A thin layer identification method for Pseudocones and its application

Abstract

The present invention belongs to the field of pseudocong identification, and specifically relates to a thin-layer identification method of pseudocong and its application. The pseudocong medicinal material thin-layer chromatography identification method of the present invention uses pseudocong control medicinal materials and vitexin-2-O-rhamnoside as controls, optimizes and establishes a thin-layer chromatography method, can accurately and quickly identify pseudocong medicinal materials, and has a simple operation method, good repeatability, and strong characteristic. By adopting the identification method of the present invention, the betel leaves that are very similar to pseudocong medicinal materials can be accurately distinguished, which greatly facilitates the application of pseudocong medicinal materials and provides a new method for the identification of pseudocong medicinal materials.

Description

Technical Field

[0001] The invention belongs to the field of pseudocong identification, and particularly relates to a pseudocong thin layer identification method and application thereof. Background Art

[0002] Pseudocong is a plant of the Piperaceae family, and its aerial part is a medicinal material. However, the current identification standards for Pseudocong medicinal materials are not perfect, and it is difficult to quickly and effectively distinguish Pseudocong medicinal materials from other similar medicinal materials, which seriously affects the application of Pseudocong medicinal materials. Therefore, developing a method for quickly and effectively identifying Pseudocong medicinal materials has important application value for the rapid and accurate identification of Pseudocong medicinal materials. Summary of the invention

[0003] The invention provides a thin layer chromatography identification method of Pseudocongol and application thereof, and can quickly identify Pseudocongol medicinal materials through thin layer chromatography.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A thin layer identification method for pseudocone comprises the following steps:

[0006] (1) Preparation of test solution

[0007] Take the test sample powder, add anhydrous ethanol and sonicate, and take the filtrate as the test solution; prepare the control medicinal material solution of Pseudoconazole in the same way; take the vitexin-2-O-rhamnoside reference substance, add anhydrous ethanol to prepare a solution containing 0.2 mg per 1 ml, as the reference solution;

[0008] (2) Thin layer chromatography detection

[0009] TLC detection of control medicinal materials: Take equal amounts of the test sample solution and the control medicinal material solution, spot them on the same silica gel thin layer plate, develop them with cyclohexane-ethyl acetate-acetone in a volume ratio of 7:2:1 as the developing solvent, take them out, dry them, spray them with sulfuric acid ethanol solution, heat them until the spots are clearly colored, and examine them under sunlight and ultraviolet light respectively;

[0010] TLC detection of reference substance: Take 2 μL of the reference substance solution, reference medicinal material solution and test substance solution, spot them on the same silica gel thin layer plate, develop them with n-butanol-formic acid-water in a volume ratio of 8:1:1, take them out, dry them, spray them with aluminum chloride test solution, heat them until the spots are clearly colored, and inspect them under ultraviolet light;

[0011] In the thin layer chromatography test of the control medicinal material, spots of the same color should appear at the corresponding positions of the test sample chromatogram as those of the control medicinal material chromatogram and the control substance chromatogram; in the thin layer chromatography test of the control substance, spots of the same color should appear at the corresponding positions of the control medicinal material chromatogram and the control substance chromatogram; if both the thin layer chromatography test of the control medicinal material and the thin layer chromatography test of the control substance meet the requirements, the test sample is Pseudocong, and if any one of the tests does not meet the requirements, the test sample is not Pseudocong.

[0012] Furthermore, the silica gel thin layer plate in step (2) is a silica gel G thin layer plate.

[0013] Furthermore, in the step (1), the volume ratio of the test sample powder mass to the added anhydrous ethanol is 1:10, and the ultrasonic time is 20 min.

[0014] Furthermore, in the control medicinal material thin layer chromatography test in step (2), 2 µL of the test sample solution and the control medicinal material solution are respectively spotted on the same silica gel thin layer plate.

[0015] Furthermore, in the step (1), the volume concentration of the sulfuric acid ethanol solution is 10%; and the wavelength of the ultraviolet lamp is 365nm.

[0016] Furthermore, in the thin layer chromatography test of the reference substance in step (2), 2 µL of the reference substance solution, the reference medicinal material solution and the test substance solution are respectively spotted on the same silica gel thin layer plate.

[0017] In the step (2), the expansion distance is 8-12 cm.

[0018] Furthermore, in the step (2), the silica gel thin layer plate is heated at 105°C.

[0019] The above-mentioned thin-layer identification method of Pseudocong is applied in identifying Pseudocong medicinal materials.

[0020] The above-mentioned thin-layer identification method of Pseudocong is applied to distinguishing Pseudocong medicinal materials and Betel leaves.

[0021] Beneficial effects of the present invention:

[0022] The thin layer chromatography identification method of the medicinal material of Pseudocong, using the Pseudocong control medicinal material and vitexin-2-O-rhamnoside as controls, optimizes and establishes a thin layer chromatography method, which can accurately and quickly identify the medicinal material of Pseudocong, has a simple operation method, good repeatability, and strong characteristic. The identification method of the present invention can accurately distinguish the Betel leaf ( Piper betle L.), which greatly facilitates the application of Pseudocong medicinal materials and provides a new method for the identification of Pseudocong medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 To develop a thin layer chromatogram for system optimization, samples: 1. piperine; 2. pellitorycin; 3. vitexin-2-O-rhamnoside; 4. JJ-4; 5. JJ-10; 6. reference herbal medicine; 7. piperine; 8. pellitorycin; 9. vitexin-2-O-rhamnoside. Inspection: lower left: daylight; upper and lower right: UV light (365 nm).

