CN112129860B - Method for determining content of coumarins in radix angelicae pubescentis - Google Patents
Method for determining content of coumarins in radix angelicae pubescentis Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides a method for determining the content of coumarin components in radix angelicae pubescentis, which is characterized in that the content of 6 coumarin components in the radix angelicae pubescentis is determined simultaneously by adopting an ionic liquid-microwave-assisted extraction combined high performance liquid chromatography-fluorescence detection method; the 6 kinds of coumarins include dihydrocarveol-beta-D-glucoside, dihydrocarveol, angelol A, dihydrocarveol acetate, osthole, and dihydrocarveol angelate. By adopting the method, the chromatographic condition and the fluorescence detection condition are reasonably selected, so that the rapid extraction and detection of 6 coumarin components in the radix angelicae pubescentis can be realized, and the method has the advantages of simplicity, convenience, high sensitivity, high analysis speed and the like, and can be used for quality control of the radix angelicae pubescentis medicinal material.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine component determination, in particular to a method for determining content of coumarin components in radix angelicae pubescentis.
Background
Radix Angelicae Pubescentis (Angelica pubescens Radix) is the dried root of Angelica gigas nakai. Pubescent angelica root is one of the most commonly used Chinese herbs. Radix angelicae pubescentis originally recorded in Shennong herbal channels, is the top grade, has the efficacies of expelling wind-damp, relieving exterior syndrome, relieving pain and the like, and is commonly used for treating wind-damp, lumbago and gonalgia, cold headache and the like. Both the Notopterygii rhizoma and the radix Angelicae Pubescentis and herba Taxilli decoction use radix Angelicae Pubescentis as the principal drug, and have effects of stopping pathogenic wind and relieving pain. The quality of the medicament has great influence on the effectiveness and safety of clinical medication.
The chemical components of radix Angelicae Pubescentis mainly comprise volatile oil and coumarin compounds, wherein the coumarin compounds are compounds containing benzophenone alpha-pyrone basic structure. The coumarin compounds are various in types, and can be divided into simple coumarin, furocoumarin, pyranocoumarin and dicumarol. The medicinal chemistry research shows that the coumarin component of radix Angelicae Pubescentis mainly comprises dihydrocarveol-beta-D-glucoside, umbelliferone, dihydrocarveol, bergapten, angelol A, dihydrocarveol acetate, isoimperatorin, osthole and dihydrocarveol angelate, and has effects of relieving pain, resisting inflammation, inhibiting platelet aggregation, and resisting thrombi. Therefore, the content determination of the coumarin components has important significance for the quality control of the radix angelicae pubescentis medicinal material.
Disclosure of Invention
The invention aims to provide a method for determining the content of coumarin components in radix angelicae pubescentis, which is used for realizing the rapid extraction and detection of 6 coumarin components in the radix angelicae pubescentis medicinal material, namely dihydrocarveol-beta-D-glucoside, dihydrocarveol, angelol A, dihydrocarveol acetate, osthole and dihydrocarveol angelate, and can be used for the quality control of the radix angelicae pubescentis medicinal material. The specific technical scheme is as follows:
the application provides a method for determining the content of coumarin components in radix angelicae pubescentis, which is characterized in that the content of 6 coumarin components in the radix angelicae pubescentis is determined simultaneously by adopting an ionic liquid-microwave assisted extraction method combined with a high performance liquid chromatography-fluorescence detection method; the 6 coumarin components comprise dihydrocarveol-beta-D-glucoside, dihydrocarveol, angelol A, dihydrocarveol acetate, osthole and dihydrocarveol angelate, and the method comprises the following steps:
(1) establishing a standard curve of 6 coumarin components;
preparing 5-10 mixed reference substance solutions containing 6 coumarin components with different known concentrations by using methanol as a solvent; wherein, the concentrations of the columbianadin-beta-D-glucoside, the columbianadin and the columbianadin are respectively 0.005-8 mug/mL, the concentrations of the columbianadin A and the columbianadin acetate are respectively 0.02-30 mug/mL, and the concentration of the osthole is 0.3-30 mug/mL;
under the same chromatographic conditionsAnd under fluorescence detection conditions, the volume V 1 Respectively injecting the mixed reference substance solution into a high performance liquid chromatograph, determining coumarin components of each chromatographic peak through fluorescence detection, and obtaining the chromatographic peak area of each coumarin component;
wherein the chromatographic conditions comprise:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: the phase A is methanol, and the phase B is water; gradient elution is carried out by adopting 40-85% of phase A and 15-60% of phase B in volume fraction; flow rate: 0.2-0.5 mL/min; column temperature: 25-35 ℃; sample volume V 1 :1-5μL；
Respectively establishing a standard curve of each coumarin component by taking the peak area of each coumarin component chromatographic peak as a vertical coordinate and the concentration of each coumarin component as a horizontal coordinate;
(2) obtaining the chromatographic peak area of a sample solution to be detected;
taking the sample to be measured with the mass M as the volume V 2 Microwave extracting the ionic liquid water solution with the concentration of 30-200 millimole per liter, and filtering to obtain a sample solution to be detected, wherein M/V 2 Is (3-30): 1mg/mL, the microwave extraction power is 50-600W, and the microwave extraction time is 1-7 minutes;
taking the volume V under the same chromatographic conditions and fluorescence detection conditions as in step (1) 1 Injecting the sample solution to be detected into a high performance liquid chromatograph, determining coumarin components of each chromatographic peak through fluorescence detection, and obtaining the chromatographic peak area of each coumarin component;
(3) determining the content of 6 coumarin components in a sample to be detected;
according to the established standard curve of each coumarin component, the concentration C of each coumarin component is respectively obtained according to the chromatographic peak area of each coumarin component in the sample solution to be detected 1 Respectively calculating the contents C of 6 coumarin components in the sample to be detected according to the following formula;
C＝C 1 ×V 2 /M。
in some embodiments of the present application, in step (2)In the method, a sample to be measured with the mass M is divided into V with the volume 2 Microwave extracting the ionic liquid water solution with the concentration of 50-150 millimole per liter, and filtering to obtain a sample solution to be detected, wherein M/V 2 Is (5-20): 1mg/mL, the microwave extraction power is 100-500W, and the microwave extraction time is 2-6 minutes.
