A quality control method for Wenjing Decoction compound preparation

Abstract

The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a quality control method for a Wenjing Decoction compound preparation. The quality control method comprises: preparing a test solution A, and based on the test solution A, using thin layer chromatography to identify moutan bark, chuanxiong, angelica and cinnamon bark; preparing a test solution B, and based on the test solution B, using thin layer chromatography to identify moutan bark, white peony root and liquorice; preparing a test solution C, and based on the test solution C, using thin layer chromatography to identify zedoary turmeric; preparing a test solution D, and based on the test solution D, using thin layer chromatography to identify ox-kiangsu; preparing a test solution E, and based on the test solution E, using thin layer chromatography to identify ginseng. This method uses the same test solution for thin layer chromatography identification of multiple medicinal flavors, significantly reducing the sample preparation procedures and costs. At the same time, it realizes the thin layer chromatography identification of zedoary turmeric in Wenjing Decoction, effectively improving the comprehensiveness of the Wenjing Decoction quality control method.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a quality control method for a Wenjing Decoction compound preparation. Background Art

[0002] Wenjing Decoction originates from Chen Ziming's Complete Prescriptions for Women in the Song Dynasty. The original text states: "Danggui, Chuanxiong, Shaoyao, Guixin, Moutan Bark, and Zedoaria, each half a liang; Ginseng, Achyranthes, and Licorice, each one liang. Grind the above ingredients into a paste. Take five qian each time, add one and a half cups of water, boil until eight parts are left, remove the residue, and drink warm." This recipe, a decoction-based powder, warms the meridians and dispels cold, unclogs the blood vessels, activates blood circulation and removes stasis, nourishes both qi and blood, and regulates both the liver and spleen. Guixin warms the meridians and unclogs the blood vessels, while ginseng warms and nourishes qi. The blood-activating and blood-stasis-removing effects of Zedoaria, Moutan Bark, and Achyranthes enhance the blood-activating, nourishing, and menstrual-regulating effects of Donggui, Chuanxiong, and Shaoyao. The combination of peony and licorice relieves pain. The entire recipe works by activating blood circulation, promoting qi circulation, and warming the meridians to dissipate blood stasis.

[0003] Classical prescriptions have undergone clinical practice and refinement, and possess significant development value. However, the numerous medicinal components and complex composition of these prescriptions make comprehensive quality control difficult. Thin-layer chromatography (TLC) is a key qualitative testing method for compound preparations in the Chinese Pharmacopoeia.

[0004] In the existing thin-layer test of Wenjing Decoction compound preparations, due to the large number of medicinal flavors that need to be tested, there are problems such as cumbersome methods and low detection efficiency. At the same time, the comprehensiveness of the existing detection methods is also poor. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the thin layer detection method of Wenjing Decoction compound preparation in the prior art, such as the complicated method, low detection efficiency and poor comprehensiveness, thereby providing a quality control method for Wenjing Decoction compound preparation.

[0006] To this end, the present invention provides a quality control method for a Wenjing Decoction compound preparation, wherein the Wenjing Decoction compound preparation is prepared from raw materials including angelica sinensis, Chuanxiong rhizome, white peony root, cinnamon bark, moutan bark, zedoary turmeric, ginseng, cypress and liquorice, and the quality control method comprises:

[0007] preparing a test solution A, and identifying Moutan Cortex, Chuanxiong Rhizome, Angelica Sinensis, and Cinnamomum cassia bark by thin layer chromatography based on the test solution A;

[0008] preparing a test solution B, and identifying white peony root and liquorice root by thin layer chromatography based on the test solution B;

[0009] preparing a test solution C, and identifying Curcuma zedoaria based on the test solution C by thin layer chromatography;

[0010] preparing a test solution D, and identifying Achyranthes bidentata by thin layer chromatography based on the test solution D;

[0011] A test solution E was prepared, and ginseng was identified based on the test solution E using thin layer chromatography.

[0012] Optionally, the preparation process of the test solution A includes: taking a test sample of the Wenjing Decoction compound preparation, adding methanol to dissolve it and then performing solid-liquid separation, taking the liquid and evaporating it to dryness, re-dissolving the obtained residue in water and then performing solid-liquid separation, taking the liquid and adding ether to extract it, taking the ether extract and evaporating it to dryness, and re-dissolving the obtained residue in acetone.

[0013] Illustratively, the preparation process of the test solution A includes: taking 1 to 2 g of the Wenjing Decoction compound preparation test sample, adding 15 to 30 ml of methanol, ultrasonically treating for 20 to 40 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 10 to 20 ml of water, filtering, and extracting the filtrate by shaking with ether 2 to 3 times, each time 10 to 20 ml, combining the ether extracts, evaporating to dryness, and adding 0.5 to 1 ml of acetone to dissolve the residue.

[0014] Optionally, the preparation process of the test solution B includes: taking the aqueous solution after ether extraction in the preparation process of the test solution A, extracting it with water-saturated n-butanol, evaporating the n-butanol extract to dryness, and re-dissolving the obtained residue with methanol.

[0015] Illustratively, the preparation process of the test solution B includes: taking the aqueous solution after ether extraction in the preparation process of the test solution A, shaking and extracting it 2 to 3 times with water-saturated n-butanol, 10 to 20 ml each time, combining the n-butanol extracts, evaporating to dryness, and adding 2 to 4 ml of methanol to dissolve the residue.

[0016] Optionally, the preparation process of the test solution C includes: taking the Wenjing Decoction compound preparation test sample, adding n-hexane for extraction, performing solid-liquid separation, taking the liquid and evaporating it to dryness, and re-dissolving the obtained residue with n-hexane.

[0017] Optionally, the preparation process of the test solution C includes: taking 5-6 g of the Wenjing Decoction compound preparation test sample, adding 40-60 ml of n-hexane, ultrasonically treating for 15-30 minutes, filtering, evaporating the filtrate to dryness, and adding 0.5-1 ml of n-hexane to dissolve the residue.

[0018] Optionally, the preparation process of the test solution D includes: taking a test sample of the Wenjing Decoction compound preparation, extracting it with methanol and water, performing solid-liquid separation, evaporating the liquid to dryness, re-dissolving the obtained residue with water, and then further extracting it with water-saturated n-butanol, evaporating the n-butanol extract to dryness, and re-dissolving the obtained residue with methanol.

[0019] Illustratively, the preparation process of the test solution D includes: taking 1-2 g of the Wenjing Decoction compound preparation test sample, adding 25-50 ml of 70-90% methanol, reflux treatment for 50-70 minutes, filtering, evaporating the filtrate to dryness, adding 25-50 ml of water to the residue, shaking and extracting 2-3 times with water-saturated n-butanol, 15-30 ml each time, combining the n-butanol extracts, shaking and washing 2-3 times with water saturated with n-butanol, 10-20 ml each time, taking the n-butanol layer extract and evaporating it to dryness, and adding 1-2 ml of methanol to dissolve it.

[0020] Optionally, the preparation process of the test solution E includes: taking the Wenjing Decoction compound preparation test sample, adding water to dissolve it, and then continuing to add water-saturated n-butanol to extract it, separating the solid and liquid and taking the liquid, adding ammonia test solution and then letting it stand to separate the layers, taking the n-butanol extract and evaporating it to dryness, and adding methanol to redissolve the obtained residue.

[0021] Illustratively, the preparation process of the test solution E includes: taking 0.5-1 g of the Wenjing Decoction compound preparation test sample, adding 15-30 ml of water to dissolve it, then adding 25-50 ml of water-saturated n-butanol and ultrasonically treating it for 20-40 min, centrifuging and taking the supernatant, adding 3-4 times the amount of ammonia test solution, shaking it well, letting it stand to separate, taking the n-butanol extract and evaporating it to dryness, and adding 1-2 ml of methanol to the residue to dissolve it.

[0022] Optionally, based on the test solution A, thin layer chromatography is used to identify the peony bark, comprising the following steps:

[0023] Take the test solution A, the peony bark control medicinal material solution, and the paeonol reference solution, respectively, and spot them on the same silica gel thin layer plate, use cyclohexane-ethyl acetate-glacial acetic acid as the developing solvent, and use aluminum chloride test solution as the color developer, and inspect under ultraviolet light;

[0024] Optionally, the quality control method further includes a step of preparing the peony bark control medicinal material solution A according to the preparation process of the test solution A; the silica gel thin layer plate includes a silica gel GF254 thin layer plate and / or a silica gel HSGF254 thin layer plate; the spotting amount of the paeonol reference solution and the test solution A is 2~4μL, and the spotting amount of the peony bark control medicinal material solution A is 1~3μL; in the developing agent, the volume ratio of cyclohexane, ethyl acetate and glacial acetic acid is (10~11):(3~4):0.2, preferably 10:3:0.2; the concentration of the aluminum chloride test solution can be 0.01g / mL; the wavelength of the ultraviolet light is 365nm.

[0025] For example, when preparing the peony bark reference medicinal material solution A, the amount of the peony bark reference medicinal material is 0.5 to 1 g. The paeonol reference substance solution is prepared by dissolving the paeonol reference substance in ethyl acetate, with 1 to 2 mg of the paeonol reference substance dissolved in 1 mL of ethyl acetate.

[0026] Optionally, when identifying Paeonia suffruticosa based on the test solution A, the quality control method further includes the steps of preparing a Wenjing Decoction Paeonia suffruticosa single negative control solution, a Wenjing Decoction Paeonia suffruticosa single positive control solution, and a Wenjing Decoction Paeonia suffruticosa and White Peony Root double negative control solution according to the preparation process of the test solution A, and the steps of spotting and inspecting each control solution.

[0027] Optionally, based on the test solution A, thin layer chromatography is used to identify Chuanxiong and Angelica sinensis, comprising the following steps:

[0028] Take the test solution A, Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, ligustilide reference solution and ligustilide A reference solution, respectively, and spot them on the same silica gel thin layer plate, use n-hexane-dichloromethane-ethyl acetate-formic acid as the developing solvent, and inspect under ultraviolet light;

[0029] Optionally, the quality control method further comprises the step of preparing the Angelica sinensis control medicinal material solution and the Chuanxiong rhizome control medicinal material solution according to the preparation process of the test solution A; optionally, the silica gel thin layer plate comprises a silica gel HSGF254 thin layer plate and / or a silica gel GF254 thin layer plate; optionally, the spotting amount of the test solution A, the Angelica sinensis control medicinal material solution, the Chuanxiong rhizome control medicinal material solution, the Ligusticum chuanxiong lactone reference solution and the Ligusticum chuanxiong lactone A reference solution is 2 to 4 μL; optionally, in the developing agent, the volume ratio of n-hexane, dichloromethane, ethyl acetate and formic acid is (8~9):(3):(1~2):0.2, preferably 8:3:1:0.2; optionally, the wavelength of the ultraviolet light is 254 nm.

[0030] Illustratively, when preparing the Angelica sinensis reference medicinal material solution, the amount of Angelica sinensis reference medicinal material used is 0.5 to 1 g. When preparing the Chuanxiong Rhizoma control medicinal material solution, the amount of Chuanxiong Rhizoma control medicinal material used is 0.5 to 1 g. The ligustilide reference substance solution is prepared by dissolving the ligustilide reference substance in methanol, with 1 to 2 mg of the ligustilide reference substance dissolved in 1 mL of methanol. The ligustilide A reference substance solution is prepared by dissolving the ligustilide A reference substance in methanol, with 1 to 2 mg of the ligustilide A reference substance dissolved in 1 mL of methanol.

[0031] Optionally, when identifying Chuanxiong and Angelica sinensis based on the test solution A, the quality control method also includes the steps of preparing Wenjing Decoction Angelica sinensis single negative control solution, Wenjing Decoction Chuanxiong single negative control solution, Wenjing Decoction Angelica sinensis Chuanxiong double negative control solution, Wenjing Decoction Angelica sinensis single positive control solution, and Wenjing Decoction Chuanxiong single positive control solution according to the preparation process of the test solution A, as well as the steps of spotting and inspecting each control solution.

[0032] Optionally, based on the test solution A, thin layer chromatography is used to identify cinnamon, comprising the following steps:

[0033] Take the test solution A, cinnamon reference medicinal material solution, and cinnamic acid reference solution, respectively, and spot them on the same silica gel thin layer plate, use petroleum ether-ethyl acetate-formic acid as the developing solvent, and inspect under ultraviolet light;

[0034] Optionally, the quality control method further includes a step of preparing the cinnamon control medicinal material solution according to the preparation process of the test solution A; the silica gel thin layer plate includes a silica gel GF254 thin layer plate and / or a silica gel HSGF254 thin layer plate; the spotting volume of the cinnamic acid reference solution and the cinnamon control medicinal material solution is 1~3μL, and the spotting volume of the test solution A is 4~6μL; in the developing solvent, the volume ratio of petroleum ether, ethyl acetate and formic acid is (8~8.5):(3~3.5):0.2, preferably 8:3:0.2; the distillation range of the petroleum ether is 60~90℃; the wavelength of the ultraviolet light is 254nm.

[0035] For example, when preparing the cinnamon reference medicinal material solution, the amount of cinnamon reference medicinal material used is 1 to 2 g. The cinnamic acid reference substance solution is prepared by dissolving the cinnamic acid reference substance in anhydrous ethanol, with 0.25 to 0.5 mg of the cinnamic acid reference substance dissolved in 1 mL of anhydrous ethanol.

[0036] Optionally, when identifying cinnamon based on the test solution A, the quality control method further includes the steps of preparing a Wenjing Decoction cinnamon single negative control solution and a Wenjing Decoction cinnamon single positive control solution according to the preparation process of the test solution A, and the steps of spotting and inspecting the control solutions.

