Thin layer identification method for schizonepeta spike and schizonepeta spike charcoal formula granules

Abstract

The invention provides a schizonepeta spike and schizonepeta spike charcoal formula granule thin-layer identification method which comprises the following steps: A) pre-treating schizonepeta spike formula granules to obtain a schizonepeta spike test solution; pre-treating the schizonepeta spike charcoal formula granules to obtain a schizonepeta spike charcoal test solution; b) pre-treating a schizonepeta spike reference medicinal material to obtain a reference medicinal material solution; c) performing thin-layer chromatography detection on the schizonepeta spike test solution, the schizonepeta spike charcoal test solution and the reference medicinal material solution, wherein a thin-layer plate is a silica gel G thin-layer plate; the developing solvent is dichloromethane-butyl acetate-formic acid; and D) placing under an ultraviolet lamp for inspection, if the schizonepeta spike test sample and the reference medicinal material solution respectively have a spot at the positions with the specific shift values of 0.48-0.58 and 0.60-0.74, taking the spot as an identification point of the schizonepeta spike formula granules, and at the same position, the schizonepeta spike charcoal formula granules do not have spots with the same color. The schizonepeta spike and schizonepeta spike charcoal formula granule thin-layer identification chromatography method established by the invention is used for rapidly, accurately and effectively identifying schizonepeta spike and schizonepeta spike charcoal formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and detection, in particular to a thin-layer identification method for schizonepeta spicate and schizonepeta spicate charcoal formula particles. Background Art

[0002] Schizonepeta spicata is the dried flower spike of Schizonepeta tenuisfolia Briq., a plant of the Lamiaceae family. It is harvested in summer and autumn when the flowers have reached their peak and the spikes are green, then freed of impurities and sun-dried. Schizonepeta spicata has the properties of dispelling wind, clearing rashes, and eliminating sores, and is used for colds, headaches, measles, rubella, and the early stages of sores. Schizonepeta spicata was first mentioned as a medicinal herb in the Song Dynasty's "Compendium of Materia Medica," which described it as "an essential medicine for treating headaches, consumptive diseases, scabies, and blood-induced wind in women." Schizonepeta spicata charcoal is a processed product of Schizonepeta spicata. After charring, its properties and efficacy undergo a qualitative change, becoming astringent and more effective at stopping bleeding. It is used for treating bloody stools, metrorrhagia, and postpartum blood loss. This suggests that certain chemical components of the two herbs may transform during the processing process.

[0003] While the decoction pieces of Schizonepeta spicata and Schizonepeta spicata charcoal can be distinguished based on their properties, their extracts and preparations exhibit similar brown to tan coloration and a slightly astringent and pungent flavor, making them easily confused. A method for distinguishing the two has not yet been reported. Therefore, this study aimed to establish a thin-layer chromatography (TLC) method for the identification of Schizonepeta spicata granules and Schizonepeta spicata charcoal granules, providing a simple, rapid, and economical method for their identification. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a thin layer identification method for Schizonepeta spicata and Schizonepeta spicata charcoal formula particles.

[0005] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that other elements may be present 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] The thin-layer chromatography identification method disclosed herein shows a spot at a ratio shift of 0.48 to 0.58 and 0.60 to 0.74, respectively, when comparing the thin-layer chromatography of the Schizonepeta spicata formula granules with that of the Schizonepeta spicata control medicinal material. These spots serve as identification points for the Schizonepeta spicata formula granules. At the same locations, the Schizonepeta spicata charcoal formula granules should not exhibit spots of the same color. This thin-layer chromatography method can rapidly and effectively identify Schizonepeta spicata and Schizonepeta spicata charcoal formula granules that have lost their medicinal piece form. The detection method is simple to operate, durable, and sensitive. It can provide a reference for the formulation and further improvement and revision of quality standards for Schizonepeta spicata and Schizonepeta spicata charcoal formula granules, and has promising application prospects.

