CN116203181A - Identification method of elsholtzia base antigen and application thereof - Google Patents
Identification method of elsholtzia base antigen and application thereof Download PDFInfo
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- CN116203181A CN116203181A CN202310418183.8A CN202310418183A CN116203181A CN 116203181 A CN116203181 A CN 116203181A CN 202310418183 A CN202310418183 A CN 202310418183A CN 116203181 A CN116203181 A CN 116203181A
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Abstract
The invention provides an identification method of elsholtzia base stock and application thereof, wherein the identification method comprises the following steps: (1) Mixing a sample to be tested with a solvent, filtering, drying the filtrate, and dissolving to obtain a sample solution; (2) Mixing the reference medicinal material with water, heating and refluxing, filtering, drying the filtrate, and dissolving to obtain a reference medicinal material sample solution; (3) dissolving the reference substance to prepare a reference substance solution; (4) Carrying out thin layer chromatography analysis on the sample solution, the reference medicinal material sample solution and the reference substance solution, and judging elsholtzia-base antigen of the sample to be detected according to the result; the elsholtzia base includes Jiang Xiangru and elsholtzia. The method provided by the invention can rapidly and effectively distinguish whether the basic source of the standard Chinese mosla herb and Chinese mosla herb decoction freeze-dried powder is Jiang Xiangru or Chinese mosla herb, is simple in operation method, and provides a new method for identifying Chinese mosla herb and products thereof.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to an identification method of elsholtzia base stock and application thereof.
Background
The Chinese pharmacopoeia of 2020 edition prescribes that herba Moslae is dry aerial part of herba Moslae (Mosla chinensis maxim.) or Jiang Xiangru (Mosla chinensis 'jiangxianglu'), which is a common medicinal material for long history of medicine in China. This herb is slightly warm and pungent in flavor and enters lung and stomach meridians. Belongs to the Chinese herbal medicine category of pungent and warm exterior-releasing drugs, and has the main functions of inducing sweat, relieving exterior syndrome, resolving dampness and harmonizing middle energizer. Can be used for treating common cold due to summer-heat and dampness, aversion to cold, fever, headache, no sweat, abdominal pain, vomiting and diarrhea, edema, and dysuria.
At present, few thin-layer chromatography identification literature reports are about elsholtzia base original identification and standard decoction freeze-dried powder thereof, in the existing literature reports, only few thin-layer chromatography identification methods about elsholtzia medicinal materials or preparations are provided, the preparation method of a sample solution is complex, diethyl ether needs to be added for reflux or evaporation, extraction and elution by using a polyamide column, the method is limited, the method cannot be directly applied to identification and base original identification of the standard decoction freeze-dried powder and the preparation thereof, and no simple, fast and thin-layer chromatography identification method for identifying the original components of the elsholtzia base original powder is yet discovered. Therefore, how to provide an identification method for identifying the standard elsholtzia, the standard elsholtzia decoction freeze-dried powder and the preparation thereof, and ensure the accuracy of the medicine base source, and the method becomes a problem to be solved urgently.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for identifying elsholtzia base stock and application thereof. The method provided by the invention can rapidly and effectively distinguish whether the basic source of the standard Chinese mosla herb and Chinese mosla herb decoction freeze-dried powder is Jiang Xiangru or Chinese mosla herb, is simple in operation method, and provides a new method for identifying Chinese mosla herb and products thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in one aspect, the invention provides a method for identifying elsholtzia base, which comprises the following steps:
(1) Mixing a sample to be tested with a solvent, filtering, drying the filtrate, and dissolving to obtain a sample solution;
(2) Mixing the reference medicinal material with water, heating and refluxing, filtering, drying the filtrate, and dissolving to obtain a reference medicinal material sample solution;
(3) Dissolving the reference substance to obtain reference substance solution;
(4) And carrying out thin layer chromatography analysis on the sample solution, the reference medicinal material sample solution and the reference substance solution, and judging elsholtzia-base antigen of the sample to be detected according to the result.
The steps (1), (2) and (3) do not distinguish the sequence;
the elsholtzia base includes Jiang Xiangru and elsholtzia.
The method can effectively distinguish whether the basic sources of the elsholtzia medicinal materials and the elsholtzia products are Jiang Xiangru or elsholtzia through carrying out thin layer chromatography detection on the samples and the reference substances together and controlling related reagent parameters, and the whole method is simple and rapid.
