Thin-layer chromatography for identification of Cinnamomum burmannii formula granules and standard decoction

Abstract

The present application relates to the technical field of pharmaceutical analysis, and in particular to a thin-layer identification method for Qingxiangteng formula granules and standard decoction, which comprises the following steps: subjecting test sample solution and control medicinal material solution to thin-layer chromatography analysis, and comparing the obtained chromatograms, wherein the developing agent is a mixed solution of chloroform, methanol and formic acid in a volume ratio of (5-7):(0.5-2):(0.4-0.6). The present application is simple to operate, free of negative interference, and clear in color development, and is specific and durable, thus being an improvement on the existing Qingxiangteng preparation identification method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a thin-layer identification method of formula granules and standard decoction of jasminum lanceolaria roxburgh. BACKGROUND

[0002] The jasminum lanceolaria roxburgh is a dry vine stem of jasminum lanceolaria roxburgh of Oleaceae, and the whole plant can be used as medicine. The medicinal material name is jasminum lanceolaria roxburgh (osteoclast wind), and the medicinal part of jasminum lanceolaria roxburgh in Hunan Province Chinese medicinal material standard and Chongqing City Chinese medicinal material quality standard is dry vine stem, and the medicinal part of jasminum lanceolaria roxburgh in Guangxi Chinese medicinal material standard is dry whole plant. It is widely distributed in mountain slope, shrub, valley dense forest below 2200 meters in Anhui, Taiwan, Fujian, Jiangxi, Hubei, Hunan, Hainan, Guangdong, Guangxi, Guizhou, Sichuan and Yunnan. It has the effects of expelling wind and removing dampness, cooling blood and detoxifying, and is often used for rheumatic arthralgia, contusion and injury, unknown sore and gynecological inflammation; it is also used for treating fever, rheumatic pain, inflammation, eye pain and the like in folk.

[0003] Thin-layer chromatography is a kind of chromatography, which is a very important experimental technology for rapid separation and qualitative analysis of a small amount of substance. It has low cost, low requirement for sample pretreatment, and wide selection range of developing agent, so it plays an important role in the qualitative analysis of drugs. For example, the 2009 edition of Hunan Province Chinese medicinal material standard records that jasminum lanceolaria roxburgh is identified by thin-layer chromatography with petroleum ether (30-60℃)-ethyl acetate (18:4) as developing agent.

[0004] However, the formula granules of jasminum lanceolaria roxburgh are prepared by water extraction, concentration, drying and granulation of single herb medicine decoction pieces, and the standard decoction is prepared by standardizing process. Compared with medicinal materials and decoction pieces, formula granules and standard decoction have lost their inherent form, and the corresponding active ingredients have also changed accordingly. For example, many water-insoluble compounds in decoction pieces are not transferred or only a small amount is transferred. Therefore, the thin-layer identification analysis of medicinal materials or decoction pieces cannot reflect the internal quality, and there is no thin-layer detection technology for formula granules or standard decoction of jasminum lanceolaria roxburgh. Therefore, it is necessary to establish a thin-layer identification method for formula granules or standard decoction of jasminum lanceolaria roxburgh, which can provide a basis for effectively controlling and more comprehensively evaluating the quality of formula granules or standard decoction of jasminum lanceolaria roxburgh. SUMMARY

[0005] The present application aims to solve at least one of the technical problems in the prior art. To this end, the present application provides a thin-layer identification method of formula granules and standard decoction of jasminum lanceolaria roxburgh, which can scientifically and reasonably analyze the quality of formula granules and standard decoction of jasminum lanceolaria roxburgh.

[0006] The first aspect of the present application provides a method for identifying Qingxiangteng formula granules and standard decoction, comprising: performing thin layer chromatography on test sample solution and control medicinal material solution, and comparing the obtained chromatograms, wherein the developing agent is a mixed solution of chloroform, methanol and formic acid in a volume ratio of (5-7):(0.5-2):(0.4-0.6).

[0007] wherein "(5-7)" can be any numerical value within the range, including but not limited to "(5-6.5)", "(5-6)", "(5.5-7)", "(5.5-6.5)", "(5.5-6)", "(6-7)", "(6-6.5)", "(6)", "(0.5-2)" can be any numerical value within the range, including but not limited to "(0.5-1.5)", "(0.5-1)", "(1-2)", "(1-1.5)", "(1)", "(0.4-0.6)" can be any numerical value within the range, including but not limited to "(0.4-0.5)", "(0.5-0.6)", "(0.5)".

[0008] It should be noted that the volume ratio of chloroform, methanol and formic acid in the developing agent of the present application can be any combination of the above-mentioned any numerical value, such as (5.5-6.5):(0.5-1.5):(0.4-0.6), 6:1:0.5.

