Thin-layer identification method for charred schizonepeta and preparation thereof
By employing solvent extraction without strong acid hydrolysis and suitable thin-layer chromatography, the problem of poor separation in the thin-layer identification method of catnip charcoal was solved, enabling effective differentiation and quality evaluation of catnip charcoal and catnip samples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGYIN TIANJIANG PHARMA
- Filing Date
- 2026-01-26
- Publication Date
- 2026-04-17
AI Technical Summary
Existing thin-layer chromatography identification methods for catnip charcoal lack effective differentiation methods, cannot accurately distinguish between samples before and after processing, and the existing methods have poor separation when applied to catnip charcoal formulation granules, which cannot meet the requirements for quality evaluation.
The test sample and control solution were prepared by solvent extraction without strong acid hydrolysis. Ethyl acetate was used for liquid-liquid partition extraction, combined with thin-layer chromatography using toluene-ethyl acetate-formic acid as the developing solvent and sulfuric acid ethanol solution as the colorimetric reagent. The samples were examined under sunlight to distinguish between charred catnip and catnip samples.
It effectively distinguishes between charred catnip and catnip samples, with good chromatographic spot separation, simple and rapid operation, and low detection cost, making it suitable for quality supervision of charred catnip and its preparations.
Smart Images
Figure CN121878100A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of quality control of traditional Chinese medicine, and in particular to a thin-layer chromatography method for the identification of charred Schizonepeta tenuifolia and its preparations. Background Technology
[0002] Catnip charcoal is a type of catnip plant belonging to the Lamiaceae family. Schizonepeta tenuifolia The dried aerial parts of *Britishia* are processed products. *Schizonepeta tenuifolia* charcoal is a processed product of *Schizonepeta tenuifolia*. The 2025 edition of the *Chinese Pharmacopoeia* does not include a thin-layer chromatography method for identifying *Schizonepeta tenuifolia* charcoal. It has been reported that the main chemical components of *Schizonepeta tenuifolia* are flavonoids, organic acids, and tannins. Raw *Schizonepeta tenuifolia* has the functions of dispelling wind and relieving exterior syndromes, clearing toxins and promoting rash eruption, and is mainly used to treat colds due to wind-cold, sore throat, and various skin diseases. Charcoal *Schizonepeta tenuifolia* has astringent and hemostatic effects and can be used for bleeding such as hematochezia, metrorrhagia, and postpartum hemorrhage. The significant difference in efficacy between the two indicates that the effective components of *Schizonepeta tenuifolia* change during the processing. *Schizonepeta tenuifolia* mainly contains phenolic acids, flavonoids, and volatile oils, among which rosmarinic acid and caffeic acid are representative phenolic acids, quercetin and hesperidin are representative flavonoids, and menthone and menthol are representative volatile oils. Modern research shows that, compared with catnip, no menthone, rosmarinic acid and menthone were detected in catnip charcoal; the changes in hesperidin and quercetin were not obvious.
[0003] There are few literature reports on existing thin-layer identification methods for nepeta charcoal, and when the existing methods are applied to nepeta charcoal formulation granules, the thin-layer spots are few and the separation is poor. They also cannot distinguish between samples before and after processing, and cannot accurately evaluate the quality. Summary of the Invention
[0004] Purpose of the invention: To address the shortcomings and defects of existing technologies, this invention provides a thin-layer chromatography method for identifying charred catnip and its preparations. This method presents the specific components of charred catnip to the greatest extent, can distinguish charred catnip from catnip samples, is simple and rapid to operate, has good chromatographic spot separation, and is low in detection cost, safe and environmentally friendly.