[0025] Figure 2 Expand the TLC of the system optimization, samples: 1. piperine; 2. JJ-4 + piperine; 3. JJ-4; 4. JJ-4 + pellitoryline; 5. pellitoryline. Inspection: lower left: daylight; upper and lower right: UV light (365 nm).

[0026] Figure 3 Expand the thin layer chromatogram of the system optimization, samples: 1. Vitexin-2-O-rhamnoside; 2. JJ-4; 3. JJ-10; 4. JJ-16.

[0027] Figure 4 Thin layer chromatograms of the optimized development system, samples: 1. Vitexin-2-O-rhamnoside; 2. JJ-4; 3. JJ-10; 4. JJ-16. Developing solvent: left: ethyl acetate-formic acid-water (6:1:1); right: n-butanol-formic acid-water (8:1:1).

[0028] Figure 5 Thin layer chromatograms of the extraction solvent investigation, samples: 1. Anhydrous ethanol extract (JJ-4); 2. Ethyl acetate extract (JJ-4); 3. Acetone extract (JJ-4); 4. Anhydrous ethanol extract (control medicinal material); 5. Ethyl acetate extract (control medicinal material); 6. Acetone extract (control medicinal material). Inspection: Left: Daylight; Right: UV light (365 nm).

[0029] Figure 6Thin layer chromatogram of extraction solvent investigation, samples: 1. Vitexin-2-O-rhamnoside; 2. Anhydrous ethanol extract (JJ-4); 3. Ethyl acetate extract (JJ-4); 4. Acetone extract (JJ-4); 5. Anhydrous ethanol extract (control medicinal material); 6. Ethyl acetate extract (control medicinal material); 7. Acetone extract (control medicinal material); 8. Vitexin-2-O-rhamnoside.

[0030] Figure 7 Thin layer chromatograms of extraction time investigation, samples: 1. 10 min (JJ-4); 2. 20 min (JJ-4); 3. 30 min (JJ-4); 4. 10 min (control herbal material); 5. 20 min (control herbal material); 6. 30 min (control herbal material). Inspection: left: daylight, right: UV light (365 nm).

[0031] Figure 8 Thin layer chromatograms of extraction time investigation, samples: 1. vitexin-2-O-rhamnoside; 2. 10 min (JJ-4); 3. 20 min (JJ-4); 4. 30 min (JJ-4); 5. 10 min (control medicinal material); 6. 20 min (control medicinal material); 7. 20 min (control medicinal material); 8. vitexin-2-O-rhamnoside.

[0032] Fig. 9 Thin layer chromatograms of the material-liquid ratio investigation, samples: 1. 1:10 (JJ-4); 2. 1:20 (JJ-4); 3. 1:30 (JJ-4); 4. 1:10 (control medicinal material); 5. 1:20 (control medicinal material); 6. 1:30 (control medicinal material). Inspection: left: daylight, right: UV light (365 nm).

[0033] Fig.10 Thin layer chromatograms of the solid-liquid ratio investigation, samples: 1. Vitexin-2-O-rhamnoside; 2. 1:10 (JJ-4); 3. 1:20 (JJ-4); 4. 1:30 (JJ-4); 5. 1:10 (control medicinal material); 6. 1:20 (control medicinal material); 7. 1:30 (control medicinal material); 8. Vitexin-2-O-rhamnoside.

[0034] Fig.11 Inspection of the spotting amount of control herbs and samples, samples: 1, 2, 3, 4: control herbs; 5, 6, 7, 8: JJ-4. Spotting amount: 1-4: 1, 2, 3, 4 μL; 5-8: 1, 2, 3, 4 μL. Inspection: left: daylight; right: UV light (365 nm).

[0035] Fig.12 Sample spotting volume inspection for the test samples: samples 1-4: vitexin-2-O-rhamnoside; 5-8: JJ-4; 9-12: control medicinal materials. Sample spotting volume: 1-4: 1, 2, 3, 4 μL; 5-8: 1, 2, 3, 4 μL; 9-12: 1, 2, 3, 4 μL.

[0036] Fig.13 Distance inspection under sunlight, samples: 1. Control medicinal material; 2. JJ-4; 3. JJ-10; 4. Control medicinal material. Left picture 100×100 mm silica gel G plate thin layer plate, right picture 100×200 mm silica gel G plate thin layer plate.

[0037] Fig.14 Distance observation was carried out under ultraviolet light (365 nm), samples: 1. control medicinal material; 2. JJ-4; 3. JJ-10; 4. control medicinal material. The left picture is a 100×100 mm silica gel G plate thin layer plate, and the right picture is a 100×200 mm silica gel G plate thin layer plate.

[0038] Fig.15 Distance observation under ultraviolet light (365 nm), samples: 1. Vitexin-2-O-rhamnoside, 2. Control herbal medicine; 3. JJ-4, 4. JJ-10, 5. Control herbal medicine. Left: 100×100 mm silica gel G plate thin layer plate, right: 100×200 mm silica gel G plate thin layer plate.

