Method for identifying notopterygium root dampness-removing decoction

Abstract

The method comprises the following steps: preparing a first test solution, a second test solution, a third test solution, a reference medicinal material solution and a reference substance solution, sampling the solutions on a silica gel G thin-layer plate, and identifying notopterygium root, radix angelicae pubescentis, ligusticum, ligusticum wallichii, divaricate saposhnikovia root and liquorice. According to the identification method of the notopterygium root-containing dampness-clearing decoction, the first test solution for simultaneously identifying notopterygium root, radix angelicae pubescentis, ligusticum and ligusticum wallichii, the second test solution for identifying divaricate saposhnikovia root and the third test solution for identifying liquorice are obtained through a specific extraction process, so that the detection efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine ingredient detection, and relates to a method for identifying Qianghuo Shengshi Decoction. BACKGROUND

[0002] Qianghuo Shengshi Decoction is composed of seven traditional Chinese medicines, i.e., Notopterygium incisum, Angelica pubescens, Ligusticum sinense, Saposhnikovia divaricata, Glycyrrhiza uralensis, Vitex negundo and Szechwan Lovage Rhizome, and has the effects of dispelling wind, overcoming dampness and relieving pain. Qianghuo Shengshi Decoction is clinically used for treating rheumatic arthritis, rheumatoid arthritis, hyperosteogeny, ankylosing spondylitis, cervical spondylosis and shoulder and back and head pain. Since the prescription has complex components and the traditional Chinese medicinal materials are greatly affected by factors such as production place, harvesting, processing and storage, in order to ensure the quality stability and reliable curative effect of Qianghuo Shengshi Decoction, a scientific identification method must be established. However, the existing technical means cannot realize the identification of effective components.

[0003] Therefore, developing a method capable of identifying Qianghuo Shengshi Decoction has become a problem to be solved by those skilled in the art. SUMMARY

[0004] In order to solve the problems in the prior art, the application provides a method for identifying Qianghuo Shengshi Decoction, and the technical scheme adopted by the application is as follows: the method comprises the following steps:

[0005] S1: petroleum ether is used to extract Qianghuo Shengshi Decoction decoction liquid and water washing is performed, the washing liquid is discarded, petroleum ether extraction water liquid and petroleum ether liquid are obtained, the petroleum ether liquid is evaporated to dryness, the residue is dissolved in ethyl acetate to obtain a first test sample solution, part of the water liquid is extracted with water-saturated n-butanol and washed with water-saturated n-butanol, the n-butanol liquid is evaporated to dryness, the residue is dissolved in methanol to obtain a second test sample solution, and part of the water liquid is extracted with ethyl acetate and washed with water, the ethyl acetate liquid is evaporated to dryness, and the residue is dissolved in methanol to obtain a third test sample solution;

[0006] S2: Qianghuo control medicinal material solution, Duhuo control medicinal material solution, Ligusticum sinense control medicinal material solution, Saposhnikovia divaricata control medicinal material solution, Glycyrrhiza uralensis control medicinal material solution, Notopterygium incisum alcohol control solution, Cnidium monnieri element control solution, Ligusticum sinense inner ester control solution, Glycyrrhiza uralensis glycoside control solution, Rhizoma Cimicifugae element control solution, 5-O-methyl Viscum ammi alcohol glycoside control solution are prepared respectively;

[0007] S3: take the first test solution, Notopterygium control drug solution, Notopterygium alcohol control solution, respectively, sample on silica gel G thin layer plate, with the first developing agent, develop, color, identify Notopterygium; take the first test solution, Notopterygium control drug solution, Notopterygium alcohol control solution, respectively, sample on silica gel G thin layer plate, with the second developing agent, develop, color, identify Notopterygium; take the first test solution, Notopterygium control drug solution, Notopterygium alcohol control solution, respectively, sample on silica gel G thin layer plate, with the third developing agent, develop, color, identify Notopterygium and Chuanxiong; take the second test solution, Notopterygium control drug solution, Notopterygium alcohol control solution, respectively, sample on silica gel G thin layer plate, with the fourth developing agent, develop, color, identify Notopterygium; take the third test solution, Notopterygium control drug solution, Notopterygium alcohol control solution, respectively, sample on silica gel G thin layer plate, with the fifth developing agent, develop, color, identify Notopterygium.

[0008] In a preferred embodiment of the present application, in step S1,

[0009] The volume ratio of Notopterygium Shengshi decoction and petroleum ether is 1:0.5-1; and / or,

[0010] When preparing the first test solution, the volume ratio of ethyl acetate and Notopterygium Shengshi decoction is 20-25:1; and / or,

[0011] When preparing the second test solution, the volume ratio of water and water-saturated n-butanol is 1:0.8-1.2; the volume ratio of water and methanol is 20-25:1; and / or,

[0012] When preparing the third test solution, the volume ratio of water and ethyl acetate is 1:0.8-1.2; the volume ratio of water and methanol is 20-25:1.

[0013] In a preferred embodiment of the present application, step S2 comprises:

[0014] Take Notopterygium control drug, Notopterygium control drug, Ligusticum control drug and Chuanxiong control drug, respectively, after reflux extraction and filtration, the filtrate is prepared according to the preparation method of the first test solution to obtain Notopterygium control drug solution, Notopterygium control drug solution, Ligusticum control drug solution and Chuanxiong control drug solution; take Notopterygium control drug, after reflux extraction and filtration, the filtrate is prepared according to the preparation method of the second test solution to obtain Notopterygium control drug solution; take Notopterygium control drug, after reflux extraction and filtration, the filtrate is prepared according to the preparation method of the third test solution to obtain Notopterygium control drug solution.

[0015] In a preferred embodiment of the present application, step S2 comprises:

[0016] Take the control sample of cnidridin, ostruthin, ligusticum lactone and glycyrrhizin respectively, and add methanol to obtain the control sample solution of cnidridin, ostruthin, ligusticum lactone and glycyrrhizin respectively; take the control sample of rhizomae cimicugae glycoside and 5-O-methyl visamminol glycoside respectively, and add ethanol to obtain the control sample solution of rhizomae cimicugae glycoside and 5-O-methyl visamminol glycoside respectively.

[0017] In a preferred embodiment of the present application, in step S3,

[0018] The first developing agent is n-hexane-toluene-ethyl acetate, and the volume ratio is preferably 1.8-2.2:1:1.8-2.2.

[0019] In a preferred embodiment of the present application, in step S3,

[0020] The second developing agent is n-hexane-toluene-ethyl acetate-acetone, and the volume ratio is preferably 3.6-4.4:1.8-2.2:1.8-2.2:0.4.

