CN115420815B - Method for measuring content of various effective components in powder and application thereof - Google Patents
Method for measuring content of various effective components in powder and application thereof Download PDFInfo
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- CN115420815B CN115420815B CN202210833255.0A CN202210833255A CN115420815B CN 115420815 B CN115420815 B CN 115420815B CN 202210833255 A CN202210833255 A CN 202210833255A CN 115420815 B CN115420815 B CN 115420815B
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- G—PHYSICS
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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
- G01N30/02—Column chromatography
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- 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)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a method for measuring the content of various effective components in a powder and application thereof, belonging to the technical field of medicine detection, wherein the method for measuring the content of various effective components in the powder comprises the steps of preparing a reference standard solution I and a sample solution containing the powder; respectively taking a reference standard solution I and a sample solution for high performance liquid chromatography detection, and respectively calculating the contents of glycyrrhizin, hesperidin, glycyrrhizic acid, naringin and apigenin in the powder by using an external standard method; the content determination method is used for researching the intestinal absorption characteristics of the powder. The content determination method of the invention is not only suitable for quality monitoring of the powder in the production process, but also can be used for researching the content determination of the effective components in the process of the absorption characteristics of the powder from the intestines.
Description
Technical Field
The invention relates to a drug detection technology, in particular to a method for measuring the content of various effective components in a powder and application thereof.
Background
The Yigong powder is also called five-flavor Yigong powder, and is prepared from 6 g of flavored dried orange peel based on 6 g of Sijunzi decoction ginseng, 6 g of bighead atractylodes rhizome, 6 g of poria cocos, and 6 g of honey-fried licorice root by a method of generating Yigong powder from Qian Ying Ji Zhi Ju Ji in North Song's famous medical science. The powder is mainly used for treating weakness of spleen and stomach, anorexia, vomiting, diarrhea, chest and epigastric fullness and distention. Ginseng is a monarch drug in the prescription, sweet and warm, and has the effects of strengthening middle-jiao and replenishing qi; atractylodis rhizoma is used as ministerial drug, and has effects of bitter and warm, invigorating qi and spleen; poria is adjuvant drug, sweet and light, and has effects of promoting water penetration and removing dampness; the honey-fried licorice root is used as a guiding drug, sweet and warm, and has the effects of tonifying middle-jiao and harmonizing stomach; dried orange peel Wen Gu, invigorating spleen and activating stomach. Wherein ginseng, bighead atractylodes rhizome and liquorice invigorate spleen and nourish stomach; poria cocos and dried orange peel combined with diuresis to alleviate edema and bighead atractylodes rhizome combined with the function of dispelling spleen dampness; ginseng and white atractylodes rhizome combined with the recipe of activating spleen and promoting transportation, and dried orange peel combined with the recipe of replenishing qi and blood without stagnation. Modern pharmacological researches have proved that the prescription can relax intestinal canal, improve gastrointestinal motility, antagonize free radical injury, and enhance metabolism and immunity.
At present, researches on the content of active ingredients in the qigong powder are relatively less, and only a content measuring method for simultaneously measuring the glycyrrhizin, the hesperidin and the glycyrrhizic acid is found to be established, so that index ingredients of the method are less, the quality of the qigong powder is difficult to comprehensively evaluate, the requirement of monitoring indexes required by gastrointestinal absorption of the qigong powder is more difficult to meet, and the absorption characteristics of the qigong powder in small intestines are not easy to analyze.
Disclosure of Invention
Aiming at the problems, the invention provides a method for measuring the content of various effective components in the powder and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for measuring the content of various active ingredients in the powder comprises the following steps:
preparing a reference standard solution I, wherein the reference standard solution I contains glycyrrhizin, hesperidin, glycyrrhizic acid, rutin naringin and apigenin;
preparing a sample solution from the powder;
respectively taking reference standard solution I and sample solution for high performance liquid chromatography detection, and respectively calculating the contents of liquiritin, hesperidin, glycyrrhizic acid, naringin and apioside glycyrrhizin by external standard method;
the elution mode of the high performance liquid chromatography detection is gradient elution;
The high performance liquid chromatography of the reference standard solution I and the sample solution respectively takes 0.05wt% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and the elution conditions are as follows:
0-10 min,83% -70% of mobile phase A,17% -30% of mobile phase B;
10-17 min,70% → 68.5% mobile phase A,30% → 31.5% mobile phase B;
17-26 min,68.5% -64% mobile phase A,31.5% -36% mobile phase B;
26-42 min,64% -61% mobile phase A,36% -39% mobile phase B;
42-46 min,61% → 0% mobile phase A,39% → 100% mobile phase B;
46-48 min,0% mobile phase A,100% mobile phase B;
48-51 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
51-54 mi,83% mobile phase A,17% mobile phase B.
Further, the concentration of glycyrrhizin in the reference standard solution I is between 1.32 and 80.96 mug/mL, the concentration of hesperidin is between 1.59 and 255.00 mug/mL, the concentration of glycyrrhizin is between 3.39 and 244.08 mug/mL, the concentration of rutin is between 3.00 and 96.00 mug/mL, and the concentration of apigenin is between 0.21 and 13.29 mug/mL;
the concentration range of the powder in the sample solution is 4-6 mg/mL;
and the reference standard solution I and the solvent of the test sample solution both contain Tyrode buffer solution.
Further, preparing a ginsenoside reference standard solution containing ginsenoside Re;
when the content of ginsenoside Re in the powder is measured, the high performance liquid chromatography of the ginsenoside reference standard solution and the sample solution respectively takes 0.05wt% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B under the condition of 203nm wavelength, and the elution conditions are as follows:
0-9 min,83% -80% mobile phase A,17% -20% mobile phase B;
9-31 min,80% → 76.5% mobile phase A,20% → 23.5% mobile phase B;
31-38 min,76.5% -76% mobile phase A,23.5% -24% mobile phase B;
38-43 min,76% → 0% mobile phase A,24% → 100% mobile phase B;
43-46 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
46-49 min,83% mobile phase A,17% mobile phase B.
Further, the concentration of ginsenoside Re in the ginsenoside reference standard solution is between 3.98 and 127.20 mug/mL;
the concentration range of the powder in the sample solution is 4-6 mg/mL;
and the ginsenoside reference standard solution and the solvent of the sample solution both contain Tyrode buffer solution.
The application of the method for measuring the content of various active ingredients in the qigong powder is to utilize the method for measuring the content of various active ingredients of the qigong powder to research the intestinal absorption characteristics of the qigong powder.
