Thin-layer identification method for ligusticum wallichii and angelica sinensis in body pain and stasis removing decoction

Abstract

The invention provides a thin-layer identification method for ligusticum wallichii and angelica sinensis in Tongtongzhi decoction, and belongs to the technical field of drug detection, the thin-layer identification method is characterized in that petroleum ether (60-90 DEG C)-ethyl acetate with the volume ratio of 8.8-9.2: 1 is used as a developing solvent, and the ligusticum wallichii and angelica sinensis in the Tongtongzhi decoction are identified by thin-layer chromatography. Compared with a high performance liquid chromatography identification method, the thin-layer identification method for the ligusticum wallichii and the angelica sinensis in the body pain and stasis removing decoction has the advantages of simple equipment, convenience in operation, small sample dosage, high analysis speed and the like.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a thin layer identification method of chuanxiong and angelica in shentongzhuyu decoction. Background Art

[0002] Shentong Zhuyu Decoction is a traditional Chinese medicine prescription, and its composition formula mainly includes the following traditional Chinese medicines: Gentiana macrophylla, peach kernel, safflower, liquorice, notopterygium root, Chuanxiong, myrrh, angelica root, aconiti lobata, cyperus rotundus, Achyranthes bidentata, earthworm. Shentong Zhuyu Decoction has the effects of promoting blood circulation and removing blood stasis, dispelling wind and dampness, warming the meridians and dispersing cold, dredge meridians and relieve pain, and replenishing blood and qi. It is often used clinically to treat symptoms such as low back and leg pain, chest pain, headache, and dysmenorrhea caused by qi stagnation and blood stasis.

[0003] Thin layer chromatography is an important chromatographic analysis technique. It is based on the principle of adsorption and uses the interaction between the stationary phase and the mobile phase to separate, qualitatively and quantitatively analyze the components in a mixture. Thin layer chromatography analysis has the advantages of simple equipment, easy operation, small sample dosage, and fast analysis speed.

[0004] However, there is no related research on the identification of the effective components of Chuanxiong and Angelica in Shentong Zhuyu Decoction by thin layer chromatography. Although there are methods for detecting Chuanxiong by thin layer chromatography and Angelica by thin layer chromatography, the ingredients in Shentong Zhuyu Decoction are complex, and the ingredients contained therein are obviously more types than those contained in Chuanxiong or Angelica. At the same time, there are many similar ingredients in Chuanxiong and Angelica, and these ingredients in Shentong Zhuyu Decoction are difficult to separate, which easily form negative interference, and it is difficult to achieve double negative without interference. Therefore, the thin layer chromatography identification method for detecting Chuanxiong directly or the thin layer chromatography identification method for detecting Angelica directly cannot detect Chuanxiong and Angelica in Shentong Zhuyu Decoction, and there will be obvious negative interference, and it is impossible to achieve double negative without interference.

[0005] Therefore, how to establish a method for detecting Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction using thin layer chromatography has far-reaching significance for the quality control of Shentong Zhuyu Decoction. Summary of the invention

[0006] In view of the above problems, the present invention provides a thin layer identification method for chuanxiong and angelica in shentongzhuyu decoction.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] The invention discloses a thin layer chromatography identification method for chuanxiong and angelica in shentong zhuyu decoction. The thin layer chromatography identification method uses petroleum ether (60-90°C)-ethyl acetate with a volume ratio of 8.8-9.2:1 as a developing solvent and adopts thin layer chromatography to identify chuanxiong and angelica in shentong zhuyu decoction.

[0009] Furthermore, in the thin layer identification process, the test solution used for identification is prepared by adding anhydrous ethanol to Shentong Zhuyu Decoction, ultrasonically treating, filtering, evaporating the filtrate, and dissolving the residue in anhydrous ethanol.

[0010] Furthermore, in the preparation of the test solution, the weight-to-volume ratio of Shentong Zhuyu Decoction to anhydrous ethanol for ultrasonic treatment was 1 g: 7.5-12.5 mL;

[0011] The weight-to-volume ratio of the Shentong Zhuyu Decoction to the anhydrous ethanol used to dissolve the residue is 1 g: 0.45-0.55 mL.

[0012] Furthermore, during the thin layer identification process, ligustilide, ligusticum chuanxiong or angelica sinensis were used as controls.

[0013] Furthermore, the thin layer chromatography was observed under a 365 nm ultraviolet lamp.

[0014] Furthermore, the thin layer identification method comprises the following steps:

[0015] S1. Prepare the test solution with Shentongzhuyu decoction;

[0016] S2. Take the test sample solution and spot it on the thin layer plate. After unfolding and drying, place it under a 365nm ultraviolet lamp for inspection.

[0017] Further, in step S2, the amount of the test solution spotted on the thin layer plate is 0.8 to 1.5 μL;

[0018] The unfolded extension distance is 5 to 20 cm.

