control substances such as quercetin 10.08mg, kaempferol 10.23mg, and isorhamnetin 10.45mg, and diluting with methanol to constant volume of 10ml to obtain concentrations of 1.008, 1.023, and 1.045 mg/ml-1Mixing the standard solutions.
(3) Preparation of test solution
Taking 0.1g of ginkgo leaf extract, precisely weighing, adding 25ml of a mixed solution of methanol-25% hydrochloric acid solution (4:1), placing in a water bath, heating and refluxing for 30 minutes, rapidly cooling to room temperature, transferring to a 50ml measuring flask, fixing the volume to the scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the ginkgo leaf extract.
(4) Specialization inspection
Taking the mixed reference solution and the folium Ginkgo extract sample solution, and determining according to the above chromatographic conditions, referring to figure 1 for HPLC chromatogram of the mixed reference solution of quercetin, kaempferol, isorhamnetin and folium Ginkgo extract.
The result shows that corresponding chromatographic peaks of quercetin, kaempferol and isorhamnetin in the chromatogram of the ginkgo leaf extract test solution correspond to the chromatographic peaks, and the retention time is respectively as follows: t1 ═ 10.57 min; t2 ═ 17.12 min; t3 ═ 18.87 min. The results show that: the chromatographic peaks of quercetin, kaempferol and isorhamnetin and other components can achieve better baseline separation, and the separation degree R is more than 1.5.
(5) Investigation of linear relationships
Collecting quercetin (1.008 mg. ml)-1) Kaempferol (1.023 mg/ml)-1) And isorhamnetin (1.045 mg/ml)-1) Mixing standard solutions, adding methanol to dilute to series of standard solutions, wherein the concentrations of quercetin are 2.016, 10.08, 50.4, 100.8, 201.6 and 403.2 μ g/ml respectively-1The kaempferol concentrations are 2.046, 10.23, 51.15, 102.3, 204.6 and 409.2 mug/ml respectively-1The isorhamnetin concentration is2.09, 10.45, 52.25, 104.5, 209.0 and 418.0. mu.g/ml-1. The measurement was performed under the chromatographic conditions in the present example, and linear regression was performed with the compound concentration as abscissa (X) and the peak area as ordinate (Y). Wherein the regression equation of quercetin is Y-19.131X-44.723, r-0.9997; the regression equation of kaempferol is 15.157X +12.556, and r is 0.9999; the regression equation of isorhamnetin is 25.4X +17.4, and r is 0.9996. The results show that: the content of quercetin, kaempferol and isorhamnetin is 2.016-403.2, 2.046-409.2, 2.09-418.0 μ g/ml-1The linear relationship within the concentration range is good. Wherein the detection limits (signal-to-noise ratio 3:1) of quercetin, kaempferol and isorhamnetin are 0.231, 0.305 and 0.206 μ g/ml respectively-1(ii) a The quantitative limits (signal-to-noise ratio of 10: l) are 0.762, 1.006 and 0.680 mu g/ml respectively-1。
(6) Precision test
Respectively and precisely absorbing 10 mu L of high, medium and low concentration quercetin, kaempferol and isorhamnetin reference substance solutions, measuring according to the chromatographic condition under 2.1, continuously and repeatedly injecting samples for 6 times, and the result is shown in Table 1, wherein the peak areas RSD of the high, medium and low concentration quercetin, kaempferol and isorhamnetin reference substances are less than 3%, which indicates that the used instrument has good precision.
TABLE 1 results of precision test investigation
(7) Repeatability test
The method of this example was followed to prepare test solutions from ginkgo biloba leaves, and 6 samples were processed in parallel, and the total flavonol content of the 6 samples was measured according to the chromatographic conditions of this example, as shown in table 2, the average content of quercetin, kaempferol, isorhamnetin, and total flavonol control samples were 56.1, 57.4, 10.6, and 124.1 μ g-1RSD are all<3%, indicating that the method has good repeatability.