[0021] In a preferred embodiment of the present application, in step S3,

[0022] The third developing agent is cyclohexane-ethyl acetate, and the volume ratio is preferably 8.5-9.5:1.

[0023] In a preferred embodiment of the present application, in step S3,

[0024] The fourth developing agent is ethyl acetate-formic acid-glacial acetic acid-water, and the volume ratio is preferably 14-16:1:1:1.8-2.2.

[0025] In a preferred embodiment of the present application, in step S3,

[0026] The fifth developing agent is ethyl acetate-anhydrous ethanol-acetone-ammonia reagent, and the volume ratio is preferably 1.8-2.2:1:1.8-2.2:1.

[0027] In a preferred embodiment of the present application, in step S3,

[0028] When identifying Qianghuo, after development, spray with color developing agent, heat until the spots are clear, and observe under ultraviolet light; and / or,

[0029] When identifying Duhuo, after development, observe under ultraviolet light; and / or,

[0030] When identifying Ligusticum and Chuanxiong, after development, spray with color developing agent, heat until the spots are clear, and observe under ultraviolet light; and / or,

[0031] When identifying Fangfeng, after development, observe under ultraviolet light; and / or,

[0032] When identifying licorice, spray it with a color developer after unfolding, heat it until the spots are clear, and then examine it under sunlight.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. The identification method of Qianghuo Shengshi Decoction of the present invention can identify the six medicinal ingredients in Qianghuo Shengshi Decoction: Qianghuo, Duhuo, Gaoben, Chuanxiong, Fangfeng, and Gancao, thus achieving comprehensive quality control.

[0035] 2. The identification method of Qianghuo Shengshi Decoction of the present invention obtains a first test solution that can be used to simultaneously identify Qianghuo, Duhuo, Gaoben, and Chuanxiong through a specific extraction process, a second test solution for identifying Fangfeng, and a third test solution for identifying Gancao, which greatly improves the detection efficiency.

[0036] 3. The identification method of Qianghuo Shengshi Decoction of the present invention has good overall separation effect, clear spots, strong specificity, and good durability. It can accurately identify Qianghuo, Duhuo, Gaoben, Chuanxiong, Fangfeng and Gancao in Qianghuo Shengshi Decoction, providing a good foundation for the quality control of Qianghuo Shengshi Decoction. Attached Figure Description

[0037] Figure 1 The image shows the thin-layer identification results of Notopterygium incisum when using silica gel G thin-layer plates produced by Qingdao Ocean Chemical Co., Ltd. in Example 2 of this invention; where 1 is the Notopterygium incisum negative test solution, 2 is the first test solution, 3 is Notopterygium incisum alcohol reference solution, and 4 is Notopterygium incisum reference medicinal material solution.

[0038] Figure 2 The image shows the thin-layer identification results of Notopterygium incisum when using silica gel G thin-layer plates produced by Yantai Chemical Industry Research Institute in Example 2 of this invention; where 1 is the Notopterygium incisum negative test solution, 2 is the first test solution, 3 is the Notopterygium incisum alcohol reference solution, and 4 is the Notopterygium incisum reference medicinal material solution.

[0039] Figure 3 The image shows the thin-layer chromatography identification results of Notopterygium incisum under low temperature conditions in Example 2 of this invention; where 1 is the Notopterygium incisum negative test solution, 2 is the first test solution, 3 is the Notopterygium incisum alcohol reference solution, and 4 is the Notopterygium incisum reference medicinal material solution.

[0040] Figure 4 The image shows the thin-layer chromatography identification results of Notopterygium incisum under high humidity conditions in Example 2 of this invention; where 1 is the Notopterygium incisum negative test solution, 2 is the first test solution, 3 is the Notopterygium incisum alcohol reference solution, and 4 is the Notopterygium incisum reference medicinal material solution.

[0041] Figure 5The image shows the results of thin-layer identification of Angelica pubescens using silica gel G thin-layer plates produced by Yantai Chemical Industry Research Institute in Example 2 of this invention; where 1 is the negative test solution of Angelica pubescens, 2 is the first test solution, 3 is the reference medicinal material solution of Angelica pubescens, and 4 is the reference solution of osthol.

[0042] Figure 6 The image shows the results of thin-layer identification of Angelica pubescens using silica gel G thin-layer plates produced by Qingdao Ocean Chemical Co., Ltd. in Example 2 of this invention; where 1 is the negative test solution of Angelica pubescens, 2 is the first test solution, 3 is the reference medicinal material solution of Angelica pubescens, and 4 is the reference solution of osthol.

[0043] Figure 7 The image shows the results of thin-layer identification of Angelica pubescens under low temperature conditions in Example 2 of this invention; where 1 is the negative test solution of Angelica pubescens, 2 is the first test solution, 3 is the reference medicinal material solution of Angelica pubescens, and 4 is the reference solution of osthol.

[0044] Figure 8 The image shows the results of thin-layer chromatography identification of Angelica pubescens under high humidity conditions in Example 2 of this invention; where 1 is the negative test solution of Angelica pubescens, 2 is the first test solution, 3 is the reference medicinal material solution of Angelica pubescens, and 4 is the osthol reference solution.

[0045] Figure 9 The image shows the thin-layer chromatography identification results of Ligusticum striatum and Ligusticum chuanxiong when using silica gel G thin-layer plates produced by Yantai Chemical Industry Research Institute in Example 2 of this invention; wherein, 1 is the negative decoction test solution of Ligusticum striatum and Ligusticum chuanxiong (3 μl), 2 is the negative decoction test solution of Ligusticum striatum and Ligusticum chuanxiong (5 μl), 3 is the negative decoction test solution of Ligusticum striatum and Ligusticum chuanxiong (7 μl), 4 is the first test solution (3 μl), 5 is the first test solution (5 μl), 6 is the first test solution (7 μl), 7 is the reference medicinal material solution of Ligusticum chuanxiong (5 μl), 8 is the reference medicinal material solution of Ligusticum striatum (5 μl), and 9 is the reference solution of Ligusticum striatum lactone (5 μl);

[0046] Figure 10 The image shows the thin-layer chromatography identification results of Ligusticum striatum and Ligusticum chuanxiong when using silica gel G thin-layer plates produced by Qingdao Ocean Chemical Co., Ltd. in Example 2 of this invention; wherein, 1 is the negative decoction test solution of Ligusticum striatum and Ligusticum chuanxiong (3 μl), 2 is the negative decoction test solution of Ligusticum striatum and Ligusticum chuanxiong (5 μl), 3 is the negative decoction test solution of Ligusticum striatum and Ligusticum chuanxiong (7 μl), 4 is the first test solution (3 μl), 5 is the first test solution (5 μl), 6 is the first test solution (7 μl), 7 is the reference medicinal material solution of Ligusticum chuanxiong (5 μl), 8 is the reference medicinal material solution of Ligusticum striatum (5 μl), and 9 is the reference solution of Ligusticum striatum lactone (5 μl);

[0047] Figure 11The image shows the thin-layer chromatography identification results of Ligusticum striatum and Ligusticum chuanxiong under low temperature conditions in Example 2 of the present invention; wherein, 1 is the negative test solution of Ligusticum striatum and Ligusticum chuanxiong, 2 is the first test solution, 3 is the reference medicinal material solution of Ligusticum chuanxiong, 4 is the reference medicinal material solution of Ligusticum striatum, and 5 is the reference solution of Ligusticum striatum lactone.

