CN113156017B - Method for simultaneously determining contents of 12 chemical components in strong dizzy-stop tablet by adopting HPLC (high performance liquid chromatography) - Google Patents
Method for simultaneously determining contents of 12 chemical components in strong dizzy-stop tablet by adopting HPLC (high performance liquid chromatography) Download PDFInfo
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Abstract
The invention discloses a method for simultaneously measuring the contents of 12 chemical components in a strong dizzy-stop tablet by adopting an HPLC method. The method can simultaneously determine the content of 12 chemical components including gastrodin, gallic acid, 5-hydroxymethyl furfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin in the strong dizzy relieving tablet. The research result shows that the method for detecting 12 chemical components has good linear relation (r is more than 0.9990) in the test range, the average sample adding recovery rate is 96.0-117.0%, and the RSD is 1.00-2.80%. The invention establishes a method for simultaneously determining the contents of 12 chemical components in the strong dizzy-fixing tablet by an HPLC method for the first time, the method is accurate and sensitive, the determination result is not interfered by other substances in medicinal materials and preparations, the specificity is strong, the qualitative and quantitative analysis of the chemical components can be carried out on the strong dizzy-fixing tablet more comprehensively, and reference and basis are provided for the quality control of the strong dizzy-fixing tablet.
Description
Technical Field
The invention relates to a method for determining the content of chemical components in a medicament by adopting an HPLC method, in particular to a method for simultaneously determining the content of 12 chemical components in a strong dizzy-stop tablet by adopting an HPLC method. The invention belongs to the technical field of medicines.
Background
The strong dizzy-stopping tablet (Chinese medicine quan-type Z61020139) is a medicine produced by Shanxi Han Wang medicine limited company, is a compound preparation composed of 5 Chinese medicines of gastrodia tuber, eucommia bark leaf, wild chrysanthemum flower and Szechuan lovage rhizome, and has excellent compatibility, and the medicines are used together to play the effects of calming endogenous wind and relieving convulsion, calming the liver and suppressing yang, and tonifying qi and invigorating blood circulation. It can be used for treating hypertension, hyperlipidemia, and vertigo. The medicine can relieve vasospasm, dilate blood vessels, reduce blood viscosity, and increase the levels of norepinephrine, dopamine and 5-hydroxytryptamine in brain tissues of patients, and has remarkable clinical curative effect.
The traditional Chinese medicine, especially the traditional Chinese medicine compound, has the characteristics of complex chemical components and multi-target synergistic drug effect, and the quality of the traditional Chinese medicine and the traditional Chinese medicine compound cannot be comprehensively reflected by the quantification of a single chemical component. The quantitative determination of the multi-index chemical components can more comprehensively and objectively evaluate the quality of the compound preparation. The legal quality standard of the strong dizzy-stopping tablet lacks the quantitative detection index of multiple chemical components reflecting the quality of the medicine.
The inventor qualitatively studies chemical components in the strong dizzy-fixation tablet by adopting a liquid chromatography-mass spectrometry technology in earlier research, and finally determines that the chemical components related to quality control and efficacy in the strong dizzy-fixation tablet mainly comprise but are not limited to 12 types by combining related literature research, comparison of reference substances, HPLC system analysis and other methods for verification. Therefore, the invention establishes a method for simultaneously measuring the contents of 12 components in the strong anti-dazzling tablet by adopting an HPLC (high performance liquid chromatography) method for the first time so as to provide theoretical and technical support for the production process and the quality control standard of the strong anti-dazzling tablet.
