Thin-layer identification method for safflower carthamus in body pain and stasis removing decoction

Abstract

The invention provides a thin-layer identification method for safflower in Tongtongzhu decoction, and belongs to the technical field of medicine detection.The thin-layer identification method comprises the steps that ethyl acetate, methyl alcohol, formic acid and water with the volume ratio being 7: (1.9-2.1): (2.9-3.1): (0.9-1.1) serve as a developing solvent, and the safflower in the Tongtongzhu decoction is identified through a thin-layer chromatography method. According to the method, the hydroxysafflor yellow A (a main identification component in safflower) in the Tongtongzhu decoction is detected by adopting the thin layer chromatography, the overall separation effect is good, spots are clear and do not interfere with one another, the specificity is high, the durability is good, the characteristics of the safflower in the Tongtongzhu decoction can be accurately displayed, and a good basis is provided for quality control of the Tongtongzhu decoction.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a thin layer identification method of safflower 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 research on the identification of the effective ingredients of safflower in Shentong Zhuyu Decoction using thin layer chromatography. Hydroxysafflor yellow A is one of the main components of safflower. Although there is a method of using thin layer chromatography to detect hydroxysafflor yellow A in safflower, due to the complex ingredients in Shentong Zhuyu Decoction, the ingredients contained in it are obviously more diverse than those contained in safflower, and Shentong Zhuyu Decoction contains ingredients with similar solubility and polarity to hydroxysafflor yellow A. Therefore, the thin layer chromatography identification method directly used to detect hydroxysafflor yellow A in safflower cannot detect hydroxysafflor yellow A in Shentong Zhuyu Decoction, and there will be obvious negative interference.

[0005] Therefore, how to establish a method for detecting safflower 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 of safflower in shentongzhuyu decoction.

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

[0008] A thin layer chromatography identification method for safflower in shentong zhuyu soup is to use ethyl acetate-methanol-formic acid-water with a volume ratio of 7:1.9-2.1:2.9-3.1:0.9-1.1 as a developing solvent, and to identify the safflower in shentong zhuyu soup by thin layer chromatography.

[0009] Furthermore, in the thin layer identification process, hydroxysafflor yellow A or safflower was used as a control.

[0010] Furthermore, the thin layer chromatography was observed under a 365 nm ultraviolet lamp.

[0011] Furthermore, the thin layer identification method of safflower in the Shentong Zhuyu Decoction comprises the following steps:

[0012] S1. Prepare the test solution with Shentongzhuyu decoction;

[0013] 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.

[0014] Furthermore, in step S1, the test solution is prepared by adding Shentong Zhuyu Decoction to an acetone aqueous solution, ultrasonically treating the solution, and filtering the solution.

[0015] Furthermore, during the preparation of the test solution, the concentration of acetone in the acetone aqueous solution was 78 vol% to 82 vol%;

[0016] The weight-to-volume ratio of the Shentong Zhuyu Decoction liquid to the acetone aqueous solution is 1 g: 4.5-5.5 mL.

[0017] Further, in step S2, the amount of the test solution spotted on the thin layer plate is 1.5 to 2.5 μL;

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

[0019] Furthermore, the thin layer chromatography identification method of safflower in the Shentong Zhuyu Decoction also includes preparing a reference solution using hydroxysafflor yellow A and identifying it using thin layer chromatography.

[0020] Furthermore, the content of hydroxysafflor yellow A in the reference solution is 0.25-0.35 mg / mL.

[0021] Furthermore, the thin layer chromatography identification method of safflower in the Shentong Zhuyu Decoction also includes preparing a safflower-deficient negative sample solution using a safflower-deficient negative control substance, and identifying it by thin layer chromatography;

[0022] The safflower-deficient negative sample solution is prepared using Radix Gentianae Macrophyllae, Rhizoma Chuanxiong, Rhizoma Pruni, Radix Glycyrrhizae, Radix Notopterygii, Myrrha Vinegar, Radix Angelicae Sinensis, Rhizoma Aconiti Lateralis Preparata, Rhizoma Cyperi, Rhizoma Cyperi and Pheretima.

