Micro-emulsion thin-layer identification method for quantitative analysis of multi-components by single marker of cold asthma Zupao

Abstract

The invention provides a multi-information thin-layer identification method for rapid'quantitative analysis of multi-components by single marker 'of cold-asthma Zupao granules, and particularly, the concept of the'quantitative analysis of multi-components by single marker' is used for reference, and the method is applied to thin-layer qualitative identification, namely, one to two easily available and low-price reference substances or substitute reference substances are adopted to identify the cold-asthma Zupao granules. A plurality of components of licorice extract, fenugreek, roses and celery seeds in a thin-layer chromatogram of the cold-asthma Zupa granules are accurately positioned and identified according to relative expansion positions in thin-layer chromatography, so that unknown components are positioned by known reference substances while multi-component quality control is performed, and equivalently, the characteristic thin-layer chromatogram of the cold-asthma Zupa granules is established. According to the method, rapid and accurate identification can be carried out, most spots are unknown components, adulteration of known components in traditional Chinese medicine preparations can be avoided, and effective quality control is achieved.

Description

Technical Field

[0001] The invention relates to a "one-test-multiple-evaluations" thin-layer identification method, in particular to a microemulsion thin-layer identification technology for traditional Chinese medicine preparations. Background Art

[0002] 1. Current Standards for Thin-Layer Identification of Hanchuanzupa Granules

[0003] Hanchuanzupa Granules is a medicine included in the "Uyghur Medicine Volume of the Drug Standards of the Ministry of Health of the People's Republic of China". It is composed of 9 medicinal ingredients: hyssop, maidenhair fern, licorice extract, fennel, celery seeds, fenugreek, rue, rose, and nettle seeds. It has the effects of suppressing cough, resolving phlegm, warming the lungs and relieving asthma. It is used to treat acute colds, coughs caused by the cold "Naizilai" and abnormal mucous asthma.

[0004] Under the current quality standard, two reference medicinal materials and three reference substances are used to identify three medicinal materials in the preparation using three thin-layer development systems, namely, rose, licorice and fenugreek in the preparation. Obviously, it is impossible to fully control the quality of Hanchuanzupa Granules. The preparation process of the test solution in the current standard is complicated, time-consuming, and uses more organic solvents; the solvents used in the three development systems are highly toxic and pollute the environment.

[0005] In addition to the current quality standards, there are few literature studies related to the quality standards of Hanchuanzupa. Parida Abilizi et al. (Parida Abilizi, Miriguli Musa, Cong Yuanyuan et al. Thin layer chromatography identification of Uyghur medicine Hanchuanzupa granules [J]. Xinjiang Traditional Chinese Medicine, 2008 (02): 42-43.) used 5 different developing agents to identify fennel, fenugreek, licorice, celery seeds, and rose in Hanchuanzupa granules. Each medicinal material required a different pretreatment method to prepare the test solution; Jin Xiaohong et al. (Jin Xiaohong, Gui Hongwu, Geng Wei. Study on the quality standard of Hanchuanzupa granules [J]. Chinese Pharmacy, 2012, 23 (15): 1405-1406.) used 2 different developing agents to identify rose and fennel in Hanchuanzupa granules. However, the spots of Hyssopus officinalis, Adiantum officinalis, celery seeds, fenugreek, Rue and Urtica dioica in the prescription were not clear after thin layer development and could not be used as the basis for qualitative identification of the preparation.

[0006] In summary, in the current standards and literature, the preparation methods of the sample solution in the thin layer chromatography identification of Hanchuanzupa granules are relatively cumbersome and time-consuming, with low identification efficiency, and it is impossible to conduct comprehensive identification and quality control of Hanchuanzupa granules; and the solvents in the development system used are highly toxic, causing certain harm to personnel and the environment. Therefore, it is urgent to develop an efficient and low-pollution thin layer identification method to achieve a more comprehensive quality control of Hanchuanzupa granules.

[0007] 2. Background of the "One Test, Multiple Evaluations" Technical Method for Hanchuan Zupa Granules

[0008] In the TLC identification of the current edition of the Chinese Pharmacopoeia and various medicinal materials / TCM preparation standards, reference medicinal materials and / or reference substances are often used to identify medicinal materials. Using reference medicinal materials for TLC identification is a feature of TLC identification of Chinese medicinal materials / TCM preparations, which can scientifically identify the ingredients contained in medicinal materials. However, in actual application, there are problems such as a small number of reference medicinal materials, difficulty in identifying authentic medicinal materials, and difficulty in obtaining them.

[0009] Only 51% of the TLC identification items of medicinal materials in the 2020 edition of the Chinese Pharmacopoeia were performed with reference medicinal materials. A large number of medicinal materials / traditional Chinese medicine preparations were mostly identified with reference materials for TLC. This method usually uses reference materials to identify a corresponding component in the medicinal material / traditional Chinese medicine preparation. There is no TLC identification of multiple spots in the TLC of the medicinal material test sample using a reference material. However, in the actual experimental process, multiple spots often appear in the TLC of the medicinal material / traditional Chinese medicine preparation, representing multiple compounds in the medicinal material / traditional Chinese medicine preparation. Therefore, identifying only one component will result in a waste of TLC information. At the same time, only identifying one or several medicinal materials in the traditional Chinese medicine compound preparation cannot meet the quality control of the compound medicinal materials to a certain extent.