A method for measuring the content of various active ingredients in the powder comprises the following steps:
preparing reference standard solutions I with different concentrations, wherein the reference standard solutions I with different concentrations contain glycyrrhizin, hesperidin, glycyrrhizic acid, naringin and apigenin;
respectively taking reference substance standard solutions I with different concentrations for high performance liquid chromatography detection, and drawing standard curves corresponding to different chemical components according to the concentrations of the different chemical components and peak areas measured by the corresponding concentrations of the corresponding chemical components;
preparing a sample solution from the powder;
detecting the sample solution by high performance liquid chromatography, substituting the peak areas of the obtained corresponding chemical components into corresponding standard curves, and respectively calculating the contents of glycyrrhizin, hesperidin, glycyrrhizic acid, naringin and apigenin in the powder;
the elution mode of the high performance liquid chromatography detection is gradient elution;
the high performance liquid chromatography of the reference standard solution I and the sample solution with different concentrations respectively takes 0.05wt% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and the elution conditions are as follows:
0-10 min,83% -70% of mobile phase A,17% -30% of mobile phase B;
10-17 min,70% → 68.5% mobile phase A,30% → 31.5% mobile phase B;
17-26 min,68.5% -64% mobile phase A,31.5% -36% mobile phase B;
26-42 min,64% -61% mobile phase A,36% -39% mobile phase B;
42-46 min,61% → 0% mobile phase A,39% → 100% mobile phase B;
46-48 min,0% mobile phase A,100% mobile phase B;
48-51 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
51-54 mi,83% mobile phase A,17% mobile phase B.
Further, the concentration of glycyrrhizin in the reference standard solution I with different concentrations is between 1.32 and 80.96 mug/mL, the concentration of hesperidin is between 1.59 and 255.00 mug/mL, the concentration of glycyrrhizin is between 3.39 and 244.08 mug/mL, the concentration of naringin is between 3.00 and 96.00 mug/mL, and the concentration of apigenin is between 0.21 and 13.29 mug/mL;
the concentration range of the powder in the sample solution is 4-6 mg/mL;
and the reference standard solution I with different concentrations and the solvent of the test sample solution both contain Tyrode buffer solution.
Further, the standard curves corresponding to the different chemical components are as follows:
The standard curve corresponding to the glycyrrhizin is Y=6830.9X+6291.7, R 2 ＝0.9990；
Hesperidin has a standard curve of y=2954.3x+10306.0, r 2 ＝0.9990；
The standard curve corresponding to glycyrrhizic acid is Y=6052.8X+5120.3, R 2 ＝0.9998；
Rutaceae naringin pairThe corresponding standard curve is y=14643x+27949.0, r 2 ＝0.9995；
The standard curve corresponding to the apigenin is Y=34315X+8109.8, R 2 ＝0.9992。
Further, preparing ginsenoside reference substance standard solutions containing ginsenoside Re at different concentrations;
when the content of ginsenoside Re in the powder is measured, the high performance liquid chromatography of the ginsenoside reference standard solution and the sample solution with different concentrations under the wavelength condition of 203nm respectively takes 0.05wt% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and the elution conditions are as follows:
0-9 min,83% -80% mobile phase A,17% -20% mobile phase B;
9-31 min,80% → 76.5% mobile phase A,20% → 23.5% mobile phase B;
31-38 min,76.5% -76% mobile phase A,23.5% -24% mobile phase B;
38-43 min,76% → 0% mobile phase A,24% → 100% mobile phase B;
43-46 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
46-49 min,83% mobile phase A,17% mobile phase B;
the concentration of ginsenoside Re in the ginsenoside reference standard solution with different concentrations is between 3.98 and 127.20 mug/mL;
The concentration range of the powder in the sample solution is 4-6 mg/mL;
the ginsenoside reference standard solutions with different concentrations and the solvent of the sample solution both contain Tyrode buffer solution;
the standard curve corresponding to the ginsenoside Re is Y=2875X+2282.3, R 2 ＝0.9992。
The application of the method for measuring the content of various active ingredients in the qigong powder is to utilize the method for measuring the content of various active ingredients of the qigong powder to research the intestinal absorption characteristics of the qigong powder.
The method for measuring the content of various effective components in the powder and the application thereof have the beneficial effects that:
the invention establishes a content determination method for detecting 6 chemical components of glycyrrhizin, hesperidin, glycyrrhizic acid, naringin, apioside and ginsenoside Re in the powder by high performance liquid chromatography, and realizes the monitoring of a plurality of index components in the powder;
in the invention, the components in the extract of the Isgong powder are complex, and a high performance liquid chromatography system is preferentially selected for development of an analysis method;
according to the content determination method, the content determination of 5 chemical components of glycyrrhizin, hesperidin, glycyrrhizic acid, naringin and apioside can be completed by adjusting the technological parameters and pretreatment conditions and scanning three detection wavelengths for the sample solution containing different chemical components through one-time sample injection; then the content of ginsenoside Re is determined by one sample injection;
The determination method can quickly, simply, accurately and reliably acquire the quality condition of the powder, solves the problem that the detection method in the prior art can only determine single components and cannot comprehensively control the quality of the powder, realizes the purposes of determining the content of 5 chemical components through scanning, sample introduction, elution and separation at different wavelengths and determining the content of ginsenoside Re through sample introduction, elution and separation at another time by preparing the reference standard solution I and the ginsenoside reference standard solution I with different concentrations at one time and the ginsenoside reference standard solution with different concentrations at one time, and further provides references for the development and quality standard formulation of the powder with classical name;
the content determination method has the advantages of strong specificity, high accuracy, high precision and good linear relation;
according to the invention, through controlling high performance liquid chromatography conditions and screening proper technological parameters, 5 chemical components of glycyrrhizin, hesperidin, glycyrrhizic acid, naringin and apioside can be completely separated by one sample injection, and other impurity peaks can be effectively separated, so that interference is avoided, and the pretreatment step of the powder is simplified; the purpose that 5 chemical components contained in the powder are all in a linear range is achieved by scanning 3 wavelengths through one sample injection;
The extraction efficiency of the effective components in the traditional Chinese medicine materials is also different due to different solvents and the use amount or different extraction time, and the invention improves the extraction efficiency of the powder by selecting proper extraction solvents, the use amount and the extraction time, thereby accurately measuring the content of 5 chemical components in the powder;
by examining different kinds of acids, chromatographic columns, temperature, flow velocity, sample injection amount and the like, the tailing phenomenon of chromatographic peaks is effectively improved, chromatographic peaks with good separation degree and peak shape are obtained, and a foundation is provided for subsequent content detection;
the method for measuring the content of 6 chemical components by taking the Tyrode buffer solution as a solvent is not only suitable for monitoring the quality of the isokinetic powder in the production process, but also can be used for researching the content measurement of the effective components in the process of the absorption characteristics of the isokinetic powder from the intestines, and the content measurement result of each chemical component is not influenced by the medium contained in the Tyrode buffer solution under the chromatographic condition of the invention.