In some embodiments of the present application, in step (2), the sample to be tested with mass M is treated with volume V 2 Microwave extracting the ionic liquid water solution with the concentration of 70-130 millimole per liter, and filtering to obtain a sample solution to be detected, wherein M/V 2 Is (5-15): 1mg/mL, the microwave extraction power is 200-400W, and the microwave extraction time is 2-5 minutes.
In some embodiments of the present application, the ionic liquid is selected from at least one of 1-dodecyl-3-methylimidazole trifluoromethylsulfonate, 1-dodecyl-3-methyl-1H-imidazole hydrogensulfate, brominated-1-dodecyl-3-methylimidazole, and brominated-1-ethyl-3-methylimidazole.
In some embodiments of the present application, the concentrations of columbiananol- β -D-glucoside, columbiananol angelate are 0.02-5 μ g/mL, respectively, the concentrations of columbinol a, columbinol acetate are 0.1-25 μ g/mL, respectively, and the concentration of osthole is 0.5-25 μ g/mL in the mixed control solution.
In some embodiments of the present application, in step (1), a mixed control stock solution containing 6 coumarin components is prepared by using methanol as a solvent, wherein the concentrations of columbiananol-beta-D-glucoside, columbiananol angelate are respectively 5-8 μ g/mL, the concentrations of columbinol A and columbinol acetate are respectively 25-30 μ g/mL, and the concentration of osthole is 25-30 μ g/mL;
and diluting the mixed reference substance stock solution with methanol to obtain the 5-10 mixed reference substance solutions containing 6 coumarin components with different known concentrations.
In some embodiments of the present application, the gradient elution is specifically: 40-40% of A in 0-5 min; 5-10 minutes, 40% -45% A; 45% -45% of A for 10-15 minutes; 45% -52% of A in 15-25 min; 25-35 minutes, 52% -52% A; 35-37 minutes, 52% -75% A; 37-42 minutes, 75% -80% A; 80-85% of A in 42-45 minutes; 45-50 minutes, 85% -85% A; 85% -40% of A in 50-52 minutes; 52-57 minutes, 40% -40% A.
In some embodiments of the present application, the fluorescence detection conditions comprise: an excitation wavelength of 325nm and an emission wavelength of 400 nm.
The method for determining the content of the coumarin components in the radix angelicae pubescentis provided by the invention adopts the combination of ionic liquid-microwave assisted extraction and high performance liquid chromatography-fluorescence detection, wherein the ionic liquid-microwave assisted extraction can quickly extract the coumarin components from the dried radix angelicae pubescentis powder, and the content of 6 coumarin components in the radix angelicae pubescentis can be simultaneously determined by reasonably selecting chromatographic conditions and fluorescence detection conditions.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other embodiments can be obtained by using the drawings without creative efforts.
FIG. 1 is a high performance liquid chromatogram of radix Angelicae Pubescentis under fluorescent detection; wherein, the A picture is a high performance liquid chromatogram of a blank solvent; b is a high performance liquid chromatogram of the mixed reference solution; the C picture is a high performance liquid chromatogram of the sample solution to be detected; wherein, each numeral symbol represents: 1. dihydrocarveol-beta-D-glucoside; 2. dihydrocarveol; 3. angelol A; 4. carvacrol acetate; 5. osthole; 6. dihydrocarveol angelate.
FIG. 2 is a graph of the relationship between total coumarin content extracted and a single factor test; wherein, the A picture is the ionic liquid species; b, the concentration of an ionic liquid aqueous solution is shown in the figure, wherein the ionic liquid is brominated-1-dodecyl-3-methylimidazole; the C figure is the amount of the medicinal materials; d, drawing microwave extraction power; and E is the microwave extraction time.