[0005] Technical solution: The thin-layer chromatography identification method for charred catnip and its preparations according to the present invention includes the following steps: (1) Preparation of test solution: Take the charred catnip sample and process it with solvent extraction without strong acid hydrolysis. The resulting extract is extracted with ethyl acetate by liquid-liquid partition extraction to prepare the test solution. (2) Preparation of control solution: Take the charred Schizonepeta tenuifolia control slices and process them with a solvent extraction method without strong acid hydrolysis. The resulting extract is extracted with ethyl acetate by liquid-liquid partition extraction to obtain the control slice solution. (3) Thin-layer chromatography identification: The reference decoction piece solution and the test sample solution are spotted on the same thin-layer plate, developed with the developing solvent, and then sprayed with a color developing agent. The plate is then examined under a fluorescent lamp to obtain a daylight thin-layer chromatogram, thus completing the identification operation. The developing solvent is toluene-ethyl acetate-formic acid.
[0006] Preferably, in step (2), the reference slices of Schizonepeta tenuifolia are decocted with water or heated under reflux, filtered, the filtrate is concentrated to near dryness, treated with methanol by sonication, evaporated to dryness, dissolved in water, and extracted with ethyl acetate by shaking. After standing and separating into layers, the ethyl acetate layers are combined, evaporated to dryness, and the residue is dissolved in an alcohol solvent to obtain the reference slice solution.
[0007] Preferably, in step (1), the catnip charcoal sample is catnip charcoal slices; the preparation of its test solution includes: taking the catnip charcoal sample, adding water to decoct or heating to reflux, filtering, concentrating the filtrate to near dryness, adding methanol for ultrasonic treatment, evaporating to dryness, dissolving in water and then adding ethyl acetate for shaking extraction, allowing to stand for layering and then combining the ethyl acetate layers, evaporating to dryness, dissolving the residue in an alcohol solvent to obtain the test solution; Alternatively, in step (1), the catnip charcoal sample is catnip charcoal formulation granules or catnip charcoal standard decoction; the preparation of its test solution includes: taking the catnip charcoal sample, adding an alcohol solvent for ultrasonic extraction, filtering, evaporating the filtrate to dryness, dissolving it in water, adding ethyl acetate for shaking extraction, allowing it to stand for layering and combining the ethyl acetate layers, evaporating to dryness, dissolving the residue in an alcohol solvent to obtain the test solution.
[0008] Preferably, when the charred catnip reference slices are decocted with water, the mass-to-volume ratio of the reference slices to water is 1:25~50 g / mL, and the decocting or reflux treatment time is 30~60 min.
[0009] Preferably, when the catnip charcoal sample is catnip charcoal slices, the mass-to-volume ratio of the catnip charcoal slices to water during decoction is 1:25~50 g / mL, and the decoction or reflux treatment time is 30~60 min. More preferably, the mass-to-volume ratio of the control slices, catnip charcoal slices, and water is 1:50 g / mL, and the decoction or reflux treatment time is 60 min.
[0010] Preferably, in step (1), when the catnip charcoal sample is catnip charcoal formulation granules or catnip charcoal standard decoction, the mass-to-volume ratio of the catnip charcoal sample to methanol during methanol extraction is 1:25~50 g / mL, the ultrasonic treatment power is 200~250 W, the frequency is 35~45 kHz, the time is 20~30 min, the ethyl acetate shaking extraction is performed 2~3 times, and the volume ratio of ethyl acetate solution to the sample aqueous solution is 1~1.2:1. More preferably, the mass-to-volume ratio of the catnip charcoal sample to methanol is 1:50 g / mL, the ultrasonic treatment power is 250 W, the frequency is 40 kHz, the time is 30 min, the ethyl acetate shaking extraction is performed 2 times, and the volume ratio of ethyl acetate solution to the sample aqueous solution is 1:1.
[0011] Preferably, the volume ratio of toluene-ethyl acetate-formic acid in the developing solvent in step (3) is 8:6~7:0.1. More preferably, the volume ratio of toluene-ethyl acetate-formic acid in the developing solvent is 8:6:0.1.
[0012] Preferably, the amount of sample spotting for the control decoction and the test sample solution in step (3) is 2 μl to 3 μl.
[0013] Preferably, the color-developing agent in step (3) is a 9-11% sulfuric acid ethanol solution, heated at 104-106°C until the spots are clear. More preferably, the color-developing agent is a 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly colored.
[0014] Preferably, the thin film in step (3) is a silicone G thin film.
