CN114166965B - Detection method of traditional Chinese medicine composition for treating dizziness - Google Patents
Detection method of traditional Chinese medicine composition for treating dizziness Download PDFInfo
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- CN114166965B CN114166965B CN202111416185.0A CN202111416185A CN114166965B CN 114166965 B CN114166965 B CN 114166965B CN 202111416185 A CN202111416185 A CN 202111416185A CN 114166965 B CN114166965 B CN 114166965B
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Classifications
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- G01N30/90—Plate chromatography, e.g. thin layer or paper 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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a detection method of a traditional Chinese medicine composition for treating dizziness, which belongs to the technical field of quality detection of traditional Chinese medicine compositions, and comprises the steps of measuring the content of loganin and paeonol in the traditional Chinese medicine composition, detecting the content of loganin by adopting a high performance liquid chromatography method and qualitatively identifying gardenia in the traditional Chinese medicine composition by adopting thin layer chromatography identification. The invention innovates and optimizes chromatographic conditions and preparation methods of test products, adopts newly designed acetonitrile-0.3% phosphoric acid aqueous solution for gradient elution, uses dual wavelengths under the same chromatographic system, and simultaneously detects the contents of various components, thereby being simple, convenient, quick, easy to operate and good in repeatability. The gardenia qualitative identification adopts a newly designed extraction method and thin-layer chromatography conditions, is simpler and faster than the existing method, is easy to operate and has good repeatability, and the fluorescence spots are very clear after color development, and the negative effect is free from interference. The evaluation index of the invention can effectively control the quality of the intermediate product and the finished product of the preparation.
Description
Technical Field
The invention belongs to the technical field of quality detection of traditional Chinese medicine compositions, and particularly relates to a detection method of a traditional Chinese medicine composition for treating dizziness.
Background
Vertigo is the most common clinical syndrome, and the incidence rate of the vertigo is increased with aging of population, so that the vertigo is widely valued by medical community at home and abroad. Vertigo is reported as the third most common symptom in outpatient service. Vertigo is a chronic problem in clinical practice due to its complex etiology, long course of disease, recurrent attacks or more persistent.
The traditional Chinese medicine composition for treating dizziness, which is aimed at in the invention, is prepared by organically cutting on the basis of the experience of treating dizziness by the ancient prescription and the proved prescription. The traditional Chinese medicine composition is prepared from radix rehmanniae, tree peony bark, uncaria, poria cocos, chinese yam, dogwood, chinese angelica, mother of pearl, chrysanthemum, ligusticum wallichii, bitter apricot seed, wild jujube seed, earthworm, pinellia tuber (prepared), gardenia, liquorice, stiff silkworm, arisaema cum bile and the like, and the efficacies of the medicines are synergistic, so that dizziness can be effectively treated.
The invention has the advantages of obvious treatment effect, no obvious toxic or side effect, convenient oral administration, short treatment course, quick response and safe and reliable clinical use.
In clinical use for many years, the traditional Chinese medicine composition for treating dizziness has the effects of nourishing liver and kidney, clearing heat and resolving phlegm, is definite and stable in curative effect, safe, free of toxic and side effects, short in treatment course and quick in effect taking, and has obvious curative effects on dizziness of different symptoms. Can be used for treating dizziness, tinnitus, deafness, heart convulsion, insomnia, numbness of hand and face, hectic fever, night sweat, excessive phlegm, chest distress, etc.
However, the quality control method of the traditional Chinese medicine composition has a larger gap from the current quality control requirements of traditional Chinese medicine products, and the stability of the quality of the therapeutic preparation cannot be effectively controlled, so that the production of the products is influenced and the quality is ensured.
Disclosure of Invention
The invention aims to provide a detection method of a traditional Chinese medicine composition for treating dizziness, so as to solve the problems.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a detection method of a traditional Chinese medicine composition for treating dizziness comprises the steps of measuring the contents of loganin and paeonol in the traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises radix rehmanniae, cortex moutan, uncaria, poria cocos, chinese yam, dogwood, angelica sinensis, mother-of-pearl, chrysanthemum, ligusticum wallichii, bitter apricot seed, spine date seed, earthworm, pinellia tuber, gardenia, liquorice, stiff silkworm and arisaema cum bile, and the detection method comprises the following steps:
(1) Detecting the content of loganin by adopting a high performance liquid chromatography method, wherein the chromatographic conditions are as follows:
filler: octadecylsilane chemically bonded silica gel;
mobile phase: acetonitrile-0.3% phosphoric acid aqueous solution;
elution mode: gradient elution, the volume ratio of acetonitrile in the mobile phase during gradient elution changes with time to ：0.01-15min,8%-12%;15-20min,8%-12%→6%-10%;20-25min,6%-10%→16%-20%;25-27min,16%-20%→35%-39%;27-38min,35%-39%;38-43min,35%-39%→8%-12%;43-48min,8%-12%;
Column temperature: 36-44 ℃;
flow rate: 1.0ml/min;
Detection wavelength: loganin 240nm and paeonol 274nm;
Theoretical plate number: calculated according to the peaks of loganin and paeonol, the peak value is not less than 3000.
