A method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations by thin layer chromatography

Abstract

The present application relates to the technical field of pharmaceutical analysis, in particular to a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations, which mainly comprises the following steps: preparation of test sample solution and control medicinal material solution, and thin layer chromatography detection, wherein the developing agent used is petroleum ether and ethyl acetate with a boiling range of 60-90 DEG C and a volume ratio of (7-11):(1-3). The present application provides a thin layer identification method for different base original Zedoary Turmeric and its preparations, which comprises the same preparation steps of test sample solution and control medicinal material solution as above, and the developing agent used is petroleum ether, acetone and ethyl acetate with a boiling range of 60-90 DEG C and a volume ratio of (93-95):(6-4):1. The method provided by the present application has the advantages of clear spots, good separation degree, strong specificity, no interference, good durability and less influence from external factors; one identification test can reflect the information of different medicinal ingredients, and the identification is comprehensive, thereby reducing the number of thin layer identification tests and improving the test efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations. BACKGROUND

[0002] Wenjing Decoction is composed of nine medicinal ingredients, i.e. Angelica sinensis, Ligusticum chuanxiong, Radix Paeoniae Alba, Cinnamomum cassia, Moutan Cortex, Curcuma zedoary, Panax ginseng, Glycyrrhiza uralensis and Cyathula officinalis. Wenjing Decoction has the effects of warming meridians, dispelling cold, promoting blood circulation, regulating menstruation and tonifying qi and blood. Wenjing Decoction is warm but not dry, and it can attack pathogenic factors without damaging healthy qi, and it can tonify without stagnation. Wenjing Decoction is effective in treating dysmenorrhea caused by cold and blood stasis, and amenorrhea caused by external invasion of cold pathogens. Wenjing Decoction is a commonly used prescription for regulating menstruation in gynecology, and it is also widely used in the treatment of functional uterine bleeding, chronic pelvic inflammation and infertility.

[0003] In Wenjing Decoction, the monarch drug is Cinnamomum cassia and Panax ginseng, which have the pharmacological effects of dilating blood vessels, acting on the central nervous system and cardiovascular system, and regulating immunity. Cinnamomum cassia has volatile oil and organic acid as active ingredients, and Panax ginseng has saponins and polysaccharides as active ingredients. The ministerial drug is Curcuma zedoary and Cyathula officinalis, which have the effects of regulating immunity, exciting uterus, anti-inflammatory and analgesic, anti-virus, anti-tumor, etc. Cyathula officinalis has steroidal components as active ingredients, and Curcuma zedoary has volatile oil as the main active ingredient. The assistant drug is Angelica sinensis, Ligusticum chuanxiong, Radix Paeoniae Alba and Moutan Cortex, which have the effects of anti-oxidation, anti-cerebral ischemia, anti-virus, anti-tumor, regulating smooth muscle, protecting liver, etc. They have volatile oil, monoterpenoid glycosides and phenolic acid as active ingredients. The ministerial drug is Glycyrrhiza uralensis, which has the effects of anti-arrhythmia, anti-inflammatory, analgesic and anti-tussive. It has triterpenes and flavonoids as active ingredients. By analyzing the components and pharmacological activities of the medicinal ingredients in Wenjing Decoction, it is found that five medicinal ingredients have volatile components, including Cinnamomum cassia, Moutan Cortex, Angelica sinensis, Ligusticum chuanxiong and Curcuma zedoary. Therefore, the identification and control of the volatile components in Wenjing Decoction can comprehensively evaluate the overall quality of the prescription.

[0004] The thin layer identification method is the main means of qualitative identification in the quality standards of traditional Chinese medicine compound preparations. The identification items are mostly arranged according to the identification of medicinal ingredients, i.e. one identification item for one medicinal ingredient in a prescription, one thin layer plate for one identification item, one development for one identification item, and one medicinal ingredient for one identification item. In addition, the treatment methods of the test solution, the control medicinal material solution and the control solution are different for each medicinal ingredient. In order to achieve the basic coverage of the thin layer chromatography identification method for the medicinal ingredients in traditional Chinese medicine compound preparations, multiple solutions need to be treated by different methods, and a large number of tests are often required, which consumes a lot of manpower and material resources. SUMMARY

[0005] Therefore, the present application aims to solve the technical problem of overcoming the defects in the prior art that multiple solutions need to be treated by different methods, and multiple thin layer plates need to be used to develop each medicinal ingredient for identification when identifying the components of Wending Decoction by thin layer chromatography, thereby providing a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wending Decoction and its preparations.

[0006] To this end, the present application provides the following technical solutions:

[0007] The present application provides a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wending Decoction and its preparations, comprising the following steps:

[0008] S1: Take the sample to be tested, dissolve it with water, extract the obtained solution with petroleum ether with a boiling range of 30-60 DEG C, separate the petroleum ether liquid, dry it, dissolve it with an organic solvent to obtain a test solution;

[0009] S2: Take the angelica control medicinal material, the chuanxiong control medicinal material and the pungent turmeric control medicinal material, respectively, add petroleum ether with a boiling range of 30-60 DEG C to extract, and prepare each control medicinal material solution;

[0010] S3: Absorb the test solution, the angelica control medicinal material solution, the chuanxiong control medicinal material solution and the pungent turmeric control medicinal material solution, respectively, and point them on the same thin layer plate, develop them with the first developing agent, take them out, dry them, place them under ultraviolet light for inspection, identify angelica and chuanxiong, spray them with the first color developing agent, heat them, and inspect them to identify turmeric;

[0011] The first developing agent is petroleum ether with a boiling range of 60-90 DEG C and ethyl acetate with a volume ratio of 7-11:1-3.

[0012] Further, the S2 step further comprises the step of taking the cortex mali control medicinal material, adding petroleum ether with a boiling range of 30-60 DEG C to extract, and preparing a cortex mali control medicinal material solution, and the S3 step further comprises the step of pointing the cortex mali control medicinal material solution on the same thin layer plate used in the S3 step before development.

[0013] Further, the thin layer chromatography method further comprises: taking the Cinnamomum cassia control medicinal material, adding petroleum ether with a boiling range of 30-60°C for extraction to prepare a Cinnamomum cassia control medicinal material solution; and sucking the test sample solution and the Cinnamomum cassia control medicinal material solution and spotting them on another thin layer plate, respectively, and developing with a first developing agent, taking out, air-drying, spraying with a second color developing agent, and observing to identify Cinnamomum cassia; optionally, the thin layer chromatography method further comprises: taking cinnamyl aldehyde control, adding an organic solvent to prepare a cinnamyl aldehyde control solution, and spotting the test sample solution, the Cinnamomum cassia control medicinal material solution and the cinnamyl aldehyde control solution on the same thin layer plate before development; optionally, the second color developing agent comprises a dinitrophenylhydrazine ethanol solution; optionally, the organic solvent comprises at least one of anhydrous ethanol, methanol and n-hexane; optionally, 0.5-1 mg of the control is contained in each 1 mL of the cinnamyl aldehyde control solution; and optionally, after taking the Cinnamomum cassia control medicinal material and adding petroleum ether with a boiling range of 30-60°C for extraction, the method further comprises the steps of ultrasonic treatment, solid-liquid separation, drying and dissolving in anhydrous ethanol.

[0014] Further, the thin layer chromatography method further comprises: taking at least one of ligustilide control, paeonol control, curcumin control, zedoary ketone control, furanodiene control, curcumol control and zedoarondienone control, adding an organic solvent to prepare a control solution, and spotting the control solution on the same thin layer plate used in S3 before development; optionally, 0.5-1 mg of the control is contained in each 1 mL of the control solution; and optionally, the organic solvent comprises at least one of anhydrous ethanol, methanol and n-hexane.

[0015] The present application provides a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wending Decoction and its preparations, wherein during the preparation of the test sample solution, no violent shaking is needed to avoid emulsification effect, and severe emulsification can affect the spot of zedoary; the test sample solution after petroleum ether extraction is not easy to be placed for too long after drying, and the test sample solution after overnight re-dissolution can affect the pink spot of zedoary; the thin layer plate after development is not easy to be placed for too long, and it is appropriate to be placed within 2 hours, and long placement can affect the other spots of angelica and chuanxiong except ligustilide which are consistent with the control medicinal material.