[0010] In the current thin-layer chromatography identification of the quality standard of Hanchuanzupa Granules: (1) the use of rose flower as a reference material and gallic acid as a reference substance is to control the quality of rose flower; (2) the use of licorice as a reference material and liquiritin as a reference substance is to control the quality of licorice extract; among the 9 medicinal materials / extracts in the compound, only 2 of them are subject to quality control, and the remaining 7 medicinal materials have no clear quality control standards; in view of the current drug quality problems in traditional Chinese medicine compound preparations, the current thin-layer chromatography identification method can no longer meet the quality control requirements of Hanchuanzupa Granules, and a more economical, convenient, intuitive, comprehensive and accurate evaluation method is urgently needed. Summary of the invention

[0011] The purpose of the present invention is to provide a thin layer identification method which can simultaneously identify licorice extract, fenugreek, celery seeds and rose medicinal materials in Hanchuanzupa granules, and is simple, fast, highly efficient, low-toxic and low-pollution, so as to control the quality of Hanchuanzupa granules.

[0012] In a first aspect of the present invention, a thin layer chromatography identification method of Hanchuanzupa granules is provided, wherein the Hanchuanzupa granules contain liquorice extract, fenugreek, rose flower, and celery seed;

[0013] The method comprises the steps of:

[0014] s1. Providing a test solution, a control medicinal material solution, a negative control solution, and an optional boiled control medicinal material solution;

[0015] Wherein, the control medicinal material solution includes a plurality of liquorice extract solution, fenugreek solution, rose solution and celery seed solution;

[0016] The boiled control medicinal material solution includes multiple boiled liquorice extract solution, boiled fenugreek solution, boiled rose solution and boiled celery seed solution;

[0017] The negative control solution includes a plurality of negative control solutions lacking liquorice extract, a negative control solution lacking fenugreek, a negative control solution lacking rose, and a negative control solution lacking celery seeds;

[0018] The control medicinal material solution and the control medicinal materials in the boiled control medicinal material solution correspond one to one; the control medicinal materials in the control medicinal material solution and the medicinal materials missing from the negative control solution correspond one to one;

[0019] Wherein, the preparation method of the test solution comprises the following steps:

[0020] (a) grinding the raw material, mixing with a lower alcohol, ultrasonically extracting, filtering, and concentrating the filtrate to obtain the test solution; the raw material is Hanchuanzupa granules;

[0021] The preparation methods of the control medicinal material solution, negative control solution, and boiled control medicinal material solution are independently the same as the above step (a), except that the raw materials are replaced with the corresponding control medicinal materials, negative control substances, or boiled control medicinal materials, and the amount of the raw materials is optionally adjusted;

[0022] s2. The test solution, control medicinal material solution, negative control solution, and any boiled control medicinal material solution are respectively applied to a polyamide film, developed under the action of a developing agent, and after drying, a color developer is applied to the film for color development, and then the film is placed under ultraviolet light for inspection;

[0023] Wherein, the developing agent is a mixed solution of microemulsion and formic acid,

[0024] The preparation method of the microemulsion comprises: (b) mixing sodium dodecyl sulfate, C3-C6 alcohol, C5-C10 alkane and water and allowing to stand to obtain the microemulsion;

[0025] The method for applying the developer comprises: (c) first spraying a solution of 2-aminoethyl diphenylborate on a polyamide film, and then spraying a polyethylene glycol 400 solution on the polyamide film.

[0026] The control medicinal material solution is not a mixed solution of multiple (2, 3 or 4) kinds of liquorice extract solution, fenugreek solution, rose solution and celery seed solution. When the method identifies two kinds of medicinal materials at the same time, the control medicinal material solution is a collection of two solutions, and two different control medicinal material solutions of two different control medicinal materials are prepared respectively. In step s2, the two different control medicinal material solutions are respectively applied on the polyamide film to form two strips. Similarly, when identifying three or four strips at the same time, three or four different control medicinal material solutions are respectively applied on the polyamide film to form three or four strips. The negative control solution and the control medicinal material solution after boiling are also the same, which are also a collection of one or more different solutions, not a mixed solution.

[0027] In another preferred embodiment, the negative control substance is a control substance obtained by removing only the corresponding control medicinal materials from the formula of Hanchuanzupa Granules. For example, in the preparation method of the negative control solution lacking licorice extract, the raw material, i.e., the negative control substance, is the formula of Hanchuanzupa Granules with only licorice extract removed. Similarly, the negative control solution lacking fenugreek, the negative control solution lacking rose, and the negative control solution lacking celery seeds, wherein the negative control substances are the formula of Hanchuanzupa Granules with only the corresponding fenugreek, rose, and celery seeds removed.

[0028] In another preferred embodiment, in step (a), the lower alcohol is selected from the group consisting of methanol, ethanol, n-propanol, n-butanol, or a combination thereof, preferably methanol.

[0029] In another preferred embodiment, in the step (a), the mass volume ratio of the Hanchuanzupa granules to the lower alcohol is 0.1-1 g / mL, preferably 0.2-0.5 g / mL.

[0030] In another preferred embodiment, in the step (a), the ultrasonic extraction time is 10-60 min, for example, 20 min.

[0031] In another preferred embodiment, in the step (a), the mass volume ratio of the Hanchuanzupa granules raw material to the concentrated liquid volume after the filtrate is concentrated is 1-10 g / mL, preferably 3-8 g / mL.

[0032] In another preferred embodiment, in the preparation methods of the control medicinal material solution, the negative control solution and the control medicinal material solution after boiling in water, the mass volume ratio of the raw material to the lower alcohol is independently 0.05-1 g / mL, preferably 0.08-0.3 g / mL.

[0033] In another preferred embodiment, in the preparation methods of the control medicinal material solution, the negative control solution and the control medicinal material solution after boiling in water, the mass volume ratio of the raw material to the volume of the concentrated solution after the filtrate is concentrated is 1-10 g / mL, preferably 1-5 g / mL.