[0010] The present invention provides a thin layer identification method for schizonepeta spicate and schizonepeta spicate charcoal formula particles, comprising:

[0011] A) taking the schizonepeta spicate granules and pre-treating them to obtain a schizonepeta spicate test solution;

[0012] Pre-treat the schizonepeta spicate charcoal granules to obtain a schizonepeta spicate charcoal test solution;

[0013] B) Extract a control medicinal material of Schizonepeta spicata by heating with water, filtering, evaporating to dryness, and refluxing the residue with ether. The solvent is evaporated from the residue, and the residue is dissolved in 70% methanol. The mixture is sonicated, filtered, and the filtrate is evaporated to dryness. The residue is dissolved in methanol, and the supernatant is passed through a C18 solid phase extraction cartridge and eluted with 30% methanol. The eluate is discarded and eluted again with methanol. The eluate is collected and the solvent is recovered to dryness. The residue is dissolved in methanol to obtain a control medicinal material solution.

[0014] C) Thin layer chromatography was performed on the control medicinal material solution using a silica gel G thin layer plate and a developing solvent of dichloromethane-butyl acetate-formic acid;

[0015] D) Place the sample under ultraviolet light for inspection. If the Schizonepeta spicata test sample and the control herbal solution each have a spot at the transfer ratio of 0.48-0.58 and 0.60-0.74, these spots will serve as identification points for the Schizonepeta spicata granules. The Schizonepeta spicata charcoal granules should not have spots of the same color at the same location.

[0016] The invention provides schizonepeta spicate and schizonepeta spicate charcoal formula granules. First, the schizonepeta spicate formula granules are pretreated to obtain a schizonepeta spicate test sample solution.

[0017] The pretreatment of the schizonepeta spicate granules of the present invention is specifically as follows:

[0018] The granules of Schizonepeta spicata were extracted by reflux with ether, the solvent was evaporated from the residue, the residue was dissolved in 70% methanol, ultrasonically treated, filtered, the filtrate was evaporated to dryness, the residue was dissolved in methanol, the supernatant was collected, passed through a C18 solid phase extraction column, eluted with 30% methanol, the eluate was discarded, and eluted again with methanol, the eluate was collected, the solvent was recovered to dryness, and the residue was dissolved in methanol to obtain a Schizonepeta spicata test solution;

[0019] In a specific embodiment of the present invention, specifically:

[0020] Take the formula granules of Schizonepeta spicata, place them in a Soxhlet extractor respectively, add an appropriate amount of ether, heat to reflux, discard the ether solution, evaporate the solvent from the residue, transfer it to a stoppered conical flask, add 70% methanol, ultrasonically treat, filter, evaporate the filtrate to dryness, add 10 ml of methanol to dissolve the residue, take 2 ml of the supernatant, pass it through a C18 (300 mg) solid phase extraction column, elute with 10 ml of 30% methanol, discard the eluate, and elute with 10 ml of methanol again, collect the eluate, recover the solvent to dryness, and dissolve the residue in 1 ml of methanol as the test solution.

[0021] The power of the ultrasound in the present invention is 300W and the frequency is 24 kHz; the heating and reflux time is 3 to 4 hours.

[0022] Specifically, the mass volume ratio of the Schizonepeta spicata granules and 70% methanol is 1.5 g:25 mL; the mass ratio of the supernatant and the C18 solid phase extraction column is 2 ml:300 mg.

[0023] The pretreatment of the schizonepeta spicate charcoal particles of the present invention is specifically as follows:

[0024] The schizonepeta spicate charcoal granules were extracted by reflux with ether, the solvent was evaporated from the residue, the residue was dissolved in 70% methanol, ultrasonically treated, filtered, the filtrate was evaporated to dryness, the residue was dissolved in methanol, the supernatant was collected, passed through a C18 solid phase extraction cartridge, eluted with 30% methanol, the eluate was discarded, and then eluted with methanol. The eluate was collected, the solvent was recovered until dry, and the residue was dissolved in methanol to obtain a schizonepeta spicate charcoal test solution. The ultrasonic power was 300W and the frequency was 24kHz. The heating reflux time was 3-4 hours. The mass-to-volume ratio of the schizonepeta spicate charcoal granules to 70% methanol was 1.5g:25mL. The volume-to-mass ratio of the supernatant to the C18 solid phase extraction cartridge was 2ml:300mg.

[0025] A control medicinal material of Schizonepeta spicata was taken, extracted by heating with water, filtered, evaporated to dryness, and the residue was extracted by reflux with ether. The solvent was evaporated from the medicinal residue, and the residue was dissolved in 70% methanol. The mixture was ultrasonically treated and filtered. The filtrate was evaporated to dryness, and the residue was dissolved in methanol. The supernatant was taken and passed through a C18 solid phase extraction column, eluted with 30% methanol, the eluate was discarded, and the mixture was eluted with methanol again. The eluate was collected and the solvent was recovered to dryness. The residue was dissolved in methanol to obtain a control medicinal material solution.

