Thin layer chromatography identification method of Amomum villosum formula granules

Abstract

The present invention provides a thin-layer chromatography (TLC) identification method for Amomum villosum formula granules, comprising the steps of preparing a test solution, preparing a control medicinal material solution, and performing TLC identification. The test solution and the control medicinal material solution are prepared using a simple and rapid preparation method, and are spotted on the same TLC plate. After development, the TLC plate is inspected under ultraviolet light to obtain a multi-information TLC of the Amomum villosum formula granules. This method uses the control medicinal material as a reference and flavonoids as the main identification component. It has high specificity and can distinguish Amomum villosum of different origins (Amomum villosum and Amomum villosum var. amomi) and identify mixed and counterfeit Amomum villosum (Amomum villosum var. longifolia).

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a thin layer identification method for Amomum villosum formula granules. Background Art

[0002] Amomum villosum is the dried mature fruit of Amomum villosum Lour., Amomum villosum Lour. var. xanthioides TLWu et Senjen, or Amomum longiligulare TLWu, all of the Zingiberaceae family. Amomum villosum is warm in nature and pungent in taste, primarily entering the spleen, stomach, and kidney meridians. It has the effects of eliminating dampness and stimulating appetite, warming the spleen and stopping diarrhea, and regulating qi and stabilizing pregnancy. Recent literature reports indicate that Amomum villosum contains bioactive components such as volatile oils, flavonoids, and polysaccharides, exhibiting antioxidant, antihypertensive, and antibacterial properties. However, due to regional differences in medication usage, other Zingiberaceae plants, such as Alpinia oxyphylla, Amomum villosum tsaoko, and Amomum longiligulare, are often used as Amomum villosum. This leads to confusion in the market regarding the source of Amomum villosum and inconsistent quality grades, seriously impacting the safety and efficacy of Amomum villosum in clinical practice.

[0003] Chinese medicine formula granules are a breakthrough in the application form of traditional Chinese medicine slices. They are made from slices of medicine, through standardized extraction, concentration, drying and granulation. Their nature, flavor, meridians and efficacy are basically consistent with the original slices, and they have the advantages of no need for decoction, easy adjustment, storage and convenient taking, and have gradually been widely used. In the 2020 edition of the Chinese Pharmacopoeia, only a thin layer identification project for volatile components in Amomum villosum was established under the Amomum villosum item. Specifically, borneol acetate was used as a reference substance, cyclohexane: ethyl acetate (22: 1) was used as a developing agent, and 5% vanillin-sulfuric acid solution was used as a color developer for thin layer identification. However, the extraction of volatile oil in this method takes a long time, and the specificity of borneol acetate is poor. Amomum villosum of different origins (Amomum villosum and Amomum villosum green shell) and Amomum villosum counterfeit (Amomum villosum longus) all contain this ingredient, and there are fewer spots after color development by this method, and the spots of the reference substance are not clear. In addition, volatile components are unstable. The ventilation and temperature of the storage environment and the exposure area of ​​the medicinal materials will affect the content of volatile components in the medicinal materials, thereby affecting the quality of the medicinal materials (Pan Hongling, Lou Yiyi, Chen Xiuxiu, et al. Study on the stability of the content of bornyl acetate in Amomum villosum [J]. Medical Herald. 2018, 37(S1): 65-66). Therefore, it is necessary to improve the specificity and accuracy of the detection and identification methods of Amomum villosum.

[0004] In addition to volatile components, Amomum villosum formula granules also contain non-volatile components. Among them, flavonoids, as water-soluble non-volatile components in Amomum villosum, have antibacterial and anti-tumor activities (Gao Linlin, Wang Qian, Zhang Jingwen, et al., Refining of flavonoids from Amomum villosum and Alpinia oxyphylla and their antibacterial and anti-tumor functions [J]. Shizhen Traditional Chinese Medicine. 2010, 21(10): 2534-2536). However, due to the low extraction rate of flavonoids in Amomum villosum and the lack of in-depth research on their pharmacological effects, the existing identification methods do not take into account the existence of such components, rarely use them as the object of quality control, and no quality control standards for them have been established. Therefore, a fast and simple thin-layer identification method using highly polar flavonoids as the main identification component is established. This method not only improves the existing identification methods of Amomum villosum, but also can distinguish Amomum villosum of different origins and identify mixed and counterfeit Amomum villosum. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] In order to solve the problems existing in the above-mentioned prior art, the present invention aims to create a thin-layer identification method for Amomum villosum formula granules, which is efficient, rapid, accurate, simple and easy to operate, has obvious identification characteristics, is easy to judge the results, has good specificity, can accurately identify Amomum villosum of different origins (Amomum villosum and Amomum villosum var. amomi), and can effectively distinguish them from common counterfeit products (Amomum villosum longifolia) in the market.