[0034] In another preferred embodiment, the method for preparing the test solution comprises the following steps:

[0035] (a) Grind 1-10 g of the raw material, mix with 5-50 mL of methanol, perform ultrasonic extraction for 10-60 min, filter, and concentrate the filtrate to 0.05-2 mL to obtain the test solution; the raw material is Hanchuan Zupa granules;

[0036] Preferably, (a) 5-10 g of the raw material is ground, mixed with 10-30 mL of methanol, ultrasonically extracted for 10-30 min, then filtered, and the filtrate is concentrated to 0.5-2 mL to obtain the test solution; the raw material is Hanchuan Zupa granules;

[0037] More preferably, (a) 6 g of the raw material is ground, mixed with 20 mL of methanol, ultrasonically extracted for 20 min, then filtered, and the filtrate is concentrated to 1 mL to obtain the test solution; the raw material is Hanchuanzupa granules.

[0038] In another preferred embodiment, the preparation methods of the control medicinal material solution, the negative control solution, and the boiled control medicinal material solution each independently comprise the following steps:

[0039] 1-8 g of the raw material is ground, mixed with 5-50 mL of methanol, and ultrasonically extracted for 10-60 min, then filtered, and the filtrate is concentrated to 0.05-2 mL to obtain the solution; the raw material is Hanchuan Zupa granules;

[0040] Preferably, (a) 2-5 g of the raw material is ground, mixed with 10-30 mL of methanol, subjected to ultrasonic extraction for 10-30 min, filtered, and the filtrate is concentrated to 0.5-2 mL to obtain the solution; the raw material is Hanchuan Zupa granules;

[0041] More preferably, (a) 3 g of the raw material is ground, mixed with 20 mL of methanol, and subjected to ultrasonic extraction for 20 min, followed by filtration, and the filtrate is concentrated to 1 mL to obtain the solution; and the raw material is Hanchuanzupa granules.

[0042] In another preferred embodiment, in step s2, 1-5 μL (eg 2 μL) of each of the test solution, control medicinal material solution, negative control solution, and optional boiled control medicinal material solution are independently taken and spotted on the same polyamide film.

[0043] In another preferred embodiment, in the step s2, the color development includes applying a color developer to the film and then heating it.

[0044] In another preferred embodiment, the heating temperature is 90-120°C, such as 100°C, 105°C, 110°C.

[0045] In another preferred embodiment, the heating time is 1-5 min, such as 2 min, 3 min.

[0046] In another preferred embodiment, in step s2, the wavelength of the ultraviolet light is 350-380 nm, such as 360 nm, 365 nm, 370 nm.

[0047] In another preferred embodiment, the step s2 comprises: respectively taking 1-5 μL of the above-mentioned test solution, control medicinal material solution, negative control solution, and optional boiled control medicinal material solution and spotting them on the same polyamide film, developing them under the action of a developing agent, taking them out, drying them, spraying a color developer on the film, heating them at 90-120° C. for 1-5 min, and then placing them under ultraviolet light with a wavelength of 350-380 nm for inspection;

[0048] Preferably, the step s2 comprises: respectively aspirating 2 μL of the above-mentioned test solution, control medicinal material solution, negative control solution, and optional boiled control medicinal material solution and spotting them on the same polyamide film, developing them under the action of a developing agent, taking them out, drying them, spraying a color developer on the film, heating them at 105°C for 2 minutes, and then inspecting them under ultraviolet light with a wavelength of 365 nm.

[0049] In another preferred embodiment, the developing agent is a mixed solution of microemulsion and formic acid in a volume ratio of 5-10:1, and the volume ratio is preferably 7-9:1.

[0050] In another preferred embodiment, in the developing agent, the water content of the microemulsion is 70-99.9 vol%, preferably 80-95 vol%, for example 90 vol%.

[0051] In another preferred embodiment, in step (b), the C3-C6 alcohol is selected from the group consisting of n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, n-hexanol, or a combination thereof, preferably n-butanol.

[0052] In another preferred embodiment, in step (b), the C5-C10 alkane is selected from the group consisting of n-pentane, n-hexane, n-heptane, n-octane, n-decane, or a combination thereof, preferably n-heptane.

[0053] In another preferred embodiment, in the step (b), the standing time is 18-48 hours, such as 24 hours, 36 hours.

[0054] In another preferred embodiment, the step (b) comprises: mixing 0.5-5 g of sodium dodecyl sulfate, 1-10 g of n-butanol, 0.1-1 g of n-heptane, and 10-60 g of water, and standing for 18-48 hours to obtain the microemulsion;

[0055] Preferably, the step (b) comprises: mixing 0.5-2 g of sodium dodecyl sulfate, 2-5 g of n-butanol, 0.1-1 g of n-heptane, and 20-50 g of water, and standing for 18-36 hours to obtain the microemulsion;

[0056] More preferably, the step (b) comprises: mixing 1.4 g sodium dodecyl sulfate, 3.15 g n-butanol, 0.5 g n-heptane and 45 g water, and letting the mixture stand for 24 hours to obtain the microemulsion.

[0057] In another preferred embodiment, in the step (c), the solution of 2-aminoethyl diphenylborate is a methanol solution of 2-aminoethyl diphenylborate.

[0058] In another preferred embodiment, in the step (c), the volume fraction of 2-aminoethyl diphenylborate is 5-30 mg / mL, preferably 10-20 mg / mL.

[0059] In another preferred embodiment, in the step (c), the solution of polyethylene glycol 400 is a methanol solution of polyethylene glycol 400.

[0060] In another preferred embodiment, in step (c), the volume fraction of polyethylene glycol 400 is 20-80 mg / mL, preferably 30-70 mg / mL.