[0048] Figure 12 The image shows the thin-layer chromatography identification results of Ligusticum striatum and Ligusticum chuanxiong under high humidity conditions in Example 2 of the present invention; wherein, 1 is the negative test solution of Ligusticum striatum and Ligusticum chuanxiong, 2 is the first test solution, 3 is the reference medicinal material solution of Ligusticum chuanxiong, 4 is the reference medicinal material solution of Ligusticum striatum, and 5 is the reference solution of Ligusticum striatum lactone.

[0049] Figure 13 This is a graph showing the identification results of the thin-layer plate of *Saposhnikovia divaricata* produced by Yantai Chemical Industry Research Institute in Example 2 of the present invention; wherein, 1 is the negative test solution of *Saposhnikovia divaricata* (3 μl), 2 is the negative test solution of *Saposhnikovia divaricata* (5 μl), 3 is the negative test solution of *Saposhnikovia divaricata* (7 μl), 4 is the second test solution (3 μl), 5 is the second test solution (5 μl), 6 is the second test solution (7 μl), 7 is the reference medicinal material solution of *Saposhnikovia divaricata* (5 μl), 8 is the 5-O-methylvisamidol glycoside reference solution (2 μl), and 9 is the cimicifuga glycoside reference solution (2 μl);

[0050] Figure 14 This is a graph showing the identification results of the thin-layer plate of *Saposhnikovia divaricata* produced by Qingdao Ocean Chemical Co., Ltd. in Example 2 of the present invention; wherein, 1 is the negative test solution of *Saposhnikovia divaricata* (3 μl), 2 is the negative test solution of *Saposhnikovia divaricata* (5 μl), 3 is the negative test solution of *Saposhnikovia divaricata* (7 μl), 4 is the second test solution (3 μl), 5 is the second test solution (5 μl), 6 is the second test solution (7 μl), 7 is the reference medicinal material solution of *Saposhnikovia divaricata* (5 μl), 8 is the 5-O-methylvisamidol glycoside reference solution (2 μl), and 9 is the cimicifuga glycoside reference solution (2 μl);

[0051] Figure 15 The image shows the identification results of the thin film of Saposhnikovia divaricata under low temperature conditions in Example 2 of the present invention; wherein, 1 is the negative test sample solution of Saposhnikovia divaricata, 2 is the second test sample solution, 3 is the reference medicinal material solution of Saposhnikovia divaricata, 4 is the reference solution of 5-O-methylvisamidol glycoside, and 5 is the reference solution of cimicifuga glycoside.

[0052] Figure 16 The image shows the identification results of the thin-layer blotting of Saposhnikovia divaricata under high humidity conditions in Example 2 of this invention; where 1 is the negative test solution of Saposhnikovia divaricata, 2 is the second test solution, 3 is the reference herb solution of Saposhnikovia divaricata, 4 is the reference solution of 5-O-methylvisamidol glycoside, and 5 is the reference solution of cimicifugoside.

[0053] Figure 17This is a thin-layer chromatography (TLC) identification result of licorice using silica gel G thin-layer plates produced by Yantai Chemical Industry Research Institute in Example 2 of the present invention; wherein, 1 is licorice negative test solution (3 μl), 2 is licorice negative test solution (5 μl), 3 is licorice negative test solution (7 μl), 4 is third test solution (3 μl), 5 is third test solution (5 μl), 6 is third test solution (7 μl), 7 is licorice reference material solution (5 μl), and 8 is glycyrrhizin reference solution (2 μl);

[0054] Figure 18 This is a thin-layer chromatography (TLC) identification result of licorice using silica gel G thin-layer plates produced by Qingdao Ocean Chemical Co., Ltd. in Example 2 of the present invention; wherein, 1 is licorice negative test solution (3 μl), 2 is licorice negative test solution (5 μl), 3 is licorice negative test solution (7 μl), 4 is third test solution (3 μl), 5 is third test solution (5 μl), 6 is third test solution (7 μl), 7 is licorice reference material solution (5 μl), and 8 is glycyrrhizin reference solution (2 μl);

[0055] Figure 19 This is a thin-layer chromatography (TLC) identification result of licorice using silica gel G thin-layer plates produced by Qingdao Dingkang Silica G Co., Ltd. in Example 2 of the present invention; wherein, 1 is licorice negative test solution (3 μl), 2 is licorice negative test solution (5 μl), 3 is licorice negative test solution (7 μl), 4 is third test solution (3 μl), 5 is third test solution (5 μl), 6 is third test solution (7 μl), 7 is licorice reference material solution (5 μl), and 8 is glycyrrhizin reference solution (2 μl);

[0056] Figure 20 The image shows the results of thin-layer chromatography identification of licorice under low-temperature conditions in Example 2 of this invention; where 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin.

[0057] Figure 21 This is a thin-layer chromatography identification result of licorice under high humidity conditions in Example 2 of the present invention; wherein, 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin.

[0058] Figure 22 The image shows the thin-layer chromatography identification results of licorice under the condition of heating at a color development temperature of 100℃ for 5 minutes in Example 2 of the present invention; wherein, 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin.

[0059] Figure 23The image shows the thin-layer chromatography identification results of licorice under the color development temperature of 105℃ and heating for 5 min in Example 2 of this invention; where 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin.

[0060] Figure 24 The image shows the thin-layer chromatography identification results of licorice under the color development temperature of 110℃ and heating for 5 min in Example 2 of the present invention; where 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin.

[0061] Figure 25 The image shows the thin-layer chromatography identification results of licorice under the color development temperature of 105℃ and heating for 3 min in Example 2 of the present invention; where 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin.

[0062] Figure 26 The image shows the thin-layer chromatography identification results of licorice under the color development temperature of 105℃ and heating for 5 min in Example 2 of this invention; where 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin.

[0063] Figure 27 The image shows the thin-layer chromatography identification results of licorice under the color development temperature of 105℃ and heating for 7 min in Example 2 of the present invention; where 1 is the negative test solution of licorice, 2 is the third test solution, 3 is the reference solution of licorice, and 4 is the reference solution of glycyrrhizin. Detailed Implementation

[0064] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0065] All raw materials used in the examples are commercially available; the ammonia test solution is commercially available concentrated ammonia solution.