[0023] The beneficial effects of the thin layer identification method of safflower in the body pain and stasis removal soup of the present invention are:

[0024] The present invention adopts thin layer chromatography to detect hydroxysafflor yellow A (the main identification component in safflower) in Shentong Zhuyu Decoction, which has good overall separation effect, clear spots without mutual interference, strong specificity and good durability, and can accurately show the characteristics of safflower in Shentong Zhuyu Decoction, providing a good basis for the quality control of Shentong Zhuyu Decoction.

[0025] The present invention can effectively separate hydroxysafflor yellow A in Shentong Zhuyu Decoction from other components with similar solubility and polarity (without negative interference) by selecting a specific developing agent, thereby obtaining a method for detecting hydroxysafflor yellow A in Shentong Zhuyu Decoction by thin layer chromatography, so as to monitor the dissolution of safflower in Shentong Zhuyu Decoction, and provide a basis for quality control of Shentong Zhuyu Decoction.

[0026] The present invention can effectively separate and detect hydroxysafflor yellow A in Shentong Zhuyu Decoction by selecting a specific development system, which greatly improves the efficiency of safflower detection in Shentong Zhuyu Decoction.

[0027] Compared with the high performance liquid chromatography identification method, the thin layer identification method of safflower 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

[0028] Figure 1 It is the thin layer identification result of Shentong Zhuyu Decoction in the first step of the system screening process in Example 1 of the present invention;

[0029] Figure 2 It is the thin layer identification result of Shentong Zhuyu Decoction in the second process of the system screening in Example 1 of the present invention;

[0030] Figure 3 It is the thin layer identification result of Shentong Zhuyu Decoction in the process of developing system optimization in Example 1 of the present invention;

[0031] Figure 4 It is the thin layer identification result of Shentong Zhuyu Decoction in the chromatographic spot investigation of Example 1 of the present invention;

[0032] Figure 5 This is the thin layer identification result of Shentong Zhuyu Decoction in the spot sample quantity investigation of Example 1 of the present invention;

[0033] Figure 6 It is the TLC identification result of the Shentong Zhuyu Decoction in Example 2 of the present invention;

[0034] Figure 7 This is the thin layer identification result using the Yinlong silica gel G plate in Example 7 of the present invention;

[0035] Figure 8 This is the thin layer identification result using Yantai Zhifu silica gel G plate in Example 7 of the present invention;

[0036] Fig. 9 This is the result of thin layer identification using Qingdao silica gel G plate in Example 7 of the present invention;

[0037] Fig.10is the thin layer identification result in Example 7 of the present invention under a high humidity environment;

[0038] Fig.11 is the thin layer identification result in low temperature environment in Example 7 of the present invention;

[0039] Figure 12 to Figure 13 This is the TLC identification result of 30 different batches of Shentong Zhuyu Decoction in Example 7 of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] Example 1 Screening of TLC Identification Method of Safflower in Shentong Zhuyu Decoction

[0042] 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:

[0043] 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.

[0044] The safflower-deficient negative dry paste powder (i.e., the safflower-deficient negative control) is made by the same preparation method as the material benchmark of the Shentong Zhuyu Decoction, with the only difference being that safflower is not added to the Chinese medicinal materials used (i.e., only Gentiana macrophylla, Ligusticum chuanxiong, peach kernel (crushed), Licorice, Notopterygium wilfordii, Myrrha vinegar, Angelica sinensis, Aconiti Lateralis preparata, Cyperus rotundus, Achyranthes bidentata and Pheretima japonica are added).