[0061] In another preferred embodiment, the step (c) comprises: first spraying a methanol solution of 2-APB with a volume fraction of 5-30 mg / mL, and then spraying a methanol solution of polyethylene glycol 400 with a volume fraction of 20-80 mg / mL;

[0062] Preferably, the step (c) comprises: first spraying a methanol solution of 2-APB with a volume fraction of 10-20 mg / mL, and then spraying a methanol solution of polyethylene glycol 400 with a volume fraction of 30-70 mg / mL;

[0063] More preferably, the step (c) comprises: first spraying a methanol solution of 2-APB with a volume fraction of 10 mg / mL, and then spraying a methanol solution of polyethylene glycol 400 with a volume fraction of 50 mg / mL.

[0064] In another preferred embodiment, the step s2 comprises: respectively aspirating 1-5 μL of the above-mentioned test solution, control medicinal material solution, negative control solution, and optional boiled control medicinal material solution, and spotting them on the same polyamide film, using a mixed solution of microemulsion with a water content of 80-95 vol% and formic acid (volume ratio of 5-10:1) as a developing agent, developing under the action of the developing agent, taking out, drying, spraying with a methanol solution of 2-APB with a volume fraction of 5-30 mg / mL, and then spraying with a methanol solution of polyethylene glycol 400 with a volume fraction of 20-80 mg / mL, heating at 90-120°C for 1-5 min, and then placing it under ultraviolet light with a wavelength of 350-380 nm for inspection.

[0065] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The thin layer identification chromatograms of the licorice extract in Hanchuanzupa granules are shown (1: licorice extract solution; 2: Hanzupa granules solution; 3: negative control solution lacking licorice extract).

[0067] Figure 2 The thin layer identification chromatograms of fenugreek in Hanchuanzupa granules are shown (1: fenugreek control medicinal material solution; 2: fenugreek control medicinal material solution after boiling in water; 3: Hanchuanzupa granule solution; 4: negative control solution lacking fenugreek).

[0068] Figure 3 The thin layer identification chromatograms of rose in Hanchuanzupa granules are shown (1: rose control medicinal material solution; 2: rose control medicinal material solution after boiling; 3: Hanchuanzupa granule solution; 4: negative control solution without rose).

[0069] Figure 4 The layer identification chromatograms of celery seeds in Hanzupa granules are shown (1: celery seeds control medicinal material solution; 2: celery seeds control medicinal material solution after boiling; 3: Hanchuanzupa granule solution; 4: negative control solution lacking celery seeds).

[0070] Figure 5The thin layer identification chromatograms of licorice extract, fenugreek medicinal materials, rose medicinal materials, and celery seed medicinal materials in Hanchuanzupa granules are shown (1: licorice extract solution; 2: negative control solution lacking licorice extract; 3: fenugreek control medicinal material solution; 4: negative control solution lacking fenugreek; 5-7: Hanchuanzupa granule solution; 8: rose medicinal material solution; 9: negative control solution lacking rose; 10: celery seed control medicinal material solution; 11: negative control solution lacking celery seed). DETAILED DESCRIPTION

[0071] After extensive and in-depth research, the inventors have discovered for the first time a fast "one test, multiple evaluations" multi-information thin-layer identification method for Hanchuanzupa granules, which can simultaneously identify licorice extract, fenugreek, rose and celery seeds in Hanchuanzupa granules in one thin-layer identification process. By using a specific thin-layer identification method with a specific developer, a color developer, etc., fast and accurate identification can be performed, while improving the identification efficiency and avoiding the possibility of adulteration with known ingredients to the greatest extent. The present invention was completed on this basis.

[0072] the term

[0073] Unless defined otherwise, all 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.

[0074] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0075] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0076] Thin layer chromatography identification method under current quality standards

[0077] The specific method is as follows:

[0078] 1) Take 3g of this product, grind it, add 30ml of ethanol, heat and reflux for 1 hour, filter, evaporate the filtrate, add 30ml of water to dissolve the residue, shake and extract with 20ml of ethyl acetate, separate the ethyl acetate solution, recover the solvent until dry, add 1ml of methanol to dissolve the residue, and use it as the test solution. Take 1g of rose flower reference medicinal material, add 20ml of water, reflux and extract for 30 minutes, filter, and prepare the reference medicinal material solution in the same way as "shake and extract with 20ml of ethyl acetate". Take the gallic acid reference substance, add methanol to prepare a solution containing 1mg per 1ml, and use it as the reference substance solution. Take 5μl of each of the above three solutions and spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-acetone-formic acid (8:3:4:1) as the developing solvent, develop, take out, dry, spray with 1% ferric chloride ethanol solution, and heat at 105℃ until the spots are clearly colored. In the test sample chromatogram, spots of the same color appear at corresponding positions in the control medicinal material chromatogram and the control sample chromatogram.

[0079] 2) Take 3g of this product, grind it, add 40ml of ether, heat and reflux for 1 hour, filter, discard the ether solution, add 30ml of methanol to the residue, heat and reflux for 1 hour, filter, recover the solvent from the filtrate until dry, add 40ml of water to the residue to dissolve it, put it in a separatory funnel, shake and extract it 3 times with water-saturated n-butanol, 20ml each time, combine the n-butanol solution, wash it 3 times with water, 20ml each time, discard the water solution, recover the solvent from the n-butanol solution until dry, add 1ml of ethanol to the residue to dissolve it as the test solution. Take another 1g of licorice control medicinal material and prepare the control medicinal material solution in the same way. Take the liquiritin reference substance again, add ethanol to make a solution containing 1mg per 1ml as the reference substance solution. Take 10 μl of each of the three solutions and spot them on the same silica gel G thin layer plate, use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample.

