Thin-layer chromatography identification method and application of bighead atractylodes rhizome and extract thereof

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and relates to a thin-layer chromatography identification method and application of bighead atractylodes rhizome and an extract thereof. The invention provides two thin-layer identification methods, one thin-layer identification method takes four components such as atractylenolide I, atractylenolide II, atractylenolide III and beta-eudesmol in bighead atractylodes rhizome as index components, and the other thin-layer identification method takes three components such as atractylenone, geranene B and beta-elemene in bighead atractylodes rhizome as index components. The two methods can be used for rapidly and accurately identifying the rhizoma atractylodis macrocephalae medicinal material, the rhizoma atractylodis macrocephalae volatile oil, the volatile oil clathrate and the rhizoma atractylodis macrocephalae clathrate related preparations, are mild in condition and easy to operate, are favorable for comprehensively improving the quality control level of rhizoma atractylodis macrocephalae related products, provide feasible reference for innovation and research and development related to rhizoma atractylodis macrocephalae in each stage, and have important industrial value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine quality control, and relates to a thin layer chromatography identification method and application of Atractylodes macrocephala Koidz. BACKGROUND

[0002] Atractylodes macrocephala Koidz. is a rhizome of Atractylodes macrocephala Koidz. of the family Compositae, which is recorded in the Pharmacopoeia of the People's Republic of China (2020 edition). It is bitter and sweet in nature, warm in property, and belongs to the spleen and stomach channels. It has the effects of invigorating the spleen and tonifying qi, drying dampness and promoting water, stopping sweating, and preventing miscarriage. It is one of the major medicinal materials in China. Pharmacological studies have shown that atractylone isolated from Atractylodes macrocephala Koidz. has the effects of anti-gastric ulcer, reducing ulcerative colitis, and anti-virus. The atractylenolide components (atractylenolide I, atractylenolide II, atractylenolide III) in Atractylodes macrocephala Koidz. are effective components for anti-inflammatory and anticancer. β-eudesmol has anti-inflammatory activity and can regulate angiogenesis. β-elemene has anticancer effect. In addition, Atractylodes macrocephala Koidz. also contains terpenes such as bigeorgiyol B and β-caryophyllene.

[0003] Thin layer chromatography, as a common chromatographic separation and identification technology, has the advantages of simple operation, good separation effect, high sensitivity, and is widely used in the qualitative identification and quality control of various traditional Chinese medicinal materials, traditional Chinese medicinal slices, and traditional Chinese patent medicines. This method uses the differences in adsorption capacity on adsorbents and solubility in developing agents of different components, so that continuous adsorption and desorption occurs during development, and each component can be eluted to different positions of the adsorbent at the same time, thereby achieving the separation of components.

[0004] At present, for the above-mentioned several effective components, the thin layer identification method of Atractylodes macrocephala Koidz. recorded in the Chinese Pharmacopoeia (2020 edition) is to use petroleum ether (60-90℃)-ethyl acetate (50:1) as the developing agent. After development, 5% vanillin sulfuric acid solution is sprayed for coloration, and the pink spots are the index component atractylone. After consulting the data, it is found that the thin layer chromatography identification index of Atractylodes macrocephala Koidz. is concentrated on atractylone, atractylenolide I, atractylenolide II, and atractylenolide III. There are articles that have disclosed a thin layer identification method which can identify the four components at the same time. However, no other chemical components in Atractylodes macrocephala Koidz. have been found as thin layer identification index, and the identification basis is not sufficient. Chinese patent document CN112305142A discloses a gradient multi-information thin layer identification method of Atractylodes macrocephala Koidz. Under multiple viewing conditions, about 20 chemical components of Atractylodes macrocephala Koidz. are detected, but no component identification is performed.

[0005] At present, there is no other component in Atractylodes macrocephala Koidz. as a thin layer identification index component, the identification basis is less, and there is no report on the application of thin layer method to Atractylodes macrocephala Koidz., Atractylodes macrocephala Koidz. extract, extract preparation inclusion complex, and inclusion complex preparation. SUMMARY

[0006] In view of the above-mentioned disadvantages of the prior art, the present application follows the purpose of being comprehensive, simple, accurate, efficient, and environmentally friendly, and provides a thin layer chromatography identification method for target components in Atractylodes rhizome samples, one of which takes four components of atractyloides lactone I, atractyloides lactone II, atractyloides lactone III and beta-eucalyptol in Atractylodes rhizome as index components, and the other takes three components of atractylodes ketone, germacrene B and beta-elemene in Atractylodes rhizome as index components. Both methods can quickly and accurately identify Atractylodes rhizome, Atractylodes rhizome volatile oil, volatile oil inclusion compound, and inclusion compound related preparations, the conditions are mild, easy to operate, and conducive to improving the quality control level of Atractylodes rhizome related products, providing a feasible reference for innovative research and development related to Atractylodes rhizome at various stages, and having important industrial value.

[0007] To achieve the above-mentioned purposes and other related purposes, the first aspect of the present application provides a thin layer chromatography identification method for target components in Atractylodes rhizome samples, comprising the following steps:

[0008] (1) providing a test sample solution: if the sample is Atractylodes rhizome medicinal material, Atractylodes rhizome powder is taken, an organic solvent is added, ultrasonic extraction is performed, filtration is performed, and the filtrate is taken to obtain the test sample solution; if the sample is Atractylodes rhizome volatile oil, Atractylodes rhizome volatile oil is taken, an organic solvent is added, the volume is adjusted, ultrasonic extraction is performed, filtration is performed, and the filtrate is taken to obtain the test sample solution; if the sample is a preparation containing Atractylodes rhizome volatile oil inclusion compound, the preparation containing Atractylodes rhizome volatile oil inclusion compound is taken, an organic solvent is added, ultrasonic extraction is performed, filtration is performed, and the filtrate is taken to obtain the test sample solution;

[0009] (2) providing a control sample solution: when the target components are atractyloides lactone I, atractyloides lactone II, atractyloides lactone III and beta-eucalyptol, atractyloides lactone I control sample solution, atractyloides lactone II control sample solution, atractyloides lactone III control sample solution and beta-eucalyptol control sample solution are prepared respectively; when the target components are atractylodes ketone, germacrene B and beta-elemene, atractylodes ketone control sample solution, germacrene B control sample solution and beta-elemene control sample solution are prepared respectively;

[0010] (3) detection: the test sample solution and the control sample solution are respectively taken and spotted on the same silica gel G thin layer plate, a developing agent is added, development is performed, air drying is performed, a color developing agent is sprayed, heating is performed until the spots are clearly colored, and under the ultraviolet lamp, the test sample chromatogram shows the same color main spots at the positions corresponding to the control sample chromatogram.

[0011] In some embodiments of the present application, the Atractylodes rhizome powder in step (1) has a particle size of ≤355 μm, and most of the particles have a particle size of 150-355 μm. The operation usually adopted is to pass the Atractylodes rhizome powder through a No. 3 sieve. When the preparation containing Atractylodes rhizome volatile oil inclusion compound is a tablet, the tablet needs to be ground into fine powder.

[0012] In some embodiments of the present application, the organic solvent in step (1) is selected from n-hexane, absolute ethanol or methanol; preferably, if the sample is Atractylodes lancea, the organic solvent is n-hexane; if the sample is Atractylodes lancea volatile oil, the organic solvent is absolute ethanol; if the sample is a preparation containing Atractylodes lancea volatile oil inclusion complex, the organic solvent is methanol.

[0013] In some embodiments of the present application, the ratio of the mass of Atractylodes lancea powder to the volume of the organic solvent added in step (1) is 1g:3-5ml, preferably 1g:4ml; the ratio of the volume of Atractylodes lancea volatile oil to the volume of the organic solvent added is 10mg:1ml; the ratio of the mass of the preparation containing Atractylodes lancea volatile oil inclusion complex to the volume of the organic solvent added is 1g:2-3ml, preferably 1g:2.5ml.

[0014] In some embodiments of the present application, the ultrasonic extraction in step (1) uses running water ultrasonic extraction, and the ultrasonic extraction time is 10-40min; preferably, if the sample is Atractylodes lancea, the ultrasonic extraction time is 20-30min, more preferably 25min; if the sample is Atractylodes lancea volatile oil, the ultrasonic extraction time is 10-20min, more preferably 15min; if the sample is a preparation containing Atractylodes lancea volatile oil inclusion complex, the ultrasonic extraction time is 20-40min, more preferably 30min.

[0015] In the present application, the filtration without special instructions is all filtered through a 0.22μm or 0.45μm microporous filter membrane.

[0016] Preferably, the preparation of the control solution in step (2) comprises: taking the control, adding an organic solvent, ultrasonic dissolving, and constant volume; preferably, the organic solvent is selected from methanol.