Drawings
FIG. 1 is a standard graph of glycyrrhizin at 254nm wavelength in example 7 of the present invention;
FIG. 2 is a graph of hesperidin standard at a wavelength of 254nm in example 7 of the present invention;
FIG. 3 is a graph of glycyrrhizic acid standard at 254nm wavelength in example 7 according to the present invention;
FIG. 4 is a graph of the naringin standard at 283nm wavelength in example 7 of the present invention;
FIG. 5 is a graph of the standard of apigenin at 360nm wavelength in example 7 of the invention;
FIG. 6 is a graph showing the Re standard of ginsenoside at 203nm wavelength in example 7 of the present invention;
FIG. 7 is a graph showing cumulative absorption of 6 chemical components in sample solutions of different concentrations in the small intestine in experimental example 8 of the present invention;
FIG. 8 is a graph showing cumulative absorption (V) -time (t) of 6 chemical components in sample solutions of different concentrations in experimental example 8 of the present invention in the small intestine;
FIG. 9 is a comparison of the relative proportions of the 6 chemical components in the sample solutions of different concentrations in Experimental example 8 of the present invention before and after absorption;
FIG. 10 is a graph showing the results of the specific experiments in Experimental example 1 of the present invention.
Detailed Description
The following description of the technical solution in the embodiments of the present invention is clear and complete. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Example 1 method for measuring content of various effective components in Isovidone powder
The embodiment adopts the powder for measuring the content of multiple components of the classical formula powder by taking the powder for freeze-drying of the powder as raw materials, and the specific content measuring method comprises the following steps:
1) Preparation of test solution and reference solution
11 Tyrode buffer preparation
Precisely weighing 8.0g of NaCl, 0.2g of KCl and 1.0g of NaHCO 3 0.05g of NaH 2 PO4, 0.1g MgCl 2 Adding 500mL of pure water to prepare solution A; precisely weighing 0.2g of CaCl 2 Adding 500mL of pure water to prepare solution B; preserving at 4 ℃. When in use, the solution A and the solution B are mixed, 1.0g of glucose is added, and the pH is regulated to 7.2-7.4, thus obtaining the Tyrode buffer solution.
12 Preparation of test sample solution
9.0g of the powder is precisely weighed (i.e. m powder=9.0 g), 100mL of Tyrode buffer solution is added, the solution is continuously stirred and is fully dissolved by ultrasound, the solution passes through a microporous filter membrane of 0.22 mu m, the obtained subsequent filtrate (i.e. the solution of the test sample) is diluted, and the solution of the test sample with the concentration of 5mg/mL (i.e. C test-Iso ＝5mg/mL)。
13 Preparation of control stock solution
Respectively taking glycyrrhizin, hesperidin, glycyrrhizic acid, naringin, apigenin and 6.36mg ginsenoside Re reference substances, precisely weighing, respectively placing into volumetric flasks, adding methanol for full dissolution and fixing volume to obtain reference substance stock solution I, wherein the concentration of the glycyrrhizin is 80.96 mug/mL, the concentration of the hesperidin is 255 mug/mL, the concentration of the glycyrrhizic acid is 244.08 mug/mL, the concentration of the rutinoside is 96 mug/mL and the concentration of the apigenin is 13.29 mug/mL;
And (3) taking a ginsenoside Re reference substance, precisely weighing, respectively placing into 5mL volumetric flasks, adding methanol for full dissolution and fixing the volume to obtain a ginsenoside reference substance stock solution, wherein the concentration of the ginsenoside Re is 127.2 mug/mL.
14 Standard solution preparation of reference substance
Precisely measuring reference stock solution I and ginsenoside reference stock solution respectively, and diluting with Tyrode buffer solution for 2 times to obtain reference standard solution I and ginsenoside reference standard solution.
2) High performance liquid chromatography detection
21 Respectively taking reference standard solution I and ginsenoside reference standard solution, and performing high performance liquid chromatography detection to obtain peak area A under different concentrations of chemical components Para-corresponding chemical composition 。
Taking glycyrrhizin as an example, namely the peak area corresponding to the glycyrrhizin in the measured reference standard solution I is A Para-glycyrrhizin Other chemical components are deduced in this way.
The high performance liquid chromatography conditions of the detection reference standard solution I are as follows:
chromatographic column: agela C18 column (4.6X105 mm,5 μm);
mobile phase: 0.05wt% phosphoric acid aqueous solution (A) -acetonitrile (B);
the elution mode is gradient elution;
elution procedure: 0-10 min,83% -70% of mobile phase A,17% -30% of mobile phase B;
10-17 min,70% → 68.5% mobile phase A,30% → 31.5% mobile phase B;
17-26 min,68.5% -64% mobile phase A,31.5% -36% mobile phase B;
26-42 min,64% -61% mobile phase A,36% -39% mobile phase B;
42-46 min,61% → 0% mobile phase A,39% → 100% mobile phase B;
46-48 min,0% mobile phase A,100% mobile phase B;
48-51 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
51-54 mi,83% mobile phase A,17% mobile phase B.
Under the condition of one sample injection, detection is carried out by utilizing different detection wavelengths, specifically: 254nm (detecting glycyrrhizin, hesperidin and glycyrrhizic acid), 283nm (detecting rutin naringin), 360nm (detecting apigenin);
sample injection amount: 10. Mu.L;
volume flow rate: 1.0mL/min;
column temperature: 30 ℃.
In addition, the same mobile phase, chromatographic column, sample injection amount, volume flow and column temperature are used for changing the elution program and detection wavelength, detecting the ginsenoside reference standard solution, and determining the content of ginsenoside Re, wherein the specific elution program is as follows:
0-9 min,83% -80% mobile phase A,17% -20% mobile phase B;
9-31 min,80% → 76.5% mobile phase A,20% → 23.5% mobile phase B;
31-38 min,76.5% -76% mobile phase A,23.5% -24% mobile phase B;
38-43 min,76% → 0% mobile phase A,24% → 100% mobile phase B;
43-46 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
46-49 min,83% mobile phase A,17% mobile phase B.