[0026] According to the present invention, the mass ratio of the Schizonepeta spicata control medicinal material to water and heating extraction is 5g:100mL; the heating is performed by boiling for 20-30 minutes; the ultrasonic power is 300W, the frequency is 24kHz, and the heating reflux time is 3-4 hours; the mass-to-volume ratio of the Schizonepeta spicata charcoal granules to 70% methanol is 1.5g:25mL; and the volume-to-mass ratio of the supernatant to the C18 solid-phase extraction cartridge is 2ml:300mg.

[0027] The present invention investigates the extraction method and finds that only the extraction method of the present invention can effectively distinguish the granules formulated with Schizonepeta spicata and the granules formulated with Schizonepeta spicata charcoal.

[0028] The Schizonepeta spicata test solution, Schizonepeta spicata charcoal test solution and control medicinal material solution were subjected to thin layer chromatography detection using a silica gel G thin layer plate;

[0029] In some embodiments of the present invention, the silica gel G thin layer plate is a prefabricated silica gel G plate produced by Tianjin Silida or Qingdao Ocean. The results show that the method has good durability and can meet the identification requirements.

[0030] In some embodiments of the present invention, the developing solvent is dichloromethane-butyl acetate-formic acid.

[0031] In some embodiments of the present invention, the volume ratio of dichloromethane-butyl acetate-formic acid is 20:3:0.1.

[0032] The present invention investigates different developing agents and finds that only the developing agent of the present invention can produce clear spots and can well identify the granules formulated with Schizonepeta spicata and the granules formulated with Schizonepeta spicata charcoal.

[0033] Unfold, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C for 5 minutes, and inspect under ultraviolet light (365nm).

[0034] In some embodiments of the present invention, the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition General Rules 0502) is used for the test. 10 μl of each of the above solutions are taken and spotted on the same silica gel G thin layer plate, and dichloromethane-butyl acetate-formic acid (20:3:0.1) is used as the developing agent. The plate is developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C for 5 minutes, and inspected under ultraviolet light (365 nm).

[0035] If the Schizonepeta spicata test sample and the control medicinal material solution each have a spot at the transfer ratio values ​​of 0.48-0.58 and 0.60-0.74, which can be used as the identification point of the Schizonepeta spicata formula granules, the Schizonepeta spicata charcoal formula granules should not have spots of the same color at the same position.

[0036] In a preferred embodiment of the present invention, the spotting volume of the thin layer chromatography test sample is 5 to 15 μL, specifically 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, and 15 μL; the spotting volume of the control medicinal material solution is 5 to 15 μL, specifically 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, and 15 μL;

[0037] The Rf values ​​of spot 1 are 0.53 and 0.54; the Rf values ​​of spot 2 are 0.67 and 0.68.

[0038] The inspection temperature of the present invention is 4°C to 35°C; specifically it can be 4°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, and 35°C.

[0039] This method has good durability under different temperatures. Compared with the reference herbal medicine atlas, the test sample atlas and the reference herbal medicine atlas show spots of the same color at corresponding positions.

[0040] The inspection humidity of the present invention is 32% rh to 75% rh. Specifically, it can be: 32% rh, 33% rh, 34% rh, 35% rh, 36% rh, 37% rh, 38% rh, 39% rh, 40% rh, 41% rh, 42% rh, 43% rh, 44% rh, 45% rh, 46% rh, 47% rh, 48% rh, 49% rh, 50% rh, 51% rh, 52% rh, 53% rh rh, 54% rh, 55% rh, 56% rh, 57% rh, 58% rh, 59% rh, 60% rh, 61% rh, 62% rh, 63% rh, 64% r h, 65% rh, 66% rh, 67% rh, 68% rh, 69% rh, 70% rh, 71% rh, 72% rh, 73% rh, 74% rh, 75% rh.

[0041] This method has good durability under different humidity conditions. Compared with the reference herbal medicine atlas, the test sample atlas and the reference herbal medicine atlas show spots of the same color at corresponding positions.