[0017] In some embodiments of the present application, the concentration of the control solution in step (2) is independently selected from 0.5-2mg / mL, preferably 1mg / mL; in some specific embodiments of the present application, the concentration of the atractylenolide I control solution, the atractylenolide II control solution, the atractylenolide III control solution, and the β-eudesmol control solution are all 1mg / mL; the concentration of the atractylodin control solution, the globulol B control solution, and the β-elemene control solution are all 1mg / mL.

[0018] In some embodiments of the present application, the sample is Atractylodes lancea (Thunb.) DC. or Atractylodes lancea (Thunb.) DC. volatile oil, and the target components are atractylochide I, atractylochide II, atractylochide III and β-eudesmol, and the sample solution is spotted in an amount of 20-40 μl, preferably 20 μl if the sample is Atractylodes lancea (Thunb.) DC., and preferably 40 μl if the sample is Atractylodes lancea (Thunb.) DC. volatile oil. If the sample is Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound or a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and the target components are atractylochide I, atractylochide II, atractylochide III and β-eudesmol, the sample solution is spotted in an amount of 4-8 μl, preferably 4 μl if the sample is Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and preferably 8 μl if the sample is a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound. If the sample is Atractylodes lancea (Thunb.) DC., and the target components are atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol, the sample solution is spotted in an amount of 20-40 μl, preferably 20 μl. If the sample is Atractylodes lancea (Thunb.) DC. volatile oil, Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound or a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and the target components are atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol, the sample solution is spotted in an amount of 2-4 μl, preferably 2 μl if the sample is Atractylodes lancea (Thunb.) DC. volatile oil or Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and preferably 4 μl if the sample is a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound.

[0019] In some embodiments of the present application, the sample is Atractylodes lancea (Thunb.) DC. or Atractylodes lancea (Thunb.) DC. volatile oil, and the target components are atractylochide I, atractylochide II, atractylochide III and β-eudesmol, and the sample solution is spotted in an amount of 20-40 μl, preferably 20 μl if the sample is Atractylodes lancea (Thunb.) DC., and preferably 40 μl if the sample is Atractylodes lancea (Thunb.) DC. volatile oil. If the sample is Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound or a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and the target components are atractylochide I, atractylochide II, atractylochide III and β-eudesmol, the sample solution is spotted in an amount of 4-8 μl, preferably 4 μl if the sample is Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and preferably 8 μl if the sample is a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound. If the sample is Atractylodes lancea (Thunb.) DC., and the target components are atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol, the sample solution is spotted in an amount of 20-40 μl, preferably 20 μl. If the sample is Atractylodes lancea (Thunb.) DC. volatile oil, Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound or a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and the target components are atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol, the sample solution is spotted in an amount of 2-4 μl, preferably 2 μl if the sample is Atractylodes lancea (Thunb.) DC. volatile oil or Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound, and preferably 4 μl if the sample is a preparation containing Atractylodes lancea (Thunb.) DC. volatile oil inclusion compound.

[0020] In some embodiments of the present application, if the sample is Atractylodes lancea (Thunb.) DC. in step (3), the detection also includes taking the medicinal material solution and spotting it on the same silica gel G thin layer plate, and the preparation method of the medicinal material solution is the same as that of the sample solution in step (1), and the amount of the control medicinal material solution spotted is 20-40 μl, preferably 20 μl.

[0021] In some embodiments of the present application, when the target components are atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol in step (3), the developing agent is selected from petroleum ether-acetone, petroleum ether-ethyl acetate, n-hexane-acetone, and preferably petroleum ether-acetone with a volume ratio of 9:1; when the target components are atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol, the developing agent is selected from petroleum ether or n-hexane, and preferably petroleum ether.

[0022] In some embodiments of the present application, in step (3), when the target component is atractyloides I, atractyloides II, atractyloides III and β-eudesmol, the color developing agent is selected from 10% sulfuric acid ethanol solution or 5% p-dimethylaminobenzaldehyde-10% sulfuric acid ethanol solution, preferably 5% p-dimethylaminobenzaldehyde-10% sulfuric acid ethanol solution; when the target component is atractylone, bigeneol B and β-elemene, the color developing agent is selected from 5% p-dimethylaminobenzaldehyde-10% sulfuric acid ethanol solution.

[0023] In some embodiments of the present application, in step (3), the temperature of the environment for development is 4-40℃. In the present application, there is no obvious difference in development effect at 4℃ (low temperature), 25℃ (room temperature) and 40℃ (high temperature), that is, the separation effect between the spots is good, and the spots are clear without tailing phenomenon, thus the application is good at different temperatures.

[0024] In some embodiments of the present application, in step (3), the humidity of the environment for development is 33%-80%. In the present application, there is no obvious difference in development effect at 33% (low humidity), 60% (normal humidity) and 80% (high humidity), that is, the separation effect between the spots is good, and the spots are clear without tailing phenomenon, thus the application is good at different humidities.

[0025] In the present application, the silica gel G thin layer plate used for thin layer chromatography development is a thin layer chromatography plate with silica gel G as the stationary phase, for example, commercially available brands include Huanghai Yantai, Shengya, Qingdao Haoyang, Qingdao Qianting Hexin, etc., and other equivalent products meeting the pharmacopoeia standards are also applicable.

[0026] The second aspect of the present application provides the use of the above-mentioned thin layer chromatography identification method of target components in Atractylodes lancea in the quality detection and quality control of Atractylodes lancea products.

[0027] The Atractylodes lancea products include Atractylodes lancea medicinal materials, Atractylodes lancea extracts and Atractylodes lancea preparations; the Atractylodes lancea medicinal materials refer to the dried rhizomes of Atractylodes lancea (Thunb.) DC. of the family Compositae, including raw Atractylodes lancea, bran-fried Atractylodes lancea, soil-fried Atractylodes lancea, charred Atractylodes lancea, etc.; the Atractylodes lancea extract is a refined product obtained by directional extraction from Atractylodes lancea medicinal materials using modern extraction and separation technologies (such as solvent extraction, steam distillation, supercritical extraction, column chromatography, etc.), and enriching specific active component groups or single components, such as Atractylodes lancea volatile oil. The Atractylodes lancea preparation is a finished pharmaceutical product which can be directly used for disease prevention or treatment, and is processed by modern pharmaceutical technology with Atractylodes lancea medicinal materials or Atractylodes lancea extracts as the main raw materials, including but not limited to one of tablet, capsule, granule, pill, powder, oral liquid, injection or external use pharmaceutical preparation, for example, Atractylodes lancea inclusion compound, Atractylodes lancea inclusion compound tablet.

[0028] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0029] 1. The method for identifying target components in the sample of Atractylodes rhizome provided by the application, one of which takes atractylodin, atractylenolide II, atractylenolide III and beta-eudesmol as index components, and the other takes atractylone, germacrene B and beta-elemene as index components, both of which can quickly and accurately identify Atractylodes rhizome, Atractylodes rhizome volatile oil, Atractylodes rhizome volatile oil inclusion compound and related preparations. The method has mild conditions and is easy to operate, which is conducive to improving the quality level of Atractylodes rhizome related products, providing reliable quality control reference for each stage of research and development, and has important industrial application value.

[0030] 2. The method for identifying target components in the sample of Atractylodes rhizome provided by the application, one of which takes atractylodin, atractylenolide II, atractylenolide III and beta-eudesmol as index components, and the other takes atractylone, germacrene B and beta-elemene as index components, both of which can quickly and accurately identify Atractylodes rhizome, Atractylodes rhizome volatile oil, Atractylodes rhizome volatile oil inclusion compound and related preparations. The method has mild conditions and is easy to operate, which is conducive to improving the quality level of Atractylodes rhizome related products, providing reliable quality control reference for each stage of research and development, and has important industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The development effect of atractylodin, atractylenolide II, atractylenolide III and beta-eudesmol in Atractylodes rhizome in different development systems, wherein (a) is the development effect of the developing agent A, (b) is the development effect of the developing agent B, (c) is the development effect of the developing agent C, (d) is the development effect of the developing agent D, (e) is the development effect of the developing agent E, and (f) is the development effect of the developing agent F under 365 nm ultraviolet light; sample A to D are atractylodin, atractylenolide II, atractylenolide III and beta-eudesmol control points in turn, E is an Atractylodes rhizome control sample, and F is an Atractylodes rhizome test sample.

[0032] Figure 2 The development effect of atractylodin, atractylenolide II, atractylenolide III and beta-eudesmol in Atractylodes rhizome in different developing agents, wherein (a) is the development effect of 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution, (b) is the development effect of 10% sulfuric acid ethanol test solution, and (c) is the development effect of 5% vanillin sulfuric acid solution; sample A to D are atractylodin, atractylenolide II, atractylenolide III and beta-eudesmol control points in turn, E is an Atractylodes rhizome control sample, and F is an Atractylodes rhizome test sample.