[0066] All instruments used in the examples are commercially available instruments.

[0067] In this embodiment, the preparation method of Qianghuo Shengshi Decoction is as follows: Take Qianghuo, Duhuo, Gaoben, Fangfeng, Gancao, Chuanxiong, and Manjingzi slices and crush them into coarse particles (all passing through a 4-mesh sieve but not a 10-mesh sieve). Weigh out the daily prescription amount of coarse powder (Qianghuo 4.13g, Duhuo 4.13g, Gaoben 2.06g, Fangfeng 2.06g, Gancao 2.06g, Chuanxiong 2.06g, and Manjingzi 1.24g), place them in a decoction pot, add 600ml of drinking water, cover, bring to a boil over high heat, and then decoct until the remaining volume of the decoction is 300ml (±10%). Filter the decoction through a 300-mesh filter cloth while hot and at normal pressure to obtain the final product.

[0068] Example 1 This example relates to the identification method of Qianghuo Shengshi Decoction.

[0069] 1.1 Preparation of the test solution

[0070] Take 40 ml of the decoction of Qianghuo Shengshi Decoction, add petroleum ether and shake to extract twice, each time adding 30 ml of petroleum ether. Combine the two petroleum ether extracts and wash twice with water, each time adding 20 ml of water. Discard the washings to obtain the aqueous extract and the petroleum ether extract. Take the petroleum ether extract, evaporate to dryness, and dissolve the residue in 2 ml of ethyl acetate to obtain the first test solution. Take a portion of the aqueous extract after petroleum ether extraction, add water-saturated n-butanol and shake to extract twice, each time adding 15 ml of water-saturated n-butanol. Combine the two n-butanol extracts and wash twice with n-butanol-saturated water, each time adding 10 ml of n-butanol-saturated water. Discard the washings, evaporate the n-butanol extract to dryness, and dissolve the residue in 1 ml of methanol to obtain the second test solution. Take a portion of the aqueous extract after petroleum ether extraction, add ethyl acetate and shake to extract twice, each time adding 15 ml of ethyl acetate. Combine the two ethyl acetate extracts, wash twice with water, each time adding 10 ml of water. Discard the washings, evaporate the ethyl acetate extract to dryness, dissolve the residue in 1 ml of methanol, and use this as the third test solution.

[0071] 1.2 Preparation of reference medicinal materials

[0072] Take 1.3g of Notopterygium incisum and Angelica pubescens reference herbs, and 0.7g of Ligusticum striatum and Ligusticum chuanxiong reference herbs, respectively. Add 100ml of drinking water to each herb, reflux for 45 minutes, filter while hot, and cool the filtrate. Take 40ml of the filtrate and extract twice with 30ml of petroleum ether each time. Combine the two petroleum ether extracts and wash twice with 20ml of water each time. Discard the washings to obtain the aqueous extract and the petroleum ether extract. Take the petroleum ether extract, evaporate to dryness, and dissolve the residue in 2ml of ethyl acetate to obtain the reference herb solutions of Notopterygium incisum, Angelica pubescens, Ligusticum striatum, and Ligusticum chuanxiong, respectively.

[0073] Take 0.8g of *Saposhnikovia divaricata* reference material, add 100ml of drinking water, reflux for 45 minutes, filter while hot, and cool the filtrate. Take 40ml of the filtrate, add petroleum ether and shake to extract twice, each time adding 30ml of petroleum ether. Combine the two petroleum ether extracts, wash twice with water, each time adding 20ml of water. Discard the washings to obtain the aqueous extract and the petroleum ether extract. Take a portion of the aqueous extract, add water-saturated n-butanol and shake to extract twice, each time adding 15ml of water-saturated n-butanol. Combine the two n-butanol extracts, wash twice with n-butanol-saturated water, each time adding 10ml of n-butanol-saturated water. Discard the washings, evaporate the n-butanol extract to dryness, dissolve the residue in 1ml of methanol to obtain the *Saposhnikovia divaricata* reference material solution.

[0074] Take 0.8g of licorice reference material, add 100ml of drinking water, reflux for 45 minutes, filter while hot, and cool the filtrate. Take 40ml of the filtrate, add petroleum ether and shake to extract twice, each time adding 30ml of petroleum ether. Combine the two petroleum ether extracts, wash twice with water, each time adding 20ml of water. Discard the washings to obtain the aqueous extract and the petroleum ether extract. Take a portion of the aqueous extract, add ethyl acetate and shake to extract twice, each time adding 15ml of ethyl acetate. Combine the two ethyl acetate extracts, wash twice with water, each time adding 10ml of water. Discard the washings, evaporate the ethyl acetate extract to dryness, and dissolve the residue in 1ml of methanol to obtain the licorice reference material solution.

[0075] 1.3 Preparation of reference solution

[0076] Take the reference standards of gentianol, osthol, ligustilide and glycyrrhizin respectively, and add methanol to prepare solutions containing 1 mg per 1 ml, thus obtaining the reference solutions of gentianol, osthol, ligustilide and glycyrrhizin.

[0077] Take cimicifugoside and 5-O-methylvisamilolol reference standards respectively, and add ethanol to prepare solutions containing 1 mg per 1 ml, thus obtaining cimicifugoside reference solution and 5-O-methylvisamilolol reference solution.

[0078] 1.4 Identification Methods

[0079] 1.4.1 Identification of Notopterygium incisum.

[0080] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2025 edition of the Chinese Pharmacopoeia, 2 μl each of the first test sample solution, the Notopterygium incisum reference material solution, and the Notopterygium incisum alcohol reference solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using n-hexane-toluene-ethyl acetate (volume ratio 2:1:2), removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ for 5 minutes, and examined under ultraviolet light (254 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference material and the reference standard.

[0081] 1.4.2 Identification of Angelica pubescens.

[0082] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2025 edition of the Chinese Pharmacopoeia, 2 μl each of the first test sample solution, the Angelica pubescens reference material solution, and the osthol reference solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using a hexane-toluene-ethyl acetate-acetone (volume ratio 4:2:2:0.4), removed, air-dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0083] 1.4.3 Identification of Ligusticum striatum and Ligusticum chuanxiong.

[0084] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2025 edition of the Chinese Pharmacopoeia, 5 μl each of the first test sample solution, the Ligusticum striatum reference material solution, the Ligusticum chuanxiong reference material solution, and the ligustilide reference standard solution were spotted separately onto the same silica gel G thin-layer plate. Cyclohexane-ethyl acetate (volume ratio 9:1) was used as the developing solvent. The plate was then removed, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ for 5 minutes, and examined under ultraviolet light (254 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal materials and the reference standards.

