Use of a developing agent in a thin layer identification method and a thin layer identification method

Abstract

The application provides an application of a developing agent in a thin-layer identification method of Fructus Aurantii Immaturus, medicinal slices, intermediates and finished products containing Fructus Aurantii Immaturus as raw materials, and the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene with a volume ratio of 19:7:2:2-15:5:1:1. According to the chemical structure and properties of each effective component of traditional Chinese medicines, the extraction principle of similar solubility is followed, and a suitable extraction solvent is adopted to simply and quickly prepare a test sample solution and a control medicinal material solution. Then, a suitable developing agent is adopted for development, and various chemical components can be well separated on a thin-layer plate according to the different adsorption, desorption, re-adsorption and re-desorption capacities of the developing agent. Then, various effective components with similar polarities present different color spots under the same viewing conditions on the same thin-layer plate, and a thin-layer chromatogram is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a thin-layer identification method for Citrus aurantium L. and Citrus sinensis Osbeck medicinal materials, decoction pieces, intermediates and finished products containing Citrus aurantium L. and Citrus sinensis Osbeck medicinal ingredients as raw materials. BACKGROUND

[0002] Citrus aurantium L. is the dried young fruit of Citrus aurantium L. and its cultivated varieties or Citrus sinensis Osbeck, which tastes bitter, pungent and sour, is warm in nature, and belongs to the meridians of the spleen and stomach. It has the effects of breaking qi and resolving accumulation, resolving phlegm and dissipating mass. It is used for accumulation and stagnation, distention and pain, post- diarrhea, constipation, phlegm and qi stagnation, chest stuffiness, and prolapse of viscera. The compounds isolated from it mainly include flavonoids, alkaloids, volatile oils and other components. The medicinal material is included in the Chinese Pharmacopoeia, and the method for thin-layer identification in the quality standard is as follows: 0.5 g of powder is added with 10 ml of methanol, ultrasonic treatment is performed for 20 minutes, filtration is performed, the filtrate is evaporated to dryness, the residue is dissolved with 0.5 ml of methanol to obtain a test solution. In addition, 0.5 mg of synephrine reference substance is added with methanol to prepare a solution containing 0.5 mg per 1 ml, which is used as a reference solution. The two solutions are tested by the thin-layer chromatography (general test 0502), 2 μl of each solution is spotted on the same silica gel G thin-layer plate, the upper solution of n-butanol-glacial acetic acid-water (4:1:5) is used as a developing agent, development is performed, the plate is taken out, dried, and sprayed with 0.5% ninhydrin ethanol solution, and heated at 105 DEG C until the spots are clear.

[0003] At present, the commonly used Citrus aurantium L. and Citrus sinensis Osbeck medicinal materials, decoction pieces, intermediates and finished products containing Citrus aurantium L. and Citrus sinensis Osbeck medicinal ingredients as raw materials mostly use reference substances as references for identification, such as synephrine, hesperidin and neohesperidin, and only one identification spot is obtained. In the quality detection of medicinal materials, these methods cannot effectively control the authenticity of the medicinal materials. Some literatures report that the reference medicinal material of Citrus aurantium L. is used as a reference, and the preparation methods of the reference medicinal material solution and the test solution are relatively complicated. Some methods use heating reflux, which can destroy the heat-sensitive components in the test solution and is relatively complicated to operate. Some methods have inconsistent preparation methods of the reference medicinal material solution and the test solution, which increases the workload in the identification process. As for the identification results, the number of identification spots in the reported methods is relatively small.

[0004] Citrus sinensis Osbeck is the dried immature fruit of Citrus aurantium L. and its cultivated varieties. The fruit is harvested when the pericarp is green in July, cut into two halves from the middle, and dried or low-temperature dried.

[0005] The identification method of Fructus Aurantii in Chinese Pharmacopoeia 2020 edition: take 0.2g of the powder, add 10ml of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate to dryness, add 5ml of methanol to dissolve as the test solution. Take naringin reference substance, neohesperidin reference substance, add methanol to prepare a mixed solution containing 0.5mg per 1ml, as the reference solution. Test by thin layer chromatography (general rule 0502), take 10ul of the above test solution and 20ul of the reference solution, respectively, and point on the same silica gel G thin layer plate, with chloroform-methanol-water (13:6:2) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105℃ for about 5 minutes, and observe under ultraviolet light (365nm). In the test chromatogram, the same color fluorescent spots appear at the corresponding positions of the reference chromatogram. This method uses hesperidin and neohesperidin as reference, and the number of identification spots is small; and the preparation method of the test solution is complicated. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide an application of a developing agent in a thin layer identification method of Fructus Aurantii medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii medicinal materials, and a thin layer identification method. The developing agent provided by the present application can separate the sample on the thin layer plate well, and 7 common information spots can be obtained after development.

[0007] The present application provides an application of a developing agent in a thin layer identification method of Fructus Aurantii medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii medicinal materials, wherein the developing agent is selected from a lower layer solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 19:7:2:2 to 15:5:1:1.

[0008] Preferably, the developing agent is selected from a lower layer solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 18:6:1.5:2 to 16:6:1:1.

[0009] Preferably, the developing agent is selected from a lower layer solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 17:6:1.5:2.

