A thin-layer identification method for Pharbitis nil samples

Abstract

The present invention relates to a thin-layer identification method for Pharbitis nil samples, including the preparation of test solution, reference crude drug solution and reference substance solution, and the thin-layer identification step. The present invention adopts a simple and rapid pretreatment method to obtain the test solution, reference crude drug solution and reference substance solution, which are respectively spotted on the same thin-layer plate, developed with a suitable developing agent, and a multi-information thin-layer chromatogram of Pharbitis nil is obtained. This method is simple and rapid to operate, has good separation of chromatographic spots, low detection cost, safety and environmental protection, and is more conducive to the quality supervision of Pharbitis nil samples.

Description

Technical Field

[0001] The present invention belongs to the field of quality control of traditional Chinese medicines, and specifically relates to a thin-layer identification method for Pharbitidis Semen samples. Background Art

[0002] Pharbitidis Semen is the seed of Pharbitis nil (L.) Choisy. or Pharbitis purpurea (L.) Voigt of the Convolvulaceae family. Pharbitidis Semen has the effects of inducing purgation for relieving constipation, eliminating phlegm and removing fluid retention, killing parasites and attacking accumulation. Modern research has clarified that the main chemical components in Pharbitidis Semen include phenolic acids, resin glycosides, terpenoids, lignans, fatty oils, etc., and have various pharmacological effects such as anti-tumor effects, purgative and diuretic effects, exciting isolated rabbit intestines, antibacterial and exciting isolated rat uterine smooth muscles, and having anthelmintic effects on Ascaris suum in pigs. Among them, phenolic acids are its representative components and are also the index components selected in the thin-layer identification method of the Chinese Pharmacopoeia. However, the preparation of test samples and the preparation of control medicinal materials in the thin-layer identification method of "Pharbitidis Semen" in Part I of the Chinese Pharmacopoeia 2020 edition are cumbersome, taking more than 8 hours, and high-performance silica gel G thin-layer plates need to be used, with a relatively high detection cost compared to ordinary silica gel G thin-layer plates. At the same time, the thin-layer chromatographic spots of Pharbitidis Semen test samples are light and fuzzy, which is prone to misjudgment. When the developing agent under the pharmacopoeia is used for the thin-layer identification of Pharbitidis Semen preparations, the spot resolution is poor, and quality evaluation cannot be accurately carried out. Some other thin-layer identification methods of Pharbitidis Semen in preparations reported in the literature (such as the study on the thin-layer chromatography identification of Pharbitidis Semen in Chenxiang Dianxian San by Zhang Hongxing et al. [J]. Chinese Journal of Ethnomedicine and Ethnopharmacy, 2013, (8); Yang Jianhong, Thin-layer chromatography identification of Pharbitidis Semen in KaiXiong ShunQi Pills [J]. Chinese Traditional Patent Medicine, 1996, 18(12).) all use toxic and harmful solvents such as benzene, and the pretreatment is cumbersome. Summary of the Invention

[0003] Problems to be Solved by the Invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a thin-layer identification method for Pharbitidis Semen samples, which is simple, rapid, has good chromatographic spot resolution, low detection cost, is safe, environmentally friendly, and has a wide range of applications.

[0005] Solutions for Solving the Problems

[0006] The present invention provides the following technical solutions:

[0007] [1] A thin-layer identification method for Pharbitidis Semen samples, which includes the following steps:

[0008] 1) Preparation of test solution:

[0009] The Pharbitidis Semen sample is selected from Pharbitidis Semen medicinal materials or Pharbitidis Semen preparations;

[0010] When the Pharbitidis Semen sample is selected from Pharbitidis Semen medicinal materials or Pharbitidis Semen decoction pieces, the preparation of the test solution includes the step of extracting the Pharbitidis Semen sample by decocting with water or refluxing with heating.

[0011] When the Pharbitidis Semen sample is selected from other preparations except Pharbitidis Semen decoction pieces, the preparation of the test solution includes the step of taking the Pharbitidis Semen sample and performing ultrasonic extraction with an alcohol solvent.

[0012] 2) Preparation of the control medicinal material solution and the reference substance solution:

[0013] Take the Pharbitidis Semen control medicinal material and prepare the control medicinal material solution by decocting with water or refluxing with heating; take the caffeic acid reference substance and prepare the reference substance solution.

[0014] 3) Thin-layer identification step: It includes spotting the control medicinal material solution, the reference substance solution and the test solution on the same thin-layer plate respectively, developing with the developing agent, and the developing agent is dichloromethane - methanol - formic acid. By volume ratio, dichloromethane - methanol - formic acid = (70 - 85):(4 - 7):1.

[0015] [2] According to the thin-layer identification method of the Pharbitidis Semen sample described in [1], wherein: when decocting the Pharbitidis Semen medicinal material, Pharbitidis Semen decoction pieces or Pharbitidis Semen control medicinal material with water in step 1), the mass - volume ratio of the Pharbitidis Semen medicinal material, Pharbitidis Semen decoction pieces or Pharbitidis Semen control medicinal material to water is 1:50 g / mL - 200 g / mL, and the treatment time of decocting with water or refluxing with heating is 15 min - 60 min.