(2) The preparation method of the traditional Chinese medicine composition test sample for treating dizziness comprises the following steps:
Taking 3 parts by weight of a traditional Chinese medicine composition, precisely weighing, placing into a conical bottle with a plug, precisely adding 50 parts by volume of methanol, sealing, weighing, performing ultrasonic treatment for 60min, taking out, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a traditional Chinese medicine composition sample solution for treating dizziness;
(3) Preparing a reference substance:
Precisely weighing the loganin and paeonol reference substances, placing into a conical bottle with a plug, adding methanol to prepare a mixed solution containing the loganin 1.5354-30.7088 mug and the paeonol 2.5010-50.0093 mug per 1ml, and preparing a mixed reference substance solution of the loganin and the paeonol; simultaneously preparing a negative control solution without dogwood and moutan bark;
(4) Precisely sucking the sample solution, reference solution and negative reference solution without Corni fructus and cortex moutan, respectively, and injecting into liquid chromatograph for measurement;
every 9g of the traditional Chinese medicine composition for treating dizziness contains cornus officinalis calculated by loganin, and the content of the cornus officinalis is not less than 0.62mg; the content of cortex moutan calculated as paeonol is not less than 0.99mg.
To further realize the present invention, the volume ratio of acetonitrile in the mobile phase at the time of the gradient elution in the step (1) is changed with time to ：0.01-15min,10%;15-20min,10%→8%;20-25min,8%→18%;25-27min,18%→37%;27-38min,37%;38-43min,37%→10%;43-48min,10%.
In order to further carry out the present invention, the column temperature in step (1) is 40 ℃.
To further carry out the invention, the power of the ultrasonic treatment in step (2) is 600W and the frequency is 40kHz.
In order to further realize the invention, the detection method also comprises the qualitative identification of the gardenia in the traditional Chinese medicine composition by adopting thin layer chromatography identification, and comprises the following steps of:
(1) Preparing a traditional Chinese medicine composition for treating dizziness into a sample solution, and simultaneously preparing a gardenoside reference substance solution and a negative reference substance solution without gardenia;
(2) Respectively taking the three solutions obtained in the step (1) to be spotted on the same silica gel G thin layer plate;
(3) The method comprises the steps of taking ethyl acetate-acetone-formic acid-water solution as a developing agent, placing a thin layer plate in a developing box for developing, taking out, airing, spraying sulfuric acid ethanol solution, heating until spots develop clearly, and inspecting under sunlight, wherein spots with the same color appear on the chromatographic positions corresponding to the control in the chromatogram of the sample to be tested, and the negative control is free from interference.
In order to further realize the invention, the qualitative identification of the gardenia in the traditional Chinese medicine composition by adopting thin layer chromatography identification comprises the following steps:
(1) Taking 10-36 parts by weight of a traditional Chinese medicine composition for treating dizziness, crushing, placing in a container, adding 35-40 parts by volume of diethyl ether, shaking for 10-20min, discarding diethyl ether, volatilizing diethyl ether from residues, adding 35 parts by volume of ethyl acetate, heating and refluxing on a water bath for 1h, cooling, filtering, evaporating filtrate to dryness, and adding 1 part by volume of ethanol into residues to dissolve to obtain a sample solution;
Adding ethanol into the gardenoside reference substance to obtain solution containing 2mg per 1ml, and preparing into gardenoside reference substance solution;
Preparing a negative sample without gardenia according to the proportion of each medicinal material in the traditional Chinese medicine composition for treating dizziness and the preparation process, and preparing a negative reference solution without gardenia by the same method;
(2) Respectively taking 5 parts by volume of each of the three solutions obtained in the step (1), and spotting the three solutions on the same silica gel G thin layer plate;
(3) The method comprises the steps of taking a solution with the volume ratio of ethyl acetate to acetone to formic acid to water of 8-12:5.5-8.5:1-3:0.2-0.7 as a developing agent, placing a thin layer plate in a developing box for developing, developing a distance of 9-11cm, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 90-105 ℃ until spots develop clearly, inspecting under sunlight, and displaying spots with the same color on the corresponding chromatographic positions of a reference substance in a chromatographic manner of a sample to be tested, wherein negative reference is free from interference.