Fig. 3 is a graph of the response of individual variables to the effect of total coumarin content.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Instrument for measuring the position of a moving object
High performance liquid chromatograph: HPLC 1200 (agilent technologies, usa); one tenth of a ten thousand electronic balance: AX205(METTLER TOLEDO, switzerland); an ultra-pure water device: Mill-Q type II (Millipore, USA); a microwave extraction instrument: anton paar Microwave Reaction System SOLV (Graz, Austria); a pulverizer (Tianjin Tester instruments, Ltd.).
Reagent
Methanol: chromatographic purity, Fisher corporation; ultrapure water: Mill-QII type, Millipore corporation;
ionic liquid: 1-octyl-3-methylimidazolium iron tetrachloride ([ C ] 8 MIM]FeCl 4 ) 1-ethyl-3-methylimidazolium ferric chloride ([ C ] 2 MIM]FeCl 3 ) 1-hexyl-3-methylimidazolium tetrafluoroborate ([ C ] 6 MIM]BF 4 ) 1-butyl-3-methylimidazolium hexafluorophosphate ([ C) 4 MIM]PF 6 ) 1-dodecyl-3-methylimidazolium trifluoromethanesulfonate ([ C ] 12 MIM]CF 3 SO 3 H) 1-dodecyl-3-methylimidazolium hydrogen sulfate ([ C ] 12 MIM]HSO 4 ) 1-dodecyl-3-methylimidazole [ C ] bromide 12 MIM]Br), bromo-1-ethyl-3-methylimidazole ([ C ] 2 MIM]Br), purchased from shanghai chengjie chemical ltd (shanghai, china); the ionic liquid is dissolved in water to prepare an ionic liquid aqueous solution with corresponding concentration.
Material
Twelve batches of radix angelicae pubescentis medicinal materials are purchased from Sichuan, Hubei and Gansu Chinese medicinal material markets, and dried crude drugs are crushed by a crusher and pass through a 50-mesh screen to obtain dried radix angelicae pubescentis powder as a sample to be detected. The standard samples of dihydrocarveol acetate, dihydrocarveol angelate and osthole are purchased from WUDSTER Biotechnology Co., Ltd, and the standard samples of dihydrocarveol, angelol A and dihydrocarveol-beta-D-glucoside are obtained by laboratory separation, and the purity is more than 98% through high performance liquid chromatography determination. The structural formula of each standard product is as follows:
chromatographic conditions
A chromatographic column: agilent Eclipse Plus C 18 (4.6X 100mm,1.8 μm); mobile phase: the phase A is methanol, and the phase B is ultrapure water; gradient elution, the elution procedure is: 40-40% of A in 0-5 min; 5-10 minutes, 40% -45% A; 45% -45% of A for 10-15 minutes; 45% -52% of A in 15-25 min; 25-35 minutes, 52% -52% A; 35-37 minutes, 52% -75% A; 37-42 minutes, 75% -80% A; 80-85% of A in 42-45 minutes; 45-50 minutes, 85% -85% A; 85% -40% of A in 50-52 minutes; 52-57 minutes, 40% -40% A; column temperature: 30 ℃; flow rate: 0.3 mL/min; sample introduction volume: 2 mu L of the solution;
conditions for fluorescence detection
Excitation wavelength: 325 nm; emission wavelength: 400 nm.
Example 1 Standard Curve establishment
1. Preparation of stock solution of mixed reference substance
Accurately weighing dihydrocarveol-beta-D-glucoside, dihydrocarveol, angelol A, dihydrocarveol acetate, osthole and dihydrocarveol angelate, respectively dissolving with methanol, and preparing stock solutions of each reference substance with concentration of 1 mg/mL. Precisely measuring each reference substance stock solution respectively to prepare a mixed reference substance stock solution containing 5 mu g/mL of dihydrocarveol-beta-D-glucoside, 5 mu g/mL of dihydrocarveol, 25 mu g/mL of angelol A, 25 mu g/mL of dihydrocarveol acetate, 25 mu g/mL of osthole and 5 mu g/mL of dihydrocarveol angelate, and placing the mixed reference substance stock solution in a refrigerator at 4 ℃ for later use.
2. Establishing a standard curve
Accurately measuring the mixed reference substance stock solution in the step 1, sequentially diluting with methanol as a solvent to obtain mixed reference substance solutions with different concentrations, and taking the mixed reference substance solutions with different concentrations, and carrying out sample injection of 2 muL for analysis to obtain liquid chromatogram maps of the reference substances with different concentrations, wherein the liquid chromatogram map of the mixed reference substance solution with the concentration of 4 is shown in a B picture in figure 1. The peak area (Y) of the analyte was taken as the ordinate, the concentration (X) of the analyte was taken as the abscissa, regression calculation was performed by the weighted least square method, the weight coefficient was 1/X, the regression equation and the correlation coefficient of each compound were obtained, the concentration of each control when S/N (signal-to-noise ratio) was 10 was taken as the limit of quantitation (LOQ), the concentration of each control when S/N was 3 was taken as the limit of detection (LOD), and the results are shown in Table 2.