[0015] In step (3), the thin-layer chromatogram shows spots of the same color at the corresponding positions as the chromatogram of the reference decoction pieces.
[0016] Principle Analysis: Based on the chemical structure and properties of the active ingredients in traditional Chinese medicine, and following the extraction principle of "like dissolves like," a suitable extraction solvent is used to easily and quickly prepare the test sample and reference medicinal material or reference decoction pieces solution. Then, using a suitable developing solvent, the various chemical components are developed, and based on their different adsorption, desorption, re-adsorption, and re-desorption capacities, they are well separated on the thin-layer plate. Furthermore, by utilizing the fact that various active ingredients with similar polarities appear as distinct color spots on the same thin-layer plate under different viewing conditions (sunlight, ultraviolet light, or colorimetric reagent), a multi-information thin-layer chromatogram is obtained.
[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention employs a simple and rapid pretreatment method to obtain the test solution and the reference herbal solution, which are then spotted separately on the same thin-layer plate. After development with a suitable developing solvent and spraying with a colorimetric reagent, the chromatogram is examined under sunlight to obtain a multi-information thin-layer chromatogram of schizonepeta charcoal. This invention establishes for the first time a method for identifying the prototype components of schizonepeta charcoal that does not rely on acid hydrolysis, thus filling the gap in the pharmacopoeia's lack of such a thin-layer detection method. Furthermore, it can distinguish between schizonepeta and schizonepeta charcoal samples, has low detection costs, and is more conducive to quality supervision. Attached Figure Description
[0018] Figure 1 This is a thin-layer chromatogram of the charred granules of the catnip formulation in Example 1 of the present invention.
[0019] Figure 2 This is a thin-layer chromatogram of the standard decoction of schizonepeta charcoal in Example 1 of the present invention.
[0020] Figure 3 This is a thin-layer chromatogram of the charred Schizonepeta tenuifolia slices in Example 1 of the present invention.
[0021] Figure 4 These are thin-layer chromatograms of different spotting amounts in Example 2 of the present invention.
[0022] Figure 5 These are thin-layer chromatograms taken at different temperatures in Embodiment 2 of the present invention.
[0023] Figure 6 This is a thin-layer chromatogram of different humidity levels in Embodiment 2 of the present invention.
[0024] Figure 7 These are thin-layer chromatograms of thin-layer plates from different brands in Embodiment 2 of the present invention.
[0025] Figure 8 This is a thin-layer chromatogram of different developing systems of the charred granules of the catnip formulation in Comparative Example 1 of this invention.
[0026] Figure 9 This is a thin-layer chromatogram of the charred catnip formulation particles in Comparative Example 1 of this invention under different inspection conditions.
[0027] Figure 10 This is a thin-layer chromatogram of the preparation methods of the charred catnip formulation particles in Comparative Example 1 of this invention, using hydrochloric acid hydrolysis and non-hydrolysis.
[0028] Figure 11 This is a thin-layer chromatogram of the charred granules of Schizonepeta tenuifolia and the control decoction pieces prepared by different methods in Comparative Example 2 of this invention.
[0029] Figure 12 This is a thin-layer chromatogram of the standard decoction of schizonepeta charcoal and the standard decoction of schizonepeta in Comparative Example 3 of this invention. Detailed Implementation
[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0031] The instruments and reagents used in this invention are as follows: Instruments: Automated thin-layer imaging system (CAMAG TLC VISUALIZER), 0.001 g balance (Mettler-Toledo, Switzerland), GKC temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.), silica gel G thin-layer plates (Aladdin, Qingdao Kangyexin Pharmaceutical Silica Gel Desiccant Co., Ltd., Yantai Xincheng Silica Gel Material Co., Ltd., Merck, Germany).
[0032] Reagents: Methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), hydrochloric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.), toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd.), cyclohexane (Sinopharm Chemical Reagent Co., Ltd.), and formic acid 88% (Shanghai Titan Technology Co., Ltd.) were all of analytical grade.
