Thin-layer identification method of fructus broussonetiae and application of thin-layer identification method

Abstract

The invention discloses a thin-layer identification method of fructus broussonetiae and application of the thin-layer identification method. Respectively dripping the reference medicinal material solution and the test solution on the same thin-layer plate, developing by using toluene-ethyl acetate-formic acid-water as a developing solvent, and inspecting; and when fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference medicinal material in the chromatogram of the sample to be tested, the sample to be tested is the fructus broussonetiae. According to the method, a proper developing agent is used for developing, direct inspection under ultraviolet light is not needed, operation errors caused by non-uniform distribution of the developing agent or environmental changes are avoided, spots are clear, the separation degree is good, the Rf value is moderate, and positive and fake products can be well distinguished; compared with a pharmacopoeia method and existing literature reports, the method is suitable for identification and application of pharmaceutical preparations such as formula granules taking water as a solvent, is good in specificity, wide in application range and low in detection cost, is more accurate and scientific by adopting consistency of reference medicinal materials for evaluation, and is more beneficial to quality supervision.

Description

Technical Field

[0001] The invention relates to a thin-layer identification method for Corydalis chinensis seeds and application thereof, and belongs to the field of quality control of traditional Chinese medicines. Background Art

[0002] Paper mulberry seeds are the dried mature fruits of Broussonetia papyrifera (L.) Vent., a plant of the Moraceae family. They are harvested in autumn when the fruits are ripe. They have the effects of nourishing the kidney and clearing the liver, improving eyesight, and promoting urination. They are used for liver and kidney deficiency, soreness of the waist and knees, asthenia, dizziness, eye film, and edema. Modern research shows that paper mulberry seeds contain a variety of chemical components such as fatty oil, amino acids, pigments, and polysaccharides, and have the effects of anti-oxidation, lowering blood lipids, enhancing immunity, anti-tumor, and protecting liver function.

[0003] The existing thin-layer identification method for Cucurbitacin is mainly the thin-layer identification method for Cucurbitacin medicinal materials included in the 2020 edition of the "Chinese Pharmacopoeia". However, this method is mainly for medicinal samples. When applied to Cucurbitacin formula granules, the thin-layer spot tailing and the Rf value are too high. It is not suitable for thin-layer identification of Cucurbitacin formula granules and cannot perform accurate quality evaluation. In addition, Liu Lina et al. used thin-layer chromatography to identify Cuscuta australis (Liu Lina, Zhou Xinbei, Ouyang Rong. Identification of adulterated products in Cuscuta australis [J]. Journal of Hunan University of Traditional Chinese Medicine, 2004, (03): 16-18.). This method was also used for medicinal material samples. The spots it showed were mainly small polar components in the medicinal materials. The chromatographic information obtained was relatively small, and it was impossible to identify the authenticity of the samples. Moreover, it was not suitable for the identification and quality evaluation of pharmaceutical preparations such as formula granules with water as the solvent. Its developing solvent system contained volatile solvents such as ether, which could easily have adverse effects on the environment. In addition, it used iodine vapor for color development, and the chromatogram obtained had poor reproducibility and short storage time, so its application was relatively limited. No accurate and effective thin-layer chromatography identification method has been reported in the existing literature, and it is difficult to effectively distinguish Cuscuta australis and its counterfeits. Moreover, these methods are only used for medicinal materials, and no reports have been made on formula granules and standard decoctions prepared after water extraction. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a thin layer identification method of paper mulberry seeds with good specificity, wide application range and low detection cost and its application.

[0005] Technical solution: To solve the above technical problems, the present invention provides a thin layer identification method of paper mulberry seeds, comprising the following steps:

[0006] (1) Preparation of test sample solution: dissolve the test sample powder in water, filter, extract with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to prepare the test sample solution;

[0007] (2) Preparation of control medicinal material solution: dissolving the Cuscuta australis control medicinal material, filtering, extracting, evaporating to dryness, and dissolving the residue in methanol to prepare the control medicinal material solution;

[0008] (3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate, develop them with toluene-ethyl acetate-formic acid-water as the developing solvent, and examine and compare the thin layer chromatograms;

[0009] (4) When a fluorescent spot of the same color appears at a corresponding position in the chromatogram of the test sample and the chromatogram of the control medicinal material, it indicates that the test sample is Cuscuta australis; the Cuscuta australis includes medicinal materials, formula granules or standard decoctions.