[0010] The present application also provides a thin layer identification method of Fructus Aurantii medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii medicinal materials, comprising the following steps:

[0011] A) mixing Fructus Aurantii medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii medicinal materials with methanol to obtain a supernatant to obtain a test solution;

[0012] B) mixing Fructus Aurantii reference medicinal materials with methanol to obtain a supernatant to obtain a reference medicinal material solution;

[0013] B) the test solution and the control medicinal material solution are spotted on the same silica gel G thin layer plate respectively, and developed with a developing agent, dried and colored, and observed under ultraviolet conditions; if the same color spots appear in the test chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified;

[0014] The developing agent is selected from the developing agents used in the above applications.

[0015] The application further provides a thin layer identification method for Fructus Aurantii Immaturus / Fructus Aurantii, medicinal pieces, intermediates and finished products containing Fructus Aurantii Immaturus / Fructus Aurantii as raw materials, comprising the following steps:

[0016] a) mixing Fructus Aurantii Immaturus / Fructus Aurantii medicinal materials, pieces, intermediates and finished products with methanol to obtain a test solution;

[0017] b) mixing Fructus Aurantii Immaturus control medicinal material with methanol to obtain a control medicinal material solution;

[0018] b) the test solution and the control medicinal material solution are spotted on the same silica gel G thin layer plate respectively, and developed with a developing agent, dried and colored, and observed under ultraviolet conditions; if the same color spots appear in the test chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified;

[0019] The developing agent is selected from the developing agents used in the above applications.

[0020] Preferably, the mixing is ultrasonic mixing.

[0021] Preferably, the coloring method is as follows:

[0022] The silica gel G thin layer plate is sprayed with an aluminum chloride ethanol test solution and then heated.

[0023] Preferably, the heating temperature is 105℃, and the heating time is 4-6 min.

[0024] Preferably, the ultraviolet conditions are that the ultraviolet wavelength is 365 nm.

[0025] Compared with the prior art, the application provides an application of a developing agent in a thin layer identification method of Fructus Aurantii Immaturi / Citri Fructus medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii Immaturi / Citri Fructus medicinal materials as raw materials, wherein the developing agent is a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 19:7:2:2 to 15:5:1:1. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A thin layer TLC diagram obtained by the method of Comparative Example 1;

[0027] Figure 2 A thin layer TLC diagram obtained by the method of Comparative Example 2;

[0028] Figure 3 A thin layer TLC diagram obtained by the method of Comparative Example 3;

[0029] Figure 4 A thin layer TLC diagram obtained by the method of Comparative Example 4;

[0030] Figure 5 A thin layer TLC diagram obtained by the method of Comparative Example 5;

[0031] Figure 6 A thin layer TLC diagram obtained by the method of Comparative Example 6;

[0032] Figure 7 A thin layer TLC diagram obtained by the method of Comparative Example 7;

[0033] Figure 8 A thin layer TLC diagram obtained by the method of Example 1;

[0034] Figure 9 A thin layer TLC diagram obtained by the method of Example 2;

[0035] Figure 10 A thin layer TLC diagram obtained by the method of Example 2;

[0036] Figure 11 A thin layer TLC diagram obtained by the method of Example 2;

[0037] Figure 12Thin layer TLC chromatogram obtained by the method of Example 2;

[0038] Figure 13 Thin layer TLC chromatogram obtained by the method of Example 2;

[0039] Figure 14 Thin layer TLC chromatogram obtained by the method of Example 2;

[0040] Figure 15 Thin layer TLC chromatogram obtained by the method of Example 3. DETAILED DESCRIPTION

[0041] The present application provides an application of a developing agent in a thin layer identification method of Fructus Aurantii Immaturi, the developing agent being selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 19:7:2:2 to 15:5:1:1.

[0042] Further, in some embodiments of the present application, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 18:6:1.5:2 to 16:6:1:1.

[0043] Further, in some embodiments of the present application, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 17:6:1.5:2;

[0044] Alternatively, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 18:6:1.5:2;

[0045] Alternatively, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 16:6:1.5:2;

[0046] Alternatively, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 17:6:1.5:1;

[0047] Alternatively, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 17:6:1:1;

[0048] Alternatively, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 17:6:1:2.

[0049] Most preferably, the developing agent is selected from a lower solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 17:6:1.5:2.

[0050] The toluene itself belongs to a toxic solvent, but when no toluene is added in the developing agent, the number of identifiable spots is small and the resolution is poor; however, when too much toluene is added, the separation effect is poor and the environment is more seriously polluted. The developing agent provided in the application can increase the number of identifiable information spots and has better resolution under the premise of small amount of toluene, and the pollution to the environment is small, thus unexpected technical effects are generated.

[0051] The application further provides a thin layer identification method for Citrus aurantium L. medicinal materials, decoction pieces, intermediates and finished products containing Citrus aurantium L. medicinal materials, which comprises the following steps:

[0052] A) mixing the Citrus aurantium L. medicinal materials, decoction pieces, intermediates and finished products containing Citrus aurantium L. medicinal materials with methanol to obtain a supernatant to obtain a test sample solution;

[0053] B) mixing the test sample solution and the control medicinal material solution on the same silica gel G thin layer plate respectively, developing with a developing agent, drying and developing color, and observing under ultraviolet conditions.

