CN114609303B - Method for extracting and detecting chemical components in Yangxin tablet by using biosurfactant and application of method - Google Patents
Method for extracting and detecting chemical components in Yangxin tablet by using biosurfactant and application of method Download PDFInfo
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- CN114609303B CN114609303B CN202210405564.8A CN202210405564A CN114609303B CN 114609303 B CN114609303 B CN 114609303B CN 202210405564 A CN202210405564 A CN 202210405564A CN 114609303 B CN114609303 B CN 114609303B
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention provides a method for extracting and detecting chemical components in a Yangxin tablet by using a biosurfactant and application thereof, belonging to the technical field of medicine detection and quality evaluation. The method is stable and reliable as shown by the result of methodology verification. In addition, the optimal extraction condition of the target analyte in the Yangxin shi tablet is optimized by using a response surface method. Meanwhile, the established method is used for measuring the content of 11 chemical components in 20 Xinshi tablets. The content measurement results show that the chemical component content varies from batch to batch. The method established by the invention follows the concept of green chemistry, takes the biosurfactant as an extraction solution instead of an organic solvent, also shows that the trehalose lipid solution as an environment-friendly extraction solvent has great application prospect in sample treatment. Meanwhile, the method established by the technical scheme has the advantages of accuracy, reliability, high sensitivity, low specific detection limit, short detection time and the like, and provides a reference basis for quality control of the Yangxin shi tablet.
Description
Technical Field
The invention belongs to the technical field of drug detection and quality evaluation, and particularly relates to a method for extracting and detecting chemical components in a Yangxin tablet by using a biosurfactant and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The Yangxin shi tablet is a traditional Chinese medicine prescription developed by Qingdao national Feng pharmaceutical industry Co.Ltd. The prescription is composed of 13 medicines, and according to the prescription compatibility principle of monarch, minister, assistant and guide, astragalus, ginseng and red sage root are taken as monarch medicines, kudzuvine root, chinese angelica, hawthorn, rhizoma corydalis and dangshen are taken as ministerial medicines, rehmannia root, coptis root, epimedium herb and lucid ganoderma are taken as adjuvant medicines, and prepared licorice is taken as guide. Clinically, the Yangxin shi tablet has obvious curative effect on coronary heart disease, angina pectoris, myocardial infarction, hyperlipidemia, hyperglycemia and other diseases. The Yangxin shi tablet contains complex chemical components including flavonoids, triterpenes, phenolic acids, alkaloids and other chemical components. At present, the research on chemical components in the Yangxin shi tablet mainly adopts an organic solvent extraction method, but the organic solvent has the defects of toxicity, flammability, environmental pollution and the like, and the green and environment-friendly extraction solvent needs to be searched for as a substitute. The existing researches show that some green extraction solvents, such as ionic liquids, eutectic solvents, surfactants and the like, can be used as substitutes of organic solvents and have been successfully used for extraction analysis of samples. Ultrasonic assisted micelle extraction and microwave assisted micelle extraction have been used to extract gingerol, etc. from ginger, and ultrasonic or microwave assisted micelle extraction of chemical components of polyphenols and flavonoids has also been reported.
Biosurfactants are produced by animals, plants and microorganisms and are amphiphilic surfactants having hydrophilic and hydrophobic groups. Types of biosurfactants include glycolipids, lipoproteins, glycoproteins, polyol lipids, and the like. Glycolipids are one of the most commonly used biosurfactants, of which rhamnolipids, sophorolipids and trehalose lipids have been extensively studied. To date, biosurfactants have been used in a variety of fields including industrial, pharmaceutical and environmental aspects. Trehalose lipids (FIG. 1) have been reported to be produced by a variety of microorganisms including Rhodococcus, arthrobacter, gordonia and Nocardia, with the Rhodococcus trehalose lipids being intensively studied. The trehalose lipids have various biological activities such as antitumor, antibacterial, antiviral, etc. Compared with the traditional organic solvent, the trehalose lipid biosurfactant is a green extraction solvent with better application prospect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for extracting and detecting chemical components in Yangxin shi tablets by using a biosurfactant and application thereof. The invention establishes a method combining efficient and green ultrasonic-assisted micelle extraction with ultra-high performance liquid chromatography (UHPLC-PDA), which is used for analyzing 11 main chemical components of puerarin, soyabean glycoside, ferulic acid, calycosin, tetrahydropalmatine, coptisine, epiberberine, jateorhizine, berberine, palmatine hydrochloride and icariine in the yangxin tablet, and has good practical application value.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for extracting and detecting chemical components in a yangxin tablet using a biosurfactant, the chemical components including puerarin, daidzin, ferulic acid, calycosin, tetrahydropalmatine, coptisine, epiberberine, jateorhizine, berberine, palmatine hydrochloride and icariine, the method comprising: placing the pretreated Yangxin tablet in a trehalose lipid solution for ultrasonic assisted micelle extraction to obtain a sample to be detected, and detecting the sample to be detected based on UHPLC-PDA.