[0019] Furthermore, the thin layer identification method also includes preparing a reference solution using ligustilide and identifying it using thin layer chromatography.

[0020] Furthermore, the content of ligustilide in the reference solution is 0.013-0.020 μL / mL.

[0021] Furthermore, the thin layer chromatography identification method further comprises using angelica to prepare an angelica reference medicinal material solution, and using thin layer chromatography for identification;

[0022] The reference solution of Chuanxiong was prepared using Chuanxiong and identified by thin layer chromatography;

[0023] The negative control substance lacking Chuanxiong was used to prepare the negative sample solution lacking Chuanxiong, and the solution was identified by thin layer chromatography; wherein the negative control substance lacking Chuanxiong was made of Radix Gentianae Macrophyllae, Semen Persicae, Carthamus tinctorius, Radix Glycyrrhizae, Radix Notopterygii, Myrrha Vinegar, Radix Angelicae Sinensis, Rhizoma Aconiti Lateralis Preparata, Rhizoma Cyperi, Radix Achyranthis Bidentatae and Pheretima;

[0024] The negative control substance lacking angelica was used to prepare the negative sample solution lacking angelica, and the solution was identified by thin layer chromatography; wherein the negative control substance lacking angelica was made of gentiana macrophylla, chuanxiong rhizome, peach kernel, safflower, liquorice, notopterygium wilfordii, vinegar myrrh, vinegar aconiti lobata, cyperus rotundus, achyranthes bidentata and earthworm;

[0025] Negative sample solutions lacking chuanxiong and angelica were prepared using negative control substances lacking chuanxiong and angelica, and identified by thin layer chromatography; the negative control substance lacking angelica was made of gentiana macrophylla, peach kernel, safflower, liquorice, notopterygium wilfordii, vinegar myrrh, vinegar aconiti lobata, cyperus rotundus, Achyranthes bidentata and earthworm.

[0026] The beneficial effects of the thin layer identification method of Chuanxiong and Angelica sinensis in the Shentong Zhuyu Decoction of the present invention are:

[0027] The present invention adopts thin layer chromatography to detect ligustilide (the main identification component in Chuanxiong and Angelica sinensis) in Shentong Zhuyu Decoction, with good overall separation effect, clear spots, no double negative interference, strong specificity, good durability, and can accurately show the characteristics of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction, providing a good basis for the quality control of Shentong Zhuyu Decoction;

[0028] The present invention can effectively separate ligustilide in Shentong Zhuyu Decoction from components with similar solubility and polarity (and double negative without interference) by selecting a specific developing agent, thereby obtaining a method for detecting ligustilide in Shentong Zhuyu Decoction by thin layer chromatography, thereby effectively monitoring the dissolution of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction, and providing a basis for quality control of Shentong Zhuyu Decoction.

[0029] The present invention can effectively separate and detect ligustilide in Shentong Zhuyu Decoction by selecting a specific development system, thereby greatly improving the detection efficiency of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction;

[0030] Compared with the high performance liquid chromatography identification method, the thin layer identification method of Chuanxiong and Angelica sinensis in the Shentong Zhuyu Decoction of the present invention has the advantages of simple equipment, convenient operation, small sample dosage, fast analysis speed and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the thin layer identification result of Shentong Zhuyu Decoction in the screening process using the development system in Example 1 of the present invention;

[0032] Figure 2 It is the thin layer identification result of Shentong Zhuyu Decoction in the developing agent screening process in Example 1 of the present invention;

[0033] Figure 3 It is the thin layer identification result of Shentong Zhuyu Decoction in the process of screening the solvent preparation method of Example 1 of the present invention;

[0034] Figure 4This is the thin layer identification result of Shentong Zhuyu Decoction in the chromatographic spot investigation process of Example 1 of the present invention;

[0035] Figure 5 It is the TLC identification result of the Shentong Zhuyu Decoction in Example 2 of the present invention;

[0036] Figure 6 This is the thin layer identification result using Yantai Zhifu silica gel G plate in Example 7 of the present invention;

[0037] Figure 7 This is the thin layer identification result using the Yinlong silica gel G plate in Example 7 of the present invention;

[0038] Figure 8 This is the result of thin layer identification using Qingdao silica gel G plate in Example 7 of the present invention;

[0039] Fig. 9 is the thin layer identification result in low temperature environment in Example 7 of the present invention;

[0040] Fig.10 is the thin layer identification result in Example 7 of the present invention under a high humidity environment;

[0041] Figure 11 to Figure 12 This is the TLC identification result of 30 different batches of Shentong Zhuyu Decoction in Example 7 of the present invention. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0043] Example 1 Screening of TLC Identification Methods of Chuanxiong and Angelica in Shentong Zhuyu Decoction

