CN110857938B - Method for simultaneously identifying lucid ganoderma and vine of multiflower knotweed in traditional Chinese medicine composition - Google Patents
Method for simultaneously identifying lucid ganoderma and vine of multiflower knotweed in traditional Chinese medicine composition Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine analysis, and discloses a method for simultaneously identifying lucid ganoderma and vine of multiflower knotweed in a traditional Chinese medicine composition. Silica gel thin layer plates with different specifications are adopted, and the detection results are basically the same, which shows that the method has good reproducibility; in the chromatogram of each test sample, spots with the same color are displayed at the positions corresponding to the chromatograms of the positive reference substances, and no interference spots exist in the chromatograms of the negative reference substances, which shows that the method has stronger specificity. Meanwhile, the method simplifies the operation process, reduces the time required by identification, improves the working efficiency and reduces the detection cost.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for simultaneously identifying lucid ganoderma and vine of multiflower knotweed in a traditional Chinese medicine composition.
Background
Traditional Chinese medicines (namely 'traditional Chinese medicine' or 'traditional Chinese medicine') are specific medicines of the traditional Chinese medicine in China, have thousands of years of application history, and play an important role in maintaining the proliferation of Chinese nationalities and the health of the public. Traditional Chinese medicines are usually composed of a plurality of different types of components, and each type of component may contain a few, a dozen, dozens or even more chemical components, so that in order to ensure the safety and effectiveness of clinical administration of traditional Chinese medicines and ensure the quality of products, detection and analysis of various components are necessary.
The lucid ganoderma and the vine of multiflower knotweed are common components in the traditional Chinese medicine composition, and the detection and analysis methods of the lucid ganoderma and the vine of multiflower knotweed in the prior art are various, for example, the Chinese journal literature, "quality control method research of allergic health granules", discloses a thin-layer chromatography identification method of caulis polygoni multiflori and ganoderma lucidum, considering that the components and property differences of the two medicinal materials are large and the identification is difficult under the same condition, the method has different pretreatment modes for the caulis polygoni multiflori and ganoderma lucidum, the finally selected developing solvent systems are different, the former adopts a benzene-ethanol system as the developing solvent, and the latter adopts a petroleum ether (60-90 ℃) ethyl formate-formic acid system as the developing solvent, so the operation is very inconvenient, especially when large-scale detection is needed, the workload is very large, and the working efficiency is low, so that a method for simultaneously detecting the lucid ganoderma and the vine of multiflower knotweed in the traditional Chinese medicine composition needs to be researched.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the problems of large detection workload and high cost caused by different detection methods of caulis polygoni multiflori and ganoderma lucidum in the traditional Chinese medicine composition, thereby providing a method for simultaneously identifying ganoderma lucidum and caulis polygoni multiflori in the traditional Chinese medicine composition.
In order to solve the technical problems, the invention provides a method for simultaneously identifying lucid ganoderma and tuber fleeceflower stem in a traditional Chinese medicine composition, which comprises the following steps:
(1) preparation of positive control solution and test solution
Respectively adding a first solvent into a ganoderma lucidum positive control product and a vine of multiflower knotweed positive control product, filtering, evaporating filtrate to dryness, and adding a second solvent to respectively serve as respective positive control medicinal material solutions;
adding the first solvent into the Chinese medicinal composition, filtering, evaporating the filtrate, and adding the second solvent to obtain a sample solution;
(2) testing by thin layer chromatography
Sucking the positive control solution and the sample solution, respectively dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent, taking out, air drying, inspecting under ultraviolet lamp and/or spraying with sulfuric acid ethanol solution, and drying until the spots are clearly developed.
Further, the positive reference substance of the lucid ganoderma is selected from at least one of lucid ganoderma reference medicinal materials or lucid ganoderma extracts; the positive control of caulis Polygoni Multiflori is selected from at least one of caulis Polygoni Multiflori control medicinal materials or caulis Polygoni Multiflori extract.
Further, the first solvent in the step (1) is one or more of methanol, ethanol and propanol; the second solvent is one or more of methanol, ethanol and propanol.