[0016] Further, in S1, the organic solvent used comprises at least one of anhydrous ethanol, methanol and n-hexane.

[0017] Further, in S1, the extraction comprises shaking extraction.

[0018] Further, in S1, the drying comprises air-drying.

[0019] Further, in S2, after adding petroleum ether with a boiling range of 30-60°C, the method further comprises the steps of ultrasonic treatment, solid-liquid separation, drying and dissolving in anhydrous ethanol.

[0020] Further, in the step S3, when identifying Angelica sinensis and Ligusticum chuanxiong, the inspection wavelength is 365nm.

[0021] Further, the first color developing agent includes 2-5% of vanillin sulfuric acid ethanol solution.

[0022] Further, in the step S3, after spraying the first color developing agent, the heating temperature is 105℃.

[0023] Further, in the step S1, 5-10g of the sample to be tested is taken, 50-100mL of water is added to dissolve it, the obtained solution is extracted with petroleum ether with a boiling range of 30-60℃ for 2-4 times, each time with 25-50mL, the petroleum ether liquid is separated, dried, and the obtained residue is dissolved in 2-5mL of anhydrous ethanol to obtain the sample solution; in the step S2, 0.5-1g of Angelica sinensis control medicinal material, 0.5-1g of Ligusticum chuanxiong control medicinal material and 0.5-1g of Curcuma zedoary control medicinal material are taken, petroleum ether with a boiling range of 30-60℃ is added, ultrasonic treatment is performed for 20-60min, filtration is performed, dried, and the obtained residue is dissolved in 2-5mL of anhydrous ethanol to prepare the control medicinal material solution.

[0024] Further, in the preparation of the Cinnamomum cassia control medicinal material solution, 0.5-1g of Cinnamomum cassia control medicinal material is taken, petroleum ether with a boiling range of 30-60℃ is added, ultrasonic treatment is performed for 20-60min, filtration is performed, dried, and the obtained residue is dissolved in 2-5mL of anhydrous ethanol to prepare the Cinnamomum cassia control medicinal material solution.

[0025] In any thin layer method provided by the present application, the Wanjing Decoction and its preparation are prepared from Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Cinnamomum cassia, Radix Moutan, Curcuma zedoary, Panax ginseng, Glycyrrhiza uralensis and Achyranthes bidentata according to conventional technical means. The Wanjing Decoction and its preparation include the following raw materials according to weight fraction: Cinnamomum cassia 1-2 parts, roasted Glycyrrhiza uralensis 2-3 parts, wine Achyranthes bidentata 2-3 parts, Panax ginseng 2-3 parts, white Paeonia lactiflora 1-2 parts, vinegar Curcuma zedoary 1-2 parts, wine Angelica sinensis 1-2 parts, Ligusticum chuanxiong 1-2 parts, and Radix Moutan 1-2 parts.

[0026] The Wanjing Decoction and its preparation are preferably at least one of the Wanjing Decoction intermediate extract, the Wanjing Decoction inclusion compound, the Wanjing Decoction reference sample water decoction, the Wanjing Decoction reference sample concentrated extract, and the Wanjing Decoction reference sample freeze-dried powder.

[0027] Preferably, the Wanjing Decoction preparation is the Wanjing Decoction reference sample freeze-dried powder obtained by decocting and freeze-drying 9 kinds of traditional Chinese medicines of Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Cinnamomum cassia, Radix Moutan, Curcuma zedoary, Panax ginseng, Glycyrrhiza uralensis and Achyranthes bidentata.

[0028] The present application provides a thin layer identification method for different base Curcuma zedoary and its preparation, which comprises the following steps:

[0029] Preparation of test sample solution: take the sample to be tested, dissolve in water, extract the obtained solution with petroleum ether with a boiling range of 30-60 DEG C, separate the petroleum ether liquid, dry, dissolve in an organic solvent to obtain the test sample solution;

[0030] Preparation of control medicinal material solution: take the Curcuma xanthorrhiza Roxb control medicinal material, Guangxi Curcuma zedoaria Rosc control medicinal material, and Curcuma wenyujin control medicinal material, respectively, add petroleum ether with a boiling range of 30-60 DEG C to extract to prepare each control medicinal material solution;

[0031] Thin layer chromatography detection: take the test sample solution, Curcuma xanthorrhiza Roxb control medicinal material, Guangxi Curcuma zedoaria Rosc control medicinal material, and Curcuma wenyujin control medicinal material, respectively, and point on the same thin layer plate, develop with the second developing agent, take out, dry, spray with the third color developing agent, heat, and observe to identify Curcuma xanthorrhiza Roxb, Curcuma wenyujin, and Guangxi Curcuma zedoaria Rosc;

[0032] The second developing agent is petroleum ether with a boiling range of 60-90 DEG C, acetone, and ethyl acetate with a volume ratio of 93-95:6-4:1.

[0033] The thin layer identification method for different base sources of Curcuma and its preparations provided in the application, wherein the Curcuma and its preparations are selected from Curcuma medicinal materials, Curcuma decoction pieces, Curcuma single formula preparations, or Curcuma compound formula preparations.

[0034] Further, take curcumin reference substance, gima ketone reference substance, furan diene reference substance, and curcuma alcohol reference substance, respectively, add an organic solvent to prepare reference substance solutions, and in the step of the thin layer chromatography detection, the step of pointing the curcumin reference substance solution, gima ketone reference substance solution, furan diene reference substance solution, and curcuma alcohol reference substance solution on the same thin layer plate before development is further included.

[0035] Further, the organic solvent used includes at least one of anhydrous ethanol, methanol, and n-hexane.

[0036] Further, the curcumin reference substance solution, gima ketone reference substance solution, furan diene reference substance solution, and curcuma alcohol reference substance solution each contain 0.5-1 mg of reference substance per 1 mL of solution.

[0037] Further, in the step of preparing the test sample solution, the extraction includes vibration extraction.

[0038] Further, in the step of preparing the test sample solution, the drying includes evaporation.

[0039] Further, in the step of preparing the control medicinal material solution, after adding petroleum ether with a boiling range of 30-60 DEG C, the steps of ultrasonic treatment, solid-liquid separation, and drying, and dissolving in anhydrous ethanol are further included.

[0040] Further, the third color developing agent comprises 2-5% of vanillin sulfuric acid ethanol solution.

[0041] Further, after spraying the third color developing agent, the heating temperature is 105 DEG C.

[0042] Further, the preparation method of the test sample solution comprises: taking 5-10g of the sample to be tested, dissolving the sample in 50-100mL of water, extracting the obtained solution with petroleum ether with a boiling range of 30-60 DEG C for 2-4 times, each time with 25-50mL of petroleum ether, separating the petroleum ether liquid, drying, and dissolving the obtained residue in 2-5mL of ethanol to obtain the test sample solution.

[0043] Further, the preparation method of the control medicinal material solution comprises: taking 0.5-1g of each of the control medicinal materials of Curcuma zedoary, Curcuma kwangsiensis and Curcuma wenyujin, adding petroleum ether with a boiling range of 30-60 DEG C, and ultrasonically treating for 20-60min, filtering, drying, and dissolving the obtained residue in 2-5mL of anhydrous ethanol to obtain the control medicinal material solution.

[0044] The technical scheme of the present application has the following advantages:

[0045] The thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations provided by the present application comprises the steps of preparing a test sample solution, preparing a control medicinal material solution, and detecting by thin layer chromatography. When preparing the test sample solution, the sample to be tested is taken, dissolved in water, and extracted with petroleum ether with a boiling range of 30-60 DEG C, and the petroleum ether liquid is separated, dried, and dissolved in an organic solvent to obtain the test sample solution. When preparing the control medicinal material solution, the control medicinal materials of Angelica sinensis, Ligusticum chuanxiong and Curcuma zedoary are taken, extracted with petroleum ether with a boiling range of 30-60 DEG C respectively, and the control medicinal material solutions are prepared. When detecting by thin layer chromatography, the test sample solution, the control medicinal material solution of Angelica sinensis, the control medicinal material solution of Ligusticum chuanxiong and the control medicinal material solution of Curcuma zedoary are taken, spotted on the same thin layer plate respectively, developed with a first developing agent, taken out, dried, placed under ultraviolet light, and observed to identify Angelica sinensis and Ligusticum chuanxiong. The first developing agent is sprayed, heated, observed, and Curcuma zedoary is identified. The developing agent used is petroleum ether with a boiling range of 60-90 DEG C and ethyl acetate with a volume ratio of (7-11):(1-3). The method provided by the present application has clear spots, good separation degree, strong specificity, no background interference, no negative control interference, good durability, and is not easily affected by the thin layer plate, temperature, humidity and spotting mode. One identification test can reflect the quality information of multiple medicinal ingredients, the identification is more comprehensive, which is more conducive to reflecting the overall quality characteristics, and the number of thin layer identification tests is reduced, the amount of reagent is reduced, time is saved, and the test efficiency is improved.

