CN106290599B - Content determination method of traditional Chinese medicine composition - Google Patents
Content determination method of traditional Chinese medicine composition Download PDFInfo
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Abstract
The traditional Chinese medicine composition adopts a UP L C method, simultaneously measures the contents of the active ingredients of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid, has simple and quick method, is suitable for the quality control of the traditional Chinese medicine composition, and provides reference for the dynamic real-time monitoring in the continuous production of the traditional Chinese medicine composition.
Description
Technical Field
The invention relates to a content determination method of a traditional Chinese medicine composition, belongs to the field of traditional Chinese medicine analysis, and particularly relates to a content determination method of each component in the traditional Chinese medicine composition.
Background
The pharmaceutical composition mainly comprises astragalus mongholicus, golden cypress, cinnamon, selfheal, glossy privet fruit and the like, aims at strengthening the spleen and tonifying the kidney, and disinhibiting water and resolving masses as treatment principles, and provides a novel traditional Chinese medicine preparation [ Supingju, Liuyi, Japanese pine, Gaoypeng, Zhaoshua, Liuyan ] for patients aiming at main symptoms of benign prostatic hyperplasia clinically, wherein the content of active ingredients in the traditional Chinese medicine composition [ A ] is determined by ultra-high performance liquid chromatography of 3 active ingredients in the traditional Chinese medicine composition [ A ] China Mediterranean Med. Neuropathy basis and clinical research (10) [ C ] Chinese medical society, 2014:3, Hohliean, Magui cloud, Jinzhi, Liu Wen Bo, ren, Yong of the traditional Chinese medicine composition of the invention [ J ] microbiological limit examination method research [ J ] modern remote education, 2014-154, 155, ] is determined by thin-layer chromatography of Shen dynasty, the contents of astragalus mongholicus, the contents in Shen, the traditional Chinese medicines [ HP 48, 8, the contents of active ingredients in traditional Chinese medicines [ HP 48, 8, H ] are determined by ultra-H-K-H-K method, H-H, H-H, H-H, H-H.
The traditional Chinese medicine composition has more components, the currently common detection method has long period and high cost, so in order to effectively control the product quality and ensure the medication safety of the public, the quality control of the traditional Chinese medicine composition is carried out, the research adopts a UP L C method to simultaneously determine the contents of 5 main effective components of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid in the traditional Chinese medicine composition, the method is simple and quick, and the reference is provided for dynamic real-time monitoring in the continuous production of the traditional Chinese medicine composition.
Disclosure of Invention
The invention aims to provide a method for detecting the content of five components simultaneously, which has short detection period and high sensitivity.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the traditional Chinese medicine composition is composed of the following raw materials, by weight, 30-150 parts of astragalus membranaceus, 7-28 parts of talc, 10-40 parts of selfheal, 7-28 parts of glossy privet fruit, 10-40 parts of lychee seeds, 1-4 parts of amber, 1.5-6 parts of cinnamon and 3-15 parts of golden cypress, and the content determination method is to simultaneously determine the content of the five components by adopting a UP L C method.
The content determination method is preferably:
A. the preparation method of the test solution comprises the following steps: weighing the content of the traditional Chinese medicine composition, weighing 0.5-3 g, precisely weighing, placing in a conical flask with a plug, precisely adding 10-75 ml of 50-100% methanol, weighing, ultrasonically treating for 10-60 minutes, cooling, weighing, supplementing the lost weight with 50-90% methanol, and shaking up to obtain the traditional Chinese medicine composition;
B. the preparation method of the reference substance solution comprises the following steps: weighing calycosin glucoside, specnuezhenoside and hydrochloric acidThe palmatine, berberine hydrochloride and rosmarinic acid reference substances are precisely weighed and dissolved in methanol to obtain the concentrations of 0.396 mg.ml respectively-1、1.156 mg•ml-1、0.572 mg•ml-1、0.74 mg•ml-1、1.578 mg•ml-1Mixing the reference stock solution to obtain;
C. chromatographic conditions are as follows: a chromatographic column: c18 column, column temperature: 35-45 ℃; the mobile phase A is acetonitrile or methanol, and the mobile phase B is: phosphoric acid aqueous solution or formic acid aqueous solution, the gradient elution ratio is: in 0-2 min, the content of the mobile phase A is 15-20%, and the content of the mobile phase B is 85-80%; 2-7 min, the content of the mobile phase A is from 20% to 30%, and the content of the mobile phase B is from 80% to 70%; in 7-10 min, the mobile phase A is 30-32%, and the mobile phase B is 70-68%; the mobile phase A is 32-35% in 10-12 min, and the mobile phase B is 68-65%; 35% of mobile phase A and 65% of mobile phase B in 12-14 min; detection wavelength: 240 nm; column temperature: 35 ℃; flow rate: 0.2 ml.min-1；
D. And (3) measuring, namely sucking the test sample solution and the reference solution by 0.2-5 mu L, injecting the test sample solution and the reference solution into a UP L C liquid phase, and calculating the content of the sample by peak area.
The content determination method is preferably: the preparation method of the test solution in the step A comprises the following steps: weighing the content of the traditional Chinese medicine composition, taking 1 g, precisely weighing, placing in a conical flask with a plug, precisely adding 25 ml of 80% methanol, weighing, ultrasonically treating for 30 minutes (power 250w, frequency 40kHz), cooling, weighing, supplementing the lost weight with 80% methanol, and shaking up to obtain the traditional Chinese medicine composition.