[0007] Solutions for solving problems

[0008] The present invention provides the following technical solutions:

[0009] [1]. The present invention provides a thin layer chromatography identification method for Amomum villosum formula granules, wherein the method comprises the preparation of a test solution, the preparation of a control medicinal material solution and thin layer chromatography identification.

[0010] The preparation of the test solution comprises taking Amomum villosum granules, dissolving them in water, extracting them with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue in an alcohol solvent;

[0011] The preparation of the control medicinal material solution comprises taking Amomum villosum as a control medicinal material, adding water to reflux extraction, filtering, extracting the filtrate with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue with an alcohol solvent;

[0012] The thin layer identification comprises taking a test sample solution and a control medicinal material solution, spotting them on a polyamide thin layer chromatography plate, developing them with methanol-glacial acetic acid-water as a developing agent, and inspecting them.

[0013] [2] The thin layer identification method according to [1], wherein the thin layer identification method further comprises the preparation of a negative control solution, the preparation of the negative control solution comprises taking negative control formula granules without Amomum villosum, and the remaining steps refer to the preparation method of the test solution; the Amomum villosum formula granules and the negative control formula granules are both in powder form.

[0014] [3] The thin layer identification method according to [1] or [2], wherein, in the preparation of the test solution, the ratio of the Amomum villosum granules to water is 1 g: (30-50) mL; the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; the extraction is performed twice or more, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); the alcohol solvent is methanol or an aqueous solution of methanol.

[0015] [4] The thin-layer identification method according to any one of the technical solutions [1] to [3], wherein, in the preparation of the control medicinal material solution, the ratio of the reference medicinal material Amomum villosum to water is 1 g: (15-25) mL; the reflux extraction time is 20-40 min; the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; the number of extractions is two or more, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); the alcohol solvent is methanol or a methanol solution.

[0016] [5] The thin layer chromatography identification method according to any one of the technical solutions [1] to [4], wherein, in the thin layer chromatography identification, the test sample solution and the control medicinal material solution are respectively spotted on the same polyamide thin layer chromatography plate.

[0017] [6]. The thin layer identification method according to any one of the technical solutions [1] to [5], wherein, in the thin layer identification, methanol-glacial acetic acid-water with a volume ratio of (5-12): (1-5): (1-5) is used as the developing agent.

[0018] [7]. The thin layer identification method according to any one of the technical solutions [1] to [6], wherein the thin layer identification further comprises the steps of unfolding and drying, spraying with a color developer, heating until the spots are clearly colored, and inspecting under ultraviolet light; preferably, the color developer is a 1% to 5% aluminum chloride solution.

[0019] [8]. The thin layer identification method according to any one of the technical solutions [1] to [7], wherein, in the thin layer identification, the spotting volume of the test solution is 1 to 4 μL, and the spotting volume of the control medicinal material solution is 1 to 10 μL.

[0020] [9] The thin-layer identification method according to any one of the technical solutions [1] to [8], wherein, in the thin-layer identification, the inspection includes comparing the chromatogram of the test sample with the chromatogram of the control medicinal material to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value.

[0021]

[10] . Application of the thin layer identification method according to any one of the technical solutions [1] to [9] in distinguishing Amomum villosum formula granules of different origins and identifying mixed and counterfeit Amomum villosum formula granules.

[0022] Effects of the Invention

[0023] The thin-layer identification method for Amomum villosum formula granules provided by the present invention can well develop and separate compounds with greater polarity in the Amomum villosum formula granules. After color development, the spots are rich and clear, with good separation. The spots are within the Rf value range specified in the Chinese Pharmacopoeia, the identification characteristics are obvious, the results are easy to judge, and the specificity is good.

[0024] In addition to volatile oil, the Amomum villosum formula granules also contain rich water-soluble components such as flavonoids. Compared with the pharmacopoeia method, the method established by the present invention greatly shortens the analysis and detection time, can quickly identify the Amomum villosum formula granules, improves the quality controllability of the identified objects, has high practical value, and the method is simple, fast, efficient and low-cost.

[0025] In addition, the thin layer identification method of the Amomum villosum formula granules of the present invention can effectively distinguish Amomum villosum of different origins (Amomum villosum and Amomum villosum green shell), and can identify its common mixed counterfeits (Amomum villosum longus) on the market. This method is easy to promote and apply, and also provides a basis for accurate clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. The drawings described below are some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0027] Figure 1 The thin layer chromatography (TLC) patterns of the preparation methods of different test solutions and different control medicinal material solutions in Example 1 of the present invention are shown (1-3: test solution preparation methods 1-3; S1-S2: control medicinal material solution preparation methods 1-2).

