Thin-layer identification method for kadsura pepper stem and preparation thereof

Abstract

The invention discloses a thin-layer identification method of kadsura pepper stem and a preparation thereof, which optimizes a preparation method of a test sample and takes dichloromethane-acetone-methanol as a developing solvent. The method is simple, convenient and rapid to operate, does not need a column chromatography elution separation step, does not damage chemical components of the kadsura pepper stem, maximally presents prototype components of the kadsura pepper stem, and is rich in chromatographic spot information; dichloromethane is used for replacing toxic trichloromethane to serve as a developing solvent, so that the safety and the environmental protection property are enhanced; evaluation is carried out by adopting the consistency of reference medicinal materials, and is more accurate and scientific.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicines, and in particular to a thin-layer identification method for Piper mesenteriae and its preparations. Background Art

[0002] Kadsura ohwi is the dried stems of Piperkadsura (Choisy) Ohwi, a plant in the Piperaceae family. Harvested in summer and autumn, the roots and leaves are removed, and the plant is sun-dried. Kadsura ohwi primarily contains lignans, volatile oils, alkaloids, flavonoids, epoxides, and other compounds.

[0003] Currently, there are few reports on the thin-layer identification of Piper meridionale. The existing thin-layer identification method for Piper meridionale is the thin-layer identification method for Piper meridionale medicinal materials in the 2020 edition of the Chinese Pharmacopoeia. This method uses Piper meridionale control medicinal materials as a reference. Column chromatography steps are used in the preparation of sample solutions and reference medicinal material solutions, and cyclohexane-ethyl acetate (1:1) is used for elution and separation. The sample preparation method is relatively cumbersome. The thin-layer identification item for Piper meridionale medicinal materials in the pharmacopoeia uses chloroform-acetone-methanol (7:1:0.5) as the developing solvent. Due to the high toxicity of chloroform, it is dangerous and has poor applicability. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a thin layer identification method for Piper mesenteriae and its preparations.

[0005] Technical solution: The thin-layer identification method of Piper mesenteriae and its preparations of the present invention comprises the following steps:

[0006] 1) Dissolve a sample of the Piper mesangiales preparation in water, then shake and extract with ethyl acetate. After stratification, evaporate the supernatant to dryness, and dissolve the residue in an alcohol solvent to obtain a Piper mesangiales preparation test solution.

[0007] 2) Take a control medicinal material or medicinal material sample of Pieris ovata, decoct it in water or heat it under reflux, filter it, and extract it by shaking with ethyl acetate solution. After standing and stratifying, take the supernatant and evaporate it to dryness. Dissolve the residue in an alcohol solvent to prepare a control medicinal material solution or medicinal material test solution;

[0008] 3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate respectively, develop them with a developing agent, and obtain a thin layer chromatogram to complete the identification operation.

[0009] Furthermore, the Piper mesangiales preparation sample in step 1) includes Piper mesangiales formula granules or standard decoction.

[0010] Furthermore, when the Piper mestolide preparation sample described in step 1 is extracted with water, the mass volume ratio of the Piper mestolide preparation sample to water is 1:50 g / mL~200 g / mL, the ethyl acetate solution is shaken for extraction 1~4 times, and the volume ratio of the ethyl acetate solution to the sample aqueous solution is 0.5:2~1.

[0011] Furthermore, when the Piper mestolide control medicinal material or medicinal material sample in step 1 is decocted with water, the mass volume of the Piper mestolide control medicinal material or medicinal material sample and water is 1:10g / mL~100g / mL, the number of times the ethyl acetate solution is shaken for extraction is 1 to 4 times, and the volume ratio of the ethyl acetate solution to the sample aqueous solution is 0.5:2~1.

[0012] Furthermore, the developing solvent in step 3) is dichloromethane-acetone-methanol.

[0013] Furthermore, the volume ratio of dichloromethane-acetone-methanol is 9:(0.8-1.2):(0.3-0.8).

[0014] Furthermore, the spotting volume of the control medicinal material solution and the test solution in step 3) is 3 μl to 8 μl.

[0015] Furthermore, the thin layer plate in step 3) is a silica gel G thin layer plate.

[0016] Furthermore, in the thin layer chromatogram in step 3), a fluorescent spot of the same color appears in the chromatogram of the test solution at a position corresponding to the chromatogram of the control medicinal material solution.