The content determination method is preferably that the chromatographic column in the step C has the model number of Phenomenex UP L C KinetexC18 with the specification of 2.1 × 100mm and 2.6 μm, or the model number of Waters Acquity UP L C ○ R Shield RP18 with the specification of 2.1 × 100mm and 1.7 μm, or the model number of ACQUITY BEH C18 with the specification of 2.1 × 100mm and 1.7 μm.
The content determination method is preferably: in the step C, the mobile phase A is methanol, and the mobile phase B is 0.1% phosphoric acid aqueous solution; or the mobile phase A is methanol, and the mobile phase B is 0.2% formic acid aqueous solution; or the mobile phase A is acetonitrile, and the mobile phase B is 0.2% formic acid aqueous solution; or the mobile phase A is acetonitrile, and the mobile phase B is 0.1% phosphoric acid water solution.
The traditional Chinese medicine composition of the content determination method preferably comprises the following raw material medicines in parts by weight:
70 parts of astragalus mongholicus, 14 parts of talcum, 21 parts of selfheal, 14 parts of glossy privet fruit, 21 parts of lychee seed, 2.1 parts of amber and 2.8 parts of cinnamon and 7 parts of golden cypress.
The traditional Chinese medicine composition of the content determination method preferably comprises the following raw material medicines in parts by weight:
30 parts of astragalus mongholicus, 7 parts of talcum, 10 parts of selfheal, 7 parts of glossy privet fruit, 10 parts of lychee seed, 1 part of amber and 1.5 parts of cortex cinnamomi, and 3 parts of cortex phellodendri.
The active ingredients of the traditional Chinese medicine composition of the content determination method are prepared by the following steps:
(1) weighing the traditional Chinese medicinal materials according to the weight proportion of the raw materials, respectively cleaning and crushing;
(2) extracting radix astragali and cortex Phellodendri with 50-70% ethanol under reflux for 0.5-2 hr for 1-3 times, filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, and concentrating to obtain fluid extract with relative density of 1.20-1.25;
(3) extracting fructus Ligustri Lucidi with 6-10 times of 70-90% ethanol under reflux for 1-3 times (1-3 hr each time), filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, and concentrating to obtain fluid extract with relative density of 1.20-1.25;
(4) soaking cortex Cinnamomi in 4-8 times of water for 1-2 hr, extracting volatile oil for 2-6 hr,
collecting volatile oil in another container, filtering water extractive solution, and collecting residue;
(5) taking selfheal, lychee seeds and the residues obtained in the step (4), adding 8-10 times of water, decocting twice, wherein the first time is 1-3 hours, the second time is 1-2 hours, filtering the extracting solution, combining, adding the aqueous solution obtained in the step (4), and concentrating into clear paste with the relative density of 1.20-1.25 for later use;
(6) pulverizing pulvis Talci and Succinum into 100 mesh fine powder;
the clear paste obtained in the step (2), the clear paste obtained in the step (3), the volatile oil obtained in the step (4), the clear paste obtained in the step (5) and the fine powder obtained in the step (6) jointly form the active ingredients of the traditional Chinese medicine composition.
The dosage form of the traditional Chinese medicine composition prepared by the content determination method is capsules, tablets, oral liquid or pills.
The Chinese medicinal composition capsule for the content determination method comprises the following steps:
(1) cleaning the above Chinese medicinal materials, pulverizing, and weighing at a certain proportion;
(2) extracting radix astragali and cortex Phellodendri with 6-12 times of 50-70% ethanol under reflux for 1-3 times, each time for 1-3 hr, filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, concentrating to obtain fluid extract with relative density of 1.20-1.25, vacuum drying at 60-70 deg.C under vacuum degree of 0.04-0.06Mpa in vacuum drying oven to obtain dry extract;
(3) extracting fructus Ligustri Lucidi with 6-10 times of 70-90% ethanol under reflux for 1-3 times (1-3 hr each time), filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, concentrating to obtain fluid extract with relative density of 1.20-1.25, vacuum drying at 60-70 deg.C under vacuum degree of 0.04-0.06Mpa in a vacuum drying oven, and keeping the dry extract;
(4) soaking cortex Cinnamomi in 4-8 times of water for 1-2 hr, extracting volatile oil for 2-6 hr,
collecting volatile oil with other container, wherein the oil yield is not less than l.0%, filtering the water extractive solution, and collecting the residue;
(5) taking selfheal, lychee seed and residue after oil extraction of cinnamon, adding 8-10 times of water, decocting twice, the first time is 1-3 hours, the second time is 1-2 hours, filtering the extracting solution, merging, adding the water solution after oil extraction of cinnamon, concentrating into clear paste with the relative density of 1.20-1.25, putting into a vacuum drying oven, drying under reduced pressure at the temperature of 60-70 ℃ and the vacuum degree of 0.04-0.06Mpa, and crushing the dry paste, the astragalus mongholicus, the golden cypress and the glossy privet fruit into 100-mesh powder for later use;
(6) pulverizing pulvis Talci and Succinum into 100 mesh powder;
(7) mixing the dry extract powder, the crude powder and starch, granulating with 80% ethanol as binder under high speed stirring, oven drying at 60-70 deg.C, and grading;
(8) sieving to obtain fine powder, spraying volatile oil, mixing with the granules, sealing, and making into capsule.