[0039] Fig.16 Thin layer plate inspection, samples: 1. Control medicinal materials; 2. JJ-4; 3. JJ-10; 4. Control medicinal materials. Thin layer plate: upper left and lower left pictures: Qingdao Bangkai Gaosi Technology Materials Co., Ltd. (G); upper middle and lower middle pictures: Qingdao Ocean Chemical Co., Ltd. (GF 254 ); upper right and lower right images: Qingdao Ocean Chemical Co., Ltd. (G). Inspection: upper left, upper middle and upper right images: sunlight; lower left, lower middle and lower right images: UV light (365 nm).

[0040] Fig.17 Thin layer plate inspection, samples: 1. Vitexin-2-O-rhamnoside, 2. Control medicinal materials; 3. JJ-4, 4. JJ-10, 5. Control medicinal materials. Thin layer plate: Left: Qingdao Bangkai Gaosi Technology Materials Co., Ltd. (G); Middle: Qingdao Ocean Chemical Co., Ltd. (GF 254 ); Right: Qingdao Ocean Chemical Co., Ltd. (G).

[0041] Fig.18TLC identification results at room temperature (25℃ / 60%), samples: left, middle: 1. control medicinal material, 2. JJ-4, 3. JJ-10, 4. control medicinal material; right: 1. Vitexin-2-O-rhamnoside, 2. control medicinal material; 3. JJ-4, 4. JJ-10, 5. control medicinal material, developing solvent: left, middle: cyclohexane-ethyl acetate-acetone (7:2:1); right: n-butanol-formic acid-water (8:1:1). Color development: left, middle: 10% sulfuric acid ethanol solution, heated at 105℃ right: aluminum chloride test solution. Inspection: left: daylight; middle, right: ultraviolet light (365 nm).

[0042] Fig.19 Thin layer identification results under high temperature and high humidity / high temperature and low humidity conditions, upper figure: 35℃ / 32%; lower figure: 35℃ / 72%. Samples: upper left, lower left, upper middle, lower middle: 1. Control medicinal materials, 2. JJ-4, 3. JJ-10, 4. Control medicinal materials; upper right, lower right: 1. Vitexin-2-O-rhamnoside, 2. Control medicinal materials; 3. JJ-4, 4. JJ-10, 5. Control medicinal materials, developing agents: upper left, lower left, upper middle, lower middle: cyclohexane-ethyl acetate-acetone (7:2:1); upper right, lower right: n-butanol-formic acid-water (8:1:1). Color development: upper left, lower left, upper middle, lower middle: 10% sulfuric acid ethanol solution, heated at 105℃; upper right, lower right: aluminum chloride test solution. Inspection: Upper left, lower left: daylight; upper middle, lower middle, upper right, lower right: UV light (365 nm).

[0043] Fig. 20 The results of thin layer identification under low temperature and high humidity / low temperature and low humidity conditions, left: 4℃ / 32%, right: 4℃ / 72%. Fig.19 same.

[0044] Fig.21 Identification results of the samples of Pseudocongmus konjac, samples: 1: control herb (JJ-16); 2~16: JJ-1~JJ-15; 17: control herb (JJ-16). Inspection: upper picture: daylight; lower picture: ultraviolet light (365 nm).

[0045] Fig. 22 Identification results of Pseudoconazole medicinal material samples, samples: 1: vitexin-2-O-rhamnoside; 2~16: JJ-1~JJ-15; 17: control medicinal material (JJ-16), 18: vitexin-2-O-rhamnoside.

[0046] Fig.23 Identification results of two materials of Pseudoconch and Betel leaves, sample: 1: Vitexin-2-O-rhamnoside; 2: Material a; 3: Material b; 4: Control herbal medicine (JJ-16). Inspection: Left: Daylight; Right: UV light (365 nm).

[0047] Fig.24 Identification results of two materials of Pseudoconazole and Betel leaves, sample: 1: vitexin-2-O-rhamnoside; 2: material a; 3: material b; 4: control medicinal material (JJ-16). DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1

[0050] (1) Preparation of test solution

[0051] Take 1.0 g of the test herb powder, add 10 mL of anhydrous ethanol, ultrasonically treat for 20 min, cool, filter, and take the concentrated filtrate to obtain the test solution. Take 1.0 g of the reference herb of Pseudoconium konjac and prepare the reference herb solution in the same way. Take the reference herb of Vitexin-2-O-rhamnoside and add anhydrous ethanol to prepare a solution containing 0.2 mg per 1 ml as the reference solution.

[0052] (2) Thin layer chromatography detection

[0053] ① Thin layer chromatography test of reference medicinal materials

[0054] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 µL of the above-mentioned control medicinal materials and test sample solutions were taken and spotted on the same silica gel G thin layer plate, developed with cyclohexane-ethyl acetate-acetone (volume ratio 7:2:1) as the developing solvent, taken out, dried, sprayed with 10% volume concentration of ethanolic sulfuric acid solution, heated until the spots were clearly colored, and examined under sunlight and ultraviolet light (365 nm), respectively.