[0080] 3) Take 5g of this product, grind it, add 30ml of methanol, ultrasonically treat it for 30 minutes, filter it, wash the residue with 20ml of methanol, combine the methanol solution, recover the solvent until dry, add 20ml of 3mol / L hydrochloric acid solution (10ml, 5ml and 5ml in sequence) to the residue to dissolve it in batches, heat it in a water bath for hydrolysis for 30 minutes, cool it, add 30ml of chloroform, heat it under reflux for 15 minutes, take the chloroform solution, add 30ml of chloroform to the water solution and treat it once in the same way, combine the chloroform solution, recover the solvent until dry, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take another diosgenin reference substance, add methanol to make a solution containing 0.5mg per 1ml, and use it as the reference substance solution. Pipette 10-15μl of the test solution and 3μl of the reference solution, spot them on the same silica gel G thin layer plate, use chloroform-methanol (20:1) as the developing agent, develop, take out, dry, spray with 5% phosphomolybdic acid ethanol test solution, and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference drug.

[0081] It can be seen that the identification method of the current quality standard requires separate identification of single medicinal materials. Different test solutions, reference solutions, different developing agents, and different color developers are required for different medicinal materials. Therefore, the steps are complicated and the operation is very inconvenient.

[0082] Method of the present invention

[0083] The “one test, multiple evaluations” method was first proposed by Wang Zhimin et al. of the China Academy of Chinese Medical Sciences in 2006 (Wang Zhimin, Gao Huimin, Fu Xuetao, et al. Methodological Study on the “One Test, Multiple Evaluations” Method for Quality Evaluation of Traditional Chinese Medicine [J]. Chinese Journal of Traditional Chinese Medicine, 2006(23):1925-1928.). This method uses a relatively easy-to-obtain and inexpensive internal reference substance to achieve simultaneous determination of the contents of multiple components. It has the characteristics of simple operation, accurate and comprehensive determination, etc. At present, the “one test, multiple evaluations” method is mostly used for quantitative determination by high performance liquid chromatography and gas chromatography, or for qualitative identification by fingerprint spectrum, and there is no literature report on its application in thin layer chromatography identification. (Gao Yuan, Wang Haili, He Lidan, et al. Study on quality control of Coptis chinensis and its preparations based on one-test-multiple-evaluation method[J]. Chinese Medicinal Materials, 2023(12):3049-3054.; Liu Min, Zhao Wen, Wan Linchun. Application of one-test-multiple-evaluation method in the detection of four components of clove[J]. Drug Evaluation, 2022, 19(20):1238-1242.; Xu Qian, Lu Meixia, Zhou Yaping, et al. HPLC fingerprint of osteomyelitis granules and one-test-multiple-evaluation method for determination of seven components[J]. Central South Pharmacy, 2023, 21(11):3030-3035.)

[0084] The present invention draws on the concept of the "one test, multiple evaluations" method and applies it to qualitative identification of thin layer chromatography, that is, using one or two easily available, low-priced reference substances or alternative reference substances, accurately locate and identify multiple components in the thin layer chromatogram of medicinal materials / traditional Chinese medicine preparations based on their relative development positions in the thin layer chromatography, and realize multi-component quality control while locating unknown components with known reference substances, which is equivalent to establishing a characteristic thin layer spectrum of medicinal materials / traditional Chinese medicine preparations. Because under the same chromatographic conditions, the positions of thin layer spots of different substances are relatively fixed, and most of the spots are unknown components, adulteration of known components in traditional Chinese medicine preparations can be avoided, and quality control of medicinal materials / traditional Chinese medicine preparations can be achieved.

[0085] The specific technical solution of the method of the present invention comprises the following steps:

[0086]

Stationary phase

[0087] [Preparation of microemulsion developing agent]: Take 1.4g of surfactant sodium dodecyl sulfate, 3.15g of n-butanol, 0.5g of n-heptane and 45g of water respectively, put them in a conical flask, mix well and leave for 24 hours to obtain the microemulsion developing agent.

[0088] [Preparation of natural product polyethylene glycol color developer]: Take diphenyl boric acid-2-aminoethyl ester (2-APB) and add methanol to make a solution containing 10 mg per 1 ml; take polyethylene glycol 400 and add methanol to make a solution containing 50 mg per 1 ml.

[0089] [Preparation of test solution]: Take 6g of Hanchuanzupa granules, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml.

[0090] [Preparation of reference medicinal material solution]: Take 3g each of licorice extract, rose, fenugreek and celery seed reference medicinal materials, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml.

[0091] [Preparation of negative control solution]: Take 3g each of the negative control samples of licorice extract, rose, fenugreek, and celery seeds, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml.

[0092] [Identification]: Pipette 1 μl of the above-mentioned control medicinal material solution, 2 μl of the test solution and the negative control solution respectively, spot them on the same polyamide film, use microemulsion (90% water content)-formic acid as the developing agent, wherein the volume ratio of microemulsion and formic acid is 9:1. Develop, take out, dry, first spray with 2-APB solution, then spray with polyethylene glycol 400 solution, heat at 105℃ for 2 minutes, and examine under ultraviolet lamp (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding position of the chromatogram of the control, and there is no spot interference at the same position of the chromatogram of the negative control solution.

[0093] The main advantages of the present invention include:

[0094] 1. For the first time, a fast "one test, multiple evaluations" multi-information thin-layer identification method for Hanchuanzupa granules was created. The test sample, control medicinal material and negative control solution were obtained by a simple and fast pretreatment method, and were spotted on the same polyamide film respectively. After unfolding, they were inspected under ultraviolet light (365nm). The 6 spot information in Hanchuanzupa granules were inspected. Different spots represent different chemical components. The spots are clear and the relative positions are fixed, which is easy to evaluate the quality. And only licorice extract can obtain 3 characteristic spots. The key is that most of these spots are unknown components, which can improve the identification efficiency while avoiding the possibility of adulteration with known components to the greatest extent. At present, no reports on multi-information thin-layer identification of Hanchuanzupa granules have been found.