In order to further realize the invention, the qualitative identification of the gardenia in the traditional Chinese medicine composition by adopting thin layer chromatography identification comprises the following steps:
(1) Taking 18 parts by weight of a traditional Chinese medicine composition for treating dizziness, crushing, placing in a container, adding 35 parts by volume of diethyl ether, shaking for 10min, discarding diethyl ether, volatilizing diethyl ether from residues, adding 35 parts by volume of ethyl acetate, heating and refluxing on a water bath for 1h, cooling, filtering, evaporating filtrate, and adding 1 part by volume of ethanol into residues to dissolve to obtain a sample solution;
Adding ethanol into the gardenoside reference substance to obtain solution containing 2mg per 1ml, and preparing into gardenoside reference substance solution;
Preparing a negative sample without gardenia according to the proportion of each medicinal material in the traditional Chinese medicine composition for treating dizziness and the preparation process, and preparing a negative reference solution without gardenia by the same method;
(2) Respectively taking 5 parts by volume of each of the three solutions obtained in the step (1), and spotting the three solutions on the same silica gel G thin layer plate;
(3) The volume ratio of ethyl acetate-acetone-formic acid-water is 10:7:2:0.5 as developing agent, placing the thin layer plate in a developing box for developing at a distance of 10cm, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of the spot is clear, and inspecting in sunlight to obtain the color spot with the same color as the color spot of the reference substance in the color spectrum of the sample, wherein the negative reference substance has no interference.
Compared with the prior art, the invention has the beneficial effects that:
The content determination in the invention is innovative and optimized in terms of chromatographic conditions and preparation methods of test products, especially mobile phases in chromatographic conditions, and adopts a newly designed acetonitrile-0.3% phosphoric acid aqueous solution for gradient elution, and under the same chromatographic system, the content of various components is detected simultaneously by using double wavelengths, so that the sample processing times are reduced, the inspection time is shortened, the working efficiency and the product quality controllability are improved, the product quality is ensured, the use amount of an organic solvent is reduced, the content of loganin and paeonol can be simultaneously determined, the detection sensitivity is improved, and the method is simple, convenient, quick, easy to operate and good in repeatability.
The qualitative identification of the gardenia adopts a newly designed extraction method and thin-layer chromatography conditions, and compared with the identification method of the gardenia in Chinese pharmacopoeia and related literature methods, the method is simple, convenient, quick, easy to operate and good in repeatability, and the fluorescence spots are very clear after color development, and negative and no interference are generated.
According to basic research of pharmacodynamics substances of traditional Chinese medicine and the integral of the drug effect of the compound preparation and the characteristics of multi-target and multi-path functions, the invention establishes new quantitative and qualitative evaluation indexes, and can effectively control the quality of intermediate products and finished products of the preparation.
Drawings
FIG. 1 is a full-wave scan of the exterior of the horse Qian Ganzi according to the present invention;
FIG. 2 is a full-wavelength ultraviolet scan of paeonol in the present invention;
FIG. 3 is a chromatogram of a loganin control of the invention (240 nm wavelength);
FIG. 4 is a graph of a paeonol control (274 nm wavelength) according to the present invention;
FIG. 5 is a chromatogram (240 nm wavelength) of a test sample prepared in example 1 of the present invention;
FIG. 6 is a chromatogram (274 nm wavelength) of a test sample prepared in example 1 of the present invention;
FIG. 7 is a chromatogram (240 nm wavelength) of a negative sample in the present invention;
FIG. 8 is a chromatogram of a negative sample of the present invention (274 nm wavelength);
FIG. 9 is a linear diagram of loganin in the present invention;
FIG. 10 is a linear diagram of paeonol in the present invention;
FIG. 11 is a thin layer chromatogram of a negative sample, a gardenoside control, and three parallel runs of example 6.
Detailed Description
The invention is further described below with reference to the drawings and the detailed description.