[0046] The application provides a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations, which comprises identification of Cortex Moutan, and the same thin layer plate can be used for identifying Angelica sinensis, Ligusticum chuanxiong and Curcuma zedoary, thereby reducing the test times of thin layer identification, saving time and being high in efficiency.

[0047] The application provides a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations, which comprises identification of Cortex Cinnamomi, and the same developing agent is used for identifying Angelica sinensis, Ligusticum chuanxiong and Curcuma zedoary, the test sample solution prepared by the same method is used, the experimental steps are reduced, and time and manpower are saved.

[0048] The application provides a thin layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations, which comprises using Ligusticum chuanxiong lactone reference substance solution, Danpi phenol reference substance solution, Cinnamaldehyde reference substance, Gima ketone reference substance, furan diene reference substance, Curcuma zedoary alcohol reference substance and Curcuma zedoary furan diene ketone reference substance solution to assist in identifying different medicinal ingredients in Wenjing Decoction and its preparations, only the corresponding reference substance solution needs to be spotted on the same thin layer plate before development, and no additional developing agent system is needed, thereby being convenient and fast.

[0049] In the application, only one developing agent is used to identify different medicinal ingredients in Wenjing Decoction and its preparations, the amount of reagent is reduced, and the operation is more convenient; and the developing agent used in the application is not layered, and can be used after being mixed in proportion, and does not need to be placed at low temperature for a long time, and is convenient to prepare. In the application, only one test sample solution is used to identify different medicinal ingredients, the test sample solution does not need to be treated separately for each medicinal ingredient, the test efficiency is improved; and only water, petroleum ether and ethanol are used when preparing the test sample solution, the reference medicinal material solution and the reference substance solution, and no easily toxic and explosive reagent is used in the whole process, and the environment is friendly.

[0050] The application provides a thin-layer identification method of different base origins of Curcuma zedoary and preparations thereof, and comprises the steps of preparing a sample solution, preparing a control medicinal material solution and detecting by a thin-layer chromatography method; when the sample solution is prepared, a sample to be detected is taken, dissolved with water, and the obtained solution is extracted with petroleum ether with a boiling range of 30-60 DEG C, the petroleum ether liquid is separated, dried, dissolved with an organic solvent, and the sample solution is obtained; when the control medicinal material solution is prepared, Curcuma zedoary control medicinal materials, Guangxi Curcuma zedoary control medicinal materials and Wenyujin control medicinal materials are taken, and are extracted with petroleum ether with a boiling range of 30-60 DEG C respectively to prepare the control medicinal material solutions; when the detection by the thin-layer chromatography method is performed, the sample solution, the Curcuma zedoary control medicinal materials, the Guangxi Curcuma zedoary control medicinal materials and the Wenyujin control medicinal materials are taken, and are spotted on the same thin-layer plate respectively, are developed with a second developing agent, are taken out, are dried, are sprayed with a third color developing agent, are heated, are observed, and the Curcuma zedoary, the Wenyujin and the Guangxi Curcuma zedoary are identified; the second developing agent is petroleum ether with a boiling range of 60-90 DEG C, acetone and ethyl acetate with a volume ratio of 93-95:6-4:1; the method provided by the application can identify different base origins of Curcuma zedoary medicinal materials or different base origins of Curcuma zedoary in Wenyujing Decoction, and is fast, convenient, time-saving and labor-saving. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0052] Figure 1 It is the result of the thin-layer chromatography method test of the Wenyujing Decoction reference sample, preparation intermediates and compound preparation samples of the application, wherein: S1 is cinnamyl aldehyde, S2 is paeonol, S3 is ligustilide, S4 is zedoary furan dienone, 1 is cortex moutan control medicinal material, 2 is angelica control medicinal material, 3 is chuanxiong control medicinal material, 4 is Curcuma zedoary control medicinal material, 5 is Wenyujing Decoction reference decoction (JY202210001), 6 is Wenyujing Decoction reference sample (JZ202210001), 7 is Wenyujing Decoction granules (220601), 8 is Wenyujing Decoction aromatic water (FXS220601), 9 is Wenyujing Decoction inclusion compound (BHW220601), 10 is Wenyujing Decoction inclusion compound (BHW220602);

[0053] Figure 2is the determination and differentiating result chart of the identification spots of the thin layer chromatography of the invention, wherein: S1 is curcumin, S2 is germacrone, S3 is furandiene, S4 is zingiberol, 1 is negative solution of vinegar-free zedoary, 2 is angelica sinensis medicinal material, 3 is Guangxi zedoary medicinal material, 4 is pungent zedoary medicinal material, 5 is the reference sample of Wenjing decoction (JZ202210004), 6 is vinegar angelica sinensis decoction (K202106001), 7 is vinegar Guangxi zedoary decoction (K20221002), 8 is vinegar pungent zedoary decoction (K20221001), 9 is Wenjing decoction granules (220601);

[0054] Figure 3 is the special property investigation result of angelica sinensis, chuanxiong rhizome, zedoary and peony in the thin layer chromatography method of Wenjing decoction and its preparation, wherein, S1 is ligustilide, S2 is paeonol, 1 is negative solution of angelica sinensis and chuanxiong rhizome, 2 is angelica sinensis medicinal material, 3 is chuanxiong rhizome medicinal material, 4 is negative solution of vinegar-free zedoary, 5 is pungent zedoary medicinal material, 6 is negative solution of peony, 7 is peony medicinal material, 8 is the reference sample of Wenjing decoction (JZ202210001), 9 is Wenjing decoction granules, 10 is zedoaryl glycol;

[0055] Figure 4 is the special property investigation result of cinnamomum cassia in the thin layer chromatography method of Wenjing decoction and its preparation, wherein: 1 is negative solution of cinnamomum cassia, 2 is the reference sample of Wenjing decoction (JZ202210001), 3 is the reference sample of Wenjing decoction (JZ202210002), 4 is the reference sample of Wenjing decoction (JZ202210003), 5 is cinnzaldehyde, 6 is cinnamomum cassia medicinal material;

[0056] Figure 5 is the test sample preparation optimization investigation result of the thin layer chromatography method of Wenjing decoction and its preparation, wherein, S is ligustilide, 1 is negative solution of angelica sinensis and chuanxiong rhizome, 2 is angelica sinensis medicinal material, 3 is chuanxiong rhizome medicinal material, 4 is negative solution of vinegar-free zedoary, 5 is pungent zedoary medicinal material, 6 is test sample solution A, 7 is test sample solution B, 8 is test sample solution C, 9 is test sample solution D;

[0057] Figures 6-10 is the developing agent optimization investigation result of the thin layer chromatography method of Wenjing decoction and its preparation, according to the volume ratio, Figure 6 the developing agent used is petroleum ether (60-90℃): ethyl acetate=(9:1), Figure 7 is n-hexane: acetone: dichloromethane=(94:5:2), Figure 8 is petroleum ether (60-90℃): acetone: ethyl acetate=(94:5:1), Figure 9 is petroleum ether (60-90℃): ethyl acetate=(17:3), Figure 10Petroleum ether (60-90℃) : ethyl acetate = (15:1), wherein: S is ligustilide, 1 is a negative solution of Angelica sinensis and Ligusticum chuanxiong, 2 is Angelica sinensis control medicinal material, 3 is Ligusticum chuanxiong control medicinal material, 4 is a negative solution of Myristicae Fructus, 5 is Myristicae Fructus control medicinal material, 6 is Wanjing Decoction reference sample (JZ202210001), 7 is Wanjing Decoction reference sample (JZ202210002), 8 is Wanjing Decoction reference sample (JZ202210003);