[0045] The above-mentioned Shentong Zhuyu Decoction material standard was used as the sample to be tested, and the thin layer identification method of safflower in Shentong Zhuyu Decoction was screened, as follows:

[0046] 1. Expand system screening

[0047] 1) Solution preparation

[0048] 11) Take 3 g of the substance standard of Shentong Zhuyu Decoction, add 15 mL of water, ultrasonically treat for 30 min, filter, adjust the pH value of the filtrate to 2.0 with dilute hydrochloric acid, extract with 10 mL of ethyl acetate, discard the water layer, wash the ethyl acetate layer with 20 mL of water, take the ethyl acetate solution, evaporate to dryness, add 2 mL of anhydrous ethanol to dissolve the residue, and use it as the test solution.

[0049] 12) Take 3 g of safflower negative dry powder and prepare safflower negative sample solution according to the same method as the above method for preparing the test solution.

[0050] 13) Take 2 g of safflower powder, add 30 mL of water and boil for 30 min, cool and filter, adjust the pH value of the filtrate to 2.0 with dilute hydrochloric acid, extract with 10 mL of ethyl acetate, discard the water layer, wash the ethyl acetate layer with 20 mL of water, take the ethyl acetate solution, evaporate to dryness, add 2 mL of anhydrous ethanol to dissolve the residue, and use it as the safflower solution.

[0051] 2) Thin layer chromatography identification

[0052] Chloroform, acetone and a 98 wt % formic acid aqueous solution in a volume ratio of 10:0.5:0.5 were mixed to obtain chloroform-acetone-98% formic acid (10:0.5:0.5), which was used as a developing agent.

[0053] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2015 edition of the Chinese Pharmacopoeia), 5 μL of the test solution, safflower negative sample solution, and safflower medicinal material solution were taken and spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate) respectively. Under the conditions of developing temperature of 24.3°C and humidity of 72.0% RH, chloroform-acetone-98% formic acid (10:0.5:0.5) was used as the developing agent. The plate was developed with a development distance of 8 cm. The plate was taken out, dried, and examined under ultraviolet light (254 nm). The results are shown in Table 1. Figure 1 . Figure 1 The strip numbers 1 to 2 in the middle represent the test sample solutions, the strip numbers 3 to 4 represent the safflower negative sample solutions, and the strip number 5 represents the safflower medicinal material solution.

[0054] Depend on Figure 1It can be seen that the chromatograms of the test solution and the chromatograms of the negative sample solution lacking safflower show spots of the same color at the corresponding positions of the chromatograms of the safflower medicinal material solution, indicating that the negative has interference (i.e., Shentong Zhuyu Decoction contains components with similar solubility and polarity to those in safflower, which cannot be separated), and the separation is poor, so this method is not feasible. Consider changing the solution preparation method and the development system agent for re-identification.

[0055] 2. Expand system screening 2

[0056] 1) Solution preparation

[0057] 11) Take 2 g of the substance base of Shentong Zhuyu Decoction, add 10 mL of 80 vol% acetone aqueous solution, ultrasonically treat for 10 min, filter, and use as the test solution.

[0058] 12) Take the safflower negative dry paste powder and prepare the safflower negative sample solution in the same way as the test solution.

[0059] 13) Take 1 g of safflower powder, add 10 mL of 80 vol% acetone aqueous solution, ultrasonically treat for 10 min, and filter to obtain a safflower solution.

[0060] 2) Thin layer chromatography identification

[0061] Take ethyl acetate, methanol, formic acid and water in a volume ratio of 7:2:3:0.4, mix them evenly, and prepare ethyl acetate-methanol-formic acid-water (7:2:3:0.4) as a developing solvent.

[0062] According to the thin layer chromatography method (General Rule 0502 of the 2015 edition of the Chinese Pharmacopoeia), 5 μL of the test solution, safflower negative sample solution and safflower medicinal material solution were taken and spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate), and the plate was developed at a temperature of 24.3°C and a humidity of 72.0% RH with ethyl acetate-methanol-formic acid-water (7:2:3:0.4) as the developing agent. The plate was developed with a development distance of 8 cm. The plate was taken out, dried and examined under ultraviolet light (365 nm). The results are as follows: Figure 2 As shown, Figure 2 The strips numbered 1 to 3 represent the test solution, safflower negative sample solution and safflower medicinal material solution respectively.