[0037] Optionally, based on the test solution C, thin layer chromatography is used to identify Curcuma zedoaria, comprising the following steps:

[0038] Take the test solution C and the control medicinal material solution of Curcuma zedoaria, spot them on the same silica gel thin layer plate, use cyclohexane-ethyl acetate-formic acid as the developing solvent and sulfuric acid ethanol solution as the color developer, heat until the spots are clearly colored, and then inspect them under ultraviolet light;

[0039] Optionally, the quality control method further includes the step of preparing the Curcuma zedoaria control medicinal material solution according to the preparation process of the test solution C; the silica gel thin layer plate includes a silica gel GF254 thin layer plate and / or a silica gel HSGF254 thin layer plate; the spotting volume of the test solution C and the Curcuma zedoaria control medicinal material solution is 15 to 25 μL; in the developing agent, the volume ratio of cyclohexane, ethyl acetate and formic acid is (9 to 9.5): (1 to 1.5): 0.2, preferably 9:1:0.2; the concentration of sulfuric acid in the sulfuric acid ethanol solution is 0.1%; the heating temperature is 100 to 110° C.; and the wavelength of the ultraviolet light is 365 nm.

[0040] Illustratively, when preparing the Curcuma zedoaria control medicinal material solution, the amount of the Curcuma zedoaria control medicinal material used is 0.25 to 0.5 g.

[0041] Optionally, when identifying Curcuma zedoaria based on the test solution C, the quality control method further includes the steps of preparing a Wenjing Decoction Curcuma single negative control solution and a Wenjing Decoction Curcuma single positive control solution according to the preparation process of the test solution C, and the steps of spotting and inspecting the control solutions.

[0042] Optionally, based on the test solution B, thin layer chromatography is used to identify white peony root and liquorice, comprising the following steps:

[0043] The test solution B, white peony root control medicinal material solution, liquorice root control medicinal material solution, paeoniflorin reference substance solution, and liquiritin reference substance solution were spotted on the same silica gel thin layer plate, developed with ethyl acetate-acetone-methanol-ammonia solution, sprayed with ethanolic sulfuric acid solution, and heated until color developed, and / or inspected under ultraviolet light;

[0044] Optionally, the quality control method further includes the step of preparing the white peony control medicinal material solution and the licorice control medicinal material solution according to the preparation process of the test solution B; the silica gel thin layer plate includes a silica gel GF254 thin layer plate and / or a silica gel HSGF254 thin layer plate; the spotting amount of the test solution B, the licorice control medicinal material solution and the liquiritin reference solution is 1~3μL; the spotting amount of the paeoniflorin reference solution is 4~6μL; the spotting amount of the white peony control medicinal material solution is 2~4μL; in the developing agent, the volume ratio of ethyl acetate, acetone, methanol and ammonia water is (5~6): (2~3): 3:2, preferably 5:2:3:2; the concentration of the ammonia water is 25~28%; the concentration of sulfuric acid in the sulfuric acid ethanol solution is 0.1%; the heating temperature is 100~110℃; the wavelength of the ultraviolet light is 365nm.

[0045] Exemplarily, when preparing the white peony root control medicinal material solution and the licorice root control medicinal material solution, the amount of the white peony root control medicinal material and the licorice root control medicinal material is 0.5 to 1 g respectively. The paeoniflorin reference substance solution is prepared by dissolving the paeoniflorin reference substance in methanol, with 2 to 3 mg of the paeoniflorin reference substance dissolved in 1 mL of methanol. The liquiritin reference substance solution is prepared by dissolving the liquiritin reference substance in methanol, with 0.5 to 1 mg of the liquiritin reference substance dissolved in 1 mL of methanol.

[0046] Optionally, when identifying white peony root and licorice based on the test solution B, the quality control method also includes the steps of preparing a Wenjing Decoction white peony root single negative control solution, a Wenjing Decoction peony bark and white peony root double negative control solution, a Wenjing Decoction lacking licorice single negative control solution, a Wenjing Decoction white peony root single positive control solution, and a Wenjing Decoction lacking licorice single positive control solution according to the preparation process of the test solution B, as well as the steps of spotting and inspecting each control solution.

[0047] Optionally, based on the test solution D, thin layer chromatography is used to identify Achyranthes bidentata, comprising the following steps:

[0048] Take the test solution D, the Achyranthes bidentata reference medicinal material solution, and the β-ecdysterone reference solution, respectively, and spot them on the same silica gel thin layer plate, use chloroform-methanol-water-formic acid as the developing solvent, and use sulfuric acid ethanol solution as the color developer, heat until the spots are clearly colored, and then inspect under ultraviolet light;

[0049] Optionally, the quality control method also includes the step of preparing the Achyranthes bidentata control medicinal material solution according to the preparation process of the test solution D; the silica gel thin layer plate includes a silica gel GF254 thin layer plate and / or a silica gel HSGF254 thin layer plate; the spotting amount of the test solution D is 1~4μL, the spotting amount of the Achyranthes bidentata control medicinal material solution is 4~7μL, and the spotting amount of β-ecdysterone is 2~4μL; in the developing agent, the volume ratio of chloroform, methanol, water and formic acid is (8~9): (2~2.5): 0.3:0.5, preferably 8:2:0.3:0.5; the concentration of sulfuric acid in the sulfuric acid ethanol solution is 0.1%; the heating temperature is 100~110℃; the wavelength of the ultraviolet light is 365nm.

[0050] For example, when preparing the Achyranthes bidentata reference medicinal material solution, the amount of Achyranthes bidentata reference medicinal material used is 1 to 2 g. The β-ecdysterone reference solution is prepared by dissolving the β-ecdysterone reference substance in methanol, with 0.2 to 0.4 mg of the β-ecdysterone reference substance dissolved in 1 mL of methanol.

[0051] Optionally, when identifying Achyranthes bidentata based on the test solution D, the quality control method also includes the steps of preparing a single negative control solution of Wenjing Decoction Achyranthes bidentata and a single positive control solution of Wenjing Decoction Achyranthes bidentata according to the preparation process of the test solution D, and the steps of spotting and inspecting the control solutions.

[0052] Optionally, based on the test solution E, thin layer chromatography is used to identify ginseng, comprising the following steps:

[0053] Take the test solution E, ginseng reference medicinal material solution, and mixed reference solution, spot them respectively on the same silica gel thin layer plate, use chloroform-anhydrous methanol-water as the developing solvent, use sulfuric acid ethanol solution as the color developer, and heat until color is developed; wherein the mixed reference solution contains ginsenoside Rb1 reference substance, ginsenoside Rb2 reference substance, ginsenoside Rc reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rg1 reference substance;

[0054] Optionally, the quality control method further includes a step of preparing the ginseng control medicinal material solution according to the preparation process of the test solution E; the silica gel thin layer plate includes a silica gel HSGF254 thin layer plate and / or a silica gel GF254 thin layer plate; the spot volume of the test solution E is 4~6μL, and the spot volume of the ginseng control medicinal material solution and the mixed reference solution is 2~4μL; in the developing solvent, the volume ratio of chloroform, anhydrous methanol and water is (9~9.5):(4.5~5):1, preferably 9:4.5:1; the concentration of sulfuric acid in the sulfuric acid ethanol solution is 0.1%; and the heating temperature is 100~110°C.

[0055] Illustratively, when preparing the ginseng control medicinal material solution, the amount of ginseng control medicinal material used is 0.5-1 g. The mixed reference solution is prepared by dissolving ginsenoside Rb1 reference substance, ginsenoside Rb2 reference substance, ginsenoside Rc reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance, and ginsenoside Rg1 reference substance in methanol, with the amount of each ginsenoside dissolved in 1 mL of methanol being 1-2 mg.

[0056] Optionally, the Wenjing Decoction compound preparation includes a solid preparation, a semisolid preparation or a liquid preparation of Wenjing Decoction. Exemplarily, the Wenjing Decoction compound preparation is a standard decoction of Wenjing Decoction.

[0057] The technical solution of the present invention has the following advantages:

[0058] 1. The quality control method for the Wenjing Decoction compound preparation provided by the present invention uses the same test solution for the thin layer chromatography identification of multiple medicinal flavors. For example, the test solution A is used for the thin layer chromatography identification of peony bark, Chuanxiong, Chinese angelica, and cinnamon, and the test solution B is used for the thin layer chromatography identification of white peony root and liquorice. This significantly reduces the sample preparation procedures and costs, significantly reduces the complexity of the quality control method, and improves the efficiency of thin layer chromatography identification. At the same time, the quality control method of the present application also realizes the thin layer chromatography identification of Curcuma zedoaria in Wenjing Decoction. Compared with the existing technology, the comprehensiveness of the quality control method of the Wenjing Decoction compound preparation is effectively improved.

[0059] 2. The quality control method for the Wenjing Decoction compound preparation provided by the present invention is simple and stable in the preparation of each test solution. In particular, the aqueous solution after ether extraction during the preparation of test solution A is directly used to prepare test solution B, which significantly shortens the sample preparation time, further improving the identification efficiency and reducing the identification cost.

[0060] 3. The quality control method for the Wenjing Decoction compound preparation provided herein utilizes cyclohexane-ethyl acetate-glacial acetic acid as the developing agent and aluminum chloride solution as the color developer when identifying Paeonia suffruticosa moutan based on test solution A, resulting in enhanced specificity and clearer imaging. In addition to paeonol, specific spots corresponding to those in the control Paeonia suffruticosa moutan can also be detected, effectively enhancing the richness and accuracy of the test results.

[0061] 4. The quality control method for the Wenjing Decoction compound preparation provided by the present invention can identify the Chuanxiong component in Wenjing Decoction by using the reference substance of ligustilide A, overcoming the defect in the prior art that ferulic acid is difficult to accurately distinguish Chuanxiong from Angelica sinensis. At the same time, it can also identify ligustilide, a common component of Chuanxiong and Angelica sinensis, resulting in richer inspection results and more accurate test results.

[0062] 5. The quality control method for the Wenjing Decoction compound preparation provided by the present invention uses a cinnamic acid reference substance to identify cinnamon in Wenjing Decoction. Cinnamic acid belongs to the phenylpropanoid compound class and has relatively stable chemical properties and is not easily volatile. The storage time of the sample does not interfere with the detection. Compared with the cinnamaldehyde in the prior art, it has better stability.

[0063] 6. The quality control method for the Wenjing Decoction compound preparation provided herein utilizes a single development system to distinguish white peony root and licorice root, using the same color developer and observing under different light sources, significantly improving detection efficiency. Under a 365nm light source, in addition to liquiritin, specific spots corresponding to those in the licorice control material can be detected, effectively increasing the richness and accuracy of the test results.

[0064] 7. The quality control method of the Wenjing Decoction compound preparation provided by the present invention is simple and rapid in preparation of the test solution, development of thin-layer chromatography, and color development when identifying Achyranthes bidentata based on the test solution D.

[0065] 8. The quality control method for the Wenjing Decoction compound preparation provided by the present invention uses chloroform-anhydrous methanol-water as the developing solvent and ethanolic sulfuric acid solution as the color developer when identifying ginseng based on test solution E. This method can effectively identify at least six ginsenoside components in Wenjing Decoction, and the degree of separation of each component is high, the color development effect is better, and the information richness of the inspection results is significantly increased.

[0066] 9. The quality control method for the Wenjing Decoction compound preparation provided by the present invention uses 10% ethanolic sulfate as a color developer or no color developer is required for the identification of paeonol, which further simplifies the entire quality control method. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0068] Figures 1-6 Part b is a diagram showing the methodological validation results in Example 1 of the present invention;

[0069] Figure 7 Part A- Figure 12 Part d is a diagram showing the methodological validation results in Example 2 of the present invention;

[0070] Figures 13-18 Part b is a diagram showing the methodological validation results in Example 3 of the present invention;

[0071] Figures 19-24 Part b is a diagram showing the methodological validation results in Example 4 of the present invention;

[0072] Figures 25-30 Part b is a diagram showing the methodological validation results in Example 5 of the present invention;

[0073] Figures 31-36 Part b is a diagram showing the methodological validation results in Example 6 of the present invention;

[0074] Figures 37-42 Part b is a diagram of the methodological verification results in Example 7 of the present invention. DETAILED DESCRIPTION

[0075] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0076] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0077] equipment:

[0078] Decoction pot (manufacturer: Kangyashun Electric Co., Ltd., model: DTL5).

[0079] Reference substances:

[0080] Paeonol reference substance (source: China Food and Drug Inspection Institute, batch number: 110708-201908);

[0081] Paeoniflorin reference substance (source: China Food and Drug Inspection Institute, batch number: 110736-202145);

[0082] Liquiritin reference substance (source: China Food and Drug Inspection Institute, batch number: 111610-201908);

[0083] Ligustilide reference substance (source: Shanghai Naiqi Biotechnology Co., Ltd., batch number: 070017-202011);

[0084] Caulis ligusticum lactone A reference substance (source: Chengdu Aifa Biotechnology Co., Ltd., batch number: A222011451);

[0085] Cinnamic acid reference substance (source: China Food and Drug Inspection Institute, batch number: 110786-201604);

[0086] β-ecdysterone reference substance (source: China Food and Drug Inspection Institute, batch number: 111638-201907);

[0087] Ginsenoside Rb1 reference substance (source: China Food and Drug Inspection Institute, batch number: 110704-202129);

[0088] Ginsenoside Rb2 reference substance (source: Shanghai Naiqi Biotechnology Co., Ltd., batch number: 180016-202109);

[0089] Ginsenoside Rc reference substance (source: Chengdu Aifa Biotechnology Co., Ltd., batch number: AF21012552);

[0090] Ginsenoside Re reference substance (source: China Food and Drug Inspection Institute, batch number: 110754-202129);

[0091] Ginsenoside Rf reference substance (source: China Food and Drug Inspection Institute, batch number: 111719-201806);

[0092] Ginsenoside Rg1 reference substance (Source: China Food and Drug Inspection Institute, batch number: 110703-202034).

[0093] Control herbs:

[0094] Moutan bark control medicinal material (source: China Food and Drug Inspection Institute, batch number: 121490-201603);

[0095] White peony root control medicinal material (source: China Food and Drug Inspection Institute, batch number: 120905-202011);

[0096] Licorice control medicinal material (Source: China Food and Drug Inspection Institute, Batch No.: 120904-202021);

[0097] Angelica sinensis control medicinal material (source: China Food and Drug Inspection Institute, batch number: 120927-201617);

[0098] Chuanxiong reference medicinal material (source: China Food and Drug Administration, batch number: 120918-201813);

[0099] Cinnamon bark as a control herbal medicine (Source: China Food and Drug Administration, Batch No. 121363-201703);

[0100] Curcuma zedoaria control medicinal material (source: Chengdu Ruifensi Biotechnology Co., Ltd., batch number: DZYC-E-003);

[0101] Achyranthes bidentata control medicinal material (source: China Food and Drug Inspection Institute, batch number: 121066-201809);

[0102] Ginseng reference medicinal material (Source: China Food and Drug Inspection Institute, batch number: 120917-201712).