[0010] Wherein, the volume of the control medicinal material solution in step (3) is 3 to 10 μl; the volume of the test solution is 3 to 10 μl.

[0011] Wherein, the development temperature in step (3) is 4 to 35°C.

[0012] Wherein, the humidity of the unfolding in step (3) is 18% to 88%.

[0013] The method also provides application of the method in distinguishing Cuscuta australis seeds from Perilla seeds and Cuscuta australis seeds.

[0014] The method also provides a method for distinguishing Cuscuta australis seeds from Perilla seeds and Cuscuta australis seeds, comprising the following steps:

[0015] (1) Preparation of test sample solution: dissolve the test sample powder in water, filter, extract with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to prepare the test sample solution;

[0016] (2) Preparation of control medicinal material solution: extract the Cuscuta australis control medicinal material, filter, extract, evaporate to dryness, and dissolve the residue in methanol to prepare the control medicinal material solution;

[0017] (3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate, develop them with toluene-ethyl acetate-formic acid-water as the developing solvent, and examine and compare the thin layer chromatograms;

[0018] (4) When a fluorescent spot of the same color appears at a corresponding position in the chromatogram of the test sample and the chromatogram of the control medicinal material, it indicates that the test sample is Cuscuta australis; otherwise, it is a counterfeit; the Cuscuta australis includes medicinal materials, formula granules or standard decoctions; the Perilla seeds and Cuscuta seeds include medicinal materials, formula granules or standard decoctions of Perilla seeds and Cuscuta seeds.

[0019] Wherein, the volume of the control medicinal material solution in step (3) is 3 to 10 μl; the volume of the test solution is 3 to 10 μl.

[0020] Wherein, the development temperature in step (3) is 4 to 35°C.

[0021] Wherein, the humidity of the unfolding in step (3) is 18% to 88%.

[0022] Principle analysis: Thin layer chromatography is a trace, fast and simple chromatographic method. According to the chemical structure and properties of each effective ingredient of traditional Chinese medicine, following the principle of similar and compatible extraction, a suitable extraction solvent is used to simply and quickly prepare the test sample and reference medicinal material solutions, and then a suitable developing agent is used for development. Due to the different polarities and adsorption capacities of various compounds, various chemical components will be well separated on the thin layer plate according to the ability of adsorption, desorption, re-adsorption and re-desorption with the selected developing agent. In addition, on the same thin layer plate, under different inspection conditions, different chemical components with similar polarities will show different color spots, thereby obtaining more information in the thin layer chromatogram.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention uses a suitable developing agent for development, and does not require a developer for direct inspection under ultraviolet light, thereby avoiding operational errors caused by uneven distribution of the developer or environmental changes. The spots are clear, the separation is good, the Rf value is moderate, and the genuine and counterfeit products can be distinguished well; 2. Compared with the pharmacopoeia method and existing literature reports, the present method is suitable for the identification and application of pharmaceutical preparations such as formula granules with water as the solvent, has good specificity, a wide range of applications, low detection cost, and uses the consistency of control medicinal materials for evaluation, which is more accurate, scientific, and more conducive to quality supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The thin layer chromatogram of the Cuscuta australis granules in Example 1 of the present invention (wherein 1 to 3 are the test sample chromatograms of Cuscuta australis granules of different batches, and S is the Cuscuta australis reference medicinal material chromatogram);

[0025] Figure 2 The thin layer chromatogram of the standard decoction of Cuscuta australis in Example 1 of the present invention (wherein, 1 to 20 are the chromatograms of the test samples of different batches of the standard decoction of Cuscuta australis, and S is the chromatogram of the reference medicinal material of Cuscuta australis);

[0026] Figure 3 The thin layer chromatogram of the Cuscuta australis medicinal material in Example 1 of the present invention (wherein, 1 to 20 are chromatograms of test samples of Cuscuta australis medicinal materials from different batches, and S is a chromatogram of the Cuscuta australis reference medicinal material);