[0054] B) mixing the test sample solution and the control medicinal material solution on the same silica gel G thin layer plate respectively, developing with a developing agent, drying and developing color, and observing under ultraviolet conditions.

[0055] The test sample solution is Citrus aurantium L. medicinal materials, decoction pieces, intermediates and finished products containing Citrus aurantium L. medicinal materials. The Citrus aurantium L. is the dried young fruit of Citrus aurantium L. and its cultivated varieties or Citrus sinensis Osbeck. The fallen fruits are collected from May to June, impurities are removed, the middle part is cut into two halves, and the smaller ones are directly dried. In the application, the Citrus aurantium L. is selected from the dried young fruit of Citrus aurantium L.

[0056] The mass-volume ratio of the test sample to methanol is preferably 1g:(15-25)ml, preferably 1g:15ml, 1g:20ml, 1g:25ml, or any value between 1g:(15-25)ml.

[0057] The mass-volume ratio of the control medicinal material to methanol in the control medicinal material solution is preferably 1g:(15-25)ml, preferably 1g:15ml, 1g:20ml, 1g:25ml, or any value between 1g:(15-25)ml.

[0058] When the test sample solution and the control medicinal material solution are prepared, the mixing is preferably ultrasonic mixing to promote the dissolution of chemical components in the medicinal materials.

[0059] Then, the mixed solution is solid-liquid separated. The method for the solid-liquid separation is not particularly limited in the present application, and the solid-liquid separation method known to those skilled in the art can be used. In the present application, centrifugation or filtration can be used.

[0060] The test sample and the solution are spotted on the same silica gel G thin layer plate, developed with a developing agent, dried and colored, and observed under ultraviolet conditions. If the same color spots appear in the test sample chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified.

[0061] The coloring method is as follows:

[0062] The silica gel G thin layer plate is sprayed with an aluminum chloride ethanol test solution and then heated.

[0063] The aluminum chloride ethanol test solution is prepared according to the aluminum chloride test solution in 8002 test solution of the Chinese Pharmacopoeia 2020 edition.

[0064] The heating temperature is 105 DEG C, and the heating time is 4-6 min, preferably 4, 5, 6, or any value between 4 and 6 min.

[0065] The ultraviolet condition is that the ultraviolet wavelength is 365 nm.

[0066] The present application also provides a thin layer identification method for Fructus Aurantii medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii medicinal materials as raw materials, comprising the following steps:

[0067] a) mixing Fructus Aurantii medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii medicinal materials as raw materials with methanol to obtain a test sample solution;

[0068] b) mixing Fructus Aurantii medicinal materials with methanol to obtain a control medicinal material solution;

[0069] b) mixing Fructus Aurantii medicinal materials with methanol to obtain a control medicinal material solution;

[0070] The test sample is Fructus Aurantii medicinal materials, decoction pieces, intermediates and finished products containing Fructus Aurantii medicinal materials as raw materials. Fructus Aurantii is the dried immature fruit of Citrus aurantium L. and its cultivated varieties of Rutaceae. The fruit is harvested in July when the peel is still green, cut into two halves from the middle, dried or dried at low temperature.

[0071] The mass-volume ratio of the test product to methanol is preferably 1g:(15-25)ml, preferably 1g:15ml, 1g:20ml, 1g:25ml, or any value between 1g:(15-25)ml.

[0072] The mass-volume ratio of the test product to methanol is preferably 1g:(15-25)ml, preferably 1g:15ml, 1g:20ml, 1g:25ml, or any value between 1g:(15-25)ml.

[0073] When preparing the test product solution and the control medicinal material solution, the mixing is preferably ultrasonic mixing to facilitate the dissolution of chemical components in the medicinal material.

[0074] Then, the mixture is solid-liquid separated. The method of solid-liquid separation in the present application is not particularly limited, and the method of solid-liquid separation known to those skilled in the art can be used. In the present application, centrifugation or filtration can be used.

[0075] The test product solution and the control medicinal material solution are spotted on the same silica gel G thin layer plate, developed with a developing agent, dried and colored, and then observed under ultraviolet conditions. If the same color spots appear in the test product chromatogram at the positions corresponding to the control medicinal material chromatogram, the sample is qualified.

[0076] The coloring method is as follows:

[0077] The silica gel G thin layer plate is sprayed with an aluminum chloride ethanol test solution and then heated.

[0078] The aluminum chloride ethanol test solution is prepared according to the aluminum chloride test solution in 8002 test solution of Chinese Pharmacopoeia 2020 edition.

[0079] The heating temperature is 105℃, and the heating time is 4-6min, preferably 4, 5, 6, or any value between 4-6min.

[0080] The ultraviolet condition is that the wavelength of ultraviolet light is 365nm.

[0081] The present application provides a method for identifying the medicinal materials, decoction pieces, intermediates and finished products containing the medicinal materials of Citrus aurantium L. and Fructus Aurantii by thin layer chromatography. The test sample and medicinal material solution are obtained by simple and convenient pretreatment method, and are spotted on the same thin layer plate, and then are observed under 365 nm after development. Seven common information spots of the medicinal materials, decoction pieces, intermediates and finished products containing the medicinal materials of Citrus aurantium L. and Fructus Aurantii are observed, which are all fluorescence spots excited by heating after adding aluminum chloride ethanol reagent. Compared with the existing thin layer identification method of the medicinal materials, decoction pieces, intermediates and finished products containing the medicinal materials of Citrus aurantium L. and Fructus Aurantii, the quality controllability of the identified substances is greatly improved, which is beneficial to quality supervision, and the method is simple, fast, efficient and low in cost.