1. Determination of the content of loganin and paeonol in a traditional Chinese medicine composition for treating dizziness:
Instrument: LC-20AB high performance liquid chromatograph (Shimadzu corporation); an ultrasonic multi-frequency cleaner (YQ 08-0007);
Reagent: acetonitrile is a chromatographic grade reagent; the water is ultrapure water; the other reagents were all analytically pure.
Control: loganin (purchased from chinese food and drug assay institute); paeonol (purchased from Chinese food and drug inspection institute) for content determination.
Test article: supplied by the company of foci pharmaceutical, inc.
Example 1:
(1) Detecting the content of loganin by adopting a high performance liquid chromatography, and testing chromatographic conditions and system adaptability:
filler: octadecylsilane chemically bonded silica gel;
mobile phase: acetonitrile-0.3% phosphoric acid aqueous solution;
elution mode: gradient elution, wherein the volume ratio of acetonitrile in the mobile phase changes with time:
Column temperature: 40 ℃;
flow rate: 1.0ml/min;
Detection wavelength: loganin 240nm (see FIG. 1) and paeonol 274nm (see FIG. 2);
theoretical plate number: calculating to be not less than 3000 according to the peaks of loganin and paeonol;
(2) The preparation method of the traditional Chinese medicine composition test sample for treating dizziness comprises the following steps:
Taking 3 parts by weight of a traditional Chinese medicine composition, precisely weighing, placing into a conical bottle with a plug, precisely adding 50 parts by volume of methanol, sealing, weighing, performing ultrasonic treatment for 60min, taking out, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a traditional Chinese medicine composition sample solution for treating dizziness;
(3) Preparing a reference substance:
Precisely weighing the loganin and paeonol reference substances, placing into a conical bottle with a plug, adding methanol to prepare a mixed solution containing the loganin 1.5354-30.7088 mug and the paeonol 2.5010-50.0093 mug per 1ml, and preparing a mixed reference substance solution of the loganin and the paeonol; simultaneously preparing a negative control solution without dogwood and moutan bark;
(4) Precisely sucking the sample solution, reference solution and negative reference solution without Corni fructus and cortex moutan, respectively, and injecting into liquid chromatograph for measurement;
every 9g of the traditional Chinese medicine composition for treating dizziness contains cornus officinalis calculated by loganin, and the content of the cornus officinalis is not less than 0.62mg; the content of cortex moutan calculated as paeonol is not less than 0.99mg.
And (3) identifying the loganin and paeonol content determination methodology:
And (3) carrying out methodological verification on the loganin and paeonol content measurement method according to the medicine quality standard analysis method verification guiding principle (the fourth edition of Chinese pharmacopoeia 2020, general rule 9101).
1. Specialization of
A negative sample without cortex moutan and Corni fructus was prepared by the preparation process of example 1, and detected according to the above chromatographic conditions, wherein the chromatogram of the test sample shows chromatographic peaks with the same retention time at the positions corresponding to the chromatogram of the control sample, and the negative control solution has no interference at the retention time. The chromatograms of the loganin and paeonol reference substances are shown in fig. 3 and 4, the chromatograms of the test substances prepared in example 1 are shown in fig. 5 and 6, and the chromatograms of the negative samples are shown in fig. 7 and 8.
The results show that: the determination method has stronger specificity.
2. Accuracy of
3.0G of the traditional Chinese medicine composition of the example 1 is precisely weighed by adopting a sample adding recovery test, 6 parts are precisely weighed, 1.4ml of a loganin reference substance solution (the concentration is 0.2258 mg/ml) and 4.2ml of a paeonol reference substance solution (the concentration is 0.2156 mg/ml) are precisely added respectively, the mixture is uniformly mixed, then the mixture is prepared according to the method of the example 1, the chromatographic condition in the content measuring method of the example 1 is measured, the recovery rate is calculated according to the following formula, the RSD is calculated, and the test results are shown in the tables 1 and 2.
Percent recovery of loganin = [ (amount of loganin measured-amount of loganin in sample)/amount of loganin added ×100%
Recovery of paeonol = [ (amount of paeonol measured-amount of paeonol in sample)/amount of paeonol added ] ×100%
Experimental results show that the method has good accuracy.
3. Repeatability test
The same batch of the Chinese medicinal composition was repeatedly measured for 6 times according to the preparation method and chromatographic conditions of the sample solution in example 1, and the test results are shown in Table 3.
The test results show that: the content determination method has good repeatability.
