A thin-layer identification method of portulaca oleracea and its preparation

Abstract

The application discloses a thin-layer identification method for Portulaca oleracea and its preparation, wherein a simple and quick pretreatment method is used to obtain a test sample solution and a control medicinal material solution, which are spotted on the same thin-layer plate, and then developed with cyclohexane-methylene chloride-ethyl acetate-formic acid as a developing agent to obtain a multi-information thin-layer chromatogram of Portulaca oleracea. The method is simple and quick in operation, and the chromatographic spot has good separation degree, is suitable for identifying Portulaca oleracea and its preparation, can distinguish true products from fake products, has the characteristics of specificity, low detection cost, safe operation and environment-friendly reagents.

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 purslane and its preparations. Background Technology

[0002] Purslane is the dried aerial part of the plant *Portulaca oleracea* L., belonging to the Portulacaceae family. It has a sour taste and cold properties, and possesses the effects of clearing heat and detoxifying, cooling the blood and stopping bleeding, and relieving dysentery. It is used for dysentery caused by heat toxins, carbuncles, boils, eczema, erysipelas, snake and insect bites, hematochezia, hemorrhoidal bleeding, and metrorrhagia. Modern pharmacological studies have found that purslane has antibacterial, antiviral, lipid-lowering, and anti-atherosclerotic effects. Its chemical components mainly include organic acids, alkaloids, flavonoids, and lignans.

[0003] The existing thin-layer chromatography (TLC) identification methods for purslane mainly refer to the TLC identification method for purslane medicinal materials included in Part I of the 2020 edition of the Chinese Pharmacopoeia. However, this method has a long pretreatment time, identifies amino acid components, has poor specificity, and contains amino acid components in many kinds of herbal medicines. In addition, the chromatographic spot separation is poor, which can easily lead to misjudgment and cannot accurately identify the substance. Other TLC identification methods for purslane in other preparations reported in the literature (Hou Yinhuan. Study on chemical composition and quality evaluation of purslane [D]. Shenyang Pharmaceutical University, 2008.; Sharina et al. Establishment of quality standards for Healing King preparations [J]. Chinese Pharmacist, 2016, 19(11):2165-2167.) all use toxic and harmful solvents such as benzene, toluene, and chloroform, which have adverse effects on the environment and the safety of laboratory personnel. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a thin-layer chromatography method and its application for the identification of purslane and its preparations.

[0005] Technical solution: The thin-layer chromatography identification method for purslane and its preparations provided by this invention includes the following steps:

[0006] (1) Prepare a purslane test solution, wherein the purslane test solution includes purslane medicinal material, purslane formula granules and purslane standard decoction;

[0007] (2) Preparation of purslane reference herb solution;

[0008] (3) Spot the purslane test solution and the purslane reference solution onto the same thin-layer plate, develop them with cyclohexane-dichloromethane-ethyl acetate-formic acid as the developing solvent, remove them and examine them under ultraviolet light.

[0009] Further, in step (1), when the purslane test sample is purslane medicinal material, the preparation process of the purslane test sample solution, or the preparation process of the purslane reference medicinal material solution in step (2) is as follows: take purslane medicinal material, add water to decoct or heat and reflux, filter, add ethyl acetate to the filtrate and shake to extract, let stand to separate the layers and combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in an alcohol solvent to obtain the final product;

[0010] Alternatively, take purslane, decoct it in water or heat it under reflux, filter it, add methanol to the residue, sonicate it, filter it, evaporate the filtrate to dryness, dissolve the residue in methanol, and you will get the product.

[0011] Furthermore, the mass-to-volume ratio of the purslane herb to water is 1:50g / mL to 200g / mL, the decoction or reflux treatment time is 15min to 60min, the number of ethyl acetate shaking extractions is 1 to 3, and the volume ratio of ethyl acetate solution to the sample aqueous solution is 0.5 to 2:1.