TABLE 2 repeatability test investigation results
(8) Stability test
The same ginkgo biloba extract test solution is taken and placed at room temperature for 0, 2, 4, 6, 8, 12 and 24 hours respectively, the results of the chromatographic condition determination given in the embodiment are shown in table 3, the average peak areas of quercetin, kaempferol and isorhamnetin corresponding to the ginkgo biloba extract are 4765.4, 3850.5 and 633.0 respectively, and the RSD is less than 2.0%, which shows that the test solution is stable in 24 hours at room temperature.
TABLE 3 stability test investigation results
(9) Sample addition recovery experiment
The average sample recovery rates of quercetin, kaempferol, isorhamnetin and total flavonol glycoside measured according to the chromatographic conditions given in this example were 98.1% (RSD ═ 2.4%), 97.3% (RSD ═ 1.8%), 101.2% (RSD ═ 1.6%) and 99.3% (RSD ═ 2.1%) respectively, indicating good recovery rates, when 9 parts of ginkgo leaf extract with known content were taken and added to the sample, respectively, the sample was treated according to the method for preparing the sample solution given in this example, which corresponds to the content of quercetin, kaempferol and isorhamnetin in the ginkgo leaf extract sample, 80%, 100% and 120%.
Example 2
Establishment of method for measuring total ginkgolic acid content in ginkgo leaf extract
(1) Chromatographic conditions
A chromatographic column: dikma Diamond Plus C18 column (5 μm, 4.6 mm. times.250 mm); gradient elution was performed according to the procedure for mobile phase in table 4 using acetonitrile containing 0.1% trifluoroacetic acid as mobile phase a and water containing 0.1% trifluoroacetic acid as mobile phase B; detection wavelength: 310 nm. The theoretical plate number is not less than 6000 calculated according to the neoacid of ginkgo.
TABLE 4 HPLC mobile phase gradient for determination of total ginkgolic acid content in ginkgo biloba leaf extract
(2) Preparation of control solutions
Precisely weighing neoacid ginkgo 8.05mg, diluting with methanol to 25m L, and making into 322.0 μ g/mL solution-1The ginkgolic acid reference solution; an appropriate amount of total ginkgolic acid reference substance is taken, and methanol is used for preparing a solution containing 20.21 mu g of total ginkgolic acid per l of total ginkgolic acid reference substance as a reference solution for positioning.
(3) Preparation of test solution
Weighing about 2g of the powder, accurately weighing, placing in a conical flask with a plug, accurately adding 10ml of methanol, weighing, ultrasonically dissolving, cooling, adding methanol to make up for the lost weight, shaking, filtering, and collecting the filtrate to obtain the sample solution.
(4) Sample assay
Precisely absorbing the test solution, the neoacid ginkgo reference solution and the total ginkgoic acid reference solution by 10 mu 1 respectively, injecting the solutions into a high performance liquid chromatograph to respectively obtain the total peak areas of the chromatographic peaks corresponding to the total ginkgoic acid reference in the test solution, determining 5 chromatographic peaks of the total ginkgoic acid in the chromatogram of the ginkgo biloba extract by using the total ginkgoic acid positioning reference solution, and calculating the content of the total ginkgoic acid by using the neoacid ginkgo reference by adopting an external standard method.
(5) Specialization inspection
Taking a ginkgo neoacid reference substance, a total ginkgo acid reference substance and a test solution, determining according to the chromatographic conditions, wherein the result shows that the ginkgo neoacid reference substance has the same chromatographic peak at the chromatographic peak position of the ginkgo neoacid reference substance, the ginkgo leaf extract test solution has the same chromatographic peak, the retention time is 8.432min, 5 chromatographic peaks can be detected in the chromatogram of the total ginkgo acid reference substance, corresponding chromatographic peaks correspond to the chromatographic peaks in the chromatogram of the ginkgo leaf extract test solution, and the retention time is respectively: t1 ═ 8.43 min; t2 ═ 8.95 min; t3 ═ 9.76 min; t4 ═ 11.23 min; t5 ═ 11.79 min. The chromatographic peaks of the total ginkgoic acid and other components can achieve better baseline separation, and are shown in figure 2.