The pretreatment method of the Yangxin tablet comprises the following steps: coating the Yangxin shi tablet, and grinding the tablet core into powder;
according to the invention, the research shows that the extraction efficiency of the effective chemical components in the Yangxin tablet can be further improved by adding formic acid into the trehalose, so that the trehalose lipid solution is a trehalose lipid solution containing 0.5-2% (preferably 1%) of formic acid. Meanwhile, by controlling the concentration of the trehalose grease, micelles can be formed more easily to wrap more chemical components, so that the concentration of the trehalose in the trehalose solution is 1-10mg/mL.
In a second aspect of the invention, there is provided the use of the above method in quality assessment of Yangxin shi tablets.
Compared with the prior art, the one or more technical schemes have the following beneficial technical effects:
The technical scheme establishes a method for combining efficient and green ultrasonic-assisted micelle extraction with UHPLC-PDA, and is successfully applied to analysis of chemical components in the Yangxin tablet. The method is stable and reliable as shown by the result of methodology verification. In addition, the optimal extraction condition of the target analyte in the Yangxin shi tablet is optimized by using a response surface method. Meanwhile, the established method is used for measuring the content of 11 chemical components in 20 Xinshi tablets. The content measurement results show that the chemical component content varies from batch to batch.
The method established by the technical scheme follows the concept of green chemistry, uses biosurfactant to replace organic solvent as extraction solution, and also shows that the trehalose lipid solution as environment-friendly extraction solvent has great application prospect in sample treatment. Meanwhile, the method established by the technical scheme has the advantages of accuracy, reliability, high sensitivity, low specific detection limit, short detection time and the like, and provides a reference basis for quality control of the Yangxin shi tablet.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a diagram showing the structure of trehalose lipids.
FIG. 2 is a block diagram of 11 compounds in the Yangxin tablet tested in accordance with the present invention; 1. puerarin 2, daidzin 3, icariin 4, calycosin 5, tetrahydropalmatine 6, coptisine 7, berberine 8, jateorhizine 9, palmatine hydrochloride 10, ferulic acid 11 and epiberberine.
FIG. 3 is a chromatogram of a mixed control solution (A) and a sample solution (B) according to an embodiment of the present invention; 1. puerarin 2, daidzein 3, ferulic acid base 4, calycosin 5, tetrahydropalmatine 6, coptisine 7, epiberberine 8, jateorhizine 9, berberine 10, palmatine hydrochloride 11 and icariine.
Fig. 4 is a single factor optimization experiment in the embodiment of the present invention, which respectively examines (a) the type of extraction solvent (b) the concentration of the extraction solvent (c) the extraction time (d) the influence of ultrasonic power (e) on the total content of the compound.
FIG. 5 is a three-dimensional (3D) response surface diagram in an embodiment of the invention: (a) interaction plot of surfactant concentration versus extraction time versus total analyte extraction (b) interaction plot of extraction time versus liquid-solid ratio versus total analyte extraction (c) interaction plot of surfactant concentration versus liquid-solid ratio versus total analyte extraction.
FIG. 6 is a UHPLC chromatogram of a blank solution of marine algae glycolipid in an embodiment of the invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
In an exemplary embodiment of the present invention, there is provided a method for extracting and detecting chemical components including puerarin, daidzin, ferulic acid, calycosin, tetrahydropalmatine, coptisine, epiberberine, jateorhizine, berberine, palmatine hydrochloride, and icariine in a yangxin tablet using a biosurfactant, the method comprising: placing the pretreated Yangxin tablet in a trehalose lipid solution for ultrasonic assisted micelle extraction to obtain a sample to be detected, and detecting the sample to be detected based on UHPLC-PDA.