[0055] ② Thin layer chromatography test of reference substances

[0056] Take 2 μL of the above-mentioned reference solution, reference medicinal material solution and test solution, respectively, and spot them on the same silica gel G thin layer plate, develop it with n-butanol-formic acid-water (volume ratio 8:1:1), take it out, dry it, spray it with aluminum chloride test solution, and heat it at 105°C until the spots are clearly colored.

[0057] In the thin layer chromatography test of the control medicinal material, spots of the same color should appear at the corresponding positions of the test sample chromatogram as those of the control medicinal material chromatogram and the control substance chromatogram; in the thin layer chromatography test of the control substance, spots of the same color should appear at the corresponding positions of the control medicinal material chromatogram and the control substance chromatogram; if both the thin layer chromatography test of the control medicinal material and the thin layer chromatography test of the control substance meet the requirements, the test sample is Pseudocong, and if any one of the tests does not meet the requirements, the test sample is not Pseudocong.

[0058] Screening and optimization of thin layer chromatography conditions

[0059] We collected 16 batches of Pseudocongium konjac samples from different sources in Hainan Province, including 1 batch of control medicinal materials (JJ-16) and 15 batches of medicinal materials samples. All samples were identified as Pseudocongium konjac of the Piperaceae family. Piper sarmentosum Roxb. Among them, 12 batches were above-ground parts and 3 batches were whole plants (JJ-9, JJ-11, and JJ-15). The samples were stored in the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences. Specific information is shown in Table 1. The samples were shade-dried or sun-dried after collection.

[0060] Table 1 List of Pseudocongium konjac sample information

[0061] serial number Latitude and longitude Collection time JJ-1 <![CDATA[E:110 o 7’ 10’’;N:19 o 43’ 51’’]]> 2023.5.11 JJ-2 <![CDATA[E:109 o 53’ 13’’;N:19 o 32’ 20’’]]> 2023.5.11 JJ-3 <![CDATA[E:109 o 32’ 36’’;N:19 o 34’ 36’’]]> 2023.5.11 JJ-4 <![CDATA[E:109 o 39’ 58’’;N:19 o 27’ 6’’]]> 2023.5.11 JJ-5 <![CDATA[E:109 o 24’ 41’’;N:19 o 28’ 36’’]]> 2023.5.11 JJ-6 <![CDATA[E:109 o 8’ 1’’;N:19 o 21’ 34’’]]> 2023.5.11 JJ-7 <![CDATA[E:109 o 19’ 41’’;N:19 o 17’ 5’’]]> 2023.5.11 JJ-8 <![CDATA[E:109 o 32’ 57’’ ;N:19 o 12’ 21’’]]> 2023.5.11 JJ-9 <![CDATA[E:109 o 33’ 56’’;N:19 o 3’ 16’’]]> 2023.5.11 JJ-10 <![CDATA[E:109 o 32’ 38’’;N:18 o 55’ 5’’]]> 2023.5.11 JJ-11 <![CDATA[E:110 o 27’ 11’’;N:19 o 54’ 7’’]]> 2023.5.11 JJ-12 <![CDATA[E:110 o 22’ 44’’;N:19 o 41’ 5’’]]> 2023.5.11 JJ-13 <![CDATA[E:110 o 26’ 32’’;N:19 o 16’ 13’’]]> 2023.5.11 JJ-14 <![CDATA[E:110 o 29’ 17’’;N:19 o 2’ 34’’]]> 2023.5.11 JJ-15 <![CDATA[E:109 o 58’ 45’’;N:18 o 35’ 7’’]]> 2023.5.11 JJ-16 <![CDATA[E:109 o 29' 40'';N: 19o 30' 46'']]> 2023.5.11

[0062] 1 Expand System Optimization

[0063] Pseudocones is a plant of the Piperaceae family. With piperine, pellitorine and vitexin-2-O-rhamnoside as reference substances, the above three components were tested by thin layer chromatography. The detection method is as follows: Take 1.0 g of the powder of this product, place it in a conical flask, add 10 mL of anhydrous ethanol, ultrasonically treat for 30 min, cool, filter, and take the filtrate as the test solution. Take 2 μL of each of the above solutions and spot them on the same silica gel G thin layer plate (100×100 mm), use "cyclohexane-ethyl acetate-acetone (7:2:1)" as the development system, develop 8 cm upward, take out, and dry. First examine under ultraviolet light (365nm), then spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight and ultraviolet light (365nm) respectively.

[0064] Preparation of reference solution: Take appropriate amount of piperine, pellitoryrine and vitexin-2-O-rhamnoside, weigh accurately, and dilute with anhydrous ethanol to obtain a reference solution with a concentration of 0.2 mg / mL.

[0065] The reference substance, reference medicinal material (JJ-16) and two batches of samples (JJ-4, 10) were prepared and identified by thin layer chromatography according to the above method. The results are shown in Figure 1, where the upper picture is the uncolored picture, and the lower picture is the picture after spraying 10% sulfuric acid ethanol solution and heating at 105℃ for color development; the lower left picture is viewed under sunlight, and the upper and lower right pictures are viewed under ultraviolet light. The results show that the piperine and pellitoryline reference substances, reference medicinal materials and samples are well developed, while vitexin-2-O-rhamnoside is not developed. Before color development, at 365 nm, the two batches of samples showed spots of the same color as piperine at the corresponding positions ( Figure 1 ), the control herb had none; after color development, the two batches of samples and the control herb showed spots of the same color at the corresponding positions at 365nm, which were suspected to be the same as those of pellitoryline ( Figure 1 , lower right).