Selection of detection wavelength: the spectral scanning of 5 compounds was performed using a PDA detector, and calycosin glucoside exhibited three absorption peaks in the PDA detection spectrum: 220nm, 249nm and 290nm, and the response values are in the order of magnitude: 220nm is more than 249nm and more than 290nm, the maximum absorption wavelength of the specnuezhenide is 226nm, except that the absorption peak at the tail end is larger, the absorption peak at 280-350nm is basically in a smooth state, and the change is not large; berberine hydrochloride and palmatine hydrochloride both have absorption peaks at about 228nm, 264nm and 347nm, and the response values are basically equivalent; rosmarinic acid exhibited two absorption peaks in the PDA detection spectrum: 220nm and 329nm, and the response values are basically equivalent. The five compounds have maximum absorption at 220nm, but because the impurity peaks are increased near the ultraviolet end in the whole chromatogram, the accurate quantification of each component is influenced, the spectral characteristics of the five compounds are comprehensively considered, the response of the five compounds at 240nm is relatively high, the interference of the impurity peaks is less, and finally the detection wavelength of taking 240nm as the five compounds is determined.
Selection of mobile phase: the investigation results of different mobile phase combinations such as acetonitrile-0.1% phosphoric acid water, acetonitrile-0.2% phosphoric acid water, acetonitrile-0.1% formic acid water system and the like show that the four mobile phases can separate the components to be detected, the peak types of the components are better, the separation degree is more than 1.5, the separation time is short, and the analysis of a high-flux sample is facilitated. When the mobile phase is acetonitrile-0.2% formic acid water for gradient elution, the separation degree of each component to be detected in the sample is better than other three components, and particularly for berberine hydrochloride and palmatine hydrochloride which are difficult to separate, the separation degree is more than 2.5.
The extraction method comprises the following steps: the experiment compares the influence of 4 solvents of 50% methanol, 70% methanol, 80% methanol and 100% methanol on the extraction rate of the components to be detected, and the result shows that the 4 solvents of 50% methanol, 70% methanol, 80% methanol and 100% methanol have higher extraction rate on the components to be detected, the extraction efficiency is highest with 80% methanol, and the impurities are relatively few; meanwhile, the ultrasonic extraction time (20 min, 30min, 40min and 60 min) is optimized, and the content of each component can be completely extracted after the ultrasonic extraction is carried out for 30 min.
The content of the traditional Chinese medicine composition is a substance to be detected of a medicinal preparation, such as: capsule contents, tablet cores with coatings removed from the tablets, pills, oral liquids, and the like.
To verify the feasibility and accuracy of the method of the invention, the following methodological verification test was performed with the capsules of example 1:
1 instruments and materials
1.1 instruments
Ultra-high performance liquid chromatography (Waters Acquity-H-Class, equipped with quaternary high pressure gradient pump, vacuum degasser, autosampler, column oven, diode array detector, Empower 3 chromatography workstation), ultrasonic cleaner (KQ-300 VDB, 250W power, 40kHz frequency, Kunshan ultrasonic instruments, Inc.), analytical balance (AT 201, METT L ERTO L EDO), Milli-Q Advantage A10 ultra-pure water system (Millipore).
Material
Calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid (the batch numbers are 111920-201102, 111926-201102, 110732-200506, 110713-201212, 111871-201001 and 110781-200613 respectively) are purchased from China institute for testing and determining food and drug;
the traditional Chinese medicine composition (with the batch numbers of 130501, 111001, 131001, 110601 and 110301 respectively) is produced by Ling pharmaceutical industry, Inc. Acetonitrile, methanol (chromatographically pure, Merk, germany), formic acid (chromatographically pure, Fisher Scientific, usa). Other reagents were analytically pure.
Method and results
2.1 chromatographic conditions
Chromatographic column Waters Acquity UP L C
Shield RP18 （2.1×100 mm，1.7μm), column temperature: 40 ℃; mobile phase: acetonitrile (a) -0.2% formic acid (B) solution, gradient elution (table 1), detection wavelength: 240 nm; flow rate: 0.2 ml/min-1(ii) a Sample introduction amount: 1μl。
Watch (A)
Gradient of mobile phase
2.2 preparation of test solutions
Weighing about 1 g of the content of the traditional Chinese medicine composition, precisely weighing, placing in a conical flask with a plug, precisely adding 25 ml of 80% methanol, weighing, carrying out ultrasonic treatment for 30 minutes (power 250w, frequency 40kHz), cooling, weighing, supplementing the lost weight with 80% methanol, and shaking uniformly to obtain the traditional Chinese medicine composition.
2.3 preparation of control solutions
Weighing appropriate amount of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride, and rosmarinic acid reference substances, precisely weighing, and dissolving with methanol to obtain concentrations of 0.396 mg/ml-1、1.156 mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing the reference stock solution to obtain the final product.
2.4 Linear relationship investigation
Precisely sucking a certain amount of reference substance stock solution to prepare working reference substance solution. And precisely sucking the working reference substance solution, and injecting the working reference substance solution into an ultra-high performance liquid chromatograph for measurement. The concentration was plotted on the abscissa and the peak area integral was plotted on the ordinate to obtain a standard curve, and the results are shown in Table 2.