22 Taking the sample solution, and respectively performing high performance liquid chromatography detection under the two chromatographic conditions to obtain peak area A of corresponding chemical components contained in the sample solution Test-corresponding chemical composition The method comprises the steps of carrying out a first treatment on the surface of the Respectively obtaining glycyrrhizin, hesperidin, glycyrrhizic acid, naringin, apigenin and ginseng contained in the sample solutionThe peak area corresponding to the saponin Re, namely A test-Liquiritigenin 、A Test-hesperidin 、A Glycyrrhizinic acid 、A test-Ruta naringin 、A Test-apigenin 、A Try-ginsenoside Re 。
According to C Test-corresponding chemical composition ＝(A Test-corresponding chemical composition ×C Para-corresponding chemical composition )/A Para-corresponding chemical composition Calculating the concentration C of each chemical component in the sample solution Test-corresponding chemical composition ；
The content of each chemical component in the powder=C Para-corresponding chemical composition ÷C test-Iso Calculating the content of each chemical component in the powder;
alternatively, the content of each chemical component in the powder=a Test-corresponding chemical composition ×C Para-corresponding chemical composition /(C test-Iso ×A Para-corresponding chemical composition ) Calculating the content of each chemical component in the powder;
taking liquiritin as an example:
firstly, calculating the concentration C of glycyrrhizin in a sample solution test-Liquiritigenin ＝(A test-Liquiritigenin ×C Para-glycyrrhizin )/A Para-glycyrrhizin ；
Then, the content of liquiritin=c in the powder is calculated Para-glycyrrhizin /C test-Iso 。
Other chemical components and so on.
Calculation method II, content of liquiritin in Iso-functional powder=A test-Liquiritigenin ×C Para-glycyrrhizin /(C test-Iso ×A Para-glycyrrhizin )。
Other chemical components and so on.
In the embodiment, the powder contains 2.61mg/g of glycyrrhizin, 10.85mg/g of hesperidin, 6.85mg/g of glycyrrhizic acid, 3.40mg/g of naringin, 0.15mg/g of apigenin and 4.64mg/g of ginsenoside Re.
Examples 2 to 5 method for measuring contents of various effective components in Isovidone powder
Examples 2 to 5 are methods for measuring the content of various effective components in the powder, and the steps are basically the same as those in example 1, except that the process parameters are different, and the specific details are shown in table 1:
table 1 list of process parameters in examples 2 to 5
The process parameters and steps of the other parts of examples 2 to 5 are the same as those of example 1.
Example 6 method for measuring content of various effective components in Isovidone powder
b1 Preparation of test solution and reference solution
b11 Tyrode buffer preparation
Tyrode buffer was prepared as in step 11) of example 1, with the same steps and amounts as in example 1.
b12 Preparation of test sample solution
Test solutions were prepared in the same manner as in example 1, step 12).
b13 Preparation of control stock solution
Control stock solution I and control stock solution of ginsenoside were prepared according to the method of step 13) in example 1, wherein the steps and amounts were the same as in example 1.
b14 Standard solution preparation of reference substances with different concentrations
Precisely measuring the reference substance stock solution I and the reference substance stock solution of ginsenoside, and respectively diluting 0, 2, 4, 8, 16 and 32 times with Tyrode buffer solution to obtain reference substance standard solution I with different concentrations and ginsenoside reference substance standard solution with different concentrations.
The concentration of glycyrrhizin in the reference standard solution I with different concentrations is 80.96 mug/mL, 40.48 mug/mL, 20.24 mug/mL, 10.12 mug/mL, 5.06 mug/mL, 1.32 mug/mL, 255 mug/mL, 127.5 mug/mL, 63.75 mug/mL, 31.88 mug/mL, 15.94 mug/mL, 1.59 mug/mL, and the concentration of glycyrrhizin is 244.08 mug/mL, 122.04 mug/mL, 61.02 mug/mL, 30.51 mug/mL, 15.26 mug/mL, 3.39 mug/mL, rutinoside is 96 mug/mL, 48 mug/mL, 24 mug/mL, 12 mug/mL, 6 mug/mL, 3 mug/mL, 13.29 mug/mL, 6.65 mug, 3.32 mug/mL, 1.84 mug/mL, 42 g, 42.83 mug/mL, respectively.
Wherein, since the concentration of the solution containing all the samples and the quantitative limit cannot be satisfied at the same time when the glycyrrhizin, hesperidin, glycyrrhizic acid and apioside are diluted 32 times, further dilution is performed to the lowest point concentration of the quantitative limit, wherein the glycyrrhizin dilution 61.3333 times, the hesperidin dilution 160.3774 times, the glycyrrhizic acid dilution 72 times and the apioside dilution 64.04819 times.
The concentrations of ginsenoside Re in the ginsenoside reference solutions with different concentrations are 127.2 mug/mL, 63.6 mug/mL, 31.8 mug/mL, 15.9 mug/mL, 7.95 mug/mL and 3.975 mug/mL respectively.
b2 High performance liquid chromatography detection
b21 Respectively taking the reference substance standard solution I with different concentrations and the ginsenoside reference substance standard solution with different concentrations, carrying out high performance liquid chromatography detection under the corresponding chromatography conditions in the embodiment 1 to obtain peak areas measured by the corresponding concentrations of the corresponding chemical components, drawing standard curves corresponding to the different chemical components according to the concentrations of the different chemical components and the peak areas measured by the corresponding concentrations of the corresponding chemical components, and taking the concentrations of the different chemical components as an abscissa (i.e. x, in mug/mL) and the peak areas measured by the corresponding concentrations of the corresponding chemical components as an ordinate (i.e. y).
Taking liquiritin as an example, when the concentration of liquiritin is 5.28 μg/mL, x=5.28 μg/mL, the peak area corresponding to the concentration is the ordinate y, and the like is similar to other chemical components.