[0012] Further, in step (1), when the purslane test sample is purslane formula granules or purslane standard decoction, the preparation process of the purslane test sample solution is as follows: take the purslane preparation test sample, dissolve or dilute it with water, extract it by shaking with ethyl acetate, let it stand to separate the layers and combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in an alcohol solvent to obtain the solution.

[0013] Alternatively, take a purslane preparation as a test sample, dissolve it in methanol or ethanol, sonicate it, filter it, evaporate the filtrate to dryness, and dissolve the residue in an alcohol solvent to obtain the final product.

[0014] Furthermore, the mass-to-volume ratio of the test sample to water is 1:20 g / mL to 50 g / mL, the ultrasonic treatment power is 250 W to 600 W, the frequency is 30 kHz to 50 kHz, the time is 15 min to 60 min, the number of times of ethyl acetate shaking extraction is 1 to 3, and the volume ratio of ethyl acetate solution to sample aqueous solution is 0.5 to 2:1.

[0015] Furthermore, the thin sheet mentioned in step (3) is a silicone sheet.

[0016] Further, the volume ratio of cyclohexane-dichloromethane-ethyl acetate-formic acid in step (3) is (3-5):(0.5-1.5):(3-5):0.1.

[0017] Furthermore, in step (3), the amount of the test solution is 2 μl to 10 μl, and the amount of the control herbal solution is 5 μl to 15 μl.

[0018] Furthermore, in step (3), the wavelength of the ultraviolet lamp is 360–370 nm.

[0019] The present invention also provides the application of the above-mentioned thin-layer identification method of purslane and its preparations in distinguishing genuine and counterfeit products.

[0020] 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 herb solution, which are then spotted separately on the same thin-layer plate. Cyclohexane-dichloromethane-ethyl acetate-formic acid is used as the developing solvent to obtain a multi-information thin-layer chromatogram of purslane. This method is simple and rapid to operate, with good chromatographic spot separation, suitable for the identification of purslane and its preparations, and can distinguish between genuine and counterfeit products. It has specificity, low detection cost, safe operation, and uses more environmentally friendly reagents. Attached Figure Description

[0021] Figure 1 This is a thin-layer chromatogram of the purslane formula granules in Example 1 of the present invention (wherein, 1 to 3 are the chromatograms of test samples of different batches of purslane formula granules, and S is the chromatogram of purslane reference medicinal material).

[0022] Figure 2 This is a thin-layer chromatogram of the purslane standard decoction in Example 1 of the present invention (wherein, 1 to 3 are the chromatograms of test samples of different batches of purslane standard decoction, and S is the chromatogram of purslane reference medicinal material).

[0023] Figure 3 This is a thin-layer chromatogram of purslane medicinal material in Example 1 of the present invention (wherein, 1 to 3 are chromatograms of test samples of different batches of purslane medicinal material, and S is the chromatogram of purslane reference medicinal material);

[0024] Figure 4 These are thin-layer chromatograms of different developing solvent ratios in Example 2 of the present invention (wherein, 1 to 3 are chromatograms of test samples of different batches of purslane medicinal materials, and S is the chromatogram of purslane reference medicinal material).

[0025] Figure 5 These are thin-layer chromatograms of different spotting amounts in Example 2 of the present invention (wherein, 1 to 4 are chromatograms of purslane formulation granules of different spotting amounts, and S1 to S4 are chromatograms of purslane reference medicinal materials of different spotting amounts).

[0026] Figure 6 These are thin-layer chromatograms taken at different temperatures in Example 2 of the present invention (where 1 to 3 are the test chromatograms of purslane formulation granules, and S is the chromatogram of purslane reference medicinal material).

[0027] Figure 7 These are thin-layer chromatograms of different humidity levels in Example 2 of the present invention (where 1 to 3 are the test samples of purslane formulation granules, and S is the reference material purslane chromatogram).

[0028] Figure 8This is a thin-layer chromatogram used in Example 3 of the present invention to distinguish between the genuine and counterfeit purslane medicinal material and the counterfeit Sedum sarmentosum medicinal material (wherein, 1 to 3 are chromatograms of test samples from different batches of Sedum sarmentosum medicinal material, 4 to 6 are chromatograms of test samples from different batches of purslane medicinal material, and S is the chromatogram of purslane reference medicinal material).