[0033] Figure 3The development effect of atractylodin, atractylenolide II, atractylenolide III and β-eudesmol in Atractylodes lancea (Thunb.) DC. medicinal materials in different sample application amounts, wherein (a) is the application of four control samples, and (b) is the application of Atractylodes lancea (Thunb.) DC. test sample; (a) in the figure, A1-A3, B1-B3, C1-C3 are atractylodin, atractylenolide II, atractylenolide III with sample application amounts of 2 μL, 4 μL, 8 μL, and D1-D3 are β-eudesmol with sample application amounts of 0.5 μL, 1 μL, 2 μL; (b) in the figure, F1-F3 are Atractylodes lancea (Thunb.) DC. medicinal material test sample application amounts of 10 μL, 20 μL, 40 μL.

[0034] Figure 4 The development effect of atractylodin, atractylenolide II, atractylenolide III and β-eudesmol in Atractylodes lancea (Thunb.) DC. medicinal materials at different temperatures, wherein (a) is normal temperature (25℃), (b) is low temperature (4℃), and (c) is high temperature (40℃) under 365nm ultraviolet; samples A-D are atractylodin, atractylenolide II, atractylenolide III and β-eudesmol control points in turn, E is Atractylodes lancea (Thunb.) DC. control sample, and F is Atractylodes lancea (Thunb.) DC. test sample.

[0035] Figure 5 The development effect of atractylodin, atractylenolide II, atractylenolide III and β-eudesmol in Atractylodes lancea (Thunb.) DC. medicinal materials at different humidities, wherein (a) is normal humidity, (b) is high humidity, and (c) is low humidity under 365nm ultraviolet; samples A-D are atractylodin, atractylenolide II, atractylenolide III and β-eudesmol control points in turn, E is Atractylodes lancea (Thunb.) DC. control sample, and F is Atractylodes lancea (Thunb.) DC. test sample.

[0036] Figure 6 The development effect of atractylodin, atractylenolide II, atractylenolide III and β-eudesmol in Atractylodes lancea (Thunb.) DC. medicinal materials in different factory thin layer plates, wherein (a)-(d) are the thin layer chromatograms of Atractylodes lancea (Thunb.) DC. medicinal materials in different factory silica gel G plates under 365nm ultraviolet; samples A-D are atractylodin, atractylenolide II, atractylenolide III and β-eudesmol control points in turn, E is Atractylodes lancea (Thunb.) DC. control sample, and F is Atractylodes lancea (Thunb.) DC. test sample.

[0037] Figure 7 The development effect of atractylodin, atractylenolide II, atractylenolide III and β-eudesmol in Atractylodes lancea (Thunb.) DC. medicinal materials in different development systems, wherein (a) and (b) are the development effects of developing agents G and H under 365nm ultraviolet; samples A-C are atractylodin, atractylenolide II and β-eudesmol control points in turn, D is Atractylodes lancea (Thunb.) DC. test sample, and E is Atractylodes lancea (Thunb.) DC. control sample.

[0038] Figure 8The development effect of atractylodin, germacrene B and β-elemene in Atractylodes rhizome medicinal materials under different color reagents, wherein (a) is 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution coloration, (b) is 10% sulfuric acid ethanol test solution coloration, (c) is 5% vanillin sulfuric acid solution coloration; sample points A~C are atractylodin, germacrene B and β-elemene control points in turn, D is Atractylodes rhizome control product, and E is Atractylodes rhizome test product.

[0039] Figure 9 The development effect of atractylodin, germacrene B and β-elemene in Atractylodes rhizome medicinal materials under different sample points, wherein sample points A1~A3 are atractylodin sample points 4 μL, 8 μL and 16 μL respectively, B1~B3 are germacrene B sample points 2 μL, 4 μL and 8 μL respectively, C1~C3 are β-elemene sample points 10 μL, 20 μL and 40 μL respectively, and D1~D3 are Atractylodes rhizome test product sample points 5 μL, 10 μL and 20 μL respectively.

[0040] Figure 10 The development effect of atractylodin, germacrene B and β-elemene in Atractylodes rhizome medicinal materials under different temperatures, wherein (a) is normal temperature (25 ℃), (b) is low temperature (4 ℃), and (c) is high temperature (40 ℃) under 365 nm ultraviolet light; sample points A~C are atractylodin, germacrene B and β-elemene control points in turn, D is Atractylodes rhizome control product, and E is Atractylodes rhizome test product.

[0041] Figure 11 The development effect of atractylodin, germacrene B and β-elemene in Atractylodes rhizome medicinal materials under different humidities, wherein (a) is normal humidity, (b) is high humidity, and (c) is low humidity under 365 nm ultraviolet light; sample points A~C are atractylodin, germacrene B and β-elemene control points in turn, D is Atractylodes rhizome control product, and E is Atractylodes rhizome test product.

[0042] Figure 12 The development effect of atractylodin, germacrene B and β-elemene in Atractylodes rhizome medicinal materials under different factory thin layer plates, wherein (a)-(d) are thin layer chromatograms of silicagel G plates of Yantai, Shengya, Qingdao Haoyang, Qingdao Qianting Heyixin and other factories under 365 nm ultraviolet light; sample points A~C are atractylodin, germacrene B and β-elemene control points in turn, D is Atractylodes rhizome control product, and E is Atractylodes rhizome test product.

[0043] Figure 13 The development effect of atractylodin, germacrene B and β-elemene in Atractylodes rhizome medicinal materials under different humidities, wherein (a) is normal humidity, (b) is high humidity, and (c) is low humidity under 365 nm ultraviolet light; sample points A~C are atractylodin, germacrene B and β-elemene control points in turn, D is Atractylodes rhizome control product, and E is Atractylodes rhizome test product.

[0044] Figure 14 The developing effect of atractylodin, germacrane B and β-elemene in the volatile oil of Atractylodes macrocephala Koidz, wherein the sample points A-C are the control points of atractylodin, germacrane B and β-elemene in turn, and D is the test sample of the volatile oil of Atractylodes macrocephala Koidz.

[0045] Figure 15 The developing effect of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eudesmol in the inclusion compound of Atractylodes macrocephala Koidz, wherein the sample points A and B are the control sample mixture and the test sample of the inclusion compound in turn, and the spots 1-4 are atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eudesmol in turn.

[0046] Figure 16 The developing effect of atractylodin, germacrane B and β-elemene in the inclusion compound of Atractylodes macrocephala Koidz, wherein the sample points A-C are the control points of atractylodin, germacrane B and β-elemene in turn, and D is the test sample of the inclusion compound.

[0047] Figure 17 The developing effect of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eudesmol in the tablet containing the inclusion compound of Atractylodes macrocephala Koidz, wherein the sample points A and B are the control sample mixture and the test sample of the inclusion compound in turn, and the spots 1-4 are atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eudesmol in turn.

[0048] Figure 18 The developing effect of atractylodin, germacrane B and β-elemene in the tablet containing the inclusion compound of Atractylodes macrocephala Koidz, wherein the sample points A-C are the control points of atractylodin, germacrane B and β-elemene in turn, and D is the test sample of the tablet. DETAILED DESCRIPTION

[0049] The present application is further described in conjunction with the following specific examples, which are intended to be illustrative of the present application and not limiting thereof.

[0050] The present application is further described in conjunction with the following specific examples, which are intended to be illustrative of the present application and not limiting thereof.

[0051] The instruments used in the examples are as follows:

[0052] XS104 electronic analytical balance (METTLER TOLEDO); XS105 electronic analytical balance (METTLER TOLEDO); SK7200HP ultrasonic cleaner (Shanghai Keduo Ultrasonic Instrument Co., Ltd.); air drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.); ATS 4 automatic sample applicator (Swiss Camag); 5810R high-speed centrifuge (Eppendorf China Co., Ltd.); dark box ultraviolet analyzer (Shanghai Weikai Instrument and Equipment Co., Ltd.); DZF-6050 vacuum drying oven (Shanghai Boxun Industry Co., Ltd.).

[0053] Thin layer consumables and reagent information used in the examples:

[0054] Silica gel G thin layer plate (manufactured by Huanghai Yantai Jiangyou Silica Gel Development Co., Ltd., Shanghai Shengyahaichem Co., Ltd., Qingdao Qianting Hexin New Material Co., Ltd., Qingdao Haoyang Chemical Co., Ltd.);

[0055] Methanol, anhydrous ethanol, sulfuric acid, p-dimethylaminobenzaldehyde, n-hexane, acetone, ethyl acetate, petroleum ether, vanillin (National Pharmaceutical Group Chemical Reagent Co., Ltd.).