TABLE 2 Linear relationship examination
2.5 precision test and stability test
And respectively and precisely absorbing the working reference substance solution, carrying out continuous sample injection for 5 times, and measuring peak areas, wherein the results of 1.46%, 1.29%, 1.18%, 1.45% and 1.41% of calycosin glucoside, specnuezhenoside, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid RSD respectively show that the precision of the instrument is good.
Precisely sucking the same test solution, respectively injecting samples for 0, 2, 4, 8, 12 and 24 hours to determine peak areas, wherein the RSDs of the peak areas of 5 compounds of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 0.961%, 1.49%, 1.29%, 1.02% and 1.31% within 24 hours of determination, which indicates that the test solution is basically stable within 24 hours.
2.6 repeatability test
The same batch of samples of the traditional Chinese medicine composition is treated by 9 parts according to the preparation method of the test solution under item 2.2, and the treated samples are injected into an ultra-high performance liquid chromatograph for determination, so that the RSD values of the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid in the samples are respectively 2.35%, 1.71%, 2.33% and 2.20%, and the method has good repeatability.
2.7 recovery test
By adopting a sample adding and recovering test method, 0.5g of the same traditional Chinese medicine composition sample with known content is precisely weighed, 9 parts are parallelly added, a certain amount of reference substance mixed solution is precisely added respectively, ultrasonic extraction and filtration are respectively carried out according to the preparation method of a test sample solution, the mixture is injected into an ultra-high performance liquid chromatograph for measurement, the recovery rate is calculated, and the acteoside glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 100.2%, 99.3%, 100.8%, 100.4% and 101.1%, and the RSD values are respectively 1.49%, 1.13%, 1.40%, 1.46% and 0.684%, which shows that the method is accurate and reliable.
2.8 sample determination
Taking a sample of the traditional Chinese medicine composition, respectively carrying out ultrasonic extraction, filtering, injecting into an ultra-high performance liquid chromatograph for determination, and recording a chromatogram according to the preparation method of the test solution; the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid were calculated at 240nm, and the results of the content measurements are shown in Table 3.
TABLE 3 measurement results of contents (mg. g)-1）
The result shows that the analysis method has the characteristics of strong specificity, high precision, accurate and stable result, good repeatability, high sensitivity and the like, and is suitable for the quality control of the traditional Chinese medicine composition.
Detailed Description
Example 1: preparation of the inventive pharmaceutical capsules
Prescription:
astragalus root 70 g talcum 14 g selfheal 21 g ligustrum japonicum 14 g
Litchi seed 21 g amber 2.1 g cinnamon 2.8 g phellodendron bark 7 g
The preparation method comprises the following steps:
(1) weighing the traditional Chinese medicinal materials according to the prescription amount, respectively cleaning and crushing;
(2) extracting radix astragali and cortex Phellodendri with 60% ethanol under reflux for 2 times, 10 times for the first time and 2 hours and 8 times for the second time for 1 hour, filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, concentrating to obtain fluid extract with relative density of 1.20-1.25, vacuum drying at 65 deg.C and 0.05Mpa in vacuum oven, and collecting the dry extract;
(3) extracting fructus Ligustri Lucidi with 8 times of 80% ethanol under reflux for 2 times (each time for 2 hr), filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, concentrating to obtain fluid extract with relative density of 1.20-1.25, vacuum drying at 65 deg.C under vacuum degree of 0.05Mpa in a vacuum drying oven, and keeping the dry extract;
(4) soaking the cinnamon in 6 times of water for 1 hour, extracting volatile oil for 4 hours, collecting the volatile oil in another container, filtering and collecting a water extracting solution, and reserving residues for later use;
(5) taking selfheal, lychee seeds and the residue obtained in the step (4), adding 9 times of water, decocting twice, decocting for 2 hours for the first time and 1.5 hours for the second time, filtering the extracting solution, combining, adding the aqueous solution obtained in the step (4), concentrating into clear paste with the relative density of 1.20-1.25, putting into a vacuum drying oven, drying under reduced pressure at the temperature of 65 ℃ and the vacuum degree of 0.05Mpa, mixing the obtained dry paste with the dry paste obtained in the step (2) and the dry paste obtained in the step (3), and crushing into 100-mesh powder for later use;
(6) pulverizing pulvis Talci and Succinum into 100 mesh powder;
(7) uniformly mixing the dry paste powder obtained in the step (5), the fine powder obtained in the step (6) and a proper amount of auxiliary materials, granulating by using 80% ethanol as a binding agent, drying and finishing granules;
(8) and (4) screening out fine powder, spraying the volatile oil obtained in the step (4), uniformly mixing with the granules, sealing, and encapsulating to obtain the capsule.
The content determination method comprises the following steps:
A. preparation of a test solution: weighing about 1 g of the content of the traditional Chinese medicine composition, precisely weighing, placing in a conical flask with a plug, precisely adding 25 ml of 80% methanol, weighing, carrying out ultrasonic treatment for 30 minutes (power 250w, frequency 40kHz), cooling, weighing, supplementing the lost weight with 80% methanol, and shaking uniformly to obtain the traditional Chinese medicine composition.
B. Preparation of control solutions: weighing appropriate amount of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride, and rosmarinic acid reference substances, precisely weighing, and dissolving with methanol to obtain concentrations of 0.396 mg/ml-1、1.156mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing the reference stock solution to obtain the final product.