4. Linearity of
Taking appropriate amounts of loganin reference substance and paeonol reference substance, precisely weighing, adding methanol to prepare mixed reference substance solutions containing loganin 30.7088 mug and paeonol 50.0093 mug per lml, respectively taking 0.5, 2, 3, 4, 5 and 10ml of mixed mother solution, and fixing the volume to 10ml by using methanol to prepare mixed reference substance solutions with 6 concentration gradients. The samples were taken under the above chromatographic conditions, and the peak areas were measured, and the measurement results are shown in Table 4. Drawing a standard curve by taking the peak area integral value as an ordinate and the reference sample injection concentration as an abscissa, and calculating a regression equation as follows:
loganin y= 14881x-4487.1 correlation coefficient r=1
Paeonol y= 49343x-846.17 correlation coefficient r=1
Experimental results show that loganin has good linear relationship in the range of 1.5354-30.7088 mug/ml, and the linear graph is shown in figure 9. Paeonol has good linear relationship in 2.5010-50.0093 μg/ml range, and the linear graph is shown in figure 10.
5. Stability of
The traditional Chinese medicine composition in example 1 is taken, a sample solution is prepared, sample injection is respectively carried out at 0, 2, 4, 8, 12 and 24 hours, the content of loganin and paeonol is measured, and the results are shown in Table 5.
Test results show that the test sample solution prepared by the method has better stability.
6. Determination of content limits
In order to determine the limit of the contents of loganin and paeonol in the traditional Chinese medicine composition, ten batches of traditional Chinese medicine compositions were tested for the contents of loganin and paeonol, and the results are shown in Table 6.
The average content of loganin in ten batches of traditional Chinese medicine compositions is 172.2927 mug/g, and the average content of paeonol is 274.0789 mug/g. Taking into account the difference of the content of medicinal materials, the loss and destruction of the components possibly occurring in the whole preparation and storage processes, the difference of the content of the administered materials in batches during the production of enterprises and the error (calculated by 40%) in the operation process. The minimum content of loganin in each pill (9 g) of the composition is as follows: 172.2927 μg/g×9g×40% = 620.2537 μg=0.62 mg, the lowest content of paeonol is: 274.0789 μg/g×9g×40% = 986.6840 μg=0.99 mg. The specified content is: the content of fructus Corni per pill is not less than 0.62mg calculated by loganin (C 17H26O10); contains cortex moutan (calculated as paeonol (C 9H10O3)) not less than 0.99mg.
Example 2:
(1) Detecting the content of loganin by adopting a high performance liquid chromatography, and testing chromatographic conditions and system adaptability:
filler: octadecylsilane chemically bonded silica gel;
mobile phase: acetonitrile-0.3% phosphoric acid aqueous solution;
elution mode: gradient elution, wherein the volume ratio of acetonitrile in the mobile phase changes with time:
Column temperature: 36 ℃;
flow rate: 1.0ml/min;
Detection wavelength: loganin 240nm and paeonol 274nm;
theoretical plate number: calculating to be not less than 3000 according to the peaks of loganin and paeonol;
(2) The preparation method of the traditional Chinese medicine composition test sample for treating dizziness comprises the following steps:
Taking 3 parts by weight of a traditional Chinese medicine composition, precisely weighing, placing into a conical bottle with a plug, precisely adding 50 parts by volume of methanol, sealing, weighing, performing ultrasonic treatment for 60min, taking out, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a traditional Chinese medicine composition sample solution for treating dizziness;
(3) Preparing a reference substance:
Taking a loganin and paeonol reference substance, precisely weighing, placing into a conical flask with a plug, adding methanol to prepare a mixed solution containing the loganin 6.1418 mug and the paeonol 10.0038 mug per 1ml, and preparing a loganin and paeonol mixed reference substance solution; simultaneously preparing a negative control solution without dogwood and moutan bark;
(4) Precisely sucking the sample solution, reference solution and negative reference solution without Corni fructus and cortex moutan, respectively, and injecting into liquid chromatograph for measurement;
every 9g of the traditional Chinese medicine composition for treating dizziness contains cornus officinalis calculated by loganin, and the content of the cornus officinalis is not less than 0.62mg; the content of cortex moutan calculated as paeonol is not less than 0.99mg.