[0082] According to the chemical structure and properties of each effective component of traditional Chinese medicine, the extraction principle of similar solubility is followed, and the suitable extraction solvent is used to simply and quickly prepare the test sample and the control medicinal material solution. Then, the suitable developing agent is used for development, and various chemical components can be well separated on the thin layer plate according to the different adsorption, desorption, re-adsorption and re-desorption capacities of the selected developing agent. Then, various effective components with similar polarity can present different color spots on the same thin layer plate under the same observation condition, and the thin layer chromatogram is obtained.

[0083] The beneficial effects are as follows:

[0084] 1. The present application uses the control medicinal material of Citrus aurantium L. as reference, compared with the single control as reference, the control medicinal material of Citrus aurantium L. can identify more spots in the same thin layer chromatography, and the present application can also be used for identifying the authenticity of medicinal materials and preventing the addition of synephrine control to unqualified Citrus aurantium L. medicinal materials to make them qualified. Compared with the control medicinal material of Citrus aurantium L. as reference in other literature reports, the preparation method of the test sample solution and the control medicinal material solution in the present application uses methanol ultrasonic, which has less effect on the chemical components in the sample than heating reflux, and the ultrasonic extraction efficiency is similar to that of heating reflux to a certain extent, but the heating reflux is more complex, and the ultrasonic treatment is more convenient. The present application only adds 10 ml of methanol for ultrasonic treatment for 20 min to obtain the supernatant, which is convenient in operation steps, short in time, high in efficiency and low in cost.

[0085] 2. The test solution in the application is the medicinal material, decoction piece, intermediate and finished product containing the medicinal material of Citrus aurantium L. and the main component to be detected is Citrus aurantium L. The main components in Citrus aurantium L. are flavonoids, alkaloids and volatile oil, among which the content of flavonoids is the highest. The flavonoids have relatively large polarity. The main components in Citrus aurantium L. include volatile oil, flavonoid glycosides and alkaloids. According to the principle of similar dissolves similar, the developer with relatively large polarity and the color developing agent of aluminum chloride ethanol solution for the development of flavonoids are selected. It is found through the previous experiment that when the developer system is chloroform-methanol-water, some spots in Citrus aurantium L. can be identified, but all the spots cannot be displayed in the same chromatogram by adjusting the proportion. Therefore, another organic solvent is selected to adjust the polarity of the developer. Through the comparison test, it is found that after the addition of toluene based on chloroform-methanol-water, the polarity of the developer is optimal and the spots are well separated. Each identifiable spot is not coincident and is separated from the origin. In the developer system, toluene is easily soluble in chloroform and methanol, slightly soluble in water and has smaller polarity than water, chloroform and methanol. After the addition of toluene, the polarity of the whole solvent is reduced, the spots originally developed to the front are separated from the front, and more spots are displayed in the range of Rf 0.2-0.9. In the developer provided by the application, the number of identifiable information spots is increased under the premise of small amount of toluene, the separation degree is better, the pollution to the environment is small, and unexpected technical effects are achieved.

[0086] 3. The lower solution of chloroform-methanol-water-toluene with the volume ratio of 17:6:1.5:2 is used as the developer to develop the medicinal material, decoction piece, intermediate and finished product containing the medicinal material of Citrus aurantium L. and Citrus aurantium L. After the spraying of aluminum chloride ethanol solution, the flavonoid components are excited to fluorescence, and under the ultraviolet 365 nm, the test sample and the control medicinal material both present seven fluorescent spots. Without any impurity removal process, the test sample is directly ultrasonically treated with methanol, and under the condition of one-time development, seven information spots are obtained by using one viewing condition, which are consistent with the thin layer chromatogram of the control medicinal material.

[0087] 4. The method has strong specificity for the identification of the medicinal material of Citrus aurantium L. and can be used for the medicinal material, decoction piece, intermediate and finished product containing the medicinal material of Citrus aurantium L. and Citrus aurantium L. In the specificity experiment, the negative sample does not interfere with the test solution.

[0088] 5. The identification method only needs the control medicinal material of Citrus aurantium L., the medicinal material, decoction piece, intermediate and finished product containing the medicinal material of Citrus aurantium L. and Citrus aurantium L. and methanol with the mass-volume ratio of 1 g:(15-25) ml, and the time is 20-30 minutes. The test sample and the control medicinal material solution can be obtained. After the addition of the developer, the development is carried out for 45-60 minutes, and the quality of the sample can be evaluated by multiple indexes. The method is simple, convenient and has high practical value.

[0089] In order to further understand the present application, the thin layer identification method of the Fructus Aurantii / Fructus Aurantii medicine, decoction pieces, intermediates and finished products containing Fructus Aurantii / Fructus Aurantii medicine are described below in combination with the comparative examples and examples, and the protection scope of the present application is not limited by the following comparative examples and examples.