[0016] [3] According to the thin-layer identification method of the Pharbitidis Semen sample described in [1], wherein: the other preparations include one or more of Pharbitidis Semen formula granules and Pharbitidis Semen standard decoctions. Preferably, when performing ultrasonic extraction of the other preparations with an alcohol solvent, the mass - volume ratio of the other preparations to the alcohol solvent is 1:30 g / mL - 200 g / mL; further, the ultrasonic treatment time is 15 min - 60 min.

[0017] [4] According to the thin-layer identification method of the Pharbitidis Semen sample described in any one of [1] - [3], wherein: the alcohol solvent is selected from lower alcohol solvents with 1 - 5 carbon atoms or their aqueous solutions. Further, the lower alcohol solvents with 1 - 5 carbon atoms are selected from methanol, ethanol or isopropanol.

[0018] [5] According to the thin-layer identification method of the Pharbitidis Semen sample described in any one of [1] - [4], wherein: the concentration of caffeic acid in the reference substance solution is 0.5 - 2 mg / mL.

[0019] [6]The thin-layer identification method of Pharbitis semen samples according to any one of the technical solutions [1] to [5], wherein: the volume ratio of dichloromethane: methanol: formic acid is 80:6:1.

[0020] [7]The thin-layer identification method of Pharbitis semen samples according to any one of the technical solutions [1] to [6], wherein: the spotting amounts of the control crude drug solution, the reference substance solution and the test solution in step 4) are 1 μL to 10 μL.

[0021] [8]The thin-layer identification method of Pharbitis semen samples according to any one of the technical solutions [1] to [7], wherein: the thin-layer identification step further includes examining under an ultraviolet lamp after development to obtain an ultraviolet thin-layer chromatogram; and / or, spraying a developer for color development and heating until the spots are clearly developed to obtain a daylight thin-layer chromatogram. Preferably, the wavelength of the ultraviolet lamp is 365 nm, the developer is phosphomolybdic acid test solution, and the heating temperature is 105-120 °C.

[0022] [9]The thin-layer identification method of Pharbitis semen samples according to any one of the technical solutions [1] to [8], wherein: the thin-layer plate is a silica gel G thin-layer plate.

[0023]

[10] The thin-layer identification method of Pharbitis semen samples according to any one of the technical solutions [1] to [9], wherein: the method further includes comparing the test chromatogram with the control crude drug chromatogram and the reference substance chromatogram to determine whether fluorescent spots of the same color are shown at the corresponding positions.

[0024] Effects of the invention

[0025] The present invention provides a thin layer identification method for the medicinal material of Pharbitidis and its preparation, and obtains a multi-information thin layer chromatogram of the Pharbitidis under two inspection conditions of sunlight and ultraviolet light. Compared with the pharmacopeia method and existing literature reports, this method is simple and fast to operate, has good chromatographic spot separation, low detection cost, safety, environmental protection, and is more conducive to quality supervision. The preparation operation of the test solution and the control medicinal material solution of the present invention is simpler and faster, and does not require extraction with petroleum ether (60-90°C) for 2 hours and defatting, and then extraction with a mixed solution of dichloromethane-methanol (3:1) for 6 hours. In some preferred embodiments of the present invention, the medicinal material or decoction piece of the Pharbitidis only needs simple water decoction for 15-60 minutes. In other preferred embodiments of the present invention, the standard decoction and formula granules only need methanol ultrasound for 15-60 minutes. The present invention greatly shortens the preparation time in the test sample preparation link, avoids the use of organic reagents such as petroleum ether and dichloromethane, and is safer and more environmentally friendly. The developing agent selected in the present invention can be used for thin-layer identification of Pharbitidis Semen medicinal materials or decoction pieces, and can also be used for thin-layer identification of other Pharbitidis Semen preparations, and the chromatographic spots are more abundant and the separation is good, which overcomes the defects of the existing thin-layer identification method, such as blurred spots of the test sample caffeic acid, less spot information of the thin-layer spectrum of the Pharbitidis Semen preparations, and poor separation. In addition, in some preferred embodiments of the present invention, the thin-layer plate of the present invention only needs an ordinary silica gel G thin-layer plate, without the need to use an expensive high-efficiency silica gel G thin-layer plate, and the effect of rich spot information and good separation can be achieved.

[0026] It should be noted that the above description does not disclose all embodiments of the present invention and all advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the following is a brief introduction to the drawings required for the specific implementation or the prior art description. The drawings described below are some implementations of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 This is a thin layer chromatogram of the Pharbitis chinensis formula granules using the developing solvent: dichloromethane-methanol-formic acid (93:9:4) and high-efficiency silica gel G thin layer plate (1-3 are chromatograms of test samples of the Pharbitis chinensis formula granules prepared by three preparation methods, S1-S3 are chromatograms of Pharbitis chinensis reference medicinal materials prepared by three preparation methods, and T is the chromatogram of the caffeic acid reference substance; the left picture is a thin layer chromatogram under ultraviolet light, and the right picture is a thin layer chromatogram under sunlight).