[0063] Depend on Figure 2 It can be seen that the test solution shows the same yellow-green fluorescent spot at the corresponding position of the chromatogram of the safflower medicinal material solution, and there is no interference in the negative, but the main spot is severely tailed, so the developer is adjusted for exploration. The chromatogram of the test solution and the main spot of the chromatogram of the safflower medicinal material solution are tailing, so the amount of sample is reduced for exploration. At the same time, the method is optimized by diluting and reducing the amount of sample.

[0064] 3. Carry out system optimization

[0065] The development system was optimized using the test solution, safflower-deficient negative sample solution and safflower medicinal material solution prepared in the development system screening step 2, as follows:

[0066] Take 1 mL of safflower medicinal material solution, add 80 vol% acetone aqueous solution to dilute to 5 mL, and obtain a control medicinal material solution.

[0067] The preparation of the test solution and safflower-deficient negative sample solution is the same as step 2.

[0068] The developing agent was adjusted by mixing ethyl acetate, methanol, formic acid and water in a volume ratio of 7:2:3:1 to obtain ethyl acetate-methanol-formic acid-water (7:2:3:1) as the developing agent.

[0069] According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 2 μL of the test solution, safflower negative sample solution and control medicinal material solution (i.e. the spot volume was adjusted to 2 μL) 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 24.3°C and a humidity of 72.0% RH, using ethyl acetate-methanol-formic acid-water (7:2:3:1) 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 3 As shown, Figure 3 The strips numbered 1 to 3 represent the test solution, safflower-deficient negative sample solution and control medicinal material solution respectively.

[0070] Depend on Figure 3 It can be seen that the test solution shows the same color fluorescent spot at the corresponding position of the chromatogram of the control medicinal material solution, and there is no interference with the negative. Therefore, ethyl acetate-methanol-formic acid-water (7:2:3:1) was determined to be used as the developing agent. However, since the spots shown in the chromatogram of the control medicinal material solution are different from those shown in the chromatogram of the test solution, the study of preparing medicinal material samples after boiling safflower medicinal materials in water was added.

[0071] 4. Chromatographic spot inspection

[0072] Add research on preparing medicinal material samples and reference materials after boiling safflower medicinal materials, as follows:

[0073] 1) Solution preparation

[0074] Take 1g of safflower powder, add 15mL of water, boil for 30min, filter, evaporate the filtrate, add 10mL of 80vol% acetone aqueous solution, ultrasonically treat for 10min, filter, take 1mL of the obtained filtrate, add 80vol% acetone aqueous solution to dilute to 5mL, and obtain the safflower boiled sample solution.

[0075] Take an appropriate amount of hydroxysafflor yellow A and add methanol to prepare a reference solution, in which the content of hydroxysafflor yellow A is 0.3 mg / mL.

[0076] The preparation of the control medicinal material solution, the test sample solution and the safflower-deficient negative sample solution is the same as in step three.

[0077] 2) Thin layer chromatography identification

[0078] According to the thin layer identification method in the development system optimization of step 3, the thin layer identification results of the reference solution, safflower boiled sample solution (safflower boiled), control medicinal material solution (safflower not boiled), test solution and safflower negative sample solution with a spot volume of 2 μL were investigated at a development temperature of 20.7°C and a humidity of 41.6% RH, and the other conditions were kept unchanged. The results are as follows Figure 4 As shown, Figure 4 The strips numbered 1 to 5 represent the reference solution, safflower boiled sample solution, reference medicinal material solution, test solution and safflower-deficient negative sample solution, respectively.

[0079] Depend on Figure 4 It can be seen that the test solution shows the same color fluorescent spots at the corresponding positions of the chromatograms of hydroxysafflor yellow A in the reference solution, safflower boiled sample solution and reference medicinal material solution, and there is no interference in the negative. And the safflower boiled sample solution only shows one spot that is the same as hydroxysafflor yellow A.