[0044] In this embodiment, the substance standard of Shentong Zhuyu Decoction is used as the sample to be tested, wherein the preparation method of the substance standard of Shentong Zhuyu Decoction is as follows:

[0045] Take 3.75g of Gentiana macrophylla, 7.50g of Chuanxiong, 11.25g of peach kernel (crushed), 11.25g of safflower, 7.50g of liquorice, 3.75g of Notopterygium wilfordii, 7.50g of vinegar myrrh, 11.25g of angelica sinensis, 7.50g of vinegar aconitifolia, 3.75g of Cyperus rotundus, 11.25g of Achyranthes bidentata and 7.50g of earthworm, put them in a casserole (capacity 3000mL), heat with open flame, add 12 times the weight of water for the first time, soak for 30min, boil over high heat and then simmer over low heat 30min, filter with 200-mesh filter cloth while hot; add 10 times the weight of water for the second time, boil over high heat, then simmer for 20min over low heat, filter with 200-mesh filter cloth while hot, combine the filtrates, and concentrate under reduced pressure (70℃, -0.08Mpa~-0.1Mpa) to a concentrate with a feed amount: concentrate volume ratio of about 1:1 (g:mL), and freeze-dry (pre-freezing temperature: -20~-45℃, cold trap temperature: -60~-70℃, vacuum degree: <100Pa) to obtain.

[0046] The negative dry paste powder lacking angelica (i.e., the negative control lacking angelica) is prepared by the same preparation method as the material benchmark of the Shentong Zhuyu Decoction, with the only difference being that angelica is not added to the Chinese medicinal materials used [i.e., only Gentiana macrophylla, Ligusticum chuanxiong, peach kernel (crushed), safflower, liquorice, Notopterygium wilfordii, vinegar myrrh, vinegar aconite, Cyperus rotundus, Achyranthes bidentata and Pheretima].

[0047] The negative dry paste powder lacking Chuanxiong (negative control lacking Chuanxiong) is prepared by the same preparation method as the material benchmark of Shentong Zhuyu Decoction, with the only difference being that Chuanxiong is not added to the Chinese medicinal materials used [i.e., only Qinjiao, peach kernel (crushed), safflower, liquorice, Qianghuo, vinegar myrrh, angelica, vinegar aconite, Cyperus rotundus, Achyranthes bidentata and earthworm are added].

[0048] The negative dry paste powder lacking Chuanxiong and Angelica sinensis (negative control lacking Chuanxiong and Angelica sinensis) is prepared by the same preparation method as the material benchmark of Shentong Zhuyu Decoction, with the only difference being that Chuanxiong and Angelica sinensis are not added to the Chinese medicinal materials used [i.e., only Gentiana macrophylla, peach kernel (crushed), safflower, liquorice, Notopterygium wilfordii, vinegar myrrh, vinegar aconite, Cyperus rotundus, Achyranthes bidentata and Pheretima japonica] are added.

[0049] The above-mentioned Shentong Zhuyu Decoction material standard was used as the sample to be tested, and the thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction was screened as follows:

[0050] 1. Start system screening

[0051] 1) Solution preparation

[0052] 11) Take 3 g of the substance standard of Shentong Zhuyu Decoction, add 30 mL of water, seal it, treat it with ultrasound for 30 min, filter it, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, add 30 mL of ether to extract twice, 30 mL each time, combine the ether solutions, evaporate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test solution.

[0053] 12) Take the Angelica sinensis negative dry paste powder and prepare the Angelica sinensis negative sample solution in the same way as the test solution.

[0054] Take the dried powder of Lack of Chuanxiong Negative and prepare the Lack of Chuanxiong Negative sample solution in the same way as the test solution.

[0055] Take the dry paste powder lacking Chuanxiong and Angelica sinensis negative, and prepare the sample solution lacking Chuanxiong and Angelica sinensis negative in the same way as the test solution.

[0056] 13) Take 3g of Angelica sinensis powder, add 30mL of water and boil for 30min, filter, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, add 30mL of ether to extract twice, 30mL each time, combine the ether solution, evaporate to dryness, add 1mL of methanol to dissolve the residue, and obtain Angelica sinensis control solution;

[0057] Take 3g of Chuanxiong decoction slice powder, add 30mL of water and boil for 30min, filter, adjust the filtrate to pH 2-3 with dilute hydrochloric acid, add 30mL of ether and extract twice, 30mL each time, combine the ether solution, evaporate to dryness, add 1mL of methanol to dissolve the residue, and obtain Chuanxiong control solution.

[0058] 2) Thin layer chromatography identification

[0059] Toluene, chloroform, ethyl acetate and glacial acetic acid in a volume ratio of 6:5:2:1 were mixed to prepare toluene-chloroform-ethyl acetate-glacial acetic acid (6:5:2:1) as a developing agent.