[0103] Preparation Example

[0104] (1) Prepare the standard decoction of Wenjing Decoction for testing as follows:

[0105] Weigh 5g each of wine-soaked angelica, Chuanxiong, cinnamon bark, vinegar-soaked zedoaria, white peony root, and coarse-grained peony bark, and 10g each of wine-soaked cypress, ginseng, and stir-fried licorice. Mix and add 1350ml of purified water. Soak for 60 minutes. Bring the decoction to a boil over high heat (500W), then reduce the heat to simmer (400W) and simmer with the lid uncovered for 50 minutes. Filter the decoction through a 150-mesh sieve, cool to room temperature, and record the volume (ml). If the volume is between 720 and 740ml, add water to the volume to 1000ml. After freeze-drying, collect the sample for the Wenjing Decoction standard decoction. The parameters for each stage of the freeze-drying process are shown in Table 1.

[0106] Table 1 Parameter settings for each stage of the freeze-drying process

[0107]

[0108] (2) According to the preparation method in item (1), when the medicinal material X is removed, a single-negative freeze-dried powder of Wenjing Decoction X is prepared; when the medicinal materials X and Y are removed, a double-negative freeze-dried powder of Wenjing Decoction X and Y is prepared, wherein X is selected from any one of the group consisting of angelica sinensis, chuanxiong rhizome, cinnamon bark, zedoaria zedoaria with vinegar, white peony root, peony bark, cypress root with wine, ginseng and stir-fried liquorice, and Y is selected from any one of the group consisting of angelica sinensis, chuanxiong rhizome, cinnamon bark, zedoaria zedoaria with vinegar, white peony root, peony bark, cypress root with wine, ginseng and stir-fried liquorice except X.

[0109] (3) According to the preparation method in item (1), a single-positive freeze-dried powder of Wenjing Decoction Z is prepared using only the medicinal material Z, wherein Z is selected from any one of the following: angelica sinensis with wine, chuanxiong rhizome, cinnamon bark, zedoaria zedoaria with vinegar, white peony root, moutan bark, cyperus rotundus with wine, ginseng and stir-fried licorice.

[0110] Example 1 Thin layer identification of peony bark in the test sample of Wenjing Decoction standard decoction

[0111] 1. Identification method

[0112] (1) Preparation of test solution A: Take 2 g of the test sample of the standard decoction of Wenjing Decoction, add 30 ml of methanol, and ultrasonically treat for 30 minutes. Filter and evaporate the filtrate to dryness. Dissolve the residue in 20 ml of water and filter. Shake the filtrate with ether twice, 10 ml each time. Combine the ether solutions, evaporate to dryness, and dissolve the residue in 1 ml of acetone to prepare the test solution A.

[0113] (2) Preparation of control medicinal material solution: Take 1 g of peony bark control medicinal material and prepare the peony bark control medicinal material solution according to the preparation method of test solution A.

[0114] (3) Preparation of reference solution: Take paeonol reference substance and add ethyl acetate to prepare a solution containing 2 mg of paeonol reference substance per 1 ml, which is used as the paeonol reference solution.

[0115] (4) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 2 μl of the above-mentioned control medicinal material solution, 3 μl of the test solution and 3 μl of the paeonol reference solution were respectively taken and spotted on the same silica gel HSGF254 thin layer plate, and cyclohexane-ethyl acetate-glacial acetic acid (10:3:0.2) was used as the developing agent. The plate was taken out, dried, sprayed with aluminum chloride test solution, dried, and examined under ultraviolet light (365 nm).

[0116] In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference medicinal material; and a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0117] 2. Methodological Validation

[0118] 1. Specificity verification

[0119] (1) Preparation of negative control solution: Take 2 g of Wenjing Decoction Mudan Cortex single negative test sample and prepare Mudan Cortex single negative control solution according to the preparation method of test sample solution A; take 2 g of Wenjing Decoction white peony root and peony root double negative test sample and prepare white peony root and peony root double negative control solution according to the preparation method of test sample solution A.

[0120] (2) Preparation of positive control solution: Take 2 g of Wenjing Decoction Mudan Cortex positive test sample and prepare Mudan Cortex positive control solution according to the preparation method of test sample solution A.

[0121] (3) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 2 μl of the above-mentioned control medicinal material solution and the single positive control solution of Paeonia suffruticosa, 3 μl of the test solution A, the single negative control solution of Paeonia suffruticosa, the double negative control solution of Paeonia lactiflora and Paeonia suffruticosa, the paeonol reference solution, and the blank solution (acetone) were respectively taken and spotted on the same silica gel HSGF254 thin layer plate. Cyclohexane-ethyl acetate-glacial acetic acid (10:3:0.2) was used as the developing solvent. The plate was taken out, dried, sprayed with aluminum chloride test solution, dried, and examined under ultraviolet light (365 nm). The results are as follows: Figure 1 shown.

[0122] exist Figure 1 In the table, 1 is the control medicinal material solution of Paeonia suffruticosa; 2 is the test solution A; 3 is the paeonol reference solution; 4 is the single positive solution of Paeonia suffruticosa; 5 is the single negative solution of Paeonia suffruticosa; 6 is the double negative solution of Paeonia lactiflora and Paeonia suffruticosa; 7 is the blank solvent (acetone). Figure 1 It can be seen that in the test sample chromatogram, the spot separation is good, and fluorescent spots of the same color appear at the corresponding positions of the control medicinal material chromatogram. Fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the paeonol reference substance. There are no interfering spots in the negative control chromatogram. The specificity of this method is good.

[0123] 2. Repeatability verification

[0124] Different experimenters prepared various solutions according to the identification method and the records under the specificity verification, and repeated the thin layer chromatography test under the specificity verification. The results are as follows: Figure 2 shown.

[0125] exist Figure 2 In the table, 1 is the control medicinal material solution of Paeonia suffruticosa suffruticosa; 2 is the test solution; 3 is the paeonol reference solution; 4 is the single positive solution of Paeonia suffruticosa suffruticosa; 5 is the single negative solution of Paeonia suffruticosa suffruticosa; 6 is the double negative solution of Paeonia lactiflora and Paeonia suffruticosa suffruticosa; 7 is the blank solvent (acetone). Figure 2 It can be seen that after measurement by different experimenters, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the paeonol reference solution and the peony bark reference medicinal material solution, with good separation and no interference from the negative control solution. The thin layer identification method has good repeatability.

[0126] 3. Durability verification

[0127] (1) Durability verification of different sample sizes

[0128] The above-mentioned paeonol reference solution (2μl, 3μl, 4μl), the above-mentioned test solution A (2μl, 3μl, 4μl), and the above-mentioned peony bark reference medicinal material solution (1μl, 2μl, 3μl) were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate, and developed according to the thin layer conditions under the identification method. The results are as follows Figure 3 shown.

[0129] exist Figure 3 In the figure, 1 is 2 μl of paeonol reference solution; 2 is 3 μl of paeonol reference solution; 3 is 4 μl of paeonol reference solution; 4 is 2 μl of test solution A; 5 is 3 μl of test solution A; 6 is 4 μl of test solution A; 7 is 1 μl of moutan bark reference solution; 8 is 2 μl of moutan bark reference solution; 9 is 3 μl of moutan bark reference solution. Figure 3 It can be seen that good detection results can be obtained at different sample sizes, indicating that this method has good durability for different sample sizes.

[0130] (2) Durability verification at different ambient temperatures

[0131] Under normal temperature and humidity (T: 23°C, RH: 42%) and low temperature and humidity (T: 6°C, RH: 42%) conditions, 3 μl of the test solution A, 3 μl of the paeonol reference solution, 2 μl of the peony bark single positive control solution, and 2 μl of the peony bark control medicinal material solution were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate, and developed according to the thin layer conditions under the identification method. The results are as follows: Figure 4Part a and Figure 4 As shown in part b, Figure 4 Part a is the test result under normal temperature and humidity conditions. Figure 4 Part b is the test result under low temperature and normal humidity conditions.

[0132] Figure 4 Part a and Figure 4 In part b, 1 is the control medicinal material solution of Paeonia suffruticosa; 2 is the test solution A; 3 is the paeonol reference solution; 4 is the single positive solution of Paeonia suffruticosa. Figure 4 Part a and Figure 4 As can be seen from part b in Figure 3, good test results can be obtained at different ambient temperatures, indicating that this method has good durability under different ambient temperatures.

[0133] (3) Durability verification in different environmental humidity

[0134] Under the conditions of room temperature and low humidity (T: 23 ℃, RH: 22%) and room temperature and high humidity (T: 23 ℃, RH: 78%), 3 μl of the test solution A, 3 μl of the paeonol reference solution, 2 μl of the peony bark single positive control solution and 2 μl of the peony bark control medicinal material solution were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate, and developed according to the thin layer conditions under the identification method. The results are as follows: Figure 5 Part a and Figure 5 As shown in part b, Figure 5 Part a is the test result under normal temperature and low humidity conditions. Figure 5 Part b is the test results under normal temperature and high humidity conditions.

[0135] Figure 5 Part a and Figure 5 In part b, 1 is the control medicinal material solution of Paeonia suffruticosa; 2 is the test solution A; 3 is the paeonol reference solution; 4 is the single positive solution of Paeonia suffruticosa. Figure 5 Part a and Figure 5 As can be seen from part b in Figure 3, good test results can be obtained under different ambient humidity conditions, indicating that this method has good durability under different ambient humidity conditions.

[0136] (4) Durability verification of different thin layer boards

[0137] Respectively take 3 μl of the above test solution A, 3 μl of the above paeonol reference solution, 2 μl of the above peony bark single positive control solution and 2 μl of the above peony bark control medicinal material solution, and spot them on different thin layer plates: Yinlong thin layer plate HSGF 254 (Yantai Chemical Industry Research Institute, 20191022), Qingdao Marine Thin Layer Plate GF 254(Made by Qingdao Ocean Chemical Co., Ltd., July 7, 2020), Yantai Jiangyou Thin Layer Board HSGF 254 (Yantai Zhifu Huangwu Silicone Silica Gel Development and Testing Plant, October 7, 2016); Then, according to the thin layer conditions under the identification method, the results show that good test results can be obtained using different thin layer plates, indicating that this method has good durability for thin layer plates of different brands.

[0138] (5) Durability verification of different developing agent ratios

[0139] 3 μl of the test solution A, 3 μl of the paeonol reference solution, 2 μl of the peony bark single positive control solution, and 2 μl of the peony bark control medicinal material solution were respectively taken and spotted on the same silica gel HSGF254 thin layer plate. The two thin layer plates were spotted in the same way; then the two thin layer plates were developed with cyclohexane-ethyl acetate-glacial acetic acid (10:4:0.2) and cyclohexane-ethyl acetate-glacial acetic acid (11:3:0.2) as the developing solvent, respectively. The results were checked as shown in the figure. Figure 6 Part a and Figure 6 As shown in part b, Figure 6 Part a in the figure is the detection result using cyclohexane-ethyl acetate-glacial acetic acid (10:4:0.2) as the developing agent. Figure 6 Part b shows the detection results using cyclohexane-ethyl acetate-glacial acetic acid (11:3:0.2) as the developing solvent.

[0140] exist Figure 6 Part a and Figure 6 In part b, 1 is the control medicinal material solution of Paeonia suffruticosa; 2 is the test solution A; 3 is the paeonol reference solution; 4 is the single positive solution of Paeonia suffruticosa. Figure 6 Part a and Figure 6 As can be seen from part b, good detection results can be obtained using different ratios of developing agents, indicating that this method has good durability for different developing agent ratios.

[0141] Example 2 Thin layer chromatography identification of white peony root and liquorice in the standard decoction of Wenjing Decoction

[0142] 1. Identification method

[0143] (1) Preparation of test solution B: Take the aqueous solution after ether extraction during the preparation of test solution A under the identification of Paeonia suffruticosa peony bark, and shake and extract it twice with water-saturated n-butanol, 15 ml each time. Combine the n-butanol extracts, evaporate to dryness, and dissolve the residue in 2 ml of methanol to prepare test solution B.

[0144] (2) Preparation of control medicinal material solution: Take 1 g of licorice control medicinal material and 1 g of white peony root control medicinal material, and prepare licorice control medicinal material solution and white peony root control medicinal material solution according to the preparation method of test solution B.

[0145] (3) Preparation of reference solution: Take the paeoniflorin reference substance, add methanol, and prepare a solution containing 2 mg of paeoniflorin reference substance per 1 ml, which is used as the paeoniflorin reference solution; take the glycyrrhizin reference substance, add methanol, and prepare a solution containing 1 mg of glycyrrhizin reference substance per 1 ml, which is used as the glycyrrhizin reference solution.

[0146] (4) Chromatographic conditions: 2 μl of each of the test solution B, licorice control medicinal material solution, and liquiritin control solution, 5 μl of peony inoside control solution, and 3 μl of white peony control medicinal material solution were taken and spotted on the same silica gel HSGF254 thin layer plate. Ethyl acetate-acetone-methanol-ammonia water (5:2:3:2) was used as the developing agent. The plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, dried, and heated in a 105°C oven until the spots were clearly colored. Then, the plates were inspected under white light and 365 light sources respectively.

[0147] In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference medicinal material; and a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0148] 2. Methodological Validation

[0149] 1. Specificity verification

[0150] (1) Preparation of negative control solution: Take 2 g of the corresponding negative lyophilized powder and prepare licorice single negative control solution, white peony single negative control solution and white peony and moutan bark double negative control solution according to the preparation method of test solution B.