Further, the volume ratio of the developing solvent in the step (2) is VCyclohexane∶VEthyl acetate∶VFormic acid＝5∶5∶0.2。
Further, the method comprises the steps of:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Adding ethanol with the volume 10-20 times of the mass of a lucid ganoderma reference medicinal material into the lucid ganoderma reference medicinal material, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 0.5-1.5 times of the mass of the lucid ganoderma reference medicinal material into the filtrate to obtain a lucid ganoderma reference medicinal material solution;
taking a caulis polygoni multiflori reference medicinal material, adding 100-120 times of ethanol by mass of the caulis polygoni multiflori reference medicinal material, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, and adding 2-6 times of ethanol by mass of the caulis polygoni multiflori reference medicinal material to obtain a caulis polygoni multiflori reference medicinal material solution;
adding ethanol with the mass being 20-40 times of the mass of the ganoderma lucidum extract into the ganoderma lucidum extract, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the mass being 0.5-1.5 times of the mass of the ganoderma lucidum extract to obtain a ganoderma lucidum extract solution;
taking a vine of multiflower knotweed extract, adding ethanol with the mass being 20-40 times of the mass of the vine of multiflower knotweed extract, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, and adding ethanol with the mass being 0.5-1.5 times of the mass of the vine of multiflower knotweed extract to obtain a vine of multiflower knotweed extract solution;
adding ethanol with the volume being 4-8 times of the mass of a negative control product without the ganoderma lucidum extract into the negative control product without the ganoderma lucidum extract, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume being 0.1-0.3 times of the mass of the negative control product without the ganoderma lucidum extract into the filtrate to serve as a negative control product solution without the ganoderma lucidum extract;
taking a negative control product without the vine of multiflower knotweed extract, adding ethanol with the volume 4-8 times of the mass of the negative control product, performing ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, and adding ethanol with the volume 0.1-0.3 times of the mass of the negative control product without the vine of multiflower knotweed extract to serve as a negative control solution without the vine of multiflower knotweed extract;
adding ethanol with the mass 4-8 times of the volume of the traditional Chinese medicine composition into the traditional Chinese medicine composition, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the mass 0.1-0.3 times of the volume of the traditional Chinese medicine composition to serve as a test solution;
the relationship between the mass and the volume is g/ml;
(2) testing by thin layer chromatography
Sucking the control medicinal material solution, the extract solution, the negative control solution and the sample solution respectively 4-6 microliters, respectively dropping on the same silica gel thin-layer plate, taking cyclohexane-ethyl acetate-formic acid with the volume ratio of 5: 0.2 as a developing agent, developing, taking out, airing, placing under an ultraviolet lamp of 366nm, and inspecting and/or spraying 10% sulfuric acid ethanol solution to dry until spots are clearly developed.
Further, the method comprises the steps of:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Adding ethanol 15 times the mass of the reference medicinal material into the reference medicinal material, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol 0.5 times the mass of the reference medicinal material to obtain a reference medicinal material solution;
taking a caulis polygoni multiflori reference medicinal material, adding ethanol with the volume 120 times of the mass of the caulis polygoni multiflori reference medicinal material, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 4 times of the mass of the caulis polygoni multiflori reference medicinal material to obtain a caulis polygoni multiflori reference medicinal material solution;
adding ethanol with the volume 30 times of the mass of the ganoderma lucidum extract into the ganoderma lucidum extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume of the mass of the ganoderma lucidum extract to obtain a ganoderma lucidum extract solution;
taking a vine of multiflower knotweed extract, adding ethanol with the volume 30 times of the mass of the vine of multiflower knotweed extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume equal to the mass of the vine of multiflower knotweed extract to obtain a vine of multiflower knotweed extract solution;
adding ethanol with the volume 6 times that of the negative control product without the lucid ganoderma extract into the negative control product without the lucid ganoderma extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 0.2 times that of the negative control product without the lucid ganoderma extract into the filtrate to be used as a negative control product solution without the lucid ganoderma extract;
taking a negative control product without the vine of multiflower knotweed extract, adding ethanol with the volume 6 times of the mass of the negative control product, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 0.2 times of the mass of the negative control product without the vine of multiflower knotweed extract to serve as a vine negative control product solution without the vine of multiflower knotweed extract;
adding ethanol 6 times the mass of the traditional Chinese medicine composition into the traditional Chinese medicine composition, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol 0.2 times the mass of the traditional Chinese medicine composition into the filtrate to obtain a test solution;
the relationship between the mass and the volume is g/ml;
(2) testing by thin layer chromatography
Sucking the control medicinal material solution, the extract solution, the negative control solution and the sample solution 5 microliter respectively, dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent at volume ratio of 5: 0.2, taking out, air drying, placing under 366nm ultraviolet lamp, inspecting and/or spraying 10% sulphuric acid ethanol solution, and drying until the spots are clear in color.