[0017] The thin-layer identification method of the present invention is used in identifying Kadura rapa and its counterfeit Schisandra chinensis heteromorpha.

[0018] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: The present invention utilizes a simple and rapid pretreatment method to obtain a test solution and a control medicinal material solution, which are then spotted on the same thin-layer plate, developed with a suitable developer, and examined under ultraviolet light to obtain a multi-information thin-layer chromatogram of Piper meridionale with clear spots and excellent separation, and can effectively distinguish Piper meridionale from common counterfeits. Compared with the pharmacopoeial method, this method is simple and rapid to operate, does not require a column chromatography elution separation step, does not destroy the chemical components of Piper meridionale, and maximizes the presentation of the prototype components of Piper meridionale, with rich chromatographic spot information. The use of dichloromethane instead of toxic chloroform as the developer enhances safety and environmental protection. The use of the control medicinal material for consistency evaluation is more accurate and scientific. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a thin layer chromatogram of the Piper mesenteriae formula granules in Example 1 of the present invention;

[0020] Figure 2 This is a thin layer chromatogram of the standard decoction granules of Kadzurea brevis in Example 1 of the present invention;

[0021] Figure 3 This is a thin layer chromatogram of different sample amounts in Example 2 of the present invention;

[0022] Figure 4 is a thin layer chromatogram at different temperatures in Example 2 of the present invention;

[0023] Figure 5 is a thin layer chromatogram of different humidity in Example 2 of the present invention;

[0024] Figure 6 is a thin layer chromatogram of different developing agent ratios in Example 2 of the present invention;

[0025] Figure 7 This is a chromatogram comparing the standard decoction of Kadzu vine and the counterfeit Schisandra chinensis heteromorpha in Example 3 of the present invention;

[0026] Figure 8 This is a chromatogram of the preparation of the test sample of Piper methysticum granules using the Chinese Pharmacopoeia method developer in Comparative Example 1 of the present invention;

[0027] Figure 9 This is a chromatogram of the preparation of the test sample of Piper mesenteriae formula granules compared with the developer of the present invention in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] The present invention adopts the following instruments and reagents:

[0030] Instruments: CAMAG TLC VISUALIZER, ME204E 1 / 10,000 balance (Mettler-Toledo), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), silica gel G thin layer plate (Qingdao Ocean Chemical Plant, Yantai Xincheng Silica Gel Material Co., Ltd., Qingdao Kangyexin Pharmaceutical Silica Gel Desiccant Co., Ltd.).

[0031] Reagents: methanol (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), chloroform (Sinopharm Chemical Reagent Co., Ltd.), acetone (Sinopharm Chemical Reagent Co., Ltd.), and dichloromethane (Sinopharm Chemical Reagent Co., Ltd.) were all of analytical grade.

[0032] The control medicinal material of Piper methysticum (121635-202104) was purchased from the China Food and Drug Inspection Institute.

[0033] Piper mesenteriae formula granules (1: 24100049; 2: 24100059; 3: 24100069), Piper mesenteriae standard decoction (1: DG2309011; 2: DG2309015; 3: DG2309016; 4: DG2309017; 5: DG2309019; 6: DG2309021; 7: DG2309022; 8: DG2309023). :DG2309025;9:DG2309028;10:DG2411001;11:DG2411002;12:DG2411003;13:DG2411005;14:DG122008036;15:DG122008037;16:DG122008038;) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0034] Example 1:

[0035] Preparation of test solution of formula granules or standard decoction: Take 0.2g of Piper mestolide formula granules or standard decoction, grind it into powder, add 20ml of water to dissolve it, shake and extract it with ethyl acetate twice, 20ml each time, combine the ethyl acetate solution, evaporate to dryness, and add 1ml of methanol to dissolve the residue as the test solution.

[0036] Preparation of control medicinal material solution: Take 2g of Pieris ovata control medicinal material, add 50ml of water, boil for 30 minutes, filter, concentrate the filtrate to about 20ml, shake and extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the control medicinal material solution;

[0037] TLC identification steps: 5 μl of the test solution and the control medicinal material solution were respectively aspirated and spotted on the same silica gel G thin layer plate. The plate was developed with dichloromethane-acetone-methanol (9:1:0.5) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm).