[0042] The invention provides a thin layer chromatography identification method for schizonepeta spicata and schizonepeta spicata charcoal formula granules, comprising the following steps: A) pre-treating schizonepeta spicata formula granules to obtain a schizonepeta spicata test solution; pre-treating schizonepeta spicata charcoal formula granules to obtain a schizonepeta spicata charcoal test solution; B) taking a schizonepeta spicata control medicinal material, adding water and heating for extraction, filtering, evaporating to dryness, adding ether to the residue for reflux extraction, evaporating the solvent from the medicinal residue, dissolving the residue with 70% methanol, ultrasonically treating, filtering, evaporating the filtrate to dryness, dissolving the residue with methanol, taking the supernatant, passing it through a C18 solid phase extraction cartridge, eluting with 30% methanol, discarding the eluate, and eluting with methanol again. The eluate is collected, the solvent is recovered until dry, and methanol is added to the residue to dissolve it to obtain a control medicinal material solution; C) the schizonepeta spicate test sample solution and the schizonepeta spicate charcoal test sample solution and the control medicinal material solution are subjected to thin layer chromatography detection, the thin layer plate is a silica gel G thin layer plate; the developing solvent is dichloromethane-butyl acetate-formic acid; D) the schizonepeta spicate test sample and the control medicinal material solution are placed under ultraviolet light for inspection. If the schizonepeta spicate test sample and the control medicinal material solution each have a spot at a ratio shift value of 0.48-0.58 and 0.60-0.74, it is used as the identification point of the schizonepeta spicate formula granules. At the same position, the schizonepeta spicate charcoal formula granules should not have spots of the same color. The thin layer identification chromatography method of schizonepeta spicate and schizonepeta spicate charcoal formula granules established by the present invention is used to quickly, accurately and effectively identify schizonepeta spicate and schizonepeta spicate charcoal formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Spot sampling inspection;

[0044] Figure 2 Exclusive inspection;

[0045] Figure 3 Inspection of different thin layer boards-Tianjin Silida;

[0046] Figure 4 Investigation of different thin layer plates-Qingdao Ocean;

[0047] Figure 5 Different development temperatures were investigated at 4°C;

[0048] Figure 6 Different development temperatures were investigated at 35°C;

[0049] Figure 7 Different development humidity test 75%;

[0050] Figure 8 Different development humidity inspection 32%;

[0051] Figure 9 Verification of samples from different batches;

[0052] Figure 10 Comparative Example 1 Method Thin Layer Atlas;

[0053] Figure 11 Comparative Example 2 Method Thin Layer Atlas;

[0054] Figure 12 Comparative Example 3 Method Thin Layer Atlas;

[0055] Figure 13 Comparative Example 4 Method Thin Layer Atlas;

[0056] Figure 14 Comparative Example 5 method thin layer spectrum (under ultraviolet light (365nm));

[0057] Figure 15 Comparative Example 5 method thin layer spectrum (under visible light). DETAILED DESCRIPTION

[0058] To further illustrate the present invention, a thin layer identification method for Schizonepeta spicata and Schizonepeta spicata charcoal particles provided by the present invention is described in detail below in conjunction with examples.

[0059] Instruments: constant temperature drying oven, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel G thin layer plate (Qingdao Ocean Chemical Plant, batch number: 20240504; Tianjin Silida Technology Co., Ltd., batch number: 240716; Merck, batch number: HX31556754).

[0060] Reagents: Methanol, n-hexane, hydrochloric acid, chloroform, ether, toluene, ethyl acetate, formic acid, n-butanol, glacial acetic acid, dichloromethane, etc. were all of analytical grade, and water was ultrapure water.

[0061] Test drugs: Nepeta spicata control medicinal material (China Food and Drug Inspection Institute, batch number: 121424-202106), Nepeta spicata formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: KL01; KL02; KL03), Nepeta spicata charcoal formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: KL07; KL08; KL09)

[0062] Example 1

[0063] Take 1.5 g each of Schizonepeta spicata granules and Schizonepeta spicata charcoal granules, place them separately in a Soxhlet extractor, add an appropriate amount of ether, heat and reflux for 4 hours, discard the ether solution, evaporate the solvent from the residue, transfer it to a stoppered conical flask, add 25 ml of 70% methanol, ultrasonically treat (power 300 W, frequency 24 kHz) for 45 minutes, filter, evaporate the filtrate to dryness, add 10 ml of methanol to dissolve the residue, take 2 ml of the supernatant, pass it through a C18 (300 mg) solid phase extraction column, elute with 10 ml of 30% methanol, discard the eluent, and elute with 10 ml of methanol again, collect the eluent, recover the solvent to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0064] Take 5 g of Schizonepeta spicata reference medicinal material, add 100 ml of water, heat and boil for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the reference medicinal material solution from the residue in the Soxhlet extractor in the same way.