[0058] Figure 11 is the optimal result of different sample application amounts of the Wanjing Decoction and its preparation thin-layer chromatography method of the application, wherein: S is ligustilide, 1 is a negative solution of Angelica sinensis and Ligusticum chuanxiong, 2 is Angelica sinensis control medicinal material, 3 is Ligusticum chuanxiong control medicinal material, 4 is a negative solution of Myristicae Fructus, 5 is Myristicae Fructus control medicinal material, 6 is Wanjing Decoction reference sample (10 μl), 7 is Wanjing Decoction reference sample (15 μl), 8 is Wanjing Decoction reference sample (20 μl), 9 is Wanjing Decoction reference sample (25 μl);

[0059] Figure 12 、 13 is the result of different temperatures of the Wanjing Decoction and its preparation thin-layer chromatography method of the application, Figure 12 the experimental temperature is 4℃, Figure 13 the experimental temperature is 30℃, wherein: S is ligustilide, 1 is a negative solution of Angelica sinensis and Ligusticum chuanxiong, 2 is Angelica sinensis control medicinal material, 3 is Ligusticum chuanxiong control medicinal material, 4 is a negative solution of Myristicae Fructus, 5 is Myristicae Fructus control medicinal material, 6 is Wanjing Decoction reference sample (JZ202210001), 7 is Wanjing Decoction reference sample (JZ202210002), 8 is Wanjing Decoction reference sample (JZ202210003);

[0060] Figure 14 、 15 is the result of different humidities of the Wanjing Decoction and its preparation thin-layer chromatography method of the application, Figure 14 the experimental humidity is 32% RH, Figure 15 the experimental humidity is 88% RH, wherein: S is ligustilide, 1 is a negative solution of Angelica sinensis and Ligusticum chuanxiong, 2 is Angelica sinensis control medicinal material, 3 is Ligusticum chuanxiong control medicinal material, 4 is a negative solution of Myristicae Fructus, 5 is Myristicae Fructus control medicinal material, 6 is Wanjing Decoction reference sample (JZ202210001), 7 is Wanjing Decoction reference sample (JZ202210002), 8 is Wanjing Decoction reference sample (JZ202210003);

[0061] Figure 16 、 17 is the result of different brands of thin-layer plates of the Wanjing Decoction and its preparation thin-layer chromatography method of the application, Figure 16 Yantai plates are used, Figure 17The used is Qingdao plate, wherein: S is ligustilide, 1 is negative solution of lack of Angelica sinensis and Ligusticum chuanxiong, 2 is Angelica sinensis control medicinal material, 3 is Ligusticum chuanxiong control medicinal material, 4 is negative solution of lack of Curcuma zedoary, 5 is Curcuma zedoary control medicinal material, 6 is Wenyjing decoction reference sample (JZ202210001), 7 is Wenyjing decoction reference sample (JZ202210002), and 8 is Wenyjing decoction reference sample (JZ202210003);

[0062] Figure 18 The table is the result of different sample application modes of the Wenyjing decoction and its preparation thin layer chromatography method, wherein: S is ligustilide, 1 is negative solution of lack of Angelica sinensis and Ligusticum chuanxiong, 2 is Angelica sinensis control medicinal material, 3 is Ligusticum chuanxiong control medicinal material, 4 is negative solution of lack of Curcuma zedoary, 5 is Curcuma zedoary control medicinal material, 6 is Wenyjing decoction reference sample (JZ202210001), 7 is Wenyjing decoction reference sample (JZ202210002), and 8 is Wenyjing decoction reference sample (JZ202210003). DETAILED DESCRIPTION

[0063] The technical solutions of the present application will be described clearly and completely in combination with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0064] In the following examples and comparative examples, the reagents not specifically stated are conventional reagents, which can be purchased from conventional reagent production and sales companies.

[0065] 1. Experimental raw materials

[0066] Cinnamyl aldehyde (110710-202022), Paeonol (110708-201908), Curcumin (110823-202107), Germacrone (111665-201906), Furanodiene (111824-202203), Zedoary alcohol (100185-201908), Cinnamomum cassia control medicinal material (121363-201703), Moutan cortex control medicinal material (121490-201603), Angelica sinensis control medicinal material (120927-202118), Ligusticum chuanxiong control medicinal material (120918-201813), Curcuma zedoary control medicinal material (120973-201304), Curcuma wenyujin control medicinal material (121304-201904) are provided by China Institute for Food and Drug Control.

[0067] Vinegar Wengjing decoction (K202106001), vinegar Guangxi Zedoary decoction (K20221002), vinegar Pong Zedoary decoction (K20221001) are provided by Guangdong Daxiang Traditional Chinese Medicine Pharmaceutical Co., Ltd. Vinegar Zedoary is made from Zedoary, typically non-limiting, the steps are as follows: take clean Zedoary, put it into the cooking container, add vinegar and appropriate amount of water to immerse, cook until the vinegar liquid is absorbed, when the white core is cut open, take it out, dry slightly, cut into thick slices, dry.

[0068] Ligustilide (PS011680), Curdlan, Zedoary furan diene ketone are provided by Chengdu Pusai Biotechnology Co., Ltd.

[0069] Guangxi Zedoary control drug material is provided by Tianjin Zhefen Pharmaceutical Technology Co., Ltd.

[0070] Wenjing decoction drug preparation is prepared by taking Angelica, Szechuan Lovage Rhizome, Paeonia, Cassia, Tree Peony, Zedoary, Ginseng, Licorice, and Cow's Knee as raw materials, adding conventional adjuvants, and following the conventional process to form a clinically acceptable tablet, capsule, pill, granule, honeyed pill, sustained-release preparation, immediate-release preparation, controlled-release preparation, oral liquid preparation or injection preparation.

[0071] The preparation methods of the following examples and experimental examples of Wenjing decoction and its preparations are as follows.

[0072] (1) Wenjing decoction dry extract powder: according to the prescription ratio, 1.25 kg of Cassia, 2.5 kg of fried Licorice, 2.5 kg of wine Cow's Knee, 2.5 kg of Ginseng, 1.25 kg of White Peony, 1.25 kg of vinegar Zedoary, 1.25 kg of wine Angelica, 1.25 kg of Szechuan Lovage Rhizome, and 1.25 kg of Tree Peony, a total of 15 kg, are sequentially added into a 300 L extraction tank in the above order. 3 The extraction tank, for the first time, 90 L of purified water is added to the extraction tank, heated to reflux extraction for 2 hours (the temperature of heating reflux is heated to 95℃ above the temperature of the medicinal liquid to boiling), while collecting 15 kg of aromatic water (the collection rate of aromatic water is 0.5 times the total weight of raw material per hour), filtering, adding 150 L of purified water to the residue, heating to reflux extraction for 1 hour (the temperature of heating reflux is heated to 95℃ above the temperature of the medicinal liquid to boiling), filtering, combining the filtrate, concentrating to a relative density of about 1.10 at 50℃ under reduced pressure, and the concentrated liquid is spray dried to obtain dry extract powder 4.8 kg.

[0073] The Zedoary negative sample, the vinegar Zedoary negative sample, the wine Angelica and Szechuan Lovage Rhizome double negative sample, the Tree Peony negative sample, and the Cassia negative sample are prepared by the same method as the Wenjing decoction dry extract powder, and the only difference in the preparation method is that the corresponding medicinal materials are missing. Dissolving the negative sample obtains the corresponding negative solution.

[0074] (2)Wenjing Decoction Aromatic Water: 9 kinds of drugs including Cinnamomum cassia 1.25 kg, roasted licorice 2.5 kg, wine yunnan 2.5 kg, ginseng 2.5 kg, white peony root 1.25 kg, vinegar atractylodes 1.25 kg, wine angelica 1.25 kg, chuanxiong 1.25 kg, and cork tree bark 1.25 kg, totaling 15 kg, were added into 300 m 3 In the extraction tank, 90 L of purified water was added for the first time, and heated reflux extraction was performed for 2 hours (the temperature of heated reflux was heated to above 95°C of the temperature of the medicinal liquid to boiling). At the same time, 15 kg of aromatic water was collected (the collection rate of aromatic water was 0.5 times the total weight of raw material per hour). A batch of Wenjing Decoction Aromatic Water was prepared, with batch number FXS220601.