[0038] Evaluation requirements: In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0039] Result analysis: Figure 1 1 to 3 correspond to the chromatograms of the test samples of three batches of Pieris glabra granules, and S is the chromatogram of the Pieris glabra reference medicinal material. Figure 2 1 to 16 correspond to the chromatograms of the test samples of 16 batches of Piper meridionale standard decoction, and S is the chromatogram of the Piper meridionale reference medicinal material.

[0040] Example 2:

[0041] The durability verification of the thin layer identification in Example 1 specifically includes:

[0042] (1) Investigation of different sample quantities:

[0043] To compare the effect of different spot sizes on spot separation, Pieris serrata formula granules (24100049) and Pieris serrata control medicinal materials were prepared according to the method described in Example 1. The samples were spotted on the same silica gel G thin layer plate, and dichloromethane-acetone-methanol (9:1:0.5) was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (365nm). The results are shown in Figure 3 , Figure 3 1 to 4 correspond to the chromatograms of 1 μL, 3 μL, 5 μL, and 8 μL of the Pieris glabra formula granules test sample, and S1 to S4 correspond to the chromatograms of 1 μL, 3 μL, 5 μL, and 8 μL of the Pieris glabra reference medicinal material. Figure 3 It can be seen that when the sample volume is 3μL~8μL, the chromatogram of the Piper mestolide formula particles and the chromatogram of the Piper mestolide reference medicinal material show clear spots at corresponding positions without other interference.

[0044] (2) Investigation at different temperatures:

[0045] Take Piper mesenteriae formula granules (24100049) and Piper mesenteriae control medicinal materials, prepare the test solution and the control medicinal material solution according to the method described in Example 1, and spot them on the same silica gel G thin layer plate, use dichloromethane-acetone-methanol (9:1:0.5) as the developing solvent, and develop them under low temperature (T:4℃, RH:47%), room temperature (T:22.5℃, RH:47%) and high temperature (T:40℃, RH:47%) conditions respectively. Take out, dry, and examine under ultraviolet light (365nm). The results are shown in Figure 4 , Figure 4 1 to 3 correspond to the chromatograms of the test sample of Piper methysticum granules, and S corresponds to the chromatogram of the reference medicinal material. Figure 4 Under different temperature conditions, the chromatograms of the Piper methysticum granules tested and the control herbs showed fluorescent spots of the same color at corresponding positions, and the separation effect was good. The experimental results show that temperature has little effect on the thin-layer chromatography identification of Piper methysticum granules, indicating that the thin-layer chromatography identification method has good durability at different temperatures.

[0046] (3) Investigation of different humidity:

[0047] Take Piper mesenteriae formula granules (24100049) and control medicinal material solution, prepare test solution and control medicinal material solution according to the method described in Example 1, spot them on the same silica gel G thin layer plate, use dichloromethane-acetone-methanol (9:1:0.5) as developing solvent, develop under different humidity conditions (RH: 18%, 47%, 88%), take out, dry, and examine under ultraviolet light (365nm). Figure 5 , Figure 51 to 3 correspond to the chromatograms of the test sample of Piper methysticum granules, and S corresponds to the chromatogram of the reference medicinal material. Figure 5 Under different humidity conditions, the chromatograms of the Piper methysticum granules tested and the control herbs showed fluorescent spots of the same color at corresponding positions, and the separation effect was good. The experimental results show that humidity has little effect on the thin-layer chromatography identification of Piper methysticum granules, indicating that the thin-layer chromatography identification method is robust to varying humidity conditions.

[0048] (4) Investigation of developing agent ratio:

[0049] Take the Kadzuvine Formula Granules (24100049) and the control medicinal material solution, and prepare the test solution and the control medicinal material solution according to the method described in Example 1. Spot them on the same silica gel G thin layer plate, and use dichloromethane-acetone-methanol (9:0.8:0.3), dichloromethane-acetone-methanol (9:1:0.5), and dichloromethane-acetone-methanol (9:1.2:0.8) as the developing solvent, develop, take out, dry, and examine under ultraviolet light (365nm). The results are shown in Figure 6 In the figure, 1 to 3 correspond to the chromatograms of the test sample of the Piper methysticum formula granules, and S corresponds to the chromatogram of the reference medicinal material. It can be seen from the figure that within the ratio range of dichloromethane-acetone-methanol 9:(0.8~1.2):(0.3~0.8), the corresponding positions of the chromatograms of the test sample of the Piper methysticum formula granules and the chromatograms of the reference medicinal material show fluorescent spots of the same color, and the separation effects are both good.