[0095] 2. Using 90% water content microemulsion-formic acid (9:1) as the developing agent and 2-APB / PEG as the coloring agent, after development and coloring, 6 spots were obtained under ultraviolet light (365nm), each with clear color and moderate Rf value. Among them, 3 spots are components in licorice extract, 1 spot is a component in fenugreek, 1 spot is a component in rose, 1 spot is a component in celery seeds, and the remaining spots are common components in medicinal materials; it can not only achieve simultaneous qualitative identification of multiple components in multiple medicinal materials, but also avoid the possibility of adulteration by adding known components.

[0096] 3. This method is simple and quick to operate. It only requires 6g of Hanchuanzupa granules, 3g of each control medicinal material, 80ml of methanol, and ultrasonic extraction for 20 minutes to obtain the medicinal material and preparation test solution. In the experiment, only one polyamide thin layer plate, 10ml of developing agent, and 60 minutes of development are required. It takes a total of 80 minutes to identify the four medicinal materials in Hanchuanzupa granules. Compared with the conventional thin layer identification method, the low cost, high efficiency, and intuitiveness of the color of each spot are not available in ordinary thin layer identification. At the same time, it has achieved the simplification of complex problems and the miniaturization of high costs, and has a high practical application value.

[0097] 4. This method uses low-toxic, environmentally friendly and low-cost microemulsion as a developing agent, and 2-APB / PEG as a color developer. Four medicinal materials were used on a thin layer plate to identify Hanchuanzupa granules containing 9 medicinal materials / extracts and conduct quality control; using microemulsion as a developing agent can greatly improve the separation effect between different components and provide more useful information; at the same time, components with different chemical structures can be separated; using 2-APB / PEG as a color developer can show richer colors of spots, making it easy to distinguish different components. 2-APB / PEG was rarely used in China before, and its principle is that 2-APB interacts with different reaction sites of flavonoids; at the same time, PEG400 reduces the detection limit and enhances fluorescence, making it easier to identify and distinguish different components.

[0098] 5. Compared with the methods in the current standards and literature, the most important innovation of the method of this application is that the preparation time of the test solution is short, the amount of solvent used is small, and it is no longer based on one medicinal material as the identification indicator, but four medicinal materials are used as indicators at the same time, which greatly improves the amount of detection information and efficiency. The method is also simpler, faster, greener and more environmentally friendly than the current standards and literature, saving costs while improving detection efficiency.

[0099] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0100] According to the preparation method of the Hanchuan Zupa Granules prescription, the individual medicinal materials contained in the prescription are subjected to the same operation, that is, boiled in water and then concentrated and dried to obtain the extracts of the corresponding medicinal materials.

[0101] Example 1

[0102] Take 6g of Hanchuanzupa granules, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml as the test solution;

[0103] Take 3g of licorice extract and operate in the same way as the control medicinal material solution;

[0104] Take 3 g of negative control sample of licorice extract and operate in the same way as the negative control solution.

[0105] 1 μl of the control drug solution, 2 μl of the test solution and the negative control solution were taken respectively, and spotted on the same polyamide film, and 90% water content microemulsion-formic acid (9:1) was used as the developing agent. After development, the film was taken out, dried, and first sprayed with 2-APB, then with polyethylene glycol 400, heated at 105°C for 2 minutes, and inspected under ultraviolet light (365nm).

[0106] The results are as follows Figure 1 As shown, it can be seen that the fluorescent spots in the red frame show that the corresponding positions of the test sample chromatogram and the control medicinal material chromatogram show spots of the same color, while the corresponding positions of the negative control solution do not have spots of the same color, indicating that the negative has no interference, indicating that the licorice extract is effectively identified.

[0107] Example 2

[0108] Take 6g of Hanchuanzupa granules, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml as the test solution;

[0109] Take 3g of fenugreek as the control medicinal material and operate in the same way as the control medicinal material solution;

[0110] Take 3 g of boiled fenugreek as a control medicinal material and perform the same procedure as above to prepare the boiled control medicinal material solution.

[0111] Take 3 g of the negative control sample lacking fenugreek and perform the same operation as the negative control solution.

[0112] 1 μl of the control medicinal material solution, 2 μl of the test solution and the negative control solution were taken separately, and spotted on the same polyamide film. 90% water content microemulsion-formic acid (9:1) was used as the developing agent. After development, the film was taken out and dried. First, 2-APB was sprayed, and then polyethylene glycol 400 was sprayed. The film was heated at 105°C for 2 minutes and examined under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding position of the chromatogram of the control sample, and there was no interference with the negative sample ( Figure 2 ).

[0113] Example 3

[0114] Take 6g of Hanchuanzupa granules, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml as the test solution;

[0115] Take 3g of rose flower as the reference medicinal material and operate in the same way as the reference medicinal material solution;

[0116] Take 3g of boiled rose flower as the reference medicinal material and perform the same operation as the boiled reference medicinal material solution;

[0117] Take 3 g of the negative control sample lacking rose and perform the same operation as the negative control solution.

[0118] 1 μl of the control medicinal solution, 2 μl of the test solution and the negative control solution were respectively taken and spotted on the same polyamide film. 90% water content microemulsion-formic acid (9:1) was used as the developing agent. After development, the film was taken out and dried. First, 2-APB was sprayed, then polyethylene glycol 400 was sprayed, and the film was heated at 105°C for 2 minutes. The film was then inspected under a UV lamp (365nm).

[0119] The results are as follows Figure 3 As shown, it can be seen that the fluorescent spots in the red frame show that the corresponding positions of the test sample chromatogram, the control medicinal material chromatogram, and the control medicinal material chromatogram after boiling in water all show spots of the same color, while the corresponding positions of the negative control chromatogram do not have spots of the same color, indicating that the negative has no interference, indicating that the rose is effectively identified.

