Thin layer identification method for formulation granules of lycopodiastrum casuarinoides and lycopodium japonicum

Abstract

A thin layer identification method for formulation granules of Lycopodiastrum casuarinoides and Lycopodium japonicum, comprising: A) taking a sample to be tested, and pre-treating to obtain a product solution for testing; B) taking a control medicinal material of Lycopodiastrum casuarinoides, and pre-treating to obtain a control medicinal material solution; C) performing thin layer chromatography on the product solution to be tested and the control medicinal material solution, a thin layer plate being a silica gel G thin layer plate, and a developing agent being cyclohexane-ethyl acetate; D) placing under an ultraviolet light for inspection. If there is a blue fluorescent spot at an Rf value of 0.31~0.464 in the chromatogram of the product solution to be tested, it is a formulation granule of Lycopodiastrum casuarinoides; if there is no blue fluorescent spot at the Rf value of 0.31~0.464, it is a formulation granule of Lycopodium japonicum. Using the thin layer chromatography method for identifying formulation granules of Lycopodiastrum casuarinoides and Lycopodium japonicum, by means of observing whether there is a fluorescent spot in a specified retention factor range in the thin layer chromatography spectrum, it is possible to quickly and effectively identify formulation granules of Lycopodiastrum casuarinoides and Lycopodium japonicum.

Description

Thin layer identification method of Shujincao and Shenjincao formula granules

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on October 25, 2023, with application number 202311397059.4 and invention name “Thin layer identification method for Shujincao and Shenjincao formula granules”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of analysis and detection, in particular to a thin layer identification method for granules of Shujincao and Shenjincao formula. Background Art

[0003] Shujincao (Shujincao) is the dried aerial part of Lycopodiastrum casuarinoides (Spring) Holub, a plant of the Lycopodiaceae family. It dispels wind and dampness, relaxes muscles and activates blood circulation. It is used for rheumatic numbness, traumatic injuries, muscle and bone pain, and menstrual irregularities.

[0004] Stretching Herb is the dried whole herb of Lycopodium japonicum Thunb, a plant in the Lycopodium family. It dispels wind and dampness, relaxes tendons, and activates joints. It is used for joint pain and difficulty flexing and extending.

[0005] Because Shujincao is primarily used clinically for rheumatic diseases, while Shenjincao is primarily used for muscle and bone injuries, the two cannot be used interchangeably. However, due to the incomplete TLC method for Shujincao granules in the current standard, there is no effective way to distinguish it from the easily confused Shenjincao granules.

[0006] Therefore, a thin layer method is needed to establish which can effectively identify Shujincao formula granules and distinguish them from its easily confused product Shenjincao formula granules.

[0007] Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a thin layer identification method for the formula granules of Shujincao and Shenjincao.

[0009] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0010] In this application, the term "and / or" describes the association relationship between associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0011] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0012] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the following processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0013] The present invention provides a thin layer identification method for Shujincao and Shenjincao formula granules, comprising:

[0014] A) Take the sample to be tested and obtain the test solution after pretreatment;

[0015] B) taking a control medicinal material of Herba Lycopodii Herba, and pre-treating it to obtain a control medicinal material solution;

[0016] C) The test solution and the control medicinal material solution were subjected to thin layer chromatography using a silica gel G thin layer plate and a cyclohexane-ethyl acetate developing solvent;

[0017] D) Check under ultraviolet light. If there are blue fluorescent spots at Rf values ​​of 0.31 to 0.464 in the chromatogram of the test sample, it is Shujincao formula granules; if there are no blue fluorescent spots at Rf values ​​of 0.321 to 0.464, it is Shenjincao formula granules.

[0018] The pretreatment in step A) of the present invention is specifically as follows: dissolving the sample in petroleum ether, heating under reflux for extraction, discarding the petroleum ether solution, decocting with water, filtering, and ultrasonically treating with methanol. After filtering, dissolving the residue in methanol to obtain a test solution.

[0019] Specifically, the mass volume ratio of the sample to be tested, water and methanol is: 1 g: 100 mL: 40 mL.

[0020] In some embodiments, the heating reflux extraction time is 2 to 3 hours; the decoction time is 1.5 to 2 hours; the ultrasonic treatment time is 30 to 40 minutes; and the boiling point of petroleum ether is 60 to 90°C.

[0021] In some embodiments, the heating reflux extraction time is 3 hours; the decocting time is 2 hours; the ultrasonic treatment time is 30 minutes; and the boiling point of petroleum ether is 60-90°C.