[0050] Example 3:

[0051] The authenticity of Pieris ovata is identified using the thin layer chromatography method in Example 1, specifically comprising:

[0052] Preparation of test solution: Take 0.2 g of standard decoction of Piper melongena and Schisandra chinensis (DG1912076, DG1912077; provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.), grind it into powder, add 20 ml of water to dissolve it, shake and extract it with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution.

[0053] TLC identification steps: 5 μl of the test solution and the control medicinal material were respectively taken and spotted on the same silica gel G thin layer plate, and developed with dichloromethane-acetone-methanol (9:1:0.5) as the developing agent. After development, the plate was taken out, dried, and examined under ultraviolet light (365 nm). Figure 6 , Figure 6Numbers 1-2 correspond to two batches of heteromorphic Schisandrae Chinensis standard decoction, and numbers 3-4 correspond to two batches of Piper meridionalis standard decoction. S corresponds to the Piper meridionalis control material. The results showed that the chromatograms of the heteromorphic Schisandrae Chinensis standard decoction (a Piper meridionalis counterfeit) and the Piper meridionalis standard decoction were clearly different. The heteromorphic Schisandrae Chinensis standard decoction (a Piper meridionalis counterfeit) did not show the same fluorescent spot at the corresponding position on the chromatogram as the Piper meridionalis control material. This identification condition can be used to distinguish genuine Piper meridionalis from counterfeit products.

[0054] Comparative Example 1:

[0055] This comparative example uses chloroform-acetone-methanol (7:1:0.5) as a thin layer developer under the entry of Piper mesenteriae in the 2020 edition of the Chinese Pharmacopoeia to investigate the preparation methods of the test solution of Piper mesenteriae formula granules (24100049), including the pharmacopoeial method and the present invention. The specific process is as follows:

[0056] Method 1 (pharmacopoeia method): Take 0.2 g of Piper mesangium wilfordii granules, add 30 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 2 ml of anhydrous ethanol to dissolve the residue, add 3 g of silica gel G, mix well, place on a water bath to evaporate the solvent, add to a silica gel G column (15 g, inner diameter of 1.5-2 cm), elute with 100 ml of a cyclohexane-ethyl acetate (1:1) mixed solution, collect the eluate, evaporate to dryness, and dissolve the residue in 2 ml of ethanol as the test solution.

[0057] Method 2 (the present invention): Take 0.2 g of Piper mesangineum granules, add 20 ml of water to dissolve it, shake and extract it with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0058] Thin layer identification steps: Referring to the pharmacopoeia method, aspirate 5μl of the test solution and spot it on the same silica gel G thin layer plate, use chloroform-acetone-methanol (7:1:0.5) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0059] See the results Figure 7 , Figure 7 Figure 1 shows the chromatogram of a sample of Piper mesenteriae granules prepared using the pharmacopoeial method, while Figure 2 shows the chromatogram of a sample of Piper mesenteriae granules prepared using the present invention. When the Piper mesenteriae granules were prepared using the pharmacopoeial method, the sample spots were less, while the sample solution prepared using the present invention produced more and clearer chromatographic spots.

[0060] Comparative Example 2:

[0061] This comparative example uses the optimized thin layer developing agent of the present invention, dichloromethane-acetone-methanol (9:1:0.5), to investigate the preparation methods of the test sample solution of Piper methysticum granules, including the pharmacopoeial method and the present invention. The specific process is as follows:

[0062] Method 1 (pharmacopoeia method): Take 0.2 g of Piper mesangium wilfordii granules, add 30 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 2 ml of anhydrous ethanol to dissolve the residue, add 3 g of silica gel G, mix well, place on a water bath to evaporate the solvent, add to a silica gel G column (15 g, inner diameter of 1.5-2 cm), elute with 100 ml of a cyclohexane-ethyl acetate (1:1) mixed solution, collect the eluate, evaporate to dryness, and dissolve the residue in 2 ml of ethanol as the test solution.