In another specific embodiment of the present invention, the pretreatment method of the yangxin shi tablet comprises: coating the Yangxin shi tablet, and grinding the tablet core into powder;
According to the invention, the research shows that the extraction efficiency of the effective chemical components in the Yangxin tablet can be further improved by adding formic acid into the trehalose, so that the trehalose lipid solution is a trehalose lipid solution containing 0.5-2% (preferably 1%) of formic acid. Meanwhile, by controlling the concentration of the trehalose lipid, micelles can be formed more easily and more chemical components are wrapped, so that the trehalose concentration in the trehalose solution is 1-10mg/mL, and more preferably 2-8mg/mL, such as 2, 4, 6, 7 and 8mg/mL.
In yet another embodiment of the present invention, the ultrasonic frequency is controlled to be 100-1000w, further controlled to be 200-600w, such as 200, 300, 400, 500 and 600w;
in yet another embodiment of the present invention, the liquid to solid ratio is controlled to be 50-250 mL/1 g, such as 50:1, 100:1, 125:1, 200:1 and 250:1 (mL/g) during the extraction process.
In yet another embodiment of the present invention, the extraction time is 5-60min, further controlled to 10-50min, such as 10, 20, 30, 40 and 50min.
In another specific embodiment of the invention, the UHPLC-PDA detects the sample to be detected by the following specific method:
in yet another embodiment of the present invention, the liquid chromatography conditions include:
Gradient elution was used, mobile phase a phase: 0.2% phosphoric acid water, mobile phase B phase: methanol;
the chromatographic column is a C18 column such as ZORBAX Eclipse XDB-C18 (2.1X100 mm,1.8-Micron, agilent); the flow rate of the mobile phase is 0.2-0.5 mL/min (preferably 0.3 mL/min); the column temperature is 25-40 ℃ (preferably 30 ℃); the sample injection amount is 1-5 mu L (preferably 2 mu L);
The gradient elution mode is specifically as follows ：：0-5min,10％-25％(B);5-8min,25％-27％(B);8-15min,27％-32％(B);15-22min,32％-60％(B);22-25min,60％-77％(B);25-26min,77％-10％(B).
The detection wavelength is controlled to 250-260nm, preferably 254nm.
In yet another embodiment of the present invention, there is provided the use of the above method in quality assessment of Yangxin shi tablets.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Examples
1 Instrument and materials
1.1 Instruments
Ultra performance liquid chromatograph Waters ACQUITY UPLC (Waters company, usa); ultrasonic cleaner (Shanghai communication instruments Co., ltd.); an electronic balance (MSA 225P-0CE-DU, sartorius, germany); ultrapure water machines (Milli-Q ACADEMIC ultrapure water systems, millipore, U.S.A.).
1.2 Reagents and reagents
1.2.1 Reagents
Ultrapure water (Millipore Co., U.S.A., milli-Q ACADEMIC); phosphoric acid (chromatographic purity, fisher, usa); formic acid (mass spectrometry purity, anaqua Chemicals Supply, usa); methanol (chromatographic purity, concotide); trehalose lipid biosurfactants (Seanbo Co., ltd.).
1.2.2 Medicinal materials and Standard substance
20 Batches of Yangxin shi tablets were provided by Qingdao Fengshi pharmaceutical Co., ltd.
Puerarin, daidzin, calycosin, corktarin and berberine are all purchased from Chengdu Dest Biotechnology Co., ltd, ferulic acid, coptisine, epiberberine and icariine are purchased from Chengdu Mandsite Biotechnology Co., ltd, and palmatine hydrochloride is purchased from Tianjin party sciences Co. All standards had a purity of greater than 98%.
2 Method
2.1 Chromatographic conditions
Chromatographic column: ZORBAX Eclipse XDB-C18 (2.1X100 mm,1.8-Micron, agilent); mobile phase: 0.2% phosphoric acid water (a) -methanol (B), gradient elution, elution gradient ：0-5min,10％-25％(B);5-8min,25％-27％(B);8-15min,27％-32％(B);15-22min,32％-60％(B);22-25min,60％-77％(B);25-26min,77％-10％(B); flow rate: 0.3mL/min; column temperature: 30 ℃; wavelength: 254nm; sample injection volume: 2. Mu.L. The chromatogram is shown in FIG. 3.