[0080] After comprehensive consideration, the thin layer identification of safflower in Shentong Zhuyu Decoction was studied only with hydroxysafflor yellow A as the reference. However, since the spot of hydroxysafflor yellow A in the reference solution was large, the spotting volume of the reference solution was adjusted to 1 μL and the experiment was repeated.

[0081] 5. Sample quantity inspection

[0082] Based on the reference solution, test solution and safflower-deficient negative sample solution prepared in step 4, the spotting amount was investigated as follows:

[0083] According to the thin layer identification method in the development system optimization of step 3, the thin layer identification results of different sample amounts were examined at a development temperature of 25.9°C and a humidity of 19.3% RH, while keeping the other conditions unchanged. The results are as follows Figure 5 As shown, Figure 5The strips numbered 1 to 5 respectively represent the reference solution, the test solution, the test solution, the test solution and the safflower-deficient negative sample solution (i.e., the strips numbered 2 to 4 all represent the test solution, the strip numbered 1 represents the reference solution, and the strip numbered 5 represents the safflower-deficient negative sample solution).

[0084] Among them, the different spotting volumes include the spotting volume of the reference solution of 1 μL, and the spotting volume of the three parallel test solutions and the safflower-deficient negative sample solution is 2 μL.

[0085] Depend on Figure 5 It can be seen that the test solution shows fluorescent spots of the same color at the corresponding positions of the hydroxysafflor yellow A chromatography in the reference solution, and there is no negative interference (that is, the safflower-deficient negative sample solution does not show spots of the same color at the corresponding positions). This method is feasible.

[0086] Example 2 A thin layer identification method of safflower in Shentong Zhuyu Decoction

[0087] S1. Prepare the test solution, safflower-deficient negative sample solution and reference solution

[0088] S11. Take 2 g of the substance base of Shentong Zhuyu Decoction, add 10 mL of 80 vol% acetone aqueous solution, treat with ultrasound for 10 min, filter and use this as the test solution.

[0089] S12. Take the safflower negative dry paste powder and prepare a safflower negative sample solution in the same way as the test solution.

[0090] S13. Take an appropriate amount of hydroxysafflor yellow A and add methanol to prepare a reference solution, wherein the content of hydroxysafflor yellow A is 0.3 mg / mL.

[0091] S2. Thin layer chromatography identification

[0092] Take ethyl acetate, methanol, formic acid and water in a volume ratio of 7:2:3:1, mix them evenly, and prepare ethyl acetate-methanol-formic acid-water (7:2:3:1) as a developing solvent.

[0093] According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 1 μL of the reference solution was taken, and 2 μL of each of the three parallel test solution and safflower-deficient negative sample solution were taken, and spotted on the same silica gel G thin layer plate (Yantai Zhifu silica gel G plate), and developed at a development temperature of 25.9°C and a humidity of 19.3% RH, using ethyl acetate-methanol-formic acid-water (7:2:3:1) as the developing agent, with a development distance of 8 cm. The plate was taken out, dried, and examined under ultraviolet light (365 nm). The results are as follows: Figure 6 As shown, Figure 6The strips numbered 5 in the figure represent the reference solution, the test solution, the test solution, the test solution and the safflower-deficient negative sample solution respectively (i.e., strips numbered 2 to 4 all represent the test solution, strip numbered 1 represents the reference solution, and strip numbered 5 represents the safflower-deficient negative sample solution).

[0094] Depend on Figure 6 It can be seen that the hydroxysafflor yellow A in the Shentong Zhuyu Decoction and the hydroxysafflor yellow A reference substance are at corresponding positions, showing fluorescent spots of the same color. The thin layer identification method of the present invention can be used for thin layer identification of safflower in the Shentong Zhuyu Decoction.