Preferably, the sample to be tested in the step (1) comprises elsholtzia medicinal materials and/or elsholtzia standard decoction freeze-dried powder.
Preferably, the ratio of the sample to be tested to the solvent in the step (1) is (0.5-2): 25-30 g/mL.
Preferably, the solvent comprises water and/or ethanol.
Preferably, the method of the mixing treatment in the step (1) comprises heating reflux or ultrasonic treatment.
Preferably, the time of the mixing treatment in the step (1) is 25-35min.
Preferably, the feed liquid ratio of the control medicinal material to water in the step (2) is 2 (25-35) g/mL.
Preferably, the heating reflux time in the step (2) is 25-35min.
Preferably, the reference substance in step (3) comprises caffeic acid and/or rosmarinic acid.
Preferably, in the thin layer chromatography analysis of step (4), the sample application amount of the test solution is 6 to 8. Mu.L.
The ratio of the sample to be tested to the solvent in the step (1) may be 0.5:25g/mL, 1:26g/mL, 1.5:28g/mL, 2:30g/mL, etc., the time of the mixing treatment in the step (1) may be 25min, 26min, 27min, 28min, 29min, 30min, etc., the ratio of the reference medicinal material to the water in the step (2) may be 2:25g/mL, 2:26g/mL, 2:27g/mL, 2:28g/mL, 2:29g/mL, 2:30g/mL, 2:31g/mL, 2:32g/mL, 2:33g/mL, 2:34g/mL, 2:35g/mL, etc., the time of the mixing treatment in the step (3) may be 25min, 26min, 27min, 28min, 29min, 30min, etc., the sample application amount may be 6 μl, 6.5 μl, 7 μl, 7.5 μl, 7 μl, or 8 μl, etc., but the values are not limited to the above values.
Preferably, in the thin layer chromatography analysis in the step (4), the developing agent includes any one or a combination of at least two of toluene, chloroform, ethyl acetate, methanol and formic acid, for example, a combination of toluene and chloroform, a combination of chloroform and ethyl acetate, or a combination of ethyl acetate and methanol, etc., but not limited to the above-listed combinations, and other non-listed combinations within the above-listed combinations are equally applicable.
Preferably, in the thin layer chromatography analysis of step (4), the developing agent is a combination of toluene, chloroform, ethyl acetate, methanol and formic acid.
The developing agent with the specific composition can effectively improve the definition and the resolution effect of chromatographic images and improve the detection accuracy through the combination of five components and the synergistic effect.
Preferably, the volume ratio of toluene, chloroform, ethyl acetate, methanol and formic acid is (1.8-2.2): (2.8-3.2): (3.8-4.2): (0.4-0.6): (0.18-0.22), wherein the specific point value in (1.8-2.2) may be 1.8, 1.9, 2, 2.1 or 2.2, etc. (2.8-3.2) may be 2.8, 2.9, 3, 3.1 or 3.2, etc. (3.8-4.2) may be 3.8, 3.9, 4, 4.1 or 4.2, etc. (0.4-0.6) may be 0.4, 0.45, 0.5, 0.55 or 0.6, etc. (0.18-0.22) may be 0.18, 0.19.21, 0.21, etc. (3.8-4.2, etc.), but the specific point value in (0.8-4.2) may be 3.8, 4.2, 4.1 or 4.2, etc. may not be the same.
On the other hand, the invention also provides the application of the identification method in the identification of the elsholtzia and the elsholtzia products.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for identifying elsholtzia base stock, which can effectively distinguish whether the base stock of elsholtzia medicinal materials and elsholtzia products is Jiang Xiangru or elsholtzia by carrying out thin layer chromatography detection on a sample and a reference substance together and controlling related reagent parameters.