[0029] Figure 9 This is a thin-layer chromatogram used in Example 3 of the present invention to distinguish between the purslane standard decoction and the counterfeit Sedum sarmentosum standard decoction (wherein, 1-3 are the test samples of different batches of Sedum sarmentosum standard decoction, 4-6 are the test samples of different batches of purslane standard decoction, and S is the purslane reference herb chromatogram).

[0030] Figure 10 These are thin-layer chromatograms of the test solutions and reference medicinal materials of purslane formula granules prepared by different methods in Comparative Example 1 of the present invention (wherein, 1 to 3 are the test chromatograms of purslane formula granules prepared by the three methods, and S1 to S3 are the chromatograms of purslane reference medicinal materials prepared by the three methods).

[0031] Figure 11 The images show thin-layer chromatograms of purslane formulation granules obtained by different identification methods in Comparative Example 2 of this invention (wherein, the left image uses the thin-layer developing solvent from the Purslane section of the 2020 edition of the Chinese Pharmacopoeia: water-saturated n-butanol-glacial acetic acid-water (4∶1∶1), and the colorimetric reagent is 0.2% ninhydrin ethanol solution; the right image uses the developing solvent reported in the literature: toluene-chloroform-ethyl formate-formic acid (5∶1∶11∶1)). Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0033] The instruments and reagents used in this invention are as follows:

[0034] Instruments: Automated thin-layer imaging system (CAMAG TLC VISUALIZER), 0.001 g balance (Mettler-Toledo), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), GKC temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.), TH-II type digitally controlled thin-layer chromatography heater (Shanghai Kezhe Biochemical Technology Co., Ltd.), high-efficiency silica gel G thin-layer plate (Qingdao Ocean Chemical Plant).

[0035] Reagents: Methanol (Shanghai Titan Technology Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), cyclohexane (Sinopharm Chemical Reagent Co., Ltd.), dichloromethane (Sinopharm Chemical Reagent Co., Ltd.), n-butanol (Sinopharm Chemical Reagent Co., Ltd.), glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd.), toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), chloroform (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethyl formate (Shanghai Titan Technology Co., Ltd.), and formic acid (Shanghai Titan Technology Co., Ltd.) were all of analytical grade.

[0036] Purslane reference material (batch number: 121598-202004) was purchased from the National Institutes for Food and Drug Control.

[0037] The test samples were: Purslane granules (batch numbers: 24060019, 24060029, 24060039), Purslane standard decoction (batch numbers: DG2202009, DG1012404008, DG1012404010), Purslane medicinal material (batch numbers: YC2202009, YC1012404008, YC1012404010), Sedum sarmentosum standard decoction (batch numbers: DG1804157, DG1807040, DG1807105), and Sedum sarmentosum medicinal material (batch numbers: YC1804157, YC1807040, YC1807105), all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0038] For experiments not specifically described in this invention, the procedures and 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.

[0039] Example 1: Thin-layer chromatography method for identifying purslane

[0040] Preparation of test solution / reference solution of medicinal material: Take 1g of purslane test / reference medicinal material, add 50ml of water, heat under reflux for 30 minutes, filter, concentrate the filtrate to about 20ml, extract twice with ethyl acetate, 20ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use as the test solution / reference solution.

[0041] Preparation of the test solution for the formulation (the preparation method for the test solution of the formulation granules and the standard decoction is the same): Take 0.5g of this product, grind it into a fine powder, add 20ml of water to dissolve it, extract it twice with ethyl acetate, 20ml 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 for the formulation.