Further, the method comprises the steps of:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Taking 2g of lucid ganoderma reference medicinal material, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain lucid ganoderma reference medicinal material solution;
taking 0.25g of caulis polygoni multiflori as a reference medicinal material, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a caulis polygoni multiflori reference medicinal material solution;
taking 1g of ganoderma lucidum extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain ganoderma lucidum extract solution;
taking 1g of caulis polygoni multiflori extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a caulis polygoni multiflori extract solution;
taking 5g of a negative control product without the ganoderma lucidum extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a negative control product solution without the ganoderma lucidum extract;
taking 5g of a negative control product without the vine of multiflower knotweed extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a negative control product solution without the vine of multiflower knotweed extract;
taking 5g of the traditional Chinese medicine composition, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a test solution;
(2) testing by thin layer chromatography
Sucking the control medicinal material solution, the extract solution, the negative control solution and the sample solution 5 microliter respectively, dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent at volume ratio of 5: 0.2, taking out, air drying, placing under 366nm ultraviolet lamp, inspecting and/or spraying 10% sulphuric acid ethanol solution, and drying until the spots are clear in color.
In the invention, the raw materials of the traditional Chinese medicine composition comprise a lucid ganoderma extract and a vine of multiflower knotweed extract.
The technical scheme of the invention has the following advantages:
1. the method for simultaneously identifying the lucid ganoderma and the vine of multiflower knotweed in the traditional Chinese medicine composition adopts the same pretreatment method for the lucid ganoderma and the vine of multiflower knotweed in the traditional Chinese medicine composition, and selects a specific developing agent system, so that various components in the lucid ganoderma and the vine of multiflower knotweed can be well separated, and the simultaneous identification of the two medicinal materials is realized. Silica gel thin layer plates with different specifications are adopted, and the detection results are basically the same, which shows that the method has good reproducibility; in the chromatogram of each test sample, spots with the same color are displayed at the positions corresponding to the chromatograms of the positive reference substances, and no interference spots exist in the chromatograms of the negative reference substances, which shows that the method has stronger specificity. Meanwhile, the method simplifies the operation process, reduces the time required by identification, improves the working efficiency and reduces the detection cost.
2. The method for simultaneously identifying the lucid ganoderma and the vine of multiflower knotweed in the traditional Chinese medicine composition provides a new idea for identifying each component of the traditional Chinese medicine composition by thin-layer chromatography, and is beneficial to improving the quality control standard of the traditional Chinese medicine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a thin-layer chromatogram for simultaneous identification of Ganoderma lucidum and caulis Polygoni Multiflori in example 1 of the present invention;
FIG. 2 is a thin-layer chromatogram for simultaneous identification of Ganoderma lucidum and caulis Polygoni Multiflori in example 2 of the present invention;
FIG. 3 is a thin-layer chromatogram of simultaneous identification of Ganoderma lucidum and caulis Polygoni Multiflori in example 3 of the present invention.
Description of reference numerals:
1-negative control containing no caulis Polygoni Multiflori extract; 2-caulis polygoni multiflori extract; 3-caulis polygoni multiflori contrast medicinal materials; 4-negative control containing no Ganoderma extract; 5-ganoderma lucidum extract; 6-ganoderma lucidum reference medicinal material; 7-9 test samples (batch numbers: 20170204-1, 20170204-2, and 20170204-3, respectively).
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In the following examples, the Chinese medicinal composition is provided by Perfect (Chinese) Inc.; negative control containing no caulis Polygoni Multiflori extract and negative control containing no Ganoderma extract are provided by Perfect (Chinese) Inc.; ganoderma extract and caulis Polygoni Multiflori extract are provided by Guangdong pharmaceutical Co., Ltd; (ii) a The Ganoderma reference medicinal material and caulis Polygoni Multiflori reference medicinal material are purchased from China food and drug testing research institute.
In the following examples, methanol, ethanol and propanol were all analytically pure and had a volume concentration of 95% or more.