Drawings
FIG. 1 is a thin layer chromatography result at a spotting amount of 10. Mu.L in example 1;
FIG. 2 shows the results of thin layer chromatography after the sample application amount was increased in example 1;
FIG. 3 is the results of thin layer chromatography of example 2;
FIG. 4 is the thin layer chromatography result of example 3;
FIG. 5 is the thin layer chromatography result of example 4;
FIG. 6 is the thin layer chromatography result of example 5;
FIG. 7 is the thin layer chromatography result of example 6;
FIG. 8 is a thin layer chromatography result at normal temperature and humidity in a durability test;
FIG. 9 is a thin layer chromatography result at normal temperature and low humidity in a durability test;
FIG. 10 is a thin layer chromatography result at normal temperature and high humidity in a durability test;
FIG. 11 shows the results of thin layer chromatography at low temperature and high humidity in the durability test.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
The embodiment provides a method for identifying elsholtzia base, which comprises the following specific steps:
preparing a medicinal material sample solution: 2g of elsholtzia medicinal material powder (sieving with a No. 2 sieve) is taken, placed in a conical flask, added with 30mL of water, heated and refluxed for 30 minutes, filtered, evaporated to dryness, and the residue is added with 1mL of ethanol to dissolve, thus obtaining the elsholtzia medicinal material sample solution.
Preparing a standard decoction freeze-dried powder sample solution: 0.5g of standard Chinese mosla herb decoction freeze-dried powder is taken, placed in a conical flask, added with 25mL of ethanol, subjected to ultrasonic treatment for 30 minutes, filtered, evaporated to dryness, and the residues are added with 1mL of ethanol to be dissolved, and respectively used as standard Chinese mosla herb decoction freeze-dried powder test solution.
Preparation of control medicinal material solution: jiang Xiangru/2 g of herba Moslae reference material is taken, placed in an conical flask, added with 30mL of water, heated and refluxed for 30 minutes, filtered, evaporated to dryness, and 1mL of ethanol is added into the residue to dissolve the residue to be used as a reference material sample solution.
Preparation of a control solution: taking caffeic acid and rosmarinic acid reference substances, adding ethanol to prepare solutions containing 1mg per 1mL respectively, and taking the solutions as reference substance solutions.
Thin layer condition: according to thin layer chromatography (0502 of Chinese pharmacopoeia 2020 edition), sucking 7 μl of each of the sample solution and the control solution, respectively, and spotting 1 μl of each of the control solution on the same silica gel G thin layer plate, spreading with toluene, chloroform, ethyl acetate, methanol, and formic acid at a volume ratio of 2:3:4:0.5:0.2 as developing agent, taking out, air drying overnight, and inspecting under ultraviolet lamp (365 nm). The results are shown in figure 1 (from left to right, the sample of herba Moslae, caffeic acid, lyophilized powder of herba Moslae standard decoction, sample of herba Moslae, herba Moslae reference, rosmarinic acid, herba Moslae reference) respectively, and fluorescent spots with the same color appear on the corresponding positions of the sample chromatogram and the reference chromatogram.
It can be found that the herba Moslae control material chromatogram has partial speckle deficiency compared with herba Moslae control material thin layer chromatogram. Then the sample solution of the herba elsholtziae control medicine is added to 30 mu L, the sample application amount of the rest reagents is unchanged, and thin layer chromatography is carried out again, and the results are shown in figure 2 (caffeic acid, the herba elsholtziae control medicine and rosmarinic acid are respectively from left to right), so that partial spot loss can still be obviously seen under the condition that the sample application amount of the herba elsholtziae control medicine is 3 times that of the herba elsholtziae control medicine, and the method is fully described to be effectively used for identifying the basic sources of the herba elsholtziae medicine and products thereof.
Example 2
The present example provides a method for identifying elsholtzia base stock, which is the same as example 1 except that chloroform, ethyl acetate, methanol and formic acid are used as developing agents in a volume ratio of 3:4:0.5:0.2.
The results are shown in figure 3 (caffeic acid, herba Moslae control, and rosmarinic acid from left to right respectively).
Example 3
The present example provides a method for identifying elsholtzia base stock, which is the same as example 1 except that toluene, ethyl acetate, methanol and formic acid are used as developing agents in a volume ratio of 2:4:0.5:0.2.
The results are shown in figure 4 (caffeic acid, herba Moslae control, and rosmarinic acid from left to right respectively).
Example 4
The present example provides a method for identifying elsholtzia base stock, which is the same as example 1 except that toluene, chloroform, methanol, and formic acid are used as developing agents in a volume ratio of 2:3:0.5:0.2.
The results are shown in figure 5 (from left to right, caffeic acid, herba Moslae control, herba Moslae standard decoction lyophilized powder, herba Moslae control, and rosmarinic acid respectively).
Example 5
The present example provides a method for identifying elsholtzia base stock, which is the same as example 1 except that toluene, chloroform, ethyl acetate, and formic acid are used as developing agents in a volume ratio of 2:3:4:0.2.