[0028] Figure 2 Shows the TLC spectra using different developing agents (1-3: Amomum villosum formula granules; S: Amomum villosum control medicinal material)

[0029] Figure 3 The TLC spectra with different sample volumes are shown (1 to 4: the sample volumes of the test solution are 1 μL, 2 μL, 3 μL, and 4 μL, respectively; 5 to 8: the sample volumes of the control medicinal material solution are 1 μL, 2 μL, 5 μL, and 10 μL, respectively)

[0030] Figure 4 Shows the thin layer chromatography (TLC) spectra at different development temperatures (1-3: Amomum villosum formula granules; S: Amomum villosum control medicinal material)

[0031] Figure 5 Shows the TLC spectra of different development humidity (1-3: Amomum villosum formula granules; S: Amomum villosum control medicinal material)

[0032] Figure 6 The TLC patterns of the specificity test are shown (1-3: Amomum villosum formula granules; 4: negative control; S: Amomum villosum control medicinal material)

[0033] Figure 7 The TLC patterns of different batches of Amomum villosum granules in Example 2 are shown (1-3: Amomum villosum granules batch numbers 19060289, 19060299, 19060309; S: Amomum villosum control medicinal material)

[0034] Figure 8 The TLC patterns of the green shell sand, Amomum villosum and Amomum villosum formula granules in Example 3 are shown (1-3: green shell sand; 4-6: Amomum villosum; 7-9: Amomum villosum; S: Amomum villosum control medicinal material)

[0035] Figure 9 The TLC analysis (TLC) patterns obtained by the thin-layer identification method specified in the Pharmacopoeia in Comparative Example 1 are shown (1-2: Amomum villosum; 3-4: Green shell sand; 5-6: Amomum villosum; T: Bornyl acetate reference substance) DETAILED DESCRIPTION

[0036] To better illustrate the present invention, numerous specific details are provided in the following detailed description. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0037] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0039] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0040] It will be understood that the singular article "a," "an," and "the," as used in the specification and claims of this application, includes plural referents unless the context clearly dictates otherwise.

[0041] In this specification, references to "one or some specific / preferred embodiments / solutions," "another or other specific / preferred embodiments / solutions," "one or another embodiment / solution," "one or another technical solution," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiment are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any appropriate manner.

[0042] The term "comprises" and any variations thereof in the specification and claims of the present invention and the accompanying drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements, but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0043] Unless otherwise specified, the term "herbal medicine" used in this article refers to unprocessed or unfinished Chinese medicine raw materials; "control medicinal materials" (or "reference medicinal materials") refer to standard medicinal materials that have completed species identification and are used to identify the samples to be tested.

[0044] Unless otherwise specified, the term "test article" used herein refers to an experimental sample used for detection or identification.

[0045] Unless otherwise specified, the term "thin layer chromatography" used in this article is a trace, rapid and simple separation and analysis method. A suitable stationary phase is coated on a glass plate, plastic or aluminum substrate to form a uniform thin layer. After the sample is spotted and developed, the relative shift value (Rf) is compared with the relative shift value of the chromatogram obtained by the same method with a suitable reference substance, and is used for drug identification, impurity inspection or content determination. This method is a very important experimental technique for rapid separation and qualitative analysis of small amounts of substances, and is also used to track the progress of reactions. It has the characteristics of short development time, strong separation ability and high sensitivity. Thin layer chromatography is one of the important inspection items for traditional Chinese medicines. By comparing the chromatographic behavior of the test sample and the reference medicinal materials and reference substances, the authenticity and quality of the medicinal materials can be judged.

[0046] Unless otherwise specified, the term "spotting" used herein refers to the process of adding drops of sample solution to be separated and identified onto a thin layer plate.

[0047] Unless otherwise specified, the term "development" used herein refers to the process in which the developing agent carries the sample components to migrate a certain distance on the thin layer plate by the capillary action of the stationary phase on the thin layer plate.

[0048] The present invention provides a thin-layer chromatography (TLC) identification method for Amomum villosum formula granules, comprising the steps of preparing a test solution, preparing a control medicinal material solution, and performing TLC identification. The test solution and the control medicinal material solution are prepared using a simple and rapid preparation method, and are spotted on the same TLC plate. After development, the TLC plate is inspected under ultraviolet light to obtain a multi-information TLC of the Amomum villosum formula granules. This method uses the control medicinal material as a reference and flavonoids as the main identification component. It has high specificity and can distinguish Amomum villosum of different origins (Amomum villosum and Amomum villosum var. amomi) and identify mixed and counterfeit Amomum villosum (Amomum villosum var. longifolia).

[0049] <Preparation of test solution>

[0050] The inventors investigated the preparation methods of the test solution and compared the final extraction results, and finally determined the preparation method of the test solution, which specifically includes taking Amomum villosum formula granules, dissolving them in water, extracting them using an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue in an alcohol solvent.