2.2 Preparation of sample solutions
Removing coating of YANGXIN tablet, grinding tablet core into powder, precisely weighing 20mg in 50mL conical flask, transferring 2.5mL trehalose lipid water solution (7 mg/mL, containing 1% formic acid) in conical flask, ultrasonic extracting (300 w,40 KHz) for 40min, centrifuging, collecting supernatant, and filtering with 0.22 μm organic microporous membrane to obtain sample solution.
2.3 Preparation of control solution
Accurately weighing 1.0mg of puerarin, soyabean glycoside, ferulic acid, calycosin glycoside, tetrahydropalmatine, coptisine, epiberberine, jateorhizine, berberine, palmatine hydrochloride and icariine standard substances, adding methanol to prepare standard substance solutions with concentration of 1mg/mL, and storing in a refrigerator at-4deg.C for use.
2.4 Single factor optimization experiments
The type of the extraction solvent, the concentration of the extraction solvent, the ultrasonic power, the extraction time and the liquid-material ratio are respectively examined to influence the total extraction amount of 11 chemical components in the Yangxin tablet. Each extraction condition was examined at the following level: type of extraction solvent (water, trehalose lipid solution, methanol, trehalose lipid solution with 1% formic acid), concentration of extraction solvent (2, 4,6,8, 10 mg/mL), ultrasonic power (200, 300, 400, 500, 600 w), extraction time (10, 20, 30, 40, 50 min), liquid-to-solid ratio (50:1, 100:1, 125:1, 200:1, 250:1 mL/g).
2.5Box-Behnken design-response surface optimization experiment
Based on a single-factor optimization experiment, an appropriate extraction range of each factor is determined, and three-factor (trehalose lipid concentration, extraction time, liquid-solid ratio) and three-level optimization experiments are designed by using a response surface method through Design Expert (version 8.0.6) software. And finally, carrying out corresponding experimental verification according to the predicted optimal extraction conditions.
3 Results and discussion
3.1 Single factor optimization experiments
3.1.1 Type of extraction solvent
The effect of water, a trehalose lipid solution, methanol and a trehalose lipid solution containing 1% formic acid on the total extraction amount of chemical components in the yangxin tablet was examined respectively. As a result, as shown in FIG. 4 (a), the trehalose lipid solution can obtain the best extraction efficiency, because it is likely to be related to the polyhydroxy structure of the trehalose lipid, making the force between it and the analyte stronger. Thus, a trehalose lipid solution containing 1% formic acid was selected as the extraction solvent.
3.1.2 Concentration of extraction solvent
In the ultrasonic assisted micelle extraction method, the concentration of the surfactant is a very important influencing factor. The Critical Micelle Concentration (CMC) is the lowest solution concentration at which the surfactant forms micelles. Studies have shown that the critical micelle concentration of the trehalose lipid biosurfactant is 250mg/L. In a single factor optimization experiment, the effect of trehalose lipid solutions with concentrations of 2,4,6,8 and 10mg/mL on the total extraction amount of chemical components was examined respectively. As a result, as shown in FIG. 4 (b), the total extraction amount of the analyte was the highest (P < 0.05) when the concentration of the trehalose lipid solution was 6mg/mL. The reason may be that the trehalose lipid solution forms micelles, which can encapsulate more chemical components, increasing their solubility in solution. Therefore, the concentration of the optimum extraction solvent is 6mg/mL.
3.1.3 Extraction time
The influence of ultrasonic extraction time on the total extraction amount of the chemical components was examined, and as the extraction time increased, the total extraction amount of the chemical components gradually increased, and when the extraction time was 40min, the total extraction amount of the chemical components reached the maximum value. When the extraction time was 50min, the total extraction amount of the chemical components was almost the same as that of 40min, and there was no increase (P > 0.05), and the result was shown in FIG. 4 (c). Therefore, the optimal extraction time is 40min.
3.1.4 Ultrasonic Power
As important influencing factors of ultrasonic extraction, the influence of ultrasonic power of 200, 300, 400, 500 and 600w on the total extraction amount of chemical components in the yangxin shi tablet is examined respectively. As a result, as shown in fig. 4 (d), the total extraction amount of the chemical components reached the maximum when the ultrasonic power was 300w, and there was no significant difference (P > 0.05) from the extraction amounts when the ultrasonic power was 400, 500, 600 w. Thus, the optimal ultrasonic power is 300w.