TABLE 1 Mixed control solutions
TABLE 2 Standard Curve, Linear Range, LOD and LOQ
Example 2 determination of extraction conditions of Angelica pubescens
1. Single factor test
The influence of the types of the ionic liquids (8 types), the concentrations of the ionic liquid aqueous solutions (50, 75, 100, 125 and 150mM), the amounts of the medicinal materials (50, 100, 150, 200 and 250mg), the microwave extraction powers (100, 200, 300, 400 and 500W) and the microwave extraction times (2, 3, 4, 5 and 6min) on the extraction of the coumarin components of the radix angelicae pubescentis is respectively researched through a single-factor test.
1.1 Effect of Ionic liquid species on extraction results
8 different types of ionic liquids are selected, 1 is 1-octyl-3-methylimidazole ferric chloride, 2 is 1-ethyl-3-methylimidazole ferric chloride, 3 is 1-hexyl-3-methylimidazole tetrafluoroborate, 4 is 1-butyl-3-methylimidazole hexafluorophosphate, 5 is bromo-1-ethyl-3-methylimidazole, 6 is bromo-1-dodecyl-3-methylimidazole, 7 is 1-dodecyl-3-methylimidazole hydrogen sulfate, and 8 is 1-dodecyl-3-methylimidazole trifluoromethylsulfonate, and the ionic liquid aqueous solutions are respectively dissolved in water to prepare different types of ionic liquid aqueous solutions with the concentration of 100 millimoles per liter (mM).
Precisely weighing 100mg of dried radix angelicae pubescentis powder, weighing 8 parts, respectively adding 10mL of ionic liquid aqueous solutions with different types and concentrations of 100mM, performing microwave extraction, wherein the microwave extraction power is 300W, the microwave extraction time is 4 minutes, filtering the obtained extraction solution by using a 0.22-micrometer filter membrane, injecting samples according to the chromatographic conditions, measuring peak areas, and calculating the content of 6 coumarin components according to the standard curve of example 1.
The results are shown in graph a of fig. 2, where the abscissa is the ionic liquid species; the ordinate is total coumarin content, calculated by the sum of the contents of 6 coumarin components (the same below). As can be seen from the diagram a in fig. 2, when the ionic liquid is any one of brominated-1-ethyl-3-methylimidazole, brominated-1-dodecyl-3-methylimidazole, 1-dodecyl-3-methylimidazole hydrogensulfate and 1-dodecyl-3-methylimidazole trifluoromethanesulfonate, the content of extracted total coumarin is high, and the extraction rate of the total coumarin is high.
1.2 Effect of concentration of Ionic liquid in aqueous solution on extraction results
The brominated-1-dodecyl-3-methylimidazole is dissolved in water to prepare aqueous solution of the brominated-1-dodecyl-3-methylimidazole with the concentration of 50mM, 75 mM, 100mM, 125 mM and 150mM respectively.
Accurately weighing 100mg of dried radix angelicae pubescentis powder, weighing 5 parts, respectively adding 10mL of the brominated-1-dodecyl-3-methylimidazole water solutions with different concentrations, performing microwave extraction, wherein the microwave extraction power is 300W, the microwave extraction time is 4 minutes, filtering the obtained extraction solution by using a 0.22-micrometer filter membrane, injecting samples according to the chromatographic conditions, measuring the peak area, and calculating the content of 6 coumarin components according to the standard curve of example 1.
The results are shown in graph B of FIG. 2, in which the concentration of the aqueous solution of bromo-1-dodecyl-3-methylimidazole is plotted on the abscissa and the total coumarin content is plotted on the ordinate. As can be seen from the graph B in FIG. 2, the concentration of the aqueous solution of bromo-1-dodecyl-3-methylimidazole from 50mM to 100mM increased the content of total coumarin extracted, and from 100mM to 150mM, the content of total coumarin extracted was substantially unchanged. The concentration of the ionic liquid aqueous solution used in the present application is 30-200mM, preferably 50-150mM, and more preferably 70-130mM, in consideration of the extraction efficiency and the test cost.
1.3 influence of the ratio of the sample quality to be measured to the volume of the ionic liquid aqueous solution on the extraction result
Respectively weighing 50mg, 100mg, 150 mg, 200 mg and 250mg of dried radix angelicae pubescentis powder, adding 10mL of 100mM aqueous solution of bromo-1-dodecyl-3-methylimidazole, performing microwave extraction with the microwave extraction power of 300W for 4 minutes, filtering the obtained extraction solution with a 0.22 mu m filter membrane, injecting samples according to the chromatographic conditions, measuring peak areas, and calculating the content of 6 coumarin components according to the standard curve of example 1.