[0027] Figure 4 The thin layer chromatograms of different sample amounts in Example 2 of the present invention (wherein, 1 to 4 are chromatograms of test samples of the Cuscuta australis formula granules with different sample amounts, and 5 to 8 are chromatograms of the Cuscuta australis control medicinal materials with different sample amounts);

[0028] Figure 5 The thin layer chromatograms developed at different temperatures in Example 2 of the present invention (where 1 to 3 are the chromatograms of the test samples of different batches of standard decoctions of Cuscuta australis, and S is the chromatogram of the Cuscuta australis reference medicinal material);

[0029] Figure 6 The thin layer chromatograms developed at different humidity in Example 2 of the present invention (wherein, 1 to 3 are the chromatograms of the test samples of different batches of standard decoctions of Cuscuta australis, and S is the chromatogram of the Cuscuta australis reference medicinal material);

[0030] Figure 7 The thin layer chromatograms for comparison of the standard decoctions of the seeds of Cuscuta australis and its counterfeit in Example 3 of the present invention (wherein, 1 to 3 are thin layer chromatograms of different batches of standard decoctions of Perilla seeds, 4 to 6 are thin layer chromatograms of different batches of standard decoctions of Cuscuta australis, 7 to 9 are thin layer chromatograms of different batches of standard decoctions of Cuscuta australis, and S is a thin layer chromatogram of a control medicinal material of Cuscuta australis);

[0031] Figure 8 The thin layer chromatograms for comparing the Cuscuta australis and unknown medicinal materials in Example 4 of the present invention (wherein, 1 to 3 are thin layer chromatograms of different batches of unknown medicinal materials, 4 to 6 are thin layer chromatograms of different batches of Cuscuta australis medicinal materials, and S is a thin layer chromatogram of Cuscuta australis reference medicinal material);

[0032] Fig. 9 The thin layer chromatograms of different preparation methods of the Cuscuta australis formula granules and the reference medicinal materials in Comparative Example 1 of the present invention (wherein, 1 to 3 are the chromatograms of the test samples of the Cuscuta australis formula granules respectively prepared by the three preparation methods, and S1 to S3 respectively correspond to the chromatograms of the Cuscuta australis reference medicinal materials prepared by the three preparation methods);

[0033] Fig.10 The thin layer chromatogram of the Cuscuta chinensis formula granules in comparative example 2 of the present invention (wherein, 1 to 3 are the test sample chromatograms of Cuscuta chinensis formula granules of different batches, and S is the Cuscuta chinensis reference medicinal material chromatogram);

[0034] Fig.11 The figure is a thin layer chromatogram comparing different developing agent systems of the standard decoction of Cuscuta australis and its counterfeit in Comparative Example 3 of the present invention (wherein, C is the standard decoction of Cuscuta australis, S is the control medicinal material of Cuscuta australis, Z is the standard decoction of Perilla seed, and T is the standard decoction of Cuscuta australis). DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0036] The instrument and reagent used in the present invention are as follows:

[0037] Instruments: automatic thin layer imager (CAMAGTLCVISUALIZER), one-tenth of a balance (Mettler-Toledo), GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.), KQ-250E ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.), silica gel H thin layer plate (Qingdao Ocean Chemical Plant, Sinopharm Chemical Reagent Co., Ltd.).

[0038] Reagents: petroleum ether (60-90°C) (Sinopharm Chemical Reagent Co., Ltd.), methanol (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), formic acid (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.).

[0039] Cuscuta australis reference material (121720-201501) was purchased from China Food and Drug Inspection Institutes.

[0040] Cuscuta australis formula granules (KL1~KL3), Cuscuta australis standard decoction (DG-C1~DG-C20), Cuscuta australis medicinal materials (YC-C1~YC-C20), as well as fake Perilla seed standard decoction (DG-Z1~DG-Z3), and fake Cuscuta australis standard decoction (DG-T1~DG-T3) were all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0041] If no specific experimental steps or conditions are specified in the present invention, the conventional experimental steps or conditions described in the literature in the art can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0042] Example 1

[0043] Preparation of test solution of formula granules: Take 0.5g of paper mulberry seed formula granules, grind them into powder, add 50ml of hot water to dissolve them, filter them, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time. Combine the ethyl acetate solutions, evaporate them to dryness, and add 1ml of methanol to dissolve the residue as the test solution.