C. Chromatographic conditions of chromatographic column Waters Acquity UP L C
Shield RP18 （2.1×100 mm，1.7μm), column temperature: 40 ℃; mobile phase: acetonitrile (a) -0.2% formic acid (B) solution, gradient elution (table 4), detection wavelength: 240 nm; flow rate: 0.2 ml/min-1(ii) a Sample introduction amount: 1μl。
The experimental results are as follows:
1. investigation of linear relationships
Precisely sucking a certain amount of reference substance stock solution to prepare working reference substance solution. And precisely sucking the working reference substance solution, and injecting the working reference substance solution into an ultra-high performance liquid chromatograph for measurement. The concentration was plotted on the abscissa and the peak area integral was plotted on the ordinate to obtain a standard curve, and the results are shown in Table 5.
TABLE 5 Linear relationship examination
2. Precision test and stability test:
and respectively and precisely absorbing the working reference substance solution, carrying out continuous sample injection for 5 times, and measuring peak areas, wherein the results of 1.46%, 1.29%, 1.18%, 1.45% and 1.41% of calycosin glucoside, specnuezhenoside, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid RSD respectively show that the precision of the instrument is good.
Precisely sucking the same test solution, respectively injecting samples for 0, 2, 4, 8, 12 and 24 hours to determine peak areas, wherein the RSDs of the peak areas of 5 compounds of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 0.961%, 1.49%, 1.29%, 1.02% and 1.31% within 24 hours of determination, which indicates that the test solution is basically stable within 24 hours.
3. Repeatability test
The same batch of samples of the traditional Chinese medicine composition is treated by 9 parts according to the preparation method of the test solution under item 2.2, and the treated samples are injected into an ultra-high performance liquid chromatograph for determination, so that the RSD values of the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid in the samples are respectively 2.35%, 1.71%, 2.33% and 2.20%, and the method has good repeatability.
4. Recovery test
By adopting a sample adding and recovering test method, 0.5g of the same traditional Chinese medicine composition sample with known content is precisely weighed, 9 parts are parallelly added, a certain amount of reference substance mixed solution is precisely added respectively, ultrasonic extraction and filtration are respectively carried out according to the preparation method of a test sample solution, the mixture is injected into an ultra-high performance liquid chromatograph for measurement, the recovery rate is calculated, and the acteoside glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 100.2%, 99.3%, 100.8%, 100.4% and 101.1%, and the RSD values are respectively 1.49%, 1.13%, 1.40%, 1.46% and 0.684%, which shows that the method is accurate and reliable.
5. Sample assay
Taking a sample of the traditional Chinese medicine composition, respectively carrying out ultrasonic extraction, filtering, injecting into an ultra-high performance liquid chromatograph for determination, and recording a chromatogram according to the preparation method of the test solution; the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid were calculated at 240nm, and the results of the content measurements are shown in Table 6.
The result shows that the analysis method has the characteristics of strong specificity, high precision, accurate and stable result, good repeatability, high sensitivity and the like, and is suitable for the quality control of the traditional Chinese medicine composition.
Example 2: preparation of the pharmaceutical tablet of the invention
Prescription:
astragalus root 30 g talcum 7 g selfheal 10 g ligustrum japonicum 7 g
Litchi seed 10 g amber 1 g cinnamon 1.5g phellodendron bark 3g
The preparation method comprises the following steps:
(1) weighing the traditional Chinese medicinal materials according to the prescription amount, respectively cleaning and crushing;
(2) extracting radix astragali and cortex Phellodendri with 60% ethanol under reflux for 2 times, 9 times for the first time and 3 hr for the second time and 8 times for the second time for 1.5 hr, filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, and concentrating to obtain fluid extract with relative density of 1.20-1.25;
(3) extracting fructus Ligustri Lucidi with 7 times of 80% ethanol under reflux for 2 times (2 hr each time), filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, and concentrating to obtain fluid extract with relative density of 1.20-1.25;
(4) soaking the cinnamon in 6 times of water for 1.5 hours, extracting volatile oil for 5 hours, collecting the volatile oil in another container, filtering and collecting the water extract, and reserving residues for later use;
(5) taking the selfheal, the lychee seeds and the residues obtained in the step (4), adding 9 times of water, decocting twice, wherein the first time is 2 hours, the second time is 1.5 hours, filtering extracting solutions, combining, adding the aqueous solution obtained in the step (4), and concentrating into clear paste with the relative density of 1.20-1.25 for later use;
(6) pulverizing pulvis Talci and Succinum into 100 mesh fine powder;
(7) and making into tablet by conventional method.
The content determination method comprises the following steps:
A. preparation of a test solution: weighing about 1 g of the content of the traditional Chinese medicine composition, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 80% methanol, weighing, carrying out ultrasonic treatment for 30 minutes (power 250w, frequency 40kHz), cooling, weighing, supplementing the lost weight with 80% methanol, and shaking uniformly to obtain the traditional Chinese medicine composition.
B. Preparation of control solutions: weighing appropriate amount of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride, and rosmarinic acid reference substances, precisely weighing, and dissolving with methanol to obtain concentrations of 0.396 mg/ml-1、1.156mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing the reference stock solution to obtain the final product.