[0085] 1.4.4 Identification of windproofing

[0086] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2025 edition of the Chinese Pharmacopoeia, 5 μl each of the second test sample solution and the Saposhnikovia divaricata reference material solution, and 2 μl each of the Cimicifuga glycoside reference solution and the 5-O-methylvisamidol glycoside reference solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-formic acid-glacial acetic acid-water (volume ratio 15:1:1:2), removed, air-dried, and examined under ultraviolet light (254 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standards.

[0087] 1.4.5 Identification of Licorice

[0088] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2025 edition of the Chinese Pharmacopoeia, 5 μl each of the third test sample solution, the licorice reference material solution, and the glycyrrhizin reference solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-anhydrous ethanol-acetone-ammonia reagent (volume ratio 2:1:2:1), removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ for 5 minutes, and examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatograms of the reference material and the reference standard.

[0089] Example 2 This example involves the methodological verification of the method of the present invention.

[0090] 2.1 Methodological Validation of Notopterygium incisum Thin-Layer Identification

[0091] 2.1.1 Specificity Verification

[0092] The preparation method of Qianghuo Yin-Yang Decoction is as follows: Take the sliced ​​herbs of Angelica pubescens, Ligusticum striatum, Saposhnikovia divaricata, Glycyrrhiza uralensis, Ligusticum chuanxiong, and Vitex trifolia, and crush them into coarse particles (all passing through a 4-mesh sieve but not a 10-mesh sieve). Weigh out the daily prescription amount of coarsely crushed herbs (Angelica pubescens 4.13g, Ligusticum striatum 2.06g, Saposhnikovia divaricata 2.06g, Glycyrrhiza uralensis 2.06g, Ligusticum chuanxiong 2.06g, and Vitex trifolia 1.24g), place them in a decoction pot, add 600ml of drinking water, cover, bring to a boil over high heat, and then decoct until the remaining volume of the decoction is 300ml (±10%). Filter the decoction through a 300-mesh filter cloth while hot and at normal pressure to obtain the final product.

[0093] The preparation method of the Notopterygium incisum negative test solution is as follows: Take 40 ml of Notopterygium incisum negative decoction, add petroleum ether and shake to extract twice, with 30 ml of petroleum ether added each time. Combine the two petroleum ether extracts, wash twice with water, with 20 ml of water added each time, and discard the washings to obtain the aqueous solution and petroleum ether solution after petroleum ether extraction. Take the petroleum ether solution, evaporate to dryness, and dissolve the residue in 2 ml of ethyl acetate to obtain the final product.

[0094] Take 2 μl each of the first test solution, the Notopterygium incisum negative test solution, the Notopterygium incisum reference material solution, and the Notopterygium incisum alcohol reference solution, and spot them separately onto the same silica gel G thin-layer plate (produced by Qingdao Ocean Chemical Plant). Use n-hexane-toluene-ethyl acetate (volume ratio 2:1:2) as the developing solvent, and develop at 26℃ and RH 30%. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light (254nm). The results are as follows. Figure 1 As shown. By Figure 1 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, and there is no negative interference, indicating that the method of the present invention has good specificity.

[0095] 2.1.2 Durability Verification

[0096] 2.1.2.1 Durability verification of thin-layer boards from different manufacturers

[0097] This embodiment is exactly the same as section 2.1.1, except that the manufacturer of the silicone G thin-film plate is different. The silicone G thin-film plate used in this embodiment is manufactured by Yantai Chemical Industry Research Institute. The experimental results using the silicone G thin-film plate produced by Yantai Chemical Industry Research Institute are as follows... Figure 2 As shown.

[0098] 2.1.2.2 Temperature durability verification

[0099] This embodiment is exactly the same as section 2.1.1, except that the temperature is 8℃. The experimental results obtained using this embodiment are as follows: Figure 3 As shown.

[0100] 2.1.2.3 Durability verification under humidity conditions

[0101] This embodiment is exactly the same as section 2.1.1, except that the humidity is 90% (RH90%). The experimental results obtained using this embodiment are as follows: Figure 4 As shown.

[0102] Depend on Figures 2-4 The results show that the brand of silica gel G thin-layer plate, the developing temperature, and the relative humidity have little impact on this method. The main spots in the chromatograms under different conditions are clear, and there is no interference from negative samples. This indicates that the method of the present invention has good robustness.

[0103] 2.2 Methodological Validation of Thin-Layer Identification of Angelica pubescens

[0104] 2.2.1 Specificity Verification

[0105] The preparation method of Angelica pubescens decoction is as follows: Take the slices of Notopterygium incisum, Ligusticum striatum, Saposhnikovia divaricata, Glycyrrhiza uralensis, Ligusticum chuanxiong, and Vitex trifolia, and crush them into coarse particles (all passing through a 4-mesh sieve but not a 10-mesh sieve). Weigh out the daily prescription amount of coarsely crushed slices (Notopterygium incisum 4.13g, Ligusticum striatum 2.06g, Saposhnikovia divaricata 2.06g, Glycyrrhiza uralensis 2.06g, Ligusticum chuanxiong 2.06g, and Vitex trifolia 1.24g), place them in a decoction pot, add 600ml of drinking water, cover, bring to a boil over high heat, and then decoct until the remaining volume of the decoction is 300ml (±10%). Filter the decoction through a 300-mesh filter cloth while hot and at normal pressure to obtain the final product.

[0106] The preparation method of the Angelica pubescens negative test solution is as follows: Take 40 ml of Angelica pubescens negative decoction, add petroleum ether and shake to extract twice, with 30 ml of petroleum ether added each time. Combine the two petroleum ether extracts, wash twice with water, with 20 ml of water added each time, and discard the washings to obtain the aqueous solution and petroleum ether solution after petroleum ether extraction. Take the petroleum ether solution, evaporate to dryness, and dissolve the residue in 2 ml of ethyl acetate to obtain the final product.

[0107] Take 2 μl each of the first test solution, the Angelica pubescens negative test solution, the Angelica pubescens reference herb solution, and the osthol reference solution, and spot them separately onto the same silica gel G thin-layer plate (produced by Yantai Chemical Industry Research Institute). Use n-hexane-toluene-ethyl acetate-acetone (volume ratio 4:2:2:0.4) as the developing solvent, and develop at 24℃ and RH 28%. Remove the plate, air dry, and examine under ultraviolet light (365 nm). The results are as follows. Figure 5 As shown. By Figure 5 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, and there is no negative interference, indicating that the method of the present invention has good specificity.