[0042] Thin-layer identification procedure: Perform the thin-layer chromatography test (General Chapter 0502 of Chinese Pharmacopoeia 2020). Take 5 μl of the test solution and 10 μl of the reference herb solution and spot them separately on the same high-performance silica gel G thin-layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:4:0.1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0043] Evaluation requirements: In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0044] Results analysis: Figure 1 Chromatograms 1-3 are chromatograms of test samples from different batches of purslane formulation granules, and S is the chromatogram of purslane reference medicinal material. Figure 2 Chromatograms of purslane standard decoctions from different batches are shown in Figures 1-3, and Figure S is the chromatogram of purslane reference material. Figure 3 Chromatograms 1-3 show the test samples of different batches of purslane medicinal materials, and S is the chromatogram of the purslane reference medicinal material. The chromatograms of purslane medicinal materials, standard decoction, and formula granules were compared with the chromatogram of the reference medicinal material. Fluorescent spots of the same color appeared at the same position, indicating that all were qualified products.

[0045] Example 2: Durability Study of Thin-Layer Identification Method for Purslane

[0046] (1) Investigation of different developing solvent ratios:

[0047] Take purslane formula granules (batch number: 24060029) and purslane reference medicinal material, and prepare test solution and reference medicinal material solution respectively according to the method described in Example 1. Take 5 μl of purslane formula granule test solution and 10 μl of purslane reference medicinal material solution respectively, and spot them on the same high-performance silica gel G thin layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (3:0.5:3:0.1), (4:1:4:0.1), (5:1.5:5:0.1), (6:2:6:0.1) as developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm).

[0048] See results Figure 4 , Figure 4 In the diagram, 1-3 correspond to the chromatograms of the purslane formula granules test sample, and S corresponds to the chromatogram of the purslane reference herb. Figure 4It was found that under different developing solvent ratios, the chromatograms of the purslane formulation granules and the control medicinal material showed fluorescent spots of the same color at corresponding positions. When the volume ratio of cyclohexane-dichloromethane-ethyl acetate-formic acid was (3–5):(0.5–1.5):(3–5):0.1, the fluorescence spot separation was better, and the chromatogram was more aesthetically pleasing. Therefore, subsequent experiments used a volume ratio of cyclohexane-dichloromethane-ethyl acetate-formic acid of 4:1:4:0.1 for further investigation.

[0049] (2) Investigation of different sample sizes:

[0050] Take purslane formula granules (batch number: 24060029) and purslane reference medicinal material, and prepare test solution and reference medicinal material solution respectively according to the method described in Example 1. Take 2 μl, 5 μl, 8 μl, 10 μl of purslane formula granule test solution and 5 μl, 8 μl, 10 μl, 15 μl of purslane reference medicinal material solution respectively, and spot them on the same high-performance silica gel G thin layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:4:0.1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm).

[0051] See results Figure 5 , Figure 5 S1-S4 correspond to the chromatograms of purslane formula granules (2 μl, 5 μl, 8 μl, and 10 μl, respectively) as test samples, and S1-S4 correspond to the chromatograms of purslane reference materials (5 μl, 8 μl, 10 μl, and 15 μl, respectively). Figure 5 It can be seen that when the sample volume of the test sample solution is 2μl to 10μl and the sample volume of the reference medicinal material solution is 5μl to 15μl, the fluorescent spots in the chromatogram of the test sample and the chromatogram of the reference medicinal material are clear at the corresponding positions, without other interference, and can correspond one-to-one.

[0052] (3) Investigation at different temperatures

[0053] Take purslane formula granules (batch number: 24060029) and purslane reference medicinal material, and prepare test solution and reference medicinal material solution respectively according to the method described in Example 1. Take 5 μl of purslane formula granule test solution and 10 μl of purslane reference medicinal material solution respectively, and spot them on the same high-performance silica gel G thin layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:4:0.1) as the developing solvent, and develop under different temperatures (T: 4℃, 19.6℃, 35℃). Remove, air dry, and examine under ultraviolet light (365nm).

[0054] See results Figure 6 , Figure 6 In the diagram, 1-3 correspond to the chromatograms of the purslane formula granules test sample, and S corresponds to the chromatogram of the purslane reference herb. Figure 6 It can be seen that under different temperature conditions, the chromatograms of the purslane formula granules and the reference herb show fluorescent spots of the same color at the corresponding positions. The experimental results indicate that temperature has no significant effect on the thin-layer identification of purslane formula granules, suggesting that this thin-layer identification method is robust to different temperatures.