[0151] (2) Preparation of positive control solution: Take 1 g of Wenjing Decoction Licorice single positive freeze-dried powder and 2 g of Wenjing Decoction White Peony single positive freeze-dried powder, and prepare Licorice single positive control solution and White Peony single positive control solution respectively according to the preparation method of test solution B.

[0152] (3) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 3 μL of the above-mentioned white peony control medicinal material solution, 5 μL of the paeoniflorin reference solution, 1 μL of the white peony positive control solution, and 2 μL of the remaining solutions were respectively taken and spotted on the same silica gel GF254 thin layer plate, and ethyl acetate-acetone-methanol-ammonia water (5:2:3:2) was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and then inspected under white light and 365nm light sources respectively. The inspection results are as follows: Figure 7 Part a and Figure 7As shown in part b, Figure 7 Part a in the figure is the inspection result under white light source. Figure 7 Part b in the figure shows the inspection result under 365nm light source.

[0153] exist Figure 7 Part a and Figure 7 In part b, 1 is the white peony root control medicinal material solution; 2 is the test solution B; 3 is the paeoniflorin reference solution; 4 is the white peony root single-positive solution; 5 is the white peony root single-negative solution; 6 is the white peony root and moutan bark double-negative solution; 7 is the licorice control medicinal material; 8 is the test solution B; 9 is the liquiritin reference solution; 10 is the licorice root single-positive solution; 11 is the licorice root single-negative solution; 12 is the blank solvent (methanol).

[0154] exist Figure 7 Part a and Figure 7 In part b, in the chromatogram of the test sample, when observed under sunlight, spots of the same color appeared at the corresponding positions on the chromatograms of the paeoniflorin reference solution and the white peony reference medicinal material solution, with good separation and no interference from the double negative control solution, which met the specificity requirements; when observed at 365 nm, spots of the same color appeared at the corresponding positions on the chromatograms of the test sample, with glycyrrhizin reference solution and the glycyrrhiza reference medicinal material solution, with good separation and no interference from the negative control solution, which met the specificity requirements.

[0155] 2. Repeatability verification

[0156] Different experimenters prepared each solution according to the identification method and the records under the specificity verification, and repeated the thin layer chromatography test under the specificity verification. Figure 8 Part a and Figure 8 As shown in part b, Figure 8 Part a in the figure is the inspection result under white light source. Figure 8 Part b in the figure shows the inspection result under 365nm light source.

[0157] exist Figure 8 Part a and Figure 8 In part b, 1 is the white peony root control medicinal material solution; 2 is the test solution B; 3 is the paeoniflorin reference solution; 4 is the white peony root single-positive solution; 5 is the white peony root single-negative solution; 6 is the white peony root and moutan bark double-negative solution; 7 is the licorice control medicinal material; 8 is the test solution B; 9 is the liquiritin reference solution; 10 is the licorice root single-positive solution; 11 is the licorice root single-negative solution; 12 is the blank solvent (methanol).

[0158] Depend on Figure 8 Part a and Figure 8As can be seen from part b, after measurement by different experimenters, in the chromatogram of the test sample, when observed under sunlight, spots of the same color appeared at the corresponding positions of the chromatograms of the paeoniflorin reference solution and the white peony reference medicinal material solution, with good separation and no interference from the double negative control solution; when observed at 365nm, spots of the same color appeared at the corresponding positions of the chromatograms of the test sample, with glycyrrhizin reference solution and the glycyrrhizin reference medicinal material solution, with good separation and no interference from the negative control solution, and the thin layer identification method has good repeatability.

[0159] 3. Durability verification

[0160] (1) Durability verification of different sample sizes

[0161] The above-mentioned white peony control medicinal material solution (2μl, 3μl, 4μl), paeoniflorin reference solution (4μl, 5μl, 6μl), test solution B (1μl, 2μl, 3μl), licorice control medicinal material solution (1μl, 2μl, 3μl), and liquiritin reference solution (1μl, 2μl, 3μl) were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate, and developed according to the thin layer conditions under the identification method. The results are as follows Figure 9 Part a and Figure 9 As shown in part b, Figure 9 Part a in the figure is the inspection result under white light source. Figure 9 Part b in the figure shows the inspection result under 365nm light source.

[0162] exist Figure 9 Part a and Figure 9 In part b, 1 is 2 μl of white peony root control medicinal material solution; 2 is 3 μl of white peony root control medicinal material solution; 3 is 4 μl of white peony root control medicinal material solution; 4 is 4 μl of paeoniflorin reference solution; 5 is 5 μl of paeoniflorin reference solution; 6 is 6 μl of paeoniflorin reference solution; 7 is 1 μl of test solution B; 8 is 2 μl of test solution B; 9 is 3 μl of test solution B; 10 is 1 μl of licorice root control medicinal material solution; 11 is 2 μl of licorice root control medicinal material solution; 12 is 3 μl of licorice root control medicinal material solution; 13 is 1 μl of liquiritin reference solution; 14 is 2 μl of liquiritin reference solution; 15 is 3 μl of liquiritin reference solution. Figure 9 Part a and Figure 9 As can be seen from part b in Figure 3, good detection results can be obtained at different sample sizes, indicating that this method has good durability for different sample sizes.

[0163] (2) Durability verification at different ambient temperatures

[0164] Under normal temperature and humidity (T: 20℃, RH: 30%) and low temperature and humidity (T: 8℃, RH: 30%) conditions, according to the sampling amount under the specificity verification item, the test solution B, the corresponding reference solution, the corresponding single positive control solution and the corresponding control medicinal material solution were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate, and developed according to the thin layer conditions. The results are as follows Figure 10 Part a of - Figure 10 As shown in part d, Figure 10 Part a is the white light source inspection result under normal temperature and humidity conditions. Figure 10 Part b is the inspection result of 365nm light source under normal temperature and humidity conditions. Figure 10 Part c is the white light source inspection result under low temperature and normal humidity conditions. Figure 10 Part d in the figure shows the inspection results using a 365nm light source under low temperature and normal humidity conditions.

[0165] exist Figure 10 Part a of - Figure 10 In part d, 1 is the white peony root control solution; 2 is the test solution B; 3 is the paeoniflorin reference solution; 4 is the white peony root single positive solution; 5 is the licorice root control solution; 6 is the test solution; 7 is the liquiritin reference solution; 8 is the licorice single positive solution. Figure 10 Part a of - Figure 10 It can be seen from part d in that good detection results can be obtained at different ambient temperatures, indicating that this method has good durability for different ambient temperatures.

[0166] (3) Durability verification in different environmental humidity

[0167] Under the conditions of room temperature and low humidity (T: 20 ℃, RH: 10%) and room temperature and high humidity (T: 20 ℃, RH: 75%), according to the sampling volume under the specificity verification item, the test solution B, the corresponding reference solution, the corresponding single positive control solution and the corresponding control medicinal material solution were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate, and developed according to the thin layer conditions.

[0168] The results are as follows Figure 11 As shown in parts a-d, Figure 11 Part a is the inspection result of white light source under normal temperature and low humidity conditions. Figure 11 Part b is the inspection result of 365nm light source under normal temperature and low humidity conditions. Figure 11 Part c is the white light source inspection result under normal temperature and high humidity conditions. Figure 11 Part d shows the inspection results under normal temperature and high humidity conditions using a 365nm light source.

[0169] exist Figure 11In parts a-d, 1 is the white peony root control solution; 2 is the test solution B; 3 is the paeoniflorin reference solution; 4 is the white peony root single positive solution; 5 is the licorice root control solution; 6 is the test solution; 7 is the liquiritin reference solution; 8 is the licorice single positive solution. Figure 11 It can be seen from parts a to d in the figure that good test results can be obtained under different ambient humidity conditions, indicating that this method has good durability under different ambient humidity conditions.

[0170] (4) Durability verification of different thin layer boards

[0171] According to the sampling volume under the specificity verification item, the test solution B, the corresponding reference solution, the corresponding single positive control solution and the corresponding control medicinal material solution are respectively aspirated and spotted on different thin layer plates: Yinlong thin layer plate HSGF 254 (Yantai Chemical Industry Research Institute, 20191022), Yantai Jiangyou Thin Layer Board HSGF 254 (Yantai Zhifu Huangwu Silicone Development and Testing Plant, October 7, 2016) and Qingdao Dingkang Thin Layer Plate G (Qingdao Dingkang Co., Ltd., May 8, 2019); then developed according to the thin layer conditions. The results show that good test results were obtained using different thin layer plates, indicating that this method has good durability for different brands of thin layer plates.

[0172] (5) Durability verification of different developing agent ratios

[0173] According to the sampling volume under the specificity verification item, the test solution B, the corresponding reference solution, the corresponding single positive control solution and the corresponding reference medicinal material solution were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate. The two thin layer plates were spotted in the same way; then the two thin layer plates were developed with ethyl acetate-acetone-methanol-ammonia water (5:3:3:2) and ethyl acetate-acetone-methanol-ammonia water (6:2:3:2) as the developing solvents. The inspection results are as follows: Figure 12 As shown in parts a-d, Figure 12 Part a shows the results of white light inspection using ethyl acetate-acetone-methanol-ammonia water (5:3:3:2) as the developing solvent. Figure 12 Part b is the result of the examination using a 365nm light source with ethyl acetate-acetone-methanol-ammonia water (5:3:3:2) as the developing solvent. Figure 12 Part c is the result of white light inspection using ethyl acetate-acetone-methanol-ammonia water (6:2:3:2) as the developing solvent. Figure 12 Part d shows the results of examination using a 365 nm light source with ethyl acetate-acetone-methanol-ammonia water (6:2:3:2) as the developing solvent.

[0174] exist Figure 12In parts a-d, 1 is the white peony root control solution; 2 is the test solution B; 3 is the paeoniflorin reference solution; 4 is the white peony root single positive solution; 5 is the licorice root control solution; 6 is the test solution; 7 is the liquiritin reference solution; 8 is the licorice single positive solution. Figure 12 As can be seen from parts a to d, good detection results can be obtained using different ratios of developing agents, indicating that this method has good durability for different developing agent ratios.

[0175] Example 3 Thin layer identification of Achyranthes bidentata in the standard decoction of Wenjing Decoction

[0176] 1. Identification method

[0177] (1) Preparation of test solution D: Take 2 g of the test sample of the standard decoction of Wenjing Decoction, add 50 ml of 80% methanol, reflux for 1 h, filter, evaporate the filtrate to dryness, dissolve the residue in 50 ml of water, extract twice with 30 ml of n-butanol saturated with water, combine the n-butanol, wash twice with 20 ml of water saturated with n-butanol, evaporate the n-butanol to dryness, and dissolve it in 1 ml of methanol to prepare the test solution D.

[0178] (2) Preparation of control medicinal material solution: Take 2 g of Achyranthes bidentata control medicinal material and prepare Achyranthes bidentata control medicinal material solution according to the preparation method of test solution D.

[0179] (3) Preparation of reference solution: Take β-ecdysterone reference solution and add methanol to prepare a solution containing 0.4 mg of β-ecdysterone reference solution per 1 ml.

[0180] (4) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 3 μl of the above-mentioned reference solution, 5 μl of the reference medicinal material solution, and 2 μl of the test solution were respectively taken and spotted on the same silica gel GF254 thin layer plate, and chloroformane-methanol-water-formic acid (8:2:0.3:0.5) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, dried, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365 nm).

[0181] In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference medicinal material; and a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0182] 2. Methodological Validation

[0183] 1. Specificity verification

[0184] (1) Preparation of negative control solution: Take 2 g of Wenjing Decoction Achyranthes single negative freeze-dried product and prepare Achyranthes single negative control solution according to the preparation method of test solution D.

[0185] (2) Preparation of positive control solution: Take 2 g of the single positive freeze-dried product of Wenjing Decoction Achyranthes bidentata and prepare the single positive control solution of Achyranthes bidentata according to the preparation method of test solution D.

[0186] (3) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 3 μl of the above-mentioned reference solution, 5 μl of the reference medicinal material solution, 2 μl each of the negative control solution, the positive control solution, the test solution D and the blank solution (methanol) were respectively taken and spotted on the same silica gel GF254 thin layer plate. Chloroform-methanol-water-formic acid (8:2:0.3:0.5) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, dried, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The results are as follows Figure 13 shown.

[0187] exist Figure 13 In the table, 1 is the Achyranthes bidentata control medicinal material solution; 2 is the test solution D; 3 is the β-ecdysterone reference solution; 4 is the Achyranthes bidentata single positive solution; 5 is the Achyranthes bidentata single negative solution; and 6 is the blank solvent (methanol). Figure 13 It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the β-ecdysterone reference solution and the Achyranthes bidentata reference medicinal material solution, the separation is good and there is no interference from the negative control solution, which meets the specificity requirements.

[0188] 2. Repeatability verification

[0189] Different experimenters prepared various solutions according to the identification method and the records under the specificity verification, and repeated the thin layer chromatography test under the specificity verification. The results are as follows: Figure 14 shown.

[0190] exist Figure 14 In the table, 1 is the Achyranthes bidentata control medicinal material solution; 2 is the test solution D; 3 is the β-ecdysterone reference solution; 4 is the Achyranthes bidentata single positive solution; 5 is the Achyranthes bidentata single negative solution; and 6 is the blank solvent (methanol). Figure 14 It can be seen that after measurement by different experimenters, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the β-ecdysterone reference solution and the Achyranthes bidentata reference medicinal material solution, with good separation and no interference from the negative control solution. The thin layer identification method has good repeatability.

[0191] 3. Durability verification

[0192] (1) Durability verification of different sample sizes

[0193] The above-mentioned β-ecdysterone reference solution (2μl, 3μl, 4μl), test solution D (1μl, 2μl, 3μl), and Achyranthes bidentata reference medicinal material solution (4μl, 5μl, 6μl) were respectively aspirated and spotted on the same silica gel HSG thin layer plate, and developed according to the thin layer conditions under the identification method. The results are as follows Figure 15 shown.