[0022] A control medicinal material of Herba Eupatorii was taken and pretreated to obtain a control medicinal material solution. The pretreatment specifically comprises: dissolving the control medicinal material of Herba Eupatorii in petroleum ether, heating under reflux for extraction, discarding the petroleum ether solution, decocting with water, filtering, and ultrasonically treating with methanol. After filtering, the residue was dissolved in methanol to obtain a control medicinal material solution.

[0023] Specifically, the mass volume ratio of the control medicinal material, petroleum ether, water and methanol is: 1g:mL:100mL:40mL

[0024] In some embodiments, the heating reflux extraction time is 1 to 2 hours; the decoction time is 1.5 to 2 hours; the ultrasonic treatment time is 30 to 40 minutes; and the boiling point of petroleum ether is 60 to 90°C.

[0025] In some embodiments, the heating reflux extraction time is 2 hours; the decocting time is 2 hours; the ultrasonic treatment time is 30 minutes; and the boiling point of petroleum ether is 60-90°C.

[0026] The test solution and the control medicinal material solution were subjected to thin layer chromatography.

[0027] Preferably, the test sample solution and the control medicinal material solution are aspirated and spotted on the same silica gel G thin layer plate, developed with a developing agent, taken out, dried, and inspected under ultraviolet light.

[0028] The ultraviolet light wavelength of the present invention is 365nm.

[0029] The thin layer plate of the present invention is a silica gel G thin layer plate, which can be selected from the tailorable thin layer chromatography plate of Tianjin Silida Technology Co., Ltd., Merck, and the prefabricated silica gel G plate of Qingdao Ocean Chemical Plant Branch.

[0030] The results showed that the blue fluorescent spot ratio shift values ​​(Rf) at 3.193-5.130 cm from the origin on the three thin-layer plates were 0.424, 0.310, and 0.354, respectively, which all met the identification requirements and were used to identify Shujincao formula granules.

[0031] Check under ultraviolet light. If there are blue fluorescent spots at Rf values ​​of 0.31-0.464 in the chromatogram of the test sample, it is the Shujincao formula granules; if there are no blue fluorescent spots at Rf values ​​of 0.31-0.464, it is the Shenjincao formula granules.

[0032] The Rf value is 0.31 to 0.464, and specifically can be 0.421, 0.437, 0.424, 0.310, 0.354, 0.417, 0.428, 0.481, 0.460, 0.468, 0.459 and 0.409.

[0033] The sample volume of the test solution of the present invention is 1 to 5 μL; the sample volume of the control medicinal material solution is 1 to 3 μL.

[0034] In some preferred embodiments of the present invention, the mass ratio of cyclohexane to ethyl acetate is 5:4; wherein the boiling point of petroleum ether is 60-90°C.

[0035] The inspection temperature of the present invention is 4° C. to 35° C. The method of the present invention has good adaptability to different temperatures, and the Shujincao formula granules show blue fluorescent spots at the ratio shift values ​​of 0.417, 0.428, and 0.481, while the Shenjincao formula granules have no spots.

[0036] The inspection humidity of the present invention is 32% rh to 75% rh. The method of the present invention has good adaptability to different humidity, and the Shujincao formula granules show blue fluorescent spots at the ratio shift values ​​of 0.584, 0.613, and 0.599, while the Shenjincao formula granules have no spots.

[0037] The sample to be tested in the present invention is the formula granules of Shujincao and / or Shenjincao.

[0038] The present invention provides a thin-layer chromatography identification method for formula granules of Shujincao and Shenjincao, comprising: A) taking a sample to be tested, pre-treating it, and obtaining a test solution; B) taking a control medicinal material of Shujincao and pre-treating it, and obtaining a control medicinal material solution; C) subjecting the test solution and the control medicinal material solution to thin-layer chromatography, using a silica gel G thin-layer plate and a cyclohexane-ethyl acetate developing solvent; D) inspecting under ultraviolet light; if a blue fluorescent spot is present at an Rf value of 0.31 to 0.464 in the chromatogram of the test sample, the granules are Shujincao formula granules; if no blue fluorescent spot is present at an Rf value of 0.31 to 0.464, the granules are Shenjincao formula granules. The thin-layer chromatography identification method for formula granules of Shujincao and Shenjincao established by the present invention uses the presence of fluorescent spots within a specified ratio shift value range as a distinguishing point to quickly and effectively distinguish the granules of Shujincao and Shenjincao formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows the spotting amount investigation;

[0040] Figure 2 shows the specificity investigation;

[0041] Figure 3 shows the inspection of different thin layer plates - Tianjin Silida.