C. Chromatographic conditions of chromatographic column Waters Acquity UP L C
Shield RP18 （2.1×100 mm，1.7μm), column temperature: 40 ℃; mobile phase: acetonitrile (a) -0.1% phosphoric acid (B) in water, gradient elution (table 7), detection wavelength: 240 nm; flow rate: 0.2 ml/min-1(ii) a Sample introduction amount: 0.5μl。
The experimental results are as follows:
1. and (3) precision test: and respectively and precisely absorbing the working reference substance solution, carrying out continuous sample injection for 5 times, and measuring peak areas, wherein the results of 1.38%, 1.07%, 1.25%, 1.37% and 1.18% of calycosin glucoside, specnuezhenoside, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid RSD respectively show that the precision of the instrument is good.
2. And (3) stability test: precisely sucking the same test solution, respectively injecting samples for 0, 2, 4, 8, 12 and 24 hours to determine peak areas, wherein the RSDs of the peak areas of 5 compounds of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 0.87%, 1.17%, 1.42%, 1.12% and 1.53% within 24 hours of determination, which indicates that the test solution is basically stable within 24 hours.
3. And (3) repeatability test: the same batch of samples of the traditional Chinese medicine composition is treated by 9 parts according to the preparation method of the test solution under item 2.2, and the treated samples are injected into an ultra-high performance liquid chromatograph for determination, so that the RSD values of the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid in the samples are respectively 1.85%, 1.72%, 2.17%, 2.32% and 2.18%, and the method has good repeatability.
4. Recovery test
By adopting a sample adding and recovering test method, 0.5g of the same traditional Chinese medicine composition sample with known content is precisely weighed, 9 parts are parallelly added, a certain amount of reference substance mixed solution is precisely added respectively, ultrasonic extraction and filtration are respectively carried out according to the preparation method of a test sample solution, the mixture is injected into an ultra-high performance liquid chromatograph for measurement, the recovery rate is calculated, and the acteoside glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 101.0%, 99.7%, 100.3%, 99.1% and 100.1%, and the RSD values are respectively 1.40%, 1.21%, 1.28%, 1.51% and 0.97%, thus the method is accurate and reliable.
5. Sample assay
Taking a sample of the traditional Chinese medicine composition, respectively carrying out ultrasonic extraction, filtering, injecting into an ultra-high performance liquid chromatograph for determination, and recording a chromatogram according to the preparation method of the test solution; the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid were calculated at 240nm, and the results of the content measurements are shown in Table 8.
The result shows that the analysis method has the characteristics of strong specificity, high precision, accurate and stable result, good repeatability, high sensitivity and the like, and is suitable for the quality control of the traditional Chinese medicine composition.
Example 3: preparation of the inventive pharmaceutical capsules
Prescription:
astragalus root 150 parts talc 28 parts prunella spike 40 parts ligustrum japonicum 28 parts
40 parts of lychee seeds, 4 parts of amber, 6 parts of cinnamon and 15 parts of golden cypress.
The preparation method comprises the following steps: making into capsule by conventional method.
The content determination method comprises the following steps:
A. preparation of a test solution: weighing about 3g of the content of the traditional Chinese medicine composition, precisely weighing, placing in a conical flask with a plug, precisely adding 75ml of 50% methanol, weighing, ultrasonically treating for 60 minutes, cooling, weighing, supplementing the lost weight with 50% methanol, and shaking uniformly to obtain the traditional Chinese medicine composition.
B. Preparation of control solutions: weighing appropriate amount of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride, and rosmarinic acid reference substances, precisely weighing, and dissolving with methanol to obtain concentrations of 0.396 mg/ml-1、1.156mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing the reference stock solution to obtain the final product.
C. The chromatographic conditions are that the chromatographic column is Phenomenex UP L C Kinetex C18Specification 2.1 × 100mm, 2.6 μm, column temperature 35 deg.C, mobile phase methanol (A) -0.1% phosphoric acid (B) solution, gradient elution (Table 9), detection wavelength 240nm, flow rate 0.2 ml/min-1(ii) a Sample introduction amount: 3μl。
TABLE 9 gradient of mobile phase
The experimental results are as follows:
1. and (3) precision test: and respectively and precisely absorbing the working reference substance solution, carrying out continuous sample injection for 5 times, and measuring peak areas, wherein the results of 1.53%, 1.27%, 1.14%, 1.62% and 1.33% of calycosin glucoside, specnuezhenoside, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid RSD respectively show that the precision of the instrument is good.
2. And (3) stability test: precisely sucking the same test solution, respectively injecting samples for 0, 2, 4, 8, 12 and 24 hours to determine peak areas, wherein the RSDs of the peak areas of 5 compounds of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 1.72%, 1.32%, 1.69%, 1.58% and 1.13% within 24 hours of determination, which indicates that the test solution is basically stable within 24 hours.
3. And (3) repeatability test: the same batch of samples of the traditional Chinese medicine composition is treated by 9 parts according to the preparation method of the test solution under item 2.2, and the treated samples are injected into an ultra-high performance liquid chromatograph for determination, so that the RSD values of the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid in the samples are 1.72%, 2.31%, 2.18%, 2.51% and 2.10% respectively, which indicates that the method has good repeatability.