[0056] Information of control and control medicinal materials in the examples:

[0057] Atractylodin I control (Anhui Xiqingguo Biological Technology Co., Ltd., batch number: 130011-221101);

[0058] Atractylodin II control (Chengdu Pusai Biological Technology Co., Ltd., batch number: PS010511);

[0059] Atractylodin III control (Anhui Xiqingguo Biological Technology Co., Ltd., batch number: 130011-221101);

[0060] Beta-eudesmol control (Anhui Xiqingguo Biological Technology Co., Ltd., batch number: 200431-221101);

[0061] Cangzhu ketone control (Shanghai Hongyong Biological Technology Co., Ltd., batch number: 030048-202106);

[0062] Big root geraniol B control (Aijie Technology, batch number: 13-JMR-24-2)

[0063] Beta-elemene control (Anhui Xiqingguo Biological Technology Co., Ltd., batch number: 230281-221101)

[0064] Atractylodes control medicinal materials (China Food and Drug Inspection Institute, batch number: 120925-202114).

[0065] Example 1: Optimization of the developing condition for simultaneous identification of four components in Atractylodes Macrocephala Koidz by thin layer chromatography

[0066] I. Experimental scheme

[0067] 1.1 Preparation of reagents

[0068] 10% sulfuric acid ethanol: 1000 mL of ethanol was placed in ice water, 57 mL of concentrated sulfuric acid was slowly added, which contained 9.5% to 10.5% sulfuric acid.

[0069] 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution: 2.5 g of p-dimethylaminobenzaldehyde solid was weighed and transferred to a conical flask, and 47.5 ml of 10% sulfuric acid ethanol was added, and it was obtained.

[0070] 1.2 Preparation of control solution

[0071] An appropriate amount of atractylenolide I, atractylenolide II, atractylenolide III, and β-eudesmol control samples were accurately weighed, ultrasonically dissolved with methanol, and diluted to a concentration of about 1 mg / mL to prepare each control solution.

[0072] 1.3 Preparation of test sample

[0073] Atractylodes Macrocephala Koidz medicinal material powder was taken, 4 times the amount of n-hexane was added, ultrasonic extraction was performed for 15 minutes, filtration was performed, and the filtrate was taken as the medicinal material (cutting) test sample solution.

[0074] The same method was used to prepare the Atractylodes Macrocephala Koidz medicinal material control solution.

[0075] 1.4 Thin layer spotting setting

[0076] According to the thin layer chromatography test (Chinese Pharmacopoeia 2020 edition four general tests 0502), 4 μL of atractylenolide I, atractylenolide II, and atractylenolide III control solution, 1 μL of β-eudesmol control solution, 20 μL of test sample solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate. Different reagents were mixed in a certain proportion as developing agent, developed, dried, sprayed with color developing agent, heated at 105°C for 10 minutes to develop color, and observed under ultraviolet lamp (365 nm); In the test sample, the same color spots were observed at the corresponding positions of atractylenolide I, atractylenolide II, atractylenolide III, and β-eudesmol control, and Atractylodes Macrocephala Koidz control medicinal material chromatogram.

[0077] 1.5 Comparison of developing effects of different developing agents and proportions

[0078] The sample was spotted on 10*10 cm yellow Yantai silica gel G thin layer plate with "1.4" sample setting, a total of 6 thin layer plates were spotted, and different developing agents were used to develop (as shown in Table 1), air dried, sprayed with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution, heated at 105°C for 10 minutes to develop color, and placed under ultraviolet lamp (365nm) for observation.

[0079] Table 1 reagents contained in different developing agent systems and their proportions

[0080]

[0081]

[0082] II. Experimental results

[0083] As shown in Table 1, the sample was spotted on 10*10 cm yellow Yantai silica gel G thin layer plate with "1.4" sample setting, a total of 6 thin layer plates were spotted, and different developing agents were used to develop (as shown in Table 1), air dried, sprayed with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution, heated at 105°C for 10 minutes to develop color, and placed under ultraviolet lamp (365nm) for observation. Figure 1 The developing agent A / B / C / D has little difference in developing effect under the same sample setting, silica gel G plate and developing condition. Among them, the combination of developing agent (a), i.e. petroleum ether-acetone system, can relatively better separate several control components, and the control medicinal materials and atractylodes rhizome test samples can be identified, therefore, the combination of petroleum ether-acetone is finally selected for further optimization investigation.

[0084] The developing agents A / E / F have different proportions of developing agents, and the developing effect has great difference. Among them, the combination of developing agent (a), i.e. petroleum ether: acetone = 9:1 system, can separate several control components, and the control medicinal materials and atractylodes rhizome test samples can be identified, therefore, the combination of petroleum ether: acetone = 9:1 is finally selected as the developing agent system for atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eudesmol in atractylodes rhizome.

[0085] III. Summary

[0086] The combination of petroleum ether and acetone is the best developing agent combination for the four components in atractylodes rhizome, and the best proportion is petroleum ether: acetone = 9:1, which can better separate atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eudesmol. The developing agent will be used for subsequent investigation of the developing condition of the four components in atractylodes rhizome.

[0087] Example 2: Optimization of developing condition for simultaneous identification of four components in atractylodes rhizome by thin layer chromatography

[0088] I. Experimental scheme

[0089] 1.1 Preparation of reagents

[0090] 10% sulfuric acid ethanol: 1000mL ethanol was placed in ice water, and 57mL concentrated sulfuric acid was slowly added, which contained 9.5%-10.5% sulfuric acid.

[0091] 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution: 2.5 g of p-dimethylaminobenzaldehyde solid was weighed into a conical flask, and 47.5 ml of 10% sulfuric acid ethanol was added, to obtain the test solution.

[0092] 5% vanillin sulfuric acid solution: 2.5 g of vanillin solid was weighed into a conical flask, and 47.5 ml of 10% sulfuric acid ethanol was added, to obtain the test solution.

[0093] 1.2 Preparation of the control solution

[0094] The control solutions of atractylodin I, atractylodin II, atractylodin III and β-eudesmol with a concentration of about 1 mg / mL were prepared according to “1.2” of Example 1.

[0095] 1.3 Preparation of the test sample

[0096] The test sample of Atractylodes lancea and the control sample of Atractylodes lancea were prepared according to “1.3” of Example 1.

[0097] 1.4 Comparison of the color development effects of different color developing agents

[0098] According to the thin layer chromatography method (General Test Methods 0502 in Chinese Pharmacopoeia 2020), 4 μL of the atractylodin I, atractylodin II and atractylodin III control solution, 1 μL of the β-eudesmol control solution, 20 μL of the test sample solution and 20 μL of the control sample solution were taken and spotted on the same silica gel G thin layer plate. Petroleum ether and acetone were mixed in a ratio of 9:1 to obtain the developing agent, and the plate was developed, dried, sprayed with 10% sulfuric acid ethanol test solution, 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution or 5% vanillin sulfuric acid solution, heated at 105°C for 10 minutes to develop color, and observed under ultraviolet light (365 nm).

[0099] II. Experimental results

[0100] The same sample spotting setup was used, and the color development effects of different color developing agents were compared by developing with petroleum ether-acetone (9:1), and it was found that Figure 2 When 5% vanillin sulfuric acid solution was used for color development, only the spots of atractylodin II and β-eudesmol could be observed, and the spots of atractylodin I and atractylodin III could not be observed. When 10% sulfuric acid ethanol test solution was used for color development, the spots of different index components were clear, but the colors were not easy to distinguish. When 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol test solution was used for color development, the spots of different index components were clear and the colors could be distinguished. Therefore, 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol was selected as the color developing agent for atractylodin I, atractylodin II, atractylodin III and β-eudesmol in Atractylodes lancea.

[0101] III. Conclusion

[0102] Comparing the color developing effects of three different color developing agents, observing the color development of four index components, i.e. atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol, with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution as the color developing agent, and then investigating the spotting amount of the color developing agent and other researches.

[0103] Example 3: Optimization of the spotting amount of the reference substance and the test substance of the four components in the Bai-Zhu medicinal material by the thin layer chromatography method

[0104] I. Experimental scheme

[0105] 1.1 Preparation of reagents

[0106] The same as “1.1” in Example 1.

[0107] 1.2 Preparation of the reference substance solution

[0108] The same as “1.2” in Example 1, and the reference substance solutions of atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol with a concentration of about 1 mg / mL were prepared.

[0109] 1.3 Preparation of the test substance

[0110] The same as “1.3” in Example 1, and the test substance of the Bai-Zhu medicinal material and the reference of the Bai-Zhu medicinal material were prepared.

