A method for identifying Elsholtzia rhizome and its application

Abstract

The present invention provides an identification method of Elsholtzia root and its application, the identification method comprising the following steps: (1) mixing a sample to be tested with a solvent, filtering, drying and dissolving the filtrate to obtain a test solution; (2) mixing a reference medicinal material with water, heating and refluxing, filtering, drying and dissolving the filtrate to obtain a reference medicinal material test solution; (3) dissolving the reference substance to prepare a reference substance solution; (4) subjecting the above-mentioned test solution, reference medicinal material test solution and reference substance solution to thin layer chromatography analysis, and judging the Elsholtzia root of the sample to be tested according to the result; the Elsholtzia root includes Elsholtzia jiangensis and Elsholtzia lichenifolia. The method provided by the present invention can quickly and effectively distinguish whether the Elsholtzia root of Elsholtzia jiangensis and the Elsholtzia lichenifolia decoction freeze-dried powder is Elsholtzia jiangensis or Elsholtzia lichenifolia, the operation method is simple, and a new method is provided for the identification of Elsholtzia jiangensis and its products.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to an identification method of Elsholtzia rhizome and application thereof. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia stipulates that Elsholtzia chinensis is the dried aerial part of Mosla chinensis Maxim. or Mosla chinensis'Jiangxiangru', a plant of the Lamiaceae family. It has a long history of medicinal use in my country and is a commonly used medicinal material. This product is slightly warm in nature and pungent in taste, and enters the lung and stomach meridians. It belongs to the category of pungent and warm antipyretic drugs, and its functions and indications are sweating and relieving the exterior, removing dampness and harmonizing the middle. It is used for summer-heat cold, aversion to cold and fever, headache without sweating, abdominal pain, vomiting and diarrhea, edema, and urinary difficulty.

[0003] At present, there are few literature reports on the identification of the origin of Elsholtzia and the thin layer chromatography identification of its standard decoction freeze-dried powder. In the existing literature reports, there are only a few thin layer identification methods for Elsholtzia medicinal materials or preparations, and the preparation method of the test solution is complicated, requiring the addition of ether to reflux or evaporate to dryness, extraction, and elution using a polyamide column. The method is limited and cannot be directly applied to the identification and origin identification of its standard decoction freeze-dried powder and its preparations, and there is no report on a simple, fast, thin layer chromatography identification method for identifying its original components. Therefore, how to provide an identification method for Elsholtzia, Elsholtzia standard decoction freeze-dried powder and its preparations to ensure the accuracy of the drug origin has become an urgent problem to be solved. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a method for identifying the origin of Elsholtzia ciliata and its application. The method provided by the present invention can quickly and effectively distinguish whether the origin of Elsholtzia ciliata medicinal materials and Elsholtzia ciliata standard decoction freeze-dried powder is Elsholtzia jiangensis or Elsholtzia schrenkiana, and the operation method is simple, providing a new method for identifying Elsholtzia ciliata medicinal materials and their products.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides a method for identifying Elsholtzia rhizome, the identification method comprising the following steps:

[0007] (1) Mixing the sample to be tested with the solvent, filtering, drying and dissolving the filtrate to obtain a test solution;

[0008] (2) mixing the control medicinal material with water, heating and refluxing, filtering, drying and dissolving the filtrate to obtain a control medicinal material test solution;

[0009] (3) dissolving the reference substance to prepare a reference substance solution;

[0010] (4) The test sample solution, the reference medicinal material test sample solution and the reference solution are subjected to thin layer chromatography analysis, and the Elsholtzia rhizome of the test sample is determined based on the results.

[0011] Steps (1), (2), and (3) are not performed in any order;

[0012] The Elsholtzia origin includes Elsholtzia jiangxiensis and Elsholtzia littoralis.

[0013] The method can effectively distinguish whether the origin of Elsholtzia ciliata medicinal materials and Elsholtzia ciliata products is Elsholtzia jiangensis or Elsholtzia littoralis by performing thin layer chromatography detection on the sample and the reference substance together and controlling relevant reagent parameters. The overall method is simple and rapid.

[0014] Preferably, the sample to be tested in step (1) comprises Elsholtzia ciliata medicinal material and / or Elsholtzia ciliata standard decoction freeze-dried powder.