[0033] The charred Schizonepeta tenuifolia reference slices, used as a working control, batch number YP1012406613, were prepared by Tianjiang Pharmaceutical Co., Ltd.
[0034] The charred slices of Schizonepeta tenuifolia, the standard decoction of Schizonepeta tenuifolia, the slices of Schizonepeta tenuifolia, and the granules of Schizonepeta tenuifolia are all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.
[0035] For experiments not specifically described in this invention, the procedures or conditions can be performed according to conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.
[0036] Example 1
[0037] Preparation of the test solution of the medicinal slices: Take 2g of charred Schizonepeta tenuifolia slices, add 50ml of water, heat under reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to the residue to dissolve, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the test solution.
[0038] Preparation of test solution for granules: Take 0.5g of this product, grind it into a fine powder, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use this as the test solution.
[0039] Preparation of standard decoction test solution: Take 0.3g of this product, grind it into a fine powder, add 20ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.
[0040] Preparation of the control herbal solution: Take 2g of charred Schizonepeta tenuifolia control herbal slices, add 50ml of water, heat under reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to the residue to dissolve, extract twice with ethyl acetate (25ml each time), combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the control herbal solution.
[0041] Thin-layer identification procedure: Perform the thin-layer chromatography test (General Chapter 0502 of Chinese Pharmacopoeia 2025). Take 2-3 μL of the test solution and the reference decoction solution and spot them separately on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (8:6:0.1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight.
[0042] Evaluation requirements: In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatogram of the reference decoction pieces.
[0043] Results analysis: Figure 1 Chromatograms 1-3 are chromatograms of test samples from different batches of charred granules of Schizonepeta tenuifolia, and S is the chromatogram of reference slices of charred granules of Schizonepeta tenuifolia. Figure 2 Chromatograms 1-17 are chromatograms of test samples from different batches of charred schizonepeta standard decoction, and S is the chromatogram of charred schizonepeta reference slices. Figure 3 Chromatograms 1-17 show the test samples of different batches of charred Schizonepeta tenuifolia slices, and S is the reference chromatogram of charred Schizonepeta tenuifolia slices. The charred Schizonepeta tenuifolia slices, granules, and standard decoctions all correspond one-to-one with the chromatograms of the reference slices.
[0044] Example 2
[0045] The robustness verification of the thin-layer identification in Example 1 was specifically performed, including: (1) Investigation of different sample sizes: Take the Schizonepeta Carbonis Formula Granules (batch number: 24100029) and the Schizonepeta Carbonis control cut crude drug, and prepare the test solution and the control cut crude drug solution respectively according to the method described in Example 1. Then take 1 μL, 2 μL, 3 μL, 4 μL of the Schizonepeta Carbonis Formula Granules test solution and 1 μL, 2 μL, 3 μL, 4 μL of the Schizonepeta Carbonis control cut crude drug solution, spot them on the same silica gel G thin layer plate, use toluene - ethyl acetate - formic acid (8∶6∶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 examine under daylight. The results are shown in Figure 4 , Figure 4 In Figure 4 , 1 - 4 respectively correspond to the chromatograms of the Schizonepeta Carbonis Formula Granules test samples of 1 μL, 2 μL, 3 μL, 4 μL, and S1 - S4 respectively correspond to the chromatograms of the Schizonepeta Carbonis control cut crude drug of 1 μL, 2 μL, 3 μL, 4 μL. It can be seen from Figure 4 that when the sample application volume is 2 μL - 3 μL, clear spots appear at the corresponding positions in the chromatograms of the Schizonepeta Carbonis Formula Granules and the control cut crude drug, without other interference.