The concentrations and peak areas of the chemical components in the reference standard solution I with different concentrations and the ginsenoside reference standard solution with different concentrations are shown in the following table:
table 2 results list of reference standard solutions I at different concentrations and ginsenoside reference standard solutions at different concentrations
The standard curves drawn by using the reference substance standard solution I with different concentrations and the ginsenoside reference substance standard solution with different concentrations are as follows:
the standard curve corresponding to the glycyrrhizin is Y=6830.9X+6291.7, R 2 =0.9990, standard curve is shown in fig. 1;
hesperidin has a standard curve of y=2954.3x+10306.0, r 2 =0.9990, standard curve is shown in fig. 2;
the standard curve corresponding to glycyrrhizic acid is Y=6052.8X+5120.3, R 2 =0.9998, standard curve is shown in fig. 3;
the standard curve corresponding to the naringin is Y=14643X+27949.0, R 2 =0.9995, standard curve is shown in fig. 4;
the standard curve corresponding to the apigenin is Y=34315X+8109.8, R 2 = 0.9992, standard curve is shown in fig. 5;
the standard curve corresponding to ginsenoside Re is Y=2875X+2282.3, R 2 Standard curve is shown in fig. 6, = 0.9992.
b22 Taking the sample solution, performing high performance liquid chromatography detection under the corresponding chromatographic conditions in the example 1 to respectively obtain peak areas corresponding to glycyrrhizin, hesperidin, glycyrrhizic acid, naringin, apigenin and ginsenoside Re in the sample solution, substituting the peak areas of corresponding chemical components into corresponding standard curves to obtain the concentrations (C Test-corresponding chemical composition )。
And then the concentration (C) of the corresponding chemical component in the sample solution Test-corresponding chemical composition ) Dividing the concentration of the isokinetic powder in the prepared test solution (C test-Iso ) Obtaining the iso-workThe content of the corresponding chemical component in the powder, that is to say the content of the corresponding chemical component in the powder = C Para-corresponding chemical composition ÷C test-Iso 。
In the case of glycyrrhizin, the peak area (A) Para-glycyrrhizin ) Substituting the obtained standard curve to obtain the concentration (C) test-Liquiritigenin )；
Content of glycyrrhizin in Iso-powder = C Para-glycyrrhizin ÷C test-Iso The calculation method of other chemical components is deduced in this way.
In the embodiment, the powder comprises 2.61mg/g of glycyrrhizin, 10.85mg/g of hesperidin, 6.85mg/g of glycyrrhizic acid, 3.40mg/g of naringin, 0.15mg/g of apigenin and 4.64mg/g of ginsenoside Re
In addition, in the practical application process, under the condition of knowing a standard curve, the test solution can be directly prepared for high performance liquid chromatography detection and then directly substituted into the standard curve for calculation. The process of repeatedly preparing the reference substance standard solution I with different concentrations and the ginsenoside reference substance standard solution with different concentrations for determination and drawing a standard curve is not needed.
Method for measuring contents of various effective components in Isovidone powder of examples 7-10
Examples 7 to 10 are methods for measuring the content of various effective components in the powder, and the steps are basically the same as those in example 6, except that the process parameters are different, and the specific details are shown in table 3:
table 3 list of process parameters in examples 7 to 10
The process parameters and steps of the other parts of examples 7 to 10 are the same as in example 6.
Example 11 application of method for measuring content of various active ingredients in Isovian powder
c1 Preparation of test and control solutions
c11 Tyrode buffer preparation
Tyrode buffer was prepared as in step 11) of example 1, with the same steps and amounts as in example 1.
c12 Preparation of test article solution
Three test solutions were prepared in the same manner as in example 1 at step 12), wherein the concentrations of the test solutions were 30mg/mL (low concentration), 90mg/mL (medium concentration) and 150mg/mL (high concentration), respectively.
c13 Rat intestinal capsule eversion experiment
Male SD rats (200+ -20 g, SPF grade) were fasted for 12h before the experiment and were free to drink water. After the rat was sacrificed by cervical removal, the small intestine was rapidly removed, and tissues such as mesentery and fat attached to the small intestine were peeled off, and the intestine was rinsed with ice-cold Tyrode buffer until no content was eluted. The small intestine is turned over by a special tool, one end of the small intestine is ligated, and the other end of the small intestine is ligated on a plastic tube, so that the small intestine forms a saccular everting intestine tube. 2mL of Tyrode buffer preheated to 37℃was injected into the intestine using a syringe as a test solution, and placed in a constant temperature bath at 37℃with Tyrode buffer. Introducing 95% O into the bath 2 /5％CO 2 After 5min of equilibration, the everting intestine was placed in test solutions of three concentrations (n=5) and timing was started, and 100 μl of the test solutions were sampled from the intestine as the absorption solution of the isokinetic powder intestinal sac at 7 time points of 15, 30, 45, 60, 90, 120, 150min, respectively, while the same volume of the blank Tyrode buffer was supplemented. The sample was placed in a 1.5mL centrifuge tube with lid and stored at-80 ℃. After the sampling is completed, the length L and the inner diameter D of the intestinal segment are measured, and the small intestine area A is calculated.
c14 Adding methanol into the absorption liquid of the Isoque powder intestinal capsules to dilute until the concentration of the Isoque powder is within a standard curve range (wherein the absorption liquid of the Isoque powder intestinal capsules corresponding to the first four time points of the low-concentration test sample solution is not diluted, and the absorption liquid of the Isoque powder intestinal capsules corresponding to the last three time points is diluted by 2 times; the absorption liquid of the Isofacial powder enteric-coated capsules corresponding to the first four time points of the medium concentration test solution is diluted by 2 times, and the absorption liquid of the Isofacial powder enteric-coated capsules corresponding to the last three time points is diluted by 4 timesThe method comprises the steps of carrying out a first treatment on the surface of the Diluting the absorption liquid of the isokinetic powder enteric-coated capsule corresponding to the first four time points of the high-concentration test solution by 10 times, diluting the absorption liquid of the isokinetic powder enteric-coated capsule corresponding to the last three time points by 20 times), vortex mixing 1min, centrifuging 12000r/min for 10min, taking supernatant, filtering, respectively performing high performance liquid chromatography detection according to the chromatographic conditions in the embodiment 1 to respectively obtain peak areas corresponding to glycyrrhizin, hesperidin, glycyrrhizic acid, rutin, apigenin and ginsenoside Re in the supernatant, substituting the peak areas of corresponding chemical components into the corresponding standard curves in the embodiment 7, and obtaining the concentration (C) of glycyrrhizin, hesperidin, glycyrrhizic acid, rutin, naringin apigenin and ginsenoside Re in the supernatant n-corresponding chemical composition )。
The concentration of glycyrrhizin, hesperidin, glycyrrhizic acid, naringin, apioside glycyrrhizin and ginsenoside Re in the supernatant (C) n-corresponding chemical composition ) The calculation can also be performed by using the external standard method in example 1, the reference standard solution I and the ginsenoside reference standard solution can be prepared, or the peak areas A corresponding to the chemical components in the reference standard solution I and the ginsenoside reference standard solution measured in example 1 can be used Para-corresponding chemical composition And its corresponding concentration C Para-corresponding chemical composition The calculation was performed according to the calculation formula in example 1.