(6) Linear relationship investigation of ginkgolic acids
The neoacid ginkgo control solution prepared in this example was diluted with methanol to a concentration of 161.0, 80.5, 40.25, 4.025, 1.006, 0.503. mu.g/mL-1Respectively absorbing 10 mul of the solution, sampling, measuring the total peak area value of each chromatographic peak, drawing a standard curve by taking the concentration x as a horizontal coordinate and the total peak area value y as a vertical coordinate, and performing linear regression to obtain a regression equation: y is 20.29x-5.4, r is 0.9997, and the results show that: the total peak area and concentration of neoic acid of semen Ginkgo are 0.503-161.0 μ g/mL-1The linear relationship within the range is good. Wherein the detection limit and the quantification limit are calculated by ginkgolic acid, and the detection limit (signal to noise ratio 3:1) is 0.167 mu g/mL-1The limit of quantitation (signal-to-noise ratio of 10: l) is about 0.503. mu.g.mL-1。
(7) Precision test
Precisely sucking 10 μ L of each of high, medium and low concentration ginkgolic acid reference substance solutions (1.006, 50.2, 200.8), measuring according to the chromatographic conditions given in this example, and continuously repeating sample injection for 6 times, wherein the results are shown in Table 5, and the peak area RSD of the high, medium and low concentration reference substance is less than 3%, which indicates that the used instrument has good precision.
TABLE 5 results of precision test investigation
(8) Repeatability test
The method of this example was used to prepare a test solution from ginkgo biloba extract, 6 portions were processed in parallel, and the total ginkgolic acid content of the 6 samples was determined according to the chromatographic conditions of this example, as shown in Table 6, the average total ginkgolic acid content was 9.686 μ g-1，RSD<3%, indicating that the method has good repeatability.
TABLE 6 repeatability test investigation results
(9) Stability test
The same ginkgo biloba extract test solution is taken and placed at room temperature for 0, 2, 4, 6, 8, 12 and 24 hours respectively, the result is determined according to the chromatographic condition under the item of 4.2.1, the result is shown in table 7, the average value of the total ginkgolic acid peak area corresponding to the ginkgo biloba extract is 34.7, and the RSD is less than 2.8 percent, which shows that the test solution is stable within 24 hours at room temperature.
Table 7 stability test investigation results
(10) Sample application recovery test
The ginkgo neoacid control solutions with the content of 80%, 100% and 120% of the total ginkgoic acid content in the ginkgo biloba extract sample are respectively added into 9 parts of the ginkgo biloba extract with the known content, the ginkgo neoacid control solutions are treated according to the preparation method of the test solution of the embodiment, and the results are measured according to the chromatographic conditions of the embodiment and are shown in table 8. The average recovery (n ═ 9) was 99.2%, and RSD < 3.0%, indicating good recovery.
Table 8 sample recovery test results (n ═ 9)
Example 3
Research on preparation process of ginkgo leaf extract
The method takes the total flavonol glycosides in the ginkgo leaf extract as an index, examines the influence of ethanol concentration, solvent dosage, extraction time and extraction frequency on the extraction rate of the total flavonoids in the ginkgo leaf in the alcohol extraction preparation process of the ginkgo leaf extract, and first examines main influencing factors in the alcohol extraction method of the ginkgo leaf extract by a single-factor experiment.
(1) Preparation of folium Ginkgo ethanol reflux extract
Accurately weighing 10g of folium ginkgo fine powder in a round-bottom flask, adding ethanol, heating, refluxing and extracting, combining extracting solutions, filtering, recovering ethanol, concentrating to a proper amount, precisely weighing 1ml, placing in a 100ml volumetric flask, and fixing the volume with methanol. Measuring the content of total flavonol glycoside in the extractive solution by high performance liquid chromatography, and calculating the extraction rate of total flavonol glycoside.
The total flavonol glycoside extraction rate (%) - (%) of the total flavonol glycoside in the extracting solution/the total flavonol glycoside content (mg) in the ginkgo leaf.
(2) Influence of ethanol concentration on extraction rate of total flavonol glycosides
Accurately weighing 10g of ginkgo leaf fine powder, respectively adding 8 times of ethanol with different concentrations, heating and refluxing for 3 times, wherein the ethanol concentrations are respectively 20%, 40%, 60% and 80%, and investigating the influence of the ethanol with different concentrations on the extraction rate of the total flavonol glycosides. The results showed that the extraction rate of total flavonol glycosides gradually increased with the increase of ethanol concentration, and increased to (85.2 ± 12.1)%, and tended to be stable when the ethanol concentration was increased to 60%, and to (87.3 ± 9.7)%, when the ethanol concentration was increased to 80%, therefore, orthogonal experiments were performed with ethanol concentrations of 60%, 70%, and 80%, and the results are shown in fig. 3.