The result is shown in figure 2C, with the abscissa representing the amount of the medicinal material and the ordinate representing the total coumarin content. As shown in the C chart of FIG. 2, when the amount of the medicinal material is 50-250mg, the ratio of M/V is 2 Is (5-25): when the concentration is 1mg/mL, the content of the extracted total coumarin is higher, and the extraction rate of the total coumarin is higher. Thus, the M/V employed in this application 2 Is (3-30): 1mg/mL, preferably M/V 2 Is (5-20): 1mg/mL, more preferably M/V 2 Is (5-15): 1 mg/mL.
1.4 influence of microwave extraction Power on extraction results
Accurately weighing 100mg of dried radix angelicae pubescentis powder, weighing 5 parts, respectively adding 10mL of 100mM aqueous solution of bromo-1-dodecyl-3-methylimidazole, performing microwave extraction with microwave extraction power of 100, 200, 300, 400 and 500W for 4 minutes, filtering the obtained extraction solution with a 0.22 mu m filter membrane, injecting sample according to the chromatographic conditions, measuring the peak area, and calculating the content of 6 coumarin components according to the standard curve of example 1.
The results are shown in graph D of FIG. 2, with microwave extraction power on the abscissa and total coumarin content on the ordinate. As can be seen from the graph D in FIG. 2, when the microwave extraction power is 100-500W, the extracted total coumarin content is higher, and the extraction rate of the total coumarin is higher, so that the microwave extraction power of the present application is selected from 50-600W, preferably 100-500W, and more preferably 200-400W.
1.5 Effect of microwave extraction time on extraction results
Accurately weighing 100mg of dried radix angelicae pubescentis powder, weighing 5 parts, respectively adding 10mL of 100mM aqueous solution of bromo-1-dodecyl-3-methylimidazole, performing microwave extraction with the microwave extraction power of 300W for 2, 3, 4, 5 and 6 minutes, filtering the obtained extraction solution with a 0.22 mu m filter membrane, injecting sample according to the chromatographic conditions, measuring the peak area, and calculating the content of 6 coumarin components according to the standard curve of example 1.
The results are shown in graph E of FIG. 2, with microwave extraction time on the abscissa and total coumarin content on the ordinate. As can be seen from the graph E in fig. 2, when the microwave extraction time is 2 to 6 minutes, the content of extracted total coumarin is high, and the extraction rate of total coumarin is high, so the microwave extraction time in the present application is selected from 1 to 7 minutes, preferably 2 to 6 minutes, and more preferably 2 to 5 minutes.
2. Response surface test
According to the single-factor test result, the influence of the method parameters on microwave extraction of coumarin components is determined by adopting a three-factor three-level Box Behnken Design (BBD) and a Response Surface Method (RSM). The BBD of the independent variables and their levels are shown in Table 3. The complete design included 12 treatments with 5 replicates at the center as shown in table 4. The total coumarin content is represented by the sum of the contents of 6 coumarin components. The total coumarin value is expressed by a quadratic polynomial mathematical model. The mathematical model is shown in equation (1).
Y＝α 0 +α 1 X 1 +α 2 X 2 +α 3 X 3 +α 4 X 12 +α 5 X 13 +α 6 X 23 +α 7 X 1 2 +α 8 X 2 2 +α 9 X 3 2 (1)
Wherein, X 1 、X 2 And X 3 Respectively representing the amount of the medicinal material, the microwave extraction power and the microwave extraction time. Alpha is alpha 0 Is the intercept of the model. Alpha is alpha n The values represent the corresponding regression coefficients.
Table 3 independent variables and levels in BBD
TABLE 4 experimental design of BBD
Analysis of variance of response surface quadratic model As shown in Table 5, the model F value (588.28) and associated lower p value for total coumarin in the experiment are shown as p<0.0001. this result confirms that the established model is meaningful when the P value is<At 0.05, the model terms may be considered meaningful. According to the principle, the models B, C, BC and A 2 、B 2 And C 2 Are considered to be significant. From the F value, the size sequence of the factors influencing the extraction of the coumarin components is as follows: microwave extraction time>Microwave power extraction>The amount of the medicinal materials. The fitting loss of the F value and the p value is respectively 4.45 and 0.0916, and further proves that the model formula can better predict the content of the total coumarin, and the established second-order model is as follows:
Y＝11.49+0.005X 1 +0.064X 2 +0.14X 3 -0.0025X 12 -0.0075X 13 -0.06X 23 -0.9X 1 2 -0.66X 2 2 -0.25X 3 2 (8)
TABLE 5 analysis of variance of response surface quadratic model
* Variance source p-value<0.05；R 2 ＝0.9987；R 2 adj＝0.9970
The response graph of the influence of the single variable on the total coumarin content is shown in fig. 3, and the optimal extraction condition is predicted to be 303.56W of microwave extraction power, 3.29min of microwave extraction time and 100.08mg of medicinal material through response surface software optimization analysis. According to the actual operation conditions of the experiment, the optimal extraction conditions are adjusted to that the microwave extraction power is 300W, the microwave extraction time is 3.3min, and the amount of the medicinal materials is 100 mg. 10mL of bromide-1-dodecyl-3-methylimidazole aqueous solution with the concentration of 100mM is used as an extraction solvent, a sample solution to be detected is prepared under the condition, the content of the extracted total coumarin is 11.47mg/g by sample injection analysis according to the chromatographic condition, and the experimental result is very close to a predicted value (11.514), so that the optimal extraction condition is reliable.