[0029] Figure 2It is the thin layer chromatogram of Pharbitidis semen formula granules using developing agent: dichloromethane - methanol - formic acid (80∶6∶1) and ordinary silica gel G thin layer plate (1 - 3 are the test sample chromatograms of Pharbitidis semen formula granules prepared by 3 different preparation methods respectively, S1 - S3 are the chromatograms of Pharbitidis semen control medicinal materials prepared by 3 different preparation methods respectively, T is the chromatogram of caffeic acid reference substance; the left figure is the thin layer chromatogram under ultraviolet light, and the right figure is the thin layer chromatogram under daylight).

[0030] Figure 3 It is the thin layer chromatogram of Pharbitidis semen formula granules in Example 1 of the present invention (1 - 3 are the test sample chromatograms of Pharbitidis semen formula granules with different batch numbers respectively, S is the chromatogram of Pharbitidis semen control medicinal material, T is the chromatogram of caffeic acid reference substance; the left figure is the thin layer chromatogram under ultraviolet light, and the right figure is the thin layer chromatogram under daylight).

[0031] Figure 4 It is the thin layer chromatogram of Pharbitidis semen standard decoction in Example 2 of the present invention (manufacturer of the thin layer plate: Qingdao Kangyexin Medicinal Silica Gel Desiccant Co., Ltd.; 1 - 3 are the test sample chromatograms of Pharbitidis semen standard decoction with different batch numbers respectively, S is the chromatogram of Pharbitidis semen control medicinal material, T is the chromatogram of caffeic acid reference substance; the left figure is the thin layer chromatogram under ultraviolet light, and the right figure is the thin layer chromatogram under daylight).

[0032] Figure 5 It is the thin layer chromatogram of Pharbitidis semen standard decoction in Example 2 of the present invention (manufacturer of the thin layer plate: Yantai Chemical Industry Research Institute; 1 - 3 are the test sample chromatograms of Pharbitidis semen standard decoction with different batch numbers respectively, S is the chromatogram of Pharbitidis semen control medicinal material, T is the chromatogram of caffeic acid reference substance; the left figure is the thin layer chromatogram under ultraviolet light, and the right figure is the thin layer chromatogram under daylight).

[0033] Figure 6 It is the thin layer chromatogram of Pharbitidis semen standard decoction in Example 2 of the present invention (manufacturer of the thin layer plate: Qingdao Ocean Chemical Factory; 1 - 3 are the test sample chromatograms of Pharbitidis semen standard decoction with different batch numbers respectively, S is the chromatogram of Pharbitidis semen control medicinal material, T is the chromatogram of caffeic acid reference substance; the left figure is the thin layer chromatogram under ultraviolet light, and the right figure is the thin layer chromatogram under daylight).

[0034] Figure 7 It is the thin layer chromatogram of Pharbitidis semen cut crude drug in Comparative Example 1 of the present invention (Pharmacopoeia method) (1 - 8 are the test sample chromatograms of Pharbitidis semen standard decoction with different batch numbers respectively, S is the chromatogram of Pharbitidis semen control medicinal material, T is the chromatogram of caffeic acid reference substance). Detailed implementation manners

[0035] To better illustrate the present invention, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that the present invention can be implemented without some of these specific details. In other instances, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail so as to highlight the gist of the present invention.

[0036] Unless otherwise defined, the technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0037] In this specification, the numerical range expressed as "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0038] In this specification, the meaning expressed by "may" includes the meanings of both performing a certain process and not performing a certain process.

[0039] It should be understood that the singular form of the article "a" (corresponding to "a", "an", and "the" in English) used in the specification and claims of the present application includes plural objects, unless otherwise clearly specified in the text.

[0040] In this specification, the "one or some specific / preferred embodiments / schemes", "another or some other specific / preferred embodiments / schemes", "one or another embodiment / scheme", "one or another technical scheme", etc. mentioned refer to the specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment, which are included in at least one of the embodiments described herein, and may exist in other embodiments or may not exist in other embodiments. Additionally, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0041] The terms "comprising" and any variations thereof in the specification and claims of the present invention and the above-mentioned drawings are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device comprising a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products, or devices.

[0042] Unless otherwise specified, the term "reference medicinal material" (or "reference drug") used herein refers to the standard medicinal material that has completed species identification and is used to identify the test sample.

[0043] Unless otherwise specified, the term "test sample" used herein refers to the experimental sample used for detection or identification.

[0044] Unless otherwise specified, the term "thin-layer chromatography" as used herein is a micro, rapid, and simple separation and analysis method. It involves coating a suitable stationary phase on a glass plate, plastic, or aluminum substrate to form a uniform thin layer. After spotting and development, the retardation factor (Rf) is compared with that of the chromatogram obtained in the same manner with a suitable reference substance to identify drugs, check for impurities, or determine the content. This method is an important experimental technique for the rapid separation and qualitative analysis of small amounts of substances and is also used to track the progress of reactions. It features a short development time, strong separation ability, and high sensitivity. Thin-layer chromatography is one of the important inspection items for traditional Chinese medicinal materials. By comparing the chromatographic behaviors of test samples and reference medicinal materials and reference substances, the authenticity and quality of the medicinal materials can be judged.

[0045] Unless otherwise specified, the term "spotting" as used herein refers to the process of adding droplets of the sample solution to be separated and identified onto the thin-layer plate.

[0046] Unless otherwise specified, the term "development" as used herein refers to the process in which the developer carries the sample components a certain distance on the thin-layer plate by capillary action of the stationary phase on the thin-layer plate.