3.1.5 Liquid to solid ratio
The ratio of the volume of the extract to the amount of solid matter is called the liquid-solid ratio. The concentration and viscosity of the solution can change along with the change of the liquid-solid ratio, thereby affecting the leaching degree of chemical components in the medicinal material. Therefore, in the single factor optimization experiment, the change of the total extraction amount of the chemical components is examined when the liquid-solid ratio is 50:1, 100:1, 125:1, 200:1, 250:1 (mL/g), respectively. As a result, as shown in FIG. 4 (e), the total amount of the chemical components eluted from the sample solution was maximized (P < 0.05) at a liquid-solid ratio of 125:1 (mL/g). Therefore, a liquid-solid ratio of 125:1 (mL/g) is the optimal extraction condition.
3.2Box-Behnken design-response surface optimization experiment
3.2.1 Experimental design
Based on the single-factor optimization experimental result, three factors including the concentration, the extraction time and the liquid-solid ratio of the trehalose lipid solution are selected as investigation objects, three levels are set for each factor, and the total extraction amount of 11 chemical components is used as an evaluation index. According to Design Expert (version 8.0.6) software, a total of 17 experiments were designed, and the experimental Design, the predicted value of the total extraction amount of the analyte and the actual experimental value are shown in table 1.
TABLE 1 response surface design, predictive and experimental values for total analyte content
Note that: concentration of A trehalose lipid solution, B extraction time, C liquid-solid ratio
3.2.2 Model fitting
And carrying out regression fitting on the experimental result to obtain a final quadratic regression model equation as follows:
Y＝+19.99+0.96A+0.17B+0.51C-0.10AB-0.24AC+0.082BC-0.88A2-0.44B2-0.58C2
In the regression equation, Y represents the predicted value of the total extraction amount of chemical components, A, B, C respectively represents the concentration, extraction time and liquid-solid ratio of the trehalose lipid biosurfactant. The fitness of the model was evaluated by analysis of variance and the results are shown in table 2. The analysis of variance result shows that the model P is less than 0.0001, the regression relation between the independent variable and the dependent variable of the regression model is obvious, the mismatch term P= 0.5946 has no obvious difference, and the correlation coefficient R 2 = 0.9932, which indicates that the regression equation of the model has better fitting degree and high reliability with experimental data. The model predicts that R 2 is 0.9646 and adjusts R 2 to 0.9632 to be more than 0.9, which shows that the model has better regression and can be used for predictive analysis of experimental data. Meanwhile, the coefficient of variation (cv=0.65%) indicates that the experimental data has higher accuracy and reliability. In combination with the above analysis, the model can be successfully used to predict the total extraction rate of chemical components in the yangxin shi tablet.
3.2.3 Response surface Experimental results analysis
The analysis of variance results shows that the P values of three influencing factors A, B, C (concentration of trehalose lipid biosurfactant, extraction time, liquid-solid ratio) are all less than 0.01, and P A>PC>PB shows that the influence intensity of the three influencing factors on Y (total extraction amount of chemical components) is as follows: a > C > B. And the quadratic terms (a 2、B2、C2) are all significant, indicating that there is not a simple linear relationship between the independent and dependent variables. In addition, the interaction term AC is remarkable, which shows that the concentration of the biosurfactant and the liquid-solid ratio have obvious influence on the total extraction amount of the chemical components.
Fig. 5 (a-c) are three-dimensional (3D) response surface plots of the effect of independent variables and their interactions on the total yield of chemical components. As can be seen from fig. 5 (a), as the concentration of biosurfactant increases from 1mg/mL to 7mg/mL, the total extraction of chemical components also increases gradually as the liquid-solid ratio remains unchanged. When the biosurfactant concentration exceeds 7mg/mL, the total extraction amount of the chemical components slightly decreases, because it may be related to the change in the structure of the biosurfactant micelle. Meanwhile, the total extraction amount of the chemical components gradually increases along with the increase of the extraction time, and the total extraction amount of the chemical components does not change obviously along with the increase of the extraction time when the extraction time exceeds 40 min. As can be seen from fig. 5 (b), the total extraction amount of the chemical components reaches the maximum value at a liquid-solid ratio of about 130:1 (mL/g), and the total extraction amount of the chemical components is not significantly changed as the liquid-solid ratio continues to increase. Finally, the optimal condition for ultrasonic assisted micelle extraction determined by the response surface optimization method is that the concentration of the trehalose lipid biosurfactant is 6.98mg/mL, the liquid-solid ratio is 133.84:1 (mL/g), the extraction time is 41.67min, and the maximum extraction amount of 11 chemical components is 20.3322mg/g.