[0075] (3) Wenjing Decoction Inclusion Compound: Wenjing Decoction Aromatic Water was prepared using the method in (2). 900 g of β-cyclodextrin was added to the aromatic water and shaken, then stirred for 2.5 hours using a homogenizer at a speed of 1500 r / min, and placed in a -2°C refrigerator for 96 hours. The supernatant was removed, and the mixture was dried at 65°C under reduced pressure for 2 hours. The powder was crushed and sieved (50 mesh) to obtain 685 g of inclusion powder. Two batches of Wenjing Decoction Inclusion Compound were prepared using raw materials from different sources, with batch numbers BHW220601 and BHW220602.

[0076] (3) Wenjing Decoction Granules: Wenjing Decoction Dry Concentrate Powder was prepared using the method in (1), and Wenjing Decoction Inclusion Compound was prepared using the method in (3). 4.8 kg of Wenjing Decoction Dry Concentrate Powder, 685 g of Wenjing Decoction Inclusion Compound, and 1.79 kg of malt dextrin were mixed evenly, and granules were prepared using dry compression granulation. The dried granules were collected in a vortex shaker and sieved with 10 mesh and 40 mesh screens to obtain Wenjing Decoction Granules. A batch of Wenjing Decoction Granules was prepared, with batch number 220601.

[0077] (4) Wenjing Decoction Reference Decoction: 1 part of Cinnamomum cassia, 2 parts of roasted licorice, 2 parts of wine yunnan, 2 parts of ginseng, 1 part of white peony root, 1 part of vinegar atractylodes, 1 part of wine angelica, 1 part of chuanxiong, and 1 part of cork tree bark were weighed according to the prescription proportion, totaling 20 g, and placed in a sand pot with 450 ml of water. Boil with a strong fire, then change to a weak fire and decoct to about 240 ml. Filter to obtain Wenjing Decoction Reference Decoction. A batch of Wenjing Decoction Reference Decoction was prepared, with batch number JY202210001.

[0078] (5)Wenjing Decoction Reference Sample (lyophilized powder): Take Angelica 1.67 g, Szechuan Lovage Rhizome 1.67 g, White Peony Root 1.67 g, Cassia Bark 1.67 g, Dark Red Root 1.67 g, Styrax 1.67 g, Ginseng 3.33 g, Fried Licorice 3.33 g, Wine-processed Milkvetch Root 3.33 g, break the above nine kinds of materials into coarse particles, put them in a 2L sand pot, add water 450ml, cover, soak for 30 minutes, use a kingfah body heating plate as the heating device, boil with strong fire (power 500W), simmer with gentle fire (power 300W) until 240ml±10ml, filter with 200 mesh, put it in the refrigerator at-80℃ for 12 hours after cooling, freeze-dry, cold trap temperature-80℃, vacuum degree less than 100pa, dry for more than 72 hours, then get it, prepare a batch of Wenjing Decoction Reference Sample (lyophilized powder). Four batches of Wenjing Decoction Reference Sample are prepared using raw materials of different sources, batch numbers are JZ202210001, JZ202210002, JZ202210003, JZ202210004 respectively.

[0079] 2. Thin layer plate

[0080] The manufacturers and batch numbers of the thin layer plates used are shown in the following table.

[0081]

[0082] Example 1

[0083] This example provides a thin layer chromatography method for identifying Wenjing Decoction preparations, which specifically includes:

[0084] Preparation of test sample solution: Take Wenjing Decoction Reference Decoction (JY202210001), Wenjing Decoction Reference Sample (JZ202210001), Wenjing Decoction Granules (220601), Wenjing Decoction Aromatic Water (FXS220601), Wenjing Decoction Inclusion Compound (BHW220601), and Wenjing Decoction Inclusion Compound (BHW220602) as test samples, and prepare the test sample solution according to the following method. Take 5g of the test sample, add 50mL of water to dissolve it, and extract the obtained solution with petroleum ether of boiling range 30-60℃ for 2 times, 25mL each time, separate the petroleum ether liquid, and evaporate it. Add 2mL of ethanol to dissolve the obtained residue to obtain the test sample solution.

[0085] Preparation of control medicinal material solution: Take 0.5g of Angelica control medicinal material, Szechuan Lovage Rhizome control medicinal material, Turmeric control medicinal material, and Dark Red Root control medicinal material, add 25mL of petroleum ether of boiling range 30-60℃, ultrasonic treat for 20min, filter, evaporate, add 2mL of anhydrous ethanol to dissolve the obtained residue to prepare the corresponding control medicinal material solution.

[0086] Preparation of the control solution: Take ligustilide control, cinnamic aldehyde control, paeonol control, zedoary alcohol furan diene ketone control, add ethanol to prepare 1 mg / mL solution, that is, the corresponding control solution.

[0087] Identification: 20 μl of the above test solution, 10 μl of the control medicinal material solution, and 4 μl of the control solution were taken and spotted on the same silica gel G thin layer plate in the form of strips. Petroleum ether (60-90°C): ethyl acetate (9:1) was used as the developing agent, and the plate was developed, removed, and dried. The plate was then examined under ultraviolet light (365 nm), and then 2% vanillin sulfuric acid ethanol solution (1→10) was sprayed on it. The plate was heated at 105°C until the spots were clearly colored, and then examined under daylight. The development of different warm meridian soup samples was observed. The results are shown in Table 1. Figure 1 .

[0088] As can be seen from the image, in the test sample chromatogram, the same color fluorescent main spot appears at the corresponding position of ligustilide control, angelica control medicinal material, and chuanmiao control medicinal material. After spraying 2% vanillin sulfuric acid ethanol (1→10) solution and heating at 105°C until the spots are clearly colored, the plate is examined under daylight. In the test sample chromatogram, the same color spot appears at the corresponding position of zedoary turmeric control medicinal material, peony bark control medicinal material, and paeonol control. In this color developing agent, the cinnamic aldehyde spot is not obvious, so a second color developing agent (dinitrosophenylhydrazine ethanol test solution) needs to be used for identification. The components in aromatic water and inclusion compound are relatively simple, only cinnamic aldehyde, paeonol, and ligustilide, so there is less spot information.

[0089] Example 2

[0090] The present embodiment provides a method for identifying different base origin turmeric medicinal materials by thin layer chromatography, comprising the following steps:

[0091] Preparation of the test solution: The test sample was prepared according to the following method using the following as the test sample: warm meridian soup reference sample (JZ202210004), warm meridian soup granules (220601), vinegar wenyujin decoction pieces (K202106001), vinegar Guangxi zedoary decoction pieces (K20221002), and vinegar pungent zedoary decoction pieces (K20221001). 5 g of the test sample was added to 50 mL of water to dissolve it. The resulting solution was extracted with petroleum ether (boiling range 30-60°C) for 2 times, 25 mL each time. The petroleum ether liquid was separated and evaporated. The resulting residue was dissolved in 2 mL of ethanol to obtain the test sample solution.

[0092] Preparation of the reference material solution: Take 0.5 g of the reference material of Curcuma zedoary, the reference material of Curcuma kwangsiensis and the reference material of Curcuma wenyujin, add 25 mL of petroleum ether with a boiling range of 30-60°C, and ultrasonically treat for 20 min. Filter, evaporate to dryness, and dissolve the residue in 2 mL of anhydrous ethanol to prepare the corresponding reference material solution.

[0093] Preparation of the reference solution: Take the appropriate amount of curcumin reference, gimaone reference, furandiene reference and zedoary alcohol reference, and add ethanol to prepare a solution containing 1 mg per 1 mL to obtain the corresponding reference solution.

[0094] Preparation of the negative solution: Take 5 g of the negative sample of Curcuma zedoary, add 50 mL of water to dissolve it, and extract the solution with petroleum ether with a boiling range of 30-60°C for 2 times, 25 mL each time. Separate the petroleum ether liquid, evaporate to dryness, and dissolve the residue in 2 mL of ethanol to obtain the negative solution of Curcuma zedoary.