The results are shown in figure 6 (from left to right, caffeic acid, herba Moslae control, rosmarinic acid, herba Moslae standard decoction lyophilized powder respectively).
Example 6
The present example provides a method for identifying elsholtzia base stock, which is the same as example 1 except that toluene, chloroform, ethyl acetate, and methanol are used as developing agents in a volume ratio of 2:3:4:0.5.
The results are shown in FIG. 7 (from left to right, caffeic acid, herba Moslae control, rosmarinic acid, herba Moslae standard decoction lyophilized powder).
It can be seen from fig. 3-7 that there are problems with caffeic acid that multiple spots, ghosts, appear, and that the main spots appear less pronounced and less clear. By combining with FIG. 1, the method can effectively improve the definition and resolution effect of the chromatographic image and improve the detection accuracy through the combination and synergistic effect of specific five developing agent components.
Durability inspection:
the process of example 1 was repeated under different conditions of temperature and humidity, the results are shown in FIGS. 8-11, wherein FIG. 8 is a normal temperature and humidity condition, the condition being 25.5℃and the relative humidity being 42%; FIG. 9 is a normal temperature and low humidity condition, with a temperature of 25.5℃and a relative humidity of 11%; FIG. 10 is a graph showing high humidity at room temperature under conditions of 25.5℃and 80% relative humidity; FIG. 11 is a low temperature high humidity, conditioned at 7deg.C, relative humidity 75%; the figure shows caffeic acid, sample solution, herba Moslae control material, and rosmarinic acid from left to right.
Under different temperature and humidity conditions, chromatographic spots of the sample solution and the control medicinal material solution are clear, and the correspondence and the separation effect are good, which indicates that the thin layer identification method has good durability to temperature and humidity.
The applicant states that the present invention describes the method of identifying elsholtzia based material and its use by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be practiced by the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. An identifying method of elsholtzia base stock, characterized in that the identifying method comprises the following steps:
(1) Mixing a sample to be tested with a solvent, filtering, drying the filtrate, and dissolving to obtain a sample solution;
(2) Mixing the reference medicinal material with water, heating and refluxing, filtering, drying the filtrate, and dissolving to obtain a reference medicinal material sample solution;
(3) Dissolving the reference substance to obtain reference substance solution;
(4) Carrying out thin layer chromatography analysis on the sample solution, the reference medicinal material sample solution and the reference substance solution, and judging elsholtzia-base antigen of the sample to be detected according to the result;
the steps (1), (2) and (3) do not distinguish the sequence;
the elsholtzia base includes Jiang Xiangru and elsholtzia.
2. The method according to claim 1, wherein the sample to be tested in step (1) comprises herba Moslae and/or herba Moslae standard decoction lyophilized powder.
3. The identification method according to claim 1 or 2, wherein the ratio of the sample to be tested to the solvent in the step (1) is (0.5-2): 25-30 g/mL;
preferably, the solvent comprises water and/or ethanol.
4. A method of authentication according to any one of claims 1 to 3, wherein the method of mixing treatment of step (1) comprises heat refluxing or ultrasonic treatment;
preferably, the time of the mixing treatment in the step (1) is 25-35min.
5. The method of any one of claims 1-4, wherein the ratio of the control drug to water in step (2) is 2 (25-35) g/mL;
preferably, the heating reflux time in the step (2) is 25-35min;
preferably, the reference substance in step (3) comprises caffeic acid and/or rosmarinic acid.
6. The method according to any one of claims 1 to 5, wherein the sample application amount of the sample solution in the thin layer chromatography in the step (4) is 6 to 8. Mu.L.
7. The method of any one of claims 1-6, wherein in the thin layer chromatography of step (4), the developing reagent comprises any one or a combination of at least two of toluene, chloroform, ethyl acetate, methanol, and formic acid.
8. The method of claim 7, wherein in the thin layer chromatography of step (4), the developing reagent is a combination of toluene, chloroform, ethyl acetate, methanol and formic acid.
9. The method of claim 8, wherein the volume ratio of toluene, chloroform, ethyl acetate, methanol and formic acid is (1.8-2.2): (2.8-3.2): (3.8-4.2): (0.4-0.6): (0.18-0.22).
10. Use of an identification method according to any one of claims 1 to 9 in the identification of elsholtzia and elsholtzia products.
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