[0065] According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the above solutions were taken and spotted on the same silica gel G thin layer plate, and developed with dichloromethane-butyl acetate-formic acid (20:3:0.1). The plate was taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C for 5 minutes, and inspected under ultraviolet light (365 nm).

[0066] Example 2

[0067] 2.2.3.1 Inspection of sampling quantity

[0068] Under the experimental conditions proposed above, 5μl, 10μl, and 15μl of the control herbal solution and the test solution were spotted on the same silica gel G thin layer plate (manufacturer: Merck). The results were shown in the table below. Figure 1 As can be seen from the figure, when the sample volume of the control herb solution and the test solution was 5-15 μl, the TLC fluorescent spots were clearly colored, confirming that the sample volume of the control herb solution and the test solution should be 5-15 μl. Compared with the spectrum of the Schizonepeta spicata control herb, the Schizonepeta spicata granules exhibit distinct spots at Rf values ​​(Rf) of 0.53 and 0.67, while the Schizonepeta spicata charcoal granules do not have the same color spots.

[0069] Figure 1 Note: 1 to 3 are the sample volumes of Schizonepeta Spicae formula granules, 5μl, 10μl, and 15μl; 4 to 6 are the sample volumes of control medicinal materials, 5μl, 10μl, and 15μl; 7 to 9 are the sample volumes of Schizonepeta Spicae charcoal formula granules, 5μl, 10μl, and 15μl.

[0070] 2.2.3.2 Specificity Investigation

[0071] According to the above test sample preparation method, the control medicinal material solution of Schizonepeta spicata, the formula granule solution of Schizonepeta spicata granule solution, the formula granule solution of Schizonepeta spicata charcoal, and the negative solution were prepared and spotted on the same silica gel G thin layer plate (manufacturer: Merck). The plate was developed according to the proposed thin layer chromatography conditions and the results were examined. Figure 2 The results showed that negative samples did not interfere with the detection of spots in the Schizonepeta spicata and Schizonepeta spicata charcoal granules, indicating that the method had good specificity. Compared with the Schizonepeta spicata control herb atlas, the Schizonepeta spicata granules had distinct spots at ratio shifts (Rf) of 0.53 and 0.67, while the Schizonepeta spicata charcoal granules did not have spots of the same color.

[0072] Figure 2Specificity investigation; Note: 1 is the negative solution; 2 is the granules of Schizonepeta spicata; 3 is the control medicinal material of Schizonepeta spicata; 4 is the granules of Schizonepeta spicata charcoal.

[0073] 2.2.3.3 Durability Investigation

[0074] 2.3.3.3.1 Comparison of different thin layer plates

[0075] Two brands of prefabricated silica gel G plates, Qingdao Ocean and Tianjin Silida, were used to conduct tests according to the proposed test methods. Figures 3-4 The results showed that both brands of TLC plates met the expected identification requirements, demonstrating the robustness of the method. Compared with the reference herb atlas of Schizonepeta spicata, the Schizonepeta spicata granules exhibited distinct spots at Rf values ​​(Rf) of 0.53, 0.67, and 0.54, 0.68, while the Schizonepeta spicata charcoal granules showed no spots of the same color.

[0076] Figure 3 Investigation of different thin layer plates - Tianjin Silida; Note: 1 is the formula granules of Schizonepeta spicata; 2 is the control medicinal material of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal.

[0077] Figure 4 Inspection of different thin layer plates - Qingdao Ocean; Note: 1 is the formula granules of Schizonepeta spicata; 2 is the control medicinal material of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal.

[0078] 2.3.3.3.2 Comparison of different development temperatures

[0079] Take the spotted thin layer plate and place it in a low temperature environment of 4℃ and a normal temperature of 35℃ for development. Figures 5-6 It can be seen that the method has good durability under different temperatures. Compared with the reference medicinal material atlas of Schizonepeta spicata, the Schizonepeta spicata granules have obvious spots at the specific transfer values ​​(Rf) of 0.53 and 0.67, while the Schizonepeta spicata charcoal granules have no spots of the same color.