[0015] Preferably, in step (1), the solid-liquid ratio of the sample to be tested to the solvent is (0.5-2):(25-30) g / mL.

[0016] Preferably, the solvent comprises water and / or ethanol.

[0017] Preferably, the mixing treatment method in step (1) includes heating under reflux or ultrasonic treatment.

[0018] Preferably, the mixing treatment time in step (1) is 25-35 minutes.

[0019] Preferably, the solid-liquid ratio of the control medicinal material to water in step (2) is 2:(25-35) g / mL.

[0020] Preferably, the heating reflux time in step (2) is 25-35 min.

[0021] Preferably, the reference substance in step (3) comprises caffeic acid and / or rosmarinic acid.

[0022] Preferably, in the thin layer chromatography analysis of step (4), the spotting volume of the test solution is 6-8 μL.

[0023] Wherein, the solid-liquid ratio of the sample to be tested and the solvent in step (1) can be 0.5:25 g / mL, 1:26 g / mL, 1.5:28 g / mL or 2:30 g / mL, etc., the mixing treatment time in step (1) can be 25 min, 26 min, 27 min, 28 min, 29 min or 30 min, etc., and the solid-liquid ratio of the control medicinal material to water in step (2) can be 2:25 g / mL, 2:26 g / mL, 2:27 g / mL, 2:28 g / mL, 2:2 The mixing time in step (3) can be 25 min, 26 min, 27 min, 28 min, 29 min or 30 min, and the spotting volume can be 6 μL, 6.5 μL, 7 μL, 7.5 μL or 8 μL, but are not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0024] Preferably, in the thin layer chromatography analysis of step (4), the developing solvent includes any one of toluene, chloroform, ethyl acetate, methanol or formic acid, or a combination of at least two thereof, such as a combination of toluene and chloroform, a combination of chloroform and ethyl acetate, or a combination of ethyl acetate and methanol, but is not limited to the combinations listed above. Other combinations not listed within the above combination range are also applicable.

[0025] Preferably, in the thin layer chromatography analysis of step (4), the developing solvent is a combination of toluene, chloroform, ethyl acetate, methanol and formic acid.

[0026] The developing agent of the above-mentioned specific composition is compounded by five components, and the synergistic effect can effectively improve the clarity and resolution effect of the chromatographic image and improve the detection accuracy.

[0027] 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) can be 1.8, 1.9, 2, 2.1 or 2.2, etc., and the specific point value in (2.8-3.2) can be 2.8, 2.9, 3, 3.1 or 3.2, etc., The specific point values ​​in (3.8-4.2) can be 3.8, 3.9, 4, 4.1 or 4.2, etc., the specific point values ​​in (0.4-0.6) can be 0.4, 0.45, 0.5, 0.55 or 0.6, etc., the specific point values ​​in (0.18-0.22) can be 0.18, 0.19, 0.2, 0.21 or 0.22, etc., but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0028] On the other hand, the present invention also provides the use of the above identification method in identifying Elsholtzia ciliata and Elsholtzia ciliata products.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The invention provides a method for identifying the origin of Elsholtzia ciliata. By performing thin layer chromatography detection on a sample and a reference substance together and controlling relevant reagent parameters, it is possible to effectively distinguish whether the origin of Elsholtzia ciliata medicinal materials and Elsholtzia ciliata products is Elsholtzia jiangensis or Elsholtzia littoralis. The overall method is simple and rapid. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the TLC result when the sample volume is 10 μL in Example 1;

[0032] Figure 2 This is the TLC result after increasing the amount of sample spotted in Example 1;

[0033] Figure 3 It is the thin layer chromatography result of Example 2;

[0034] Figure 4 It is the thin layer chromatography result of Example 3;

[0035] Figure 5 is the thin layer chromatography result of Example 4;

[0036] Figure 6 is the thin layer chromatography result of Example 5;

[0037] Figure 7 is the thin layer chromatography result of Example 6;

[0038] Figure 8 It is the result of TLC at normal temperature and humidity in the durability study;

[0039] Fig. 9 It is the result of TLC at room temperature and low humidity in the durability study;

[0040] Fig.10 It is the result of TLC at room temperature and high humidity in the durability study;

[0041] Fig.11 It is the result of TLC under low temperature and high humidity in durability investigation. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0043] Example 1

[0044] This embodiment provides a method for identifying the base of Elsholtzia ciliata, and the specific steps are as follows:

[0045] Preparation of the medicinal material test solution: Take 2 g of Elsholtzia ciliata powder (passed through No. 2 sieve), place it in a conical flask, add 30 mL of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of ethanol to the residue to dissolve it, and use it as the Elsholtzia ciliata test solution.