[0047] Unless otherwise specified, the term "test sample" as used herein refers to the experimental sample used for detection or identification. Unless otherwise specified, the term "reference medicinal material" (or "reference drug") as used herein refers to the standard medicinal material that has completed species identification and is used to identify the sample to be tested.

[0048] A thin-layer identification method for Pharbitis nil and its preparations provided by the present invention, based on the chemical structures and properties of the active ingredients in traditional Chinese medicine, follows the extraction principle of similar compatibility, uses a suitable extraction solvent, and simply and quickly prepares the test sample and reference medicinal material solutions. Then, with a suitable developer for development, various chemical components will be well separated on the thin-layer plate according to their different abilities of adsorption, desorption, re-adsorption, and re-desorption with the selected developer. With the help of various active ingredients with similar polarities, on the same thin-layer plate, under different inspection conditions (daylight, ultraviolet lamp, or color development with a color reagent), different color spots will appear, obtaining a multi-information thin-layer chromatogram.

[0049] The thin-layer identification method for Pharbitis nil samples provided by the present invention includes the preparation of the test sample solution, the preparation of the reference medicinal material solution and the reference substance solution, and the thin-layer identification step.

[0050] <Preparation of the test sample solution>

[0051] The preparation of the test sample solution in the present invention is simple and fast. Depending on the type of Pharbitis nil samples, different methods for preparing the test sample solution are used in the present invention.

[0052] The Pharbitidis Semen samples described in the present invention are selected from Pharbitidis Semen medicinal materials or Pharbitidis Semen preparations. "Medicinal materials" refer to raw materials of traditional Chinese medicine that have not been processed or made into finished products; "preparations" refer to forms of traditional Chinese medicine that are made suitable for medical or preventive applications before clinical use, such as tablets, injections, aerosols, pills, powders, ointments, etc. In the present invention, Pharbitidis Semen preparations particularly include one or more of Pharbitidis Semen cut crude drugs, standard decoctions, and formula granules. "Cut crude drugs" refer to traditional Chinese medicine that is processed into a certain shape according to needs for convenient use and can be directly used in traditional Chinese medicine clinical practice; "standard decoctions" are single-herb cut crude drug water decoctions prepared by standardized processes under the guidance of traditional Chinese medicine theory and based on clinical applications, with reference to modern extraction methods; "formula granules" are granules made from single-herb cut crude drugs after being processed according to traditional standards and then extracted and concentrated for use in traditional Chinese medicine clinical prescriptions. In some specific embodiments of the present invention, in order to achieve more complete extraction, pretreatment can be performed on Pharbitidis Semen samples such as Pharbitidis Semen medicinal materials, for example, sieving after pulverization (such as through a No. 2 sieve).

[0053] In some specific embodiments of the present invention, when the Pharbitidis Semen sample is selected from Pharbitidis Semen medicinal materials or Pharbitidis Semen decoction pieces, water decoction or heating under reflux is used for extraction to obtain a test solution. In some specific embodiments of the present invention, the Pharbitidis Semen sample is taken, subjected to water decoction or heating under reflux treatment, filtered, and the filtrate is concentrated to dryness, and the residue is dissolved in an alcohol solvent to obtain a test solution. Heating under reflux generally refers to a method of extracting medicinal materials with volatile organic solvents such as ethanol or water, heating and distilling the leaching solution, where the volatile solvent or water is distilled out and then condensed and repeatedly flows back into the extractor to extract the medicinal materials. In this way, the process is repeated until the effective components are completely extracted by reflux. This method uses less solvent and the extraction is relatively more complete. Preferably, in some specific embodiments of the present invention, water heating under reflux is used for the extraction of Pharbitidis Semen medicinal materials or Pharbitidis Semen decoction pieces. In some preferred embodiments of the present invention, if water decoction or water heating under reflux is used, the mass-volume ratio of Pharbitidis Semen medicinal materials or Pharbitidis Semen decoction pieces to water is 1:50 g / mL to 200 g / mL, more preferably 1:80 to 150 g / mL, and the treatment time of water decoction or heating under reflux is 15 min to 60 min, and further preferably 20 min to 40 min. The filtration method after extraction is not limited and can be any conventional filtration means in the art, such as filtration with filter paper, cotton, etc. The filtrate obtained after filtration needs to be concentrated, and it can be concentrated by evaporation in a water bath, heating and concentrating with a rotary evaporator, or by vacuum evaporation. For the method of water decoction, in some specific embodiments of the present invention, after water decoction, filtration is carried out, and then the filtrate is evaporated to dryness, and the residue is dissolved in an alcohol solvent. For the method of water heating under reflux, the method of ultrasonic treatment with an organic solvent can also be combined to make the extraction more complete. In some specific embodiments of the present invention, the Pharbitidis Semen medicinal materials or Pharbitidis Semen decoction pieces are taken, added with water, heated under reflux, filtered, the filtrate is concentrated to nearly dryness, added with an alcohol solvent, ultrasonically treated, filtered, the filtrate is evaporated to dryness, and the residue is dissolved in an alcohol solvent. Preferably, the heating under reflux treatment time is 20 min to 40 min, and the ultrasonic treatment time is 30 to 50 min. The medicinal materials are first heated under reflux with water, which is equivalent to simulating the production and extraction process of standard decoctions or granules, making the chemical components obtained by extraction closer to those of standard decoctions and granules. The alcohol solvents mentioned in each step of the present invention are each independently selected from lower alcohol solvents with 1 to 5 carbon atoms or their aqueous solutions. Further, the lower alcohol solvents with 1 to 5 carbon atoms are selected from methanol, ethanol or isopropanol. Preferably, the alcohol solvent is selected from methanol or an aqueous methanol solution, and further preferably from methanol.