Disclosure of Invention
The invention aims to establish a method for simultaneously measuring the contents of 12 chemical components in a strong dizzy-fixation tablet by adopting an HPLC method, the method is quick, accurate, efficient and sensitive, the measurement result is not interfered by other substances in medicinal materials and preparations, the specificity is strong, and the quantitative detection and analysis can be comprehensively carried out on the strong dizzy-fixation tablet.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to a method for simultaneously determining the content of 12 chemical components in a strong dizzy-relieving tablet by adopting an HPLC (high performance liquid chromatography) method, wherein the 12 chemical components comprise gastrodin, gallic acid, 5-hydroxymethyl furfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin, and the method comprises the following steps:
(1) Preparation of test solution
Taking 5-10 test samples, removing film coat, grinding, precisely weighing 0.4g, placing in 5-50mL measuring flask, adding appropriate amount of diluted ethanol, ultrasonic treating for 15-45min, cooling, adding diluted ethanol to desired volume, shaking, filtering, and collecting filtrate;
(2) Preparation of control solutions
Accurately weighing appropriate amount of gastrodin, gallic acid, 5-hydroxymethyl furfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin as reference substances, and adding methanol to obtain mixed reference substance solution;
(3) Preparation of single medicinal material and negative sample solution
Respectively preparing single medicinal material samples of the gastrodia elata, the eucommia ulmoides leaves, the wild chrysanthemum flowers and the ligusticum wallichii and negative simulation samples lacking the gastrodia elata, the eucommia ulmoides and the ligusticum wallichii, and the eucommia ulmoides leaves, the wild chrysanthemum flowers and the ligusticum wallichii by adopting the processes of water decoction, ethanol reflux extraction, concentration and reduced pressure drying, and preparing corresponding negative control solutions according to the method in the step (1);
(4) HPLC method for measuring content of 12 chemical components
A chromatographic column: lamdo Stamsil C 18 Columns (250X 4.6mm,5 μm); mobile phase acetonitrile (a) -0.05% phosphoric acid waterSolution (B), gradient elution (0-5min, 5-15min, 7% A, 15-30min, 7-12% A, 30-90min, 12-20% A, 20-50% A); detection wavelength: 280nm; sample introduction amount: 5 mu L of the solution; volume flow rate: 0.8mL/min; column temperature: 30 ℃;
(5) Preparation of Linear regression equation
Respectively and precisely absorbing a proper amount of the mixed reference substance solution, gradually diluting the mixed reference substance solution into a series of standard solutions with different mass concentrations, carrying out sample injection measurement according to the chromatographic conditions in the step (4), and carrying out linear regression by taking the mass concentration as a horizontal coordinate and taking a peak area as a vertical coordinate to obtain a linear regression equation;
(6) Determination of 12 chemical component contents in test solution
And (4) absorbing the sample solution to be tested, carrying out sample injection measurement according to the chromatographic conditions in the step (4), obtaining the chromatogram of the 12 chemical components in the sample solution, determining the corresponding peak areas, and respectively substituting the peak areas of the 12 chemical components into the linear regression equation, thus obtaining the content of the 12 chemical components in the sample solution.
In the method of the present invention, preferably, the time of the ultrasonic treatment in step (1) is 15-30min, the power is 250W, and the frequency is 40kHz.
In the method of the present invention, preferably, in step (2), each 1mL of the mixed control solution contains 1.045mg of gastrodin, 1.002mg of gallic acid, 0.185mg of 5-hydroxymethyl furfural, 0.41mg of p-hydroxybenzyl alcohol, 0.102mg of neochlorogenic acid, 0.1mg of chlorogenic acid, 0.135mg of vanillic acid, 0.136mg of p-hydroxybenzaldehyde, 0.109mg of pinoresinol diglucoside, 0.108mg of sophoricoside, 22mg of ligustilide, and 0.111mg of linarin.
In the method of the present invention, preferably, the diluted ethanol in the step (1) and the step (2) is 45% to 55% (v/v) ethanol solution.
The research result proves that the content of 12 chemical components in the strong dizzy-fixing sheet is measured by using the detection method of the invention, the linear relation of the 12 chemical components in the test range is good (r is more than 0.9990), the average sample-adding recovery rate is 96.0-117.0%, and the RSD is 1.00-2.80%.