[0060] According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 3 μL of the test solution, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution were respectively spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate), and the plate was developed at a developing temperature of 26°C and a humidity of 73.0% RH with toluene-chloroform-ethyl acetate-glacial acetic acid (6:5:2:1) as the developing agent. The plate was developed at a developing distance of 8 cm, taken out, dried, and examined under an ultraviolet lamp (365 nm). The results are as follows: Figure 1 shown. Figure 1 The strips numbered 1 to 6 represent the test solution, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution, respectively.

[0061] Depend on Figure 1 It can be seen that each spot R fThe value is large, and the test solution shows the same fluorescent spot at the corresponding position of the chromatogram of the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution. There is a certain negative interference. This method is not feasible. Consider replacing the developing agent for re-identification.

[0062] 2. Developing agent screening

[0063] The developing agent screening was performed using the test solution prepared in the development system screening of step 1, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution, and the Chuanxiong control solution, as follows:

[0064] Petroleum ether (60-90°C) and ethyl acetate were mixed in a volume ratio of 9:1 to prepare petroleum ether (60-90°C)-ethyl acetate (9:1) as a developing solvent.

[0065] According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 3 μL of the test solution, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution were respectively spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate), and the plate was developed at a development temperature of 26.8°C and a humidity of 77.0% RH with petroleum ether (60-90°C)-ethyl acetate (9:1) as the developing agent. The plate was developed with a development distance of 8 cm, taken out, dried, and examined under an ultraviolet lamp (365 nm). The results are as follows: Figure 2 As shown, Figure 2 The strips numbered 1 to 6 represent the test solution, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution, respectively.

[0066] Depend on Figure 2 It can be seen that the test solution shows the same fluorescent spots at the corresponding positions of the chromatograms of the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution, and there is a certain negative interference. This method is not feasible, and it is considered to change the solution preparation method.

[0067] 3. Solution preparation method screening

[0068] 1) Solution preparation

[0069] 11) Take 2 g of the substance base of Shentong Zhuyu Decoction, add 20 mL of anhydrous ethanol, seal, ultrasonicate for 20 min, filter, evaporate the filtrate, add 1 mL of anhydrous ethanol to the residue to dissolve it, and use it as the test solution.

[0070] 12) Take the Angelica sinensis negative dry paste powder and prepare the Angelica sinensis negative sample solution in the same way as the test solution.

[0071] Take the dried powder of Lack of Chuanxiong Negative and prepare the Lack of Chuanxiong Negative sample solution in the same way as the test solution.

[0072] Take the dry paste powder lacking Chuanxiong and Angelica sinensis negative, and prepare the sample solution lacking Chuanxiong and Angelica sinensis negative in the same way as the test solution.

[0073] 13) Take 2 g of Angelica sinensis powder, add 20 mL of anhydrous ethanol, plug, ultrasonicate for 20 min, filter, evaporate the filtrate to dryness, add 1 mL of anhydrous ethanol to the residue to dissolve it, and obtain an Angelica sinensis control solution; wherein the Angelica sinensis powder has not been boiled.

[0074] Take 2g of Chuanxiong slice powder, add 20mL of anhydrous ethanol, seal, ultrasonicate for 20min, filter, evaporate the filtrate to dryness, add 1mL of anhydrous ethanol to the residue to dissolve, and obtain Chuanxiong control solution; wherein the Chuanxiong slice powder is not boiled in water.

[0075] 2) Thin layer chromatography identification

[0076] Take petroleum ether (60-90°C) and ethyl acetate in a volume ratio of 9:1, mix them evenly, and prepare petroleum ether (60-90°C)-ethyl acetate (9:1) as a developing solvent.

[0077] According to the thin layer chromatography method (General Rule 0502 of the 2015 edition of the Chinese Pharmacopoeia), 3 μL of the test solution, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution were respectively spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate), and the plate was developed at a development temperature of 22.8°C and a humidity of 73.0% RH with petroleum ether (60-90°C)-ethyl acetate (9:1) as the developing agent. The plate was developed at a development distance of 8 cm, taken out, dried, and examined under an ultraviolet lamp (365 nm). The results are as follows: Figure 3 As shown, Figure 3 The strips numbered 1 to 6 represent the test solution, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Chuanxiong, the negative sample solution lacking Chuanxiong and Angelica sinensis, the Angelica sinensis control solution and the Chuanxiong control solution, respectively.

[0078] Depend on Figure 3 It can be seen that the test solution shows the same fluorescent spots at the corresponding positions of the chromatograms of the Angelica sinensis control solution and the Chuanxiong rhizome control solution, and there is no interference in the double negatives, so this method is feasible.