[0090] In the following comparative examples and examples, the specific sources of the samples are as follows:

[0091] ①The Fructus Aurantii control medicine was purchased from China Institute for Food and Drug Control, batch number: 120936-201606;

[0092] ②Fructus Aurantii medicines 1-4 were the same batch of samples, which were purchased from Xingan County, Ji'an City, Jiangxi Province;

[0093] ③Fructus Aurantii Xiebai Guizhi Decoction samples 1-6 were laboratory-made Fructus Aurantii Xiebai Guizhi Decoction; the specific preparation method is as follows:

[0094] Preparation method: take Fructus Aurantii 9g, Magnolia officinalis 12g, soak in 1000ml water for 45min, boil with strong fire and then decoct with gentle fire for 30min, filter, add Fructus Trichosanthis 24g, Allium bakeri 25g, Cinnamomum cassia 3.1g (soak the three medicines in 1.1 times the amount of water for 45min), boil with strong fire and then decoct with gentle fire for 15min, filter to obtain Fructus Aurantii Xiebai Guizhi Decoction. According to the above preparation method, 6 samples were prepared.

[0095] ④Fructus Aurantii Xiebai Guizhi Decoction preparation samples 1-3: granule samples 1-3 prepared by one-step granulator after concentration of the above Fructus Aurantii Xiebai Guizhi Decoction samples 1-3;

[0096] ⑤Negative sample: lack of Fructus Aurantii medicine, decoct the obtained decoction according to the above Fructus Aurantii Xiebai Guizhi Decoction decoction process;

[0097] ⑥The Citrus reticulata control medicine was purchased from China Institute for Food and Drug Control, batch number: 120969-201109;

[0098] ⑦Naringin control was purchased from China Institute for Food and Drug Control, batch number: 110722-201111;

[0099] ⑧Weiguihe Capsules, Xiangshayangwei Pills (Wanxi Pharmaceutical), Weisu Granules and Huaijiao Pills were all purchased from Keiyi Pharmacy in Pidu District, Chengdu City, and the specific ingredients are shown in the following table:

[0100] Table 1

[0101]

[0102]

[0103] 8 Synephrine reference substance was purchased from China Institute for Drug Control, batch number: 110727-201809.

[0104] Comparative Example 1

[0105] 1) Test sample: Citrus aurantium L. reference material, Citrus aurantium L. Xiebai Guizhi Decoction sample 1

[0106] 2) Treatment method:

[0107] Precisely take 10 ml of Citrus aurantium L. Xiebai Guizhi Decoction sample 1, evaporate to dryness, add 5 ml of methanol to dissolve, and take the supernatant to obtain the test solution.

[0108] Add 0.5 g of Citrus aurantium L. reference material to 10 ml of methanol, ultrasonic for 20 min, take the supernatant to obtain the reference material solution.

[0109] 3) Test scheme

[0110] According to the thin layer chromatography method (General Test 0502 of Chinese Pharmacopoeia), take 2 μl of the above two solutions respectively, and point them on the same silica gel G thin layer plate. Use chloroform-methanol-water (13:6:2) lower layer solution as developing agent, develop, take out, dry, spray with aluminum chloride test solution (prepared according to the aluminum chloride test solution in General Test 8002 of Chinese Pharmacopoeia 2020 edition), heat at 105℃ for 5 min, and observe under ultraviolet 365 nm. The results are shown in Figure 1 . Figure 1 From left to right in Figure 1 are: Citrus aurantium L. reference material, Citrus aurantium L. Xiebai Guizhi Decoction sample 1. As can be seen from Figure 1 , the number of identifiable spots is small, and the spot separation degree is low.

[0111] Comparative Example 2

[0112] Test sample: Citrus aurantium L. reference material, Citrus aurantium L. Xiebai Guizhi Decoction sample 1, negative sample;

[0113] Among them, the treatment method of Citrus aurantium L. reference material and Citrus aurantium L. Xiebai Guizhi Decoction is the same as Comparative Example 1,

[0114] The treatment method of the negative sample is: the negative sample is 10 ml, evaporated to dryness, added with 5 ml of methanol to dissolve, and the supernatant was taken to obtain the test solution.

[0115] According to the method of Comparative Example 1, the developing agent is chloroform-methanol-water (18:6:2) lower layer solution.

[0116] The results are shown in Figure 2 . Figure 2 From left to right in Figure 2 are: Citrus aurantium L. reference material, Citrus aurantium L. Xiebai Guizhi Decoction sample 1, negative sample. As can be seen from Figure 2 , the number of identifiable spots is small.

[0117] Comparative Example 3

[0118] The test samples were: Citrus aurantium L. control medicinal material, Xiebai Guizhi Tang sample 1, Xiebai Guizhi Tang preparation sample 1, and negative sample.

[0119] The processing method of the Citrus aurantium L. control medicinal material, negative sample, and Xiebai Guizhi Tang sample 1 was as described in Comparative Example 1.

[0120] The processing method of the Xiebai Guizhi Tang preparation sample 1 was as follows: 0.5 g of Xiebai Guizhi Tang preparation sample 1 was added to 10 ml of methanol, and ultrasonic treatment was performed for 20 minutes. The supernatant was obtained to obtain the sample solution.