[0044] Preparation of standard decoction test solution: Take 0.3 g of standard decoction of Cucurbitacin, add 50 ml of hot water to dissolve it, filter it, and extract the filtrate by shaking with ethyl acetate twice, 40 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.

[0045] Preparation of the test solution of medicinal materials: Take 2g of Cuscuta australis, add 50ml of water, boil for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1ml of methanol to dissolve the residue as the test solution.

[0046] Preparation of control medicinal solution: Take 2g of Cuscuta australis control medicinal material, add 50ml of water, boil for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1ml of methanol to the residue to dissolve it as the control medicinal solution.

[0047] Thin layer identification steps: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 3 μl of the test sample solution and 5 μl of the control medicinal material solution were respectively aspirated and spotted on the same silica gel H thin layer plate. The upper layer solution of toluene-ethyl acetate-formic acid-water (6:5:1:0.5, v / v) was used as the developing agent. The plate was developed, taken out, dried, and inspected under ultraviolet light (365 nm).

[0048] 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.

[0049] Result analysis: Figure 1 1 to 3 are the chromatograms of the test samples of different batches of Cuscuta australis formula granules (batch numbers: KL1 to KL3), and S is the chromatogram of Cuscuta australis reference medicinal material (development temperature 18.5°C, humidity 31%). Figure 2 In the figure, 1 to 10 and 11 to 20 are the chromatograms of the test samples of the standard decoction of Cuscuta australis (batch numbers: DG-C1 to DG-C20), and S is the chromatogram of the Cuscuta australis reference medicinal material (development temperature 26°C, humidity 53%). Figure 3 1-10, 11-20 are the test sample chromatograms of Cuscuta australis medicinal materials (batch numbers: YC-C1-YC-C20), and S is the Cuscuta australis reference medicinal material chromatogram (development temperature 24°C, humidity 77%). Cuscuta australis granules, standard soup, and medicinal materials all correspond to the Cuscuta australis reference medicinal material chromatograms one by one.

[0050] Example 2

[0051] The durability verification of the thin layer identification in Example 1 specifically includes:

[0052] Preparation of test solution of formula granules: Take 0.5g of paper mulberry seed formula granules, grind them into powder, add 50ml of hot water to dissolve them, filter them, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time. Combine the ethyl acetate solutions, evaporate them to dryness, and add 1ml of methanol to dissolve the residue as the test solution.

[0053] Preparation of control medicinal solution: Take 2g of Cuscuta australis control medicinal material, add 50ml of water, boil for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1ml of methanol to the residue to dissolve it as the control medicinal solution.

[0054] (1) Investigation of different spotting amounts: Take Cuscuta chinensis formula granules (batch number: KL1) and Cuscuta chinensis control medicinal materials, and prepare test solution and control medicinal material solution according to the method described in Example 1, respectively. Take 1 μl, 3 μl, 5 μl, 10 μl of Cuscuta chinensis formula granules test solution and 1 μl, 3 μl, 5 μl, 10 μl of Cuscuta chinensis control medicinal material solution, respectively, and spot them on the same silica gel H thin layer plate. Use toluene-ethyl acetate-formic acid-water (6:5:1:0.5) upper layer solution as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm).

[0055] Results Figure 4 , Figure 4 1 to 4 correspond to the chromatograms of 1μl, 3μl, 5μl, and 10μl of the test sample of Cuscuta australis granules, and 5 to 8 correspond to the chromatograms of 1μl, 3μl, 5μl, and 10μl of the Cuscuta australis reference medicinal material. Figure 4 It can be seen that when the sample volume of Cuscuta australis granules and control medicinal materials is 3-10 μl, the chromatograms of Cuscuta australis granules and control medicinal materials show main spots of the same color at the same position without other interference.