[0080] Figure 5 Different development temperatures were investigated at 4°C. Note: 1 is the granules of Schizonepeta spicata formula; 2 is the control medicinal material of Schizonepeta spicata; 3 is the granules of Schizonepeta spicata charcoal formula;

[0081] Figure 6 Different development temperatures were investigated at 35°C; Note: 1 is the formula granules of Schizonepeta spicata; 2 is the control medicinal material of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal.

[0082] 2.3.3.3.2 Comparison of different expansion humidity

[0083] Take the spotted thin layer plate and place it in 32% and 75% humidity environment for development. Figures 7-8It can be seen that the method has good durability under different humidity conditions. Compared with the atlas of the control medicinal material Schizonepeta spicata, the Schizonepeta spicata granules have obvious spots at the specific transfer values ​​(Rf) of 0.53 and 0.67, while the Schizonepeta spicata charcoal granules have no spots of the same color. Figure 7 Different humidity levels were tested at 75%; Note: 1 is the formula granules of Schizonepeta spicata; 2 is the control medicinal material of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal.

[0084] Figure 8 Different humidity levels were investigated at 32%; Note: 1 is the formula granules of Schizonepeta spicata; 2 is the control medicinal material of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal.

[0085] 2.3.3.4 Verification

[0086] Three batches of schizonepeta spicate granules and three batches of schizonepeta spicate charcoal granules were used to verify the thin layer chromatography method. The experimental results are shown in Figure 6 The results showed that compared with the atlas of the control medicinal material Schizonepeta spicata, the Schizonepeta spicata formula granules had obvious spots at the ratio transfer values ​​(Rf) of 0.53 and 0.67, while the Schizonepeta spicata charcoal formula granules had no spots of the same color. Figure 9 Verification of samples from different batches; Note: 1-3 are Schizonepeta spicata formula granules KL01, KL02, and KL03; 4 is Schizonepeta spicata control medicinal material; 5-7 are Schizonepeta spicata charcoal formula granules KL07, KL08, and KL09.

[0087] Example 3 Determination of the Rf value of the identification point of the schizonepeta spicate and schizonepeta spicate charcoal formula granules

[0088] 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 Schizonepeta spicata and Schizonepeta spicata charcoal granules should be within the range of 10%, with the specified values ​​being 0.53 and 0.67.

[0089] Table 1 Summary of methodological investigation ratios

[0090]

[0091]

[0092] Comparative Example 1

[0093] Take 1g of Schizonepeta spicata granules and 1g of Schizonepeta spicata charcoal granules, grind them into powder, add 25ml of 75% ethanol respectively, ultrasonically treat for 30 minutes, filter, and extract the filtrate with petroleum ether (60-90°C) by shaking twice, 30ml each time, combine the petroleum ether liquid, evaporate to dryness, and add 1ml of petroleum ether (60-90°C) to the residue to dissolve it as the test solution. Take another 1g of Schizonepeta spicata control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 10μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (10:1:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). The results are as follows Figure 10 shown.

[0094] Figure 10 Comparative Example 1 TLC analysis; Note: 1 is Schizonepeta Spike charcoal granules; 2 is Schizonepeta Spike charcoal granules; 3 is Schizonepeta Spike control. The results show that using the TLC identification method described above, the spots on Schizonepeta Spike charcoal granules and Schizonepeta Spike charcoal granules are identical, making the two indistinguishable.

[0095] Comparative Example 2:

[0096] Take 1g of Schizonepeta spicata granules and 1g of Schizonepeta spicata charcoal granules, grind, add 25ml of water, sonicate for 30min, filter, and extract the filtrate by shaking with ethyl acetate twice, 30ml each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1ml of ethyl acetate to prepare the test solution. Take another 2g of Schizonepeta spicata control medicinal material, add 50ml of water, decoct for 30 minutes, filter, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 15μl of each of the three solutions was taken and spotted on the same silica gel G thin layer plate. Use toluene-ethyl acetate-formic acid (10:4:0.1) as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0097] The results are as follows Figure 11 shown. Figure 11 Comparative Example 2 method thin layer chromatography; Note: 1 is the reference medicinal material of Schizonepeta spicata; 2 is the formula granules of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal.