[0051] In some specific embodiments of the present invention, the Amomum villosum granules are crushed and ground into a fine powder, passed through a No. 2 sieve, and then dissolved in water. In the present invention, using water as the extraction solvent can maximize the extraction of water-soluble components such as flavonoids. The present invention does not particularly limit the amount of water used for extraction. To ensure extraction efficiency and better extract the components to be identified, the ratio of the Amomum villosum granules to water can be 1g:(30-50mL); preferably 1g:(35-45mL); and more preferably 1g:40mL.

[0052] After dissolving in water, an organic solvent is used for extraction. Compared with extraction methods such as ultrasonic treatment, shaking extraction is simpler and easier. The organic solvent used can be one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate. In some specific embodiments of the present invention, ethyl acetate is preferred. In order to make the extraction more complete and the extraction effect better, the number of extractions is more than two times, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); preferably 1:1. The ethyl acetate liquid is then combined and concentrated to dryness. The present invention has no particular restrictions on the method of concentration to dryness. In some specific embodiments of the present invention, it can be carried out by vacuum distillation and drying. After evaporation, an alcohol solvent is added to the residue. In some specific embodiments of the present invention, the alcohol solvent is selected from methanol, an aqueous solution of methanol, or ethanol, an aqueous solution of ethanol, etc., preferably methanol or an aqueous solution of methanol. Considering solubility, methanol is more preferred. From the perspective of dissolution efficiency, in some specific embodiments of the present invention, the usage ratio of Amomum villosum formula granules to alcohol solvent is (0.5-1) g: (1-2) mL, preferably (0.5-1) g: (0.8-1.5) mL, and more preferably 0.5 g: 1 mL.

[0053] <Preparation of Control Medicinal Material Solution>

[0054] The preparation method of the control medicinal material solution of the present invention comprises taking Amomum villosum as a control medicinal material, adding water for reflux extraction, filtering, extracting the filtrate with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue with an alcohol solvent.

[0055] In some specific embodiments of the present invention, the method of adding water and refluxing is selected to treat the Amomum villosum reference medicinal material based on Amomum villosum because water extraction is closer to the traditional decoction method of Chinese medicine than alcohol extraction (methanol, ethanol, etc.), and can fully extract the water-soluble components in the medicinal material and better characterize the clinical quality of the medicinal material; the present invention has no particular restriction on the amount of water used for water reflux extraction. In order to ensure the efficiency of the extraction and better extract the components to be identified, the ratio of the Amomum villosum reference medicinal material to water can be 1g: (15-25mL); preferably 1g: (17-23mL); more preferably 1g: 20mL.

[0056] To facilitate subsequent extraction, a filtration step is preferably performed after water reflux extraction. The present invention does not specifically limit the filtration method and can be any conventional filtration method in the art, such as filtration with filter paper or cotton. After filtration, the filtrate is extracted with an organic solvent. The organic solvent used can be one or more of ether, dichloromethane, chloroform, carbon tetrachloride, or ethyl acetate. In some specific embodiments of the present invention, ethyl acetate is preferred. To ensure more complete extraction and better extraction results, the extraction is performed two or more times, and the volume ratio of organic solvent to water used in each extraction is (1-2):(1-1.5); preferably 1:1. The ethyl acetate solutions are then combined and concentrated to dryness. The present invention does not specifically limit the method of concentration to dryness. In some specific embodiments of the present invention, the filtration can be carried out by vacuum distillation. After evaporation to dryness, an alcoholic solvent is added to the residue. In some specific embodiments of the present invention, the alcoholic solvent is selected from methanol, an aqueous solution of methanol, or ethanol, an aqueous solution of ethanol, etc., preferably methanol or an aqueous solution of methanol. Methanol or an aqueous solution of methanol is more preferred due to solubility. Considering the dissolution efficiency, in some specific embodiments of the present invention, the usage ratio of Amomum villosum reference medicinal material to alcohol solvent is (1-1.5) g: (1-2) mL, preferably 1 g: 1 mL.

[0057] In addition, in some preferred embodiments of the present invention, the thin layer identification method of the present invention also includes the preparation of a negative control solution. The preparation of the negative control solution of the present invention includes taking negative control formula particles that do not contain Amomum villosum. The rest of the preparation method refers to the above <Preparation of the test solution>.

[0058] <Thin layer identification step>

[0059] The thin layer identification step of the present invention comprises taking a test sample solution and a control medicinal material solution, spotting them on a polyamide thin layer chromatography plate respectively, developing them with methanol-glacial acetic acid-water as a developing agent, drying them after development, spraying them with a color developer, heating them until the spots are clearly colored, and inspecting them under ultraviolet light.