[0111] 1.4 Investigation of the spotting amount of the reference substance and the test substance of the Bai-Zhu medicinal material

[0112] 2 μL, 4 μL and 8 μL of the atractylenolide I, atractylenolide II and atractylenolide III reference substance solutions and 0.5 μL, 1 μL and 2 μL of the β-eudesmol reference substance solution were taken and spotted on the same 10*20 silica gel G thin layer plate, 10 μL, 20 μL and 40 μL of the Bai-Zhu medicinal material test substance were taken and spotted on the same 10*10 silica gel G thin layer plate, petroleum ether and acetone were mixed in a ratio of 9:1 as the developing agent, development, air drying, spraying with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, heating at 105°C for 10 minutes to develop color, and then observing under the ultraviolet lamp (365 nm), and comparing the development effects of different spotting amounts.

[0113] II. Experimental results

[0114] From the above results, it can be seen that the spotting amount of the reference substance and the test substance of the four components in the Bai-Zhu medicinal material can be optimized by the thin layer chromatography method. Figure 3It can be seen that the same developing agent, silica gel G plate and color developing conditions, different amounts of the same sample have obvious differences in the developing effect. When the sample amount of the atractylenolide I reference solution is 4-8 μL, the color of each spot is clear. In the subsequent methodological study, the sample amount of the atractylenolide I reference solution is 4 μL. When the sample amount of the atractylenolide II and atractylenolide III reference solutions is 2-8 μL, the color of each spot is clear. In the subsequent methodological study, the sample amount of the atractylenolide II and atractylenolide III reference solutions is 4 μL. When the sample amount of the β-eudesmol reference solution is 1-2 μL, the color of each spot is clear. In the subsequent methodological study, the sample amount of the β-eudesmol reference solution is 1 μL. When the sample amount of the atractylodes medicinal material test sample is 20-40 μL, the color of each spot is clear. In the subsequent methodological study, the sample amount of the test sample is 20 μL.

[0115] Three, Summary

[0116] Through the sample amount investigation of the atractylenolide I, atractylenolide II, atractylenolide III, β-eudesmol reference solutions, and the atractylodes medicinal material test sample, it can be seen that the sample amounts of the atractylenolide I, atractylenolide II, atractylenolide III, β-eudesmol, and the test sample are 4-8 μL, 2-8 μL, 2-8 μL, 1-2 μL, and 20-40 μL, respectively, and the color of each spot is clear. In the subsequent methodological study, the sample amounts of the atractylenolide I, atractylenolide II, atractylenolide III, β-eudesmol, and the test sample are 4 μL, 4 μL, 4 μL, 1 μL, and 20 μL, respectively.

[0117] Example 4: Methodological investigation of simultaneously identifying four components of atractylodes medicinal material by thin layer chromatography

[0118] One, Experimental scheme

[0119] 1.1 Preparation of reagents

[0120] The same as "1.1" of Example 1.

[0121] 1.2 Preparation of reference solutions

[0122] The same as "1.2" of Example 1, and the concentrations of the prepared atractylenolide I, atractylenolide II, atractylenolide III, and β-eudesmol reference solutions are about 1 mg / mL.

[0123] 1.3 Preparation of test samples

[0124] The same as "1.3" of Example 1, and the atractylodes medicinal material test sample and the atractylodes medicinal material control are prepared.

[0125] 1.4 Investigation of durability

[0126] 1.4.1 Investigation of temperature durability

[0127] Take 4 μL of atractylodin, 4 μL of atractylodin II, 4 μL of atractylodin III, 1 μL of β-eudesmol, 20 μL of atractylodes control drug and 20 μL of atractylodes test sample, respectively, and spot them on a yellow sea Yantai silica gel G thin layer plate. Take petroleum ether-acetone as developing agent in a ratio of 9:1. Develop at room temperature (25°C), low temperature (4°C) and high temperature (40°C), respectively. Take out, dry, spray with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol, heat at 105°C for 10 minutes to develop color, and place under ultraviolet lamp (365nm) to observe. Compare the developing effects at different temperatures.

[0128] 1.4.2 Humidity resistance investigation

[0129] Take 4 μL of atractylodin, 4 μL of atractylodin II, 4 μL of atractylodin III, 1 μL of β-eudesmol, 20 μL of atractylodes control drug and 20 μL of atractylodes test sample, respectively, and spot them on a yellow sea Yantai silica gel G thin layer plate. Take petroleum ether-acetone as developing agent in a ratio of 9:1. Develop at room temperature (25°C), low temperature (4°C) and high temperature (40°C), respectively. Take out, dry, spray with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol, heat at 105°C for 10 minutes to develop color, and place under ultraviolet lamp (365nm) to observe. Compare the developing effects at different temperatures.

[0130] Note: The normal laboratory control humidity is 60% RH, saturated ammonium chloride solution (25°C, 80% relative humidity); saturated magnesium chloride solution (25°C, 33% relative humidity). Place the saturated solution in a desiccator, and place the developing cylinder and hygrometer in the desiccator.

[0131] 1.4.3 Investigation of resistance of silica gel G plates from different manufacturers

[0132] Take 4 μL of atractylodin, 4 μL of atractylodin II, 4 μL of atractylodin III, 1 μL of β-eudesmol, 20 μL of atractylodes control drug and 20 μL of atractylodes test sample, respectively, and spot them on a yellow sea Yantai silica gel G thin layer plate. Take petroleum ether-acetone as developing agent in a ratio of 9:1. Develop, take out, dry, spray with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol, heat for 10 minutes to develop color (set the temperature at 105°C for yellow sea Yantai, and set the temperature at 80°C for Shengya, Qingdao Haoyang, and Qingdanganting Hexin), and place under ultraviolet lamp (365nm) to observe. Compare the developing and color developing effects of thin layer plates from different manufacturers.

[0133] II. Experimental results

[0134] 2.1 Temperature resistance investigation

[0135] From Figure 4It can be seen that under the same sample setting, developing agent and color developing conditions, the developing effect of different temperatures has no obvious difference, that is, the separation effect between the spots is good, the spots are clear, and there is no tailing phenomenon, so it has good durability to different temperatures.

[0136] 2.2 Relative humidity durability investigation

[0137] From Figure 5 It can be seen that under the same sample setting, developing agent and color developing conditions, the developing effect of different humidity has no obvious difference, that is, the separation effect between the spots is good, the spots are clear, and there is no tailing phenomenon, so it has good durability to different humidity.

[0138] 2.3 TLC manufacturer durability investigation

[0139] From Figure 6 It can be seen that under the same sample setting, developing agent and color developing conditions, the developing effect of different manufacturers has no obvious difference, that is, the separation effect between the spots is good, and there is no tailing phenomenon, and the spots of individual manufacturers are not clear. Therefore, the TLC silica gel G plate prepared by different manufacturers has good durability.

[0140] Three, summary

[0141] Through the temperature and humidity durability and the durability of silica gel plates from different manufacturers, it can be seen that the TLC conditions are stable and feasible, and can simultaneously and rapidly identify 4 components of atractylenolide I, atractylenolide II, atractylenolide III and β-eudesmol.

[0142] Example 5: Optimization of developing conditions for simultaneously identifying 3 index components of atractylodes rhizome by thin layer chromatography

[0143] I. Experimental scheme

[0144] 1.1 Preparation of reagents

[0145] The same as "1.1" of Example 1.

[0146] 1.2 Preparation of reference substance solution

[0147] 10 mg of artactylenolide reference substance powder, 5 mg of big root geranial B reference substance solution, and 20 mg of β-elemene reference substance solution were weighed and accurately weighed, and then transferred to a volumetric flask. Methanol was used for ultrasonic dissolution, and then diluted to constant volume to prepare each reference substance solution with a concentration of about 1 mg / mL.

[0148] 1.3 Preparation of test sample

[0149] The same as "1.3" of Example 1, atractylodes rhizome test sample and atractylodes rhizome control were prepared.

[0150] 1.4 TLC sample setting

[0151] The sample solution and the control drug material solution were each 10 μL, and the different reagents were mixed according to a certain proportion to form a developing agent. After development, drying, spraying with a color developing agent, heating at 105 ℃ for 10 minutes to develop color, and placing under a UV lamp (365 nm) for observation, the same color spots were observed in the sample solution at the corresponding positions of the atractylodin, germacrene B and β-elemene control solutions, and the atractylodes control drug material.

[0152] 1.5 Comparison of the developing effects of different developing agents

[0153] The sample solution and the control drug material solution were each 10 μL, and the different reagents were mixed according to a certain proportion to form a developing agent. After development, drying, spraying with a color developing agent, heating at 105 ℃ for 10 minutes to develop color, and placing under a UV lamp (365 nm) for observation, the same color spots were observed in the sample solution at the corresponding positions of the atractylodin, germacrene B and β-elemene control solutions, and the atractylodes control drug material.