[0054] In some specific embodiments of the present invention, when the Pharbitidis Semen sample is selected from other Pharbitidis Semen preparations except Pharbitidis Semen decoction pieces, such as Pharbitidis Semen formula granules and standard decoctions, the preparation of the test solution includes the step of taking the Pharbitidis Semen sample and performing ultrasonic extraction with an alcohol solvent. Preferably, the mass-volume ratio of the other preparation to the alcohol solvent is 1:30 g / mL to 200 g / mL; more preferably, the mass-volume ratio is 1:40 g / mL to 100 g / mL. The power and frequency of ultrasonic have little influence on the present invention. In some preferred embodiments of the present invention, the duration of ultrasonic extraction is 20 min to 60 min, the power is 250 W to 300 W, and the frequency is 35 kHz to 45 kHz. In some specific embodiments of the present invention, take the Pharbitidis Semen sample, perform ultrasonic extraction with an alcohol solvent, filter, concentrate the filtrate to dryness, dissolve the residue with an alcohol solvent to obtain the test solution. The filtration and concentration methods are the same as the previous content. The alcohol solvent is also separately selected from lower alcohol solvents with 1 to 5 carbon atoms or their aqueous solutions. Further, the lower alcohol solvent with 1 to 5 carbon atoms is selected from methanol, ethanol or isopropanol. Preferably, the alcohol solvent is selected from methanol or methanol aqueous solution, and more preferably from methanol.

[0055] <Preparation of control crude drug solution and reference substance solution>

[0056] In the present invention, the control crude drug solution is prepared by decocting or heating under reflux the control crude drug of Pharbitidis Semen; the reference substance solution is prepared by taking caffeic acid reference substance. In some specific embodiments of the present invention, the preparation of the control crude drug solution is the same as the preparation method of the Pharbitidis Semen sample selected from Pharbitidis Semen in the above <Preparation of test solution>. In some other specific embodiments of the present invention, take caffeic acid reference substance, dissolve it with an alcohol solvent to prepare the reference substance solution. The definition of the alcohol solvent is the same as before. Preferably, the concentration of caffeic acid in the reference substance solution is 0.5 to 2 mg / mL, and further 0.8 to 1.5 mg / mL.

[0057] <Thin layer identification step>

[0058] The thin-layer identification step of the present invention includes spotting the control medicinal material solution, reference substance solution, and test sample solution on the same thin-layer plate respectively, and developing with a developing agent. The developing agent is dichloromethane - methanol - formic acid. By volume ratio, dichloromethane - methanol - formic acid = (70 - 85):(4 - 7):1. The specific selection of the volume ratio in the present invention helps to obtain a thin-layer chromatogram of Pharbitis nil samples with rich information and clear and abundant spots. If the developing agent with the volume ratio of the present invention is not used, for example, dichloromethane - methanol - formic acid (93∶9∶4) disclosed in the pharmacopoeia is used, problems such as less spot information and poor resolution will occur. In some preferred embodiments of the present invention, dichloromethane - methanol - formic acid = (72 - 82):(5 - 7):1, and more preferably, dichloromethane - methanol - formic acid = 80∶6∶1. The thin-layer plate of the present invention is preferably a silica gel G thin-layer plate, which can achieve the effects of rich spot information and good resolution, and has a lower cost compared with the high-efficiency silica gel G thin-layer plate. In some specific embodiments of the present invention, the spotting amounts of the control medicinal material solution, reference substance solution, and test sample solution are 1 μL - 10 μL. Further, the preferred spotting amounts of the test sample solution and the control medicinal material solution are 3 μL - 8 μL, and the preferred spotting amount of the reference substance solution is 1 - 2 μL. In some specific embodiments of the present invention, the thin-layer identification step further includes examining under an ultraviolet lamp after development to obtain an ultraviolet thin-layer chromatogram; and / or, spraying a color-developing agent to develop color, and heating until the spots are clearly developed to obtain a daylight thin-layer chromatogram. In some preferred embodiments of the present invention, the wavelength of the ultraviolet lamp is 365 nm, and the color-developing agent is preferably a phosphomolybdic acid test solution. Compared with ferric chloride and aluminum chloride color-developing agents, the spots are clearer when using the phosphomolybdic acid test solution. Further, the phosphomolybdic acid test solution can be a 5% - 10% phosphomolybdic acid ethanol solution. For the preparation method of the color-developing agent, it can be prepared by a conventional method, for example, adding 10 g of phosphomolybdic acid to 100 mL of absolute ethanol. For the heating temperature, it is preferably 105 - 120 °C, for example, it can be 110 °C. The present invention can obtain a thin-layer chromatogram of Pharbitis nil with rich information under both daylight and ultraviolet light inspection conditions. The spots obtained by the two inspection methods are basically the same. In some specific embodiments of the present invention, the daylight or ultraviolet light inspection method can be optionally selected.