Example 1
Taking the traditional Chinese medicine composition, a negative control product without a vine of multiflower knotweed extract, 5g of each negative control product without lucid ganoderma, 1g of lucid ganoderma extract and 1g of vine of multiflower knotweed extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate in a water bath to dryness, and adding 1mL of ethanol for dissolving to respectively serve as a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 on the general rule of 2015 edition of pharmacopoeia of the people's republic of China, 5 mu L of each solution is absorbed, the solution is respectively spotted on the same silica gel G thin-layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), cyclohexane-ethyl acetate-formic acid (the volume ratio is 5: 0.2) is used as a developing agent, the developing solution is taken out, dried in the air, sprayed with 10% sulfuric acid ethanol test solution, and dried at 105 ℃ until the color development is clear. The results are shown in FIG. 1, the chromatogram of the test sample and the extract shows the main spots with the same color at the same position as the chromatogram of the control material, and the chromatogram of the negative control sample does not show the main spots with the same color at the same position as the chromatogram of the control material.
Example 2
Taking the traditional Chinese medicine composition, a negative control product without a vine of multiflower knotweed extract, 5g of each negative control product without lucid ganoderma, 1g of lucid ganoderma extract and 1g of vine of multiflower knotweed extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate in a water bath to dryness, and adding 1mL of ethanol for dissolving to obtain a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015, 5 mu L of each solution is absorbed, respectively dropped on the same silica gel G thin layer plate (20cm x10cm, MACHEREY-NAGEL company, Germany), and the cyclohexane-ethyl acetate-formic acid (volume ratio is 5: 0.2) is taken as a developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, and dried at 105 ℃ until the color development is clear. The results are shown in FIG. 2, the chromatogram of the test solution and the extract solution showed the main spots of the same color at the same position as the chromatogram of the control solution, and the chromatogram of the negative control solution showed no main spots of the same color at the same position as the chromatogram of the control solution.
Example 3
Taking the traditional Chinese medicine composition, the negative control product without the caulis polygoni multiflori extract, the negative control product without the ganoderma lucidum, 5g of each of the ganoderma lucidum extract, 1g of the ganoderma lucidum extract and 1g of the caulis polygoni multiflori extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate in a water bath to dryness, and adding 1mL of ethanol for dissolving to obtain a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015, 5 mu L of each solution is absorbed, respectively dropped on the same silica gel G thin layer plate (20cm x10cm, Germany Merck company), cyclohexane-ethyl acetate-formic acid (the volume ratio is 5: 0.2) is used as a developing agent, developed, taken out, dried in the air, sprayed with 10% sulfuric acid ethanol test solution, and dried at 105 ℃ until the color is clear. The results are shown in fig. 3, the chromatogram of the test sample and the extract showed the main spots with the same color at the same position as the control material, and the chromatogram of the negative control sample showed no main spots with the same color at the same position as the control material.
Example 4
Taking the traditional Chinese medicine composition, a negative control product without a vine of multiflower knotweed extract, 5g of each negative control product without lucid ganoderma, 1g of lucid ganoderma extract and 1g of vine of multiflower knotweed extract, adding 20mL of methanol, carrying out ultrasonic treatment for 40 minutes, filtering, evaporating filtrate in a water bath to dryness, and adding 1.5mL of propanol for dissolving, wherein the filtrate is respectively used as a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015 edition, the above solutions are respectively absorbed by 4 mu L, respectively dropped on the same silica gel G thin layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), and cyclohexane-ethyl acetate-formic acid (volume ratio is 5: 0.2) is used as a developing agent, developed, taken out, dried and inspected under an ultraviolet lamp of 366 nm. The result shows that the chromatogram of the test sample and the extract has main spots with the same color at the same position as the chromatogram of the control drug, and the chromatogram of the negative control sample has no main spots with the same color at the same position as the chromatogram of the control drug.