[0095] Embodiment 3~6 Thin layer identification method of safflower in Shentong Zhuyu Decoction

[0096] Examples 3 to 6 are respectively a thin layer identification method for safflower in shentong zhuyu soup. According to the optimal thin layer identification conditions given in Example 2, the range of conditions that can achieve better identification of hydroxysafflor yellow A in shentong zhuyu soup is further explored to obtain the thin layer identification methods in Examples 3 to 6. The thin layer identification methods of safflower in shentong zhuyu soup 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:

[0097] Table 1 List of process parameters in Examples 3 to 6

[0098]

[0099]

[0100] 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 that hydroxysafflor yellow A in Shentong Zhuyu Decoction can be better identified, no drawings are added here), and they will not be repeated here.

[0101] Example 7 Methodological Investigation

[0102] This example conducts a methodological investigation on the thin layer identification method of safflower in Shentong Zhuyu Decoction, as follows:

[0103] 1. Investigation of Silicone G Thin Layer Sheets from Different Manufacturers

[0104] Three sets of parallel test solution, safflower negative sample solution and reference solution were prepared according to the method in Example 2, and the thin layer identification method in step S2 of Example 2 was used to examine the thin layer identification results of silica gel G thin layer plates from different manufacturers at a development temperature of 25.9°C and a humidity of 19.3%RH, while keeping the other conditions unchanged. The results are shown in FIG. Figures 7 to 9 . Figures 7 to 9The strips numbered 1 to 5 respectively represent the reference solution, three sets of parallel test solution and safflower-deficient negative sample solution (i.e., strips numbered 2 to 4 all represent the test solution, strip numbered 1 represents the reference solution, and strip numbered 5 represents the safflower-deficient negative sample solution).

[0105] Among them, the silica gel G thin layer plates from different manufacturers are: Yinlong silica gel G plate (manufacturer: Yantai Weiqi Chemical Products Co., Ltd.), Yantai Zhifu silica gel G plate (manufacturer: Yantai Jiangyou Silicone Development Co., Ltd.), and Qingdao silica gel G plate (manufacturer: Qingdao Ocean Chemical Co., Ltd.).

[0106] Depend on Figures 7 to 9 It can be seen that the thin layer identification method of safflower in the Shentong Zhuyu Decoction of the present invention is not affected by the silica gel G thin layer plate manufacturer.

[0107] 2. Investigation of different development temperatures and humidity

[0108] Prepare the test solution, safflower-deficient negative sample solution and reference solution according to the method in Example 2.

[0109] Using the above test solution, safflower negative sample solution, safflower medicinal material solution and reference solution, according to the thin layer identification method in step S2 of Example 2, the thin layer identification results were examined at a development temperature of 22.4°C and a humidity of 78.0%RH (high humidity environment), keeping the other conditions unchanged. The results are shown in FIG. Fig.10 . Fig.10 The strips numbered 1 to 5 respectively represent the reference solution, the test solution, the test solution, the test solution and the safflower-deficient negative sample solution (i.e., the strips numbered 2 to 4 all represent the test solution, the strip numbered 1 represents the reference solution, and the strip numbered 5 represents the safflower-deficient negative sample solution).

[0110] Using the above test solution, safflower-deficient negative sample solution and reference solution, according to the thin layer identification method in step S2 of Example 2, the thin layer identification results were investigated at a development temperature of 4.5°C and a humidity of 44.0%RH (low temperature environment), keeping the other conditions unchanged. The results are shown in Fig.11 . Fig.11 The strips numbered 1 to 5 respectively represent the reference solution, the test solution, the test solution, the test solution and the safflower-deficient negative sample solution (i.e., the strips numbered 2 to 4 all represent the test solution, the strip numbered 1 represents the reference solution, and the strip numbered 5 represents the safflower-deficient negative sample solution).

[0111] By comparison Figure 6 , Fig.10 and Fig.11 It is found that the thin layer identification method of safflower in the Shentong Zhuyu Decoction of the present invention is not affected by the development temperature and humidity.

[0112] 3. Multi-batch verification

[0113] 30 different batches of Shentong Zhuyu Decoction material standards were taken respectively, and 30 different test solutions were prepared according to the method in Example 2. Hydroxysafflor yellow A was taken according to the method in Example 2 to prepare a reference solution.