[0098] The results showed that using the above-mentioned thin layer identification method, the spots of the granules of Schizonepeta spicata and the granules of Schizonepeta spicata charcoal were consistent and the two could not be distinguished.

[0099] Comparative Example 3:

[0100] Take appropriate amounts of Schizonepeta spicata granules and Schizonepeta spicata charcoal granules, grind them finely, take 1g, add 25ml of 75% ethanol, ultrasonicate for 30 minutes, filter, and extract twice with petroleum ether (60-90°C) by shaking, each time with 30ml. Combine the petroleum ether solutions, evaporate to dryness, and dissolve the residue in 1ml of petroleum ether (60-90°C) to prepare the test solution. Separately, take 1g of Schizonepeta spicata control medicinal material, add 40ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and add 25ml of 75% ethanol to the residue to prepare the control medicinal material solution in the same manner. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the above solutions were taken and spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (10:1:0.1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365 nm).

[0101] The results are as follows Figure 12 shown. Figure 12 Comparative Example 3 method thin layer chromatography; Note: 1 is the reference medicinal material of Schizonepeta spicata; 2 is the formula granules of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal.

[0102] The results showed that using the above-mentioned thin layer identification method, the spots of the granules of Schizonepeta spicata and the granules of Schizonepeta spicata charcoal were consistent and the two could not be distinguished.

[0103] Comparative Example 4:

[0104] Take an appropriate amount of Schizonepeta spicata granules and Schizonepeta spicata charcoal granules, grind them finely, and take approximately 1g. Add 50ml of methanol and sonicate for 30 minutes. Filter and evaporate the filtrate to dryness. Dissolve the residue in 20ml of water. Shake and extract three times with 20ml of water-saturated n-butanol. Combine the n-butanol solutions and wash once with an equal volume of ammonia test solution. Take the n-butanol solution, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 1.0g of Schizonepeta spicata control medicinal material and prepare a control medicinal material solution using the same method. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of the test solution and the control medicinal material solution were taken and spotted on the same silica gel G plate, and ethyl acetate-petroleum ether (60℃~90℃)-methanol-concentrated ammonia water (9:2:3:1) was used as the developing solvent. The plate was developed in an upward direction. The plate was taken out, dried, sprayed with 2% vanillin in 10% sulfuric acid ethanol solution and heated at 105℃ until the spots were clearly colored.

[0105] The results are as follows Figure 13 shown. Figure 13 Comparative Example 4 TLC analysis; Note: 1 is the reference herb, Schizonepeta spicata; 2 is the Schizonepeta spicata granules; 3 is the Schizonepeta spicata charcoal granules. The results show that using the TLC identification method described above, the spots on the Schizonepeta spicata granules and the Schizonepeta spicata charcoal granules are identical, making the two indistinguishable.

[0106] Comparative Example 5:

[0107] Take an appropriate amount of Schizonepeta Spike formula granules and Schizonepeta Spike charcoal formula granules, grind them finely, take about 1g, add 25ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take another 1g of Schizonepeta Spike control medicinal material and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 5μl of each of the above solutions and spot them on the same silica gel GF254 thin layer plate, use toluene-butyl acetate-formic acid (10:2:0.1) as the developing agent, develop, take out, dry, spray with 6% sulfuric acid ethanol solution, heat until the spots are clearly colored, and inspect under ultraviolet light (365nm) and visible light.

[0108] The results are as follows Figure 14 、 15 shown. Figure 14 Comparative Example 5 method thin layer chromatography (under ultraviolet light (365nm)); Note: 1 is the reference medicinal material of Schizonepeta spicata; 2 is the formula granules of Schizonepeta spicata; 3 is the formula granules of Schizonepeta spicata charcoal; Figure 15 Comparative Example 5 TLC (visible light) analysis; Note: 1 is a control herb of Schizonepeta spicata; 2 is a granule of Schizonepeta spicata; 3 is a granule of Schizonepeta spicata charcoal. The results show that using the TLC identification method described above, the spots on the granules of Schizonepeta spicata and the granules of Schizonepeta spicata charcoal are identical, making it impossible to distinguish the two.