[0095] Identification: Take 20 μL of each of the above test sample solutions, 20 μL of the negative solution of Curcuma zedoary, 20 μL of the solution of the decoction piece of Curcuma zedoary, 10 μL of each of the solutions of the reference materials of Curcuma zedoary, Curcuma kwangsiensis and Curcuma wenyujin, and 4 μL of each of the reference solutions of curcumin, gimaone, furandiene and zedoary alcohol, and spot them on the same silica gel G thin layer plate. Use petroleum ether (60-90°C)-acetone-ethyl acetate (94:5:1) as the developing agent, develop, remove, air dry, spray with 2% vanillin sulfuric acid ethanol solution (1→10), heat at 105°C until the spots develop clearly, and observe under a daylight lamp. The results are shown in Table 1. Figure 2 .

[0096] As can be seen from the images, in the test samples of the decoction piece of Curcuma zedoary and the Wenjing Decoction prepared from Curcuma zedoary, there are two main spots at the positions corresponding to the reference material of Curcuma zedoary, which are pink and dark blue spots, respectively. This method can distinguish Curcuma zedoary, Curcuma wenyujin and Curcuma kwangsiensis from each other, and the main spot information will be different. Therefore, this thin layer identification method can be used to identify Curcuma zedoary from different origins.

[0097] Example 3

[0098] This example provides a thin layer chromatography method for identifying the Wenjing Decoction, and verifies its specificity, including the following steps:

[0099] Preparation of test solution: The test solution was prepared according to the following method using the three batches of Angelica sinensis and Ligusticum chuanxiong reference samples (JZ202210001, JZ202210002, JZ202210003) as the test sample. 5 g of the test sample was dissolved in 50 mL of water. The resulting solution was extracted with petroleum ether (30-60℃) for 2 times, 25 mL each time. The petroleum ether extract was collected and evaporated to dryness. The residue was dissolved in 2 mL of ethanol to obtain the test solution.

[0100] Preparation of reference material solution: 0.5 g of Angelica sinensis reference material, Ligusticum chuanxiong reference material, Curcuma phaeocaulis reference material, Paeonia suffruticosa reference material, and Cinnamomum cassia reference material were added to 25 mL of petroleum ether (30-60℃), and ultrasonically treated for 20 min. The mixture was filtered and the residue was evaporated to dryness. The residue was dissolved in 2 mL of anhydrous ethanol to obtain the corresponding reference material solution.

[0101] Preparation of reference solution: The ligustilide reference, cinnamyl aldehyde reference, and paeonol reference were dissolved in ethanol to obtain a solution containing 1 mg / mL of each reference, which was the corresponding reference solution.

[0102] Preparation of negative solution: The negative samples were prepared according to the preparation method of the test solution using the Angelica sinensis and Ligusticum chuanxiong double-negative sample, Curcuma phaeocaulis negative sample, Paeonia suffruticosa negative sample, and Cinnamomum cassia negative sample as the negative sample to obtain the negative solution.

[0103] Identification: 20 μl of the Angelica sinensis and Ligusticum chuanxiong double-negative solution, Paeonia suffruticosa negative solution, Curcuma phaeocaulis negative solution, and test solution of the Wanjing Decoction reference sample (batch number: JZ202210001), 10 μl of the Paeonia suffruticosa reference material solution, Angelica sinensis reference material solution, Ligusticum chuanxiong reference material solution, and Curcuma phaeocaulis reference material solution, and 4 μl of the paeonol and ligustilide reference solution were spotted on the same silica gel G thin layer plate. Petroleum ether (60-90℃): ethyl acetate (9:1, by volume) was used as the developing agent. After development, the plate was taken out, dried, and examined under ultraviolet light (365 nm). Then, 2% vanillin sulfuric acid ethanol solution (1→10) was sprayed on the plate, which was heated at 105℃ until the spots were clearly colored. The plate was then examined under daylight. The results are shown in Table 1. Figure 3 .

[0104] Cinnamon negative solution, test solution of the Wanjing Decoction reference sample (JZ202210001, JZ202210002, JZ202210003), 10 μl of the Cinnamomum cassia reference material solution, and 4 μl of the cinnamyl aldehyde reference solution were spotted on the same silica gel G thin layer plate. Petroleum ether (60-90℃): ethyl acetate (9:1, by volume) was used as the developing agent. After development, the plate was taken out, dried, and sprayed with dinitrosophenylhydrazine ethanol solution. The plate was then examined under daylight. The results are shown in Table 2. Figure 4 .

[0105] from Figure 3 As can be seen from the chromatogram, the test sample chromatogram shows a fluorescent main spot of the same color at the corresponding positions as the chromatograms of ligustilide, angelica sinensis, and chuanxiong rhizome reference materials, with no interference from the negative test solution. When sprayed with a 2% vanillin-sulfuric acid ethanol (1→10) solution and heated at 105℃ until the spots are clearly visible, and examined under a fluorescent lamp, the test sample chromatogram shows spots of the same color at the corresponding positions as the chromatograms of turmeric, peony bark, and paeonol reference materials, with no interference from the negative test solution. From... Figure 4 As can be seen from the chromatogram, the test sample showed spots of the same color at the corresponding positions as the chromatograms of cinnamon reference material and cinnamaldehyde reference standard, and the negative test sample solution showed no interference. This method has good specificity for identifying Angelica sinensis, Ligusticum chuanxiong, Curcuma zedoaria, Paeonia suffruticosa, and cinnamon.

[0106] Experimental Example 1

[0107] In this experimental example, four identical Wenjing Decoction reference samples (JZ20221004) were used as test samples and prepared according to four different test sample preparation methods, as follows.

[0108] (1) Test group 1: Take 5g of the test sample, add 50mL of water to dissolve it, extract the obtained solution twice with petroleum ether with a boiling range of 30-60℃, 25mL each time, separate the petroleum ether liquid, evaporate to dryness, add 2mL of ethanol to dissolve the residue to obtain test sample solution A.

[0109] (2) Control group 1: Take 5g of the sample to be tested, add 50ml of water, sonicate for 30min, cool, and extract the aqueous solution twice with petroleum ether (30-60℃), 25ml each time. Separate the petroleum ether solution, evaporate overnight, and dissolve the residue in 2ml of anhydrous ethanol to obtain test solution B.

[0110] (3) Control group 2: Take 5g of the sample to be tested, add 50ml of petroleum ether (30~60℃), sonicate for 30min, filter, evaporate to dryness, dissolve the residue in 2ml of anhydrous ethanol to obtain test solution C.

[0111] (4) Control group 3: Take 5g of the sample to be tested, add 50ml of petroleum ether (30~60℃), soak overnight, sonicate for 30min, filter, evaporate to dryness, dissolve the residue in 2ml of anhydrous ethanol to obtain test solution D.

[0112] Preparation of reference herb solutions: Take 0.5g each of Angelica sinensis, Ligusticum chuanxiong, and Curcuma zedoaria as reference herbs, add 25mL of petroleum ether with a boiling range of 30-60℃, sonicate for 20min, filter, evaporate to dryness, dissolve the residue in 2mL of anhydrous ethanol to obtain the corresponding reference herb solutions.

[0113] Preparation of the control solution: Take ligustilide control solution, add ethanol to prepare a solution containing 1 mg per 1 mL, that is, the corresponding control solution is obtained.

[0114] Preparation of the negative solution: Take the negative samples of Angelica sinensis and Ligusticum chuanxiong, and negative sample of Fructus torxici as the negative samples, and prepare the negative solution according to the preparation method of the test solution.

[0115] Identification: Take 20 μl of each of the above test solutions A, B, C and D, 20 μl of the negative solutions of Angelica sinensis and Ligusticum chuanxiong, 20 μl of the negative solution of Fructus torxici, 4 μl of the ligustilide control solution, 10 μl of each of the control medicinal material solutions of Angelica sinensis, Ligusticum chuanxiong and Fructus torxici, and point them on the same silica gel G thin layer plate. Take petroleum ether (60-90°C) : ethyl acetate (9:1) as the developing agent, develop, take out, dry, and observe under the ultraviolet light (365 nm). Then spray with 2% vanillin sulfuric acid ethanol solution (1→10), heat at 105°C until the spots develop clearly, and observe under the daylight lamp. The results are shown in Table 1. Figure 5 .