[0120] Example 4

[0121] Take 6g of Hanchuanzupa granules, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml as the test solution;

[0122] Take 3g of celery seed as the control medicinal material and operate in the same way as the control medicinal material solution;

[0123] Take 3g of boiled celery seeds as a reference medicinal material and perform the same operation as the boiled reference medicinal material solution;

[0124] Take 3 g of the negative control sample lacking celery seeds and perform the same operation as the negative control solution.

[0125] 1 μl of the control medicinal solution, 2 μl of the test solution and the negative control solution were respectively taken and spotted on the same polyamide film. 90% water content microemulsion-formic acid (9:1) was used as the developing agent. After development, the film was taken out and dried. First, 2-APB was sprayed, then polyethylene glycol 400 was sprayed, and the film was heated at 105°C for 2 minutes. The film was then inspected under a UV lamp (365nm).

[0126] The results are as follows Figure 4 As shown, it can be seen that the fluorescent spots in the red frame show that the corresponding positions of the test sample chromatogram, the control medicinal material chromatogram, and the control medicinal material chromatogram after boiling in water all show spots of the same color, while the corresponding positions of the negative control chromatogram do not have spots of the same color, indicating that the negative has no interference, indicating that celery seeds are effectively identified.

[0127] Example 5

[0128] Take 6g of Hanchuanzupa granules, grind them into powder, add 20ml of methanol, extract by ultrasonic for 20 minutes, filter, and concentrate the filtrate to 1ml as the test solution;

[0129] Take 3g of licorice extract, rose, fenugreek, and celery seed respectively, and operate in the same way as the control medicinal solution;

[0130] Take 3g each of negative control samples lacking licorice extract, rose, fenugreek, and celery seeds, and operate in the same way as negative control solutions.

[0131] 1 μl of the above-mentioned control medicinal material solution, 2 μl of the test solution and the negative control solution were respectively taken and spotted on the same polyamide film. 90% water content microemulsion-formic acid (9:1) was used as the developing agent. After development, the film was taken out and dried. First, 2-APB was sprayed, then polyethylene glycol 400 was sprayed, and the film was heated at 105°C for 2 minutes. The film was inspected under a UV lamp (365nm).

[0132] In the chromatogram of the test sample, the same color fluorescent spots appear at the corresponding positions in the chromatogram of the respective control medicinal materials, and there is no interference with the negative control ( Figure 5 ).

[0133] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A thin layer chromatography identification method for Hanchuanzupa granules, wherein the Hanchuanzupa granules contain licorice extract, fenugreek, rose, and celery seeds; The method comprises the steps of: s1. Providing a test solution, a control medicinal material solution, a negative control solution, and an optional boiled control medicinal material solution; in, The control medicinal material solution includes multiple types of liquorice extract solution, fenugreek solution, rose solution and celery seed solution; The boiled control medicinal material solution includes multiple boiled liquorice extract solution, boiled fenugreek solution, boiled rose solution and boiled celery seed solution; The negative control solution includes a plurality of negative control solutions lacking liquorice extract, a negative control solution lacking fenugreek, a negative control solution lacking rose, and a negative control solution lacking celery seeds; The control medicinal material solution and the control medicinal materials in the boiled control medicinal material solution correspond one to one; the control medicinal materials in the control medicinal material solution and the medicinal materials missing from the negative control solution correspond one to one; Wherein, the preparation method of the test solution comprises the following steps: (a) grinding the raw material, mixing with a lower alcohol, ultrasonically extracting, filtering, and concentrating the filtrate to obtain the test solution; the raw material is Hanchuanzupa granules; The preparation methods of the control medicinal material solution, negative control solution, and boiled control medicinal material solution are independently the same as the above step (a), except that the raw materials are replaced with the corresponding control medicinal materials, negative control substances, or boiled control medicinal materials, and the amount of the raw materials is optionally adjusted; s2. The test solution, control medicinal material solution, negative control solution, and any boiled control medicinal material solution are respectively applied to a polyamide film, developed under the action of a developing agent, and after drying, a color developer is applied to the film for color development, and then the film is placed under ultraviolet light for inspection; Wherein, the developing agent is a mixed solution of microemulsion and formic acid, The preparation method of the microemulsion comprises: (b) mixing sodium dodecyl sulfate, C3-C6 alcohol, C5-C10 alkane and water and allowing to stand to obtain the microemulsion; The method for applying the developer comprises: (c) first spraying a solution of 2-aminoethyl diphenylborate on a polyamide film, and then spraying a polyethylene glycol 400 solution on the polyamide film.

2. The method according to claim 1, characterized in that The step (a) has one or more characteristics selected from the following group: (1) The lower alcohol is selected from the group consisting of methanol, ethanol, n-propanol, n-butanol, or a combination thereof, preferably methanol; (2) The mass volume ratio of the Hanchuanzupa granules to the lower alcohol is 0.1-1 g / mL, preferably 0.2-0.5 g / mL; (3) The ultrasonic extraction time is 10-60 min, for example 20 min; (4) The mass volume ratio of the concentrated solution volume after the filtrate is concentrated is 1-10 g / mL, preferably 3-8 g / mL.

3. The method according to claim 1, characterized in that The preparation method of the test solution comprises the following steps: (a) Grind 1-10 g of the raw material, mix with 5-50 mL of methanol, perform ultrasonic extraction for 10-60 min, filter, and concentrate the filtrate to 0.05-2 mL to obtain the test solution; the raw material is Hanchuan Zupa granules; Preferably, (a) 5-10 g of the raw material is ground, mixed with 10-30 mL of methanol, ultrasonically extracted for 10-30 min, then filtered, and the filtrate is concentrated to 0.5-2 mL to obtain the test solution; the raw material is Hanchuan Zupa granules; More preferably, (a) 6 g of the raw material is ground, mixed with 20 mL of methanol, ultrasonically extracted for 20 min, then filtered, and the filtrate is concentrated to 1 mL to obtain the test solution; the raw material is Hanchuanzupa granules.