[0055] (4) Investigation of different humidity levels

[0056] Take purslane formula granules (batch number: 24060029) and purslane reference medicinal material, and prepare test solution and reference medicinal material solution respectively according to the method described in Example 1. Take 5 μl of purslane formula granule test solution and 10 μl of purslane reference medicinal material solution respectively, and spot them on the same high-performance silica gel G thin layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:4:0.1) as the developing solvent, and develop under different humidity conditions (RH: 18%, 58.1%, 88%). Remove, air dry, and examine under ultraviolet light (365nm).

[0057] See results Figure 7 , Figure 7 In the diagram, 1-3 correspond to the chromatograms of the purslane formula granules test sample, and S corresponds to the chromatogram of the purslane reference herb. Figure 7 It was found that under different humidity conditions, the chromatograms of the purslane formula granules and the reference medicinal material showed fluorescent spots of the same color at the corresponding positions. The experimental results indicate that humidity has no significant effect on the thin-layer identification of purslane formula granules, demonstrating the good durability of this thin-layer identification method under different humidity conditions.

[0058] Example 3: Identification of Purslane and its Preparations from Counterfeits

[0059] (1) Identification of Purslane medicinal material and its counterfeit medicinal material (Sedum sarmentosum).

[0060] Take Sedum sarmentosum, Portulaca oleracea (test sample), and Portulaca oleracea (reference sample), and prepare test sample solutions and reference sample solutions respectively according to the method described in Example 1. Take 10 μl of each of the Sedum sarmentosum test sample solution, Portulaca oleracea test sample solution, and Portulaca oleracea (reference sample) solution and spot them on the same high-performance silica gel G thin-layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:4:0.1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0061] See results Figure 8 , Figure 8 Chromatography 1-3 correspond to the chromatograms of the *Sedum sarmentosum* sample, 4-6 correspond to the chromatograms of the *Portulaca oleracea* sample, and S corresponds to the chromatogram of the *Portulaca oleracea* reference sample. Figure 8It can be seen that the fluorescent spots of the purslane medicinal material correspond one-to-one with those of the purslane control material. The upper part of the thin-layer chromatogram of the Sedum sarmentosum material has a yellow fluorescent spot, which is inconsistent with the spot of the purslane control material. Therefore, this thin-layer identification is effective in distinguishing between purslane medicinal material and the counterfeit Sedum sarmentosum material.

[0062] (2) Differentiation between standard purslane decoction and counterfeit standard decoction (Sedum sarmentosum).

[0063] Take the standard decoction of Sedum sarmentosum, the standard decoction of Portulaca oleracea, and the reference herb of Portulaca oleracea, and prepare the test solution and the reference herb solution respectively according to the method described in Example 1. Take 5 μl of each of the test solution of the standard decoction of Sedum sarmentosum and the test solution of the standard decoction of Portulaca oleracea, and 10 μl of each of the reference herb solution of Portulaca oleracea, and spot them on the same high-performance silica gel G thin layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:4:0.1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0064] See results Figure 9 , Figure 9 Chromatograms 1-3 correspond to the chromatograms of the standard decoction of *Sedum sarmentosum*, 4-6 correspond to the chromatograms of the standard decoction of *Portulaca oleracea*, and S corresponds to the chromatogram of the reference herb *Portulaca oleracea*. Figure 9 It can be seen that the fluorescent spots of the standard decoction of purslane correspond one-to-one with those of the purslane reference material. The upper part of the thin-layer chromatogram of the standard decoction of Sedum sarmentosum has a yellow fluorescent spot, which is inconsistent with the spot of the purslane reference material. Therefore, this thin-layer identification is sufficient to effectively distinguish between the standard decoction of purslane and the counterfeit standard decoction of Sedum sarmentosum.