3. Preparation of sample solution to be tested
Preparing a sample solution to be detected according to the extraction conditions determined by the response surface test, which specifically comprises the following steps:
precisely weighing 100mg of dried radix Angelicae Pubescentis powder, and adding 10mL of brominated-1-dodecyl-3-methylimidazole ([ C ] with a concentration of 100 millimole per liter (mM) 12 MIM]Br), performing microwave extraction with the microwave extraction power of 300W for 3.3 minutes, and filtering the obtained extraction solution with a 0.22 mu m filter membrane to obtain a sample solution to be detected.
Example 3 determination of content of coumarin components in Angelica pubescens
Taking 12 batches of radix angelicae pubescentis medicinal material powder, 3 parts of each, weighing 100mg of each, obtaining a sample solution to be detected according to the preparation method of the sample solution to be detected in embodiment 2, and determining according to the chromatographic conditions and the fluorescence detection conditions to obtain the high performance liquid chromatogram of each coumarin component in the radix angelicae pubescentis sample, wherein the high performance liquid chromatogram of the sample solution to be detected in the batch of Sichuan-1 is shown as a graph C in figure 1, and a graph A in figure 1 is the high performance liquid chromatogram of a blank solvent without the sample to be detected. Standard curves according to the coumarin components in Table 2Respectively determining the concentration C corresponding to the peak area of each coumarin component in the radix angelicae pubescentis sample 1 Respectively calculating the contents of 6 coumarin components in the sample to be detected according to the following formula; calculating the total content of coumarin according to the total content of 6 coumarin components; the results are reported in tables 6-1 and 6-2.
Content of each coumarin component
TABLE 6-1 quantitative analysis of coumarin component of radix Angelicae Pubescentis in twelve batches (n ═ 3)
And (3) annotation: extracting radix Angelicae Pubescentis according to the method of' 2015 edition Chinese pharmacopoeia
TABLE 6-2 twelve batches of radix Angelicae Pubescentis coumarin component quantitative analysis results (n ═ 3)
Note that: extracting radix Angelicae Pubescentis according to the method of' 2015 edition Chinese pharmacopoeia
In 2015 edition of Chinese pharmacopoeia, the extraction method of the sample solution to be detected is ultrasonic extraction for 30 minutes, and compared with the ultrasonic extraction method in Chinese pharmacopoeia, the extraction time (3.3 minutes) of the ionic liquid combined with the microwave-assisted extraction method is obviously shortened; the content of the total coumarins extracted from the radix angelicae pubescentis by the extraction method is 11.50mg/g (the sum of the contents of 6 coumarin components), which is also superior to the total coumarins extracted by the extraction method in the Chinese pharmacopoeia 2015 edition; the extraction method for extracting the coumarin component from the radix angelicae pubescentis by combining the ionic liquid with the microwave assistance is simple, convenient and rapid.
Example 4 precision test
Preparing mixed reference substance solutions with low, medium and high concentrations, continuously injecting sample for 6 times according to the chromatographic conditions, and calculating the Relative Standard Deviation (RSD) of precision within a day; sampling 3 concentrations continuously for 3 days, 6 needles each time, and calculating the daytime precision RSD value. The precision results are shown in table 7, the relative standard deviation ranges of the precision in the day and the time of the dihydrocarveol-beta-D-glucoside, the dihydrocarveol, the angelol A, the dihydrocarveol acetate, the cnidium lactone and the dihydrocarveol angelate are 0.26-4.76% and 0.55-4.89%, and the accuracy ranges are 96.76-103.83% and 96.66-104.08%. The results of these tests show that the established method is accurate (RSD < 5%).
TABLE 7 precision within and during day (n ═ 6)
Example 5 repeatability and stability test
Taking the same batch of radix angelicae pubescentis samples, preparing 6 parts of sample solutions to be detected in parallel according to the preparation method of the sample solutions to be detected in the embodiment 2, and carrying out continuous sample injection analysis. The results are shown in table 8, and the RSD values for 6 component contents are all less than 5%, indicating that the method is good in repeatability.
And taking the same sample solution to be detected, and respectively carrying out sample injection analysis for 0, 2, 4, 6, 8, 10, 12 and 24 hours to examine the stability of the sample. The results are shown in Table 8, and the RSD values of 6 components are less than 5%, which indicates that the sample stability is better.