[0056] (2) Investigation at different temperatures: Take Cuscuta chinensis formula granules (batch number: KL1) and Cuscuta chinensis control medicinal materials, and prepare test solution and control medicinal material solution according to the method described in Example 1, respectively. Take 3 μl of the Cuscuta chinensis formula granules test solution and 5 μl of the Cuscuta chinensis control medicinal material, respectively, and spot them on the same silica gel H thin layer plate. Use toluene-ethyl acetate-formic acid-water (6:5:1:0.5) upper layer solution as the developing agent, and develop at low temperature (T: 4°C), room temperature (T: 18.5°C), and high temperature (T: 35°C) conditions, respectively. Take out, dry, and inspect under ultraviolet light (365nm).

[0057] Results Figure 5 , Figure 5 1 to 3 correspond to the chromatograms of the test samples of different batches of Cuscuta australis granules, and S corresponds to the chromatogram of the Cuscuta australis reference medicinal material. Figure 5 It can be seen that under different temperature conditions, the chromatograms of the formula granules of Cuscuta australis and the control medicinal materials show the same main spots of the same color at the same position, and the separation effect is good. The experimental results show that different temperatures have no significant effect on the thin layer identification of the formula granules of Cuscuta australis, indicating that the thin layer identification method has good durability at different temperatures.

[0058] (3) Investigation at different humidity levels: Take Cuscuta australis formula granules (batch number: KL1) and Cuscuta australis control medicinal materials, and prepare test solution and control medicinal material solution according to the method described in Example 1, respectively. Take 3 μl of the Cuscuta australis formula granules test solution and 5 μl of the Cuscuta australis control medicinal material, respectively, and spot them on the same silica gel H thin layer plate. Use toluene-ethyl acetate-formic acid-water (6:5:1:0.5) upper layer solution as the developing agent, and develop under different humidity conditions (RH: 18%, 31%, 88%), respectively. Take out, dry, and inspect under ultraviolet light (365 nm).

[0059] Results Figure 6 , Figure 6 1 to 3 correspond to the chromatograms of the test samples of different batches of Cuscuta australis granules, and S corresponds to the chromatogram of the Cuscuta australis reference medicinal material. Figure 6 It can be seen that under different humidity conditions, the chromatograms of the formula granules of Cuscuta australis and the control medicinal materials show the same main spots of the same color at the same position, and the separation effect is good. The experimental results show that different temperatures have no significant effect on the thin layer identification of the formula granules of Cuscuta australis, indicating that the thin layer identification method has good durability under different humidity.

[0060] Example 3

[0061] The authenticity of paper mulberry seeds is identified by the thin layer identification method in Example 1, which specifically includes:

[0062] Preparation of test solution: Take 0.3 g of standard decoction of Cuscuta australis and standard decoction of pseudo products (Perilla seed and Cuscuta seed) respectively, add 50 ml of hot water to dissolve, filter, and extract the filtrate by shaking with ethyl acetate twice, 40 ml each time, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 ml of methanol as the test solution.

[0063] Thin layer identification steps: Pipette 3μl of the test sample solution and 5μl of the control medicinal material solution, respectively spot them on the same silica gel H thin layer plate, use the upper layer solution of toluene-ethyl acetate-formic acid-water (6:5:1:0.5) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0064] Results Figure 7 , Figure 7 1 to 3 correspond to different batches of counterfeit (Perilla seed) standard decoction (batch number: DG-Z1 to DG-Z3), 4 to 6 correspond to different batches of counterfeit (Dodder seed) standard decoction (batch number: DG-T1 to DG-T3), 7 to 9 correspond to Cuscuta australis standard decoction (batch number: DG-C1 to DG-C3), and S corresponds to Cuscuta australis control medicinal material. Figure 7It can be seen that in the chromatogram of the test sample, the spots of the standard decoction of Cuscuta australis and the control medicinal material of Cuscuta australis correspond one to one, and there are obvious differences in the main spots between the counterfeit standard decoctions of Perilla frutescens and Cuscuta australis and the standard decoction of Cuscuta australis. Compared with the standard decoction of Cuscuta australis, the counterfeit standard decoction of Perilla frutescens showed green fluorescent spots at Rf value 0.80 and blue fluorescent spots at Rf value 0.40; compared with the standard decoction of Cuscuta australis, the counterfeit standard decoction of Cuscuta australis showed green fluorescent spots at Rf value 0.70 and Rf value 0.25. This method can be used to distinguish Cuscuta australis and its counterfeit products.