[0065] Comparative Example 1:

[0066] This comparative study investigated different methods for preparing the test and control herbal solutions, as detailed below:

[0067] (1) Investigation of different preparation methods of purslane formula granules (batch number: 24060029) test sample solution:

[0068] Method 1: Take 0.5g of this product, grind it into a fine powder, add 20ml of methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to obtain the test solution.

[0069] Method 2: Take 0.5g of this product, grind it into a fine powder, add 20ml of ethanol, sonicate (600W power, 40kHz frequency) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol to obtain the test solution.

[0070] Method 3: Take 0.5g of this product, grind it into a fine powder, add 20ml of water to dissolve it, extract it twice with ethyl acetate, 20ml 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.

[0071] (2) Investigation of different preparation methods of purslane reference herb solution:

[0072] Method 1: Take 1g of purslane reference material, add 20ml of methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the reference material solution.

[0073] Method 2: Take 1g of purslane reference material, add 50ml of water, heat under reflux for 30 minutes, filter, concentrate the filtrate to near dryness, add 20ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the reference material solution.

[0074] Method 3: Take 1g of purslane reference material, add 50ml of water, heat under reflux for 30 minutes, filter, concentrate the filtrate to about 20ml, extract twice with ethyl acetate, 20ml each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1ml of methanol, and use as the reference material solution.

[0075] Perform the thin-layer chromatography test (General Chapter 0502 of Chinese Pharmacopoeia 2020). Take 5 μl of the test solution and 10 μl of the reference herb solution and spot them separately on the same high-performance silica gel G thin-layer plate. Use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:4:0.1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0076] Results analysis: Figure 10 Chromatograms S1-S3 are the test samples of purslane granules prepared by the three corresponding methods, and chromatograms of purslane reference materials prepared by the three corresponding methods. Figure 10 It can be seen that the fluorescent spots of the samples prepared by the three test solution preparation methods are clear and the separation effect is good.

[0077] The first method for preparing the reference herb solution had excessive pigment, which affected the visibility of the fluorescent spots. Methods two and three, on the other hand, showed clearer chromatographic spots and better separation. To minimize the influence of pigment, considering the convenience of spotting, and ensuring consistency between the preparation methods of the test sample solution and the reference herb solution, method three was selected as the optimal treatment method for the purslane formula granules test sample, and method three was also selected as the optimal treatment method for both the purslane reference herb and the test sample.

[0078] Comparative Example 2:

[0079] This comparative example compares different developing solvents for thin-layer chromatography, as detailed below:

[0080] (1) The developing solvent for thin-layer chromatography under the Portulaca oleracea section of the 2020 edition of the Chinese Pharmacopoeia (General Chapter 0502).

[0081] Take 1-2 μl each of the purslane test sample (formula granules) solution and the control herbal solution prepared by the method in Example 1, and spot them separately on the same silica gel G thin layer plate. Use water-saturated n-butanol-glacial acetic acid-water (4:1:1) as the developing solvent, develop, remove, air dry, spray with 0.2% ninhydrin ethanol solution, and heat at 110°C until the spots are clearly visible.

[0082] (2) The thin-layer chromatography test reported in the literature (Hou Yinhuan. Study on chemical composition and quality evaluation of purslane [D]. Shenyang Pharmaceutical University, 2008.; Sharina et al. Establishment of quality standard of Healing King preparation [J]. Chinese Pharmacist, 2016, 19(11):2165-2167.) used 5 μl of the purslane test sample (formula granules) solution and 10 μl of the control herbal solution prepared by the method in Example 1, and spotted them on the same silica gel G thin-layer plate. Toluene-chloroform-ethyl formate-formic acid (5∶1∶11∶1) was used as the developing solvent. The plate was saturated for 25 minutes, developed, and the development distance was 14 cm. The plate was then removed, dried, and examined under ultraviolet light (365 nm).