[0108] 2.2.2 Durability Verification

[0109] 2.2.2.1 Durability verification of thin-layer boards from different manufacturers

[0110] This embodiment is identical to section 2.2.1, except that the manufacturer of the silicone G thin-film plate is different. The silicone G thin-film plate used in this embodiment is manufactured by Qingdao Haiyang Chemical Co., Ltd. The experimental results using the silicone G thin-film plate manufactured by Qingdao Haiyang Chemical Co., Ltd. are as follows: Figure 6 As shown.

[0111] 2.2.2.2 Temperature durability verification

[0112] This embodiment is exactly the same as section 2.2.1, except that the temperature is 8℃. The experimental results obtained using this embodiment are as follows: Figure 7 As shown.

[0113] 2.2.2.3 Durability verification under humidity conditions

[0114] This embodiment is exactly the same as section 2.2.1, except that the humidity is 90%. The experimental results obtained using this embodiment are as follows: Figure 8 As shown.

[0115] Depend on Figures 5-8 The results show that the brand of silica gel G thin-layer plate, the developing temperature, and the relative humidity have little impact on this method. The main spots in the chromatograms under different conditions are clear, and there is no interference from negative samples. This indicates that the method of the present invention has good robustness.

[0116] 2.3 Methodological Validation of Thin-Layer Chromatography Identification of Ligusticum striatum and Ligusticum chuanxiong

[0117] 2.3.1 Specificity Verification

[0118] The preparation method of the decoction of Ligusticum striatum and Ligusticum chuanxiong is as follows: Take the slices of Notopterygium incisum, Angelica pubescens, Saposhnikovia divaricata, Glycyrrhiza uralensis, and Vitex trifolia, and crush them into coarse particles (all passing through a 4-mesh sieve but not a 10-mesh sieve). Weigh out the daily prescription amount of coarsely crushed slices (Notopterygium incisum 4.13g, Angelica pubescens 4.13g, Saposhnikovia divaricata 2.06g, Glycyrrhiza uralensis 2.06g, and Vitex trifolia 1.24g), place them in a decoction pot, add 600ml of drinking water, cover, bring to a boil over high heat, and then decoct until the remaining volume of the decoction is 300ml (±10%). Filter the decoction through a 300-mesh filter cloth while hot and at normal pressure to obtain the final product.

[0119] The preparation method of the negative test solution of Ligusticum striatum and Ligusticum chuanxiong is as follows: Take 40 ml of the negative decoction of Ligusticum striatum and Ligusticum chuanxiong, add petroleum ether and shake to extract twice, with 30 ml of petroleum ether added each time. Combine the two petroleum ether extracts, wash twice with water, with 20 ml of water added each time, discard the washings, and obtain the aqueous solution and petroleum ether solution after petroleum ether extraction. Take the petroleum ether solution, evaporate to dryness, and dissolve the residue in 2 ml of ethyl acetate to obtain the final product.

[0120] Three aliquots of the first test solution (3 μl, 5 μl, and 7 μl respectively), three aliquots of the Ligusticum striatum and Ligusticum chuanxiong negative test solution (3 μl, 5 μl, and 7 μl respectively), 5 μl of Ligusticum striatum reference material solution, 5 μl of Ligusticum chuanxiong reference material solution, and 5 μl of Ligusticum lactone reference solution were spotted separately onto the same silica gel G thin-layer plate (produced by Yantai Chemical Industry Research Institute). Cyclohexane-ethyl acetate (volume ratio 9:1) was used as the developing solvent, and the plate was developed at 26℃ and RH 32%. The plate was then removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ for 5 minutes, and examined under ultraviolet light (254 nm). The results are as follows. Figure 9 As shown. By Figure 9 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, and there is no negative interference, indicating that the method of the present invention has good specificity.

[0121] 2.3.2 Durability Verification

[0122] 2.3.2.1 Durability verification of thin-layer boards from different manufacturers

[0123] This embodiment is identical to section 2.3.1, except that the manufacturer of the silicone G thin-film plate is different. The silicone G thin-film plate used in this embodiment is manufactured by Qingdao Haiyang Chemical Co., Ltd. The experimental results using the silicone G thin-film plate manufactured by Qingdao Haiyang Chemical Co., Ltd. are as follows: Figure 10 As shown.

[0124] 2.3.2.2 Temperature durability verification

[0125] This embodiment is exactly the same as section 2.3.1, except that: first, the temperature is 8℃; second, only one sample of the Ligusticum striatum negative test solution and the first test solution is taken, with a volume of 5 μl for each. The experimental results of the detection using this embodiment are as follows: Figure 11 As shown.

[0126] 2.3.2.3 Durability verification under humidity conditions

[0127] This embodiment is exactly the same as section 2.3.1, except that: first, the humidity is 90%; second, only one sample of both the Ligusticum striatum negative test solution and the first test solution is taken, with a volume of 5 μl each. The experimental results of the detection using this embodiment are as follows: Figure 12 As shown.

[0128] Depend on Figures 9-12 The results show that the sample volume, silica gel G thin-layer plate brand, development temperature, and relative humidity have little impact on this method. The main spots in the chromatograms under different conditions are clear, and there is no interference from negative samples. This indicates that the method of the present invention has good robustness.

[0129] 2.4 Methodological Validation of Windproof Thin-Layer Identification

[0130] 2.4.1 Specificity Verification

[0131] The preparation method of Fangfeng Yin-Yang Decoction is as follows: Take Qianghuo, Duhuo, Gaoben, Gancao, Chuanxiong, and Manjingzi decoction pieces and crush them into coarse particles (all passing through a 4-mesh sieve but not a 10-mesh sieve). Weigh out the daily prescription amount of coarse particles (Qianghuo 4.13g, Duhuo 4.13g, Gaoben 2.06g, Gancao 2.06g, Chuanxiong 2.06g, and Manjingzi 1.24g), place them in a decoction pot, add 600ml of drinking water, cover, bring to a boil over high heat, and then decoct until the remaining volume of the decoction is 300ml (±10%). Filter the decoction through a 300-mesh filter cloth while hot and at normal pressure to obtain the final product.

[0132] The preparation method of the negative test solution for Saposhnikovia divaricata is as follows: Take 40 ml of Saposhnikovia divaricata decoction, add petroleum ether and shake to extract twice, with 30 ml of petroleum ether added each time. Combine the two petroleum ether extracts, wash twice with water, with 20 ml of water added each time, and discard the washings to obtain the aqueous solution and petroleum ether solution after petroleum ether extraction. Take half of the aqueous solution, add water-saturated n-butanol and shake to extract twice, with 15 ml of water-saturated n-butanol added each time. Combine the two n-butanol extracts, wash twice with n-butanol-saturated water, with 10 ml of n-butanol-saturated water added each time, and discard the washings. Evaporate the n-butanol solution to dryness, and dissolve the residue in 1 ml of methanol to obtain the final product.