[0060] In some specific embodiments of the present invention, there is no special restriction on the material of the thin layer plate, and silica gel thin layer plates, polyamide thin layer plates, ordinary prefabricated plates, high-efficiency prefabricated plates, etc. can be selected. Since the identification components of the present invention are water-soluble components with high polarity such as flavonoids, in order to fully separate them, polyamide thin layer chromatography plates are preferred; further, in order to facilitate inspection and reduce the differences caused by the stationary phase, it is preferred to take the test solution and the control medicinal material solution and spot them on the same polyamide thin layer chromatography plate respectively; in some specific embodiments of the present invention, the spotting volume of the test solution is 1-4 μL, preferably 2 μL; the spotting volume of the control medicinal material solution is 1-10 μL, preferably 3-6 μL, and more preferably 5 μL; if the spotting volume is too low, the spot separation is relatively poor and the spots cannot be clearly displayed; the present invention uses methanol-glacial acetic acid-water as the developing agent for development, which is conducive to the good separation of various chemical components of the Amomum villosum formula granules and the Amomum villosum control medicinal materials, especially the flavonoid components, on the thin layer plate. In some preferred embodiments of the present invention, in order to obtain better separation, the developing agent of the present invention is preferably calculated in a volume ratio of methanol: glacial acetic acid: water = (5-12): (1-5): (1-5), further (7-10): (1-3): (1-3), and further 9:1:1.

[0061] The present invention does not particularly limit the temperature during development; it can be carried out at 20-40°C or 3-8°C, and different temperatures have little effect on the separation effect. The present invention also does not particularly limit the relative humidity during development; it can be carried out at a relative humidity of 18% to 88%, and different relative humidities have little effect on the separation effect. In some specific embodiments of the present invention, the thin layer color developer is selected from aluminum chloride solution, and the concentration of the aluminum chloride solution is preferably 1% to 5%, more preferably 2% to 4%, and most preferably 3%, which can achieve better color development effect than a 5% vanillin-sulfuric acid solution.

[0062] Afterwards, heating is carried out, and the present invention is not particularly limited to the mode of heating. It can be carried out using a conventional method in the art, and preferably, heating is carried out by hot air drying; the temperature of heating is 100-110 DEG C, preferably 105 DEG C. The present invention is inspected under ultraviolet lamp (365nm), and compared with inspection under sunlight, the spot color development under ultraviolet light is clear and colorful. In some specific embodiments of the present invention, the test sample chromatogram is compared with the control medicinal material chromatogram, and it is judged whether the same fluorescent spot is shown at one or more positions with the same Rf value. Specifically, the thin layer chromatogram of the Amomum villosum formula granules of Amomum villosum based on Amomum villosum and the Amomum villosum control medicinal material of Amomum villosum based on Amomum villosum, shows the same spot at Rf value 0.2; the Amomum villosum formula granules of green shell sand are not obvious at Rf value 0.2, and at Rf value 0.35, the Amomum villosum formula granules of Amomum villosum based on Amomum villosum, the Amomum villosum control medicinal material of Amomum villosum based on Amomum villosum and the Amomum villosum formula granules of green shell sand all show the same fluorescent spot, but the long sequence Amomum villosum formula granules lack corresponding spots here.

[0063] In other specific embodiments of the present invention, the thin-layer chromatography identification step may further include spotting a test sample solution, a control medicinal material solution, and a negative control solution onto a polyamide thin-layer chromatography plate, developing the plate with methanol-glacial acetic acid-water, air-drying the plate after development, spraying the plate with a color developer, heating the plate until the spots are clearly colored, and inspecting the plate under ultraviolet light. The results show that the test sample chromatogram displays the same fluorescent spots at corresponding positions in the control medicinal material chromatogram, while the negative control exhibits no interference.

[0064] By using the thin-layer identification method of the Amomum villosum formula granules provided by the present invention, a chromatogram of the test sample can be obtained that is consistent with the spots in the chromatogram of the control medicinal material. The separation and point characteristics in the chromatogram are good, the development time is short, and the inspection is clear, which can better control the quality of the Amomum villosum formula granules and distinguish their origin and authenticity.

[0065] Example

[0066] The technical solutions of the present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to explain and illustrate the present invention, and are not intended to limit the scope of protection of the present invention.

[0067] <Instruments and reagents>

[0068] Instruments: CAMAH TLC VISUALIZER, METTLERTOLEDO balance, KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), polyamide film (Luqiao Sijia Biochemical Plastic Factory, Taizhou City, Zhejiang Province).

[0069] Reagents: ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd.), methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), aluminum chloride (Sinopharm Chemical Reagent Co., Ltd.) were all of analytical grade, and water (pure water) was used.