[0083] Results analysis: Figure 11 The figures correspond to two different developing systems: the left figure shows a developing solvent of water-saturated n-butanol-glacial acetic acid-water (4:1:1), and the right figure shows a developing solvent of toluene-chloroform-ethyl formate-formic acid (5:1:11:1). When using the pharmacopoeia-based developing system, sample pretreatment is time-consuming, chromatographic spot resolution is poor, and misjudgment is easily caused, making accurate quality evaluation impossible. The developing systems reported in the literature all use toxic and harmful solvents such as toluene and chloroform, which are not environmentally friendly, have high Rf values, and provide limited spot information (only two spots), which is detrimental to the quality evaluation of purslane.

Claims

1. A thin-layer chromatography method for the identification of purslane and its preparations, characterized in that: Includes the following steps: (1) Prepare a purslane test solution, wherein the purslane test solution includes purslane medicinal material, purslane formula granules and purslane standard decoction; The preparation process of the purslane test solution is as follows: When the purslane test sample is purslane medicinal material, take the purslane test sample, add water to decoct or heat to reflux, filter, add ethyl acetate to the filtrate and shake to extract, let stand to separate the layers, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in an alcohol solvent to obtain the test sample; Alternatively, take purslane as a test sample, decoct it in water or heat it under reflux, filter it, add methanol to the residue, sonicate it, filter it, evaporate the filtrate to dryness, dissolve the residue in methanol, and you will get the test sample. When the purslane test sample is a purslane preparation test sample, i.e. purslane formula granules or purslane standard decoction, take the purslane preparation test sample, dissolve or dilute it with water, extract it with ethyl acetate by shaking, allow it to stand and separate into layers, combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in an alcohol solvent to obtain the final product. Alternatively, take a purslane preparation as a test sample, dissolve it in methanol or ethanol, sonicate it, filter it, evaporate the filtrate to dryness, and dissolve the residue in an alcohol solvent to obtain the final product; (2) Preparation of purslane reference herb solution; the preparation process is as follows: Take purslane medicinal material, decoct it with water or heat it under reflux, filter it, add ethyl acetate to the filtrate and shake to extract it, let it stand to separate the layers and combine the ethyl acetate layers, evaporate it to dryness, dissolve the residue in an alcohol solvent to obtain the product; Alternatively, take purslane medicinal material, decoct it in water or heat it under reflux, filter it, add methanol to the residue, sonicate it, filter it, evaporate the filtrate to dryness, dissolve the residue in methanol, and you will get the product. (3) Spot the purslane test solution and the purslane reference solution onto the same silica gel G thin layer plate, develop them with cyclohexane-dichloromethane-ethyl acetate-formic acid as the developing solvent, remove them and examine them under ultraviolet light; The volume ratio of cyclohexane-dichloromethane-ethyl acetate-formic acid is (3-5):(0.5-1.5):(3-5):0.

1.

2. The thin-layer chromatography method for identifying purslane and its preparations according to claim 1, characterized in that: When the purslane reference herb solution or the purslane sample is purslane herb, the mass-to-volume ratio of the purslane herb to water is 1:50 g / mL to 200 g / mL, the decoction or reflux treatment time is 15 min to 60 min, the number of ethyl acetate shaking extractions is 1 to 3, and the volume ratio of ethyl acetate solution to the sample aqueous solution is 0.5 to 2:

1.

3. The thin-layer chromatography identification method for purslane and its preparations according to claim 1, characterized in that: When the purslane test sample is a purslane preparation test sample, the mass-to-volume ratio of the test sample to water is 1:20 g / mL to 50 g / mL, the ultrasonic treatment power is 250 W to 600 W, the frequency is 30 kHz to 50 kHz, the time is 15 min to 60 min, and the number of ethyl acetate shaking extractions is 1 to 3.

4. The thin-layer chromatography method for identifying purslane and its preparations according to claim 1, characterized in that: The amount of the test solution to be spotted in step (3) is 2 μl to 10 μl, and the amount of the control herbal solution to be spotted is 5 μl to 15 μl.

5. The thin-layer chromatography identification method for purslane and its preparations according to claim 1, characterized in that: In step (3), the wavelength of the ultraviolet lamp is 360-370nm.

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