[0079] However, when preparing the Angelica sinensis control solution and the Chuanxiong rhizome control solution in the above method, the Angelica sinensis decoction slice powder and the Chuanxiong decoction slice powder were not boiled in water, while the Angelica sinensis and Chuanxiong rhizome in the Shentong Zhuyu Decoction dry paste powder were boiled in water. Therefore, the Angelica sinensis control medicinal material solution boiled in water and the Chuanxiong rhizome control medicinal material solution boiled in water were added for comparison.

[0080] Meanwhile, since the spots in the chromatograms of the Angelica sinensis control solution and the Chuanxiong rhizome control solution were relatively large, the spotting amounts of the Angelica sinensis control solution and the Chuanxiong rhizome control solution were adjusted.

[0081] 4. Chromatographic spot inspection

[0082] 1) Solution preparation

[0083] 11) Take 2 g of the substance base of Shentong Zhuyu Decoction, add 20 mL of anhydrous ethanol, seal, ultrasonicate for 20 min, filter, evaporate the filtrate, add 1 mL of anhydrous ethanol to the residue to dissolve it, and use it as the test solution.

[0084] 12) Take the Angelica sinensis negative dry paste powder and prepare the Angelica sinensis negative sample solution in the same way as the test solution.

[0085] Take the dried powder of Lack of Chuanxiong Negative and prepare the Lack of Chuanxiong Negative sample solution in the same way as the test solution.

[0086] Take the dry paste powder lacking Chuanxiong and Angelica sinensis negative, and prepare the sample solution lacking Chuanxiong and Angelica sinensis negative in the same way as the test solution.

[0087] 13) Take 2 g of Angelica sinensis powder, add 20 mL of anhydrous ethanol, plug, ultrasonicate for 20 min, filter, evaporate the filtrate to dryness, add 1 mL of anhydrous ethanol to the residue to dissolve it, and obtain an Angelica sinensis control solution; wherein the Angelica sinensis powder has not been boiled.

[0088] Take 2g of Chuanxiong slice powder, add 20mL of anhydrous ethanol, seal, ultrasonicate for 20min, filter, evaporate the filtrate to dryness, add 1mL of anhydrous ethanol to the residue to dissolve, and obtain Chuanxiong control solution; wherein the Chuanxiong slice powder is not boiled in water.

[0089] 14) Take 2 g of Angelica sinensis powder, add 30 mL of water, boil for 30 min, filter, add 20 mL of anhydrous ethanol, plug, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, add 1 mL of anhydrous ethanol to dissolve the residue, and obtain an Angelica sinensis control medicinal material solution;

[0090] Take 2g of Chuanxiong decoction slice powder, add 30mL of water, boil for 30min, filter, add 20mL of anhydrous ethanol, plug, ultrasonically treat for 20min, filter, evaporate the filtrate to dryness, add 1mL of anhydrous ethanol to the residue to dissolve it, and obtain Chuanxiong control medicinal material solution.

[0091] 15) Take an appropriate amount of ligustilide and add methanol to prepare a reference solution, in which the content of ligustilide is 0.015 μL / mL.

[0092] 2) Thin layer chromatography identification

[0093] Take petroleum ether (60-90°C) and ethyl acetate in a volume ratio of 9:1, mix them evenly, and prepare petroleum ether (60-90°C)-ethyl acetate (9:1) as a developing solvent.

[0094] According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 15 μL of Angelica sinensis control medicinal material solution (Angelica sinensis boiled in water), 1 μL of Angelica sinensis control solution (Angelica sinensis not boiled in water), 5 μL of Chuanxiong control medicinal material solution (Chuanxiong boiled in water), 1 μL of Chuanxiong control solution (Chuanxiong not boiled in water), 1 μL of negative sample solution lacking Chuanxiong and Angelica sinensis, 1 μL of negative sample solution lacking Angelica sinensis, 1 μL of negative sample solution lacking Chuanxiong, 1 μL of reference solution and 1 μL of test solution were spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate), and the plate was developed at a development temperature of 24.3°C and a humidity of 19.3% RH with petroleum ether (60-90°C)-ethyl acetate (9:1) as the developing agent. The plate was developed with a development distance of 8 cm. The plate was taken out, dried in the air, and examined under an ultraviolet lamp (365 nm). The results are as follows: Figure 4 As shown, Figure 4 The strips numbered 1 to 9 represent angelica control medicinal material solution, angelica control solution, Chuanxiong control medicinal material solution, Chuanxiong control solution, negative sample solution lacking Chuanxiong and angelica, negative sample solution lacking angelica, negative sample solution lacking Chuanxiong, reference solution and test solution, respectively.