Example 5
Taking the traditional Chinese medicine composition, a negative control product without a vine of multiflower knotweed extract, 5g of each negative control product without lucid ganoderma, 1g of lucid ganoderma extract and 1g of vine of multiflower knotweed extract, adding 40mL of propanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating filtrate in a water bath to dryness, and adding 0.5mL of methanol for dissolving to respectively serve as a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 on the general rules of the national pharmacopoeia of the people's republic of China 2015 edition, 6 mu L of each solution is absorbed, respectively dropped on the same silica gel G thin-layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), cyclohexane-ethyl acetate-formic acid (volume ratio is 5: 0.2) is used as a developing agent, developed, taken out, dried, inspected under an ultraviolet lamp of 366nm, sprayed with 10% sulfuric acid ethanol test solution, and dried at 105 ℃ until the color development is clear. The result shows that the chromatogram of the test sample and the extract has main spots with the same color at the same position as the chromatogram of the reference drug, the chromatogram of the negative reference sample has no main spots with the same color at the same position as the chromatogram of the reference drug, and the result of the 366nm ultraviolet lamp inspection is consistent with the result of the spraying of 10% sulfuric acid ethanol reagent and the drying at 105 ℃.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. It is not necessary or necessary to exhaustively enumerate all embodiments herein, and obvious variations or modifications can be made without departing from the scope of the invention.
Claims (5)
1. A method for simultaneously identifying lucid ganoderma and tuber fleeceflower stem in a traditional Chinese medicine composition is characterized by comprising the following steps:
(1) preparation of positive control solution and test solution
Respectively adding a first solvent into a ganoderma lucidum positive control and a vine of multiflower knotweed positive control, filtering, evaporating filtrate to dryness, and adding a second solvent to respectively serve as respective positive control solutions;
adding the first solvent into the Chinese medicinal composition, filtering, evaporating the filtrate, and adding the second solvent to obtain a sample solution;
(2) testing by thin layer chromatography
Sucking the positive control solution and the sample solution, respectively dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent, taking out, air drying, placing under ultraviolet lamp, inspecting and/or spraying with sulfuric acid ethanol solution, and drying until the spots are clearly developed;
the volume ratio of the developing solvent in the step (2) is VCyclohexane：VEthyl acetate：VFormic acid＝5：5：0.2；
The first solvent in the step (1) is one or more of methanol, ethanol and propanol; the second solvent is one or more of methanol, ethanol and propanol;
the raw materials of the traditional Chinese medicine composition comprise a lucid ganoderma extract and a vine of multiflower knotweed extract.
2. The method according to claim 1, wherein the positive control of Ganoderma lucidum is selected from at least one of a control material or an extract of Ganoderma lucidum; the positive control of caulis Polygoni Multiflori is selected from at least one of caulis Polygoni Multiflori control medicinal materials or caulis Polygoni Multiflori extract.
3. Method according to claim 1 or 2, characterized in that it comprises the following steps:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Adding ethanol with the volume 10-20 times of the mass of a lucid ganoderma reference medicinal material into the lucid ganoderma reference medicinal material, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 0.5-1.5 times of the mass of the lucid ganoderma reference medicinal material into the filtrate to obtain a lucid ganoderma reference medicinal material solution;
taking a caulis polygoni multiflori reference medicinal material, adding 100-120 times of ethanol by mass of the caulis polygoni multiflori reference medicinal material, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, and adding 2-6 times of ethanol by mass of the caulis polygoni multiflori reference medicinal material to obtain a caulis polygoni multiflori reference medicinal material solution;
adding ethanol with the mass being 20-40 times of the mass of the ganoderma lucidum extract into the ganoderma lucidum extract, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the mass being 0.5-1.5 times of the mass of the ganoderma lucidum extract to obtain a ganoderma lucidum extract solution;
taking a vine of multiflower knotweed extract, adding ethanol with the mass being 20-40 times of the mass of the vine of multiflower knotweed extract, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, and adding ethanol with the mass being 0.5-1.5 times of the mass of the vine of multiflower knotweed extract to obtain a vine of multiflower knotweed extract solution;
adding ethanol with the volume being 4-8 times of the mass of a negative control product without the ganoderma lucidum extract into the negative control product without the ganoderma lucidum extract, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume being 0.1-0.3 times of the mass of the negative control product without the ganoderma lucidum extract into the filtrate to serve as a negative control product solution without the ganoderma lucidum extract;
taking a negative control product without the vine of multiflower knotweed extract, adding ethanol with the volume 4-8 times of the mass of the negative control product, performing ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, and adding ethanol with the volume 0.1-0.3 times of the mass of the negative control product without the vine of multiflower knotweed extract to serve as a negative control solution without the vine of multiflower knotweed extract;
adding ethanol with the mass 4-8 times of the volume of the traditional Chinese medicine composition into the traditional Chinese medicine composition, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the mass 0.1-0.3 times of the volume of the traditional Chinese medicine composition to serve as a test solution;
the relationship between the mass and the volume is g/ml;
(2) testing by thin layer chromatography
And (3) sucking 4-6 microliters of the control medicinal material solution, the extract solution, the negative control substance solution and the test sample solution respectively, and respectively dropping the solutions on the same silica gel thin-layer plate in a volume ratio of 5: 5: 0.2 cyclohexane-ethyl acetate-formic acid as developing agent, developing, taking out, air drying, placing under 366nm ultraviolet lamp for inspection and/or spraying 10% sulphuric acid ethanol solution for drying until the spots are clearly developed.