And calculating the cumulative absorption capacity, the absorption rate constant, the apparent permeability coefficient and the content proportion at different time points by using a conventional calculation method of the everting intestinal sac method.
c21 Cumulative absorption quantity Q)
The cumulative absorption Q (μg) is calculated as follows:
wherein V is 0 To add the volume of blank Tyrode buffer, V 1 Volume of the Isofacial powder enteric-coated absorption liquid taken at each time point, C n-corresponding chemical composition The actual concentrations of the respective chemical components measured for each time point.
As shown in fig. 7, the Q values of the following 6 chemical components showed a continuous increase with an increase in t after administration of test solutions of different concentrations, and all had a time dependence. The Q values of glycyrrhizin, hesperidin, naringin and ginsenoside Re increase with increasing concentration, and have concentration dependence. Wherein, the Q value of the hesperidin is highest, and the second is glycyrrhizic acid, ginsenoside Re, rutin naringin, and glycyrrhizin, and the content of the apigenin is lowest, which is consistent with the lower content of the apigenin in the powder.
Since there is a difference in the cumulative absorption amounts of 6 chemical components, the absorption rate constant Ka, the cumulative absorption rate V, and the apparent permeability coefficient Papp are further used to study the absorption law.
c22 Absorption rate constant
The absorption rate constant Ka (. Mu.g.min) was calculated from the Q-t curves of 6 chemical components in the small intestine -1 ·cm -2 ) The calculation formula is ka=k/a, where K is the slope of the correlation regression analysis of Q and t, and a is the intestinal area. The data were analyzed using Graph pad prism 8.0.2 software and the data were compared between groups using One-way analysis (One-way analysis of variance, ANOVA) with the following table:
table 4 6 chemical components Q-t curve related parameters @n＝5)/>
As shown in Table 4, the Ka values of the respective components increased with increasing concentrations of the powder, wherein the Ka values of glycyrrhizin, hesperidin, naringin and ginsenoside Re were significantly different in the three concentrations of the test sample solutions (P < 0.05), and exhibited a linear increase (R 2 > 0.95), indicating that the absorption of these 4 components is passive. The Ka values of the apioside and the glycyrrhizic acid have no significant difference between the medium concentration and the high concentration of the test sample solution and are increased in a nonlinear way, which indicates that the apioside and the glycyrrhizic acid penetrate in the small intestine Active transport exists. R2 > 0.9 of the Q-t curve of 6 chemical components in the medium-concentration and high-concentration test sample solutions shows that the 6 chemical components are linearly absorbed in the intestinal absorption process of the medium-concentration and high-concentration test sample solutions, and the absorption rate accords with zero-order absorption rate, namely, the absorption rate is not influenced by concentration differences at two ends of the small intestinal wall.
c23 Cumulative absorption rate)
Cumulative absorption V (%) = (Q/V) of 6 chemical components in small intestine was calculated 0 )/C 0 Q is the accumulated absorption quantity of the medicine within t time, V 0 To add the volume of blank Tyrode liquid, C 0 Is the initial concentration of each chemical component in the bath.
The cumulative drug absorption rate is the ratio of the concentration of drug in the intestinal capsule after a period of administration to the concentration of drug administered, and can be used to characterize the absorption capacity of the small intestine for drug. Referring to fig. 8, the results show that the cumulative absorption rate of each component increases with time after administration of the different concentrations of the drug solution; the decrease trend was observed with increasing drug concentration before 90 min. Except for glycyrrhizin and ginsenoside Re, the Vmax of other components appears at medium concentration of 150 min. The average V value of ginsenoside Re is 58.07%, and the average V value of hesperidin is 35.32%. As can be seen from FIG. 8, the small intestine absorbs ginsenoside Re best, and then glycyrrhizin, glycyrrhizic acid, naringin, apigenin and hesperidin.
c24 Apparent permeability coefficient)
Calculation of apparent permeability coefficients Papp=dQ/dt×1/A×1/C for 6 chemical Components 0 Q is cumulative absorption (. Mu.g), dQ/dt (. Mu.g/s) is absorption rate, C 0 (μg/mL) is the initial concentration of the corresponding compound in the crude drug solution in the bath, A (cm) 2 ) For the area of the intestinal canal taken, the specific results are given in the following table:
table 5 apparent permeability coefficients of 6 chemical components in test sample solutions (n=5, ×10 -5 cm/s)
| Compounds of formula (I) | 30mg/mL | 90mg/mL | 150mg/mL |
| Liquiritigenin | 0.39±0.19 | 0.72±0.28 | 0.37±0.17 |
| Hesperidin | 0.29±0.16 | 0.56±0.25 | 0.50±0.45 |
| Glycyrrhizic acid | 0.65±0.39 | 0.88±0.48 | 0.53±0.22 |
| Rutaceae naringin | 0.55±0.50 | 0.82±0.46 | 0.43±0.40 |
| Apiose isoliquiritigenin | 0.45±0.16 | 0.88±0.30 | 0.88±0.41 |
| Ginsenoside Re | 0.65±0.43 | 0.78±0.42 | 0.79±0.35 |
As shown in Table 5, P of other ingredients except apioside and ginsenoside Re app The values show a decreasing trend in the high-concentration test sample solution, and P of apioside isoliquiritigenin and ginsenoside Re app The values were not significantly different in the medium and high concentration test solutions (P > 0.05), and it was speculated that transport proteins on the intestinal membrane in the high concentration test solutions were substantially saturated with transport of these 6 chemical components. Due to P app The value is 1X 10 -6 ～1×10 -5 cm/s is the drug with moderate absorption, P app ＞1×10 -5 cm/s is a drug with good absorption, P of these 6 chemical components app The values are all 1X 10 -6 ～1×10 -5 cm/s and therefore belong to the medium-absorption range.
c25 Ratio of) content
The chemical components in the traditional Chinese medicine compound are complex and often take effect comprehensively by a plurality of components, so the total mass of 6 chemical components is set to be 1, and the small intestine absorption liquid (Q value of 6 chemical components) under different concentration conditions and the traditional Chinese medicine of the original medicine are compared with each other in a comparative example. As shown in fig. 9, the proportions of the ingredients in the powder change before and after absorption. In the test solution, the content sequence is hesperidin > glycyrrhizic acid > ginsenoside Re > rutin naringin > glycyrrhizin > apigenin and isoliquiritigenin. In the absorption process, the ratio of the rutin naringin and the ginsenoside Re is increased after absorption; the ratio of hesperidin decreases after absorption; the proportion of glycyrrhizic acid after absorption in the low-concentration and medium-concentration test sample solutions is increased, and the proportion of glycyrrhizic acid after absorption in the high-concentration test sample solutions is reduced; the ratio of the liquiritin after being absorbed in the low-concentration and high-concentration test sample solution is increased, and the ratio of the liquiritin after being absorbed in the medium-concentration test sample solution is reduced; the content of apioside isoliquiritigenin does not change greatly. The results show that the proportion of 6 chemical components of the qigong powder is changed after the powder is absorbed by the small intestine, and the effect can be influenced.