4. The method according to claim 1, characterized in that The preparation methods of the control medicinal material solution, the negative control solution, and the boiled control medicinal material solution each independently have one or more characteristics selected from the following group: (1) In the preparation method of the control medicinal material solution, the negative control solution, and the control medicinal material solution after boiling, the mass volume ratio of the raw material to the lower alcohol is independently 0.05-1 g / mL, preferably 0.08-0.3 g / mL; (2) In the preparation methods of the control medicinal material solution, negative control solution, and boiled control medicinal material solution, the mass volume ratio of the raw material phase to the volume of the concentrated solution after the filtrate is concentrated is 1-10 g / mL, preferably 1-5 g / mL.

5. The method according to claim 1, characterized in that The preparation methods of the control medicinal material solution, the negative control solution, and the boiled control medicinal material solution each independently comprise the following steps: 1-8 g of the raw material is ground, mixed with 5-50 mL of methanol, and ultrasonically extracted for 10-60 min, then filtered, and the filtrate is concentrated to 0.05-2 mL to obtain the solution; the raw material is Hanchuan Zupa granules; Preferably, (a) 2-5 g of the raw material is ground, mixed with 10-30 mL of methanol, subjected to ultrasonic extraction for 10-30 min, filtered, and the filtrate is concentrated to 0.5-2 mL to obtain the solution; the raw material is Hanchuan Zupa granules; More preferably, (a) 3 g of the raw material is ground, mixed with 20 mL of methanol, and subjected to ultrasonic extraction for 20 min, followed by filtration, and the filtrate is concentrated to 1 mL to obtain the solution; and the raw material is Hanchuanzupa granules.

6. The method according to claim 1, characterized in that The step s2 has one or more features selected from the following group: (1) 1-5 μL (e.g., 2 μL) of each of the test solution, control medicinal material solution, negative control solution, and optional boiled control medicinal material solution are independently taken and spotted on the same polyamide film; (2) In the step s2, the color development includes heating the film after applying the developer, the heating temperature is 90-120° C., and the heating time is 1-5 min; (3) In step s2, the wavelength of the ultraviolet light is 350-380nm.

7. The method according to claim 1, characterized in that The developing agent has one or more characteristics selected from the following group: (1) The developing agent is a mixed solution of microemulsion and formic acid in a volume ratio of 5-10:1, preferably 7-9:1; (2) In the developing agent, the water content of the microemulsion is 70-99.9 vol%, preferably 80-95 vol%, for example 90 vol%; (3) In step (b), the C3-C6 alcohol is selected from the group consisting of n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, n-hexanol, or a combination thereof, preferably n-butanol; (4) In step (b), the C5-C10 alkane is selected from the group consisting of n-pentane, n-hexane, n-heptane, n-octane, n-decane, or a combination thereof, preferably n-heptane; (5) In step (b), the standing time is 18-48 hours, for example 24 hours, 36 hours.

8. The method according to claim 1, characterized in that The step (c) has one or more characteristics selected from the group consisting of: (1) In the step (c), the solution of 2-aminoethyl diphenylborate is a methanol solution of 2-aminoethyl diphenylborate; (2) In the step (c), the volume fraction of 2-aminoethyl diphenylborate is 5-30 mg / mL, preferably 10-20 mg / mL; (3) In the step (c), the solution of polyethylene glycol 400 is a methanol solution of polyethylene glycol 400; (4) In step (c), the volume fraction of polyethylene glycol 400 is 20-80 mg / mL, preferably 30-70 mg / mL.

9. The method according to claim 1, characterized in that The step (b) comprises: mixing 0.5-5 g of sodium dodecyl sulfate, 1-10 g of n-butanol, 0.1-1 g of n-heptane and 10-60 g of water, and standing for 18-48 hours to obtain the microemulsion; Preferably, the step (b) comprises: mixing 0.5-2 g of sodium dodecyl sulfate, 2-5 g of n-butanol, 0.1-1 g of n-heptane, and 20-50 g of water, and standing for 18-36 hours to obtain the microemulsion; More preferably, the step (b) comprises: mixing 1.4 g of sodium dodecyl sulfate, 3.15 g of n-butanol, 0.5 g of n-heptane and 45 g of water, and standing for 24 hours to obtain the microemulsion; The step (c) comprises: first spraying a methanol solution of 2-APB with a volume fraction of 5-30 mg / mL, and then spraying a methanol solution of polyethylene glycol 400 with a volume fraction of 20-80 mg / mL; Preferably, the step (c) comprises: first spraying a methanol solution of 2-APB with a volume fraction of 10-20 mg / mL, and then spraying a methanol solution of polyethylene glycol 400 with a volume fraction of 30-70 mg / mL; More preferably, the step (c) comprises: first spraying a methanol solution of 2-APB with a volume fraction of 10 mg / mL, and then spraying a methanol solution of polyethylene glycol 400 with a volume fraction of 50 mg / mL.

10. The method according to claim 1, characterized in that The step s2 comprises: aspirating 1-5 μL of the above-mentioned test solution, control medicinal material solution, negative control solution, and optional boiled control medicinal material solution respectively and spotting them on the same polyamide film, using a mixed solution of microemulsion with a water content of 80-95 vol% and formic acid (volume ratio of 5-10:1) as a developing agent, developing under the action of the developing agent, taking out, drying, spraying with a methanol solution of 2-APB with a volume fraction of 5-30 mg / mL, and then spraying with a methanol solution of polyethylene glycol 400 with a volume fraction of 20-80 mg / mL, heating at 90-120°C for 1-5 minutes, and then placing it under ultraviolet light with a wavelength of 350-380 nm for inspection.

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