[0066] To further determine whether the above-mentioned characteristic spots exist in the sample, a control substance is added to the sample for development under the current development system. The results are shown in Figure 2 , where the upper picture is a non-colorized picture, and the lower picture is a picture after spraying 10% sulfuric acid ethanol solution and heating at 105℃ for color development; the lower left picture is an inspection under sunlight, and the upper and lower right pictures are inspections under ultraviolet light. The results showed that after adding the reference substance to the sample, the thin layer characteristics displayed were different from the thin layer characteristics without adding the reference substance, indicating that piperine and pellitoryrine are not suitable as thin layer identification characteristics. However, under this development system and color development conditions, the identification characteristics of the control medicinal materials and samples are obvious, and the characteristic spot development effect is good, so it is suitable as a chromatographic condition for thin layer identification of Pseudoconium.

[0067] In addition, Pseudocones contain a relatively high amount of vitexin-2-O-rhamnoside. Under the above-mentioned development system, the vitexin-2-O-rhamnoside reference spot failed to develop. This component belongs to the flavonoid glycosides. The detection method for this component is as follows: Take 1.0 g of the powder of this product, place it in a conical flask, add 10 mL of anhydrous ethanol, ultrasonically treat it for 30 min, cool it, filter it, and take the filtrate as the test solution. Pipette 2 μL of each of the above solutions and spot them on the same silica gel G thin layer plate (100×50 mm), use "ethyl acetate-formic acid-water (8:1:1)" as the developing agent, develop it, take it out, and dry it. Spray it with aluminum chloride test solution, wait for the ethanol to evaporate, and then inspect it under ultraviolet light (365nm). The results are shown in Figure 3 The results showed that in the chromatogram of the test sample, the same color fluorescent spots appeared at the corresponding positions of the chromatogram of the reference sample and the reference medicinal material, but the development distance of vitexin-2-O-rhamnoside was shorter, indicating that the polarity of the development system was smaller. After the sample was spotted on a silica gel G thin layer plate (100×50 mm), the development system was adjusted and the samples were separated by ethyl acetate-formic acid-water (6:1:1) ( Figure 4 left image) and "n-Butanol-Formic Acid-Water (8:1:1)" ( Figure 4The results showed that both development systems can make vitexin-2-O-rhamnoside have a good development distance. In the chromatogram of the test sample, the same color fluorescent spots appeared at the corresponding positions of the chromatogram of the reference sample and the chromatogram of the reference medicinal material. Among them, the development effect of the "n-butanol-formic acid-water (8:1:1)" system was better, and the spots were clearer. Therefore, "n-butanol-formic acid-water (8:1:1)" was initially selected as the development system that reflects the characteristics of vitexin-2-O-rhamnoside for the next step of research.

[0068] In summary, two expansion systems, "cyclohexane-ethyl acetate-acetone (7:2:1)" and "n-butanol-formic acid-water (8:1:1)", were preliminarily established, which can reflect the overall characteristics of Pseudoconazole and the characteristics of vitexin-2-O-rhamnoside, respectively.

[0069] 2 Investigation of the preparation method of test solution

[0070] 2.1 Investigation of extraction solvent

[0071] Take 1.0 g of each of three samples of Pseudoconium konjac (JJ-4) and three reference medicinal materials (JJ-16), accurately weigh them, put them into a conical flask, add 10 mL of anhydrous ethanol, ethyl acetate, and acetone, respectively, and ultrasonically treat for 30 min. Cool, filter, and take the filtrate to obtain the product;

[0072] 2 µL of the control drug solution and the test solution were taken respectively, spotted on a silica gel G thin layer plate (100×100 mm), and cyclohexane-ethyl acetate-acetone (7:2:1) was used as the developing solvent. The plate was developed up to 8 cm, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were clearly colored, and placed in sunlight ( Figure 5 Left) and UV light (365nm) ( Figure 5 The results show that the spot characteristics of each solvent extract are similar and the difference is not significant.

[0073] 2 µL of the reference solution of vitexin-2-O-rhamnoside, the reference medicinal material solution and the test solution were taken respectively, spotted on a silica gel G thin layer plate (100×100 mm), developed 8 cm upward with n-butanol-formic acid-water (8:1:1) as the developing solvent, taken out, dried, sprayed with aluminum chloride test solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The results are shown in Figure 6 The results showed that the anhydrous ethanol extract and the acetone extract showed the same color fluorescent spots at the corresponding positions of the reference substance chromatogram and the reference medicinal material chromatogram, but the spots of the anhydrous ethanol extract were clearer and brighter; the spots of the ethyl acetate extract were not obvious. Considering the two opening systems comprehensively, it is more appropriate to choose anhydrous ethanol as the extraction solvent.