4. Recovery test
1.5g of the same traditional Chinese medicine composition sample with known content is precisely weighed by adopting a sample adding and recycling test method, 9 parts of the sample are parallel, a certain amount of reference substance mixed solution is precisely added respectively, ultrasonic extraction and filtration are respectively carried out according to the preparation method of a test sample solution, the sample solution is injected into an ultra-high performance liquid chromatograph for measurement, the recycling rate is calculated, and the acteoside glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 100.8%, 98.9%, 101.0%, 99.6% and 100.1%, and the RSD values are respectively 1.84%, 1.35%, 1.57%, 1.49% and 1.92%, thus the method is accurate and reliable.
5. Sample assay
Taking a sample of the traditional Chinese medicine composition, respectively carrying out ultrasonic extraction, filtering, injecting into an ultra-high performance liquid chromatograph for determination, and recording a chromatogram according to the preparation method of the test solution; the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid were calculated at 240nm, and the results of the content measurements are shown in Table 10.
The result shows that the analysis method has the characteristics of strong specificity, high precision, accurate and stable result, good repeatability, high sensitivity and the like, and is suitable for the quality control of the traditional Chinese medicine composition.
Example 4: preparation of the inventive medicinal pill
Prescription:
astragalus root 30 g talcum 28 g selfheal 10 g ligustrum japonicum 28 g
Litchi seed 10 g amber 4 g cinnamon 1.5g phellodendron bark 15 g
The preparation method comprises the following steps: making into pill by conventional method.
The content determination method comprises the following steps:
A. preparation of a test solution: weighing the content of the traditional Chinese medicine composition, precisely weighing about 0.5g of the content, placing the content in a conical flask with a plug, precisely adding 10ml of methanol, weighing, ultrasonically treating for 10 minutes, cooling, weighing, supplementing the weight loss with methanol, and shaking up to obtain the traditional Chinese medicine composition.
B. Preparation of control solutions: weighing appropriate amount of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride, and rosmarinic acid reference substances, precisely weighing, and dissolving with methanol to obtain concentrations of 0.396 mg/ml-1、1.156mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing the reference stock solution to obtain the final product.
C. Chromatographic conditions are as follows: chromatographic columnIs ACQUITY BEH C18 with specification of 2.1 × 100mm, 1.7 μm, column temperature of 45 deg.C, mobile phase of methanol (A) -0.2% phosphoric acid (B) solution, gradient elution (Table 11), detection wavelength of 240nm, flow rate of 0.2 ml/min-1(ii) a Sample introduction amount: 1μl。
The experimental results are as follows:
1. and (3) precision test: and respectively and precisely absorbing the working reference substance solution, carrying out continuous sample injection for 5 times, and measuring peak areas, wherein the results of 1.05%, 1.32%, 1.28%, 1.47% and 1.55% of calycosin glucoside, specnuezhenoside, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid RSD respectively show that the precision of the instrument is good.
2. And (3) stability test: precisely sucking the same test solution, respectively injecting samples for 0, 2, 4, 8, 12 and 24 hours to determine peak areas, wherein RSDs of the peak areas of 5 compounds of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 1.35%, 1.39%, 1.78%, 1.66% and 1.57% within 24 hours of determination, which indicates that the test solution is basically stable within 24 hours.
3. And (3) repeatability test: the same batch of samples of the traditional Chinese medicine composition is treated by 9 parts according to the preparation method of the test solution under item 2.2, and the treated samples are injected into an ultra-high performance liquid chromatograph for determination, so that the RSD values of the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid in the samples are respectively 1.75%, 1.81%, 2.03%, 2.58% and 2.24%, which indicates that the method has good repeatability.
4. Recovery test
By adopting a sample adding and recovering test method, 0.25g of the same traditional Chinese medicine composition sample with known content is precisely weighed, 9 parts are parallelly added, a certain amount of reference substance mixed solution is precisely added respectively, ultrasonic extraction and filtration are respectively carried out according to the preparation method of a test sample solution, the mixture is injected into an ultra-high performance liquid chromatograph for measurement, the recovery rate is calculated, and the acteoside glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid are respectively 99.8%, 99.9%, 101.3%, 98.6% and 100.0%, and the RSD values are respectively 1.56%, 1.29%, 1.36%, 1.44% and 1.85%, thus the method is accurate and reliable.
5. Sample assay
Taking a sample of the traditional Chinese medicine composition, respectively carrying out ultrasonic extraction, filtering, injecting into an ultra-high performance liquid chromatograph for determination, and recording a chromatogram according to the preparation method of the test solution; the contents of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid were calculated at 240nm, and the results of the content measurements are shown in Table 12.
The result shows that the analysis method has the characteristics of strong specificity, high precision, accurate and stable result, good repeatability, high sensitivity and the like, and is suitable for the quality control of the traditional Chinese medicine composition.