[0009] According to some preferred embodiments of the present application, the volume ratio of chloroform, methanol and formic acid is (5.5-6.5):(0.5-1.5):(0.4-0.6).

[0010] According to some more preferred embodiments of the present application, the volume ratio of chloroform, methanol and formic acid is 6:1:0.5.

[0011] According to some embodiments of the present application, the preparation step of the test sample solution comprises: taking Qingxiangteng formula granules or standard decoction, adding an alcohol solvent, ultrasonic treatment, filtration, evaporating the filtrate to dryness, and dissolving the residue in an alcohol solvent to obtain the test sample solution.

[0012] According to some embodiments of the present application, in the preparation step of the test sample solution, the mass-volume ratio of Qingxiangteng formula granules or standard decoction to alcohol solvent is 1g:30-60mL; preferably 1g:30-50mL; more preferably 1g:35-45mL.

[0013] According to some embodiments of the present application, the preparation step of the control medicinal material solution comprises: adding an alcohol solvent to Qingxiangteng control medicinal material, ultrasonic treatment, filtration, evaporating the filtrate to dryness, and dissolving the residue in an alcohol solvent to obtain the control medicinal material solution.

[0014] According to some embodiments of the present application, the mass-volume ratio of the control medicinal material to the alcohol solvent in the preparation step of the control medicinal material solution is 1g:5-20mL; preferably 1g:5-15mL.

[0015] According to some embodiments of the present application, the alcohol solvent is methanol and / or ethanol, preferably methanol.

[0016] According to some embodiments of the present application, the sample volume of the thin layer chromatography is 2-10μL.

[0017] According to some embodiments of the present application, the thin layer plate is a silica gel G thin layer plate.

[0018] According to some embodiments of the present application, the thin layer chromatography further comprises air-drying after development and color development under a 365nm ultraviolet lamp.

[0019] According to some embodiments of the present application, the environmental temperature during development is 4-30℃, and / or the environmental humidity during development is 40%-90%.

[0020] According to some embodiments of the present application, the comparison step is to compare the chromatogram of the test product solution with that of the control medicinal material solution, and determine whether the color and color development position of the fluorescent spots are the same.

[0021] According to some embodiments of the present application, in the test product chromatogram, compared with the control medicinal material chromatogram, the same color spots are shown at the corresponding positions with Rf values of 0.44-0.50, 0.63-0.71 and 0.89-0.97.

[0022] In the second aspect of the present application, the above method is provided for use in identifying Qingxiangteng formula granules and standard decoction.

[0023] Advantages:

[0024] The thin layer chromatography identification method provided by the present application is suitable for identifying Qingxiangteng formula granules and standard decoction by improving the formula of the developing agent, overcoming the limitations of the existing Qingxiangteng identification standard in identifying Qingxiangteng formula granules and standard decoction. The present application is simple to operate, has no negative interference, and has clear and distinguishable color development; it is specific and durable, and is an improvement over the existing Qingxiangteng preparation identification method. From the analysis of the thin layer chromatogram, the spots of the control medicinal material are almost consistent with those of the formula granules, indirectly indicating that during the process of decoction, drying and granulation, the chemical components in the medicinal material are not decomposed and destroyed, but most of them are transferred to the granules. The method of the present application can be used for quality supervision of Qingxiangteng formula granule preparation process, and ensures the clinical efficacy of the preparation.

[0025] Other features and advantages of the present application will be set forth in the description that follows, and in part will be apparent from the description, or can be learned by practice of the application.

[0026] In the present application, the term:

[0027] Unless otherwise defined, the term "control medicinal material" as used herein refers to a standard medicinal material that has been identified by species and is used to identify a test sample.

[0028] Unless otherwise defined, the term "test sample" as used herein refers to an experimental sample used for detection or identification. BRIEF DESCRIPTION OF DRAWINGS

[0029] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description that follows, including the figures that illustrate at least one embodiment of the application, and, while showing several embodiments, the application should not be limited to only these embodiments.