[0121] Test scheme: according to the method of Comparative Example 1, the developing agent was chloroform-methanol-water-acetone (19:6:2:1) lower solution, and the results are shown in Table 2. Figure 3 . Figure 3 In Table 2, from left to right were Citrus aurantium L. control medicinal material, Xiebai Guizhi Tang sample 1, Xiebai Guizhi Tang preparation sample 1, and negative sample. It can be seen from Table 2 that the number of identifiable spots was small, and the spot separation degree was low. Figure 3

[0122] Comparative Example 4

[0123] The test samples were the same as in Comparative Example 3, i.e., Citrus aurantium L. control medicinal material, Xiebai Guizhi Tang sample 1, Xiebai Guizhi Tang preparation sample 1, and negative sample.

[0124] The sample processing method is described in Comparative Example 3.

[0125] Test scheme: according to the method of Comparative Example 1, the developing agent was chloroform-methanol-water-n-hexane (18:6:2:2) lower solution, and the results are shown in Table 3. Figure 4 . Figure 4 In Table 3, from left to right were Citrus aurantium L. control medicinal material, Xiebai Guizhi Tang sample 1, Xiebai Guizhi Tang preparation sample 1, and negative sample. It can be seen from Table 3 that the polarity of the developing agent was small, some spots did not spread from the origin, and the number of identifiable spots was small. Figure 4

[0126] Comparative Example 5

[0127] The test samples were: synephrine reference substance, Citrus aurantium L. medicinal material 1, Citrus aurantium L. medicinal material 2, Citrus aurantium L. medicinal material 3, and Citrus aurantium L. medicinal material 4. Citrus aurantium L. medicinal material 1, Citrus aurantium L. medicinal material 2, Citrus aurantium L. medicinal material 3, and Citrus aurantium L. medicinal material 4 were all medicinal material powders.

[0128] Sample processing method: 0.5 g of each of Citrus aurantium L. medicinal material 1, Citrus aurantium L. medicinal material 2, Citrus aurantium L. medicinal material 3, and Citrus aurantium L. medicinal material 4 was added to 10 ml of methanol, and ultrasonic treatment was performed for 20 minutes. The filtrate was evaporated to dryness, 0.5 ml of methanol was added to dissolve the residue, and the resulting solution was used as the test sample solution 1-4.

[0129] ​​Take synephrine reference substance, add methanol to prepare a solution containing 0.5 mg per 1 ml as a reference solution.

[0130] Test scheme: The chromatogram (developing agent: n-butanol-glacial acetic acid-water (4:1:5) upper solution) of the method in Chinese Pharmacopoeia 2020 edition, 2 μl of synephrine reference substance solution, Citrus aurantium L. medicinal material 1, Citrus aurantium L. medicinal material 2, Citrus aurantium L. medicinal material 3, Citrus aurantium L. medicinal material 4, and 1-4 of the test sample solution were spotted on the same silica gel G thin layer plate, and developed with the above developing agent, taken out, dried, sprayed with 0.5% ninhydrin ethanol solution, heated at 105°C until the spots were clear, and the thin layer TLC diagram under daylight, the results are shown in Figure 5 . Figure 5 From left to right in Figure 6 are Citrus aurantium L. reference medicinal material, Citrus aurantium L. medicinal material 1, Citrus aurantium L. medicinal material 2, Citrus aurantium L. medicinal material 3, and Citrus aurantium L. medicinal material 4. Figure 5 It can be seen from

[0131] Comparative Example 6

[0132] Test samples: Citrus aurantium L. reference medicinal material, Citrus aurantium L. Xiebai Guizhi Decoction sample 1, negative sample;

[0133] The sample treatment method is the same as that of Comparative Example 2;

[0134] The test scheme is according to the method of Comparative Example 1; the type of developing agent is changed.

[0135] The developing agent is a mixed solution of ethyl acetate, methanol and water with a volume ratio of 100:17:13, and the results are shown in Figure 6 . Figure 6 From left to right in Figure 6 are Citrus aurantium L. reference medicinal material, Citrus aurantium L. Xiebai Guizhi Decoction sample 1, and negative sample.

[0136] It can be seen from Figure 6 that the developing agent of the method provided in Comparative Example 6 is too large in polarity, resulting in a large Rf value of the spot and poor separation degree of the spot.

[0137] Comparative Example 7

[0138] Test samples: Citrus aurantium L. reference medicinal material, Citrus aurantium L. medicinal material 1, Citrus aurantium L. Xiebai Guizhi Decoction preparation sample 1, Citrus aurantium L. Xiebai Guizhi Decoction preparation sample 2, and Citrus aurantium L. Xiebai Guizhi Decoction preparation sample 3;

[0139] The sample treatment method is the same as that of Comparative Example 3;

[0140] The test scheme is according to the method of Comparative Example 1; the type of developing agent is changed.

[0141] The developing agent is a mixed solution of chloroform, methanol, water and toluene with a volume ratio of 17:6:1.5:20, and the results are shown in Figure 7 .Figure 7 From left to right in turn are: reference material of Fructus Aurantii, Fructus Aurantii 1, decoction preparation sample 1 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, decoction preparation sample 2 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, decoction preparation sample 3 of Fructus Aurantii, Baizhi, Xiebai and Guizhi.

[0142] It can be seen that the developing agent of the method provided by Comparative Example 7 is too small in polarity, and part of the spots are not developed away from the origin. There are only 4 spots of the same color in the same position that are shown by the reference material and the preparation sample, and two spots are not separated. Figure 7 Example 1

[0143] The test samples are: decoction samples 1-6 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, and reference material of Fructus Aurantii.