[0074] 2.2 Extraction time investigation

[0075] Take 1.0 g of each of 3 samples of Pseudoconium konjac (JJ-4) and 3 reference medicinal materials (JJ-16), accurately weigh them, put them into a conical flask, add 10 mL of anhydrous ethanol, and ultrasonically treat them for 10, 20, and 30 min respectively, cool them, filter them, and take the filtrate to obtain the product;

[0076] 2 µL of the control drug solution and the test solution were taken respectively, spotted on a silica gel G thin layer plate (100×100 mm), developed with cyclohexane-ethyl acetate-acetone (7:2:1) as the developing solvent, and developed 8 cm upward. The plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were clearly colored, and placed in sunlight ( Figure 7 left) and UV light (365 nm) ( Figure 7 View it below.

[0077] 2 µL of the reference solution of vitexin-2-O-rhamnoside, the reference medicinal material solution and the test solution were taken respectively, spotted on a silica gel G thin layer plate (100×100 mm), developed 8 cm upward with n-butanol-formic acid-water (8:1:1) as the developing solvent, taken out, dried, sprayed with aluminum chloride test solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). The results are shown in Figure 8 The results showed that there was no significant difference in the spot size after ultrasonic treatment for 10, 20, and 30 min, and the spots of the sample treated with ultrasonic treatment for 20 min were more round. In summary, ultrasonic extraction for 20 min was selected to ensure uniform sample extraction.

[0078] 2.3 Investigation of material-liquid ratio

[0079] Take 1.0 g of each of three samples of Pseudoconium konjac (JJ-4) and three reference medicinal materials (JJ-16), accurately weigh them, put them into a conical flask, add anhydrous ethanol at a solid-liquid ratio of 1:10, 1:20 and 1:30, respectively, ultrasonically treat for 20 min, cool, filter, and take the filtrate to obtain;

[0080] 2 µL of the control medicinal material solution and the test solution were taken, respectively, and spotted on a silica gel G thin layer plate (100×100 mm), and developed with cyclohexane-ethyl acetate-acetone (7:2:1) as the developing solvent, and then developed 8 cm upward. The plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were clearly colored, and examined under sunlight and ultraviolet light (365 nm). 2 µL of the vitexin-2-O-rhamnoside reference solution, the control medicinal material solution, and the test solution were taken, respectively, and developed with n-butanol-formic acid-water (8:1:1) as the developing solvent. The plate was taken out, dried, sprayed with aluminum chloride test solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365 nm). The results are shown in Fig. 9 , 10 The results showed that the spots of each extract were clearly visible, and the spots were clearer when the solid-liquid ratio was 1:10. Therefore, the optimal solid-liquid ratio was set at 1:10.

[0081] In summary, the preparation method of the test solution is as follows: weigh about 1.0 g of the crude powder of the product, accurately weigh it, put it into a stoppered conical flask, add 10 mL of anhydrous ethanol, ultrasonically treat it for 20 min, cool it, filter it, and take the filtrate to obtain the solution.

[0082] 2.3 Sampling quantity investigation

[0083] Prepare the reference solution according to "2.1", and prepare the test solution and reference medicinal material solution according to the preparation method of the test solution determined in "2.2". Take 1, 2, 3, and 4 μL of the reference medicinal material and the test solution, respectively, and spot them on the same silica gel G thin layer plate (100×100 mm), develop 8 cm upward with cyclohexane-ethyl acetate-acetone (7:2:1) as the developing solvent, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and inspect them under sunlight and ultraviolet light (365 nm). Take 1, 2, 3, and 4 μL of the reference solution of vitexin-2-O-rhamnoside and 1, 2, 3, and 4 μL of the test solution respectively on the same silica gel G thin layer plate (100×200 mm), develop 8 cm upward with n-butanol-formic acid-water (8:1:1) as the developing solvent, take out, dry, spray with aluminum chloride test solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365nm). The results are shown in Fig.11 , 12 The results showed that when the sample volume was 1-4 μL, the chromatographic spots of each sample were clear and appeared at the same chromatographic position as the control medicinal material. The chromatographic effect was best when the sample volume was 2 μL. Therefore, the sample volume of the reference solution was set to 2 μL, and the sample volume of the test solution and the control medicinal material solution was set to 2 μL.

[0084] 2.4 Durability test

[0085] 2.4.1 Distance survey

[0086] Prepare the reference solution according to "2.1", and prepare the test solution and reference medicinal material solution according to the test solution preparation method determined in "2.2". Take 2 μL of the above test solution and reference medicinal material solution, respectively, and spot them on silica gel G thin layer plates of different specifications (100×100 mm, 100×200 mm), develop them with cyclohexane-ethyl acetate-acetone (7:2:1) as the developing solvent, and develop them 8 cm or 12 cm upward. Take them out, dry them, spray them with 10% sulfuric acid ethanol solution, heat them until the spots are clearly colored, and inspect them under sunlight and ultraviolet light (365 nm). Take 2 μL of the reference solution and the test solution, respectively, and spot them on different specifications of silica gel G thin layer plates (100×100 mm, 100×200 mm), develop them 8 cm or 12 cm upward with n-butanol-formic acid-water (8:1:1) as the developing solvent, take them out, dry them, spray them with aluminum chloride test solution, heat them at 105℃ until the spots are clearly colored, and examine them under ultraviolet light (365 nm). The results are shown in Fig.13 , 14 , 15. When the development distance was 8-12 cm, the chromatographic spots of each sample were clear and the identification characteristics were obvious, indicating that the method was feasible when the chromatographic condition was 8-12 cm.