TABLE 2 analysis of variance of BBD-RSM regression model
Note that: concentration of A trehalose lipid solution, B extraction time, C liquid-solid ratio
* Indicating a level of significance P < 0.01
3.2.4 Verification test
According to the optimal extraction conditions predicted by the response surface optimization method, 2.5mL of trehalose lipid biosurfactant solution with concentration of 7mg/mL is used for ultrasonic extraction for 40min, and experimental verification is carried out. The result shows that the total extraction amount of the chemical components is 20.57+/-0.01 mg/g, and the error from the predicted value is only 0.01%, which shows that the model is reasonable and can be used for predicting the total extraction amount of the chemical components in the Yangxin tablet.
3.3 Methodological verification
3.3.1 Properties
As shown in fig. 3 and 6, there was no significant interference in the trehalose lipid blank solution chromatograms during the retention time of the analyte; in the sample solution chromatogram, all chemical components have good separation degree. Therefore, the method has higher specificity and can be used for analyzing chemical components in the Yangxin shi tablet.
3.3.2 Linearity and sensitivity
And (3) taking the standard substance solution under the item of 2.3, preparing a mixed standard substance solution containing the compounds at proper concentrations, and sequentially diluting to obtain a standard curve solution. And (3) analyzing according to the condition under the term of 2.1, and drawing a standard curve by taking the standard solution concentration of the compound as an X axis and the peak area as a Y axis. The correlation coefficient r 2 of the linear regression equation is more than or equal to 0.9995, which shows that 11 chemical components have good linear relation in the respective linear range. The detection Limit (LOD) of the 11 chemical components is in the range of 0.017-0.306, and the quantification Limit (LOQ) is in the range of 0.071-1.158 μg/mL. Regression equations, linear ranges, LOQ and LOD for the 11 chemical components are shown in table 3.
3.3.3 Precision
Three concentration levels (low, medium, high) of mixed standard solutions were used to evaluate the daily, diurnal precision of 11 chemical components. As shown in table 4, at three concentration levels, the peak area RSD values of 11 chemical components were all 3.24% or less (n=6). The method has good precision.
3.3.4 Repeatability
Taking the same batch of Yangxin tablet samples, preparing six test sample solutions in parallel according to the test sample preparation method under the item of 2.2, and analyzing according to the chromatographic condition of 2.1. As shown in Table 4, the RSD values of the peak areas of the 11 compounds were all less than 1.65%, indicating that the method was good in reproducibility.
3.3.5 Stability
Mixed standard solutions at three concentration levels (low, medium, high), were analyzed at room temperature at 0, 2, 4, 6, 8, 12, 24h, respectively. As shown in table 4, the peak area RSD values for 11 analytes at three concentration levels were all below 1.99% (n=6), indicating good stability of the analytes at room temperature for 24 hours.
3.3.6 Sample recovery
Precisely weighing 10mg of Yangxin tablet powder, and adding the standard substance in a mode of 1:1. The addition amount of each chemical component standard substance is 12 mug/mL of puerarin, 2.5 mug/mL of daidzein, 0.5 mug/mL of ferulic acid, 0.3 mug/mL of calycosin, 5 mug/mL of tetrahydropalmatine, 7 mug/mL of coptisine, 4 mug/mL of epiberberine, 6 mug/mL of jateorhizine, 29 mug/mL of berberine, 8 mug/mL of palmatine hydrochloride and 2 mug/mL of icariine respectively. Both the labeled and unlabeled samples were analyzed under the "2.1" chromatographic conditions, and the results are shown in table 5, with an average sample recovery of 11 chemical components ranging from 95.1% to 104%, and corresponding RSD values of less than 2.81%.
3.4 Content determination
The ultrasonic assisted micelle extraction and ultra-high performance liquid chromatography method established in the research is applied to the determination of the content of 11 chemical components in 20 Xinshi tablets. As shown in Table 6, the total content of 11 chemical components in each of 20 Xuezhen tablets was 18.00mg/g or more, the lowest was 18.27.+ -. 0.08mg/g (batch 0520027), and the highest was 20.57.+ -. 0.01mg/g (batch 0520006). As can be seen from the table, the same analyte has a large difference in chemical content between batches, such as icariin. Meanwhile, in the same batch, the content of different analytes is different, the berberine content is higher, and the content of the calycosin is lower.