[0070] The control medicinal material of Amomum villosum (Yangchunsha) (batch number: 120985-201406) was purchased from the China Food and Drug Inspection Institute; Amomum villosum (Yangchunsha) formula granules (batch numbers: 19060289, 19060299, 19060309), Amomum villosum (Green Shell Sand) formula granules (batch number: 18050099), and Amomum villosum longifolia formula granules (batch number: 21110149) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0071] Example 1: Construction of Thin Layer Identification Method for Amomum villosum Formula Granules

[0072] Investigation of the preparation methods of test solution and control medicinal material solution

[0073] <Preparation of test solution>

[0074] Method 1: Take 0.5 g of Amomum villosum formula granules (batch number: 19060299), grind it into powder, add 20 ml of methanol, ultrasonically treat it for 20 minutes, filter it, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0075] Method 2: Take 0.5 g of Amomum villosum formula granules (batch number: 19060299), grind it into powder, add 20 ml of ethanol, ultrasonically treat it for 20 minutes, filter it, evaporate the filtrate to dryness, and add 1 ml of ethanol to dissolve the residue as the test solution.

[0076] Method 3: Take 0.5 g of Amomum villosum granules (batch number: 19060299), grind it into powder, add 20 ml of water to dissolve it, shake and extract it with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0077] <Preparation of Control Medicinal Material Solution>

[0078] Method 1: Take 1 g of Amomum villosum (Amomum villosum) as a control medicinal material, add 20 ml of ethyl acetate, ultrasonically treat for 20 minutes, filter, evaporate the filtrate to dryness, and add 1 ml of ethyl acetate to dissolve the residue as the control medicinal material solution.

[0079] Method 2: Take 1 g of Amomum villosum (Amomum villosum) as a control medicinal material, add 20 ml of water, heat and reflux for 30 minutes, filter, and extract the filtrate by shaking with ethyl acetate twice, 20 ml each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 ml of methanol as the control medicinal material solution.

[0080] <Preparation of Negative Control Solution>

[0081] Take 0.5 g of negative control medicinal material lacking Amomum villosum (Amomum villosum) and prepare a negative control solution according to "Method 3" in <Preparation of Test Solution>.

[0082] <Thin layer identification>

[0083] The test solution and control medicinal material solution prepared according to the above three test solution preparation methods and the two control medicinal material solution preparation methods were spotted on the same polyamide film, and developed with methanol-glacial acetic acid-water (9:1:1). The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results are as follows. Figure 1 .

[0084] The results showed that the chromatogram obtained by method 3 in the preparation method of the test solution had clear spots and a good separation effect; the chromatogram obtained by method 2 in the preparation method of the control medicinal material solution was more consistent with the particle spots.

[0085] Investigation of thin layer identification method

[0086] <Study of Developing Agent>

[0087] Take Amomum villosum formula granules (batch number: 19060299) and Amomum villosum (Amomum villosum) control medicinal material and prepare the test solution and control medicinal material solution according to the above test solution preparation method 3 and control medicinal material solution preparation method 2, respectively, and spot them on the same polyamide film. Use methanol-glacial acetic acid-water (9:1:1) and ethyl acetate-methanol-water (8:4:0.5) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride solution, blow dry with hot air, and examine under ultraviolet light (365nm). The results are as follows Figure 2 , where the left side is the chromatogram obtained using methanol-glacial acetic acid-water (9:1:1) as the developing solvent, and the right side is the chromatogram obtained using ethyl acetate-methanol-water (8:4:0.5) as the developing solvent.

[0088] The results showed that when the developing solvent was methanol-glacial acetic acid-water (9:1:1), the spots in the chromatograms of the test sample and the control medicinal material were clearer and the separation was better. Therefore, methanol-glacial acetic acid-water (9:1:1) was selected as the developing solvent.

[0089] <Inspection of Sample Quantity>

[0090] Take Amomum villosum formula granules (batch number: 19060299) and Amomum villosum (Amomum villosum) control medicinal material and prepare test solution and control medicinal material solution according to the above test solution preparation method 3 and control medicinal material solution preparation method 2, respectively. Spot 1μL, 2μL, 3μL, and 4μL of the test solution, and spot 1μL, 2μL, 5μL, and 10μL of the control medicinal material solution on the same polyamide film, use methanol-glacial acetic acid-water (9:1:1) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride solution, blow dry with hot air, and examine under ultraviolet light (365nm). The results are as follows Figure 3 .

[0091] The results showed that when the spot volume of the test solution was 2 μL and the spot volume of the control medicinal material solution was 5 μL, the spots at the corresponding positions in the test sample chromatogram and the control medicinal material chromatogram clearly corresponded to each other without other interference.