[0065] Example 4

[0066] The thin layer chromatography identification method in Example 1 is used to identify the seeds of Cuscuta australis and unknown medicinal materials, specifically including:

[0067] Preparation of test solution: Take 2 g of Cucurbitacin and unknown medicinal materials respectively, add 50 ml of water, boil for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 40 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.

[0068] Thin layer identification steps: Pipette 3μl of the test sample solution and 5μl of the control medicinal material solution, respectively spot them on the same silica gel H thin layer plate, use the upper layer solution of toluene-ethyl acetate-formic acid-water (6:5:1:0.5) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0069] Results Figure 8 , Figure 8 1 to 3 correspond to different batches of unknown medicinal materials (batch numbers: 1 to 3), 4 to 6 correspond to different batches of Cuscuta australis medicinal materials (batch numbers: YC-C1 to YC-C3), and S corresponds to the Cuscuta australis control medicinal material. Figure 8 It can be seen that in the chromatogram of the test sample, the spots of the Cuscuta chinensis medicinal material and the Cuscuta chinensis control medicinal material correspond one to one, and there are obvious differences in the main spots between the unknown medicinal material and the Cuscuta chinensis medicinal material. Compared with the Cuscuta chinensis medicinal material, the unknown medicinal material has more blue fluorescent spots at the Rf value of 0.75 and one less blue-green fluorescent spot at the Rf value of 0.30. This method can be used to well identify Cuscuta chinensis and unknown medicinal materials.

[0070] Comparative Example 1:

[0071] This comparative example investigates different preparation methods of the test sample and the control medicinal material using the developing agent of the present invention and their thin layer chromatography effects. The specific process is as follows:

[0072] (1) Investigation of different preparation methods of test solution of Cuscuta australis granules:

[0073] 1) Take 0.5 g of Cuscuta australis granules (batch number: KL1), grind them into powder, add 25 ml of methanol, treat with ultrasound for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0074] 2): Take 0.5g of Cuscuta australis granules (batch number: KL1), grind them into powder, add 50ml of hot water to dissolve them, filter them, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time. Combine the ethyl acetate solutions, evaporate them to dryness, and add 1ml of methanol to dissolve the residue as the test solution.

[0075] 3): Take 0.5g of Cuscuta australis granules (batch number: KL1), grind them into powder, add 25ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 40ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 40ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution.

[0076] Other conditions are the same as in Example 1.

[0077] (2) Investigation of different preparation methods of Cuscuta australis reference medicinal material solution:

[0078] S1: Take 2 g of Cuscuta australis reference medicinal material, add 50 ml of petroleum ether (60-90°C), seal, ultrasonically treat for 30 minutes, filter, discard the petroleum ether liquid, repeat the operation 3 times, evaporate the solvent from the residue, add 50 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and dissolve the residue in 1 ml of methanol as the reference medicinal material solution.

[0079] S2: Take 2 g of Cuscuta australis reference medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 50 ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to the residue to dissolve, and use as the reference medicinal material solution.

[0080] S3: Take 2g of Cuscuta australis reference medicinal material, add 50ml of water, boil for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time. Combine the ethyl acetate solutions, evaporate to dryness, and add 1ml of methanol to the residue to dissolve it as the reference medicinal material solution.

[0081] Other conditions are the same as in Example 1.

[0082] According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 3 μl of each of the three test sample solutions and 5 μl of each of the three control medicinal material solutions were respectively spotted on the same silica gel H thin layer plate, and the upper layer solution of toluene-ethyl acetate-formic acid-water (6:5:1:0.5) was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0083] Result analysis: Fig. 9 S1 to S3 correspond to the chromatograms of the test samples of the Cuscuta chinensis formula granules prepared by the three preparation methods, and S1 to S3 correspond to the chromatograms of the Cuscuta chinensis control medicinal materials prepared by the three preparation methods. When the Cuscuta chinensis formula granules were prepared by methods one and three, the sample spots were blurred, and the chromatographic spots were clearer and had better separation when method two was used; when the control medicinal materials were prepared by method three, the background interference was less, and they corresponded one to one with the spots of method two of the Cuscuta chinensis formula granules.