[0095] Depend on Figure 4 It can be seen that the same fluorescent spots appear at the corresponding positions of the chromatograms of the test solution, Angelica sinensis control medicinal material solution, Angelica sinensis control solution, Chuanxiong control medicinal material solution, Chuanxiong control solution and reference solution, and there is no interference in the double negative. The chromatograms of Angelica sinensis Chuanxiong slices, and the solutions prepared by boiling and not boiling Chuanxiong slices can all show ligustilide, but the spot color intensity of ligustilide in the boiled slices is weaker than that of the unboiled slices. Therefore, ligustilide can be used as a reference for the thin layer identification of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction, and this method is feasible.

[0096] Example 2 A thin layer identification method of Chuanxiong and Angelica in Shentong Zhuyu Decoction

[0097] S1. Prepare test solution and reference solution

[0098] S11. Take 2 g of the substance base of Shentong Zhuyu Decoction, add 20 mL of anhydrous ethanol, seal, treat with ultrasound for 20 min, filter, evaporate the filtrate to dryness, add 1 mL of anhydrous ethanol to the residue to dissolve it, and use it as the test solution.

[0099] S12. Take the Angelica sinensis negative dry paste powder and prepare the Angelica sinensis negative sample solution in the same way as the test solution.

[0100] Take the dried powder of Lack of Chuanxiong Negative and prepare the Lack of Chuanxiong Negative sample solution in the same way as the test solution.

[0101] Take the dry paste powder lacking Chuanxiong and Angelica sinensis negative, and prepare the sample solution lacking Chuanxiong and Angelica sinensis negative in the same way as the test solution.

[0102] S13. Take 2 g of Angelica sinensis powder, add 30 mL of water, boil for 30 min, filter, add 20 mL of anhydrous ethanol to the filtrate, seal, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, add 1 mL of anhydrous ethanol to the residue to dissolve it, and obtain an Angelica sinensis control medicinal material solution.

[0103] Take 2g of Chuanxiong decoction slice powder, add 30mL of water, boil for 30min, filter, add 20mL of anhydrous ethanol to the filtrate, plug, ultrasonically treat for 20min, filter, evaporate the filtrate to dryness, add 1mL of anhydrous ethanol to the residue to dissolve, and obtain Chuanxiong control medicinal material solution.

[0104] S14. Take an appropriate amount of ligustilide and add methanol to prepare a reference solution, in which the content of ligustilide is 0.015 μL / mL.

[0105] S2. Thin layer chromatography identification

[0106] Take petroleum ether (60-90°C) and ethyl acetate in a volume ratio of 9:1, mix them evenly, and prepare petroleum ether (60-90°C)-ethyl acetate (9:1) as a developing solvent.

[0107] According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 15 μL of Angelica sinensis control medicinal material solution, 5 μL of Chuanxiong control medicinal material solution, 1 μL of negative sample solution lacking Chuanxiong and Angelica sinensis, 1 μL of negative sample solution lacking Angelica sinensis, 1 μL of negative sample solution lacking Chuanxiong, 1 μL of test solution and 1 μL of reference solution were respectively spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate), and the plate was developed at a development temperature of 24.3°C and a humidity of 19.3% RH with petroleum ether (60-90°C)-ethyl acetate (9:1) as the developing agent, with a development distance of 8 cm. The plate was taken out, dried, and examined under an ultraviolet lamp (365 nm). The results are as follows: Figure 5 As shown, Figure 5 The strips numbered 1 to 7 represent, respectively, Angelica sinensis control medicinal material solution, Chuanxiong rhizome control medicinal material solution, reference substance solution, test substance solution, negative sample solution lacking Chuanxiong rhizome and Angelica sinensis, negative sample solution lacking Angelica sinensis, and negative sample solution lacking Chuanxiong rhizome.

[0108] Depend on Figure 5It can be seen that the same fluorescent spots are shown at the corresponding positions of the chromatograms of the test solution, the reference Chinese angelica solution, the reference Chinese chuanxiong solution and the reference solution, and the double negatives are without interference. The thin layer identification method of the present invention can be used to identify the Chinese chuanxiong and Chinese angelica in the Shentong Zhuyu Decoction.

[0109] Examples 3 to 6 Thin-layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction

[0110] Examples 3 to 6 are respectively a thin layer identification method for Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction. According to the optimal thin layer identification conditions given in Example 2, the range of conditions that can achieve better identification of ligustilide in Shentong Zhuyu Decoction is further explored to obtain the thin layer identification methods in Examples 3 to 6. The thin layer identification methods of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction in Examples 3 to 6 are basically the same as those in Example 2, and the only difference is that some process parameters are different, as shown in Table 1 for details:

[0111] Table 1 List of process parameters in Examples 3 to 6

[0112]

[0113] The contents and results of other parts of Examples 3 to 6 are the same as those of Example 2 (since other embodiments of the present invention have been able to fully demonstrate the ability to better identify the ligusticum lactone in Shentong Zhuyu Decoction, no drawings are added here), and they will not be repeated here.