[0087] 2.4.2 Investigation of thin layer plates

[0088] Prepare the reference solution of 2-O-rhamnoside according to item "2.1", and prepare the test solution and reference medicinal material solution according to the preparation method of the test solution determined in item "2.2". Take 2 μL of the above test solution and reference medicinal material solution respectively, and apply them to silica gel G or GF 254 On the thin layer plate (all 100×100 mm), cyclohexane-ethyl acetate-acetone (7:2:1) was used as the developing agent to develop 8 cm upward, then taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were clearly colored, and inspected under sunlight and ultraviolet light (365 nm). Take 2 μL of the above reference solution and test solution, respectively, and spot on different specifications of silica gel G thin layer plates (100×100 mm), n-butanol-formic acid-water (8:1:1) was used as the developing agent to develop 8 cm upward, taken out, dried, sprayed with aluminum chloride test solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm). The results are shown in Fig.16 , 17 The sample, reference solution and reference medicinal material showed spots of the same color at the corresponding positions on the chromatograms. The chromatographic spots on the silica gel G thin layer plates from the three manufacturers were clear, with good separation and development, and obvious identification characteristics, indicating that the identification conditions can be used on thin layer plates from different manufacturers.

[0089] 2.4.3 Conduct environmental investigation

[0090] Prepare the reference solution of vitexin according to item "2.1", and prepare the test solution and reference medicinal material solution according to the preparation method of the test solution determined in item "2.2". Take 2 μL of the above test solution and reference medicinal material solution respectively, and spot them on different silica gel G thin layer plates (all 100×100 mm), use cyclohexane-ethyl acetate-acetone (7:2:1) as the developing solvent, and develop 8 cm upward under ① room temperature and relative humidity of 60%; ② 4℃~10℃ (low temperature), relative humidity of 32% and 72%; ③ 35℃, relative humidity of 32% and 72%, respectively. Take out and dry; spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and inspect them under sunlight and ultraviolet light (365 nm). Take 2 μL of the reference solution and the test solution, respectively, and spot them on different silica gel G thin layer plates (all 100×100 mm), use n-butanol-formic acid-water (8:1:1) as the developing solvent, and develop them under ① room temperature and relative humidity of 60%; ② 4℃~10℃ (low temperature), relative humidity of 32% and 72%; ③ 35℃, relative humidity of 32% and 72%, respectively. Take out, dry, spray with aluminum chloride test solution, heat at 105℃ until the spots are clearly colored, and inspect under ultraviolet light (365 nm). Results are shown in Fig.18 , 19 20. Under room temperature conditions, the chromatographic separation system is stable, and spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal materials and the reference substance, and the spots are clear; under high temperature and high humidity and high temperature and low humidity conditions, spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal materials and the reference substance, and the spots are clear and the identification effect is good; under low temperature and high humidity and low temperature and low humidity conditions, the spots drift slightly, but it does not affect the spot identification; this indicates that the identification conditions have good durability to environmental conditions.

[0091] In summary, the TLC identification method has good durability and can be used for the qualitative identification of Pseudocong. The final TLC identification method of Pseudocong is: take 1.0 g of the powder of this product, add 10 mL of anhydrous ethanol, ultrasonically treat for 20 minutes, cool, filter, and take the filtrate to obtain the test solution. Take 1.0 g of Pseudocong control medicinal material and prepare the control medicinal material solution in the same way. Take the reference material of vitexin-2-O-rhamnoside and add anhydrous ethanol to prepare a solution containing 0.2 mg per 1 ml as the reference solution.

[0092] TLC detection of control medicinal materials: According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 µL of the control medicinal materials and the test sample solution were taken and spotted on the same silica gel G thin layer plate, and developed with cyclohexane-ethyl acetate-acetone (7:2:1) as the developing agent (upward development 8-12 cm), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were clearly colored, and examined under sunlight and ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material.

[0093] TLC detection of reference substance: Take 2 μL of the reference substance solution, reference medicinal material solution and test substance solution, spot them on the same silica gel G thin layer plate, develop them with n-butanol-formic acid-water (8:1:1) as the developing solvent (development up 8-12 cm), take them out, dry them, spray them with aluminum chloride test solution, and heat them at 105°C until the spots are clearly colored. In the chromatogram of the test substance, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

[0094] If both of the above tests are met, the test sample is a false cone; if any one of them is not met, the test sample is not a false cone.

[0095] 2.5 Identification of Pseudocongium konjac samples

[0096] The 16 batches of samples collected were identified according to the TLC identification method finally determined under "2.4" (JJ-16 was used as the control herbal medicine). The results are shown in Fig.21 , 22 In the chromatographic test of the control medicinal material, cyclohexane-ethyl acetate-acetone (7:2:1) was used as the developing solvent and the upper direction was 8 cm. The 15 batches of samples showed spots of the same color at the corresponding positions of the chromatographic test of the control medicinal material ( Fig.21 ); In the thin layer chromatography test of the reference substance, n-butanol-formic acid-water (8:1:1) was used as the developing solvent and the upper layer was developed for 8 cm. The chromatograms of 15 batches of samples showed spots of the same color at the corresponding positions of the reference substance and the reference medicinal material (JJ-16) ( Fig. 22 ). In the chromatogram of the test sample, the spots are clear and the identification characteristics are obvious, indicating that all 15 batches of samples are genuine Pseudo-cong. The above results are consistent with the previous identification results.