[0194] exist Figure 15 In the table, 1 is 4 μl of Achyranthes bidentata control medicinal material solution; 2 is 5 μl of Achyranthes bidentata control medicinal material solution; 3 is 6 μl of Achyranthes bidentata control medicinal material solution; 4 is 1 μl of test solution; 5 is 2 μl of test solution; 6 is 3 μl of test solution; 7 is 2 μl of β-ecdysterone reference solution; 8 is 3 μl of β-ecdysterone reference solution; 9 is 4 μl of β-ecdysterone reference solution. Figure 15 It can be seen that good detection results can be obtained at different sample sizes, indicating that this method has good durability for different sample sizes.

[0195] (2) Durability verification at different ambient temperatures

[0196] Under normal temperature and humidity (T: 25℃, RH: 52%) and low temperature and humidity (T: 6℃, RH: 52%) conditions, according to the sampling amount under the specificity verification item, the above-mentioned test solution D, β-ecdysterone reference solution, Achyranthes bidentata single positive control solution and Achyranthes bidentata control medicinal material solution were respectively aspirated and spotted on the same silica gel HSG thin layer plate, and developed according to the thin layer conditions under the identification method item. The results are as follows Figure 16 Part a and Figure 16 As shown in part b, Figure 16 Part a is the inspection result under normal temperature and humidity conditions. Figure 16 Part b shows the inspection results under low temperature and normal humidity conditions.

[0197] exist Figure 16 Part a and Figure 16 In part b, 1 is the Achyranthes bidentata control medicinal material solution; 2 is the test solution; 3 is the β-ecdysterone reference solution; 4 is the Achyranthes bidentata single positive solution; 5 is the Achyranthes bidentata single negative solution; 6 is the blank solvent (methanol). Figure 16 As can be seen from parts a and b, good test results can be obtained under different ambient temperatures, indicating that this method has good durability under different ambient temperatures.

[0198] (3) Durability verification in different environmental humidity

[0199] Under the conditions of room temperature and low humidity (T:25℃, RH:16%) and room temperature and high humidity (T:25℃, RH:75%), according to the sampling amount under the specificity verification item, the above-mentioned test solution D, β-ecdysterone reference solution, Achyranthes bidentata single positive control solution and Achyranthes bidentata control medicinal material solution were respectively aspirated and spotted on the same silica gel HSG thin layer plate, and developed according to the thin layer conditions under the identification method item. The results are as follows Figure 17 Part a and Figure 17 As shown in part b, Figure 17 Part a in the middle is the inspection result under normal temperature and low humidity conditions. Figure 17 Part b shows the inspection results under normal temperature and high humidity conditions.

[0200] exist Figure 17 Part a and Figure 17 In part b, 1 is the Achyranthes bidentata control medicinal material solution; 2 is the test solution; 3 is the β-ecdysterone reference solution; 4 is the Achyranthes bidentata single positive solution; 5 is the Achyranthes bidentata single negative solution; 6 is the blank solvent (methanol). Figure 17 As can be seen from parts a and 17b, good test results can be obtained under different ambient humidity conditions, indicating that this method has good durability under different ambient humidity conditions.

[0201] (4) Durability verification of different thin layer boards

[0202] According to the sampling volume under the specificity verification item, the above-mentioned test solution D, β-ecdysterone reference solution, Achyranthes bidentata single positive control solution and Achyranthes bidentata reference medicinal material solution were respectively aspirated and spotted on different thin layer plates: Yantai Chemical Thin Layer Plate HSGF 254 (Yantai Institute of Chemical Industry, 20220325), Merck Thin Layer Plate G (Germany, HX98929621), Yantai Jiangyou Thin Layer Plate HSGF 254 (Yantai Jiangyou Silicone Development Co., Ltd., 20161007); then developed according to the thin layer conditions. The results show that good test results can be obtained using different thin layer plates, indicating that this method has good durability for different brands of thin layer plates.

[0203] (5) Durability verification of different developing agent ratios

[0204] According to the sampling volume under the specificity verification item, the above-mentioned test solution D, β-ecdysterone reference solution, Achyranthes bidentata single positive control solution and Achyranthes bidentata reference medicinal material solution were respectively aspirated and spotted on the same silica gel HSGF254 thin layer plate. The two thin layer plates were spotted in the same way; then the two thin layer plates were developed with chloroform-methanol-water-formic acid (9:2:0.3:0.5) and chloroform-methanol-water-formic acid (8:2.5:0.3:0.5) as the developing solvents respectively. The inspection results are as follows: Figure 18As shown in parts a and b, Figure 18 Part a shows the results of the test using chloroform-methanol-water-formic acid (9:2:0.3:0.5) as the developing solvent. Figure 18 Part b shows the inspection results using chloroform-methanol-water-formic acid (8:2.5:0.3:0.5) as the developing solvent.

[0205] exist Figure 18 Part a and Figure 18 In part b, 1 is the Achyranthes bidentata control medicinal material solution; 2 is the test solution; 3 is the β-ecdysterone reference solution; 4 is the Achyranthes bidentata single positive solution; 5 is the Achyranthes bidentata single negative solution; 6 is the blank solvent (methanol). Figure 18 Part a and Figure 18 As can be seen from part b, good detection results can be obtained using different ratios of developing agents, indicating that this method has good durability for different developing agent ratios.

[0206] Example 4 Thin-layer identification of ginseng in the standard decoction of Wenjing Decoction

[0207] 1. Identification method

[0208] (1) Preparation of test solution E: Take 0.5 g of the test sample of the standard decoction of Wenjing Decoction, add 15 ml of water to dissolve it, then add 25 ml of water-saturated n-butanol and ultrasonicate for 30 min. Centrifuge, take the supernatant, add 3 times the amount of ammonia test solution, shake well, let it stand to separate, evaporate the n-butanol solution to dryness, and add 1 ml of methanol to dissolve the residue, which is used as the test solution E.

[0209] (2) Preparation of control medicinal material solution: Take 0.5 g of ginseng control medicinal material and prepare ginseng control medicinal material solution according to the preparation method of test solution E.

[0210] (3) Preparation of reference solution: Take ginsenoside Rb1 reference, ginsenoside Rb2 reference, ginsenoside Rc reference, ginsenoside Re reference, ginsenoside Rf reference and ginsenoside Rg1 reference, add methanol to make a mixed solution containing 1 mg of each per ml, and obtain the mixed reference solution.

[0211] (4) According to the thin layer chromatography method (General Rule 0502), 3 μL of the above-mentioned control medicinal material solution and mixed reference substance solution and 5 μL of the test solution were respectively spotted on the same silica gel HSGF254 thin layer plate, and chloroform-anhydrous methanol-water (9:4.5:1) solution was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored.

[0212] In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference medicinal material; and a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0213] 2. Methodological Validation

[0214] 1. Specificity verification

[0215] (1) Preparation of negative control solution: Take 0.5 g of Wenjing Decoction ginseng single negative test sample and prepare ginseng single negative control solution according to the preparation method of test sample solution E.

[0216] (2) Preparation of positive control solution: Take 0.5 g of Wenjing Decoction ginseng single positive test sample and prepare ginseng single positive control solution according to the preparation method of test sample solution E.

[0217] (3) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 3 μL of the above-mentioned control medicinal material solution, positive control solution and mixed reference solution, 5 μL of the test solution, negative control solution and blank solution (methanol) were taken and spotted on the same silica gel HSGF254 thin layer plate, and chloroform-anhydrous methanol-water (9:4.5:1) solution was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. The results are as follows: Figure 19 shown.

[0218] exist Figure 19 In the table, 1 is the ginseng control medicinal material solution; 2 is the test solution; 3 is the mixed reference solution; 4 is the ginseng negative control solution; 5 is the ginseng positive control solution; and 6 is the blank solvent (methanol). Figure 19 It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the ginseng control medicinal material solution and the mixed reference solution, with good separation, and there is no interference from the ginseng negative control solution, which meets the specificity requirements.

[0219] 2. Repeatability verification

[0220] Different experimenters prepared various solutions according to the identification method and the records under the specificity verification, and repeated the thin layer chromatography test under the specificity verification. The results are as follows: Figure 20 shown.

[0221] exist Figure 20 In the table, 1 is the ginseng control medicinal material solution; 2 is the test solution; 3 is the mixed reference solution; 4 is the ginseng negative control solution; 5 is the ginseng positive control solution; and 6 is the blank solvent (methanol). Figure 20It can be seen that after measurement by different experimenters, spots of the same color appeared at the corresponding positions of the chromatogram of the test sample and the chromatogram of the ginseng control medicinal material solution and the ginseng mixed reference solution, with good separation, and no interference from the ginseng negative control solution. The thin layer identification method has good repeatability.

[0222] 3. Durability verification

[0223] (1) Durability verification of different sample sizes

[0224] Respectively draw the above-mentioned ginseng reference medicinal material solution (2μl, 3μl, 4μl), test solution (4μl, 5μl, 6μl), and mixed reference solution (2μl, 3μl, 4μl) and spot them on the same high-performance silica gel G thin layer plate. Develop, color and examine according to the chromatographic conditions under the identification method. The results are as follows. Figure 21 shown.

[0225] exist Figure 21 In the equation, 1 is 2 μl of ginseng control medicinal material solution; 4 is 4 μl of test solution; 7 is 2 μl of ginseng mixed reference solution; 2 is 3 μl of ginseng control medicinal material solution; 5 is 5 μl of test solution; 8 is 3 μl of ginseng mixed reference solution; 3 is 4 μl of ginseng control medicinal material solution; 6 is 6 μl of test solution; and 9 is 4 μl of ginseng mixed reference solution. Figure 21 It can be seen that good detection results can be obtained at different sample sizes, indicating that this method has good durability for different sample sizes.

[0226] (2) Durability verification at different ambient temperatures

[0227] Under normal temperature and humidity (T: 19°C, RH: 32%) and low temperature and humidity (T: 6°C, RH: 32%) conditions, according to the sampling amount under the specificity verification item, the above-mentioned control medicinal material solution, ginseng mixed reference solution, test solution E, ginseng negative control solution and ginseng positive control solution were respectively aspirated and spotted on the same high-efficiency silica gel G thin layer plate, and developed, colored and inspected according to the chromatographic conditions under the identification method item. The results are as follows: Figure 22 Part a and Figure 22 As shown in part b, Figure 22 Part a is the inspection result under normal temperature and humidity conditions. Figure 22 Part b shows the inspection results under low temperature and normal humidity conditions.

[0228] exist Figure 22 Part a and Figure 22 In part b, 1 is ginseng control medicinal material solution; 2 is test solution; 3 is ginseng mixed reference solution; 4 is ginseng negative control solution; 5 is ginseng positive control solution. Figure 22It can be seen from parts a and b that good test results can be obtained at different ambient temperatures, indicating that this method has good durability for different ambient temperatures.

[0229] (3) Durability verification in different environmental humidity

[0230] Under the conditions of room temperature and low humidity (T: 19 ℃, RH: 24%) and room temperature and high humidity (T: 19 ℃, RH: 84%), according to the sampling amount under the specificity verification item, the above-mentioned control medicinal material solution, ginseng mixed reference solution, test solution E, ginseng negative control solution and ginseng positive control solution were respectively aspirated and spotted on the same high-efficiency silica gel G thin layer plate, and developed, developed and inspected according to the chromatographic conditions under the identification method item. The results are as follows Figure 23 Part a and Figure 23 As shown in part b, Figure 23 Part a in the middle is the inspection result under normal temperature and low humidity conditions. Figure 23 Part b shows the inspection results under normal temperature and high humidity conditions.

[0231] exist Figure 23 Part a and Figure 23 In part b, 1 is ginseng control medicinal material solution; 2 is test solution; 3 is ginseng mixed reference solution; 4 is ginseng negative control solution; 5 is ginseng positive control solution. Figure 22 Part a and Figure 23 As can be seen from part b, good test results can be obtained under different ambient humidity conditions, indicating that this method has good durability under different ambient humidity conditions.

[0232] (4) Durability verification of different thin layer boards

[0233] According to the sampling volume under the specificity verification item, the above-mentioned control medicinal material solution, ginseng mixed reference solution, test solution E, ginseng negative control solution and ginseng positive control solution were respectively aspirated and spotted on different thin layer plates: Merck high-efficiency silica gel G thin layer plate (Merck Group, HX00928621), Chemical Yinlong silica gel HSGF 254 Thin layer plate (Yantai Chemical Industry Research Institute, 20191022), Yantai Jiangyou Silicone HSGF 254 Thin layer plates (Yantai Zhifu Huangwu Silicone Silica Gel Development and Testing Plant, October 7, 2016) were then developed according to the thin layer conditions. The results show that good test results were obtained using different thin layer plates, indicating that this method has good durability for different brands of thin layer plates.

[0234] (5) Durability verification of different developing agent ratios

[0235] According to the sampling volume under the specificity verification item, the above-mentioned control medicinal material solution, ginseng mixed reference solution, test solution E, ginseng negative control solution and ginseng positive control solution were respectively aspirated and spotted on the same thin layer plate. The same method was used to spot two thin layer plates; then the two thin layer plates were developed with chloroform-anhydrous methanol-water (9:5:1) and chloroform-anhydrous methanol-water (9.5:4.5:1) as the developing solvent respectively. The inspection results are as follows: Figure 24 Part a and Figure 24 As shown in part b, Figure 24 Part a shows the results of the test using chloroform-anhydrous methanol-water (9:5:1) as the developing solvent. Figure 24 Part b shows the results of the analysis using chloroform-anhydrous methanol-water (9.5:4.5:1) as the developing solvent.

[0236] exist Figure 24 Part a and Figure 24 In part b, 1 is ginseng control medicinal material solution; 2 is test solution; 3 is ginseng mixed reference solution; 4 is ginseng negative control solution; 5 is ginseng positive control solution. Figure 24 Part a and Figure 24 As can be seen from part b, good detection results can be obtained using different ratios of developing agents, indicating that this method has good durability for different developing agent ratios.

[0237] Example 5 Thin layer chromatography identification of Chuanxiong and Angelica in the standard decoction of Wenjing Decoction

[0238] 1. Identification method

[0239] (1) Preparation of test solution: Take the test solution A under the identification of Paeonia suffruticosa.