[0042] Figure 4 Investigation of different thin layer plates - Merck;

[0043] Figure 5 shows different thin layer plate surveys - Qingdao Ocean;

[0044] Figure 6 shows different temperatures -4°C;

[0045] Figure 7 shows different temperatures -25°C;

[0046] Figure 8 shows different temperatures -35°C;

[0047] Figure 9 shows different humidity levels -32%;

[0048] Figure 10 shows different humidity levels -51%;

[0049] Figure 11 shows different humidity levels -75%;

[0050] Figure 12 shows the multi-batch verification results;

[0051] FIG13 is a graph showing the results of Comparative Example 1 of the present invention;

[0052] Figure 14 is a diagram showing the results of the method for comparative example 2;

[0053] FIG15 is a diagram showing the results of the comparative example 2 method. DETAILED DESCRIPTION

[0054] In order to further illustrate the present invention, the thin layer identification method of the Shujincao and Shenjincao formula granules provided by the present invention is described in detail below with reference to the examples.

[0055] Water bath, Soxhlet extractor, hot plate, thin layer imaging system: CAMAG TLC Visualizer, silica gel G thin layer plate (Qingdao Ocean Chemical Plant, batch number: 20180527, Tianjin Silida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353)

[0056] Petroleum ether (60-90°C), methanol, cyclohexane, and ethyl acetate were all of analytical grade, and water was ultrapure water (prepared in the laboratory).

[0057] Shujincao control medicinal material (China Food and Drug Inspection Institute, batch number: 380003-202104), Shujincao formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2302078, 2302079, 2302080); Shenjincao formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2002001; 2002002; 2002003).

[0058] Example 1

[0059] Preparation of test solution

[0060] Take 1g of this product, grind it into powder, add an appropriate amount of petroleum ether (60-90℃), place it in a Soxhlet extractor, heat and reflux for 3 hours, discard the petroleum ether, evaporate the residue, add 100ml of water and boil for 2 hours, filter, evaporate the filtrate to dryness, add 40ml of methanol to the residue, ultrasonicate for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 1μl of the test solution and 1μl of the control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate. Use cyclohexane-ethyl acetate (5:4) as the developing solvent, develop, remove, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a fluorescent spot of the same color appears.

[0061] Preparation of reference substances and reference medicinal material solutions

[0062] Take 2g of medicinal material powder, add appropriate amount of petroleum ether (60-90℃), place in Soxhlet extractor, heat and reflux for 2 hours, discard the petroleum ether liquid, evaporate the medicinal residue, add 100ml of water and boil for 2 hours, filter, evaporate the filtrate to dryness, add 40ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, and add 1ml of methanol to dissolve the residue as the control medicinal material solution.

[0063] Example 2 Methodological Investigation

[0064] 2.1 Sampling quantity inspection

[0065] Under the experimental conditions proposed above, 1μl, 3μl, 5μl, 10μl, 15μl, and 20μl of the control and test solutions of Shujincao were spotted on the same silica gel G thin layer plate. The fluorescent spots on the thin layer chromatography were clearly colored. The spot sizes for the control and test solutions were 1-3μl and 1-5μl, respectively. The spot with the same color at a relative shift (Rf) of 0.421 can be used as the identification point for the Shujincao formula granules.

[0066] 2.2 Specificity Investigation

[0067] Following the above test sample preparation method, a control solution of Shujincao herbal medicine, a solution of Shujincao formula granules, and a negative solution were prepared. Thin-layer chromatography identification experiments were performed according to the proposed chromatographic conditions. The results are shown in Figure 2. As can be seen from the figure, the negative sample did not interfere with the Shujincao formula granules test sample, indicating that this method has good specificity and can be used to identify Shujincao formula granules.

[0068] 2.3 Durability Investigation

[0069] 2.3.1 Comparison of different thin layer plates

[0070] Cuttable thin-layer chromatography plates from Tianjin Silida Technology Co., Ltd., prefabricated silica gel G plates from Merck and Qingdao Ocean Chemical Plant were tested according to the proposed test method (see Figures 3-5). The results show that the relative shifts (Rf) of the blue fluorescent spots at 3.193-5.130 cm from the origin on the three thin-layer plates were 0.424, 0.310, and 0.354, respectively, all meeting the identification requirements. This was used to identify Shujincao formula granules.