Experimental example 1 methodology investigation
The content determination method of various effective components in the experimental example IsoChase powder is studied, and the specificity, accuracy, precision, detection limit and quantitative limit and durability of the content determination method are mainly studied.
1. Specialization of
Taking the IsoChan powder enteric-coated absorption liquid prepared in the example 11, adding methanol for dilution according to the method in the example 11, taking the reference substance standard solution I, the ginsenoside reference substance standard solution, the test substance solution and the Tyrode buffer solution prepared in the example 1, respectively vortex mixing 1min, and centrifuging for 10min at 12000r/min to obtain corresponding liquid to be detected;
the corresponding solutions to be detected are respectively detected and analyzed according to the chromatographic conditions in the embodiment 1, and the results are shown in fig. 10, wherein A and B are chromatograms of a reference standard solution I and a ginsenoside reference standard solution, C and D are chromatograms of an isokinetic enteric-coated absorption solution, E and F are chromatograms of a sample solution, G and H are chromatograms of Tyrode buffer solution, and in the figures, the peak 1 is glycyrrhizin, the peak 2 is rutin, the peak 3 is hesperidin, the peak 4 is apiose isoglycyrrhizin, the peak 5 is glycyrrhizic acid, and the peak 6 is ginsenoside Re.
As can be seen from fig. 10, the reference standard solution I, the reference standard solution of ginsenoside, the absorption liquid of the isokinetic enteric capsule and the solution of the test sample have absorption peaks at the same positions, the separation degree of each peak is greater than 1.5, and no interference peak is generated at the same positions of the blank absorption liquid, which indicates that the method has good specificity.
2. Precision, stability and repeatability test
Taking the reference substance standard solution I prepared in the example 1 and the ginsenoside reference substance standard solution, and continuously sampling for 6 times in the same day according to the chromatographic conditions in the example 1; and then sampling is carried out 3 times a day, sampling is carried out continuously for 3 days, peak areas of the main components are recorded, and the precision in the daytime and the daytime is calculated, and the results are shown in Table 6.
The same sample solutions prepared in example 1 were taken and left at room temperature for 0, 2, 4, 6, 12 and 24 hours, and then subjected to detection analysis according to the chromatographic conditions in example 1, respectively, and peak areas were recorded, and the results are shown in Table 6.
Taking the sample solution prepared in the example 1, vortex mixing 1min, centrifuging 12000r/min for 10min, respectively carrying out detection analysis on the obtained repetitive test solution according to the chromatographic conditions in the example 1, and repeatedly sampling for 6 times, wherein the results are shown in Table 6.
TABLE 6 precision, stability and repeatability test resultsn＝5)/>
The result shows that the daily precision RSD value is between 0.37 and 2.19 percent, and the daily precision RSD value is between 1.52 and 2.76 percent, which indicates that the precision of the instrument meets the requirement. The stability RSD value of each chemical component is between 1.36 and 2.96 percent, which shows that the 6 chemical components are stable within 24 hours at room temperature. The repeatability of each chemical component is 1.09-2.64%, which shows that the method has good repeatability and meets the measurement requirement.
3. Sample addition recovery test
Taking the sample solution prepared in the example 1, mixing by vortex for 1min and centrifuging for 10min at 12000r/min to obtain a recovery experiment solution to be detected;
detecting and analyzing the recovery experiment liquid to be detected according to the chromatographic conditions in the embodiment 1 respectively to obtain the corresponding concentration of each chemical component in the recovery experiment liquid to be detected;
preparing a reference substance mixed solution with the same concentration as the chemical components in the liquid to be tested according to the corresponding concentration of the chemical components in the liquid to be tested in the recovery experiment (namely, the corresponding concentration of the chemical components in the reference substance mixed solution is equal to the corresponding concentration of the chemical components in the liquid to be tested in the recovery experiment);
the same volume of reference substance mixed solution is added into the liquid to be tested in the recovery experiment, after the mixture is uniformly mixed, detection and analysis are respectively carried out according to the chromatographic conditions in the example 1, six groups of parallel experiments are carried out according to the method, and average values of detection amount and recovery rate are respectively calculated, wherein the specific results are shown in the table 7.
TABLE 7 sample recovery results [ (]n＝5)
As can be seen from Table 7, the sample recovery rate ranges from 98.07% to 101.48%, and the RSD ranges from 0.92% to 2.41%, so that the measurement requirements of the sample recovery test are met.
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (10)
1. The method for measuring the content of various effective components in the powder is characterized by comprising the following steps of:
preparing a reference standard solution I, wherein the reference standard solution I contains glycyrrhizin, hesperidin, glycyrrhizic acid, rutin naringin and apigenin;
preparing a sample solution from the powder;
the reference standard solution I and the solvent of the sample solution both contain Tyrode buffer solution;
respectively taking reference standard solution I and sample solution for high performance liquid chromatography detection, and respectively calculating the contents of liquiritin, hesperidin, glycyrrhizic acid, naringin and apioside glycyrrhizin by external standard method;
the elution mode of the high performance liquid chromatography detection is gradient elution;
the high performance liquid chromatography of the reference standard solution I and the sample solution respectively takes 0.05wt% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and the elution conditions are as follows:
0-10 min,83% -70% of mobile phase A,17% -30% of mobile phase B;
10-17 min,70% → 68.5% mobile phase a,30% → 31.5% mobile phase B;
17-26 min,68.5% -64% mobile phase A,31.5% -36% mobile phase B;
26-42 min,64% -61% mobile phase A,36% -39% mobile phase B;
42-46 min,61% → 0% mobile phase a,39% → 100% mobile phase B;
46-48 min,0% mobile phase A,100% mobile phase B;
48-51 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
51-54 min,83% of mobile phase A,17% of mobile phase B;
the column used was an Agela C18 column, 4.6X1250 mm,5 μm.