Example 3:
(1) Detecting the content of loganin by adopting a high performance liquid chromatography, and testing chromatographic conditions and system adaptability:
filler: octadecylsilane chemically bonded silica gel;
mobile phase: acetonitrile-0.3% phosphoric acid aqueous solution;
elution mode: gradient elution, wherein the volume ratio of acetonitrile in the mobile phase changes with time:
Column temperature: 44 ℃;
flow rate: 1.0ml/min;
Detection wavelength: loganin 240nm and paeonol 274nm;
theoretical plate number: calculating to be not less than 3000 according to the peaks of loganin and paeonol;
(2) The preparation method of the traditional Chinese medicine composition test sample for treating dizziness comprises the following steps:
Taking 3 parts by weight of a traditional Chinese medicine composition, precisely weighing, placing into a conical bottle with a plug, precisely adding 50 parts by volume of methanol, sealing, weighing, performing ultrasonic treatment for 60min, taking out, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a traditional Chinese medicine composition sample solution for treating dizziness;
(3) Preparing a reference substance:
Taking a loganin and paeonol reference substance, precisely weighing, placing into a conical flask with a plug, adding methanol to prepare a mixed solution containing the loganin 6.1418 mug and the paeonol 10.0038 mug per 1ml, and preparing a loganin and paeonol mixed reference substance solution; simultaneously preparing a negative control solution without dogwood and moutan bark;
(4) Precisely sucking the sample solution, reference solution and negative reference solution without Corni fructus and cortex moutan, respectively, and injecting into liquid chromatograph for measurement;
every 9g of the traditional Chinese medicine composition for treating dizziness contains cornus officinalis calculated by loganin, and the content of the cornus officinalis is not less than 0.62mg; the content of cortex moutan calculated as paeonol is not less than 0.99mg.
2. Qualitative identification of gardenia in a traditional Chinese medicine composition for treating dizziness:
example 4:
(1) Taking 10g of a traditional Chinese medicine composition for treating dizziness, pulverizing, placing into a container, adding 35ml of diethyl ether, shaking for 10min, removing diethyl ether, volatilizing diethyl ether from residues, adding 35ml of ethyl acetate, heating and refluxing on a water bath for 1h, cooling, filtering, evaporating filtrate to dryness, and adding 1ml of ethanol into residues to dissolve to obtain a sample solution;
Adding ethanol into the gardenoside reference substance to obtain solution containing 2mg per 1ml, and preparing into gardenoside reference substance solution;
Preparing a negative sample without gardenia according to the proportion of each medicinal material in the traditional Chinese medicine composition for treating dizziness and the preparation process, and preparing a negative reference solution without gardenia by the same method;
(2) Respectively taking 5ml of each of the three solutions obtained in the step (1), and spotting the three solutions on the same silica gel G thin layer plate;
(3) The method comprises the steps of taking a solution with the volume ratio of ethyl acetate to acetone to formic acid to water being 8:5.5:1:0.2 as a developing agent, placing a thin layer plate in a developing box for developing, developing the thin layer plate by a distance of 9cm, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 90 ℃ until spots develop clearly, viewing under sunlight, and displaying spots with the same color on the corresponding chromatographic positions of a reference substance in the chromatogram of a sample to be tested, wherein negative control is undisturbed.
Example 5:
(1) Taking 36g of a traditional Chinese medicine composition for treating dizziness, pulverizing, placing into a container, adding 40ml of diethyl ether, shaking for 20min, removing diethyl ether, volatilizing diethyl ether from residues, adding 35ml of ethyl acetate, heating and refluxing on a water bath for 1h, cooling, filtering, evaporating filtrate to dryness, and adding 1ml of ethanol into residues to dissolve to obtain a sample solution;
Adding ethanol into the gardenoside reference substance to obtain solution containing 2mg per 1ml, and preparing into gardenoside reference substance solution;
Preparing a negative sample without gardenia according to the proportion of each medicinal material in the traditional Chinese medicine composition for treating dizziness and the preparation process, and preparing a negative reference solution without gardenia by the same method;
(2) Respectively taking 5ml of each of the three solutions obtained in the step (1), and spotting the three solutions on the same silica gel G thin layer plate;
(3) The method comprises the steps of taking a solution of ethyl acetate, acetone, formic acid and water with the volume ratio of 12:8.5:3:0.7 as a developing agent, placing a thin layer plate in a developing box for developing, taking out the thin layer plate with the developing distance of 11cm, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots develop clearly, viewing under sunlight, and displaying spots with the same color on the corresponding chromatographic positions of a reference substance in the chromatogram of a sample to be tested, wherein negative control is undisturbed.