[0059] In some specific embodiments of the present invention, the method of the present invention further includes comparing the test sample chromatogram with the control medicinal material chromatogram and the reference substance chromatogram to determine whether fluorescent spots of the same color are shown at corresponding positions. Preferably, in the test sample solution chromatogram, at the corresponding positions of the control medicinal material chromatogram and the reference substance chromatogram, fluorescent spots or spots of the same color are shown.

[0060] The thin-layer identification method of Pharbitis nil samples provided by the present invention is simple, rapid, has good chromatographic spot resolution and spot formation, short development time, clear inspection, low detection cost, safety, and environmental protection, and can better control the quality of Pharbitis nil samples.

[0061] Examples

[0062] The technical solutions in the present invention will be further described below in conjunction with specific examples.

[0063] The instruments and reagents used in the present invention are as follows:

[0064] Instruments: Automatic TLC Imager (CAMAG TLC VISUALIZER), ten-thousandth balance (METTLER TOLEDO, Switzerland), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), electronic temperature-controlled electric furnace (Jiangyin Poly Scientific Instruments Co., Ltd.), GKC thermostatic water bath (Nantong Huatai Experimental Instruments Co., Ltd.), silica gel G TLC plates (Qingdao Marine Chemical Factory), high-efficiency silica gel G TLC plates (Qingdao Marine Chemical Factory), silica gel G TLC plates (Yantai Chemical Industry Research Institute), silica gel G TLC plates (Qingdao Kangyexin Medicinal Silica Gel Desiccant Co., Ltd.).

[0065] Reagents: Methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), dichloromethane (Sinopharm Chemical Reagent Co., Ltd.), formic acid (Sinopharm Chemical Reagent Co., Ltd.), petroleum ether (60-90 °C) (Sinopharm Chemical Reagent Co., Ltd.), sulfuric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.) are all of analytical grade; water is purified water.

[0066] Caffeic acid reference substance (batch number: 110885-201703), Pharbitidis Fructus reference crude drug (batch number: 121024-201606) are all purchased from the National Institutes for Food and Drug Control.

[0067] Pharbitidis Fructus formula granules (batch numbers: 21010019, 21010029, 21010039), Pharbitidis Fructus standard decoction (batch numbers: DG122102022, DG122102023, DG122102024), Pharbitidis Fructus decoction pieces (batch numbers: YP1903023, YP1903024, YP1903025, YP1911076, YP1911077, YP1911078, YP912034, YP1912035) are prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0068] For those not specifying specific experimental steps or conditions in the present invention, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For those reagents or instruments not specifying the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0069] Preparation methods of test samples and control crude drug solutions and investigation of developing solvents

[0070] 1: Take 0.4 g of Pharbitidis Fructus formula granules (batch number: 21010029), grind them finely, add 25 mL of methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the test solution.

[0071] 2: Take 0.4 g of Pharbitidis Fructus formula granules (batch number: 21010029), grind them finely, add 25 mL of ethanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol to obtain the test solution.

[0072] 3: Take 0.4 g of Pharbitidis Fructus formula granules (batch number: 21010029), grind them finely, add 25 mL of a dichloromethane - methanol (3:1) mixed solution, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of the dichloromethane - methanol (3:1) mixed solution to obtain the test solution.

[0073] S1: Take 0.2 g of the reference crude drug of Pharbitidis Fructus, add 25 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the reference crude drug solution.

[0074] S2: Take 0.2 g of the reference crude drug of Pharbitidis Fructus, add 25 mL of water, heat under reflux for 30 minutes, filter, concentrate the filtrate to nearly dryness, add 25 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the reference crude drug solution.

[0075] S3: Take 0.2 g of the reference crude drug of Pharbitidis Fructus, place it in a Soxhlet extractor, add an appropriate amount of petroleum ether (60 - 90 °C), heat under reflux for 2 hours, discard the petroleum ether solution, dry the medicinal residues by volatilizing the solvent, add a dichloromethane - methanol (3:1) mixed solution for extraction for 6 hours, recover the solvent to 1 mL to obtain the reference crude drug solution.

[0076] Take caffeic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL as the reference substance solution. According to the thin - layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) for testing, absorb 5 μL of each of the above 3 test solutions and 3 reference crude drug solutions and 1 μL of the reference substance solution, spot them on a thin - layer plate, and develop separately with a high - efficiency silica gel G plate and dichloromethane - methanol - formic acid (93:9:4) and a common silica gel G plate and dichloromethane - methanol - formic acid (80:6:1). Develop, take out, air - dry, examine under an ultraviolet lamp (365 nm); spray with phosphomolybdic acid test solution, heat at 110 °C until the spots are clearly visible, and examine under daylight. The results are shown in Figure 1 and Figure 2 .