[0114] Example 7 Methodological Investigation

[0115] This example investigates the methodological aspects of the thin layer chromatography identification of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction, as follows:

[0116] 1. Investigation of Silicone G Thin Layer Sheets from Different Manufacturers

[0117] According to the method in Example 2, Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, reference solution, test solution (test solution was prepared in triplicate), negative sample solution lacking Chuanxiong and Angelica sinensis, negative sample solution lacking Angelica sinensis, and negative sample solution lacking Chuanxiong were prepared, and the above solutions were used to investigate silica gel G thin layer plates from different manufacturers, as follows:

[0118] According to the thin layer identification method in step S2 of Example 2, the thin layer identification results of the silica gel G thin layer plates from different manufacturers were examined at a development temperature of 21.7°C and a humidity of 19.0% RH. The other conditions were kept unchanged. The thin layer identification results of the silica gel G thin layer plates from different manufacturers are shown in FIG. Figures 6 to 8 . Figures 6 to 8The strip numbers 1 to 9 represent the angelica control medicinal material solution, Chuanxiong control medicinal material solution, reference solution, test solution, test solution, test solution, negative sample solution lacking Chuanxiong and angelica, negative sample solution lacking angelica, and negative sample solution lacking Chuanxiong, that is, strip numbers 4 to 6 represent three parallel test solution.

[0119] Among them, the silica gel G thin layer plates from different manufacturers are: Yantai Zhifu silica gel G plate (manufacturer: Yantai Jiangyou Silica Gel Development Co., Ltd.), Yinlong silica gel G plate (manufacturer: Yantai Weiqi Chemical Products Co., Ltd.), and Qingdao silica gel G plate (manufacturer: Qingdao Ocean Chemical Co., Ltd.).

[0120] Depend on Figures 6 to 8 It can be seen that the thin layer identification method of Chuanxiong and Angelica sinensis in the Shentong Zhuyu Decoction of the present invention is not affected by the manufacturer of the silica gel G thin layer plate.

[0121] 2. Investigation of different development temperatures and humidity

[0122] Using the Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, reference solution, test solution (the test solution was prepared in triplicate), Chuanxiong and Angelica sinensis negative sample solution, Angelica sinensis negative sample solution, and Chuanxiong negative sample solution prepared in step 1 from different manufacturers' silica gel G thin layer plates, different development temperatures and humidities were investigated, as follows:

[0123] 1) Low temperature environment investigation

[0124] According to the thin layer identification method in step S2 of Example 2, the thin layer identification results when the development temperature is 2.8°C and the humidity is 43.0%RH (i.e., low temperature environment) are examined, and the other conditions are kept unchanged. The results are as follows: Fig. 9 As shown, Fig. 9 The strip numbers 1 to 9 represent the angelica control medicinal material solution, Chuanxiong control medicinal material solution, reference solution, test solution, test solution, test solution, negative sample solution lacking Chuanxiong and angelica, negative sample solution lacking angelica, and negative sample solution lacking Chuanxiong, that is, strip numbers 4 to 6 represent three parallel test solution.

[0125] 2) High humidity environment investigation

[0126] According to the thin layer identification method in step S2 of Example 2, the thin layer identification results when the development temperature is 21.6°C and the humidity is 77.6%RH (i.e., high humidity environment) are examined, and the other conditions are kept unchanged. The results are as follows: Fig.10 As shown, Fig.10The strip numbers 1 to 9 represent the angelica control medicinal material solution, Chuanxiong control medicinal material solution, reference solution, test solution, test solution, test solution, negative sample solution lacking Chuanxiong and angelica, negative sample solution lacking angelica, and negative sample solution lacking Chuanxiong, that is, strip numbers 4 to 6 represent three parallel test solution.

[0127] By comparison Figure 6 , Fig. 9 and Fig.10 It is found that the thin layer identification method of Chuanxiong and Angelica sinensis in the Shentong Zhuyu Decoction of the present invention is not affected by the development temperature and humidity.

[0128] 3. Multi-batch verification

[0129] 30 different batches of Shentong Zhuyu Decoction material standards were taken respectively, and 30 different test sample solutions were prepared according to the method in Example 2. Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution and reference product solution were also prepared according to the method in Example 2.