[0097] 7.2.6 Identification of Pseudoconch and Betel Leaves

[0098] Take Pseudocones ( Piper sarmentosum Roxb.) and Piper betel ( Piper betleL.), randomly numbered material a and material b, and identified the two sample materials according to the thin layer identification method finally determined under "2.4". Vitexin-2-O-rhamnoside and reference medicinal material (JJ-16) were used as controls. The sample volume of each sample was 2 μL, and they were spotted on the same silica gel G thin layer plate (100×100 mm), and developed with cyclohexane-ethyl acetate-acetone (7:2:1) and n-butanol-formic acid-water (8:1:1) as the developing solvents for 8 cm upward. The results are shown in Fig.23 and 24 . In the chromatogram of the test sample, with cyclohexane-ethyl acetate-acetone (7:2:1) as the developing solvent, the material a and the reference medicinal material (JJ-16) chromatogram showed spots of the same color at the corresponding positions, and the material b showed spots different from the chromatogram of the reference medicinal material (JJ-16); with n-butanol-formic acid-water (8:1:1) as the developing solvent, the material a and the reference substance and the reference medicinal material (JJ-16) chromatogram showed spots of the same color at the corresponding positions, and the spots were clear and the identification characteristics were obvious, while the material b and the reference substance and the reference medicinal material (JJ-16) chromatogram did not show spots of the same color at the corresponding positions. Therefore, material a is the medicinal material of Pseudocong, and material b is Piper betel.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A thin layer identification method for Pseudocones, characterized in that: The following steps are involved: (1) Preparation of test solution Take the test sample powder, add anhydrous ethanol and sonicate, and take the filtrate as the test solution; prepare the control medicinal material solution of Pseudoconazole in the same way; take the reference substance of Vitexin-2-O-rhamnoside, add anhydrous ethanol to prepare a solution containing 0.2 mg per 1 ml, as the reference solution; (2) Thin layer chromatography TLC detection of control medicinal materials: Take equal amounts of the test sample solution and the control medicinal material solution, spot them on the same silica gel thin layer plate, develop them with hexane-ethyl acetate-acetone in a volume ratio of 7:2:1 as the developing solvent, take them out, dry them, spray them with sulfuric acid ethanol solution, heat them until the spots are clearly colored, and examine them under sunlight and ultraviolet light respectively; TLC detection of reference substance: separately take the above-mentioned reference substance solution, reference medicinal material solution and test substance solution, spot them on the same silica gel thin layer plate, develop them with n-butanol-formic acid-water with a volume ratio of 8:1:1 as the developing solvent, take them out, dry them, spray them with aluminum chloride test solution, and heat them until the spots are clearly colored; In the thin layer chromatography test of the control medicinal material, spots of the same color should appear at the corresponding positions of the test sample chromatogram as those of the control medicinal material chromatogram and the control substance chromatogram; in the thin layer chromatography test of the control substance, spots of the same color should appear at the corresponding positions of the control medicinal material chromatogram and the control substance chromatogram; if both the thin layer chromatography test of the control medicinal material and the thin layer chromatography test of the control substance meet the requirements, the test sample is Pseudocong, and if any one of the tests does not meet the requirements, the test sample is not Pseudocong.

2. A thin layer identification method for Pseudocones as claimed in claim 1, characterized in that: The silica gel thin layer plate in step (2) is a silica gel G thin layer plate.

3. A thin layer identification method for Pseudoconazole as claimed in claim 1, characterized in that: In the step (1), the volume ratio of the test sample powder mass to the added anhydrous ethanol is 1:10, and the ultrasonic time is 20 minutes.

4. A thin layer identification method for Pseudocones as claimed in claim 1, characterized in that: In the control medicinal material thin layer chromatography test of step (2), 2 μL of the control medicinal material solution and the test sample solution are respectively spotted on the same silica gel thin layer plate.

5. A thin layer identification method for Pseudocones as claimed in claim 1, characterized in that: In the step (2), the volume concentration of the sulfuric acid ethanol solution is 10%; and the wavelength of the ultraviolet lamp is 365nm.

6. A thin layer identification method for Pseudocones as claimed in claim 1, characterized in that: In the step (2) of thin layer chromatography of the reference substance, 2 μL of the reference substance solution, the reference medicinal material solution and the test substance solution are respectively spotted on the same silica gel thin layer plate.

7. A thin layer identification method for Pseudoconazole as claimed in claim 1, characterized in that: In the step (2), the expansion distance is 8-12 cm.

8. A thin layer identification method for Pseudocones as claimed in claim 1, characterized in that: In the step (2), the silica gel thin layer plate is heated at 105°C.

9. Use of the thin-layer identification method of Pseudocongol as claimed in any one of claims 1 to 8 in identifying Pseudocongol medicinal materials.

10. Use of the thin-layer identification method of Pseudocongol as claimed in any one of claims 1 to 8 in distinguishing Pseudocongol medicinal materials and Betel leaves.

Images