[0092] <Expansion of Temperature>

[0093] Take the test solution (batch number: 19060299) and the control medicinal material solution, respectively, and spot them on the same polyamide film. Use methanol-glacial acetic acid-water (9:1:1) as the developing solvent, develop at 35℃ and 4℃ respectively (relative humidity is 58%), take out, dry, spray with 3% aluminum chloride solution, blow dry with hot air, and examine under ultraviolet light (365nm). The results are as follows Figure 4 , wherein the left side is the chromatogram obtained when the temperature is developed at 35°C, and the right side is the chromatogram obtained when the temperature is developed at 4°C.

[0094] The results show that under different development temperature conditions, the same color fluorescent spots appear at the corresponding positions in the chromatograms of the test sample and the control medicinal material, and the separation effect is good. The experimental results show that the development temperature has little effect on the thin layer chromatography identification results of Amomum villosum (Amomum villosum) formula granules, indicating that the thin layer chromatography identification method is robust to different development temperatures.

[0095] <Expanding humidity>

[0096] Take the test solution (batch number: 19060299) and the control medicinal material solution, respectively, and spot them on the same polyamide film. Use methanol-glacial acetic acid-water (9:1:1) as the developing solvent, develop them under the conditions of relative humidity of 18% and 88% respectively (temperature of 21.1℃), take them out, dry them, spray them with 3% aluminum chloride solution, blow them dry with hot air, and examine them under ultraviolet light (365nm). The results are as follows Figure 5 , wherein the left side is the chromatogram obtained when the temperature is developed under the condition of relative humidity of 18%, and the right side is the chromatogram obtained when the temperature is developed under the condition of relative humidity of 88%.

[0097] The results show that under different development humidity conditions, the same color fluorescent spots appear at the corresponding positions in the chromatograms of the test sample and the control medicinal material, and the separation effect is good. The experimental results show that development humidity has little effect on the thin-layer chromatography identification results of Amomum villosum (Amomum villosum) formula granules, indicating that the thin-layer chromatography identification method is robust to different development humidity conditions.

[0098] <Specificity test>

[0099] Take the test solution (batch number: 19060299), the control medicinal material solution and the negative control solution, and spot them on the same polyamide film. Use methanol-glacial acetic acid-water (9:1:1) as the developing agent, develop, remove, dry, spray with 3% aluminum chloride solution, blow dry with hot air, and examine under ultraviolet light (365nm). Figure 6 .

[0100] The results showed that the chromatograms of the test sample and the control herb showed fluorescent spots of the same color at the corresponding positions, and the negative control showed no interference, indicating that the TLC identification method has good specificity.

[0101] <Determination of Thin Layer Identification Method>

[0102] According to the above investigation results, the thin layer identification method of Amomum villosum formula granules is determined as follows:

[0103] Take 0.5g of Amomum villosum granules, grind them into powder, add 20ml of water to dissolve them, shake and extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0104] Separately, take 1g of Amomum villosum as a control medicinal material, add 20ml of water, heat under reflux for 30 minutes, filter, and add 20ml of ethyl acetate to the filtrate. Prepare a control medicinal material solution in the same manner. According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 2μL of the test solution and 5μL of the control medicinal material solution were spotted on the same polyamide film. Develop with methanol-glacial acetic acid-water (9:1:1). Remove, air-dry, spray with 3% aluminum chloride solution, blow dry with hot air, and examine under ultraviolet light (365nm). 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.

[0105] Example 2: Validation of the Thin Layer Identification Method for Different Batches of Amomum villosum Granules

[0106] Different batches of Amomum villosum formula granules (batch numbers: 19060289, 19060299, 19060309) were taken and prepared into test solution according to the above method for preparing test solution. The control medicinal material Amomum villosum was taken and prepared into control medicinal material solution according to the above method for preparing control medicinal material. The samples were spotted on the same polyamide film and developed with methanol-glacial acetic acid-water (9:1:1). The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results were as follows. Figure 7 .

[0107] The results showed that the chromatograms of the test samples and the control medicinal materials made from different batches of Amomum villosum granules showed fluorescent spots of the same color at corresponding positions, indicating that the thin-layer chromatography identification method of the present invention can well develop and separate the compounds in different batches of Amomum villosum granules. After color development, the spots are rich and clear, the separation is good, and the identification results are easy to judge.

[0108] Example 3: Differentiation of Amomum villosum granules of different origins and identification of counterfeit Amomum villosum

[0109] Separately, Amomum villosum (Yangchunsha) formula granules (batch number: 19060299), Amomum villosum (Green Shell Sand) formula granules (batch number: 21110149) and Amomum villosum longifolia formula granules (batch number: 21110149) were taken according to the above-mentioned preparation method of the test solution to prepare the test solution. Separately, Amomum villosum (Yangchunsha) control medicinal material was taken and the control medicinal material solution was prepared according to the above-mentioned preparation method of the control medicinal material. The samples were spotted on the same polyamide film, and methanol-glacial acetic acid-water (9:1:1) was used as the developing agent. The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results are as follows: Figure 8 .