[0154] Table 2 Reagents contained in different developing agent systems and their proportions

[0155]

[0156] II. Experimental results

[0157] It can be seen that the developing agents A and B can better separate the three index components. Figure 7

[0158] III. Summary

[0159] The developing effects of different developing agents were compared, and petroleum ether or n-hexane was the best developing agent for the three components in atractylodes rhizome, which could better separate atractylodin, germacrene B and β-elemene. Since the polarity of petroleum ether is slightly lower than that of n-hexane, petroleum ether will be used as the developing agent for the color developing condition investigation of the three index components in atractylodes rhizome in the future.

[0160] Example 6: Optimization of color developing conditions for simultaneously identifying three index components in atractylodes rhizome by thin layer chromatography

[0161] I. Experimental scheme

[0162] 1.1 Preparation of reagents

[0163] The same as “1.1” in Example 2.

[0164] 1.2 Preparation of control solution

[0165] The same as “1.2” in Example 5, and the control solutions of atractylodin, germacrene B and β-elemene with a concentration of about 1 mg / mL were prepared. ​

[0166] 1.3 Preparation of the test sample

[0167] The test sample of Atractylodes rhizome and the control sample of Atractylodes rhizome were prepared according to “1.3” of Example 1.

[0168] 1.4 Comparison of color development effects of different color developing agents

[0169] According to the test of thin layer chromatography (General Test Methods in Chinese Pharmacopoeia 2020, Volume 4, 0502), 4, 4 and 10 μL of atractylone, longesquiphylene B and β-elemene control sample solutions, 10 μL of the test sample solution and 10 μL of the control sample solution were taken and spotted on the same silica gel G thin layer plate, petroleum ether was used as the developing agent, and then the plate was developed, dried, sprayed with 10% sulfuric acid ethanol solution, 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution or 5% vanillin sulfuric acid solution, heated at 105°C for 10 minutes to develop color, and then observed under ultraviolet light (365 nm).

[0170] II. Experimental results

[0171] The same sample spotting setup was used, petroleum ether was used for development, and the color development effects of different color developing agents were compared. Figure 8 It can be seen that when 5% vanillin sulfuric acid solution is used for color development, the spots can be clearly displayed, and the spot of β-elemene is blurred; when 10% sulfuric acid ethanol solution is used for color development, only the spots of atractylone and longesquiphylene B can be developed, and the spot of β-elemene cannot be developed; when 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution is used for color development, the spots of different index components are clear and the colors can be distinguished, therefore, 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution is selected as the color developing agent for atractylone, longesquiphylene B and β-elemene in Atractylodes rhizome.

[0172] III. Summary

[0173] The color development effects of three different color developing agents were compared, and the three index components of atractylone, longesquiphylene B and β-elemene were observed. When 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution is used for color development, the sample spotting amount of the color developing agent will be investigated in the subsequent research.

[0174] Example 7: Optimization of sample spotting amount of control sample and test sample of three index components in Atractylodes rhizome by thin layer chromatography

[0175] I. Experimental scheme

[0176] 1.1 Preparation of reagents

[0177] The same as “1.1” of Example 1

[0178] 1.2 Preparation of control sample solution

[0179] As in "1.2" of Example 5, reference solutions of atractylone, geraniol B and β-elemene with a concentration of about 1 mg / mL were prepared.

[0180] 1.3 Preparation of the test sample

[0181] As in "1.3" of Example 1, a sample of Atractylodes macrocephala and a control sample of Atractylodes macrocephala were prepared.

[0182] 1.4 Investigation on the sampling quantity of reference standard and Atractylodes macrocephala test sample

[0183] Take 4 μL, 8 μL, and 16 μL of atractylone reference solution, 2 μL, 4 μL, and 8 μL of geraniol B reference solution, and 5 μL, 10 μL, and 20 μL of β-elemene and 10 μL, 20 μL, and 40 μL of Atractylodes macrocephala test solution, and spot them on the same 10*20 silica gel G thin-layer plate. Use petroleum ether as the developing solvent, develop, air dry, spray with 5% p-dimethylaminobenzaldehyde and 10% sulfuric acid ethanol test solution, heat at 105℃ for 10 minutes until color development, and examine under a UV lamp (365 nm) to compare the development effect of different spotting amounts.

[0184] II. Experimental Results

[0185] Depend on Figure 9 It can be seen that, under the same developing solvent, silica gel G plate, and colorimetric conditions, the developing effect varies significantly with different spotting volumes. When the spotting volume of atractylone reference solution was 4–16 μL, each spot showed clear color; therefore, the spotting volume of atractylone reference solution was subsequently set at 4 μL for methodological investigation. Similarly, when the spotting volume of geraniol B reference solution was 4–8 μL, each spot showed clear color; therefore, the spotting volume of geraniol B reference solution was subsequently set at 4 μL for methodological investigation. When the spotting volume of β-elemene reference solution was 10–40 μL, each spot showed clear color; therefore, the spotting volume of β-elemene reference solution was subsequently set at 10 μL for methodological investigation. When the spotting volume of Atractylodes macrocephala medicinal material test sample was 10–20 μL, each spot showed clear color; therefore, the spotting volume of the test sample was subsequently set at 20 μL for methodological investigation.

[0186] III. Summary

[0187] By examining the spotting amounts of atractylone, geraniol B, β-elemene reference standards, and the test sample of Atractylodes macrocephala, it was found that the spots were clearly visible when the spotting amounts of atractylone, geraniol B, β-elemene, and the test sample were 4–16 μL, 4–8 μL, 10–40 μL, and 10–20 μL, respectively. In subsequent methodological studies, spotting amounts of atractylone 4 μL, geraniol B 4 μL, β-elemene 10 μL, and the test sample 10 μL can be used.

[0188] Example 8: Methodological investigation of simultaneous identification of three indicator components in Atractylodes macrocephala by thin-layer chromatography

[0189] II. Experimental Protocol

[0190] 1.1 Preparation of Reagents

[0191] Same as "1.1" of Example 1.

[0192] 1.2 Preparation of Control Solutions

[0193] Same as "1.2" of Example 5, control solutions of atractylodin, globulol B and β-elemene were prepared at a concentration of about 1 mg / mL.

[0194] 1.3 Preparation of Test Samples

[0195] Same as "1.3" of Example 1, test samples of Atractylodes lancea and control samples of Atractylodes lancea were prepared.

[0196] 1.4 Investigation of Stability

[0197] 1.4.1 Investigation of Temperature Stability

[0198] 4 μL of atractylodin, 4 μL of globulol B, 10 μL of β-elemene, 10 μL of control samples of Atractylodes lancea and 10 μL of test samples of Atractylodes lancea were spotted on a yellow Yantai silica gel G thin layer plate, and developed with petroleum ether as the developing agent at room temperature (25°C), low temperature (4°C) and high temperature (40°C) respectively, removed, air dried, sprayed with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol, heated at 105°C for 10 minutes to develop color, placed under a UV lamp (365 nm) for observation, and the developing effects at different temperatures were compared.

[0199] 1.4.2 Investigation of Humidity Stability

[0200] 4 μL of atractylodin, 4 μL of globulol B, 10 μL of β-elemene, 10 μL of control samples of Atractylodes lancea and 10 μL of test samples of Atractylodes lancea were spotted on a yellow Yantai silica gel G thin layer plate, and developed with petroleum ether as the developing agent at room temperature (25°C) under normal humidity (60% RH), high humidity (80% RH) and low humidity (33% RH) respectively, removed, air dried, sprayed with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol, heated at 105°C for 10 minutes to develop color, placed under a UV lamp (365 nm) for observation, and the developing effects at different temperatures were compared.

[0201] Note: The normal laboratory controlled humidity is 60% RH, saturated ammonium chloride solution (25°C, 80% relative humidity); saturated magnesium chloride solution (25°C, 33% relative humidity), the saturated solution was placed in a desiccator, and the developing cylinder and the hygrometer were placed in the desiccator.

[0202] 1.4.3 Investigation of Stability of Silica Gel G Plates from Different Manufacturers

[0203] Respectively take 4 μL of atractylone, 10 μL of β-elemene, 10 μL of Bai Zhi control medicinal materials and Bai Zhi medicinal material test sample, and point on different manufacturers (Huanghai Yantai, Shengya, Qingdao Ocean, Qingdao Qianting Hexin) silica gel G thin layer plate, with petroleum ether as developing agent, develop, take out, dry, spray with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, heat for 10 minutes to develop color (Huanghai Yantai set temperature 105℃, Shengya, Qingdao Ocean, Qingdao Qianting Hexin set temperature 80℃), and place under ultraviolet lamp (365nm) to observe, and compare the developing and color developing effects of different manufacturers' thin layer plates.