4. Method according to claim 1 or 2, characterized in that it comprises the following steps:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Adding ethanol 15 times the mass of the reference medicinal material into the reference medicinal material, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol 0.5 times the mass of the reference medicinal material to obtain a reference medicinal material solution;
taking a caulis polygoni multiflori reference medicinal material, adding ethanol with the volume 120 times of the mass of the caulis polygoni multiflori reference medicinal material, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 4 times of the mass of the caulis polygoni multiflori reference medicinal material to obtain a caulis polygoni multiflori reference medicinal material solution;
adding ethanol with the volume 30 times of the mass of the ganoderma lucidum extract into the ganoderma lucidum extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume of the mass of the ganoderma lucidum extract to obtain a ganoderma lucidum extract solution;
taking a vine of multiflower knotweed extract, adding ethanol with the volume 30 times of the mass of the vine of multiflower knotweed extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume equal to the mass of the vine of multiflower knotweed extract to obtain a vine of multiflower knotweed extract solution;
adding ethanol with the volume 6 times that of the negative control product without the lucid ganoderma extract into the negative control product without the lucid ganoderma extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 0.2 times that of the negative control product without the lucid ganoderma extract into the filtrate to be used as a negative control product solution without the lucid ganoderma extract;
taking a negative control product without the vine of multiflower knotweed extract, adding ethanol with the volume 6 times of the mass of the negative control product, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol with the volume 0.2 times of the mass of the negative control product without the vine of multiflower knotweed extract to serve as a vine negative control product solution without the vine of multiflower knotweed extract;
adding ethanol 6 times the mass of the traditional Chinese medicine composition into the traditional Chinese medicine composition, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding ethanol 0.2 times the mass of the traditional Chinese medicine composition into the filtrate to obtain a test solution;
the relationship between the mass and the volume is g/ml;
(2) testing by thin layer chromatography
Sucking 5 microliters of the control medicinal material solution, the extract solution, the negative control substance solution and the test sample solution respectively, and respectively dropping the solutions on the same silica gel thin-layer plate in a volume ratio of 5: 5: 0.2 cyclohexane-ethyl acetate-formic acid as developing agent, developing, taking out, air drying, placing under 366nm ultraviolet lamp for inspection and/or spraying 10% sulphuric acid ethanol solution for drying until the spots are clearly developed.
5. Method according to claim 1 or 2, characterized in that it comprises the following steps:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Taking 2g of lucid ganoderma reference medicinal material, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain lucid ganoderma reference medicinal material solution;
taking 0.25g of caulis polygoni multiflori as a reference medicinal material, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a caulis polygoni multiflori reference medicinal material solution;
taking 1g of ganoderma lucidum extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain ganoderma lucidum extract solution;
taking 1g of caulis polygoni multiflori extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a caulis polygoni multiflori extract solution;
taking 5g of a negative control product without the ganoderma lucidum extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a negative control product solution without the ganoderma lucidum extract;
taking 5g of a negative control product without the vine of multiflower knotweed extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a negative control product solution without the vine of multiflower knotweed extract;
taking 5g of the traditional Chinese medicine composition, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a test solution;
(2) testing by thin layer chromatography
Sucking 5 microliters of the control medicinal material solution, the extract solution, the negative control substance solution and the test sample solution respectively, and respectively dropping the solutions on the same silica gel thin-layer plate in a volume ratio of 5: 5: 0.2 cyclohexane-ethyl acetate-formic acid as developing agent, developing, taking out, air drying, placing under 366nm ultraviolet lamp for inspection and/or spraying 10% sulphuric acid ethanol solution for drying until the spots are clearly developed.
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