(3) Influence of extraction frequency on total flavonol glycoside extraction rate
Accurately weighing 10g of folium Ginkgo fine powder, adding 8 times of 60% ethanol, heating, reflux extracting, and examining the influence of different extraction times (1 time, 2 times, 3 times, 4 times) on the extraction rate of total flavonol glycosides. The experimental results show that when 60% ethanol is used for extracting the total flavonol glycosides from ginkgo leaves, the extraction rate is basically stable after more than 3 times of extraction, and the extraction rates of 3 times of extraction and 4 times of extraction are respectively (87.2 +/-9.4)% and (89.2 +/-10.1)%, and the results are shown in figure 4. In order to save the extraction time, the number of times of extraction of ginkgo biloba leaves is selected to be 3.
(4) Preparation process for optimizing ginkgo leaf extract in orthogonal test
The single factor investigation result shows that the main factors influencing the yield of the ginkgo leaf extract comprise the concentration of ethanol, the dosage of solvent and the extractionTime, three levels for each of the above three factors, using L9 (3)4) Table orthogonal experiments were performed as shown in table 9.
TABLE 9 factor level table
Table 10 orthogonal experimental design table and results
TABLE 11 evaluation index Quadrature experiment ANOVA results
F0.05(2，2)＝19.0、F0.01(2，2)＝99.0
The results of the anova showed that the main factor of the three factors of ethanol concentration (A), ethanol volume (B) and refluxing time (C) in the ethanol refluxing process of ginkgo leaves was ethanol concentration (A), and the best extraction process was A2B2C2, as shown in tables 10-11. Weighing folium Ginkgo powder, reflux-extracting with 70% ethanol 8 times the weight of folium Ginkgo for 3 times, each time for 2 hr, mixing extractive solutions, filtering, recovering ethanol, and vacuum drying to obtain folium Ginkgo extract.
(5) Verification of folium ginkgo ethanol reflux process conditions
The ethanol reflux process verification experiment is carried out according to the preferable experimental conditions of the orthogonal analysis experiment, the experiment is carried out for three times, the extraction rate of the total flavonol glycosides in the reflux liquid is measured, and the measurement results are shown in table 12. And (4) conclusion: the ethanol reflux process optimized in the orthogonal experiment is stable and reliable, and can be used for preparing the ginkgo leaf extract.
TABLE 12 results of the validation experiment of the orthogonal experiment
Example 4
Sample assay for Ginkgo biloba leaf extract
(1) The method for measuring the content of total flavonol glycosides in the extract of example 1 was used to calculate the extraction yield of total flavonol glycosides.
The total ginkgolic acid content of ginkgo biloba extract was determined by weighing 3 samples of ginkgo biloba extract according to the method of example 2, and the results are shown in table 13. The total content of ginkgolic acid in 3 batches of ginkgo leaf extract samples is not higher than 10 mug/g.
Table 13 determination of total ginkgolic acid content in ginkgo biloba extract samples (n ═ 3)
(2) Thin-layer chromatography identification of ginkgo leaf extract
Taking folium Ginkgo extract sample 0.2g, adding n-butanol 15ml, placing in water bath, warm soaking for 15min, shaking, cooling, filtering, evaporating filtrate, dissolving residue with ethanol 2ml to obtain sample solution. Another control extract solution is prepared from folium Ginkgo 0.2g by the same method. According to thin layer chromatography general rule (0502), sucking 1 μ l of the two solutions, respectively dropping on the same silica gel G thin layer plate with sodium carboxymethylcellulose solution containing 4% sodium acetate as adhesive, spreading with ethyl acetate-butanone-formic acid-water (5:3:1:1) as developing agent, removing, air drying, spraying with 3% aluminum trichloride ethanol solution, and inspecting under ultraviolet lamp (365 nm). In the thin layer chromatography of the 3 test samples, the same color of fluorescent spot appeared at the corresponding position of the control extract chromatogram, as shown in FIG. 5.