Example 6:
(1) Taking 18g of a traditional Chinese medicine composition for treating dizziness, pulverizing, placing in a container, adding 35ml of diethyl ether, shaking for 10min, removing diethyl ether, volatilizing diethyl ether from residues, adding 35ml of ethyl acetate, heating and refluxing on a water bath for 1h, cooling, filtering, evaporating filtrate to dryness, and adding 1ml of ethanol into residues to dissolve to obtain a sample solution;
Adding ethanol into the gardenoside reference substance to obtain solution containing 2mg per 1ml, and preparing into gardenoside reference substance solution;
Preparing a negative sample without gardenia according to the proportion of each medicinal material in the traditional Chinese medicine composition for treating dizziness and the preparation process, and preparing a negative reference solution without gardenia by the same method;
(2) Respectively taking 5ml of each of the three solutions obtained in the step (1), and spotting the three solutions on the same silica gel G thin layer plate;
(3) The volume ratio of ethyl acetate-acetone-formic acid-water is 10:7:2:0.5 as developing agent, placing the thin layer plate in a developing box for developing at a distance of 10cm, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of the spot is clear, and inspecting in sunlight to obtain the color spot with the same color as the color spot of the reference substance in the color spectrum of the sample, wherein the negative reference substance has no interference.
The thin layer chromatograms of examples 4-6 are shown in FIG. 11.
Claims (6)
1. A detection method of a traditional Chinese medicine composition for treating dizziness comprises radix rehmanniae, cortex moutan, ramulus Uncariae cum Uncis, poria, rhizoma Dioscoreae, corni fructus, radix Angelicae sinensis, concha Margaritifera, flos Chrysanthemi, rhizoma Ligustici Chuanxiong, semen Armeniacae amarum, semen Ziziphi Spinosae, lumbricus, rhizoma Pinelliae, fructus Gardeniae, glycyrrhrizae radix, bombyx Batryticatus and Arisaema cum bile, and is characterized by comprising the following steps:
(1) Detecting the content of loganin by adopting a high performance liquid chromatography method, wherein the chromatographic conditions are as follows:
filler: octadecylsilane chemically bonded silica gel;
mobile phase: acetonitrile-0.3% phosphoric acid aqueous solution;
elution mode: gradient elution, the volume ratio of acetonitrile in the mobile phase during gradient elution changes with time to ：0.01-15min,8%-12%;15-20min,8%-12%→6%-10%;20-25min,6%-10%→16%-20%;25-27min,16%-20%→35%-39%;27-38min,35%-39%;38-43min,35%-39%→8%-12%;43-48min,8%-12%;
Column temperature: 36-44 ℃;
flow rate: 1.0ml/min;
Detection wavelength: loganin 240nm and paeonol 274nm;
theoretical plate number: calculating to be not less than 3000 according to the peaks of loganin and paeonol;
(2) The preparation method of the traditional Chinese medicine composition test sample for treating dizziness comprises the following steps:
Taking 3 parts by weight of a traditional Chinese medicine composition, precisely weighing, placing into a conical bottle with a plug, precisely adding 50 parts by volume of methanol, sealing, weighing, performing ultrasonic treatment for 60min, taking out, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a traditional Chinese medicine composition sample solution for treating dizziness;
(3) Preparing a reference substance:
Precisely weighing the loganin and paeonol reference substances, placing into a conical bottle with a plug, adding methanol to prepare a mixed solution containing the loganin 1.5354-30.7088 mug and the paeonol 2.5010-50.0093 mug per 1ml, and preparing a mixed reference substance solution of the loganin and the paeonol; simultaneously preparing a negative control solution without dogwood and moutan bark;
(4) Precisely sucking the sample solution, reference solution and negative reference solution without Corni fructus and cortex moutan, respectively, and injecting into liquid chromatograph for measurement;
every 9g of the traditional Chinese medicine composition for treating dizziness contains cornus officinalis calculated by loganin, and the content of the cornus officinalis is not less than 0.62mg; contains cortex moutan (calculated as paeonol) not less than 0.99mg;
the detection method also comprises the qualitative identification of the gardenia in the traditional Chinese medicine composition by adopting thin layer chromatography identification, and comprises the following steps of:
(1) Preparing a traditional Chinese medicine composition for treating dizziness into a sample solution, and simultaneously preparing a gardenoside reference substance solution and a negative reference substance solution without gardenia;
(2) Respectively taking the three solutions obtained in the step (1) to be spotted on the same silica gel G thin layer plate;
(3) The method comprises the steps of taking ethyl acetate-acetone-formic acid-water solution as a developing agent, placing a thin layer plate in a developing box for developing, taking out, airing, spraying sulfuric acid ethanol solution, heating until spots develop clearly, and inspecting under sunlight, wherein spots with the same color appear on the chromatographic positions corresponding to the control in the chromatogram of the sample to be tested, and the negative control is free from interference.