[0133] Take three aliquots of the second test solution (3 μl, 5 μl, and 7 μl respectively), three aliquots of the Saposhnikovia divaricata negative test solution (3 μl, 5 μl, and 7 μl respectively), 5 μl of Saposhnikovia divaricata reference herb solution, 2 μl of Cimicifuga glycoside reference solution, and 2 μl of 5-O-methylvisamidol glycoside reference solution, and spot them separately onto the same silica gel G thin-layer plate (produced by Yantai Chemical Industry Research Institute). Use ethyl acetate-formic acid-glacial acetic acid-water (volume ratio 15:1:1:2) as the developing solvent, and develop at 27℃ and RH 30%. Remove, air dry, and examine under ultraviolet light (254 nm). The results are as follows. Figure 13 As shown. By Figure 13 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, and there is no negative interference, indicating that the method of the present invention has good specificity.

[0134] 2.4.2 Durability Verification

[0135] 2.4.2.1 Durability verification of thin-layer boards from different manufacturers

[0136] This embodiment is identical to section 2.4.1, except that the manufacturer of the silicone G thin-film plate is different. The silicone G thin-film plate used in this embodiment is manufactured by Qingdao Haiyang Chemical Co., Ltd. The experimental results using the silicone G thin-film plate manufactured by Qingdao Haiyang Chemical Co., Ltd. are as follows: Figure 14 As shown.

[0137] 2.4.2.2 Temperature durability verification

[0138] This embodiment is exactly the same as section 2.4.1, except that: first, the temperature is 8℃; second, only one sample of the negative test solution and the second test solution is taken, and the volume of each is 5μl. The experimental results obtained using this embodiment are as follows: Figure 15 As shown.

[0139] 2.4.2.3 Durability verification under humidity conditions

[0140] This embodiment is exactly the same as section 2.4.1, except that: first, the humidity is 90%; second, only one sample of the windproof negative test solution and the second test solution is taken, and the volume of each is 5 μl. The experimental results obtained using this embodiment are as follows: Figure 16 As shown.

[0141] Depend on Figures 13-16 The results show that the sample volume, silica gel G thin-layer plate brand, development temperature, and relative humidity have little impact on this method. The main spots in the chromatograms under different conditions are clear, and there is no interference from negative samples. This indicates that the method of the present invention has good robustness.

[0142] 2.5 Methodological Validation of Licorice Thin-Layer Chromatography Identification

[0143] 2.5.1 Specificity Verification

[0144] The preparation method of licorice negative-natured decoction is as follows: Take the slices of Notopterygium root, Angelica pubescens root, Ligusticum striatum root, Saposhnikovia divaricata root, Ligusticum chuanxiong rhizome, and Vitex trifolia fruit, and crush them into coarse particles (all passing through a 4-mesh sieve but not a 10-mesh sieve). Weigh out the daily prescription amount of coarsely crushed slices (Notopterygium root 4.13g, Angelica pubescens root 4.13g, Ligusticum striatum root 2.06g, Saposhnikovia divaricata root 2.06g, Ligusticum chuanxiong rhizome 2.06g, Vitex trifolia fruit 1.24g), place them in a decoction pot, add 600ml of drinking water, cover, bring to a boil over high heat, and then decoct until the remaining volume of the decoction is 300ml (±10%). Filter the decoction through a 300-mesh filter cloth while hot and at normal pressure to obtain the final product.

[0145] The preparation method of the licorice negative test solution is as follows: Take 40 ml of licorice negative decoction, add petroleum ether and shake to extract twice, with 30 ml of petroleum ether added each time. Combine the two petroleum ether extracts, wash twice with water, with 20 ml of water added each time, and discard the washings to obtain the aqueous solution and petroleum ether solution after petroleum ether extraction. Take half of the aqueous solution, add ethyl acetate and shake to extract twice, with 15 ml of ethyl acetate added each time. Combine the two ethyl acetate extracts, wash twice with water, with 10 ml of water added each time, and discard the washings. Evaporate the ethyl acetate solution to dryness, and dissolve the residue in 1 ml of methanol to obtain the final product.

[0146] Take three aliquots of the third test sample solution (3 μl, 5 μl, and 7 μl respectively), three aliquots of the licorice negative test sample solution (3 μl, 5 μl, and 7 μl respectively), 5 μl of licorice reference material solution, and 2 μl of glycyrrhizin reference solution, and spot them separately onto the same silica gel G thin-layer plate (produced by Yantai Chemical Industry Research Institute). Use ethyl acetate-anhydrous ethanol-acetone-ammonia test solution (volume ratio 2:1:2:1) as the developing solvent, and develop at 27℃ and RH 35%. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ for 5 minutes, and examine under sunlight. The results are as follows. Figure 17 As shown. By Figure 17 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, and there is no negative interference, indicating that the method of the present invention has good specificity.

[0147] 2.5.2 Durability Verification

[0148] 2.5.2.1 Durability Verification of Thin-Layer Laminates from Different Manufacturers

[0149] This embodiment is identical to section 2.5.1, except that the manufacturers of the silicone G thin-film plates are different. The silicone G thin-film plates used in this embodiment are from two different manufacturers: Qingdao Haiyang Chemical Co., Ltd. and Qingdao Dingkang Silicone Co., Ltd. The experimental results using the silicone G thin-film plates produced by Qingdao Haiyang Chemical Co., Ltd. are as follows: Figure 18 As shown, the experimental results of using silicone G thin-layer plates produced by Qingdao Dingkang Silicone Co., Ltd. are as follows. Figure 19 As shown.

[0150] 2.5.2.2 Temperature durability verification

[0151] This embodiment is exactly the same as section 2.5.1, except that: first, the temperature is 8°C; second, only one sample of the licorice negative test solution and the third test solution is taken, each with a volume of 5 μl. The experimental results obtained using this embodiment are as follows: Figure 20 As shown.

[0152] 2.5.2.3 Durability verification under humidity conditions

[0153] This embodiment is exactly the same as section 2.5.1, except that: first, the humidity is 90%; second, only one sample of the licorice negative test solution and the third test solution is taken, each with a volume of 5 μl. The experimental results obtained using this embodiment are as follows: Figure 21 As shown.

[0154] 2.5.2.4 Durability verification of color development temperature

[0155] This embodiment is exactly the same as section 2.5.1, except that: first, only one sample of the licorice negative test solution and the third test solution is taken, each with a volume of 5 μl; second, the color development temperatures are 100℃, 105℃, and 110℃, respectively. The experimental results obtained using this embodiment are as follows: Figures 22-24 As shown.