[0116] The test solution A has more chromatographic spot information and better separation. There are no spots of Angelica sinensis and Ligusticum chuanxiong in the test solutions B, C and D which are not treated according to the test solution treatment method in the present application, and the spot information of Fructus torxici is less. Therefore, it is determined that the test solution treatment method in the present application is the best treatment method. At the same time, attention should be paid to the fact that the drying of the test solution should not be too long, which will affect the amount of spot information.

[0117] Experimental Example 2

[0118] This experimental example provides five thin layer chromatography methods for identifying Wenjing Decoction preparations, which differ in the use of different developing agents.

[0119] (1) Test group 2: Preparation of the test solution: Take three batches of Wenjing Decoction reference samples (JZ202210001, JZ202210002 and JZ202210003) as the test samples, and prepare the test solution according to the following method. Take 5 g of the test sample, add 50 mL of water to dissolve it, and extract the obtained solution with petroleum ether with a boiling range of 30-60°C for 2 times, 25 mL each time. Separate the petroleum ether liquid, dry it, and dissolve the obtained residue in 2 mL of ethanol to obtain the test solution.

[0120] Preparation of the control medicinal material solution: Take 0.5 g of each of the control medicinal materials of Angelica sinensis, Ligusticum chuanxiong and Fructus torxici, add 25 mL of petroleum ether with a boiling range of 30-60°C, and ultrasonically treat for 20 min. Filter, dry, and dissolve the obtained residue in 2 mL of anhydrous ethanol to prepare the corresponding control medicinal material solution.

[0121] Preparation of the reference solution: take ligustilide reference substance, add ethanol to prepare a solution containing 1 mg per 1 mL, and then the corresponding reference solution is obtained.

[0122] Preparation of the negative solution: take the negative samples of lack of Angelica sinensis and Ligusticum chuanxiong, and lack of turmeric, and prepare the negative solution according to the preparation method of the test solution.

[0123] Identification: take 20 μl of each of the three batches of Wanjing decoction reference sample test solution, 20 μl of each of the negative solutions of lack of Angelica sinensis and Ligusticum chuanxiong, 4 μl of ligustilide reference solution, 10 μl of each of the Angelica sinensis reference medicinal material solution, Ligusticum chuanxiong reference medicinal material solution, and turmeric reference medicinal material solution, and point them on the same silica gel G thin layer plate, take petroleum ether (60-90 ℃) : ethyl acetate (9:1) as the developing agent, develop, take out, dry, and observe under the ultraviolet light (365 nm), then spray with 2% vanillin sulfuric acid ethanol solution (1→10), heat at 105 ℃ until the spots develop clearly, and observe under the sunlight. The results are shown in Figure 6 .

[0124] (2) Control group 4: the difference between this control group and the test group 2 is only that the developing agent used is n-hexane, acetone, and dichloromethane in a volume ratio of 94:5:2. The results are shown in Figure 7 .

[0125] (3) Control group 5: the difference between this control group and the test group 2 is only that the developing agent used is petroleum ether (60-90 ℃), acetone, and ethyl acetate in a volume ratio of 94:5:1. The results are shown in Figure 8 .

[0126] (4) Control group 6: the difference between this control group and the test group 2 is only that the developing agent used is petroleum ether (60-90 ℃) and ethyl acetate in a volume ratio of 17:3. The results are shown in Figure 9 .

[0127] (5) Control group 7: the difference between this control group and the test group 2 is only that the developing agent used is petroleum ether (60-90 ℃) and ethyl acetate in a volume ratio of 15:1. The results are shown in Figure 10 .

[0128] It can be seen from Figures 6-10 that in the tests of control groups 4-7, which do not use the developing agent specified in the present application, the developing distance is shorter, the spot separation effect is poorer, and there is negative interference of other components at one spot of turmeric. In the test of the test group 2, which uses the developing agent specified in the present application, the developing distance is longer, the spot separation effect is better, and there is no negative interference of other components at one spot of turmeric. Figure 6As can be seen, under ultraviolet light (365nm), five spots were separated, one of which was a ligustilide reference standard present in both Angelica sinensis and Ligusticum chuanxiong; the remaining two spots belonged to Angelica sinensis, and one to Ligusticum chuanxiong. After development, under fluorescent light, multiple spots were separated, with the top two pink and dark blue spots belonging to Curcuma zedoaria. There was no negative interference, the spots provided the most information, and the separation was optimal. Therefore, the developing solvent specified in this invention is determined to be the optimal developing solvent component.

[0129] Experimental Example 3

[0130] 1. Investigation of sample size

[0131] Following the preparation method given in Example 3, the following solutions were prepared: a reference sample solution for Wenjing Decoction, a double negative solution for Angelica sinensis and Ligusticum chuanxiong lacking alcohol, a negative solution for Curcuma zedoaria lacking vinegar, a reference medicinal material solution for Angelica sinensis, Ligusticum chuanxiong, and Curcuma zedoaria, and a reference solution for ligustilide.

[0132] Identification: Take 20 μl each of the double-negative solutions of Angelica sinensis and Ligusticum chuanxiong (lacking alcohol), and the negative solution of Curcuma zedoaria (vinegar-processed), 10 μl, 15 μl, 20 μl, and 25 μl of the test sample solution, 10 μl each of the reference solutions of Angelica sinensis, Ligusticum chuanxiong, and Curcuma zedoaria, and 4 μl of the ligustilide reference solution, and spot them separately onto the same silica gel G thin-layer plate. Develop the plate using a petroleum ether (60–90℃):ethyl acetate ratio of 9:1 (v / v). Remove the plate, air dry, and examine under ultraviolet light (365 nm). Then spray with 2% vanillin-sulfuric acid ethanol solution (1→10), heat at 105℃ until the spots are clearly visible, and examine under daylight. Examine the development of different sample amounts. Results are shown below. Figure 11 .

[0133] from Figure 11 As can be seen, the spots are clear and the chromatographic effect is good regardless of the amount of sample applied. Specifically, in the image obtained using this method, five spots can be seen separated under ultraviolet light (365nm), one of which is the ligustilide reference standard found in both Angelica sinensis and Ligusticum chuanxiong, the remaining two spots belong to Angelica sinensis, and one spot belongs to Ligusticum chuanxiong; after color development, multiple spots can be seen separated under fluorescent light, among which the top two pink and dark blue spots belong to Curcuma zedoaria.

[0134] 2. Conduct an investigation into temperature.

[0135] Following the preparation method given in Example 3, the following were prepared: the reference sample test solution of Wenjing Decoction (JZ202210001, JZ202210002, JZ202210003), the double negative solution of Angelica sinensis and Ligusticum chuanxiong lacking alcohol, the negative solution of Curcuma zedoaria lacking vinegar, the reference medicinal material solution of Angelica sinensis, Ligusticum chuanxiong, and Curcuma zedoaria, and the reference solution of ligustilide.

[0136] Identification: Take 20 μl of the negative solution of Angelica sinensis and Ligusticum chuanxiong, 20 μl of the negative solution of Styrax tonkinensis, 10 μl of each of the control drug material solutions of Angelica sinensis, Ligusticum chuanxiong and Styrax tonkinensis, 4 μl of the control solution of ligustilide, and spot them on the same silica gel G thin layer plate. Use petroleum ether (60-90 ℃) : ethyl acetate (9:1 by volume) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm). Then spray with 2% vanillin sulfuric acid ethanol solution (1→10), heat at 105 ℃ until the spots develop clear color, and examine under daylight. Investigate the developing conditions at different temperatures (4 ℃, room temperature, 30 ℃), and the results are shown in Table 1. Figure 12 , 13 .

[0137] As can be seen from the images, different temperatures have certain effects on the Rf values of the spots, but there are no obvious differences in separation effect and spot display, indicating that the method has good durability at environmental temperatures of 4-30 ℃.

[0138] 3. Investigation of developing humidity

[0139] Prepare the reference sample test solution of Wenzhi Decoction (JZ202210001, JZ202210002, JZ202210003), the negative solution of Angelica sinensis and Ligusticum chuanxiong, the negative solution of Styrax tonkinensis, the control drug material solutions of Angelica sinensis, Ligusticum chuanxiong and Styrax tonkinensis, and the control solution of ligustilide according to the preparation method given in Example 3.