[0046] (2) Investigation at different temperatures: Take the Schizonepeta Carbonis Formula Granules (batch number: 24100029) and the Schizonepeta Carbonis control cut crude drug, and prepare the test solution and the control cut crude drug solution respectively according to the method described in Example 1. Then take 2 μL of the Schizonepeta Carbonis Formula Granules test solution and 2 μL of the Schizonepeta Carbonis control cut crude drug solution, spot them on the same silica gel G thin layer plate, use toluene - ethyl acetate - formic acid (8∶6∶0.1) as the developing agent, and develop respectively under low temperature (T: 4 °C, RH: 50.1%), normal temperature (T: 23.6 °C, RH: 50.1%) and high temperature (T: 40 °C, RH: 50.1%) conditions. Take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly colored, and examine under daylight. The results are shown in Figure 5 , Figure 5 In Figure 5 , 1 - 3 respectively correspond to the chromatograms of the Schizonepeta Carbonis Formula Granules test samples, and S corresponds to the chromatogram of the Schizonepeta Carbonis control cut crude drug. It can be seen from Figure 5 that under different temperature conditions, spots of the same color appear at the corresponding positions in the chromatograms of the Schizonepeta Carbonis Formula Granules test samples and the control cut crude drug, and the separation effect is good. The experimental results show that temperature has little influence on the thin layer identification of the Schizonepeta Carbonis Formula Granules, indicating that this thin layer identification method has good durability for different temperatures.
[0047] (3) Investigation at different humidities: Take Herba Schizonepetae Carbonisata formula granules (batch number: 24100029) and Herba Schizonepetae Carbonisata control cut pieces, and prepare test solution and control cut piece solution respectively according to the method described in Example 1. Then take 2 μL of the test solution of Herba Schizonepetae Carbonisata formula granules and 2 μL of the control cut piece solution of Herba Schizonepetae Carbonisata, spot them on the same silica gel G thin layer plate, use toluene - ethyl acetate - formic acid (8∶6∶0.1) as the developing agent, develop under different humidity conditions (RH: 18%, 50.1%, 88%) respectively, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly colored, and examine under daylight. The results are shown in Figure 6 , Figure 6 In Figure 6 , 1 - 3 respectively correspond to the chromatogram of the test product of Herba Schizonepetae Carbonisata formula granules, and S corresponds to the chromatogram of Herba Schizonepetae Carbonisata control cut pieces. It can be seen from Figure 6 that under different humidity conditions, the spots of the same color appear at the corresponding positions of the chromatogram of the test product of Herba Schizonepetae Carbonisata formula granules and the chromatogram of the control cut pieces, and the separation effect is good. The experimental results show that humidity has little influence on the thin layer identification of Herba Schizonepetae Carbonisata formula granules, indicating that this thin layer identification method has good durability for different humidity.
[0048] (4)Investigation of different brands of thin layer plates: Take Herba Schizonepetae Carbonisata formula granules (batch number: 24100029) and Herba Schizonepetae Carbonisata control cut pieces, and prepare test solution and control cut piece solution respectively according to the method described in Example 1. Then take 2 μL of the test solution of Herba Schizonepetae Carbonisata formula granules and 2 μL of the control cut piece solution of Herba Schizonepetae Carbonisata, spot them on the same thin layer plates of silica gel G of different brands (Qingdao Marine, Aladdin, Yantai Xincheng), use toluene - ethyl acetate - formic acid (8∶6∶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 examine under daylight. The results are shown in Figure 7 , Figure 7 In Figure 7 , 1 - 3 respectively correspond to the chromatogram of the test product of Herba Schizonepetae Carbonisata formula granules, and S corresponds to the chromatogram of Herba Schizonepetae Carbonisata control cut pieces. It can be seen from Figure 7 that under different brands of silica gel G thin layer plates, the spots of the same color appear at the corresponding positions of the chromatogram of the test product of Herba Schizonepetae Carbonisata formula granules and the chromatogram of the control cut pieces, and the separation effect is good. The experimental results show that humidity has little influence on the thin layer identification of Herba Schizonepetae Carbonisata formula granules, indicating that this thin layer identification method has good durability for different humidity.
[0049] Comparative Example 1: In this comparative example, different developing agent systems were compared using Herba Schizonepetae Carbonisata formula granules. The specific process is as follows: Preparation of the test solution: Take 0.5g of schizonepeta charcoal granules, grind them finely, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use as the test solution.
[0050] Preparation of the control decoction solution: Take 2g of charred Schizonepeta tenuifolia control decoction, add 50ml of water, heat under reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to the residue to dissolve, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the control decoction solution.