[0240] (2) Preparation of control medicinal material solution: Take 0.5 g of Angelica sinensis control medicinal material and 0.5 g of Ligusticum chuanxiong control medicinal material, and prepare Angelica sinensis control medicinal material solution and Ligusticum chuanxiong control medicinal material solution according to the preparation method of test solution A.

[0241] (3) Preparation of reference solution: Take the ligustilide reference standard and add methanol to prepare a solution containing 1 mg of ligustilide reference standard per 1 ml, thus obtaining the ligustilide reference standard solution; take the ligustilide A reference standard and add methanol to prepare a solution containing 1 mg of ligustilide A reference standard per 1 ml, thus obtaining the ligustilide A reference standard solution.

[0242] (4) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 3 μL of each of the test solution A, reference medicinal material solution, ligusticum lactone A reference solution, and ligusticum lactone reference solution were taken and spotted on the same silica gel HSGF254 thin layer plate, and the plate was developed with n-hexane-dichloromethane-ethyl acetate-formic acid (8:1:3:0.2) solution as the developing solvent. The plate was taken out, dried, and inspected under ultraviolet light (254 nm).

[0243] In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference medicinal material; and a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0244] 2. Methodological Validation

[0245] 1. Specificity verification

[0246] (1) Preparation of negative control solution: Take 2.0 g of Wenjing Decoction Angelica sinensis single negative freeze-dried powder and prepare Angelica sinensis single negative control solution according to the preparation method of test solution A; take 2.0 g of Wenjing Decoction Chuanxiong single negative freeze-dried powder and prepare Chuanxiong single negative control solution according to the preparation method of test solution A; take 2.0 g of Wenjing Decoction Angelica sinensis and Chuanxiong double negative freeze-dried powder and prepare Angelica sinensis and Chuanxiong double negative control solution according to the preparation method of test solution A.

[0247] (2) Preparation of positive control solution: Take 1.0 g each of Wenjing Decoction Angelica sinensis single positive freeze-dried powder and Wenjing Decoction Chuanxiong single positive freeze-dried powder, and prepare Angelica sinensis single positive control solution and Chuanxiong single positive control solution according to the preparation method of test solution A.

[0248] (3) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 3 μL of each of the test solution A, control medicinal material solution, negative control solution, positive control solution, ligustilide A reference solution, ligustilide reference solution, and blank solution (acetone) were taken and spotted on the same silica gel HSGF254 thin layer plate. The plate was developed with n-hexane-dichloromethane-ethyl acetate-formic acid (8:1:3:0.2) solution as the developing solvent. The plate was taken out, dried, and examined under ultraviolet light (254 nm). The results were as follows: Figure 25 shown.

[0249] exist Figure 25 In the table, 1 is the control solution of Chinese Angelica sinensis; 2 is the control solution of Chuanxiong Rhizoma; 3 is the test solution; 4 is the control solution of Ligusticum chuanxiong lactone; 5 is the control solution of Ligusticum chuanxiong lactone A; 6 is the single negative control solution of Chinese Angelica sinensis in wine; 7 is the single negative control solution of Chuanxiong Rhizoma; 8 is the double negative control solution of Chinese Angelica sinensis and Chuanxiong in wine; 9 is the single positive control solution of Chinese Angelica sinensis in wine; 10 is the single positive control solution of Chuanxiong; 11 is the blank solvent (methanol). Figure 25It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the Angelica sinensis control medicinal material, Chuanxiong control medicinal material, Ligusticum chuanxiong lactone reference material and Ligusticum chuanxiong lactone reference material, with good separation, and there is no interference with the double negative control solution of Angelica sinensis and Chuanxiong wine, which meets the specificity requirements.

[0250] 2. Repeatability verification

[0251] Different experimenters prepared various solutions according to the identification method and the records under the specificity verification, and repeated the thin layer chromatography test under the specificity verification. The results are as follows: Figure 26 shown.

[0252] exist Figure 26 In the table, 1 is the control solution of Chinese Angelica sinensis; 2 is the control solution of Chuanxiong Rhizoma; 3 is the test solution; 4 is the control solution of Ligusticum chuanxiong lactone; 5 is the control solution of Ligusticum chuanxiong lactone A; 6 is the single negative control solution of Chinese Angelica sinensis in wine; 7 is the single negative control solution of Chuanxiong Rhizoma; 8 is the double negative control solution of Chinese Angelica sinensis and Chuanxiong in wine; 9 is the single positive control solution of Chinese Angelica sinensis in wine; 10 is the single positive control solution of Chuanxiong; 11 is the blank solvent (methanol). Figure 26 It can be seen that after measurement by different experimenters, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal materials of Angelica sinensis, Ligusticum chuanxiong, Ligusticum chuanxiong lactone reference material and Ligusticum chuanxiong lactone reference material, with good separation, and no interference from the double negative control solution of Angelica sinensis and Ligusticum chuanxiong. The thin layer identification method has good repeatability.

[0253] 3. Durability verification

[0254] (1) Durability verification of different sample sizes

[0255] 2 μl, 3 μl and 4 μl of the above-mentioned reference medicinal material solution of Angelica sinensis, reference medicinal material solution of Chuanxiong, reference medicinal material solution of Ligusticum chuanxiong A and reference solution of Ligustilide and test solution were respectively taken and spotted on the same silica gel HSGF. 254 On the thin layer plate, develop and examine according to the chromatographic conditions. The results are as follows. Figure 27 shown.

[0256] exist Figure 27In the figure, 1 is 2μl of Angelica sinensis control medicinal material solution; 2 is 3μl of Angelica sinensis control medicinal material solution; 3 is 4μl of Angelica sinensis control medicinal material solution; 4 is 2μl of Chuanxiong Rhizoma control medicinal material solution; 5 is 3μl of Chuanxiong Rhizoma control medicinal material solution; 6 is 4μl of Chuanxiong Rhizoma control medicinal material solution; 7 is 2μl of test solution; 8 is 3μl of test solution; 9 is 4μl of test solution; 10 is 2μl of ligustilide reference solution; 11 is 3μl of ligustilide reference solution; 12 is 4μl of ligustilide reference solution; 13 is 2μl of ligustilide A reference solution; 14 is 3μl of ligustilide A reference solution; 15 is 4μl of ligustilide A reference solution. Figure 27 It can be seen that good detection results can be obtained at different sample sizes, indicating that this method has good durability for different sample sizes.

[0257] (2) Durability verification at different ambient temperatures

[0258] Under normal temperature and humidity (T: 22℃, RH: 45%) and low temperature and humidity (T: 6℃, RH: 45%) conditions, according to the sampling amount under the specificity verification item, the above-mentioned Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, Ligusticum chuanxiong lactone A reference solution, Ligusticum ligusticum lactone reference solution, test solution, Angelica sinensis single negative control solution, Chuanxiong single negative control solution, Angelica sinensis and Chuanxiong double negative control solution, Angelica sinensis single positive control solution, Chuanxiong single positive control solution were respectively aspirated and spotted on the same thin layer plate, and developed, developed and inspected according to the chromatographic conditions under the identification method item. The results are as follows: Figure 28 Part a and Figure 28 As shown in part b, Figure 28 Part a is the inspection result under normal temperature and humidity conditions. Figure 28 Part b shows the inspection results under low temperature and normal humidity conditions.

[0259] exist Figure 28 Part a and Figure 28 In part b, 1 is the control solution of Chinese Angelica sinensis; 2 is the control solution of Chuanxiong; 3 is the test solution; 4 is the control solution of Ligusticum chuanxiong lactone; 5 is the control solution of Ligusticum chuanxiong lactone A; 6 is the single negative control solution of Chinese Angelica sinensis in wine; 7 is the single negative control solution of Chuanxiong; 8 is the double negative control solution of Chinese Angelica sinensis and Chuanxiong in wine; 9 is the single positive control solution of Chinese Angelica sinensis in wine; 10 is the single positive control solution of Chuanxiong. Figure 28 Part a and Figure 28 As can be seen from part b, good test results can be obtained at different ambient temperatures, indicating that this method has good durability under different ambient temperatures.

[0260] (3) Durability verification in different environmental humidity

[0261] Under the conditions of room temperature and low humidity (T: 21 ℃, RH: 21%) and room temperature and high humidity (T: 21 ℃, RH: 83%), according to the sampling amount under the specificity verification item, the above-mentioned Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, Ligusticum chuanxiong lactone A reference solution, Ligusticum ligusticum lactone reference solution, test solution, Angelica sinensis single negative control solution, Chuanxiong single negative control solution, Angelica sinensis and Chuanxiong double negative control solution, Angelica sinensis single positive control solution, Chuanxiong single positive control solution were respectively aspirated and spotted on the same thin layer plate, and developed, developed and examined according to the chromatographic conditions under the identification method item. The results are as follows: Figure 29 Part a and Figure 29 As shown in part b, Figure 29 Part a in the middle is the inspection result under normal temperature and low humidity conditions. Figure 29 Part b shows the inspection results under normal temperature and high humidity conditions.

[0262] exist Figure 29 Part a and Figure 29 In part b, 1 is the control solution of Chinese Angelica sinensis; 2 is the control solution of Chuanxiong; 3 is the test solution; 4 is the control solution of Ligusticum chuanxiong lactone; 5 is the control solution of Ligusticum chuanxiong lactone A; 6 is the single negative control solution of Chinese Angelica sinensis in wine; 7 is the single negative control solution of Chuanxiong; 8 is the double negative control solution of Chinese Angelica sinensis and Chuanxiong in wine; 9 is the single positive control solution of Chinese Angelica sinensis in wine; 10 is the single positive control solution of Chuanxiong. Figure 29 Part a and Figure 29 As can be seen from part b, good test results can be obtained under different ambient humidity conditions, indicating that this method has good durability under different ambient humidity conditions.

[0263] (4) Durability verification of different thin layer boards

[0264] According to the sampling volume under the specificity verification item, the above-mentioned Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, Yangchuanoliactone A reference solution, Ligusticum chuanxiong reference solution, test solution, Angelica sinensis single negative control solution, Chuanxiong single negative control solution, Angelica sinensis and Chuanxiong double negative control solution, Angelica sinensis single positive control solution, Chuanxiong single positive control solution were respectively aspirated and spotted on different thin layer plates: Qingdao Ocean Silica Gel GF 254 Thin layer plate (manufactured by Qingdao Ocean Chemical Co., Ltd., 20210108), Chemical Yinlong Silica Gel HSGF 254 Thin layer plate (Yantai Chemical Industry Research Institute, 20191022), Yantai Jiangyou Silicone HSGF 254 Thin layer plates (Yantai Zhifu Huangwu Silicone Silica Gel Development and Testing Plant, October 7, 2016) were then developed according to the thin layer conditions. The results show that good test results were obtained using different thin layer plates, indicating that this method has good durability for different brands of thin layer plates.

[0265] (5) Durability verification of different developing agent ratios

[0266] According to the sampling volume under the specificity verification item, the above-mentioned Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, Ligusticum chuanxiong lactone A reference solution, Ligusticum ligusticum lactone reference solution, test solution, Angelica sinensis single negative control solution, Chuanxiong single negative control solution, Angelica sinensis and Chuanxiong double negative control solution, Angelica sinensis single positive control solution, Chuanxiong single positive control solution were respectively aspirated and spotted on the same thin layer plate, and two thin layer plates were spotted in the same way; then the two thin layer plates were developed with n-hexane-dichloromethane-ethyl acetate-formic acid (9:3:1:0.2) and n-hexane-dichloromethane-ethyl acetate-formic acid (8:3:2:0.2) as developing agents respectively. The inspection results are as follows: Figure 30 Part a and Figure 30 As shown in part b, Figure 30 Part a shows the results of the test using n-hexane-dichloromethane-ethyl acetate-formic acid (9:3:1:0.2) as the developing solvent. Figure 30 Part b shows the results of the examination using n-hexane-dichloromethane-ethyl acetate-formic acid (8:3:2:0.2) as the developing solvent.

[0267] exist Figure 30 Part a and Figure 30 In part b, 1 is the control solution of Chinese Angelica sinensis; 2 is the control solution of Chuanxiong; 3 is the test solution; 4 is the control solution of Ligusticum chuanxiong lactone; 5 is the control solution of Ligusticum chuanxiong lactone A; 6 is the single negative control solution of Chinese Angelica sinensis in wine; 7 is the single negative control solution of Chuanxiong; 8 is the double negative control solution of Chinese Angelica sinensis and Chuanxiong in wine; 9 is the single positive control solution of Chinese Angelica sinensis in wine; 10 is the single positive control solution of Chuanxiong. Figure 30 Part a and Figure 30 As can be seen from part b, good detection results can be obtained using different ratios of developing agents, indicating that this method has good durability for different developing agent ratios.

[0268] Example 6 Thin layer chromatography identification of cinnamon bark in the standard decoction of Wenjing Decoction

[0269] 1. Identification method

[0270] (1) Preparation of test solution: Take the test solution A under the identification of Paeonia suffruticosa.

[0271] (2) Preparation of control medicinal material solution: Take 2 g of cinnamon control medicinal material and prepare cinnamon control medicinal material solution according to the preparation method of test solution A.

[0272] (3) Preparation of reference solution: Take cinnamic acid reference solution and add anhydrous ethanol to make a solution containing 0.5 mg of cinnamic acid reference solution per 1 ml.

[0273] (4) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 2 μl of the above-mentioned reference solution and reference medicinal material solution and 5 μl of the test solution were respectively taken and spotted on the same silica gel HSGF254 thin layer plate. Petroleum ether (60~90℃)-ethyl acetate-formic acid (8:3:0.2) was used as the developing solvent. The plate was developed, taken out, dried, and inspected under ultraviolet light (254nm).

[0274] In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference medicinal material; and a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0275] 2. Methodological Validation

[0276] 1. Specificity verification

[0277] (1) Preparation of negative control solution: Take 2 g of Wenjing Decoction cinnamon single negative test sample and prepare cinnamon single negative control solution according to the preparation method of test sample solution A.

[0278] (2) Preparation of positive control solution: Take 1 g of Wenjing Decoction cinnamon single positive test sample and prepare cinnamon single positive control solution according to the preparation method of test solution A.