Claims (4)
1. A content determination method of a traditional Chinese medicine composition comprises the following raw material medicines, by weight, 70 parts of astragalus membranaceus, 14 parts of talc, 21 parts of selfheal, 14 parts of glossy privet fruit, 21 parts of lychee seed, 2.1 parts of amber, 2.8 parts of cinnamon and 7 parts of golden cypress, and the content determination method is to simultaneously determine the content of five components, namely calycosin glucoside, specnuezhenoside, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid in the traditional Chinese medicine composition by adopting an UP L C method, and is characterized by comprising the following steps:
A. weighing the content of the traditional Chinese medicine composition, grinding, taking 1 g, precisely weighing, placing in a conical flask with a plug, precisely adding 25 ml of 80% methanol, weighing, ultrasonically treating for 30 minutes, cooling, weighing, supplementing the lost weight with 80% methanol, and shaking uniformly to obtain the traditional Chinese medicine composition;
B. the preparation method of the reference substance solution comprises the following steps: weighing appropriate amount of calycosin glucoside, specnuezhenide, palmatine hydrochloride, berberine hydrochloride, and rosmarinic acid reference substances, precisely weighing, and dissolving in methanol to obtain mixed reference substance stock solution;
C. chromatographic conditions of chromatographic column Waters Acquity UP L C Shield RP18 with specification of 2.1 × 100mm and 1.7μm, column temperature: 40 ℃; the mobile phase A is acetonitrile, the mobile phase B is 0.2% formic acid water solution, and the gradient elution ratio is as follows: in 0-2 min, the content of the mobile phase A is 15-20%, and the content of the mobile phase B is 85-80%; 2-7 min, the content of the mobile phase A is from 20% to 30%, and the content of the mobile phase B is from 80% to 70%; in 7-10 min, the mobile phase A is 30-32%, and the mobile phase B is 70-68%; the mobile phase A is 32-35% in 10-12 min, and the mobile phase B is 68-65%; 35% of mobile phase A and 65% of mobile phase B in 12-14 min; detection wavelength: 240 nm; flow rate: 0.2 ml.min-1；
D. And (3) measuring, namely respectively sucking 1 mu L of the test sample solution and the control solution, injecting the test sample solution and the control solution into a UP L C liquid phase, and calculating the content of the sample according to the peak area.
2. The content determination method according to claim 1, wherein the active ingredients of the Chinese medicinal composition are prepared by the steps of:
(1) weighing the traditional Chinese medicinal materials according to the weight proportion of the raw materials, respectively cleaning and crushing;
(2) extracting radix astragali and cortex Phellodendri with 50-70% ethanol under reflux for 0.5-2 hr for 1-3 times, filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, and concentrating to obtain fluid extract with relative density of 1.20-1.25;
(3) extracting fructus Ligustri Lucidi with 6-10 times of 70-90% ethanol under reflux for 1-3 times (1-3 hr each time), filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, and concentrating to obtain fluid extract with relative density of 1.20-1.25;
(4) soaking cortex Cinnamomi in 4-8 times of water for 1-2 hr, extracting volatile oil for 2-6 hr,
collecting volatile oil in another container, filtering water extractive solution, and collecting residue;
(5) taking selfheal, lychee seeds and the residues obtained in the step (4), adding 8-10 times of water, decocting twice, wherein the first time is 1-3 hours, the second time is 1-2 hours, filtering the extracting solution, combining, adding the aqueous solution obtained in the step (4), and concentrating into clear paste with the relative density of 1.20-1.25 for later use;
(6) pulverizing pulvis Talci and Succinum into 100 mesh fine powder;
the clear paste obtained in the step (2), the clear paste obtained in the step (3), the volatile oil obtained in the step (4), the clear paste obtained in the step (5) and the fine powder obtained in the step (6) jointly form the active ingredients of the traditional Chinese medicine composition.
3. The content determination method according to claim 1, wherein the formulation of the Chinese medicinal composition is capsule, tablet, oral liquid or pill.
4. The assay method according to claim 3, wherein said capsule is comprised of the steps of:
(1) cleaning the above Chinese medicinal materials, pulverizing, and weighing at a certain proportion;
(2) extracting radix astragali and cortex Phellodendri with 6-12 times of 50-70% ethanol under reflux for 1-3 times, each time for 1-3 hr, filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, concentrating to obtain fluid extract with relative density of 1.20-1.25, vacuum drying at 60-70 deg.C under vacuum degree of 0.04-0.06Mpa in vacuum drying oven to obtain dry extract;
(3) extracting fructus Ligustri Lucidi with 6-10 times of 70-90% ethanol under reflux for 1-3 times (1-3 hr each time), filtering the extractive solutions, mixing, recovering ethanol under reduced pressure, concentrating to obtain fluid extract with relative density of 1.20-1.25, vacuum drying at 60-70 deg.C under vacuum degree of 0.04-0.06Mpa in a vacuum drying oven, and keeping the dry extract;
(4) soaking cortex Cinnamomi in 4-8 times of water for 1-2 hr, extracting volatile oil for 2-6 hr,
collecting volatile oil with other container, wherein the oil yield is not less than l.0%, filtering the water extractive solution, and collecting the residue;
(5) taking Prunellae Spica, semen litchi and residue of cortex Cinnamomi after oil extraction, adding 8-10 times of water, decocting twice, the first time for 1-3 hr and the second time for 1-2 hr, filtering extractive solutions, mixing, adding cortex Cinnamomi, and extracting
Concentrating the oily water solution into fluid extract with relative density of 1.20-1.25, vacuum drying at 60-70 deg.C under vacuum degree of 0.04-0.06Mpa, and pulverizing the dry extract, radix astragali, cortex Phellodendri, and fructus Ligustri Lucidi into 100 mesh powder;
(6) pulverizing pulvis Talci and Succinum into 100 mesh powder;
(7) mixing the dry extract powder, the crude powder and starch, granulating with 80% ethanol as binder under high speed stirring, oven drying at 60-70 deg.C, and grading;
(8) sieving to obtain fine powder, spraying volatile oil, mixing with the granules, sealing, and making into capsule.
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