[0030] Figure 1 is a thin layer chromatogram of different sample amounts and specificity investigation of the present application, from left to right, points 1-7 represent 1. Qingxiangteng negative control (5 μl); 2. Qingxiangteng control medicinal material (2 μl); 3. Qingxiangteng control medicinal material (5 μl); 4. Qingxiangteng control medicinal material (10 μl); 5. Qingxiangteng granules 230452 (2 μl); 6. Qingxiangteng granules 230452 (5 μl); 7. Qingxiangteng granules 230452 (10 μl);

[0031] Figure 2 is a thin layer chromatogram of different humidity investigation of the present application, from left to right, points 1-4 represent 1. Qingxiangteng control medicinal material (5 μL); 2-4. Qingxiangteng formula granules 230452, 230453, 230454 (5 μL each) in turn;

[0032] Figure 3 is a thin layer chromatogram of different temperature investigation of the present application, from left to right, points 1-5 represent 1. Qingxiangteng control medicinal material (5 μL); 2. Qingxiangteng negative control (5 μl); 3-5. Qingxiangteng formula granules 230452, 230453, 230454 (5 μL each) in turn;

[0033] Figure 4 is a thin layer chromatogram of different thin layer plate investigation of the present application, from left to right, points 1-5 represent 1. Qingxiangteng control medicinal material (5 μL); 2-4. Qingxiangteng formula granules 230452, 230453, 230454 (5 μL each) in turn; 5. Qingxiangteng negative control (5 μl);

[0034] Figure 5is the thin layer chromatogram of the formula granules of the control example 1, from left to right, points 1-2 represent, in turn, as follows: 1. control medicinal material of lindera aggregata; 2. formula granules of lindera aggregata;

[0035] Figure 6 is the thin layer chromatogram of the formula granules of the control example 2, from left to right, points 1-5 represent, in turn, as follows: 1. negative control of lindera aggregata (5 μl); 2. control medicinal material of lindera aggregata (5 μL); 3-5. formula granules of lindera aggregata 230452, 230453, 230454 (5 μL each), in turn;

[0036] Figure 7 is the thin layer chromatogram of the formula granules of the control example 3, from left to right, points 1-5 represent, in turn, as follows: 1. control medicinal material of lindera aggregata (5 μL); 2-4. formula granules of lindera aggregata 230452, 230453, 230454 (5 μL each), in turn; 5. negative control of lindera aggregata (5 μl);

[0037] Figure 8 is the thin layer chromatogram of the formula granules of the control example 4, from left to right, points 1-5 represent, in turn, as follows: 1. negative control of lindera aggregata (5 μl); 2. control medicinal material of lindera aggregata (5 μL); 3-5. formula granules of lindera aggregata 230452, 230453, 230454 (5 μL each), in turn;

[0038] Figure 9 is the thin layer chromatogram of the formula granules of the control example 5, from left to right, points 1-5 represent, in turn, as follows: 1. control medicinal material of lindera aggregata (5 μL); 2-4. formula granules of lindera aggregata 230452, 230453, 230454 (5 μL each), in turn; 5. negative control of lindera aggregata (5 μl);

[0039] Figure 10 is the thin layer chromatogram of the formula granules of the control example 6, from left to right, points 1-5 represent, in turn, as follows: 1. negative control of lindera aggregata (5 μl); 2. control medicinal material of lindera aggregata (5 μL); 3-5. formula granules of lindera aggregata 230452, 230453, 230454 (5 μL each), in turn. DETAILED DESCRIPTION

[0040] The concept and the technical effects generated by the present application will be described below in conjunction with the examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples, and other examples obtained by the person skilled in the art on the basis of the examples of the present application without creative labor, all belong to the protection scope of the present application.

[0041] Qingxiangteng Dispensing Granules were provided by Hunan Chunguang Jiu Hui Modern Chinese Medicine Co., Ltd. (batch numbers 230452, 230453, 230454).

[0042] Example 1

[0043] The present example provides a thin layer identification method for Qingxiangteng Dispensing Granules, for the qualitative identification of Qingxiangteng Dispensing Granules. The identification method comprises the following steps:

[0044] Preparation of the test solution: 0.5 g of Qingxiangteng Dispensing Granules was finely ground, 20 mL of methanol was added, and the mixture was ultrasonically treated for 30 minutes. The filtrate was evaporated to dryness, 2 mL of methanol was added to dissolve the residue, and the solution was used as the test solution.

[0045] Preparation of the control medicinal material solution: 2 g of Qingxiangteng control medicinal material was added to 20 mL of methanol, and the mixture was ultrasonically treated for 30 minutes. The filtrate was evaporated to dryness, 2 mL of methanol was added to dissolve the residue, and the solution was used as the control medicinal material solution.

[0046] 5 μL of the control medicinal material solution and the test solution were each spotted on the same silica gel GF254 thin layer plate, chloroform-methanol-formic acid (volume ratio of 6:1:0.5) was used as the developing agent, and the plate was developed, removed, and air-dried. The plate was then observed under a 365 nm fluorescent light (temperature of 25 °C and humidity of 65%).