[0144] The sample processing method is as described in Comparative Example 1.

[0145] The test scheme is as follows: according to the method of Comparative Example 1, the developing agent is the lower solution of chloroform-methanol-water-toluene (17:6:1.5:2), and the results are shown in Table 1.

[0146] .In Table 1, from left to right in turn are: decoction samples 1-3 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, reference material of Fructus Aurantii, decoction samples 4-6 of Fructus Aurantii, Baizhi, Xiebai and Guizhi. It can be seen that the 7 identifiable spots are well separated; all the spots are developed away from the origin, and there are 7 spots of the same color in the same position that are shown by the reference material and the decoction samples, and the separation degree is good; the Rf values from bottom to top are: 0.21, 0.32, 0.57, 0.61, 0.78, 0.82, 0.90. Figure 8 Figure 8 Example 2 Figure 8 The test samples are: reference material of Fructus Aurantii, decoction sample 1 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, decoction preparation sample 1 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, and negative sample.

[0147] The processing method is the same as that of Comparative Example 3

[0148] The test scheme is as follows: according to the method of Comparative Example 1,

[0149] The developing agents are as follows:

[0150] (1) the lower solution of chloroform-methanol-water-toluene (18:6:1.5:2), and the results are shown in Table 2.

[0151] .In Table 2, from left to right in turn are: reference material of Fructus Aurantii, decoction sample 1 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, decoction preparation sample 1 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, and negative sample. It can be seen that the separation degree of the spots is good, there are 7 identifiable spots, and the negative sample does not interfere.

[0152] Figure 9 Figure 9 In Table 3, from left to right in turn are: reference material of Fructus Aurantii, decoction sample 1 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, decoction preparation sample 1 of Fructus Aurantii, Baizhi, Xiebai and Guizhi, and negative sample. It can be seen that the separation degree of the spots is good, there are 7 identifiable spots, and the negative sample does not interfere. Figure 9

[0153] ​​(2) The lower layer solution of chloroform - methanol - water - toluene (16:6:1.5:2), and the results are shown in Figure 10 . Figure 10 Among them, from left to right are: the control medicinal material of Fructus Aurantii Immaturus, sample 1 of Zhishi Xiebai Guizhi Decoction, preparation sample 1 of Zhishi Xiebai Guizhi Decoction, and negative sample; from Figure 10 it can be seen that there are 7 distinguishable spots, the spots are basically separated, and the negative has no interference.

[0154] (3) The lower layer solution of chloroform - methanol - water - toluene (17:6:1.5:1), and the results are shown in Figure 11 . Figure 11 Among them, from left to right are: the control medicinal material of Fructus Aurantii Immaturus, sample 1 of Zhishi Xiebai Guizhi Decoction, preparation sample 1 of Zhishi Xiebai Guizhi Decoction, and negative sample; from Figure 11 it can be seen that there are 7 distinguishable spots, the spots are basically separated, and the negative has no interference.

[0155] (4) The lower layer solution of chloroform - methanol - water - toluene (17:6:1.5:2), and the results are shown in Figure 12 . Figure 12 Among them, from left to right are: the control medicinal material of Fructus Aurantii Immaturus, sample 1 of Zhishi Xiebai Guizhi Decoction, preparation sample 1 of Zhishi Xiebai Guizhi Decoction, and negative sample; from Figure 12 it can be seen that the resolution of the spots is good, there are 7 distinguishable spots, the negative has no interference, and the spots identified in the decoction sample and the preparation sample are consistent.

[0156] (5) The lower layer solution of chloroform - methanol - water - toluene (17:6:1:1), and the results are shown in Figure 13 . Figure 13 Among them, from left to right are: the control medicinal material of Fructus Aurantii Immaturus, sample 1 of Zhishi Xiebai Guizhi Decoction, preparation sample 1 of Zhishi Xiebai Guizhi Decoction, and negative sample; from Figure 13 it can be seen that there are 7 distinguishable spots, the spots are basically separated, and the negative has no interference.

[0157] (6) The lower layer solution of chloroform - methanol - water - toluene (17:6:1:2), and the results are shown in Figure 14 . Figure 14 Among them, from left to right are: the control medicinal material of Fructus Aurantii Immaturus, sample 1 of Zhishi Xiebai Guizhi Decoction, preparation sample 1 of Zhishi Xiebai Guizhi Decoction, and negative sample. From Figure 14 it can be seen that there are 7 distinguishable spots, the spots are basically separated, and the negative has no interference.

[0158] Example 3

[0159] 1. Preparation of sample solution

[0160] Control medicinal material solution: Precisely weigh 0.5 g of the control medicinal material of Fructus Aurantii Immaturus, add 10 ml of methanol, ultrasonically treat for 20 min, filter, and take the continuous filtrate to obtain.

[0161] The test sample is: Zhishixieba Guizhi Decoction Sample 1, Weisu Granules, Weitai and Capsules, Huaijiao Pills, Xiangshayangwei Pills (manufactured by Wanxi Pharmaceutical)

[0162] Precisely take 10 ml of Zhishixieba Guizhi Decoction Sample 1, evaporate to dryness, add 5 ml of methanol to dissolve, and take the supernatant to obtain;

[0163] The test sample solution: precisely take 1 g of the above Chinese medicine, add 10 ml of methanol, ultrasonically treat for 20 min, filter, and take the filtrate to obtain.