3.5 Method comparison
At present, most of analysis of chemical components in Yangxin shi tablets adopts an organic solvent extraction method and a high performance liquid chromatography method for analysis. There are studies on establishing fingerprint spectra of 20 Xuexin tablets by HPLC-DAD method and content determination of 6 components therein. The extraction solvent is 70% methanol solution, ultrasonic extraction is carried out for 1h, and chromatographic separation time is 75min. The method for extracting and separating is compared with the reported method, the method for extracting and separating is less in amount of samples and extracting solvents, shorter in extracting and separating time and higher in sensitivity, and organic solvents are not used in the sample treatment process, so that the analysis concept of green chemistry is met. The trehalose lipid biosurfactant is used for replacing an organic solvent for extraction, and has the advantages of low toxicity and environment friendliness. Therefore, the ultrasonic assisted micelle extraction combined with the ultra-high performance liquid chromatography established in the research is an efficient and green extraction and separation method.
In conclusion, the research establishes a method for combining efficient and green ultrasonic-assisted micelle extraction with UHPLC-PDA, and is successfully applied to analysis of chemical components in the Yangxin tablet. The method is stable and reliable as shown by the result of methodology verification. In addition, the optimal extraction condition of the target analyte in the Yangxin shi tablet is optimized by using a response surface method. Meanwhile, the established method is used for measuring the content of 11 chemical components in 20 Xinshi tablets. The content measurement results show that the chemical component content varies from batch to batch. The method established by the research follows the concept of green chemistry, takes biosurfactant as an extraction solution instead of an organic solvent, and simultaneously shows that the trehalose lipid solution as an environment-friendly extraction solvent has great application prospect in sample treatment.
It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the spirit and scope of the technical solutions of the present invention.
Claims (12)
1. A method for extracting and detecting chemical components in a yangxin tablet by using a biosurfactant, wherein the chemical components comprise puerarin, daidzin, ferulic acid, calycosin, tetrahydropalmatine, coptisine, epiberberine, jateorhizine, berberine, palmatine hydrochloride and icariine, and the method is characterized by comprising the following steps: placing the pretreated Yangxin tablet in a trehalose lipid solution for ultrasonic assisted micelle extraction to obtain a sample to be detected, and detecting the sample to be detected based on UHPLC-PDA;
the trehalose lipid solution is a trehalose lipid solution containing 0.5-2% formic acid;
The concentration of the trehalose lipid in the trehalose lipid solution is 1-10 mg/mL;
The ultrasonic frequency is controlled to be 100-1000w;
The UHPLC-PDA detection specific method for the sample to be detected comprises the following steps: the liquid chromatography conditions included:
Gradient elution was used, mobile phase a phase: 0.2% phosphoric acid water, mobile phase B phase: methanol;
the chromatographic column adopts a C18 column;
The gradient elution mode is specifically as follows ：0-5min,10%-25%B;5-8min,25%-27%B;8-15min,27%-32%B;15-22min,32%-60%B;22-25min,60%-77%B;25-26min,77%-10%B.
2. The method of claim 1, wherein the pretreatment method of the yangxin shi tablet comprises: the Yangxin shi tablet is coated and the tablet core is ground into powder.
3. The method of claim 1, wherein the trehalose lipid solution is a trehalose lipid solution containing 1% formic acid; the concentration of the trehalose lipid in the trehalose lipid solution is 2-8 mg/mL.
4. The method of claim 1, wherein the ultrasound frequency is controlled to be 200-600w.
5. The method of claim 1, wherein the liquid to solid ratio is controlled to be 50-250 ml/1 g.
6. The method of claim 1, wherein the extraction time is 5-60 minutes.
7. The method of claim 6, wherein the extraction time is 10-50 minutes.
8. The method of claim 1, wherein the mobile phase flow rate is 0.2-0.5 mL/min; the column temperature is 25-40 ℃; the sample injection amount is 1-5 mu L.
9. The method of claim 8, wherein the mobile phase flow rate is 0.3 mL/min; the column temperature is 30 ℃; the sample injection amount was 2. Mu.L.
10. The method of claim 1, wherein the detection wavelength is controlled to be 250-260nm.
11. The method of claim 10, wherein the detection wavelength is 254nm.
12. Use of the method according to any one of claims 1-11 for quality assessment of yangxin shi tablets.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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