[0279] (3) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 2 μl of the above-mentioned reference solution, reference medicinal material solution, positive control solution and blank solution (acetone), 5 μl of the negative control solution and test solution were respectively spotted on the same silica gel HSGF254 thin layer plate, and petroleum ether (60~90℃)-ethyl acetate-formic acid (8:3:0.2) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (254nm). The results are as follows: Figure 31 shown.

[0280] exist Figure 31 In the table, 1 is the cinnamon reference medicinal material solution; 2 is the test solution; 3 is the cinnamic acid reference solution; 4 is the cinnamon negative solution; 5 is the cinnamon single positive solution; and 6 is the blank solvent (acetone). Figure 31 It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the cinnamic acid reference solution and the cinnamon reference medicinal material solution. The separation is good and there is no interference from the negative control solution, which meets the specificity requirements.

[0281] 2. Repeatability verification

[0282] Different experimenters prepared various solutions according to the identification method and the records under the specificity verification, and repeated the thin layer chromatography test under the specificity verification. The results are as follows: Figure 32 shown.

[0283] exist Figure 32 In the table, 1 is the cinnamon reference solution; 2 is the test solution; 3 is the cinnamic acid reference solution; 4 is the cinnamon negative solution; 5 is the cinnamon single positive solution; and 6 is the blank solvent (acetone). Figure 32 It can be seen that after measurement by different experimenters, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the cinnamic acid reference solution and the cinnamon reference medicinal material solution. The separation was good and there was no interference from the negative control solution. The thin layer identification method had good repeatability.

[0284] 3. Durability verification

[0285] (1) Durability verification of different sample sizes

[0286] The above-mentioned cinnamic acid reference solution (1μl, 2μl, 3μl), test solution (4μl, 5μl, 6μl), and cinnamon reference medicinal material solution (1μl, 2μl, 3μl) were respectively aspirated and spotted on the same silica gel HSGF. 254 On the thin layer plate, expand and inspect according to the thin layer conditions. The results are as follows Figure 33 shown.

[0287] exist Figure 33 In the figure, 1 is 4 μl of test solution; 2 is 5 μl of test solution; 3 is 6 μl of test solution; 4 is 1 μl of cinnamic acid reference solution; 5 is 2 μl of cinnamic acid reference solution; 6 is 3 μl of cinnamic acid reference solution; 7 is 1 μl of cinnamon reference solution; 8 is 2 μl of cinnamon reference solution; 9 is 3 μl of cinnamon reference solution. Figure 33 It can be seen that good detection results can be obtained at different sample sizes, indicating that this method has good durability for different sample sizes.

[0288] (2) Durability verification at different ambient temperatures

[0289] Under normal temperature and humidity (T: 22.5℃, RH: 33%) and low temperature and humidity (T: 6℃, RH: 33%) conditions, according to the sampling amount under the specificity verification item, the above-mentioned test solution, reference solution, single positive control solution and reference medicinal material solution were respectively aspirated and spotted on the same thin layer plate, and developed, developed and inspected according to the chromatographic conditions under the identification method item. The results are as follows: Figure 34 Part a and Figure 34 As shown in part b, Figure 34 Part a is the inspection result under normal temperature and humidity conditions. Figure 34 Part b shows the inspection results under low temperature and normal humidity conditions.

[0290] exist Figure 34 Part a and Figure 34In part b, 1 is the cinnamon reference medicinal material solution; 2 is the test solution; 3 is the cinnamic acid reference solution; 4 is the cinnamon single positive solution. Figure 34 Part a and Figure 34 As can be seen from part b, good test results can be obtained at different ambient temperatures, indicating that this method has good durability for different ambient temperatures.

[0291] (3) Durability verification in different environmental humidity

[0292] Under the conditions of room temperature and low humidity (T: 22℃, RH: 26%) and room temperature and high humidity (T: 22℃, RH: 78%), according to the sampling amount under the specificity verification item, the above-mentioned test solution, reference solution, single positive control solution and reference medicinal material solution were respectively aspirated and spotted on the same thin layer plate, and developed, developed and inspected according to the chromatographic conditions under the identification method item. The results are as follows Figure 35 Part a and Figure 35 As shown in part b, Figure 35 Part a in the middle is the inspection result under normal temperature and low humidity conditions. Figure 35 Part b shows the inspection results under normal temperature and high humidity conditions.

[0293] exist Figure 35 Part a and Figure 35 In part b, 1 is the cinnamon reference medicinal material solution; 2 is the test solution; 3 is the cinnamic acid reference solution; 4 is the cinnamon single positive solution. Figure 35 Part a and Figure 35 As can be seen from part b, good test results can be obtained under different ambient humidity conditions, indicating that this method has good durability under different ambient humidity conditions.

[0294] (4) Durability verification of different thin layer boards

[0295] According to the sampling volume under the specificity verification item, the above-mentioned test solution, reference solution, single positive control solution and reference medicinal material solution are respectively aspirated and spotted on different thin layer plates: Yinlong thin layer plate HSGF 254 (Yantai Chemical Industry Research Institute, 20191022), Qingdao Marine Thin Layer Plate GF 254 (Made by Qingdao Ocean Chemical Co., Ltd., July 7, 2020), Yantai Jiangyou Thin Layer Board HSGF 254 (Yantai Zhifu Huangwu Silicone Development and Testing Plant, October 7, 2016); Then, the test was carried out according to the thin layer conditions. The results show that good test results were obtained using different thin layer plates, indicating that this method has good durability for different brands of thin layer plates.

[0296] (5) Durability verification of different developing agent ratios

[0297] According to the sampling volume under the specificity verification item, the above-mentioned test solution, reference solution, single positive control solution and reference medicinal material solution were respectively aspirated and spotted on the same thin layer plate. The same method was used to spot two thin layer plates; then the two thin layer plates were developed with petroleum ether (60~90℃)-ethyl acetate-formic acid (8.5:3:0.2) and petroleum ether (60~90℃)-ethyl acetate-formic acid (8:3.5:0.2) as the developing solvent respectively. The inspection results are as follows: Figure 36 Part a and Figure 36 As shown in part b, Figure 36 Part a is the inspection result using petroleum ether (60~90℃)-ethyl acetate-formic acid (8.5:3:0.2) as the developing solvent. Figure 36 Part b shows the inspection results using petroleum ether (60~90℃)-ethyl acetate-formic acid (8:3.5:0.2) as the developing solvent.

[0298] exist Figure 36 Part a and Figure 36 In part b, 1 is the cinnamon reference medicinal material solution; 2 is the test solution; 3 is the cinnamic acid reference solution; 4 is the cinnamon single positive solution. Figure 36 Part a and Figure 36 As can be seen from part b, good detection results can be obtained using different ratios of developing agents, indicating that this method has good durability for different developing agent ratios.

[0299] Example 7 Thin layer identification of Curcuma zedoaria in the standard decoction of Wenjing Decoction

[0300] 1. Identification method

[0301] (1) Preparation of test solution C: Take 4 g of the test sample of the standard decoction of Wenjing Decoction, add 50 ml of n-hexane, treat with ultrasound for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 0.5 ml of n-hexane to prepare test solution C.

[0302] (2) Preparation of control medicinal material solution: Take 0.25 g of Curcuma zedoaria control medicinal material and prepare the Curcuma zedoaria control medicinal material solution according to the preparation method of test solution C.

[0303] (3) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 20 μl of the control medicinal material solution and the test sample solution were taken and spotted on the same silica gel HSGF254 thin layer plate respectively. Cyclohexane-ethyl acetate-formic acid (9:1:0.2) was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365 nm).

[0304] In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions in the chromatogram of the control medicinal material.

[0305] 2. Methodological Validation

[0306] 1. Specificity verification

[0307] (1) Preparation of negative control solution: Take 4 g of Wenjing Decoction Curcuma zedoariae single negative freeze-dried powder and prepare Curcuma zedoariae single negative control solution according to the preparation method of test solution C.

[0308] (2) Preparation of positive control solution: Take 2 g of the single positive test sample of Wenjing Decoction Curcuma zedoariae and prepare the single positive control solution of Curcuma zedoariae according to the preparation method of test sample solution C.

[0309] (3) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 20 μl of the above-mentioned control medicinal material solution, test solution and negative control solution, 5 μl of the positive control solution and blank solution (n-hexane) were taken and spotted on the same silica gel HSGF254 thin layer plate, and cyclohexane-ethyl acetate-formic acid (9:1:0.2) was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The results are as follows Figure 37 shown.

[0310] exist Figure 37 In the figure, 1 is blank solvent (n-hexane); 2 is positive solution of Curcuma zedoaria; 3 is test solution; 4 is control solution of Curcuma zedoaria; 5 is negative solution of Curcuma zedoaria. Figure 37 It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the Curcuma zedoaria control medicinal material solution, the separation is good and there is no interference from the negative control solution, which meets the specificity requirements.

[0311] 2. Repeatability verification

[0312] Different experimenters prepared various solutions according to the identification method and the records under the specificity verification, and repeated the thin layer chromatography test under the specificity verification. The results are as follows: Figure 38 shown.

[0313] exist Figure 38 In the figure, 1 is blank solvent (n-hexane); 2 is positive solution of Curcuma zedoaria; 3 is test solution; 4 is control solution of Curcuma zedoaria; 5 is negative solution of Curcuma zedoaria. Figure 38 It can be seen that after measurement by different experimenters, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the Curcuma zedoaria control medicinal material solution, with good separation and no interference from the negative control solution. The thin layer identification method has good repeatability.

[0314] 3. Durability verification

[0315] (1) Durability verification of different sample sizes

[0316] The above test solution (15μl, 20μl, 25μl) and the control drug solution of Curcuma zedoaria (15μl, 20μl, 25μl) were respectively spotted on the same silica gel HSGF254 thin layer plate, developed and inspected according to the thin layer conditions. The results are as follows Figure 39 shown.

[0317] exist Figure 39 In the equation, 1 is 15 μl of the control solution of Curcuma zedoaria; 2 is 20 μl of the control solution of Curcuma zedoaria; 3 is 25 μl of the control solution of Curcuma zedoaria; 4 is 15 μl of the test solution; 5 is 20 μl of the test solution; and 6 is 25 μl of the test solution. Figure 39 It can be seen that good detection results can be obtained at different sample sizes, indicating that this method has good durability for different sample sizes.

[0318] (2) Durability verification at different ambient temperatures

[0319] Under normal temperature and humidity (T: 24℃, RH: 33%) and low temperature and humidity (T: 7℃, RH: 33%) conditions, according to the sampling amount under the specificity verification item, the above-mentioned test sample solution, single positive control solution and control medicinal material solution were respectively aspirated and spotted on the same thin layer plate, and developed, developed and inspected according to the chromatographic conditions under the identification method item. The results are as follows Figure 40 Part a and Figure 40 As shown in part b, Figure 40 Part a is the inspection result under normal temperature and humidity conditions. Figure 40 Part b shows the inspection results under low temperature and normal humidity conditions.

[0320] exist Figure 40 Part a and Figure 40 In part b, 1 is the single positive solution of Curcuma zedoaria; 2 is the test solution; 3 is the control medicinal material solution of Curcuma zedoaria. Figure 40 Part a and Figure 40 As can be seen from part b, good test results can be obtained at different ambient temperatures, indicating that this method has good durability under different ambient temperatures.

[0321] (3) Durability verification in different environmental humidity

[0322] Under the conditions of room temperature and low humidity (T: 24℃, RH: 26%) and room temperature and high humidity (T: 24℃, RH: 78%), according to the sampling amount under the specificity verification item, the above-mentioned test sample solution, single positive control solution and control medicinal material solution were respectively aspirated and spotted on the same thin layer plate, and developed, developed and inspected according to the chromatographic conditions under the identification method item. The results are as follows Figure 41 Part a and Figure 41 As shown in part b, Figure 41 Part a in the middle is the inspection result under normal temperature and low humidity conditions. Figure 41 Part b shows the inspection results under normal temperature and high humidity conditions.

[0323] exist Figure 41 Part a and Figure 41 In part b, 1 is the single positive solution of Curcuma zedoaria; 2 is the test solution; 3 is the control medicinal material solution of Curcuma zedoaria. Figure 41 Part a and Figure 41 As can be seen from part b, good test results can be obtained under different ambient humidity conditions, indicating that this method has good durability under different ambient humidity conditions.

[0324] (4) Durability verification of different thin layer boards

[0325] According to the sampling volume under the specificity verification item, the above-mentioned test solution, single positive control solution and control medicinal material solution are respectively aspirated and spotted on different thin layer plates: Yinlong thin layer plate HSGF 254 (Yantai Chemical Industry Research Institute, 20191022), Qingdao Marine Thin Layer Plate GF 254 (Made by Qingdao Ocean Chemical Co., Ltd., July 7, 2020), Yantai Jiangyou Thin Layer Board HSGF 254 (Yantai Zhifu Huangwu Silicone Development and Testing Plant, October 7, 2016); Then, the test was carried out according to the thin layer conditions. The results show that good test results were obtained using different thin layer plates, indicating that this method has good durability for different brands of thin layer plates.

[0326] (5) Durability verification of different developing agent ratios

[0327] According to the sampling volume under the specificity verification item, the above-mentioned test solution, single positive control solution and control medicinal material solution were respectively aspirated and spotted on the same thin layer plate. The same method was used to spot two thin layer plates; then the two thin layer plates were developed with cyclohexane-ethyl acetate-formic acid (9.5:1:0.2) and cyclohexane-ethyl acetate-formic acid (9:1.5:0.2) as the developing solvent respectively. Check the results as follows Figure 42 Part a and Figure 42 As shown in part b, Figure 42 Part a in the middle shows the inspection results using cyclohexane-ethyl acetate-formic acid (9.5:1:0.2) as the developing solvent. Figure 42 Part b shows the results of the analysis using cyclohexane-ethyl acetate-formic acid (9:1.5:0.2) as the developing solvent.

[0328] exist Figure 42 Part a and Figure 42 In part b, 1 is the single positive solution of Curcuma zedoaria; 2 is the test solution; 3 is the control medicinal material solution of Curcuma zedoaria. Figure 42 Part a and Figure 42 As can be seen from part b, good detection results can be obtained using different ratios of developing agents, indicating that this method has good durability for different developing agent ratios.