[0130] According to the thin layer chromatography identification method of step S2 in Example 2, 30 different test sample solutions, Angelica sinensis reference medicinal material solutions, Ligusticum chuanxiong reference medicinal material solutions and reference solution were subjected to thin layer chromatography identification. The results are shown in FIG. Figure 11 to Figure 12 . Fig.11 The middle strip number 8 represents the control medicinal material solution of Angelica sinensis, the strip number 9 represents the control medicinal material solution of Chuanxiong, the strip number 10 represents the reference solution, and the strip numbers 1 to 7 and 11 to 18 represent 15 of the 30 different test solutions respectively; Fig.12 The middle strip number 26 represents the Angelica sinensis control medicinal material solution, the strip number 27 represents the Chuanxiong control medicinal material solution, the strip number 28 represents the reference substance solution, and the strip numbers 19 to 25 and 29 to 36 represent the other 15 of the 30 different test solution solutions.

[0131] Depend on Figure 11 to Figure 12 It can be seen that under the same developing solvent conditions, the test solutions prepared from multiple batches of Shentong Zhuyu Decoction can show the same blue fluorescent spots at the corresponding positions of the chromatograms of Angelica sinensis slices, Ligusticum chuanxiong slices and Ligusticum chuanxiong lactone.

[0132] In summary, the development conditions in the thin layer identification method of Chuanxiong and Angelica sinensis in the Shentong Zhuyu Decoction of the present invention are good in durability, strong in specificity, clear in main spots, and without interference in double negatives.

[0133] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A thin layer identification method for Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction, characterized in that: The thin layer identification method uses petroleum ether-ethyl acetate with a volume ratio of 8.8-9.2:1 as a developing solvent, and adopts thin layer chromatography to identify Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction.

2. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to claim 1, characterized in that: In the thin layer identification process, the test solution used for identification is prepared by adding anhydrous ethanol to Shentong Zhuyu Decoction, ultrasonically treating it, filtering it, evaporating the filtrate, and dissolving the residue in anhydrous ethanol.

3. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to claim 2, characterized in that: In the preparation of the test solution, the weight-to-volume ratio of Shentong Zhuyu Decoction to anhydrous ethanol for ultrasonic treatment was 1 g: 7.5-12.5 mL; The weight-to-volume ratio of the Shentong Zhuyu Decoction to the anhydrous ethanol used to dissolve the residue is 1 g: 0.45-0.55 mL.

4. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to any one of claims 1 to 3, characterized in that: During the TLC identification process, ligustilide, ligusticum chuanxiong or angelica were used as controls.

5. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to any one of claims 1 to 3, characterized in that: The TLC was observed under UV light at 365 nm.

6. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to any one of claims 1 to 3, characterized in that: The thin layer identification method comprises the following steps: S1. Prepare the test solution with Shentongzhuyu decoction; S2. Take the test sample solution and spot it on the thin layer plate. After unfolding and drying, place it under a 365nm ultraviolet lamp for inspection.

7. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to claim 6, characterized in that: In step S2, the sample solution is spotted on the thin layer plate in an amount of 0.8 to 1.5 μL; The unfolded extension distance is 5 to 20 cm.

8. The thin layer chromatography identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to any one of claims 1 to 3 and 7, characterized in that: The thin layer identification method also includes preparing a reference solution using ligustilide and identifying it using thin layer chromatography.

9. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to claim 8, characterized in that: The content of ligustilide in the reference solution was 0.013-0.020 μL / mL.

10. The thin layer identification method of Chuanxiong and Angelica sinensis in Shentong Zhuyu Decoction according to claim 1, 2, 3, 7 or 9, characterized in that: The thin layer identification method also includes preparing an angelica reference medicinal material solution using angelica sinensis and identifying it by thin layer chromatography; The reference solution of Chuanxiong was prepared using Chuanxiong and identified by thin layer chromatography; The negative control substance lacking Chuanxiong was used to prepare the negative sample solution lacking Chuanxiong, and the solution was identified by thin layer chromatography; wherein the negative control substance lacking Chuanxiong was made of Radix Gentianae Macrophyllae, Semen Persicae, Carthamus tinctorius, Radix Glycyrrhizae, Radix Notopterygii, Myrrha Vinegar, Radix Angelicae Sinensis, Rhizoma Aconiti Lateralis Preparata, Rhizoma Cyperi, Radix Achyranthis Bidentatae and Pheretima; The negative control substance lacking angelica was used to prepare the negative sample solution lacking angelica, and the solution was identified by thin layer chromatography; wherein the negative control substance lacking angelica was made of gentiana macrophylla, chuanxiong rhizome, peach kernel, safflower, liquorice, notopterygium wilfordii, vinegar myrrh, vinegar aconiti lobata, cyperus rotundus, achyranthes bidentata and earthworm; Negative sample solutions lacking chuanxiong and angelica were prepared using negative control substances lacking chuanxiong and angelica, and identified by thin layer chromatography; the negative control substance lacking angelica was made of gentiana macrophylla, peach kernel, safflower, liquorice, notopterygium wilfordii, vinegar myrrh, vinegar aconiti lobata, cyperus rotundus, Achyranthes bidentata and earthworm.

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