[0046] Preparation of test solution of standard decoction lyophilized powder: take 0.5 g of lyophilized powder of standard decoction of Elsholtzia jiangxiensis, put it in a conical flask, add 25 mL of ethanol, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate to dryness, add 1 mL of ethanol to dissolve the residue, and use them as test solution of standard decoction lyophilized powder.

[0047] Preparation of control medicinal material solution: Take 2 g of Elsholtzia jiangensis / Elsholtzia littoralis control medicinal material, place it in a conical flask, add 30 mL of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of ethanol to the residue to dissolve it, and use it as the control medicinal material test solution.

[0048] Preparation of reference solution: Take caffeic acid and rosmarinic acid reference substances, add ethanol to make solutions containing 1 mg per 1 mL, as reference solutions.

[0049] Thin layer conditions: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 General Rules 0502), 7 μL of the test solution and the reference drug solution, and 1 μL of the reference solution were taken and spotted on the same silica gel G thin layer plate, using toluene: chloroform: ethyl acetate: methanol: formic acid in a volume ratio of 2:3:4:0.5:0.2 as the developing solvent, developing, taking out, drying overnight, and inspecting under ultraviolet light (365nm). Results are shown in Figure 1 (From left to right are Elsholtzia ciliata medicinal material test sample, caffeic acid, Elsholtzia ciliata standard decoction freeze-dried powder test sample, Elsholtzia ciliata medicinal material test sample, Elsholtzia ciliata control medicinal material, rosmarinic acid, Elsholtzia ciliata control medicinal material). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference sample.

[0050] It can be found that compared with the thin-layer chromatogram of the Elsholtzia chinensis control medicinal material, some spots are missing. Then, the sample volume of the Elsholtzia chinensis control medicinal material solution was increased to 30μL, and the sample volumes of other reagents remained unchanged. TLC was performed again, and the results are as follows: Figure 2 As shown (from left to right, caffeic acid, Elsholtzia jiangensis control material, Elsholtzia littoralis control material, and rosmarinic acid), it can be found that when the amount of Elsholtzia littoralis control material is 3 times that of Elsholtzia jiangensis control material, some spots are still obviously missing, which fully demonstrates that this method can be effectively used to identify the origin of Elsholtzia jiangensis and its products.

[0051] Example 2

[0052] This embodiment provides a method for identifying Elsholtzia ciliata. The specific steps are consistent with those of Embodiment 1 except that chloroform:ethyl acetate:methanol:formic acid in a volume ratio of 3:4:0.5:0.2 is used as a developing solvent.

[0053] Results Figure 3 (From left to right: caffeic acid, Elsholtzia jiangshanica control material, Elsholtzia lichen control material, and rosmarinic acid).

[0054] Example 3

[0055] This embodiment provides a method for identifying Elsholtzia ciliata. The specific steps are the same as those of Embodiment 1 except that toluene:ethyl acetate:methanol:formic acid is used as a developing solvent in a volume ratio of 2:4:0.5:0.2.

[0056] Results Figure 4 (From left to right: caffeic acid, Elsholtzia jiangshanica control material, Elsholtzia lichen control material, and rosmarinic acid).

[0057] Example 4

[0058] This embodiment provides a method for identifying Elsholtzia ciliata. The specific steps are consistent with those of Embodiment 1 except that toluene: chloroform: methanol: formic acid is used as a developing solvent in a volume ratio of 2:3:0.5:0.2.

[0059] Results Figure 5 (From left to right, they are caffeic acid, Elsholtzia jiangshanica control medicinal material, Elsholtzia littoralis control medicinal material, Elsholtzia littoralis standard decoction freeze-dried powder, Elsholtzia littoralis control medicinal material, and rosmarinic acid).

[0060] Example 5

[0061] This embodiment provides a method for identifying Elsholtzia ciliata. The specific steps are consistent with those of Embodiment 1 except that toluene: chloroform: ethyl acetate: formic acid in a volume ratio of 2:3:4:0.2 is used as a developing solvent.