[0051] Different developing systems: 1) Toluene-acetone-methanol-formic acid (6∶3∶0.5∶2.5), examined under ultraviolet light (365nm); 2) Cyclohexane-ethyl acetate-formic acid (6∶4∶0.1), with 5% vanillic acid and 10% sulfuric acid ethanol solution as the color reagent, examined under sunlight; 3) Toluene-ethyl acetate-formic acid (6∶4∶0.1), with 10% sulfuric acid ethanol solution as the color reagent, examined under sunlight; 4) The developing solvent of this invention: toluene-ethyl acetate-formic acid (8∶6∶0.1), with 10% sulfuric acid ethanol solution as the color reagent, examined under sunlight. The test solution of the schizonepeta charcoal formulation granules and the control decoction piece solution prepared by the above method were spotted onto a silica gel G thin-layer plate, developed under the different thin-layer identification conditions described above, sprayed with the specified color reagent, and examined under the specified conditions.
[0052] Results analysis: Figure 8 ① to ④ correspond to three different developing systems. ① The thin-layer speckle tailing phenomenon is severe, and the separation is poor, so it is not suitable for identifying the charred granules of *Schizonepeta tenuifolia*. When using developing system ②, the speckle separation is poor. When using developing system ③, the speckle shift value is low, and the specks cannot be completely separated. When using developing system ④, i.e., the developing system in this invention, the separation of the granules is good, the speckle information is rich, and they correspond one-to-one with the control slices.
[0053] Comparative Example 2: This comparative example uses nepeta charcoal granules to compare different inspection conditions. The specific process is as follows: Preparation of the test solution: Take 0.5g of schizonepeta charcoal granules, grind them finely, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use as the test solution.
[0054] Preparation of the control decoction solution: Take 2g of charred Schizonepeta tenuifolia control decoction, add 50ml of water, heat under reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to the residue to dissolve, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the control decoction solution.
[0055] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2025 Edition). Take 2 μL of each of the above three test solutions and 2 μL of each of the two reference decoction pieces solutions, and spot them on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (8:6:0.1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm) and sunlight respectively.
[0056] Results Analysis: Based on Figure 9 Under ultraviolet light (365nm), the spots were blurry and the separation was poor; under sunlight, the separation of the formula granules was good, the spot information was rich and corresponded one-to-one with the control decoction pieces.
[0057] Comparative Example 3: This comparative example used schizonepeta charcoal granules to compare the differences in thin-layer chromatography between hydrolyzed and non-hydrolyzed samples under hydrochloric acid hydrolysis. The specific procedure is as follows: Preparation of the test solution: 1) Take 0.5g of schizonepeta charcoal granules, grind them finely, add 25ml of 20% hydrochloric acid, heat under reflux for 1 hour, cool, centrifuge, take the supernatant, and extract twice with ethyl acetate, 25ml each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution. 2) Take 0.5g of schizonepeta charcoal granules, grind them finely, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25ml of water, and extract twice with ethyl acetate, 25ml each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution.
[0058] Preparation of the control decoction solution: Take 2g of charred Schizonepeta tenuifolia control decoction, add 50ml of water, heat under reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to the residue to dissolve, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the control decoction solution.
[0059] Results Analysis: Based on Figure 101 is the chromatogram of the test sample of the charred granules of Nepeta cataria prepared by hydrolysis with hydrochloric acid, 2 is the chromatogram of the test sample of the charred granules of Nepeta cataria prepared without hydrolysis with hydrochloric acid, and S corresponds to the above-mentioned control decoction solution. The results show that the spots after hydrolysis with hydrochloric acid are less than those of the non-hydrolyzed sample, and the chromatographic spot information is less.
[0060] Comparative Example 4: This comparative example compares the same developing solvent system, namely, the preparation method of the test sample and control decoction piece solution using the developing solvent of this invention, for the detection of schizonepeta charcoal formulation granules. The specific process is as follows: (1) Preparation methods of test samples of different catnip charcoal formulations: 1) Take 0.5g of schizonepeta charcoal granules, grind them into a fine powder, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.