2. The method for detecting a traditional Chinese medicine composition for treating dizziness according to claim 1, wherein: the volume ratio of acetonitrile in the mobile phase at the time of the gradient elution in the step (1) of the determination of the contents of loganin and paeonol changes with time to ：0.01-15min,10%;15-20min,10%→8%;20-25min,8%→18%;25-27min,18%→37%;27-38min,37%;38-43min,37%→10%;43-48min,10%.
3. The method for detecting a traditional Chinese medicine composition for treating dizziness according to claim 2, wherein: the column temperature in the step (1) of the content determination of the loganin and the paeonol is 40 ℃.
4. The method for detecting a traditional Chinese medicine composition for treating dizziness according to claim 3, wherein: the power of the ultrasonic treatment in the step (2) of the content measurement of the loganin and the paeonol is 600W, and the frequency is 40kHz.
5. The method for detecting a traditional Chinese medicine composition for treating dizziness according to claim 4, wherein the method comprises the following steps: the qualitative identification of the gardenia in the traditional Chinese medicine composition by adopting thin-layer chromatography identification comprises the following steps:
(1) Taking 10-36 parts by weight of a traditional Chinese medicine composition for treating dizziness, crushing, placing in a container, adding 35-40 parts by volume of diethyl ether, shaking for 10-20min, discarding diethyl ether, volatilizing diethyl ether from residues, adding 35 parts by volume of ethyl acetate, heating and refluxing on a water bath for 1h, cooling, filtering, evaporating filtrate to dryness, and adding 1 part by volume of ethanol into residues to dissolve to obtain a sample solution;
Adding ethanol into the gardenoside reference substance to obtain solution containing 2mg per 1ml, and preparing into gardenoside reference substance solution;
Preparing a negative sample without gardenia according to the proportion of each medicinal material in the traditional Chinese medicine composition for treating dizziness and the preparation process, and preparing a negative reference solution without gardenia by the same method;
(2) Respectively taking 5 parts by volume of each of the three solutions obtained in the step (1), and spotting the three solutions on the same silica gel G thin layer plate;
(3) The method comprises the steps of taking a solution with the volume ratio of ethyl acetate to acetone to formic acid to water of 8-12:5.5-8.5:1-3:0.2-0.7 as a developing agent, placing a thin layer plate in a developing box for developing, developing a distance of 9-11cm, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 90-105 ℃ until spots develop clearly, inspecting under sunlight, and displaying spots with the same color on the corresponding chromatographic positions of a reference substance in a chromatographic manner of a sample to be tested, wherein negative reference is free from interference.
6. The method for detecting a traditional Chinese medicine composition for treating dizziness according to claim 5, wherein the method comprises the following steps: the qualitative identification of the gardenia in the traditional Chinese medicine composition by adopting thin-layer chromatography identification comprises the following steps:
(1) Taking 18 parts by weight of a traditional Chinese medicine composition for treating dizziness, crushing, placing in a container, adding 35 parts by volume of diethyl ether, shaking for 10min, discarding diethyl ether, volatilizing diethyl ether from residues, adding 35 parts by volume of ethyl acetate, heating and refluxing on a water bath for 1h, cooling, filtering, evaporating filtrate, and adding 1 part by volume of ethanol into residues to dissolve to obtain a sample solution;
Adding ethanol into the gardenoside reference substance to obtain solution containing 2mg per 1ml, and preparing into gardenoside reference substance solution;
Preparing a negative sample without gardenia according to the proportion of each medicinal material in the traditional Chinese medicine composition for treating dizziness and the preparation process, and preparing a negative reference solution without gardenia by the same method;
(2) Respectively taking 5 parts by volume of each of the three solutions obtained in the step (1), and spotting the three solutions on the same silica gel G thin layer plate;
(3) The volume ratio of ethyl acetate-acetone-formic acid-water is 10:7:2:0.5 as developing agent, placing the thin layer plate in a developing box for developing at a distance of 10cm, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of the spot is clear, and inspecting in sunlight to obtain the color spot with the same color as the color spot of the reference substance in the color spectrum of the sample, wherein the negative reference substance has no interference.
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