Compared with the prior art, the invention has the beneficial effects that:
the invention establishes a method for simultaneously determining the contents of 12 chemical components in the strong dizzy-fixing tablet by an HPLC method for the first time, the method is accurate and sensitive, the determination result is not interfered by other substances in medicinal materials and preparations, the specificity is strong, the quantitative detection and analysis can be comprehensively carried out on the strong dizzy-fixing tablet, and reference and basis are provided for the quality control of the strong dizzy-fixing tablet.
Drawings
FIGS. 1A-K are HPLC chromatograms of components;
wherein, 1: gastrodine; 2: gallic acid; 3, 5-hydroxymethylfurfural; 4: p-hydroxybenzyl alcohol; 5: neochlorogenic acid; 6: chlorogenic acid; 7: vanillic acid; 8: p-hydroxybenzaldehyde; 9: pinoresinol diglucoside; 10: sophoricoside; 11: ligustilide; 12: linarin.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.
Example 1
1. Material
1.1 instruments LC-2010CHT high performance liquid chromatograph (Shimadzu corporation, japan), LCLabsolution chromatography workstation (Shimadzu corporation, japan), BSA2202S type electronic balance (sartorius, germany), pipette gun (Eppendorf, germany), milli-Q Integral 5 ultra pure water integrated system (Millipob corporation); KQ-500E ultrasonic cleaner (ultrasonic instruments, inc., 250W, 40kHz).
The 1.2 reagents and 6 batches of strong dizzy-fixing tablets with medicines are provided by Shaanxi Hanwang pharmaceutical industry GmbH with the numbers 007101, 024102, 019101, 016101, 021101 and 026102. Gastrodin (HR 16313B1 with purity of 98.0% or more), 5-hydroxymethylfurfural (HH 248894198 with purity of 98.0% or more), p-hydroxybenzyl alcohol (H21D 6Q7813 with purity of 98.0% or more), neochlorogenic acid (HR 20422B1 with purity of 98.0% or more), p-hydroxybenzaldehyde (HA 060208198 with purity of 98.0% or more), pinoresinol diglucoside (HS 0870W2 with purity of 98.0% or more), ligustilide (HL 09355198 with purity of 98.0% or more) and other reference products are purchased from Gastrongy photo-biological science and technology Limited company; chlorogenic acid (110753-201415, the purity is more than or equal to 96.20%), linarin (111528-201300, the purity is more than or equal to 95.1%), vanillic acid (110776-200402), sophoricoside (111695-200501) and other reference substances are purchased from China food and drug testing institute; gallic acid (CHB 180114, purity greater than or equal to 98.0%) reference was purchased from Kyohima Biotech, inc.; acetonitrile and methanol are both chromatographically pure, fisher corporation, USA; the rest reagents are analytically pure; the water is ultrapure water.
2. Method and results
2.1 chromatographic conditions column: lamdo Stamsil C 18 Columns (250X 4.6mm,5 μm); mobile phase acetonitrile (a) -0.05% aqueous phosphoric acid solution (B), gradient elution (0-5min, 5-7% a, 5-15min, 7% a, 15-30min, 7-12% a, 30-90min, 12-20% a, 20-50% a); detection wavelength: 280nm; sample introduction amount: 5 mu L of the solution; volume flow rate: 0.8mL/min; column temperature: at 30 ℃.
2.2 preparing test solution 10 tablets, removing the film coat, grinding, precisely weighing 0.4g, placing in a 5mL measuring flask, adding appropriate amount of 50% (v/v) diluted ethanol, performing ultrasonic treatment for 30min (power 250W, frequency: 40 kHz), cooling, adding 50% (v/v) diluted ethanol to constant volume to scale, shaking up, filtering, and taking the subsequent filtrate.
2.3 preparation of reference solution A proper amount of gastrodin, gallic acid, 5-hydroxymethyl furfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin reference is accurately weighed, and pure methanol is added to prepare a mixed reference solution containing 1.045mg of gastrodin, 1.002mg of gallic acid, 0.185mg of 5-hydroxymethyl furfural, 0.41mg of p-hydroxybenzyl alcohol, 0.102mg of neochlorogenic acid, 0.1mg of chlorogenic acid, 0.135mg of vanillic acid, 0.136mg of p-hydroxybenzaldehyde, 0.109mg of pinoresinol diglucoside, 0.108mg of sophoricoside, 22mg of ligustilide and 0.111mg of linarin in per 1 mL.