(3) Determination of moisture
Taking a proper amount of ginkgo leaf extract sample, paving the ginkgo leaf extract sample in a flat weighing bottle which is dried to constant weight, precisely weighing the ginkgo leaf extract sample until the thickness is not more than 5mm, opening a bottle cap, drying the ginkgo leaf extract sample at the temperature of 100-105 ℃ for 5h, covering the ginkgo leaf extract sample, moving the ginkgo leaf extract sample into a dryer, cooling the ginkgo leaf extract sample for 30 minutes, precisely weighing the ginkgo leaf extract sample, drying the ginkgo leaf extract sample at the temperature for 1h, cooling the ginkgo leaf extract sample, and weighing the ginkgo leaf extract sample until the difference between. The water content of the test article was calculated based on the weight loss and found to be not higher than 5%.
The invention adopts single factor to primarily screen the concentration of the extraction solvent and the extraction times, and determines that the ethanol concentration range for ethanol reflux extraction is between 60 and 80 percent, and the extraction times are 3 times. Orthogonal experimental design was performed using an L9(34) orthogonal experimental chart, and it was determined that the optimum process was extraction with 8 times of 70% ethanol for 3 times, each for 2 hours. The optimized process will be further validated by a scale-up pilot experiment for use in industrial production.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A method for measuring the content of total flavonol glycosides and total ginkgolic acid in a ginkgo leaf extracting solution is characterized by comprising the steps of measuring the content of the total flavonol glycosides in a ginkgo leaf extract and the content of the total ginkgolic acid in the ginkgo leaf extract,
the method for measuring the content of the total flavonol glycosides in the ginkgo leaf extract comprises the following steps:
taking folium Ginkgo extract, preparing into sample solution with methanol hydrochloric acid solution, measuring total flavonol glycoside content in the sample solution by HPLC,
the chromatographic conditions for detecting the content of the total flavonol glycosides are as follows: a chromatographic column: dikma Diamonsil Plus C18 column with methanol-0.4% phosphoric acid solution (50:50) as mobile phase, detection wavelength: 360 nm;
the method for measuring the content of the total ginkgoic acid in the ginkgo leaf extract comprises the following steps:
taking ginkgo leaf extract, preparing a test solution by using methanol, measuring the content of total ginkgoic acid in the test solution by using HPLC,
the chromatographic conditions for detecting the total ginkgoic acid content are as follows: a chromatographic column: a Dikma Diamonsil Plus C18 chromatography column; performing gradient elution by using acetonitrile containing 0.1% of trifluoroacetic acid as a mobile phase A and water containing 0.1% of trifluoroacetic acid as a mobile phase B; detection wavelength: 310 nm.
2. The method for measuring the content of total flavonol glycosides and total ginkgolic acid in ginkgo leaf extract as claimed in claim 1, wherein the preparation method of the test solution for measuring the content of total flavonol glycosides is as follows:
precisely weighing folium Ginkgo extract, adding mixed solution of methanol-25% hydrochloric acid solution (4:1), heating and refluxing in water bath, rapidly cooling to room temperature, transferring to a measuring flask, adding methanol to desired volume, shaking, filtering, and collecting filtrate to obtain test solution for detecting total flavonol glycoside content.
3. The method for measuring the content of total flavonol glycosides and total ginkgolic acid in ginkgo leaf extract as claimed in claim 2, wherein the preparation method of the test solution for measuring the content of total flavonol glycosides is as follows:
taking 0.1g of ginkgo leaf extract, precisely weighing, adding 25ml of a mixed solution of methanol-25% hydrochloric acid solution (4:1), placing in a water bath, heating and refluxing for 30 minutes, rapidly cooling to room temperature, transferring to a 50ml measuring flask, fixing the volume to a scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution for detecting the content of the total flavonol glycosides.
4. The method for determining the content of total flavonol glycosides and total ginkgolic acid in ginkgo biloba extract according to claim 1, wherein when the content of total flavonol glycosides is detected, the specification of a Dikma Diamond Plus C18 chromatographic column is as follows: 5 μm, 4.6 mm. times.250 mm.