[0164] 2. Determination method

[0165] According to the thin layer chromatography method (General Rule 0502 of Chinese Pharmacopoeia), take 2 μl of the above solution respectively, and point on the same silica gel G thin layer plate. Take the lower layer solution of chloroform-methanol-water-toluene (17:6:1.5:2) as the developing agent, develop, take out, dry, spray with aluminum chloride ethanol solution, heat at 105°C for 5 min, and observe under ultraviolet 365 nm. In the test sample chromatogram, the same color spots appear at the positions corresponding to the control medicinal material chromatogram.

[0166] The experimental results are shown in Table 1. Figure 15 .The left graph of FIG. 1 is the original test graph, and the right graph is the graph after marking on the original test graph. Figure 15

[0167] Figure 15 In FIGS. 1 and 2, from left to right, they are: Zhishi control medicinal material, Zhishixieba Guizhi Decoction Sample 1, Weisu Granules, Weitai and Capsules, Huaijiao Pills, and Xiangshayangwei Pills (manufactured by Wanxi Pharmaceutical)

[0168] The results show that: Zhishixieba Guizhi Decoction Sample 1, Weisu Granules, and Huaijiao Pills show 7 same color spots at the same position in the chromatogram of the Zhishi control medicinal material solution; Weitai and Capsules show 5 same color spots at the same position in the chromatogram of the Zhishi control medicinal material solution; and Xiangshayangwei Pills (manufactured by Wanxi Pharmaceutical) show 6 same color spots at the same position in the chromatogram of the Zhishi control medicinal material solution. This experiment shows that this method can identify Zhishi or Zhikui medicinal materials in most Chinese medicines on the market through multiple characteristic spots.

[0169] The above is only the preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. The application of an unfolding agent in the TLC identification method of Fructus Aurantii Immaturus, its medicinal slices, intermediates and finished products containing Fructus Aurantii Immaturus as raw materials, characterized in that, The developing agent is selected from the lower layer solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 19:7:2:2~15:5:1:1, and the Citrus aurantium fruit-containing medicinal flavor is at least one of the intermediate and finished product selected from Weisu granules, Weitai and capsule, Huaijiao pill, Xiangsha Yangwei pill, and Citrus aurantium fruit Xiebai Guizhi decoction.

2. Use according to claim 1, characterized in that, The developing agent is selected from the lower layer solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 18:6:1.5:2~16:6:1:

1.

3. Use according to claim 1, characterized in that, The developing agent is selected from the lower layer solution of a mixed solution of chloroform, methanol, water and toluene in a volume ratio of 17:6:1.5:

2.

4. A method for identifying the medicinal material, decoction piece, intermediate and finished product containing the medicinal material of Citrus aurantium by thin layer chromatography, characterized in that, The method comprises the following steps: A) mixing the Citrus aurantium fruit medicinal material, decoction piece, intermediate and finished product containing the Citrus aurantium fruit medicinal flavor with methanol to obtain a supernatant, thereby obtaining a test sample solution; the intermediate and finished product containing the Citrus aurantium fruit medicinal flavor is at least one of Weitai and capsule, Xiangsha Yangwei pill, and Citrus aurantium fruit Xiebai Guizhi decoction; mixing the Citrus aurantium fruit control medicinal material with methanol to obtain a supernatant, thereby obtaining a control medicinal material solution; B) spotting the test sample solution and the control medicinal material solution on the same silica gel G thin layer plate respectively, developing with a developing agent, drying, coloring, and observing under ultraviolet conditions; if the same color spots appear in the test sample chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified; the developing agent is the developing agent used in the application of any one of claims 1-3; the coloring method is: spraying aluminum chloride ethanol reagent solution on the silica gel G thin layer plate and then heating.

5. A method for identifying the medicinal material, decoction piece, intermediate and finished product containing the medicinal material of Fructus Aurantii by thin layer chromatography, characterized in that, The method comprises the following steps: a) mixing the Citrus aurantium fruit medicinal material, decoction piece, intermediate and finished product containing the Citrus aurantium fruit medicinal flavor with methanol to obtain a supernatant, thereby obtaining a test sample solution; the intermediate and finished product containing the Citrus aurantium fruit medicinal flavor is at least one of Weitai and capsule, Xiangsha Yangwei pill, and Citrus aurantium fruit Xiebai Guizhi decoction; mixing the Citrus aurantium fruit control medicinal material with methanol to obtain a supernatant, thereby obtaining a control medicinal material solution; b) spotting the test sample solution and the control medicinal material solution on the same silica gel G thin layer plate respectively, developing with a developing agent, drying, coloring, and observing under ultraviolet conditions; if the same color spots appear in the test sample chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified; the developing agent is the developing agent used in the application of any one of claims 1-3; the coloring method is: spraying aluminum chloride ethanol reagent solution on the silica gel G thin layer plate and then heating.

6. The thin layer discrimination method according to claim 4 or 5, characterized in that, The mixing is ultrasonic mixing.

7. The thin layer discrimination method according to claim 4 or 5, characterized by, The heating temperature is 105°C, and the heating time is 4-6 min.

8. The thin layer discrimination method according to claim 4 or 5, characterized by, The ultraviolet condition is that the ultraviolet wavelength is 365 nm.

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