[0156] 2.5.2.5 Durability verification of color development time

[0157] This embodiment is exactly the same as section 2.5.1, except that: first, only one sample of the licorice negative test solution and the third test solution is taken, each with a volume of 5 μl; second, the color development times are 3 min, 5 min, and 7 min, respectively. The experimental results obtained using this embodiment are as follows: Figures 25-27 As shown.

[0158] Depend on Figures 18-27 The results show that the sample volume, silica gel G thin-layer plate brand, development temperature, relative humidity, color development temperature, and color development time have little impact on this method. Chromatograms under different conditions all show clear main spots and no negative interference. This indicates that the method of this invention has good robustness.

Claims

1. A method for identifying Notopterygium for dampness granules decoction, characterized in that, Comprising: S1: the Notopterygium Dampness-Relieving Granules decoction is extracted with petroleum ether and washed with water, the washings are discarded, and a petroleum ether extract and a water extract are obtained; The petroleum ether extract is evaporated to dryness, the residue is dissolved in ethyl acetate to obtain a first test sample solution; part of the water extract is extracted with water-saturated n-butanol and washed with n-butanol saturated water, the n-butanol extract is evaporated to dryness, the residue is dissolved in methanol to obtain a second test sample solution; part of the water extract is extracted with ethyl acetate and washed with water, the ethyl acetate extract is evaporated to dryness, and the residue is dissolved in methanol to obtain a third test sample solution; S2: Notopterygium control drug solution, Angelica control drug solution, Ligusticum control drug solution, Chuanxiong control drug solution, Saposhnikovia control drug solution, Glycyrrhiza control drug solution, Notopterygium alcohol control solution, Cnidium monnieri element control solution, Ligusticum lactone control solution, Glycyrrhizin control solution, Rhizoma Coptidis glycoside control solution, 5-O-methyl Vismia alcohol glycoside control solution are prepared respectively; S3: the first test sample solution, Notopterygium control drug solution, Notopterygium alcohol control solution are respectively spotted on silica gel G thin layer plate, developed with the first developing agent, colored, and Notopterygium is identified; the first test sample solution, Angelica control drug solution, Cnidium monnieri element control solution are respectively spotted on silica gel G thin layer plate, developed with the second developing agent, colored, and Angelica is identified; the first test sample solution, Ligusticum control drug solution, Chuanxiong control drug solution, Ligusticum lactone control solution are respectively spotted on silica gel G thin layer plate, developed with the third developing agent, colored, and Ligusticum and Chuanxiong are identified; the second test sample solution, Saposhnikovia control drug solution, Rhizoma Coptidis glycoside control solution, 5-O-methyl Vismia alcohol glycoside control solution are respectively spotted on silica gel G thin layer plate, developed with the fourth developing agent, colored, and Saposhnikovia is identified; the third test sample solution, Glycyrrhiza control drug solution, Glycyrrhizin control solution are respectively spotted on silica gel G thin layer plate, developed with the fifth developing agent, colored, and Glycyrrhiza is identified.

2. The authentication method according to claim 1, characterized in that, In step S1, the volume ratio of Notopterygium Dampness-Relieving Granules decoction to petroleum ether is 1:0.5-1; and / or, when preparing the first test sample solution, the volume ratio of ethyl acetate to Notopterygium Dampness-Relieving Granules decoction is 20-25:1; and / or, when preparing the second test sample solution, the volume ratio of water extract to water-saturated n-butanol is 1:0.8-1.2; the volume ratio of water extract to methanol is 20-25:1; and / or, when preparing the third test sample solution, the volume ratio of water extract to ethyl acetate is 1:0.8-1.2; the volume ratio of water extract to methanol is 20-25:

1.

3. The authentication method according to claim 1, characterized by, Step S2 comprises: Notopterygium control drug, Angelica control drug, Ligusticum control drug and Chuanxiong control drug are taken respectively, the filtrate after reflux extraction is filtered, and Notopterygium control drug solution, Angelica control drug solution, Ligusticum control drug solution and Chuanxiong control drug solution are prepared according to the preparation method of the first test sample solution; Saposhnikovia control drug is taken, the filtrate after reflux extraction is filtered, and Saposhnikovia control drug solution is prepared according to the preparation method of the second test sample solution; Glycyrrhiza control drug is taken, the filtrate after reflux extraction is filtered, and Glycyrrhiza control drug solution is prepared according to the preparation method of the third test sample solution.

4. The authentication method according to claim 1, characterized by, Step S2 comprises: Take the control sample of Ziyol, Osthole, Ligusticum Chalcone and Liquiritin respectively, and add methanol to obtain the control sample solution of Ziyol, Osthole, Ligusticum Chalcone and Liquiritin respectively; take the control sample of Rhizoma Cimicifugae Glycoside and 5-O-methyl Vismi Alcohol Glycoside respectively, and add ethanol to obtain the control sample solution of Rhizoma Cimicifugae Glycoside and 5-O-methyl Vismi Alcohol Glycoside respectively.

5. The authentication method according to any one of claims 1 to 4, characterized in that, In step S3, The first developing agent is n-hexane-toluene-ethyl acetate, and the volume ratio thereof is preferably 1.8-2.2:1:1.8-2.

2.

6. The authentication method according to any one of claims 1 to 4, characterized by, In step S3, The second developing agent is n-hexane-toluene-ethyl acetate-acetone, and the volume ratio thereof is preferably 3.6-4.4:1.8-2.2:1.8-2.2:0.

4.

7. The method of any one of claims 1 to 4, wherein, In step S3, The third developing agent is cyclohexane-ethyl acetate, and the volume ratio thereof is preferably 8.5-9.5:

1.

8. The authentication method according to any one of claims 1 to 4, characterized by, In step S3, The fourth developing agent is ethyl acetate-formic acid-glacial acetic acid-water, and the volume ratio thereof is preferably 14-16:1:1:1.8-2.

2.

9. The authentication method according to any one of claims 1 to 4, characterized by, In step S3, The fifth developing agent is ethyl acetate-anhydrous ethanol-acetone-ammonia solution, and the volume ratio thereof is preferably 1.8-2.2:1:1.8-2.2:

1.

10. The authentication method according to claim 1, characterized by, In step S3, When identifying Rhizoma Notopterygii, after development, spray with color developing agent, heat until the spots are clear, and then observe under ultraviolet light; and / or, When identifying Rhizoma Angelicae Pubescentis, after development, observe under ultraviolet light; and / or, When identifying Ligusticum and Chuanxiong, after development, spray with color developing agent, heat until the spots are clear, and then observe under ultraviolet light; and / or, When identifying Saposhnikovia, after development, observe under ultraviolet light; and / or, When identifying Radix Glycyrrhizae, after development, spray with color developing agent, heat until the spots are clear, and then observe under sunlight.

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