[0084] Comparative Example 2:

[0085] This comparative example adopts the developing agent and inspection method under the item of Cuscuta australis in the 2020 edition of the Chinese Pharmacopoeia to detect the effect of Cuscuta australis formula granules. The specific process is as follows:

[0086] Preparation of test solution: Take 0.5 g of paper mulberry seed granules, grind them into powder, add 50 ml of hot water to dissolve them, filter them, and extract the filtrate by shaking with ethyl acetate twice, 40 ml each time. Combine the ethyl acetate solutions, evaporate them to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0087] Preparation of control medicinal solution: Take 2g of Cuscuta australis control medicinal material, add 50ml of water, boil for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1ml of methanol to the residue to dissolve it as the control medicinal solution.

[0088] Thin layer identification steps: Pipette 3μl of the test sample solution and 5μl of the control medicinal material solution, respectively spot them on the same silica gel H thin layer plate, use the upper layer solution of toluene-ethyl acetate-formic acid-water (6:5:1:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ for several minutes, and inspect under ultraviolet light (365nm).

[0089] Evaluation requirements: In the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the reference sample chromatogram.

[0090] Result analysis: Fig.10 1 to 3 correspond to the chromatograms of the test samples of different batches of Cuscuta australis formula granules (batch numbers: KL1 to KL3), and S corresponds to the chromatogram of the Cuscuta australis control medicinal material. The results show that the TLC identification spectra obtained by this method have high Rf values, few spots and different colors, so the TLC identification method in this comparative example is not suitable for the identification of Cuscuta australis formula granules.

[0091] Comparative Example 3:

[0092] This comparative example uses standard decoctions of Cuscuta australis and its counterfeit to compare the effects of different developer systems. The specific process is as follows:

[0093] Preparation of test solution: Take 0.3 g of standard decoction (Curcuma pachyrhizoma, Cuscuta australis or Perilla frutescens), add 50 ml of hot water to dissolve it, filter it, and extract the filtrate by shaking with ethyl acetate twice, 40 ml each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue with 1 ml of methanol as the test solution.

[0094] Preparation of control medicinal solution: Take 2g of Cuscuta australis control medicinal material, add 50ml of water, boil for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 40ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1ml of methanol to the residue to dissolve it as the control medicinal solution.

[0095] Different developing systems: ① Toluene-n-butanol-formic acid (10:5:1.3), the color developer is 10% sulfuric acid ethanol solution, and the examination is carried out under ultraviolet light (365nm); ② The thin layer developing agent in the literature (Liu Linna, Zhou Xinbei, Ouyang Rong. Identification of adulterated substances in paper mulberry seeds [J]. Journal of Hunan University of Traditional Chinese Medicine, 2004, (03):16-18.): n-hexane-ethyl ether-glacial acetic acid (7:3:0.1), the color developer is iodine vapor, and the examination is carried out under sunlight; ③ The developing agent of the present invention: the upper layer solution of toluene-ethyl acetate-formic acid-water (6:5:1:0.5), and the examination is carried out under ultraviolet light (365nm).

[0096] The test sample solution and the control medicinal material solution were aspirated and spotted on the silica gel H thin layer plate respectively. The plates were developed using the above three developing agents respectively and examined after color development under the corresponding color development conditions (10% sulfuric acid ethanol solution, examined under ultraviolet light (365nm); examined under sunlight after iodine vapor fumigation; examined under ultraviolet light (365nm) without color developer).

[0097] Result analysis: Fig.11 The thin-layer chromatograms correspond to three different development systems. When development system ① is used, the spot separation degree is poor in the standard decoction chromatogram of Cuscuta australis (DG-C1), the reference medicinal material chromatogram of Cuscuta australis (S), and the standard decoction chromatogram of the fake Perilla seed (DG-Z1~DG-Z2), and the Rf value is high.