2.4 preparation of single medicinal material and negative sample solution by water decoction, ethanol reflux extraction, concentration and reduced pressure drying processes, respectively preparing single medicinal material samples of rhizoma Gastrodiae, eucommiae cortex, folium Eucommiae, flos Chrysanthemi Indici, and rhizoma Ligustici Chuanxiong and negative simulation samples of rhizoma Gastrodiae, eucommiae cortex and rhizoma Ligustici Chuanxiong, eucommiae cortex and folium Eucommiae, flos Chrysanthemi Indici and rhizoma Ligustici Chuanxiong, and preparing corresponding negative control solution by the method under item 2.2.
2.5 the specificity test accurately absorbs the test solution, the mixed reference solution, the single medicinal material solution and the negative reference solution respectively, and the analysis is carried out according to the chromatographic condition under the item of '2.1', and the result is shown in figures 1A-K, which shows that the method has no interference in the negative reference and strong specificity.
2.6 Linear relationship examination precisely absorbs a proper amount of mixed reference solution prepared under the item 2.3, gradually dilutes the mixed reference solution into a series of 6 standard solutions ( series concentration 1,2,3,4,5, 6) with different mass concentrations, and samples are injected and measured under the chromatographic condition under the item 2.1. Linear regression was performed with mass concentration as abscissa (X) and peak area as ordinate (Y), see table 1. The results show that the linear relation of each component in the respective mass concentration range is good, and r is more than or equal to 0.9990.
TABLE 1 Linear relationship of ingredients
2.7 precision test A mixed control solution (series concentration 3) was precisely aspirated, and sample introduction was performed continuously for 6 times under the chromatographic condition of "2.1" to determine the peak area. As a result, RSD values of peak areas of gastrodin, gallic acid, 5-hydroxymethylfurfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide, and linarin were 1.0636%, 1.4784%, 1.5964%, 0.6490%, 1.0980%, 0.5572%, 1.5793%, 0.6774%, 0.6928%, 1.5932%, 1.7577%, and 1.7235%, respectively, indicating good instrument precision.
2.8 repeatability test 6 parts of the sample (test sample number 1) are precisely weighed, the sample solution is prepared according to the method under item 2.2, sample introduction and measurement are carried out according to the chromatographic condition under item 2.1, the chromatogram of 12 chemical components in the sample solution is obtained, corresponding peak areas are determined, and the peak areas of the 12 chemical components are respectively substituted into the linear regression equation, so that the content of the 12 chemical components in the sample solution can be obtained. As a result, the RSD values of the contents of gastrodin, gallic acid, 5-hydroxymethyl furfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin are respectively determined as follows: 0.89%, 0.73%, 1.39%, 0.78%, 1.32%, 2.05%, 1.57%, 1.59%, 1.5600%, 1.86%, 1.61%, 0.97%; the average contents are respectively: 1.4518, 0.3287, 0.1021, 0.2073, 0.5049, 0.2288, 0.0309, 0.0124, 0.0734, 0.0361, 45.7028, 0.1014mg/g, indicating that the process is very reproducible.
2.9 stability test A mixed control solution (series concentration 3) was precisely aspirated and measured by injection at 0, 2, 4,6, 8, 10, 24, 36h, respectively, under the chromatographic conditions of "2.1". As a result, RSDs of peak areas of gastrodin, gallic acid, 5-hydroxymethylfurfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide, and linarin were measured to be 1.02%, 1.87%, 1.58%, 0.60%, 1.35%, 0.51%, 1.90%, 1.22%, 0.87%, 1.42%, 1.56%, and 1.59%, respectively, indicating that the stability of the sample solution was good within 36 hours.