[0071] The results are shown in Figure 3, which shows different thin layer plate inspections - Tianjin Silida. Among them: 1 is the control herbal solution of Shujincao; 2-4 are the formula granule solutions of Shujincao.

[0072] Figure 4: Different thin layer plate inspection - Merck; where: 1 is the control medicinal material solution of Shujincao; 2-4 are the formula granule solutions of Shujincao.

[0073] Figure 5 shows the investigation of different thin layer plates - Qingdao Ocean; 1 is the control medicinal material solution of Shujincao; 2-4 are the formula granule solutions of Shujincao.

[0074] 2.3.2 Comparison of different temperatures

[0075] After spotting, the thin-layer plates were developed at 4°C, 25°C, and 35°C. Figures 6-8 show that this method is well adapted to different temperatures, with blue spot ratio shifts (Rf) of 0.417, 0.428, and 0.481, respectively. This method was used to identify Shujincao granules.

[0076] Figure 6 shows different temperatures of -4°C; 1 is the control herbal solution of Shujincao; 2-4 are the formula granule solutions of Shujincao.

[0077] Figure 7 shows different temperatures of -25°C, where 1 is the control herbal solution of Shujincao; 2-4 are the formula granule solutions of Shujincao.

[0078] Figure 8 shows different temperatures of -35°C, where 1 is the control herbal solution of Shujincao; 2-4 are the formula granule solutions of Shujincao.

[0079] 2.3.3 Comparison of different humidity levels

[0080] After spotting, thin-layer plates were developed at 32%, 51%, and 75% humidity, as shown in Figures 9-11. As can be seen from the figures, the method has good adaptability to different temperatures, with blue spot ratio shift values ​​(Rf) of 0.584, 0.613, and 0.599, respectively. This method was used to identify Shujincao formula granules.

[0081] Figure 9 shows different humidity levels -32%; 1 is a control herbal solution of Shujincao; 2-4 are herbal granule solutions of Shujincao.

[0082] Figure 10 shows different humidity levels - 51%; 1 is a control herbal solution of Shujincao; 2-4 are herbal granule solutions of Shujincao.

[0083] Figure 11 shows different humidity levels - 75%; Figure 1 is a control herbal solution of Shujincao; Figures 2-4 are herbal granule solutions of Shujincao.

[0084] 2.3.4 Verification

[0085] Three batches of Shujincao formula granules were subjected to thin layer identification verification. The ratio shift value (Rf) of the blue fluorescent spot at 5.03 cm away from the origin was 0.409. The experimental results are shown in Figure 12.

[0086] Figure 12 shows the results of multiple batches of verification; among them, 1 is the control medicinal material solution of Shujincao; 2-4 are the formula granule solutions of Shujincao.

[0087] 2.4 Distinguishing between Shujincao Formula Granules and Shenjincao Formula Granules

[0088] Solutions of Shujincao and Shenjincao granules were prepared using the above test sample preparation method. Thin-layer chromatography identification tests were performed. The blue fluorescent spot ratio shift (Rf) of the Shujincao granules at a distance of 5.2 cm from the origin was 0.591, while the Shenjincao granules had no spots at this location. The results are shown in Figure 13, where 1 and 2 represent Shujincao granules, and 3 and 4 represent Shenjincao granules. This figure demonstrates that this method can be used to distinguish between Shujincao and Shenjincao granules.

[0089] 2.5 Determination of the identification point Rf of Shujincao formula granules and Shenjincao formula granules

[0090] The data of various ratio transfer values ​​in the thin layer chromatography methodology investigation are summarized and the results are shown in Table 1. Based on the summary results, it is determined that the identification spot ratio transfer value (Rf) of Shujincao formula granules should be within the range of 10%, and the specified value is 0.482.

[0091] Table 1 Summary of methodological investigation ratios

[0092] Comparative Example 1

[0093] Take 2g of this product, grind it into powder, add 40mL of water and boil for 3 hours, filter, evaporate the filtrate to dryness, add 20mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2mL of methanol to prepare the test solution. Separately, take 1g of the control herb Shujincao and 2g of the negative control product without the Shujincao. Prepare the control herb solution and negative control product solution in the same manner. Take 5μL of each solution and spot it on the same silica gel G thin layer plate, develop it with cyclohexane-ethyl acetate (8:5), remove it, and air dry. Inspect under ultraviolet light (365nm). No red spots appear in the chromatogram of the test product at the corresponding position in the chromatogram of the control herb, indicating no interference with the negative control. The results are shown in Figure 14. Figure 14 is the result of Comparative Example 1 of the present invention. Where: 1-2 are the control herb solutions; 3-4 are the solutions of the Shujincao formula granules; 5 is the negative control solution. The results showed that using the above-mentioned thin layer identification method, the spots of the control medicinal material of Shujincao were inconsistent with the finished formula granules, and the two could not be identified.