TABLE 8 repeatability and stability
EXAMPLE 6 sample recovery test
Precisely weighing 50mg of the same batch of radix angelicae pubescentis samples, precisely adding a certain amount of mixed reference solution, preparing according to the preparation method of the sample solution to be detected in the embodiment 2, carrying out sample injection according to the chromatographic conditions to obtain peak areas, and calculating the content of each component, wherein each sample is parallel for 6 times. Meanwhile, the unlabeled 50mg sample was operated in parallel, and the peak area was measured and the content was calculated. The recovery rate is calculated by (measured amount-original amount)/additive standard amount multiplied by 100 percent, the result is shown in the table 9, the average recovery rate of 6 coumarins is between 96.80 percent and 103.50 percent, and the relative standard deviation is less than 5 percent, which indicates that the method is accurate and reliable.
TABLE 9 recovery rate of samples (n ═ 6)
Document 1 (Wangmuiri, Tanshu, Lilong Yun, etc., determination of HPLC fingerprint of radix Angelicae Pubescentis and 4 coumarin components. J. pharmaceutical analysis, 2018,38(06):955 + 963.) discloses ultrasonic extraction of a radix Angelicae Pubescentis sample solution to be detected, wherein the ultrasonic extraction time is 20 minutes, and an extraction solvent is methanol; determining the content of coumarin components by high performance liquid chromatography-diode array detection (HPLC-DAD) for 100 min; wherein the limit of the quantification of the columbianadin is 0.115 mu g/mL, and the limit of the detection is 0.028 mu g/mL; the limit of the osthole quantification is 0.148 mug/mL, and the limit of the osthole detection is 0.044 mug/mL; the limit of quantification of the columbianadin is 0.264 mug/mL, and the limit of detection is 0.069 mug/mL.
Document 2 (bin, zhou' an, yan foam, etc., high performance liquid chromatography fingerprint spectrum research of coumarin components of radix angelicae pubescentis medicinal materials, university of traditional Chinese medicine of anhui, 2019,38(01): 87-92) discloses ultrasonic extraction of a radix angelicae pubescentis sample solution to be detected, wherein the ultrasonic extraction time is 30 minutes, and an extraction solvent is a mixed solution of methanol and dichloromethane; the content of coumarin components is determined by a high performance liquid chromatography-diode array detection method (HPLC-DAD), and the detection time is 70 minutes.
Document 3 (jia meng, zhao hua cong, chua shuhui, etc., HPLC fingerprint and quality evaluation research of multi-component quantitative radix angelicae pubescentis decoction pieces, proceedings of nanjing university of traditional Chinese medicine, 2020,36(01):88-93.) discloses ultrasonic extraction of a radix angelicae pubescentis sample solution to be detected, wherein the ultrasonic extraction time is 30 minutes, and the extraction solvent is 70% methanol; and (3) determining the content of the coumarin components by adopting a high performance liquid chromatography-diode array detection method (HPLC-PDA), wherein the detection time is 95 minutes.
The present application was compared with the extraction method and detection method of documents 1 to 3, see table 10. As can be seen from table 10, the extraction solvent used in the present application is an ionic liquid, while the organic solvents in documents 1 to 3 are all organic solvents, and the ionic liquid used in the present application is more environmentally friendly than the organic solvents. The method for extracting the coumarin components by using microwaves is adopted, the extraction time is only 3.3 minutes, the extraction time of documents 1 to 3 is 20 to 30 minutes, and the extraction method enables the preparation of the sample solution to be detected to be rapid, simple, convenient and efficient. The detection time of documents 1 to 3 is 70 to 100 minutes, while the detection time of the application is only 57 minutes, the application obviously shortens the detection time, and the radix angelicae pubescentis sample detection becomes rapid and efficient. The quantitative limit and the detection limit of the dihydrooroselol, the osthole and the dihydrooroselol angelate detected by the application are respectively 0.01 mu g/mL, 0.003 mu g/mL, 0.4 mu g/mL, 0.13 mu g/mL, 0.01 mu g/mL and 0.003 mu g/mL; compared with the literature 1, the detection method (HPLC-FLD) has higher sensitivity. Therefore, the method has the advantages of simplicity, convenience, high efficiency, environmental protection, high sensitivity, high analysis speed and the like.
TABLE 10 comparison of the methods of the present application with those of references 1 to 3
The method adopts ionic liquid-microwave assisted extraction combined with a high performance liquid chromatography-fluorescence detection method, simultaneously determines the content of 6 coumarin components including dihydrocarveol-beta-D-glucoside, dihydrocarveol, angelol A, dihydrocarveol acetate, osthole and dihydrocarveol angelate in the radix angelicae pubescentis, realizes the rapid extraction and detection of the coumarin components in the radix angelicae pubescentis, and has the advantages of simplicity, convenience, high sensitivity, high analysis speed and the like.