2.10 sample application recovery test about 0.2g of the test sample (test sample number 1), 6 parts in total, were weighed precisely, and the mixed control solutions (according to table 2) were added precisely, prepared according to the method under "2.2", and the sample application was measured, the average sample application recovery was 96.31% -116.81%, and the RSD was 1.33% -2.78%, as shown in table 2.
TABLE 2 sample recovery for 12 chemistries (n = 6)
2.11 sample content determination 0.4g of sample is precisely weighed, a sample solution is prepared according to the method under item 2.2, sample introduction determination is carried out according to the chromatographic condition under item 2.1, a chromatogram of 12 chemical components in the sample solution is obtained, corresponding peak areas are determined, and the peak areas of the 12 chemical components are respectively introduced into the linear regression equation, so that the content of the 12 chemical components in the sample solution can be obtained. As a result, the contents (mg/g) of gastrodin, gallic acid, 5-hydroxymethylfurfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin were determined, and the results are shown in Table 3.
Table 3 results of chemical component content measurement in six batches of strong anti-glare tablets (mg/g, n = 3)
3. Discussion of the related Art
3.1 selection of chromatographic conditions in the section of Chinese pharmacopoeia 2020 edition, the detection wavelengths of gastrodin, chlorogenic acid, pinoresinol diglucoside, linarin and the like have larger difference, and the polarity difference of 12 chemical components detected by the experiment is considered to be larger, so a diode array detector is adopted to scan a strong dizzy-fixing sheet sample in the wavelength range of 200-400 nm, and a spectrogram is acquired.
3.2 the extraction solvent is selected according to the standard of Chinese pharmacopoeia, and the extraction solvent can be selected from but not limited to water, 45-55% (v/v) ethanol solution and 60-100% methanol solution for extraction. Experiments show that 45-55% (v/v) ethanol solution has high extraction rate, more peaks and better separation degree, so 45-55% (v/v) ethanol solution is selected as extraction solvent for extraction, and 50% (v/v) ethanol solution is the best.
3.3 the ultrasonic extraction time of the test sample treatment method has influence on the extraction rate of chemical components; but may alternatively be, but not limited to, 15-45min.
The research compares two methods of heating reflux and ultrasonic extraction, considers that volatile components such as ligustilide, gastrodin and the like are easily decomposed by heating, and has literature reports [15，16] The content of gastrodin will decrease when heated to above 60 deg.C, and new substances will be generated, so ultrasonic extraction method is selected.
3.4 the index components determine the selection of the index chemical components, and the monarch, minister, assistant and guide compatibility theory of the traditional Chinese medicine and the effectiveness, testability and specificity of the compound functional components are comprehensively considered. Tianma in Qiangli Dingxuan tablets is the monarch drug, so the drug should be considered first; the whole formula has the effects of reducing blood pressure, reducing blood fat and relieving dizziness, so possible functional components and index components in the formula are mainly searched and detected. The indicative components are typical components which have higher content in the traditional Chinese medicine, can represent similar components with similar structures and effects, are suitable for measuring the content of multi-index components by a one-measurement multi-evaluation method, and achieve the aim of quality control, such as gastrodin in gastrodia elata, linarin in wild chrysanthemum and the like.
Linarin is a specific flavonoid component in wild chrysanthemum, so the 12 components are selected as indexes in the invention. In the early stage of the invention, the chemical components in the strong dizzy-fixation tablet are qualitatively researched by adopting a liquid chromatography-mass spectrometry technology, and the main 12 chemical components related to quality control and efficacy in the strong dizzy-fixation tablet are finally determined by combining related literature research, comparison of reference substances, HPLC system analysis and other methods for verification, so that the 12 chemical components are used as determination indexes.
The method for simultaneously determining the contents of 12 chemical components such as gastrodin, gallic acid and the like is quick, accurate, efficient and sensitive, the determination result is not interfered by other substances in medicinal materials and preparations, the specificity is strong, and the powerful dizzy-fixing tablet can be comprehensively and quantitatively detected and analyzed, so that a theoretical basis is provided for the quality control of the powerful dizzy-fixing.