[0114] According to the thin layer chromatography identification method of step S2 in Example 2, 30 different test solutions of the above-mentioned reference solution were subjected to thin layer chromatography identification. The identification results are shown in FIG. Figure 12 to Figure 13 . Fig.12 The middle strip number 9 represents the reference solution, and strip numbers 1 to 8 and 10 to 16 represent 15 of the 30 different test solutions; Fig.13 The middle strip number 25 represents the reference solution, and strip numbers 17 to 24 and 26 to 32 represent the other 15 of the 30 different test solutions.

[0115] Depend on Figure 12 to Figure 13 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 yellow-green fluorescent spot at the corresponding position of the chromatogram of the hydroxysafflor yellow A reference substance.

[0116] In summary, the development conditions of the thin layer identification method of safflower in the Shentong Zhuyu Decoction of the present invention are good in durability, strong in specificity, and the main spots are clear.

[0117] 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 of safflower in shentong zhuyu decoction, characterized in that: The thin layer chromatography identification method of safflower in the Shentong Zhuyu Decoction is to use ethyl acetate-methanol-formic acid-water with a volume ratio of 7:1.9-2.1:2.9-3.1:0.9-1.1 as a developing solvent, and to identify the safflower in the Shentong Zhuyu Decoction by thin layer chromatography.

2. The thin layer identification method of safflower in Shentong Zhuyu Decoction according to claim 1, characterized in that: In the thin layer identification process, hydroxysafflor yellow A or safflower was used as a control.

3. The thin layer identification method of safflower in the Shentong Zhuyu Decoction according to claim 1 or 2, characterized in that: The TLC was observed under UV light at 365 nm.

4. The thin layer identification method of safflower in the Shentong Zhuyu Decoction according to claim 1 or 2, characterized in that: The thin layer identification method of safflower in the Shentong Zhuyu Decoction comprises the following steps: S1. Prepare the test solution by taking Shentong Zhuyu 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.

5. The thin layer identification method of safflower in Shentong Zhuyu Decoction according to claim 4, characterized in that: In step S1, the test solution is prepared by adding acetone aqueous solution to Shentong Zhuyu Decoction, ultrasonically treating, and filtering.

6. The thin layer identification method of safflower in Shentong Zhuyu Decoction according to claim 5, characterized in that: During the preparation of the test solution, the concentration of acetone in the acetone aqueous solution is 78 vol% to 82 vol%; The weight-to-volume ratio of the Shentong Zhuyu Decoction liquid to the acetone aqueous solution is 1 g: 4.5-5.5 mL.

7. The thin layer identification method of safflower in Shentong Zhuyu Decoction according to claim 4, characterized in that: In step S2, the sample solution is spotted on the thin layer plate in an amount of 1.5 to 2.5 μL; The unfolded extension distance is 5 to 20 cm.

8. The thin layer identification method of safflower in Shentong Zhuyu Decoction according to claim 4, characterized in that: The thin layer chromatography identification method of safflower in the Shentong Zhuyu Decoction also includes preparing a reference solution using hydroxysafflor yellow A and identifying it by thin layer chromatography.

9. The thin layer identification method of safflower in Shentong Zhuyu Decoction according to claim 8, characterized in that: The content of hydroxysafflor yellow A in the reference solution is 0.25-0.35 mg / mL.

10. The thin layer identification method of safflower in Shentong Zhuyu Decoction according to claim 1 or 2, characterized in that: The thin layer chromatography identification method of safflower in Shentong Zhuyu Decoction also includes preparing a safflower-deficient negative sample solution using a safflower-deficient negative reference substance, and identifying it by thin layer chromatography; The safflower-deficient negative sample solution is prepared using Radix Gentianae Macrophyllae, Rhizoma Chuanxiong, Rhizoma Pruni, Radix Glycyrrhizae, Radix Notopterygii, Myrrha Vinegar, Radix Angelicae Sinensis, Rhizoma Aconiti Lateralis Preparata, Rhizoma Cyperi, Rhizoma Cyperi and Pheretima.

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