[0140] Identification: Take 20 μl of the negative solution of Angelica sinensis and Ligusticum chuanxiong, 20 μl of the negative solution of Styrax tonkinensis, 10 μl of each of the control drug material solutions of Angelica sinensis, Ligusticum chuanxiong and Styrax tonkinensis, 4 μl of the control solution of ligustilide, and spot them on the same silica gel G thin layer plate. Use petroleum ether (60-90 ℃) : ethyl acetate (9:1 by volume) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm). Then spray with 2% vanillin sulfuric acid ethanol solution (1→10), heat at 105 ℃ until the spots develop clear color, and examine under daylight. Investigate the developing conditions at different humidities (32% RH, 88% RH). The results are shown in Table 2. Figure 14 , 15 .

[0141] As can be seen from the images, different humidities have certain effects on the Rf values of the spots, but there are no obvious differences in separation effect and spot display, indicating that the method has good durability at humidities of 32% RH-88% RH.

[0142] 4. Investigation of different thin layer plates

[0143] The reference sample of Wanjing decoction was prepared according to the preparation method given in Example 3 to prepare the test solution (JZ202210001, JZ202210002, JZ202210003), the negative solution of lack of Angelica sinensis and Ligusticum chuanxiong, the negative solution of lack of Atractylodes lancea, the control medicinal material solution of Angelica sinensis, Ligusticum chuanxiong and Atractylodes lancea, and the control solution of ligustilide.

[0144] Identification: 20 μl of the negative solution of lack of Angelica sinensis and Ligusticum chuanxiong, the negative test solution of Atractylodes lancea, and the test solution were taken, 10 μl of the control medicinal material solution of Angelica sinensis, Ligusticum chuanxiong and Atractylodes lancea, and 4 μl of the control solution of ligustilide were taken, and they were spotted on the same silica gel G thin layer plate. Petroleum ether (60-90°C): ethyl acetate (9:1) was used as the developing agent, and the plate was developed, taken out, dried, and observed under ultraviolet light (365 nm). Then, 2% vanillin sulfuric acid ethanol solution (1→10) was sprayed, heated to 105°C until the spots developed color clearly, and observed under daylight. The development of different brands of thin layer plates (Yantai plate, Qingdao plate) was investigated, and the results are shown in Table 1. Figure 16 、 17 .

[0145] From Figure 16 (Yantai plate), Figure 17 (Qingdao plate), Figure 1 (Merck plate) combined with Example 1,

[0146] 5. Investigation of different spotting methods

[0147] The reference sample of Wanjing decoction was prepared according to the preparation method given in Example 3 to prepare the test solution (JZ202210001, JZ202210002, JZ202210003), the negative solution of lack of Angelica sinensis and Ligusticum chuanxiong, the negative solution of lack of Atractylodes lancea, the control medicinal material solution of Angelica sinensis, Ligusticum chuanxiong and Atractylodes lancea, and the control solution of ligustilide.

[0148] Identification: 20 μl of the negative solution of lack of Angelica sinensis and Ligusticum chuanxiong, the negative test solution of Atractylodes lancea, and the test solution were taken, 10 μl of the control medicinal material solution of Angelica sinensis, Ligusticum chuanxiong and Atractylodes lancea, and 4 μl of the control solution of ligustilide were taken, and they were spotted on the same silica gel G thin layer plate. Petroleum ether (60-90°C): ethyl acetate (9:1) was used as the developing agent, and the plate was developed, taken out, dried, and observed under ultraviolet light (365 nm). Then, 2% vanillin sulfuric acid ethanol solution (1→10) was sprayed, heated to 105°C until the spots developed color clearly, and observed under daylight. The development of different brands of thin layer plates (Yantai plate, Qingdao plate) was investigated, and the results are shown in Table 1. Figure 18 .

[0149] The circular dot spotting method has no significant effect on the Rf value of each spot, and the separation effect and spot display have no significant difference, indicating that the method has good durability under different spotting methods.

[0150] Obviously, the above examples are only examples for clearly illustrating, but not limitation of the embodiments. Other different forms of changes or variations can be made on the basis of the above description for those skilled in the art. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations, characterized in that, Includes the following steps: S1: Take the sample to be tested, dissolve it in water, extract the resulting solution with petroleum ether with a boiling range of 30~60℃, separate the petroleum ether solution, dry it, dissolve it in an organic solvent to obtain the test solution; The organic solvent includes at least one of anhydrous ethanol and methanol; S2: Take Angelica sinensis, Ligusticum chuanxiong, and Curcuma zedoaria as reference materials, extract them with petroleum ether at a boiling range of 30-60℃, sonicate them, separate them into solid and liquid, dry them, and dissolve them in anhydrous ethanol to prepare solutions of each reference material. S3: Take the test solution, Angelica sinensis reference medicinal material solution, Ligusticum chuanxiong reference medicinal material solution, and Curcuma zedoaria reference medicinal material solution, and spot them separately on the same silica gel G thin layer plate. Develop with the first developing solvent, remove, air dry, and examine under ultraviolet light to identify Angelica sinensis and Ligusticum chuanxiong. Spray with the first color developer, heat, examine, and identify Curcuma zedoaria; The first colorimetric reagent comprises a 2-5% vanillin-sulfuric acid ethanol solution; The first developing solvent is petroleum ether or ethyl acetate with a boiling range of 60-90℃ and a volume ratio of 7-11:1-3.

2. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to claim 1, characterized in that, Step S2 also includes taking peony bark reference material, extracting it with petroleum ether at a boiling range of 30-60℃, ultrasonicating it, filtering it, evaporating it, and dissolving it in anhydrous ethanol to obtain a peony bark reference material solution. Step S3 also includes spotting the peony bark reference material solution onto the same silica gel G thin-layer plate used in step S3 before development.

3. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to claim 1, characterized in that, The thin-layer chromatography method further includes: taking cinnamon reference material, extracting it with petroleum ether at a boiling range of 30-60℃, ultrasonicating it, filtering it, evaporating it to dryness, dissolving it in anhydrous ethanol to prepare a cinnamon reference material solution; taking the test solution and the cinnamon reference material solution, spotting them separately on another silica gel G thin-layer plate, developing them with the first developing solvent, removing them, drying them, spraying them with the second colorimetric solvent, examining them, and identifying cinnamon.

4. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to claim 3, characterized in that, The thin-layer chromatography method further includes: taking cinnamaldehyde reference standard, adding organic solvent to prepare cinnamaldehyde reference standard solution, and spotting it, the test solution and cinnamon reference material solution on the same silica gel G thin-layer plate before development.

5. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to claim 3, characterized in that, The second colorimetric reagent comprises a dinitrophenylhydrazine ethanol solution.

6. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to claim 1, characterized in that, The thin-layer chromatography method further includes: taking at least one of the following reference standards: ligustilide, paeonol, curcumin, gemmaconazole, furandiene, turmeric alcohol, and turmeric furandiene, adding an organic solvent to prepare a reference solution. Before development, step S3 further includes spotting the reference solution onto the same silica gel G thin-layer plate used in step S3.

7. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to claim 6, characterized in that, Each 1 mL of the reference solution contains 0.5 to 1 mg of reference standard.

8. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to claim 1, characterized in that, In step S1, the extraction includes shaking extraction; And / or, in step S1, drying includes evaporation; And / or, in step S3, the inspection wavelength for identifying Angelica sinensis and Ligusticum chuanxiong includes 365nm; And / or, in step S3, after spraying the first color developer, the heating temperature is 105°C.

9. The thin-layer chromatography method for simultaneously identifying different medicinal ingredients in Wenjing Decoction and its preparations according to any one of claims 1 to 8, characterized in that, Step S1 includes taking 5-10g of the sample to be tested, adding 50-100mL of water to dissolve it, extracting the obtained solution with petroleum ether with a boiling range of 30-60℃ by shaking 2-4 times, 25-50mL each time, separating the petroleum ether solution, evaporating it to dryness, and dissolving the obtained residue with 2-5mL of anhydrous ethanol to obtain the test solution. And / or, step S2 includes taking 0.5-1g each of Angelica sinensis reference material, Ligusticum chuanxiong reference material and Curcuma zedoaria reference material, adding petroleum ether with a boiling range of 30-60℃, ultrasonically treating for 20-60 minutes, filtering, evaporating to dryness, dissolving the residue in 2-5mL of anhydrous ethanol to prepare reference material solutions respectively.

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