[0077] Result analysis: Figure 1 - Figure 2Among them, 1-3 are the chromatograms of the test samples of Pharbitidis semen formula granules prepared by 3 corresponding preparation methods respectively, S1-S3 are the chromatograms of the corresponding control medicinal materials of Pharbitidis semen prepared by 3 preparation methods respectively, and T is the chromatogram of caffeic acid reference substance. The left figure is the thin-layer chromatogram under ultraviolet light, and the right figure is the thin-layer chromatogram under daylight. Among them Figure 1 The thin-layer plate is a high-efficiency silica gel G thin-layer plate, and the developing agent is dichloromethane-methanol-formic acid (93:9:4). Figure 2 The thin-layer plate is a common silica gel G thin-layer plate, and the developing agent is dichloromethane-methanol-formic acid (80:6:1). From Figure 1 and Figure 2 By comparison, it can be seen that when using the developing agent dichloromethane-methanol-formic acid (93:9:4), there is less spot information and poor resolution. When the test sample preparation of Pharbitidis semen formula granule sample adopts Method 3 (that is, the test sample preparation method of Pharbitidis semen medicinal material slices in the pharmacopoeia), the operation takes more than 8 hours, and there is less spot information of the sample, indicating that this test sample preparation method is not applicable to Pharbitidis semen formula granules and is only suitable for the medicinal materials and slices of Pharbitidis semen.

[0078] Example 1

[0079] Take 0.4 g of Pharbitidis semen formula granules, grind them finely, add 25 mL of methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to make a test sample solution. Separately take 0.2 g of the control medicinal material of Pharbitidis semen, add 25 mL of water, heat under reflux for 30 minutes, filter, concentrate the filtrate to nearly dry, add 25 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to make a control medicinal material solution. Then take caffeic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL as the reference substance solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) test, absorb 5 μL of the above-mentioned test sample solution and control medicinal material solution and 1 μL of the reference substance solution respectively, and spot them on the same silica gel G thin-layer plate. Use dichloromethane-methanol-formic acid (80:6:1) as the developing agent, develop, take out, dry in air, and examine under an ultraviolet lamp (365 nm); spray with phosphomolybdic acid test solution, heat at 110 °C until the spots are clearly developed, and examine under daylight. The results are shown in Figure 3 .

[0080] Result analysis: Figure 3 Among them, 1-3 correspond to the chromatograms of the test samples of Pharbitidis semen formula granules with batch numbers 21010019, 21010029, and 21010039 respectively, S is the chromatogram of the control medicinal material of Pharbitidis semen, and T is the chromatogram of caffeic acid reference substance. The left figure is the thin-layer chromatogram under ultraviolet light, and the right figure is the thin-layer chromatogram under daylight. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the control medicinal material and the reference substance, fluorescent spots or spots of the same color appear.

[0081] Example 2

[0082] Take 0.3 g of the standard decoction of Pharbitidis Semen, grind it finely, add 25 mL of methanol, ultrasonically treat for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the test solution. Separately, take 0.2 g of the control medicinal material of Pharbitidis Semen, add 25 mL of water, heat under reflux for 30 minutes, filter, concentrate the filtrate to nearly dryness, add 25 mL of methanol, ultrasonically treat for 40 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the control medicinal material solution. Further, take caffeic acid reference substance, prepare a solution containing 1 mg per 1 mL with methanol to obtain the reference substance solution. According to the thin-layer chromatography method (General Principle 0502 of the Chinese Pharmacopoeia 2020 Edition), take 5 μL of each of the above-mentioned test solution and control medicinal material solution, and 1 μL of the reference substance solution, respectively spot them on the same silica gel G thin-layer plate, use dichloromethane-methanol-formic acid (80:6:1) as the developing solvent, develop, take out, dry in air, examine under an ultraviolet lamp (365 nm); spray with phosphomolybdic acid test solution, heat at 110 °C until the spots develop clearly, and examine under daylight. This example was developed on ordinary silica gel G thin-layer plates produced by 3 manufacturers respectively. The results are shown in Figure 4 - Figure 6 .

[0083] Result analysis: Figure 4 - Figure 6 In Figures 1-3, they respectively correspond to the test chromatograms of the standard decoction of Pharbitidis Semen with batch numbers DG122102022, DG122102023, and DG122102024, and S is the chromatogram of the control medicinal material of Pharbitidis Semen. The left figure is the thin-layer chromatogram under ultraviolet light, and the right figure is the thin-layer chromatogram under daylight. In the test chromatogram, at the positions corresponding to the chromatograms of the control medicinal material and the reference substance, fluorescent spots or spots of the same color appear. Ordinary silica gel G thin-layer plates from different manufacturers can reproduce the thin-layer chromatogram of Pharbitidis Semen of the present invention.