[0061] 2) Take 0.5g of the charcoal formulation granules of Schizonepeta tenuifolia, grind them into a fine powder, add 25ml of water to dissolve them, and extract them twice with ethyl acetate, 25ml each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution.
[0062] 3) Take 0.5g of schizonepeta charcoal granules, grind them finely, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.
[0063] (2) Preparation methods of different charred catnip control decoction pieces: S1: Take 2g of charred Schizonepeta tenuifolia reference material, add 50ml of water, heat under reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to the residue to dissolve, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the reference decoction solution.
[0064] S2: Take 2g of charred Schizonepeta as a reference herb, add 25ml of methanol, sonicate for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 25ml of water to dissolve the residue, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use as the reference herb solution.
[0065] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2025 Edition). Take 2 μL of each of the above three test solutions and 2 μL of each of the two reference decoction pieces solutions, and spot them on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (8:6:0.1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight.
[0066] Results analysis: Figure 11 Chromatograms 1-3 show the test samples of the charred granules of *Schizonepeta tenuifolia* prepared by the three corresponding methods, while S1-S2 show the reference slices of *Schizonepeta tenuifolia* prepared by the two corresponding methods. When the test samples of the charred granules of *Schizonepeta tenuifolia* were prepared using methods 1 and 2, the sample spots were either blurred or few, while the spots were clear and round when prepared using method 3. When the reference slices were prepared using method 1, the spots were well separated, abundant, and clear, and the spots of the granules prepared by method 3 corresponded one-to-one.
[0067] Comparative Example 5: This comparative example compares the same developing solvent system, specifically comparing the thin-layer chromatographic differences between the standard decoction of Schizonepeta tenuifolia using the developing solvent of this invention and the standard decoction of processed Schizonepeta tenuifolia charcoal. The specific process is as follows: Preparation method of test sample: Take 0.3g of standard decoction of Schizonepeta or standard decoction of Schizonepeta charcoal, grind finely, add 20ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use as the test sample solution.
[0068] Preparation of the control decoction solution: Take 2g of charred Schizonepeta tenuifolia control decoction, add 50ml of water, heat under reflux for 1 hour, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to the residue to dissolve, extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the control decoction solution.
[0069] Results analysis: Figure 12 In the figures 1-3, the test samples of the corresponding processed Schizonepeta charcoal standard decoction are shown respectively; in the figures 4-6, the test samples of the corresponding Schizonepeta standard decoction are shown respectively; in the figure S, the chromatogram of the Schizonepeta charcoal reference decoction is shown. The thin-layer chromatogram spots of the Schizonepeta charcoal standard decoction and the Schizonepeta standard decoction do not correspond, which proves that the standard decoctions prepared after the Schizonepeta decoction is charred are quite different. This thin-layer method can distinguish the two well.
[0070] Comprehensive Analysis: This invention provides a thin-layer chromatography (TLC) method for distinguishing between charred and processed *Schizonepeta tenuifolia* (Jingjie) and its preparations, as well as raw and processed samples. The sample preparation process is simple and rapid. The developing solvent used in this invention can be used for TLC identification of both raw and processed *Schizonepeta tenuifolia* charred slices and preparations, resulting in richer chromatographic spots and better separation. This TLC method overcomes the deficiency of existing pharmacopoeia entries lacking TLC identification for *Schizonepeta tenuifolia* charred samples. It can also distinguish between *Schizonepeta tenuifolia* and processed *Schizonepeta tenuifolia* charred samples, thus differentiating between raw and processed samples, and can serve as a means of testing for raw and processed samples.