[0047] Investigation scheme

[0048] (1) Spotting amount and specificity investigation

[0049] According to the steps in Example 1, Qingxiangteng control medicinal material and Qingxiangteng Dispensing Granules (batch number: 230452) were each prepared into a control medicinal material solution and a test solution.

[0050] In addition, a Qingxiangteng negative control solution was prepared according to the following steps: 0.5 g of dextrin was added to 20 mL of methanol, and the mixture was ultrasonically treated for 30 minutes. The filtrate was evaporated to dryness, 2 mL of methanol was added to dissolve the residue, and the solution was used as the negative control solution.

[0051] 5 μL of the negative control solution, 2 μL, 5 μL, and 10 μL of the test solution, and 2 μL, 5 μL, and 10 μL of the control medicinal material solution were each spotted on the same silica gel G254. Chloroform-methanol-formic acid (volume ratio of 6:1:0.5) was used as the developing agent, and the plate was developed for a distance of about 12 cm, removed, air-dried, and observed under a 365 nm light (temperature of 26 °C and humidity of 65%). The observation results are shown in Table 1.

[0052] The results are shown in Table 1. Figure 1As shown, the negative control has no interference, indicating that the thin layer identification method has good specificity. When the sample solution and the control drug solution are 2 μL to 10 μL, the chromatogram of Qingxiangteng formula granules and the chromatogram of the control drug are both clear spots at the corresponding positions.

[0053] (2) Humidity investigation

[0054] According to the steps in Example 1, the present application respectively develops the silica gel G254 plate under the conditions of 42% and 72% humidity, takes out, air dries, and observes under 365 nm fluorescence (temperature is 26°C). The observation results are shown in the following table.

[0055] As shown in the following table, different humidity has little effect on the thin layer identification results of Qingxiangteng granules, and the relative humidity conditions in the environment are both ideal. Figure 2

[0056] (3) Temperature investigation

[0057] According to the steps in Example 1, the present application respectively develops the silica gel G254 plate under the conditions of 4-10°C and 26°C, takes out, air dries, and observes under 365 nm fluorescence (humidity is 65%). The observation results are shown in the following table.

[0058] As shown in the following table, different temperature conditions (4-10°C and 26°C) have little effect on the thin layer identification results of Qingxiangteng granules. Figure 3

[0059] (4) Investigation of thin layer plate

[0060] According to the steps in Example 1, the present application investigates the silica gel GF254 plates from Qingdao Silicon Creation Fine Chemical Co., Ltd. and Qingdao Yumingyuan Silica Gel Reagent Co., Ltd., respectively develops with chloroform-methanol-formic acid (volume ratio 6:1:0.5) solution, takes out, air dries, and observes under 365 nm fluorescence (temperature is 25°C, humidity is 46%). The observation results are shown in the following table.

[0061] As shown in the following table, different brands of thin layer plates have no significant effect on the thin layer identification results of Qingxiangteng granules, indicating that the thin layer identification method has good durability. Figure 4

[0062] In the above investigation experiments, the Rf values of each spot in the sample thin layer chromatogram are shown in the following table 1.

[0063] Table 1

[0064]

[0065] Comparative Example 1

[0066] ​​​​The comparative example provides a thin layer identification method of Qingxiangteng formula granules for qualitatively identifying Qingxiangteng formula granules. The thin layer chromatography method recorded in the 2009 edition of Hunan Province Chinese medicinal material standard is used to identify Qingxiangteng formula granules, and the method is as follows:

[0067] Take Qingxiangteng formula granules 2 g, add methanol 20 ml, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate, add methanol 2 ml to dissolve as the test solution. Another 2 g of Qingxiangteng control drug is prepared into a control drug solution in the same way. According to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502), 5 μL of the above two solutions are taken and spotted on the same silica gel G thin layer plate, with petroleum ether (30-60℃)-ethyl acetate (18:4) as the developing agent, and then developed, taken out, dried, and observed under ultraviolet light (365 nm). In the test sample chromatogram, the same colored fluorescent spots appear at the corresponding positions of the control drug chromatogram.

[0068] The results are shown in Table Figure 5 The spots of the control drug are obvious, and there are no spots in Qingxiangteng formula granules, indicating that the method recorded in the current Hunan Province Chinese medicinal material standard cannot well reflect the quality of Qingxiangteng formula granules; therefore, this method is not applicable to the thin layer chromatography identification of Qingxiangteng formula granules.