[0063] Method 2 (the present invention): Take 0.2 g of Piper mesangineum granules, add 20 ml of water to dissolve it, shake and extract it with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0064] Thin layer identification steps: Referring to the optimized thin layer method of the present invention, 5 μl of the test solution was aspirated and spotted on the same silica gel G thin layer plate, and developed with dichloromethane-acetone-methanol (9:1:0.5) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm).

[0065] See the results Figure 8 , Figure 8 Figure 1 shows the chromatogram of a sample of Piper mesenteriae granules prepared using the pharmacopoeial method, while Figure 2 shows the chromatogram of a sample of Piper mesenteriae granules prepared using the present invention. When the Piper mesenteriae granules were prepared using the pharmacopoeial method, the sample spots were less, while the sample solution prepared using the present invention produced more and clearer chromatographic spots.

[0066] Comprehensive Analysis: The thin-layer chromatography identification method for Piper meridionalis and its preparations provided by the present invention simplifies and accelerates sample preparation, eliminating the need for tedious steps such as column chromatography and elution with cyclohexane-ethyl acetate (1:1) separation. Furthermore, the sample solution yields more abundant and distinct chromatographic spots, better demonstrating the completeness of the chemical composition of the Piper meridionalis preparations. Furthermore, the developer provided by the present invention uses dichloromethane instead of chloroform, which is less toxic and provides better chromatographic separation results for Piper meridionalis preparations.

Claims

1. A thin layer identification method for Piper mesenteriae and its preparations, characterized in that: The steps include: 1) Dissolve a sample of the Piper mesangiales preparation in water, then shake and extract with ethyl acetate. After stratification, evaporate the supernatant to dryness, and dissolve the residue in an alcohol solvent to obtain a Piper mesangiales preparation test solution. 2) Take a control medicinal material or medicinal material sample of Pieris ovata, decoct it in water or heat it under reflux, filter it, and extract it by shaking with ethyl acetate solution. After standing and stratifying, take the supernatant and evaporate it to dryness. Dissolve the residue in an alcohol solvent to prepare a control medicinal material solution or medicinal material test solution; 3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate respectively, develop them with a developing agent, and obtain a thin layer chromatogram to complete the identification operation.

2. The thin layer identification method of Piper mesenteriae and its preparation according to claim 1, characterized in that: The Piper mesangineum preparation sample in step 1) includes Piper mesangineum formula granules or standard decoction.

3. The thin layer identification method of Piper mesenteriae and its preparation according to claim 1, characterized in that: When the Piper mestolide preparation sample is extracted with water in step 1), the mass volume ratio of the Piper mestolide preparation sample to water is 1:50 g / mL to 200 g / mL, the ethyl acetate solution is shaken and extracted 1 to 4 times, and the volume ratio of the ethyl acetate solution to the sample aqueous solution is 0.5:2 to 1.

4. The thin layer identification method of Piper mesenteriae and its preparation according to claim 1, characterized in that: When the Pieris ovata control medicinal material or medicinal material sample described in step 1 is decocted with water, the mass volume of the Pieris ovata control medicinal material or medicinal material sample and water is 1:10g / mL~100g / mL, the ethyl acetate solution is shaken and extracted 1 to 4 times, and the volume ratio of the ethyl acetate solution to the sample aqueous solution is 0.5:2~1.

5. The thin layer identification method of Piper mesenteriae and its preparation according to claim 1, characterized in that: The developing solvent in step 3) is dichloromethane-acetone-methanol.

6. The thin layer identification method of Piper mesenteriae and its preparation according to claim 5, characterized in that: The volume ratio of dichloromethane-acetone-methanol is 9:(0.8~1.2):(0.3~0.8).

7. The thin layer identification method of Piper mesenteriae and its preparation according to claim 1, characterized in that: The spotting volume of the control medicinal material solution and the test solution described in step 3) is 3 μl to 8 μl.

8. The thin layer identification method of Piper mesenteriae and its preparation according to claim 1, characterized in that: In the step 3), the thin layer plate is a silica gel G thin layer plate.

9. The thin layer identification method of Piper mesenteriae and its preparation according to claim 1, characterized in that: In the thin layer chromatogram of step 3), a fluorescent spot of the same color appears in the chromatogram of the test solution at a position corresponding to the chromatogram of the control medicinal material solution.

10. Use of the thin-layer identification method according to any one of claims 1 to 9 in identifying Kadura kadzuvine and its counterfeit Schisandrae chinensis.

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