[0329] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A quality control method for a Wenjing Decoction compound preparation, wherein the Wenjing Decoction compound preparation is prepared from raw materials including angelica sinensis, Chuanxiong rhizome, white peony root, cinnamon bark, moutan bark, zedoary turmeric, ginseng, cypress and liquorice, characterized in that: The quality control method includes: preparing a test solution A, and identifying Moutan Cortex, Chuanxiong Rhizome, Angelica Sinensis, and Cinnamomum cassia bark by thin layer chromatography based on the test solution A; preparing a test solution B, and identifying white peony root and liquorice root by thin layer chromatography based on the test solution B; preparing a test solution C, and identifying Curcuma zedoaria based on the test solution C by thin layer chromatography; preparing a test solution D, and identifying Achyranthes bidentata by thin layer chromatography based on the test solution D; Prepare a test solution E, and identify ginseng by thin layer chromatography based on the test solution E, The preparation process of the test solution A comprises: taking a test sample of the Wenjing Decoction compound preparation, dissolving it in methanol, and then performing solid-liquid separation, evaporating the liquid, re-dissolving the obtained residue in water, and then performing solid-liquid separation, extracting the liquid with ether, evaporating the ether extract, and re-dissolving the obtained residue in acetone; the preparation process of the test solution B comprises: taking the aqueous solution after ether extraction in the preparation process of the test solution A, extracting it with water-saturated n-butanol, evaporating the n-butanol extract, and re-dissolving the obtained residue in methanol; Based on the test solution B, thin layer chromatography was used to identify white peony root and liquorice, comprising the following steps: The test solution B, white peony control medicinal material solution, licorice control medicinal material solution, paeoniflorin reference solution, and liquiritin reference solution are respectively spotted on the same silica gel HSGF254 thin layer plate, using ethyl acetate-acetone-methanol-ammonia water as a developing solvent, spraying with sulfuric acid ethanol solution, heating until color is developed, and inspecting under ultraviolet light; the quality control method also includes the step of preparing the white peony control medicinal material solution and the licorice control medicinal material solution according to the preparation process of the test solution B; wherein the spotted amount of the test solution B, licorice control medicinal material solution, and liquiritin reference solution is 1-3 μL; the spotted amount of the paeoniflorin reference solution is 4-6 μL; the spotted amount of the white peony control medicinal material solution is 2-4 μL; in the developing solvent, the volume ratio of ethyl acetate, acetone, methanol, and ammonia water is (5-6): (2-3): 3:2; the concentration of the ammonia water is 25-28%; and the wavelength of the ultraviolet light is 365 nm.

2. The quality control method according to claim 1, characterized in that The preparation process of the test solution C comprises: taking a test sample of the Wenjing Decoction compound preparation, extracting it with n-hexane, performing solid-liquid separation, evaporating the liquid to dryness, and re-dissolving the obtained residue with n-hexane; The preparation process of the test solution D comprises: taking a test sample of the Wenjing Decoction compound preparation, extracting it with methanol and water, performing solid-liquid separation, evaporating the liquid to dryness, re-dissolving the resulting residue with water, and then further extracting it with water-saturated n-butanol, evaporating the n-butanol extract to dryness, and re-dissolving the resulting residue with methanol; The preparation process of the test solution E includes: taking a test sample of the Wenjing Decoction compound preparation, adding water to dissolve it, and then continuously adding water-saturated n-butanol to extract it, separating the solid and liquid and taking the liquid, adding an ammonia test solution and then letting it stand to separate, taking the n-butanol extract and evaporating it to dryness, and adding methanol to redissolve the obtained residue.

3. The quality control method according to claim 1, characterized in that: Based on the test solution A, thin layer chromatography was used to identify the peony bark, comprising the following steps: Take the test solution A, the peony bark control medicinal material solution, and the paeonol reference solution, respectively, and spot them on the same silica gel thin layer plate, use cyclohexane-ethyl acetate-glacial acetic acid as the developing agent, and use aluminum trichloride test solution as the color developer, and inspect under ultraviolet light.

4. The quality control method according to claim 3, characterized in that: The quality control method further includes the step of preparing the Moutan Cortex control medicinal material solution according to the preparation process of the test solution A.

5. The quality control method according to claim 3, characterized in that: In the process of identifying Paeonia suffruticosa murraina by thin layer chromatography, the spot volume of paeonol reference solution and test solution A was 2~4μL, and the spot volume of Paeonia suffruticosa murraina reference medicinal material solution was 1~3μL.

6. The quality control method according to claim 3, characterized in that: In the process of identifying Paeonia suffruticosa murrill by thin layer chromatography, the volume ratio of cyclohexane, ethyl acetate and glacial acetic acid in the developing solvent is (10-11): (3-4): 0.

2.

7. The quality control method according to claim 6, characterized in that: In the process of identifying Paeonia suffruticosa moutan by thin layer chromatography, the volume ratio of cyclohexane, ethyl acetate and glacial acetic acid in the developing solvent is 10:3:0.

2.

8. The quality control method according to claim 3, characterized in that: In the process of identifying the peony bark by thin layer chromatography, the wavelength of the ultraviolet light is 365nm.

9. The quality control method according to claim 1, characterized in that: Based on the test solution A, thin layer chromatography was used to identify Chuanxiong and Angelica sinensis, comprising the following steps: Take the test solution A, Angelica sinensis control medicinal material solution, Chuanxiong control medicinal material solution, Ligusticum chuanxiong lactone reference solution and Ligusticum chuanxiong lactone A reference solution, respectively, and spot them on the same silica gel thin layer plate, use n-hexane-dichloromethane-ethyl acetate-formic acid as the developing agent, and inspect under ultraviolet light.

10. The quality control method according to claim 9, characterized in that: In the process of identifying Chuanxiong and Angelica sinensis by thin layer chromatography, the quality control method further includes the step of preparing the Angelica sinensis control medicinal material solution and the Chuanxiong control medicinal material solution according to the preparation process of the test solution A.

11. The quality control method according to claim 9, characterized in that: In the process of identifying Chuanxiong and Angelica sinensis by thin layer chromatography, the sample volume of test solution A, Angelica sinensis reference medicinal material solution, Chuanxiong reference medicinal material solution, ligustilide reference solution and ligustilide A reference solution was 2-4 μL.

12. The quality control method according to claim 9, characterized in that: In the process of identifying Chuanxiong and Angelica sinensis by thin layer chromatography, the volume ratio of n-hexane, dichloromethane, ethyl acetate and formic acid in the developing solvent is (8~9): (3): (1~2): 0.

2.

13. The quality control method according to claim 12, characterized in that: In the process of identifying Chuanxiong and Angelica sinensis by thin layer chromatography, the volume ratio of n-hexane, dichloromethane, ethyl acetate and formic acid in the developing solvent is 8:3:1:0.

2.

14. The quality control method according to claim 9, characterized in that: In the process of identifying Chuanxiong and Angelica sinensis by thin layer chromatography, the wavelength of the ultraviolet light is 254nm.

15. The quality control method according to claim 1, characterized in that: Based on the test solution A, thin layer chromatography was used to identify cinnamon, comprising the following steps: Take the test solution A, cinnamon reference medicinal material solution, and cinnamic acid reference solution, respectively, and spot them on the same silica gel thin layer plate, use petroleum ether-ethyl acetate-formic acid as the developing solvent, and examine under ultraviolet light.

16. The quality control method according to claim 15, characterized in that: In the process of identifying cinnamon by thin layer chromatography, the quality control method further includes the step of preparing the cinnamon control medicinal material solution according to the preparation process of the test solution A.

17. The quality control method according to claim 15, characterized in that: In the process of identifying cinnamon by thin layer chromatography, the sample volume of cinnamic acid reference solution and cinnamon reference medicinal material solution is 1~3μL, and the sample volume of test solution A is 4~6μL.

18. The quality control method according to claim 15, characterized in that: In the process of identifying cinnamon by thin layer chromatography, the volume ratio of petroleum ether, ethyl acetate and formic acid in the developing solvent is (8-8.5): (3-3.5): 0.

2.

19. The quality control method according to claim 18, characterized in that: In the process of identifying cinnamon by thin layer chromatography, the volume ratio of petroleum ether, ethyl acetate and formic acid in the developing solvent is 8:3:0.

2.

20. The quality control method according to claim 15, characterized in that: In the process of identifying cinnamon by thin layer chromatography, the distillation range of the petroleum ether is 60-90°C.

21. The quality control method according to claim 15, characterized in that: In the process of identifying cinnamon bark by thin layer chromatography, the wavelength of the ultraviolet light is 254nm.

22. The quality control method according to claim 2, characterized in that: Based on the test solution C, thin layer chromatography was used to identify Curcuma zedoaria, comprising the following steps: Take the test solution C and the control medicinal material solution of Curcuma zedoaria, respectively, and spot them on the same silica gel thin layer plate, use cyclohexane-ethyl acetate-formic acid as the developing solvent and sulfuric acid ethanol solution as the color developer, heat until the spots are clearly colored, and then inspect under ultraviolet light.

23. The quality control method according to claim 22, characterized in that: In the process of identifying Curcuma zedoaria by thin layer chromatography, the quality control method further includes the step of preparing the Curcuma zedoaria control medicinal material solution according to the preparation process of the test solution C.

24. The quality control method according to claim 22, characterized in that: When using thin layer chromatography to identify Curcuma zedoaria, the spotting volume of the test solution C and the Curcuma zedoaria control medicinal material solution was 15-25 μL.

25. The quality control method according to claim 22, characterized in that: In the process of identifying Curcuma zedoaria by thin layer chromatography, the volume ratio of cyclohexane, ethyl acetate and formic acid in the developing solvent is (9-9.5): (1-1.5): 0.

2.

26. The quality control method according to claim 22, characterized in that: In the process of identifying Curcuma zedoaria by thin layer chromatography, the volume ratio of cyclohexane, ethyl acetate and formic acid in the developing solvent is 9:1:0.

2.

27. The quality control method according to claim 22, characterized in that: In the process of identifying Curcuma zedoaria by thin layer chromatography, the heating temperature is 100-110°C.

28. The quality control method according to claim 22, characterized in that: In the process of identifying Curcuma zedoaria by thin layer chromatography, the wavelength of the ultraviolet light is 365nm.

29. The quality control method according to claim 1, characterized in that: In the process of identifying white peony root and liquorice by thin layer chromatography, the heating temperature is 100-110°C.

30. The quality control method according to claim 1, characterized in that In the process of identifying white peony root and liquorice by thin layer chromatography, the volume ratio of ethyl acetate, acetone, methanol and ammonia water in the developing solvent is 5:2:3:

2.

31. The quality control method according to claim 2, characterized in that: Based on the test solution D, thin layer chromatography was used to identify Achyranthes bidentata, comprising the following steps: Take the test solution D, Achyranthes bidentata reference medicinal material solution, and β-ecdysterone reference solution, respectively, and spot them on the same silica gel thin layer plate, use chloroform-methanol-water-formic acid as the developing agent, and use sulfuric acid ethanol solution as the color developer. Heat until the spots are clearly colored and then inspect under ultraviolet light.

32. The quality control method according to claim 31, characterized in that In the process of identifying Achyranthes bidentata by thin layer chromatography, the quality control method further includes the step of preparing the Achyranthes bidentata control medicinal material solution according to the preparation process of the test solution D.

33. The quality control method according to claim 31, characterized in that: In the process of identifying Achyranthes bidentata by thin layer chromatography, the spotting volume of the test solution D was 1~4μL, the spotting volume of the Achyranthes bidentata control medicinal material solution was 4~7μL, and the spotting volume of β-ecdysterone was 2~4μL.

34. The quality control method according to claim 31, characterized in that In the process of identifying Achyranthes bidentata by thin layer chromatography, the volume ratio of chloroform, methanol, water and formic acid in the developing solvent is (8-9): (2-2.5): 0.3: 0.

5.

35. The quality control method according to claim 34, characterized in that: In the process of identifying Achyranthes bidentata by thin layer chromatography, the volume ratio of chloroform, methanol, water and formic acid in the developing solvent is 8:2:0.3:0.

5.

36. The quality control method according to claim 31, characterized in that In the process of identifying Achyranthes bidentata by thin layer chromatography, the heating temperature is 100-110°C.

37. The quality control method according to claim 31, characterized in that In the process of identifying Achyranthes bidentata by thin layer chromatography, the wavelength of the ultraviolet light is 365nm.

38. The quality control method according to claim 2, characterized in that: Based on the test solution E, thin layer chromatography was used to identify ginseng, comprising the following steps: Take the test solution E, ginseng control medicinal material solution, and mixed reference solution, and spot them on the same silica gel thin layer plate respectively, use chloroform-anhydrous methanol-water as the developing agent, and use sulfuric acid ethanol solution as the color developer, and heat until color is developed; wherein the mixed reference solution contains ginsenoside Rb1 reference substance, ginsenoside Rb2 reference substance, ginsenoside Rc reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rg1 reference substance.

39. The quality control method according to claim 38, characterized in that The quality control method further includes the step of preparing the ginseng control medicinal material solution according to the preparation process of the test solution E.

40. The quality control method according to claim 38, wherein: The spotting volume of the test solution E is 4~6μL, and the spotting volume of the ginseng control medicinal material solution and the mixed reference solution is 2~4μL.

41. The quality control method according to claim 38, wherein: In the developing solvent, the volume ratio of chloroform, anhydrous methanol and water is (9-9.5): (4.5-5):

1.

42. The quality control method according to claim 41, characterized in that In the developing solvent, the volume ratio of chloroform, anhydrous methanol and water is 9:4.5:

1.

43. The quality control method according to claim 38, characterized in that In the process of identifying ginseng by thin layer chromatography, the heating temperature is 100-110°C.

44. The quality control method according to any one of claims 1 to 43, characterized in that: The Wenjing Decoction compound preparation includes a solid preparation, a semisolid preparation or a liquid preparation of Wenjing Decoction.

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