[0062] Results Figure 6 (From left to right: caffeic acid, Elsholtzia jiangensis reference material, Elsholtzia lichen reference material, rosmarinic acid, Elsholtzia lichen standard decoction freeze-dried powder).

[0063] Example 6

[0064] This embodiment provides a method for identifying Elsholtzia ciliata. The specific steps are consistent with those of Embodiment 1 except that toluene: chloroform: ethyl acetate: methanol in a volume ratio of 2:3:4:0.5 is used as a developing solvent.

[0065] Results Figure 7(From left to right: caffeic acid, Elsholtzia jiangensis reference material, Elsholtzia lichen reference material, rosmarinic acid, Elsholtzia lichen standard decoction freeze-dried powder).

[0066] from Figure 3-7 It can be found that there are multiple spots and double images of caffeic acid, and the main spots are not obvious and clear. Figure 1 It can be found that the present application can effectively improve the clarity and resolution of the chromatographic image and improve the detection accuracy by compounding five specific developing agent components and acting synergistically.

[0067] Durability test:

[0068] The method in Example 1 was repeated under different temperature and humidity conditions. The results are as follows Figure 8-11 As shown, Figure 8 Normal temperature and humidity conditions, 25.5°C, relative humidity 42%; Fig. 9 It is a normal temperature and low humidity situation, the conditions are 25.5℃ and relative humidity 11%; Fig.10 Normal temperature and high humidity, the conditions are 25.5℃ and relative humidity 80%; Fig.11 The condition is low temperature and high humidity, with the temperature at 7°C and relative humidity at 75%. From left to right in the figure are caffeic acid, test solution, Elsholtzia jiangsu control medicinal material, and rosmarinic acid.

[0069] Under different temperature and humidity conditions, the chromatographic spots of the test solution and the control medicinal material solution were clear, and the correspondence and separation effects were good, indicating that the thin layer identification method has good durability to temperature and humidity.

[0070] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the identification method of the Elsholtzia rhizome and its application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0071] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0072] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for identifying the origin of Elsholtzia ciliata, characterized in that: The identification method comprises the following steps: (1) Mixing the sample to be tested with the solvent, filtering, drying and dissolving the filtrate to obtain a test solution; (2) mixing the control medicinal material with water, heating and refluxing, filtering, drying and dissolving the filtrate to obtain a control medicinal material solution; (3) dissolving the reference substance to prepare a reference substance solution; (4) performing thin layer chromatography analysis on the test sample solution, the control medicinal material solution and the reference substance solution, and determining the Elsholtzia rhizome of the test sample according to the results; Steps (1), (2), and (3) are not performed in any order; The Elsholtzia rhizome includes Elsholtzia jiangensis and Elsholtzia lichenifolia; In step (1), the sample to be tested is Elsholtzia medicinal material and / or Elsholtzia standard decoction freeze-dried powder; the sample to be tested is Elsholtzia medicinal material, and the solvent is water; the sample to be tested is Elsholtzia standard decoction freeze-dried powder, and the solvent is ethanol; The reference substances in step (3) are caffeic acid and rosmarinic acid; In the thin layer chromatography analysis of step (4), the developing solvent is a combination of toluene, chloroform, ethyl acetate, methanol and formic acid, and 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); the thin layer chromatography analysis is carried out using a silica gel G thin layer plate.

2. The identification method according to claim 1, characterized in that: The solid-liquid ratio of the sample to be tested to the solvent in step (1) is (0.5-2):(25-30) g / mL.

3. The identification method according to claim 1, characterized in that: The mixing treatment method in step (1) includes heating under reflux or ultrasonic treatment.

4. The identification method according to claim 1, characterized in that: The mixing time in step (1) is 25-35 minutes.

5. The identification method according to claim 1, characterized in that: The solid-liquid ratio of the control medicinal material to water in step (2) is 2:(25-35) g / mL.

6. The identification method according to claim 1, characterized in that: The heating reflux time in step (2) is 25-35 minutes.

7. The identification method according to claim 1, characterized in that: In the thin layer chromatography analysis of step (4), the spotting volume of the test solution is 6-8 μL.

8. Use of the identification method according to any one of claims 1 to 7 in identifying Elsholtzia ciliata medicinal materials and Elsholtzia ciliata standard decoctions.

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