5. The method for determining the contents of total flavonol glycosides and total ginkgolic acid in ginkgo biloba leaves extract as claimed in claim 1, wherein the theoretical plate number is not less than 8000 calculated according to the peak of quercetin when the content of total flavonol glycosides is determined.
6. The method for determining the content of total flavonol glycosides and total ginkgolic acid in ginkgo leaf extract as claimed in claim 1, wherein the method for calculating the extraction rate of total flavonol glycosides when detecting the content of total flavonol glycosides is as follows:
the total flavonol glycoside extraction rate (%) - (%) of the total flavonol glycoside in the extracting solution/the total flavonol glycoside content (mg) in the ginkgo leaf.
7. The method for measuring the content of total flavonol glycosides and total ginkgolic acid in ginkgo leaf extract as claimed in claim 1, wherein the preparation method of the test solution for measuring the content of total ginkgolic acid is as follows:
precisely weighing folium Ginkgo extract, adding methanol, weighing, dissolving with ultrasound, cooling, adding methanol to supplement the weight loss, shaking, filtering, and collecting the filtrate to obtain the sample solution.
8. The method for determining the content of total flavonol glycosides and total ginkgolic acid in ginkgo biloba extract according to claim 1, wherein the number of theoretical plates is not less than 6000 calculated according to neoginkgolic acid when the content of total ginkgolic acid is detected.
9. The method for determining the content of total flavonol glycosides and total ginkgolic acid in ginkgo biloba extract according to claim 1, wherein the HPLC mobile phase gradient for determining the content of total ginkgolic acid in the ginkgo biloba extract is as follows: within 0-30 minutes, the content of the mobile phase A is 75-90%, the content of the mobile phase B is 25-10%, within 30-35 minutes, the content of the mobile phase A is 90%, within 10% of the mobile phase B, within 35-36 minutes, the content of the mobile phase A is 90-75%, within 10-25% of the mobile phase B, within 36-45 minutes, the content of the mobile phase A is 75%, and the content of the mobile phase B is 25%.
10. The method for determining the content of total flavonol glycosides and total ginkgolic acid in ginkgo biloba extract as claimed in claim 1, wherein when the content of total ginkgolic acid is detected,
absorbing a test solution, a ginkgo neoacid reference solution and a total ginkgoic acid reference solution, injecting the test solution, the ginkgo neoacid reference solution and the total ginkgoic acid reference solution into a high performance liquid chromatograph, respectively obtaining total peak areas of chromatographic peaks corresponding to the total ginkgoic acid reference in the test solution, determining 5 chromatographic peaks of the total ginkgoic acid in a chromatogram of a ginkgo leaf extract by using the total ginkgoic acid positioning reference solution, and calculating the content of the total ginkgoic acid by using the ginkgo neoacid reference by using an external standard method;
wherein the concentration of the neoacid reference solution is 322.0 μ g/mL-1(ii) a The total ginkgoic acid reference solution refers to: a solution containing 20.21 μ g of total ginkgolic acid per 1ml prepared from methanol.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315058A (en) * | 2017-05-04 | 2017-11-03 | 黑龙江珍宝岛药业股份有限公司 | A kind of method of total ginkgoic acid in detection ginkgo biloba succi |
| CN111983077A (en) * | 2020-08-18 | 2020-11-24 | 贵州益佰制药股份有限公司 | Detection method of ginkgo dipyridamole injection preparation |
-
2020
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315058A (en) * | 2017-05-04 | 2017-11-03 | 黑龙江珍宝岛药业股份有限公司 | A kind of method of total ginkgoic acid in detection ginkgo biloba succi |
| CN111983077A (en) * | 2020-08-18 | 2020-11-24 | 贵州益佰制药股份有限公司 | Detection method of ginkgo dipyridamole injection preparation |
Non-Patent Citations (1)
| Title |
|---|
| 詹益兴, 科学技术文献出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115219623A (en) * | 2022-07-14 | 2022-10-21 | 山东宜岛康制药有限公司 | Method for measuring content of total flavonol glycosides and terpene lactones in ginkgo leaf granules |
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