Claims (4)
1. A method for simultaneously determining the content of 12 chemical components in a Qiangli Dingxuan tablet by adopting an HPLC method is characterized in that the 12 chemical components comprise gastrodin, gallic acid, 5-hydroxymethyl furfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin, and the method comprises the following steps:
(1) Preparation of test solution
Taking 5-10 tablets of the sample, removing the film coat, grinding, precisely weighing 0.4g, placing in a 5mL measuring flask, adding appropriate amount of diluted ethanol, performing ultrasonic treatment for 15-45min, cooling, adding diluted ethanol to constant volume to scale, shaking, filtering, and taking the subsequent filtrate;
(2) Preparation of control solutions
Accurately weighing appropriate amount of gastrodin, gallic acid, 5-hydroxymethyl furfural, p-hydroxybenzyl alcohol, neochlorogenic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, pinoresinol diglucoside, sophoricoside, ligustilide and linarin reference substance, and adding methanol to obtain mixed reference substance solution;
(3) Preparation of single medicinal material and negative sample solution
Respectively preparing single medicinal material samples of the gastrodia elata, the eucommia ulmoides leaves, the wild chrysanthemum flowers and the ligusticum wallichii and negative simulation samples of the rhizoma gastrodiae, the eucommia ulmoides and the ligusticum wallichii, the eucommia ulmoides and the eucommia ulmoides leaves, the wild chrysanthemum flowers and the ligusticum wallichii by adopting the processes of water decoction, ethanol reflux extraction, concentration and reduced pressure drying, and preparing corresponding negative control solutions according to the method in the step (1);
(4) HPLC method for measuring content of 12 chemical components
A chromatographic column: lamdo Stamsil C 18 A column, 250X 4.6mm,5 μm; mobile phase acetonitrile A-0.05% phosphoric acid water solution B, gradient elution: 0 to 5min,5 to 7% A;5 to 15min,7% by weight A; 15-30min, 7% -12% A; 30-90min, 12% -20% of A; 90-100min, 20% -50% A; detection wavelength: 280nm; sample introduction amount: 5 mu L of the solution; volume flow rate: 0.8mL/min; column temperature: 30 ℃;
(5) Preparation of Linear regression equation
Respectively and precisely absorbing appropriate amount of the mixed reference substance solution, gradually diluting the mixed reference substance solution into a series of standard solutions with different mass concentrations, carrying out sample injection measurement according to the chromatographic conditions in the step (4), and carrying out linear regression by taking the mass concentration as a horizontal coordinate and taking a peak area as a vertical coordinate to obtain a linear regression equation;
(6) Determination of 12 chemical component contents in test solution
And (4) absorbing the sample solution to be tested, carrying out sample injection measurement according to the chromatographic conditions in the step (4), obtaining the chromatogram of the 12 chemical components in the sample solution, determining the corresponding peak areas, and respectively substituting the peak areas of the 12 chemical components into the linear regression equation, thus obtaining the content of the 12 chemical components in the sample solution.
2. The method of claim 1, wherein the sonication in step (1) is carried out for a period of 15-30min at a power of 250W and a frequency of 40kHz.
3. The method of claim 1, wherein the mixed control solution of step (2) contains 1.045mg of gastrodin, 1.002mg of gallic acid, 0.185mg of 5-hydroxymethylfurfural, 0.41mg of p-hydroxybenzyl alcohol, 0.102mg of neochlorogenic acid, 0.1mg of chlorogenic acid, 0.135mg of vanillic acid, 0.136mg of p-hydroxybenzaldehyde, 0.109mg of pinoresinol diglucoside, 0.108mg of sophoricoside, 22mg of ligustilide and 0.111mg of linarin per 1 mL.
4. The method of claim 1, wherein the dilute ethanol in steps (1) and (2) is a 45% -55% v/v ethanol solution.
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