[0084] Comparative Example 1

[0085] In this comparative example, the thin-layer chromatography detection of Pharbitidis Semen slices was carried out by the method in Part I of the Chinese Pharmacopoeia 2020 Edition. The specific process is as follows:

[0086] Take 1 g of the powder of Pharbitidis Semen slices, place it in a Soxhlet extractor, add an appropriate amount of petroleum ether (60-90 °C), heat under reflux for 2 hours, discard the petroleum ether solution, dry the residue by evaporation of the solvent, add a mixed solution of dichloromethane-methanol (3:1) for extraction for 6 hours, recover the solvent to 5 mL to obtain the test solution. Separately, take 1 g of the reference crude drug of Pharbitidis Semen and prepare a reference crude drug solution in the same method. Then take caffeic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL as the reference substance solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) for testing, absorb 20 μL of the test solution and the reference crude drug solution respectively, and 3 μL of the reference substance solution, spot them on the same high-performance silica gel G thin-layer plate respectively, use dichloromethane-methanol-formic acid (93:9:4) as the developing agent, develop, take out, dry in air, spray with phosphomolybdic acid test solution, and heat at 110 °C until the spots are clearly developed. The results are shown in Figure 7 。

[0087] Result analysis: Figure 7 Among them, 1-8 are the chromatograms of the test solutions of Pharbitidis Semen slices (batch numbers: YP1903023, YP1903024, YP1903025, YP1911076, YP1911077, YP1911078, YP912034, YP1912035), S is the chromatogram of the reference crude drug of Pharbitidis Semen, and T is the chromatogram of caffeic acid reference substance. At the positions corresponding to the spots of the caffeic acid reference substance on the chromatograms of the test solution and the reference crude drug, the spots are blurred and lighter in color, which is likely to cause misjudgment.

[0088] The above embodiments are only used to illustrate several implementation schemes of the present invention, and the description is relatively specific and detailed, but it should not be understood as imposing any limitation on the scope of the present invention. It should be clear that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A thin-layer identification method for Pharbitis nil samples, characterized in that: It includes the following steps: 1) Preparation of test solution: The Pharbitidis Semen sample is selected from Pharbitidis Semen medicinal materials or Pharbitidis Semen preparations; When the Pharbitidis Semen sample is selected from Pharbitidis Semen medicinal materials or Pharbitidis Semen decoction pieces, the preparation of the test solution includes the step of extracting the Pharbitidis Semen sample by heating under reflux with water. For the method of heating under reflux with water, take Pharbitidis Semen medicinal materials or Pharbitidis Semen decoction pieces, add water, heat under reflux, filter, concentrate the filtrate nearly to dryness, add an alcohol solvent, perform ultrasonic treatment, filter, evaporate the filtrate to dryness, and dissolve the residue in an alcohol solvent; When the Pharbitidis Semen sample is selected from other preparations except Pharbitidis Semen decoction pieces, the preparation of the test solution includes the step of ultrasonically extracting the Pharbitidis Semen sample with an alcohol solvent; the other preparations include one or more of Pharbitidis Semen formula granules and Pharbitidis Semen standard decoctions; The alcohol solvent is selected from methanol, ethanol, aqueous solution of methanol or aqueous solution of ethanol; 2) Preparation of control medicinal material solution and reference substance solution: Take the Pharbitidis Semen control medicinal material and prepare the control medicinal material solution by decocting with water or heating under reflux; take caffeic acid reference substance and prepare the reference substance solution; 3) Thin-layer identification step: It includes spotting the control medicinal material solution, reference substance solution and test solution on the same thin-layer plate respectively, developing with a developing agent, the developing agent is dichloromethane - methanol - formic acid, by volume, dichloromethane - methanol - formic acid = 80:6:1; the thin-layer plate is a silica gel G thin-layer plate.

2. The TLC identification method of the Pharbitis nil sample according to claim 1, wherein: When the Pharbitidis Semen medicinal materials, Pharbitidis Semen decoction pieces or Pharbitidis Semen control medicinal material are heated under reflux with water, the mass - volume ratio of Pharbitidis Semen medicinal materials, Pharbitidis Semen decoction pieces or Pharbitidis Semen control medicinal material to water is 1:50 g / mL - 200 g / mL, and the treatment time of heating under reflux with water is 15 min - 60 min.

3. The thin-layer identification method of the Pharbitis nil sample according to claim 1, wherein: When the other preparations are ultrasonically extracted with an alcohol solvent, the mass - volume ratio of the other preparations to the alcohol solvent is 1:30 g / mL - 200 g / mL.

4. The TLC identification method of the Pharbitis nil sample according to claim 3, characterized in that: The ultrasonic treatment time is 15 min - 60 min.

5. The TLC identification method for Pharbitis nil samples according to any one of claims 1 to 4, characterized in that: The concentration of caffeic acid in the reference substance solution is 0.5 - 2 mg / mL.

6. The TLC identification method of the Pharbitis nil sample according to any one of claims 1 to 4, characterized in that: In the step 3), the spotting amounts of the control medicinal material solution, reference substance solution and test solution are 1 μL - 10 μL.

7. The TLC identification method for the Pharbitis nil sample according to any one of claims 1 to 4, characterized in that: The thin-layer identification step further includes the steps of examining under an ultraviolet lamp after development to obtain an ultraviolet thin-layer chromatogram; and / or, spraying with a developer for color development and heating until the spots are clearly developed to obtain a daylight thin-layer chromatogram.

8. The TLC identification method of the Pharbitis nil sample according to claim 7, characterized in that: The wavelength of the ultraviolet lamp is 365 nm, the developer is phosphomolybdic acid test solution, and the heating temperature is 105 - 120 °C.

9. The TLC identification method of the Pharbitis Semen sample according to any one of claims 1 to 4, characterized in that: The method further includes comparing the test sample chromatogram with the control medicinal material chromatogram and the reference substance chromatogram to judge whether fluorescent spots of the same color are shown at the corresponding positions.

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