[0094] Comparative Example 2

[0095] Grind 2g of this product, add 5mL of ammonia solution, and stir evenly. Then add 20mL of chloroform, heat under reflux for 2 hours, filter, and place the filtrate in a separatory funnel. Add 20mL of 2% sulfuric acid solution and shake. Adjust the pH of the acid-water layer to 8-9 with sodium hydroxide solution, transfer to a separatory funnel, and extract with chloroform twice, 10mL each time. Concentrate the chloroform extract to 2mL, and use this as the test solution. Separately, prepare the control and negative control solutions by the same method using 2g of the control herb and 2g of the negative control without the herb. 10μL of each solution was spotted onto the same silica gel G thin-layer plate, developed with chloroform-methanol-acetone-ammonia solution (40:5:5:2), removed, and air-dried. Color development was achieved with a mixture of equal parts of iodine-potassium iodide and potassium bismuth iodide. Inspect under sunlight. The test sample chromatogram showed a yellow-brown spot at the corresponding position in the control herb chromatogram, with no interference from the negative control. The results are shown in Figure 15.

[0096] Figure 15 is a graph showing the results of the method of Comparative Example 2. 1 is a negative control solution; 2 is a control herbal solution of Shujincao; 3-5 are herbal granule solutions of Shujincao.

[0097] The results showed that using the above-mentioned thin layer identification method, the color spots of the Shujincao formula granules were too few and not beautiful enough, so the two could not be distinguished.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A thin layer identification method for the formula granules of Shujincao and Shenjincao, comprising: A) taking the sample to be tested, and obtaining the test solution after pretreatment; B) taking a control medicinal material of Herba Lysimachiae and pretreating it to obtain a control medicinal material solution; C) The test solution and the control medicinal material solution are subjected to thin layer chromatography, the thin layer plate is a silica gel G thin layer plate, and the developing solvent is cyclohexane-ethyl acetate; D) Check under ultraviolet light. If there is a blue fluorescent spot at the Rf value of 0.31-0.464 in the chromatogram of the test product, it is the Shujincao formula granules; If there are no blue fluorescent spots at the Rf value of 0.31-0.464, it is the granules of the herb.

2. The method according to claim 1, characterized in that The pretreatment in step A) is specifically as follows: dissolving the sample in petroleum ether, heating under reflux for extraction, discarding the petroleum ether solution, boiling in water, filtering, and ultrasonically treating with methanol; after filtering, dissolving the residue in methanol to obtain a test solution.

3. The method according to claim 2, characterized in that The mass volume ratio of the sample to be tested, water and methanol is: 1g:100mL:40mL; The time of the heating reflux extraction is 2 to 3 hours; the time of the decoction is 1.5 to 2 hours; the time of the ultrasonic treatment is 30 to 40 minutes; and the boiling point of the petroleum ether is 60 to 90°C.

4. The method according to claim 1, characterized in that: Step B) The pretreatment is specifically as follows: dissolving the control medicinal material of Shujincao with petroleum ether, heating under reflux for extraction, discarding the petroleum ether solution, decocting with water, filtering, and ultrasonically treating with methanol. After filtering, dissolving the residue with methanol to obtain a test solution; The time of the heating reflux extraction is 1 to 2 hours; the time of the decoction is 1.5 to 2 hours; the time of the ultrasonic treatment is 30 to 40 minutes; and the boiling point of the petroleum ether is 60 to 90°C.

5. The method according to claim 1, characterized in that The sample volume of the test solution is 1 to 5 μL; the sample volume of the control medicinal material solution is 1 to 3 μL.

6. The method according to claim 1, characterized in that Step D) the mass ratio of cyclohexane to ethyl acetate is 5:

4.

7. The method according to claim 1, characterized in that The wavelength of the ultraviolet light in step E) is 365 nm.

8. The method according to claim 1, characterized in that Step E) Check the temperature to be between 4°C and 35°C.

9. The method according to claim 8, characterized in that Step E) The humidity is checked to be 32% rh to 75% rh.

10. The method according to claim 1, characterized in that Step A): the sample to be tested is the granules of Herba Lycopodii and / or Herba Lycopodii.

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