Claims
1. A method for identifying Schizonepeta Herb charcoals and preparations thereof by thin layer chromatography, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the charred sample of catnip and process it with solvent extraction method without strong acid hydrolysis. The obtained extract is extracted with ethyl acetate by liquid-liquid partition extraction to prepare the test solution. (2) Preparation of control solution: Take the charred Schizonepeta tenuifolia control slices and process them using a solvent extraction method without strong acid hydrolysis. The resulting extract is extracted with ethyl acetate by liquid-liquid partition extraction to obtain the control slice solution. (3) Thin-layer chromatography identification: The reference decoction piece solution and the test sample solution are spotted on the same thin-layer plate, developed with the developing solvent, and then sprayed with a color developing agent. The plate is then examined under a fluorescent lamp to obtain a daylight thin-layer chromatogram, thus completing the identification operation. The developing solvent is toluene-ethyl acetate-formic acid.
2. The method of claim 1, wherein, In step (2), the reference slices of Schizonepeta tenuifolia were decocted with water or heated under reflux, filtered, the filtrate was concentrated to near dryness, methanol was added for ultrasonic treatment, evaporated to dryness, dissolved in water, and then extracted with ethyl acetate by shaking. After standing and separating into layers, the ethyl acetate layers were combined, evaporated to dryness, and the residue was dissolved in an alcohol solvent to obtain the reference slice solution.
3. The method of claim 1, wherein, In step (1), the charred catnip sample is charred catnip slices; The preparation of the test solution includes: taking a sample of catnip charcoal, adding water and boiling or heating under reflux, filtering, concentrating the filtrate to near dryness, adding methanol and sonicating, evaporating to dryness, dissolving in water and then adding ethyl acetate for extraction by shaking, allowing the layers to stand and separate, combining the ethyl acetate layers, evaporating to dryness, dissolving the residue in an alcohol solvent to obtain the test solution; Alternatively, in step (1), the catnip charcoal sample is catnip charcoal formulation granules or catnip charcoal standard decoction; the preparation of its test solution includes: taking the catnip charcoal sample, adding an alcohol solvent for ultrasonic extraction, filtering, evaporating the filtrate to dryness, dissolving it in water, adding ethyl acetate for shaking extraction, allowing it to stand for layering and combining the ethyl acetate layers, evaporating to dryness, dissolving the residue in an alcohol solvent to obtain the test solution.
4. The method as claimed in claim 2, wherein the said method is a TLC method for identification of catmint charcoal and its preparations. When the reference herb charcoal is decocted with water, the mass-to-volume ratio of the reference herb to water is 1:25~50 g / mL, and the decocting or reflux treatment time is 30~60 min.
5. The method as claimed in claim 3, wherein the said method is a TLC method for identification of catmint charcoal and its preparations. When the charred catnip sample is charred catnip slices, the mass-to-volume ratio of the charred catnip slices to water during decoction is 1:25~50 g / mL, and the decoction or reflux treatment time is 30~60 min.
6. The method as claimed in claim 3, wherein the said method is a TLC method for identification of catmint charcoal and its preparations. In step (1), when the catnip charcoal sample is catnip charcoal formulation granules or catnip charcoal standard decoction, when the catnip charcoal sample is extracted with methanol, the mass-volume ratio of the catnip charcoal sample to methanol is 1:25~50g / mL, the ultrasonic treatment power is 200~250W, the frequency is 35~45kHz, the time is 20~30min, the number of times the ethyl acetate is shaken is 2~3, and the volume ratio of ethyl acetate solution to the sample aqueous solution is 1~1.2:
1.
7. The method as claimed in claim 1, wherein the said method is a TLC method for identification of catmint charcoal and its preparations. The volume ratio of toluene-ethyl acetate-formic acid in the developing solvent in step (3) is 8:6~7:0.
1.
8. The thin-layer chromatography identification method for charred catnip and its preparations according to claim 1, characterized in that, In step (3), the sample amounts of the control decoction and the test sample solution are 2 μl to 3 μl.
9. The thin-layer chromatography identification method for charred catnip and its preparations according to claim 1, characterized in that, The colorimetric agent in step (3) is a 9-11% sulfuric acid ethanol solution, heated at 104-106°C until the spots are clear.
10. The thin-layer chromatography identification method for charred catnip and its preparations according to claim 1, characterized in that, The thin-layer plate mentioned in step (3) is a silicone G thin-layer plate.