[0204] II. Experimental results

[0205] 2.1 Temperature durability investigation

[0206] It can be seen that under the same sample setting, developing agent and color developing conditions, the developing effects of different temperatures have no obvious difference, that is, the separation effect between the spots is good, and the spots are relatively clear without tailing phenomenon, so it has good durability to different temperatures. Figure 10 2.2 Relative humidity durability investigation

[0207] It can be seen that under the same sample setting, developing agent and color developing conditions, the developing effects of normal humidity and low humidity environment have no obvious difference, that is, the separation effect between the spots is good, and the spots are relatively clear without tailing phenomenon, but the separation effect of β-elemene in the test sample is poor under high humidity environment, so it has good durability to low humidity environment and poor durability to high humidity environment.

[0208] Figure 11 2.3 Thin layer manufacturer durability investigation

[0209] It can be seen that under the same sample setting, developing agent and color developing conditions, the developing effects of different manufacturers have no obvious difference, that is, the separation effect between the spots is good, and the spots are relatively clear without tailing phenomenon. Therefore, it has good durability to the thin layer silica gel G plates prepared by different manufacturers.

[0210] Figure 12

[0211] III. Conclusion

[0212] Through the temperature and humidity durability investigation and the silica gel plate durability investigation of different manufacturers, it can be seen that the thin layer condition only has poor separation effect of β-elemene in the test sample under high humidity environment, and can simultaneously and rapidly identify atractylone, bigenol and β-elemene and other three components under other environments.

[0213] Example 9: Simultaneous identification of four components and three index components in Bai Zhi volatile oil by thin layer chromatography

[0214] I. Experimental scheme ​​​

[0215] 1.1 Reagent Preparation

[0216] Same as "1.1" in Example 1.

[0217] 1.2 Preparation of reference solution

[0218] As in "1.2" of Example 1, reference solutions of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, β-eucalyptol, atractylone, geraniol B and β-elemene with a concentration of about 1 mg / mL were prepared.

[0219] 1.3 Preparation of the test sample

[0220] Accurately weigh 50mg of Atractylodes macrocephala volatile oil, dilute to 5ml with anhydrous ethanol, extract by ultrasonication for 15 minutes, filter, and collect the filtrate to obtain the product.

[0221] 1.4 Thin-layer chromatography identification of four components in Atractylodes macrocephala volatile oil

[0222] Take 4 μL of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, 1 μL of β-eucalyptol reference solution, and 40 μL of Atractylodes macrocephala volatile oil test sample, and spot them on the same 10*10 silica gel G thin-layer plate. Use a 9:1 mixture of petroleum ether and acetone as the developing solvent. Develop the plate, air dry it, spray it with 5% p-dimethylaminobenzaldehyde and 10% sulfuric acid ethanol test solution, heat it at 105℃ for 10 minutes until color development, and examine it under a UV lamp (365nm). Compare the developing effects of different spotting amounts.

[0223] 1.5 Thin-layer chromatography identification of three components in the volatile oil of Atractylodes macrocephala

[0224] Take 4 μL of atractylone, geraniol B, 10 μL of β-elemene, and 2 μL of Atractylodes macrocephala volatile oil samples respectively and spot them on the same 10*10 silica gel G thin layer plate. Use petroleum ether as the developing solvent, develop, air dry, spray with 5% p-dimethylaminobenzaldehyde and 10% sulfuric acid ethanol solution, heat at 105℃ for 10 minutes until color development, and examine under a UV lamp (365nm). Compare the development effects of different spotting amounts.

[0225] II. Experimental Results

[0226] 2.1 Thin-layer chromatography identification of four components in Atractylodes macrocephala volatile oil

[0227] Depend on Figure 13 It can be seen that the spots of Atractylodes macrocephala volatile oil are clear under this thin-layer condition, indicating good applicability.

[0228] 2.2 Thin-layer chromatography identification of three components in Atractylodes macrocephala volatile oil

[0229] Depend on Figure 14 It can be seen that the spots of Atractylodes macrocephala volatile oil are clear under this thin-layer condition, indicating good applicability.

[0230] Three, Summary

[0231] Seven components in volatile oil of Atractylodes Macrocephala Koidz. can be identified by the two methods, and the spots are clear.

[0232] Example 10: Simultaneous identification of four components and three index components in inclusion complex of volatile oil of Atractylodes Macrocephala Koidz. by thin layer chromatography

[0233] I. Experimental scheme

[0234] 1.1 Preparation of reagents

[0235] The same as "1.1" of Example 1.

[0236] 1.2 Preparation of control solution

[0237] A certain amount of Atractylodes lactone I, Atractylodes lactone II, Atractylodes lactone III and β-eudesmol control powder was accurately weighed, dissolved in methanol to prepare a control mixture solution with a concentration of about 1 mg / mL.

[0238] The preparation of atractylone, longibombykol B and β-elemene control solution was the same as "1.2" of Example 5.

[0239] 1.3 Preparation of test sample

[0240] 2g of inclusion complex of volatile oil of Atractylodes Macrocephala Koidz. was weighed, dissolved in 5ml of methanol, ultrasonically extracted for 30 minutes, filtered, and the filtrate was obtained.

[0241] 1.4 Identification of four components in inclusion complex by thin layer chromatography

[0242] 4μL of Atractylodes lactone I, Atractylodes lactone II, Atractylodes lactone III, 1μL of β-eudesmol control solution and 4μL of inclusion complex test sample were taken respectively and spotted on the same 10*10 silica gel G thin layer plate, petroleum ether and acetone were mixed in a ratio of 9:1 as developing agent, developed, air dried, sprayed with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol reagent, heated at 105℃ for 10 minutes to develop color, placed under ultraviolet lamp (365nm) for observation, and the developing effects of different sample amounts were compared.

[0243] 1.5 Identification of three components in inclusion complex by thin layer chromatography

[0244] 4μL of atractylone, longibombykol B and 10μL of β-elemene were taken respectively, 2μL of inclusion complex test sample was taken, and they were spotted on the same 10*10 silica gel G thin layer plate, petroleum ether was used as developing agent, developed, air dried, sprayed with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol reagent, heated at 105℃ for 10 minutes to develop color, placed under ultraviolet lamp (365nm) for observation, and the developing effects of different sample amounts were compared.

[0245] II. Experimental results

[0246] 2.1 Identification of 4 components in Baizhu volatile oil inclusion complex by TLC

[0247] It can be seen that the spots of Baizhu volatile oil inclusion complex are clear under the TLC conditions, and the suitability is good. Figure 15

[0248] 2.2 Identification of 3 components in Baizhu volatile oil inclusion complex by TLC

[0249] It can be seen that the spots of Baizhu volatile oil inclusion complex are clear under the TLC conditions, and the suitability is good. Figure 16

[0250] Three, Summary

[0251] Through the two methods, 7 components in Baizhu volatile oil can be identified, and the spots are clear.

[0252] Example 11: Simultaneous identification of 4 components and 3 index components in Baizhu volatile oil inclusion complex preparation by TLC

[0253] I. Experimental scheme

[0254] 1.1 Preparation of reagents

[0255] The same as "1.1" of Example 1.

[0256] 1.2 Preparation of control solution

[0257] The same as "1.2" of Example 10, and the control mixed solution of Baizhu lactone I, Baizhu lactone II, Baizhu lactone III and β-eudesmol was prepared with a concentration of about 1 mg / mL, and each control solution of artactone, globulol B and β-elemene was prepared.

[0258] 1.3 Preparation of test sample

[0259] Grind the Baizhu volatile oil inclusion complex tablets into fine powder. If the proportion of Baizhu volatile oil inclusion complex in the tablets is about 50%-60%, take 2 g of tablet fine powder, dissolve in 5 ml of methanol, ultrasonic extraction for 30 minutes, filter, and take the filtrate.

[0260] 1.4 Identification of 4 components in Baizhu volatile oil inclusion complex tablets by TLC

[0261] Take 4 μL of Baizhu lactone I, Baizhu lactone II, Baizhu lactone III, 1 μL of β-eudesmol control solution, and 8 μL of tablet test sample, respectively, and point them on the same 10*10 silica gel G thin layer plate. Mix petroleum ether and acetone in a ratio of 9:1 as developing agent, develop, air dry, spray with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, heat at 105°C for 10 minutes to develop color, and observe under ultraviolet lamp (365 nm), and compare the development effects of different sample amounts. ​​

[0262] 1.5 Thin-layer chromatography identification of three indicator components in Atractylodes macrocephala volatile oil inclusion complex tablets

[0263] Take 4 μL of atractylone, geraniol B, 10 μL of β-elemene, and 4 μL of tablet sample respectively, and spot them on the same 10*10 silica gel G thin-layer plate. Use petroleum ether as the developing solvent, develop, air dry, spray with 5% p-dimethylaminobenzaldehyde and 10% sulfuric acid ethanol solution, heat at 105℃ for 10 minutes until color development, and examine under a UV lamp (365nm). Compare the development effects of different spotting amounts.