[0110] The results showed that the chromatogram of the Amomum villosum (Yangchunsha) formula granules had a distinct spot at Rf value 0.2, while the chromatogram of the Amomum villosum (Green Shell Sand) formula granules had no distinct spot at Rf value 0.2, indicating that this thin-layer identification method can distinguish Amomum villosum granules of different origins. Compared with the chromatogram of the Amomum villosum (Yangchunsha) formula granules, the chromatogram of the Long-sequence Amomum villosum formula granules lacked the corresponding spot at Rf value 0.35, indicating that this identification method can distinguish Amomum villosum and its counterfeit (Long-sequence Amomum villosum).

[0111] Comparative Example 1: Thin layer identification method specified in the Pharmacopoeia

[0112] Take the essential oil under [Determination of Content], add ethanol to make a solution containing 20μL per 1ml, as the test solution. Take the borneol acetate reference substance, add ethanol to make a solution containing 10μL per 1ml, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 1μL of each of the above two solutions and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate (22:1) as the developing solvent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clearly colored. The results are as follows Figure 9 .

[0113] The results showed that although the same purple-red spots appeared in the chromatogram of the test sample at the corresponding position in the chromatogram of the reference sample, the chromatographic spots were not clear. In addition, since the Amomum villosum formula granules of different origins (Amomum villosum and Amomum villosum with green shell) and the Amomum villosum counterfeit (Amomum villosum with long sequence) all contained borneol acetate, it was impossible to distinguish the Amomum villosum formula granules of different origins using it as a reference sample, nor could it distinguish Amomum villosum from the common mixed counterfeit (Amomum villosum with long sequence) in the market.

[0114] The above examples are merely intended to illustrate several embodiments of the present invention. Although the descriptions thereof are relatively specific and detailed, they are not to be construed as limiting the scope of the present invention. It should be understood that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A thin layer detection method for Amomum villosum formula granules, characterized in that: The method includes the preparation of a test solution, the preparation of a control medicinal material solution and thin layer detection. The preparation of the test solution comprises taking the Amomum villosum granules, dissolving them in water, extracting them with ethyl acetate, separating the organic phase and concentrating it to dryness, and dissolving the residue in an alcohol solvent; The preparation of the control medicinal material solution comprises taking Amomum villosum reference medicinal material of Amomum villosum base, adding water for reflux extraction, filtering, extracting the filtrate with ethyl acetate, separating the organic phase and concentrating to dryness, and dissolving the residue with an alcohol solvent; The thin layer chromatography test comprises taking the test sample solution and the control medicinal material solution, spotting them on a polyamide thin layer chromatography plate, developing them with methanol-glacial acetic acid-water in a volume ratio of 9:1:1 as a developing agent, and inspecting them. The thin layer test also includes drying after unfolding, spraying with a color developer, heating until the spots are clearly colored, and inspecting under ultraviolet light; The color developer is an aluminum chloride solution with a concentration of 1% to 5%.

2. The thin layer detection method according to claim 1, characterized in that: The thin layer detection method also includes the preparation of a negative control solution, which includes taking negative control formula particles that do not contain Amomum villosum, and the remaining steps refer to the preparation method of the test solution; the Amomum villosum formula particles and the negative control formula particles are both powdered.

3. The thin layer detection method according to claim 1 or 2, characterized in that: In the preparation of the test solution, the ratio of the Amomum villosum formula granules to water is 1 g: (30~50) mL; the number of extractions is more than two times, and the volume ratio of the organic solvent to water used in each extraction is (1~2): (1~1.5); the alcohol solvent is methanol or an aqueous solution of methanol.

4. The thin layer detection method according to claim 1 or 2, characterized in that: In the preparation of the control medicinal material solution, the dosage ratio of the Amomum villosum control medicinal material to water is 1 g: (15-25) mL; the reflux extraction time is 20-40 min; the number of extractions is more than two times, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); the alcohol solvent is methanol or an aqueous solution of methanol.

5. The thin layer detection method according to claim 1 or 2, characterized in that: In the thin layer chromatography test, the test sample solution and the control medicinal material solution are respectively spotted on the same polyamide thin layer chromatography plate.

6. The thin layer detection method according to claim 1 or 2, characterized in that: In the thin layer test, the spot volume of the test solution is 1~4μL, and the spot volume of the control medicinal material solution is 1~10μL.

7. The thin layer detection method according to claim 1 or 2, characterized in that: In the thin layer test, the inspection includes comparing the chromatogram of the test sample with the chromatogram of the reference medicinal material to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value.

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