[0069] Comparative examples 2-6

[0070] Comparative examples 2-6 provide a thin layer identification method of Qingxiangteng formula granules for qualitatively identifying Qingxiangteng formula granules. The difference between comparative examples 2-6 and example 1 is only the developing agent, wherein:

[0071] The developing agent in comparative example 2 is: chloroform:methanol (volume ratio of 6:1);

[0072] The developing agent in comparative example 3 is: chloroform:formic acid (volume ratio of 6:0.5);

[0073] The developing agent in comparative example 4 is: methanol:formic acid (volume ratio of 1:0.5);

[0074] The developing agent in comparative example 5 is: chloroform:methanol:formic acid (volume ratio of 3:1:0.5);

[0075] The developing agent in comparative example 6 is: chloroform:methanol:formic acid (volume ratio of 1:1:1).

[0076] Respectively take 5uL of negative control solution, control medicinal material solution, test solution (230452, 230453, 230454 three batches) respectively, and point on the same silica gel GF254 thin layer plate, respectively, and use the developing agent in Comparative Example 2-6 to develop, take out, and air dry; under 365nm fluorescence (temperature is 25℃, humidity is 65%) inspection.

[0077] The detection results in Comparative Example 2-6 correspond to Figures 6 to 10 respectively.

[0078] Figure 6 In (corresponding to Comparative Example 2), the control medicinal material solution and the test solution still show three spots on the thin layer plate, but the positions are different from Example 1 (Rf values are smaller, and the original position is not run off) and the clarity is also reduced.

[0079] Figure 7 In (corresponding to Comparative Example 3), the control medicinal material solution and the test solution also show three spots on the thin layer plate, but the positions are quite different from Example 1 and the clarity is significantly reduced.

[0080] Figure 8 In (corresponding to Comparative Example 4), the development fails, and the approximate reason can be obtained: the polarity adjustment of the developing agent is not suitable, and the developing agent needs to contain chloroform.

[0081] Figure 9 In (corresponding to Comparative Example 5), the tailing phenomenon is serious, which makes the boundary between the spots blurred, and the detection result is difficult to judge.

[0082] Figure 10 In (corresponding to Comparative Example 6), the result is slightly improved compared with Comparative Example 4, but the development also fails, and the approximate reason can be obtained: it is more beneficial to development to ensure that the developing agent contains a higher proportion of chloroform.

[0083] The above experimental results show that the method has strong specificity, and the durability (test product and control medicinal material different sample amount and negative control, low temperature, normal temperature, low humidity, high humidity and silica gel GF254 thin layer plate of different manufacturers) test proves that the method is suitable for the detection of Qingxiangteng formula granules and standard decoction thin layer identification. When the components in the developing agent of the application are omitted or the volume ratio range of each component is changed, the identification result will be significantly affected.

[0084] The above embodiments of the application are described in detail in combination with the drawings, but the application is not limited to the above embodiments, and various changes can be made within the knowledge range of ordinary skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for thin layer identification of the formula granules of Evolvulus aurius L. and standard decoction, characterized in that, The test sample solution and the control medicinal material solution are subjected to thin layer chromatography analysis, and the obtained chromatograms are compared, wherein the developing agent is a mixed solution of chloroform, methanol and formic acid in a volume ratio of 5-7:0.5-2:0.4-0.6; The preparation step of the test sample solution comprises: taking the Qingxiangteng formula granules or standard decoction, adding methanol, ultrasonic treatment, filtration, evaporating the filtrate to dryness, and dissolving the residue in methanol to obtain the test sample solution. The thin layer chromatography analysis is performed on a silica gel GF254 plate.

2. The method of claim 1, wherein, The mass-volume ratio of the Qingxiangteng formula granules or standard decoction to methanol is 1g:30-60mL.

3. The method of claim 1, wherein, The preparation step of the control medicinal material solution comprises: adding methanol to the Qingxiangteng control medicinal material, ultrasonic treatment, filtration, evaporating the filtrate to dryness, and dissolving the residue in methanol to obtain the control medicinal material solution.

4. The method of claim 3, wherein, In the preparation step of the control medicinal material solution, the mass-volume ratio of the control medicinal material to methanol is 1g:5-20mL.

5. The method of claim 1, wherein, The spotting amount of the thin layer chromatography analysis is 2-10μL.

6. The method of claim 1, wherein, The thin layer chromatography analysis further comprises air-drying after development and developing under a 365nm ultraviolet lamp.

7. The method of claim 6, wherein, The development environment temperature is 4-30℃, and / or the development environment humidity is 40%-90%.

8. The method of claim 1, wherein, The comparison step is: comparing the chromatograms of the test sample solution and the control medicinal material solution to determine whether the color and developing position of the fluorescent spots are the same.

9. The method according to any one of claims 1-8 for identifying Qingxiangteng formula granules or standard decoction.

Images