2. The method for determining the content of various active ingredients in the powder according to claim 1, wherein the concentration of glycyrrhizin in the reference standard solution I is 1.32-80.96 mug/mL, the concentration of hesperidin is 1.59-255.00 mug/mL, the concentration of glycyrrhizin is 3.39-244.08 mug/mL, the concentration of naringin is 3.00-96.00 mug/mL and the concentration of apiose isoliquiritigenin is 0.21-13.29 mug/mL;
the concentration range of the powder in the sample solution is 4-6 mg/mL.
3. The method for measuring the content of various effective components in the powder according to claim 1 or 2, wherein a ginsenoside reference standard solution containing ginsenoside Re is prepared;
the ginsenoside reference standard solution and the solvent of the sample solution both contain Tyrode buffer solution;
when the content of ginsenoside Re in the powder is measured, the high performance liquid chromatography of the ginsenoside reference standard solution and the sample solution respectively takes 0.05wt% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B under the condition of 203nm wavelength, and the elution conditions are as follows:
0-9 min,83% -80% of mobile phase A,17% -20% of mobile phase B;
9-31 min,80% → 76.5% mobile phase A,20% → 23.5% mobile phase B;
31-38 min,76.5% -76% mobile phase A,23.5% -24% mobile phase B;
38-43 min,76% → 0% mobile phase A,24% → 100% mobile phase B;
43-46 min,0% →83% mobile phase A,100% →17% mobile phase B;
46-49 min,83% mobile phase A,17% mobile phase B;
the column used was an Agela C18 column, 4.6X1250 mm,5 μm.
4. The method for determining the content of various effective components in the powder according to claim 3, wherein the concentration of ginsenoside Re in the ginsenoside reference standard solution is 3.98-127.20 μg/mL;
the concentration range of the powder in the sample solution is 4-6 mg/mL.
5. The application of the method for measuring the content of various active ingredients in the powder is characterized in that the intestinal absorption characteristics of the powder are studied by using the method for measuring the content of various active ingredients in the powder according to any one of claims 1 to 4.
6. The method for measuring the content of various effective components in the powder is characterized by comprising the following steps of:
preparing reference standard solutions I with different concentrations, wherein the reference standard solutions I with different concentrations contain glycyrrhizin, hesperidin, glycyrrhizic acid, naringin and apigenin;
Respectively taking reference substance standard solutions I with different concentrations for high performance liquid chromatography detection, and drawing standard curves corresponding to different chemical components according to the concentrations of the different chemical components and peak areas measured by the corresponding concentrations of the corresponding chemical components;
preparing a sample solution from the powder;
the reference standard solution I with different concentrations and the solvent of the test sample solution both contain Tyrode buffer solution;
detecting the sample solution by high performance liquid chromatography, substituting the peak areas of the obtained corresponding chemical components into corresponding standard curves, and respectively calculating the contents of glycyrrhizin, hesperidin, glycyrrhizic acid, naringin and apigenin in the powder;
the elution mode of the high performance liquid chromatography detection is gradient elution;
the high performance liquid chromatography of the reference standard solution I and the sample solution with different concentrations respectively takes 0.05wt% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and the elution conditions are as follows:
0-10 min,83% -70% of mobile phase A,17% -30% of mobile phase B;
10-17 min,70% → 68.5% mobile phase a,30% → 31.5% mobile phase B;
17-26 min,68.5% -64% mobile phase A,31.5% -36% mobile phase B;
26-42 min,64% -61% mobile phase A,36% -39% mobile phase B;
42-46 min,61% → 0% mobile phase a,39% → 100% mobile phase B;
46-48 min,0% mobile phase A,100% mobile phase B;
48-51 min,0% → 83% mobile phase A,100% → 17% mobile phase B;
51-54 min,83% of mobile phase A,17% of mobile phase B;
the column used was an Agela C18 column, 4.6X1250 mm,5 μm.
7. The method for determining the content of various effective components in the powder according to claim 6, wherein the concentration of glycyrrhizin in the standard solution I of the reference substances with different concentrations is 1.32-80.96 μg/mL, the concentration of hesperidin is 1.59-255.00 μg/mL, the concentration of glycyrrhizin is 3.39-244.08 μg/mL, the concentration of naringin is 3.00-96.00 μg/mL, and the concentration of apigenin is 0.21-13.29 μg/mL;
the concentration range of the powder in the sample solution is 4-6 mg/mL.
8. The method for determining the content of a plurality of active ingredients in the powder according to claim 6 or 7, wherein the standard curves corresponding to the different chemical ingredients are as follows:
the standard curve corresponding to the glycyrrhizin is Y=6830.9X+6291.7, R 2 =0.9990；
Hesperidin has a standard curve of y=2954.3x+10306.0, r 2 =0.9990；
The standard curve corresponding to glycyrrhizic acid is Y=6052.8X+5120.3, R 2 =0.9998；
The standard curve corresponding to the naringin is Y=14643X+27949.0, R 2 =0.9995；
The standard curve corresponding to the apigenin is Y=34315X+8109.8, R 2 =0.9992。
9. The method for measuring the content of various effective components in the powder according to claim 6 or 7, wherein the standard solutions of ginsenoside reference substances with different concentrations of ginsenoside Re are prepared;
when the content of ginsenoside Re in the powder is measured, the high performance liquid chromatography of the ginsenoside reference standard solution and the sample solution with different concentrations under the wavelength condition of 203nm respectively takes 0.05wt% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and the elution conditions are as follows:
0-9 min,83% -80% of mobile phase A,17% -20% of mobile phase B;
9-31 min,80% → 76.5% mobile phase A,20% → 23.5% mobile phase B;
31-38 min,76.5% -76% mobile phase A,23.5% -24% mobile phase B;
38-43 min,76% → 0% mobile phase A,24% → 100% mobile phase B;
43-46 min,0% →83% mobile phase A,100% →17% mobile phase B;
46-49 min,83% mobile phase A,17% mobile phase B;
the chromatographic column used was an Agela C18 chromatographic column, 4.6X1250 mm,5 μm;
the concentration of ginsenoside Re in the ginsenoside reference standard solution with different concentrations is 3.98-127.20 mug/mL;
The concentration range of the powder in the sample solution is 4-6 mg/mL;
the ginsenoside reference standard solutions with different concentrations and the solvent of the sample solution both contain Tyrode buffer solution;
the standard curve corresponding to the ginsenoside Re is Y=2875X+2282.3, R 2 =0.9992。
10. The application of the method for measuring the content of various active ingredients in the powder is characterized in that the intestinal absorption characteristics of the powder are studied by using the method for measuring the content of various active ingredients in the powder according to any one of claims 6 to 9.
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