All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (6)
1. A method for determining the content of coumarin components in radix angelicae pubescentis is characterized in that the content of 6 coumarin components in radix angelicae pubescentis is determined simultaneously; the 6 coumarin components comprise dihydrocarveol-beta-D-glucoside, dihydrocarveol, angelol A, dihydrocarveol acetate, osthole and dihydrocarveol angelate, and the method comprises the following steps:
(1) establishing a standard curve of 6 coumarin components;
preparing 5-10 mixed reference substance solutions containing 6 coumarin components with different known concentrations by using methanol as a solvent; wherein the concentrations of columbianadin-beta-D-glucoside, columbianadin and columbianadin are respectively 0.005-8 μ g/mL, the concentrations of columbinol A and columbinol acetate are respectively 0.02-30 μ g/mL, and the concentration of osthole is 0.3-30 μ g/mL;
under the same chromatographic condition and fluorescence detection condition, the volume V 1 Respectively injecting the mixed reference substance solution into a high performance liquid chromatograph, determining coumarin components of each chromatographic peak through fluorescence detection, and obtaining the chromatographic peak area of each coumarin component;
wherein the chromatographic conditions comprise:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: the phase A is methanol, and the phase B is water; gradient elution is carried out by adopting 40-85% of phase A and 15-60% of phase B in volume fraction; flow rate: 0.2-0.5 mL/min; column temperature: 25-35 ℃; sample volume V 1 :1-5μL；
Respectively establishing a standard curve of each coumarin component by taking the peak area of each coumarin component chromatographic peak as a vertical coordinate and the concentration of each coumarin component as a horizontal coordinate;
wherein the gradient elution is specifically: 40-40% of A in 0-5 min; 5-10 minutes, 40% -45% A; 45% -45% of A for 10-15 minutes; 45% -52% of A in 15-25 min; 25-35 minutes, 52% -52% A; 35-37 minutes, 52% -75% A; 37-42 minutes, 75% -80% A; 80-85% of A in 42-45 minutes; 45-50 minutes, 85% -85% A; 85% -40% of A in 50-52 minutes; 52-57 minutes, 40% -40% A;
(2) obtaining the chromatographic peak area of a sample solution to be detected;
taking the sample to be measured with the mass M as V 2 Microwave extracting the ionic liquid water solution with the concentration of 30-200 millimole per liter, and filtering to obtain a sample solution to be detected, wherein M/V 2 Is (3-30): 1mg/mL, the microwave extraction power is 50-600W, and the microwave extraction time is 1-7 minutes;
taking the volume V under the same chromatographic conditions and fluorescence detection conditions as in step (1) 1 Injecting the sample solution to be detected into a high performance liquid chromatograph, determining coumarin components of each chromatographic peak through fluorescence detection, and obtaining the chromatographic peak area of each coumarin component;
wherein the ionic liquid is selected from at least one of 1-dodecyl-3-methylimidazole trifluoromethyl sulfonate, 1-dodecyl-3-methyl-1H-imidazole hydrogen sulfate, brominated-1-dodecyl-3-methylimidazole and brominated-1-ethyl-3-methylimidazole;
(3) determining the content of 6 coumarin components in a sample to be detected;
according to the established standard curve of each coumarin component, the standard curve is measuredThe chromatographic peak area of each coumarin component in the sample solution is respectively used for obtaining the concentration C of each coumarin component 1 Respectively calculating the contents C of 6 coumarin components in the sample to be detected according to the following formula;
C＝C 1 ×V 2 /M。
2. the method according to claim 1, wherein in step (2), the sample to be tested having mass M is measured in volume V 2 Microwave extracting the ionic liquid water solution with the concentration of 50-150 millimole per liter, and filtering to obtain a sample solution to be detected, wherein M/V 2 Is (5-20): 1mg/mL, the microwave extraction power is 100-500W, and the microwave extraction time is 2-6 minutes.
3. The method according to claim 1, wherein in step (2), the sample to be tested having mass M is measured in volume V 2 Microwave extracting 70-130 millimole/L ionic liquid water solution, filtering to obtain sample solution, wherein M/V 2 Is (5-15): 1mg/mL, the microwave extraction power is 200-400W, and the microwave extraction time is 2-5 minutes.
4. The method according to any one of claims 1 to 3, wherein the concentrations of columbiananol- β -D-glucoside, columbiananol angelate are 0.02-5 μ g/mL, respectively, columbinol A, columbinol acetate are 0.1-25 μ g/mL, respectively, and osthole is 0.5-25 μ g/mL, respectively, in the mixed control solution.
5. The method according to any one of claims 1 to 3, wherein in step (1), a stock solution of a mixed control containing 6 kinds of coumarins is prepared using methanol as a solvent, wherein the concentrations of columbiananol- β -D-glucoside, columbinol angelate are 5 to 8 μ g/mL, respectively, the concentrations of columbinol A and columbinol acetate are 25 to 30 μ g/mL, respectively, and the concentration of osthole is 25 to 30 μ g/mL;
and (3) diluting the mixed reference substance stock solution by using methanol to obtain the 5-10 mixed reference substance solutions containing 6 coumarin components with different known concentrations.
6. The method of any one of claims 1-3, wherein the fluorescence detection conditions comprise: an excitation wavelength of 325nm and an emission wavelength of 400 nm.
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