[0109] 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 Schizonepeta spicata and Schizonepeta spicata charcoal granules, comprising: A) taking the schizonepeta spicate granules and pre-treating them to obtain a schizonepeta spicate test solution; Pre-treat the schizonepeta spicate charcoal granules to obtain a schizonepeta spicate charcoal test solution; B) Extract a control medicinal material of Schizonepeta spicata by heating with water, filtering, evaporating to dryness, and refluxing the residue with ether. The solvent is evaporated from the residue, and the residue is dissolved in 70% methanol. The mixture is sonicated, filtered, and the filtrate is evaporated to dryness. The residue is dissolved in methanol, and the supernatant is passed through a C18 solid phase extraction cartridge and eluted with 30% methanol. The eluate is discarded and eluted again with methanol. The eluate is collected and the solvent is recovered to dryness. The residue is dissolved in methanol to obtain a control medicinal material solution. C) Thin layer chromatography was performed on the control medicinal material solution using a silica gel G thin layer plate and a developing solvent of dichloromethane-butyl acetate-formic acid; D) Place the sample under ultraviolet light for inspection. If the Schizonepeta spicata test sample and the control herbal solution each have a spot at the transfer ratio of 0.48-0.58 and 0.60-0.74, these spots will serve as identification points for the Schizonepeta spicata granules. The Schizonepeta spicata charcoal granules should not have spots of the same color at the same location.

2. The method according to claim 1, characterized in that The pretreatment of the schizonepeta spicate granules in step A) is specifically as follows: The granules of Schizonepeta spicata are subjected to ether reflux extraction, the solvent is evaporated from the residue, the residue is dissolved in 70% methanol, ultrasonically treated, filtered, the filtrate is evaporated to dryness, the residue is dissolved in methanol, the supernatant is taken, passed through a C18 solid phase extraction column, eluted with 30% methanol, the eluate is discarded, and eluted again with methanol, the eluate is collected, the solvent is recovered to dryness, and methanol is added to dissolve the residue to obtain a Schizonepeta spicata test solution; the power of the ultrasonic treatment is 300W and the frequency is 24 kHz; and the heating reflux time is 3 to 4 hours.

3. The method according to claim 2, characterized in that Step A) The mass volume ratio of the Schizonepeta spicata granules and 70% methanol is 1.5 g:25 mL; the mass volume ratio of the supernatant and C18 solid phase extraction cartridge is 2 ml:300 mg.

4. The method according to claim 1, wherein The pretreatment of the schizonepeta spicate charcoal particles is specifically as follows: The granules of Schizonepeta spicata charcoal are extracted by reflux extraction with ether, the solvent is evaporated from the residue, the residue is dissolved in 70% methanol, ultrasonically treated, filtered, the filtrate is evaporated to dryness, methanol is added to the residue to dissolve it, the supernatant is taken, passed through a C18 solid phase extraction column, eluted with 30% methanol, the eluate is discarded, and then eluted with methanol, the eluate is collected, the solvent is recovered to dryness, and methanol is added to the residue to dissolve it to obtain a Schizonepeta spicata charcoal test solution; the power of the ultrasound is 300W and the frequency is 24kHz; the heating reflux time is 3 to 4h; the mass volume ratio of the Schizonepeta spicata charcoal granules and 70% methanol is 1.5g:25mL; the mass ratio of the supernatant and the C18 solid phase extraction column is 2ml:300mg.

5. The method according to claim 1, wherein The sample spotting volume of the thin layer chromatography test sample is 5 to 15 μL, and the sample spotting volume of the control medicinal material solution is 5 to 15 μL.

6. The method according to claim 1, characterized in that In step C), the volume ratio of dichloromethane-butyl acetate-formic acid is 20:3:0.

1.

7. The method according to claim 1, characterized in that Step D) The inspection includes: spraying with 10% sulfuric acid ethanol solution, heating at 105° C. for 5 minutes, and inspecting under an ultraviolet lamp; the wavelength of the ultraviolet lamp is 365 nm.

8. The method according to claim 1, characterized in that The silica gel G thin layer plate in step D) is a prefabricated silica gel G plate produced by Tianjin Silida or Qingdao Ocean.

9. The method according to claim 8, characterized in that Step D) checking the temperature to be 4° C. to 35° C.; and checking the humidity to be 32% rh to 75% rh.

10. The method according to claim 1, characterized in that Step D) The Rf values ​​of spot 1 are 0.53 and 0.54; the Rf values ​​of spot 2 are 0.67 and 0.68.

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