[0098] There are fewer spots in the chromatogram (DG-T1~DG-T2) and the Rf value is low; when the development system ② is used, the spots in the standard decoction chromatogram of Cuscuta australis (DG-C1) and the control medicinal material chromatogram of Cuscuta australis (S) are tailing, and cannot be distinguished from the counterfeit standard decoction chromatogram of Perilla seed (DG-Z1~DG-Z2), and almost no spots can be seen in the standard decoction chromatogram of Cuscuta australis (DG-T1~DG-T2); when the developing agent system of the present invention, that is, the development system ③, is used, the fluorescent spots are relatively abundant and clear, and the Rf value is moderate, which can effectively distinguish Cuscuta australis (C) and its counterfeit Perilla seed (Z) and Cuscuta australis (T) at the same time; and there is no need to spray a color developer, and the operation is fast and simple.

[0099] Comprehensive analysis: The thin-layer identification method of Cuscuta australis and its preparation provided by the present invention is simple and fast in sample preparation operation. The developing agent in the present invention can be used for thin-layer identification of Cuscuta australis medicinal materials and thin-layer identification of Cuscuta australis preparations. The chromatographic spots are more abundant, the separation is good, and the genuine and fake products can be distinguished, which overcomes the problem of poor separation of sample spots in existing thin-layer identification methods and inability to accurately identify the authenticity. At the same time, the method of the present invention does not need to spray a color developer, and the simple operation can achieve the effect of clear spot display and good separation.

Claims

1. A thin layer identification method for paper mulberry seeds, characterized in that: The following steps are involved: (1) Preparation of test sample solution: dissolve the test sample powder in water, filter, extract with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to prepare the test sample solution; (2) Preparation of control medicinal material solution: dissolving the Cuscuta australis control medicinal material, filtering, extracting, evaporating to dryness, and dissolving the residue in methanol to prepare the control medicinal material solution; (3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate, develop them with toluene-ethyl acetate-formic acid-water as the developing solvent, and examine and compare the thin layer chromatograms; (4) When a fluorescent spot of the same color appears at a corresponding position in the chromatogram of the test sample and the chromatogram of the control medicinal material, it indicates that the test sample is Cuscuta australis; the Cuscuta australis includes medicinal materials, formula granules or standard decoctions.

2. The thin layer identification method according to claim 1, characterized in that: The volume of the control medicinal material solution in step (3) is 3 to 10 μl; the volume of the test solution is 3 to 10 μl.

3. The thin layer identification method according to claim 1, characterized in that: The development temperature in step (3) is 4 to 35°C.

4. The thin layer identification method according to claim 1, characterized in that: The humidity of the development in step (3) is 18% to 88%.

5. Use of the method according to any one of claims 1 to 4 in distinguishing Cuscuta australis seeds from Perilla seeds and Cuscuta australis seeds.

6. A method for distinguishing Cuscuta australis seeds from Perilla seeds and Cuscuta australis seeds, characterized in that: The following steps are involved: (1) Preparation of test sample solution: dissolve the test sample powder in water, filter, extract with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to prepare the test sample solution; (2) Preparation of control medicinal material solution: extract the Cuscuta australis control medicinal material, filter, extract, evaporate to dryness, and dissolve the residue in methanol to prepare the control medicinal material solution; (3) Take the control medicinal material solution and the test sample solution and spot them on the same thin layer plate, develop them with toluene-ethyl acetate-formic acid-water as the developing solvent, and examine and compare the thin layer chromatograms; (4) When a fluorescent spot of the same color appears at a corresponding position in the chromatogram of the test sample and the chromatogram of the control medicinal material, it indicates that the test sample is Cuscuta australis; otherwise, it is a counterfeit; the Cuscuta australis includes medicinal materials, formula granules or standard decoctions; the Perilla seeds and Cuscuta seeds include medicinal materials, formula granules or standard decoctions of Perilla seeds and Cuscuta seeds.

7. The thin layer identification method according to claim 6, characterized in that: The volume of the control medicinal material solution in step (3) is 3 to 10 μl; the volume of the test solution is 3 to 10 μl.

8. The thin layer identification method according to claim 6, characterized in that: The development temperature in step (3) is 4 to 35°C.

9. The thin layer identification method according to claim 6, characterized in that: The humidity of the development in step (3) is 18% to 88%.

Images