[0264] II. Experimental Results

[0265] 2.1 Thin-layer chromatography identification of four components in Atractylodes macrocephala volatile oil inclusion complex tablets

[0266] Depend on Figure 17 It can be seen that the inclusion complex tablets of Atractylodes macrocephala volatile oil showed clear spots under this thin-layer condition, indicating good applicability.

[0267] 2.2 Thin-layer chromatography identification of three indicator components in Atractylodes macrocephala volatile oil inclusion complex tablets

[0268] Depend on Figure 18 It can be seen that the inclusion complex tablets of Atractylodes macrocephala volatile oil showed clear spots under this thin-layer condition, indicating good applicability.

[0269] III. Summary

[0270] These two methods can identify seven components in Atractylodes macrocephala volatile oil inclusion complex tablets, and the spots are relatively clear, which is quite innovative.

[0271] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A thin-layer chromatography method for identifying target components in Atractylodes macrocephala samples, characterized in that, Includes the following steps: (1) Provide the test solution: If the sample is Atractylodes macrocephala, take Atractylodes macrocephala powder, add organic solvent, extract by ultrasonication, filter, and take the filtrate; if the sample is Atractylodes macrocephala volatile oil, take Atractylodes macrocephala volatile oil, add organic solvent, make up to volume, extract by ultrasonication, filter, and take the filtrate; if the sample is a preparation containing Atractylodes macrocephala volatile oil inclusion complex, take the preparation containing Atractylodes macrocephala volatile oil inclusion complex, add organic solvent, extract by ultrasonication, filter, and take the filtrate. (2) Provide reference solutions: When the target components are atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eucalyptol, prepare reference solutions for atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eucalyptol respectively; when the target components are atractylone, geraniol B and β-elemene, prepare reference solutions for atractylone, geraniol B and β-elemene respectively. (3) Detection: Take the test solution and the reference solution separately and spot them on the same silica gel G thin layer plate. Add the developing solvent, develop, dry, spray with the color developing agent, heat until the spots are clearly visible, and examine under ultraviolet light. In the chromatogram of the test sample, the same color main spot appears at the corresponding position as in the chromatogram of the reference sample.

2. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 1, characterized in that, Step (1) includes one or more of the following features: (a) The particle size of the Atractylodes macrocephala powder is ≤355μm; when the preparation containing the Atractylodes macrocephala volatile oil inclusion complex is a tablet, the tablet needs to be ground into a fine powder; (b) The organic solvent is selected from n-hexane, anhydrous ethanol, or methanol; (c) The mass ratio of Atractylodes macrocephala powder to the volume of organic solvent added is 1g:3-5ml; the volume ratio of Atractylodes macrocephala volatile oil to organic solvent added is 10mg:1mL; the mass ratio of preparations containing Atractylodes macrocephala volatile oil inclusion complex to the volume of organic solvent added is 1g:2-3ml. (d) The ultrasonic extraction is performed using running water ultrasound, and the ultrasonic extraction time is 10 to 40 minutes.

3. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 1, characterized in that, Step (2) includes one or more of the following features: (a) The preparation of the reference solution includes: taking the reference standard, adding an organic solvent, sonicating to dissolve, and making up to volume; (b) The concentration of the reference solution is independently selected from 0.5 to 2 mg / mL.

4. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 3, characterized in that, Step (2) includes one or more of the following features: (a1) The organic solvent is selected from methanol; (b1) The concentration of the reference solution is 1 mg / mL.

5. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 1, characterized in that, Step (3) includes one or more of the following features: (a) The spotting volume of atractylodes lactone I reference solution is 4–8 μl, the spotting volume of atractylodes lactone II reference solution is 2–8 μl, the spotting volume of atractylodes lactone III reference solution is 2–8 μl, the spotting volume of β-eucalyptol reference solution is 1–2 μl, the spotting volume of atractylone reference solution is 4–16 μl, the spotting volume of geraniol B reference solution is 4–8 μl, and the spotting volume of β-elemene reference solution is 10–40 μl. (b) If the sample is Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil, and the target components are atractylodes macrocephala lactone I, atractylodes macrocephala lactone II, atractylodes macrocephala lactone III and β-eucalyptol, the sample volume of the test solution is 20-40 μl; (c) If the sample is an inclusion complex of Atractylodes macrocephala volatile oil or a preparation containing an inclusion complex of Atractylodes macrocephala volatile oil, and the target components are atractylodes macrocephala lactone I, atractylodes macrocephala lactone II, atractylodes macrocephala lactone III and β-eucalyptol, the sample volume of the test solution is 4 to 8 μl. (d) If the sample is Atractylodes macrocephala and the target components are atractylone, geraniol B, and β-elemene, the sample volume of the test solution should be 10-20 μl. (e) If the sample is Atractylodes macrocephala volatile oil, Atractylodes macrocephala volatile oil inclusion complex or preparation containing Atractylodes macrocephala volatile oil inclusion complex, and the target component is atractylone, geraniol B, or β-elemene, the sample volume of the test solution is 2 to 4 μl. (f) The ambient temperature for the deployment is 4–40°C; (g) The ambient humidity for the unfolding process is 33% to 60%.

6. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 5, characterized in that, Includes one or more of the following characteristics: (a1) The spotting volume of atractylodes lactone I reference solution was 4 μl, the spotting volume of atractylodes lactone II reference solution was 4 μl, the spotting volume of atractylodes lactone III reference solution was 4 μl, the spotting volume of β-eucalyptol reference solution was 1 μl, the spotting volume of atractylone reference solution was 4 μl, the spotting volume of geraniol B reference solution was 4 μl, and the spotting volume of β-elemene reference solution was 10 μl. (b1) If the sample is Atractylodes macrocephala, and the target components are atractylodes macrocephala I, atractylodes macrocephala II, atractylodes macrocephala III and β-eucalyptol, the sample volume of the test solution is 20 μl; (b2) If the sample is Atractylodes macrocephala volatile oil and the target components are atractylodes macrocephala lactone I, atractylodes macrocephala lactone II, atractylodes macrocephala lactone III and β-eucalyptol, the sample volume of the test solution is 40 μl; (c1) If the sample is a volatile oil inclusion complex of Atractylodes macrocephala, and the target components are atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eucalyptol, the sample volume of the test solution is 4 μl; (c2) If the sample is a preparation containing Atractylodes macrocephala volatile oil inclusion complex, and the target components are atractylodes macrocephala lactone I, atractylodes macrocephala lactone II, atractylodes macrocephala lactone III and β-eucalyptol, the sample volume of the test solution is 8 μl; (d1) If the sample is Atractylodes macrocephala and the target components are atractylone, geraniol B, and β-elemene, the sample volume of the test solution is 20 μl. (e1) If the sample is Atractylodes macrocephala volatile oil and the target components are atractylone, geraniol B, and β-elemene, the sample volume of the test solution is 2 μl. (e2) If the sample is a volatile oil inclusion complex of Atractylodes macrocephala, and the target components are atractylone, geraniol B, and β-elemene, the sample volume of the test solution is 2 μl. (e3) If the sample is a preparation containing Atractylodes macrocephala volatile oil inclusion complex, and the target components are atractylone, geraniol B, and β-elemene, the sample volume of the test solution is 4 μl.

7. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 1, characterized in that, In step (3), when the target components are atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eucalyptol, the developing agent is selected from petroleum ether-acetone, petroleum ether-ethyl acetate, and n-hexane-acetone; when the target components are atractylone, geraniol B, and β-elemene, the developing agent is selected from petroleum ether or n-hexane.

8. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 7, characterized in that, The volume ratio of petroleum ether to acetone is 9:

1.

9. The thin-layer chromatography identification method for the target component in a *Atractylodes macrocephala* sample as described in claim 1, characterized in that, In step (3), when the target components are atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and β-eucalyptol, the colorimetric reagent is selected from 10% sulfuric acid ethanol solution or 5% p-dimethylaminobenzaldehyde-10% sulfuric acid ethanol solution; when the target components are atractylone, geraniol B, and β-elemene, the colorimetric reagent is selected from 5% p-dimethylaminobenzaldehyde-10% sulfuric acid ethanol solution.

10. The use of the thin-layer chromatography identification method for the target component in Atractylodes macrocephala samples as described in any one of claims 1 to 9 in the quality inspection and quality control of Atractylodes macrocephala products.

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